Encyclopedia of Microbiology, Third Edition [3ed.] 012373939X, 9780123739391, 9780123739445, 0123739446, 9781780344690, 1780344694

Table of contents :
Content: Applied microbiology : Agro/food --
Applied microbiology : industrial --
Archaea --
Bacteria --
Cell structure chemical composition --
Environmental microbiology and ecology --
Evolution and systematics --
Fungi --
Genetics, Genomics --
History and culture --
History and culture, (and biographies) --
Mutualism and commensalism --
Pathogenesis --
Physiology --
Protists --
Public issues --
Techniques --
Viruses.

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PREFACE

Nearly a decade separates this edition from its predecessor. During this time, progress in Microbiology has been staggering in both quantity and importance. Whereas this is a cause for satisfaction to both practitioners and the consumers of this science, it does accentuate the ancient problem of what to include in a work such as this one. The solution that has presented itself is not new: decide on what can be said to be the most important and central subjects to this science and ask authorities to write about the field of their expertise. The topics chosen for this edition are not novel but their treatment mirrors the changes in Microbiology. Several themes are becoming more dominant in this science. Among them are the following grand realizations:

• • •

A large proportion of the biosphere is microbial and has a profound influence on nearly all aspects of this planet’s metabolism, including its geology, cycles of matter, and even its meteorology. Microbes have been the sole, ergo, the dominant force in the first 80% or so of life on Earth and have played a key role in the evolution of all life forms. Practically all human activities have a microbial component, including health and food production.

Whereas these developments involve more and more fields of specialization, microbiology has become more unified. Common molecular mechanisms of adhesion to surfaces, quorum sensing, signal transduction, constructing communities, or injecting proteins directly into host cells are found in dissimilar organisms that represent a broad spectrum of microbial life. Microbiologists now tend to speak a common language. Nowadays, a marine microbiologist can easily talk to one studying human pathogens and a food microbiologist can converse effortlessly with one studying evolution. Those studying the microbial world share not just a genomic database but, more profoundly, the realization that microorganisms make use of similar capabilities for diverse purposes. The most significant outward change in this edition has been its conversion to the Web. This has advantages over the printed versions, including the ability to search for topics and subtopics, linking to the references, and avoiding the backbreaking task of carrying it around. The volumes of the previous edition weighed in toto about 15 Kg, these would have topped 20 Kg! We are grateful to many colleagues who made suggestions and provided help to us. Special thanks go to Mark Jensen, who made highly skillful contributions to our editing process. Moselio Schaechter

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EDITOR-IN-CHIEF

Moselio Schaechter San Diego State University San Diego, CA, USA

EDITOR-IN-CHIEF

Professor Schaechter did his graduate work at the University of Kansas and the University of Pennsylvania. He worked on the biology of rickettsiae at Walter Reed Army Institute of Research, and was a postdoctoral fellow for two years in Copenhagen, in the laboratory of Ole Maaløe. Professor Schaechter’s research interest concerned various aspects of the regulation of bacterial growth. He discovered the existence of polyribosomes in bacteria and was among the first to elucidate aspects of polyribosome metabolism and the role of the cell membrane in DNA synthesis and chromosome segregation. Professor Schaechter spent most of his career at Tufts University in Boston, MA, where he chaired the department of Molecular Biology and Microbiology for 23 years. Since 1995 he has resided in San Diego, California, where he teaches and continues to write books and a blog, ‘‘Small Things Considered.’’ He has written nine books, including several textbooks and reference works, and he has served as president of the American Society of Microbiology and in many advisory capacities to agencies and organizations.

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SECTION EDITORS

Sandra L. Baldauf Uppsala University Uppsala, Sweden

Allen I. Laskin Laskin/Lawrence Associates Somerset, NJ, USA

John A. Baross University of Washington Seattle, WA, USA

Bruce R. Levin Emory University Atlanta, GA, USA

David C. Baulcombe University of Cambridge Cambridge, UK

Thomas M. Schmidt Michigan State University East Lansing, MI, USA

Robert Haselkorn University of Chicago Chicago, IL, USA

William C. Summers Yale University School of Medicine New Haven, CT, USA

David A. Hopwood John Innes Centre Norwich, UK

James F. White, Jr. Rutgers University New Brunswick, NJ, USA

John L. Ingraham Professor Emeritus University of California Davis, CA, USA

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APPLIED MICROBIOLOGY: AGRO/FOOD Contents Agrobacterium and Plant Cell Transformation Aquaculture Beer/Brewing Dairy Products Fermented Foods Food Spoilage, Preservation and Quality Control Forest Products: Biotechnology in Pulp and Paper Processing Insecticides, Microbial Pesticides, Microbial Water, Drinking Wine

Agrobacterium and Plant Cell Transformation P J Christie, University of Texas Medical School at Houston, Houston, TX, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Overview of Infection Process Ti Plasmid Chromosomally Encoded Virulence Genes T-DNA Processing

Glossary autoinducer An acylhomoserine lactone secreted from bacteria, which, under conditions of high cell density, passively diffuses across the bacterial envelope and activates transcription. border sequences 25-bp direct, imperfect repeats that delineate the boundaries of T-DNA. conjugation Transfer of DNA between bacteria by a process requiring cell-to-cell contact. conjugative pilus An extracellular filament encoded by a conjugative plasmid involved in establishing contact

VirB/D4 System, a Member of the Type IV Secretion Family Substrate Transfer Through the Plant Cell Agrobacterium Host Range and Genetic Engineering Conclusions Further Reading

between plasmid-carrying donor cells and recipient cells. mobilizable plasmid Conjugal plasmid that carries an origin of transfer (oriT) but lacks genes coding for its own transfer across the bacterial envelope. T-DNA Segment of the Agrobacterium genome transferred to plant cells. transconjugant A cell that has received a plasmid from another cell as a result of conjugation. transfer intermediate A nucleoprotein particle composed of a single-strand of the DNA destined for

1

2 Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

export and one or more proteins that facilitate DNA delivery to recipient cells. type IV secretion system A conserved family of macromolecular translocation systems evolutionarily

related to conjugation systems for translocating DNA or protein effector molecules between prokaryotic cells or to eukaryotic hosts.

Abbreviations

NLS OD OM SR T-DNA Ti TMS TrIP T4S VBTs

AAI ABC AHL CP Dtr GFP GGI IM Mpf

autoinducer ATP-binding cassette acylhomoserine lactone coupling protein DNA transfer and replication green fluorescent protein gonococcal genetic island inner-membrane mating pair formation

Defining Statement Agrobacterium tumefaciens transfers oncogenic DNA (TDNA) to susceptible plant cells, causing formation of tumors called Crown galls. This is a multistage infection process involving sensory recognition of specific plant signals, attachment to the plant host, induction of a virulence regulon, and T-DNA processing, transfer, and integration into the plant genome.

Introduction Agrobacterium tumefaciens is a Gram-negative soil bacterium with the ability to infect plants through a process that involves delivery of a specific segment of its genome to the nuclei of susceptible plant cells. The transferred DNA (T-DNA) is a discrete region of the bacterial genome delimited by 23 base pair (bp) direct repeats carried by the tumor-inducing (Ti) plasmid. The T-DNA is important for infection because it codes for genes that, when expressed in the plant cell, disrupt plant cell growth and division events. However, this oncogenic DNA can be excised from the transferred DNA and replaced by virtually any gene of interest for A. tumefaciens-mediated engineering of a wide array of plant species. The discovery that A. tumefaciens is a natural and efficient DNA delivery vector spawned an entire new industry of plant genetic engineering, which today has many diverse goals ranging from crop improvement to the use of plants as ‘pharmaceutical factories’ for high-level production of biomedically important proteins. Because of the dual importance of Agrobacterium as a plant pathogen and as a DNA delivery system, an extensive literature has

nuclear localization sequences overdrive outer membrane substrate receptor transferred DNA tumor-inducing transmembrane segments transfer DNA immunoprecipitation type IV secretion VirB2-interacting proteins

emerged describing numerous aspects of the infection process and the myriad of ways this organism has been exploited for plant genetic engineering. Here, I will summarize recent findings pertaining to the mechanistic details of vir gene induction, T-DNA processing and transfer, and T-DNA movement and integration in the plant host.

Overview of Infection Process Agrobacterium species are commonly found in a variety of environments including cultivated and nonagricultural soils, plant roots, and even plant vascular systems. Despite the ubiquity of Agrobacterium species in soil and plant environments, only a small percentage of isolates are pathogenic. Two species are known to infect plants by delivering DNA to susceptible plant cells. A. tumefaciens is the causative agent of crown gall disease, a neoplastic disease characterized by uncontrolled cell proliferation and formation of unorganized tumors. Agrobacterium rhizogenes induces formation of hypertrophies with a hairy-root appearance referred to as ‘hairy-root’ disease. The pathogenic strains of both the species possess large plasmids Ti and Ri, respectively, that encode most of the genetic information required for DNA transfer to susceptible plant cells. The basic infection process is similar for both the species, although the gene composition of the transferred DNA differs, and, therefore, the outcome of the infection. Agrobacterium has been widely viewed as the only bacterial genus capable of transferring genes to plants, but in fact other members of the alphaproteobacteria can transform plants when carrying an Agrobacterium Ti plasmid. The plant symbionts Rhizobium sp. NGR234,

Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation Agrobacterium tumefaciens

3

Plant cell Phenolics, Sugars, acidic pH

VirA VirG

Signal recognition Activation of vir genes

pTi

Vir regulon

Wound Nucleus

Cell-cell attachment

T-DNA VirD1 VirD2 5′ 3′

3′ 5′

VirE2, VirF, VirD5, VirE3

T-DNA processing

T-DNA integration VirB/D4 T4S System

VirD2 T-strand

Translocation-competent nucleoprotein

VirE2

Contact-dependent translocation

VirD2

VirF, VirD5, VirE3

Figure 1 Overview of Agrobacterium tumefaciens infection process. Upon activation of the VirA/VirG two-component signal transduction system by signals released from wounded plant cells, a single-strand transferred DNA (T-DNA) is processed from the Ti plasmid and delivered as a nucleoprotein complex (T-complex) to plant nuclei. Expression of T-DNA genes in the plant results in loss of cell growth control and tumor formation (see text for details).

Sinorhizobium meliloti and Mesorhizobium loti, were found to transfer T-DNA, albeit inefficiently, into the chromosomes of tobacco, Arabidopsis, and rice plants. This discovery highlights the importance of the Ti-plasmidencoded virulence (vir) genes and certain conserved chromosomal loci among these alphaproteobacteria for infection. Here, I will focus on Agrobacterium-mediated transformation as a model for understanding the requirements for interkingdom DNA transfer. Agrobacterium-mediated transformation can be depicted as a multistage process involving (1) sensory perception of plant signals and induction of virulence genes, (2) establishment of physical contact between A. tumefaciens and the plant host, (3) processing of T-DNA and protein effectors for translocation, (4) translocation across the bacterial envelope via a dedicated secretion channel, (5) movement of substrates through the plant cell cytoplasm to the nucleus, (6) integration of T-DNA into the plant genome, and (7) expression of T-DNA genes (see Figure 1). With the exception of attachment, early stages of infection are mediated by genes encoded by the Ti plasmid.

border sequences. The T-DNA harbors genes that are expressed exclusively in the plant cell. Transcription of T-DNA in the plant cell produces 39 polyadenylated RNA typical of eukaryotic RNA message that is translated in the cytoplasm. The second region of the Ti plasmid involved in infection harbors the genes responsible for sensory recognition of plant signals, T-DNA processing for transfer, and substrate transfer across the bacterial envelope. Two additional regions of the Ti plasmid code for functions that are not essential for the T-DNA transfer process per se, but are nevertheless intimately associated with the overall infection process. One of these regions harbors genes involved in catabolism of novel amino acid derivatives termed opines that A. tumefaciens induces plants to synthesize as a result of T-DNA transfer. The other encodes Ti plasmid transfer functions for distributing copies of the Ti plasmid and its associated virulence factors to other A. tumefaciens cells by a process termed as conjugation. Intriguingly, a novel regulatory cascade involving chemical signals released both from the transformed plant cells and the infecting bacterium serves to activate conjugative transfer of the Ti plasmid among A. tumefaciens cells residing in the vicinity of the plant tumor.

Ti Plasmid Ti plasmids range in size from 180 to as many as 800 kilobases (kb). Two regions of the Ti plasmid contribute to infection (Figure 2). The first is the T-DNA, typically a segment of 20–35 kb delimited by 25-bp directly repeated

T-DNA The T-DNA is delimited by DNA repeats termed as border sequences (Figure 2). Flanking one border is a

4 Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation Auxins Plant: cytokinins opines RBO

LB

T-DNA movement virE

D

T-DNA

T-DNA processing/transport virD T-DNA processing & recruitment virC vir activator virG

Tra

Ti plasmid processing

Vir region Translocation virB

Ti Opine catabolism

Sensor kinase virA

Opine uptake & degradation

Trb Rep Ti plasmid replication

Ti plasmid conjugation

Figure 2 Regions of the Ti plasmid that contribute to infection (vir region and T-DNA), cell survival in the tumor environment (opine catabolism), and conjugal transfer of the Ti plasmid to recipient agrobacteria (tra and trb). The various contributions of the vir gene products to T-DNA transfer are listed. T-DNA, delimited by 25-bp border sequences (blue boxes; RB, right border; LB, left border) codes for biosynthesis of auxins, cytokinins, and opines in the plant. OD, overdrive sequence (red box) that enhances VirD2-dependent processing at the T-DNA border sequences.

sequence termed as overdrive that functions to stimulate the T-DNA-processing reaction. All DNA between the border sequences can be excised and replaced with genes of interest, and A. tumefaciens will still efficiently transfer the engineered T-DNA to plant cells. This shows that the border sequences are the only cis elements required for T-DNA transfer to plant cells. Additionally, genes encoded on the T-DNA play no role in the movement of T-DNA to plant cells. The T-DNA genes instead code for synthesis of enzymes within transformed plant cells. Oncogenes synthesize enzymes involved in the synthesis of two plant growth regulators, auxins and cytokinins. Production of these plant hormones results in a stimulation in cell division and a loss of cell growth control leading to the formation of characteristic crown gall tumors. Other enzymes catalyze the synthesis of novel amino acid derivatives termed as opines. The pTiA6 plasmid, for example, carries two T-DNA’s that code for genes involved in synthesis of octopines – a reductive condensation product of pyruvate and arginine. Other Ti plasmids carry T-DNAs that code for nopalines, derived from -ketoglutarate and arginine, and still others code for different classes of opines. Plants cannot metabolize opines. However, the Ti plasmid carries opine catabolism genes that are responsible for the active transport of opines and their degradation, thus providing a source of carbon and nitrogen for the bacterium. The ‘opine concept’ was developed to rationalize the finding that A. tumefaciens evolved as a pathogen by acquiring the ability to transfer DNA to

plant cells. According to this concept, A. tumefaciens adapted a DNA conjugation system for interkingdom DNA transport to incite opine synthesis in its plant host. The cotransfer of oncogenes ensures that transformed plant cells proliferate, resulting in enhanced opine synthesis. The tumor, therefore, is a rich chemical environment favorable for growth and propagation of the infecting A. tumefaciens. Of further interest, a given A. tumefaciens strain generally catabolizes only those opines that it incites plant cells to synthesize. This ensures a selective advantage of the infecting bacterium over other A. tumefaciens strains that are present in the vicinity of the tumor.

Opine Catabolism The regions of Ti plasmids involved in opine catabolism code for three functions related to opine catabolism. The first is a regulatory function controlling expression of opine transport and catabolism genes. For the octopine catabolism region of plasmid pTiA6, the regulatory protein is OccR, a member of the family of LysR transcription factors. OccR positively regulates expression of the occ genes involved in octopine uptake and catabolism by inducing a bend in the DNA at the OccR-binding site. Octopine modulates OccR regulatory activity by altering both the affinity of OccR for its target site and the angle of the DNA bend. The regulatory protein for the nopaline catabolism region of plasmid pTiC58 is

Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

AccR. In contrast to OccR, AccR functions as a negative regulator of acc genes involved in nopaline catabolism. Several other genes transcribed from a single promoter specify functions for opine transport and catabolism. At the proximal end of the operon are transport genes mediating opine-specific binding and uptake. Typically, one or more of these genes encode proteins homologous to energy-coupling proteins found associated with the socalled ATP-binding cassette (ABC) superfamily of transporters. The ABC transporters are ubiquitous among bacterial and eukaryotic cells, and provide a wide variety of transport functions utilizing the energy of ATP hydrolysis to drive the transport reaction. At the distal end of the operon are genes whose products cleave opines to their parent compounds for use as carbon and nitrogen sources for the bacterium.

Ti Plasmid Conjugation The Ti plasmid transfer (tra and trb) functions direct the conjugative transfer of the Ti plasmid to bacterial recipient cells (Figure 2). The transfer genes of conjugative plasmids code for DNA-processing factors and a translocation system. The Ti plasmid transfer system is related in sequence and function to other plasmid transfer systems, as well as dedicated protein translocation systems. These systems are now classified as type IV secretion (T4S) systems (see below). A regulatory cascade activates Ti plasmid transfer under conditions of high cell density (Figure 3). This regulatory cascade initiates when A. tumefaciens imports

Agrobacterium

VirA VirG Vir gene induction T-DNA

opines released from plant cells. For the octopine pTiA6 plasmid, OccR acts in conjunction with octopine to activate transcription of the occ operon. Although the majority of the occ operon codes for octopine transport and catabolism functions, the distal end of the occR operon encodes a gene for a transcriptional activator termed TraR. TraR is related to LuxR, an activator shown nearly 20 years ago to regulate synthesis of an acylhomoserine lactone (AML) termed as autoinducer. Cells that synthesize autoinducer molecules secrete these molecules into the environment. At low cell densities, autoinducer is in low concentration, whereas at high cell densities this substance accumulates in the surrounding environment and passively diffuses back into the bacterial cell to activate transcription of a defined set of genes. In the case of A. tumefaciens, the autoinducer is an N-3-(oxo-octonoyl)-L-homoserine lactone termed Agrobacterium autoinducer (AAI). AAI acts in conjunction with TraR to activate transcription of the Ti plasmid tra genes as well as traI whose product mediates synthesis of AAI. Therefore, synthesis of TraR under conditions of high cell density creates a positive-feedback loop whereby a TraR–AAI complex induces transcription of TraI, which, in turn, results in enhanced synthesis of more AAI. This regulatory cascade, involving opinemediated expression of traR and TraR-AAI mediated expression of Ti plasmid transfer genes under conditions of high cell density, has the net effect of enhancing Ti plasmid transfer in the environment of the plant tumor. This complex regulatory system likely evolved to maximize the number of Ti-plasmid-carrying bacterial cells in the vicinity of the plant wound site.

Phenolics

Transformed plant cell

Sugars low pH

T-DNA transfer

Tra Ti plasmid transfer

Nucleus OccR + Opine

Ti

T-DNA Auxins cytokinins

Trb TraR + AAI

AAI

5

TraR + Tral

Opines Opine catabolism

Uncontrolled cell proliferation

Tumors Autoinducer (AAI)

Cell growth/division

Figure 3 A schematic of chemical signaling events between Agrobacterium and the transformed plant cell. Signals released from wounded plant cells initiate the infection process leading to tumor formation. Opines released from wounded plant cells activate opine catabolism functions for growth of infecting bacteria. Opines also activate synthesis of TraR for autoinducer (AAI) synthesis. TraR and AAI at a critical concentration activate the Ti plasmid conjugation functions (see text for details).

6 Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

AAI-mediated activation of Ti plasmid transfer is also negatively controlled. For example, TraR activity is antagonized by two proteins, TraM and TrlR. TraM interacts with the C-terminus of TraR, which inactivates TraR and disrupts TraR–DNA complexes. TrlR is a truncated form of TraR that suppresses TraR activity through formation of inactive heterodimers. In addition, an AAI signal turnover system is composed of attJ, a regulatory gene, and attM whose product is an N-acylhomoserine lactone-lactonase that hydrolyzes the lactone ring of AAI. Lactonase production suppresses AAI-dependent expression of conjugation genes as a means of fine-tuning plasmid transfer in response to changes in cell growth and density. vir Genes The Ti plasmid carries a 35-kb region harboring a number of operons involved in T-DNA transfer (Figure 1). Some operons have a single open reading frame, while others code for up to 11 open reading frames. The products of the vir region direct events within the bacterium that must precede export of a copy of the T-DNA to plant cells: (1) a VirA/VirG two-component regulatory system induces expression of the vir genes in response to perception of plant-derived signals, (2) VirC and VirD proteins process T-DNA into a nucleoprotein particle for delivery to plant nuclei, and (3) a T4S system composed of VirB proteins and VirD4 translocates the T-DNA transfer intermediate and effector proteins across the bacterial envelope (Figure 2). Infection is initiated when bacteria sense and respond to an array of signals, including specific classes of plant phenolic compounds, aldose monosaccharides, low PO4, and an acidic pH that are present at a plant wound site (Figure 1). The VirA/VirG signal transduction system together with ChvE, a periplasmic sugar-binding protein, mediates recognition of plant phenolics and sugars. VirA was one of the first described of what now is recognized as a very large family of sensor kinases identified in bacteria and more recently in eukaryotic cells. The members of this protein family are typically integral membrane proteins with an N-terminal extracytoplasmic domain. Upon sensory perception, the kinase autophosphorylates at a conserved histidine residue, then transferring the phosphate group to a conserved aspartate residue on the second component of this transduction pathway, the response regulator. The phosphorylated response regulator coordinately activates transcription of several operons whose products mediate a specific response to the inducing environmental signal. For the A. tumefaciens vir system, the response regulator is VirG, and phosphorylated VirG activates transcription of six essential vir operons as well as a number of other Ti plasmid and chromosomally encoded operons whose products are probably important for

infection of certain plant species or under certain environmental conditions. The VirA/VirG two-component system also activates expression of the repABC genes responsible for replication of the Ti plasmid. Plant signals thus enhance Ti plasmid copy number and, consequently, virulence potential upon perception of environmental conditions favorable for interkingdom DNA transfer. VirA senses most of the plant-derived signals listed above. The most important signal molecules are phenols that carry an o-methoxy group. The type of substitution at the para position distinguishes strong inducers such as acetosyringone from weaker inducers such as ferulic acid and acetovanillone. A variety of aldose monosaccharides, including glucose, galactose, arabinose, and the acidic sugars D-galacturonic acid and D-glucuronic acid, strongly enhance vir gene induction. The inducing phenolic compounds as well as the monosaccharides are secreted intermediates of biosynthetic pathways involved in cell wall repair. As such, the presence of these compounds is a general feature of most plant wounds and likely contributes to the extremely broad host range of A. tumefaciens. VirA functions as a homodimer, and a model that VirA interacts directly with inducing molecules that diffuse across the outer membrane (OM) into the periplasm is supported by genetic experiments though direct evidence for signal binding is lacking. Sugarmediated inducing activity occurs via an interaction between sugars and the periplasmic sugar-binding protein ChvE. In turn, ChvE sugar interacts with the periplasmic domain of VirA to induce a conformational change that increases the sensitivity of VirA to phenolic inducer molecules. A periplasmic domain of VirA is also implicated in recognition of acidic pH, though the physical mechanism of pH perception is unknown. On the basis of recent crystallographic analysis of CheY, a homologue of VirG, phosphorylation of this family of response regulators is thought to induce a conformational change. Phospho-VirG activates transcription of the vir genes by interacting with a cis- acting regulatory sequence (TNCAATTGAAAPy) called the vir box located upstream of each of the vir promoters. Interestingly, both nonphosphorylated and phosphorylated VirG bind to the vir box, indicating that a phosphorylation-dependent conformation is necessary for a productive interaction with components of the transcription machinery.

Chromosomally Encoded Virulence Genes Most studies of the A. tumefaciens infection process have focused on the roles of Ti plasmid genes in T-DNA transfer, but several essential and ancillary chromosomal genes also contribute to A. tumefaciens virulence. Although mutations in these genes are often pleiotropic, they generally function to regulate vir gene

Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

expression or mediate attachment to plant cells. This latter activity will be described in the section titled ‘Attachment to plant cells’.

Regulators of vir Gene Expression At least three groups of chromosomal genes activate or repress vir gene expression. As described above, the periplasmic sugar-binding protein ChvE complexed with any of a wide variety of monosaccharides induces conformational changes in VirA allowing it to interact with phenolic inducers. chvE mutants are severely compromised for T-DNA transfer, but they also show defects in chemotaxis toward sugars. ChvE thus appears to play a dual role in the infection process, by promoting bacterial chemotaxis toward nutrients and by enhancing the efficiency of opine-encoding T-DNA to plant cells. A second locus codes for Ros, a novel prokaryotic zinc finger protein that transcriptionally represses certain vir operons. As described below, the VirC and VirD operons contribute to the T-DNA processing reaction. Although the promoters for these operons are subject to positive regulation by the VirA/VirG transduction system, they are also negatively regulated by the Ros repressor. Ros binds to a 9-bp inverted repeat, the ros box residing upstream of these promoters. In the presence of plant signals, Ros repression is counteracted by the VirA/VirG induction system, but in the absence of plant signals, Ros binding to the virC and virD promoters prevents the T-DNA-processing reaction. In addition to repressing expression of T-DNA processing genes in the absence of a suitable plant host, Ros prevents premature expression of the T-DNA oncogenes in the bacterium. Finally, a two-component regulatory system, distinct from the VirA/VirG system, senses environmental signals and mounts a behavioral response by modulating gene expression. ChvG is the sensor kinase and ChvI is the response regulator. Null mutations in genes for these proteins block vir gene induction or growth of cells at an acidic pH of 5.5. The molecular basis underlying the effect of the ChvG and ChvI proteins on vir gene expression is presently unknown.

T-DNA Processing One of the early events following attachment to plant cells and activation of vir gene expression in response to plant signals involves the processing of T-DNA into a form that is competent for transfer across the bacterial cell envelope and translocation through the plant plasma

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membrane, cytosol, and nuclear membrane. The prevailing view strongly supported by molecular and genetic data is that T-DNA is transferred as a nucleoprotein particle composed of a single-stranded DNA molecule (T-strand) covalently attached to a nicking enzyme (see below).

Roles of VirD2 Relaxase in T-DNA Processing and Transfer It is now widely accepted that DNA-processing reactions associated with T-DNA transfer are equivalent to those mediating bacterial conjugation. In the generalized reaction, a set of proteins termed as the DNA transfer and replication (Dtr) proteins assemble at an origin-of-transfer (oriT) sequence to generate a nucleoprotein complex termed as the relaxosome. One component of the relaxosome, the relaxase, cleaves and remains covalently associated with the 59 end of the DNA strand destined for transfer (T-strand). The T-strand is unwound from its template by a strand displacement reaction, generating the translocation-competent relaxase-T-strand substrate. In A. tumefaciens, the VirD2 relaxase generates nicks at oriT-like sequences located in the T-DNA border repeats. VirD2 remains covalently bound to the 59 phosphoryl end of the nicked T-DNA via conserved tyrosine residue Tyr-29. Purified VirD2 catalyzes cleavage of oligonucleotides bearing a T-DNA nick site. However, an ancillary protein, VirD1, is essential for nicking in vitro when the nick site is present on a supercoiled, doublestranded plasmid. In addition to oriT nicking, the relaxase component of the conjugative transfer intermediate is thought to participate in translocation of substrate DNA by supplying a signal motif recognizable by the transport machinery. VirD2 and other relaxases carry a motif at their extreme C termini that is devoid of secondary structure and rich in positively charged amino acids, particularly arginines. This motif is also present at the C-termini of protein substrates of the VirB/D4 T4S system and, as expected, mutations in the signal motif of one such substrate, VirF, block translocation. The charged motif likely confers recognition of the substrate by the secretion channel, as suggested by evidence that the VirD2-T-strand complex, as well as another protein substrate, VirE2, interact with the VirD4 substrate receptor (SR). Moreover, when the C-terminal fragment of VirE2 is fused to the green fluorescent protein (GFP), it mediates binding of the reporter protein to VirD4 in living cells. Early studies supplied evidence for 59-39 unidirectional transfer of the T-strand, which is also compatible with the notion that the relaxase serves to pilot the attached T-strand through the secretion channel.

8 Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

Roles of Ancillary Processing Factors in T-DNA Processing and Transfer Although VirD2 catalyzes nicking of T-DNA substrates in vitro, border cleavage in vivo requires accessory proteins including VirD1, VirC1, and VirC2 proteins. Early studies showed that VirC1 binds the overdrive sequence located next to the right border repeat sequences of octopine-type Ti plasmids. A recent quantitative analysis established that both VirC1 and VirC2 are required for synthesis of as many as 50 copies of the T-DNA transfer intermediate per cell within a 24-h induction period. A mutation in an invariant Lys residue in the Walker A nucleotide triphosphate binding motif of VirC1 (VirC1K15Q) abolished the stimulatory effect of VirC1 on T-strand production, suggesting that VirC1’s activity is regulated by ATP binding or hydrolysis. VirC1 is related to the ParA/MinD family of ATPases, which mediate partitioning of chromosomes and plasmids during cell division. Very interestingly, VirC1 localizes at cell poles, which is also the site of VirB/D4 machine assembly (see below). Besides stimulating the conjugative processing reaction, polar-localized VirC1 supplies another important function to stimulate substrate translocation. It recruits the VirD2-T-strand nucleoprotein particle to the VirB/D4 transfer machine. Such stimulatory functions associated with conjugation have not been described previously for other ParA/MinD homologues, but note that many mobile elements – both integrated and extrachromosomal – encode ParA/MinD homologues.

In future studies, it will be interesting to determine whether these proteins provide VirC1-like functions to couple processed DNA substrates with their cognate transfer machines.

VirB/D4 System, a Member of the Type IV Secretion Family A. tumefaciens translocates the T-complex as well as effector proteins through a dedicated secretion channel assembled from 11 VirB subunits and VirD4. The VirB proteins are termed as the mating pair formation (Mpf) proteins, and VirD4 the SR, also termed as the coupling protein (T4CP). As discussed above for the Dtr-processing factors, the VirB and VirD4 proteins are related in sequence and function to subunits of conjugation systems, further underscoring the notion that A. tumefaciens adapted an ancestral conjugation system to deliver effector macromolecules to plants during infection. The VirB/D4 system and other conjugation machines of Gram-negative and -positive bacteria are members of a large family of translocation systems termed as T4S systems (Figure 4). In addition to the conjugation machines, the T4S family encompasses two other subfamilies. One, termed as the ‘DNA uptake and release’ systems, function independently of contact with a target cell to take up DNA from the extracellular milieu, as exemplified by the Helicobacter pylori ComB competence system, or to

Conjugation T-DNA

VirB

B4

B1 B2 B3

IVA

R388 RP4α F

B5

B8

B6

B9

B10

B11

D4

B7

IncW IncP IncF

Effector translocation

IVA

H. pylori R. prowazekii Brucella spp. B. pertussis

Cag VirB Ptl

DNA uptake/release

IVA

H. pylori

Com

N. gonorrhoeae

Figure 4 Alignment of genes encoding related components of the T4S systems. Of the 11 VirB proteins, those encoded by virB2 through virB11 and virD4 are essential for T-complex transport to plant cells. Ancestrally related conjugation systems mediate interbacterial transfer of DNA. Effector translocation systems function to secrete proteins to eukaryotic cells during the course of infection by many medically important bacterial pathogens. A third subfamily of T4S systems, designated as the DNA uptake/release systems, take up DNA from the extracellular milieu or release DNA to the environment.

Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

release DNA to the milieu, as exemplified by a chromosomally encoded F-like transfer system carried on the gonococcal genetic island (GGI) of Neisseria gonorrhoeae. As with the conjugation machines, these systems promote genetic exchange and, therefore, also represent potential mechanisms for transfer of survival traits during infection. The third subfamily, the ‘effector translocator’ systems, play indispensable roles in the infection processes of many prominent pathogens of plants and mammals. These machines can be viewed as ‘injectisomes’, reminiscent of the needle complexes elaborated by type III secretion (T3S) machines, because they deliver their substrates through direct contact with the eukaryotic target cell. The list of pathogens dependent on effector translocators for disease progression includes at least two phytopathogens, A. tumefaciens and Burkholderia cepacia, plant symbionts such as S. meliloti, and several pathogens of mammals including H. pylori, Legionella pneumophila, and Brucella and Bartonella species. Bordetella pertussis uses an effector translocator as well, but this system functions as a true exporter to deliver its toxin substrate to the extracellular milieu. Related systems of several additional pathogens are also implicated in the trafficking of substrates to eukaryotic cells, and thus the list of T4S effector translocators continues to grow.

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The T4S systems are classified on the basis of extensive sequence similarities with subunits of conjugation machines (Figure 4). Although these systems are functionally versatile in terms of the substrates and target cells to which substrates are delivered, they share a number of common structural and functional features that distinguish them from other known bacterial translocation systems.

VirB/D4 Type IV System: Machine Architecture The VirB/D4 system is composed of two surface structures – a secretion channel and a conjugative pilus (Figure 5). At this time, there is no high-resolution structure for either structure. Nevertheless, fairly comprehensive architectural models of the T4S system can be generated through topological, structural, and interaction studies of machine subunits. These studies have supplied evidence for at least three stable subassemblies of VirB/D4 components. Energy subcomplex – VirD4, VirB4, VirB11

VirD4, VirB11, and VirB4 are the three energetic components of the VirB/D4 T4SS. Each of these subunits possesses a characteristic nucleoside triphosphate binding site (Walker A) motif required for substrate translocation. B5 B2

VirB5 VirB2

VirB8

OM

VirB7 VirB9

B9

VirB10

P VirB4

VirB11 VirD4

Core complex ATP

B5 B8

VirB1

VirB6

B7 B3

B10

B1

B10

VirB2 VirB3 VirB5

ATP

B6 B4

CM

B11 D4

ATP Substrate receptor Figure 5 Topologies, structures, and cellular localizations of the VirB/D4 T4S subunits. The Agrobacterium tumefaciens VirB/D4 T4S system localizes at the cell poles and is postulated to assemble as a transenvelope complex through which substrates pass to the cell surface. The three ATPases energize machine assembly and substrate transfer and a stable ‘core’ complex nucleates machine assembly. All of the VirB proteins are required to build the T pilus; the VirB proteins plus VirD4 are required for substrate secretion. The T pilus is sloughed from the cell surface and is not essential for DNA or protein translocation.

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Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

Mutations in the Walker A motifs invariably abolish substrate translocation, strongly indicating that ATP binding drives machine assembly or function. Structures of soluble domains of two VirD4-like proteins have now been solved by X-ray crystallography, one of TrwC encoded by plasmid R388 and one of Escherichi coli FtsK. TrwC presents as six equivalent protomers assembled as a spherical particle of overall dimensions 110 A˚ in diameter and 90 A˚ in height. The overall structure bears a striking resemblance to the F1-ATPase 3 3 heterohexamer, whereas the structure of the soluble domain closely resembles DNA ring helicases and other proteins such as FtsK that translocate along ss- or dsDNA. The FtsK structure is slightly larger with an outer diameter of 120 A˚ and a central annulus of 30 A˚. The predicted structure is a dodecamer composed of two hexamers stacked in a head-to-head arrangement. As shown by electron microscopy imaging, dsDNA runs through the FtsK annulus, providing a structural view of a previously described ATP-dependent translocase activity. VirD4, therefore, functions as a receptor for the T-DNA and protein substrates of the VirB/D4 T4S system, and it might also function as an inner-membrane (IM) translocase, though this needs to be explored. VirB11 is a member of a large family of ATPases associated with systems dedicated to secretion of macromolecules. Purified homologues TrbB, TrwC, H. pylori HP0525, and Brucella suis VirB11 assemble as homohexameric rings discernible by electron microscopy, and the last two also by X-ray crystallography. These structures present as double-stacked rings formed by the N- and C-terminal halves and a central cavity of 50 A˚ in diameter. VirB11 associates peripherally but tightly with the IM of A. tumefaciens, and there is some evidence for ATP regulation of membrane binding. The role of VirB11 in T4S is still fundamentally unknown. VirB4 subunits are large IM proteins with consensus Walker A and B nucleoside triphosphate-binding domains. A combination of experimental studies and computer modeling has yielded a topology model depicting VirB4 as predominantly cytoplasmic with possible periplasmic loops, one near the N-terminus and a second just Nterminal to the Walker A motif. As with VirB11, the contribution VirB4 to machine assembly and function is unknown. Core subcomplex – VirB6, VirB7, VirB8, VirB9, VirB10

Five VirB proteins are implicated as forming a ‘core’ transenvelope structure on the basis of phylogenetic relationships, and cell localization and protein–protein interaction data. VirB6 is highly hydrophobic with five predicted transmembrane segments (TMS) and a cytoplasmic C-terminus. A large central periplasmic loop, designated loop P2, is now known to play an important

role in substrate translocation (see below). VirB6 interacts with two OM proteins, VirB7 lipoprotein and VirB9, and probably also with the other VirB ‘core’ subunits. VirB6 exerts stabilizing effects on other VirB subunits, and it colocalizes with VirD4 and the T pilus at the cell poles of A. tumefaciens. The available data are consistent with a proposal that VirB6 assembles as a central component of the secretion channel mediating substrate transfer across the IM. The VirB7 lipoprotein forms a disulfide bridge with VirB9, and the heterodimer sorts to the OM where it exerts stabilizing effects on other machine subunits. VirB8 and VirB10 are bitopic IM subunits. Recently, structures of periplasmic fragments of both the subunits were solved by X-ray crystallography. Over its length, VirB10 shares several structural features with TonB, including a small N-terminal cytoplasmic domain, a single TMS, a Prorich region, and a region of sequence conservation at the C-terminal end. For TonB, the Pro-rich motif contributes to a rigid, extended structure in the periplasm that might permit simultaneous contacts with partner subunits at the IMs and OMs. Similarly, A. tumefaciens VirB10 interacts with the IM subunits VirB8, VirD4, and VirB4, and with the OM-associated VirB7–VirB9 heterodimer. Intriguingly, VirB10 also functionally resembles TonB by linking energy at the IM to the assembly or gating of the T4S channel for substrate translocation (see below).

T pilus subcomplex – VirB2, VirB5, VirB7

The T4S systems involved in conjugation elaborate pili for establishing contact between plasmid-bearing donor cells and recipient cells. Electron microscopy studies have demonstrated the presence of long filaments approximately 10 nm in diameter on the surfaces of A. tumefaciens cells induced for expression of the virB genes. These filaments are absent from the surfaces of mutant strains defective in expression of one or more of the virB genes. Furthermore, the interesting observation was made that cells grown at room temperature rarely possess pili, whereas cells grown at 19  C possess these structures in abundance. This finding correlates nicely with previous findings that low temperature stimulates the virB-dependent transfer of substrates to plants. All of the VirB proteins, but not VirD4, are required for the assembly of the T pilus, which is composed of the VirB2 pilin protein. VirB2 bears both sequence and structural similarity to the TraA pilin subunit of the F plasmid and to the TrbB subunit of plasmid RP4. VirB2, like TraA and RP4, is processed from a 12-kDa propilin to a 7.2kDa mature protein. Furthermore, both VirB2 and TrbB undergo an unusual head-to-tail cyclization reaction, resulting in a cyclic polypeptide that accumulates in the IM. VirB2 polymerizes as the T pilus, and VirB7 lipoprotein and VirB5 associate at unspecified locations along the T pilus.

Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

Dynamics of T4S System Machine Assembly and Function

to the integral IM proteins VirB6 and VirB8. • Transfer VirB11 delivers the T-DNA substrate to the polytopic

Recent studies have begun to describe dynamic properties of T4S systems. These studies have exploited a combination of cytological and biochemical approaches to understand how secretion substrates are recruited to the T4S machine and the route of substrate translocation. Recruitment of secretion substrates to the VirB/D4 T4S machine

As noted above, ParA/MinD-like VirC1 functions to recruit the processed T-complex to the polar-localized VirD4 SR. Independent of VirC1, however, VirD4 can recruit a protein effector, VirE2, to the cell pole. Whether other protein substrates can also interact directly with VirD4 or require a mediator or coupling factor is not known. Definition of the T-DNA translocation pathway by TrIP

An assay termed transfer DNA immunoprecipitation (TrIP) was developed to trace the path of a DNA substrate through the T4S channel. TrIP, adapted from the chromatin immuoprecipitation assay, involves formaldehyde treatment of intact cells to cross-link channel subunits to the T-DNA substrate as it exits the cell, disruption of the cells, solubilization of membranes, and immunoprecipitation to recover channel subunits. The presence of T-DNA substrate in the immunoprecipitates is then detected by PCR amplification. With the TrIP assay, it was shown that the substrate forms close contacts with 6 of the 12 VirB/D4 components, VirD4, VirB11, polytopic VirB6, bitopic VirB8, VirB2 pilin, and VirB9. Analyses of various T4S mutants with the TrIP assay enabled formulation of a sequentially and spatially ordered translocation pathway for the T-DNA substrate. This pathway provides the first glimpse of how the T4S channel might be configured across the cell envelope (Figure 5). The steps in the pathway are as follows: recruitment. The T-DNA substrate binds • Substrate VirD4 and it does so independently of other VirB

•

proteins, establishing that VirD4 is the T-DNA receptor. A VirD4 Walker A mutant also retains T-DNA as well as protein substrate receptor activity, suggesting that binding of both types of substrates occurs independently of ATP energy. Transfer to the VirB11 hexameric ATPase. Next, VirD4 transfers the T-DNA substrate to the VirB11 ATPase. This early transfer step also proceeds independently of ATP energy, as deduced by the finding that VirD4 or VirB11 Walker A mutations support substrate transfer. However, VirD4 cannot transfer the substrate to VirB11 in the absence of certain ‘core’ VirB proteins, suggesting that these core components are important for productive communication between VirD4 and VirB11.

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•

VirB6 and bitopic VirB8 proteins. VirB6 mutational studies identified a central periplasmic loop, termed P2, which is important for VirB6 binding to the DNA substrate. Other domains were implicated in regulating subsequent substrate translocation steps. Substrate transfer from VirB11 to VirB6 and VirB8 also require additional ‘core’ subunits, possibly important for VirB11 binding to these latter channel subunits, as well as the energetic contributions of VirB4, a third ATPase of this secretion system. Transfer to the periplasmic and OM-associated proteins VirB2 and VirB9. VirB2 and VirB9 comprise the distal end of the T-DNA translocation pathway. As noted above, VirB2 polymerizes as the T pilus. Although it is formally possible that the T-DNA substrate moves through the lumen of the pilus to the plant cell, this probably is not the case because certain mutations block pilus production without affecting substrate translocation. In strains producing the ‘uncoupling’ mutant proteins, the cellular form of VirB2 is still required for substrate transfer. Thus, VirB2 might be a component of the secretion channel extending through the periplasm and, possibly, the OM. Several T4S subunits, including VirB3, VirB5, and VirB10, are required for this step of substrate transfer, but they do not form detectable interactions with the T-DNA. Therefore, VirB3, VirB5, and VirB10 are probably not channel subunits per se, but rather contribute to the structural integrity of the channel.

Energetics of DNA translocation: VirB10, a TonB-like ATP energy sensor subunit

Assembly and function of the conjugation machines requires both proton motive force and ATP energy. In A. tumefaciens, the bitopic protein VirB10 interacts with VirD4 and was shown to undergo a structural transition in response to ATP utilization by VirD4 and VirB11. VirB10 also interacts with the OM-associated VirB7–VirB9 heterodimer or multimer by a mechanism requiring ATP energy use by VirD4 and VirB11. Accompanying formation of the transenvelope VirD4–VirB10–VirB7–VirB9 complex, the T-DNA substrate translocates from the IM portion of the secretion channel composed of VirB6 and VirB8 to that in the periplasm composed of VirB2 and VirB9. These findings suggest that VirB10 supplies a function similar to that described for the TonB energy transducer proteins. While TonB senses an IM electrochemical gradient, VirB10 senses IM ATP energy. In both the cases, however, IM energy is converted into a mechanical force required for a latter stage of machine biogenesis. VirB10 might transduce IM energy to mediate

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Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

formation or opening of a VirB7–VirB9 channel complex to allow passage of the DNA substrate to the cell surface.

range and can infect different plant cell types than reported previously.

Spatial Positioning of the VirB/D4 T4S System

Attachment to Plant Cells

A. tumefaciens cells have been shown to via their cell poles to abiotic and biotic surfaces. As noted above, the VirB/ D4 T4S system also localizes at cell poles. In recent studies, six VirB proteins – VirB1, VirB5–VirB7, VirB9, and VirB10 – were shown to depend on production of VirB8 for polar localization, whereas VirB3, VirB4, and VirB11 were found to localize at cell poles independently of VirB8. The VirB4 and VirB11 ATPases are not required for polar targeting of other VirB proteins, and VirB4 and VirB11 Walker A mutants display WT localization patterns, suggesting that nucleation of the VirB proteins at the cell pole does not require ATP energy. At this time, therefore, at least three protein complexes have been shown to localize at cell poles independently of each other: (1) the relaxosome bound at T-DNA border sequences that consists of VirD1, VirD2, VirC1, and VirC2; (2) the VirD4 SR; and (3) the VirB channel complex. Adding to this picture, the Ti plasmid itself localizes at or near the cell poles of A. tumefaciens vegetative cells. It will be interesting in future work to identify the underlying molecular basis for polar targeting of these various protein complexes, and also to understand how these machine complexes coordinate their activities in space and time to mediate translocation of T-DNA and protein substrates to target cells.

A. tumefaciens must bind plant cells to deliver T-DNA across the plant plasma membrane. Recent evidence indicates that there are at least two binding events that may act sequentially or in tandem. The first is encoded by chromosomal loci and occurs even in the absence of the Ti plasmid genes. This binding event directs bacterial binding to many plant cells independently of whether or not the bacterium is competent for exporting T-DNA or the given plant cell is competent for the receipt of T-DNA. The second binding event is mediated by the virB-encoded T pilus. Binding via the chromosomally encoded attachment loci is a two-step process in which bacteria first attach loosely and nonspecifically to the plant cell surface. This is a nonsaturable and aggregation-like mode of interaction reversible by washing with a buffered salt solution. Next, the bacteria attach more specifically in a tight and saturable interaction that is resistant to washing. A series of genes designated attachment (att) genes were implicated in mediating the latter mode of attachment, though this has been questioned because the att genes reside on a 542 kb plasmid, pAtC58, which has been shown to be dispensable for virulence. Another set of genes designated as cel direct the synthesis of cellulose fibrils that emanate from the bacterial cell surface. These fibrils are implicated in attachment of A. tumefaciens to specific sites on the plant cell surface. Binding is saturable, suggestive of a limited number of attachment sites on the plant cell, and binding of virulent strains can also be prevented by the attachment of avirulent strains. Efficient attachment of bacteria to plant cells also requires the products of three chromosomal loci, chvA, chvB, and exoC (pscA). All three loci are involved in synthesis of transport of a cyclic -1,2 glucan molecule. Mutations in these genes are pleiotropic, suggesting that -1,2 glucan synthesis is important for the overall physiology of A. tumefaciens. Periplasmic -1,2 glucan plays a role in equalizing the osmotic pressure between the inside and outside of the cell. It has been proposed that loss of this form of glucan may indirectly disrupt virulence by reducing the activity or function of cell surface proteins. Interestingly, chv mutants accumulate low levels of VirB10, one of the proposed components of the Tcomplex transport system (see ‘The VirB/D4 System, a Member of the Type IV Secretion Family’), suggesting that -1,2 glucan might influence T-DNA export across the bacterial envelope by contributing to transporter assembly. Recent genomic studies have begun to identify possible plant proteins involved in attachment. A collection of

Substrate Transfer Through the Plant Cell The delivery of T-DNA and protein substrates to plant cells requires productive contact between A. tumefaciens and a susceptible plant cell. A. tumefaciens commonly infects plants at wound sites, giving rise to a widely held view that wounding establishes important preconditions for infection. During wounding, plants release cell wall constituents and such molecules are potent vir gene inducer molecules. Wounding potentially also creates portals of entry through damaged cell walls, and stimulates cell replication and division reactions considered to be important for T-DNA integration. However, it is now known that A. tumefaciens can deliver T-DNA to unwounded plant tissues, dispelling the notion that wounding is an essential prerequisite for transformation. The most visible manifestation of A. tumefaciens transformation is the production of plant tumors, yet transformation of unwounded tissues typically does not incite tumor formation. Many transformation events, therefore, might be phenotypically silent, raising the intriguing possibility that A. tumefaciens actually has a much broader host

Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

mutations in Arabidopsis thaliana were generated that render the host plant recalcitrant to Agrobacterium transformation (rat mutants). Some of these mutations disrupt attachment of Agrobacterium and thus might map to surface receptors. One such mutation is in the promoter of a gene encoding arabinogalactan, a probable cell wall constituent. Another candidate receptor is a vitronectin-like protein found in detergent extracts of plant cell walls. Attachment-proficient A. tumefaciens cells bind radioactive vitronection, whereas attachment-deficient cells do not bind this molecule. Intriguingly, human vitronectin and antivitronectin antibodies both inhibit the binding of A. tumefaciens to plant cells. Yet other candidate receptors identified to date include a rhicadhesin-binding protein, a cellulose synthase-like protein, and several proteins shown to bind the VirB2 pilin protein. These proteins, designated VirB2-interacting proteins (VBTs), might mediate binding of the T pilus to the plant cell surface. Substrate Movement Through the Plant Cytosol and Integration into the Host Genome The VirD2–T-strand complex is only one of several substrates delivered to plant cells through the VirB/D4 T4S system. The others identified to date include the VirE2, VirE3, VirF, and VirD5 proteins. VirE2 is a single-stranded DNA-binding protein required for transformation, whereas the other translocated substrates function to enhance the efficiency of transformation. VirE2 is exported separately from the VirD2–T-strand particle, but upon transfer to the plant VirE2 binds cooperatively to the T-strand, forming a VirD2–T-strand–VirE2 particle termed the T-complex. The T-complex, composed of a 20-kb T-strand capped at its 59 end with a 60-kDa endonuclease and an estimated 600 molecules of VirE2 along its length, is a large nucleoprotein complex of an estimated size of 50  106 Da. Evidence exists that VirD2 and VirE2 protect the T-strand from plant nucleases and also facilitate T-complex movement along a microtubule network. VIP1, a protein shown to interact with VirE2, is postulated to function as a molecular link between the T-complex and microtubule track system. VirD2 also has been shown to interact with several members of a family of proteins termed cyclophilins. The postulated role for cyclophilins in this interaction is to supply a chaperone function at some stage during T-complex trafficking to the nucleus. A. tumefaciens has been demonstrated to transport DNA to representatives of prokaryotes, yeasts, and plants. Cyclophilins are ubiquitous proteins found in all of these cell types and, therefore, may be of general importance for A. tumefaciens-mediated DNA transfer. Additionally, both VirD2 and VirE2 carry nuclear localization sequences (NLS) that contribute to delivery of the T-complex to the nuclear pore. VirD2 was shown to interact with AtKAPa, a member of a conserved family of importin/ karyopherin proteins that are known to bind NLS and

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mediate nuclear import. Correspondingly, VIP1 is postulated to mediate nuclear import of VirE2. These different plant proteins, thus, might act synergistically or redundantly to mediate movement of the T-complex through the plant cytoplasm and nuclear pore. Interestingly, VirE3, another translocated substrate, mimics the function of VIP1 in mediating VirE2 nuclear import, possibly explaining how A. tumefaciens can transform nonplant species such as yeast and human cells that lack VIP1. Once inside the nucleus, the T-complex must be delivered to its site of integration in the host chromatin. Plant proteins, including CAK2M and TATA box-binding proteins that bind VirD2, VIP1 that binds VirE2, and core histones that bind VIP2, may be important for chromatin targeting of the T-complex. T-DNA integrates into the plant nuclear genome by a process termed ‘illegitimate’ recombination. According to a current model, T-DNA invades at nicks or gaps in the plant genome possibly generated as a consequence of active DNA replication. The invading ends of the single-stranded T-DNA are proposed to anneal via short regions of homology to the unnicked strand of the plant DNA. Once the ends of T-DNA are ligated to the target ends of plant DNA, the second strand of the T-DNA is replicated and annealed to the opposite strand of the plant DNA. Both VirD2 and VirE2 have been implicated in contributing to the TDNA integration step, but the molecular details of this reaction are not known. However, another translocated substrate, VirF, has been shown to possess an F-box domain and interact with several members of the ASK protein family, which are plant homologues of yeast Skp1 proteins. F-box and Skp1 are conserved components of E2 ubiquitin ligases that mediate protein destabilization. VirF was shown to destabilize a VIP1–VirE2 complex and thus might play a role in uncoating the T-DNA prior to or during integration into the host genome. Clearly, movement of T-complexes and integration of T-DNA into the plant genome is a complex multistep process involving specific binding of plant factors with bacterial effector proteins. However, note that all characterized effectors identified to date participate in some way to the movement of T-DNA through the plant cell or its integration into the plant genome. Whether the armament of translocated effectors includes proteins whose functions are unrelated to T-DNA movement and instead involved in disruption of plant physiological processes to promote the overall infection process is an interesting question for further study.

Agrobacterium Host Range and Genetic Engineering One of the most appealing features of the A. tumefaciens DNA transfer system for genetic engineering is its extremely broad host range. Agrobacterium has long been

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Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

known to transform a wide range of gymnosperms and dicotyledonous plant species of agricultural importance. Additionally, during the past two decades protocols have been developed for transformation of monocotyledonous plant species including rice, wheat, and maize. So far, most of the effort in developing these transformation protocols has been directed toward improvement of crop traits. Increasingly, however, the A. tumefaciens gene delivery system is being used to (1) isolate and characterize novel plant genes through T-DNA tagging, (2) deliver foreign DNA to specific sites in the plant genome, and (3) genetically engineer nonplant organisms. Intriguingly, Agrobacterium is now known to transform many nonplant species including other prokaryotes, yeast, and many other fungi, and human cells. And now, with the discovery that other alphaproteobacterial species including Rhizobium sp. NGR234, S. meliloti, and M. loti also deliver DNA substrates to plant target cells, the potential exists that the host range of eukaryotic cell types transformable by bacteria can be broadened even further. Nuts and Bolts of Genetic Engineering A. tumefaciens is readily manipulated such that plasmids carrying foreign genes of interest are easily introduced into appropriate bacterial strains for delivery to plants. Typically, strains used for gene delivery are ‘disarmed’, that is, deleted of oncogenic T-DNA, but still harboring intact Ti plasmid and chromosomal vir genes. Foreign genes destined for delivery to plants are generally cloned onto a plasmid that carries a single T-DNA border sequence or two T-DNA border sequences that flank various restriction sites for cloning as well as an antibiotic resistance gene to select for transformed plant cells. If the plasmid carries a single border sequence, the entire plasmid is delivered to plants, and surprisingly A. tumefaciens is capable of delivering in excess of 180-kb of DNA to plants. If the plasmid carries two border sequences, only the DNA bounded by T-DNA borders is delivered to plants. The frequency of stable transformation is often very high, well-exceeding frequencies achieved by other gene delivery methods. For example, cocultivation of A. tumefaciens with regenerating protoplasts of certain plant species can result in transformation of up to one half of the protoplasts. However, with protoplast transformation there is often a significant reduction in the number of transgenic, fertile plants recovered during selective regeneration of transformed protoplasts. For certain species, protoplasts can be transformed but are recalcitrant to regeneration into intact plants. Consequently, other transformation methods have relied on transformation of plant tissues such as excised leaves or root sections. In the case of monocot species such as maize, immature embryos are the preferred starting material for A. tumefaciens-mediated DNA

transfer. For rice, success has been achieved with callus tissue induced from immature embryos. Additional factors such as plant genotype, the type and age of plant tissue, the kinds of vectors and bacterial strains, and the types of selectable genes delivered to plant cells all influence the transformation efficiencies. For rice and corn, most of these parameters have been optimized so that now the delivery of foreign DNA to these crop plants is a routine technique. In addition to the need to identify transformable and regenerable plant tissues, a number of varieties of a given species often need to be screened to identify the susceptible varieties. A large variation in transformation efficiencies is often observed depending on which cell line is being tested. This underscores the notion that interkingdom DNA transfer is a complex process dependent on a genetic interplay between A. tumefaciens and host cells. Fortunately, many of the agronomically important species are readily transformable, but further efforts are needed to overcome present obstacles impeding efficient transformation of other species of interest. T-DNA Tagging A. tumefaciens is increasingly used to characterize and isolate novel plant genes by an approach termed T-DNA tagging. Several variations to this methodology exist depending on the desired goals. For example, because insertions are generally randomly distributed throughout the plant genome, T-DNA is widely used today as a mutagen for isolating plant genes with novel phenotypes. If the mutagenic T-DNA carries a bacterial origin of replication, the mutated gene of interest can easily be recovered in bacteria by suitable molecular techniques. Further, if the T-DNA is engineered to carry a selectable or scorable gene near one of its ends, insertion downstream of a plant promoter will permit characterization of promoter activity. Conversely, if the T-DNA is engineered to carry on outward reading promoter, insertion can result in a modulation of gene expression with potentially interesting phenotypic consequences. Finally, the discovery that A. tumefaciens can transform fungal species of interest means that all approaches developed for plants now can be applied to the characterization of fungi. Homologous or Site-Specific Recombination Although random T-DNA insertion is a boon to investigators interested in characterizing plant or fungal genes, it is an undesired event for plant genetic engineering. In addition to the potential result that T-DNA will insert into an essential gene, insertion is often accompanied by rearrangements of flanking sequences, which further

Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

enhances the chances that the insertion will have undesired consequences. Ideally, T-DNA could be delivered to a restricted number of sites in the plant genome. Recent progress toward this goal has involved the use of the bacteriophage P1 Cre/lox system for site-specific integration in the plant genome. The Cre site-specific recombinase catalyzes strand exchange between two lox sites, which, for P1, results in circularization of the P1 genome upon infection of bacterial cells. For directed T-DNA insertion, both the plant and the T-DNA are engineered to carry lox sequences and the plant is also engineered to express the Cre protein. Upon entry of T-DNA into the plant cell, Cre was shown to catalyze the site-specific integration of T-DNA at the plant lox site. The frequency of directed insertion events is low compared to random insertion events, but further manipulation of this system should enhance its general applicability. Gene Transfer to Yeast and Fungi The successful transfer of DNA to yeast depends on the presence of stabilizing sequences such as a yeast origin of replication sequence or a telomere, or regions of homology between the transferred DNA and the yeast genome for integration by homologous recombination. When the T-DNA lacks any extensive regions of homology with the Saccharomyces cerevisiae genome, it integrates at random positions by illegitimate recombination reminiscent of T-DNA integration in plants. The transformation of filamentous fungi with A. tumefaciens is an exciting advancement. A. tumefaciens was shown to efficiently deliver DNA to fungal protoplasts as well as fungal conidia and hyphal tissue. This discovery extends well beyond academic interest because the simplicity and high efficiency make this gene delivery system an extremely useful tool for the genetic manipulation and characterization of fungi. This DNA transfer system is especially valuable for species such as the mushroom Agaricus bisporus that are recalcitrant to transformation by other methods. It is also of interest to consider that both A. tumefaciens and many fungal species exist in the same soil environment, raising the possibility that A. tumefaciensmediated gene transfer to fungi may not be restricted solely to the laboratory bench.

Conclusions The early discovery that the oncogenes can be excised from T-DNA and replaced with genes of interest paved the way for the fast-growing industry of plant genetic engineering. Today, a large amount of information has been assembled about the A. tumefaciens infection process. This information has been used to successfully

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manipulate the T-DNA transfer system both to enhance its efficiency and to broaden the range of transformable plants and other organisms. Furthermore, this information has often established a conceptual framework for initiating or extending studies of other pathogenic and symbiotic relationships. The discovery that secreted chemical signals comprise the words for a dynamic dialog between A. tumefaciens and plant cells as well as other A. tumefaciens cells has stimulated a global effort to identify extracellular signals and characterize the cognate signal transduction systems in many bacterial systems. The discovery of T-DNA transport itself supplied a mechanistic explanation for how horizontal gene transfer impacts the evolution of genomes of higher organisms. This discovery also established a precedent for interkingdom transport of virulence factors by bacterial pathogens. Indeed, just in the last decade, studies have revealed that numerous pathogens employ interkingdom transport to deliver a wide array of effector proteins to plant and animal hosts. Interkingdom macromolecular translocation is mediated either by the T4S systems, which are ancestrally related to conjugation systems, or by the T3S systems, ancestrally related to flagellar systems. Both T3S and T4S systems translocate substrates via processes dependent on cell-tocell contact and, in some cases, elaboration of an extracellular filament or pilus. For the future, it is clear that studies of all the various aspects of the A. tumefaciens infection process will continue to spawn new applications for this novel DNA transfer system and yield new insights about the evolution and function of pathogenic mechanisms that are broadly distributed in nature. See also: Adhesion, Microbial; Conjugation, Bacterial; Horizontal Transfer of Genes between Microorganisms; Pili, Fimbriae; Plasmids, Bacterial; Quorum-Sensing in Bacteria; Rhizosphere

Further Reading Broothaerts W, Michell HJ, Weir B, et al. (2005) Gene transfer to plants by diverse species of bacteria. Nature 433: 629–633. Cascales E and Christie PJ (2003) The versatile bacterial type IV secretion systems. Nature Reviews Microbiology 1: 137–150. Cascales E and Christie PJ (2004) Definition of the type IV secretion pathway for a DNA substrate. Science 304: 1170–1173. Christie PJ (2004) Type IV secretion: The Agrobacterium VirB/D4 and related conjugation systems. Biochimica et Biophysica ACTA 1694: 219–234. Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski S, and Cascales E (2005) Biogenesis, architecture, and function of bacterial type IV secretion systems. Annual Review of Microbiology 59: 451–485. Citovsky V, Kozlovsky SV, Lacroix B, et al. (2007) Biological systems of the host cell involved in Agrobacterium infection. Cellular Microbiology 9: 9–20. Fuqua WC, Winans SC, and Greenberg EP (1996) Census and consensus in bacterial ecosystems: The LuxR-LuxI family of quorum-sensing transcriptional regulators. Annual Review of Microbiology 50: 727–751.

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Applied Microbiology: Agro/Food | Agrobacterium and Plant Cell Transformation

Gelvin SB (2003) Agrobacterium-mediated plant transformation: The biology behind the ‘‘gene-jockeying’’ tool. Microbiology and Molecular Biology Reviews 67: 16–37. McCullen CA and Binns AN (2006) Agrobacterium and plant cell interactions and activities required for interkingdom macromolecular transfer. Annual Review of Cell and Developmental Biology 22: 101–127. Tzvira R and Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: Biology and biotechnology. Current Opinion in Biotechnology 17: 147–154.

White CE and Winans SC (2007) Cell-cell communication in the plant pathogen Agrobacterium tumefaciens. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 362: 1135–1148. Yeo HY and Waksman G (2004) Unveiling molecular scaffolds of the type IV secretion system. Journal of Bacteriology 186: 1919–1926. Zhu J, Oger PM, Schrammeijer B, Hooykaas PJJ, Farrand SK, and Winans SC (2000) The bases of crown gall tumorigenesis. Journal of Bacteriology 182: 3885–3895.

Aquaculture H O Halvorson†, University of Massachusetts Boston, Boston, MA, USA R Smolowitz, Marine Biological Laboratory, Woods Hole, MA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Seafood Farming Bacteria-Covered Surfaces Bacterial Populations of Animals and Plants

Glossary aquaculture Farming or husbandry of aquatic plants or animals. biofilm This is a matrix of microcolonies of microorganisms with water channels in between and bacterial extracellular polysaccharides, glycoproteins, and proteins. chemotherapeutics The branch of therapeutics that deals with the treatment of disease by means of chemical substances or drugs.

Abbreviations EPS

extracellular polymeric substances

Defining Statement This article describes the role of microorganisms in aquaculture. Bacteria, viruses, and fungi all can cause diseases of fish. Bacteria form biofilms that can lead to biofouling. Some bacteria can protect fish from infection from other pathogens, while others recycle fish nutrients nitrogen and phosphate in the environment.

Seafood Farming It is unthinkable today that we could feed the world population by the ancient technique of hunting and gathering. Seventy percent of the world’s conventional commercial fish species are now fully exploited, overexploited, depleted, or recovering from depletion. This dramatic crash in world fisheries production has led to problems in food distribution, balance of payments, employment, and ecological depletion. The result has been an increase in aquaculture to maintain the y

Deceased.

Microbial Diseases Environmental Factors Recirculating Systems Further Reading

epizootics A term denoting a disease that attacks a large number of animals simultaneously (similar to an epidemic among humans). microflora The part of the population consisting of individuals that are too small to be clearly distinguished without the use of a microscope. pathogen Any disease-producing organism. zoonotic An infection or infestation shared in nature by man and lower vertebrate animals.

INAD PCB

investigational new animal drug polychlorinated biphenyl

worldwide demand for seafood. Today, farmed fish (finfish and shellfish) supply one-third of the seafood that people eat worldwide and that fraction is increasing. Aquaculture is defined as the ‘farming or husbandry of aquatic plants or animals’. Aquaculture has a long history. Some 4000 years ago aquaculture played an important role in China. A bas-relief discovered in an Egyptian tomb shows tilapia being cultivated in ancient Egypt about 2500 BC. The Japanese were rearing oysters as long ago as 2000 BC. In 475 BC Fan Li produced the first authentic study on aquaculture in which he described the potential of spawning carp in captivity in China. A 1000 years later, polyculture was introduced. During the Middle Ages aquaculture evolved in central and occidental Europe and by 1850 aquaculture was well established in North America. Microorganisms play a critical role in aquaculture. The exploration of the biological resources of the oceans and freshwaters for aquaculture requires not only an awareness of the biochemistry, genetics, physiology, and anatomy of plants, animals and microorganisms but also an understanding of how they interact with each other. Further, microorganisms play a major role in water quality – a critical factor in successful and sustainable aquaculture.

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Bacteria-Covered Surfaces In nutrient-limited ecosystems bacteria attach to surfaces and start the formation of a biofilm. The biofilm is a matrix of microcolonies of microorganisms with water channels in between and bacterial extracellular polysaccharides, glycoproteins, and proteins. Microbial biofilms are dynamic systems. Eventually, community growth becomes limited by the availability of substrate. Species composition may range from low to high density. The communities are subject to fluctuating environmental conditions, which influence the overall structure of the biofilm. Attached microorganisms have an enhanced ability to scavenge nutrients from the bulk water and the substratum, as well as the ability to concentrate metals. The initial adhesion is generally reversible. The unfolding of binding molecules and extracellular polymeric substances (EPS) leads to strong anchoring of adhering organisms, followed by coadhesion, coaggregation, growth, and finally detachment. EPS consist of varying proportions of carbohydrates, proteins, nucleic acids, lipids/carbohydrates, and humic acids. The significance of biofilms is that owing to their smaller dimensions, diffusional mass transport of macromolecules is many times greater than that of microorganisms. Quorum sensing systems (bacterial communication systems), in both Gram-positive and Gram-negative bacteria, have led to the development of a new field of microbiology – bacterial cell signaling – and community behavior, which regulate, among other systems, biofilms. Cell density is ‘sensed’ through the production of cell signaling molecules, which, after reaching a critical concentration, start a signal transduction cascade, leading to the expression of a number of target genes. Therefore, quorum sensing allows bacteria to organize and function as groups. Biofilms have been shown to facilitate bacterial survival under a variety of environmental stresses, including antibiotics and disinfectants. In some cases biofilms have adverse effects including energy wastage, heat transfer resistance, decreased life of equipment, safety problems, biodeterioration, biofouling, and contamination of water supplies. Detachment of biofilms can also lead to significant problems. Aquaculture by its nature is intensive and one of its biggest logistical problems is biofouling. When you tether a cage full of live fish and nutrients, other sea life is attracted like a magnet and there is very little fish farmers can do to stop it. Fouling by algae, barnacles, and other sea life is one of the most costly problems faced by the aquaculture industry.

Bacterial Populations of Animals and Plants In aquaculture, bacteria present in the aqueous environment quickly colonize animals and plants. This microflora leads to either disease epizootics or commensal

relationships. Understanding the complex interactions between the cultured organisms and bacterial communities is critical for aquaculture. This is particularly true in rearing of animals. Eggs are kept in tanks with a microflora that differs from the natural aquatic environment. After fertilization, the eggs become heavily overgrown with bacteria. The bacterial flora of the water, the egg epiflora, and the microflora of the feed colonize fish larvae. The result is the formation of an indigenous microflora or the first steps in a disease process. Beneficial bacteria (probiotics) that have been used successfully in the animal industry to displace pathogens by competitive processes are now being used to control pathogens in aquaculture. One example is in the production of shrimp, which is beset by disease primarily caused by the luminous Vibrio harvevi. In Indonesia, probiotic strains selected for their inhibitory effect eliminated luminous Vibrio from shrimp ponds. Another example is the survival of halibut larvae, which is improved by incubation with commensal, nonpathogenic bacteria, such as Lactobacillus plantarum or apathogenic strains of Vibrio salmonicida and Vibrio iliopisacrius.

Microbial Diseases Bacteria, viruses, and other pathogens are a natural part of any ecosystem and aquatic organisms have coevolved with them. Even when healthy, aquatic organisms may harbor many of these organisms, which their bodies fight off successfully. Disease is one of the most important problems and challenges confronting the aquaculture industry. Fish diseases do not occur as a single caused event but are the end result of interactions of the disease, the fish, and the environment. In intensive culture, handling, crowding, transporting, drug treatments, undernourishment, fluctuating temperatures, and poor water quality continuously affect fish. These conditions impose considerable stress on the homeostatic mechanisms of fish, rendering them susceptible to a wide variety of pathogens. Many of the same opportunistic pathogens have minimal effects in the natural environment. In addition, the transportation of fish for use in aquaculture has the potential to transport exotic pathogens, which can have a serious impact on native strains of the same species with less resistance to those pathogens. The most severe disease problems in aquaculture are caused by bacteria (Table 1). Specific bacterial pathogens are responsible for specific disease problems. Gram-negative bacteria are the most frequent cause of disease in finfish, whereas Gram-positive bacteria are the most common cause in crustaceans. Viral diseases (Table 2) cause serious problems in aquaculture, requiring quarantine and destruction of the

Applied Microbiology: Agro/Food | Aquaculture

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Table 1 Bacterial diseases of aquaculture Disease

Pathogen

Hosts

Acinetobacter disease Bacterial kidney disease Bacterial gill disease

Acinetobacter sp. Renihacierium salmonirwn Flavobacterium branchiophila Cytophaga spp. Myxobacteriums sp. Vibrio anguillarum Pseudomonas fluorescens Clostridium botulinum Flexibacter psychrophilus Vibrio salmonicida Flexibacter columnaris Edwardsiella tarda Yersinia ruckeri Edwardsielln ictaluri Eubacterium tarantellus Leucothrix Flavobacterium sp. Aeromonas sp. Flexibacter sp. Pseudomonas sp. Mycobacterium spp. Flavobacterium spp. Aeromonas salmonicida Aerococcus viridans var. homari Aeromonas spp. Nocardia asteroides Pasteurella piscicida Carnobacterium piscicola Sporocytophaga sp. Vibrio Pseudomonas Aeromonas Streptococcus spp. Streptoverticillum salmonis Vibrio spp.

Atlantic salmon, channel catfish eggs Salmonids, sable fish, Pacific herring Salmonids Salmon Most species American oyster Most species Salmonids Salmonids Atlantic salmon Many freshwater fish, marine flatfish Numerous species Salmonids, numerous warmwater fishes, crayfish Catfish, tilapia Striped mullet Shrimp and prawns Numerous species

Bacillary necrosis Bacterial septicemia Botulism Cold water disease Cold water vibriosis Columnaris Edwardardsiella septicemia Enteric redmouth Enteric septicemia Eubacterial meningitis Filamentous bacterial disease Fin rot

Fish tuberculosis Flavobacteriosis Fununculosis (and other diseases) Gaffkaemia Motile aeromonas septicemia Nocardiosis Pasteurellosis Pseudokidney disease Saltwater columnaris Shell disease caused by chitinolytic bacteria Streptococcal disease Streptomycosis Vibriosis

Most species Marine fishes Numerous freshwater fishes, marine fish susceptible American lobster Most fish species, also frogs, tunics, snakes Most species Striped bass, white perch, yellowtail Salmonids Young salmonids Shrimp, prawns, crawfish, and crustaceans

Many species Salmonids Most marine species

Table 2 Viral diseases in aquaculture in North America Disease

Pathogen

Hosts

Koi herpesvirus Infectious salmon anemia virus Epizootic hematopoietic necrosis White spot syndrome virus Infectious hematopoietic necrosis virus Viral nervous necrosis Infectious pancreatic necrosis virus

Cyprinid capio Orthomyxovirus Iridovirus White spot baculovirus Rhabdoviridae Nodavirus Birnaviridae

Carp Atlantic salmon Teleost fishes Penaeid shrimp Salmon, trout, sea bass Hatchery-reared groupers Various cultivated fish species

infected stock. Viral disease in endemic wild stock are formidable obstacles to net-pen aquaculture in open waters and introduction of ‘carrier’ animals into an artificial aquaculture system can cause mortalities of over 90% in susceptible stacks. Fungal infections (Table 3) are frequently encountered when the water quality is poor, or when there is

stress, inadequate nutrition, and skin trauma, providing a port of entry for molds. Fungus is then a secondary invader of skin wounds. Saprolegnia, one of the most common water molds encountered, occurs following handling, crowding, heavy feeding rates, and high organic loads. The reduction of stress is the most significant factor in reducing Saprolegniosis.

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Table 3 Fungal diseases in aquaculture Pathogen

Disease

Host

Aphanomyces astaci Branchiomycces demigrans Exophiala sp.

Crayfish plague Branchiomycosis, branchiomyces infection, gill rot Fungal infection, cerebral mycetoma

Fusarium solani (and other species) Icthyohponas hoferi Lagenidium callinectes

Burn spot and black gill disease in crustaceans, fungal infections in fish Ichthyophonosis, swinging disease Fungal egg disease

Crayfish Large mouth bass, northern pike, trench, and striped bass Trout, cod, dogfish, shark, seahorse, flounder, mummichog, triggerfish, and others Shark, carp, crustaceans

Saprolegnia sp. (S. parasitica– S. diclina compex) Sirolpidium zoophthorum

Saprolegniosis, oomycete infection, water mold infection Larval mycosis

Fish Diseases and Human Health Some bacteria found associated with or causing disease in fish are of special zoonotic concern for people, especially aquaculturists or fish processors. Infection usually occurs through previously suffered cuts or other wounds on the hands and arms, especially if appropriate sanitary measures are not followed when handling fish. Immunocompromised people are especially at risk for infection. Aquatic bacteria with recognized zoonotic potential are Aeromonas sp., Pseudomonas sp., Edwardsella sp., Streptococcus sp., Vibrio sp., Erysipelothrix rhusiopaathae, and Mycobacterium sp. (aquatic species such as marinum). Mycobacterial infections are of special importance since they can occur in fish even when held in good-quality water and can cause chronic joint infections in nonimmunocompromised fish handlers. Control Measures and Chemotherapeutics The first and best method of controlling and preventing disease in fish is to provide good-quality water and nutritious food. Many potential disease-causing bacteria are normally present in the water, or in the biofilm on the tank, or the fish. If the aquatic organisms are stressed or on a poor nutritional plane, and/or there is a bloom of one of these bacteria in the tank due to poor water quality, infection and resulting disease can occur. Other important preventive methods involve upkeep of the facility. Tanks and nets must be cleaned using a disinfectant between uses. Floors and other surfaces within the facility should be routinely cleaned. Footbaths should be used to disinfect shoes to prevent spread of disease into the facility from another location. Sanitary measures should not only be used to maintain the facility, but should also be a part of any routine carried out in the facility. The use of gloves and proper care of wounds as well as routine hand washing are important in

Cold water marine fish Crustacean egg masses and larval lobsters and crabs Compromised or immunosuppressed fish, fish eggs, amphibians Hatchery-reared larval bivalves

preventing infections in people who work with marine organisms. The use of chemotherapeutics in fish, as in land-based animals, is extremely controversial. Bacterial resistance to antibiotics has developed in animal systems in which antibiotics are routinely used. Resistant bacteria may be carried on food products and could infect the consumer or may infect the handler directly. Additionally, antibiotic and chemical residues in the aquacultured food are potentially a problem for the people who consume the product both because of potential toxicity of the compound (or its deriviatives) and because of development of resistance of the bacterial flora in the consumer’s digestive system. At present, because of these concerns, there are very limited numbers of chemical/antibiotic treatments approved for use in aquacultured fish by the USDA (http://www.usda.gov/ wps/portal/usdahome). Even the treatments that are approved for some fish are not approved for others. The USDA has established the Joint Committee on Aquaculture, which establishes INADs (investigational new animal drugs) to examine and eventually approve appropriate therapeutics for use on aquacultured animals. Drug development is expensive and pharmaceutical companies are not willing to invest the money necessary to directly approve drugs for use in aquacultured animals. So, INADs are one of the few methods by which a new drug can be approved for use in aquaculture. No treatments are approved for use in mollusks primarily because they are cultured widely dispersed in open water and cannot be dosed using food containing chemotherapeutics, as can fish. Since ornamental fish are not consumed, a variety of medications can be used ‘off-label’ if a client/patient/veterinary relationship exists before treatment begins. The American Veterinary Medical Association has published standards of practice for veterinarians to follow when considering the ‘off-label’ use of any drugs on aquacultured, or any aquatic animal (www.avma.org/).

Applied Microbiology: Agro/Food | Aquaculture

Environmental Factors Aquaculture, freshwater or marine, is dependent upon good water quality to sustain maximal fish growth. Ammonia and nitrate are toxic to fish. NH3 should be kept at levels below 0.05 mg l–1. Nitrite (NO2) should be kept below 0.5 mg l–1. Aerobic bacteria play a key role in detoxifying ammonia. Nitrosomonas bacteria convert NH3 to NO2, and Nitrobacter bacteria convert NO2 to NO3. For every milligram of ammonia converted about 5 mg of oxygen is consumed. An additional 5 mg of oxygen is required to satisfy the oxygen demand of the bacteria involved. Thus, reduced concentrations of dissolved oxygen may contribute to increased concentrations of ammonia, nitrate, and phosphate in the water column. The gradual accumulation of nutrients (including nitrogen and phosphorous) and organic biomass, accompanied by increased levels of production, brings about a process called eutrophication. The consequences are severalfold. Species such as Cyanobacteria change and some strains of algae accumulate, leading to reduced water quality. Oxygen levels drop, leading to anoxic bottoms, massive fish kills, and excessive production of phytoplankton and/or macroscopic plants, creating aesthetic problems. Phosphate, which is an essential element for life, can be detrimental to the biosphere in high concentrations. Since the discovery in 1988 by L. Liebermann that yeast contain granules (volutin) composed of high-polymer polyphosphates, a diverse group of microorganisms have been reported to effectively take up inorganic phosphate from the medium and convert them into biopolymers. Polyphosphates have been associated with the capsule in Neisseria gonorrhoeae, outside the plasma membrane, or as long-chain cytoplasmic reserves. In some microorganisms, these granules can account for as much as 25% of the weight of the organism and the process can be highly efficient in phosphate removal. For example, in the A/O process of activated sludge treatment, involving alternate anaerobic and aerobic cycles, a bacterial population (largely composed of a typical soil bacterium – Acinetobacter lwoffi) that accumulates polyphosphate even under extremely low phosphate concentrations is enriched. Since the consortium in the A/O process denitrifies ammonium as well as removes phosphate from the environment, it provides attractive possibilities for aquaculture. A variation of this has already been incorporated into the sequencing batch reactor for recirculating aquaculture systems. These diverse microorganisms contain highly efficient transport systems for phosphate uptake. The effects of discharge of aquaculture effluents in receiving waters are mainly the increase of suspended solids and nutrients and the fall in dissolved oxygen content. Algal blooms,

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especially of toxic species produced by high levels of nutrients, can cause environmental hazards, including fish kills. Numerous toxic compounds in the environment inhibit aquaculture. The toxic compounds of greatest concern have been polychlorinated biphenyls and mercury. Other potentially toxic metals such as lead, zinc, copper, arsenic, mercury, beryllium, barium, cadmium, chromium, nickel, and selenium have been identified. Chlorinated benzenes, chlorinated ethenes, polychlorinated biphenyls (PCBs), as well as other organohalides form a major group of environmental pollutants that inhibit aquaculture. Members of the class Chloroflexi (Dehalococcoides sp.) of microorganisms dechlorinate these compounds under anaerobic conditions. Mercury is a highly toxic metal that can cause genetic abnormalities and damage to the brain, kidneys, and liver. The three major kinds of organic mercury are phenyl mercury, methoxy mercury, and alkyl mercury such as methyl mercury. The latter is the most common and the most dangerous. The most serious consequence of methyl mercury poisoning involves its effect on the central nervous system. Methanogenic bacteria as well as certain molds in sediments are capable of converting all other forms of mercury into methyl mercury under both aerobic and anaerobic conditions.

Recirculating Systems Recirculating aquaculture systems represent a new way to farm fish. Instead of the traditional method of growing fish outdoors, this system rears fish at high densities, in indoor tanks with a ‘controlled’ environment. Recirculating systems filter and clean the water for recycling through fish culture tanks. Water is typically recirculated when there is a specific need to minimize water replacement, to maintain water quality conditions which differ from the supply water, or to compensate for an insufficient water supply. There are innumerable designs for recirculating systems and most will work effectively if they accomplish aeration, removal of particulate matter, biological filtration to remove waste ammonia and nitrite, and buffering of pH. These processes can be achieved by biofilters. These are living filters composed of a medium (corrugated plastic sheets, beads, or sand grains) upon which a film of bacteria grows. The bacteria provide the waste treatment by removing pollutants. The two primary water pollutants that need to be removed are (1) fish waste (toxic ammonia compounds) excreted into the water and (2) uneaten fish feed particles. The biofilter is the site where beneficial bacteria remove (detoxify) fish excretory products, primarily ammonia. Reoxygenating the culture water as it returns to the fish tank is crucial. Oxygen is the first limiting factor in

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recirculating aquaculture systems and with less than the required levels most fish and other aquatic organisms will die in a very short period of time. It is also critical that biofilters have access to adequate oxygen. Biofilters are homes to nitrifying bacteria, which are aerobic (use oxygen during respiration). Furthermore, nitrification, the conversion of ammonia to nontoxic nitrate by the bacteria, cannot occur without the presence of oxygen. See also: Adhesion, Microbial; Biofilms, Microbial; Food and Waterborne Illnesses; Fungal Infections, Systemic; Heavy Metal Pollutants: Environmental and Biotechnological Aspects; Nitrogen Cycle; Phosphorus Cycle

Further Reading Allisson DG, Gilbert P, Lappim-Scott HM, and Wilson M (eds.) (2001) Community Structure and Cooperation in Biofilm. Cambridge, UK: Cambridge University Press. Austin B and Austin DA (1986) Bacterial Fish Pathogens. New York, NY: Halsted Press, John Wiley & Sons. Avault JW Jr (1995) Understanding bacteria and bacterial diseases in aquaculture. Aquaculture 21: 68–76. Boghen AD (1995) Cold-Water Aquaculture in Atlantic Canada. Sackville, NB: The Tribune Press Ltd.

Hoole D, Bucke D, Burgess P, and Wellby I (2001) Diseases of Carp and Other Cyprinid Fishes, p. 264. Oxford, England: Fishing News Books, Blackwell Science. Irvine RL and Ketchum LH (1968) Sequencing batch reactors for biological wastewater treatment. CRC Critical Reviews in Environmental Control 18: 255–294. Laws EA (1993) Aquatic Pollution. Hoboken, NJ: John Wiley & Sons, Inc. Noga EJ (1996) Fish Disease Diagnosis and Treatment, p. 367. Missouri: Mosby, St. Louis. Saint-Erne N (2001) Advanced Koi Care for Veterinarians and Professional Koi Keepers, p. 194. Glendale, Arizona: Erne Enterprises. Torriani-Gorini A, Rothman FG, Silver S, Wright A, and Yagil E (1987) Phosphate Metabolism and Cellular Regulation in Microorganisms. Washington, DC: American Society for Microbiology. Watts JEM, Fagervold SK, Miller GS, Milliken CE, May HD, and Sower KR (2004) Microbial reductive dechlorination of organochlorde pollutants in the marine environment. Marine Biotechnology 6: S378–S383. Wildgoose WH (2001) BSAVA Manual of Ornamental Fish, p. 304, 2nd edn. Spain: Grafos, Barcelona. Woo PTK and Bruno DW (1999) Fish Diseases and Disorders, vol. 3, Viral, Bacterial and Fungal Infections, p. 874. New York, NY: CABI Publishing.

Relevant Websites http://www.avma.org/ – AVMA http://www.usda.gov/wps/portal/usdahome – USDA

Beer/Brewing M A Harrison, University of Georgia, Athens, GA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction History of Brewing Brewing Beer Properties

Glossary bottom-fermenting yeasts Yeasts used to produce the type of beer known as lager. chill-proofing Use of proteases to prevent haze development when beer is chilled to refrigerated temperatures. hops The dried flowers of the female Humulus lupulus plant that contribute flavor and antibacterial compounds to beer.

Defining Statement In the brewing of beer, grains are converted through fermentation to produce a desirable beverage with distinct sensory characteristics. This article outlines the steps involved in brewing beer and discusses the variations that can be done to produce the vast variety of beers available.

Introduction Beer is defined in the Bavarian Purity Law of Germany, as a fermented alcoholic beverage made of malted cereals, water, hops, and yeast. This is the classical definition and has been enforced in Germany since the sixteenth century. Many countries, however, now allow additional substances to be used in this product. For instance, various enzymes and antifoaming agents are used by some brewers during the fermentation process. Others supplement expensive barley malt with unmalted cereals such as corn, rice, or wheat, which contribute to beer flavor while reducing processing costs. Beer is generally subdivided into lagers and ales based on its geographic origin and history (Table 1). For centuries the only beer known to all brewers was ale. During the fermentation of this beer, a thick yeast foam rose to the surface. All too frequently the beer went sour, especially during warmer

Properties of Brewing Yeasts Spoilage Problems of Beer Spoilage Control Other Types of Beer Processing Further Reading

malt Major raw material used in brewing that provides the appropriate substrate and enzymes needed to yield wort. top-fermenting yeasts Yeasts used to produce the type of beer known as ale. wild yeasts Yeasts that are present in the brewing process that were not introduced purposely nor tolerated for a specific purpose during brewing. wort Liquid that remains after mash is strained, containing soluble fermentable compounds.

months. In the 1800s, German brewers observed that if beer could be kept at colder temperatures by brewing in the colder months or by using natural ice or caves, the beer soured less frequently. They also noticed that the yeast settled to the bottom of casks in the cold. This cold-storage method gave birth to ‘lager’ beer in Germany. Lager beers are now traditionally fermented cool and aged cold for several weeks to several months. Ales, on the contrary, ferment at warmer temperatures and are aged only for days. The yeast rises to the surface and is skimmed. Ales have remained the beer of choice in England. The yeast used in lagers and ales have adapted over time to ferment optimally at the temperatures used to produce the respective beer types. Classic examples of German lager beers are Ma¨rzen, Pilsener, Dortmunder, and Bock beer. All these beers are malty with a balance of hoppiness (bitterness). Color can vary from light (Helles) to dark (Dunkel). They usually contain 4.5–5% w/v alcohol, with bock containing 6% w/v. Examples of English ales are bitter, pale ale, porter, and stout. Bitter and pale ale are characterized by a copper color, full body, and elevated bitterness. Porter and stout are dark in color, contain roasted or burnt malts, and may or may not be bitter. Examples of German ales are Weizenbier (wheat beer), Alt, and Ko¨lsch. There are other beer styles that do not clearly fit into the ale or lager category. Most of these beers are Belgian in origin and are spontaneously fermented, for example, Lambic.

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Table 1 Major beer types Types Ales and stouts Alt or altbier Barley wine Bitter ale Brown ale or mild ale Cream ale Imperial stout India pale ale Ko¨lsch Lambic Porter Saison Stout Sweet stout Weizenbier or wheat beer Lagers Bock Dortmunder Dunkel Helles Malt liquor Ma¨rzen Pilsener Rauchbier Schwarzbier Vienna Zwickelbier

Country of origin

Germany England England England USA England England Germany Belgium England Belgium Ireland England Germany Germany Germany Germany Germany USA Germany Czech Republic Germany Germany Austro-Hungarian Germany

History of Brewing Evidence shows that brewing of beer was a popular practice in Mesopotamia before 3500 BCE. Beers offered people a flavorful alternative to drinking water, were often thought to possess therapeutic properties, and were safer to drink than water in some instances because they were less likely to harbor waterborne pathogens due to the fermentation process. Brewing beer, became popular in other areas of the Middle East, including Ancient Egypt and Israel. Brewing practices had spread to Rome during Caesar’s reign and into other parts of Europe. Over the centuries, it was discovered that the addition of hops and other spices would improve the flavor of beer. Since that time, hops have become essential in beer due largely to their contribution to the flavor and stability of beer. As Europeans settled in North America they brought brewing practices with them. During the sixteenth and seventeenth centuries, breweries were established in Virginia and New England. Barley did not prosper in the New England climate, so a variety of unusual ingredients were fermented, including pumpkins, maple sugar, persimmons, and apples. Two factors contributed to the further development of the North American brewing industry. Pennsylvania was found to be a good barley and hop production area, and the immigration of Germanic and Dutch brewmasters bolstered the American industry.

During this entire period, brewing was basically a hitor-miss process. Individuals experienced in brewing recognized that using old brewing vessels yielded better products than new ones. It is now realized that the cracks, crevices, and pores present in the older vessels, but lacking in the newer ones, harbored the yeasts and bacteria responsible for the fermentation. Several theories were developed in the nineteenth century in an attempt to explain the changes that occur during fermentation. Much of the debate centered on the issue of whether fermentation was a purely chemical process or a biological process. The issue was largely settled by Louis Pasteur in the 1860s and 1870s, when he published reports (e.g., ‘Etudes sur la Biere’) concluding that fermentation was due to the actions of yeast. In 1883, Emil Christian Hansen established the method of using pure yeast cultures to produce beer at the Carlsberg Brewery in Copenhagen, Denmark. He had demonstrated previously that the culture used in brewing was often a mixed culture and that the metabolism of wild yeasts caused many of the defects in improperly processed beer. Over the next several years, the practice of using pure yeast cultures to produce beer became more widely accepted. The purpose and function of yeast enzymes in the fermentation process was shown by Buchner in 1897. It was found that cell-free extracts contained enzymes that could ferment sugars. Today beer remains a popular and profitable beverage. While beer is consumed worldwide, on a per capita basis the leading beer consuming countries are the Czech Republic, Ireland, Germany, Austria, and the United Kingdom. While US per capita beer consumption in 2002 was about half of that consumed by the leading European countries, it was greater than the volume of milk and bottled water consumed in the country. In that same year, beer sales in the United States exceeded $60 billion. While US beer production is dominated by three major breweries, there has been an explosion in the number of small breweries producing the product, often using traditional methods. These smaller operations are typically labeled as microbreweries.

Brewing The conversion of cereals into beer is not a direct process. The cereals used in beer production do not contain sufficient quantities of fermentable sugars. These cereals must first undergo modification during the malting and mashing steps to yield carbohydrates that yeast can convert during the fermentation step into ethyl alcohol and carbon dioxide. Freshly produced beer can then be aged for flavor development before it undergoes finishing steps which can include filtering, pasteurization, and packaging. Each of these steps is examined more closely in the following sections and are shown in Figure 1.

Applied Microbiology: Agro/Food | Beer/Brewing

Barley Water Steep (10–20 °C, 48 h) Malting Germinate (15–20 °C, 24–48 h)

Kiln dry malt (50–110 °C, 24–48 h)

Malt milling Adjuncts Mashing (40–50 °C, 1–2 h, then 65–70 °C, 1–2 h, then to ~75 °C)

Mashing

Wort recovery Hops Wort boiling (100 °C, 0.5–2 h) Wort processing

Ale: S. cerevisiae (18–27 °C for 5–7 d)

Wort clarifying & cooling

Fermentation Lager: S. pastorianus (6–15 °C for 7–12 d) Yeast recovery Fermentation & conditioning Maturation (time and temps vary)

Cold conditioning (–2 to 0 °C, 3–4 d)

Finishing (pasteurization, filtration, packaging, etc.) Finishing Finished beer Figure 1 Brewing process.

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Applied Microbiology: Agro/Food | Beer/Brewing

Ingredients The basic ingredients in beer are water, malted cereals, hops, and yeast. Water comprises 90–95% of the content of finished beer, and its quality can influence the flavor of beer. Barley is the most common cereal used in the Americas and Europe to produce malt, although small volumes of beer are made from other cereal grains. Better beers are produced with clean barley that was properly dried after harvest (to about 10–12% moisture content). Overheating barley during drying can lead to its becoming unacceptable for malting, since its germination potential is adversely affected. Barley contains a high starch content suitable for conversion to fermentable carbohydrates and a sufficient protein content to support yeast growth and contribute to forming beer foam. In addition, it contributes unique flavor components. Hops are the dried flowers from the female hop (Humulus lupulus) plant and contribute flavor and antibacterial compounds to beer. Yeasts are the predominant fermentation organisms used to make beer worldwide. In some instances, bacteria may contribute certain characteristics to some regional beers. Malt adjuncts, such as corn, rice, wheat, sorghum grain, soybeans, cassava, potatoes, sugars, and syrups, may be used in some formulations. The adjuncts are all starch- or sugar-containing substrates that contribute fermentable carbohydrates. They also contribute flavor characteristics to produce distinctive varieties of beers. Although enzymes other than those in the malt and those contributed by the yeast are not needed to produce beer, some brewers use additional enzymes to impart desired characteristics to their product.

Malting The main objective of malting is to produce an ample supply of enzymes that degrade starch, proteins, and other components of grain. The subsequent enzymatic changes provide fermentable sugars from starch and substances needed to support yeast growth (e.g., amino acids and fatty acids) from the other substrates. Malt also is a major contributor to the final color and body characteristics of the finished beer. To produce malted barley, barley grains are first steeped in 10–15  C aerated water and then germinated at 15–20  C for 3–7 days. During this time the moisture content increases to approximately 45%. After the barley germinates, the sprouts are removed, leaving a medium rich with -amylase, -amylase, proteases, and their respective substrates. The malt is dried and kilned, under controlled conditions that remove the water without inactivating the desired enzymes, to approximately 5% moisture and ground. The flavor of the finished beer can be influenced by the amount of nonenzymatic browning and heatgenerated flavor compounds that are formed during the

drying process. Dark beers are made using darker, more flavorable malt while less roasted malt is used to brew paler beers. Grinding exposes the starchy endosperm of the grain, which makes the carbohydrates more available.

Mashing During the mashing step, most of the nonsoluble, unfermentable carbohydrates and proteins are hydrolyzed into soluble fermentable materials, by the enzymes present in the malt. To accomplish this, the ground malt is mixed with water and placed into a mash tun. To enhance -amylase action during the initial mashing period, the temperature of the mixture is maintained between 40 and 50  C. After a period of time, the temperature of the mix is increased to 65–70  C to enhance -amylase activity. Within a few hours, the process is complete, and the temperature is increased to at least 75  C to inactivate the enzymes. While the amylases are active, they degrade the starch contributed by the grain. Dextrins are produced by the action of the -amylase. The -amylase splits off the disaccharide, maltose, from the amylose portion of the starch. The products that result from the action of the two amylases known as dextrins also undergo additional enzymatic changes. Branch linkages of the amylopectin portion of starch are broken by de-branching enzymes while amyloglucoside removes single glucose residues from the dextrins. Alterations in the color are also noted (changing from light to dark amber) during mashing. The normal pH of malt is approximately 5.8 and is not acidic enough for optimum enzyme activity. To achieve optimum activity, the pH can be reduced to approximately 5.2 for lager production. The pH is adjusted to be more acidic for ale production. Adjustment of the acidity, if desired, can be accomplished by addition of acid, usually lactic acid, or by bacterial fermentation. Although the lactic acid bacteria usually are undesirable contaminants, Lactobacillus delbrueckii has been used to accomplish this pH reduction in the past. This thermophilic bacterium converts sugars to lactic acid efficiently at temperatures of 42–51  C. Because the variety of microorganisms that can grow at these temperatures is small, it is easier to maintain a pure culture bacterial fermentation as well as to reduce the possibility of contamination by other microbes. Processors may find, however, that the bacterial modification requires much greater supervision and, if not controlled, may contribute to beer spoilage. After the naturally occurring and any added enzymes are inactivated, the solids settle out, leaving the wort. Wort contains the soluble compounds including the fermentable sugars and longer, nonfermentative oligosaccharides and is separated from the solids before it is

Applied Microbiology: Agro/Food | Beer/Brewing

transferred into the brew kettle. The spent grain can be used in animal feed. The blend of grains used and the degree of enzymatic activity will influence the composition of the wort. These differences are some of the factors contributing to the characteristics noted in beers from different breweries. Wort Processing, Hop Addition, and Kettle Boil After the wort is transferred into a heating tank, called the brew kettle, hops are added to the wort before the mixture is boiled. The boiling of the wort serves several functions: (1) nearly all the microorganisms remaining after mashing are killed, (2) inactivates enzymes remaining after mashing, (3) enhances extraction of essential oils and resins from the hops, (4) substances responsible for cloudiness are precipitated, (5) enhances color development, (6) undesirable volatiles are removed, and (7) water is evaporated and the wort is concentrated. The wort is boiled for 1–2 h. Hops provide flavor and aroma compounds as well as some compounds, which possess antimicrobial activity. Bitterness is the principal flavor of note. Among the compounds extracted from the hops are essential oils humulone (-bitter acid), lupulone (-bitter acid), and tannin. Important flavor characteristics are contributed by the oils humulone and lupulone. In addition, humulone and lupulone have some antimicrobial properties. The wort is then separated from the spent hops, cooled rapidly, and placed into a fermentation vessel. The spent hops may be used as fertilizer.

Fermentation At this point in the process, the wort is inoculated with brewers’ yeast and fermented. During fermentation, the yeast produces alcohol, carbon dioxide, and some additional flavor constituents. The inoculation step is also called other names, such as pitching and seeding. The fermentation room must be maintained in a clean manner to reduce possible contamination problems, and it should be kept at a constant temperature and humidity to maintain the desired growth rate for the yeast. Fermentation vats can be glassed lined or constructed of

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wood, stainless steel, or aluminum. Wooden vats pose problems in cleaning and disinfecting that are not experienced with vessels made of any of the other materials. The strain of yeast used depends on what type of beer is desired. Lagers are produced using bottom-fermenting yeasts, whereas ales are produced using top-fermenting yeasts. These yeasts have traditionally been referred to as Saccharomyces pastorianus (formerly Saccharomyces carlsbergensis) and Saccharomyces cerevisiae, respectively. Many brewers consider these as separate species, although fungal taxonomists do not recognize them as distinct species (Table 2). Nevertheless, they do behave slightly different during the fermentation process. The temperature of fermentation for lagers produced by the bottom-fermenting yeasts is usually in the range of 6–15  C and takes 2–7 days. During fermentation, the yeasts tend to flocculate and settle to the bottom of the fermentation vat. These yeasts can be collected from the bottom of the vat for reuse in subsequent fermentations. By varying the fermentation temperature, slightly different versions of lagers can be produced. The fermentation process is exothermic so the fermentation tanks must be cooled. Ales are traditionally produced using top-fermenting yeasts and incubation temperatures of 18–27  C for 5–7 days. These yeasts tend to form small clumps of cells that are carried to the top of the fermenting liquid and adsorbed to bubbles of carbon dioxide. These yeast cells can be collected from the surface for reuse with the next fermentation batch. Since the mid-1800s, the use of bottom-fermenting yeasts in closed fermentation vessels has increased worldwide. A corresponding decrease has occurred for topfermenting yeasts in open fermentation vessels. In recent times the advantages of the closed fermenter have given rise to ‘bottom-fermenting’ ale strains. These strains maintain the ale flavor characteristics and can be harvested from the bottom, eliminating the need for an open vessel and the risk of contamination. Regardless of the type of beer made, a rapid decrease in pH during the fermentation will increase its stability and decrease potential problems of contamination. After fermentation, the pH of most lagers decreases from approximately 5.2–5.3 to approximately 4.1–4.2, and it

Table 2 Comparison of Saccharomyces cerevisiae used to make ales and Saccharomyces pastorianus used to make lager yeasts Characteristic

Saccharomyces cerevisiae

Saccharomyces pastorianus

Flocculation

Clumps or flocs entrap CO2; lower density floc; floc rises to the surface of the liquid in the fermenter 15  C >30  C 40  C Some

Higher density floc; floc settles to the bottom of the fermenter 7 C 4.1 and 500 CFU Elevated sewage contamination

This scale is being evaluated for practical use in 2007. A few ‘real-time’ viable cell count methods have been developed and tested in recent years. These methods rely on using ‘vital’ stains to stain ‘live’ cells or ATP detection of live cells. All these methods need careful sample preparation, filtration, careful selection of dyes and reagents, and instrumentation. Usually the entire system is quite costly. However, they can provide one shift results (300 CFU/m3

Total counts for food surfaces (knives, dishes, chopping blocks, etc.)

Ranges

Acceptable Intermediate Not acceptable

0–10 CFU/cm2 10–100 CFU/cm2 >100 CFU/cm2

Source: Al-Dagal and Fung (1993) for air samples. Fung, et al. (1995) for food contact surfaces.

12 log CFU g1. Fecal materials of humans and animals have 11 log CFU g1. Thus, it is not possible to have counts of 13 or 14 log CFU g1 in foods and other materials. Table 1(b) is for total count of air samples and total counts for food contact surfaces such as knives, dishes, and chopping blocks. The air quality is low or acceptable when the count is 0–100 CFU/m3; intermediate or marginally acceptable when the count is 100–300 CFU/m3; and too high and needs corrective action when the count is >300 CFU/m3. Note that in Singapore the air quality is considered not acceptable when it is 500 CFU/m3. The food contact surface is considered low or acceptable when the count is 0–10 CFU/cm2; intermediate or marginally acceptable when the count is 10–100 CFU/cm2; and high or not acceptable when the count is >100 CFU/cm2. The above guides are for total counts and do not include identification of potential pathogens in foods such as Salmonella, S. aureus, and C. perfringens. To appreciate the influence of food composition and storage environments on microbial growth potential in foods, which will affect food spoilage, food preservation,

Intrinsic Parameters of Foods Intrinsic parameters of foods are the inherent characteristics of the food itself, which will have great influence on the microorganisms in the food itself. These six parameters are pH, moisture content, oxidation–reduction potential (Eh), nutrient content, antimicrobial constituents, and biological structures as a part of the food itself that will affect the growth, death, and spoilage potential of microorganism in the food. The pH is the negative log of the dissociated hydrogen ion concentration in the food. It can be measured by a pH meter. The demarcation pH of acid and basic solution is pH 7.00 for water but for food, the demarcation of pH food microbiology is pH 4.5. Foods above pH 4.5 are considered as basic foods and foods below pH 4.5 are considered acidic foods. Basic foods (above pH 4.5) include dairy products (butter, buttermilk, milk, cream, and cheese), which have a pH range of 4.5–6.5; meat and poultry (beef, ham, veal, ground beef, and meat, chicken, shrimp, etc.), which have a pH range of 4.5–7.0; fish and shellfish (fish, clams, crabs, oysters, tuna, salmon, etc.), which have a pH range of 4.8–7.0; some vegetables (asparagus, beans, broccoli, cabbage, carrot, celery, onions, potato, pumpkin, spinach, lettuce, sweet corn, etc.); and some fruits (bananas, figs, melons, and watermelons), which have a pH range of 4.6–7.3. Acidic foods (below pH 4.5) include some vegetables (rhubarb, tomatoes, beets, egg plants, etc.) and some fruits (apples, grapefruits, limes, oranges, plums, grapes, etc.), which have a pH range of 1.8–4.5. Buffering capacity of food is very important relative to microbial growth. For example, meat has high buffering capacity and will resist pH drop unless the microorganisms have sugar to metabolize and produce lactic acid to reduce the pH to a lower level as in the case of fermented sugars. In the case of vegetables, which have low buffering capacity, during the first stage of fermentation, lactic acid bacteria although in low numbers will utilize the carbohydrate in the plant materials and rapidly reduce pH such that the predominant Enterobacteriaceae will die off and lactic acid bacteria will continue to grow to make a successful sauerkraut fermentation. This is an example of microbial succession in food fermentation. Moisture content of foods is defined by total amount of water in the food as well as the water activities of the food. Total water in a food can be obtained by measuring the percentage of total water in food by weight. But not all water is available for microorganisms to use, such as bound water, a better measurement is water activities (Aw) of the food in the environment. This parameter is

Applied Microbiology: Agro/Food | Food Spoilage, Preservation and Quality Control

defined by the ratio of the water vapor pressure of food substrate to the vapor pressure of pure water at the same temperature Aw ¼ p/po, where p is the vapor pressure of solution and po the vapor pressure of solvent (usually water). This concept is related to relative humidity (RH) in the following way: RH ¼ 100  Aw. Pure water has an Aw of 1.00, 1m NaCl has an Aw of 0.9823 and 5m NaCl has an Aw of 0.9174. The Aw of most fresh foods is above 0.99. Minimum Aw for growth of spoilage bacteria is 0.90, spoilage yeast is 0.88, and spoilage molds is 0.80. For special organisms such as salt-loving bacteria (Halophilic bacteria) the Aw is 0.75, dry-loving mold (xerophilic mold) is 0.61, and yeast surviving high osmotic pressure (osmophilic yeast) is 0.6. Minimum Aw of some important microorganisms are presented as follows: Clostridium botulinum, Type E Pseudomonas spp. E. coli Bacillus subtilis Vibrio parahaemolyticus Rhizopus stolonifer Trichosporon pullulans S. aureus Penicillium patulum Aspergillus glaucus Saccharomyces rouxii

0.97 0.97 0.96 0.95 0.94 0.93 0.91 0.86 0.81 0.70 0.62

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Enterobacteriaceae (including Salmonella, Escherichia, Shigella, Arizona, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus, Providencia, and Yersinia), Lactobacillus, Listeria, Staphylococcus, Vibrio, and yeast. Poising capacity is the resistance of food to change the oxidation and reduction potential of foods. For example, the inside of a piece of steak has high poising capacity with low oxygen penetration and ground beef has low poising capacity as it has high oxygen penetration potential. A detailed discussion on anaerobic fermentation was made by Erickson and Fung in Handbook of Anaerobic Fermentation.

Nutrient Content of Food All living things, including microorganisms, need water, source of energy, source of nitrogen, vitamins and related growth factors, and mineral to survive. The composition of the foods will dictate the ability of certain microorganisms to survive and thrive. Water is necessary for all life forms. In food microbiology, the following is the list of organisms according to their need of water (in ascending order): mold, yeast, Gram-negative bacteria, and Grampositive bacteria. The source of energy comes from proteins, starch, polysaccharides, fat sugars, alcohols, and amino acids. Some organisms can break down large molecules such as starch and cellulose, fat, and muscle tissues and then utilize simple essential molecules for metabolism. Other needs a variety of simple nutrients to grow such as the fastidious lactic acid bacteria.

Aw activities in food can be changed rapidly by the environmental conditions during packaging and storage. Antimicrobial Agents

Oxidation-Reduction Potential (O/R, Eh) Basically, this is the amount of oxygen available for microorganisms to grow. Microorganisms are classified as aerobic organisms (need oxygen to grow), facultative anaerobic organisms (can grow in the presence or absence of oxygen), and anaerobic organisms (can grow only in the absence of oxygen). Eh of an environment can be measured by an instrument similar to a pH meter and expressed in millivolts (mV). An environment with þ200 mV and higher is considered as aerobic and an environment with 200 mV and lower is considered as anaerobic environment. Microorganisms vary greatly in their requirement of oxygen and all three categories of microorganisms are important in food microbiology. Typical aerobic microorganisms in food microbiology are Pseudomonas, Acetobacter, Bacillus, Micrococcus, Corynebacterium, and molds, in general. Typical anaerobic microorganisms are Clostridium, Bacteroides, Propionibacterium, Eubacterium, and methanogenic rumen bacteria. Facultative anaerobes constitute a very large group of organisms, which includes the entire family of

Foods from different sources naturally harbor some antimicrobial agents as a part of the chemistry of the food materials. For example, milk has lactenin that prevents the growth of Gram-positive bacteria and eggs have lysozyme that also prevents the growth of Gram-positive bacteria. Cranberry has benzoic acid that prevents the growth of fungi; hence it is not possible to make cranberry wine from natural cranberry juice. Cloves have eugenol that prevents growth of bacteria in general. Spices are rich in antimicrobial agents, for example, garlic has allicin, cinnamon has cinnamaldehyde and eugenol, cloves have eugenol, sage has thujone and cineole, and oregano has thymol and carvacrol. Books have been written specifically related to antimicrobial agents such as Natural Food Antimicrobial Systems by Naidu and Antimicrobials in Foods by Davidson and Branen. Biological structures of foods also play an important part as an intrinsic parameter of food as an intact natural barrier to microbial invasion of the food. Damage of tests of seed, outer covering of fruits, shell of nuts, hides of animals, shells of eggs, and so on or breakage of biological structure can easily allow microbial attachment to the food involved.

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Applied Microbiology: Agro/Food | Food Spoilage, Preservation and Quality Control

Extrinsic Parameters of Foods Temperature of Storage Food-borne microorganisms are grouped as follows. Psychrotrophs are organisms can grow below 10  C and down to about 1  C, but usually prefer to grow at about 21  C. These organisms are responsible for spoilage of refrigerated foods such as cold stored meat, fish, poultry, and vegetables. There are many psychrotrophs but the most important one is Pseudomonas as 90% of psychrotrophs isolated from cold stored food belongs to this genus. Many yeast and mold can also grow at refrigerated temperature. Mesophiles are organisms that can grow best in human body temperature at around 37  C. They can grow well at 15–45  C with optimum at 37  C. These organisms will spoil nonrefrigerated foods kept at normal room temperature. Microorganisms will grow fast and will spoil most food within 4 h. Also this is the temperature range in which pathogenic bacteria such as E. coli O157:H7, Salmonella, Shigella, Listeria, C. botulinum, S. aureus, and Campylobacter can grow to large numbers and produce food-borne infection and food-borne intoxication in humans, which can lead to sickness and even death. Thermophiles are those organisms that can grow above 45  C with optimal temperature of growth at 55–65  C. These organisms are more related to high-temperature storage and treatment of foods such as canned foods. For food safety reasons, the rule is to keep hot food hot at above 140  F or 60  C and cold food cold at below 40  F or 4  C. Note that these temperature values are rounded for ease of memorization and recall for food professionals.

Relative Humidity of Environment It is almost common sense that dry foods when put into moist environment will cause food to gain moisture and when moist foods are put into dry environment will cause food to be dehydrated. Thus humidity of the storage environment and packaging units must be carefully monitored to prevent undesirable changes in food composition caused by moisture gain or loss. In this regard, the temperature of storage is also closely monitored along with RH control. The presence and concentration of gases in the environment also greatly influence the growth and death of microorganisms as well as the quality of the stored and packaged food. Various combinations of carbon dioxide, nitrogen, hydrogen, and air have been intensively studied to maximize optimum quality of many food commodities. Some examples include the following: steaks stored in 100% CO2 had significantly lower bacterial count 16–27 days after slaughter compared to steaks stored in 100% nitrogen or 100% oxygen or in air. When lamb chops were stored at 1  C in 80% oxygen þ20% nitrogen, or

80% oxygen þ20% CO2, or 80% nitrogen or hydrogen þ20% CO2, psychrotrophic organisms decreased successively when compared to those stored in air. Gas environments definitely influence the growth of many organisms in many food products. Time of Storage The length of time of storage is also considered as an extrinsic parameter of foods, because the longer the storage time the more likely microorganisms may metabolize the food involved. There is no question that intrinsic and extrinsic parameters of food in combination will influence the food spoilage, food preservation, and even potential for food-borne infection and intoxication.

Spoilage of Vegetables Modern Food Microbiology by Jay and colleagues is particularly good for a discussion on these topics. It has been estimated that about 25% of all fruits and vegetables harvested for human consumption is lost through microbial spoilage by one or more of 250 market diseases. Bacteria, yeast, mold, and viruses have all been implicated as agents of spoilage. As a matter of fact, the first virus studied in detail was the tobacco mosaic virus, which infected the tobacco leaves. Plants contain a great variety of carbohydrates such as polysaccharides (pentosan, hexosans such as cellulose, starch, xylans, etc.), oligosaccharides (stachyose, raffinose, etc.), disaccharides (maltose, sucrose, cellobiose, trehalose, etc.), monosaccharides (glucose, galactose, fructose, etc.), sugar alcohols (glycerol, sorbitol, etc.), sugar acids (uronic acid, ascorbic acid, etc.), esters (tannins), organic acids (citric, tartartic, oxalic, lactic, etc.), proteins (albumins, globulins, gluten, prolamines, amino acids), lipids (fatty acids, phospholipids, glycolipids, etc.), nucleic acids (purine and pyrimidine), vitamins (fat soluble: A, D, E, K; water soluble: thiamine, niacin, riboflavin, etc.), minerals (Na, K, Ca, Mg, Mn, Fe, etc.), water, and others (alkaloids, porphyrins, aromatics, etc.). In general, the contents of vegetables are: water about 88%, carbohydrate 8.6%, proteins 1.9%, fat 0.3%, and ash 0.84%. These nutrient compositions are suitable for growth of a variety of bacteria, yeast, and mold. Bacteria spoilage include bacterial soft rot, particularly caused by Erwinia carotovora and Pseudomonas marginalis due to break down of pectins giving rise to soft, mushy consistency, bad odor, and water-soaked appearance. Affected vegetables include asparagus, onions, garlic, green beans, carrots, celery, parsley, beets, lettuce, spinach, cabbage, cauliflower, broccoli, radishes, tomatoes, cucumbers, peppers, and others. Other bacteria spoilage conditions include bacterial blight of celery by Pseudomonas apii, bacterial zonate spot of cabbage and lettuce by Pseudomonas cichorii, angular

Applied Microbiology: Agro/Food | Food Spoilage, Preservation and Quality Control

leaf spot by Pseudomonas laychrymans; and bacterial leaf spot of broccoli and cauliflower by Pseudomonas maculicola. Fungal agents. Gray mold rot by Botrytis cinerea, which produces a gray mycelium on high humidity and warm temperature and affects asparagus, onions, garlic, green beans, lima beans, wax beans, carrots, parsnips, celery, tomatoes, lettuce, cabbage, brussels sprouts, turnips, cucumber, pepper, and so on. The mold can enter fruits and vegetables through the unbroken skin or through cuts and cracks. Sour rot is mainly caused by Geotrichum candidum and affects asparagus, garlic, green beans, lima beans, wax beans, endives, lettuce, cabbage, radishes, tomatoes, and so on. Rhizopus soft rot is caused by R. stolonifer and affects green beans, lima beans, wax beans, carrots, sweet potatoes, cabbage, cauliflower, rutabagas, cucumbers, pumpkins, watermelons, tomatoes, and others. The spores and mycelia of sour rot and Rhizopus soft rot molds can be carried by the fruit fly Drosophila melanogaster from decaying fruits and vegetables, and from healthy plants with broken or intact fruits and vegetables. Phytophora rot by Phytophora spp. in the field cause blight and fruit rot and affects asparagus, onions, garlic, cantaloupes, eggplants, pepper, and others.

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Pseudomonas, Salmonella, Serratia, Staphylococcus, Streptococcus, Vibrio, Yersinia, and so on.

Mold Alterniaria, Aspergillus, Cladosporium, Fusarium, Geotrichum, Mucor, Penicillium, Rhizopus, Sporotrichuim, and so on.

Yeast Candida, Debaryomyces, Rhodotorula, Saccharomyces, Torula, Trichosporon, and so on.

Fresh Meat Line of Defense Line of defense by live animal-skin, hair, hide, gastric juice, bile, alkaline conditions, and immune system.

Antemortem

Spoilage of Fruits The average water content in fruits is 84.9%, carbohydrates 13.2%, protein 0.88%, fat 0.53%, and ash 0.46%. Along with vitamins and other organic compounds, fruits are excellent material for microbial growth. Because the pH level of fruits is relatively low and more acidic, yeast and mold have more competitive edge than bacteria. Citrus fruit are more likely to be spoiled by yeast and mold than by bacteria. It is very common to see refrigerated fruits such as oranges, lime, and lemon spoiled by Penicillium, Mucor, Aspergillus, yeast, and so on. Microorganisms can spoil fruits in the fields as well.

Spoilage of Fresh and Processed Meats, Poultry, Egg, and Seafood Bacteria, yeast, and mold are all involved in the spoilage of these products. More than 39 genera of bacteria, 20 genera of mold, and 12 genera of yeast have been reported to be present on these products and many are spoilage organisms. For comprehensive account of these organisms, readers are advised to look into the reference list of Food Microbiology books presented in this section. More familiar genera are listed in this section. Bacteria Acinotobacter, Aeromonas, Bacillus, Brochothrix, Campylobacter, Citrobacter, Coryneforms, Enterobacter, Escherichia, Flavobacterium, Lactobacillus, Leuconostoc, Micrococcus, Moraxella, Pediococcus,

Microbial counts on surface animal can be as high as 5–6 log CFU/cm2 depending on the environment of the animal such as in the field, in mud, in the barn, and so on. These are a combination of aerobic and anaerobic microorganisms. Before birth, the fetus in the mother’s womb is germ-free (gnotobiotic). Once the fetus or baby goes through the birth canal, it is contaminated by various microorganisms present in the mother. Fecal materials of animals have 11 log CFU g1 and mainly anaerobes. Deep tissues of healthy animals are essentially sterile. Interestingly enough, fatigue of animal before slaughtering had a direct effect on the spoilage of the meat after processing. Fatigue of the animal during round-ups, transportation, struggling with other animals, and herding into the slaughter facilities will result in (1) decrease in gastric juice levels, thus less acidic, (2) diminished peristalsis – fecal materials will not move fast, (3) acceleration of blood flow, (4) blood drained from shocked animal is less than the amount drained from unshocked animal, and (5) most importantly, the pH of muscles in fatigued animal is higher (less acidic) than that in unfatigued animal. Meat has about 1% glycogen, which is a source of lactic acid during glycolysis. When an animal is under stress, the glycogen will be converted into lactic acid during abnormal increases of metabolism. The lactic acid is absorbed and further degraded in the animal system. Meat from unfatigued animal has glycogen left and this glycogen will be metabolized to lactic acid through glycolysis and will result in meat having a pH 5.6. The pH of meat from fatigued animal, which has very low glycogen to convert into lactic will be around 6.4.

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Applied Microbiology: Agro/Food | Food Spoilage, Preservation and Quality Control

Slaughter of Animal and Processing These activities on the animal and in the meat will cause a ‘chain of contamination’. Source of microbial contamination include stick knife, scald tank water, skinning process, dehairing and defeathering, evisceration, wash water, air, and workers processing the animal and meat cuts and products. Events in the chilling room (about 15  C) include freeing and reduction of carcass body heat, firming the meat, delaying undesirable microbial growth, delaying chemical changes, and delaying meal shrinkage.

Cutting and Storage These operations are the main sources of microorganisms in and on meat surfaces. Properly processed meat surfaces will be about 3 log CFU/cm2. Cutting of meat will spread the microorganisms to other cuts of the meat during fabrication. If meat is kept anaerobic the predominating organisms are C. perfringens, C. sporogenes, and lactic acid bacteria. If the meat is in aerobic conditions, invariable Pseudomonas will take over particularly in cold stored conditions under 10  C. During cold storage, spoilage will occur on the surfaces of whole or half carcasses. As soon as the meat is cut the surface will be contaminated with microorganism but internal meat muscle will remain sterile. Ground meat, in contrast, will have about 1 million bacteria per gram because of the spreading of microorganisms as well as introducing air into the product through the grounding process. For dry meat products, usually surface spoilage is by molds such as Thamnidium, Sporotrichum, and Botrytis. For cold, aerobic stored meat, the spoilage organism is most likely to be Pseudomonas. For vacuum-packed stored meat, the spoilage will be lactic acid bacteria and if the meat is not chilled properly the organisms will be C. perfringens and C. sporogenes. By the time one can smell spoilage, the meat becomes unsuitable for consumption. There are a great variety of methods to detect spoilage of meats, poultry, and seafood as follows. Chemical methods

Measurement of hydrogen sulfide, mercaptans, noncoagulable nitrogen, di- and trimethylamines, tyrosine, indole, nitrate reduction, creatinine content, dye-reducing capacity, ATP level, lactic acid, color changes, and so on. Physical methods

pH changes, refractive index of muscle juices, electrical conductivity, surface tension, impedance change, micro calorimetry, viscosity, and so on. Direct microbiological methods

Total aerobic bacteria, yeast and mold counts, total anaerobic counts, differential counts of target microorganisms

such as Enterobacteriaceae, coliform, fecal coliform, enterococci, lactic acid bacteria, C. perfringens, and so on.

Spoilage of Fresh Meat Cold stored beef carcasses: A low temperature of 4  C and prolonged storage favor fungi growth. Spoilage by molds involved include: Thamndium, Mucor, Rhizopus which develop ‘whiskers’, Cladosporium (‘Black’ spot), Penicillium (green patches), Sporotricum, and Chrysosporium (white spot). Yeasts involved include Candida lipolytica and C. zayloanoides, torulopsis, and Rhototorula. Ground beef and hamburger are exclusively spoiled by bacteria including Pseudomonas, Acinebacter, Moraxella, Alcaligenes, and Aeromonas. Bacteria isolated from beef, lamb, pork, and fresh sausage include Moraxella, Acinetobacter, Pseudomonas fragi, Pseudomonas fluorescens, Pseudomonas putida, and ‘Pseudomonads’, which is a group of polarly flagellated Gram-negative rods not easily identified as Pseudomonas.

Spoilage of Vacuum-Packaged Meats and Processed Meat Spoilage of long-term cold storage vacuum-packaged meat was mainly by lactic acid bacteria and Brochotothrix thermosphacta. Cooked or partially cooked meat with high pH is spoiled by Yersinia enterocolitica, Streptococcus liquefaciens, Alteromonas putrefaciens, and Lactobacillus. Spoilage of frankfurters, bologna, sausage, and luncheon meats are characterized by sliminess, souring, and greening. Sliminess can be caused by yeast, Lactobacillus, Streptococcus, and B. thermosphacta. Souring can be from growth of lactobacilli, streptococci, and related organisms, and greening can be caused by lactobacilli and Leuconostoc. These organisms produce peroxides that act upon cured meat pigments and produce the green color. Spoilage of bacon and cured ham is caused by a variety of molds such as Aspergillus, Alterniaria, Fusarium, Mucor, Rhizopus, and Penicillium. Bacteria such as Streptococcus, Lactobacillus, and Micrococcus are also involved. Cured ham can be spoiled by souring due to fermentation of sugar in the curing solutions. Organisms involved include Acinetobacter, Bacillus, Lactobacillus, Proteus, and Pseudomonas. Moldiness of cured ham is by a variety of common mold on the surface.

Spoilage of Poultry and Egg One of the major concerns in poultry and egg is Salmonella. Almost all chicken and poultry flocks tested harbored one or more of the 2300þ serotypes of Salmonella. Recently, there is a major change in naming of the genus Salmonella. Now there is only one species of Salmonella, which is Salmonella enterica and all serotypes are now named as

Applied Microbiology: Agro/Food | Food Spoilage, Preservation and Quality Control

serovar of S. enterica. For example, the classic organism Salmonella typhimurium is now named as S. enterica subsp. enterica serotype Typhimurium. Note also that the ‘species’ name ‘Typhimurim’ is capitalized whereas in the old form the species name is not capitalized. As the new long name is inconvenient, some authors use the long form once in an article and then use the old name in the subsequent instances but the species is now capitalized: Salmonella Typhimurium. As fresh poultry products are usually stored aerobically and in cold temperature, without a doubt, Pseudomonas is the most important bacterium in spoilage of poultry meat. Other bacteria include Acinotobacter, Flavobacterium, Streptococcus, and Corynebacterium. Pseudomonas is a psychrotrophic bacterium and can grow below 10  C with the production of odor, slime, pigment, and even fluorescence on poultry meat surfaces. One good demonstration is to put a piece of spoiled, slimy, moist chicken piece under UV light in a box and one can see the chicken ‘glow’ in the dark. At that point, the count of Pseudomonas is about 10 log CFU/cm2 of the chicken meat. Yeasts involved with poultry spoilage include Candida, Rhodotorula, and Torula. Egg is discussed separately because it has different issues in spoilage. An intact egg has a waxy shell, which protects it from microbial invasion. When the shell is cracked, microbial invasion will occur easily and spoil the egg. There is also an inner membrane, which reduces the chance of microbial invasion. In the white of the egg there are lysozymes, which can kill Gram-positive bacteria; conalbumen, which binds iron, is necessary for bacterial growth; and avidin, which binds biotin and makes this important compound unavailable for bacterial growth. Also the pH is very high, at 9.3, which also retards microbial growth. The egg shell has many pores. Microorganisms usually cannot enter the shell easily through the normal pores. However, when an egg is washed the pores will be filled with water and microbes can migrate into the eggs easily. Thus the water used for washing eggs must be chlorinated to kill microbes adequately to reduce spoilage and Salmonellosis. In some countries, merchants are still selling eggs without refrigeration, a practice that seems strange to people from the United States. Microorganisms found in eggs include Pseudomonas, Achromobacterium, Proteus, Salmonella, and others. Spoiled eggs are easy to detect because of the obnoxious odor of hydrogen sulfide produced by some members of the Enterobacteriaceae as well as Pseudomonas. Because of the problem of Salmonellosis, much effort internationally has been made to reduce or eliminate Salmonella from all poultry and egg products. One important area of research is competitive exclusion technology or the Nurmi concept. This idea is to inoculate good bacteria into newly hatched chicks so that Salmonella, Campylobacter, and other pathogenic bacteria cannot be established in the already saturated lining of the intestine of the chick. In the past 30 years much research has been conducted with some encouraging results. Some companies are actually selling live ‘good cultures’ for this

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purpose. In another development, scientists are even trying to use the same concept on cattle using good E. coli to out compete E. coli O157:H7. One group even tried to develop nonpathogenic E. coli O157:H7 to compete with the pathogenic strain. The research is continuing.

Spoilage of Fish and Shellfish Both salt-water and fresh-water fish contain high levels of protein and other nitrogenous compounds. The safety of fish and shellfish depends greatly on the water in which they were reared and harvested. It is, therefore, of great importance to monitor the pollution level of sea water, fresh water, pond and hatchery water, and so on, where these commodities are reared or obtained. In addition, the sanitation of ships and boats and equipment used for catching and processing fish and shellfish must be monitored. Fish muscle has a high level of protein and nonprotein nitrogen. The latter compounds are much easier for microbes to utilize, which is the reason why fish meat spoils much faster than beef, which has low nonprotein nitrogen. Another important fact is that fish muscle has no carbohydrates, thus there is no production of lactic acid in fish meat as opposed to beef meat in which some lactic acid is produced by glycolysis. Common microorganisms found in fish include Pseudomonas, Achromobacter, Alcaligenes, Bacillus, and coryneforms and coliform. Crustaceans include shrimp, lobster, crab, and crayfish, which have about 0.5% carbohydrates and high free amino acid. The spoilage pattern is similar to fish and the most important organism is Pseudomonas along with Moraxella and Proteus. Mollusks include oyster, clam, squid, and scallop and they have 1–3% of carbohydrate in the meat, and free amino acids such as arginine, aspartic acid, and glutamic acid. The spoilage potential is thus different from crustaceans. A drop in pH in the meat of mollusks can indicate spoilage because of fermentation of microbes of the carbohydrate. For oyster meat, a pH of 6.2–5.9 is good, pH of 5.8 is ‘off ’, pH of 5.7–5.5 is musty, and pH of 5.2 and below is sour or putrid. Another important aspect of mollusks is the fact that they filter water through their system; by doing so they can concentrate microbes including pathogenic bacteria and viruses. A variety of bacteria have been isolated from spoiled oysters, which included Serratia, Pseudomonas, Proteus, Clostridium, Bacillus, Escherichia, Enterobacter, and Lactobacillus. Sugar has low Aw, and high osmotic pressure. Leuconostoc mesenteroides can form slime in sugar solution. Molds can grow on top of the product and absorb moisture from air. Spices can have many microorganisms up to 6 log CFU g1. Radiation and ethylene oxide can sterile spices effectively.

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Applied Microbiology: Agro/Food | Food Spoilage, Preservation and Quality Control

Beer unpasteurized (Draft and unfiltered) can be spoiled by Pediococcus cerevisiae and Acetobacter aceti. Opened and leftover beer can be spoiled by the same organisms. Wine can be spoiled by A. aceti, which oxidizes alcohol to vinegar in the presence of air. Nut meat has high fat and low Aw, spoiled mainly by oxidation, chemical deterioration, and lipolytic bacteria. Soft drinks are usually acidic at pH 3.5 and will be spoiled by yeasts and molds. Pickled food such as sauerkraut can be spoiled by bacteria, yeast, mold, or processing steps. Soft kraut is spoiled by excessive pressing of the cabbage and excess fermentation time. Dark kraut is spoiled by mold and plant enzymes. Pink kraut is spoiled by growth of yeast and slime formation is because of Lactobacillus plantarum. Fermented pickles need to absorb salt in the processing step. If the cucumber is too large (more than 4 in. long) salt may not be absorbed fast enough resulting in gas production by Enterobacter and yeast causing floaters and bloaters. Soft pickles are caused by Penicillium and Fusarium enzymes. Black pickles are bacteria that produce hydrogen sulfide. Spoilage of canned foods can result from chemical or microbiological processes or a combination of both. Because the threat of botulism caused by C. botulinum is very severe, canned goods showing spoilage should never be opened or tasted. The rule is to heat the can and the food to 100  C for 10 min and discard the materials because this time and temperature will destroy botulin toxin, which is the most toxic compound produced by a biological system. It has been estimated that 1 pure ounce of the botulin toxin can kill 200 million people! Spoilage of cans can be the result of under-process recontamination by cooling water, leaky seams, physical stress, improper lining of enamel in the can, and extreme acidity of food. Flat sour spoilage has no pressure inside the can caused by Bacillus stearothermophilus, which forms acid but no gas. Flipper and springer have slight pressure. When one squeezes the side of the can, the top and or bottom can slightly yield. When one pokes the top or the bottom the other side will yield. Soft swell is when, with one hard push, the can forms a dent. Hard swell is when one cannot dent this swollen can. TA cans are spoiled by thermophilic anaerobe (Clostridium thermosaccharolyticum), which does not produce hydrogen sulfide but forms hydrogen and carbon dioxide from low and medium acidic foods. PA cans are spoiled by C. sporogenes due to putrefactive anaerobic (mesophilic) organisms with the production of hydrogen sulfide, ammonia, indole, carbon dioxide, and hydrogen generated in the can, which causes the swell. Sulfur stinker spoilage is by Clostridium nigrificans, which produces black hydrogen sulfide compounds and odor. Advanced gas spoilage will cause the can to leak and burst. In glass containers, one can see cloudiness and feel the swell on the cap. Spoilage by nonspore formers

is caused by mild heat treatment and under-processing or leakage. Byssachlamys fulva is a heat-resistant mold that can survive mild heat treatment of cans and cause spoilage.

Microbiology of Food Preservation To reduce and/or prevent food spoilage by microorganisms, we need to use various methods to preserve our food supplies from microbial activities. There must be a balance of killing microorganisms with preservation methods and the quality of the foods being treated as well as the economy related to the treatment methods. To prepare food for processing, one needs to use wholesome food for the treatment. At times, people tend to use lower quality of food for preservation and sell better quality food for higher price. That is not a recommended practice. Food commodities for preservation first need to be cleansed with water to remove unwanted food particles, soil, insects, chemicals, and other extraneous matters. After that, workers need to peel, trim, and sort the food materials for quality control and assurance. One of the important procedures in food processing is blanching. Boiling raw materials for 1 min in water during blanching will inactivate enzymes, enhance/fix green color for plant materials, reduce the number of microorganisms usually by about 10–50%, reduce food volume by displacement of entrapped air, and so on.

Food Preservation by Drying and Dehydration Drying or dehydration is one of the oldest methods in food processing. Originally, human beings used the sun to dry food and later used the process of dehydration by fire, hot air, and so on. Sun drying is an uncontrolled removal of water as the procedure depends on the availability of sun energy. It requires a large amount of space to spread out the food in thin layers horizontally or vertically and is inexpensive but subject to contamination by insects, rodents, birds, large and small animals, and human beings. Dehydration is removal of water from food under controlled conditions of temperature, humidity, air flow, and so on. It has economy of space and good sanitation, but it is far more expensive than sun drying. Foods involved include raisins, figs, nuts/grains, meats, fruits, and fish. The purposes for drying food are to prevent microbial growth and chemical deterioration, ease of storage, packing, and transportation. Some of the types of dryers are (1) heated air as the drying medium; (a) cabinet dryer in which hot air rises through the food and carries the moisture from the food in a cabinet; (b) tunnel dryer in which foods are placed on a conveyer belt and travel through a hot cannister (tunnel) where hot air removes

Applied Microbiology: Agro/Food | Food Spoilage, Preservation and Quality Control

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moisture from the food as they pass through the tunnel; and (c) kiln dryer is similar to cabinet dryer but in a much larger scale. (2) Heat transfer through solid surface: (a) drum dryer. Products flow over the surface of heated stainless-steel drums. The products are dried and are scraped off with stationary blades, for example milk, fruits, vegetable juice, purees, and cereal; (b) vacuum shelf drying – uses lower temperature. As food is placed on heat shelves, a vacuum is applied which will allow moisture to leave the food at a lower temperature thus protecting the flavor and color of the food.

meat, milk, etc.), refrigeration (0–2  C and 5–7  C for perishable and semi-perishable foods), and freezing (150 30–630

95–180 768–3740 3806–6476 ND 2200a 1400

0.4–0.75 0.2–6.7 0.2–2.1 ND 26a 7.2–18.5

0.02 0.1–0.2 0.006–0.06 ND 0.5–0.6 1.8–2.0

7200b

7200c

78c

5–10b

a

Values for 99.9% inactivation at pH 6–9. 99% inactivation at pH 7 and 25  C. 90% inactivation at pH 7 and 25  C. ND – No data. All CT values are for 99% inactivation at 5  C except where noted.

b c

Applied Microbiology: Agro/Food | Water, Drinking

Chlorine Table 2 shows that enteric viruses (represented by poliovirus type 1) are more resistant to inactivation by chlorine than are bacteria (represented by E. coli), and protozoan cysts are nearly two orders of magnitude more resistant than the enteric viruses. Chloramines Comparison of chloramines with chlorine for disinfection of microorganisms (Table 2) shows that, in general, for all types of microorganisms, CT values for chloramines are higher than CT values for free chlorine species. Chlorine dioxide Chlorine dioxide CT values in Table 2 show that at pH 7.0, ClO2 is not as strong a bactericide and virucide as HOCl. However, as the pH is increased, the efficiency of ClO2 for inactivation of viruses increases. CT data for protozoan cyst inactivation is not available. Ozone Overall, comparison of CT values for ozone with those for chlorine and ClO2 indicates that ozone is a much more effective biocide than the other disinfectants. E. coli is about tenfold (1 log10) more sensitive to ozone (Table 2) than poliovirus type 1. Giardia muris cysts are about tenfold more resistant to ozone than poliovirus type 1. Since ozone is a powerful oxidant, it reacts rapidly with both microorganisms and organic solutes and is very useful as a primary disinfectant. The order of microbial disinfectant efficiency is O3 > ClO2 > HOCl > OCl > NH2Cl > NHCl2 > RNHCl (organic chloramines). However, for technical reasons, practical handling considerations, cost and effectiveness, the frequency of use of disinfectants by utilities in the United States is generally chlorine >> chloramines > O3 > ClO2. Ultraviolet Light Sensitivity of the various microbial groups to ultraviolet light is similar to that for chemical disinfectants. Enteric bacteria are most sensitive, followed by enteric viruses; protozoan cysts are the least sensitive. Organisms that are sublethally injured by UV light exposure may, under appropriate conditions, be able to repair the damage (i.e., photoreactivation or dark repair). Ranges of UV doses required for 99.9% inactivation of microorganisms of concern in drinking water are: bacteria, 7–167 mJ cm2; viruses, 16–167 mJ cm2; protozoan oo(cysts), 11–119 mJ cm2.

Distribution Systems Description

Water transmission and distribution systems are needed to deliver water to the consumers. In 2006, the US National Academy of Sciences published a report, ‘‘Drinking Water Distribution Systems: Assessing and Reducing Risks’’ that

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recommended the following measures to control waterborne pathogens entering the distribution system. 1. Storage facilities should be inspected on a regular basis. 2. Better sanitary practices are needed during installation, repair, replacement, and rehabilitation of distribution system infrastructure. 3. Water residence times in pipes, storage facilities, and premise plumbing should be minimized. 4. Positive water pressure should be maintained. 5. Distribution system monitoring and modeling are critical to maintaining hydraulic integrity. 6. Microbial growth and biofilm development in distribution systems should be minimized. 7. Residual disinfectant choices should be balanced to meet the overall goal of protecting public health. 8. Standards for materials used in distribution systems should be updated to address their impact on water quality, and research is needed to develop new materials that will have minimal impacts. 9. Although it is difficult and costly to perform, condition assessment of buried infrastructure should be a top priority for utilities. 10. Cross-connection control should be in place for all water utilities. 11. Where feasible, surge protection devices should be installed. 12. Prior to distribution, the quality of treated water should be adjusted to minimize deterioration of water quality. Distribution systems represent the major investment of a municipal water works and consist of large mains that carry water from the source or treatment plant, service lines that carry water from the mains to the buildings or properties being served, and storage reservoirs that provide water storage to meet demand fluctuations, for firefighting use, and to stabilize water pressure. The branch and loop (or grid) are the two basic configurations for most water distribution systems. The layout of a branch system is similar to that of a tree branch, with smaller pipes branching off from larger pipes throughout the area served. This system, or a derivative of it, is normally used to supply rural areas where water demand is relatively low and long distances must be covered. Disadvantages of this configuration are the possibility that a large number of customers will be without service should a main break occur, and the potential water quality problems in parts of the system resulting from the presence of stagnant water. System flushing should be accomplished at regular intervals to reduce the possibility of water quality problems. The loop configuration currently is the most widely used distribution system design. Good design practices for smaller systems call for feeder mains to form a loop approximately 1 mi (1600 m) in radius around the center

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Applied Microbiology: Agro/Food | Water, Drinking

of the town with additional feeder loops according to the particular layout and geography of the area to be served. The area inside and immediately surrounding the feeder loops should be gridded with connecting water mains on every street. The most commonly used pipes for water mains are ductile iron, pre-stressed concrete cylinders, polyvinyl chloride (PVC), reinforced plastic, steel, and asbestos cement. Microbiology

Microbiologically, water distribution systems are interesting bacterial ecosystems that present a real challenge to the water utilities in terms of maintaining good-quality water with low bacterial densities. The construction characteristics, operation, and maintenance of a water distribution system provide ample opportunities for microbial recontamination of the treated water during distribution. Pipe joints, valves, elbows, tees, and other fittings as well as the vast amount of pipe surface provide both changing water movement and stagnant areas where bacteria can attach and colonize. Water distribution systems are susceptible to cross-connections that may allow entry of pathogens into the system. A cross-connection is any direct connection between the drinking water distribution system and any nonpotable fluid or substance. Biofilms in water distribution systems

Bacteria found in water distribution systems can be classified into indigenous (autochthonous) and exogenous (allochthonous) populations. The indigenous organisms are well-adapted biofilm-forming bacteria that represent a stable ecosystem that is difficult to eradicate. The exogenous bacteria are contaminants that are transported into the system by a variety of mechanisms. The development of a permanent biofilm in the distribution system occurs because the bacteria find physical and chemical conditions conducive to colonization and growth at the solid surface/water interface. These conditions include an ample supply of nutrients (AOC) for growth, a relatively stable temperature, and some degree of protection from exposure to harmful chemicals such as the disinfectant(s) used to treat the water. When an adequate disinfectant residual is maintained in the water throughout a distribution system, growth of bacteria is usually well controlled and the density of bacteria in the bulk water traveling through the pipes will remain low – in the range of 10 mmol l–1), which would be sufficient to sustain slow growth, did inhibit further development. Response to C-Signal As mentioned in the previous section, C-signal manages cell movement to build the fruiting body. It also initiates the expression of many C-signal-dependent genes, and it initiates spore formation. It coordinates these processes in time and in space, using the signal transduction circuit shown in Figure 20. Each of the several processes indicated in the figure has been shown to have their own threshold C-signal level, which implies different thresholds for different genes. C-signal on the surface of the signal donor cell interacts with a receptive cell by direct contact between motile cells, as mentioned in Figure 17. Thus, C-signal transmission conveys the fact that the signaling cells are in end-to-end contact with each other, and most probably they are at high cell density. Due to the positive feedback in the C-signaling circuit controlled by the act operon indicated in Figure 20, progressively larger numbers of C-signal molecules are displayed on the signal donor cell as development proceeds. The level of FruAP in the signal recipient cell tracks the donor signal level (Figure 20). FruA Figure 20 shows that activated FruA has two kinds of activities. First, it signals the frizzilator to regulate cell movement, as discussed in the previous section. Second, FruA, or FruAP, also activates expression of many developmentally regulated genes. Consequently, fruA mutants have abnormal aggregation, they fail to form

A-signal

Cell movement, reversal, streaming FruA

High

Sporulation genes FruA~P act ABCDE

Aggregation genes

dev operon

Ω7536

Waves Low

csgA

C-signal on cell surface

C-signal sensor (hypothetical)

Figure 20 C-signal transduction pathway and gene expression. (An expansion of Figure 17.) The boxed gradient represents the level of FruAP required to activate a particular C-signal-dependent gene.

242

Bacteria | Myxococcus

fruiting bodies, or to differentiate spores. Transcription of the fruA gene itself is induced early in development by the A-signal, as indicated in the figure. The fruA gene belongs to expression group (ii), and the FruA protein can be detected in cells at about 6 h of development. FruA protein is a DNA-binding response regulator that is a member of the Fix J subfamily, and it has a helix-turn-helix motif. Following C-signal transmission, FruA protein is activated by a posttranslational modification, which is necessary for aggregation and sporulation. The modification is likely to be phosphorylation of the aspartate-59 equivalent (FixJ residue number) in FruA. dev Operon The C-signal regulates expression of the dev operon via FruAP, which acts as a transcription factor for the operon (Figure 20). The dev operon consists of five genes of which only the latter three, devTRS, have been characterized. Mutants defective in devTRS are able to aggregate, but they fail to sporulate. Expression of dev is negatively autoregulated as well as spatially restricted to the fruiting body; peripheral rods do not express dev. According to Viswanathan and coworkers, this regulatory pattern results from several positive elements located between 500 bp upstream of the promoter and 580 bp downstream of the transcriptional start site. The 35 and 10 of the promoter resemble those recognized by A RNA polymerase, but there is very little expression of dev during growth. In addition, a negative element is located between þ219 and þ280 that is responsible for the observed negative autoregulation. Finally, there is evidence for interactions between upstream and downstream regulatory elements that suggest DNA looping. Binding sites for FruA have been found in the dev promoter that explain its FruA-dependence. Recently, a consensus site for FruA has been proposed. Expression of the sporulation gene marked by the Tn5 lac insertion 7536 is dependent on the dev operon. Since that insertion mutant aggregates normally yet fails to sporulate, it can be placed in the sporulation pathway downstream of dev (Figure 20). The morphological differentiation of myxospores occurs once aggregation is complete. Nevertheless, M. xanthus can bypass the multicellular steps of fruiting body construction and proceed directly to sporulation when certain substances that interfere with peptidoglycan turnover are added to a growing culture of vegetative cells. For example, glycerol or dimethylsulfoxide added to growing cells induce more than 90% of them to become spores. This conversion of a rod-shaped cell to a spherical spore is much faster, more synchronous, and more efficient than in fruiting body sporulation. Thus, a cell intrinsic capacity for sporulation awaits activation by the dev operon. Just as fruiting body development initiates -galactosidase production from the

7536 reporter, glycerol does so as well, suggesting that

glycerol induction enters the sporulation branch after the dev operon and before 7536. Binding sites for FruA have also been found in the act promoter, the fdgA promoter, the dofA promoter, and the

4400 promoter. It is thought that those and other developmentally regulated genes would be expressed when the level of FruAP had risen to the level appropriate to each gene. The various FruAP levels are represented by the gradient in Figure 20. More than 50 genes appear to encode EBP transcription factors in the M. xanthus genome. S. cellulosum, another myxobacterium whose genome has recently been completed has 76 in its 13 Mb genome. Remarkably, EBPs constitute similar proportions of their total gene content. Might this be a general property of the myxobacteria? Many EBPs are components of signal transduction circuits that regulate transcription in response to environmental cues. A-signal, discussed above, is an example. EBPs have a common domain organization with a central AAA-ATPase domain responsible for ATP hydrolysis and interaction with 54, a C-terminal DNA-binding domain, and an Nterminal sensory domain that regulates the ATPase activity of the central domain in response to stimuli, shown in Figure 21. The N-terminal sensory domain is the most variable from one EBP to another, and two distinct groups of N-terminal sequences have thus far been recognized in M. xanthus. Most often, a two-component response regulator receiver domain is found in that position. In addition, a forkhead-associated (FHA) domain is found as the Nterminal sensory domain in 13 EBPs. The FHA domains of the 13 are shown aligned with the prototypical RAD53 FHA1 from Saccharomyces cerevisiae in Figure 21. The FHA domain was deleted from the Mx4885 EBP in M. xanthus, and the mutant was found to have a deficiency in raising the level of FruAP after C-signaling. In addition, aggregation was delayed for a day, sporulation was decreased by more than 99%, and several developmentally regulated genes were not expressed. Clearly, the FHA domain is necessary for the function of this EBP. FHA is a phospho-threonine recognition domain indicating that the EBP interacts with proteins that are themselves regulated by reversible protein phosphorylation by the action of serine threonine protein kinases (STPKs) and phosphatases. The simplest signal transduction pathway involving an FHAEBP protein would be a direct interaction between the EBP and a cognate STPK. The STPK, having been autophosphorylated in response to a particular stimulus, would then phosphorylate the EBP, and activate it to initiate transcription. This suggested pathway remains to be experimentally verified. M. xanthus encodes 99 STPKs and S. cellulosum encodes 317. These two and presumably the other myxobacteria have forged a novel link between 54-dependent developmental gene expression and signal transduction pathways involving STPKs. The general importance of STPK in

Bacteria | Myxococcus Forkhead sensory domain

σ 54 interactionATPase domain

N

243

DNA binding domain C

Figure 21 Domain organization of enhancer binding proteins (EBPs) with forkhead-associated (FHA) DNA-binding domains. The FHA domains are thought to recognize phospho-threonine residues in proteins and to interact with specific ones. Reproduced with permission from Jelsbak L, Givskov M, and Kaiser D (2005) Enhancer-binding proteins with a forkhead-associated domain and the 54 regulon in Myxococcus xanthus fruiting body development. Proceedings of the National Academy of Sciences of the United States of America 102: 3010–3015. Copyright (2005) National Academy of Sciences, U.S.A.

M. xanthus became clear in 1992, when more than 26 STPK genes had been found by Southern blot hybridization by Sumiko Inouye and her colleagues. Expression of STPK no. 1 (Pkn1) had been shown in developing cells but not in growing cells, and Pkn1 deletion mutants made small, less compact fruiting bodies. Deletion of the STPK genes has shown that at least one-fifth of them are essential for fruiting body development and sporulation. Eighty-three of the M. xanthus STPK genes appear to have arisen by duplication in the delta-proteobacterial lineage that gave rise to the myxobacteria with their enhanced sensory complexity.

development, surface motility, and cell-to-cell signaling. The genome of M. xanthus is quite large (9.14 Mb), and gene duplication followed by divergence appear to have been the major contributors to a large genome size. Over 1500 lineage-specific duplications were identified within the M. xanthus genome, representing over 15% of the total genes. Expansion of the genome was not random as the genes most closely associated with cell-to-cell signaling, small molecule sensing, and integrative transcription control were selectively amplified. M. xanthus devotes almost 9% of its genes to the production of secondary metabolites, but those genes were not found among the lineagespecific duplications.

Conclusions

See also: Dictyostelium; Biofilms, Microbial; Ecology, Microbial; Food Webs, Microbial; Low-Nutrient Environments; Nutrition, Microbial; Quorum-Sensing in Bacteria

Myxobacteria are one of nature’s explorations of survival benefits to be gained from a communal life. These singlecelled, soil-dwelling prokaryotes move and feed in predatory groups. Myxococcus also constructs species-specific multicellular structures called fruiting bodies and differentiates spores within them for survival in the long term. Sporulation and growth alternate according to nutrient (prey) availability. Nutrient limitation initiates fruiting body development, whereas nutrient availability leads spores to germinate and supports vegetative growth. Cooperation between cells during development is orchestrated by the cell-to-cell exchange of soluble and contact-mediated extracellular signals. A complex signaling network controls cell movement and regulates gene expression. The ecological success of myxobacteria is reflected in an estimated density of a million cells per gram of cultivated soil and in the fact that most of their 40 species can be isolated from soils around the earth. Thus, M. xanthus is an attractive model for bacterial

Further Reading Collins RF and Derrick JP (2007) Wza: A new structural paradigm for outer membrane secretory proteins. Trends in Microbiology 15: 96–100. Dworkin M and Kaiser D (eds.) (1993) Myxobacteria II. Washington, DC: American Society for Microbiology. Goldman BS, Nierman WC, Kaiser D, et al. (2006) Evolution of sensory complexity recorded in a myxobacterial genome. Proceedings of the National Academy of Sciences of the United States of America 103: 15200–15205. Gronewold TMA and Kaiser D (2001) The act operon controls the level and time of C-signal production for M. xanthus development. Molecular Microbiology 40: 744–756. Igoshin O, Goldbetter A, Kaiser D, and Oster G (2004) A biochemical oscillator explains the developmental progression of myxobacteria. Proceedings of the National Academy of Sciences of the United States of America 101: 15760–15765. Kaiser D (2004) Signaling in myxobacteria. Annual Review of Microbiology 58: 75–98.

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Lobedanz S and So¨gaard-Andersen L (2003) Identification of the C-signal, a contact-dependent morphogen coordinating multiple developmental responses in Myxococcus xanthus. Genes & Development 17: 2151–2161. Morett E and Segovia L (1993) The 54 bacterial enhancer-binding protein family: Mechanics of action and phylogenetic relationship of their functional domains. Journal of Bacteriology 175: 6067–6074. Sager B and Kaiser D (1994) Intercellular C-signaling and the traveling waves of Myxococcus. Genes & Development 8: 2793–2804.

Viswanathan P, Ueki T, Inouye S, and Kroos L (2007) Combinatorial regulation of genes essential for Myxococcus xanthus development involves an upstream response regulator and a downstream LysRtype regulator. Proceedings of the National Academy of Sciences of the United States of America 104: 7969–7974. Wolgemuth C, Hoiczyk E, Kaiser D, and Oster G (2002) How myxobacteria glide. Current Biology: CB 12: 369–377. Yu R and Kaiser D (2007) Gliding motility and polarized slime secretion. Molecular Microbiology 63: 454–467.

Pseudomonas A Zago, Northwestern University, Chicago, IL, USA S Chugani, University of Washington, Seattle, WA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Human Pathogens Plant Pathogens Environmental Aspects of Pseudomonas

Glossary biofilms Aggregation of microorganisms embedded in an adhesive and protective matrix. hypersensitive response A plant defense mechanism against pathogenic microorganism. operon A transcriptional unit encoding one or more genes transcribed from the same promoter to produce a single messenger RNA (mRNA).

Abbreviations

HCH 2-DE ABC AHL AlgL AP ASD CF CFTR CTX Dab DAP ECF EF-2 EPS ExoS FAS HR INA

-hexachlorocyclohexane two-dimensional gel electrophoresis ATP-binding cassette acyl-homoserine lactone alginate lyase alkaline protease aspartate -semialdehyde dehydrogenase cystic fibrosis cystic fibrosis transmembrane conductance regulator cytotoxin 2,4-diaminobutyrate diaminopimelic acid extracytoplasmic function eukaryotic translation factor-2 exopolysaccharides exoenzyme S factor for activating exoenzyme S hypersensitive response ice-nucleation-active

Defining Statement The genus Pseudomonas represents a physiologically and genetically diverse group with a great ecological

Genetic and Molecular Tools Used to Study Pseudomonas Biotechnology Further Reading

opportunistic organism An organism that is generally harmless and becomes pathogenic in an immunocompromised host. sigma factor A protein that helps the RNA polymerase core enzyme to recognize the promoter at the start of a gene. transcriptional regulation Mechanisms that regulate the expression of a specific gene or operon.

IR IS IVET LPS LTTR mRNA NRPS OMP ORF PAH PCB PLC PLC-H PMI-GMP PQS PVD SOD TCE Vfr

inverted repeat sequence insertion sequence in vivo expression technology lipopolysaccharide LysR-type responsive transcriptional regulators messenger RNA nonribosomal peptide synthetases outer-membrane protein open reading frame polycyclic aromatic hydrocarbon polychlorinated biphenyls phospholipases C hemolytic phopholipase C phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase Pseudomonas quinolone signal pyoverdine superoxide dismutase trichloroethane Virulence factor regulator

significance. The article describes this widely investigated and ubiquitous group of bacteria, their pathogenesis in humans, the complex interactions with plants, and their active metabolism in the environment.

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Bacteria | Pseudomonas

Introduction Pseudomonads are ubiquitous microorganisms, found in all major natural environments and in intimate association with plants and animals. Their wide distribution reflects a remarkable physiological and genetic adaptability. In humans, they are opportunistic pathogens, found in lungs of cystic fibrosis (CF) patients, in people with eye infections, in burn victims, and in AIDS patients. Pathogenicity is due to the secretion of a large number of toxins, which weaken or allow evasion of the host immune system, enabling the bacteria to survive. In plants, they cause disease by producing hypersensitive response (HR), resulting in leaf and root tissue damage. In soils, they detoxify environmentally hazardous compounds, such as aromatic compounds, halogenated derivatives, and various recalcitrant organic residues. The genus Pseudomonas includes Gram-negative, non-spore-forming, rod-shaped bacteria, motile by means of one or more polar flagella. They are obligate aerobes, but some species can grow in anaerobic conditions in the presence of nitrate. Catalase positive, they metabolize sugars oxidatively and none is fermentative or photosynthetic. The size of Pseudomonas is about 1–5 mm long and about 0.5–1.0 mm wide. The genome sizes vary from 3 to 7 Mb with a substantial genome size polymorphism within each species and a GC content ranging from 58 to 70 mol%. The family Pseudomonadaceae is classified, on the basis of rRNA:DNA hybridization analysis and 16S rRNA sequencing, into five rRNA groups. The current genus of Pseudomonas is restricted to the rRNA group I, which belongs to the subclass of the Proteobacteria. This genus contains mostly fluorescent Pseudomonas spp. as well as few nonfluorescent species. The rRNA group II Pseudomonads belong to the genera Burkholderia and Ralstonia, organisms of rRNA group III that are now classified in the family Comamonadaceae, while the rRNA groups IV and V form the genera Brevundimonas and Stenotrophomonas, respectively.

Human Pathogens Pseudomonas aeruginosa is an opportunistic human pathogen capable of colonizing and infecting virtually any tissue. Since this microorganism is ubiquitous in nature, most human hosts counteract the infectious process effectively via the innate immune system. All clinical cases of P. aeruginosa infection are associated with a compromised host defense. The three most common human diseases caused by P. aeruginosa are bacteremia in severe burn victims, chronic lung infection in cystic fibrosis (CF) patients, and acute ulcerative keratitis in coal miners, farmers, and users of extended wear contact lenses. P. aeruginosa also causes osteomyelitis and urinary tract

infections. This pathogen produces many factors that promote adherence to host cells and mucins, damage host tissues, elicit inflammation, and disrupt defense mechanisms. Investigation of these bacterial virulence factors has provided understanding of P. aeruginosa pathogenesis at the molecular and cellular level. Virulence Factors and Pathogenicity Flagella

Pseudomonas is motile by a single, polar flagellum and exhibits chemotaxis to favorable molecules, such as sugars. The flagella of P. aeruginosa have been associated with virulence since nonflagellated mutants do not readily establish infection in animal models and demonstrate reduced invasion of cultured corneal epithelial cells. Strains of P. aeruginosa express either an a- or b-type flagellum. This classification is primarily based on the apparent size of the flagellin subunit (encoded by the fliC gene) and its antigenicity. The a-type flagellins are heterogeneous and are divided into various subgroups, whereas the b-type flagellins are homogeneous. The a-type fliC open reading frame (ORF) varies in length between 1164 and 1185 bp, with the subunit size ranging from 45 to 52 kDa. The b-type fliC open reading frame is 1467 bp in length and encodes a 53 kDa size protein. The N- and C-terminal sequences of both these flagellins are nearly identical, whereas the central region is variable. The a-type flagellins undergo glycosylation, whereas the b-type flagellins are phosphorylated at tyrosine residues. Such modifications are unique among the prokaryotic flagella, which are often methylated at the lysine residue. The phosphorylated flagellin protein is believed to serve as a signal for intact flagellin export from cytoplasm to the flagellar assembly apparatus. Expression of flagellar motility requires more than 40 genes controlled by a hierarchical transcriptional regulation organized in four levels. Class I genes are constitutively expressed and include the transcriptional regulator fleQ and the alternative sigma factor fliA (28). FleQ directly or indirectly regulates the expression of the majority of flagellar gene promoters with the exception of fliA (rpoF). FliA, a member of the alternative sigma factors 28, regulates transcription of the major flagellin subunit (fliC). Transcription of fleQ gene is controlled by 70 and is repressed by virulence factor regulator (Vfr). Class II genes include the two-component regulatory system FleSR and require FleQ and RpoN (54) for their transcriptional activation. FleR and RpoN regulate expression of class III genes. The anti-sigma factor FlgM binds and inhibits FliA. When FlgM is secreted through the hookbasal body rod structure assembled by class II and III genes, FliA is free to activate expression of class IV genes. Pili

The type IV pili of P. aeruginosa or N-methyl-phenylalanine (NMePhe) pili are an important cell-associated

Bacteria | Pseudomonas

virulence factor that plays a crucial role in mediating bacterial adherence and colonization of mucosal surfaces and a flagella-independent method of surface translocation known as twitching motility. Pili also serve as the receptors for bacteriophage. The pili are long polar filaments consisting of homopolymers of a 15–18 kDa protein, called pilin, which is encoded by the pilA gene. PilA is first synthesized as a prepilin, which then undergoes processing during its export to produce the final pilin subunit. After cleavage, the leader peptide of the newly generated N-terminus undergoes methylation. There are three other accessory genes, designated pilBCD, which are required for the biogenesis of pili. These genes are located adjacent to the pilin structural gene. The pilD gene encodes the prepilin peptidase and the methylase that process the prepilin protein. A multimeric outer membrane protein (PilQ) forms gated pores in the outer membrane, through which the pilus is thought to extrude. In P. aeruginosa, the pilQ gene is located in an operon that includes four other genes (pilMNOPQ) also required for pili assembly, twitching motility, and phage sensitivity. The pilin protein is retained in the outer membrane of the cell before its assembly into the intact pilus. Pilin filaments have a diameter of 5.2 nm, with an average length of 2.5 nm, and the subunits are arranged in a helical array, which forms a hollow cylindrical structure. The pilin C-terminal 12–17 semiconserved amino acid residues are exposed at the tip of the pilus and bind to the asialo-GM1 and asialo-GM2 on epithelial cells. Transcription of PilA in P. aeruginosa is controlled by the RpoN-dependent, two-component regulatory system PilR/PilS. PilS is a transmembrane protein located at the pole of the cell, which responds to a signal that remains to be identified. Twitching mobility in P. aeruginosa is also controlled by the sensor–regulator pair FimS/AlgR. Since fimS and algR mutants lack extracellular pili and FimS and AlgR do not affect PilA expression, these two proteins must be involved in the regulation of some other aspects of the system. In addition, twitching motility is also controlled by Vfr, which may bind cAMP and cGMP, widely utilized by P. aeruginosa for global physiological regulation. Lipopolysaccharide

The lipopolysaccharide (LPS) produced by P. aeruginosa is a key factor in virulence, protects the bacterial cells from host defense, and mediates entry into eukaryotic cells. It is a typical Gram-negative bacterial LPS, with a basic lipid A structure formed by an N- and O-acylated diglucosamine biphosphate backbone that anchors the LPS molecule into the outer leaflet of the bacterial outer membrane. Lipid A binds to a core oligosaccharide region, which can be divided into inner and outer core. The inner core oligosaccharide consists of two 3-deoxy-D-manno-oct-2-ulosonic acid residues (KdoI and KdoII) and two L-glycero-D-manno-heptose residues (HepI and HepII). The latter can be phosphorylated in three

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major sites and 7-O-carbamoylated on HepII. LPS phosphorylation, which occurs only in the inner core, is essential for bacterial viability and it is associated with intrinsic resistance to some antibiotics. The outer core contains an N-alanylated galactosamine residue, three D-glucose residues, and one L-rhamnose residue. The core structure is linked to the O-antigen, which is responsible for conferring serogroup specificity and diverges in 11 chemical variants (N-acyl derivatives of various amino sugars). The LPS outer core binds to cystic fibrosis transmembrane conductance regulator (CFTR) host receptor to mediate bacterial internalization into epithelial cells. This interaction activates NF-B nuclear translocation; however, a lag in the immune response allows P. aeruginosa to establish itself in the lungs. P. aeruginosa strains may be either LPS smooth (expressing many long O side chains) or LPS rough (expressing few, short, or no O side chains). Although resistance to serum is conferred by the smooth phenotype, LPS-rough isolates unable to produce O-antigen predominate in CF lungs, which suggest that LPS-mediated serum resistance is not essential for survival of P. aeruginosa in CF lungs. Alginate

In the human pathogen P. aeruginosa, alginate is an important virulence factor during infection of human epithelia. Alginate is a linear nonrepetitive copolymer of -D-mannuronic acid linked to its C5 epimer -Lguluronic acid via (1–4) glycosidic bonds. The production of extensive amounts of alginate confers a mucoid phenotype and is associated with the formation of biofilms. In CF patients, P. aeruginosa can colonize the lungs and contribute significantly to the disease. Since alginate production confers protection to P. aeruginosa from the local immune response and from antibiotics treatment, appearance of mucoid strains in CF lungs leads to a chronic infection and progressive decline in pulmonary function. Alginate biosynthetic pathway (Figure 1) is well characterized and consists of a cluster of 12 genes (algD, 8, 44, KEGXLIJFA). In this cluster, algA and algD encode enzymes involved in the precursor synthesis. AlgA is the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (PMI-GMP) that acts at the first and third steps of the alginate synthesis. PMI scavenges the fructose-6-phosphate from the metabolic pool and diverts it to the alginate synthetic pathway. The resulting mannose-6-phosphate is directly converted to mannose-1phosphate by the phosphomannomutase AlgC. This enzyme is also involved in rhamnolipid and LPS synthesis, which reflects its genomic localization outside of the alginate biosynthesis cluster and its own promoter regulation. Finally, mannose-1-phosphate is converted to GDP-mannose by AlgA GMP. GDP-mannose is further oxidized to GDP mannuronic acid by GDP-mannose dehydrogenase encoded by algD. Once GDP mannuronic acid is

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Bacteria | Pseudomonas (a)

LPS

F6P AlgA

M6P

M1P AlgA

AlgC

GDP Man

GDP Rha

LPS

AlgD

GDP – ManA

Alg Alg 8 44

Inner membrane

M Ac

Alg I AlgF

Alg J

Alg Alg Alg K G X

Periplasmic space AlgL

AlgE

Outer membrane

Ac

Ac

Ac

M M M M G M M M M M (b) algD

algP

alg8

algQ

alg44

algK

algR

algZ

algE

algG

algC

algX

algB

algL

algI

algJ

algF

algT mucA mucB mucC

algA

mucD

Figure 1 (a) Pseudomonas aeruginosa alginate biosynthesis. Fructose-6-phosphate (F6P), obtained from the metabolic pool, is converted to GDP-mannuronic acid (GDP-ManA), which provides mannuronate residues (M) for polymerization. Occasionally, guluronate residues (G) are incorporated via epimerization of mannuronate residues by the AlgG protein. Mannuronic acid residues of bacterial alginate are partially O-acetylated by the membrane complex formed by AlgF, AlgJ, and AlgI proteins. The dashed arrows indicate enzymatic steps leading to lipopolysaccharide (LPS) synthesis. Abbreviation used: M6P, mannose-6-phosphate; M1P, mannose-1-phosphate; GDP-man, GDP-mannose; GDP-rha, GDP-rhamnose; and Ac, O-acetyl groups. (b) The organization of the alginate gene clusters. The alginate genes are clustered at three locations in the P. aeruginosa chromosome. The arrows above the genes represent the direction of transcription. Reproduced from May T and Chakrabarty AM (1994) Pseudomonas aeruginosa: Genes and enzymes of alginate synthesis. Trends in Microbiology 2: 151–157.

synthesized in the cytosol, the membrane-associated alg8 and alg44 gene products form an alginate-polymerizing complex that catalyze alginate polymerization and transfer through the inner membrane. After polymerization, some of the mannuronate residues are epimerized to guluronate residues by a C-5-epimerase (AlgG). This polymer is randomly acetylated by the membrane complex formed by algF, algJ, and algI gene products. AlgG is supposed to interact with AlgK and AlgX to form a scaffold in the periplasm that protects the growing alginate polymer from degradation by the alginate lyase (AlgL). Alginate lyase is important for the bacteria in detaching from the cell surfaces to spread to new habitats

and in generating short oligosaccharides, which are used as primers for new alginate chains. After acetylation, the alginate polymer is transported out of the cell through the outer membrane porin called AlgE. The regulation of alginate biosynthesis is based on a complex transcriptional control and the extracytoplasmic function (ECF) sigma factor 22 is at the apex of a hierarchy of regulators that control genes for alginate production. 22 encoded by algT (also known as algU) is required for the activation of the algD promoter, which controls the alginate biosynthetic operon (algD-algA). AlgT induces expression of other regulators binding the algD promoter, including AlgB, AlgR, and AlgZ. AlgR also

Bacteria | Pseudomonas

regulates alginate production through algC by binding to its promoter. Moreover, AlgT positively regulates its own transcription by binding to the promoter of the algT-mucABCD operon. The mucA and mucB gene products inhibit 22 activity by sequestering it from RNA polymerase. Mutations in these two genes cause deregulation of AlgT and conversion to mucoidy as observed in strains of P. aeruginosa infecting CF lungs. MucD (AlgW) is a protease under the control of the 22-independent promoter located in mucC and it is supposed to destabilize the MucA–22 interaction. Synthesis of alginate is a response to several environmental stimuli, such as osmolarity, nitrogen limitation, phosphate limitation, dehydration or stress conditions, and antibiotics.

Siderophores

Siderophores are iron(III) chelators secreted by aerobic bacteria in response to iron limitation. Iron is essential for their metabolism and it is frequently not readily available to bacteria because it is mainly in an insoluble ferric form or host species actively withhold iron from the infecting bacteria. Consequently, siderophore production is crucial for the success of bacteria and results in more virulent infections. After binding iron from the environment, siderophores are selectively recognized by high-affinity receptors on the bacterial cell surface. Pyoverdines (PVDs) are complex siderophores produced by fluorescent Pseudomonas, although some species can also synthesize additional siderophores such as pyochelin or quinolobactin, or can acquire iron bound to exogenous chelators, including heterologous siderophores. PVDs consist of a conserved fluorescent dihydroxyquinoline (chromophore) bound to a side chain, generally a dicarboxylic acid or an amide, and to a strain-specific peptide of 6–12 amino acids that is essential for recognition from the corresponding receptor. More than 50 PVD structures have been elucidated to date and this structural diversity and the receptor specificity have formed the basis for a Pseudomonas spp. typing system called siderotyping. Both the chromophores and the peptide chains are synthesized by nonribosomal peptide synthetases (NRPSs) that enable peptide bond formation between amino acids that cannot be incorporated through ribosomal synthesis. These large enzymes have a modular organization, with each module (about 1000 amino acids) catalyzing the incorporation of one amino acid into a peptide. Each module or cassette typically contains activation, thiolation, and condensation protein domains, which charge, bind, and connect a specific amino acid to a growing peptide. The number and order of the modules usually corresponds to the amino acid number and sequence in the peptide product. The synthesis of the peptide backbone precedes chromophore maturation,

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but events leading to the maturation of the PVD chromophore have not yet been fully characterized. The pyoverdine-related genes and their chromosomal organization are best known for P. aeruginosa PAO1. This strain has four genes, pvdL, pvdI, pvdJ, and pvdD, which encode peptide synthetase enzymes and determine the order of amino acids in the PVD peptide. PvdH is the enzyme that generates 2,4-diaminobutyrate (Dab) for incorporation into the PVD precursor peptide, and PvdA and PvdF are the enzymes that catalyze the synthesis of fOHOrn. Other biosynthetic genes (pvcABCD) are involved in the chromophore formation. PvdQ is a periplasmic acylase essential for PVD production and release in the milieu, which suggests that an acyl group retains the PVD precursor at the membrane to facilitate the interaction with the membrane-associated biosynthetic machinery. The hypothesis is that a PVD precursor is synthesized in the cytoplasm, transported to the periplasm, perhaps through the ABC transporter PvdE, and secreted out of the cell by a not yet clarified mechanism. The pvd region was identified as the most divergent alignable locus in the genome of several P. aeruginosa strains, reflecting the strain-specific diversity of PVD structure. In this region, the outer membrane pyoverdine receptor fpvA is the most divergent gene, which is not generated by intratype recombination and shows evidence of positive selection. Adjacent to fpvA, the genes pvdE, pvdI, pvdJ, and pvdD are also highly divergent and probably coevolve to maintain mutual specificity. The first regulator of siderophores synthesis and uptake to be described was the repressor protein Fur (ferric uptake regulator), which requires ferrous iron for DNA binding. The Fur–iron complexes bind the promoters of iron uptake genes, so the transcription of these genes is shut off in iron-repleted cells. In iron-starved cells the apo-Fur cannot bind DNA and so iron uptake genes are transcribed. However, an additional level of regulation involves sigma factors and enables bacteria to respond to the presence of specific siderophores in the environment as well as to levels of intracellular iron. The ECF sigma factor PvdS in P. aeruginosa binds to an iron starvation (IS) consensus sequence that is found upstream of several genes or gene clusters involved in PVD synthesis, regulation, and transport. This sequence is also present in the promoters of genes regulated by the PvdS homologues PfrI in Pseudomonas putida, PbrA in Pseudomonas fluorescens, and PsbS in Pseudomonas B10. PVDs have several important roles in Pseudomonas biology, including production of virulence factors and development of biofilms. Pyoverdine controls production of two secreted virulence factors of P. aeruginosa, exotoxin A and PrpL protease, through a signaling system involving the receptor FpvA, the antisigma factor FpvR, and the ECF sigma factor PvdS.

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Bacteria | Pseudomonas

Other virulence Factors Secreted by P. aeruginosa In addition to LPS, alginate, and siderophores, P. aeruginosa employs a multitude of extracellular virulence factors to successfully cause diverse acute infections or to persist as a chronic colonizer. P. aeruginosa proteases are crucial for many aspects of pathogenesis on mucosa. They destroy the structural integrity of the host cells by degrading the structural proteins of the extracellular matrix, such as elastin, laminin, and collagen. Moreover, they degrade components of the complement system, human immunoglobulins and serum alpha proteins. At least three proteases have been characterized: an elastase (PE), a general protease (LasA), and one alkaline protease (AP). The PE is a zinc-protease, encoded as a pre-pro protein of 53 kDa by the lasB gene. This protein is translocated to the periplasm, where it undergoes autoproteolysis to generate the 18 kDa and 33 kDa peptides, which interact noncovalently. The active 33 kDa elastase is secreted to the extracellular environment by the type II secretion system. The lasB gene is regulated in a quorum sensing-dependent manner. The LasA protease can cleave elastin, hydrolyze -casein, and lyse Staphylococci. The lasA gene encodes a 41 kDa precursor that is subsequently cleaved to produce the 22 kDa active protein. This protease is active over a broad pH range and is considered a major virulence factor. P. aeruginosa AP is encoded by the aprA gene. Secretion of this protease requires three accessory proteins, AprD, AprE, and AprF. These three proteins form a complex that spans the periplasm to allow for secretion of the protease to the external environment in a single step. Exotoxin A (encoded by toxA) is the most toxic protein produced by Pseudomonas. In a manner akin to that of diphtheria toxin, this protein catalyzes ADP ribosylation of the eukaryotic translation factor-2 (EF-2) to form ADP-ribosyl-EF-2, which results in inhibition of host cell protein synthesis. The toxA gene is expressed at higher levels when the environmental iron levels are low and is governed by the iron-responsive Fur protein. Another extracellular toxin is the acidic cytotoxin (CTX) of 25 kDa that forms pores in the lipid layers of leukocytes. CTX is encoded by a lysogenic phage that is 35 kb in size and is integrated at the attP site on the chromosome. This toxin is localized in the bacterial periplasmic space in an inactive or weakly active form, but is converted to the active form by proteases. Exoenzyme S (ExoS) is a 43 kDa protein encoded by exoS and is similar to YopE of Yersinia enterocolitica. It has ADP phosphorylation activity toward eukaryotic proteins such as Vimentin, H-Ras, and K-Ras types of GTPbinding proteins. The ADP ribosylation step requires a eukaryotic protein called FAS (factor for activating exoenzyme S). The exoT gene encodes another FAS-dependent ADP-ribosylating enzyme called ExoT. Both ExoS and

ExoT possess cytotoxic activity and are secreted by the type III secretion system upon host cell contact. Both the exoS and exoT genes are positively regulated by the transcriptional regulator ExsA. P. aeruginosa host invasiveness and ability to cause tissue damage is linked to its complex extracellular lipolytic system including at least two phospholipases C (PLC), one outer membrane-bound esterase and one secreted lipase. The 26 kDa secreted lipase is active against a broad range of triglycerides with fatty acyl chain lengths varying from C6 to C8 and it is stereoselective for sn-1 of the triglyceride. It is encoded as a 29 kDa precursor by the lipA gene. The pre-lipase is secreted by the Sec-dependent secretion pathway into the periplasm, where it interacts with the membrane-bound lipase-specific foldase Lif. While oxidoreductases catalyze the disulfide bond formation of LipA, Lif assists the correct conformational folding of LipA. The resulting mature 26 kDa form of the lipase is then secreted to the external environment via the type II secretion pathway. The lipase operon lipAlif is under the control of the 54-dependent promoter regulated by the two-component system LipR/LipQ. The transcription of the lipRQ operon is activated by the quorum sensing activator RhlR. The sensor kinase LipQ may also be activated by so far unknown environmental stimuli or by periplasmic signals including misfolded or nonsecretable enzymes. A 55 kDa esterase tightly bound to the outer membrane has been identified in lipase-negative deletion mutants of P. aeruginosa. It preferentially hydrolyzes long-chain acyl thioesters or oxyesters. In addition, two types of phospholipase C have been characterized in P. aeruginosa, the hemolytic phopholipase C (PLC-H) and the nonhemolytic phopholipase C (PLC-N). PLC-H is a 78 kDa heat-labile protein that hydrolyzes phosphatidylcholine in erythrocyte membranes; it is also active on sphingomyelin. Its production is regulated by the plc operon including the structural gene plcH and plcR1 and plcR2, the last two encoding proteins that modify PLC-H after translation. PLC-N is a 73 kDa protein that acts on phosphatidylcholine and phosphatidylserine. The location of the plcN gene in the chromosome is quite distant from that of PLC-H. The synthesis of both PLC-H and PLC-N is stimulated under low-phosphate and aerobic conditions. The synergistic effect of lipases and PLC leads to hydrolysis of the lung surfactant dipalmitoylphosphatidylcholine, which causes tissue damage and inflammation. Release of choline leads to the accumulation of betaine, which acts as an osmoprotectant, thereby enhancing bacterial survival within host tissues. P. aeruginosa produces glycolipid biosurfactants, called rhamnolipids, which can, at high concentrations, disrupt intercellular junctions in epithelia. Rhamnolipids are also implicated in the maintenance of fluid channels in mature biofilms. The production of rhamnolipids is under quorum sensing control.

Bacteria | Pseudomonas

Bacteriocins, called pyocins, are secreted into the environment and play a significant role in the ecological dominance of this species by promoting the lysis of competing bacteria. However, production of pyocins is more frequent in clinical situations than in the environment, which suggests a role of these molecules in human diseases. Three groups of pyocins, R, F, and S, have been characterized. The R and F types are rod-like particles resembling a bacteriophage tail. The S-type pyocins are the most abundant low-molecular-weight pyocins, including S1, S2, S3, and AP41. S1 and S2 pyocins are able to inhibit phospholipid synthesis of target bacteria under iron-limited conditions, whereas AP41 possesses endonuclease activity and induces the synthesis of other pyocins (R2 and S2) as well as induction of phage. The mode of action of S3 remains to be clarified. Each S-type pyocin consists of two proteins associated in a complex. Only the large protein shows bactericidal activity, while the small protein protects the host cell from the killing activity of the bigger component. Pyocins structural genes are chromosome-located and their transcription is activated by PrtN. This positive regulator binds to the conserved P box sequences located 60–100 bp upstream of RBS of pyocins genes. Pyocin synthesis is induced by DNA damaging agents, which increase expression of the DNA repair protein RecA that cleaves the repressor protein PtrR and liberates the expression of activator PrtN. Pyocin typing has been an epidemiological tool to discriminate one P. aeruginosa strain from another to follow its propagation in nosocomial infections. Two types of sugar-binding proteins called lectins are among the set of virulence factors produced by P. aeruginosa. PA-1L is a lectin specific to D-galactose and its derivatives; PA-11L shows specificity for L-fucose and D-mannose. These lectins help the bacteria to adhere to host cells and stimulate an inflammatory response by inducing the host immune system to produce cytokines such as IL-1 and IL-6. Fluorescent Pseudomonas, including P. aeruginosa, produce and secrete phenazines, which are heterocyclic, redox-active compounds toxic to competing bacteria. The most studied phenazines are pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxylic acid. There is evidence to suggest that phenazines can penetrate biological membranes and alter cytokine production and signaling pathways in cultured airway epithelia. Also, a recent report indicates that pyocyanin is capable of regulating the expression of a set of P. aeruginosa genes. P. aeruginosa Infections in Cystic Fibrosis CF is an autosomal recessive disorder caused by mutations in the CFTR (CF transmembrane conductance regulator) gene. These mutations result in defective chloride transport across epithelia. The airways of individuals with CF

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are susceptible to recurrent bacterial infections, and the opportunistic pathogen P. aeruginosa can chronically colonize the lungs of CF patients despite aggressive antibiotic treatment. It has been hypothesized that P. aeruginosa in the CF lung may exist as biofilms wherein bacteria are organized in a self-produced polymeric matrix. The biofilm mode of lifestyle may be responsible for the antibiotic resistance of the bacteria in CF. Chronic persistence of P. aeruginosa in CF is often accompanied by bacterial adaptations that involve the repression of certain virulence factors. For example, unlike environmental strains, isolates from chronic CF infections often show a repression of flagellin and pilin expression and repression of the type III secretion system. Sustained repression may lead to mutations that result in a permanent adaptation to the unique environmental niche of the airways. P. aeruginosa Virulence in Burn Infections and Keratitis P. aeruginosa contributes to burn wound infection with many virulence factors. Pili and flagella are responsible for its adherence and particularly for its dissemination throughout the whole organism. Dissemination is also dependent upon production of elastases and proteases, which destroy the host physical and immune barriers that normally might inhibit the spread of the infection. Disruption of lasR regulatory gene that controls the QS response blocks the dissemination from the initial site of infection. QS is responsible for the regulation of several virulence factors, including elastases and biofilm formation. However, the block of dissemination caused by lasR knockout is not caused specifically by inactivation of elastases, suggesting the critical role of other virulence factors in bacterial dissemination. The cell-associated and secreted virulence factors that contribute to the pathogenesis of wound infections are responsible for invasion and destruction of the cornea in microbial keratitis. Another factor that contributes to this destructive process is a continuous recruitment of polymorphonuclear neutrophils in the corneal tissue triggered by the recognition of P. aeruginosa flagella or LPS by epithelial cells toll-like receptors. In addition to its role in this inflammatory response, P. aeruginosa LPS is a ligand for the epithelial CFTR receptor. The interaction LPS– CFTR receptor causes internalization of Pseudomonas by the corneal basal epithelium where bacteria replicate. Some Unique Behaviors Exhibited by Pseudomonas Antibiotic resistance

P. aeruginosa exhibits an intrinsic resistance to antibiotics and has a demonstrated ability to acquire genes encoding resistance determinants. This resistance is mainly due to

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Bacteria | Pseudomonas

production of -lactamases, diminished membrane permeability to antibiotics, or upregulation of efflux pumps. Unfortunately, multiple mechanisms of resistance can accumulate in some strains leading to the development of multiply resistant strains. P. aeruginosa major defense mechanism against the -lactam group of antibiotics (penicillins, cephalosporins, monobactams, and carbapenems) is the production of a variety of -lactamases. Despite these lactamases, imipenem resistance in P. aeruginosa is commonly generated via a mutational loss of a 54 kDa outer-membrane protein (OMP), usually known as OprD (or the D2 porin). Furthermore, structural modifications of the outer membrane, such as absence of 2-hydroxylaurate, presence of 4-aminoarabinose, and increase of palmitate, are responsible for resistance to colistin. However, efflux pump systems are the major cause of multidrug resistance and the most commonly observed pump system in P. aeruginosa is the MexAB-OprM, which consists of a pump (MexB) connected to the outer membrane by the linker lipoprotein MexA and the exit portal OprM. P. aeruginosa uses this upregulated MexAB system to export quinolones, penicillines, and cephalosporins, while the upregulated MexXY-OprM efflux pump is responsible for aminoglycoside resistance. Quinolone resistance is attributable not only to efflux pumps but also to mutations of gyrA and parC genes encoding topoisomerases II and IV, respectively. Few treatment options are available for multidrugresistant P. aeruginosa: cefepime and amikacin might be active against some strains, otherwise polymyxins remain the most effective agent alone or in combination with one or more of the following: a carbapenem, aminoglycoside, quinolone, or -lactam. Response to oxidative stress

Pseudomonas respond to both endogenous (aerobic growth) or exogenous (anaerobic or in macrophage) oxidative stress (superoxide anion O2 ) by producing iron- or manganesecontaining superoxide dismutase (SOD) metalloenzymes. The dismutases catalyze the disproportionation of O2 to H2O2 and O2. The organism also possesses catalases that remove the toxic H2O2 product. The SOD A (encoded by sodA) is a 23 kDa dimer, which uses manganese as a cofactor, and the SOD B (encoded by sodB) is iron-dependent and also functions as a dimer. Mucoid strains have been observed to possess higher manganese SOD activity. Cell–cell signaling in P. aeruginosa

Bacteria employ a mechanism of cell–cell signaling called quorum sensing to coordinately regulate gene expression in response to changes in population density. The quorum sensing circuit was first characterized in the marine bacterium Vibrio fischeri and includes genes encoding the signal synthase, LuxI, and the signal receptor, LuxR. P. aeruginosa utilizes two homologous acyl-homoserine

lactone (AHL) quorum sensing systems to regulate the expression of a large number of genes including virulence factor genes and genes involved in biofilm development. These two systems are the LasR-LasI and RhlR-RhlI systems. LasR is a transcriptional activator that responds to the product of the LasI synthase, N-3-(oxododecanoyl)homoserine lactone (3OC12-HSL). At sufficient environmental concentrations of this AHL signal, a number of genes are activated, including rhlR, which codes for the N-butyrylhomoserine lactone (C4-HSL) receptor, and rhlI, which codes for the C4-HSL signal generator. RhlR and C4-HSL activate many other genes. Transcriptome analyses studies indicate that P. aeruginosa regulates over 300 genes in a quorum-dependent manner. The set of quorum-controlled genes includes those coding for the virulence factors pyocyanin, hydrogen cyanide, elastase, and AP. Analysis of the P. aeruginosa genome revealed a gene coding for a homologue of LasR and RhlR but no additional genes coding for LasI and RhlI homologues. This ‘orphan receptor’ termed QscR responds to the product of the LasI synthase, 3OC12-HSL, and controls a set of genes that partially overlap the Las-Rhl quorum regulon. As might be expected in a versatile bacterium inhabiting diverse habitats, the elements of the two quorum sensing systems are controlled by other factors. In addition to the AHL quorum sensing systems, P. aeruginosa utilizes another low-molecular-weight hydrophobic molecule, 2-heptyl-3-hydroxy-4-quinolone, referred to as the Pseudomonas quinolone signal (PQS) for intercellular communication. This signal functions as a coinducer for the transcriptional regulator PqsR to activate the expression of multiple virulence genes and its own synthesis. PQS is one of several quinolones and quinolines made by P. aeruginosa. Remarkably, PQS, released in membrane vesicles, which, in addition to signaling to P. aeruginosa cells, shows potent antibacterial activity against the Gram-positive bacterium Staphylococcus aureus. Secretion System in Pseudomonas Pseudomonads rely on several pathways for the secretion of toxins, hydrolytic enzymes, and proteins important for virulence. These include the type I, II, III, IV, and VI pathways. The type I secretion (ABC exporter) system utilizes three proteins, an ATP-binding cassette (ABC) protein, a membrane fusion protein, and an outer membrane protein for the secretion of virulence factors to the extracellular environment. The type II secretion system functions in conjunction with the Sec or Tat transport systems for protein transport across the inner membrane. This system allows the transport of proteins across both the inner and outer membranes in a single step. In P. aeruginosa, the Xcp and the Hxc type II secretion systems allow for the extracellular release of elastase,

Bacteria | Pseudomonas

lipases, exotoxin A, and alkaline phosphatase. The proteins of the Xcp secretion system share several features with proteins involved in the assembly of type IV pili that are required for twitching motility, adherence to host cells, and biofilm formation. The type III secretion system allows the direct injection of toxins into host cells via a secretion complex that spans the bacterial cell envelope and penetrates the host cell membrane. Recent studies indicate the presence of a type VI secretion system in P. aeruginosa and suggest a role for it in the direct injection of virulence factors into host cells.

Plant Pathogens Several Pseudomonas species cause plant diseases in some of the most commercially important crops. Among these species, Pseudomonas syringae is the most widespread and best studied. P. syringae is taxonomically subdivided into more than 50 pathovars (pathological variants), which are typically distinguished by plant host range. Although the P. syringae species as a whole causes plant diseases on a multitude of hosts, individual P. syringae pathovars typically have a limited host range of one to a few plant species. Symptoms of diseases caused by P. syringae include leaf spots, fruit spots, and cankers on woody hosts. P. syringae diseases are currently mainly managed through the use of bactericides and through host resistance in certain crops. However, the continued expansion of understanding of host–pathogen interactions is expected to foster the utilization of host resistance in many more disease pathosystems. P. syringae pathogens utilize an impressive array of virulence factors such as effectors, toxins, and phytohormones to incite disease symptoms. The most important pathogenicity determinant is the presence of a type III secretion system, which is encoded by genes present in the hrp pathogenicity island. hrp (hypersensitive response (HR) and pathogenicity) genes were discovered in the early 1980s as genes affecting the ability of strains to elicit a HR in the reporter tobacco plant. The HR was later found to be a plant disease resistance response initiated after the intracellular recognition of pathogen effector proteins delivered via type III secretion. Like most other bacterial plant pathogens, P. syringae encodes a type III secretion system that consists of a Hrp pilus, a long syringe-like structure that must traverse the plant cell wall and that enables delivery of effector proteins directly into plant host cells. Development of microarrays and subsequent bioinformatic and functional genomic analyses have enabled the identification of the hrp regulon and the complete effector repertoire of a single P. syringae strain. These studies have established that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor, which recognizes the ‘hrp box’ motif in the

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promoter of the hrp operons and effector genes. hrp induction requires hrpS and hrpR. Since hrpL is under the control of a 54-dependent promoter in RpoN-dependent manner, it has been hypothesized that the heterodimer HrpR–HrpS interacts with RpoN and promotes hrpL transcription. HrpRS transcription is positively regulated by the GacS/GacA system, a highly conserved bacterial regulatory system that controls the expression of many cellular functions. However, it is not clear which signal is sensed by GacS and how GacA regulates hrpRS and rpoN transcription. In addition, HrpA, a major component of the type III pilus, acts as a positive regulator on hrpRS transcription by a mechanism that remains to be clarified. In this complex regulatory network, a negative control is played by the ATP-dependent Lon protease that degrades the HrpR protein and by the HrpV protein, which acts as an anti-activator of HrpS. Exopolysaccharides (EPS) and toxins allow P. syringae to cope with environmental condition and host response. P. syringae produces at least two EPS, levan, a -(2,6) polyfructan, and alginate. The latter is widely produced during plant infection and is responsible for lesions having a typical water-soaked appearance. EPS chelate heavy metals, such as copper, increasing tolerance to toxic pesticides and resistance to dessication. Like P. aeruginosa, most strains of P. syringae are normally nonmucoid and alginate production is activated by stress stimuli. The biosynthesis of alginate in P. syringae is similar to that described for P. aeruginosa and the arrangement of the alginate structural genes is conserved, although in P. syringae muc D transcription is not dependent on AlgT; muc D has its own promoter and it is not cotranscribed with the algT–muc operon. Interestingly, mucC has not been found in P. syringae. P. syringae produces several toxins different in structure and origin, which are not required for its pathogenicity but enhance P. syringae virulence, causing plant lesions and facilitating bacterial invasion and spreading in the plant. Syringomycin and syringopeptins are cyclic lipodepsinonapeptide phytotoxins secreted by P. syringae pv. siringae. Syringomycin targets the plasma membrane of the host cells, and disrupts the ion transport and membrane electrical potential, causing cytolysis. This necrotic toxin is synthesized by NRPSs. The genes dedicated to syringomycin biosynthesis (syrB1, syrB2, syrC, and syrE), secretion (syrD), and regulation (syrP) are organized in a gene cluster (syr) on the chromosome of P. syringae pv. siringae. Syringomycin production is modulated by both nutritional factors and plant signal molecules, such as phenolic glycosides, although the mechanism responsible for transduction of these signals to the syr transcriptional apparatus is still under investigation. Syringopeptins represent another class of lipodepsipeptide phytotoxins synthesized by a different set of biosynthetic genes organized in the syp gene cluster. Syp-syr genes are coregulated and respond to the same environmental stimuli. Another

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Bacteria | Pseudomonas

interesting phytotoxin is the polyketide coronatine, which structurally mimics the phytohormone jasmonic acid. This hormone regulates fruit abscission and senescence in higher plants. Coronatine causes chlorosis, induces hypertrophy, and inhibits root elongation. Recent studies suggest that coronatine might enable P. syringae to colonize the leaf interior by counteracting the plant defensive closure of the stomata. In addition to phytotoxins, Pseudomonas secretes amidases and pectate lyase, which play an important role in Pseudomonas infection of plants and fruits and destroys the appearance and quality and commercial value of the produce. Many pathovars of P. syringae also possess an epiphytic phase as part of their life cycle. Growth as an epiphyte on plant leaf surfaces enables the buildup of population size, which seems to be a requirement for pathogenesis. The leaf surface or phyllosphere is a habitat that is exposed to various environmental stress factors, and desiccation and exposure to ultraviolet radiation may be the most important. Strategies of tolerance or avoidance of stress are two possible fitness strategies of foliar pathogens and colonists in response to environmental stress. In P. syringae, traits that facilitate survival in response to environmental stress such as motility, tolerance to ultraviolet radiation, or EPS production are important to epiphytic population size. This organism commonly forms aggregates on leaf surfaces; these aggregates may form at nonrandom sites of carbon source deposition on leaves. Aggregates are important for phyllosphere survival and appear important in population increases, resulting in ingress into leaves and eventual pathogenesis. Biological control strategies aimed at reducing epiphytic populations have proven successful in some pathosystems. The ability of some P. syringae strains to serve as ice nuclei and nucleate ice formation may be an important factor in plant wounding and ingress of bacteria into plant tissues. In the absence of an ice nucleus, purified water can supercool to temperatures far below 0  C; icenucleation-active (INA) P. syringae cells can catalyze ice formation at relatively warm temperatures of 2 to 5  C. Ice formed in susceptible plant tissues can rapidly propagate; following thawing, this injured tissue is then susceptible to infection. Most P. syringae strains contain extrachromosomal plasmids, and many of these are related, belonging to the pPT23A plasmid family. P. syringae plasmids encode various traits beneficial to epiphytic growth and/or virulence. Examples include type III effectors, chemotaxis receptors, ultraviolet radiation tolerance genes, toxin biosynthesis gene clusters, and genes encoding indole acetic acid biosynthesis. Many P. syringae plasmids are also conjugative. The importance of gene transfer in the evolution of P. syringae is indicated by the evolutionary relationships

of various pathovar strains. The observation that closely related strains can belong to different pathovars with distinct host ranges implies the transfer of effectors conferring host range alterations between strains. Genome sequencing has provided the raw material for determining answers to various questions concerning pathogenesis and host range in P. syringae. Since 2003, the genome sequences of P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a have been published. In addition, genome sequences from plant-associated P. fluorescens strains and P. aeruginosa PA14 have also been published. Comparative genomics will facilitate the understanding of host range determinants and the understanding of pathogenesis in this organism. Such studies will hopefully lead to novel disease control methods in plants either through host resistance or through targeting important virulence determinants in the pathogen.

Environmental Aspects of Pseudomonas Degradation of Organic Compounds Pseudomonas are widespread in many natural environments, where they carry out a variety of biochemical conversions and mineralize organic carbon. They metabolize a large number of natural organic compounds, including aromatic hydrocarbons and their derivatives. The enzymes involved in the degradation of these compounds are generally plasmid-encoded and have low substrate specificity. These two features allow rapid evolution of new metabolic pathways for the degradation of toxic synthetic compounds (xenobiotics), such as highly chlorinated aromatics used as pesticides, herbicides, or by-products released into the environment by industrial processes. Pseudomonas degrade chlorinated aromatic hydrocarbons

Pseudomonas can utilize a wide variety of chlorinated aromatics as sole source of carbon and energy. The processes involved in the degradation of these recalcitrant compounds are well studied and have been developed in pollution control. The degradative pathway of chlorinated benzenes is initiated by dioxygenases that produce chlorinated dihydrodiol intermediates, which are subsequently converted into the corresponding chlorocatechols by dihydrodiol dehydrogenases. The resulting chlorocatechols are oxidized by chlorocatechol dioxygenases, causing either ortho-cleavage to chloromuconic acid or meta-cleavage to 2-hydroxy-6-chlorocarbonil muconic acid. Chloromuconic acids are metabolized further to intermediates of the Krebs cycle.

Bacteria | Pseudomonas

Chlorocatechols are toxic to bacterial cells, therefore the regulation of the expression of these catabolic genes is very important for cell survival. These degradative pathways are usually regulated by LysR-type responsive transcriptional regulators (LTTRs), which are typically divergently transcribed from the structural genes. LTTRs are DNA-binding proteins that bind approximately 50–60 bp upstream of the genes they regulate. The presence of an inducer molecule, which is usually a catabolic intermediate of the pathway being regulated, alters the binding pattern and results in transcriptional activation. Examples of biodegradative pathways regulated by LTTRs in Pseudomonas include the chromosomally encoded catechol degradative catBCA operon and the pheBA operon (Figure 2), which allows the growth of the P. putida strain PaW85 on phenol. Both the catBCA and pheBA operons are regulated by CatR. Other examples include the 3-chlorocatechol degradative clcABD operon, regulated by ClcR, and the 1,2,4-trichlorobenzoate degradative tcbCDEF pathway in Pseudomonas sp. strain P51, regulated by TcbR. In general, the genes that allow Pseudomonas to degrade aromatic compounds are likely recruited from preexisting catabolic pathways. The nature of the environments dictates to a large extent the mode of evolution of the new degradative pathways in microorganisms. Polychlorinated biphenyl catabolism in recombinant Pseudomonas strains

Genes encoding polychlorinated biphenyls (PCBs)degrading enzymes (bph) have been identified and isolated from several Pseudomonas species. PCBs, such as DTT, are toxic pollutants present in great abundance in the ecosystem, and bioremediation by soil bacteria has been extensively investigated in the last few decades. The catabolism of PCBs generally proceeds by the incorporation of both the atoms of oxygen (O2) at the 2 and 3 positions of the least chlorinated ring, followed by 1,2-meta-cleavage of the molecule. PCBs are finally converted to a five-carbon aliphatic acid (2-hydroxypenta2,4-dienoate), further degraded to chlorobenzoate, which accumulates in the growth medium. This dead-end product of the PCBs degradation can inhibit the bacterial growth and consequently slow down PCB biodegradation. To circumvent this limitation and to utilize Pseudomonas in bioremediation, recombinant strains are constructed by transferring the bph genes into Pseudomonas strains capable of utilizing several CBAs. Metabolism of benzene, methylbenzene, and naphthalene by Pseudomonas

Pseudomonas have the potential to degrade hydrocarbons that range in size from a single benzene, toluene, and xylene) to polycyclic (e.g., naphthalene). BTX aromatic compounds

aromatic ring (e.g., aromatics (benzene,

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toluene, and isomeric xylenes) usually occur together in gasoline and diesel oil. Pseudomonas degrade monoalkyl and dialkyl benzenes by different pathways, which include the oxidative attack on the aromatic ring and the formation of alkyl catechols, which are substrates for ring fission, or by the oxidation of alkyl substituents, which lead to the formation of aromatic carboxylic acids, further oxidized to dihydroxylated ring fission substrates. Subsequent conversion to the central metabolism intermediates proceeds through the meta-cleavage. Naphthalene and its substituted derivatives are commonly found in crude oil and oil products. Naphthalene metabolism has been widely investigated in Pseudomonas as a model to understand the degradation of more complex polycyclic aromatic hydrocarbons (PAHs). PAHs are toxic and carcinogenic compounds so widely distributed in the environment to motivate the study of the microbial metabolism of these compounds to develop bioremediation technologies. Pseudomonas metabolizes naphthalene to salicylate, which is then converted to catechol, followed by ortho- or meta-cleavage to TCA cycle intermediates. In P. putida NAH7 plasmid, the genes encoding the enzymes involved in the naphthalene upper pathway and lower pathway are organized into two operons, nah and sal, respectively. Both the operons are turned on by NahR, a 36 kDa polypeptide and a salicylate-dependent transcription activator. These catabolic pathways are mainly encoded on large plasmids such as the well-studied TOL plasmid pWWO, which is responsible for toluene and xylenes catabolism in P. putida and the naphthalene catabolic plasmid NAH7. These plasmids are generally conjugative, have low copy number, and undergo rearrangement and shuffling. Degradation of alkanes and cycloalkanes in Pseudomonas

P. putida (oleovorans) can grow on n-alkanes by virtue of the alkane hydrolase system, which catalyzes the first step of alkane degradation, the oxidation of the methyl group to alcohol. The alkane hydrolase system consists of a membrane-bound alkane hydroxylase (alkB), a soluble electron transport system consisting of two rubredoxins, and a NADH-dependent rubredoxin reductase (encoded by alkG, alkF, and alkT, respectively). This system is investigated in great detail because of its industrial application in the production of fine chemicals, such as fatty acids, alcohols, and epoxides. The alk genes are mapped on its large catabolic OCT plasmid, which confers Pseudomonas the ability to degrade soluble short-chain alkanes, such as pentane, hexane, heptane, and octane, which are toxic for the environment and are produced by petroleum refineries. Other interesting degradative activities in Pseudomonas are directed toward cycloalkanes, such as camphor or the highly toxic and persistent insecticide -hexachlorocyclohexane

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Bacteria | Pseudomonas (a)

OH COOH

COOH

Cl

Phenol pheA

Phenol monooxygenase

Benzoate benABCD

3-Chlorobenzoate benABCD

Cl

OH Dioxygenase I

OH

OH 3-Chlorocatechol

OH Catechol catA, pheB

COO– – COO Muconate lactonizing cis,cis-muconate enzyme I catB

COO– COO– Muconate lactonizing 2-chloro-cis,cis-muconate enzyme II Cl

clcB

O

O COO–

Muconolactone isomerase

COO– C=O

Cl

C=O Muconolactone catC

O

O –

Hydrolase I

Dioxygenase II

clcA

COO C=O β-Ketoadipate enol-lactone

COO– C=O Dienelactone

pcaD

Hydrolase II

clcD

O

O

clcE

COO– COO–

β-Ketoadipate

COO– COO– Maleylacetate

Succinate and acetyl CoA

(b) catR

ORF1

clcR

catB

pheB

clcA

catC

pheA

clcB

catA

ORF2

clcD

Figure 2 Enzymes and intermediates of the benzoate, phenol, and 3-chlorobenzoate degradation (a) and their genetic organization (b). Pseudomonas putida uses a modified -ketoadipate pathway to degrade 3-chlorocatechol. The genes for the regulatory proteins CatR and ClcR are divergently transcribed from the catBCA and clcABD operons that they regulate. The pheBA operon is regulated by CatR.

Bacteria | Pseudomonas

( HCH). P. putida PpG1, originally isolated by enrichment culture with D-camphor, carries the CAM plasmid, which encodes the enzymes necessary for D- or L-camphor degradation. Camphor is first converted to 5-exo-hydroxy camphor by a monoxygenase system consisting of three enzymes encoded by camA, camB, and camC genes. 5-exo-hydroxy camphor is then dehydrogenated to form 2,5-diketo camphane by F-dehydrogenase encoded by gene camD. These genes are organized in the camDCAB operon, which is negatively regulated by the product of the regulatory gene camR. CamR is located upstream of CamD and it is divergently transcribed. In the absence of camphor, CamR inhibits the expression of camDCAB and autorepresses the camR gene by binding to the operator between the regulator gene camR and the camDCAB operon. This inhibition is released in presence of the inducer camphor. P. aeruginosa ITRC-5, isolated by selective enrichment on HCH, can mineralize this insecticide. HCH catabolic pathway has been comprehensively characterized for Sphingomonas paucimobilis UT26. This chlorinated insecticide is metabolized by the enzymes encoded by linA, linB, linC, linD, linE, and linF genes to -ketoadipate, which is subsequently mineralized. Two or more copies of these genes are present in P. aeruginosa ITRC-5, which suggests that HCH is degraded by Pseudomonas through a similar enzymatic pathway. Pseudomonas take part in the natural process of lignin mineralization

Several members of the Pseudomonaceae have the ability to degrade lignin and the phenolic monomers, such as trans-ferulic, p-coumaric, and vanillic acid, which occur abundantly in the environment from the biodegradation of lignin accomplished predominantly by white-rot fungi. These products are utilized as a unique source of carbon and energy by Pseudomonas. The investigation of their degradative ability toward these lignin monomeric components is very important for bioremediation of pollutants, such as the chlorinated forms of vanillate, which are liberated in vast quantities into the environment by the wood pulp bleaching process. Other environmental pollutants degraded by Pseudomonas

1. Nylon: Nylon is a polymer of 6-aminohexanoate (Ahx), widely used in the textile industry. During the polymerization, some molecules fail to polymerize and remain as oligomers and linear dimers (Ald) or undergo head-to-head condensation to form cyclic dimers (Acd). These nylon by-products are industrial waste products released in the environment. Pseudomonas sp. NK87 can grow on these compounds as sole source of carbon and nitrogen. This strain

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produces two hydrolases, Acd hydrolase and Ald hydrolases, encoded by nylA and nylB genes, respectively. These genes occur on catabolic plasmids present in Pseudomonas sp. NK87 and have evolved from other bacteria. 2. Trichloroethane: Trichloroethane (TCE) is widely used as degreasing agent, dry cleaning fluid, fumigant, and cleanser. Such wide use of TCE has caused it to become an environmental pollutant, especially in soils and groundwater. It is known to cause anemia and kidney and liver damage in humans. P. putida is capable of degrading TCE by producing a toluene dioxygenase. This enzyme converts TCE to glyoxylate or formate that are further metabolized. Metal Resistance Pseudomonas is resistant to a number of toxic metal ions, such as mercury, arsenic, cadmium, copper, chromium, and silver. Most of the resistant genes are plasmid-encoded and, occasionally, the regulatory genes are present on the chromosome. Mercury resistance

Mercury is a toxic heavy metal. Resistant Pseudomonas species carry mercury-resistant (mer) determinants encoded on mobile genetic elements. The simplest mer determinants have been identified on transposon Tn501 in P. aeruginosa, where they are organized in the merTPAD operon. MerR is located upstream of the merTPAD and it is divergently expressed. MerR and MerD are involved in the regulation of the expression of the structural genes. MerR works as a mer operon inducer in the presence of Hg(II) and as a repressor in the absence of mercury salts. The mer operon encodes the transport proteins MerT and MerP; MerP is a periplasmic protein thought to scavenge Hg(II) in the periplasmic compartment to pass it to the inner membrane transporter MerT. From MerT, toxic Hg(II) is passed to mercuric reductase MerA. This NADPH-dependent cytoplasmic flavoenzyme detoxifies Hg(II) to volatile Hg0. Copper resistance

Copper (Cu) is a major micronutrient and it is a constituent of metalloenzymes and proteins involved in electron transport and redox reactions. However, it is extremely toxic at supraoptimal concentrations and it is also known to produce toxic free radicals. Two forms of Cu, Cu(I) and Cu(II), are normally found in bacteria. The well-studied copper resistance in P. syringae is due to a mechanism that involves copper binding and sequestration by plasmid-encoded proteins (copABCD). CopD is an inner membrane protein, which interacts with the outer membrane-associated protein CopB via the periplasmic proteins, CopC and CopA. The CopB, CopA, CopC, and

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Bacteria | Pseudomonas

CopD proteins form a copper transport unit. CopS is a membrane-embedded copper-sensing protein and CopR is a DNA-binding protein, which activates the cop operon transcription. CopR and CopS form the two-component signal transduction system for sensing the levels of copper and regulating the cop operon. In addition, copper tolerance in P. fluorescens and P. aeruginosa has been shown to be affected by a chromosome-encoded P1-type ATPase, which functions as an exporter of copper.

Silver resistance

Plasmids encoding silver resistance have been found in Pseudomonas strains isolated from silver mine or industrial sludge and in P. aeruginosa isolated from a patient in a burn unit after the topical use of silver compounds. However, silver resistance mechanism is not characterized yet.

Genetic and Molecular Tools Used to Study Pseudomonas Plasmids

Cadmium resistance

Cadmium is very toxic to bacteria even when present in low concentrations, since it damages the cells by binding to essential respiratory enzymes, inducing oxidative stress or inhibiting DNA repair. P. putida produces a lowmolecular-weight protein, which chelates cadmium, thereby reducing its toxicity. It also encodes a cadmium-transporting ATPase (CadA), an efflux system that confers resistance by reducing cadmium intracellular concentration. A cadmium and zinc efflux mechanism (czr), which is a cation–proton antiporter, rather than a cation-transport ATPase, was identified in the chromosome of P. aeruginosa.

Arsenic resistance

Arsenic is a top priority pollutant present in many ecosystems mainly in two oxidation states, arsenite [As(III)] and arsenate [As(V)]. Although some microorganisms can utilize arsenic, it generally is toxic to most bacteria. Arsenic resistance in Pseudomonas is due in part to the ars genes. Ars-mediated resistance involves As(V) reduction to As(III) via a cytoplasmic reductase (ArsC), and the As(III) is then extruded by a membrane-associated ArsB efflux pump.

The utilization of nonenteric bacteria for basic and applied molecular research has resulted in the need for well-characterized vector systems for such microorganisms. The cloning vectors developed for this purpose are generally broad host range vectors and allow the use of different species, including Escherichia coli, as intermediary host. General type cloning vector

There are many different broad-host-range vectors available for gene cloning in Pseudomonas. The majority of them are constructed based on existing replicons, such as RSF1010, RK2, or PRO1600, and inserting improved antibiotic resistance markers and additional cloning sites. Vectors such as pDSK509, pDSK519, and pRK415 are RSF1010-based vectors with the MCS from pUC19, kanamycin, or tetracycline resistance genes and the lacZ gene for easy screening of recombinant clones in E. coli. The pUC18/19 adaptation vectors, pUCP18 and pUCP19, were generated by introducing a pRO1600derived stabilizing fragment into a pUC18/19 nonessential region to allow maintenance of these plasmids in Pseudomonas species. Many of these modified vectors are self-transmissible, whereas some have to be mobilized by triparental mating using a helper strain supporting mob functions in trans.

Chromium resistance

Special purpose cloning vectors

Cr(III) and Cr(VI) are the most stable and abundant oxidative forms of chromium in nature and they are toxic to microorganisms. Cr(VI) is usually present as the oxyanion chromate, which crosses the biological membrane by means of the sulfate uptake pathway. Inside the cell, Cr(VI) is easily reduced to Cr(III), releasing free radicals that cause oxidative stress. P. putida minimize the toxic effect of chromate by means of a plasmid-encoded NADH-dependent reductase (ChrR). This flavin mononucleotide-binding protein reduces Cr(VI) to Cr(III) and, by an additional mechanism, reduces quinones, providing protection against free radicals generated by Cr(VI) reduction. P. aeruginosa ChrA protein is a chromate efflux pump, which represents an efficient mechanism of chromate resistance.

The expression of cloned genes in the host organism is often used to confirm the coding potential of a DNA fragment. However, large limitations are encountered when expressing cloned Pseudomonas genes in heterologous hosts, such as E. coli. These are primarily due to the differences in codon usage as well as due to variations in the structure of gene promoters. To overcome these problems, many laboratories have designed broad host-range expression vectors suitable for analysis of Pseudomonas genes. These vectors contain regulable promoters, such as the T7 promoter (PT7) or the E. coli lac operon-based promoters Plac, Ptac, and Ptrc. The first generation of controlled expression vectors pUCP18/19 offered regulated expression from Plac, and two derivatives of pUCP19, pUCPKS and pUCPSK, were generated to

Bacteria | Pseudomonas

allow expression from PT7. A further development were the pBluescript-derived vectors, pBSP II SK(-) and pBSP II KS(-), which allow the controlled production of plasmid-encoded proteins from PT7 and Plac. The pMMB family of expression vectors have been constructed using broad host-range vectors and the hybrid trp-lac (tac) promoter. A drawback of these controlled expression vectors in environmental applications is the high cost of the IPTG inducer. An alternative RSF1010-derived vector has been described that is based on Pm and Pu promoters of the P. putida TOL plasmid pWWO and the xylS gene, the product of which together with the coinducer benzoate positively regulates the Pseudomonas promoters. For a quantitative analysis of the role and function of promoters in Pseudomonas species, gene fusion vectors containing the reporter genes aphC or xylE from RSF1010 or TOL plasmids, respectively, have been constructed. The first vector allows positive selection of promoters by selecting for streptomycin-resistant colonies, while the second allows for screening by catechol substrate. Other promoter probe vectors have been developed to identify in vivo-induced genes in Pseudomonas species with the in vivo expression technology (IVET), a method used to identify functions of ecological relevance or important for bacterial virulence and/or pathogenicity. Ivi genes can be identified by their ability to express a promoterless selection marker gene that is essential for survival in vivo. IVET system requires a strain that is a null mutant for the essential function encoded by the selection marker gene, an IVET plasmid carrying the promoterless selection marker gene, and a reporter gene. Several biosynthetic loci are essential for bacterial growth and have been exploited in the IVET systems. They include genes essential for the synthesis of purine and pyrimidine (purA, purEK, pyrBC) or for the synthesis of diaminopimelic acid (DAP), a component of the cell wall peptidoglycan (asd). ASD (aspartate -semialdehyde dehydrogenase) is an essential enzyme in the biosynthesis of diaminopimelate, but it also plays a part in the biosynthesis of lysine, methionine, and threonine. As reporter genes, lacZY for -galactosidase and uidA for -glucuronidase have been incorporated into Pseudomonas IVET plasmids. Transposable Elements Transposable elements, including insertion sequences (ISs) and transposons, are common in various Pseudomonas species. The ISs have the capability of integrating into different sites of the genome in different bacteria since they contain the genetic determinants for transposition and short inverted repeat sequences (IRs) at both ends. During such an event, foreign genes are recruited by replicon fusion and insertional activation.

259

Composite transposons can carry catabolic genes or antibiotic resistance markers flanked by two copies of similar ISs in direct or inverted orientation. However, transposons are also important tools for genetic analysis in Pseudomonas. They are used for generating gene disruption that is nonleaky and is linked to a selectable marker, or to deliver and stably incorporate genes in the chromosome for applications where plasmid cannot be readily maintained (e.g., environmental release). Recently, miniTn5 transposons are being used in several applications, including gene regulation studies and construction of strains for bio-remediation. These new delivery vehicles allow for single-copy chromosomal insertions and overcome the drawbacks of plasmid-based constructs, for example, high copy number or supercoiling, which interfere with promoter regulation studies, and antibiotic selection, which is not feasible with environmental release. However, these mini-transposons insert randomly in the chromosome and position effects cannot be easily controlled. To overcome this problem, the sitespecific integration-proficient mini-CTX vectors were developed for P. aeruginosa. These vectors allow the insertion of gene cassettes at a defined location, the phage attachment 30 bp attB sequence, located at 2.94 Mb on the chromosome. Insertion at this naturally evolved phage integration site does not cause a mutant phenotype and does not compromise bacterial fitness. These miniCTX vectors have been used in P. aeruginosa for gene expression from T7 and lac promoters or for promoter studies using lac and lux-based reporter genes. Ongoing genome sequencing projects are identifying possible attB sites in other Pseudomonas species to develop a wider use of this tool. Proteomics and Microarrays Pseudomonas genome sequencing projects have been completed for some species (P. aeruginosa, P. fluorescens, P. stutzeri, P syringae, P. mendocina, P. entomophila) and others are in progress. This genomic information is being complemented by global analysis of proteins expressed. This analysis is based on fractionation methods (e.g., cellular localization), protein identification and protein expression patterns comparison. The latter, mainly based on twodimensional gel electrophoresis (2-DE) and multidimensional liquid chromatography, allows investigation of differential protein expression in response to one or more environmental or genetic variations. This comparative analysis has been used to detect differences among P. aeruginosa in chronic CF and in environmental isolates or among P. aeruginosa antibiotic-resistant and susceptible isolates. A proteomic study has been applied to investigate P. putida KT2440 biodegradation mechanisms by generating a proteome reference map for this strain grown in mineral salt medium and glucose as the only carbon source

260

Bacteria | Pseudomonas

and comparing it to protein expression patterns after growth on different organic compounds. The availability of sequenced genomes offers the possibility to develop DNA microarrays to investigate Pseudomonas environmental adaptation and pathogenesis. DNA microarrays are a tool to measure the simultaneous expression of thousands of genes in a single hybridization assay. Functional analysis of gene expression using microarray technology has been exploited to examine the global QS response and to characterize biofilm-regulated genes and antibiotic resistance of bacteria in the biofilm. Microarray studies have been extensively carried out in P. aeruginosa for which DNA chips have started to be commercially available. However, with increasing number of Pseudomonas strains being sequenced, microarrays can be tailored to study contaminant remediation in the environment or rizosphere colonization.

Biotechnology Pseudomonas strains and their products have been used in large-scale biotechnological applications. P. aeruginosa PR3 is used in the conversion of surplus soybean oil to new value-added oxygenated products, including a compound with antifungal properties in controlling rice blast disease. Frostban is an ice minus P. syringae, used commercially to prevent ice nucleation in strawberry and potato fields. P. putida is used as a biocontrol agent for the Fusarium wilt pathogen to control black root rot disease of tobacco. Thermostable lipases from P. fluorescens are used in the food and leather industry. Polyesters produced by P. oleovorans are used in special plastics. Pseudomonas biosurfactants are used in emulsification, phase separation, emulsion stabilization, and viscosity reduction. See also: Antibiotic Resistance; Biofilms, Microbial; Biotransformations; Exotoxins; Genome Sequence Databases: Annotation; Iron Metabolism; Lipopolysaccharides (Endotoxins); Metabolic

Reconstruction; Plant Pathogens and Disease: Newly Emerging Diseases; Plasmids, Bacterial; Polysaccharides, Microbial; Quorum-Sensing in Bacteria; Transposable Elements

Further Reading Bonomo RA and Szabo D (2006) Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa. Clinical Infectious Diseases 43(Suppl 2): 49–56. Bender CL, Alarco´n-Chaidez F, and Gross DC (1999) Pseudomonas syringae phytotoxins: Mode of action, regulation, and biosynthesis by peptide and polyketide synthetases. Microbiology and Molecular Biology Reviews 63(2): 266–292. Goodman AL and Lory S (2004) Analysis of regulatory networks in Pseudomonas aeruginosa by genomewide transcriptional profiling. Current Opinion in Microbiology 7: 39–44. Juhas M, Eberl L, and Tu¨mmler B (2005) Quorum sensing: The power of cooperation in the world of Pseudomonas. Environmental Microbiology 7(4): 459–471. Lyczak JB, Cannon CL, and Pier GB (2000) Establishment of Pseudomonas aeruginosa infection: Lessons from a versatile opportunist. Microbes and Infection 2: 1051–1060. Michel-Briand Y and Baysse C (2002) The pyocins of Pseudomonas aeruginosa. Biochimie 84: 499–510. Nouwens AS, Walsh BJ, and Cordwell SJ (2003) Applications of proteomics to Pseudomonas aeruginosa. Advances in Biochemical Engineering/Biotechnology 83: 117–140. Pier GB (2007) Pseudomonas aeruginosa lipopolysaccharide: A major virulence factor, initiator of inflammation and target for effective immunity. International Journal of Medical Microbiology 297: 277–295. Ramsey DM and Wozniak DJ (2005) Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis. Molecular Microbiology 56(2): 309–322. Ravel J and Cornelis P (2003) Genomics of pyoverdine-mediated iron uptake in pseudomonads. Trends in Microbiology 11(5): 195–200. Rosenau F and Jaeger K-E (2000) Bacterial lipases from Pseudomonas: Regulation of gene expression and mechanism of secretion. Biochimie 82: 1023–1032. Schweizer HP, Hoang TT, Propst KL, Ornelas HR, and KarkhoffSchweizer RR (2001) Vector design and development of host systems for Pseudomonas. In: Setlow JK (ed.) Genetic Engineering, vol. 23, pp. 69–81. Kluwer Academic/Plenum Publishers. Spiers AJ, Buckling A, and Rainey PB (2000) The causes of pseudomonas diversity. Microbiology 146: 2345–2350. Visca P, Imperi F, and Lamont IL (2006) Pyoverdine siderophores: From biogenesis to biosignificance. Trends in Microbiology 15(1): 22–30.

Rhizobia K D Noel, Marquette University, Milwaukee, WI, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Nitrogen Fixation in Legumes and Its Global Significance Phylogeny and Taxonomy Rhizobial Host Ranges Development of the Symbiosis

Glossary bacteroids Nitrogen-fixing rhizobia in the central zone of a legume nodule. classical rhizobia Bacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium (Ensifer), Mesorhizobium, and Azorhizobium. infection thread The tubular structure through which rhizobia invade the developing legume nodule. legumes Members of the Leguminoseae, the third largest family of flowering plants, traditionally distinguished by their fruit morphology (seed pods) and divided into the subfamilies Caesalpinioideae, Mimosoideae, and Papilionoideae. nitrogen fixation The reduction of molecular nitrogen (N2) to ammonia (NH3). nitrogenase The enzyme system that catalyzes the reduction of N2 to ammonia within prokaryotic organisms.

Abbreviations EPS

Physiology of Rhizobial Symbiotic Nitrogen Fixation Rhizobial Genes and Components Required in Symbiosis Rhizobial Genomes and Genetics Other Properties of the Rhizobia Further Reading

nodule The organ on legume roots, and sometimes stems, induced and invaded by rhizobia. Nod factor A lipochitooligosaccharide molecule that incites nodule formation and root hair curling on susceptible legumes. plasmid Bacterial DNA molecules that are smaller than the chromosome(s), ranging in size from 1000 to 2 000 000 nucleotides, and generally dispensable for basal bacterial growth. symbiont One of the partner organisms in a symbiosis. symbiosis The intimate association of two organisms of different species, classically divided into three categories: mutualism (both organisms benefit), commensalism (one organism benefits), and parasitism (one organism benefits and the other is harmed).

LPS

lipopolysaccharide

exopolysaccharides

Defining Statement Rhizobia are diverse Gram-negative members of the Proteobacteria that fix nitrogen inside root and stem nodules, which they incite on leguminous plants. All are facultative symbionts, and as a group they are successful soil bacteria. They have diverse metabolic capabilities, but most often couple versatile heterotrophy with aerobic, microaerobic, and anaerobic respiration.

Nitrogen Fixation in Legumes and Its Global Significance Nitrogen fixation is the reduction of atmospheric nitrogen (N2) to ammonia (NH3). The nitrogen atoms in N2 are triply bonded to each other, and this molecule is very inert chemically. Abiotic chemical conversions of N2 either cannot occur (are disfavored energetically) or occur at exceedingly low rates under normal ambient conditions.

261

262

Bacteria | Rhizobia

Only rare intense bursts of energy such as lightning provide sufficient activation energy and highly reactive molecules that allow formation of other compounds from N2. Even with suitable catalysts, high temperatures and pressures are required to form ammonia from N2 (as in the industrial Haber–Bosch process first commercialized about a century ago). In this context, biological nitrogen fixation is truly remarkable. It provides almost the only natural entry into living systems from the huge reservoir of nitrogen in the atmosphere. Nitrogen fixation occurs only in prokaryotes. The minority of prokaryotes capable of it are termed diazotrophs, and rhizobia are the best-known symbiotic diazotrophs. In the rhizobia–legume symbioses, this reaction occurs within nodules, organs that develop only in the presence of rhizobia (Figure 1). On some watertolerant legumes, nodules develop on stems, but much more commonly they appear on roots. Other important symbiotic diazotrophs include Frankia, an actinomycete genus whose species incite root nodules on at least 23 genera of plants in eight families of the rosid I clade of the dicotyledonous angiosperms (Figure 2), and cyanobacteria, which form nitrogen-fixing symbioses with lower plants and fungi (in lichens). Some animals, such

(b) (a)

as those which feed on wood, also enter into symbiosis with diazotrophs. The relationship between rhizobia and legumes is the best-known, best-understood mutualistic symbiosis between a prokaryote and a eukaryote. It is clear that the legumes benefit as they are allowed vigorous growth in nitrogen-deficient soil. This symbiosis is undoubtedly a major factor in the great success of legumes as a plant family. In contrast, some scientists have questioned whether the bacteria benefit or, at least, how they benefit. Nevertheless, the increased soil population of specific rhizobia after the cultivation of a compatible legume crop seems an obvious indicator that the symbiosis provides a reproductive benefit that should promote evolution of the species. The nitrogen fixed by rhizobia in legumes contributes more than half of the nitrogen fixed biologically on land. It is estimated that 100 Tg (1012 g) N per year is fixed on land in the absence of human agriculture, mainly by legume and other symbioses. Nitrogen fixed in agricultural legumes adds another 45 Tg N per year. Sparing the use of nitrogen fertilizer through agricultural nitrogen fixation in nodulated legumes is of great consequence. Man-made ammonia by the Haber–Bosch process fixes well over 100 Tg N per year but consumes fossil fuel, accounting for at least 1% per year of total human energy expenditure globally. Another economic and ecological problem with man-made fertilizers is that they are used in excess to achieve maximum crop yields and the excess leaches into watersources, creating problems in inland aquatic ecosystems and sometimes massive plumes of

(c) CAESALPINIOIDEAE

Leguminoseae Mimosa

MIMOSOIDEAE

Aeschynomene

Figure 1 (a) Clusters of nodules on bean (Phaseolus vulgaris) roots. The white striations are lenticels, which appear as the nodules mature. Elsewhere the nodules have a dark reddish hue due to the presence of leghemoglobin. (b) A slice through a nodule after it had been chemically fixed, dehydrated, and embedded in resin. It is oriented in this panel such that the bottom shows the attachment to the root. Bacteroid-containing cells in the large central zone have been stained intensely by toluidine blue. About half the plant cells in this zone are smaller and lack bacteroids (are not darkly stained). Vascular bundles are in the periphery within the nodule cortex that surrounds this central zone. The overall anatomy is typical of the determinate nodules of the Phaseoleae tribe. (The image of panel (b) is courtesy of Kathryn VandenBosch.) (c) An enlargement of the central zone showing plant cells packed with bacteroids, along with interspersed vacuolated cells free of bacteroids. These plant cells are marked by darkly staining spheroid starch grains. ª 2008 Dale Noel. Published by Elsevier Inc. All rights reserved.

Rosid I Rosids Dicots

Lotus Sesbania Trifolieae, Fabeae

PAPILIONOIDEAE

Phaseoleae Angiosperms

Figure 2 Gross outlines of legume classification for the purposes of this article. At the bottom left the concentric ovals indicate first that the legumes belong to the Rosid I clade of the Rosids. The Rosids in turn are a clade within the Eudicots (‘dicots’) of the angiosperms, the flowering plants. Genus Parasponia is in Rosid I outside of the legume family. The branchings on the right side are meant to give a rough sense of the divergence among the various groups of legumes specifically mentioned in the text. Four genera (in italics), three tribes, and the three subfamilies (in capital letters) are indicated. ª 2008 Dale Noel. Published by Elsevier Inc. All rights reserved.

Bacteria | Rhizobia

eutrophic pollution in river deltas. From this perspective, switching more crop production to practices based on nitrogen fixation by legumes is highly desirable. Rhizobia and legumes are important in agriculture and related commerce. Certain legumes are very important agricultural crops, as forage, animal feedstock, food directly consumed by humans, and commodities processed into a large number of end products. Legumes are estimated to supply about 20% of worldwide food protein. Soils deficient in the appropriate rhizobia can be inoculated with that specific rhizobia to enhance nodulation and the overall yield of a legume crop. Gross revenue generated by the inoculant industry was estimated at about $150 million in 2004. Whether inoculated or not, the total amount of nitrogen fixed by rhizobia in legume crops in the United States alone was valued at more than $2000 million, based on the price of the equivalent amount of nitrogen fertilizer in 2005. Sometimes, an objective of planting legumes is to improve the nitrogen content of the soil for the benefit of other crops. In this respect, it is important to realize that growing legume plants do not leak appreciable amounts of fixed nitrogen into the soil. Soil nitrogen is materially improved by cultivated legumes only when the legume plants and seeds are plowed into the soil, rather than harvested or eaten directly by livestock.

Phylogeny and Taxonomy Taxonomy and phylogeny of bacteria is a dynamic topic; any current classification scheme is subject to future revision. The genera of rhizobia are currently divided into the groups shown in Table 1.

263

The term ‘rhizobia’ defines a group of bacteria based on one complex property, the inciting of legume nodules inside which nitrogen is fixed. The rationale for considering these bacteria as a group is based on the great interest in and the importance of this property, rather than believing that they all belong to one monophyletic evolutionary group. On the contrary, rhizobia are polyphyletic and much more diverse than what had been supposed even a decade ago. The history of the ongoing revisions in their classification, and the accompanying consternation among nontaxonomists, illustrates the overall revolution in bacterial taxonomy within the past three decades. However, the realization that rhizobia did not belong collectively to a single, simple taxon had emerged, though gradually, much earlier. The early history of studying root nodules climaxed in 1888 when Beijerinck became the first to isolate a bacterium capable of nodulating a legume, naming the isolate Bacillus radicicola. Frank in 1889 decided that all bacteria isolated from legume nodules would be assigned the name Rhizobium leguminosarum. The type strain for the genus and species was isolated from pea nodules. Subsequently, after E. B. Fred and colleagues proposed the cross-inoculation concept, the taxonomy of the genus Rhizobium was, for many years, based on the host specificity of nodulation. Cross inoculation is a test for whether an isolate from one plant species incites nodules on another plant species. The taxonomic concept was that the rhizobia fell into groups defined according to the plants nodulated by each group. This concept was very useful in agriculture, but, gradually, it was appreciated that this classification scheme and the cross-inoculation concept often were very poor predictors of the true relatedness and diversity of these bacteria. The first departure from this taxonomy

Table 1 Classification of Rhizobia Proteobacterial

Species

division

Genus

Number

Representatives

Representative hosts

Alpha

Rhizobium

16

Bradyrhizobium

7

Sinorhizobium (Ensifer)

11

Azorhizobium Mesorhizobium (Allorhizobium) Methylobacterium Devosia Ochrobactrum Phyllobacterium Burkholderia Cupriavidus (Ralstonia)

2 11 1 1 1 1 1 5 2

leguminosarum etli tropici japonicum elkanii meliloti frediia caulinodans loti undicola nodulans neptuniae lupinus lupinii phymatum taiwanensis

Pea, clover, bean Bean Bean, Leucaena Soybean, cowpea Soybean Alfalfa Soybean, cowpea Sesbania Lotus spp. Neptunia Crotalaria Spp. Neptunia Lupinus Trifolium and Lupinus Mimosa Mimosa

Beta a

The host range of some strains is extremely broad.

264

Bacteria | Rhizobia

was based on growth rate in culture (‘fast growers’ vs. ‘slow growers’), with the slow growers being assigned to a new genus, Bradyrhizobium. Since the 1980s, rhizobial taxonomy has been based on nucleotide sequences, mainly of the DNA encoding ribosomal RNA, mirroring the efforts of bacteriologists in general to create a taxonomy reflective of true phylogenetic relatedness. Recently, the availability of total genomic sequences has supported the basic genetic relatedness deduced in this way. By 2006, 48 species of rhizobia had been classified among six genera whose names include the root word ‘rhizobium’ (Table 1). In addition, at least 11 Proteobacterial species outside these more ‘classical’ genera are capable of eliciting nitrogen-fixing nodules on legumes. These latter species are found in four genera in the alpha division of the Proteobacteria and two genera in the beta division of the Proteobacteria (Table 1). Discovery of legume-nodulating bacteria among the beta Proteobacteria is the most recent and the most dramatic indication of the breadth of distribution of this property among diverse bacteria. The current taxonomy predicts what physiological characterizations have borne out: aside from entering into symbiosis with legumes, the different groups of rhizobia exhibit great diversity in other properties. The dynamic nature of current bacterial taxonomy is illustrated by the case of Sinorhizobium meliloti. This species has a long history of study and was one of the first species to be included within ‘Rhizobium’ by the cross-inoculation concept. About two decades ago, it became apparent that this species did not belong to a genus whose type species was R. leguminosarum. Because of its close genetic relationship with the type species of the genus Sinorhizobium, S. fredii, this well-known bacterium was renamed S. meliloti. Very recently, it has been discovered that this genus should include bacteria previously assigned to the genus Ensifer. Because Ensifer as a genus name preceded the naming of Sinorhizobium, it is likely that S. meliloti will undergo another name change to Ensifer meliloti. The matter is still under debate at the time of this writing, but it appears that most taxonomists have adopted the new name. It is likely that some of the organisms now classified as Bradyrhizobium and Rhizobium will eventually be reclassified under different genus names. Both are very diverse genera according to the current norm in bacterial classification. They include rhizobia more closely related to bacteria in different genera than to other rhizobia within the genus to which they are presently assigned. Likewise, certain species currently assigned to other genera probably should be moved to Rhizobium or Bradyrhizobium, if progress toward a rational taxonomy is to be continued. A controversial example, which has implications for certain species grouped within the venerable genus Agrobacterium and some currently assigned to Rhizobium, involves the

single species of the genus currently named Allorhizobium. In Table 1 this latter genus name is in parentheses because this species and possibly several closely related Rhizobium species are likely to undergo reclassification.

Rhizobial Host Ranges Legumes are a very successful family of plants, known in botany as the Fabaceae or Leguminosae. In traditional taxonomy it is the third largest family within the angiosperms (flowering plants), with more than 700 genera and 20 000 species. This family is divided into three subfamilies: Caesalpinioideae, Mimosoideae, and Papilionoideae (Figure 2). About 20% of the known legume species have been studied to determine whether they nodulate. All three subfamilies have members that form nitrogenfixing nodules, but most of the well-known symbioses are in the Papilionoideae. It should be noted that there are legumes that, despite extensive examination in some cases, have never been documented as nodulating. In surveys among the Caesalpinioideae, 10% of the genera were observed to nodulate. Nodulation is much more common among Mimosoideae (about 80% of the genera surveyed) and Papilionoideae (about 90% of the genera surveyed). Historically, great attention has been placed on the host range of the rhizobia, and any description of a species generally has included its known host range. Some rhizobia have very narrow host ranges, composed of only one or a few closely related legume species, whereas others have much broader host ranges. One Sinorhizobium (Ensifer) strain, NGR234, can nodulate legumes belonging to at least 112 genera that include representatives of all three subfamilies. Representative hosts of various rhizobial species are listed in Table 1. Host range is a complex issue with many caveats. Obviously any statement regarding host range is limited by the number of potential hosts that have been tested. In addition, it should be strictly qualified according to the symbiotic parameters that have been observed. A strain may incite nodules, but may not provide nitrogen fixation. Hence, the host range of nitrogen fixation (‘effective’ symbiosis) is narrower than the host range of nodulation. Within a rhizobial species, different strains can have clearly different host ranges. For instance, some strains of R. leguminosarum nodulate pea (‘biovar’ viciae), whereas others nodulate clover (bv. trifolii) or bean (bv. phaseoli). These bacteria are all assigned to the same species because of the close homologies of their chromosomes, whereas, in this species, host range is specified by genes on plasmids. A given strain can be converted from one host range to another by replacing one plasmid with another. This was one type of observation that persuaded geneticists that cross inoculation was not a proper way to define species. Adding further complication is that these

Bacteria | Rhizobia

genetically defined host ranges overlap to differing degrees. Certain bv. viciae strains elicit some degree of nodulation on beans, and the quality of the nodulation can depend on the variety or race of the bean plant. The converse is also true. There are variant individuals within a host species that resist nodulation by a rhizobial strain that does well on most other races, cultivars, and varieties of that host. This type of incompatibility can be straindependent and sometimes has been traced to presence or absence of specific genes in the rhizobial strain. Given that rhizobial taxonomy is now based on divergence of ubiquitous chromosomal genes, such as those for ribosomal RNA, and that the genetics of symbiotic host specificity allows variation of host range among the strains of one species, one might ask whether there are any correlations between rhizobial taxonomic groups (species and genera) and host taxonomic groups. Table 1 implies that there are such correlations, and, indeed there are many inconclusive overall trends of this sort. For example, among legumes in the genus Mimosa (in the subfamily Mimosoideae), a majority of the reported nodule isolates belong to the beta Proteobacteria. Nevertheless, some of the beta Proteobacterial rhizobia that do well on Mimosa were isolated first from legumes in the subfamily Papilionoideae. Moreover, Bradyrhizobia also are commonly isolated from Mimosa. Therefore, this correlation is not a tight one. Another generalization is that, among highly diverged ‘temperate’ legume tribes within the Papilionoideae (including Trifolieae and Fabeae among others), ‘fast-growing’ rhizobia of the alpha Proteobacteria predominate. However, such rhizobia are also found as symbionts elsewhere among the legume family. Particular species have been observed to deviate from the general trend shown by the other species within a genus or tribe of rhizobia or legumes. For example, among isolates of Phaseolus vulgaris (common bean) nodules, fast growers predominate, but for other genera of the tribe Phaseoleae and even other species of the genus Phaseolus, Bradyrhizobia usually predominate. A related evolutionary question is whether symbiotic organisms, particularly in mutualisms, evolve toward high specificity for each other as a way to maximize the efficiency of interaction. There is no convincing evidence to support this possibility as the overall tendency among the rhizobia. However, there are instances in which the host genus and the bacterial species are mainly restricted to each other. One well-documented example is the legume genus Trifolium (clover) and R. leguminosarum bv. trifolii. Within this host–bacterial group, there is further restriction in that some of the bacterial strains nodulate and fix nitrogen only on certain Trifolium species. Does such specificity provide selective advantage, or is it an evolutionary accident? Judging by whether specialists give better nitrogen fixation than generalists, the overall evidence is mixed. Although the most promiscuous rhizobial

265

strain, NGR234, is a poor symbiont on many of its hosts, there are many instances in which, for example, a Bradyrhizobium strain is very efficient on several disparate hosts. Parasponia in the Ulmaceae (elm) family is the only known nonlegume genus that is nodulated by bacteria that also nodulate legumes. Strains within several different groups of rhizobia, including the aforementioned Sinorhizobium spp. strain NGR234, have been found to nodulate this host, but its most effective known symbioses are with certain bacteria currently classified within the genus Bradyrhizobium. It is striking how often members of this latter genus, or closely related bacteria, are found as the dominant symbionts of legumes in the Caesalpinioideae and Mimosoideae, as well as in the diverse taxa of the Papilionoideae.

Development of the Symbiosis As a general rule, legumes develop nodules in nature only in the presence of the appropriate rhizobia. There is now a basic understanding at the cellular and molecular levels regarding how nodules develop. The paradigm described in the following sections has been established by studies with a relatively few legume–rhizobia combinations, those involving certain agronomically important legumes and some closely related model legumes. All are from the subfamily Papilionoideae and fall into just four of the many diverse tribes within this subfamily. They include soybeans, common beans, and cowpea in the tropical tribe Phaseoleae and alfalfa, clover, and peas in the temperate tribes Trifolieae and Fabeae (Figure 2). The model organisms are Medicago truncatula in the same genus as alfalfa in the Trifolieae and Lotus japonicus within a separately-diverged tribe, the Loteae. These hosts exhibit what is sometimes called the root hair-infection type of nodule development, which is observed in the majority of legume–rhizobia symbioses. After presenting a summary of this type of development, deviations shall be discussed. Development Involving Nod Factors and Root Hair Infection Formation of the nodule primordium

Legume roots and germinating seedlings exude flavonoid compounds that induce specific rhizobia in the vicinity to produce and secrete a glycolipid, the Nod factor (Figure 3). This rhizobial compound then elicits two important responses in the plant: the curling of root hairs as they develop and the triggering of cell division in root cortex cells to form the nodule primordium. The primordium further generates cells that differentiate to become the nodule. In the Phaseoleae, this cell division initiates in the outer cortex. In the Trifolieae and Fabeae, it begins

266

Bacteria | Rhizobia

R3 O O R4

O R2 CH2 O

O R1 CH2 O

OH CH2 O OO R7 1–3

OO R5 N

R6

NH O

Fatty acyl

O R8 NH O

CH3

CH3

Fatty acyl variations:

O

Strains with nodA only

O 1-3

0-1

0-1

0-1

0-1

Strain-specific nodAFE alleles

Figure 3 Structures of the Nod factors. These signal molecules are produced by rhizobia and trigger nodule development in the legume host. The backbone of the structure is three to five units of N-acetylglucosamine whose linkages are catalyzed by the enzyme encoded by gene nodC. NodB protein catalyzes removal of the acetyl group from the glucosamine at the nonreducing end (leftmost in this figure), and NodA catalyzes the addition of a fatty acyl group to the deacetylated nitrogen. In many species NodA adds the predominant fatty acids made for phospholipid synthesis. Other species have NodE (a lipid desaturase) and NodF (an acyl carrier protein), which produce and deliver acyl groups with additional unsaturated groups whose location depends on the particular combination of nodAFE alleles. Almost every possible position is substituted in one or another of the numerous variants of this structure. These positions are indicated as R1–R8 in the drawing. Substitutions at R1 and R2 seem to be most common. Examples (and the specific genes required) are additions of fucosyl (nodZ), sulfyl (nodH), acetyl (nodX) moieties at R1; and acetyl (nodL) and carbamyl (nodU) at R2. The fucosyl at R1 can itself be modified by 2-O-methyl (noeI) or 4-O-acetyl (nolL) additions. A given structure has only a few of these possible modifications. For instance, the predominant structure in Sinorhizobium meliloti 1021 (and the first Nod-factor structure determined) has four glucosamine units, a fatty acyl with two unsaturated bonds, and sulfation at position R1. ª 2008 Dale Noel. Published by Elsevier Inc. All rights reserved.

within the inner cortex near the root pericycle. In the latter cases, the cell division generates a permanent meristem that leads to elongated indeterminate nodules, whereas the former leads to transient meristematic activity and determinate nodules (Figure 1). Lotus nodule development has elements of these two extremes; the primordium develops deeply in the cortex but the meristem is transient and determinate nodules result. Recent progress with the plant genetics and cell biology of nodule formation has provided insight into the plant response to Nod factors. The earliest responses that have been documented are a rapid calcium flux into the root hair cell and the resulting fluxes of other ions that cause plasma membrane depolarization. About 10–15 min later, dramatic oscillations in cytosolic calcium, called calcium spiking, occur. By analyzing plant nodulation mutants, plant genes required for nodule primordium formation have been ordered into a developmental pathway according to the

relative time at which the proteins specified by the genes appear to act. The first genes in the pathway encode presumptive Nod-factor receptors whose amino acid sequences align with those of a family of proteins that bind molecules resembling the oligochitin backbone of the Nod factors. Mutations in these genes block all responses to Nod factors including root hair curling. Mutations in some of the other genes affect actions downstream in the pathway; they block the calcium spiking completely or partially. Further downstream in the pathway is a nuclear calcium/calmodulindependent kinase. Null mutants in this protein block further progress to nodule primordium. More interesting are mutants in which this protein is constitutively active and that form nodule primordia in the absence of Nod factor. Further downstream in the pathway is a cytokinin receptor with cytokinin-dependent kinase activity. Mutants in which this protein is constitutively active also form nodule primordia in the absence of Nod factor. Hence, plant genetics suggests that formation of nodule primordia is a series of events involving at first mainly calcium fluxes as secondary signals to activate downstream protein phosphorylation events and, later, activation of further protein phosphorylation by the important plant hormone cytokinin. How this pathway connects with regulation of transcription factors and the proteins that carry out visible organogenesis remains to be elucidated. These latter proteins include nodulins, proteins that are highly upregulated in nodules. A large number of such proteins have been identified. Infection

Coincident with this organogenesis is the second major developmental program, the infection process whereby the rhizobia enter the developing nodule. Nod factor is believed to potentiate this second process by causing the development of curled, and often otherwise deformed, root hairs. Generally, the infection starts from a tightly curled portion of the root hair or where a curled root hair is folded against the main body of the root hair cell (Figure 4). From a colony of rhizobia seemingly trapped within the folds of the root hair, there develops a narrow tubular structure known as the infection thread. As seen in Figure 4(a), on certain plants, several infection threads arise from the same rhizobial colony. At the core of the infection thread are rhizobial cells in linear files. Surrounding the bacterial colony is a tubular plant wall. This wall is itself surrounded by a tubular plant plasma membrane, which at all times and locations separates the bacteria from the plant cytoplasm through which the infection thread develops. At the growing tip of the infection thread, one end of the leading bacterium is in close proximity to the plant membrane (Figure 4). Everywhere else the bacterial colony is surrounded by the plant wall. Growth of the infection thread is thought to derive basically from two influencing factors: bacterial growth and growth of the surrounding plant membrane. According to this idea, bacterial cell fission and

Bacteria | Rhizobia

267

(b)

(a)

Infection thread concept BC Plant cytoplasm RH

Infection thread

IT Vesicles and activities needed for infection thread growth

NP

Matrix material

Rhizobia Thread wall Plasma membrane

Figure 4 (a) Infection site on the surface of a bean root. This is typical anatomy on plants in the Phaseoleae, in which infections usually arise in a curled root hair appressed against the body of the root hair cell. At the stage captured in this unfixed hand slice, the developing nodule has just become apparent as a slight bump emerging from the root. RH, curled root hair; BC, bacterial colony trapped within the curl of the root hair; IT, infection threads; at least four infection threads have emerged from the bacterial colony. NP, nodule primordium; dividing cells in the outer cortex just below the infected root hair are becoming organized into the primordium from which the nodule will develop. (b) Infection thread concept. The thread is growing in the rightward direction. The bacterium on the right represents bacteria near the tip of the infection thread that can grow and reproduce, whereas the one on the left represents the rest of the colony that is encased in matrix material. The membrane that surrounds the thread grows by accretion of membrane vesicles that converge on the tip of the infection thread. They are guided to that spot by the cytoskeleton. One hypothesis is that this region of convergence is signaled by plant receptors that bind to ligands on the bacterial surface. The narrow dimensions of this region where such ligand–receptor interactions are possible in turn determine the linear character of infection thread elongation. ª 2008 Dale Noel. Published by Elsevier Inc. All rights reserved.

enlargement provide the basic driving force for the process. Observations indicate that the bacteria divide only near the growing tip of the infection thread, whereas the rest of the bacterial colony is enmeshed in a matrix of polymers deriving from both the plant and the bacteria. This enmeshed portion of the colony provides a solid anchor for the push from the dividing bacterial cells. As the bacterial colony pushes into a plant cell, the plant plasma membrane invaginates to surround the bacteria. The narrow tubular nature of its subsequent growth hypothetically arises from membrane vesicles in the plant cytosol fusing only at the tip of the infection thread membrane. Not only membrane lipids and proteins, but also the material and enzyme machinery for extending the thread wall inside the growing membrane are delivered by these vesicles. The shape and dimensions achieved in synthesizing this tubular wall are the third, and the permanent, influencing factor on the size and orientation of the infection thread. Formation of the bacteroid zone and nodule maturation

The infection thread branches and spreads the infection in all three dimensions. As the infection thread penetrates deeper into the layers of cells in the developing nodule, the last stage of infection begins: bacterial release into certain plant cells that become filled with rhizobia. During this endocytosis at the unwalled tips of infection threads (or from enlarged unwalled areas known as infection droplets) the

bacteria get surrounded with plasma membrane from the plant. The bacteria differentiate into nitrogen-fixing cells known as bacteroids, and the surrounding plant membrane becomes the specialized peribacteroid membrane. The resulting cellular units consisting of bacteroids and the surrounding peribacteroid membranes are often termed symbiosomes. Depending on the host, a symbiosome contains one or several bacteroids. Up to 10 000 bacteroids are densely packed within one infected cell. Bacteroids are generally rod-shaped. However, they may take on different shapes as determined by the plant host. Bradyrhizobial bacteroids in peanut nodules are spherical, whereas the same strain on cowpea forms rodshaped bacteroids. Clover and pea bacteroids have pleomorphic or club shape, and they are very sensitive to osmotic stress. It is this type of bacteroid that has led to the erroneous generalization that bacteroids are terminal cell forms incapable of reproduction. On the contrary, bacteroids from many plants very efficiently form colonies on appropriate agar media. In the nodules of the Phaseoleae, Fabeae, and Trifolieae, not all of the cells in the infected region of the maturing nodule fill with bacteria. The others, the uninfected cells, differentiate to carry out steps in the nitrogen assimilation pathway that are downstream of the initial steps carried out within the infected cells. The bacteriafilled cells assume a distinctly cooperative metabolism and chemical environment that allows nitrogen fixation in the bacteria, as well as assimilation of the fixed nitrogen

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transported from the bacteria on the plant side. This physiology is outlined in the section titled ‘Physiology of rhizobial symbiotic nitrogen fixation’ below. Integration and regulation of nodule development

Rhizobial mutants defective in infection have revealed that nodule differentiation depends on infection. The degree to which this is true varies from one host to another. Some hosts (e.g., pea) form almost no nodule tissue unless infection is sustained beyond the root hair. Others (e.g., alfalfa or bean) can undergo organogenesis in response to noninfective rhizobial mutants, but exhibit anatomy and cytological differentiation that are quite distinct or incomplete compared with normal nodule development. Researchers working with some of these plants (e.g., bean and Lotus spp.) refer to the resulting structures as pseudonodules to emphasize the underdevelopment and deviation from normal development. Although pseudonodules result from infections on bean that do not progress into cell layers below the root hair, almost normal anatomy results if infection stops only a few cell layers further, well short of the stage at which bacteria are released from infection threads. Nodule development is controlled by the plant according to at least two factors. One factor is whether the legume senses nitrogen limitation. Both development of nodules and feeding of the rhizobia to allow nitrogen fixation are energy-intensive processes. Perhaps because of the energy cost, the plant does not enter into symbiosis if soil has an excess of fixed nitrogen. Plants also repress nodule formation on some root sites according to whether other nodules are already forming nearby on the root. Nodule development generally initiates only in immature portions of the root where root hairs are just emerging. Nodules developing in older portions of the root will suppress nodule formation in younger portions. This is sometimes called autoregulation of nodule development. Autoregulation leads to optimal spacing of nodules that prevents wasteful unchecked nodule formation, but at a certain distance allows further nodulation as the plant grows and its need for nitrogen increases.

Deviations from the Foregoing Model Infection not in root hairs; crack entry

Some legumes do not have root hairs but still nodulate. Even those that do form root hairs may not have infection via root hairs. One alternate mechanism is that rhizobia enter at cracks in the root epidermis at the base of lateral root emergence. This process is sometimes referred to as crack entry. On certain legumes, the bacteria enter at other types of wounds, cracks, or, on some plants, by penetration between the cells of initially intact epidermal surfaces.

No symbiosomes; fixation threads

In some legumes (notably, but not limited to the Caesalpinioideae) the bacteria are not released from infection threads; that is, symbiosomes are not formed. Instead, as nodules mature, the infection threads enlarge greatly within plant cells inside the nodule cortex, allowing bacterial proliferation inside this engorged thread. In this state, nitrogen fixation occurs within the bacteria, and the fixed nitrogen presumably is transferred into the surrounding cytoplasm. Such structures are known as fixation threads. Infections between cell walls, not as threads that penetrate plant cells

In some legumes, infection threads that travel through plant cells are not observed. Instead, the infection is channeled between plant cells until the point at which bacteria enter cells in large numbers within either symbiosomes or fixation threads. Some crack-entry infections are of this type; but, on some plants, infections may start between cells and later develop true infection threads. In perhaps 25% of all nodulating legumes, infections are not observed in root hairs, and true infection threads do not develop. All plant cells in the central infected zone fill with bacteroids

In the paradigm above, it is considered very important for nitrogen assimilation by the plant that development give rise to specialized uninfected cells in the central, nitrogen-fixing zone of the nodule. Such cells are thought to channel the nitrogen-containing carbon compounds to the vasculature so that the nitrogen can be transported to the shoot. In plants that use ureide compounds for this purpose (soybeans, beans, and cowpea), the final steps of synthesizing the transport compounds occur in these specialized uninfected cells. However, this division of labor is not found uniformly in all legumes; there are tribes of legumes in which all the cells in the central nitrogenfixing zone are infected. Nod factor not needed

Nod factor synthesis is directed by rhizobial nod genes (see ‘Rhizobial genes and components required in symbiosis’). Until recently, all rhizobia surveyed for nod genes had them. However, two Bradyrhizobial strains (BTAi1 and ORS278) that nodulate two species of Aeschynomene, a genus in the Papilionoideae not closely allied with the commonly studied agricultural ones (Figure 2), do not harbor nod genes. Another Bradyrhizobial strain that nodulates these two species does have nod genes. However, mutation of its nod genes does not prevent nodulation of these plants, whereas nod mutations do prevent its nodulation of certain other species of Aeschynomene. These latter species cannot be nodulated by strains BTAi1 and ORS278. It is very interesting that these strains lack identifiable nod

Bacteria | Rhizobia

genes, but thus far the lack of this requirement in these particular symbioses cannot be correlated with any marked difference in the nodulation process. Aeschynomene species support infection by crack entry, without true infection threads, but rhizobial nod mutants on other hosts with these characteristics have failed to nodulate.

Stem nodules

Certain water-tolerant legumes such as Aeschynomene and Sesbania (Figure 2) form nodules on stems and on roots. The processes of nodule formation on the stems in these two plants exhibit many differences, but in both cases infections start where adventitious roots emerge from the stem, in a variation of the theme of crack entry. The two exceptional Aeschynomene species mentioned above do not require nod genes of the rhizobia for either stem or root nodulation. However, Sesbania rostrata does not nodulate on stems or roots if its rhizobial partner lacks nod genes. Interestingly, under some conditions on this latter plant, root nodules also start by infections in curled root hairs. The same bacterium (Azorhizobium caulinodans) is accepted by the plant under all these variants of nodulation, although a wide variety of other rhizobia also may nodulate the roots. Parasponia

Nodulation of the nonlegume genus Parasponia by rhizobia has many of the above ‘deviant’ properties. The rhizobia enter between epidermal cells and, as the bacteria penetrate deeper, true infection threads may form. Nitrogen fixation occurs in fixation threads. One additional property distinguishes Parasponia nodules from all known legume nodules. In the mature nodule, the infected cells, those that harbor the large fixation threads, are in the periphery (the nodule cortex) and vasculature develops in the center as it does in roots. This is the reverse of one property that appears to be true of all legume nodules: the infected nitrogen-fixing cells are central, with the vasculature being peripheral. In this respect, Parasponia nodule development is like that of the nodules of hosts of the actinorhizal bacterial genus Frankia. The presence of nod genes has been confirmed in rhizobia that infect Parasponia in all cases examined for this property. When the nod genes of one of these strains were mutated, it could not nodulate Parasponia or its legume hosts siratro and cowpea. In all of these cases, it is the plant that determines these differences in nodule development; the differences assort with membership in different legume taxonomic groups. In contrast, a given rhizobial strain (usually a Bradyrhizobium strain) can nodulate and fix nitrogen on plants showing different modes of nodule development and infection, for example, crack-entry on peanut and root hair infection on cowpea. Whether the set of bacterial

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genes and components required is any different on these different hosts is for the most part undetermined. Although the issues of infection threads and fixation threads have been addressed in recent sampling of the Caesalpinioideae, the issues of root hair infection and the requirement for Nod factor have hardly been addressed at all. It is important to do so, to assess whether or not the evolution of Nod factor was central to the early evolution of the symbiosis. Many of the variations in nodule development appear to be exclusively controlled by the plant, but Nod factor obviously required evolution involving the bacteria. Similarly, it would be revealing to investigate the requirements for bacterial polysaccharides (see ‘Rhizobial genes and components required in symbiosis’ and ‘Other properties of the rhizobia’) in the symbioses of the Caesalpinioideae. In general, the theory of nodule development would benefit from investigating mutants of the rhizobia that form symbioses with properties that deviate from those of the paradigm described above.

Physiology of Rhizobial Symbiotic Nitrogen Fixation Nitrogen fixation in legume nodules occurs within the bacteria inside specialized plant cells in the central region of mature nodules. Where they are carrying out nitrogen fixation, rhizobia are known as bacteroids, a term that has already been introduced in connection with nodule development. Functioning bacteroids must have the enzyme machinery for carrying out nitrogen fixation, encoded by nif genes, and the specialized catabolic and respiratory components that supply energy and electrons for the nitrogen fixation reaction, encoded by fix genes. In addition, they must have a compatible cell surface and transport systems for the exchange of metabolites that characterize the symbiosis. Once again, it should be emphasized that what is known is mainly from studies of the important agricultural legumes in the Papilionoideae. The enzyme dinitrogenase catalyzes the ultimate reaction of nitrogen fixation: N2 þ 8Hþ þ 8 electrons ! 2NH3 þ H2

The electrons are delivered at high energy potential to dinitrogenase from internal reductants by a second enzyme known as dinitrogenase reductase in a reaction that requires the hydrolysis of two ATP to two ADP and two phosphates for each electron transferred. Dinitrogenase and dinitrogenase reductase traditionally are considered as a complex known as nitrogenase. Although other nitrogenases called alternative nitrogenases exist in nature, to the extent surveyed, all nitrogen fixation in rhizobia is by the classical, and most widespread, dinitrogenase containing molybdenum. The

Bacteria | Rhizobia

Transport to shoot

Sucrose

O2

Ureides Uninfected cells Asparagine, purines

Glucose

Lgb·O2

PEP

Lgb

CO2

Glutamine Glutamate

?

Atp

Dct Amino Acids?

NH4+ ATP NH3

CO2

H2O ~ Δμ H+ ADP + Pi

O2 cbb3

Malate

bc1

270

e–

Krebs

e– N2

Bacteroid N2

Peribacteroid membrane

Figure 5 Coordination of plant and bacteroid metabolism. The leftmost third of this diagram depicts carbon metabolism. Sucrose in the infected plant cell is catabolized to malate, which enters the bacteroid via the Dct transport system and is oxidized by the Krebs cycle and ancillary reactions to yield CO2 and ‘electrons’ (e), mainly in the form of NADH. The middle third of the diagram depicts nitrogen fixation and assimilation. In some manner the electrons from catabolism reduce ferredoxins that in turn donate the electrons to nitrogenase for the reduction of N2 to NH3. Most of the fixed nitrogen may be excreted from the bacteroid as ammonium (NH4 þ ), but some may be incorporated into organic compounds diverted from the Krebs cycle to form amino acids that are transported to the plant cytosol (question marks in the diagram). The plant assimilates the nitrogen into nitrogen-rich carbon molecules such as asparagine and ureides (allantoin and allantoic acid) for transport in the xylem to the shoot. ATP for nitrogen fixation is generated by microaerobic respiration (rightmost third of the diagram). It is presumed that electrons enter the electron transport chain mainly as NADH from the Krebs cycle that reduces ubiquinone, whose reduction of oxygen is mediated by the bc1 and cbb3 respiratory complexes. Leghemoglobin (Lgb) in the plant cytosol maintains low free oxygen concentrations but mediates high oxygen flux to the bacteroids, where the high affinity of the cbb3 oxidase pulls oxygen flux toward the bacteroid membrane. The electrochemical gradient of protons (
mHþ) generated by the electron transport chain drives ATP synthesis via the bacteroid ATP synthase (Atp). ª 2008 Dale Noel. Published by Elsevier Inc. All rights reserved.

immediate source of electrons for this reaction in at least some rhizobia appears to be a ferredoxin. Although the exact connection of this ferredoxin to the rest of bacteroid metabolism is still not clear, electrons for nitrogen fixation are provided by oxidation of dicarboxylates from the plant, and the energy (ATP) is generated by aerobic respiration (Figure 5).

Oxygen Concentration, Microaerobic Metabolism, and Bacteroid Regulation As nodule organogenesis and infection reach the mature stage at which nitrogen fixation begins, a marked gradient in oxygen concentration is generated across the nodule. In the central region where nitrogen fixation occurs, the infected cells’ free oxygen concentration is 5–30 n mol l1, whereas a typical saturating aerobic concentration is 250 mmol l 1. One factor that causes this extreme decrease in oxygen is the high population of rhizobia in the central zone of the nodule. Rhizobia are obligate respirers and use oxygen in preference to other terminal electron acceptors. Another factor appears to be the creation of oxygen

diffusion barriers as the nodule differentiates, documented to exist particularly in the determinate nodules of the Phaseoleae. The anatomy responsible for attenuating oxygen diffusion appears to be an intercellular layer in the apoplastic spaces in the periphery of the nodule cortex. Importantly, this layer appears to be subject to regulation that somehow changes the underlying physical attributes according to physiological cues. A third important factor is leghemoglobin, an oxygen-binding plant protein present in high concentrations in the infected cells of the central nodule zone. Up to 30% of the soluble plant protein of a nodule is leghemoglobin, which is responsible for the characteristic red color of active nodules. As an oxygen-binding protein, it buffers the free oxygen to a low concentration consistent with its dissociation constant. At the same time, like myoglobin in animal muscle cells, leghemoglobin facilitates oxygen diffusion at a high rate within the bacteroid-containing cells, even though the free-oxygen concentration is very low. The nearly complete consumption of this oxygen by rhizobial respiration prevents poisoning of nitrogenase, both enzymes of which are irreversibly inhibited by oxygen.

Bacteria | Rhizobia

Because the concentration of free oxygen in the bacteroid environment is low , aerobic respiration by bacteroids requires a cytochrome oxidase with very high affinity for oxygen, the cbb3 enzyme that is found in many Proteobacteria. It is encoded by genes named fixNOPQ in rhizobia. Another essential component in this symbiotic microaerobic respiratory chain is cytochrome bc1, whereas cytochrome bc1 is not required by rhizobia for aerobic growth ex planta in media and conditions commonly used. The low oxygen concentration provides the cue for regulation of fix and nif genes in the nodule, mainly through its effects on activity and synthesis of central regulatory proteins such as NifA, FixLJ, and others. Indeed, unlike for bacteria that fix nitrogen nonsymbiotically, nitrogen status within bacteroids appears to be largely irrelevant in regulating symbiotic nitrogen fixation. The logic of symbiotic regulation is developmental. Nitrogenase and other enzyme systems for bacteroid metabolism are induced when development and infection have provided the optimum environment and plant components for nitrogen fixation by the bacteria and assimilation by the plant. Oxygen concentration is a simple cue for signalling that the proper stage has been achieved. Regulation by low oxygen implies that these bacteroid metabolic pathways actually might not be unique to bacteroids, and that they can be induced ex planta under low oxygen. In general, this is indeed the case. Because the degree of induction of a particular nif or fix gene cluster often is much higher in the nodule than under microaerobic conditions ex planta, it is possible that additional factors are at play in the nodule. Bacteroid Carbon Metabolism, Host Contributions, and Nitrogen Assimilation The plant supplies the bacteroids with carbon compounds, whose oxidation in the bacteria releases electrons for the reduction of nitrogen and makes possible the microaerobic respiration that generates ATP needed to drive nitrogen fixation. Inasmuch as rhizobia generally can use glucose as carbon source, it might be supposed that the sucrose provided to roots from shoot photosynthesis would be simply broken down to glucose for the bacteroids. Such is not the case, however. Instead, glucose and fructose in the nodules are catabolized to phosphoenolpyruvate and then carboxylated to form dicarboxylic acids. Bacteroid proliferation and nitrogen fixation are highly dependent on the ability of bacteroids to utilize dicarboxylic acids of the Krebs cycle (malate and succinate). This was suspected first based on physiological studies that tested the availability of different compounds to the bacteroids and their ability to utilize these and other potential carbon sources. Confirmation came with the isolation of rhizobial mutants defective in transport of dicarboxylates (Dct mutants). Such mutants

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typically fail in nodule development at the point of bacterial release from infection threads; as a consequence, the central zone of the nodule where nitrogen fixation should occur is very sparsely infected by such mutants. Another carbon compound that is relatively abundant inside legume nodules is inositol. However, rhizobial mutants unable to utilize inositol do not show the severe defects of Dct mutants. Another property of bacteroids, nonessential for nitrogen fixation but perhaps evolutionarily important, is the tendency to store excess carbon as polyhydroxybutyrate or glycogen or both. Production of these storage compounds begins in the infection thread and may provide ready fuel for the energy-demanding period of rapid bacteroid proliferation. Nitrogenase activity produces ammonium. It is generally accepted that a significant portion of the ammonium is transported directly out of the bacteroid. However, there are many indirect indications that some portion of the fixed nitrogen first is incorporated into a carbon compound within the bacteroid and transported as an amino acid into the plant cytoplasm (Figure 5). In the mature nodule, the bacteroids are not growing or reproducing. Hence they have little need for fixed nitrogen. Therefore, whether transported as ammonium or as amino acid, almost all of the nitrogen fixed by the bacteroids is ultimately assimilated by the plant to provide for the biosynthesis of nitrogen compounds needed during plant growth. This assimilation results in glutamine as an early product within the plant cells harboring bacteroids (Figure 5). Generally, the nitrogen is passed from glutamine to other metabolites, resulting in a few abundant N-rich compounds, such as asparagine and ureides, for transport of the nitrogen via the xylem. Just as the plant controls whether nodules will form, it also can inhibit nitrogen fixation by the bacteroids in response to sudden influxes of fixed nitrogen (e.g., nitrate) in the root environment. It appears that the underlying mechanism starves the bacteria of carbon or oxygen or both. This inhibition is reversible. For at least a week the inhibited nodules and their bacteroids retain full capacity such that, once the exogenous fixed nitrogen is used up or removed, nitrogen fixation activity is rapidly restored to levels observed before inhibition.

Rhizobial Genes and Components Required in Symbiosis Nodulation Aside from recently discovered exceptions mentioned earlier, the capability to incite nodules on host legumes has been shown to depend on rhizobial Nod factors (Figure 3), whose key role in triggering nodule development have been delineated already. The production of these glycolipids (lipooligochitosaccharides) is specified

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by rhizobial nod, nol, and noe genes (collectively referred to as nod genes). Genes nodA, nodB, and nodC specify the Nod factor basic structure – the N-acetylglucosamine oligomer (nodC) and the fatty acyl adduct (nodA and nodB) (Figure 3). nodIJ genes are generally found in nod clusters as well; they are presumed to effect transport of the Nod factors out of the rhizobial cell. Other nod genes provide strain-dependent features to Nod-factor structure (Figure 3) that are presumed to contribute to host specificity of nodulation, efficiency of eliciting nodule initiation, and perhaps refinement of multiple roles of the Nod factor in promoting nodule development. For instance, different alleles of the nodE gene specify different unsaturated fatty acids attached as the lipid on the Nod factor. The nodH gene of S. meliloti specifies a sulfation of the Nod factor that is required for nodulation of Medicago species (including alfalfa). Mutation of the nodL gene of R. leguminosarum bv. viciae strains strongly decreases its nodulation of some of its normal hosts but has little effect on others. Summed over all rhizobial strains, the number of genes associated with nod genes and shown (or suggested) to play an accessory role in nodulation is impressive, having run the entire alphabet on nod, nol, and almost on noe. As many as half of these genes may not affect Nod factor structure, but instead affect nodulation in other ways. The best characterized is nodO, which encodes a protein postulated to form cation-selective pores in the plant plasma membrane and thereby complement the plant response to Nod factors. It is dispensable for nodulation by wild-type R. leguminosarum bv. viceae in the laboratory, but is essential in strains that are mutated in nodE. Transcription of the common and accessory nod genes is controlled by nodD genes. NodD proteins bind to nucleotide sequences called ‘Nod boxes’ upstream of the transcription start site of the various nod operons. In the presence of the flavonoids of the rhizobial host, NodD activates transcription. Certain other exuded plant compounds, aside from flavonoids, also have been shown to trigger nod induction. The nod genes help to explain the host range of a rhizobial strain. The first factor is the combination of the rhizobial nodD and plant flavonoids; generally, they must be compatible to induce the other nod genes. The second factor is the Nod factor structure. A given plant generally responds only to a certain spectrum of structures. For example, modifications of Nod factor by previously mentioned genes nodE, nodH, and nodL can have important effects on the way in which legumes are nodulated by a given strain. In a given strain a mixture of Nod-factor structures is generally observed. This mixture can be relatively simple (e.g., in S. meliloti) or extremely complex (e.g., in Sinorhizobium spp. NGR234, whose plethora of Nod factors contributes to its broad host range). Interestingly,

it is often found that the best-known host of a strain does not require what appears to be a distinctive modification. Even though Nod factor is essential for nodule formation on almost all legumes studied, it is important to realize that additional factors can be required to elicit observable nodule tissue. For instance, pea roots form hardly any nodule tissue if an R. leguminosarum strain is incapable of exopolysaccharide production. Soybean’s response to a Bradyrhizobium japonicum mutant lacking lipopolysaccharide (LPS) O-antigen is similarly restricted. In this respect, polysaccharides and perhaps other molecules besides the Nod factor are crucial in determining the host range. Homologous nod genes are found in the rhizobia of all known taxa, including those in the beta Proteobacteria. According to phylogeny based on rRNA sequences, the two strains without nod genes are located deeply within an outlying branch of the Bradyrhizobia. Whether or not nod was central to the earliest legume nodulation in evolution, it is obviously a hugely successful invention as deduced from its near ubiquity among studied rhizobia. Yet it seems to be confined to rhizobia (i.e., to legume nodulation). The presumptive coevolution of Nod factors, plant receptors, and downstream plant responses are subjects currently becoming amenable to research and may have implications for engineering other plants to enter into this type of symbiosis.

Infection Infection of course requires growing rhizobia capable of reproduction. Any mutation that prevents growth in the rhizosphere or infection thread will arrest infection. Bacterial auxotrophs may have problems on some symbiotic combinations but not on others. Presumably, this relates to whether the nutrients are available in sufficient quantities to the bacterium in the environment of the infection. It also may depend on the particular transport systems available and the pathways for scavenging biosynthetic end products, intermediates, and derivative compounds. On almost all hosts, rhizobial purine auxotrophs fail early in infection. Whether this arises from a special role for the purine biosynthesis pathway in symbiosis has not been established. In most symbioses that have been studied, a bacterialsurface polysaccharide, often more than one type, is required for successful completion of the infection process. Examples include LPS O-antigens, cyclic glucans, acidic exopolysaccharides (EPS), and ‘K-type’ capsular polysaccharides. None of these is individually required at any particular stage in all symbioses. Over the years of studying the agricultural legumes, a generalization has emerged regarding whether EPS is required in symbiosis. The Phaseoleae do not seem to require them, whereas infection of the Trifolieae and Fabeae

Bacteria | Rhizobia

generally are very limited unless the bacterium is capable of producing its major acidic EPS. The structural basis for this requirement is best understood for the S. meliloti–alfalfa symbiosis. The main EPS of S. meliloti is known as succinoglycan, the bulk of which is produced by the bacterium ex planta as long polymers of a repeat unit composed of eight sugars modified on some residues by acetyl, pyruvyl, and succinyl groups. However, in symbiosis, the active form is believed to be the succinylated oligomers (monomers, dimers, and trimers) of the octasaccharide repeat unit. Perhaps it acts as a diffusible signal compound but its exact role is unknown. One clue might be that high production of either a different EPS (galactoglycan) or a K-type capsular polysaccharide can substitute for succinoglycan on some hosts of S. meliloti. Many investigators speculate that EPS and other polysaccharides somehow suppress host-defense responses that otherwise would arrest rhizobial infection. On legumes in all of these tribes, Rhizobium and Bradyrhizobium symbionts are required to produce LPS Oantigen. On hosts in the Trifolieae and Fabeae, this requirement occurs late in the infection process, whereas on the Phaseoleae the requirement comes about very early. Oantigen-containing LPS from A. caulinodans is proposed to act as a diffusible signal in nodule development on Sesbania. In several of the symbioses, the LPS has been shown to undergo changes during the interaction with the plant. In some cases, the same plant compounds that trigger nod induction also trigger changes in LPS. Sinorhizobium fredii strains may undergo the greatest change. Strain NGR234 produces an entirely new O-antigen in response to a nod inducer. As proposed for EPS, LPS O-antigens possibly suppress host defenses. The phenotypes of deficient mutants suggest that they also may serve as ligands for plant receptors in sustaining infection and/or for endocytotic release at the end of infection. There has been limited study of the requirement for these polysaccharides on hosts highly diverged from the agricultural Papilionoideae legumes. Requirement of both LPS O-antigen and EPS in the infection of some legumes in the Mimosoideae have been inferred from singular mutants of certain rhizobial symbionts.

Nitrogen Fixation and Oxygen-Responsive Regulation Rhizobial nif genes are those that are homologous to genes required for synthesis and function of nitrogenase in nonsymbiotic bacteria. For instance, nifKD genes encode the protein subunits of dinitrogenase, and nifH encodes the polypeptide of nitrogenase reductase. nifBEN genes specify the proteins needed to produce the iron–molybdenum cofactor of dinitrogenase. In addition, as in all Proteobacterial nitrogen-fixing bacteria, the regulatory gene nifA is invariably present.

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fix genes are additional genes needed for symbiotic nitrogen fixation. For instance, the cbb3 cytochrome oxidase that has high affinity for oxygen is encoded by genes fixNOQP. This operon is generally followed on the DNA by the fixGHIS operon, whose function has not been established in detail but is believed to be in the transport of copper and perhaps its insertion into the cbb3 enzyme. Other fix genes generally present are fixABCX, whose function still has not been established. One or more of the regulatory genes fixLJ and fixK usually are present as well. Although usually not termed fix genes, the dct genes fit the definition of fix genes noted above. They are needed for uptake of most of the carbon supplied by the plant, which is in the form of Krebs-cycle dicarboxylates, mainly succinate and malate (Figure 5). All rhizobia in which they have been mutated become deficient in nitrogen fixation (as well as bacteroid proliferation). Also of utmost importance are the genes for the enzymes of the Krebs cycle and associated reactions needed to catabolize the carbon supplied by the plant. Interestingly, in certain rhizobia, some of the enzymes of the Krebs cycle can be eliminated by mutation without severely affecting nitrogen fixation. One explanation is that pathway shunts can bypass a given step(s). Even though the enzyme for this step is normally present and active, this metabolic plasticity compensates if the activity is lost. Such plasticity may have evolved to allow rapid adaptation to changing conditions of carbon and oxygen supply. Another explanation is that not all steps in the Krebs cycle are needed if ammonium is incorporated into one of the metabolites and then exported into the plant cytoplasm (Figure 5). It has been stated already that symbiotic nitrogen fixation is regulated according to oxygen concentration. In all systems the NifA protein is a positive regulator that activates the transcription of other nif genes. In classical rhizobia this protein is inhibited directly by oxygen. Transcription of the nifA gene also is controlled according to oxygen concentration. In Sinorhizobium, this control is exerted via proteins FixL and FixJ. FixL is an oxygen sensor that under low oxygen activates FixJ, which is a transcriptional activator of nifA and certain other genes. In Bradyrhizobium, the corresponding control is via RegS and RegR, which respond to respiratory electron flow to activate transcription of nifA. In both Bradyrhizobium and Sinorhizobium, the other fix genes are controlled by FixK protein; transcription of fixK is controlled according to oxygen concentration, through regulation by FixL and FixJ.

Rhizobial Genomes and Genetics Genomes The total nucleotide sequences of the genomes of seven strains have been determined at the time of this writing. They represent four genera of the ‘classical’ rhizobia:

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Rhizobium (R. leguminosarum 3841 and R. etli CFN42), Sinorhizobium (S. meliloti 1021), Mesorhizobium (M. loti MAFF303099), and Bradyrhizobium (B. japonicum 110 and the photosynthetic Bradyrhizobial strains BTAi1 and ORS278 mentioned earlier). All of these rhizobial strains have larger than average bacterial genomes, ranging from 6.5 to 9.1 Mb (1 Mb ¼ 106 nucleotides). Plasmids

Plasmids are bacterial DNA molecules that are smaller than the chromosome(s). Generally, they are dispensable for bacterial growth at least under some conditions. Instead, they typically encode properties that allow growth or otherwise give the bacteria selective advantages under niche-specific conditions. In some rhizobia (e.g., Rhizobium and Sinorhizobium species) nod, nif, and fix genes are found on a plasmid termed the symbiotic plasmid. This plasmid is distinct in each strain, having a different size and different additional genes that have no apparent role in symbiosis. These other genes generally outnumber the genes devoted to the symbiosis. In Bradyrhizobium and Mesorhizobium species, the nod, nif, and fix genes are found on the chromosome. In some of the rhizobia, a significant portion of the genome is contained on plasmids. Plasmids larger than 1 Mb arbitrarily are termed megaplasmids. S. meliloti has two of them, which account for almost half of its 6.7 Mb genome. Plasmid A is 1.35 Mb and plasmid B is1.68 Mb. These plasmids are larger than the entire genomes of many obligately symbiotic bacteria and even some freeliving bacteria. Plasmid A is the typical symbiotic plasmid with nod, nif, and fix genes, whereas plasmid B has genes for exopolysaccharides required in the symbioses of this species. In the two Rhizobium strains whose entire genomic nucleotide sequences have been determined, the plasmids are smaller, but there are more of them. Strain R. leguminosarum 3841 has 12 plasmids that together account for about 40% of the 7.8 Mb genome. Strain R. etli CFN42 has six plasmids that account for about one third of the 6.5 Mb total genome. In both of these strains, one of the plasmids is a typical symbiotic plasmid carrying the nod, nif, and fix genes, as well as genes not required in the symbiosis. Duplicated genes

The classical rhizobia have a high incidence of duplicated genes, genes that are not merely homologous, but sometimes have near identity of nucleotide sequence. This creates a problem in analyzing gene function by mutation. Often mutating a gene has no effect because there is an isofunctional duplicated gene (paralog) compensating for its loss. Potentially, a new function can evolve in one of the paralogs of each set of duplicated genes. Therefore, rhizobia appear to have a very rich potential for evolution of new functions. Whether they are atypical in this respect

among bacteria is not clear, but they were among the first bacteria in which a high rate of duplications was recognized and studied in detail. Mobile Genetic Elements One explanation for the diversity of the rhizobia is that genes for symbiosis can be transferred from bacteria of one species to those of another species. Studies of genetic sequences strongly support this notion. Emerging examples are the rhizobia belonging to the beta Proteobacteria. Studies so far indicate that their nod and fix genes have been acquired more than once in evolution and from different sources within the rhizobia of the alpha Proteobacteria. Although sequence divergence indicates that these events happened long ago, studies among the rhizobia of the alpha Proteobacteria document that genetic transfers of clusters of symbiotic genes are occurring at readily detectable rates all the time in the soil. Most studies have documented transfers among strains in the same species, but transfers between genera have been reported as well. With the modern techniques of microbial ecology, it is possible to detect and isolate ‘nonsymbiotic’ rhizobia, rhizobia whose genomes belong to a particular rhizobial species, but lack nod, nif, and fix genes. Moreover, it has been possible to demonstrate that within a given period of time, such strains introduced into the soil had picked up symbiotic gene clusters from rhizobia that were indigenous or also introduced into the soil at the same or different times. Such transfers have been demonstrated in the laboratory as well. In retrospect, these observations are not at all surprising. Rhizobia harbor types of mobile genetic elements that are known to foster such transfers in other bacteria, and, indeed, they provide some of the best case studies for such genetic mobility in nature. Although the emphasis in rhizobia is almost always on symbiotic properties, such exchanges between rhizobia and other bacteria are not limited to symbiotic gene clusters. It is very common for bacteria to acquire in this way genes that allow growth in special niches that are not always encountered by the species. The spread of pathogenesis, antibiotic resistance, and utilization of aromatic carbon compounds are notable examples found in other bacteria. The common transfer agents and mechanisms include conjugative plasmids, conjugative symbiosis islands, specialized transduction of genes on proviruses, and insertion elements that border genetic islands and translocate them to new DNA. Studies of the rhizobia have demonstrated at least three of these mechanisms: mobilization via plasmids, symbiosis islands, and translocations mediated by insertion elements. In the genera containing symbiotic gene clusters on plasmids (e.g., Rhizobium, Ensifer (Sinorhizobium)), plasmid-mediated conjugation appears to be a main mechanism of the spread of symbiotic determinants and changes in host specificity.

Bacteria | Rhizobia

Even when the symbiotic plasmid is not a conjugative plasmid, it is mobilized by another indigenous plasmid that is conjugative. Examples include the changes in biovars among R. leguminosarum mentioned in the section titled ‘Rhizobial host ranges’. In Bradyrhizobium and Mesorhizobium, symbiotic genes are instead found on the chromosome in genetic clusters known as symbiotic islands. They can be recognized in Bradyrhizobium strains but in most cases the conjugative transfer elements that moved them into the strains have become inactive during evolution. However, in Mesorhizobium loti the transfer functions are still active. This species has been a model for demonstrating the transfer of symbiotic islands in both the field and the lab.

Genetic Analysis of Rhizobia Rhizobia have many natural agents for achieving genetic change. They have the conjugative elements noted above, and, like other forms of life, rhizobia are afflicted by viruses and insertion elements. These agents have been very important in shaping the genomes of present-day strains, but, except for a few viruses, they have not been exploited as tools for genetic analysis. Instead most such tools were developed for use in other Proteobacteria and then adapted for use in rhizobia. One example has been the extremely useful transposon Tn5, which is the workhorse mutagen for obtaining null mutations in the initial study of a particular rhizobial property. In rhizobia, Tn5 inserts itself in a relatively random manner at sites throughout the genome, generally at only one location per mutant strain. The resulting insertions are very stable (rarely revert), and, very importantly, Tn5 and its manmade derivatives carry various antibiotic-resistance genes that tag genes for selecting a mutation with no other selectable phenotype in matings and cloning. Broad- and narrow-host conjugative plasmids isolated from other bacteria also have been key genetic tools. They work as vectors to shuttle DNA between Escherichia coli (or sometimes other bacteria) and rhizobia. The narrow-host plasmids can be transferred to rhizobia, but do not replicate in them. They serve to introduce transposons or to force molecular recombination between transferred rhizobial DNA and resident DNA in the recipient rhizobial genome. This has allowed replacement of resident wildtype DNA with mutant alleles constructed in vitro. The broad-host plasmids do replicate in rhizobia and allow multiple copies of introduced DNA to be maintained in the recipient rhizobia for testing genetic complementation. Such plasmids can be used to transfer DNA between rhizobia as well. In studies of some rhizobia, notably S. meliloti and R. leguminosarum, generalized transducing phage (viruses) have been developed for this same purpose.

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Other Properties of the Rhizobia Common Traits Generalizations about rhizobia can be made, with the caveat that there will be exceptions among a group this diverse. All characterized rhizobia are Gram-negative rods that are catalase- and ‘oxidase’-positive. They have flagellar motility and exhibit chemotaxis. All of the classical rhizobia can carry out hexose catabolism by the Entner–Duodoroff pathway. In keeping with the catabolism needed in symbiosis, all have complete Krebs cycles and associated anaplerotic reactions so that organic acids can be used for growth. Considered obligate respirers, all can use oxygen, and most also use at least nitrate as the terminal electron acceptor. Rhizobia grow well microaerobically. A key enzyme for this growth, the cbb3 cytochrome oxidase, is an example of a function encoded by fix genes important in symbiosis but also induced under microaerobic conditions that rhizobia may encounter in the soil (and freshwater environments) in the absence of the plant. The cbb3 oxidase is widely distributed among related Proteobacteria that are not known to form symbioses with eukaryotes, for instance, the purple phototrophs. A point to be emphasized is that rhizobial populations persist indefinitely in the soil as part of the overall bacterial population, and they do so regardless of the presence of the host plant. Therefore, they have capabilities that allow growth in this environment. Like ‘pseudomonad’ soil bacteria, individual rhizobial strains that have been studied in the laboratory are extremely versatile heterotrophs, able to use a plethora of carbon sources, including sugars, organic acids, amino acids, and phenolics. Total genomic nucleotide sequences bear this out. The genomes are consistently large relative to the average bacterium and have a wealth of annotated genes devoted to diverse catabolic pathways and transmembrane transport systems. They utilize diverse nitrogen sources as well, including molecular nitrogen (mainly in symbiosis), ammonia, nitrate, nitrite, and amino acids. Aside from versatile heterotrophy, they all seem proficient at biosynthesis of amino acids, nucleotides, and other small molecules needed for growth. Certain vitamins, in contrast, greatly stimulate rhizobial growth. Hence, they can be grown in the laboratory on a wide variety of simple defined minimal media with one carbon source, a nitrogen source, salts, and certain vitamins. They also do well on defined and undefined rich media. How well they do depends on the genus and the composition of the particular medium. In general, the classical rhizobia require a relatively high calcium concentration for growth. Rhizobia are commonly obtained as clonal isolates from nodules by requiring colony formation on agar containing yeast extract and mannitol. As a link to older literature that

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categorized rhizobia according to growth rate, the ‘fast growers’ on this medium include the classical genera Rhizobium and Sinorhizobium. Bradyrhizobium strains are ‘slow growers,’ and Mesorhizobium strains are said to exhibit intermediate growth rates. If there are legume symbionts that do not grow on yeast extract–mannitol medium, they would have been missed in most studies because of the presumption that legume isolates will grow on this medium. Polysaccharides Historically, there has been great interest in the surface polysaccharides produced by the rhizobia, because of the likelihood that interactions between these bacteria and legume cells, as well as other biological entities, involve molecules on the rhizobial surfaces. However, the full spectrum of polysaccharides produced by any rhizobial strain has never been identified completely. Genome annotations indicate a staggering number of gene clusters apparently devoted to polysaccharides. Long before their genetics were known, certain polysaccharides, including many EPSs that are important in symbiosis, were studied biochemically because they were the most abundant of a particular class of polysaccharides. In other cases, a polysaccharide was first studied as a result of mutations that caused a deficiency in symbiosis, which was traced to a deficiency in that polysaccharide. Genes for LPS O-antigens, cyclic glucans, and some capsular polysaccharides have been discovered in this way. The functions of these polysaccharides, aside from requirements in symbiosis, have, for the most part, undergone little scrutiny. Hypothetical roles include protection against desiccation, environmental toxins, and plant defenses. They may help evade viruses, but there are cases where viruses have evolved to use them as receptors for the first step in infection. Rhizobia, particularly in the genus Rhizobium, produce impressive amounts of exopolysaccharide on carbon-rich media such as yeast-mannitol. Within a few days after appearing on inverted agar plates containing this medium, colonies will drip copious slime onto the Petri dish cover even if stored at 4  C. Outside the symbiosis, EPSs are regulated according to exogenous carbon/nitrogen ratios; that is, excess carbon leads to high EPS production. Much of the total production occurs after the net growth of a culture or colony has ceased. The structures of several rhizobial EPSs have been determined. Each genus makes a structurally distinct major EPS, whereas within a genus, the most abundant EPS in all species may be very similar. At the time of this writing, the only rhizobial LPS whose structure has been determined fully is Rhizobium etli CFN42. Almost all of the genes required for its synthesis have been identified as well. This and other Rhizobium LPSs follow the well-known model of LPS modular

structure found in the enteric bacteria. Lipid A, with some features distinctive to a particular genus and others shared among other alpha Proteobacteria, anchors the molecule in the membrane. It is attached to a branched core oligosaccharide that is conserved among R. leguminosarum and R. etli, and to this core is attached an O-antigen whose structure varies among strains within the species. It is suspected that the LPS of many other rhizobial genera follow this model. In contrast, the less-characterized structure of the LPS of Sinorhizobium species indicates some deviation from this model, at least when grown under normal laboratory conditions, in that the majority of LPS molecules do not carry a distal O-antigen and the immunodominant portion of the structure is what researchers call the core oligosaccharide.

Notable Traits Found in Some Rhizobia In view of the phylogenetic diversity of these bacteria, it is not surprising that various rhizobial species have capabilities that appear to be uncharacteristic of the majority. For instance, A. caulinodans and Burkholderia phymatum can grow ex planta with N2 fixation as the sole nitrogen source. Several other rhizobia have been shown to induce nif genes and even to produce functional nitrogenase, but indefinite growth dependent solely on nitrogen fixation has not been observed otherwise. The impact in nature of this nonsymbiotic nitrogen fixation is unclear, but is probably trivial compared with the fixation by these bacteria inside legume nodules. Many rhizobia appear to harbor ribulose bisphosphate carboxylase and perhaps other enzymes of the Calvin cycle. A few have been demonstrated to grow autotrophically, particularly with hydrogen as the lithotrophic energy source. As mentioned above, there are also certain Bradyrhizobia that appear to be capable of phototrophy. Methylobacterium nodulans is described to be the only rhizobial species capable of growth on certain one-carbon compounds including methanol, although it is not clear how many legume nodule isolates have been tested for this property. S. meliloti survives remarkably well under very dry conditions, an exceptional property that is exploited in using it as a commercial inoculant of alfalfa. This property may be shared among other Sinorhizobium (Ensifer) species. The survival is not due to spore formation. None of the rhizobia is known to form spores, and other classical rhizobia used in agriculture must be carefully maintained at certain moisture levels to assure sufficient viability at the time of inoculation of designated crops. The basis of this property in S. meliloti is under active investigation in parallel with long-term projects aimed at increasing the desiccation tolerance of other agronomically important rhizobia.

Bacteria | Rhizobia

Rhizobia are not particularly acid tolerant. S. meliloti is among the most acid-sensitive, showing growth inhibition at pH values below 6. Among the most acid tolerant are Rhizobium tropici and some Bradyrhizobia, which survive at pH 4.5. Population Biology The most basic issues in microbial ecology are to recognize the diversity and to enumerate the population under study. These are challenging objectives with all bacteria. With regard to rhizobia, one difficulty already mentioned is that rhizobia in the soil are in a dynamic genetic situation whereby they lose and pick up gene clusters that confer composite properties such as the one that we use to define them, the ability to incite nodules and fix nitrogen in association with legumes. Traditionally, soil rhizobia have been counted by using legume hosts as a means of selecting them from the notoriously diverse microbiota of the soil. Most probable numbers are calculated from the incidence of nodules arising from more and more dilute extracts of a particular sample of soil. This method is a reasonable measure of the most competitive rhizobia capable of nodulating the host that is used to trap them. It misses completely all those rhizobia that cannot do so, for example, rhizobia for other hosts and the aforementioned strains that have lost symbiotic capability. This is only one of many challenges in making absolute enumerations and enumerations relative to other bacteria (which themselves are subject to similar caveats). Therefore, the advent of new technologies, chiefly based on genetic probes, for identifying and enumerating bacteria in natural populations is revolutionizing the study of the ecology of rhizobia, just as it is of other bacteria. With these cautionary realities having been stated, it has been estimated through traditional sampling of soils that Rhizobium, Sinorhizobium, and Bradyrhizobium represent between 0.1% and 8% of the total population in bulk and rhizosphere soil. In view of the tremendous variety of bacteria that may inhabit the soil, even the lower of these numbers is significant. As might be expected, studies show that one of the most important factors determining the relative populations of rhizobia is whether a legume host has been cultivated in that soil. Generally, repeated cultivation of a particular legume will greatly enhance the populations of bacteria capable of nodulating it, relative to other rhizobia.

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Therefore, rhizobia effectively compete with other bacteria in sustaining their soil populations. Also interesting in a basic sense, and very important to the inoculant industry, is that rhizobia of course compete with one another for the habitat of the legume nodule. In a given field setting, there are ‘dominant’ strains, strains that appear to inhabit the nodules of the particular host in higher numbers than other strains. An example is the so-called serogroup 123 of B. japonicum that competes well with inoculants and other indigenous strains for nodulation of soybean in the Midwestern region of the United States. The mechanisms involved in this important property are not yet elucidated. They are likely to be multiple and complex, involving not only innate differences in the early interactions with the host legume, but also coping with the particular and varying soil conditions, such as pH, viruses, antibiotics, predation, temperature, desiccation, and available nutrients. All figures in this article are ª 2008 Dale Noel. Published by Elsevier Inc. All rights reserved. See also: Endosymbionts and Intracellular Parasites; Nitrogen Cycle; Rhizosphere

Further Reading Dixon R and Kahn D (2004) Genetic regulation of biological nitrogen fixation. Nature Reviews Microbiology 2: 621–631. Fred EB, Baldwin IL, and McCoy E (1932) Root Nodule Bacteria and Leguminous Plants. Madison: University of Wisconsin Press. Patriarca EJ, Tate R, Ferraioli S, and Iaccarino M (2004) Organogenesis of legume root nodules. International Review of Cytology 234: 201–262. Prell J and Poole P (2006) Metabolic changes of rhizobia in legume nodules. Trends in Microbiology 14: 161–168. Spaink HP (2000) Root nodulation and infection factors produced by rhizobial bacteria. Annual Review of Microbiology 54: 257–288. Spaink HP, Kondorosi A, and Hooykaas PJJ (eds.) (1998) The Rhizobiaceae: Molecular Biology of Model Plant-Associated Bacteria. Dordrecht: Kluwer Academic Publishers. Sprent JI (2001) Nodulation in Legumes. Kew: Royal Botanic Gardens. Sprent JI (2007) Evolving ideas of legume evolution and diversity: A taxonomic perspective on the occurrence of nodulation. New Phytologist 174: 11–25. Triplett EW (ed.) (2000) Prokaryotic nitrogen fixation: A Model System for the Analysis of a Biological Process. Wymondham: Horizon Scientific Press. Willems A (2006) The taxonomy of rhizobia: An overview. Plant and Soil 287: 3–14.

Spirochetes D A Haake, University of California at Los Angeles, Los Angeles, CA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Overview Treponema Borrelia

Glossary chemotaxis The movement along a chemical concentration gradient either toward or away from a chemical stimulus. commensal An organism participating in a relationship in which that species derives benefit while the other is unaffected. microbiome The entourage of associated microflora in a host.

Abbreviations BSA DHS EMJH HisK IS LBRF LD Lig LPS MCPs

Bovine serum albumin downstream homology sequence Ellinghausen–McCullough–Johnson–Harris histidine kinase sensors insertion sequence louse-borne RF Lyme disease Leptospira immunoglobulin-like repeat lipopolysaccharide methyl-accepting chemotaxis proteins

Defining Statement Spirochetes are ancient bacteria that comprise one of the major phyla within the eubacterial kingdom. Their unique morphology and rotational motility are distinguishing features that allow rapid microscopic identification. Spirochetes are widely distributed in nature as free-living bacteria, as metabolic symbionts of insects, and as commensals and parasites of animals.

Overview The spirochetes form one of the major phyla of the kingdom of Eubacteria. The depth of the spirochetal branch of the bacterial tree of life is indicated by the fact that phylum

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Brachyspira Leptospira Conclusions Further Reading

parasite An organism participating in a relationship in which that species derives benefit while the other is harmed. pathogenesis The process by which a disease occurs. saprophyte An organism that grows on and derives its nourishment from dead or decaying organic matter. symbiont An organism participating in a relationship in which both species derive benefit.

Msp NADH Omps PCR PD PDD RF TBRF UHS VSH

major sheath protein nicotinamide adenine dinucleotide outer membrane proteins polymerase chain reaction pocket depth papillomatous digital dermatitis relapsing fever tick-borne RF upstream homology sequence virus of Serpulina hyodysenteriae

Spirochaetes has a single class and a single order. As shown in Figure 1, the order Spirochaetales is divided into three families, Spirochaetaceae, Serpulinaceae, and Leptospiraceae. The first family, Spirochaetaceae, includes a complex group of organisms that have adapted to diverse niches. At one extreme, there are a large number of freeliving Spirochaeta organisms that can be cultivated from virtually any moist, nutrient-rich environment. At the other extreme is the obligate parasite, T. pallidum, which relies on the activities of a single animal host, man, for its survival and dissemination. In between these two extremes are the commensal, parasitic, and symbiotic organisms with life cycles involving insects, animals, or both. The second family, Serpulinaceae, contains a single genus, Brachyspira, and a more narrowly focused lifestyle involving residence in the lower intestinal tracts of animals. The third family,

Bacteria | Spirochetes Free living

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Escherichia coli 0157 Leptospira interrogans

Spirochaeta aurantia Leptospira biflexa Leptospira borgpetersenii Treponema azotonutricum

Treponema denticola Borrelia burgdorferi Treponema pallidum

Host dependent

Borrelia garinii Borrelia afzelii

0

1

2 3 4 Genome size (Mb)

5

Figure 2 Comparative genome sizes of spirochetes. Free-living spirochetes, including Leptospira and Spirochaeta spp., have genomes that rival the size of E. coli. Host-dependent spirochetes, such as T. pallidum and Borrelia spp., have some of the smallest known genome sizes. Treponemes that live in the complex environments of the oral cavity (T. denticola) and termite gut (T. azotonutricum) have intermediate-sized genomes.

Figure 1 Taxonomic organization of the spirochetes. Three families of spirochetes have been defined. Family Spirochaetaceae includes the free-living Spirochaeta spp., the parasitic Borrelia, and the commensal, parasitic, and symbiotic Treponema spp. Family Serpulinaceae are bacteria that colonize the lower intestinal tracts of mammals. Family Leptospiraceae includes both free-living nonpathogens and organisms that are able to invade animal reservoir hosts.

Leptospiraceae, includes both environmental saprophytes (e.g., L. biflexa) and animal parasites (e.g., L. interrogans) that cycle between bodies of freshwater and their preferred reservoir host. Comparison of genome sizes indicates that life outside the host is much more genetically challenging than a life of host dependence. Free-living organisms such as Spirochaeta aurantia and L. biflexa have relatively large genomes relative to Escherichia coli (Figure 2). In contrast, adaptation of spirochetes to a commensal or parasitic lifestyle has resulted in genomic contraction. For example, Leptospira borgpetersenii and L. interrogans evolved from a common ancestor that had the ability to survive in both nature and the mammalian host, whereas L. borgpetersenii has become an obligate parasite of cattle that requires direct transmission from animal to animal. As a result, the L. borgpetersenii genome has become 16% smaller and remains in a process of decay, with 12% of its genes as nonfunctional pseudogenes. Treponema denticola has an

intermediate-sized genome, perhaps related to the fact that although it is found only in animal hosts, it competes for nutrients in the complex oral microbial community. The Borrelia spp. and T. pallidum have the smallest genomes, with chromosomes only 1 Mb in size, which is consistent with their host dependence and lack of a freeliving phase of their life cycle. Spirochetes are defined by their unique morphology and rotational motility. Most spirochetes are helical coils – the one exception being Borrelia burgdorferi, which is actually a flat wave. Spirochetes are expert swimmers that are entertaining to watch by dark field microscopy. In low-viscosity liquids, spirochetes appear to spin in place. Increasing the viscosity by the addition of methylcellulose allows spirochetes to bore through the medium at a high rate of speed. The observer is quickly led to an understanding of how their screw-like movements would impart invasive properties to spirochetal pathogens. The organs of motility are flagella anchored near each end of the cell. Spirochete flagella are sometimes referred to as ‘endoflagella’ because they are subsurface structures, wrapping around the protoplasmic cell cylinder, as shown in Figure 3, instead of extending out beyond the surface of the cell as in all other flagellated bacteria. In at least one case, spirochete flagella also determine cell shape. B. burgdorferi mutants lacking the flaB flagellar filament protein are rod-shaped rather than wavy. Spirochetes differ in flagellar number and length. Leptospira have a single flagellum at each end of the cell that extends only a short distance along the length of the cell. In contrast, Cristispira spp. have bundles of over a 100 flagella at each end. Chemotaxis allows bacteria to swim toward attractants, such as nutrients, and away from repellants by controlling the rotational direction of the flagellar motor. Flagella can rotate in a clockwise or counterclockwise direction. Sensory

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(a)

Extroverts

E. coli 0157

L. interrogans L. borgpetersenii Hisk MCP STYK GGDEF EAL HD-GYP AC3 ACIV RRs

T. denticola B. garinii B. burgdorferi B. afzelii

Introverts

T. pallidum

(b) EF

CW

OM

EF: Endoflagellum CW: Cell wall OM: Outer membrane BB: Basal body

BB

Figure 3 Spirochetal architecture. Spirochetes share a unique structure and motility strategy in which the endoflagella are inserted at opposite poles and wrap around the protoplasmic cylinder. (a) Electron micrograph of Leptospira showing a single endoflagellum at one end of the cell. (b) Schematic diagram showing endoflagellar location relative to the outer membrane and cell wall. Reproduced from Holt SC (1978) Anatomy and chemistry of spirochetes. Microbiological Reviews 42: 114–160.

proteins called methyl-accepting chemotaxis proteins (MCPs) control the directional switch in the flagellar motor. Clockwise rotation causes a cell to tumble (stop), counterclockwise rotation causes a cell to run (go). Spirochetes are unique in having flagella at each end. Effective spirochete movement involves flagellar rotation in the counterclockwise direction at the leading end and in the clockwise rotation at the trailing end. If the flagella at alternate ends are rotating in the same direction, spirochetes will flex in place rather than spin. It is not known how spirochetes coordinate the flagella at their alternate ends, or how MCPs orient spirochete movement. In any case, spirochetes are clearly adept at chemotaxis. For example, B. burgdorferi are able to find their way into a capillary tube containing N-acetylglucosamine, a sugar required for cell wall biosynthesis. Sensory proteins are used not only for chemotaxis but also for the regulation of gene expression. Spirochetes vary widely in terms of the number of sensory proteins they have. As mentioned previously, life outside the host is challenging and ‘extroverts’ (organisms with a free-living stage) have far more sensory proteins than ‘introverts’ that never leave the host. For example, L. interrogans, which lives both inside and outside a mammalian host, has 10 times the number of sensory proteins as T. pallidum, an obligate human parasite. Figure 4 shows the correlation between lifestyle and numbers of sensory proteins. The unique spirochetal architecture has both Gramnegative and Gram-positive features. Like Gram-negative bacteria, spirochetes are ‘diderms’, or double-membrane bacteria. However, the spirochetal outer membrane is

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Figure 4 Comparison of spirochetal lifestyles and sensory transduction genes. Spirochetes have a wide variety of sensory transduction genes including histidine kinase sensors (HisK), methyl-accepting chemotaxis proteins (MCP), and so on. Spirochete ‘extroverts’ that live outside the host have a much greater number of sensory transduction proteins per genome than host-dependent ‘introverts’.

much more fluid and labile than the outer membrane of Gram-negative organisms. In typical enteric Gram-negative bacteria, the outer membrane is supported by, and closely associated with, the underlying peptidoglycan cell wall. In contrast, the spirochetal cell wall is more closely associated with the inner, or cytoplasmic, membrane than the outer membrane (Figure 5), a feature of Gram-positive bacteria. Another important difference between the outer membranes

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Figure 5 Spirochete cross-section. Elements of spirochetal architecture include (a) outer membrane; (b) periplasm; (c and d) peptidoglycan cell wall; (e) cytoplasmic membrane; (f) cytoplasm; (g) nuclear material; and (h) endoflagella. Note that the endoflagella are subsurface structures and that the cell wall is more closely associated with the cytoplasmic membrane than with the outer membrane. Reproduced from Holt SC (1978) Anatomy and chemistry of spirochetes. Microbiological Reviews 42: 114–160.

Bacteria | Spirochetes

of most Gram-negative bacteria and those of treponemes and Borrelia is a lack of lipopolysaccharide (LPS). Leptospires have LPS, but there are significant structural differences between leptospiral and E. coli LPS such that human Toll-like receptor 4 is unable to bind to leptospiral LPS. The lack of recognizable LPS allows spirochetes to function as ‘stealth pathogens’ that are able to invade and persist in the bloodstream and in tissues of the body without detection by the early warning system of innate immunity. Protein export pathways of spirochetes resemble those of other bacteria. The Sec pathway for exporting proteins with signal peptides across the cytoplasmic membrane is conserved. Genes encoding enzymes that process signal peptides are present in spirochete genomes, but their specificities are clearly unique because prediction algorithms such as Psort and LipoP frequently do not apply to spirochetal signal peptides. Computer recognition of signal peptides of spirochetal lipoproteins requires the development of spirochete-specific training sets and algorithms (e.g., SpLip). Upon reaching the periplasmic face of the cytoplasmic membrane, spirochetal lipoproteins are shuttled to the outer membrane via the Lol pathway. Here again, rules that apply for E. coli lipoproteins have been altered for spirochetal lipoproteins such that retention of spirochetal lipoproteins in the cytoplasmic membrane involves negatively charged amino acids after the N-terminal cysteine and export to the outer membrane is by default. Membrane fractionation and ultrastructure studies demonstrate three types of spirochetal outer membrane proteins (Omps), namely transmembrane porin-like molecules, lipoproteins, and peripheral (nonintegral) membrane proteins. All spirochetes have Omp85 homologues for assembly and insertion of Omps. Transmembrane Omps are required for transport functions and both transmembrane and surface lipoprotein Omps have been shown to be involved in host–pathogen interactions.

Treponema The genus Treponema includes a broad diversity of parasitic and commensal species, most of which exist in complex bacterial communities. A notable exception is highly invasive obligate human pathogen, T. pallidum, subspecies pallidum, the agent of syphilis. T. pallidum, syphilis, and its history are covered in ‘Sexually transmitted diseases’ and ‘Syphilis, historical’ of the current edition of Encyclopedia of Microbiology. In passing, it should be mentioned that T. pallidum has two other subspecies and a sister species that cause nonvenereal skin infections of humans (see Figure 1). T. pallidum subspecies pertenue causes yaws, T. pallidum subspecies endemicum causes endemic syphilis (bejel), and T. carateum causes pinta. Another member of this species group is Treponema paraluiscuniculi, the agent of venereal spirochetosis of rabbits, which has an overall

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genome sequence similarity of 98.6–99.3% with T. pallidum subspecies pallidum. It should also be mentioned that a number of additional Treponema species have been isolated from the intestinal tracts of animals including cows (Treponema bryantii and Treponema saccharophilum) and pigs (Treponema succinifaciens). In this section, we will cover the oral treponemes, the organisms that cause papillomatous digital dermatitis (PDD) of cattle, and the termite gut treponemes that contribute to the digestion of cellulose. Oral Treponemes Oral treponemes were some of the first bacteria described in the writings and drawings of Antonie van Leeuwenhoek, the father of Microbiology. In 1676, when examining a dental plaque from the mouth of an old man, van Leeuwenhoek found ‘‘an unbelievably great company of living animalcules, a-swimming more nimbly than any I had ever seen up to this time. The biggest sort. . .bent their body into curves in going forwards. . .’’ Today, anyone with a dark field microscope can repeat van Leeuwenhoek’s experiment. If the sample is taken from the periodontal space (located between the tooth and the gum) of a patient with gum disease, it is likely that spirochetes will be observed to be the predominant bacterial forms. A remarkable diversity of oral treponeme morphologies is present in the mouth, with a broad variety of diameters, lengths, wavelengths, amplitudes, and numbers of endoflagellae. Sizes range from 0.1 to 0.4 mm in diameter and from 5 to 20 mm in length. The microbial community of the mouth, referred to as the oral ‘microbiome’, is estimated to include upward of 500 different bacterial species. Given the complex environment in which they live, it is not surprising that it has been relatively difficult to isolate and cultivate oral treponemes. Like most bacteria that live in the periodontal space, most oral treponemes are strict anaerobes. However, some treponemes, such as T. denticola, can tolerate low concentrations of oxygen. Treponemes are intrinsically resistant to rifampin, which makes it possible to use rifampin-containing culture medium to exclude other bacteria and select for treponemes. Ten species of oral treponemes have now been isolated, allowing more detailed studies of their morphologies and metabolic requirements. However, more detailed enumeration of oral treponeme diversity has become available through polymerase chain reaction (PCR)-based cloning and sequencing of bacterial 16S rRNA sequences. In one important oral microbiome study of healthy and periodontitis subjects, five novel treponemal species were found for every one that had been cultivated. Nearly 25% (49/215) of the new oral bacterial species discovered were treponemes! The oral diversity of phylum Spirochaetes is exceeded only by the phylum Firmicutes, which includes the streptococci. On the basis of these

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Bacteria | Spirochetes Oral cluster 1

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molecular studies, ten phylogenetic groups of oral treponemes in two clusters have now been defined (Figure 6). Several lines of evidence implicate treponemes as oral pathogens. Although treponemes can be found in small numbers in the mouths of healthy individuals, their numbers and diversity are strongly correlated with the severity of chronic and aggressive forms of periodontitis and their numbers are diminished with clinical treatment. Two species that have been associated with periodontitis are T. denticola and Treponema lecithinolyticum. Both gingivitis and periodontitis are extremely common inflammatory gum diseases. The distinction is that while gingivitis is reversible, periodontitis involves erosion of the dental ligament that attaches the tooth to the supporting bone at the base of the periodontal pocket (Figure 7). Peridontitis affects 50% of the US population over 30 years of age and is the leading cause of tooth loss. Although treponemes are typically found at the base of the periodontal pocket in association with other pathogenic organisms, such as Porphyromonas gingivalis and Tannerella forsythia, immunofluorescence microscopy shows that the treponemes are the most invasive organisms, typically invading the epithelial cells at the leading edge of the invasion process. Treponemes also appear to be involved in endodontal (root canal) infections; Treponema maltophilum DNA was detected in 50% of root canal samples using 16S rDNA-based PCR methods.

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Figure 6 Phylogenetic tree of the treponemes. Comparison of treponeme 16S rRNA sequences shows segregation into three relatedness clusters: two clusters of oral treponemes and a cluster of termite gut treponemes. Note that T. pallidum, the agent of syphilis, is related to the first cluster of oral treponemes.

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Figure 7 Schematic representation of health, gingivitis, and periodontitis. The periodontal pocket depth (PD) is increased in gingivitis due to tissue swelling associated with inflammation. In periodontitis, the PD is further increased due to the loss of the tissue attachment to the root of the tooth (AL: attachment loss). Periodontitis is further characterized by the loss of supporting alveolar bone. Treponemes are typically found at the base of the periodontal pocket. Reproduced from Kinder-Haake S, et al. (2006) Periodontal diseases. In: Lamont et al. (eds.) Oral Microbiology & Immunology, ISBN-13: 9781555812621.

Several pathogenetic mechanisms have been identified by which oral treponemes cause disease. By virtue of their motility, chemotaxis, and narrow diameter, spirochetes are able to slip between epithelial cells and invade the subepithelial layers of the gum tissue. Although treponemes do not make Gram-negative LPSs, they do elaborate a variety of glycolipids and lipoproteins that stimulate innate inflammatory pathways. T. denticola expresses a

Bacteria | Spirochetes

serine protease, dentilisin, which digests host extracellular matrix proteins, including fibronectin, laminin, and fibrinogen. Dentilisin activates host matrix metalloproteinases, and together with dentilisin these enzymes serve to alter and eventually degrade the barriers that prevent invasion by other periodontal bacteria. Exposure to T. denticola causes cytoskeletal rearrangements that disrupt normal host cell functions. These cytotoxic effects are probably caused by the T. denticola release of Msp, the major sheath protein. Msp is a porin-like molecule that appears to insert into host cell membranes and trigger intracellular calcium fluxes, which are believed to damage epithelial cell barriers and impair the clearance of invaded bacteria by polymorphonuclear leukocytes. PDD Treponemes PDD is a polymicrobial infection of the soft tissue adjacent to the hoofs of cattle. Affected animals have painful ulcers referred to as heel warts or footwarts. Since it was first described in the 1970s, PDD has spread throughout the world, including most herds in the United States, and is now the leading cause of lameness in dairy cattle. The spread of PDD is related to industrial-scale dairy practices where cattle are continuously kept in barns or feedlots on moist surfaces and not allowed to graze. Cultures of PDD lesions show a mixed population of anaerobic bacteria including a number of spirochetes that are closely related to oral treponemes such as T. denticola, Treponema medium, and Treponema vincentii. Immunofluorescence studies of biopsies of PDD lesions reveal invasion of treponemes into the soft tissues of foot, not unlike the invasion of treponemes observed in the periodontium of the mouth. Termite Gut Treponemes Diversity

Although the volume of the termite hindgut can be as small as one microliter, the diversity of treponemal phylotypes and morphotypes within a single termite rivals that of the human mouth. Microscopy of the termite hindgut contents reveals treponemes ranging from 0.1 to 1 mm in diameter and from 3 to 100 mm in length, with a variety of cell wavelengths and amplitudes (Figure 8). Some of the larger forms can have 100 or more periplasmic flagella. Many termite gut treponemes are individual cells, whereas others are ectosymbionts of protozoa, functioning as their motility organelles. Recent metagenomic analysis of the total hindgut microbiota of arboreal wood-feeding Nasutitermes termites revealed that 68% of the genetic material was treponemal in origin. Termite treponemes form a distinct cluster within the treponeme phylogenetic tree (Figure 6). In addition to several named species, ten termite Treponema groups have been defined. The Spirochaeta species, Spirochaeta stenostrepta and Spirochaeta caldaria, were isolated

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Figure 8 Phase contrast micrograph of termite gut treponemes. A variety of treponeme sizes and morphologies are present in the termite gut. Note the treponemal appendages attached to the hypermastigote protozoan, Trichonympha Agilis. Reproduced from Breznak JA (2006) In: Radolf JD and Lukehart SA (eds.) Pathogenic Treponema: Molecular and Cellular Biology, ISBN: 1-904455-10-7.

from water as free-living organisms and named before their 16S sequences were known, but fall within the termite treponeme cluster and should be considered Treponema species likely to have been released from animals or insects. Metabolism

Unlike the commensal or pathogenic treponemes of the oral cavity, termite gut treponemes are symbionts, benefitting their termite hosts by contributing to the digestion of woody plant material. Like most host-dependent spirochetes, termite gut treponemes were difficult to isolate. Eventually, the first termite gut treponemes to be grown in pure culture were isolated from the California dampwood termite, Zootermopsis angusticollis, and assigned to the new species, Treponema primitia. T. primitia is an anaerobe, and was grown in sealed containers. In the process of working with the T. primitia cultures, it was discovered that a vacuum had developed in the headspace of the T. primitia cultures. This observation led to the finding that T. primitia could convert H2 and CO2 gases to acetate. Acetate was known to be a major source of energy and carbon for termites. The treponemes were found to reduce single carbon CO2 to two-carbon acetate molecules via a well-known acetyl-CoA pathway, thus providing nutrients to the termite that would otherwise have escaped in a gaseous form. Treponemes were subsequently found to benefit termite metabolism in other ways. Cellulose is high in energy but relatively poor in nitrogen required for the formation of amino acids. A second species, the aptly named Treponema azotonutricum, was found to be able to convert significant amounts of atmospheric N2 to ammonium using its unique dinitrogenase reductase activity. Until recently, it was not known to what extent termite gut treponemes participated in other aspects of wood

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Bacteria | Spirochetes

polysaccharide digestion. Before CO2 and H2 are formed, cellulose and xylan must be hydrolyzed to hexose and pentose oligomers, respectively, which are in turn fermented to metabolic intermediates. The metagenomic analysis referred to previously revealed a rich diversity of treponemal cellulase and hemicellulase genes. Researchers demonstrated some of the predicted enzymatic activities in the termite gut lumen proteome. Genome sequencing efforts are currently under way to further elucidate the role of termite gut treponemes in cellulose digestion. The ability to convert cellulose to energy without releasing CO2 has led to hopes that the enzymatic activities of termite gut treponemes can be harnessed for the production of green energy, in the form of termite farms, treponemal soups, or as recombinant organisms functionalized with termite gut treponeme genes.

Borrelia Morphology and Metabolism Borrelia spp. are divided into two large genetic groups: the relapsing fever (RF) Borrelia and the Lyme disease (LD)related Borrelia. This article covers the RF Borrelia. The LD Borrelia are covered in ‘Lyme disease’ of the current edition of Encyclopedia of Microbiology. Borrelia vary from 8 to 30 mm in length and from 0.2 to 0:5 mm in width, with the RF Borrelia tending to be shorter and wider than the LD-related Borrelia. Borreliae exhibit the unique rotational motility of spirochetes powered by endoflagella. The RF borreliae have 15–30 endoflagella, whereas the LD-related borreliae have 7–11 endoflagella. Unlike the endoflagella of other spirochetes, the endoflagellae of Borrelia lack sheaths. The other distinguishing morphological characteristic of the Borrelia is the lack of cytoplasmic tubules. Borrelia are obligate parasites with a life cycle that alternates between arthropod vectors and mammalian hosts. Despite their host dependence, many Borrelia spp. have been cultivated using nutritionally rich media, similar to tissue culture media, including many amino acids and vitamins. Glucose is required and is metabolized via the Embden–Meyerhof glycolytic pathway. Borrelia require exogenous N-acetylglucosamine for cell wall synthesis, presumably because this chitin component is constitutively available in ticks. Bovine serum albumin (BSA) is provided as a source of long-chain fatty acids for membrane biosynthesis. The bane of Borrelia researchers is the variable ability of different lots of BSA to support Borrelia growth. Although Borrelia make superoxide dismutase and are able to tolerate low levels of oxygen, they are oxygen sensitive. One possible explanation for the lotto-lot variability of BSA is that polyunsaturated fatty acids supplied by certain lots of BSA appear to be the target of reactive oxygen species, resulting in damage to Borrelia membranes.

Epidemiology and Phylogeny The Borrelia life cycle involves alternating parasitism of arthropod vectors and mammalian hosts. The aptly named Borrelia recurrentis is the only one of the RF Borrelia transmitted by the human body louse (Pediculus humanus) and is historically the most important of the RF Borrelia. Numerous plagues of RF have been recorded, dating back at least as far as the time of Hippocrates. Associations with war, famine, and displaced populations resulting in poverty and overcrowding were well known, but the specific association with body lice was not recognized until 1907. The twentieth century witnessed devastating epidemics of louse-borne RF (LBRF). In the aftermath of the Russian revolution, there were 13 million cases of LBRF in Russia and Eastern Europe, resulting in 5 million deaths. Because humans are the only mammalian host of LBRF and its vector, outbreaks can be effectively aborted by the treatment of clothes and bed linins with insecticides or simply by heating to at least 55  C (130 F) for 5 min. LBRF has been eradicated everywhere except for isolated areas of Ethiopia and neighboring countries involved in war (Sudan, Eritrea, and Somalia). Aside from B. recurrentis, all other RF Borrelia are transmitted by ticks and have nonhuman animal host reservoirs. These tick-borne forms of RF are considered endemic zoonoses and are found worldwide. Most (but not all) Borrelia causing tick-borne RF (TBRF) are transmitted by the Argasidae family of soft-body ticks, whereas the LD-related Borrelia are transmitted by the Ixodidae family of hard-body ticks. Because of the close relationship between TBRF Borrelia species and their tick vectors, the names of the Borrelia species derive from the species of tick vectors that transmit them: Borrelia hermsii is transmitted by Ornithodoros hermsi, Borrelia parkeri is transmitted by Ornithodoros parkeri. The distinction between different types of ticks is important because their different feeding strategies dictate the circumstances under which humans are likely to encounter the Borrelia they carry. Soft-body ticks are nocturnal feeders that seek out sleeping animals by following carbon dioxide and temperature gradients. Although large in size, they have a painless bite and their soft bodies have a distensible stomach that allows them to feed rapidly (15–90 min), drop off, and disappear before being recognized. The large blood meal allows soft-body ticks to live for up to 15 years between feedings, while retaining viable Borrelia in their midgut. Ornithodoros species of soft-body ticks may transmit Borrelia to their progeny, a process referred to as ‘transovarial transmission’. The frequency of transovarial transmission of Borrelia to tick progeny varies greatly between tick species. The frequency of transovarial transmission is high in Ornithodoros turicata, low in O. hermsi, and does not occur in O. parkeri.

Bacteria | Spirochetes

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rRNA and ileT tRNA genes. The distributions of the two B. hermsii genomic groups overlap geographically, indicating that migratory animals, such as birds, may play a role in dissemination. B. hermsii has been found in the bloodstream of a dead owl, and is phylogenetically related to B. anserina, the agent of avian spirochetosis (Figure 9). Other New World TBRF Borrelia differs from B. hermsii in their geographical distribution. B. turicatae occurs in the southwestern United States and northern Mexico. Although B. turicatae has not been isolated from humans, evidence strongly implicates it as the cause of TBRF in spelunkers in Texas. B. parkeri isolates from ticks in the coastal regions of California and Baja California have been implicated as a cause of human disease, but the evidence is circumstantial. Recently, the 16S sequence of a related Borrelia species was obtained from the argasid bat tick, Carios kelleyi, from an attic in Iowa. There is the potential for human disease given the close phylogenetic relationship with human pathogens, the cohabitation of C. kelleyi in homes and the willingness of C. kelleyi to feed on humans. Borrelia coriaceae is transmitted by soft-body Ornithodoros ticks and its reservoir in North America is the black-tailed deer. Human infection with B. coriaceae has not been described, but it is believed to cause abortion in cattle. Borrelia mazzottii and Borrelia venezuelensis have been described in Central and South America, but their 16S rRNA sequences and relatedness to other New World TBRF Borrelia are unknown. Some New World TBRF species are transmitted by hard-body ticks. Like B. burgdorferi, B. miyamotoi is found in

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Hard-body ticks seek out their blood meal during the day and feed for longer periods of time (typically days). The smaller stomach size also requires more frequent feedings. Blood meals are required for a hard-body tick to mature from larvae to nymph, from nymph to adult, and then for the adult to reproduce. Hard-body ticks are mentioned here because there are two notable exceptions to the rule that TBRF Borrelia are transmitted by soft-body ticks: B. miyamotoi is transmitted by Ixodes species (wood ticks) and Borrelia lonestari is transmitted by Amblyomma americanum (the lone star tick). 16S rRNA sequences of RF Borrelia spp. separate phylogenetically into the following three relatedness groups: Old World RF Borrelia, New World TBRF Borrelia, and the B. hermsii/B. anserina group (Figure 9). B. hermsii is the most common agent of human TBRF in North America and is endemic to the coniferous forests of the western United States and southern British Columbia from 3000 to 9000 feet in elevation. The incidence of TBRF peaks in July and August when vacationers visit rustic cabins in mountainous locations that are inaccessible during the winter. B. hermsii achieves high blood densities for prolonged periods of time in pine squirrels (Tamiasciurus spp.), which serves to facilitate transmission to other ticks. Chipmunks and some rodents may also become infected but with lower blood densities and for shorter periods of time than in pine squirrels. Two distinct genomic groups of B. hermsii have been described based on sequencing the intergenic spacer region between the 16S

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Figure 9 Phylogenetic tree of the relapsing fever Borrelia. 16S rRNA sequences of Old World Borrelia spp., including B. recurrentis, the agent of louse-borne relapsing fever, cluster in the lower right section of the tree. Sequences of New World Borrelia spp. cluster in the upper section of the tree. Sequences from B. hermsii and the bird-associated B. anserina cluster in the lower left section of the tree.

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Ixodes ticks and Peromyscus leucopus, the white-footed mouse. B. lonestari is carried by A. americanum, the lone star tick, which is widely distributed in North America and is known to transmit ehrlichiosis and tularemia. The ability of B. lonestari to infect humans is unknown. Among Old World TBRF Borrelia, Borrelia duttonii is the species that is genetically most similar to B. recurrentis, and they probably share a common ancestor. B. duttonii and the related species, Borrelia crocidurae, are transmitted by soft-body ticks and are important causes of TBRF in Sub-Saharan Africa. A number of other Old World TBRF Borrelia have been described in the Middle East, Caucasus, and central Asia, but 16S rRNA sequences are available for only a couple of these species: Borrelia persica and Borrelia hispanica, found in Israel and Spain, respectively. Molecular Pathogenesis and Disease The molecular mechanisms of antigenic variation that are the hallmark of RF have been best described in B. hermsii. In the tick, the major B. hermsii surface protein is the variable tick protein, which presumably facilitates tick– spirochete interactions. In response to temperature changes during the blood meal, B. hermsii switches expression to the variable protein locus located on the expression plasmid. As the bacteria begin to reach high densities in the bloodstream of the infected animal, the host mounts an antibody response to the protein encoded by the gene in the variable protein expression locus. Clearance of bacteria by variable protein-specific antibody is eventually followed by the emergence of bacteria that have undergone a recombinational event on the expression plasmid involving the insertion of genes encoding any one of 12 variable small proteins or 15 variable large proteins. It had long been observed that there was a bias toward a patterned sequence of variable protein gene insertion events. Recently, an explanation for the pattern was explained by the upstream homology sequence (UHS) and downstream homology sequence (DHS) of the variable genes. The probability of a subsequent gene being inserted into the variable protein expression locus was related to the homology of its UHS with the gene currently in the locus and the distance from the end of the new gene to its DHS. A programed succession of surface proteins enables RF Borrelia to repeatedly emerge at high levels in the bloodstream (Figure 10). The ability to repeatedly emerge into the bloodstream is advantageous to the bacteria, because it favors acquisition by blood-feeding arthropods. However, such a high density of bacteria is very hazardous to their animal host, because it evokes such an intense immune response to the foreign antigens. Bouts of LBRF and TBRF differ in their intensity and in the number of relapses. LBRF tends to recur less often,

Figure 10 Micrograph of blood containing relapsing fever Borrelia. Variation in surface antigens enables relapsing fever Borrelia to reach high levels in the bloodstream, often achieving densities as high as 106–107 bacteria per milliliter. Spirochetes appear as dark wavy forms. Reproduced from Figure 1 in Schwan et al., Tick-borne Relapsing Fever Caused by Borrelia hermsii, Montana. Emerging Infections Diseases 2003; 9(9): 1151–4.

but the episodes are much more severe, with a mortality rate of 4–40%. After a typical incubation period of 7 days, patients experience sudden onset of fever, rigors, headache, muscle pain, and lethargy. In LBRF, most patients have liver and spleen enlargement, while cough and symptoms of meningitis are common. Nerve palsies, paralysis, seizures, and coma may occur in severe cases. The most common causes of death in LBRF are arrhythmias of the heart, brain hemorrhage, and liver failure. LBRF during pregnancy frequently results in miscarriage. The mortality rate in TBRF is typically much lower, that is, 2–5%. Nevertheless, TBRF due to B. turicate, B. duttonii, and B. crocidurae are frequently associated with debilitating neurologic symptoms not seen with other forms of TBRF. RF Borrelia are susceptible to a broad range of antibiotics. LBRF can be successfully treated with a single dose of tetracycline. -Lactam antibiotics such as penicillin are typically avoided in LBRF because they may result in the sudden lysis of large amounts of bacterial antigens, which can precipitate the Jarisch–Herxheimer reaction, a paradoxical worsening of symptoms with severe chills, fever, and potentially life-threatening shock. Patients should be observed for 2 h after the initiation of antibiotics in case there is a need for resuscitation with intravenous fluids.

Brachyspira The second major grouping within the order Spirochaetales are the Brachyspira, which are intestinal spirochetes classified within the family, Serpulinaceae. Brachyspira are large,

Bacteria | Spirochetes

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agar and hemolysins are believed to be important virulence factors. The organism is difficult, but not impossible, to eradicate from farms. In Scandinavia, where the use of antibiotics is strictly controlled, few herds are infected by B. hyodysenteriae. In most countries, antibiotic supplementation of feed is used to suppress the B. hyodysenteriae problem, and infection rates are often over 30%. However, antibiotic resistance is growing and new strategies for prevention and control of B. hyodysenteriae infection are urgently needed. The genus Brachyspira is now populated with a number of commensal and pathogenic species, which have been isolated from the intestinal tracts of a variety of animal hosts. Species with predilections for pigs, humans, and birds are clustered on a phylogenetic tree from their 16S sequences (Figure 11). Brachyspira suanatina is the name proposed for an organism that is related to, but genetically distinct from, B. hyodysenteriae by 16S rRNA sequence analysis. B. suanatina has been isolated from both pigs and mallard ducks, is -hemolytic, and can cause disease in experimentally infected pigs. B. intermedia is a third pig isolate found in the same genetic cluster with B. hyodysenteriae and B. suanatina, and may cause disease under certain circumstances. Nondysenteric porcine diarrhea due to intestinal spirochetosis has been linked to B. pilosicoli, which has also been associated with disease in chickens and humans (see below). B. innocens and B. murdochii are considered to be commensals occasionally isolated from healthy pigs. Brachyspira species are also important in the poultry industry. Diarrhea and egg production problems in chickens have been attributed to B. alvinipulli, B. intermedia, and B. pilosicoli. As in pigs, B. innocens and B. murdochii, and a

B. murd

loosely coiled spirochetes ranging in size from 2 to 13 mm in length and from 0.2 to 0:4 mm in width. Brachyspira are able to grow under strict anaerobic conditions, but small amounts of oxygen can increase growth efficiency. The nox gene, encoding NADH (nicotinamide adenine dinucleotide) oxidase, is required for oxygen tolerance. Inactivation of the nox gene increases oxygen sensitivity 100-fold. Brachyspira are cultivated anaerobically on blood agar at 37  C and selective media are typically used for primary isolation of organisms from stool specimens. The Brachyspira have undergone a series of changes in nomenclature. The isolation of the swine dysentery agent was originally described in the early 1970s and referred to as Treponema hyodysenteriae. In the 1990s, DNA–DNA hybridization and partial 16S sequence data indicated that the T. hyodysenteriae organism had little genetic relatedness to the treponemes and was assigned its own genus, Serpula, which was quickly reclassified as Serpulina to avoid confusion with a previously named fungal genus. However, Serpulina eventually gave way to Brachyspira when it was realized that Serpulina hyodysenteriae was related to Brachyspira aalborgi, which had been isolated from humans with intestinal spirochetosis in Aalborg, Denmark, in the early 1980s. The prefix Brachy, deriving from the Greek word for ‘short’, was used as a descriptive term because the Danish isolates were only 2–6 mm in length. Brachyspira hyodysenteriae is an important worldwide problem for the pig industry. Outbreaks with mortality rates of up to 50% occur in naive herds. The infection is a true dysentery, causing inflammatory and hemorrhagic disease of the colon. B. hyodysenteriae is -hemolytic on sheep blood

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Figure 11 Phylogenetic tree of the Brachyspira. Relatedness tree of 16S rRNA sequences of Brachyspira spp., including B. hyodysenteriae, the agent of swine dysentery. B. aalborgi and B. pilosicoli cause intestinal spirochetosis in humans in developed and developing countries, respectively.

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third species, B. pulli, appear to be nonpathogenic for chickens. Chronic watery diarrhea owing to human intestinal spirochetosis has been linked to two species, B. aalborgi and B. pilosicoli, with the latter being associated with intestinal disease in pigs and chickens. High prevalence rates of B. pilosicoli carriage have been found in aboriginal populations living in poor sanitary conditions with high levels of animal exposure. In contrast, B. aalborgi occurs more frequently in developed countries, typically in AIDS patients with chronic diarrhea. The pathogenic potential of Brachyspira for humans is controversial. Biopsies show palisades of Brachyspira lining the surface of colonic epithelial cells, which is likely to impair function (Figure 12). B. pilosicoli is associated with watery diarrhea and has been isolated from the bloodstream of sick patients. Efforts are ongoing to sequence the genomes of B. hyodysenteriae (3.2 Mb) and B. pilosicoli (2.45 Mb). The overall structure of the B. hyodysenteriae genome is likely to be relatively unstable due to the presence of the interesting Virus of S. hyodysenteriae (VSH-1) prophage. Upon induction with mitomycin, VSH-1 functions as a general transduction agent, transferring random 7.5 kb fragments of B. hyodysenteriae DNA between bacteria. B. hyodysenteriae is an attractive organism for research on microbial pathogenesis because of the availability of techniques for targeted gene inactivation. In 1992, researchers at the University of Utrecht reported the first successful homologous recombination in a spirochete, inactivating the B. hyodysenteriae tlyA gene encoding a putative hemolysin. The tlyA mutant had reduced hemolytic activity on blood agar plates, and virulence was attenuated in mouse challenge studies. Subsequently, a number of additional candidate hemolysin genes have been identified,

Figure 12 Scanning electron micrograph showing palisades of Brachyspira exhibiting end-on attachment to the luminal surface of colonic epithelial cells. Marker bar ¼ 2 mm. Reproduced from Hampson DJ and Stanton TB (eds.) (1997) Intestinal Spirochaetes in Domestic Animals and Humans, ISBN: 0-85199-140-8.

and it is likely that B. hyodysenteriae -hemolytic activity is multifactorial. As in other spirochetes, the Brachyspira outer membrane is decorated with membrane proteins. The nomenclature proposed for Brachyspira membrane proteins includes the initials of the species name (Bh for B. hyodysenteriae), the type of protein (lp for lipoprotein and mp for membrane protein), and the predicted molecular mass of the mature protein. So the family of B. hyodysenteriae 29.7 kDa lipoproteins formerly referred to as BmpB and BlpA should now be referred to as Bhlp29.7a, Bhlp29.7b, and so on. Bhlp29.7a has been shown to be lipidated, is a component of the B. hyodysenteriae outer membrane proteome, and is recognized by sera from infected pigs, indicating expression during infection. Omps expressed during infection are of great interest as potential vaccines and serodiagnostic antigens.

Leptospira Morphology and Metabolism Leptospira derives from the Greek leptos (thin) and Latin spira (coiled). Aptly named, the leptospires are among the thinnest bacteria known: a mere 0.1 mm in diameter and 6–12 mm in length (Figure 13). Leptospires are righthanded helices, with 18 or more coils per cell, frequently forming hooks at one or both ends. Hooks at both ends gave rise to the species name L. biflexa, and a hook at one end was believed to look like a question mark, leading to the name L. interrogans. The hooks are due to a single endoflagellum at each end of the cell. In liquids, viable leptospires are continuously in motion. In semisolid (0.2% agarose) conditions, leptospires can be observed by dark field microscopy to remain motionless for periods of time, with occasional corkscrew-like movements. This resting state may, in part, explain the ability of leptospires to persist in the environment. Most leptospires are able to remain motile for months in distilled water, and their survival can be significantly prolonged by addition of a substrate such as agarose.

Figure 13 Transmission electron micrograph of Leptospira sp. showing characteristic helical morphology and a single endoflagellum at each end of the cell. Magnification 30,000. Shadowed electron micrograph obtained by Annabella Chang and used with permission from Ben Adler, Microbiology Department, Monash University, Australia.

Bacteria | Spirochetes

Several different leptospiral growth media have been developed. The standard culture medium is Ellinghausen– McCullough–Johnson–Harris (EMJH) medium, which provides long-chain fatty acids in the form of tween (polysorbate) as an energy and carbon source, several divalent cations (Ca2þ, Mg2þ, Zn2þ, and Mn2þ), iron, and vitamins (thiamin and cobalamin). EMJH medium contains BSA, which is believed to function by preventing fatty acid oxidation, which is toxic for spirochetes, and by providing additional trace nutrients. BSA is expensive and batch-tobatch variability in its ability to support leptospiral growth is a major problem. Serum is not required for EMJH medium but is often added to promote growth. Another problem with BSA-containing media is that due to prior concerns, many countries, including the United States, require any bovine products such as BSA to be autoclaved before import. A non-BSA containing leptospiral medium that can be used as an alternative transport medium is modified Kortoff medium, which consists of peptone, salts, and 8–10% heat-inactivated slightly hemolyzed rabbit serum. The optimum growth temperature is 30  C.

spectrum are the pathogens, including L. interrogans, which are able to produce lethal infection in a variety of mammals, including humans. Species with intermediate pathogenicity, such as Leptospira fainei, can be isolated from clinical specimens, but cause minimal or no disease. Leptospires can also be classified serologically, and over 250 different named serovars have been described. Serovars are classified into one of 28 different serogroups on the basis of antigenic cross-reactivity. Serovar specificity appears to be driven by the carbohydrate structure of LPS side chains, a dominant antigen on the leptospiral surface. There is limited correlation between the genetic and serologic classification systems, with serologically identical strains occurring in multiple species. The phylogenetic and antigenic diversity of Leptospira species reflects their ability to adapt to a variety of different environmental niches. Leptospires have been isolated from most animal species (including, reptiles and amphibians) and natural bodies of freshwater wherever the effort has been made. Presumably, leptospires represent an ancient branch of the bacterial family tree that has coevolved with vertebrates. In reservoir host animals, leptospires have developed a unique commensalist strategy. Organisms with the capacity to infect animals typically reside in the lumen of the proximal renal tubule and are shed into the environment in the urine. The fluid within the proximal tubule is a nutritionally rich filtrate of serum and is yet an immunologically protected site; kidney sections of infected rats show little

Phylogeny A number of different Leptospira species have been described with a range of pathogenic potentials (Figure 14). On one end of the spectrum are the nonpathogenic saprophytic species, including L. biflexa and Leptospira wolbachii, which are unable to cause infection. On the other end of the

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Figure 14 Phylogenetic tree of the Leptospira. Comparison of leptospiral 16s rRNA sequences shows segregation into three relatedness clusters: Nonpathogens (lower left), pathogens (lower right), and organisms with intermediate pathogenicity (upper section).

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or no inflammatory reaction surrounding infected tubules. By not subjecting their host to any detrimental effects, this arrangement effectively affords leptospires a ‘free ride’ for the life of the animal host. The life cycle of the organism is completed when organisms released into the environment encounter a new host through adhesion, vascular invasion, and dissemination to the kidney. Genome sequences provide insight into differences between the various leptospiral lifestyles. The free-living saprophytic nonpathogen, L. biflexa, has a genome size of 3.96 Mb, with a relatively high coding density, and an abundance of signal transduction genes, enabling it to respond to the unpredictable environmental stresses found outside the host. In contrast, L. interrogans has a biphasic lifestyle and seems equally at home in the aquatic environment and in the mammalian host. The 63% of L. interrogans genes shared with L. biflexa consist of essential housekeeping genes and genes important in survival outside the host. The remaining 37% of L. interrogans genes are presumed to be important for life within the mammalian host. On the other end of the spectrum is L. borgpetersenii, which has evolved into an obligate parasite of cattle. Infection occurs through direct contact with carrier animals, L. borgpetersenii has limited survival outside the host, and is difficult to culture. This host dependence is reflected in the erosion of the L. borgpetersenii genome; many of its genes are lost or inactivated by mutations or transposon insertions. L. borgpetersenii has only about half the signal transduction genes that L. biflexa has, confirming the ‘locked-in’ nature of its host dependency.

Pathogenesis To acquire the ability to invade and colonize the mammalian host, L. interrogans has acquired a large array of novel genes. Some of these pathogen-specific genes are known to encode Omps such as the porin, OmpL1, and a number of lipoproteins, some of which are involved in host–pathogen interactions. An essential host–pathogen interaction that distinguishes leptospiral pathogens from saprophytes is serum resistance. Leptospiral serum resistance is mediated, at least in part, by LenA, an outer membrane lipoprotein found exclusively in leptospiral pathogens. LenA binds Factor H, a complement regulatory protein that prevents the alternative pathway of complement from damaging host cell membranes. Leptospires (and other spirochetes) coat their surfaces with Factor H to avoid the bactericidal effects of complement. Pathogenic leptospires coat their surfaces with additional host factors using proteins belonging to the Lig (Leptospira immunoglobulin-like repeat) family. Leptospiral pathogens, but not the saprophytes, have between one and three Lig proteins. Ligs are very large (112–220 kDa) proteins

containing a series of 12–13 immunoglobulin-like repeats, some of which mediate high affinity binding to multiple host proteins, including fibronectin and fibrinogen. Interactions with host proteins are facilitated by the induction of Lig expression in response to levels of osmolarity (300 mOsm) found in host tissues. Lig expression by leptospires grown in EMJH medium, which has low osmolarity (67 mOsm), is poor. Addition of salt (or any other osmotically active molecule) to EMJH medium rapidly induces Lig expression. In this way, leptospires in aquatic environments are saved the metabolic expense of not expressing Lig proteins until they are needed. Acquisition of virulence genes was essential in the evolution of leptospires from free-living to pathogenic organisms. Genes appear to have been horizontally transferred from a variety of sources. For example, the major outer membrane lipoprotein, LipL32, is highly conserved among leptospiral pathogens and is believed to mediate interactions with extracellular matrix proteins of the host. The lipL32 gene does not occur in the nonpathogens, its closest homologue is found in the marine bacterium Pseudoalteromonas tunicata. Horizontal genetic transfer also occurs between leptospiral pathogens; 20% of ompL1 genes are mosaics containing fragments of multiple leptospiral lineages. However, permissiveness for gene acquisition is a double-edged sword – the genomes of leptospiral pathogens have much higher numbers of insertion sequence (IS) elements than the nonpathogens. The IS elements contain transposon genes that, once they infect the genome, mediate IS element proliferation and gene disruption. IS elements appear to be a major mechanism of genome erosion in L. borgpetersenii. Transposons are now being put to good use in leptospiral research – leptospiral pathogens had been much more difficult to transform than the nonpathogens, which had been a major impediment in leptospiral pathogenesis research. Now, however, the mariner tranposon has been found to be useful for manipulating the genome of leptospiral pathogens – hundreds of single-gene knockout mutations have been generated in L. interrogans strains. It is hoped that testing these mutants in animal models will lead to the identification of new leptospiral virulence genes and vaccines.

Epidemiology and Disease Leptospirosis epidemiology has traditionally been carried out by serotyping isolates or examining the serologic response of infected patients. However, serologic approaches are fraught with problems, including the frequent observation that patient’s antibody responses may not be specific for the infecting serovar. Genetic tools provide more accurate molecular approaches for tracking the epidemiology of leptospirosis. 16S sequencing can be used for species identification and is less cumbersome than DNA–DNA

Bacteria | Spirochetes

hybridization. Differentiation of strains has been performed by multilocus sequence typing using PCR primers for 11 housekeeping genes scattered across the leptospiral genome. Approaches such as these reveal that rat-associated strains of L. interrogans are frequently the cause of leptospirosis outbreaks in urban settings. Rats are found wherever people live, and wherever the studies have been carried out, urban rats are found to have a high leptospirosis carriage rate in their kidneys. The prevalence of leptospiral carriage among rats is probably the reason that leptospirosis is the most widespread zoonosis known. Leptospirosis occurs less frequently in Westernized countries because housing standards tend to exclude rats from human living spaces. However, in developing countries with poor housing standards, leptospirosis outbreaks occur regularly in urban settings after heavy rainfall and flooding. Leptospirosis infections range in severity from selflimited flu-like illness to multiorgan system failure and death. After an incubation period of 5–14 days, there is the onset of fever, myalgia, headache, abdominal pain, nausea, and vomiting. In tropical regions where leptospirosis typically occurs, these early nonspecific symptoms can be confused with dengue fever or malaria. When observed, conjunctival suffusion (scleral redness without discharge) can be a distinguishing sign of leptospirosis. During this initial septicemic phase, spirochetes can be recovered from the blood and spinal fluid. Formation of agglutinating antibody leads to the clearance of organisms and, in milder cases, to a temporary resolution of symptoms. However, a second, immune phase of the disease may follow, with milder fever, headache, and vomiting. In more severe infections, the initial phase progresses rapidly to jaundice and renal failure, known as Weil’s syndrome, with a mortality rate of 10%. The renal failure due to leptospirosis is a unique form of kidney dysfunction associated with high urine output and low serum potassium levels. At this stage, complications can be avoided with the replacement of fluids and electrolytes. If, on the other hand, dehydration occurs and renal failure ensues, access to peritoneal or hemodialysis is essential for survival. Certain strains of L. interrogans cause acute lung involvement with shortness of breath due to airspace hemorrhage and a much higher mortality rate of 50%. Pathogenic leptospires are highly susceptible to common antibiotics including doxycycline and ampicillin and it is likely that antibiotic therapy given at the first signs of infection would significantly reduce morbidity and mortality. However, currently available diagnostic tests have relatively low sensitivity during early infection and patient populations at highest risk typically have poor access to medical care. Recent studies show that most patients with early infection have antibodies to the Lig proteins. What is needed is a diagnostic test that is portable, easy to use, does not require electricity, and has a long shelf life at room temperature. Whole-cell vaccines

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are used widely in domestic animals, including dogs, pigs, and cattle. A similar vaccine has been found to be effective in humans, but is generally not available because of concerns regarding side effects and a relatively short duration of immunity. A preventative approach for adventure travelers participating in water sports in areas with a history of leptospirosis is weekly doxycycline, which has been shown to be effective in US soldiers undergoing jungle training in Panama. Doxycycline is not appropriate for children or pregnant women and may cause photosensitivity or gastrointestinal side effects. An alternative approach recommended by some travel experts is weekly azithromycin, which has a better safety profile, but has not been rigorously tested for efficacy.

Conclusions Spirochetes are widely distributed in nature as free-living bacteria, as metabolic symbionts of insects, and as commensals and parasites of animals. Spirochaeta spp. isolated from natural bodies of water are related by 16S rRNA sequence analysis to treponemes found in the oral cavity and in the digestive tracts of termites. Borrelia spp. also have the ability to colonize the digestive tracts of insects, in this case ticks and lice, which serve as vectors for transmission to animal host reservoirs. Brachyspira spp. colonize digestive tracts of animals either as commensals or as parasites. Leptospira spp. exist as free-living organisms or cycle between the aquatic environment and animal host reservoirs via their renal tubules. The diversity of spirochete lifestyles demonstrates the functional versatility of their unique morphology and mechanism of motility. See also: Lyme Disease; Sexually Transmitted Diseases; Syphilis, Historical

Further Reading Barbour AG, Dai Q, Restrepo BI, Stoenner HG, and Frank SA (2006) Pathogen escape from host immunity by a genome program for antigenic variation. Proceedings of the National Academy of Sciences of the United States of America 103: 18290–18295. Barbour AG and Hayes SF (1986) Biology of Borrelia species. Microbiological Reviews 50: 381–400. Charon NW and Goldstein SF (2002) Genetics of motility and chemotaxis of a fascinating group of bacteria: The spirochetes. Annual Review of Genetics 36: 47–73. Cullen PA, Haake DA, and Adler B (2004) Outer membrane proteins of pathogenic spirochetes. FEMS Microbiology Reviews 28: 291–318. Dworkin MS, Schoemaker PC, Fritz CL, Dowell ME, and Anderson DE (2002) The epidemiology of tick-borne relapsing fever in the united states. The American Journal of Tropical Medicine and Hygiene 66: 753–758. Ellen RP and Galimanas VB (2005) Spirochetes at the forefront of periodontal infections. Periodontology 38: 13–32. Faine S, Adler B, Bolin C, and Perolat P (1999) Leptospira and Leptospirosis, 2nd edn. Melbourne, Australia: MedSci.

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Galperin MY (2005) A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts. BMC Microbiology 5: 35. Haake DA (2000) Spirochetal lipoproteins and pathogenesis. Microbiology 146: 1491–1504. Holt SC (1978) Anatomy and chemistry of spirochetes. Microbiological Reviews 42: 114–160. Levett PN (2001) Leptospirosis. Clinical Microbiology Reviews 14: 296–326.

Paster BJ, Boches SK, Galvin JL, et al. (2001) Bacterial diversity in human subgingival plaque. Journal of Bacteriology 183: 3770–3783. Radolf JD and Lukehart SA (eds.) (2006) Pathogenic Treponema – Molecular and Cellular Biology, Norfolk, England: Caister Academic Press. Warnecke F, Luginbu¨hl P, Ivanova N, et al. (2007) Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite. Nature 450: 560–565.

Staphylococcus A F Gillaspy and J J Iandolo, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Taxonomy Cellular Structure Molecular Structure

Glossary biofilm Sessile microbial communities consisting of an accumulated mass of bacteria, extracellular matrix molecules, and secreted bacterial products that aid in adherence to biopolymers and other solid surfaces. capsule Polysaccharide outermost layer of the cell. Production imparts a viscous slimy look to colonies. cell wall One of the outer layers of the bacterial cell that protects the cell from osmotic perturbations and provides mechanical protection to the fragile cellular membrane.

Coagulase-Negative Versus Coagulase-Positive Staphylococci Pathogenesis and Disease Virulence Factors Further Reading

endonuclease Enzymes that have specific binding sites of varying complexities at which they sever phosphodiester bonds of DNA. MSCRAMM (Microbial surface components recognizing adhesive matrix molecules) Proteins involved in the establishment of infection via the initial attachment of bacteria to host molecules such as elastin, collagen, fibrinogen, and fibronectin. plasmid Autonomously replicating small extrachromosomal DNA molecules. superantigen Any of a number of proteins that elicit a massive T-cell receptor V-restricted primary response.

Abbreviations CDC CHEF cMRSA CoNS IFN- MHC II MRSA

Centers for Disease Control clamped homogeneous electrophoretic field community-acquired MRSA coagulase-negative staphylococci interferon- major histocompatibility complex class II methicillin-resistant Staphylococcus aureus

MSCRAMM NCBI PIA PVL RFLP SE SSSS

microbial surface components recognizing adhesive matrix molecule National Center for Biotechnology Information production of a slime substance Panton-Valentine leukocidin restriction fragment length polymorphism staphylococcal enterotoxin staphylococcal scalded-skin syndrome

Defining Statement

Taxonomy

The staphylococci are important bacterial pathogens that can infect both animals and humans and are responsible for numerous hospital- and community-acquired infections yearly. Staphylococcal infections result in a significant burden both economically and clinically due to several factors, including increasing antibiotic resistance and lack of effective vaccines.

The genus Staphylococcus is defined in Bergey’s Manual of Determinative Bacteriology as a member of the family Micrococcaceae. According to the most recent approved list from Bergey’s Manual (8th ed.), there are presently 41 recognized species of staphylococci (Table 1), 18 of which are indigenous to or have been shown to colonize humans; the remaining species have been isolated from

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Table 1 Currently recognized staphylococcal species S. arlettae S. auricularisa S. capraea S. caseolyticus S. cohniia S. delphini S. equorum S. fleurettii S. haemoyticusa S. hyicus S. kloosii S. lugdunensisa S. muscae S. pasteuria S. piscifermentans S. pulvereri S. saprophyticus S. sciuria S. simulansa S. vitulinusa S. xylosusa

a

S. aureus S. capitisa S. carnosus S. chromogenes S. condimenti S. epidermidisa S. felis S. gallinarum S. hominisa S. intermedius S. lentus S. lutrae S. nepalensis S. pettenkoferia S. pseudintermedius S. saccharolyticusa S. schleiferia S. simiae S. succinus S. warneria

a Denotes species that are either indigenous to humans or have been found to colonize humans.

various animal (including one primate species), plant, or food specimens. Staphylococci are Gram-positive cocci 0.7–1.2 mm in size that occur singly and in pairs in liquid media and in clusters when grown on solid media. Over the years, they have been characterized by their variety of colonial, morphological, and biochemical activities that have resulted in description of several biotypes of variable stability. They are aerobic or facultatively anaerobic, catalase-positive, and capable of generating energy by respiratory and fermentative pathways. These organisms are nutritionally fastidious with complex nitrogen requirements. Most species require several amino acids, vitamins (thiamine and niacin), and uracil (to grow anaerobically) for growth. In complex, nutritionally complete growth media, the organism is able to grow at generation times of 20 min, a rate comparable to that of E. coli. At the molecular level, the genus can be distinguished from other members of the Micrococcaceae by the low GC content of its DNA which ranges from 30 to 38%, the presence of teichoic acid in their cell wall, and their ability to tolerate extremely low water activities. Staphyloccocus aureus is routinely cultured at aw of 0.88 (15% NaCl) and has been reported to grow at water activity levels as low as 0.83 (saturated NaCl solution). Anaerobically, tolerance to low aw is less with the limit at 0.90. The normal habitat of these organisms is the skin, skin glands, and mucous membranes of warm-blooded animals. However, staphylococci can be isolated from a variety of sources that include soil, dust, air, water, and food and dairy products. As a result they present a food and water public health hazard as well as an infection risk.

Cellular Structure Cell Wall The cell wall of Staphylococcus is a thick, electron-dense structure that provides great mechanical support to the cell. It is composed of a giant polymer consisting of peptidoglycan complexed with teichoic acid and other surface proteins described elsewhere in this article. The cell wall is a heteropolymer consisting of glycan chains cross-linked through short peptides. The repeating unit in the glycan backbone is -1,4-N-acetylglucosamine and N-acetylmuramic acid (muramic acid). About 60% of the N-acetylmuramic acid residues are O-acetylated. The high level of O-acetylation makes the staphylococcal cell wall resistant to lysozyme digestion, therefore making this genus unique from most other bacteria. In fact, lysis of the cell wall under laboratory conditions is only efficient when the staphylococcal endopeptidase, lysostaphin, is added to cultured cells. Lysostaphin is produced by S. simulans and specifically cleaves the pentaglycine crossbridges found in the staphylococcal peptidoglycan. In S. aureus, adjacent polypeptides are cross-linked by pentaglycine crossbridges between the "-amino of lysine and C-terminal D-alanine, whereas in other species, the composition of the crossbridge is variable. The carboxy group of muramic acid is substituted by an oligopeptide that contains alternating L- and D-amino acids (L-alanine, D-glutamine, L-lysine, D-alanine, D-alanine). The teichoic acid component is linked to the D-alanine component of the mucopeptide by - or -glycosidic linkage through N-acetyl-D-glucosamine. In S. aureus, the teichoic acid backbone is ribitol based whereas in S. epidermidis it is glycerol based. In other species, glycerol teichoic acids are more common than their ribitol counterparts. These highly charged immunogenic cell wall components have been found to play a role in S. aureus nasal colonization, biofilm formation, and susceptibility to vancomycin and other glycopeptides.

Capsule Capsule production has been shown to occur in vivo and in vitro for both S. epidermidis and S. aureus. For S. epidermidis, there have been at least three different capsular polysaccharide types identified in the literature. In contrast, 11 capsular polysaccharide serotypes have been described for the highly virulent species, S. aureus. The most common occurring serotypes among clinical isolates are types 5 and 8. The main components of the capsules are N-acetylaminouronic acids and N-acetylfucosamine. The genes for capsule production have been identified and are located in a single operon. The production of a capsule renders the staphylococci resistant to host defenses such as opsonization and phagocytosis. However, antibodies to capsular

Bacteria | Staphylococcus

polysaccharides can neutralize the antiphagocytic properties of the capsule and opsonize the cell for phagocytosis. Opsonization has made the capsule a prime target in the search for an effective staphylococcal vaccine. Several staphylococcal species, particularly S. epidermidis and S. aureus, have been shown to produce biofilms, and capsule production has been shown to be of particular importance during biofilm formation by aiding in adherence to biomaterials such as indwelling medical devices. A trademark of staphylococcal biofilm formation is the production of a slime substance (PIA), which is a polysaccharide composed of -1,6-linked N-acetylglucosamines with partially deacetylated residues giving them a positive charge. Once bacterial cells are within this slime, they are protected from the host’s immune defenses and are resistant to other treatments such as antibiotics. PIA is not the only component that has been shown to contribute to biofilm production under certain conditions, and research in this area has become very popular in the past several years. S. epidermidis is the primary cause of catheter-associated staphylococcal infections and is a primary producer of biofilms. S. aureus biofilms have also been shown in patients with diseases such as osteomyelitis and endocarditis. Biofilm production in the staphylococci has been shown to be multifactorial and appears to be a bacterial survival mechanism when cells are exposed to sublethal levels of antibiotics and/or other stressful environmental conditions (i.e., limited nutrients, changes in temperature, oxygen limitation). It is also important to note that large phenotypic variations in biofilm production exist, with some isolates incapable of biofilm formation regardless of culture condition and others being hyperproducers under a specific set of conditions. Continued focus on understanding all of the components

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involved will give us more insight into chronic staphylococcal infections, especially those involving medical devices, and may lead to more efficient treatment of these diseases.

Molecular Structure Genome Presently, the genomes of 18 different members of the genus Staphylococcus have been completely sequenced. This number includes 14 strains of S. aureus, 2 strains of S. epidermidis, 1 strain of S. haemolyticus, and 1 of S. saprophyticus. The genomic data for each of these projects are freely available on the World Wide Web at the National Center for Biotechnology Information (NCBI) website. In all cases, the genome is circular and ranges from 2.49 Mb (S. epidermidis) to 2.9 Mb pairs (S. aureus strains). Table 2 shows a summary of the information obtained from the 18 completed staphylococcal genome projects. Consistent with the Micrococcaceae family, all 18 of the isolates have a low GC content (33%). The sequenced strains also exhibit a high amount of diversity within this genus, with some species and strains having acquired resistance to the antibiotics methicillin and/or vancomycin and some containing extrachromosomal elements called plasmids that most often contain additional genes that can contribute to pathogenesis. Among the sequenced isolates, the number of plasmids present varies from 0 to 3 and the size of these elements varies as well. Genetic and sequence data available indicate that in addition to the normal complement of housekeeping genes, the chromosome contains many accessory genetic elements that are not necessary for growth under laboratory conditions.

Table 2 Completed staphylococcal genome project Organism

Genome size

Plasmid(s) size

%GC

# of predicted genes

MRSA/VRSA

S. aureus strain MW2 S. aureus strain Mu50 S. aureus strain N315 S. aureus strain NCTC8325 S. aureus strain RF122 S. aureus strain COL S. aureus strain MRSA252 S. aureus strain MSSA476 S. aureus strain USA300 – FPR3757 S. aureus strain JH1 S. aureus strain JH9 S. aureus strain Newman S. aureus strain Mu3 S. aureus strain USA300 TCH1516 S. epidermidis strain ATCC12228 S. epidermidis strain RF62A S. haemolyticus strain JCSC1435 S. saprophyticus strain ATCC15305

2.82 Mb 2.9 Mb 2.84 Mb 2.82 Mb 2.74 Mb 2.81 Mb 2.9 Mb 2.79 Mb 2.91 Mb 2.9 Mb 2.9 Mb 2.9 Mb 2.9 Mb 2.9 Mb 2.49 Mb 2.64 Mb 2.68 Mb 2.57 Mb

N/A 25.1 kb 24.6 kb N/A N/A 4.44 kb N/A N/A 37 kb, 4.4 kb, 3.13 kb 30.4 kb 30.4 kb N/A N/A 27.0 kb N/A 27.3 kb N/A 38.5 kb, 16.3 kb

32.82% 32.84% 32.8% 32.86% 32.77% 32.81% 32.8% 32.85% 32.69% 32.0% 32.9% 32% 32% 32% 32.09% 32.14% 32.79% 33.18%

2632 2748 2623 2892 2589 2787 2744 2619 2691 2870 2816 2687 2776 2802 2419 2665 2678 2514

MRSA MRSA MRSA N/A MRSA MRSA MRSA N/A MRSA MRSA, VRSA MRSA, VRSA N/A MRSA, VRSA MRSA MRSA MRSA MRSA MRSA

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Bacteria | Staphylococcus

level of protein similarity is 61.4% across the entire group. Of course, the level of similarity is slightly higher when the same type of comparison is done within species (85–92%) but the overall similarity at the genus level is still highly significant. For perspective, the same comparison done with S. aureus NCTC8325 and B. subtilis showed only 59% overall protein similarity. In addition to large pathogenicity islands the staphylococcal genome (especially S. aureus) typically contains multiple instances of transposable elements such as Tn551, insertion sequences IS256, IS257, IS1181, and others. S. aureus Tn551 and its close relative Tn917 have been extensively utilized to generate knockout mutations in staphylococci, many of which have been mapped and localized near other genes. The primary strain of S. aureus used for genetic manipulation and gene discovery is NCTC8325 and its derivatives. Also known as PS47, this is the propagating strain for the typing bacteriophage ø47 and is a member of phage group III. It is routinely used to generate batches of bacteriophage used for typing purposes and is lysogenized by three temperate phage: ø11, ø12, and ø13. A derivative (8325-4) that has been cured of all demonstrable phage was originally developed as the prototype strain whose genome was used to build a circular map based on genetic and physical parameters. With the advancements in whole-genome sequencing technology this map is no longer the primary resource used for mapping and typing experiments. However, in the absence of sufficient genomic information, this type of mapping is still used to examine new S. aureus isolates and to determine basic lineage information for these isolates.

2616530

2872769 Chromosome Staphylococcus epidermidis RP62A

Chromosome_final Staphylococcus aureus subsp. aureus USA300–FPR3757

In S. aureus strains, pathogenicity islands SaPI1 and SaPI2 have been described and have been shown to encode virulence factors such as the toxic shock syndrome toxin gene, staphylococcal enterotoxin B, and as well as resistance to methicillin. In strains that do not contain SaPI1 or SaPI2 there are no allelic counterparts for these genes. The genes and overall genomic organization of S. aureus bear a striking resemblance to the genome of Bacillus subtilis such that the organism has been called a morphologically degenerate form of Bacillus. In addition, the genomic organization of the S. aureus strains sequenced thus far has been shown to be highly similar. Specifically, there is a genomic ‘backbone’ common to all S. aureus strains containing varying numbers of genes that may contribute to antibiotic resistance, tissue tropism, and virulence. Comparison of the two sequenced S. epidermidis strains showed that the overall genomic organization is similar, with two relatively small areas of inversion apparent when the two genomes are aligned to each other at the nucleotide level. Figure 1 shows alignments at the nucleotide level for two representative S. aureus strains as well as the two S. epidermidis strains. Alignment of S. haemolyticus and S. saprophyticus to S. aureus, S. epidermidis, or to one another showed little or no conservation of gene order. However, at the level of protein content, members of the genus are highly similar and this is illustrated graphically in Figure 2. Specifically, when the translated sequences for several of the sequenced staphylococcal genomes are compared (at a minimum cutoff level of >¼ 40% similarity) to one another using S. aureus NCTC8325 as the reference strain (since this strain has the largest number of predicted open reading frames), the

2298215

1723661

1149108

574554

0 1

564273

1128545

1692817

2257089

Chromosome Staphylococcus aureus NCTC 8325

2821361

2093224

1569918

1046612

523306

0 1

499856

999712

1499567

1999423

2499279

Chromosome Staphylococcus epidermidis ATCC 12228

Figure 1 The left panel shows alignment of S. aureus strains NCTC8325 and USA300 at the nucleotide level. The solid red line indicates that the two strains are virtually identical in genomic organization. Right panel shows the alignment of S. epidermidis strains ATCC12228 and RP62A. The break in the red line and the shorter green lines show that although the two strains are overall highly similar in organization, there are some differences. Differences are believed to be due to the presence or absence of such things as bacteriophage and pathogenecity islands.

Bacteria | Staphylococcus

Figure 2 Comparison of proteins encoded within 13 members of the genus Staphylococcus. Completed genomes used in the above comparison are listed starting with the outermost circle: S. aureus strain NCTC8325, S. aureus strain COL, S. aureus strain USA300-FPR3757, S. aureus strain MW2, S. aureus strain Mu50, S. aureus strain MSSA476, S. aureus strain N315, S. aureus strain MRSA252, S. aureus strain RF122, S. haemolyticus strain JCSC1435, S. epidermidis strain ATCC12228 and S. epidermidis strain RP62A. Using NCTC8325 as the reference strain resulted in a total number of 2892 predicted proteins used for the comparision. Of the 2892 proteins used, 1775 were found to be present in all other genomes, with 2756 present in at least another genome and only 136 not found in any other of the comparison genomes.

Plasmids As mentioned above, the staphylococci are also endowed with a generous array of plasmids ranging in size from a few kilobases to 40–50 kb. In fact, there are typically one or more plasmids found in most staphylococcal clinical isolates, including both S. aureus and the coagulasenegative staphylococci. The inability of two plasamids to coexist in a single host indicates that they have the same replication control functions and are incompatible and assigned to the same incompatibility group (inc group). Thus far, there are 15 recognized incompatibility groups (inc 1–15), but the replication characteristics of some staphylococcal plasmids remain undetermined and they are still unclassified. The 15 inc groups have been further divided into three main classes (I, II, and III) with a fourth class that contains the pSK639 family of plasmids. The four groups are based on both physical and genetic organization as well as functional characteristics. Some staphylococcal plasmids are cryptic but most carry resistance determinants and some carry other virulence factors such as toxins. Group I plasmids are relatively small

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plasmids (typically ¼ 65 years), African Americans, and males. Interestingly, the group at lowest risk included young people ranging from 5 to 17 years of age. The study concluded that invasive MRSA affects certain populations disproportionately and that it is a major public health problem primarily related to health care. The researchers also stress the point that MRSA should no longer be confined to healthcare institutions, and that the incidence of cMRSA will most likely continue to increase. There are multiple reasons that the staphylococci, especially S. aureus, have become antibiotic-resistant including overuse of antibiotics in humans, the presence of antibiotics in food and water supplies, and mutation and/or exchange of genes within the genus. Unnecessary prescriptions for antibiotics is one of the main sources contributing to staphylococcal (and other microorganisms) developing resistance. Decades of excessive antibiotic use for colds, flu, and other viral infections that do not respond to these drugs results in low-level exposure to these compounds by normal bacterial flora found in the host. This repeated exposure to sublethal concentrations results in the elimination of the majority of the resident bacteria and selects for spontaneously occurring mutants that are resistant to the antibiotic. Over time, the presence of the antibiotic has no effect on the organism and therefore they are resistant to killing by the drug when it is prescribed for actual treatment. Prescription drugs are not the only source of antibiotics. In the United States, antibiotics can be found in animal feeds especially for beef cattle, pigs, and chickens. The same antibiotics then find their way into municipal water systems when the runoff from feedlots contaminates streams and groundwater. However, antibiotics given in the proper doses to sick animals do not appear to produce resistant

Bacteria | Staphylococcus

bacteria. Even appropriate antibiotic use can contribute to the increase in drug-resistant bacteria because they may not destroy every organism within the population. Bacteria evolve rapidly; so those that survive treatment with one antibiotic are soon capable of resisting others. Bacteria mutate much more quickly than new drugs can be produced, which makes it possible for a given organism to become resistant to all available treatment options. In addition to mutation of genes, the staphylococci are adept at gene transfer via mobile genetic elements such as plasmids, phage, and transposons. By carrying and transferring antibiotic-resistant genes via these mechanisms, antibiotic resistance has become rampant within this group. Although there has not yet been a strain of S. aureus identified that is resistant to all of the available antibiotics, there are many that are resistant to the majority of drugs currently available. In fact, it is relatively uncommon for any given clinical isolate to be resistant to only one drug and almost all that are isolated are resistant to penicillin, which was the original drug used to treat most bacterial infections. More importantly, there are now multiple strains that have acquired resistance to what used to be the drug of last resort (vancomycin). Using vancomycin and other more aggressive forms of treatment contribute to the economic burden endured by the patient and hospital, and these types of treatments are also more physically demanding for the patient. For example, vancomycin is generally given in multiple intravenous doses over several weeks, which requires additional specialized care and a longer stay within the hospital. The drug itself often makes the individual feel even worse than before treatment began, with potential side effects including, but not limited to, kidney failure, temporary or permanent hearing loss, neutropenia, anaphylaxis, pain and inflammation at the injection site, severe stomach pain, diarrhea, and fever or chills.

Virulence Factors Staphylococci produce a wide array of extracellular and cell surface proteins with a large number of these encoded on plasmids and other accessory elements. Because of this, different strains have been shown to exhibit a variable array of toxins, enzymes, and other factors. A list of some of these exoproteins is presented in Table 3. The extracellular proteins are subdivided into cell surface-oriented proteins and soluble proteins. All the exoproteins are translated as precursor proteins with signal peptides, which are removed at secretion. In addition, the cell surface proteins also possess a characteristic amino sequence motif (LPXTG) at the C-terminus, which precedes the membrane spanning region and serves as an anchor, linking the protein to the cell wall peptidoglycan. Both types of exoproteins are secreted by Type II secretory

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Table 3 Extracellular proteins of Staphylococcus aureus Protein Hemolysins  Alpha toxin a  Beta toxin  Gamma toxin a  Delta toxin  Panton-Valentine leukocidin

Gene locus

Chromosome Chromosome Chromosome Chromosome Chromosome/pathogenicity island

Enterotoxins  SEA  SEB  SEC  SED  SEE  SEG  SEI

Enzymes and other toxins Lipasea Nucleasea V8 Proteasea Esterase Coagulase Cell wall hydrolase Hyaluronadase Staphylokinase Protein A Serine proteasesa Zinc metalloproteinase aureolysina Phospholipase C Leukotoxins Leukocidins Exfoliative toxin A Exfoliative toxin B Toxic shock syndrome toxin-1 MSCRAAMs Clumping factors Fibronectin-binding proteins A/B Fibrinogen-binding protein Collagen-binding protein Elastin binding proteina Extracellular matrix-binding proteinsa Intercellular adhesion proteinsa

Bacteriophage/chromosome Chromosome/pathogenicity island Plasmid Plasmid Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Bacteriophage/chromosome Chromosome Chromosome/genomic island Chromosome Chromosome Chromosome/genomic island Chromosome/pathogenecity island Chromosome Plasmid Chromosome/pathogenicity island Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome

a

Denotes virulence factors that are also present in S. epidermidis.

mechanisms involving the SecYEG pathway. With the exception of certain bacteriocins, there is no evidence to date that extracellular proteins are secreted by other secretory pathways. Soluble Exoproteins These include a wide array of toxins and enzymes such as food poisoning enterotoxins, exfoliative toxins, toxic

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shock syndrome toxin, hemolysins, coagulase proteases, lipases, and other enzymes. The exfoliative toxins are proteolytic and attack the epidermis of susceptible animals. Exfoliative toxins cause blistering skin diseases known as bullous impetigo and staphylococcal scaldedskin syndrome (SSSS). Three isoforms of exfoliative toxins (ETA, ETB, and ETD) have been identified in virulent strains of S. aureus and four isoforms have been shown to exist in the animal pathogen S. hyicus. Clinical manifestation of SSSS is typically seen in neonates and has been termed scalded skin syndrome due to the symptoms that occur culminating in areas of raw, red skin that resembles that of a first-degree burn. The enterotoxins have been shown to act at the interface between the stratum granulosum and stratum spinosum of the epidermis, thereby resulting in characteristic exfoliation of the skin. In other words, there is a loss of keratinocyte cell– cell adhesion in the epidermis. It was initially difficult to determine the mechanism of action for these toxins because purified forms of both ETA and ETB showed no direct protease activity toward multiple targets. However, recent studies have shown that the three isoforms of exfoliative toxins, ETA, ETB, and ETD, are glutamate-specific serine proteases and that ETA and ETB specifically cleave a protein in the epidermal interface called desmogelin 1. ETD has been shown to be encoded within a pathogenecity island and to play a slightly different role in the development of bullous impetigo and scalded skin syndrome disease. The hemolysins are membrane-damaging proteins whose activity is mediated through pore formation or lipolytic action. The most studied of the hemolysins is -toxin, which is toxic to a wide range of mammalian cells and is highly hemolytic for rabbit erythrocytes. It is also dermonecrotic and neurotoxic and is produced by almost all strains of S. aureus. -toxin is made in high concentrations, particularly by animal strains of S. aureus, and is highly hemolytic for sheep erythrocytes but not rabbit red blood cells. This toxin exhibits phosphorylase C activity that requires magnesium but it has a limited range of activity due to specificity for sphingomyelin and lysophosphatidyl choline. The role of -toxin in disease is not well understood but the fact that it is made at much higher levels in animal strains suggests that it may provide an advantage in animal hosts as opposed to humans. Another example of an S. aureus pore-forming toxin is the Panton–Valentine leukocidin (PVL), which is a cytotoxin that causes leukocyte destruction and tissue necrosis. PVL is encoded on a bacteriophage and has been associated with both staphylococcal skin and pulmonary infections. PVL-containing strains have also been isolated at a higher frequency from patients with severe cMRSA pneumonia and the presence of the toxin is much higher in strains that carry the SCCmecIV cassette than in strains that do not.

Another group of toxins that play a significant role in staphylococcal disease are the enterotoxins. The enterotoxins are responsible for the clinical manifestations of staphylococcal food poisoning and a septic shock-like illness. There are five major classical types of staphylococcal enterotoxins (SEs), and several new SEs or SE-like toxins have recently been identified. Ingestion of these toxins leads to severe gastroenteritis with emesis, nausea, and diarrhea. In addition, the SEs are resistant to extreme heat and are stable over a wide pH range besides being resistant to degradation by a variety of proteases. They are also classified as superantigens, a characteristic that is described in more detail below. Several of the soluble exoproteins including enterotoxins, exfoliative toxins, and toxic shock toxin are superantigens due to their ability to stimulate mitogenic activity and cytokine production for a wide array of T-lymphocyte haplotypes. They are able to activate specific sets of T-lymphocytes by binding to major histocompatibility complex class II (MHC II) proteins. They bind to the variable region of the T-cell receptor -chain. The activated cells proliferate and release cytokines/lymphokines, interferon- (IFN-), and interleukins. Because they exhibit this broad-based activity, they have been called superantigens. This activity is suspected to enhance virulence by suppressing the hosts response to staphylococcal antigens produced during infection. In addition to the factors discussed above, there are many staphylococcal enzymes such as lipase, nuclease, and proteases, and all are presumed to enhance invasiveness through tissue destruction. Cell Surface Proteins A major class of cell surface proteins are adhesins termed MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). These molecules comprise the main adhesins of the organism and include collagenbinding protein, fibronectin-binding proteins, fibrinogenbinding protein, elastin-binding protein, clumping factor, and the matrix adhesin factor. There may be as many as 12 other surface proteins that contain membrane anchor domains and potentially qualify as MSCRAMMs. A second group of cell surface proteins includes nuclease and protein A. Staphylococcal nuclease is a thermally stable endonuclease able to withstand boiling for 30 min without significant loss of activity. Protein A is able to bind to the nonantigenic Fc fragment of immunoglobulin G, causing the complex to precipitate. Its role in virulence is believed to be in escape from immune surveillance. Regulatory Mechanisms The expression of extracellular proteins is largely under the influence of a master genetic circuit called agr

Bacteria | Staphylococcus

(accessory gene regulator). This signaling arm of the operon (AgrBDCA) is activated by a quorum-sensing mechanism that depends upon the accumulation of an activating octomeric peptide (processed from the AgrD precursor by AgrB). The peptide triggers increased expression of the entire operon via an integral signal transduction pathway (AgrC, AgrA), upregulating production of the octomer and activating a second promoter that produces an unique regulatory molecule, RNAIII. RNAIII is the effector molecule for regulated protein expression. It is neither translated nor does it bind to the promoter regions of regulated genes. It presumably interacts with other genes, in an unknown way, as both a positive and negative regulator of exoprotein gene expression. RNAIII is required for the expression of soluble exoproteins and represses the expression of cell surface proteins. Because a threshold level of octapeptide is required for activation, RNAIII is not expressed until late in growth. Therefore, cell surface proteins are produced early, presumably to allow the organism to attach and colonize. The RNAIII-induced activation of soluble proteins genes results in a necrotic effect, allowing the organism to invade deeper tissues and become bacteremic. A second locus, sarA, modulates expression of the agr locus by binding to the promoter region of AgrBDCA. The sarA locus is transcribed from three different promoters (sarP1, P2, and P3) that are active at different times during growth. The major regulatory molecule encoded by sarA is a 14.5 kDa protein that has been shown to bind to the promoter regions for fibronectin-binding protein A, the collagen adhesion, protein A, and agr. It is reported that the sarA locus plays a role in transcription of over 100 different gene targets either by direct binding of upstream promoter regions or indirectly due to its effects on other regulatory loci including agr. Several sar homologues have been identified including SarR, SarS, SarU, and SarY that play a role in regulation of SarA and other factors. With the continued efforts aimed at wholegenome sequencing of staphylococcal isolates, many other regulatory systems that are not discussed in detail here have been identified and characterized. These include Rot, SaeRS, SrrAB, ArlRS, and LytRS. Another important level of regulation of virulence is regulation of gene expression in response to environmental factors. It has been demonstrated that depending on the environment that the staphylococci encounter they can adapt by altering gene expression for a variety of systems. This is not unusual for bacteria and in fact the main environmental response system that has been described for S. aureus and S. epidermidis is the sigma B pathway. Sigma factors are bacterial proteins that enable specific binding of RNA polymerase to promoter regions

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within the DNA. The staphylococcal sigma B response is similar to that described for other Gram-positive pathogens such as B. subtilis. Sigma B is an alternative sigma factor that is activated under environmentally stressful conditions such as high salt levels, presence of ethanol, energy depletion, and low pH. Comprehensive studies in S. aureus have shown that sigma B regulates gene expression of some factors by directly binding to a specific site upstream of promoters and also by indirectly affecting upstream factors to gene expression. Some of the virulence factors that have been shown to be affected due to sigma B expression include coagulase, fibronectin-binding protein B, biofilm formation, and -toxin. A change in resistance levels to some antibiotics that affect the bacterial cell wall has also contributed to overexpression of sigma B. It is important to note that some strains of S. aureus have a mutation in the sigma B activator (RsbU) that renders them sigma B defective. These strains are also capable of growing under environmentally stressful conditions, which suggests that there are additional systems that have yet to be identified and that must play a role in virulence factor expression. See also: Adhesion, Microbial; Biofilms, Microbial; Chromosome, Bacterial; DNA Sequencing and Genomics

Further Reading Crossley KB and Archer GL (1997) The Staphylococci in Human Disease. New York: Churchill Livingstone, Inc. Feng Y, Chen CJ, Su LH, Hu S, Yu J, and Chiu CH (2008) Evolution and pathogenesis of Staphylococcus aureus: Lessons learned from genotyping and comparative genomics. FEMS Microbiology Reviews 32: 1–15. Heilmann C and Peters G (2006) Biology and pathogenecity of Staphylococcus epidermidis. In: Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, and Rood JI (eds.) Gram-positive Pathogens, 2nd edn., pp. 560–571. Washington, DC: ASM Press. Iandolo JJ (1989) Genetic analysis of extracellular toxins of Staphylococcus aureus. Annual Review of Microbiology 43: 375–402. Klevens RM, Morrison MA, Nadle J, et al. (2007) Invasive methicillinresistant Staphylococcus aureus infections in the United States. Journal of the American Medical Association 298: 1763–1771. Lee CY (2001) Capsule production. In: Honeyman AL, Friedman H, and Bendinelli M (eds.) Staphylococcus aureus Infection and Disease, pp. 35–48. New York: Kluwer Academic/Plenum Publishers. Leung DYM, Huber BT, and Schlievert PM (1997) Historical perspective of superantigens and their biological activities. In: Leung DYMB, Huber T, and Schlievert PM (eds.) Superantigens; Molecular Biology, Immunology and Relevance to Human Disease, pp. 1–14. New York: Marcel Dekker, Inc. Novick RP (2003) Autoinduction and signal transduction in the regulation of staphylococcal virulence. Molecular Microbiology 48: 1429–1449. Tenover FC and Gorwitz RJ (2006) The epidemiology of Staphylococcus infections, pp. 526–534. In: Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, and Rood JI (eds.) Gram-positive Pathogens, 2nd edn., pp. 381–412. Washington, DC: ASM Press.

Streptococcus Pneumoniae R Sa´-Lea˜o, Universidade de Lisboa, Lisboa, Portugal, Universidade Nova de Lisboa, Oeiras, Portugal A Tomasz, The Rockefeller University, New York, NY, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Guides to the S. pneumoniae Literature Pneumococcus as a Pathogen and as a Model Microbe for Molecular Biology Burden of Pneumococcal Disease The Natural Reservoir of S. pneumoniae Stages in Pneumococcal Pathogenesis: Virulence Factors and Host Defense Human Intervention

Abbreviations CbpA CibABC CSP DCC EUROPNEUMO IPD ISPPD

LytA

choline-binding protein A competence induced bacteriocin competent stimulating peptide day care center European Meeting on the Molecular Biology of the Pneumococcus invasive pneumococcal disease International Symposium on Pneumococci and Pneumococcal Disease autolysin A

Defining Statement The primary (if not only) natural habitat of Streptococcus pneumoniae on this planet is the nasopharynx of preschoolage children, and antibiotics and vaccines not only combat pneumococcal disease but also drive the evolution of drug-resistant and novel capsular types of this species. In this sense, humans are not only targets but also evolutionary partners of S. pneumoniae as well.

Impact of the Seven-Valent Pneumococcal Conjugate Vaccine on Pneumococcal Disease and Carriage Day Care Center Studies Genetic Exchange In Vivo Genome Sequencing Pneumococcal Cell Wall: Composition, Structure and Mechanisms of Replication during Cell Division PostScript Further Reading

MLST NanA NET Pal PBP PFGE Ply PMN PspA SrtA

multilocus sequence typing neuraminidase neutrophil extracellular trap pneumococcal bacteriophage lytic enzyme penicillin-binding protein pulsed-field gel electrophoresis pneumolysin polymorphonuclear leucocytes pneumococcal surface protein A sortase A

Biology of the Pneumococcus) and those interested in pneumococcal disease (ISPPD: International Symposium on Pneumococci and Pneumococcal Diseases). The two groups meet in alternate years at various worldwide locations. The last meeting of EUROPNEUMO was in April 2007 in Lisbon, Portugal – organized by Hermı´nia de Lencastre and Alexander Tomasz. The latest meeting of ISPPD was in June 2008 in Reykjavik, Iceland, organized by Ingileif Jonsdottir.

Guides to the S. pneumoniae Literature Useful guides to the rapidly expanding literature on various aspects of the microbiology and infectious diseases of Streptococcus pneumoniae may be found in books listed at the end of this article which cover contributions to the field in the early and the more recent era. Periodic updates on progress are also available through informal meetings of two groups of scientists: those interested primarily in pneumococcal molecular biology (EUROPNEUMO: European Meeting on the Molecular

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Pneumococcus as a Pathogen and as a Model Microbe for Molecular Biology Pneumococcus is altogether an amazing cell. Tiny in size, simple in structure, frail in make-up, it possesses physiological functions of great variety, performs biochemical feats of extraordinary intricacy and, attacking man, sets up a stormy disease so often fatal that it must be reckoned as one of the foremost causes of human death (Benjamin White, 1938 in: The Biology of Pneumococcus)

Bacteria | Streptococcus Pneumoniae

S. pneumoniae was first described in 1881 by Pasteur and Sternberg in independent observations. In the same decade, this Gram-positive pathogen with lancet-shaped cells that grow in most media in pairs or short chains of ‘diplococci’ was recognized as a major cause of infections that included pneumonia, meningitis, otitis media, and endocarditis. S. pneumoniae routine identification is done through the alpha hemolysis surrounding colonies obtained on blood agar, negative reaction with catalase, susceptibility to optochin, and solubility of the bacteria in bile salts. Most S. pneumoniae isolates are shielded by a polysaccharide capsule that hinders phagocytosis. At least 91 different capsules have been described, and serologic typing (serotyping) remains one of the most frequently used methods for the characterization of pneumococcal isolates. Since its discovery, this bacterium has been the subject of intensive studies as a cause of major and often lifethreatening human infections. While the primary aim of these studies was the control of pneumococcal disease, the same efforts have also lead to seminal scientific discoveries in the laboratory which included the identification of the pneumococcal polysaccharide antigens as vaccines, the ability of capsular polysaccharides to induce antibodies, the discovery of bacterial gene transfer which led to the identification of the ‘transforming principle’ (later named DNA) as the genetic material. Efforts to degrade the capsular polysaccharide surrounding the pneumococcus have led to the first use of an ‘enrichment culture’. The remarkable and rapid dissolution of pneumococci by bile led to the identification of the first bacterial autolytic enzyme. Pneumococcus was among the first pathogens in which the therapeutic efficacy of the newly discovered penicillin was tested. The role of the polysaccharide capsule providing resistance against phagocytosis was identified, and studies on the rapid fluctuations in the pneumococcal capacity to take up DNA from the medium and undergo genetic change has led to the identification of the first bacterial quorum sensing factor. Thus, efforts to understand and control pneumococcal disease went hand in hand with some of the fundamental discoveries of molecular biology. Interestingly, many of these phenomena first discovered in the in vitro world of the microbiology laboratory were subsequently identified as major factors driving the evolution of new pneumococcal lineages in the real life world of pneumococcal colonization, infection, and disease. The most promising – and ambitious – current efforts to understand the impact of antibiotics or vaccines on the pneumococcal colonization, infection, and disease in humans are directed toward combining carefully designed epidemiological studies with the characterization of pneumococcal isolates by high resolution molecular techniques developed in the molecular biology laboratory.

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Burden of Pneumococcal Disease In the preantibiotic era, pneumococcal pneumonia was so common and fatal that it was termed as the ‘‘old man’s friend’’ and the ‘‘captain of the men of death’’ by William Osler. In the late 1990s, before the introduction of the first pneumococcal seven-valent conjugate vaccine in the United States, data from the Centers for Disease Control and Prevention estimated the annual frequency of pneumococcal infections as 3000 cases of meningitis, 50 000 cases of bacteremia, 500 000 cases of pneumonia, and 7 million cases of otitis media and an estimated mortality of about 40 000 deaths per year. While no similar dependable estimates are available from the less-developed countries of the world, evaluation of the impact of a nine-valent conjugate vaccine in one randomized control trial conducted in Africa clearly indicated that pneumococcal disease is a major contributor to the mortality of children in African countries. In Gambia, 77% (95% CI, 51–90%) efficacy against vaccine-type invasive pneumococcal disease (IPD) was found with a 50% (95% CI, 21–69%) efficacy against all types of IPD. Furthermore, a 16% (95% CI, 3–28%) decrease in all-cause mortality was found among vaccinated children. In South Africa, the efficacy of the ninevalent pneumococcal conjugate vaccine against IPD among HIV-negative children was 83% (95% CI, 39–97%) and corresponding figures were 65% (95%CI, 24–86%) among HIV-positive children. A report in 2007 from the WHO estimated that 1.6 million people continue to die every year due to pneumococcal disease including 0.7–1 million children aged less than 5 years, the majority living in developing countries, where the pneumococcal conjugate vaccine is not available. The burden of disease associated with the elderly in these countries remains to be defined. In addition to young children and the elderly, individuals of all ages infected with HIV are at a substantial higher risk of serious pneumococcal infection. For example, the risk of pneumococcal pneumonia is 25-fold higher among HIV-infected people compared to HIV-uninfected people. The incidence of pneumococcal invasive disease among HIV-positive children is 9–43 times higher than among HIV-negative children and the rates of IPD among HIV-positive adults are 6–343 times higher than among HIV-negative adults.

Pneumococcal Infection and Viral Disease In the era of preparedness for an anticipated new influenza or bird flu pandemic, the well-documented contribution of pneumococci in the mortality associated with flu becomes increasingly important.

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Bacteria | Streptococcus Pneumoniae

Several lines of evidence have highlighted that secondary infections by pneumococci in patients with viral respiratory disease can have devastating consequences. Studies conducted during the 1918 influenza epidemic, which is estimated to have led to at least 40–50 million deaths, demonstrated that an important fraction of the deaths took place 2 weeks after the onset of influenza symptoms, suggesting that superinfection by a common bacterial respiratory pathogen had occurred. Direct evidence supporting the role of pneumococcal secondary infection leading to fatal pneumonia has been described. In a double-blind, randomized, placebo-controlled trial of a nine-valent pneumococcal conjugate vaccine in South Africa, it was found that the vaccine prevented 31% of virus-associated pneumonia in hospitalized children, suggesting that an important fraction of virusassociated pneumonia among hospitalized children was attributable to bacterial coinfection that could be prevented by bacterial vaccines. Experiments on modeling viral–bacterial infection in animals showed that if a mouse model was challenged with a nonlethal dose of influenza virus and approximately 7 days later was challenged with pneumococcus, 100% mortality occurred. This effect was specific to viral infection preceding bacterial infection. Together these data strongly suggest that pneumococcal vaccination could have a beneficial role in preventing influenza-associated mortality in the advent of a new influenza pandemic.

The Natural Reservoir of S. pneumoniae Humans are not only the target of diseases caused by S. pneumoniae but are also the primary ecological reservoir of this bacterial pathogen – although two anecdotic studies have found carriage of pneumococci by horses and isolation from wild chimpanzees. Thus, interventions to combat pneumococcal disease such as the introduction of antibiotics or vaccines also impact on the human nasopharyngeal flora of pneumococci selecting for drugresistant lineages and strains with less common capsular types. In this sense, humans are also evolutionary partners of this microbe and many – if not all – of the genetic events that allow these bacteria to borrow pieces of foreign DNA to remodel their penicillin-sensitive enzymes, acquire mobile elements that confer antimicrobial resistance to different classes of antibiotics, or undergo capsular switches to evade the action of vaccines targeting the capsule most likely occur in pneumococcal populations that inhabit the human nasopharynx, more specifically, the nasopharynx of preschool-age children. The latter, for reasons not fully understood, are the primary carriers of this bacterial species. For these reasons,

the day care centers (DCCs) in which preschool-age children are now recruited in many of the countries of the developed world have become major foci of epidemiological studies of pneumococci. Indeed, several studies have shown that children of preschool age are the major reservoir of pneumococci and by school time a spontaneous decrease in carriage occurs. The mechanism of extensive colonization in infancy and the loss of carriage with age are not well understood. It is also well established that in infancy the most dominant capsular types colonizing the nasopharynx are typically of the serogroup 6, 9, 14, 19, and 23 in countries across the world. This commonality of serotypes colonizing the young host contrasts with the welldocumented and sometimes extensive differences in the serotypes of pneumococci that most frequently cause invasive disease in various parts of the world. The mechanism of geographic variation in serotype abundance and their change in time is not known. An important source of problems contributing to the difficulties of interpretation of many aspects of pneumococcal epidemiology is the way pneumococcal colonization has been routinely assayed in the overwhelming majority of the studies conducted so far. In most studies, a single colony recovered on blood agar plates from the nasopharyngeal swabs is assumed to represent the entire colonizing flora. However, simultaneous carriage of multiple strains of pneumococci in the nasopharynx has been known for several decades and was documented in early studies conducted in the 1930s and 1940s, which used mouse inoculation assays to detect the strains. As these methods were very labor-intensive and expensive they have been abandoned. More recent studies in which a number of colonies were picked from the primary blood agar plates and were characterized clearly showed that the nasopharyngeal flora is heterogeneous: it may consist of more than one strain of pneumococci; some representing the majority, others – often present with lower frequencies – may be of completely different serotypes and molecular type (Table 1). Certain pneumococcal serotypes such as serotypes 1 and 5 that can be recovered from disease sites but have seldom been seen in the nasopharynx may represent such minority residents in the nasopharyngeal flora. The same minority clones may be the source of the novel pneumococci emerging after the introduction of the conjugate pneumococcal vaccine. Multiple pneumococcal carriage is apparently more abundant among populations with high pneumococcal carriage rates such as children from Papua New Guinea, Gambia, or Australian Aborigines. Multiple carriage rates in the range of 20–30% have been reported among these populations. Among other children, typical rates of multiple carriage have been in the range of 5–10%.

Bacteria | Streptococcus Pneumoniae

307

Table 1 Properties of multiple isolates obtained from nasopharyngeal samples containing two strains of S. pneumoniae

Sample

Isolate code

A A A A A A A B B B B C C C C C C C D D D D D D

106 106-1 106-2 106-3 106-4 106-5 106-6 325 325-1 325-2 325-3 448 448-1 448-2 448-3 448-4 448-5 448-6 541 541-1 541-2 541-3 541-4 541-5

Serotype

MIC (mg ml 1) to penicillin

Antibiotype (resistant to)

PFGE type

PFGE-lytA (kb)

Addition mitomycin C

Phage DNA

comC allele

11 6B 11 11 11 11 6B 19F NT NT NT 19A 19A 23F 19A 19A 19A 19A 6B 19A 19A 6B 6B 6B

0.016 0.023 0.006 0.008 0.008 0.008 0.016 0.047 0.5 0.75 1 0.094 0.064 2 0.094 0.094 0.094 0.064 0.023 0.023 0.023 0.016 0.012 0.008

E, Cc E, Cc, Te E, Cc E, Cc E, Cc E, Cc E, Cc, Te – – – – SXT SXT C, Te, SXT SXT SXT SXT SXT Te, SXT SXT SXT Te, SXT Te, SXT Te, SXT

SSS M SSS SSS SSS SSS M H TTT TTT TTT D D A D D D D M UUU UUU M M M

85 280, 110, 90 85 85 85 85 280, 110, 90 90, 35 170, 90, 40, 30 170, 90, 40, 30 170, 90, 40, 30 235, 90, 80 235, 90, 80 230, 100, 40 235, 90, 80 235, 90, 80 235, 90, 80 235, 90, 80 280, 90, 60 90 90 280, 90, 60 280, 90, 60 280, 90, 60

No lysis Lysis

Yes

comC1 comC1

No lysis ND

comC2.1 comC2.1

Lysis

Yes

comC1

Lysis

Yes

comC2.2

Lysis No lysis

Yes

comC1 comC1

C, chloramphenicol; E, erythromycin; Cc, clindamycin; Te, tetracycline; SXT, sulfamethoxazole-trimethoprim. ª American Society for Microbiology. Reproduced from Sa´-Lea˜o R, Tomasz A, Santos Sanches I, and de Lencastre H (2002) Pilot study of the genetic diversity of the pneumococcal nasopharyngeal flora among children attending day care centers. Journal of Clinical Microbiology 40: 3577–3585.

Stages in Pneumococcal Pathogenesis: Virulence Factors and Host Defense Virulence in pneumococci is multifactorial and involves complex and multiple interactions with the host. Well over 300 genes have been implicated in virulence in at least one animal model of pneumococcal infection. A genome-wide screen – by signature-tagged mutagenesis – of the reference strain TIGR4 for genes essential for lung infection in an animal model identified close to 400 genes. Individual putative virulence factors appear to play a variety of roles – both ‘aggressive’ as well as ‘defensive’ at various stages of pneumococcal infections. Some of the pneumococcal virulence factors, such as the capsular polysaccharide, the major autolysin LytA, the intracellular toxin pneumolysin (Ply), and the sortase A (SrtA), are present in virtually all pneumococcal isolates. Other important virulence determinants, such as the recently described pilus operon, also play important roles in virulence but these determinants seem to be associated with specific clones of pneumococci. Secreted DNAase appears to have a role in escaping the neutrophil extracellular traps (NETs) and pneumococcal resistance to the widely spread cationic antimicrobial peptides appears to involve incorporation

of D-alanine esters into the pneumococcal cell wall teichoic acids. Host factors ‘matching’ in numbers the virulence genes of the pneumococcus have also been identified using a variety of models. These factors cover a wide range of functions: pattern recognition elements that are components of the innate immune system as well as host defense factors more specific for the invading pneumococcus such as the scavenger receptor involved with lung defense or the generation of adaptive immunity directed primarily against the capsular polysaccharide.

Capsular Polysaccharides While rare S. pneumoniae isolates free of the capsule do exist the overwhelming majority of clinical strains express one capsular polysaccharide attached to the pneumococcal cell wall. Nonencapsulated strains, for instance, strains R6 or R36A, which are most frequently used in laboratory studies on S. pneumoniae, are completely avirulent in animal models of disease. The role of the capsular polysaccharide in pneumococcal virulence appears to be the inhibition of complement-mediated opsonophagocytosis by macrophages or polymorphonuclear leucocytes (PMN). Except

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Bacteria | Streptococcus Pneumoniae

for the capsular polysaccharide types 3 and 37, all the other polysaccharide capsules appear to be covalently linked to the pneumococcal cell wall. Pneumococci have an enormous genetic repertoire to produce – potentially – as many as 91 chemically different polysaccharide chains. A unique feature of the structure of capsular loci is that typespecific genes are flanked by common determinants, thus allowing for a relatively easy exchange of genetic determinants within this region of the pneumococcal chromosome. The frequent ‘capsular switch’ that can appear spontaneously or is driven by vaccine pressure among clinical isolates appears to be the consequence of a relatively easy genetic change at the capsular loci. In such cases, isolates that share common genetic backgrounds (defined by multilocus sequence typing (MLST) or pulsed-field gel electrophoresis (PFGE) profile) appear to differ only in the type of capsule at their surface. Cell Walls in Virulence The unique choline component of the wall and membrane teichoic acid appears to perform multiple roles both in the physiology of the pneumococcus and also as an interactive component with the host. The species of S. pneumoniae is unique in that it requires choline as an essential nutrient for growth. Recently, pneumococcal strains in which this auxotrophic requirement for choline is lost have been isolated. In one of these isolates point mutations in one of the choline utilization genes – tacF – have been identified as the mechanism responsible for the cholineindependent growth. TacF, a teichoic acid flippase, was proposed to catalyze the transfer of teichoic acid chains across the pneumococcal plasma membrane. A second choline-independent strain was recovered from a heterologous genetic cross in which Streptococcus oralis, a bacterial species that contains choline in its teichoic acid but does not require choline for growth, served as the DNA donor and the recipient was the R6 strain of S. pneumoniae. The mechanism of choline independence in this strain called R6Cho appears to be different from the mechanism identified in the other choline-independent mutants. Recent studies have shown that pneumococcal constructs capable of growing without choline and expressing the capsular polysaccharide type 2 on their surface have severely reduced virulence potential in several animal models of pneumococcal disease and are also inhibited from colonizing the nasal epithelium of mice. The mechanism of this striking impact of the teichoic acid choline units on pneumococcal virulence is not understood to date. Pneumococcal cell wall components were shown to induce the production of preinflammatory cytokines both in the murine intraperitoneal models and in the rat and rabbit models of meningeal disease. Structural features of the peptidoglycan involved with the recognition

by the innate immunity system have been identified in the Drosophila model. At least two pneumococcal cell wallmodifying enzymes: PGDA, a peptidoglycan glucosamine deacetylase and PCE, a phosphoryl choline esterase, were shown to play roles in virulence as indicated by the impact of inactivation of the corresponding genetic determinant on virulence. Regulation of Virulence The expression of capsular polysaccharides appears to be regulated: contact with epithelial cells was shown to cause suppression in the amounts of capsule, which can subsequently increase once the bacteria have reached the blood stream. Different modalities of growth. Recent studies have shown that pneumococci can grow in two different modalities: as planktonic cells typical of bacteremic infections and as biofilms typical of meningitis. Different sets of genetic determinants matching these two different growth styles of pneumococci are expressed. Opaque versus transparent phenotype. In 1994, Weiser and colleagues described that pneumococci could undergo phase variation, that is, changes in properties of the cell surface that could lead to two different colony phenotypes: opaque and transparent. Spontaneous, reversible variation between the two colony phenotypes with a frequency ranging from 10 3 to 10 6 per generation has been described. This frequency appears to be independent of in vitro growth conditions. The same authors showed that the opaque phenotype was associated with increased amounts of capsular polysaccharide and pneumococcal surface protein A (PspA), whereas transparent variants were associated with increased amounts of choline-binding protein A (CbpA) and autolysin A (LytA). Opaque colonies were described as dome shaped and large contrasting with transparent colonies, which were umbilicated, suffered quicker autolysis, and had a higher efficiency of natural transformation. Cultures obtained from opaque colonies had decreased binding to serum C-reactive protein, decreased opsonophagocytosis, and increased virulence in systemic infections. By contrast, transparent colonies had increased adherence, and colonized more efficiently in an animal model of carriage. More recently, DNA microarray strategies have identified an additional number of genes that appear to have differential regulation between isogenic pairs of strains displaying opaque or transparent phenotypes. In particular, the transparent phenotype was associated with increased production of neuraminidase (NanA). Studies on the fatty acid composition of pairs of opaque and transparent colonies found a lower degree of unsaturated fatty acids in the opaque variants. A single large study has attempted to examine the prediction that opaque variants are associated with

Bacteria | Streptococcus Pneumoniae

increased pathogenesis by looking at the colony phenotypes displayed by a large collection of invasive disease isolates with a low number of in vitro passages. This collection of 304 isolates included representatives of ten serotypes displaying genetically diverse backgrounds. The authors confirmed that the opaque phenotype dominated among invasive disease isolates but also noted a previously unreported association between serotype and colony phenotype. This observation led them to suggest that the association between the opaque phenotype with particular serotypes might contribute in part to explain the observed differences in the invasive disease potential of certain serotypes. Contribution of Genotype and Serotype to the ‘Invasive Disease Potential’ The introduction of molecular typing techniques for the characterization of S. pneumoniae recovered from disease and from colonization sites combined with extensive epidemiological studies has initiated efforts to better define the contribution of serotype versus molecular type to the ‘invasive’ potential of a pneumococcal strain. The frequent representation of the so-called pediatric serotypes (primarily 6B, 14, 19F, and 23F) in the nasopharyngeal flora of children clearly provides an increased opportunity for strains expressing these serotypes to invade during periods of decreased host defense. Thus, while assigning a true ‘invasive disease potential’ to a strain, the odds ratios expressing the frequency of recovering the particular strain from colonization versus infection sites must be taken into account. The relative contribution of serotype versus genotype to the invasive disease potential is a currently unsettled issue. Although it is relatively consensual that the capsular type expressed is extremely important for the disease potential of a particular strain, there are studies suggesting that the genetic background of that strain is also important, which, after all, is in agreement with the finding of several genetic determinants essential for full virulence of pneumococci. A further potential problem contributing to the difficulties of interpretation of these empirical estimates of invasive disease potential of serotypes and clones of pneumocooci is the way pneumococcal colonization has been traditionally studied ignoring multiple colonization (discussed above). The relative contribution of the host versus the pathogen to the occurrence of pneumoccal infection is debatable but clearly the host plays a very important role. Recent studies have started to identify genetic polymorphisms in the determinants implicated in the host immune response including some that are associated with susceptibility to IPD and others that confer a protective effect to it.

309

Human Intervention There have been at least three important interventions by humans that radically altered the in vivo landscape and epidemiology of S. pneumoniae: (1) the introduction of antibiotics into therapeutic practice, (2) the introduction of the conjugate antipneumococcal vaccine, and (3) the proliferation of DCCs in the developed part of the world. The introduction of antibiotics did not change the incidence of pneumococcal infections; it contributed significantly to the control and outcome of pneumococcal disease but has also led to the emergence of antibioticresistant strains. The introduction of a conjugate antipneumococcal vaccine in the United States had major impact on the incidence of IPD. However, the rate of pneumococcal carriage did not decrease and the frequency of antibiotic-resistant strains among colonizing pneumococci has initially decreased but is now increasing due to the expansion of pneumococcal nonvaccine capsular serotypes that are also resistant to antibiotics. The institution of day care has emerged as a unique epidemiological entity in which many children of preschool age are cohorted. The high carriage rate of respiratory pathogens, immunological status, and typical child behavior, together with the frequent occurrence of viral respiratory diseases and the extensive and often imprudent use of antimicrobial agents among this age group, are the most likely reasons why attendance at DCCs has become a risk factor for both carriage and infection by antibiotic-resistant strains of pneumococci.

Antibiotic Resistance and Insights Provided by Molecular Typing The first penicillin-resistant strain of S. pneumoniae that appeared in the clinical environment and invoked comments in the infectious diseases literature was the strain isolated from the throat of a healthy child in the mid 1960s in a remote village, Agunganak in Papua New Guinea. Although initially the possibility of geographic spread of the penicillin-resistant pneumococcus was judged remote, this prediction was soon contradicted by the massive outbreak of pneumococcal disease in South African hospitals in 1977, which was caused by multidrug-resistant strains of this bacterium. Between the early 1980s and the late 1990s reports on the detection and increase both in frequency and in antibiotic resistance level of drug-resistant strains of S. pneumoniae have appeared in increasing numbers and the antibioticresistant pneumococcus has become a global phenomenon that began to make effective chemotherapy sometimes problematic.

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Bacteria | Streptococcus Pneumoniae

Global Spread of a Few Pandemic Clones of Penicillin-Resistant Pneumococci Introduction of pneumococcal typing techniques such as PFGE or MLST has clearly shown that among the very large number of lineages of resistant pneumococci a handful of highly epidemic clones emerged and achieved massive and often pandemic spread. The most outstanding of these is Spain23F ST81 originally called the ‘Spanish/USA clone’ usually expressing serotype 23F and carrying resistance to penicillin, tetracycline, and chloramphenicol and often to erythromycin and sulfamethoxazole–trimethoprim. An apparent intercontinental transfer of this clone from Southern Europe to the United States was demonstrated. This clone was subsequently identified in numerous national and international surveillance studies, both as a powerful colonizer and also as a strain capable of causing the entire spectrum of pneumococcal diseases among both adults and children. Similar importation of a multidrug-resistant pneumococcal clone, the penicillin-resistant clone Spain6B ST90 presumably from Southern Europe to Iceland was demonstrated in the early 1990s. This so-called ‘Icelandic’ clone expressing serotype 6B and resistance to penicillin, tetracycline, chloramphenicol, erythromycin, sulfamethoxazole–trimethoprim, and occasionally to third-generation cephalosporins as well, quickly spread in Iceland and within 3 years of its detection was shown to be responsible for close to 20% of all pneumococcal disease in that country. A third genetic lineage France9V ST156 originally referred to as the ‘French/Spanish’ clone carries resistance to penicillin and tetracycline and occasionally to sulfamethoxazole–trimethoprim and typically exists in two capsular serotypes: 9V or 14. This clone was shown to have spread in Europe, Latin America, USA, Canada, and Asian countries (reviewed at the Pneumococcal Molecular Epidemiology Network (PMEN), website available at www.sph.emory.edu/PMEN/). The introduction and widespread use of molecular typing techniques has led to the establishment of an international depository and ‘clearing’ house for characterized S. pneumoniae clones, the so-called PMEN. This platform has also become useful in providing a uniform set of rules to name new clonal lineages, register both their molecular and epidemiological characteristics, serotypes, isolation dates and sites and clinical sources as well (for more information visit www.sph.emory.edu/ PMEN/). Despite the fact that penicillin resistant strains have been isolated from countries all over the world, their incidence varies widely from country to country and from one geographic site to another. The impact of antibiotic resistance on chemotherapy varies with the particular infection. Even low-level

resistance to antibiotics requires change in chemotherapy in meningitis because of the low penetration of the cerebral spinal fluid by this class of antibiotics and because of the need for bactericidal concentrations. On the other hand, penicillin therapy was shown to remain effective in pneumococcal pneumonia caused by penicillin-resistant strains with MIC values as high as 1–2 mg ml 1.

Penicillin Resistance: Genes and Phenotypes The mechanism of penicillin resistance in pneumococcal clinical isolates is based on the remodeling of several of the genetic determinants – pbp genes that encode for proteins (penicillin-binding proteins, PBPs) that catalyze various stages in the pneumococcal cell wall synthesis. The process of remodeling involves recombinational events with fragments of pbp genes imported from heterologous species most often from Streptococcus mitis. These ‘mosaic’ pbp genes produce PBPs with decreased affinity for penicillin, which is the ultimate basis of the increased penicillin MIC values. Examination of the cell wall chemical structure in penicillin-resistant clinical isolates showed additional profound abnormalities in these bacteria, specifically the increased representation of branched muropeptide components in their cell wall peptidoglycan. A genetic followup of these studies identified the new determinants, murM and murN, responsible for the biosynthesis and attachment of the two amino acid components that form the muropeptide branches. The murM and murN genes of resistant strains showed clear evidence of mosaicism, indicating the presence of DNA sequences of heterologous origin. Most interestingly, inactivation of murM caused a complete loss of penicillin resistance, which could be recovered in appropriate complementation experiments. A detailed biochemical mechanism of the synthesis of pneumococcal muropeptides and a mode of action for MurM and MurN proteins was recently described. The critical nature of cell wall chemistry for the penicillin-resistant phenotype was further documented by the recent identification of yet another cell wall-modifying enzyme: a muramic acid O-acetylase. Inactivation of the structural gene named adr caused loss of penicillin MIC value, similar to the case of strains in which MurM was inactivated. It seems that genetic determinants in addition to the mosaic pbp genes producing the low-affinity penicillin targets are also essential for optimizing the resistant phenotype. This scenario is reminiscent of the mechanism identified in methicillin-resistant Staphylococcus aureus, in which high-level resistance requires not only the central resistance determinant mecA, but a number of additional ‘auxiliary determinants’ as well in the genetic background of the bacteria.

Bacteria | Streptococcus Pneumoniae

Genetic Diversity among Penicillin-Resistant and Penicillin-Susceptible Pneumococci Causing Invasive Disease Several studies have compared the genotypes of penicillin-resistant (MIC higher than 1 mg ml 1) versus penicillin-susceptible isolates of pneumococci expressing the same serotypes and recovered from invasive disease. Studies with strains isolated from children in South American countries are illustrative of the common findings of such studies. The major and striking difference among the resistant and susceptible isolates was the relative genetic homogeneity of most resistant isolates, more than 80% of which were shown to belong to one of two pandemic penicillin-resistant clones: Spain23F ST81 and/ or France9V/14 ST156 (Figure 1). In contrast, penicillin2.9% (Clone D from Chile)

16.3% (20 different PFGE types)

Type 14 Type 23F

55.2% (Clone B) SP-3

(25.6% (Clone A) SP-1

Figure 1 Pie chart showing the isolation percentages in Latin America of two penicillin-resistant pandemic clones versus all other clones from among 172 penicillin-resistant invasive isolates. Note that the two clone types A and B are responsible for 81% of all pneumococcal infections. Clone A (serotype 23F) refers to the Spain23F ST81 clone and clone B refers to the France9V ST156 clone. Tomasz A, Corso A, Severina EP, et al. (1998) Molecular epidemiologic characterization of penicillin-resistant Streptococcus pneumoniae invasive pediatric isolates recovered in six Latin-American countries – an overview. Microbial Drug Resistance 4: 195–207.

Type 14

311

susceptible isolates expressing the same serotypes and recovered from similar disease sites showed great genetic diversity (Figure 2). A similar – inverse – relationship between genetic diversity and penicillin MIC value has already been described in other epidemiological studies. These observations may reflect the expensive ‘fitness’ cost associated with the mechanism of pneumococcal penicillin resistance, which may be compatible only with a limited number of genetic backgrounds available in this bacterial species.

Impact of the Seven-Valent Pneumococcal Conjugate Vaccine on Pneumococcal Disease and Carriage In 2000, a seven-valent pneumococcal conjugate vaccine (PCV7 targeting serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F) was licensed in the United States and soon after in many other countries worldwide. The vaccine was intended for children younger than 2 years of age. This vaccine was formulated to target the serotypes that cover over 80% of the cases of IPD among children younger than 5 years of age in the United States. Expected coverage rates in other countries, particularly in developing countries, are lower. Following introduction of PCV7 in the United States, a reduction in the incidence of IPD occurred not only among young children but also in all other age groups due to a substantial indirect herd effect. In particular, a sharp decrease in IPD caused by vaccine types was observed, which was accompanied by a modest increase in IPD caused by nonvaccine types. By 2003 the total incidence of IPD among children 100 mm s1) faster than cells with peritrichous flagella (60  C) H. acidophilum OA S. acidocaldarius OH S. solfataricus OH S. metallicus OA S. tokodaii OH

    

þ   þ þ

    

Carbon assimilation (a) Mesophiles (temperature optima 20–40  C) At. ferrooxidans OA L. ferrooxidans OA Fm. acidiphilum OH At. thiooxidans OA Thiomonas spp. FA Acidiphilium spp. OH A. acidophilum FA Acidocella spp. OH Acidobacterium spp. OH Fp. acidiphilum OH

   þ þ

(Continued )

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Environmental Microbiology and Ecology | Extremophiles: Acidic Environments

Table 2 (Continued)

Metallosphaera spp. Sulfurococcus spp. A. infernus Ac. ambivalens Ac. brierleyi Sg. azoricus Ss. ohwakuensis

Carbon assimilation

Fe2þ oxidation

Fe3þ reduction

S0 oxidation

S0 reduction

FA FA OA OA FA OA FA

    þ  

      

þ þ þ þ þ  

  þ þ þ þ þ

a

Alicyclobacillus spp. include species that are facultatively autotrophic and obligately heterotrophic, and vary in terms of their dissimilatory transformations of iron and sulfur. Note: OA, obligate autotroph; FA, facultative autotroph; OH, obligate heterotroph. Genera abbreviations: At., Acidithiobacillus; L., Leptospirillum; Fm., Ferrimicrobium; A., Acidiphilum; Sb., Sulfobacillus; Fp., Ferroplasma; Am., Acidimicrobium; Fx., Ferrithrix; Acd., Acidicaldus; H., Hydrogenobaculum; S., Sulfolobus; Ac., Acidianus; Sg., Stygiolobus; Ss., Sulfurisphaera.

thermophiles, with temperature optima of 40–60  C; and (3) extreme thermophiles, with temperature optima of 60–80  C. While some acidophiles (strains of At. ferrooxidans and Acidiphilium) have been demonstrated to be active at very low (100  C) abyssal environments around submarine vents is maintained at close to neutral by the strong buffering capacity of seawater, precluding extensive colonization by acidophiles. As with neutrophilic prokaryotes, extremely thermophilic acidophiles are mostly Archaea while mesophiles are predominantly Bacteria. The majority of moderate thermoacidophiles are also Bacteria, and mostly Grampositives, while most known Gram-negative acidophilic bacteria grow best at below 40  C. There are exceptions to this general trend. Indeed, the most thermophilic acidophilic bacteria known – the sulfur-oxidizing autotroph Hydrogenobaculum acidophilum, which grows at up to 70  C, and the heterotroph Acidicaldus organivorans, which grows at up to 65  C – are both Gram-negative. The ability to tolerate elevated concentrations of protons (strictly speaking, hydronium ions; H3Oþ) is obviously what defines an acidophile. While there is no official cutoff pH value that delineates whether an organism is or is not an acidophile, the generally accepted view

is that, as a group, these can be divided into extreme acidophiles that have pH optima for growth at pH < 3, and moderate acidophiles that have pH optima of between 3 and 5. As can be anticipated, the most extremely acidic environments have less potential biodiversity than those that are moderately acidic. The number of prokaryotes that are known to grow at pH < 1 is relatively small and includes some Gram-positive bacteria (e.g., Sulfobacillus spp.), Gram-negative bacteria (e.g., Leptospirillum spp. and At. thiooxidans), and Archaea (e.g., Ferroplasma spp.) that oxidize iron and/or sulfur. The most acidophilic of all currently known life-forms is, however, a heterotrophic archaeon, Picrophilus. Two species are known, Picrophilus oshimae and Picrophilus torridus, both of which have optima pH for growth of 0.7, and grow in synthetic media poised at pH  0. These ‘hyperacidophiles’ are also thermophilic, with optimum temperatures for growth at 60  C.

Physiological Versatility in Acidophilic Prokaryotes: Specialized and Generalist Microorganisms Acidophiles as a group are highly versatile and are able to utilize a wide variety of energy sources (solar and inorganic and organic chemicals), grow in the presence or complete absence of oxygen, and at temperatures of between 4 and 96  C. However, individual species display very different degrees of metabolic versatility. On the one end of this spectrum are members of the genus Leptospirillum. Three species are known: Leptospirillum ferrooxidans, Leptospirillum ferriphilum, and Leptospirillum ferrodiazotrophum. All grow as highly motile curved rods and spirilli, and species and strains vary in temperature and pH characteristics. All three species, however, appear to use only one energy source – ferrous iron. Because of the high redox potential of the ferrous/ferric couple (see ‘Aerobic and anaerobic acidophiles’), these Bacteria, by necessity, have to use molecular oxygen as

Environmental Microbiology and Ecology | Extremophiles: Acidic Environments

an electron acceptor, restricting them to being active only in aerobic environments. All three species fix carbon dioxide (but not organic carbon) and two of the three (L. ferrooxidans and L. ferrodiazotrophum) are also able to fix molecular nitrogen. Leptospirillum spp. are, therefore, highly specialized acidophiles. Their metabolic limitations appear, however, to be compensated by their abilities to outcompete other iron-oxidizing bacteria in many natural and anthropogenic environments, such as stirred tank bioreactors used to bioleach or biooxidize sulfide ores. This is achieved, at least in part, by their greater affinities for ferrous iron and greater tolerance of ferric iron than most other iron oxidizers. At. ferrooxidans is, in contrast, a more generalist bacterium. Initially it was described as an obligate aerobe that obtains energy by oxidizing ferrous iron, elemental sulfur, sulfide, and RISCs, and fixes CO2 as its sole source of carbon. The first hint of a more extensive metabolic potential was in a report by Thomas Brock and John Gustafson in 1976 who showed that the bacterium could couple the oxidation of elemental sulfur to the reduction of ferric iron, though it was not confirmed at the time whether this could support growth of the acidophile in the absence of oxygen, though the free energy of the reaction (
G ¼ –314 kJ mol–1; eqn [6]) suggested that this might be the case. S þ 6Fe3þ þ 4H2 O ! HSO4 – þ 6Fe2þ þ 7Hþ

ð6Þ

Later, Jack Pronk and colleagues at Delft University showed conclusively that At. ferrooxidans is, indeed, a facultative anaerobe and can grow anaerobically by ferric iron respiration using not only sulfur as electron donor, but also formic acid (which can also be used as sole energy source under aerobic conditions). The finding that this acidophile can use formic acid, although somewhat unexpected, does not imply that it is capable of heterotrophic as well as autotrophic growth, as C1 compounds, such as formate and methanol, are also used by other autotrophic prokaryotes. About the same time, it was discovered that some strains of At. ferrooxidans (including the type strain) can use hydrogen as an energy source, but that bacteria cultivated on hydrogen are less acidophilic than when grown on sulfide ores. It was shown later that hydrogen oxidation could also be coupled to ferric iron reduction by some At. ferrooxidans isolates. The most generalist of all acidophiles are, however, Sulfobacillus spp. These Gram-positive bacteria can grow as chemolithotrophs, heterotrophs, or mixotrophs in aerobic or anaerobic environments. Although there are no reports of Sulfobacillus spp. using hydrogen, they can (unlike At. ferrooxidans) use a variety of organic compounds (such as glucose and glycerol) as carbon and energy sources, though their capacities for heterotrophic

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growth are more limited than Alicyclobacillus spp. (related acidophilic Firmicutes, some of which can also oxidize ferrous iron and sulfur). Acidophilic Eukaryotic Microorganisms Extremely acidophilic organisms are exclusively microbial. While some angiosperms, such as Juncus bulbosus and Eriophorum angustifolium, can grow in highly acidic (pH < 3) ponds and lakes, their root systems grow in sediments where the pH is usually significantly higher than the water body itself. Many eukaryotic microorganisms that may be found in extremely low pH environments are acid-tolerant rather than truly acidophilic and may grow equally well, or better, in higher pH waters. All known phototrophic acidophiles are eukaryotic, and both mesophilic and moderately thermophilic species are known. Some photosynthetic acidophiles are also capable of heterotrophic growth in the absence of light, provided that a suitable carbon source is available. Microalgae that can live in highly acidic environments include genera of Chlorophyta, such as Chlamydomonas acidophila and Dunaliella acidophila; Chrysophyta, such as Ochromonas sp.; and Euglenophyta, such as Euglena mutabilis (Figure 4). Some diatoms, including several Eunotia spp., have also been found to colonize extremely acidic waters. A filamentous alga, identified from its morphology as Zygnema and confirmed from biomolecular analysis to be Zygnema circumcarinatum, has been found in abundance on surface streamer growths in an extremely acidic (pH  2.7) metal-rich stream draining a mine adit in southwest Spain. Four species of thermoacidophilic Rhodophyta have been described. Of these, Galderia spp. (Galderia sulfuraria and Galderia maxima) can grow as heterotrophs, while Cyanidioschyzon merolae and the original strain of C. caldarium are strict autotrophs. One C. caldarium-like isolate has been reported to grow in synthetic media poised as low as pH 0.2. Chlorella-like microalgae have also been detected in acidic geothermal waters. Many species of yeasts and fungi can tolerate moderate or even extreme acidity. Truly acidophilic fungi are, however, less common, though these include some remarkable species, such as Acontium velatum and Scytalidium acidophilum, both of which are copper-tolerant mitosporic fungi that can grow at pH values of below 0.5. Among the most commonly encountered yeasts in metal-rich acidic waters are Rhodotorula spp., while some Cryptococcus spp. and Trichosporon dulcitum are also acidophilic yeasts. Novel acidophilic fungal isolates (proposed name Acomyces richmondensi) have been isolated from warm (30–50  C), extremely acidic (pH 0.8–1.38), and iron/ zinc/copper/arsenic-contaminated waters within the Richmond mine at Iron Mountain, California. Microscopic animal life-forms may also be found in acidic environments. The most biodiverse of these appear to be protozoa (Figure 4). Phagotrophic flagellates

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Environmental Microbiology and Ecology | Extremophiles: Acidic Environments

(a)

(b)

(c)

(d)

Figure 4 Scanning electron micrographs of eukaryotic acidophiles: (a) a Eutreptia-like flagellate protozoan, grazing on Leptospirillum ferrooxidans; (b) a Cinetochilum-like ciliate protozoan, grazing on Acidithiobacillus ferrooxidans; (c) a Vahlkampfia-like amoeboid protozoan; (d) a bundle of Euglena mutabilis (an acidophilic microalga) with individual cells arrowed. The scale bar represents 5 mm in micrographs (a)–(c), and 10 mm in micrograph (d).

(Eutreptia), ciliates (Urotricha, Vorticella, Oxyticha, and Cinetochilum), and amoeba (Vahlkampfia) have all been encountered in acidic mine waters, and some have also been grown in acidic media in the laboratory. Multicellular animal life-forms are relatively uncommon, though rotifers (such as Cephalodella hoodi and Cephalodella gibba) have occasionally been identified in acidic mine waters. The two most acidophilic species of known rotifers appear to be Elosa woralii and Brachionus sericus, though the latter can also grow at neutral pH in vitro. The pioneering crustacean Chydorus sphaericus has also been observed in the pelagic community of acid mine lakes in Germany, though it is acid-tolerant rather than acidophilic, with a pH range of 3.2–10.6.

Interactions Between Acidophilic Microorganisms The study of microbial ecology involves not only understanding the impact of the environment on microorganisms (and vice versa) but also examining how microorganisms

interact with each other. Along with increasing awareness of the biodiversity and complexity of life in extremely acidic environments have come fresh insights into the wide range of microbial interactions that occur within them. In some cases, such as grazing by phagotrophic protozoa on acidophilic bacteria, the interaction may be readily observed, though more often it is more clandestine. Mutualistic Interactions Mutualistic interactions are where both partners derive some benefit from their association. One way in which this occurs in extremely acidic environments is via redox transformations and transfer of iron and/or sulfur between prokaryotes. As noted in the section titled ‘Biodiversity of extreme acidophiles’, ferrous iron is an energy source that is widely used by acidophilic Bacteria and some acidophilic Archaea, while ferric iron can act as a highly effective alternative electron acceptor to oxygen in low pH environments. Juxtaposition of aerobic and microaerobic/anaerobic environments can lead to rapid cycling of iron between the two zones. This is aided by the fact that, in contrast to most

Environmental Microbiology and Ecology | Extremophiles: Acidic Environments

environments, ferric iron is soluble at pH < 2.5 and is more readily utilized as an electron sink as soluble Fe3þ than when present in its various amorphous and crystalline forms. The importance of iron cycling has been illustrated in major acidic environments such as the Rio Tinto, and also demonstrated in vitro. Obviously, an extraneous energy source is required for iron cycling to perpetuate. In acidic environments, this may be organic carbon, originating as exudates and lysates from primary producers (phototrophs and chemolithotrophs) that act as electron donors for ironreducing acidophiles. Cycling of iron may involve more than one species (e.g., the iron-oxidizer At. ferrooxidans and the iron-reducer Acidiphilium) or a single species (e.g., of Sulfobacillus). The situation with sulfur transformations is less clear, due in part to the relative paucity in the knowledge of bacterial sulfate/sulfur reduction in lowtemperature acidic environments, and the far greater insolubility of some reduced sulfur compounds (metal sulfides and elemental sulfur) than ferric iron at extremely low pH, which limits their free diffusion. Sulfate produced by aerobic sulfur-oxidizing acidophiles (such as Acidithiobacillus and Thiomonas spp.) can diffuse into underlying sediments and act as a terminal electron acceptor for any acidophilic/acidtolerant SRB present. These generate sulfide, which, at low pH, is present almost exclusively as gaseous H2S. The presence in the sediments of soluble metals, such as copper, that form very insoluble sulfides, results in the rapid removal of H2S, even at very low pH. However, if, as is often the case, the dominant soluble chalcophilic metal present is (ferrous) iron, the lower solubility of the sulfide mineral (FeS) means that it does not form until the pH has risen to 5. If the sediment pH is 0.1 MPa to maintain a liquid environment. Most marine hyperthermophiles are present in deep-sea hydrothermal vent sites where the in situ pressure is 20–45 MPa (pressure increases 0.1 MPa, or 1 times atmospheric pressure, for every 10 m of water depth). Pressure effects on hyperthermophiles are generally favorable for growth at high temperatures. Relative to low pressures (0.1–3 MPa), the maximum growth temperature increases 2–6  C for Pyrococcus, Thermococcus, and Desulfurococcus species when incubated at in situ pressures. For other hyperthermophiles, although their optimum growth temperature does not increase with pressure, their rate of growth does increase significantly at elevated pressure. For M. jannaschii, hyperbaric pressure significantly increases its growth rate at 86  C but does not increase its optimum growth temperature. However, the maximum temperature for CH4 formation increases from 92  C at 0.8 MPa to 98  C at 25 MPa. Likewise, methanogenesis rates are higher at higher pressures.

ore material that frequently contaminates samples. An in situ incubator containing interior melting point standards was deployed on top of a black smoker at Guaymas Basin (Vent 1). Microbial colonization was observed where a 125  C sensor had melted but not a neighboring 140  C sensor, although exposure to 125  C may have been transient. In a sulfide ore deposit from a deep-sea hydrothermal vent site, the highest concentrations of ether lipids and intact fluorescent cells were found in mineral layers consisting primarily of anhydrite and ZnFe sulfides, which suggests that their temperatures were between 100 and 140  C. Field evidence for life above 100  C is circumstantial due to the difficulties of obtaining accurate temperature measurements on the spatial scales necessary, the temporal variations in temperature at a given site, the uncertain origin of the biomass analyzed, and the absence of direct microbial activity measurements. These results are speculative rather than conclusive, and await further detailed analyses for verification and identification of indigenous microorganisms and their metabolic traits.

Laboratory Studies on Natural Microbial Assemblages

Metabolism and Growth

Natural assemblages of microorganisms collected from deep-sea hydrothermal vent sites have also been studied under controlled conditions in the laboratory. Petroleumrich sediment cores from Guaymas Basin showed maximum sulfate reduction activities at 90  C with additional activity between 105 and 110  C. The hyperthermophilic sulfate reducer Archaeoglobus profundus was cultured from these same sediment samples, which has a maximum growth temperature of 90  C and most likely is responsible for the 90  C sulfate reduction activity peak. The organisms responsible for sulfate reduction between 100 and 110  C are unknown. A natural assemblage of microorganisms collected from high-temperature black smoker fluids was incubated in solid gel material (GELRITE) that is stable at temperatures up to 120  C. Colonies of microorganisms formed in the gel at 115 and 120  C at 7 and 27 MPa. The largest colonies (0.5 mm diameter) formed at 27 MPa, which again demonstrates the positive influence of pressure on the growth of microorganisms at high temperatures. Growth also may have been enhanced by the presence of a solid matrix to which cells attach themselves.

CO2 and Acetate Assimilation The assimilation of CO2 by autotrophs is often associated with the Calvin cycle. However, this pathway is generally absent in thermophilic bacteria and is completely absent in archaea. Alternative CO2 assimilation pathways are generally used in these organisms and include the coenzyme A (CoA) pathway, the reductive citric acid cycle, the 3-hydroxypropionate cycle, and the 4-hydroxybutyrate cycle. These pathways were discovered in part by studying anaerobic thermophiles. Acetate assimilation can occur using the citramalate cycle, the glyoxylate shunt, or by reversing a portion of the acetyl-CoA pathway. Thermophilic bacteria and archaea use all of these pathways for inorganic carbon assimilation in some capacity. An archaea-specific version of the acetyl-CoA pathway is used by Euryarchaeota with CH4 production as added steps to the pathway in methanogens (Figure 1). Crenarchaeota use the reductive citric acid cycle, which is lacking in most hyperthermophilic Euryarchaeota, as well as the 3-hydroxypropionate, the 4-hydroxybutyrate, and citramalate cycles. The pathway for CO2 assimilation in the Pyrodictiaceae is largely unknown and likely represents a novel autotrophic pathway.

Field Observations Analyses for biomolecules and intact cells have been performed on exiting hydrothermal fluids, sulfide ore deposits, and sediment and rock core samples. The primary difficulty has been determining what proportion of the material originated from seawater and cooler sulfide

Acetyl-CoA pathway and methanogenesis

Moorella thermoacetica and Moorella thermoautotrophica (formerly Clostridium thermoaceticum and C. thermoautotrophicum) are facultative autotrophs and obligately anaerobic spore formers. When grown on glucose, these organisms produce

Environmental Microbiology and Ecology | Extremophiles: Hot Environments

Pyrobaculum islandicum Thermoproteus neutrophilus Sulfolobus solfataricus Acidianus ambivalens Stygiolobus azoricus Stetteria hydrogenophila Ignicoccus pacificus Pyrolobus fumarii Pyrodictium occultum

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Thermoproteaceae reductive citric acid cycle

Sulfolobaceae Mixed reductive citric acid cycle/ 3-hydroxypropionate cycle/ 4-hydroxybutyrate cycle

Desulfurococcaceae 4-hydroxybutyrate cycle

Pyrodictiaceae unknown pathway, possibly involves rubisco Geoglobus ahangari Archaeoglobus fulgidus

Archaeoglobaceae Archaeal acetyl-CoA pathway

Ferroglobus placidus Methanococcus jannaschii Methanothermus fervidus Methanopyrus kandleri

Methanogens Mixed archaeal acetyl-CoA pathway/ Methanogenesis pathway

0.02

Figure 1 Phylogeny of chemolithoautotrophic archaea based on 16S rRNA sequence homologies and the CO2 assimilation pathways for each family or group of organisms.

three molecules of acetate per glucose and little CO2. This led to the suggestion that glucose is first oxidized to two molecules each of acetate and CO2, followed by the production of a third acetate from the two CO2 molecules. 14 CO2 and 13CO2 incubation experiments confirmed that both carbons of acetate are labeled during growth. Incubation with 13C-formate led to the labeling of the methyl group in acetate and suggested that formate is an intermediate in the pathway. The isolation of the autotrophic acetogen Acetobacterium woodii confirmed CO2 assimilation can occur by means other than by the Calvin cycle, which is now known as the acetyl-CoA pathway (or Wood–Ljungdahl pathway). In addition to these thermophilic bacteria, an archaeal version of the pathway is used by autotrophic members of the Archaeoglobaceae and by all methanogens growing on H2 and CO2. The acetyl-CoA pathway reduces and condenses two molecules of CO2 to form one molecule of acetyl-CoA (Figure 2). One CO2 molecule is reduced in a series of reduction steps to a methyl group ligated to a C1 carrier. In bacteria, the C1 carriers are tetrahydrofolate and coenzyme E; in archaea, they are methanofuran and tetrahydromethanopterin. Another difference between the pathways in bacteria and archaea is the use of a soluble 5-deazaflavin called coenzyme F420 by archaea that carries two electrons but only one hydrogen. The enzymes in bacteria and archaea that catalyze these steps are conserved functionally but have little-to-no sequence homologies between them. The key enzyme in the pathway is carbon monoxide dehydrogenase/acetyl-CoA

synthase, which is a highly conserved protein across superkingdoms. This enzyme reduces the second CO2 molecule to a carbon monoxyl group, ligates it to the methyl group from the C1 carrier, and then adds CoA to form acetyl-CoA. Methanogens use three additional enzymes (methyl-MPT:CoM methyltransferase, methylCoM reductase, and heterodisulfide reductase) to dispose of electrons during their anaerobic respiration using methyl-MPT as their terminal electron acceptor (Figure 2).

Reductive citric acid cycle

Like M. thermoacetica and M. thermoautotrophica, it was shown in the photosynthetic green sulfur bacterium Chlorobium limicola, the purple sulfur bacterium Chromatium vinosum, and the purple nonsulfur bacterium Rhodospirillum rubrum that CO2 assimilation occurs by a pathway other than the Calvin cycle. The concept of the reductive citric acid cycle (Figure 3) had its origin in the discovery of ferredoxindependent reductive carboxylation reactions: pyruvate synthase and 2-oxoglutarate (formerly -ketoglutarate) synthase. They are driven by the strong reducing potential of reduced ferredoxin and complement the irreversible NADþ-dependent pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase reactions. Other key findings were enzymes that produce oxaloacetate and acetyl-CoA from citrate and complement the irreversible citrate synthase step in the oxidative citric acid cycle. In C. limicola and Desulfobacter hydrogenophilus, citrate

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Environmental Microbiology and Ecology | Extremophiles: Hot Environments Bacteria

Archaea CO2

CO2 1

NADPH + H+ NADP+

Fdox formyl-MFR MPT

HCOOH 2

MFR + Fdred

8

THF + ATP

9

ADP + Pi

MFR

formyl-MPT

formyl-THF 10

H 2O methenyl-MPT

3

H 2O methenyl-THF

F420-H2 F420

NADPH + H+ 4

NADP methylene-THF Fdred 5 Fdox

+

CoE THF

methyl-CoE

12

methylene-MPT F420-H2 13 F420

methyl-THF 6

H2 11

CoMSH 7

acetyl-CoA

2H

CoASH CoE + CO2

+

7

methyl-MPT CoMSH CoASH MPT 14 MPT + CO2

acetyl-CoA

methyl-CoM CoBSH

16

H2

2 H+

15

CoBS-SCoM CH4

Figure 2 The acetyl-CoA pathways in bacteria and archaea. The enzymes are as follows: 1, formate dehydrogenase; 2, formyl-THF synthetase; 3, methenyl-THF cyclohydrolase; 4, methylene-THF dehydrogenase; 5, methylene-THF reductase; 6, methyltransferase; 7, carbon monoxide dehydrogenase; 8, formyl-MF dehydrogenase; 9, formyl-MF:MPT formyltransferase; 10, methenyl-MPT cyclohydrolase; 11, F420-dependent methylene-MPT dehydrogenase; 12, F420-independent methylene-MPT dehydrogenase; 13, methylene-MPT reductase; 14, methyl-MPT:CoMSH methyltransferase; 15, methyl-CoM reductase; and 16, heterodisulfide reductase. THF, tetrahydrofolate; MF, methanofuran; MPT, tetrahydromethanopterin; CoE, coenzyme E; CoM, coenzyme M; CoB, coenzyme B; Fd, electron carrier ferredoxin; and F420, electron carrier coenzyme F420. The dashed lines show the enzyme steps that are unique to methanogenesis.

cleavage is accomplished in a single step by ATP citrate lyase (eqn [1]): citrate þ ATP þ CoA ! acetyl-CoA þ oxaloacetate þ ADP þ Pi

½1

This protein is the citrate cleavage enzyme that is most commonly associated with the reductive citric acid cycle. In the thermophilic bacterium Hydrogenobacter thermophilus TK-6, citrate cleavage is catalyzed in two steps by citrylCoA synthetase (eqn [2]) and citryl-CoA lyase (eqn [3]): citrate þ ATP þ CoA ! citryl-CoA þ ADP þ Pi

½2

citryl-CoA ! oxaloacetate þ acetyl-CoA

½3

The first evidence for the reductive citric acid cycle in archaea came from the study of thermoacidophile Acidianus brierleyi (formerly Sulfolobus brierleyi). Pulse labeling of autotrophically grown A. brierleyi with 14CO2 showed the formation of labeled malate, citrate, aspartate, and glutamate. Initially all of the citric acid cycle enzymes and pyruvate synthase were measured in cell extracts of

A. brierleyi grown autotrophically. However, the lack of ATP citrate lyase activity and the presence of 3-hydroxypropionate cycle activities led to the suggestion that the organism uses this latter pathway for CO2 assimilation. It was since shown that autotrophically grown A. brierleyi does possess ATP citrate lyase activity but it requires covalent modification by acetylation for activity. Therefore, the organism appears to use a combination of the reductive citric acid cycle and the 3-hydroxypropionate cycle for CO2 assimilation. The hyperthermophilic archaeon Thermoproteus neutrophilus was grown autotrophically and pulse labeled with 14 C- and 13C-succinate, yielding labeled malate, glutamate, and aspartate. This and the presence of all of the activities of the citric acid cycle enzymes, pyruvate synthase, and ATP citrate lyase suggest that it also uses the reductive citric acid cycle for CO2 assimilation. The presence of pyruvate synthase, 2-oxoglutarate synthase, and ATP citrate lyase activities in the hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically suggests that this organism likewise uses this pathway.

Environmental Microbiology and Ecology | Extremophiles: Hot Environments CO2

Pi

Phosphoenolpyruvate

Oxaloacetate 15

AMP + Pi 14 ATP Pyruvate

NAD(P)H + H+ + CO2

ATP + CO2 ADP + Pi Oxaloacetate 13

Fdox + CoA

16

135

12 Fdred + CO2

+

NAD(P)

AMP + PPi ATP + CoA Acetate

Acetyl-CoA 10 Oxaloacetate

1

11 CoA

9

NADH + H+ Malate

Citrate Acetyl-CoA ATP + + ADP + Pi CoA 8

NAD+

2 18

CoA

Oxaloacetate

7

H2O

17

Fumarate

Acetyl-CoA + H2O

Isocitrate

Glyoxylate

6

FADH2

NADP+

3 FAD Succinate

2-oxoglutarate

NADPH + CO2 + H+

5

4

Fdox + CoA

ATP + CoA ADP + Pi

Succinyl-CoA

Fdred + CO2

Figure 3 The citric acid cycle, the glyoxylate shunt, and related enzymes. The enzymes are as follows: 1, malate dehydrogenase; 2, fumarase; 3, fumarate reductase/succinate dehydrogenase; 4, succinyl-CoA synthetase; 5, 2 oxoglutarate synthase; 6, isocitrate dehydrogenase; 7, aconitase; 8, either ATP citrate lyase (one step) or citryl-CoA synthase and citryl-CoA lyase (two steps); 9, citrate lyase; 10 AMP-forming acetyl-CoA synthetase; 11, citrate synthase; 12, pyruvate synthase; 13, pyruvate carboxykinase; 14, phosphoenolpyruvate synthetase; 15, phosphoenolpyruvate carboxylase; 16, malic enzyme; 17, isocitrate lyase; and 18, malate synthase. Fd, electron carrier ferredoxin. Copyright ª American Society for Microbiology, Journal of Bacteriology, vol.188, pp. 4350–4355, 2006.

However, for both T. neutrophilus and P. islandicum, it was suggested that the ATP citrate lyase activities are too low to account for all of the CO2 assimilated. It was subsequently shown for P. islandicum that acetylated citrate lyase (eqn [4]) and AMP-forming acetyl-CoA synthase (eqn [5]) activities increase significantly in cells grown autotrophically relative to those grown heterotrophically and are higher than the ATP citrate lyase activities measured. citrate ! oxaloacetate þ acetate acetate þ CoA þ ATP ! acetyl-CoA þ AMP þ PPi

½4 ½5

Therefore, there appears to be a third mechanism for citrate cleavage in thermophiles that requires covalent modification by acetylation.

All 8 of the citric acid cycle enzymes are present within 20 of the 46 archaeal genome sequences currently available (Figure 4). The complete cycle is found in all organisms within the Thermoproteales, the Sulfolobales, and Aeropyrum pernix in the Crenarchaeota and in all Halobacteriales (i.e., extreme halophiles) and Thermoplasmales in the Euryarchaeota. Only a portion of the cycle is found in the Thermococcales, the Archaeoglobales, the Desulfurococcales (except A. pernix), and all methanogens. The distributions of the citric acid cycle enzymes in both archaeal phyla and the acetyl-CoA pathway enzymes (only in Euryarchaeota) suggest that the last common archaeal ancestor contained the complete citric acid cycle and that portions of the cycle were lost in the methanogens, Archaeoglobales, and Thermococcales, and currently it only serves to produce

136

Environmental Microbiology and Ecology | Extremophiles: Hot Environments

Euryarchaeota

Halobacteriales

Thermoplasmales Methanomicrobiales

Methanobacteriales Archaeoglobales Methanococcales

Methanosarcinales 0.05

Cenarchaeales Thermococcales Methanopyrales

Korarchaeota Thermoproteales Desulfurococcales

Sulfolobales

Crenarchaeota

Figure 4 A 16S rRNA tree of the archaea. Those taxonomic orders that contain all of the genes that encode for citric acid cycle enzymes are shown in green, whereas those with only a subset of these genes are in red. Uncultivated orders are shown as unfilled groups. The star indicates the location of the root of the tree. The tree was constructed as described previously. The scale bar represents 0.05 changes per nucleotide. Adapted by permission from Macmillan Publishers Ltd: Schleper C, Jurgens G, and Jonuscheit M (2005) Genomic studies of uncultivated Archaea, Nature Reviews, vol. 3, pp. 479–488. Copyright 2005.

intermediates for some biosynthesis reactions in these organisms.

3-hydroxypropionate cycle

Chloroflexus aurantiacus is a thermophilic green nonsulfur bacterium that is a facultative photoautotroph and an anaerobe. Cultures grown photoautotrophically with H2 and CO2 lack ribulose-1,5-bisphosphate carboxylase/ oxygenase (RubisCO) and ribulose-5-phosphate kinase (formerly phosphoribulokinase) activities, which are the key enzymes of the Calvin cycle. They also lack ATP citrate lyase and 2-oxoglutarate synthase activities that are necessary for the reductive citric acid cycle. However, C. aurantiacus secretes 3-hydroxypropionate during phototrophic growth, which suggests that it is an intermediate of CO2 assimilation. This led to the discovery of the 3-hydroxypropionate cycle where two molecules of CO2 are assimilated to form glyoxylate (Figure 5). The carboxylation reactions are catalyzed at two points in the cycle by a single bifunctional enzyme called acetyl-CoA/propionyl-CoA carboxylase. Many of the enzymes in the cycle overlap with those of the citric

acid cycle to form malate, which is then split to form glyoxylate and regenerate acetyl-CoA. As mentioned previously, the lack of ATP citrate lyase activity in the thermoacidophilic archaeon A. brierleyi and the presence of acetyl-CoA carboxylase and propionylCoA carboxylase activities led to the suggestion that this organism uses the 3-hydroxypropionate cycle for CO2 assimilation. Acetyl-CoA carboxylase and propionylCoA carboxylase activities were also measured in autotrophically grown cell extracts from Sulfolobus metallicus and Acidianus infernus. The subsequent discovery of ATP citrate lyase activity in A. brierleyi after acetylation and the activities of both the citric acid and 3-hydroxypropionate cycles suggest that this organism uses a combination of these pathways for CO2 assimilation. 4-hydroxybutyrate cycle

Ignicoccus species are hyperthermophilic obligately autotrophic archaea that belong to the Desulfurococcaceae. Ignicoccus pacificus and Ignicoccus islandicus lack ATP citrate lyase, 2-oxoglutarate synthase, carbon monoxide dehydrogenase, and acetyl-CoA/propionyl–CoA carboxylase activities that are indicative of the reductive citric acid

Environmental Microbiology and Ecology | Extremophiles: Hot Environments

137

NADP+ + CoA Malonate semialdehyde NADPH + H+ NADP+ NADPH + H+ 3 2

Malonyl-CoA

3-hydroxypropionate

ADP + Pi

CoA + ATP 1

ATP + CO2

4

ADP + Pi

Acetyl-CoA

3-hydroxypropionyl-CoA

12 13

5

Glyoxylate + ADP + Pi ATP + CoA Malate

Acrylyl-CoA NADPH + H+ 6

11

NADP+ Propionyl-CoA

Fumarate FADH2

1

10

ATP + CO2 ADP + Pi

FAD Succinate

Methylmalonyl-CoA 9

7 8

ATP + CoA Succinyl-CoA ADP + Pi

Figure 5 The 3-hydroxypropionate cycle. The enzymes are as follows: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase; 3, 3-hydroxypropionate dehydrogenase; 4, 3-hydroxypropionyl-CoA hydrolase; 5, acrylyl-CoA hydratase; 6, acrylyl-CoA dehydrogenase; 7, methylmalonyl-CoA epimerase; 8, methylmalonyl-CoA mutase; 9, succinyl-CoA synthetase; 10, succinate dehydrogenase; 11, fumarase; 12, malyl-CoA synthetase; and 13, malyl-CoA lyase.

cycle, the acetyl-CoA pathway, and the 3-hydroxypropionate cycle, respectively. It was shown that Ignicoccus hospitalis assimilates CO2 in two steps using pyruvate synthase and phosphoenolpyruvate carboxylase (Figure 6), and many of the enzymes are the same as those used in the reductive citric acid cycle. However, because the organism lacks 2-oxoglutarate synthase, it reduces succinyl-CoA in two enzymatic steps to form 4-hydroxybutyrate and eventually acetoacetyl-CoA. In the final step, acetoacetyl-CoA is cleaved to form two molecules of acetyl-CoA, one of which brings the cycle back to its starting point, yielding the net formation of one acetyl-CoA. Interestingly, the thermoacidophilic archaeon Metallosphaera sedula, which is a close relative of Sulfolobus and Acidianus species, uses a mixture of the 3-hydroxypropionate and 4-hydroxybutyrate cycles. CO2 is assimilated using acetyl-CoA/propionyl-CoA carboxylase, as is found in the 3-hydroxypropionate cycle. However, instead of forming glyoxylate and acetyl-CoA from succinyl-CoA as is found in the 3-hydroxypropionate cycle, M. sedula converts succinyl-CoA into two molecules of acetyl-CoA via 4-hydroxybutyrate using the same enzymes found in the

4-hydroxybutyrate cycle. Therefore, across all of the autotrophic Crenarchaeota (with the possible exception of the Pyrodictiaceae), CO2 assimilation often involves a mixture of the reductive citric acid cycle, the 3-hydroxypropionate cycle, and the 4-hydroxybutyrate cycle, and many of the same enzymes (i.e., malate dehydrogenase, fumarase, succinate dehydrogenase/fumarate reductase, succinyl-CoA synthetase, pyruvate synthase, PEP synthetase, and PEP carboxylase) are used, suggesting that there is an evolutionary relationship between these CO2 assimilation pathways. Other possible CO2 assimilation pathways

Hyperthermophilic archaea belonging to the Pyrodictiaceae may possess novel pathways for CO2 assimilation. Autotrophically grown P. fumarii, P. abyssi and P. occultum grown on yeast extract with H2 and CO2 all had pyruvate synthase activity but lacked 2-oxoglutarate synthase activity and generally lacked other enzymes of the citric acid cycle needed for the 3-hydroxypropionate and 4hydroxybutyrate cycles. P. abyssi and P. occultum also lack carbon monoxide dehydrogenase/acetyl-CoA synthase activity. Therefore, these organisms do not appear to use

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Environmental Microbiology and Ecology | Extremophiles: Hot Environments

AMP + Pi ATP + H2O

Phosphoenolpyruvate

HCO3– Pi 3

2

Pyruvate

Oxaloacetate NADH + H+ + 2 MVred

2 Fdox 1

2 Fdred + CO2

4 5 6

NAD+ + 2 MVox

Acetyl-CoA

Succinate

Acetyl-CoA

ATP + CoA 14

7

ADP + Pi

CoA

Succinyl-CoA

Acetoacetyl-CoA NADH + H+

8

13

NAD+

2 MVred 2 MVox + CoA

Succinic semialdehyde

(S)-3-hydroxybutyryl-CoA

NAD(P)H + H+

FADH2

9

12

NAD(P)+ FAD Crotonyl-CoA

4-hydroxybutyrate 10

11

ATP + CoA 4-hydroxybutyryl-CoA

ADP + Pi

ATP + CoA

AMP + PPi

Figure 6 The 4-hydroxybutyrate cycle. The enzymes are as follows: 1, pyruvate synthase; 2, phosphoenolpyruvate synthetase; 3, phosphoenolpyruvate carboxylase; 4, malate dehydrogenase; 5, fumarase; 6, fumarate reductase; 7, succinyl-CoA synthetase; 8, succinyl-CoA reductase; 9, succinate semialdehyde reductase; 10, 4-hydroxybuturyl-CoA synthetase; 11, 4-hydroxybutyryl-CoA dehydratase; 12, crotonyl-CoA hydratase; 13, 3-hydroxybutyryl-CoA dehydrogenase; and 14, acetoacetyl-CoA -ketothiolase.

the acetyl-CoA pathway, the reductive citric acid cycle, the 3-hydroxypropionate cycle, or the 4-hydroxybutyrate cycle for CO2 assimilation. In P. abyssi and P. occultum, there are low levels (5–15 nmol min1 mg1 cell protein) of RubisCO activity, which is the key enzyme of the Calvin cycle. For P. abyssi, RubisCO activity increases approximately twofold per 10  C temperature increase, as expected for most enzymes, and activity requires strictly anoxic and reducing conditions. The product of the reaction is 3-phosphoglycerate. However, ribulose-5-phosphate kinase activity is not measured, suggesting that CO2 assimilation does not occur via the standard Calvin cycle. Similarly, RubisCO activity is present in several Euryarchaeota including methanogens, the hyperthermophilic heterotrophs in the Thermococcaceae, and Archaeoglobus fulgidus. All lack ribulose-5-phosphate kinase activity. Ribulose-1,5bisphosphate is generated in M. jannaschii using 5phospho-D-ribose-1-pyrophosphate (PRPP) as a substrate, which is an intermediate in nucleotide biosynthesis. A similar pathway is found in T. kodakaraensis where adenosine monophosphate is used as the starting material

instead of PRPP. First the adenine in AMP is replaced with a phosphate to form ribose-1,5-bisphosphate, then an isomerase converts this to ribulose-1,5-bisphosphate. Although pathways for CO2 assimilation via RubisCO seem to exist in Euryarchaeota, they do not appear to contribute significantly, if at all, to CO2 assimilation. Acetate catabolism

Acetate is potentially an important carbon source in hightemperature environments. It is a common metabolite formed by heterotrophs during the breakdown of organic material and is the primary end product of acetogens. The most common pathway of acetate catabolism in bacteria is via the glyoxylate shunt. First, acetate and CoA are combined to form acetyl-CoA by acetyl-CoA synthase using the energy of ATP and forming AMP þ PPi (Figure 3). Then some of the acetyl-CoA condenses with oxaloacetate to form citrate and enters the citric acid cycle. Isocitrate is formed from citrate. At this point, the fate of the carbon varies. Some isocitrate remains within the citric acid cycle for biosynthesis reactions. The remainder is cleaved into succinate and glyoxylate by isocitrate

Environmental Microbiology and Ecology | Extremophiles: Hot Environments

Citramalate

2

139

Mesaconate CoA

Acetate

1

3

Pyruvate

Mesaconyl-CoA

NAD(P)H + H+ + CO2

4

12

NAD(P)+

Malate

3-methylmalyl-CoA

FADH2 10 11

5

Glyoxylate FAD Succinate ATP + CoA ADP + Pi

Propionyl-CoA 9

6

ATP + CO2 ADP + Pi

Succinyl-CoA

Methylmalonyl-CoA 7 8

Figure 7 The citramalate cycle. The enzymes are as follows: 1, citramalate synthase; 2, citramalate dehydratase; 3, mesaconyl-CoA synthetase; 4, mesaconyl-CoA hydratase; 5, 3-methylmalyl-CoA lyase; 6, propionyl-CoA carboxylase; 7, methylmalonyl-CoA epimerase; 8, methylmalonyl-CoA mutase; 9, succinyl-CoA synthetase; 10, succinate dehydrogenase; 11, fumarase; and 12, malic enzyme.

lyase. The succinate then re-enters the citric acid cycle pool of intermediates. Glyoxylate is then combined with a second molecule of acetyl-CoA to form malate by malate synthase. Malate can either enter the citric acid cycle pool for biosynthesis reactions or be used to form pyruvate using the malic enzyme. The key enzymes of the glyoxylate shunt are isocitrate lyase and malate synthase, and both of these as well as the complete citric acid cycle are found in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Other thermophilic organisms lack isocitrate lyase and pyruvate synthase activities, the two most common means for biosynthesis from acetyl-CoA, when grown on acetate. In these cases, acetate catabolism is accomplished using the citramalate cycle (Figure 7). Acetate (or acetyl-CoA) combines with pyruvate to form citramalate by citramalate synthase. After a series of enzyme reactions, 3-methylmalonyl-CoA is cleaved to form propionylCoA and glyoxylate. The glyoxylate can be used to form malate using acetyl-CoA and malate synthase. Propionyl-CoA is carboxylated and eventually forms intermediates of the citric acid cycle using enzymes found in the 3-hydroxypropionate and citric acid cycles. Pyruvate is recycled from an intermediate in the citric acid cycle. The citramalate cycle was first described in

purple bacteria, which grow best on acetate when H2, CO2, and low levels of either pyruvate or organic compound are added to the growth medium. Similarly, the hyperthermophilic archaeon P. islandicum increases its citramalate synthase and 3-methylmalyl-CoA lyase activities when grown on acetate relative to autotrophic and heterotrophic growth, suggesting that is uses the citramalate cycle in part for acetate metabolism. It grew best on acetate when H2 and low levels of yeast extract (0.001%) were added to the medium. The biochemical overlap between the citric acid cycle, the glyoxylate shunt, the 3-hydroxypropionate cycle, and the citramalate cycle highlights the importance of viewing these and other pathways holistically because it is likely that they do not always operate completely independent of the others. Furthermore, these pathways each perform multiple cellular functions for the cell. Some or all of the citric acid cycle is involved in all of the pathways listed above and also functions for energy production and biosynthesis reactions such as amino acid synthesis. Portions of the 3-hydroxypropionate cycle are also used for propanoate metabolism and the steps of the citramalate cycle are also used for leucine biosynthesis. These overlaps may have significant evolutionary implications and their study is important to understand the natural history of catabolic and anabolic pathways.

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Heterotrophy The majority of high-temperature microorganisms are heterotrophs or facultative autotrophs (Table 1). Not surprisingly, the most common organic compounds catabolized at high temperatures are carbohydrates and peptides. However, some thermophiles and hyperthermophiles also oxidize low-molecular-weight organic acids and many thermophiles catabolize hydrocarbons as sources of carbon and electrons. Carbohydrate metabolism

Carbohydrate metabolism can be divided into four categories: uptake, hydrolysis, glycolysis, and gluconeogenesis. The enzymes for gluconeogenesis are found in most organisms, including autotrophs, whereas those for uptake, hydrolysis, and glycolysis vary. Many of the hyperthermophilic enzymes involved in glycolysis and gluconeogenesis are biochemically and phylogenetically unique to either hyperthermophiles or archaea (Figure 8). For example, glucokinase and phosphofructokinase from P. furiosus are ADP-dependent rather than ATPdependent and do not show any sequence similarity with their ATP-dependent counterparts in mesophilic bacteria. Glyceraldehyde-3-phosphate is oxidized to 3-phosphoglycerate in a single enzyme step with concomitant reduction of ferredoxin rather than NADþ and is catalyzed by the unique tungsten-containing protein glyceraldehyde-3-phosphate oxidoreductase. Both NAD(P)þ-dependent glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase are present and are homologous to their counterparts in mesophilic bacteria, but they are used for gluconeogenesis rather than for glycolysis in P. furiosus. Furthermore, fructose-1,6bisphosphate aldolase, fructose-1,6-bisphosphatase, and phosphoglucose isomerase are all unique to either hyperthermophiles or archaea. Three membrane-bound sugar-binding proteins have been characterized from P. furiosus that are specific for maltose/trehalose (MalE), maltose/maltodextrin (MBP), and cellobiose (CbtA). These demonstrate the specificity and coordination of carbohydrate uptake in hyperthermophiles. The MalE binding, permease (MalFG), and ATP-binding transporter (MalK) proteins from Thermococcus litoralis and the operon encoding these proteins were characterized as well as the regulatory protein (TrmB) for this operon, and showed that the sequences, function, and regulation of ATP-binding cassette (ABC)type transport systems in hyperthermophiles are similar to those found in mesophiles. The malEFGK operon in P. furiosus is flanked by insertion sequences but is absent in P. abyssi and Pyrococcus horikoshii, suggesting lateral gene transfer from other organisms. Using proteomics, MalE and CbtA were identified in the membrane cellular fraction of P. furiosus grown on a mixture of maltose and

peptides, and MalE is one of the most abundant proteins within the membrane. The genes for MBP and CbtA in P. furiosus are likewise part of an ABC-type operon but lack flanking insertion sequences and are found in P. abyssi and P. horikoshii. Furthermore, the maltodextrin uptake operons in P. furiosus and Pyrobaculum aerophilum contain the gene encoding for the sugar hydrolase amylopullulanase (apu), whereas the cellobiose operon in P. furiosus is next to and shares a putative promoter region with the -mannosidase gene (bmn), thus demonstrating a tight coupling between sugar uptake and hydrolysis in these organisms. P. furiosus amylopullulanase is an extracellular glycosylase that is active at temperatures up to 140  C. This demonstrates that some proteins are stable well above 110  C and that the biogenic impact of P. furiosus in its native environment extends beyond its maximum growth temperature as its extracellular enzymes ‘forage’ for growth substrates. Peptide metabolism

Peptide metabolism can be divided into three categories that are functionally similar to those found in carbohydrate metabolism: uptake, hydrolysis, and peptidolysis (Figure 9). Unlike the sugar ABC transport system, little is known about the peptide ABC transport system in hyperthermophiles. Using proteomics, a putative membrane dipeptide-binding protein was highly abundant in the membrane fraction of P. furiosus cells grown on tryptone and maltose along with the MalE-binding protein. Up to 13 protease activity bands are observed in gelatin-containing zymograms from P. furiosus cell extracts, demonstrating the large suite of proteases available with the cells. Four of the 13 predicted transaminases in P. furiosus were shown to have varying degrees of specificity for amino acids, although each uses 2-oxoglutarate as the amine group acceptor. The glutamate produced from the transamination reaction is recycled back to 2-oxoglutarate by glutamate dehydrogenase with concomitant reduction of NADPþ in P. furiosus. Hyperthermophiles and archaea produce up to four ferredoxin-linked 2-keto acid oxidoreductases that decarboxylate the acid, pass electrons to ferredoxin, and ligate CoA to the remaining compound (Figure 8). Three of these (IOR, VOR, and OGOR) are unique to archaea. The coenzyme is then cleaved, forming an organic acid with the phosphorylation of ADP to ATP, which is the only substrate-level phosphorylation step within the peptidolysis pathway. P. furiosus growth on maltose was compared with its growth on peptides using growth kinetics, metabolite analyses, enzyme activities, and DNA microarray analyses. Based on growth rates, P. furiosus grows better on peptides than on maltose. As expected, the primary organic acid produced when cultures are grown on maltose is acetate (Figure 7), whereas growth on peptides yields a fairly

Environmental Microbiology and Ecology | Extremophiles: Hot Environments

141

Starch H2O

1

Maltodextrin 4

Maltose

2

4

3

Extracellular 5

Cytoplasm

5

ATP

ATP

ADP

Maltodextrin

ADP

Maltose

Glycogen 24

8

6

ADP-glucose

Glucose ADP

Maltodextrin

9

7

ATP Glucose-1-P

Glucose-6-P

Maltodextrin

PPi

23

AMP 22

10

Fructose-6-P ADP Pi 11 12 AMP Fructose-1,6-BP 13

DHAP

G-3-P 14

15

Fdox Fdred

ATP ADP

3-PG

NAD+ + Pi NADH

16

1,3-BPG 17

18

2-PG 19

H2O PEP

20

ADP ATP

AMP + Pi 21 ATP Pyruvate

Figure 8 Starch hydrolysis, uptake, and glycolysis via the modified Embden–Meyerhof pathway and steps for gluconeogenesis. The enzymes are as follows: 1, amylopullulanase; 2, maltose/maltodextrin-binding protein; 3, maltose/trehalose-binding protein; 4, sugar transport permease; 5, sugar transport ATPase; 6, -amylase; 7, -glucan phosphorylase; 8, -glucosidase; 9, glucokinase; 10, glucose-6-phosphate isomerase; 11, fructose-6-phosphate kinase; 12, fructose-1,6-bisphosphatase; 13, fructose-1,6-bisphosphate aldolase; 14, triosephosphate isomerase; 15, glyceraldehydes-3-phosphate:ferredoxin oxidoreductase; 16, glyceraldehydes-3phosphate dehydrogenase; 17, 3-phosphoglycerate kinase; 18, 3-phosphoglycerate mutase; 19, enolase; 20, pyruvate kinase; 21, phosphoenolpyruvate synthetase; 22, phosphoglucomutase; 23, ADP-glucose synthase; and 24, glycogen synthase. Fd, the electron carrier ferredoxin.

even mixture of acetate, phenylacetate, (iso)butyrate, and isovalerate (Figure 9). The activities of glutamate dehydrogenase, 2-oxoglutarate oxidoreductase, indolepyruvate oxidoreductase, isovalerate oxidoreductase, formaldehyde oxidoreductase, aldehyde oxidoreductase, acetyl-CoA synthetase I, glyceraldehydes-3-phosphate dehydrogenase, and cytoplasmic hydrogenase were all significantly higher when P. furiosus cultures were grown on peptides. Conversely, the activities of glyceraldehydes-3-phosphate oxidoreductase, acetolactate synthase, and -amylase were higher when cultures were grown on maltose. In

both cases, these enzymes appear to follow their proposed physiological functions. Formaldehyde oxidoreductase, aldehyde oxidoreductase, and glyceraldehyde:ferredoxin oxidoreductase are tungsten-containing enzymes and explain in part why hyperthermophiles generally have a tungsten requirement for growth. Respiration The consumption of electron donors and the reduction of terminal electron acceptors are among the primary

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Environmental Microbiology and Ecology | Extremophiles: Hot Environments Protein H 2O

1

Oligopeptides 4

2

Dipeptides 4

3

Extracellular 5

Cytoplasm

5

ATP

ADP

ATP

Oligopeptides

ADP

Dipeptides 7

6

Amino acid

α -ketoglutarate

Oligopeptide 8

NADPH 9

NADP+

Glutamate

α-keto acids

Pyruvate 10

Fdox Fdred

Acetyl-CoA

14

ADP ATP

Acetate

Branched chain 11

Fdox Fdred

Branched chain acyl-CoAs 14

ADP ATP

Branched chain acids

Aromatic 12

Fdox Fdred

Aryl-CoAs

14

2-oxoglutarate 13

Fdox Fdred

Succinyl-CoA

ADP ATP

Aryl acids

Figure 9 Peptide hydrolysis, uptake, and peptidolysis in archaea. The enzymes are as follows: 1, pyrolysin; 2, oligopeptide-binding protein; 3, dipeptide-binding protein; 4, peptide transport permease; 5, peptide transport ATPase; 6, intracellular protease; 7, prolidase; 8, amino acid aminotransferase; 9, glutamate dehydrogenase; 10, pyruvate:ferredoxin oxidoreductase; 11, -ketoisovalerate:ferredoxin oxidoreductase; 12, indolepyruvate:ferredoxin oxidoreductase; 13, 2-oxoglutarate:ferredoxin oxidoreductase; and 14, acyl-CoA synthetase. Fd, the electron carrier ferredoxin.

means that microorganisms have of altering the chemistry of their environment. Although several compounds can serve as electron donors for hyperthermophiles, the most common compounds used for this purpose in geothermal environments are H2, organic compounds, and reduced sulfur compounds. Hydrogen is typically oxidized on the membrane by a hydrogenase where electrons then enter the electron transport pathway. Organic compounds are oxidized as described in the section titled ‘Carbohydrate metabolism’ and ‘Peptide metabolism’ and result in the production of reduced ferredoxin and NADH. Respiration is a series of exergonic redox reactions within the cytoplasmic membrane that are coupled with proton translocation across the membrane, which forms an electrochemical gradient (Figure 10). This proton motive force is then used to generate ATP from ADP and phosphate using a membrane-bound ATP synthase. The canonical electron transport chain through the

membrane typically begins with the oxidation of NADH by a membrane-bound NADH:quinone oxidoreductase and the direct reduction of a quinone. Often electrons from the quinone are transferred to a cytochrome c by a quinol:cytochrome c oxidoreductase, typically a bc1 complex. Electrons from either the quinones or the cytochrome are then passed to a terminal reductase that reduces the terminal electron acceptor. The marvelous aspect of respiration in bacteria and archaea is that the system is modular, and individual components (e.g., terminal reductases) can be exchanged with changes in environmental conditions and electron acceptor availability. Homologues of NADH:quinone oxidoreductases are found in the genome sequences of most thermoacidophilic and hyperthermophilic archaea. The catalytic (NuoD) and quinone-binding (NuoH) subunits are conserved except for NuoH in methanogens, but all archaea lack

Environmental Microbiology and Ecology | Extremophiles: Hot Environments

2 H+

3 H+

4 H+ 4 2 e– 2

1

NAD(P)H + H+

2 H+ NAD(P)+

143

–

2e

–

e

5

3

e–

Cytoplasm

Xox + 2 H+

Xred

6

3 H+ ADP + Pi ATP

Figure 10 Membrane electron transport pathway. The components are as follows: 1, NADH:quinone oxidoreductase; 2, quinone; 3, quinol:cytochrome c oxidoreductase (bc1 complex); 4, cytochrome c; 5, generic terminal reductase; and 6, Hþ-translocating ATP synthase.

the NuoAEFGJK subunits found in bacteria, which includes the NADH binding, flavin, and iron–sulfur cluster containing subunits (NuoEFG). Membranesoluble archaeal electron carriers have been found in the hyperthermophiles P. islandicum and Pyrobaculum organotrophum, in the thermoacidophile S. solfataricus, and in the mesophilic methanogen Methanosarcina mazei. Menaquinones were found in the two Pyrobaculum species whereas two novel sulfur-containing quinonelike compounds were observed in S. solfataricus. The methanogen uses a 2-hydroxyphenazine derivative called methanophenazine, which could be reduced by a membrane-bound F420 dehydrogenase and oxidized by the membrane-bound enzyme heterodisulfide reductase. The use of methanophenazine by methanogens may explain the absence of the quinone-binding subunit in their NADH:quinone oxidoreductase. The presence of bc1 complexes in hyperthermophilic and thermoacidophilic archaea is relatively scarce. They are found in some Pyrobaculum, Aeropyrum, Sulfolobus, and Acidianus species, all organisms with some capacity for aerobic growth. Homologues of membrane-bound Hþtranslocating ATP synthase are found in the genome sequences of all thermoacidophilic and hyperthermophilic archaea. The catalytic subunits (AtpAB) are conserved, but all archaea lack the AtpGHJK subunits found in bacteria. Reduction of sulfur compounds

The reduction of elemental sulfur is one of the most common traits of thermoacidophiles and hyperthermophiles (Table 1). Elemental sulfur is the terminal electron acceptor for neutrophilic heterotrophs from marine environments (e.g., Pyrococcus and Thermococcus) and terrestrial environments (e.g., Thermoproteus and Pyrobaculum), for chemolithoautotrophs from marine environments (e.g., Pyrodictium), and for some thermoacidophiles from terrestrial environments (e.g., Acidianus). This is not surprising given the abundance of sulfur compounds in their native geothermal environments. Environmental conditions

significantly influence the form of sulfur available for respiration. Above pH 5, sulfide anion (HS-) is a nucleophile that reacts with the elemental sulfur ring (S8) forming polysulfide (S42 and S52). Above pH 7 and 75  C, elemental sulfur disproportionates into thiosulfate and sulfide (S8 þ 6H2O ! 2S2O32 þ 4HS þ 8Hþ). Pyrodictium and Acidianus species couple H2 oxidation with elemental sulfur reduction. They grow at pH 5-8 and pH 1-4, respectively, suggesting that Pyrodictium uses polysulfide whereas Acidianus uses S8. In Pyrodictium, H2 oxidation is coupled directly to sulfur/polysulfide reduction in a membrane-bound multienzyme complex with both hydrogenase and sulfur reductase activities. It contains Fe, Ni, Cu, acid-labile sulfur and hemes b and c but lacks Mo and W. Quinones were required for activity in the P. brockii complex but not in the P. abyssi complex, suggesting that the complete electron transport chain is contained within the latter complex. Dissimilatory sulfur reductase from Acidianus ambivalens is a heterotrimer with a 110 kDa catalytic subunit containing a molybdo-bismolybdopterin guanine dinucleotide (MGD) cofactor and one Fe-S center, an Fe-S electron transfer subunit, and a membrane anchor. The catalytic subunit contains a twin arginine (Tat) signal peptide sequence, suggesting that it faces the outside of the cell. Mo, but not W, was found in the solubilized membrane. Sulfolobus quinone is used to shuttle electrons from a membrane-bound hydrogenase to the sulfur reductase. The hyperthermophilic archaeon P. furiosus can reduce elemental sulfur when it is separated from the cells by a porous barrier and can use polysulfide as the electron acceptor. Pyrococcus and Thermococcus differ from Pyrodictium and Acidianus in that they do not appear to use a membrane-bound sulfur/polysulfide reductase for sulfur respiration, nor do they appear to use quinones or cytochromes as electron carriers. Instead, P. furiosus uses a soluble NAD(P)H- and CoA-dependent sulfur reductase whose gene expression increases up to sevenfold when cultures are shifted from growth without elemental sulfur to growth with sulfur. The enzyme is a homodimeric

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flavoprotein. The mechanism for generating a proton motive force is unknown. Dissimilatory sulfate, thiosulfate, and sulfite reduction is found in several hyperthermophilic archaea (Table 1). Sulfate is reduced in three steps (SO42 ! APS ! SO32 ! S2) by three enzymes localized in the cytoplasm. The ATP sulfurylase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a homodimer and activates sulfate using ATP, yielding adenosine phosphosulfate (APS) and pyrophosphate. This and the ATP sulfurylase from the thermophilic bacterium Thermus contain a zinc site that is absent in mesophiles, suggesting that it may be related to thermostability at higher temperatures. Dissimilatory APS reductase from A. fulgidus is a heterodimer that contains FAD and two Fe-S clusters. Dissimilatory sulfite reductase from A. fulgidus has an 2 2 structure and contains siroheme iron, nonheme iron, and acid-labile sulfide. It uses six electrons to reduce sulfite to sulfide. Our understanding of the source of electrons for these reductases and their relationship with the development of a proton motive force is at a rudimentary level. Dissimilatory thiosulfate reduction occurs on the membrane producing sulfite and sulfide, and then the sulfite is reduced in the cytoplasm to sulfide as described above. The amount of thiosulfate reductase in the membrane fraction of P. islandicum cultures increased dramatically in thiosulfate-grown cultures relative to those grown on elemental sulfur and iron. Like the sulfur/polysulfide reductases described above, the thiosulfate reductase in P. islandicum is predicted to be a membrane-bound heterotrimer with MGD and Fe-S cofactors. Dissimilatory sulfite reductase from P. islandicum has biochemical properties that are nearly identical to those of A. fulgidus.

grow without the addition of tungstate; however, concentrations above 0.7 mmol l1 led to a fourfold decrease in dissimilatory nitrate reductase activity. Therefore, tungsten does not replace molybdenum in this metalloenzyme as it does in other thermophiles but apparently is required by other enzymes in the organism. Variations in tungstate concentrations had no effect on nitrite reductase and NO reductase activities. NO reductase from P. aerophilum is homomeric, contains derivatives of heme b, and uses menaquinone as an electron donor. Denitrification to N2O was also measured in Ferroglobus placidus. Oxygen

The majority of thermophiles and especially hyperthermophiles are anaerobes, due in large part to the insolubility of O2 in water at high temperatures and the lack of fluid contact with O2. However, there are several organisms that are obligate aerobes, microaerophiles, or facultative anaerobes (Table 1). As expected, these organisms are generally found in geothermal environments such as in hot springs that interface with oxic environments. Aerobic respiration generally requires electrons carried by cytochrome c that are passed to O2 via cytochrome c oxidase. Various forms of this enzyme are found in Aeropyrum and Pyrobaculum whereas quinol oxidases are found in Sulfolobus and Acidianus. Pyrobaculum oguniense has both cytochrome a and cytochrome o containing heme-copper oxidases. The bc1 complex and the cytochrome o-containing oxidase are present in the membranes of cells grown aerobically and anaerobically whereas the cytochrome a-containing oxidase is only present in aerobically grown cells. The two oxidases have different affinities for O2 and are specialized for microaerophilic and aerobic growth. Metal compounds

Reduction of nitrogen compounds

Denitrification is found in a limited number of hyperthermophilic archaea (Table 1). Nitrate is reduced in four steps (NO3 ! NO2 ! NO ! N2O ! N2) by four enzymes. In contrast to denitrifying bacteria, all four denitrifying enzymes in the hyperthermophilic archaeon P. aerophilum are membrane bound and use menaquinol as an electron donor. Dissimilatory nitrate reductase from P. aerophilum is a heterotrimer that consists of a 146 kDa catalytic subunit with an MGD cofactor and one Fe-S center, an electron transfer subunit with four Fe-S centers, and a membrane anchor with biheme b and quinol-oxidizing capability. Like the sulfur reductase in A. ambivalens, the catalytic subunit contains a twin arginine (Tat) signal peptide sequence, suggesting that it faces the outside of the cell, which is unlike bacterial nitrate reductases that face the cytoplasm. If so, this would significantly influence the manner in which P. aerophilum generates a proton motive force when grown on nitrate. Cultures did not

Two forms of ferric iron are generally used for growth of bacteria and archaea: soluble Fe(III) that is chelated with citrate and insoluble Fe(III) oxide hydroxide (FeO). Several hyperthermophiles grow on FeO whereas only Pyrobaculum and Geoglobus grow on Fe(III) citrate. Often the end product of FeO reduction is insoluble magnetic iron. P. islandicum also can reduce U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). Frequent research questions with mesophilic dissimilatory iron-reducing bacteria are whether they are able to reduce FeO without direct mineral contact and whether polyheme c-type cytochromes are required. The two most commonly studied iron-reducing bacteria are Shewanella and Geobacter. Both require polyheme c-type cytochromes for iron reduction. Shewanella can grow without direct FeO contact by producing an extracellular electron shuttle whereas Geobacter requires direct contact unless a soluble mediator is provided. P. aerophilum and Pyrobaculum arsenaticum can grow without direct FeO contact whereas

Environmental Microbiology and Ecology | Extremophiles: Hot Environments

P. islandicum and Pyrobaculum calidifontis require direct contact. Genome sequence analyses show that P. aerophilum, P. islandicum, and P. arsenaticum lack polyheme c-type cytochromes whereas P. calidifontis contains a cytochrome with eight predicted hemes that is highly homologous to those found in Shewanella and Geobacter. Growth of P. aerophilum and P. islandicum on Fe(III) citrate and FeO is favored at pHs slightly above neutral and at reduction potentials that are above 220 mV. In contrast, growth of P. islandicum on thiosulfate and elemental sulfur is favored at slightly acid pHs and at low reduction potentials (570 mV). Growth of P. aerophilum on nitrate is favored at neutral pH and at reduction potentials above 220 mV. H2 production

The anaerobic catabolism of organic compounds often yields low molecular weight organic compounds (e.g., acetate) and H2. Although common, H2 production (Eo9 ¼ 410 mV) by most bacteria is easily inhibited due to their use of NADH (Eo9 ¼ 320 mV) as the electron donor for the redox reaction (thermodynamically, the midpoint potential (Eo9) of the electron donor should ideally be more negative than that of the electron acceptor). Therefore, this process often requires the presence of a H2 syntroph such as a methanogen in order to keep H2 at low partial pressure. In contrast, Pyrococcus and Thermococcus readily produce H2 as their primary metabolite when grown in the absence of elemental sulfur, their preferred terminal electron acceptor, without a H2 syntroph. The electron donor for H2 production in P. furiosus is ferredoxin (Em,95  C ¼ 471 mV), making the reaction more energetically favorable. The hydrogenase from P. furiosus is membrane bound and receives electrons directly from ferredoxin. The reaction is coupled directly with proton translocation across the membrane and the development of a
and a
pH. ATP synthesis on the membrane was likewise shown to be linked to H2 production. P. furiosus also has two cytoplasmic hydrogenases that use NADH as the electron donor, which are upregulated when cultures are grown without sulfur.

Relationship between Organisms and their Environment The high temperatures and geochemistry found in terrestrial and marine geothermal sites are unique. Volcanically derived gases and products from water– rock reactions support chemolithoautotrophic-based microbial communities in what has been termed the deep, hot biosphere. Endolithic microbial communities are pervasive in these environments and likely contribute significantly to subsurface biomass production, which

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may constitute a significant portion of the total biomass on the planet. The subsurface biosphere is a largely unknown and untapped natural resource. Thermophiles and hyperthermophiles inhabit these environments and serve as model organisms for microbial processes that occur at high in situ temperatures. Although known hyperthermophiles may comprise only a small minority of the total microbial population in a geothermal environment, their metabolisms are likely reflections of the kinds of processes occurring within them. Because they are typically not found in nongeothermal background fluids, they can serve as tracers of in situ chemical and physical conditions within geothermal environments. Before one can use these organisms as models of biogeochemical processes in geothermal environments, there are a number of fundamental questions that must be addressed related to the relationship between hightemperature organisms and their environment. For example, what are the physical and chemical constraints on metabolic processes? Are different forms of thermophile and hyperthermophile metabolism spatially and temporally segregated on the basis of fluid chemistry? Clearly, the presence of thermoacidophiles, thermoneutrophiles, and thermoalkaliphiles shows how pH can influence microbial distributions and metabolisms, but can these types of changes be observed on a finer scale even within the same organism? What are the different ways in which organisms assimilate CO2 or respire a given compound? Are these differences rooted in environmental factors that favor one metabolism over another? Many hyperthermophiles have a requirement for tungsten to meet the needs of certain enzymes found in central metabolic pathways. Are there other unique cofactors used by these organisms? What do these mean with respect to the natural history of these organisms? In conclusion, extremophiles from hot environments have moved from mere curiosity to a group of organisms that have significant medical and biotechnological applications and are useful for the study of the evolution and biochemistry of metabolic pathways and the biogeochemistry of geothermal environments. Many thermophiles and most hyperthermophiles belong to the Archaea, which is the third superkingdom of life for which there is still much to be learned. Because physiology and ecology go hand in hand, the continued study of hightemperature organisms from these two perspectives should expand our appreciation for these organisms and the function they have in nature.

See also: Archaea (overview); Autotrophic CO2 Metabolism; Deep Sub-Surface; Deep-Sea Hydrothermal Vents; High-Pressure Habitats; Iron Metabolism; Methanogenesis

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Further Reading Adams MWW (1999) The biochemical diversity of life near and above 100 C in marine environments. Journal of Applied Microbiology 85: 108S–117S. Brock TD (1978) Thermophilic Microorganisms and Life at High Temperatures. New York: Springer-Verlag. Buchanan BB and Arnon DI (1990) A reverse KREBS cycle in photosynthesis: Consensus at last. Photosynthesis Research 24: 47–53. Cabello P, Rolda´n MD, and Moreno-Vivia´n C (2004) Nitrate reduction and the nitrogen cycle in Archaea. Microbiology 150: 3527–3546. Daniel RM, van Eckert R, Holden JF, Truter J, and Cowan DA (2004) The stability of biomolecules and the implications for life at high temperatures. In: Wilcock WSD, DeLong EF, Kelley DS, Baross JA, and Cary SC (eds.) The Subseafloor Biosphere at Mid-Ocean Ridges. Geophysical Monograph Series, vol. 144, pp. 25–39. Washington, DC: American Geophysical Union Press. Holden JF and Daniel RM (2004) The upper temperature limit for life based on hyperthermophile culture experiments and field observations. In: Wilcock WSD, DeLong EF, Kelley DS, Baross JA, and Cary SC (eds.) The subseafloor biosphere at mid-ocean ridges. Geophysical Monograph Series, vol. 144, pp. 13–24. Washington, DC: American Geophysical Union Press.

Kletzin A (2007) Metabolism of inorganic sulfur compounds in archaea. In: Garrett RA and Klenk HP (eds.) Archaea: Evolution, Physiology, and Molecular Biology, pp. 261–274. Malden, MA: Blackwell Publishing. Petsko GA (2001) Structural basis of thermostability in hyperthermophilic proteins, or ‘‘there’s more than one way to skin a cat.’’ Methods in Enzymology 334: 469–478. Scha¨fer G, Engelhard M, and Mu¨ller V (1999) Bioenergetics of the Archaea. Microbiology and Molecular Biology Reviews 63: 570–620. Schleper C, Jurgens G, and Jonuscheit M (2005) Genomic studies of uncultivated Archaea. Nature Reviews 3: 479–488. Stetter KO (1990) Extremophiles and their adaptation to hot environments. FEBS Letters 452: 22–25. Verhees CH, Kengen SWM, Tuininga JE, et al. (2003) The unique features of glycolytic pathways in Archaea. Biochemistry Journal 375: 231–246. Woese CR, Kandler O, and Wheelis ML (1990) Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of Sciences of the United States of America 87: 4576–4579. Wood HG and Ljungdahl LG (1991) Autotrophic character of the acetogenic Bacteria. In: Shively JM and Barton LL (eds.) Variations in Autotrophic Life, pp. 201–250. New York: Academic Press.

Food Webs, Microbial E B Sherr and B F Sherr, Oregon State University, Corvallis, OR, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Understanding Microbial Food Webs Components and Pathways Microbes in Aquatic Food Webs Marine Pelagic Habitats Benthic Habitats

Glossary gross growth efficiency (GGE) Percentage of ingested food (I) that is converted into microbial biomass (Y ¼ yield), that is GGE ¼ Y/I, expressed in units of volume, dry weight, carbon, nitrogen, or energy. Carbon-based GGEs are typically on the order of 10–50% for bacteria and phagotrophic protists. microbial loop Amended model of pelagic food webs in which heterotrophic bacteria and their protistan grazers consume a large part of primary production, mostly via uptake of dissolved organic matter (DOM) by the bacteria, and in the process account for a large share of elemental recycling. microzooplankton Phagotrophic planktonic organisms in the general size range of 20–200 mm; includes larger-sized heterotrophic protists, notably ciliates and heterotrophic dinoflagellates. mixotrophy Mixture of trophic modes, generally combining photosynthesis with the ingestion of particles. phagotrophy Mode of feeding by which particles, usually other microbial cells, are ingested.

Abbreviations CFB DMS

Cytophaga–Flavobacteria–Bacteriodes dimethyl sulfide

Role of Microbial Food Webs in Biogeochemical Cycling Food Resource for Metazoans Modeling Microbial Food Webs Chemical Interactions between Microbes Spatial Structure of Microbial Food Webs Further Reading

picoplankton Planktonic organisms less than 2 mm in size; includes most heterotrophic bacteria as well as unicellular cyanobacteria and some small eukaryotic cells. primary producers Microbes that grow, or produce biomass, autotrophically, by photosynthesis or chemosynthesis. top-down/bottom-up controls Factors controlling biomass stocks and rate of biomass production of a particular group of organisms in an ecosystem. Top-down controls are usually due to predation by higher trophic levels; bottom-up controls refer to availability of resources, for example, inorganic nutrients, organic substrates, or prey cells, required for growth. trophic cascade Top-down control of predatory protists that results in their normal prey populations being relieved from grazing mortality and growing at a faster rate. trophic level or compartment Assemblage of organisms responsible for a major function in a food web, for example, primary production, decomposition, consumption of primary producers, consumption of heterotrophic bacteria.

GGE NPZ TEP

gross growth efficiency nutrient–phytoplankton–zooplankton transparent exopolymer particles

Defining Statement

Introduction

In natural ecosystems, microbial food webs consist of predator–prey interactions of unicellular prokaryotes and eukaryotes. In this article, we focus on the structure, and ecological and biogeochemical importance, of microbial food webs in aquatic ecosystems, and particularly in the oceans.

In his book The Ecological Theater and the Evolutionary Play, limnologist G. Evelyn Hutchinson proposed that the environment provides the stage for the drama of evolution of species. If so, microbes are the stage hands, ceaselessly building and remolding the set. For a long

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time in Earth’s history, microbes were the only actors as well, carrying out in microscale the script of production, predation, and dissolution; in other words, microbial food webs. Microbial food webs are similar in some ways to the familiar lynx and hare predator–prey interactions of nature shows. In other ways, they are not. A notable difference is that microbial food webs have an enormously important role in decomposing the plant carbon and all of the other components feces, dead bodies, and so forth, of the macroscopic world. Most microbes are single prokaryotic or eukaryotic cells, although some form either filamentous chains or colonies of single cells, and fungi produce multicellular fruiting bodies. Microbes form multispecies communities, and thus food webs, throughout the biosphere, including some habitats where multicellular life cannot exist. In natural systems, a large proportion of prokaryotic, or bacterial, cells present in an environment may be relatively inactive, or dormant, but able to start growing when conditions are favorable. Unicellular eukaryotes, or protists, may also form resting stages. Microbial ecologists refer to the total complement of microbes, both active and inactive, in a habitat as the microbial assemblage. The microbial community refers to the subset of microbes that are actively growing and metabolizing at any one time. Microbial food webs are organized into trophic levels, or compartments, depending on their function. Primary producers make up the first level, or bottom, of a food web. Decomposing organisms, heterotrophic bacteria, and fungi grow on nonliving organic matter. Phagotrophic protists can feed on single or multiple compartments of a food web, depending on the consumers’ size and feeding capability. The combined activities of microbial communities result in large-scale cycling of bioactive elements – carbon, nitrogen, phosphorus, sulfur, and trace metals – in ecosystems.

Understanding Microbial Food Webs The concept of systems ecology was crucial to understanding the role of microbes in ecosystems. The text Fundamentals of Ecology by Eugene and Howard Odum, first published in 1953, established the systems approach, which focused on ecosystem function rather than on specific populations, and followed the flows of elements such as carbon or nitrogen, or of energy, either solar energy or energy from the respiration of organic compounds, through food web compartments. Initial formulations were linear food chains, from primary producers, which captured solar energy and produced organic matter from carbon dioxide and other inorganic compounds, to primary and secondary consumers; for example, grass to antelopes to lions. Decomposing organisms, heterotrophic bacteria and fungi, were known to be important components of

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ecosystems, but did not comfortably fit into such a food chain. Early studies of the roles of heterotrophic microbes in ecosystems focused on the rates of respiration of organic carbon to carbon dioxide and on regeneration of bioactive elements from organic compounds into inorganic compounds such as ammonium and phosphate, which algae and higher plants could use for growth. The notion of trophic interactions between different groups of microbes was developed during research on marine ecosystems during the 1970s and 1980s. Microbial ecologists found that microbial plankton were responsible for the bulk of respiration in the sea. They also highlighted the role of small flagellated protists as consumers of bacteria in seawater. Bacterivorous flagellates kept bacterial stocks in check and at the same time regenerated much of the nitrogen and phosphorus accumulated in bacterial cells. Subsequent work showed that the bacterivorous flagellates were in turn grazed by larger-sized protists, establishing a microbial food chain that resulted in virtually all of the organic carbon used by bacteria being recycled back to carbon dioxide and inorganic nutrient compounds. Similar microbial food chains, from bacteria to flagellates to larger protists, were found in freshwater ecosystems and in benthic habitats in marine and freshwater environments. Further research on microbes in aquatic ecosystems showed that this microbial food chain, termed the microbial loop, was too simplistic. Both small flagellates and larger protists also fed on autotrophic cells, including photosynthetic bacteria, and on algae of all sizes. Mixotrophic phytoflagellates could ingest bacteria. Viruses infected and lysed both bacterial and algal cells, causing a short circuit of the microbial loop. A more sophisticated view of a complex microbial food web that included autotrophic, mixotrophic, and heterotrophic microbes and formed the basic food resource for metazoans such as copepods and larvae of pelagic and benthic animals emerged. Microbial food webs in terrestrial systems are simpler in that primary production is carried out by large multicellular plants, and microbes are limited to either decomposition of plant material or feeding on decomposing microbes. In this article, we will focus on the microbial food webs of aquatic ecosystems, and particularly of the ocean.

Components and Pathways Aquatic microbial food webs consist of producer and consumer compartments. Examples of microbial producers and consumers in aquatic food webs visualized via epifluorescence microscopy are shown in Figure 1. Epifluorescence microscopy is a method used by microbial ecologists to inspect cells that are either autofluorescent by virtue of their pigments, such as chlorophyll or phycobilins or made fluorescent by

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staining with dyes that bind to organic compounds in microbial cells and fluoresce at selective wavelengths. In Figure 1, autofluorescent cells fluoresce red, while heterotrophic prokaryotes and phagotrophic protists fluoresce blue due to added DAPI stain.

Microbial species also include a large size range (Figure 2). Most planktonic bacteria are about half a micron in size. The largest-sized phytoplankton and protists are 100–200 mm in length. Cell size is important in microbial food webs since most, although not all,

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(b)

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Figure 1 Examples of autotrophic and heterotrophic microbes in microbial food webs, visualized by epifluorescence microscopy with DAPI staining and sized using an image analysis system consisting of a Cooke Sensicam QE CCD camera with Image Pro Plus software mated to an Olympus BX61 Microscope with a universal fluorescence filter set. Red color indicates autofluorescence of photosynthetic pigments, blue color indicates DAPI staining of nonfluorescent cells and cytoplasm; the brightest blue staining occurs in the nucleus of the cells. (a) A mixed species bloom of diatoms, major algal producers in aquatic ecosystems, in the western Arctic Ocean, scale bar ¼ 50 mm. (b) Planktonic prokaryotes stained with DAPI in water collected in a Georgia salt marsh estuary, scale bar ¼ 2 mm. (c) Bacterivorous nanoflagellates, probably choanoflagellates, in a decaying diatom bloom in the western Arctic Ocean, scale bar ¼ 10 mm. (d) Two heterotrophic gyrodinium-type dinoflagellates with red-fluorescent food vacuoles full of picoplankton-sized autotrophic cyanobacteria and picoeukaryotes from the western Pacific Ocean off the coast of Oregon, USA, scale bar ¼ 20 mm.

Primary producers PLANKTON and NEKTON Virioplankton

FEMTO0.02–0.2 µm

PICO0.2–2 µm

Microbial consumers NANO2–20 µm

MICRO20–200 µm

MESO0.2–20 mm

Multicellular consumers MACRO2–20 cm

MEGA20–>200 cm

Centimeter nekton

Decimeter to meter nekton

Bacterioplankton Phytoplankton Protozooplankton Metazooplankton Nekton

Figure 2 Distribution of different taxonomic-trophic compartments of plankton in a spectrum of size fractions, with a comparison of size ranges of zooplankton and nekton. Solid rectangles denote size of most organisms in each size group, bars denote approximate minimum/maximum size range of group. Blue bars, heterotrophic microbes; green bar, autotrophic microbes (phytoplankton); purple bars, animals. Figure is updated from a figure published by John Sieburth and colleagues in an article in Limnology and Oceanography in 1978.

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phagotrophic protists feed on organisms smaller than themselves. The smallest microbial cells are less than 2 mm in diameter. This size category, which includes most aquatic prokaryotes, heterotrophic and autotrophic Bacteria and Archaea, and the smallest eukaryotic cells, is termed picoplankton. Nanoplankton, cells 2–20 mm in size, includes most species of flagellates, autotrophic, heterotrophic, and mixotrophic, along with some smallersized nonflagellated green algae and diatoms and the smallest species of dinoflagellates and ciliates. Microplankton, cells and chains of cells 20–200 mm long, covers the larger-sized phytoplankton, mainly single cells and chains of diatoms and larger species of photosynthetic dinoflagellates, and the larger-sized phagotrophic protists, ciliates, and heterotrophic dinoflagellates. Phagotrophic protists in the plankton greater than about 20 mm are termed microzooplankton and are major consumers, or grazers, of phytoplankton in marine and freshwater systems. Viruses that occur in all aquatic systems and are less than 0.2 mm, or 200 nm, are categorized as femptoplankton. At the bottom level of microbial food webs are the primary producers, usually photosynthetic bacteria and algae. However, in hypoxic habitats in the water column and the benthos, primary producers may be chemosynthetic bacteria that are able to gain energy by the oxidation of reduced chemicals such as sulfide, reduced iron, or methane. Such bacteria form the base of food webs at hot springs on land and at hydrothermal vents and methane seeps on the seafloor. Trophic level in aquatic food webs is not easily segregated according to the size of the microorganism, since individual species of both heterotrophic and autotrophic microbes occur across the entire range of microbial size categories. In the open ocean and in large lakes, less than 2-mm-sized coccoid-shaped cyanobacteria can be important primary producers. In coastal and shallow water systems, massive blooms of microplankton-sized algae, either diatoms or dinoflagellates, can occur. To make matters more complicated, some species of eukaryotic microbes in all size ranges are mixotrophic. Species of autotrophic flagellates, or phytoflagellates, in both picoplankton and nanoplankton size ranges are capable of ingesting heterotrophic bacteria and even small phototrophic cells. It is likely that most photosynthetic dinoflagellates are also phagotrophic, preying on both autotrophic and heterotrophic protists. Some ciliates temporarily hold chloroplasts from their algal prey just below the cell membrane and use sugars produced by the chloroplasts for supplemental nutrition. One species of marine ciliate, Mesodinium rubrum, has taken this form of mixotrophy to the extreme. Its captured chloroplasts have retained DNA from the original cryptophyte algal prey and are capable of division along with the host ciliate. Mesodinium, unlike other ciliates, is thus primarily

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autotrophic and can even form blooms in the ocean, although the ciliate is still capable of phagotrophy. There are other types of symbiotic mixotrophy in which one type of microbe lives in or on, and contributes to the metabolism of, another microbe. Examples include nitrogen-fixing cyanobacteria that live in diatom and heterotrophic dinoflagellate host cells, helping them survive in nitrogen-poor environments, and chemosynthetic sulfur-oxidizing Bacteria that live on the cell membranes of benthic ciliates, which essentially farm the chemosynthetic bacteria as their main food resource. Although trophic interactions between microbes in aquatic food webs are more complicated than trophic interactions in macroscopic food webs, the cast of characters in microbial food webs is less so. Most water column, or pelagic, food webs in both the ocean and lakes have a consistent assortment of major groups of microbes with similar trophic roles. Taxonomic groups of microbes appear to be much more uniformly distributed in the ocean, and in lakes, compared to the distribution of species of, for example, copepods and fish, in aquatic systems. Of course, this apparent uniformity may only be a result of the lack of knowledge of genetic differences among strains of distinct microbial species in different habitats. The general taxonomic groups of microbes in aquatic food webs, and their functions, are listed below.

Microbes in Aquatic Food Webs Heterotrophic prokaryotes: Most of these are less than 1-mm-sized species in the domain Bacteria and live by assimilating dissolved organic compounds from water or by degrading nonliving detrital organic matter. Species in the domain Archaea are also present everywhere in the sea and in freshwater habitats. There are four groups of Archaea in the marine pelagic environment; the most abundant of these are Marine Group 1 Archaea in the Crenarchaeota, the same subdomain as sulfur-oxidizing Archaea living in hot springs or in hydrothermal vents. The other marine Archaea (Marine Groups 2, 3, and 4) are in the Euryarchaeota and include methanogenic and halophytic prokaryotes. Very little is known about the modes of metabolism of most of the marine Archaea. Some Marine Group I Archaea have been found to assimilate amino acids. However, archaeal cells are most abundant in the sea at depths of 200–4000 m, where there is little organic carbon. It has been established that the cold-temperature Crenarchaeota present in the ocean can gain energy for growth by oxidizing ammonium. A member of the Marine Group I Archaea isolated from a marine aquarium tank has been shown to grow chemoautotrophically in culture by oxidizing ammonium and assimilating carbon dioxide.

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Microscopic and flow cytometric methods used to enumerate heterotrophic prokaryotes in aquatic systems are based on fluorescent staining of cells, which does not distinguish between species of Bacteria and Archaea. For this reason, the general term bacteria, which has long been used by microbial ecologists to mean heterotrophic prokaryotes, is assumed to include cells in both domains. Bacterioplankton refers to heterotrophic prokaryotic cells suspended in seawater or freshwater. Most strains of aquatic bacteria are in the bacterial phylum Proteobacteria. In the sea, marine bacteria are typically strains of alpha-Proteobacteria, which includes the most abundant open ocean ribotype, the SAR-ll clade, and of gamma-Proteobacteria, which includes fast-growing opportunistic strains of the genera Pseudomonas and Vibrio. In freshwater habitats and in some coastal and estuarine environments, strains of beta-Proteobacteria are abundant. In eutrophic and benthic habitats, heterotrophic Bacteria grow on surfaces, forming colonies and biofilms that are an important food resource for small invertebrates. Bacteria adapted to attach to and grow on surfaces are phylogenetically diverse, and commonly include ribotypes in the Cytophaga–Flavobacteria– Bacteriodes (CFB) group as well as in other groups of Bacteria. Autotrophic prokaryotes: Coccoid cyanobacteria, photosynthetic Bacteria less than 2 mm in diameter; are ubiquitous in marine and freshwater systems. There are two major groups of these picocyanobacteria: orange-fluorescing Synechococcus spp., which have chlorophyll a and phycobiliprotein accessory pigments, and red-fluorescing Prochlorococcus spp., which have modified chlorophyll pigments, divinyl chlorophyll a and divinyl chlorophyll b as the main accessory pigment. Prochlorococcus spp. are smaller in size than Synechococcus spp., and are typically abundant in open ocean habitats, while Synechoccocus spp. are most abundant in nearshore to outer continental shelf waters. Synechococcus spp. are also abundant in the water column of lakes, while Prochlorococcus spp. are predominantly marine. Filamentous cyanobacteria are common in polluted freshwaters and hot springs. In the ocean, the filamentous cyanobacteria Trichodesmium spp. form blooms in subtropical regions and are globally important nitrogen fixers. Non-oxygen-producing, bacteriochlorophyll-containing Bacteria, which require a source of reduced compounds to grow, can be significant primary producers in lakes and marine systems that have subsurface anoxic water masses rich in sulfide. In addition, many strains of heterotrophic bacteria living in oxic aquatic habitats have been found to contain either bacteriochlorophyll or bacteriorhodopsin pigments, which may be used to generate extra ATP by using energy harvested from light. In benthic habitats, chemosynthetic Bacteria can support both microbial and macroscopic food webs. These autotrophs include free-living single-celled and

filamentous strains of sulfur-oxidizing Bacteria, such as species of Beggiatoa and Thiospirillum, and symbiotic sulfur-oxidizing Bacteria living in or on both single-celled and multicellular eukaryotes. Examples are the sulfuroxidizing Bacteria that grow on the benthic ciliate Zoothamnium niveum, which lives at the oxic–anoxic interface in sandy marine sediments and provides its bacterial crop with both sulfide from below and oxygen from above, and the symbiotic sulfur-oxidizing Bacteria that grow in special organs of gutless hydrothermal vent tube worms, providing the worms with all of their nutrition. At methane seeps on the seafloor, methane-oxidizing Bacteria grow in the sediments and in the gill tissues of seep mussels, providing most of the primary production for these ecosystems. Autotrophic eukaryotes: Also termed algae, singlecelled photosynthetic eukaryotes are the most significant primary producers both in the sea and in lakes. Algal cells are diverse both in size and in taxonomic diversity. The smallest algal cells are 0.8 mm-diameter marine Ostreococcus spp. and 1–2 mm diameter Micromonas spp, both abundant in the open ocean. There is a great diversity of algal species in the nanoplankton size range. Most of these are golden brown-pigmented, flagellated chrysophytes and prymnesiophytes, orange-pigmented cryptophytes, and green-pigmented prasinophytes, although nonflagellated chlorophytes and diatoms also occur in this size range. Algae larger than 20 mm are less abundant than smaller-sized phytoplankton, but at times form dense blooms in coastal waters or in lakes. Bloomforming algae greater than 20 mm are typically diatoms or autotrophic dinoflagellates. Many flagellated algae, including chloroplast-bearing nanoflagellates and dinoflagellates, can ingest other microbial cells. Some species of algae capable of ingesting bacteria cannot grow in the absence of prey. Heterotrophic eukaryotes: These microbes were known as protozoa, and researchers often still use that term. However, many species of heterotrophic eukaryotes are close kin to photosynthetic species. The word Protozoa, which means first animal life in Greek, is thus not an appropriate label for these microbes, and we prefer to use the term heterotrophic protist. Heterotrophic protists, which do not have chloroplasts, are as ubiquitous and as diverse as autotrophic protists, the algae. There are some protist lineages: the bodonids, the choanoflagellates, and the kinetoplastids, that do not have any chloroplastcontaining species, and are strictly phagotrophic. The choanoflagellates are of particular interest to molecular geneticists as they are the group of single-celled protists most closely related to multicellular animals. Phagotrophic protists have the potential to be major predators in microbial food webs because they are in the same general size range as their microbial prey, bacteria, algae, and other heterotrophic protists (Figure 2), and

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because protist growth rates are on the same temporal scale, hours to days, as those of their prey. The high rate of metabolism of these small, unicellular predators also facilitates carbon and energy flux through ecosystems. The smallest heterotrophic protists are 1- to 2-mmsized flagellated species, which occur in several protist groups, including the chrysophytes and bodonids. Heterotrophic protists 2–20 mm in size, mainly nanoflagellates (e.g., Figure 1(c)), are very diverse taxonomically and are major consumers of picoplankton and smallersized nanoplankton cells in aquatic systems. Some species of ciliates and heterotrophic dinoflagellates are also less than 20 mm. Phagotrophic protists larger than 20 mm are predominately ciliates and non-chloroplast-containing dinoflagellates, Examples of these protists are shown in Figure 3. This size class of phagotrophic protist, termed microzooplankton, is abundant in the sea and in lakes, and these protists are major consumers of phytoplankton and of heterotrophic nanoflagellates. Planktonic ciliates are mainly spherical or conical spirotrichs, cells with cilia grouped around an oral end. One subgroup of spirotrichous ciliates, the tintinnids, build species-specific (a)

(d)

(b)

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houses, or loricae, in which they live, serving as a protective shelter (Figure 3(b)). Heterotrophic dinoflagellates may have rigid cellulosic plating, or armor, which gives them a distinctive shape (e.g., the cell in Figure 3(d)) and makes it difficult, though not impossible, for them to ingest prey cells directly. Many armored dinoflagellates instead feed externally by extruding a hollow tube into, or a pseudopodial veil around, their algal prey. The dinoflagellate injects digestive enzymes into the prey cells and sucks the digested prey cytoplasm back into itself by these feeding structures. Most species of heterotrophic dinoflagellate are nonarmored, and have an elastic cell membrane that allows them to ingest algal prey up to a size equal to, or even greater than, the dinoflagellate, often greatly distending the dinoflagellate cell in the process (Figures 3(e) and 3(f )). In benthic habitats, where the fluid environment interacts with surfaces such as grains of sand, clay particles, or organic detritus, the protist community is dominated by surface-feeding hymenostome ciliates similar to Paramecium spp., whose main food is bacteria. Some of these are very long and thin, adapted to move through

(c)

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Figure 3 Examples of aquatic phagotrophic protists in the 20–200 mm, or microzooplankton, size class. Cells were preserved and stained with iodine-based acid Lugol solution, which gives them a brown color, visualized via light microscopy, and sized and photographed using an image analysis system consisting of a Cooke Sensicam QE CCD camera with Image Pro Plus software mated to an Olympus BX61 Microscope. (a) Strombidium sp. ciliate, consumer of nanoplankton-sized microbial prey from the western Arctic Ocean; (b) Tintinnopsis sp. tintinnid ciliate in an aggregated lorica, consumer of nanoplankton-sized prey from the western Arctic Ocean; (c) Haptorid ciliate, species unknown, a predatory ciliate that feeds on cells as large as itself from the western Arctic Ocean; (d) Protoperidinium sp. armored heterotrophic dinoflagellate that feeds on large cells including diatoms using an extracellular pseudopodial feeding veil from the Oregon upwelling system of the western Pacific Ocean; note flagellum trailing behind the cell; (e) Gyrodinium sp. nonarmored heterotrophic dinoflagellate associated with a diatom bloom in the western Arctic Ocean; (f) Gyrodinium sp. nonarmored heterotrophic dinoflagellate distended from an ingested chain of the diatom Thalassiosira sp., associated with a diatom bloom in the Oregon upwelling system. All scale bars ¼ 20 mm.

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narrow spaces between sediment particles, and some have stiff ventral cilia that allow them to scrape bacterial biofilms off particle surfaces. Amoebae and amoeboid flagellates are also common in benthic environments and on detrital particles in the water column. Phagotrophic protists are important components of microbial food webs in extreme habitats such as sea ice, solar salterns with salinities as high as 150 parts per thousand, and the deep ocean.

Marine Pelagic Habitats The oceans, which cover more than two-thirds of the earth’s surface, provide large and variable habitats for microbial food webs. In the euphotic zone, the upper lighted depths of the sea, approximately from the surface to 50–200 m depending on the region and season, photosynthetic microbes, both prokaryotic and eukaryotic, provide a continuous source of organic carbon and prey cells for heterotrophic bacteria and protists. At subeuphotic depths, where there is insufficient sunlight for photosynthesis, heterotrophic microbes live on organic carbon sinking down from the euphotic zone. At these depths, the microbial food web regenerates most of the sinking organic carbon back to carbon dioxide and inorganic nutrients. The largest oceanic habitats are the slowly rotating gyres at the center of each of the great ocean basins. These subtropical open ocean regions have warm surface waters and low amounts of inorganic nutrients for phytoplankton growth. They are oligotrophic or nutrient limited. Phytoplankton cells are small-sized, dominated by cyanobacteria and small algal species, and the total biomass of primary producers is low. Microbial food webs are complex, with small heterotrophic protists and mixotrophic flagellated algae ingesting heterotrophic bacteria and cyanobacteria, slightly larger protists feeding on nanoalgae and on bacterivorous flagellates, and the largest protists feeding on algal cells and on bacterivorous protists. Most of the primary production is respired in this multicompartment microbial food web, with little remaining for macroscopic marine life. At the edge of continents, the oceans cover continental shelves of varying depths and widths. Continental shelf waters are dynamic, and occur in subtropical, temperate, and polar regions. Most shelf systems are mesotrophic, with higher biomass of phytoplankton and of other microbes compared to the oligotrophic gyres. Upwelling or injection of nutrient-rich subsurface water frequently occurs at the edge of the shelf, resulting in eutrophic conditions with high phytoplankton biomass and production. Some regions, such as the narrow continental shelves of the Pacific Northwest of the United States, Northwest Africa, and Peru and Chile in South America,

have seasonal or persistent upwelling of nutrient-rich seawater extending to the coast, which results in massive phytoplankton blooms, usually of species of diatoms. Phytoplankton blooms also occur each spring in temperate and polar regions due to increase in day length from winter to summer. Much of the organic matter produced in these mass phytoplankton blooms sinks down to subsurface depths or to the sediment. Some of the bloom production is utilized by heterotrophic bacteria and by microzooplankton-sized protists capable of preying on large algal cells and chains of cells (e.g., the dinoflagellate in Figure 3(f)). At the margins of the oceans are shallow nearshore and estuarine habitats influenced by inputs of freshwater from rivers or land runoff and by interactions between the water column and the sediments. Depending on their geographic location and local conditions, nearshore and estuarine systems may be oligotrophic, mesotrophic, or eutrophic. Agriculture and other human activity has resulted in a large increase in the amount of plant nutrients such as nitrate and phosphate carried by rivers and by land runoff to nearshore marine systems. As a result of enhanced phytoplankton growth due to these nutrients, microbial food webs in some nearshore regions respire enough organic matter to deplete the water of oxygen, resulting in hypoxic or anoxic dead zones. A classic example is the large and growing zone of low oxygen water off the Mississippi River in the Gulf of Mexico. Hypoxic and anoxic marine habitats also occur naturally beneath persistent upwelling regions and in some enclosed fjords and basins, for example, the Black Sea. Most of the pelagic habitat of the ocean is subsurface, below 200 m. Only a small fraction of primary production in the euphotic zone sinks out to depths below 200 m. Microbes that live below that depth are much less abundant than in the euphotic zone and must adapt to highly stressful conditions of low food, cold temperatures, and high pressure. Both Bacteria and Archaea, as well as heterotrophic flagellates, occur at depth in the sea. Coldtemperature Crenarchaeota are relatively more abundant compared to Bacteria in this habitat. Currently, not much is known about how deep ocean bacteria survive, or about deep ocean microbial food webs.

Benthic Habitats Microbial food webs in aquatic sediments are shaped by the characteristics of benthic habitats. In sediments, microscale structure results in large changes in environmental conditions, for example, redox potential, oxygen and nutrient concentrations, over millimeter to centimeter spatial scales. Particles, both inorganic and organic, are a dominant component of the benthic environment; thus, interactions with particles, for example,

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attachment to, and grazing on, surfaces, are of major importance for microbes living in and on sediments. Benthic ecosystems are mainly heterotrophic; the microbial food web is based on input of organic matter that settles to the bottom as a result of primary production carried out in the water column. Exceptions are hydrothermal vents and methane seeps, where chemosynthetic sulfur-oxidizing Bacteria or methanotrophic Bacteria form the basis of local food webs. Where the input of organic matter to sediments is high relative to the supply of oxygen, suboxic/anoxic habitats dominate in the benthos. Depth zones in the sediment reflect sequential utilization of compounds as electron acceptors with depth: oxygen, nitrate, sulfate, and carbon dioxide. Food webs in anoxic environments are shorter compared to food webs in oxic environments due to lower growth efficiencies of anaerobic microbes. The fine-scale habitat differentiation in benthic habitats, in terms of water chemistry, oxidizing/reducing conditions, and sediment texture, yields a high diversity of potential niches for microbes. Since particle surfaces predominate in the benthos, microbial biofilms are prevalent on both organic detrital particles and inorganic grains of sand or clay. Microbial exopolymers, high molecular weight polysaccharide or mucopolysaccharide secretions, are copiously made by benthic bacteria and microalgae such as diatoms. Exopolymers create a microenvironment around a microbial cell, buffering it from rapid environmental changes in pH, salinity, dessication, or nutrient regimes. The depth to which oxygen is present in sediments depends both on sediment composition: loose sandy sediments allow oxygen to penetrate to a greater depth compared to compact clay sediments, and on the amount of organic matter reaching the sediments: the more organic matter, the greater the rate of oxygen utilization. In coastal waters, usually only the sediment–water interface and the upper few millimeters or centimeters is near oxygen saturation. Oxygen is supplied mainly by diffusion from the overlying water; however, in shallow sediments, some oxygen may be provided by microalgal photosynthesis, and in all sediments, bioturbation by invertebrates results in local oxidizing zones in the top few centimeters, or deeper, in the sediment. In marine unperturbed sediments there is a standard sequence of redox zones, compounds used as electron acceptors, and associated metabolic processes of microbes with depth (Figure 4). In the upper layer of the sediment in contact with overlying waters, respiration of oxygen by prokaryotes and protists occurs. Where overlying water is rich in nitrate, anaerobic nitrate respiration to nitrite by prokaryotes and some protists, or denitrification to nitrogen gas by denitrifying Bacteria, dominates when oxygen concentration is depleted. Deeper in the sediment, both oxygen and nitrate are exhausted, but the interstitial

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Sediment Microbial processes in a marine sediment profile surface ----------------------------------------------------------Zone 1. Oxic, oxygen present Aerobic respiration of organic carbon, nitrification (oxidation of ammonium and nitrite), sulfide oxidation, and Methane oxidation. ----------------------------------------------------Zone 2. Hypoxic, low oxygen, nitrate present Nitrate respiration of organic carbon, denitrification (special case of nitrate respiration), some fermentation, and methane oxidation ----------------------------------------------------Zone 3. Upper anoxic, no oxygen, sulfate present Sulfate respiration of low molecular weight organic compounds, fermentation, and Methane oxidation using sulfate as the electron acceptor ----------------------------------------------------Zone 4. Lower anoxic, no oxygen, no sulfate Methanogenesis and fermentation

Figure 4 Sequence of redox zones and associated microbial processes with depth in an idealized sediment profile. Moving down from the sediment surface the sequence is as follows: (Zone 1) oxic zone: high oxygen concentration at the sediment surface; aerobic respiration, sulfide oxidation, nitrification, and methane oxidation; (Zone 2) hypoxic zone: low oxygen and measurable nitrate concentration; anaerobic nitrate-based respiration, denitrification, some fermentation, and methane oxidation; (Zone 3) upper anoxic zone: no oxygen but sulfate present, sulfate respiration, sulfide formation, fermentation, and methane oxidation using sulfate as the electron acceptor; (Zone 4) lower anoxic zone: no oxygen or sulfate, methanogenesis using carbon dioxide as the electron acceptor and fermentation.

seawater is still rich in sulfate. In this zone, sulfate-respiring Bacteria grow on hydrogen and fatty acids produced by anaerobic fermenting microbes. The end product of sulfate respiration is sulfide, which builds up in anoxic marine habitats, producing a characteristic rotten egg smell. Still deeper, sulfate is depleted and microbial metabolism is mainly based on methanogenesis by Archaea and fermentation by Bacteria and protists. In the anoxic zones of both marine sediments and oxygen-depleted water masses, sulfate-respiring Bacteria and methanogenic Archaea compete for the metabolites of fermenting bacteria: hydrogen and low molecular weight organic compounds, particularly acetate. Sulfatereducing Bacteria are better competitors and can grow at lower hydrogen concentrations than can methanogens. Thus in marine anoxic habitats, sulfate reducers outcompete methanogens in zones where there are significant concentrations of sulfate. Because sulfate respirers and methanogens can utilize only low molecular weight organic substrates, fermenting microbes are primarily responsible for degradation of particulate detritus and high molecular weight organic compounds in anaerobic sediments. Two major chemoautotrophic processes occur in marine sediments and water columns where there is an interface between oxic and anoxic habitats. Nitrification occurs when oxygen and ammonium are present together.

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The two phylogenetically distinct groups of nitrifying Bacteria produce energy for carbon fixation by the respective oxidation of ammonium to nitrite and nitrite to nitrate. When oxygen and sulfide are present together, sulfur-oxidizing Bacteria produce energy for carbon fixation by oxidation of reduced sulfur compounds. This is the major source of fixed carbon at hydrothermal vents. The microbial food web of most marine sediments and anoxic water masses consists mainly of detrital organic matter consumed by heterotrophic prokaryotes, which in turn are consumed by bacterivorous protists. However, in coastal and intertidal marine sediments where there is sufficient light, benthic algae are a component of food webs. Ciliates are abundant in benthic habitats and consume both bacteria and algae; the roles of heterotrophic flagellates and amoebae in sediments are less well known. Anaerobic metabolism is inherently less energetically efficient than is aerobic metabolism. Fermenting organisms typically grow with a gross growth efficiency (GGE) of substrate use of 10%, while aerobic microbes can transform 40% of assimilated organic carbon into biomass. Any process that serves to enhance the low growth efficiencies of anaerobic organisms would give a competitive edge to such organisms. Anaerobic ciliates are characteristic of both marine and freshwater anoxic habitats. These protists generate energy by fermentation of organic compounds obtained by ingesting other microbes, primarily bacteria. In these ciliates, the fermentative processes resulting in oxidation of pyruvate and production of hydrogen occur in unique organelles, hydrogenosomes. Hydrogenosomes appear to be modified mitochondria that have lost the electron transport system. Many species of anaerobic ciliates are full of endosymbiotic prokaryotes. When excited by blue light, the cells fluoresce blue-green, a characteristic of methanogenic Archaea. The observation of methane generation in these protists, along with molecular genetic analysis of the endosymbionts, has confirmed that they are in fact methanogens. The cytoplasm of the fermenting ciliate is a microhabitat with high abundance of hydrogen, acetate, and carbon dioxide – waste products of the host and substrates for methanogens. This is of particular significance in marine anoxic habitats, where high concentrations of seawater sulfate foster the growth of anaerobic sulfate-respiring Bacteria, which outcompete nonsymbiotic methanogens for available hydrogen and fatty acids. In turn, the endosymbiotic methanogens make the metabolism of the ciliate more efficient by decreasing fermentation waste products in the cell and by serving as a food resource for the ciliate. This unique microbial collaboration is a classic case of syntropy, literally feeding together, in which two organisms grow in a mutually beneficial, intimate association.

Role of Microbial Food Webs in Biogeochemical Cycling Microbes can be viewed as the chemical engineers of the biosphere. Biogeochemical cycles of carbon, nitrogen, and sulfur cannot occur without specific metabolic capabilities of various groups of prokaryotes. For many of the cycles of bioactive elements, interactions of both prokaryotic and eukaryotic species in microbial food webs are required for completion of the pathways of the elements. Conversion of organic carbon, organic nitrogen, and organic phosphorus into inorganic compounds, namely, carbon dioxide, ammonium, and phosphate, is facilitated by consumption of prokaryotic and eukaryotic prey cells by phagotrophic protists. The dominant degradation pathway of organic matter produced by autotrophic microbes, that is, primary production, in aquatic ecosytems is assimilation and respiration by heterotrophic prokaryotes, both Bacteria and Archaea. Prokaryotes utilize organic matter in many forms and steps in aquatic food webs: in the water column as dissolved organic matter released by growing algae, as nonliving particulate organic matter, or organic detritus, produced during decaying phytoplankton blooms and as waste products of protist and metazoan consumers, and in sediments from the settling of organic particles. Part of the primary production assimilated by prokaryotes is regenerated via cellular catabolism back to inorganic compounds, carbon dioxide, ammonium, and phosphate, and part is converted into cell biomass. The proportion of organic carbon assimilated by microbes that is used in anabolic processes to produce more cell biomass is termed the gross growth efficiency (GGE) (Figure 5). For prokaryotic microbes, the GGE is simply the amount of cell biomass produced as a fraction of the total amount of organic carbon assimilated. The rest of the assimilated carbon is respired to carbon dioxide. This growth efficiency is sometimes termed bacterial growth efficiency (BGE). Although the theoretical maximum BGE is 67%, in nature the community BGE is much lower, and generally ranges between 10 and 40%. A major problem with ascertaining the actual BGE of the community of growing cells in the natural environment is that a variable portion of cells in the prokaryotic assemblage are dead or dormant, which makes it difficult to scale assimilation and growth rates measured for the total assemblage to just the active community. The relative fractions of assimilated organic nitrogen and phosphate that are released as inorganic compounds by prokaryotic metabolism depend on the elemental ratios of carbon, nitrogen, and phosphate in the organic matter on which the microbial cells are growing. Elemental C:N:P ratios of phytoplankton are variable and depend on factors such as the availability of inorganic nitrogen and

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Gross growth efficiency = yield/substrate, or yield/(yield + respiration/excretion) Yield – 10–40%

Substrate – 100 %

Amount of carbon or other element, e.g., nitrogen or phosphorus, assimilated as organic matter or ingested as a prey cell

Amount of element used in anabolic processes to make more cell biomass

Respiration/excretion – 60–90%

Amount of element respired in catabolic processes to make ATP for biosynthesis or unused and excreted back to the environment

Figure 5 Diagram showing gross growth efficiency as cell biomass yield as a proportion of either total substrate or prey biomass assimilated or as a portion of the sum of yield plus amount of ingested food either respired or excreted.

phosphorus in the environment, species composition, and growth state of the phytoplankton. The classic C:N:P atom ratio of phytoplankton in the sea is 106C:16N:1P, and the ratio between carbon and nitrogen is 6–7:1. This is known as the Redfield ratio, after the oceanographer Alfred C. Redfield who proposed it as an explanation of why the general elemental ratio between nitrate and phosphate in the sea was about 16:1. However, bacterial cells have a higher requirement for both nitrogen and phosphorus, and a C:N ratio of 4–5:1. Prokaryotic cells also have a higher biomass-specific concentration of iron and other trace metals compared to eukaryotic cells. Thus prokaryotic cells tend to sequester nitrogen and phosphorus and metal ions, which are only released back to the environment by predation or by viral lysis. Ingestion and digestion of other microbial cells by phagotrophic protists is of vital importance in complete regeneration of the elements fixed by phytoplankton into organic compounds back to inorganic compounds that can be reutilized by autotrophs for further primary production. In both marine and freshwater systems, phagotrophic protists, both flagellates and ciliates, are major consumers of prokaryotic cells. Phagotrophic protists also are major consumers of algal cells, even the large-sized phytoplankton characteristic of mass blooms. The carbon-based growth efficiency of phagotrophic protists is about 40% of ingested prey biomass. Protists release undigested components of ingested prey as dissolved and particulate organic matter, as well as metabolic waste products as dissolved inorganic compounds such as ammonium and phosphate. Thus protistan grazing provides organic and inorganic substrates for further growth of their prey, both heterotrophic bacteria and autotrophic cells. Protists have much higher biomass-specific rates of nutrient excretion

than do larger-sized zooplankton, and they regenerate nutrient elements bound up both in bacteria and in phytoplankton. Thus protist consumption of microbial cells is a major process in regeneration of nitrogen and phosphorus compounds in aquatic systems. The capacity of many species of autotrophic flagellates to phagocytize gives these phytoplankters an advantage in the acquisition of nutrients in a chemically dilute environment. Mixotrophic algae ingest bacteria and eukaryotic prey to gain both organic substrates and inorganic nutrients. In oceanic systems in which iron is a limiting micronutrient, consumption of iron-rich bacterial cells is an adaptive strategy for phagotrophic algae. Bacterivorous flagellates may also experience iron limitation, and thus ingestion of prokaryotic prey with high iron concentrations can be important to heterotrophic as well as to autotrophic protists.

Food Resource for Metazoans Microbial production forms the base of aquatic food webs. A variable, and at times large, part of the production consumed by aquatic animals is direct consumption of algae. Prokaryotic biomass is a food resource for some animals, for example, rotifers and cladoceran zooplankton such as Daphnia spp. in brackish coastal systems and in lakes, and deposit feeding worms in benthic habitats. However, phagotrophic protists play a significant role in channeling microbial, both prokaryotic and algal, production at the base of the food web to higher trophic levels. In addition, phagotrophic protists consume other heterotrophic protists. Species of heterotrophic dinoflagellates and ciliates have been shown in culture to readily ingest heterotrophic

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flagellates as well as phytoplankton prey. This trophic link in aquatic microbial food webs, although in theory quite important, has, to date, received surprisingly little attention. There has been debate about the quantitative significance of trophic transfers involving protists; but there is no doubt that heterotrophic protists represent food for a variety of other consumers. The largest body of studies on this subject deals with ciliates and heterotrophic dinoflagellates as food for mesozooplankton. In regions of the ocean where most phytoplankton are less than 5 mm, which is too small for most multicellular zooplankton to capture, protists may be a primary source of food for copepods and other zooplankters. For example, in an oligotrophic atoll lagoon in the tropical Pacific Ocean, phytoplankton biomass was dominated by coccoid cyanobacteria and algal cells less than 3 mm. Grazing rate assays showed that the major pathway of carbon flow in this food web was from phytoplankton to phagotrophic protists, which formed the main food resource for copepods in the lagoon. Even in mesotrophic systems characterized by diatom blooms, phagotrophic protists can serve as an important trophic link between phytoplankton and mesozooplankton. Heterotrophic dinoflagellates, which are rich in fatty acids and sterols, represent a high-quality food for copepods and enhance their rate of reproduction. Phagotrophic protists in the plankton can also serve as a significant food resource for filter-feeding benthos such as oysters.

Modeling Microbial Food Webs Conceptual, or box, models, such as the ones shown in Figures 6 and 7, and simulation models, which put the

flows between the boxes of conceptual models on a mathematical basis, are a standard approach to understanding how the various components of ecosystems function interactively. The first quantitative models of pelagic food webs, dating from the 1940s, were simple nutrient– phytoplankton–zooplankton (NPZ) simulations based on transfers of nitrogen between an inorganic nutrient (nitrate plus ammonium) compartment and phytoplankton and zooplankton compartments. Phytoplankton production was dependent on nutrient availability, and zooplankton consumption of phytoplankton and regeneration of phytoplankton nitrogen back into inorganic nitrogen as ammonium depended on phytoplankton production. The microbial food web, including both heterotrophic prokaryotes and protists, was either ignored or put into an extra organic detritus compartment in the model to account for nitrogen regeneration from decaying phytoplankton cells or zooplankton fecal pellets. After subsequent research findings proved that heterotrophic microbes played significant and central roles in aquatic food webs, microbes began to be formally included in model diagrams and simulations. The first formulation was to add a decomposing microbial food chain composed of detrital organic matter, heterotrophic prokaryotes, bacterivorous flagellates, and larger protists that fed on the flagellates to the standard, or classic, food chain of phytoplankton to zooplankton to larger consumers (Figure 6). This microbial food chain was termed the microbial loop, and its role in the overall food web was to respire a large fraction, 50% or more, of overall phytoplankton production into carbon dioxide and to regenerate nitrogen and phosphorus nutrients for further primary production.

Classic food chain Phytoplankton

Regeneration of N and P

Herbivorous zooplankton

Fish, etc

Nonliving organic matter Heterotrophic bacteria

Respiratory loss of organic carbon

Bacterivorous flagellates Larger protists

CO2

Microbial loop Figure 6 Initial conceptualization of the place of microbes in aquatic food webs based on the box model diagram of the microbial loop concept, redrawn from Figure 1 of Ducklow (1983). In this conceptualization, a linear microbial food chain of heterotrophic bacteria to bacterivorous protists to larger protists is added on to the classic phytoplankton to zooplankton to higher consumers food chain. The role of the microbial loop is viewed in this concept as mainly a sink for primary production and a major pathway of regeneration of inorganic nutrients.

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Prokaryotic and eukaryotic autotrophs

Bacterivory (Mixotrophy)

Herbivory

Herbivory

Organic and inorganic substrates

Heterotrophic prokaryotes

Bacterivory

Smaller phagotrophic protists

Larger phagotrophic protists

N and P regeneration

Multicellular consumers

viral lysis

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Food for zooplankton

Figure 7 Current conceptual model of the major compartments and roles of the microbial food web in aquatic ecosystems. In this conceptualization, the microbial loop is embedded in a larger microbial food web that includes additional pathways of mixotrophy by phytoflagellates, consumption of phytoplankton by phagotrophic protists, viral lysis of both bacteria and phytoplankton, and phagotrophic protists as an important food resource, along with phytoplankton, for multicellular zooplankton.

Further work showed that the heterotrophic microbial food chain was embedded in a much larger, more complex microbial food web in which protists of all sizes fed on autotrophic prey and in which viral lysis of prokaryotes and algal cells at times acted to short-circuit the microbial loop (Figure 7). Recent simulation models of pelagic food webs have explicitly included microzooplankton as consumers of bacteria and phytoplankton, and as food for mesozooplankton. The proportion of phytoplankton carbon that flows through a multistep microbial food web versus a shorter phytoplankton–mesozooplankton food chain has implications for the capacity of marine ecosystems to sequester organic carbon or to efficiently produce fish biomass. Two theoretical scenarios have been proposed in which pelagic systems characterized by an active microbial food web will export less organic carbon compared to systems in which activity of heterotrophic bacteria and protists is relatively low. The first scenario is termed a microphagous food web, dominated by phagotrophic protists consuming prokaryotic and small algal cells, and the second scenario a macrophagous food web in which large-sized algae such as diatoms are consumed by copepods. To a large extent the factor that determines the degree to which pelagic food webs are microphagous versus macrophagous is the proportion of plankton biomass that consists of heterotrophic bacteria and phytoplankton cells less than about 5 mm. In an empirical test of the theory, it was found that in the St. Laurence River estuary in Canada export flux from the water column was the same during both the spring diatom bloom with a small microbial food web and the postbloom with a larger, more dynamic microbial food

web. However, the nature of the sinking material was different. During the spring bloom the vertical flux was primarily in the form of organic aggregates consisting primarily of sedimenting phytoplankton cells; during the postbloom period the major flux was in the form of fecal pellets from omnivorous copepods feeding on heterotrophic protists. Obviously, food web structure alone is not always a good predictor of the quality or quantity of sinking organic carbon. One must also include trophic flux studies coupled with hydrodynamic measurements across time and space. It is also important that studies on microbial food webs include data on all the major components of a pelagic ecosystem, not just the microbes. Since the ocean is the largest reservoir of inorganic carbon that freely exchanges with the atmosphere, understanding the influence of microbial food webs on the ability of the ocean to store atmospheric carbon dioxide is an important research theme for biological oceanographers. In modeling microbial food webs whether microbial stocks and rates of biomass production are controlled by bottom-up or by top-down processes is of critical importance. Bottom-up processes are characterized by availability of requirements for growth, for example, inorganic nutrients and light for phytoplankton and quantity and quality of organic substrates for heterotrophic prokaryotes. Top-down processes are mortality processes, mainly due to predation but also including viral lysis, as well as cell death due to unfavorable environmental conditions. An enduring question for aquatic microbial ecologists is why bacterial abundances range over only about 1.5 orders of magnitude in the euphotic zone of the

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ocean, from several hundred thousand cells per milliliter to one or two million cells per milliliter; while phytoplankton biomass, as measured by concentration of the main photosynthetic pigment chlorophyll a, varies over 3 orders of magnitude, from 0.05 mg chl a l1 in oligotrophic open ocean gyres to 30–50 mg chl a l1 in coastal phytoplankton blooms. This results in bacterioplankton biomass being equal to or even greater than phytoplankton biomass in oligotrophic open ocean systems where most primary producers are prokaryotes or small sized algae, while in coastal marine systems, bacterial biomass is often much less than phytoplankton biomass. One reason for the disparity may be that at the lower end of their abundance range, heterotrophic prokaryote cells enter into a metabolic condition termed starvation survival and persist without dying or disappearing, while at the upper end during phytoplankton blooms, bacteria may either be inhibited from growing to high biomass or have enhanced rates of mortality, that is top-down controls, from grazing and viral lysis when bacterial abundances exceed a threshold abundance of about a million cells per milliliter. A model that can predict whether marine bacterial communities were controlled mainly by bottom-up or by top-down processes has been proposed. Researchers assumed that in natural environments, when bacterial abundances were close to the carrying capacity of the local environment, bacterial growth rates would be high and variable, and mainly limited by the availability of organic substrate, or bottom-up-controlled. However, when bacterial abundances were far from the carrying capacity of the local environment, growth rates would tend to be lower, and bacterial growth was likely to be limited by mortality processes, or top-down-controlled. Thus, if there is a negative relationship between bacterial growth rate and abundance, it should indicate that the bacterial abundances are near the carrying capacity of the local environment, and that the bacterial community is limited by substrate availability. However, if bacterial abundance and growth rate are not related, it suggests that bacteria are top-down-controlled, with a small range of possible growth rates. This idea was tested by comparing environmental data on bacterioplankton abundance and assemblage growth rates collected from various open ocean habitats. The empirical data sets in fact showed a negative relationship between bacterial growth rate and bacterial abundance in eutrophic regions, indicating bottom-up or resource control, but no relationship between bacterioplankton abundance and growth rate in oligotrophic regions, suggesting top-down or mortality control. Experiments were also carried out in the oligotrophic systems in which water was screened through 0.8mm-pore-sized filters to remove bacterivorous protists, the main source of mortality. In most cases the bacterial growth rate in screened water increased compared to

growth in the presence of phagotrophic protists, confirming top-down regulation. The main processes controlling the abundances and growth rates of phagotrophic protists in aquatic ecosystems are less well understood. Comprehensive data sets on protist abundance and in situ growth rates are lacking. Mortality processes are likely to be important in determining the abundances and biomass of populations of protists. Top-down control of heterotrophic protists in aquatic food webs results in trophic cascades in which enhanced mortality of a trophic group of heterotrophic protists, for example, nanoflagellates that preferentially consume bacteria and less than 5-mm-sized phytoplankton, increases the growth rate of the prey. Factors that set up tropic cascades include variations in intrinsic growth rates of different classes of grazing protists and dependence of protist growth rate on prey abundance. In natural systems, phagotrophic protists are typically food-limited, and they are poised to rapidly increase their growth rate when they encounter higher prey abundance. Another factor is how the available prey in a microbial food web is partitioned among various groups of grazing protists, particularly by differences in selection of prey by size, as different populations of protist grazers in a system tend to have different prey size preferences. Finally, copepods and other zooplankton can exert a strong top-down control on protists larger than about 10 mm. Trophic cascade effects have been studied by manipulation experiments in which marine or freshwater samples are treated to exclude predators, and effects of the manipulation on growth of various groups of microbes in a food web are followed over time. As an example, an experiment that demonstrated trophic cascades in a microbial food web was carried out in the northern Baltic Sea. To remove various groups of phagotrophic protists, separate volumes of seawater were filtered to yield four size fractions: a less than 0.8 mm fraction that only included heterotrophic bacteria and autotrophic picoplankton, a less than 5 mm fraction that included heterotrophic bacteria, small-sized phytoplankton, and small-sized heterotrophic flagellates, a less than 10 mm fraction that also included 5–10 mm phytoplankton and flagellates, and a less than 90 mm fraction that included most components of the microbial food web but excluded metazoan grazers. The fractionated water samples were incubated in situ in dialysis bags with a molecular weight cutoff of 12–14 kDa, which allowed dissolved nutrients and organic substrates to pass in and out of the incubation bags. The development of the plankton community in the various size-fractioned water samples was followed over 8 days. The results showed that both picoplankton and nanoflagellates were top-down-controlled. Removal of all protists via the 0.8 mm filtration or inclusion of larger-sized protists in

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the less than 90 mm fraction increased the net growth rates of heterotrophic bacteria and picoplankton, while removal of larger protists by 5 mm or 10 mm filtration led to enhanced growth rates of bacterivorous nanoflagellates and decreased growth rates of picoplankon. The experiment suggested omnivory among phagotrophic protists, which led to trophic cascades in the food web.

Chemical Interactions between Microbes Microbial cells, including prokaryotes, algae, and heterotrophic protists, have patterns of behavioral response to environmental cues that affect food web interactions. Many of these cues are specific chemicals dissolved in the aqueous medium or on surfaces of other cells or nonliving particles. Motile bacterial cells can sense gradients of chemical compounds in their environment and respond by moving up or down the gradient. A large portion of prokaryotic strains cultured from seawater are motile, and it is postulated that this motility allows bacterial cells to move toward microsites of high substrate concentration, for example, phytoplankton cells or rich organic particles. The process of chemoreception is based on bacteria having specific recognition sites on their cell membranes for a particular substance. Escherichia coli, for instance, has at least 20 chemoreceptor sites that allow the bacterium to respond positively, that is, be attracted to, or negatively, that is, be repelled by, various chemicals. Generally, aerobic bacteria move toward higher concentration of small molecular weight organic substrates such as amino acids and sugars and toward higher oxygen concentration. Aerobic bacteria move away from metabolic poisons, for example, sulfide and heavy metal cations. Bacteria can sense changes in chemical concentration over time as they swim through concentration gradients. Motile bacteria respond to changes in their environment by changing their swimming behavior or direction. For bacteria having two or more flagella, the normal swimming behavior is straight-line swimming runs with the flagella operating synchronously and occasional tumbles with the flagella flailing in opposite directions, which allows the cell to orient to a new straight-line direction. An increase in concentration of an attractant results in a shift to straight-line swimming alone, while a decrease in concentration of an attractant results in dramatic increase in tumbling, leading to frequent changes of direction to facilitate the search for a more favorable concentration of substrate. Conversely, increasing the concentration of a repellent chemical results in increased tumbling, and decreasing the repellent concentration leads to straightline swimming. A bacterium that has only a single flagellum can change in rotational direction of the flagellum, so

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in this case the cell only moves in forward or reverse direction, and does not tumble. An example of bacterial behavior based on chemosensory response can be observed in the aggregation of bacteria under a coverslip: strains of bacteria that prefer a high oxygen tension will congregate at the edges of the slip, while microaerobic bacteria will remain in the middle. Motile marine bacteria have been shown to aggregate around organic-rich particles and to track the path of a phytoflagellate, presumably by chemosensory response to organic compounds emanating from the particle or algal cell. This ability allows motile bacteria to aggregate around detrital particles or algal cells, forming locally elevated prey concentrations for bacterivorous protists. Chemosensory response is thus also important in the feeding behavior of phagotrophic protists. Marine bacterivorous flagellates exhibit chemoattraction to amino acids and to bacterial cells, as has been demonstrated. Marine ciliates whose preferred prey is algae exhibit strong positive chemosensory response to some species of algae, and neutral or negative response to other prey species. Species of heterotrophic dinoflagellates have also been found to move toward algal cells and algal cell lysate, and to preferentially prey on some strains of algae. A modeling exercise designed to estimate the advantage conferred on a phagotrophic protist that is able to chemically detect prey cells showed that this capacity conserves energy used to search for food, and would confer the greatest advantage when prey are scarce. Bacterial and algal cells may deter protist predation directly by producing chemicals that are either toxic or unpalatable to phagotrophic protists. The pigment violacein, a bacterial secondary metabolite, inhibited predation by nanoflagellates on bacteria containing the pigment. Ingestion of just one to three violacein-containing bacterial cells was found to cause rapid death of the flagellate cells. The heterotrophic dinoflagellate Oxyrrhis marina was shown to have a much lower feeding rate on one particular strain of the coccolithophorid algae Emiliania huxleyi compared to a second strain of E. huxleyi. In the presence of the dinoflagellate, the strain of E. huxleyi that the dinoflagellate avoided produced a concentration of dimethyl sulfide (DMS) and acrylic acid that was an order of magnitude higher than in the second strain of E. huxleyi. DMS was found to inhibit predation of algal prey by several other species of phagotrophic protists, and appears to be a chemical deterrent to protist feeding. The deterrent mechanism is not known. The biochemical mechanism for chemosensory response in both prokaryotic and eukaryotic microbes is based on chemoreceptor sites associated with the cell membrane. The chemical compound being sensed binds to a specific chemoreceptor site, triggering signal transduction, the release of secondary messenger compounds within the cell, which results in change in motility, for

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example, direction and/or speed, or in ingestion of a prey cell by a protist. Such cellular processes are obviously important for survival and growth of aquatic bacteria in terms of locating microsites of higher substrate concentration and for aquatic protists in determining prey location, selection, capture success, and rates of ingestion. Understanding the extent to which cell surface chemoreceptor binding and consequent signal transduction pathways operate in bacterial and protist chemosensory behavior is vital to a predictive understanding of the structure and function of marine food webs.

Spatial Structure of Microbial Food Webs Initially, the environment encountered by aquatic microorganisms was perceived as being largely fluid, relatively homogeneous, and governed by diffusive processes; and it was assumed that microbes suspended in water were more or less uniformly distributed. Research on how chemosensory behavior of motile microbes can result in microbial aggregations, combined with observations of patchy distribution of phytoplankton, detrital organic matter, and high molecular weight colloidal dissolved organic substances, has led to the understanding that microbial food webs in fact occur in a nonuniform structured habitat. Phytoplankton and bacteria release polymeric material directly, other polymeric material is produced during feeding and egestion by protists and zooplankton. Biopolymer gels form when polymer chains are hydrated and cross-linked or aggregated, resulting in a three-dimensional network. These polymeric microgels then aggregate to form gel-like sheets, strings, and webs that provide surfaces, form barriers against diffusion, and furnish refuges against predators. Detrital particles and microbial cells are interspersed in these gel webs. The interactions of bacteria with the organic matter continuum from dissolved organic compounds to large particles, and the behavioral response of microbes to the patchy distribution of these particles, create microscale features, hotspots of microbial activity, and food web interactions, with distinctive natures and intensities of biogeochemical transformations. The larger organic particles formed in this way are termed marine snow because these aggregated particles, which are usually greater than 0.5 mm in diameter, strongly resemble snowflakes when seen in the water. The basic glue holding marine snow particles together is thought to consist of fibrillar, long-chain polysaccharide polymers termed transparent exopolymer particles (TEP). Marine snow particles are initially colonized by heterotrophic prokaryotes that produce extracellular enzymes that hydrolyze the organic matrix of the particle into low molecular weight dissolved organic compounds, for example, monomers or small polymers of sugars and amino acids.

The colonizing bacteria produce more of these organic substrates than can be assimilated; thus, a plume of organic material is released from the particle that attracts motile bacteria. Higher abundances of bacteria in turn attract bacterivorous flagellates, which may then attract larger protists and zooplankton. The organic particle and its plume may thus become the focus of a complex food web. Marine snow particles can sink at speeds greater than 100 m in a day, allowing them to travel from the surface to the subsurface ocean within a matter of days. As they sink, the organic material in the particles is continually degraded by microbes. Any organic material that reaches the sea floor is either consumed by benthic organisms or incorporated into sediments. Sinking of marine snow particles is important to the ocean’s capacity to sequester atmospheric carbon dioxide. Biofilms, which, as mentioned previously, form on surfaces, are similar to suspended organic particles in that they are essentially an organic polymer gel with living microorganisms embedded in it. Biofilms form on most abiotic surfaces in aquatic systems and are sites of enhanced microbial activity. The polysaccharides produced by surface-growing microbes act to cement sediment particles together and represent a food resource for benthic animals. The microbial species colonizing the biofilm and the organic composition of the matrix largely determine the physical properties of the biofilm. Once attached to a surface, bacteria begin to produce extracellular polysaccharides. The amount of biopolymer produced can exceed the mass of the bacterial cell by a factor of 100 or more. The gel is mostly water, but has properties that influence the transport of materials at the surface of the biofilm. Changes in transport rates can create unique niches within the biofilm for the proliferation of a variety of microbial species. Bacterial extracellular polysaccharides also tend to absorb cations and organic molecules from the overlying water. The reduced diffusion rates of substrates within a biofilm serve to create localized concentration gradients and the possibility of well-defined spatial relationships between individual bacterial cells. These gradients may result in suboxic and even anoxic regions in the interior of these structures, contributing to the coexistence and metabolic interaction of both aerobic and anaerobic microbes.

See also: Deep Sub-Surface; Ecology, Microbial; Freshwater Habitats; Low-Nutrient Environments; Marine Habitats; Sediment Habitats, including Watery

Further Reading Azam F, Fenchel T, Field JG, Meyer-Reil RA, and Thingstad F (1983) The ecological role of water-column microbes in the sea. Marine Ecology Progress Series 10: 257–263.

Environmental Microbiology and Ecology | Food Webs, Microbial Ducklow HW (1983) Production and fate of bacteria in the oceans. BioScience 33: 494–501. Fenchel T and Finlay BJ (1995) Ecology and Evolution in Anoxic Worlds. New York: Oxford University Press. Gasol J, Pedros-Alio C, and Vaque D (2002) Regulation of bacterial assemblages in oligotrophic plankton systems: Results from experimental and empirical approaches. Antonie van Leeuwenhoek 81: 435–452. Kirchman D (ed.) (2000) Microbial Ecology of the Oceans.. New York: Wiley-Liss. Legendre L and Le Fevre J (1995) Microbial food webs and the export of biogenic carbon in oceans. Aquatic Microbial Ecology 9: 69–77. Matz C, Deines P, Boenigk J, et al. (2004) Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates. Applied and Environmental Microbiology. 70: 1593–1599. Pohnert G, Steinke M, and Tollrian R (2007) Chemical cues, defence metabolites, and the shaping of pelagic interspecific interactions. Trends in Ecology and Evolution 22: 198–204. Pomeroy LR (1974) The ocean’s food web, a changing paradigm. BioScience 24: 499–504.

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Rivkin RB, Legendre L, Deibel D, et al. (1996) Vertical flux of biogenic carbon in the ocean: Is there food web control? Science 272: 1163–1166. Sakka A, Legendre L, Gosselin M, and Delesalle B (2000) Structure of the oligotrophic planktonic food web under low grazing of heterotrophic bacteria: Takapoto Atoll, French Polynesia. Marine Ecology Progress Series 197: 1–17. Samuelsson K and Andersson A (2003) Predation limitation in the pelagic microbial food web in an oligotrophic aquatic system. Aquatic Microbial Ecology 30: 239–250. Sherr EB and Sherr BF (2008) Understanding roles of microbes in marine pelagic food webs: A brief history. In: Kirchman D (ed.) Advances in Microbial Ecology of the Oceans, pp. 27–44. Hoboken N.J.: Wiley-Blackwell. Sieburth JMcN, Smetacek V, and Lenz J (1978) Pelagic ecosystem structure: Heterotrophic compartments of the plankton and their relationship to plankton size fractions. Limnology and Oceanography 23: 1256–1263. Wolfe GV (2000) The chemical defense ecology of marine unicellular plankton: Constraints, mechanisms, and impacts. Biological Bulletin 198: 225–244.

Freshwater Habitats L G Leff, Kent State University, Kent, OH, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Overview Freshwater Wetlands Lakes

Glossary allochthonous Originating from outside the system; produced outside the system. autochthonous Originating within the system; produced inside the system. benthos The bottom of an aquatic environment below the water column; the sediments. dimictic A lake that stratifies and turns over twice per year. epilimnion The upper water layer of a stratified lake. eutrophic A lake or other aquatic environment with large amounts of nutrients that leads to high primary production. hyphomycetes The dominant type of freshwater fungi.

Abbreviations AOA CPOM DNRA

ammonia-oxidizing archaea coarse particulate organic matter dissimilatory nitrate reduction to ammonium

Defining Statement Freshwater environments, such as wetlands, lakes, streams, and rivers, are critical components of society and support diverse and complex microbial communities. There is great variation among freshwater habitats, with hydrology, nutrient inputs, water level and movement, the role of allochthonous inputs, and other factors, all influencing the ecology of microorganisms.

Overview Freshwater environments are ubiquitous and critical to human success as sources of drinking water, conduits for waste materials, aesthetical and recreational purposes,

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Streams and Rivers Comparison Among Freshwater Habitats Further Reading

hypoliminion The bottom region (below the thermocline) of the water column of a stratified lake. hyporheos The area of water below the surface water of a river or stream. mixotrophs Protozoa that are photosynthetic and also exhibit phagocytosis of prey. oligotrophic A lake or other aquatic environment with small amounts of nutrients that limits primary production. periphyton Community of organisms growing attached to surfaces in aquatic environments. P/R ratio The ratio between primary production and respiration in a system. thermocline Area of rapid temperature change with increasing depth in a lake.

DOC DOM FPOM RCC

dissolved organic carbon dissolved organic matter fine particulate organic matter River Continuum Concept

and so on. The great diversity in form and function of freshwater systems results in significant differences in microbial community structure and processes. Water flow, water depth, light level, nutrient inputs, pH, plant communities, and numerous other factors all influence the microbial ecology of freshwater (aquatic) ecosystems. The presence of water dictates several key common properties of these environments, as do the interactions with the surrounding terrestrial environment within the watershed. Water provides a means for dispersal of organisms, transport of materials, and structural support while at the same time limiting light availability as depth increases, altering temperature regimes, and impacting oxygen concentrations. Some freshwater environments are fueled predominantly by allochthonous fixation of C, whereas others are dominated by autochthonous C

Environmental Microbiology and Ecology | Freshwater Habitats

fixation. At the same time, microbial cells may enter aquatic ecosystems from outside sources and intermingle with autochthonous organisms. Freshwater habitats are profoundly impacted by anthropogenic disturbance such as alterations in hydrology, land use, disposal of waste materials, contamination with fertilizers, heavy metals, xenobiotic organic compounds, and introduction of exotic species. Global climate change also has great potential impacts on freshwater resources through increases in carbon dioxide, elevated temperatures, an increase in sea level, and alterations in precipitation and snow melt seasonal patterns. Some of these are predicted to impact the hydrology of freshwater systems whereas others may directly impact biota. The combination of these effects is predicted to alter freshwater habitats and thus there is the potential for the microbial ecology of the systems to also change. The three main types of freshwater habitats are discussed below and include wetlands, lakes, and lotic ecosystems (streams and rivers). Wetlands are arguably the least studied of these systems relative to the structure of the microbial community, although the functional role of microorganisms in wetlands and other freshwater habitats is well established. For each freshwater habitat type, the nature of the hydrology, geographic location and climate, and origin contribute to the large variations seen in ecosystem properties. The role of allochthonous versus autochthonous sources of organic compounds, nutrients, and microbial cells also varies among ecosystem type along with the contribution of plant detritus to the food web.

Freshwater Wetlands Wetland Types and Properties The nature of freshwater wetlands varies based on hydrology, origin of the wetland, plant community composition, and other features; these variations in turn affect the microbial communities of the environment. This complexity also limits the number of generalities than can be drawn about the microbial ecology of these ecosystems. Wetlands serve as environments; that may ameliorate environmental contaminants and manage flooding. At the same time, wetlands are highly threatened environments. For example, the percentage of wetlands in the continental United States is estimated to have declined by about 50% since presettlement times. Wetland loss in Europe as well as in parts of Asia is believed to be much greater. The importance and vulnerability of freshwater wetlands lead to regulations in many countries regarding the use of and destruction of wetlands. This, in turn, contributes to the issues associated with the definition of a wetland. From a basic point of view, wetlands are shallow aquatic communities dominated by plants in which

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detritus plays a central role in the food web. From a standpoint of delineation, the definition of wetlands is more complex and has been the subject of great debate. Overall, wetlands, for purposes of this article, can be defined as systems with hydrophytes (wetland-type plants), hydric soils, and, during the growing season, periods of standing surface water or water-saturated soils. The duration and depth of standing water vary greatly among wetlands and, within a wetland, seasonally and from year to year. The hydrology, origin, and geographic location of the wetland are major determinants of plant community structure, which in turn influences the microbial communities. Wetlands include those on the fringe of larger bodies of water, such as around the Great Lakes of the United States, prairie potholes, marshes (including freshwater tidal marshes), swamps, riparian zones, and floodplains along rivers, bogs, playas, and billabongs. Each wetland is connected to the surrounding terrestrial environments in a variety of ways and they can also be connected to marine or other freshwater systems. For example, nutrients from a fringing wetland can be transported into lakes linked seasonally by water. Wetland environments can be stressful and exhibit large seasonal changes in hydrology including periodic drying. This effect, coupled with the development of areas of low oxygen concentration, low redox potential, and the wide array of different types of organic compounds, which in some cases accumulate in the system, defines the microbial ecology of the wetland. Human use of wetlands is also an important determinant of wetland function and biogeochemistry. Wetlands serve as sources of flood abatement, carbon sequestration, and phytoremediation and support a rich and unique flora and fauna. Wetlands are also created and used by humans for the production of food, such as rice, fish, and crayfish. Overall, critical features of wetlands that affect the ecology of microorganisms are as follows: role of plants and detritus in the food web. • Important Critical role of hydrology, which varies greatly among • wetlands of different types. variation in the extent of hydration of soils. • Seasonal human impact on wetland extent, nature, and • Profound hydrology. of new wetlands, perhaps with different eco• Creation logical properties, to replace wetlands lost through human use or to serve as sites of food production.

Microbial Processes The importance of microbial processes in wetlands manifests itself in the biogeochemistry of these systems and the central role that detritus decomposition plays in the food web. Dissolved organic matter (DOM) is a major

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temporally and, thus, the optimal conditions for growth of a particular microorganism may not always be present. The C cycle of wetlands is characterized by a mixture of anaerobic and aerobic processes occurring at different locations or at different times. Often these processes can occur in spatially close proximity to each other in different portions of the soil. Fermentation is a common process in wetlands and appears to play a critical role in providing suitable organic compounds for other anaerobes. Methanogenesis in wetlands tends to be better studied than fermentation and can result in the release of bubbles of methane gas (referred to as swamp gas). Methane emission from wetlands globally is quite large and is of particular interest because of the role of methane in global climate change and the possibility of management of methane entrance into the atmosphere. Quantification of methane production by wetlands reveals that there is substantial variation among wetland types and geographic locations. Properties of the N cycle in wetland are highly variable depending on plant community type and hydrology. The sediments of wetlands play a critical role in the N cycle. N can be a limiting nutrient in wetland soil and depending on the N pool, N fixation will be more or less important. For example, the floating fern, Azolla, has symbiotic nitrogen-fixing cyanobacterium (Anabaena). In wetlands created for rice cultivation the Azolla–Anabaena may help rice production. In addition, in some cases, wetland-fringing plants, such as alder (Alnus), may host nitrogen fixers that can impact the wetland N cycle. It also appears that nitrogen fixation does occur in association with wetland plants (such as some grasses); however, the organisms responsible for this activity defy cultivation and thus the amount of information on them is still relatively limited.

component of the organic matter pool and is made available to higher trophic levels by microorganisms. In some wetlands, often referred to as dystrophic, dissolved organic carbon (DOC) concentrations can be substantial and ‘stain’ the water brown to black because of large amounts of humic substances. These wetlands, such as bogs, are often characterized by unique plant communities, including, in some cases, the occurrence of Sphagnum, formation of peat, and low pH. Inundation with water creates anaerobic conditions in wetland soils, providing the opportunity for alternative metabolic processes. The strong seasonality in the hydrology of many types of wetlands creates circumstances where there is flooding and corresponding chances for anoxia in lower layers of the soil at some times of the year, but not at other times (Figure 1). This creates seasonal patterns in key microbial processes, such as methanogenesis and denitrification. In denitrification, a dissimilatory process, microorganisms reduce nitrate (or nitrite) to dinitrogen rather than using oxygen as the electron acceptor. Also, under anaerobic conditions some archaea, known as methanogens, can produce methane when grown on carbon dioxide and hydrogen or when growing on acetate or other simple organic compounds. Lastly, dissimilatory sulfate reduction may occur in anaerobic areas; during this process sulfate is reduced to hydrogen sulfide. Plants play a seminal role in determination of microbial processes in wetlands, including their impact on soil conditions, the detrital pool, and the oxygen status of the soil. Plant roots lose oxygen (from air-filled tissue in wetland plants called aerenchyma) to the surrounding soil altering the otherwise anaerobic status of the soils. The occurrence of oxygen is variable both spatially and

Organic substrate [e– donor] +

–3

Relative concentration

NH4 or PO4 SO–2 4

H2S

NO3– O2

Oxygen reduction

CH4

Fe+2 Mn+2

Nitrate reduction

Methanogenesis

Iron reduction

Manganese reduction

Sulfate reduction

Time Figure 1 Temporal changes in the availability of alternative electron acceptors in wetland soil. Reproduced from Reddy KR and D’Angelo EM (1994) Soil processes regulating water quality in wetlands. In: Mitsch WJ (ed.) Global Wetlands: Old World and New, pp. 309–324. Amsterdam: Elsevier.

Environmental Microbiology and Ecology | Freshwater Habitats

Denitrification is a potent source of N loss (as N2) because of the extensive amounts of organic matter and anaerobic zones. The large amounts of N applied to landscapes can work its way into wetlands and perhaps these ecosystems can serve as N sinks in the global N cycle. Denitrification in some wetlands may be limited by low pH, thus loss of N in this fashion, in some wetlands, is more restricted than others. However, generally, denitrification results in a major loss of N from wetlands that exceeds inputs from nitrogen fixation. Nitrification plays a critical role in the removal of ammonium from the soil, which tends to accumulate because of ammonification. In nitrification, ammonia is oxidized to nitrite, which is in turn oxidized to nitrate; these reactions are performed by ammonia-oxidizing and nitrite-oxidizing bacteria. In addition, ammonia-oxidizing archaea (AOA) have been discovered in marine environments, soil, and wastewater treatment facilities; although the number of studies is limited, AOA have also been detected in freshwater systems. Aerobic zones supportive of nitrification occur in the topmost layer of soil as well as in aerobic patches caused by plants in the rhizosphere. In some wetlands, ammonium is converted into ammonia, which can be lost to the atmosphere, and generally ammonium diffusion rates through wetland soil are a limiting process. In acidic wetlands, nitrification may be slower relative to other wetland types. Bacteria that oxidize ammonium anaerobically (anammox) are present in freshwater wetlands, although their role in the N cycle of these systems is largely unexplored. In addition, the occurrence and importance of dissimilatory nitrate reduction to ammonium (DNRA) has been demonstrated in freshwater wetland sediments. Sulfur transformations also occur in wetlands, although S is generally not a limiting nutrient and thus it is less frequently studied in freshwater wetlands. One manifestation of these transformations is the distinctive rotten egg smell from the release of hydrogen sulfide when anaerobic wetland sediments are disturbed. Compared with marine wetlands, sulfide emissions from freshwater wetlands are much lower because S concentrations in marine systems greatly exceed those of freshwater systems. Sulfides produced by sulfate reduction can subsequently be oxidized by autotrophs. Sulfides that are not oxidized are toxic to plants and can precipitate with metals. Unlike N and S, phosphorus lacks alternate oxidation states but is often a limiting nutrient in freshwater ecosystems. Some wetlands, such as bogs, are exceptionally nutrient-limited whereas others receive P input from fertilizer and other sources. Both organic and inorganic P contribute to the P pool and insoluble P complexes that affect bioavailability can be formed. For example, P can complex with iron or calcium or attach to clay or peat. In particular, the sorption of P to clay can form complexes, which may be carried into wetlands. When this material

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sediments, it helps make P available to the wetland plant community and allows the wetland to retain P.

Microbial Communities As compared to other freshwater systems, the amount of information on microbial community structure in freshwater wetlands is relatively limited. Although microbemediated processes are widely studied, as described above, the underlying communities responsible for a particular function are unknown to varying degrees with more information available on methanogens, methanotrophs, and denitrifying bacteria, for example, relative to other functional groups, such as nitrogen fixers. Although the role of fungi as decomposers of plant tissue is widely known, there is much less information available on the mycorrhizae associated with wetland plants. However, the potential importance of mycorrhizae in the success of plants may be particularly relevant in wetlands that are nutrient-limited, such as bogs and fens, and can also be important for phytoremediation. Although the number of investigations on bacterial diversity and community structure in freshwater wetlands is relatively limited, studies have revealed that for the wetlands examined Proteobacteria (-, -, and - as well as the -Proteobacteria, which are perhaps not as well represented in lakes and streams), Acidobacteria, and Verrucomicrobia are major constituents. Acidobacteria are a widespread but largely understudied group because of limitations in our ability to culture these organisms. Similarly, there are few cultures from the group Verrucomicrobia with some of the major subgroups in this taxon having virtually no cultured representatives. Verrucomicrobium and other prosthecate bacteria are among the most well-known members of this widespread and understudied group. Among the archaea, the occurrence of methanogens in wetlands is the most often studied, especially in light of the role of methane as a gas contributing to global climate change. In anoxic wetland soils, methanogenesis is a critical biogeochemical process and varies seasonally; the availability of both suitable substrates and nutrients impacts methanogen function. The nearby occurrence of methanotrophs in oxic areas and their oxidation of methane greatly impact methane release. The role and diversity of protozoa in wetlands are largely unknown. Generally, the impact of protozoa as bacterial consumers is great in aquatic environments and less so in soil. There is at least some evidence that the protozoan community composition can be very different from that of other freshwater habitats. The number of protozoa and consequently their impact on bacterial numbers fluctuates seasonally in temperate wetlands.

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Anthropogenic Disturbance Human impacts on wetlands are varied and, among these, perhaps the most important is the vast global destruction of wetlands. This leads to the phenomenon of wetland construction whereby new wetlands are created to replace those lost due to conversion to other uses (agriculture, construction, etc.). How well these created wetlands duplicate natural conditions and function as desired is variable and debatable. Hydrological modifications, to reduce flooding for example, are also common and considering the key role of hydrology in wetland microbial ecology undoubtedly greatly impact microbial processes. Wetlands are also used for various agricultural and horticultural activities, such as peat mining and production of plants, such as rice. Like other freshwater habitats, the impact of humans on water quality can translate into these environments. One manner in which some environmental issues may be addressed is through phytoremediation. Phytoremediation may be accomplished in either natural or constructed wetlands and potentially can remove xenobiotic compounds from water systems. Critical features for the success of phytoremediation in wetland include the redox potential, extent of above- and below-ground plant biomass, and hydrology. Invasive species represent one area in which the effects on microbial communities in wetlands remain largely unknown. Of particular importance are changes in native plant communities associated with the invasion of exotics, such as reed canary grass (Phalaris arundinacea), purple loosestrife (Lythrum salicaria), and alligator weed (Alternanthera philoxeroides). The role of plant communities in wetlands is a critical defining feature and thus alteration of the plant communities toward potentially nearly monospecific stands of exotic species is of great concern. Native plants are often outcompeted by exotic species of plant, perhaps resulting in a loss of the microflora associated with the native plants (such as mycorrhizae), as well as an alteration in many other factors, such as soil moisture, quality, and nature of the detrital pool. In general, invasive plants are known to alter C, N, and water in soil, such as by altering N availability and fixation, and are different from native species in terms of productivity, growth form, chemistry, and so on. Although the role of these differences in wetlands is less well studied than in terrestrial environments, the potential for a substantial change in wetland microorganisms is clear.

Lakes Properties and Types Like wetlands, lakes originate in various ways and the nature of their origin, hydrology, and morphometry impact environmental conditions and biotic functions. Basin

morphometry is of particular importance because of the limitations that depth imposes on macrophyte success, the extent of the littoral zone, and the impacts on the relative contributions of allochthonous and autochthonous production. Lake processes are very much dependent on the properties of the surrounding watershed, as in other freshwater ecosystems. Lake age is also highly variable and depends on the lake type. In many ways, human impacts are poorly understood and not always recognized because so many key processes are hidden beneath the water column and thus unseen by the lay person. One of the most critical determinants of the microbial ecology of a lake is it trophic status, which ranges from eutrophic to oligotrophic (with various modifications of the terms, such as hypereutrophic). The trophic status of the lake profoundly influences the food web, biogeochemistry, and other processes. Oligotrophic lakes are characterized by low nutrient concentrations and low primary production (as reflected in the low abundance of phytoplankton in the lake). Eutrophic lakes, in contrast, are characterized by high nutrient concentrations and correspondingly high primary production. There is a gradient of trophic status between these two conditions with lakes in the middle of the gradient referred to as mesotrophic. The trophic status is the main determinant of the number, biomass, production, and diversity of microorganisms because of the differences above and their corresponding impacts on oxygen concentration. Lakes, or lentic ecosystems, are also characterized by their stratification and mixing properties. In temperate zones, many lakes are dimictic with thermal stratification in winter and summer and spring and fall turnover events. In some regions, there are lakes with permanent stratification called meromictic lakes, such as those where salt and freshwater combine, or those that are particularly deep, whereas other lakes never stratify. Amictic lakes are permanently ice covered, such as lakes found in Antarctica. Other lakes are monomictic with stratification once per year or polymictic with mixing and stratification multiple times throughout the annual cycle. The pattern of stratification and mixing depends on latitude and lake properties. Seasonal stratification in temperate areas occurs because inputs and outputs of heat occur primarily at the surface of the lake. In such temperate zones, the heating of surface waters in summer creates a warm, circulating surface water pool (the epilimnion) with colder, undisturbed water below the thermocline in the hypolimnion (Figure 2). As fall begins, the surface water cools and at some point reaches the same temperature as the underlying water; this results in a loss of density differences and as this surface water becomes cooler and denser it sinks, causing the layers to mix or turn over. In a dimictic lake that is productive (eutrophic) during times of thermal stratification (Figure 2), the hypolimnion can be depleted of oxygen through biological

Epilimnion

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status of macrophytes in the lake; whether it is • The algal-dominated or macrophyte-dominated. stratification and mixing patterns over the course • The of a year. Microbial Processes

n

limnio

Meta

Hypolimnion

Depth

Photic zone

Environmental Microbiology and Ecology | Freshwater Habitats

May be anoxic seasonally especially in eutrophic lakes

4 °C Temperature Figure 2 The water layers and temperature profile in a typical seasonally stratified lake. The thermocline refers to the plane with the greatest decrease in temperature with depth.

activity. This happens when this layer is separated from and not mixing with the overlying water; biological activity in this confined layer resulting from decomposition can be great enough to deplete oxygen. This creates microbiologically important gradients with depth that can result in some interesting juxtapositions of biogeochemical processes as described below. Another main way of categorizing lakes is their vegetation with macrophyte-dominated systems versus algaldominated systems representing alternative stable states. The dominance of macrophytes versus algae greatly influences the trophic interactions and paths of C transfer. The two alternative states that can occur in shallow eutrophic lakes are (1) clear water with abundant macrophytes and (2) less clear water because of phytoplankton abundance without abundant macrophytes. The clear water state is often associated with desirable fish and invertebrate communities, whereas the turbid water state tends to have lower diversity and undesirable algal blooms. These alternate states represent a shift in the lake community and may result from competition with nutrients, light limitation and turbidity, allelopathy, or other factors. If the food web of a lake (such as by removal of macrophytes) or P concentrations are altered, it is possible to shift the state of a lake. Overall, important lake properties that influence the microbial ecology of the ecosystem are as follows: trophic status of the lake (as determined by con• The centrations of N and P) and its point along the oligotrophic : eutrophic gradient.

morphometry and size of the lake, which influ• The ences the size and relative contribution of the littoral zone and allochthonous inputs.

Both stratification and trophic status are major determinants of what microbial processes happen and where they happen. Stratification in a eutrophic lake, in particular, can ultimately lead to large seasonal anoxic zones, which greatly impact microbial processes as well as macrofauna. Development of these so-called ‘dead zones’ is mediated by the microbial consumption of oxygen and significantly impacts lake quality as it may limit fish community success. The nature and extent of stratification as well as nutrient availability creates obvious differences in C and nutrient cycles among different lakes. C cycle

Taken from a simplistic point of view, lake food webs include both a grazer ‘food chain’ and a microbial ‘food chain’ or microbial loop (Figure 3). The relative amount of C transferred through these two components varies from lake to lake and within a lake (e.g., nearshore vs. offshore). The dichotomy between these two paths is also reflective of the role of detritus in the system with the microbial loop reliant on the extensive dissolved portion of the detrital pool. Although the role of viruses in the microbial loop of lakes is perhaps not as well studied as it

Viruses

Phytoplankton

Dissolved organic matter

Zooplankton

Bacterioplankton

Macrofauna

Protozoa

Figure 3 Stylized and simplified food web in the water column of a lake illustrating paths of C transfer in grazer and microbial food webs; green arrows indicate grazing whereas brown arrows indicate production and use of detritus in the form of dissolved organic matter (DOM). Note within the protozoa box, multiple trophic transfers may occur. Viral infections may contribute to the DOM pool via lysis of algal or bacterial cells.

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is in marine systems, there is ample evidence demonstrating the occurrence of phage-infected bacteria and viruses in lakes. There is less information about the potentially large impact that bacteriophage and algal viruses have on the microbial loop and the contribution of virus activity to the pool of DOM. The occurrence of anoxic areas in the hypolimnion and benthos creates opportunities for the use of alternative electron acceptors at the appropriate depths within a lake. These processes can affect the C cycle; for example, methane is generated in these anoxic areas by anaerobic archaea and then can be utilized by methanotrophs in the overlying oxic area (the epiliminion). Similarly, fermentation occurs in the anaerobic zones, resulting in a variety of small organic compounds for use by other microbes. On macrophytes, extensive biofilms (often in limnology, such biofilms are referred to as periphyton) can form, which may serve as competitors for nutrients with phytoplankton. These biofilms are complex structurally and feature diatoms interconnected by the extracellular matrix as well as cyanobacteria and heterotrophic bacteria. Some components of this assemblage penetrate into the macrophyte tissue whereas others are more loosely associated. Within these biofilms, release of extracellular organic compounds by algae supports bacterial growth, and bacteria, in turn, release carbon dioxide and degradative enzymes. The surface area of macrophytes in a lake can be extensive depending on its state, depth, and relative littoral zone size. Thus, the role of these macrophyteassociated biofilms in nutrient acquisition may also be great.

N cycle

The N pool in lakes includes both inorganic and organic components that are utilized and transformed by microorganisms. Cyanobacteria, such as Anabaena and Aphanizomenon, are important nitrogen fixers, especially, but not always, those that produce heterocysts. For example, members of the Oscillatoriaceae are also common and important in lakes but lack the ability to produce heterocysts. N-fixing cyanobacteria are a valuable surface for attachment of bacteria and the heterocysts may create microzones of low oxygen concentration, creating specialized bacterial niches. There is limited information on the importance of heterotrophs, such as Azotobacter, in nitrogen fixation in lakes. This is perhaps a result of the assumption that this process might be limited by the availability of labile organic compounds. Nitrification in lakes occurs in aerobic zones and thus may be limited in the deeper sediments. In addition, specific dissolved organic compounds (such as tannins) may inhibit nitrification. Denitrification is important in areas with oxygen depletion (although it can also occur aerobically) such as the sediments of littoral zones of lakes and the hypolimnion of stratified eutrophic lakes. The

ability to denitrify is widespread among different types of bacteria, including Pseudomonas, Bacillus, and Achromobacter. P cycle

Each elemental cycle in the lake is intertwined with other cycles; this is illustrated, in particular, for the P cycle. Lake sediments are a major source of P in lentic ecosystems and P is often a limiting nutrient; additions of N and P lead to eutrophication, which greatly impacts biotic processes. Most of the P in a lake is contained in the biomass and in organic compounds and total P concentrations vary considerably from hypereutrophic to ultraoligotrophic lakes. Because P is often a limiting nutrient, much attention has been focused on internal loading from the sediments, the process by which sediment P is released into the water column. Mobilization of P is facilitated by bacteria such as Pseudomonas and Chromobacterium but chemical processes are dominant. Specifically, complexation with metals plays a major role in the lake P cycle; for example, phosphate can be released from ferric phosphate under anoxic conditions. Formation of iron sulfide associated with sulfate reduction can reduce the complexation of phosphate by iron. Microbial Communities The amount of information on lake microbial community structure has increased dramatically over the years. Prokaryotic communities have been studied in a variety of lakes of different sizes and trophic status. These studies have revealed information about the dominant bacterial taxa as well as differences between lake and marine communities. The water column microbial community is distinctive in function and also in structure from the benthic community. Depending on the water depth, the benthos may be a viable habitat for phototrophs. Often the benthos may be anaerobic (especially below the sediment surface) even if the overlying water is oxygenated, providing opportunities for methanogens and dentrifyers. Phototrophic prokaryotes fill a particular series of niches in the lake environment based on their physiological needs and pigments used. Cyanobacteria are abundant and can grow to nuisance levels, especially in eutrophic lakes with abundant P. The gas vesicles produced by cyanobacteria contribute to their buoyancy and ability to maintain position in the water column, which ultimately enhances their success. Many of these cyanobacteria can fix nitrogen, contributing to formation of undesirable blooms, which have negative impacts on water quality. Microcystis, which is abundant worldwide, produces microcystins that are of great concern for water quality from a drinking water standpoint and for the success of aquatic animals. The hepatotoxins produced by Microcystis aeruginosa have damaging effects on the livers of mammals,

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and control of this organism can be an important goal of lake management. In many countries around the world, occurrence of toxin-producing cyanobacteria in lakes is of concern. Cyanotoxins produced include hepatotoxins, neurotoxins, dermatoxins, and endotoxins, which negatively impact water quality. Efforts are made to control the blooms of cyanobacteria by biomanipulation (increasing grazing on the bacteria, top-down control) and by decreasing nutrient availability (bottom-up control). The introduction of exotic species of cyanobacteria that produced toxins, such as Cylindrospermopsis raciborskii, can compound the problems associated with these organisms by introducing new types of undesirable algae into lakes. Beyond the cyanobacteria, green sulfur, purple sulfur, and purple nonsulfur bacteria can occupy niches in lakes. Their niches occur in stratified lakes in which the anoxic hypolimnion has adequate levels of light to support their growth and, for the sulfur bacteria, hydrogen sulfide in the concentrations needed. The occurrence of these ideal conditions creates relatively narrow bands in the water column where these organisms are abundant certain times of the year in temperate dimictic lakes. Factors including oxygen concentrations, temperature, and light penetration are determinants of the vertical stratification of different types of photoautotrophs in lakes. The importance and perhaps diversity of nonoxygenic phototrophs is greater in lakes with permanent stratification; for example, in permanently frozen lakes of Antarctica or in meromictic lakes in other regions. In the case of bacteria, overall, -Proteobacteria appear to be the dominant group in freshwater, making the community of freshwater systems distinct in some ways from marine systems. The -Proteobacteria include the freshwater ammonia-oxidizing bacteria and are common in high-nutrient conditions and biofilms. In large oligotrophic lakes, there is increasing evidence for the importance of Actinobacteria. -Proteobacteria are found in peak numbers, in many cases, under more oligotrophic conditions, and can be correlated with chlorophyll concentration and have a preference for labile organic matter. In addition, the -Proteobacteria are abundant as are the Cytophaga–Flavobacterium–Bacteroides cluster and Verrucomicrobia. The Cytophaga–Flavobacterium are specialized for utilization of high-molecular weight organic compounds and thus may exhibit spatiotemporal patterns of abundance related to the occurrence of molecules of this type. The composition of the bacterial community and relative importance of each of these major groups can vary considerably among lakes as related to variations in trophic status, pH, temperature, and nutrient concentrations. Similarly, there are often differences in the relative abundances of major groups between the hypolimnion and the epilimnion in stratified lakes as well as between

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different portions of lakes (e.g., areas that are shallower and close to riverine inputs might have a different community composition than offshore sites). Fungi perhaps play a less important role in many lakes than other microorganisms because the role of plant detritus in some lakes is less significant. Aquatic fungi are important in littoral zones and in the degradation of macrophyte tissue. Many fungi that are encountered on plant material may be of allochthonous origin having colonized the tissue prior to submergence. However, the overall contribution of C from littoral zones and macrophytes is highly variable among lakes depending on size and morphometry. In many lakes, pelagic processes and in particular algal–bacterial coupling may be the central component of the microbial food web. Algae in lakes have been studied for many years and are responsible typically for most of the C fixed in many lakes, especially those that are large and too deep for growth of rooted macrophytes. Algal communities are diverse and different species fill varying ecological roles based on differences in motility, habitat preferences, and palatability to aquatic animals. Algae can be directly consumed by grazers and support bacterial growth via release of exudates (Figure 3). In addition to prokaryotic algae, diatoms, green algae, chrysophytes, and dinoflagellates are common lake flora, whereas some important marine groups (red and brown algae) are not as common in freshwater lakes. The relative balance between the prokaryotic algae (cyanobacteria) and the eukaryotic algae depends on many factors including light and nutrient availability. The stoichiometric ratio between N and P is a particularly critical factor. Protozoa play an important role in the microbial loop of lakes and are major consumers of bacteria. There is tremendous diversity of freshwater protozoa, including organisms that are mixotrophs (those that are photosynthetic and also consume prey, such as bacteria). There are large spatiotemporal changes in the distribution and abundance of different species, which is facilitated by the ability of some species to form cysts to persist undesirable conditions. There is vast morphological and functional diversity among freshwater protozoa. Overall, from an ecological standpoint, the smaller heterotrophic flagellates may be the most important bacterial grazers.

Anthropogenic Disturbance Human impacts on lakes include those that are direct and those that are indirect. Indirectly, humans impact hydrology and nutrient loading into lakes whereas anthropogenic disturbance also has direct effects through invasive species. Other anthropogenic disturbances of concern that may impact microbial ecology include changes in lake water level, decreasing pH from acid precipitation, input of

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gasoline additives from boating, heavy metal pollution (such as mercury), dredging, and so on. Invasive species in lakes range from fish to invertebrates to plants to zooplankton. Examples include the zebra mussel (Dreissena polymorpha), water hyacinth (Eichhornia crassipes), and Eurasian watermilfoil (Myriophyllum spicatum). There are numerous other examples of invasive species including the widespread practice of stocking of fish into lakes for fishing purposes. The role of exotic species in altering the food web and microbial processes in some cases is largely unknown, whereas in other cases the impacts are at least partially explored. For example, the zebra mussel has invaded areas such as the Great Lakes in North America and greatly alters the environment by clearing the water while filter feeding and depositing materials in sediments through feces and pseudofeces. This increases water clarity and decreases pelagic phytoplankton biomass. This alteration can correspondingly impact the role of the microbial loop in the food web; for example, they may alter the diversity and abundance of protozoa. In addition, these mussels have negative impacts on native bivalves and alter the community of benthic invertebrates. Lakes are managed to enhance fish productivity, alter the type of vegetation present, reduce undesirable algae, make water bodies more suitable for boating and/or swimming, and improve water clarity. Many lake management strategies are focused on controlling the loading of nutrients into the system. Lake management practices include aeration, biological control, treatment with alum, and treatment with algaecides. Management of undesirable biological features may take top-down or bottom-up approaches. Top-down control includes grazer removal by the addition of planktivorous fish, whereas bottom-up control includes inactivation of P and decreasing external or internal nutrient loading. Species-specific lake management, such as control of a specific undesirable cyanobacterium via cyanophages, is an emerging area.

Streams and Rivers Properties of Lotic Ecosystems Lotic systems (streams and rivers) are characterized by water with a unidirectional flow and are classified based on ‘size’ as represented by stream order. Essentially, as illustrated in Figure 4, the joining of two first-order streams creates a second-order stream, the joining of two secondorder streams creates a third order, and so on. The processes at any site along the system are greatly influenced by upstream processes, as described, for example, in the River Continuum Concept (RCC), as well as by processes in the watershed. Water movement imposes a persistent and critical force on the biota, increasing the importance of the benthos of the ecosystem and limiting stratification in contrast to what is typically the case in lakes.

1st order 1st order 1st order 2nd order

1st order

1st order 2nd order

3rd order Figure 4 Schematic illustrating manner in which stream order is determined.

The RCC illustrates the upstream–downstream connection and the role that riparian vegetation structure can play. Although streams in nature may not follow the RCC model, because of human alterations, like reservoirs, and other factors, the notion of this profound connectivity among parts, longitudinal changes, and gradients of biological processes are conveyed by the model. In addition to the longitudinal and lateral connections, there are vertical connections with streams and the underlying water in a zone called the hyporheos. The extent of the hyporheos varies based on geomorphology and other features. A unique fauna can develop in these areas, in addition to communities of organisms that live in both the surface and the subsurface. Biofilms formed in the interstitial spaces can be a potent site for microbial activity and create additional niches for microorganisms. Hyporheic processes depend on the redox potential of the surface and underlying waters, and the occurrence of anaerobic hyporheic zones in proximity to aerobic surface substrates can create conditions in a relatively narrow spatial scale for a variety of oxidation and reduction reactions. The occurrence of upwelling and downwelling areas, and whether the hyporheic water is oxic or anoxic, determines the biogeochemistry of the system. In temperate streams, autumnal leaf inputs are a major source of organic matter for the food web. The quality of this leaf material as a biological resource varies among species based on structural properties and chemical constituents. These leaf materials are conditioned by microorganisms, especially bacteria and aquatic hyphomycetes, and can then be consumed by macroinvertebrates, functionally defined as shredders (Figure 5). The fecal production and fragmentation resulting from the feeding of these invertebrates result in the generation of fine particulate organic matter (FPOM). This FPOM is then available to another functional group of invertebrates, the collectors. Disturbance via flooding is another major feature of streams that can have varying impacts on the biota depending on the seasonality, magnitude, and duration of the event. Stream life is adapted to the flowing water conditions, but

Environmental Microbiology and Ecology | Freshwater Habitats

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Terrestrial

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Leaching

DOM

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cu oc

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e ak

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Figure 5 Decomposition of leaf material in a stream depicting microbial colonization, consumption by macroinvertebrates shredders, and the generation of fine particulate organic matter (FPOM). Reproduced from Cummins KW and Klug MJ (1979) Feeding ecology of stream invertebrates. Annual Review of Ecology and Systematics 10: 147–172.

water velocity and depth drastically increase during flooding, causing organisms to be dislodged. In addition, benthic materials can be entrained in the water column and be transported, resulting in scouring of surfaces and loss of biofilm. Flooding also connects the stream proper to its floodplain and the inputs of allochthonous materials jumps (including microorganisms, nutrients, and organic compounds). Overall, lotic ecosystem features that are critical to microbial ecology are as follows: unidirectional flow of water. • The The high degree of mixing resulting in a lack of stra• tification and high aeration. Strong interconnection to the surrounding watershed • and terrestrial environments. role of the benthos as a habitat for micro• Important organisms and the site of nutrient uptake.

significance of allochthonous inputs of microor• The ganisms, organic compounds, nutrients, and particles. to humans as water sources and transpor• Importance ters of waste water. of humans on the environment through sewage, • Impact industrial pollutants, agricultural runoff, reservoir construction, removal of woody debris, channelization, and so on. Microbial Processes Food webs and the C cycle

As in other freshwater environments, DOM is the dominant fraction of the detrital pool. DOM is made available to higher trophic levels through the microbial loop (Figure 6); bacteria utilize the DOM and are in turn

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Leaching Coarse particulate organic matter

Dissolved organic matter

Leaf/microbe complex

Fungi

Bacteria

Protozoa

Shredder macroinvertebrates

Meiofauna

Fine particulate organic matter

Collector/filterer macroinvertebrates

Figure 6 Basic illustration of C transfers in a stream where the coarse particulate organic matter (CPOM) is leaves.

consumed by protozoa. Although rates of consumption per individual are much higher for ciliates than flagellates, small flagellates are the predominant protozoan predator of bacteria. Protozoa can then be eaten by meiofauna, such as nematodes and benthic copepods. Another critical process in the C cycle is the degradation of coarse particulate organic matter (CPOM) such as leaves and wood. Aquatic hyphomycetes play a central role in the degradation of CPOM and can penetrate into the tissue of decomposing leaves, facilitating colonization by bacteria. There is evidence of competition as well as of facilitation among bacteria and fungi on leaves decomposing in streams. As noted above, these conditioned leaves are then palatable to leaf-shredding invertebrates. There are clear patterns of temporal succession within the microbial community of leaves with some fungal and bacterial species serving as early colonizers and others appearing later in the process. Conditioning of leaf material by microorganisms facilitates consumption by macroinvertebrates that assimilate various fractions of the microbial/leaf assemblage (Figure 6). In addition, leaf-eating invertebrates play host to a variety of microorganisms; for example, the cecum of the crane fly larva, Tipula, boasts large numbers of bacteria. The importance of algal production, and thus autochthonous C sources, varies greatly and light limitation is crucial. Both shading from riparian vegetation and turbidity can limit light and restrict algal production. Many low-order streams with well-developed canopies are considered heterotrophic because the P/R ratios are less than 1. In this case, the food web is dependent on C fixed in the terrestrial environment representing allochthonous

sources of organic compounds. In contrast, the RCC predicts that the P/R ratio will reach its maximum at middle orders. The P/R ratio is also expected potentially to be greater than 1 in open canopy streams, especially those with abundant nutrients and suitable substrates for algal attachment. Algal–bacterial coupling, which is widely observed in marine and lentic ecosystems, can also be evident in streams. Although the absolute contribution of autochthonous organic C to the overall DOC pool may be limited, this fraction generally has higher lability than the allochthonous fraction. The contribution of breakdown products of plant structural materials (lignocellulose) to the allochthonous fraction is high and these products can be highly recalcitrant. The basis for the observed algal– bacterial coupling has two components, namely, growth of bacteria on algal-released organic compounds and use of the algal cells as a physical substrate for attachment. Although the role of viruses in streams is largely unexplored, they potentially can alter C flow through the microbial loop by attacking bacteria and other organisms. Clearly, in other aquatic environments bacteriophage can be an important component of the microbial loop, and the abundances of viruses observed in lotic ecosystems suggests that this is also the case in flowing waters.

N cycle

One characteristic of nutrient relations in streams is the spiraling manner with which nutrients as well as bacterial cells are transported (Figure 7). The nutrient spiraling concept describes the process by which an atom that is released into the water column travels some distance downstream before being taken up again. The spiraling length varies depending on many stream properties such as retentiveness of the channel and water velocity. Transport of nutrients in this manner is experimentally

Water flow

Compounds/ cells

Benthos Figure 7 Schematic illustrating the manner in which cells and chemical compounds are transported some distance downstream before uptake into the benthos (via attachment, sorption, biological assimilation, etc.). Materials are subsequently released creating a spiral-like pattern.

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evaluated by measuring the uptake length of material released into the water column. In addition to biological processes, geochemical processes impact N dynamics in lotic ecosystems. Reactive sites in the benthos can result in chemical transformation and sorption of nutrients. This impacts nutrient availability to the biota, as well as transport downstream. Nitrogen, in particular, has been the subject of numerous studies in stream ecology because of its potential to serve as a limiting nutrient, influxes of N from fertilizer, and role in water quality. The quantity of N is highly variable among streams based on influxes from outside sources and biological processes in the riparian zone. Both assimilatory and dissimilatory reactions occur in the N cycle in streams and the role of particular N transformations is variable. Both inorganic and organic N are important N-cycle participants, although most emphasis has been placed on inorganic N. Sorption of ammonium to sediments can be high and, correspondingly, ammonium concentrations in water of rivers and streams tend to be low. The benthos create circumstances in which ammonification, denitrification, and nitrification can take place within a comparatively narrow fraction of the benthos and there can be steep redox potential gradients (Figure 8). Depending on the redox potential, the hyporheos can be potent sites of denitrification. Denitrification in the soil of the riparian zone can also contribute to the stream N cycle. N enters streams from both upstream, terrestrial, and groundwater sources where it is subjected to various microbial transformations (Figure 9). Processes are

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dominated by benthic components and are highly dependent on the oxygen gradient within the sediment. P cycle

Phosphorous in aquatic ecosystems is often a limiting nutrient and the form of P greatly impacts biological activity. Often soluble reactive phosphate is measured and represents a readily assimilated form; dissolved organic P is also present but is used more slowly. Sorption and desorption of phosphate to sediments (particularly fine particles) act to control the concentration of inorganic P in the water in streams and rivers. In addition, P concentrations can increase greatly after flooding, especially with input from runoff or sewage. Microbial Communities Microbial communities in streams are a complex mixture of cells from allochthonous locations and those produced within the stream proper. Distinguishing among these components is quite problematic and the relative contribution of the fractions depends greatly on local events, such as rainfall, flooding, and so on. Biofilms with complex three-dimensional structures form on stream surfaces including wood, leaves, rocks, and smaller particles. These biofilms are critical habitats for microbial production in lotic ecosystems and consist of complex mixtures of cells of different types embedded in a matrix of extracellular polysaccharide. The matrix is interspersed with channels serving as transport venues and plays host to a mixture of autotrophic and

Atmosphere

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Water table Surface water

Periphyton uptake

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NH+4 Detritus decomposition Ammonification

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Fixation

High O2

Physical adsorption

Low NH+4

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Denitrification Nitrate reduction

High NH4+

Figure 8 Interplay between aerobic and anaerobic zones and N transformations in the benthos of a stream. Reproduced from Triska FJ, Jackamn AP, Duff JH, and Avanzino RJ (1994) Ammonium sorption to channel and riparian sediments: A transient storage pool for dissolved inorganic nitrogen. Biogeochemistry 26: 67–83.

Environmental Microbiology and Ecology | Freshwater Habitats

Riparian vegetation

Litter inputs

Atmospheric N2

Stream water DON Import from upstream

NH3

Assimilation

NO3 N2

Sediment surface

NH3 Interstitial water

Assimilation

NO3

Cyanobacteria and microbial populations Benthic algae

Decomposition

NO2

DON NH3 NO3

NO3

Biota

Assimilation

N2 Denitrification

Nitrogen fixation

Export to downstream

Nitrification

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O2 concentration

NH3 Excretion

Nitrogen fixation Particulate organic matter DecompNH3 and associated osition accummicrobes Excretion ulation

NO2 NO3

Groundwater DON, NO3 Figure 9 Schematic of the nitrogen cycle in a stream; DON, dissolved organic nitrogen. Reproduced from Allan JD (1995) Stream Ecology: Structure and Function of Running Waters. London: Chapman and Hall.

heterotrophic microorganisms. Water velocity is a major determinant of biofilm thickness in streams and physical disruption can ‘reset’ the community by clearing the surface of many of biofilm residents. The nature and activity of the biofilm may vary depending on substrate type and various terms are applied to biofilms on different surfaces, such as epilithon for biofilms on rocks, epiphyton for biofilms on macrophytes, and epixylon for biofilms on wood. Within the biofilm, various niches occur and there are opportunities for many types of biotic interactions, such as predation and competition. The biofilm creates an opportunity for cells to maintain position in the flowing water and to accumulate resources in transport. Of particular importance is the ability of the biofilm to aide in retention of enzymes released from the cells to degrade polymers. Activity of enzymes in biofilms and other habitats, including phenol oxidase -D-glucosidase phosphatases, endo-cellulase, cellobiohydrolase, and aminopeptidase, has often been measured to assess the potential for enzymatic degradation. Typically, assays rely on chromogenic or fluorogenic substrates. Although these methods do not allow the researcher to determine the organism that

produced the enzyme, they do provide measures of specific activity and degradative potentials. Like nutrients, microbial cells are also transported along the length of the stream; this process, called information spiraling, builds on the nutrient spiraling concept described above. A variety of processes influence the release of cells from surfaces, including the activities of animals, physical disruption, sloughing, and natural dispersal properties of the species. The uptake length of bacterial cells has been measured in streams through experimental addition of bacteria. Bacterial communities in streams, as in lakes, are numerically dominated by Proteobacteria, particularly the -Proteobacteria. Although the diversity of bacteria in streams and rivers is less studied than marine or lake environments, we do know that the Proteobacteria, Actinobacteria, and Cytophaga–Flavobacterium cluster are common in lotic ecosystems. Some bacterial groups may perform specialized roles in nutrient cycling or in degradation of particular organic compounds. For example, the actinomycetes participate in the decomposition of leaf material in streams along with other types of bacteria and the hyphomycetes.

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Although there are increasingly good data that describe the bacterial community composition in streams, in which non-culture-based approaches replace the more traditional culture-dependent methods, there are still limitations in our ability to connect structure and function. In streams and rivers (as well as other freshwater habitats), the number of bacterial cells in the community that are active (such as those that are undergoing respiration) is often a small percentage of the total number of cells. This may be a particularly important component in lotic ecosystems because of the inputs of large numbers of allochthonous cells. Although these cells do not contribute necessarily to ecological functions, they are still detected via many of the widely used methods. The contribution of archaea to lotic ecosystems is largely unknown beyond the role of methanogens. Relative to bacteria and other systems, there is very limited information about the abundance, diversity, and activity of archaea in streams and rivers. Fungal communities in streams, as well as other freshwater environments, are dominated by aquatic hyphomycetes, which are identified based on the structure of their conidia. Hyphomycetes are not a specific taxonomic group, rather they are a heterogeneous group of fungi. Hyphomycetes are one type of microsporic fungi and produce conidia directly from hyphae or via conidiophores. The group can be further subdivided and, in streams, much focus is on the so-called aquatic hyphomycetes, a polyphyletic group, that play an established role in the decomposition of leaf material. Common genera of hyphomycetes found include Anguillospora, Tetrachaetum, Tetracladium, Clavatospora, Goniopila, Helicromyces, Lemonniera, and Heliscella. Algal communities in streams can be light-limited (either by shading of riparian vegetation or by turbidity) and nutrient-limited and their contribution to the C in streams varies based on these limitations. Attachment to surfaces is another critical need as phytoplankton generally develops only in large rivers. Diatoms are both diverse and abundant in streams particularly attached to rocks and cobbles and are generally the most abundant algae in stream biofilms. The distribution of diatom species varies predictably among locations with different water velocities. In addition, the growth form of filamentous green algae (i.e., Cladophora) varies with velocity. Algal stream communities are greatly impacted by light, temperature, herbivory, water velocity, substrate type, and nutrient availability. This latter includes silica, which is essential for diatoms; however, in lotic ecosystems, silica is generally found in adequate supplies. Diatoms, which include genera such as Achnanthes, Gomphonema, and Navicula, can be more successful under low light conditions than other algae; this is a likely contributor to their success in streams. In terms of nutrient limitations, autotrophs in streams are generally thought to be P limited not N (although exceptions to this trend occur).

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Protozoa in streams are in many ways less well studied than in lakes. It is known that protozoa play a major role in the microbial loop in streams. Small flagellates are considered to be the most important consumers among the protozoa in streams and can greatly alter bacterial abundance. Anthropogenic Disturbance Human impact on streams is profound and has greatly altered the structure and function of streams and rivers globally. These impacts in turn impact the role of the microbial community and essential biogeochemical processes. Introduction of sewage effluent and fecal waste from human/animal activities into lotic systems is widespread and serves as a source of microorganisms, nutrients, organic compounds, as well as various pollutants used by humans and disposed of in sewage. The input of allochthonous microorganisms in this manner has critical impacts on water quality, human health, and the spread of water-borne diseases. Inputs of organic compounds and nutrients from sewage alter the resource availability to the biota and can, in some cases, result in reduction of oxygen concentrations in the water. Often these inputs are monitored using biological indicators of sewage contamination such as enumerating fecal coliforms. In addition, other fecal-associated organisms and viruses may be monitored to assess different types of risks, like Cryptosporidium (from cattle feces) or fecal streptococci (Gram-positive cocci) from human sewage effluent. To reduce the risk of contamination of water by pathogens, treatment of effluent is often carried out, in particular using chlorination. Chlorination does have associated health risks through the production of trichloromethanes from natural-occurring dissolved organic compounds and alternative strategies, and dechlorination can be used to minimize these risks. Humans also alter the physical structure of streams through channelization for navigation purposes, removal of woody debris, straightening of channels and removing of meanders, dam and reservoir construction, and so on. Such impacts have gone on for many years and thus have had long-term consequences on stream and river function. In particular, water residence time is altered by these activities, which, in turn, alters transport of materials and uptake lengths. For some alterations, such as addition of reservoirs, residence time is greatly increased creating unexpected lentic regions in the middle of otherwise ‘normal’ river continua. At these discontinuities, material in transport can drop out of the water column and pelagic processes can replace benthic processes in relative importance. Acid mine drainage has a specific impact on streams and has huge impacts on the microbial ecology of the

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system. The problems start with the mining of coal that is mixed with pyrite. Iron and sulfur oxidation leads to a decrease in pH and favors the growth of specific types of microorganisms, which further this oxygen and maintain the low pH. This greatly lower pH has profound negative impacts on all other forms of stream life.

Comparison Among Freshwater Habitats Hydrology, the potential for oxygen depletion, whether organic C is primarily of allochthonous or autochthonous origin, and the role of plant detritus are among the major determinants of variations among freshwater ecosystems in microbial processes. These variations coupled with the relatively limited number of studies on some aspects of ecology of microorganisms in freshwater habitats limits the number of generalities that have emerged. A critical limitation on our ability to draw generalities about microbial ecology of freshwater habitats is the relatively limited number of studies performed on certain aspects of the topic. This fact, coupled with the limited coverage of the broad range of diversity of ecosystems, suggests that we have yet to reveal many of the underlying tendencies that may unite freshwater habitats. It is not known whether specific locations that have been studied in some detail are representative of the greater array of locations that have never been studied. Overall, major determinants of the structure and function of the microbial community in freshwater habitats are the occurrence and importance of plant detritus, the nature of the hydrology of the system, and the occurrence of oxic and anoxic zones. The potential for drastic spatial and temporal changes in critical factors, such as

redox potential, creates opportunities for a diversity of different microbial niches and interconnectivities. This creates interesting spatiotemporal juxtapositions of complementary microbial processes over comparatively small scales. Each microbe-mediated process can be altered by anthropogenic disturbances, which are all too common in freshwater systems because of the proximity to human populations, strong connections to the surrounding landscape, and vital importance for human success. See also: Algal Blooms; Biofilms, Microbial; Food Webs, Microbial; Nitrogen Cycle; Sediment Habitats, including Watery

Further Reading Allan JD (1995) Stream Ecology: Structure and Function of Running Waters. London: Chapman and Hall. Bodelier PLE, Frenzel P, Drake HL, et al. (2006) Ecological aspects of microbes and microbial communities inhabiting the rhizosphere of wetland plants. In: Verhoeven JTA, Beltman B, Booink R, and Whigham DF (eds.) Wetlands and Natural Resource Management. Berlin: Springer-Verlag. Leff LG (2002) Stream microbiology. In: Bitton G (ed.) Encyclopedia of Environmental Microbiology. New York: Wiley and Sons. Mitsch WJ and Gosselink JG (2000) Wetlands, 3rd edn. New York: Wiley and Sons. Newbold JD, Elwood JW, O’Neil RV, and Van Winkle W (1981) Measuring nutrient spiraling in streams. Canadian Journal of Fisheries and Aquatic Sciences 38: 860–863. Sigee DC (2005) Freshwater Microbiology. New York: Wiley and Sons. Vannote RL, Minshall GW, Cummins KW, Sedell JR, and Cushing CE (1980) The river continuum concept. Canadian Journal of Fisheries and Aquatic Sciences 37: 130–137. Wetzel RG (2001) Limnology, 3rd edn. San Diego: Academic Press. Zwart G, Crump BC, Kamst-van Agterveld MP, Hagen F, and Han SK (2002) Typical freshwater bacteria: An analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquatic Microbial Ecology 28: 141–155.

Heavy Metals Cycle (Arsenic, Mercury, Selenium, others) C Rensing, University of Arizona, Tucson, AZ, USA B P Rosen, Wayne State University, School of Medicine, Detroit, MI, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction 33 As75 29 Cu63

Glossary arsenic The name ‘arsenic’ comes from the Persian word ‘zarnikh’, which means ‘yellow orpiment (As2S3)’. The atomic number of this metalloid is 33, and its average mass is 74.92, with a single naturally abundant isotope As75. It is present in the earth’s crust at 1 ppm, which is 0.1% of the abundance of phosphorus. Its two biologically relevant oxidation states are As(III) (arsenite) and As(V) (arsenate). Arsenic compounds have no biological roles and are only toxic. Arsenate is toxic because of its chemical similarity to phosphate. Sugar and nucleotide arsenates are less stable than phosphates. Arsenite and trivalent organoarsenicals are considerably more toxic than pentavalent arsenicals. Arsenite forms bonds with sulfur thiolates. Intracellularly, arsenite is probably complexed with glutathione (GSH), which can deplete GSH pools, raising the intracellular redox potential and result in formation of free radicals. Arsenite binds much more strongly to vicinal cysteine pairs in small molecules such as lipoic acid and enzymes such as pyruvate dehydrogenase and is therefore a potent inhibitor of oxidative metabolism. metalloid Metalloids are located in the periodic table on either side of the zigzag line that distinguishes metals from nonmetals. Metalloids such as arsenic are semiconductors. They have both metallic and nonmetallic properties. As(V) in arsenate has nonmetallic properties similar to P(V) in phosphate. As(III) has metallic properties, interacting as a soft metal with soft ligand such as thiols in enzymes. Selenium, while not formally a metalloid, Se(IV) has chemical and toxicological properties similar to those of the metalloid As(III) and is often considered in the same category. transition metal Transition metals are defined as having partly filled d or f shells. A broader definition also includes elements with partly filled d or f shells in any of their commonly occurring oxidation states. The resulting 56 transition elements can be categorized into three main groups: (1) lanthanide elements, (2) actinide elements, and (3) the d-block or main transition elements. The latter is of importance to this article

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Mn55 Se80 Further Reading

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because it includes Cu and Mn and all of them have partly filled 3d shells. Transition metals can conduct heat and electricity well and they can form alloys with one another and other metallic elements. They usually show variable valence and because of their partially filled shells form some paramagnetic compounds. copper Copper has a single s electron outside filled 3d shells and occurs mainly in two oxidation states, Cu(I) and Cu(II). Copper with an atomic number of 29 has an average atomic mass of 63.55 and is very similar in electron structure and characteristics to Ag and Au. Copper has important biological functions in many but not all organisms. Today copper is extensively used in applications such as in piping and electronics but was already an important resource in antiquity. It was named aes Cyprium because it was mined in Cyprus and later simplified to cuprum. manganese Manganese is a widely distributed metal constituting about 0.085% of the earth’s crust. It has the atomic number 25 and an average atomic weight of 54.94. In its physical and chemical properties Mn is most similar to Fe. The most common and stable oxidation states in nature are Mn(II), Mn(III), and Mn(IV). Mn(II) is a required trace element for all organisms functioning as a cofactor for a number of enzymes, including oxidoreductases, hydrolases, isomerases, ligases, and Mn-containing superoxide dismutase (SOD). The origin of the name manganese is somewhat confusing since two unrelated black minerals from Magnesia in Greece were called magnes: one being the magnetic magnetite and the other magnesia is manganese dioxide. selenium The name of the element comes from the Greek ‘selene’ (moon) because of the lustrous sheen of the metal. Selenium has an atomic number of 34 and average atomic weight of 78.96, with two major naturally abundant isotopes: 78 (23.6%) and 80 (49.7%). Its abundance in the earth’s crust is 0.05 ppm. Se(IV) (selenite) and Se(VI) (selenate) are the two major oxidation states. Elemental selenium is relatively nontoxic. Hydrogen selenide (H2Se) and other selenium

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compounds are extremely toxic. Selenium inhibits enzymes in similar reactions to those of arsenic and is sometimes used as an arsenic antidote. In lower amounts, selenium is a required micronutrient and

antioxidant. It is incorporated into antioxidant enzymes in both prokaryotes and eukaryotes primarily as selenocysteine and, to a lesser degree, as selenomethionine.

Abbreviations

MRP

AQP CCA c-Cyts DMA DMS DMSO EXAFS FMN Grx GSH LMW PTPase MCO MMA

aquaporin chromated copper arsenate c-type cytochromes dimethylarsenate dimethyl sulfide dimethyl sulfoxide extended X-ray absorption fine structure spectroscopy flavin mononucleotide glutaredoxin glutathione low molecular weight protein tyrosine phosphatases multicopper oxidase monomethylarsenate

Defining Statement Microbial enzymes are significantly involved in transforming metal(loid)s in the environment. Recent advances in identification and characterization of As-, Mn-, Cu-, and Se-transforming enzymes and remaining challenges are described.

Introduction Life may have first originated in deep oceanic hydrothermal vents that were rich in metals, such as arsenic, lead, copper, manganese, and zinc. Microorganisms not only had to deal with the toxic effects of metal(loid)s but also were actively involved in transforming metal-(loid)s, thereby shaping the environment. This ancient environmental challenge was a driving force for the evolution of mechanisms for metal ion homeostasis and detoxification. For example, copper is an essential nutrient that serves as a cofactor for numerous enzymes and yet is cytotoxic because of its role in generating reactive oxygen species and other reactive compounds. Organisms in all kingdoms of life have evolved elaborate copper homeostatic mechanisms to take advantage of copper chemistry while preventing unwanted side

NADPH N2OR PHM PQQ RNAP SOD Tat TMAO Tmp Trx UNICEF WHO

multidrug resistance-associated protein nicotinamide adenine dinucleotide phosphate N2O reductase peptidylglycine-alphahydroxylating monooxygenase pyrroloquinoline RNA polymerase superoxide dismutase twin-arginine translocation trimethylarsine oxide trimethyl purine methylase thioredoxin United Nations Children’s Fund World Health Organization

reactions. The same principle holds true for other essential metals. The four metal(loid)s discussed here are all toxic in excess but can perform biologically important roles, for example, as terminal electron acceptor. Arsenic (As) is an abundant, ubiquitous, and dynamic trace element that is involved in a variety of microbial metabolic processes including several detoxification and energy conservation pathways. The environmental processes contributing to As fate and transport have been studied intensively during the last decade, in part due to global water quality crises arising from As-contaminated drinking water. For example, arsenic in the water supply in southern and western Bangladesh and the adjacent regions of India has triggered a health catastrophe (http://bicn.com). Manganese oxides are often produced by bacteria and fungi and affect many biological processes including carbon fixation, photosynthesis, and scavenging of reactive oxygen species. In addition, manganese minerals are often utilized as terminal electron acceptor. Finally, selenocysteine incorporated into proteins as the 21st amino acid makes selenium a required trace element in many organisms. However, the higher valence states of Se(VI) and Se(IV) are toxic at elevated concentrations, and microorganisms are involved in transformations such as reduction and methylation making the products less toxic.

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others) 33

As75

The Arsenic Biogeocycle An overview of the arsenic biogeocycle is shown in Figure 1. Although arsenic is relatively rare in the Earth’s crust, ranking twentieth in abundance, it is widespread and found in high concentration in association with other minerals such as arsenides, sulfides, and sulfosalts with other metals. The free metal is uncommon, and arsenic occurs primarily in three oxidation states: arsenates (As(V)), arsenites (As(III)), and arsenides (As(-III)). Volcanic activity and hydrothermal sources are major contributors to human exposure to arsenic. For example, the Yellowstone Caldera, the largest volcanic system in North America, discharges massive amounts of arsenic from geysers and fumaroles. Some of the arsenic is in the form of insoluble sulfides, but soluble arsenic can be 4 mmol l1, one of the highest concentrations of soluble arsenic in the world. There is abundant microbial life in these streams and pools in spite of these extraordinary amounts of arsenic. The arsenic begins as the reduced form, arsenite, but is rapidly oxidized biologically by microorganisms with arsenite oxidases. Further from the source, the arsenite can be reduced by microorganisms with respiratory arsenate reductases. Some of the arsenic is volatilized, and organisms that methylate the arsenic to trimethylarsine have been

identified. Mining, copper smelting, and burning of coal also introduce arsenic into the environment. Arsenopyrite (FeAsS), the most common mineral form of arsenic, is often associated with other elements, including gold and copper. This can complicate bioleaching of ores using organisms such as thiobacilli because the sulfuric acid produced by the bacteria to dissolve the gold also dissolves the arsenic, which is toxic to the organism. Arsenic derived from an ancient Roman copper mine is also a major environmental contaminant in the Rio Tinto in southwestern Spain. Despite the millimolar concentrations of arsenic in the river, organisms proliferate, including highly arsenic resistance fungi. Anthropogenic sources of arsenic include herbicides, insecticides, rodenticides, wood preservatives, animal feeds, paints, dyes, and semiconductors. Some contain inorganic arsenic such as chromated copper arsenate (CCA), which has been used for many decades to treat wood against attack by fungi and insects. If the wood is not sealed, the arsenic can find its way into human water and food supply. Both inorganic and organic arsenicals are used for agriculture and animal husbandry. During the last century, arsenic acid (H3AsO4), sold as Desiccant L10 by Atochem/Elf Aquitaine, was euphemistically called ‘harvest aid for cotton’ because it was used to defoliate cotton to allow planting of the next cotton crop. While it is no longer used agriculturally, the inorganic arsenic

Atmospheric arsenic

Arsenic in drinking water

DMA, TMA DMA(III) DMA(V)

Methylation

Reductase As(III)

MMA(III)

As(V) As(III)

MMA(V) Oxidase

207

As(V)

Arsenobetaine Arsenocholine Arsenosugar Arsenolipid

Figure 1 The arsenic geocycle. See text for details. Reproduced from Bhattacharjee H and Rosen BP (2007) Microbial arsenic metabolism. In: Nies DH and Silver S (eds.) How Bacteria Handle Heavy Metals, pp. 371–406. Heidelberg: Springer.

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remains in fields throughout the southern United States. That land is now used for planting rice, and grocery store rice from those states constitutes the largest non-seafood source of arsenic in the American diet. The sodium and calcium salts of monomethylarsenate (MMA) and dimethylarsenate (DMA) are currently widely used as herbicides and pesticides. The sole active ingredient in Weed-B-Gone Crabgrass Killer, which is sold in neighborhood lawn and garden stores, is calcium MMA. DMA and MMA are also widely used as fungicides on golf courses in Florida, and the resulting arsenic enters the water supply of Florida municipalities. MMA is also degraded to arsenate and arsenite, presumably by microorganisms. During the Vietnam War, the United States sprayed DMA (in the form of the ‘rainbow herbicide’ Agent Blue) on rice paddies and other crops in Vietnam to eliminate the food supply. Some bacteria can use DMA as a source of carbon, with demethylation to inorganic arsenic. Organic arsenicals are also used as growth enhancers and feed supplements in animal husbandry. For example, Roxarsone (4-hydroxy-3-nitrophenylarsonic acid) is fed to half a billion chickens each year in Maryland alone to control coccidial intestinal parasites and is excreted unaltered. The manure is used as fertilizer for crops, and, during composting, the roxarsone is degraded anaerobically, at least in part by bacteria such as clostridia, into inorganic arsenic, which contaminates water supplies. The use of organic arsenicals as feed supplements also has the potential to spread antibiotic resistance. Arsenic contamination of human water and food supplies is a worldwide problem, but the worst situation is in West Bengal, India, and Bangladesh, where tens of millions of inhabitants are exposed to dangerous levels of arsenic. While this arsenic is from natural sources and is not anthropogenic, it is an unintended consequence of human intervention for humanitarian reasons. To help prevent the spread of infectious diseases such as cholera and malaria, the United Nations Children’s Fund (UNICEF) recommended and financed the construction of tube wells for water for drinking and irrigation in lieu of surface waters that carried infectious organisms. While this practice reduced the incidence of infections, the well water has dissolved arsenic concentrations that exceed the World Health Organization’s (WHO) maximal allowable level of 10 ppb by 10- to 100-fold, leading to skyrocketing rates of cancer and other arsenic-related disorders. Where the arsenic in well water comes from is still somewhat controversial. Most of the arsenic is immobilized in bedrock, so how it entered the water supply was unclear. Recent data suggest that the irrigation of crops allows organic material to percolate downward, providing anaerobic organisms with carbon and reducing potential to reduce and solubilize arsenic-containing minerals. Indeed, the location of the immobilized arsenic may

actually be in a higher stratum than originally thought, such that the arsenic actually enters the wells from above. What is becoming clear, however, is that human agriculture coupled with microbial activity is related to this environmental catastrophe. This emphasizes the substantial role that microbes play in cycling arsenic between oxidized and reduced forms and between inorganic and organic species. Arsenate is taken up by microbes, primarily via phosphate transport systems. Intracellular arsenate is reduced to arsenite, which is then extruded out of the cell, through either channels or active efflux systems. Bacteria can also use arsenate as a terminal electron acceptor in anaerobic respiration, generating arsenite as the product. Their action in arsenate-rich sediments can lead to arsenic contamination of ground water. Arsenite-oxidizing microbes utilize the reducing power from As(III) oxidation to gain energy for cell growth. Soil bacteria can also methylate arsenite to the gas trimethylarsine, returning soil arsenic to the atmosphere. Marine microorganisms convert inorganic arsenicals to various water- or lipid-soluble organic arsenicals, including di- and trimethylated arsenic derivatives, arsenocholine, arsenobetaine, arsenosugars, and arsenolipids. Metabolism of arsenobetaine by marine microbes completes the arsenic cycle in marine ecosystems. Arsenic Uptake Systems Inorganic arsenic has two biologically important oxidation states, pentavalent (As(V)) and trivalent (As(III)). Since arsenic is only a toxic element and has no nutritional or metabolic role, both trivalent and pentavalent inorganic arsenic are taken into cells adventitiously by uptake systems for other compounds. In solution, pentavalent inorganic arsenic is arsenate (H3AsO4), an analogue of phosphate, and bacteria take up arsenate via phosphate transporters. In Escherichia coli, both phosphate transporters, Pit and Pst, take up arsenate, with the Pit system being the major system. Solid, trivalent arsenic (As2O3 or arsenic trioxide) dissolves to form the undissociated acid, As(OH)3, at neutral pH. With a pKa of 9.2, it would be present in significant amounts as the anion arsenite only in alkaline environments (which could be important for the arsenic biology of alkalophiles). Even so, inorganic As(III) is usually called arsenite in the literature, so this term will be used interchangeably with arsenic trioxide. Two pathways for uptake of trivalent metalloids As(III) and Sb(III) have been identified in prokaryotes and eukaryotes. The E. coli glycerol facilitator, GlpF, was the first identified uptake system for As(III) (and Sb(III)). Bacterial GlpF was also the first member of the aquaporin (AQP) superfamily to be identified, even before the identification of the human water channel Aqp1, which led to the award of the Nobel Prize in Chemistry to Peter Agre in 2003. The superfamily has two branches, the classical aquaporins,

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others)

Human AQP9

Human AQP3 Human AQP10 Mouse AQP7 E. coli GlpF

As Aquaglyceroporins

Rat AQP9

209

5Å

S. cerevisiae Fps1p Human AQP2 Human AQP5

Rat AQP6 Human AQP1

Aquaporins

Human AQP0

3Å

Human AQP4 Human AQP8 Figure 2 Members of the aquaporin superfamily conduct As(OH)3. Left: The aquaporin superfamily is very large, and only selected members are shown. The lower branch (left) includes classical aquaporins that conduct only water. The upper branches are aquaglyceroporins from bacteria, yeast, and mammals. Of those, Escherichia coli GlpF, Saccharomyces cerevisiae Fps1p, and mammalian AQP7 and AQP9. Right: Space-filling models of As(OH)3 (top) and water (bottom) next to cross-sections of the X-ray crystal structure of AQP1 (bottom) and GlpF (top) at the narrowest point of the channel (diameter indicated).

which are water channels with small channel openings and aquaglyceroporins with channels large enough for molecules as big as glycerol (Figure 2). Most bacteria have GlpF homologues for glycerol uptake that also allow inadvertent arsenite entry, rendering them sensitive to arsenite. Eukaryotic microbes similarly have AQPs that conduct uptake of As(III), for example Fps1p, the yeast homologue of GlpF, conducts arsenite uptake in Saccharomyces cerevisiae. Another eukaryotic microbe, the human pathogen Leishmania major, takes up As(III) via aquaglyceroporin LmAQP1. It also takes up the related metalloid Sb(III) by LmAQP1, which is relevant to the treatment of leishmaniasis. The pentavalent antimonial drug Pentostam is reduced, at least in part by macrophage to Sb(III), the active form of the drug. In the phagolysosome of the infected macrophage, the Leishmania amastigote takes up the activated drug by LmAQP1. Surprisingly, some organisms have ars operons encoding an aquaglyceroporin homologue termed AqpS. Obviously, this arsenic-inducible AQP is related to arsenic resistance and did not evolve for glycerol uptake. This presents a quandary: How does a channel that can conduct solutes only down a concentration gradient confers resistance when homologues actually make cells sensitive? The answer appears to be that AqpS does, indeed, render the cells sensitive to arsenite but confer arsenate resistance in conjunction with the ArsC arsenate reductase, about which more is discussed below. As

arsenate enters the cell, it is reduced to arsenite. Lacking an arsenite efflux system, the cell substitutes the AqpS, which allows the internally generated arsenite to flow down its concentration gradient out of the cell. Another group of S. cerevisiae permeases that adventitiously facilitate arsenite uptake is the Hxt glucose transporter permease family. S. cerevisiae has 18 hexose transporters, Hxt1p to Hxt17p, Gal2p, and two glucose sensors, Snf3p and Rgt2p, and a number of them transport arsenite. While no Hxt takes up As(III) as rapidly as Fps1p, in the aggregate about 75% of the arsenite gets into yeast by Hxts and about 25% by Fps1p when glucose is absent from the medium. In the presence of glucose, which competitively inhibits arsenite uptake by Hxts, most of the arsenite gets in by Fps1p. It is not known whether bacterial glucose permeases or other sugar transporters also catalyze arsenite uptake. A glpF deletion of E. coli still takes up about 20% of the arsenite compared to wild type, suggesting the presence of one or more as yet unidentified arsenite uptake systems.

Arsenic Respiration Respiratory arsenate reductases

As discussed above, arsenate reduction plays an important role in arsenic geochemistry and may lead to arsenic contamination of drinking water supplies. Under anaerobic growth, the reduction of arsenate as a terminal electron acceptor by respiratory enzymes generates

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energy, with the production of arsenite. For example, Chrysiogenes arsenatis, uses acetate as the electron donor for arsenate respiration by an arsenate reductase enzyme, ArrAB. The C. arsenatis arsenate reductase is located in the periplasm and is a dimer composed of 87 kDa ArrA and 29 kDa ArrB subunits. ArrAB couples to the respiratory chain and provides energy for oxidative phosphorylation. ArrA is a Mo/Fe protein homologous to dimethyl sulfoxide (DMSO) reductases. ArrB probably has an [4Fe–4S] cluster that transfers electrons to the Mo cofactor of ArrA. ArrA from Shewanella sp. strain ANA-3 has a Tat (twin-arginine translocation) motif for translocation to the periplasm. ArrAB from Bacillus selenitireducens strain MLS10 is a heterodimer of 150 kDa composed of ArrA (110 kDa) and ArrB (34 kDa), again with a putative Tat signal in the gene for ArrA. In contrast to the energy-generating ArrAB arsenate reductases, cytosolic ArsC arsenate reductases are involved in arsenic detoxification (see below), and some bacteria such as Shewanella sp. strain ANA-3 have both. The arrAB genes are expressed anaerobically at nanomolar arsenate or arsenite. Only when environmental arsenic builds up to toxic levels is the ars resistance operon expressed both aerobically and anaerobically. Arsenite oxidases

Arsenite oxidases are found in both heterotrophic and chemoautotrophic bacteria. Arsenite oxidases have been cloned from -proteobacteria, heterotrophic Hydrogenophaga sp. strain NT-14 and chemolithoautotrophic bacterium, NT26. The latter two use the reducing power from As(III) oxidation for growth, suggesting a bioenergetic function for arsenite oxidases. The arsenite oxidase genes of Agrobacterium tumefaciens are regulated by a two-component sensor kinase system, composed of the aoxS (sensor) and aoxR (response regulator) genes. AoxR does not contain an identifiable As(III)-binding site, so what is actually sensed by AoxR is not known. The best characterized arsenite oxidase enzyme is from the soil bacterium Alcaligenes faecalis. The A. faecalis arsenite oxidase is a 100 kDa dimer of a large and a small subunit. The large 825-residue catalytic subunit is structurally related to DMSO reductases and is homologous to the ArrA subunit of the respiratory arsenate reductases. The small subunit (134) is homologous to Rieske proteins. The oxidase is bound to the periplasmic surface of the inner membrane by an N-terminal transmembrane helix of the small subunit.

Arsenic Detoxification Regulation of ars operons

Expression of ars genes is controlled by ArsR regulatory proteins, which are members of the ArsR/SmtB family of metal(loid)-responsive repressors. The first identified

members were the 117-residue As(III)/Sb(III)-responsive ArsR repressor of the arsRDABC operon of plasmid R773 and the 122-residue Zn(II)-responsive SmtB repressor. Over 200 homologues have been identified in Gram-positive and Gram-negative bacteria and in archaea. These include proteins that respond to As(III)/Sb(III) (ArsR), Pb(II)/Cd(II)/ Zn(II) (CadC), Cd(II)/Pb(II) (CmtR), Zn(II) (SmtB and ZiaR), and Co(II)/Ni(II) (NmtR). The well-characterized members (and by extrapolation, probably all) are homodimers that bind the operator/promoter DNA in the absence of inducing metal ion, repressing transcription. Upon binding metal, they dissociate from the DNA, resulting in derepression. Given the diversity of the ArsR/SmtB family, how can As(III)-responsive repressors be recognized? It is reasonable to assume that a homologue within or adjacent to an ars operon or to arsenic resistance genes is an ArsR repressor. Even though the arsenic-responsive repressors are all homologues, comparison of several sequences shows that As(III)-binding sites arose independently at spatially distinct locations in their structures. The best characterized is the R773 ArsR. Each ArsR subunit has a metal-binding site formed by Cys32, Cys34, and Cys37. The distance between As(III) and each of the three cysteine thiolates is 2.25 A˚, as determined by extended X-ray absorption fine structure spectroscopy (EXAFS). From crystallography of small molecule As(III)-thiol compounds, the sulfur-to-sulfur distances can be predicted to be 3.5 A˚. A structural model of the R773 ArsR aporepressor (Figure 3(a)) was constructed on the crystal structure of the homologous CadC repressor (Figure 3(c)). The As(III) (inducer)-binding site is located in the first (3) helix of the helix–loop–helix DNA-binding domain. In this model, the sulfur atoms of the three cysteines are linearly arrayed along the 3 helix, with more than 10 A˚ from Cys32 to Cys37, which is considerably more than the 2.25 A˚ from the EXAFS data. To bring the cysteine thiolates that close to each other, binding of As(III) must induce a large conformational change that breaks the helix (Figure 3(b)), resulting in dissociation of ArsR from the operator/promoter site and transcription of the resistance genes. Another ArsR that has a completely different As(III)binding site is found in an arsenic resistance operon from Acidithiobacillus ferrooxidans. AfArsR lacks the R773 C32VC34DLC37 sequence in the DNA-binding site. Instead, it has three cysteine residues, Cys95, Cys96, and Cys102, that are not present in the R73 ArsR. EXAFS results show that these three cysteine residues form a three-coordinate As(III)-binding site. DNA binding studies indicate that binding of As(III) to these cysteine residues produces derepression. From a homology model built on the CadC structure, the As(III)-binding sites in AfArsR are located at the ends of antiparallel C-terminal

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others)

37

34

Cys32 Cys34

As(III)

Cys37 Cys34

Cys37

Cys32

211

32

37 34

32

B: R773 ArsR + As(III)

A: R773 ArsR

Cys11 Cys7

Cys58

Cys60

Cys60 Cys58

Cys7 Cys11

C: CadC

Cys16 Cys15 Cys55

Cys95 Cys96

Cys56 Cys15 Cys95 Cys96

Cys16

D: CgArsR1

E: AfArsR

Figure 3 Structural models of three ArsR As(III)-responsive repressors. Shown are structures of (a) R773 ArsR, (d) Acidithiobacillus ferrooxidans ArsR, and (e) Corynebacterium glutamicum ArsR1, all of which were modeled on the 1.9 A˚ CadC crystal structure (e). The CadC dimer is shown as a ribbon diagram with secondary structural units N-1-2-3- 1-4-5- 2- 3-6-C. One As(III)- or Cd(II)binding site in each repressor is circled. In the R773 and A. ferrooxidans ArsRs, the intrasubunit As(III)-binding sites are formed by three cysteine residues in each subunit. In the C. glutamicum ArsR1 and CadC repressors, the intersubunit metal-binding sites are composed of one or two residues from each monomer. The cysteine residues that form the As(III)- or Cd(II)-binding sites are represented as sticks with the sulfurs as yellow balls. (b) On the left is the R773 ArsR 3 helix of the DNA-binding site from (a). On the right is a model of the 3 helix after binding As(III), as deduced from EXAFS data. This suggests that derepression by As(III) binding is the result of distortion of the 3 helix, resulting in ArsR dissociation from the operator/promoter DNA.

helices in each monomer that form a dimerization domain (Figure 3(d)). Thus the As(III)-S3-binding sites in AfArsR and R773 ArsR are located in spatially distinct positions in their three-dimensional structures, which implies that their mechanism of derepression by As(III) differs. A third ArsR with yet another and completely different As(III)-binding site is encoded by an ars operon from Corynebacterium glutamicum ATCC 13032. EXAFS results show that CgArsR1 binds As(III) in a three-coordinate sulfur environment to Cys15, Cys16, and Cys55. These three cysteine residues do not correspond to As(III) ligands in either of the other two ArsRs, nor do they correspond to metal binding residues in CadC or SmtB. However, from homology modeling, CgArsR1 has the N-terminal extension characteristic of CadC repressors (Figure 3(e)). In CadC, the Cd(II)-binding site is composed of two cysteine residues from the N terminus of one subunit and two cysteine residues from the first (4) helix of the DNAbinding site of the other subunit. This suggests that the

CgArsR1 inducer-binding site is formed by binding As(III) between Cys15 and Cys15 of one subunit and Cys55 from the other subunit. Even though it resembles the intrasubunit Cd(II)-binding site of CadC, the location of the three cysteines in CgArsR1 is different from the four cysteines CadC, indicating that the two metal-binding sites are not derived from a common ancestor. Moreover, the As(III)binding sites of R773 ArsR, AfArsR, and CgArsR1 likewise did not evolve from a common ancestral-binding site but appear to be the result of three independent and relatively recent evolutionary events, building on the same backbone repressor protein. Secondary arsenite efflux carrier proteins: ArsB and Acr3

Even though the most common extracellular form of arsenic under aerobic conditions is arsenate, in the highly reducing environment found inside cells, arsenite, the more toxic form, predominates. Thus, the majority of

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the detoxification mechanisms have arisen to cope with arsenite, and, most commonly, these are efflux systems. In bacteria, at least two different families of arsenite permeases have evolved, ArsB and Acr3. The first identified arsenite efflux protein, ArsB, is encoded by the R773 arsRDABC operon. ArsB is widespread in bacteria and archaea. It has 12 membrane-spanning segments, similar to many carrier proteins, and functions as an As(OH)3/ Hþ antiporter coupled to the proton motive force. ArsB can also associate with the ArsA ATPase to form a pump that confers a higher level of arsenite resistance than ArsB alone. Thus, the Ars efflux system exhibits an unusual and unique dual mode of energy coupling that depends on its subunit composition. Members of the Acr3 family are similarly widespread in bacteria, archaea, and fungi. Many Acr3s have been annotated as ArsBs, which confuses the literature – Acr3 and ArsB show no significance sequence similarity. The first Acr3s to be identified were a bacterial one encoded by the Bacillus subtilis ars operon located in the skin (sigK intervening) element and a fungal Acr3p encoded by the S. cerevisiae acr123 gene cluster.

Arsenite efflux pumps: ArsAB ATPases and multidrug resistance-associated proteins ArsAB ATPases

Cells of E. coli expressing the chromosomally encoded ArsB are resistant to moderate levels of arsenite, while cells expressing the R773 arsRDABC operon produce the ArsAB ATP-coupled efflux pump and are resistant to much higher levels of arsenite. The different levels of resistance are because ArsB is a secondary transporter that uses the proton motive force, while the ArsAB ATPase uses ATP to drive active transport of As(III) against much higher gradients than ArsB alone. The ArsA ATPase has been extensively studied at the biochemical and structural levels (Figure 4).

MRPs

ABC transporters are ubiquitous in nature. The multidrug resistance-associated protein (MRP) subfamily catalyzes ATP-coupled extrusion of drug conjugates and other solutes. S. cerevisiae Ycf1p (yeast cadmium factor) is a close homologue of the human MRP1 and catalyzes the vacuolar sequestration glutathione (GSH) conjugates of Cd(II) or As(III). Thus far, bacterial MRP homologues that confer arsenic resistance have not been identified. ArsD: an As(III) chaperone

Metallochaperones are proteins found in all kingdoms that buffer cytosolic metals and deliver them to protein targets such as metalloenzymes and extrusion pumps. For example, the S. cerevisiae chaperone Atx1 transfers copper to Ccc2p, a trans-Golgi Cu(I)–ATPase that is required for incorporation of copper into the multicopper oxidase (MCO) Fet3p. Until recently, no arsenic chaperones had been identified. However, those ars operons that contain both arsD and arsA, such as the arsRDABC operon of plasmid R773, always have the two genes adjacent to each other, suggesting that they might have interrelated functions. Recently, the product of the arsD gene, a 120residue polypeptide that is a functional homodimer, was shown to be a chaperone that delivers As(III) to the ArsAB As(III)-translocating ATPase. The presence of ArsD, in addition to the ArsAB pump, gives cells a clear growth advantage over cells with only the ArsAB pump (such as in an arsD deletion). Expression of ArsD increases the ability of the ArsAB pump to extrude As(III). ArsD and ArsA physically interact with transfer of As(III) from the chaperone to the pump. In doing so, the affinity of ArsA for As(III) is increased several orders of magnitude. In the absence of ArsD, the ArsAB pump gives resistance to concentrations of arsenite found only in the highly contaminated parts of the world, but ArsD makes the ArsAB pump more effective at much lower concentrations of arsenic such as those found more widely environmentally. Arsenate reductases: ArsCS and Acr2s

A2 NBD

A2

A1 NBD

A1 NBD

A1 A1

A2

MBD

Figure 4 Structure of R773 ArsA ATPase. Left: The overall structure of ArsA is shown as a ribbon diagram. Mg2þADP is bound to each of the two NBDs in the A1 and A2 halves of ArsA, while three Sb(III) is bound at the single MBD. Right: A view of the molecular surface of ArsA showing the relative positions of the A1 and A2 halves and details of ADP bound in the NBD1.

In contrast to the periplasmic respiratory arsenate reductases, cytosolic arsenate reductases are usually (but not always) encoded by ars operons or in fungi in arsenic gene clusters, and confer resistance to arsenate. There are three independent and unrelated families of arsenate reductases. One family of arsenate reductase that includes the chromosomal E. coli and plasmid R773 ArsCs uses glutaredoxin (Grx) and GSH as reductants. The crystal structure of the enzyme has been determined with and without bound substrates and products (Figure 5(a)). R773 ArsC is related to Spx of B. subtilis, a transcriptional repressor that interacts with the C-terminal domain of RNA polymerase (RNAP) -subunit and is essential for growth under disulfide stress. The second family includes the S. aureus plasmid pI258 ArsC and the B. subtilis

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others)

213

of arsenate reductases is found primarily in eukaryotic microorganisms and includes S. cerevisiae Acr2p and LmACR2 from the protozoan L. major. The structure of the Leishmania enzyme has been solved recently (Figure 5(c)). These eukaryotic arsenate reductases are related to the catalytic domain of the Cdc25 cell cycle protein tyrosine phosphatase. From a comparison of the three structures, which show no similarity to each other, it is clear that these arsenate reductases arose by convergent evolution.

A: R773 ArsC

Cys-75

Cys-12

C: LmACR2

Cys-10

B: pI258 ArsC

Arsenite methylases: ArsM

Cys82S-S89Cys

Members of every kingdom have the ability to methylate arsenic. The series of reactions, called the Challenger Pathway, alternates reduction of pentavalent to trivalent arsenicals with oxidative methylations (Figure 6). The pentavalent species are monomethylarsenate (MMA(V)), dimethylarsenate (DMA(V)), and trimethylarsine oxide (TMAO(V)) and the trivalent are MMA(III), DMA(III), and TMA(III). Humans and some other mammals methylate inorganic arsenic and excrete methylated species such as DMA(V) and, to a lesser extent, monomethylated MMA(V) in the urine, leading to the proposal that methylation is a detoxification process. However, the trivalent intermediates in the pathway, MAs(III), DMAs(III), and TMAs(III), are considerably more toxic than inorganic arsenate or arsenite, suggesting that methylation may activate inorganic arsenic to more toxic metabolites.

Figure 5 Arsenate reductase structures. The structure of R773 and pI258 ArsC are shown identifying their secondary structural elements. The catalytic cysteines at the active sites of either enzymes complexed with As(V) are also indicated. The Leishmania LmACR2 has a similar active site as the human Cdc25a.

chromosomal ArsC, both of which use thioredoxin (Trx) as reductant. (Again, like ArsB, unrelated arsenic resistance proteins have unfortunately been given the same name.) The structure of this ArsC has been determined in various conformations that describe the catalytic cycle (Figure 5(b)). This bacterial family of arsenate reductases is related to low molecular weight protein tyrosine phosphatases (LMW PTPase). The third family

O– As(V) HO-As = O OH

HO-As-OH As(III) OH SAM

O–

MMA(V) H3C-As = O OH

H3C-As-OH MMA(III) OH SAM

ArsM Reduction Oxidative methylation

O–

DMA(V) H3C-As = O CH3

H3C-As-OH

DMA(III)

CH3 SAM

CH3

TMAO(V)

H3C-As = O CH3

H3C-As-CH3

TMA(III)

CH3

Figure 6 The Challenger Pathway of arsenic methylation. In each step a pentavalent arsenical is reduced to a trivalent arsenical (red), which is then oxidatively methylated with S-adenosylmethionine to form the pentavalent form (blue). The overall scheme involves four reductive steps and three methylations to form the gas TMA(III) from inorganic As(V). Reproduced from Bhattacharjee H and Rosen BP (2007) Microbial arsenic metabolism. In: Nies DH and Silver S (eds.) How Bacteria Handle Heavy Metals, pp. 371–406. Heidelberg: Springer.

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Whether arsenic methylation is a detoxification processes in mammals is an unresolved question. In contrast, it is clear that bacterial methylation detoxifies arsenic. Bacteria and fungi produce volatile and toxic arsines, but, until recently, the biochemical basis and physiological role of arsenic methylation in microorganisms were not known. There are a large number of genes for homologous of the human arsenic methylase in bacteria and archaea. Those that are controlled by an arsR gene have been termed arsM and their protein product as ArsM (arsenite S-adenosylmethyltransferase). The arsM gene (accession number NP_948900.1) from the soil bacterium Rhodopseudomonas palustris for the 283-residue ArsM (29 656 Da) was cloned and expressed in an arsenic hypersensitive strain of E. coli. Expression of arsM conferred As(III) resistance in E. coli in the absence of any other ars genes, demonstrating that methylation is sufficient to detoxify arsenic. ArsM converted As(III) into DMA(V), TMAO(V), and TMA(III). While TMA(III) is more toxic than arsenite, it is a gas and volatilizes as it is being formed so that it does not accumulate in the cells or media. Since homologues of ArsM are widespread in every kingdom, microbialmediated transformation may have a significant impact on the global arsenic cycle.

ArsH NADPH + H+ NADP+

FMN FMNH2

H2O O2

Figure 7 Structure of the ArsH NADPH–FMN oxidoreductase. On the top is shown the 1.8 A˚ crystal structure of the ArsH tetramer. The monomers A, B, C, and D are colored red, green, blue, and yellow, respectively. Bottom is shown the NADPH– FMN oxidoreductase reaction that ArsH catalyzes with the generation of H2O2. 29

An NADPH–FMN reductase: ArsH

ArsH is widely distributed in bacteria and found sparsely in fungi, plants, and archaea. From its sequence, it shows conserved domains related to the NADPH (nicotinamide adenine dinucleotide phosphate) dependent flavin mononucleotide (FMN) reductase class of proteins. ArsH appears to confer resistance to arsenicals, although this is controversial, and the mechanism is not clear. ArsH from Yersinia enterocolitica or from the IncH12 plasmid R478 appears to confer resistance to both arsenite and arsenate. However, A. ferrooxidans and Synechocystis ArsHs do not seem to participate in arsenic resistance. The chromosomal ars operon of the legume symbiont Sinorhizobium meliloti Rm1021 displays a cluster of four genes: arsR, aqpS, arsC, and arsH. Inactivation of the S. meliloti arsH gene results in increased As(III) sensitivity. Overexpression of ArsH in
ars strain of S. meliloti confers resistance to trivalent arsenicals, showing that ArsH does not need the other ars gene products for resistance, indicating that it has a novel mechanism that does not require AqpS and ArsC activities. The protein has been purified and shown to catalyze azo dye reduction and H2O2 formation. Its crystal structure has been solved, showing it to be a tetramer with an FMN-binding site in each subunit (Figure 7).

Cu63

Introduction Copper speciation and availability in the environment is critical for a number of biogeochemical cycles exemplified here by nitrogen. Key steps in nitrogen cycling are redox reactions carried out by metalloenzymes of denitrifiers, nitrifiers, and nitrogen fixers. It has been shown that denitrifying bacteria are affected by copper limitation because copper is required in nitrous oxide reductase and some nitrite reductases (nirK). This is of importance since nitrous oxide (N2O) is a major greenhouse gas contributing to global warming. For example, the respiratory decomposition of organic matter in marine and freshwater environments leads to an almost complete exhaustion of available oxygen, stimulating the use of alternative terminal electron acceptors. Under oxygen-limiting conditions, denitrifying bacteria can thus reduce nitrate (NO–3), in a stepwise fashion, to nitrite (NO–2), nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2). Most significantly, denitrification is the largest sink of oceanic fixed nitrogen and is estimated to account for up to 67% of total global denitrification. It is therefore not surprising that many genomes of representative, recently sequenced marine microorganisms contain genes involved in denitrifications. Genes for nitrous oxide reduction have been found in diverse marine

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others)

bacteria including Roseobacter denitrificans, Marinobacter aquaeolei, Silicibacter pomeroyi, and Stappia aggregata, to name a few. In addition, agricultural land contributes significantly to the net increase in atmospheric N2O through denitrification and nitrification. Several rhizobia such as Bradyrhizobium japonicum USDA110 and S. meliloti are capable of denitrification including the last biochemical step mediated by N2O reductase (N2OR). Recently, it has been shown that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N2O. However, several groups have reported that cultivation of legume crops often enhances N2O emissions from fields of alfalfa, soybean, white clover, and Bengal gram. We believe this apparent paradox can in part be explained by lack of copper bioavailability in these soils and therefore an inability to assemble a catalytically active N2OR enzyme. Microorganisms can actively reduce and oxidize copper. Oxidation of copper has not been coupled to energy generation but, in all cases studied, is a copper resistance mechanism. Likewise, reduction of oxidized copper minerals, while thermodynamically feasible, as terminal electron acceptor has not been observed. In contrast, reduction of cupric ion to cuprous ion as a prerequisite for copper uptake is widespread. Oxidation of Cuprous Copper by Multicopper Oxidases MCOs couple the one-electron oxidation of substrate(s) to full reduction of molecular oxygen to water by employing a functional unit formed by three types of copper-binding sites with different spectroscopic and functional properties. Type 1 blue copper (T1) is the primary electron acceptor from the substrate, while a trinuclear cluster formed by type 2 copper and binuclear type 3 copper (T2/T3) is the oxygen-binding and oxygen-reduction site. Prominent bacterial enzymes involved in metal transformations include CueO involved in copper resistance by oxidizing Cu(I) to Cu(II) and CumA from Pseudomonas putida MnB1 and GB-1 responsible for manganese oxidation of Mn(II) to Mn(III)/(IV). CueO, a bacterial multicopper oxidase, protects periplasmic enzymes from copper induced damage and possesses laccase-like activity. CueO has antioxidant activity by inhibiting the Cu(I)-induced Fenton reaction due to removal of the primary reactants to drive the Fenton reaction (eqn [1]) and hydroperoxide-dependent lipid peroxidation (eqn [2]). CuðIÞ þ H2 O2 ! CuðIIÞ þ?OH þ OH –

½1

CuðIÞ þ LOOH ! CuðIIÞ þ LO? þ OH –

½2

Cu(I) oxidation could be shown not only for CueO but also for Fet3 from yeast. In addition, the oxidation of

215

catecholate siderophores like enterobactin by CueO was shown to protect cells by preventing reduction of Cu(II) to Cu(I) and subsequent oxidative damage. The activity of CueO is regulated by copper. Deletion of an extra helical region near the type 1 copper site converts CueO into a laccase with better access for bulkier substrates. As described later, CumA and other Mn-oxidizing MCOs might also oxidize siderophores. CueO is a 53 kDa periplasmic protein that confers copper tolerance in E. coli under aerobic conditions. CueO is capable of oxidizing a wide variety of substrates, including 2,6-dimethylphenol, enterobactin (a catecholate siderophore found in E. coli), and Fe(II). Intriguingly, very little oxidase activity occurs in the absence of additional copper in solution. Cu(I) is an excellent CueO substrate, with higher activity than that described for homologues Fet3p and ceruloplasmin, suggesting this is the in vivo target of the protein. The crystal structure of CueO was obtained at 1.4 A˚ resolution, which is by far the highest resolution achieved for this structural family and sufficient for defining the structures of trapped reaction intermediates (Figure 8). The CueO structure resembles those of laccase, ascorbate oxidase and ceruloplasmin, although the oligomeric state and addition of extra protein modules differ among these proteins. The CueO-fold consists of three azurin-like copper-binding domains connected by linker peptides. The trinuclear copper center (TNC, T2 þ T3 coppers) lies between domains 1 and 3, while the T1 copper occupies the azurin-like position in domain 3. Two aspects of CueO differ from other MCOs. First, CueO requires a fifth copper adjacent to the T1 site for activity (labeled rCu in Figure 8). This copper atom is labile and so copper concentration regulates activity. Our working hypothesis is that the fifth copper-binding site is for binding Cu(I) as substrate and Cu(II) for oxidation of other substrates such as enterobactin. Second, CueO displays a methionine-rich helix that lies over the T1 site and may function by sequestering additional Cu(I) atoms. Methionine-rich regions are found in numerous proteins involved in copper homeostasis, leading to the suggestion that such regions are involved in copper ligation; however, the functional role of such sites has not been resolved for any of these proteins. There is preliminary data demonstrating that at least two additional Cu(I) atoms can bind to the methionine-rich site. Identification of reaction intermediates in MCOs has been hampered by low resolution and insufficient spectroscopic analyses of crystalline complexes. Two MCO structures have been published, containing unusual electron density at or near the trinuclear copper centers. A 2.4-A˚ resolution structure of laccase from Melanocarpus albomyces was interpreted as having a dioxygen molecule bridging the T3 copper atoms in the trinuclear center. A 2.45-A˚ resolution structure of the endospore coat laccase

216

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others) (a)

(b) Cu3 T2Cu

Cu2

T1Cu M441 D439 M355 rCu D360

Figure 8 CueO structure. (a) Five-copper electron transfer path. (b) Overall fold with copper atoms shown as blue spheres and methionines as ball-and-stick. The methionine-rich helix is in the lower right corner.

from B. subtilis contained unexplained electron density between two histidine ligands of the T3 copper atoms, but not bound to the copper atoms, which was interpreted as an oxygen molecule. Although intriguing, the resolutions of both structures are modest and spectroscopic analyses of the complexes were not undertaken, making identification of the unexplained electron density difficult. Better resolution has been achieved in other, nonMCO, copper proteins. Side-on Cu–NO coordination was reported for nitrite reductase in a 1.3-A˚ structure with corresponding EPR data supporting this interpretation. End-on dioxygen binding to copper has also been reported for the enzyme peptidylglycine-alphahydroxylating monooxygenase (PHM); however, even at 1.85 A˚ resolution this interpretation is not unambiguous and spectroscopic data were not reported. Reduction and Acquisition of Cupric Copper Copper minerals containing cupric copper could potentially function as terminal electron acceptor and be reduced to Cu(I) or Cu(0). However, this has not been reported yet. In contrast, Cu(II) has to be reduced in order to be taken up as Cu(I). Yeast is probably the best studied microorganism regarding copper uptake. Copper uptake in Saccharomyces cerevisiae is mediated by separate highand low-affinity systems. High-affinity copper uptake requires plasma membrane reductases to reduce Cu(II) to Cu(I). This reduction is mediated by the same plasma membrane reductases, Fre1 and Fre2, that are involved in iron uptake. Once reduced to Cu(I), the ion is taken up by separate high-affinity transporter proteins encoded by the

CTR1 and CTR3 genes. High-affinity copper uptake is energy-dependent and specific for Cu(I) over other metals. A third, lower affinity system for copper uptake has also been detected in yeast, but neither the biochemical properties nor the gene(s) responsible for this activity is known. 25

Mn55

Introduction In a variety of environments, manganese is cycled between the main oxidation states, Mn(II), Mn(IV), and also Mn(III). These states are capable of influencing chemical gradients in environments such as the coastal ocean. Mn oxides have been found to be strong environmental oxidants able to influence the speciation of other redoxsensitive metals and metalloids such as Cu and As. Manganese can perform these roles because bacterial processes enable rapid cycling. It is therefore not surprising that much work has gone into attempts to understand the molecular mechanisms of manganese reduction and oxidation. Redox-active proteins involved in electron transfer to and from Mn(hydr)oxides are localized to the outer membrane in Gram-negative bacteria and include c-type cytochromes (c-Cyts) and MCOs. In addition, Mn-oxidizing activity has also been found on the spore surface of Gram-positive bacteria. In this article, we intend to understand the mechanisms and enzymes involved in these transformations. These processes are still not well understood. Other aspects such as mineralization have been extensively covered in recent reviews.

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others)

Oxidation of Mn(II) to Mn(III, IV) The capability to oxidize Mn(II) to Mn(III, IV) has been detected in bacteria and fungi. In bacteria a MCO is almost always an essential part of Mn(II) oxidation. Bacillus SG-1, Pedomicrobium ACM3067, Leptothrix discophora, and P. putida all have MCO genes, which when disrupted the organisms no longer oxidize Mn(II). However, as described in more detail below, the MCO often is part of a complex and the activity of the bacterial MCO alone is not sufficient for Mn(II) oxidation. In the systems that have been well-studied using genetics, Mn(II) oxidation seems to require an MCO, but only in the case of Bacillus SG-1 has the connection between the MCO gene and the enzyme activity been shown. Here, the MCO MnxG is thought to oxidize Mn(II) ! Mn(III) ! Mn(IV) in sequential oneelectron steps. In other microbes such as P. putida MnB1 and GB-1, it might work a bit differently. The MCO CumA is also required for Mn oxidation to take place but other proteins are also involved. In addition, Mn(II) is oxidized to Mn(III) spontaneously only in the presence of siderophores and probably stoichiometrically 1:1 Mn(II):siderophore. In the presence of sufficient Fe and therefore the absence of siderophores, Mn(II) is rapidly oxidized to Mn(IV), probably via a Mn(III) intermediate. At limiting but not depleted, Fe the reaction slows since Mn(III):PVD complex can accumulate. Since Mn oxides also form at this time, the Mn(III):PVD complex could be oxidized by the MnOx or by another enzyme that acts either on the Mn(III) or by oxidizing the PVD and releasing Mn(III), which would be disproportionate to Mn(II) þ Mn(IV). MCOs have been shown to oxidize catecholate siderophores such as enterobactin. Pyoverdin is a mixed catecholate and hydroxamate siderophore, so this reaction is possible. It is not known whether PVD or some other molecule is also acting as a carrier. Briefly, it appears that the reason behind this might be that the reaction occurs as a sequence of two enzymatically mediated one-electron transfer reactions. In Erythrobacter, SD-21 the enzyme oxidizes Mn(II) ! Mn(III). At this point, it is not known whether the organism also produces a siderophore that can oxidize Mn(II). However, a recent report suggests in E. SD21, a copper-dependent MCO did not stimulate Mn(II) oxidation activity but rather by pyrroloquinoline (PQQ). Even more surprising is that PQQ could rescue a non-Mn(II)oxidizing P. putida MnB1 insertional mutant of the anthranilate synthase gene. How these proteins and molecules act together to oxidize Mn(II) is not known but one could envision an electron pathway from the respiratory chain in cytoplasmic membrane proteins such as MCOs located at the outer membrane.

217

In fungi, MCOs such as laccases often have Mn(II) to Mn(III) oxidation activity. In addition, fungi can also oxidize Mn(II) to Mn(III) by heme-containing Mn peroxidase. However, a recent paper describes the complete oxidation of Mn(II) to Mn(IV). Mn-oxidizing activity and the genes responsible are widespread among different bacterial groups indicating these genes must have an important function. At this point this is unknown, but rather it appears that this metabolic activity seems to be accidental since there have been no reports of Mn-dependent induction of any of the genes thought to be responsible for Mn(II) oxidation. However, this ‘accidental’ activity has significant environmental impact. For example, the Mn oxides present in desert varnish are largely catalyzed by bacterial activity.

Reduction of Mn(IV) In anaerobic environments insoluble minerals such as Mn(IV) can be used as terminal electron acceptor by a number of bacteria and archaea. Dissimilatory reduction of Mn(III, IV) usually require multiheme c-Cyts often located on the outer membrane facing the extracellular space. However, the molecular mechanisms are completely unknown. Aspects about the molecular details of this process have been extensively studied in Shewanella and Geobacter species. Since Mn(IV) is insoluble, the actual process is a mineral–microbe interaction. There are a number of possibilities on how electrons can be transferred from the microbe to the mineral (Figure 9). or more proteins located at the cell surface • One directly interacts with the mineral and transfers elec-

• • •

trons, usually originating from the electron transport chain, to the substrate. This has been observed with outer membrane multiheme c-Cyts such as OmcA from S. oneidensis. A small soluble electron shuttle such as phenazine picks up electrons from the cell and delivers them to the substrate. The electrons could be transferred at the inner or outer membrane. Electrically conductive pili (geopili) could directly transfer electrons from the cells to the substrate. Substrates are bound by a ligand such as a chelator and brought to the cell for reduction.

It is clear that at this point many of the proposed mechanisms have not been proven. For example, a MCO OmpB was shown to be involved in anaerobic iron (and probably Mn) reduction in Geobacter sulfurreducens. What role this enzyme plays is unclear since MCOs usually require oxygen to oxidize their substrates.

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Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others)

red.

e–

red.

ES

S

red.

S

e–

genomic databases for the presence of selenoproteins have yielded an ever-growing diversity, most of them being redox proteins containing catalytic selenocysteines.

ox.

e–

ox.

ES ox.

S

red.

red. e–

Figure 9 Four potential strategies for extracellular Mn respiration. The substrate is shown as a solid mineral in the center (e.g., a Mn (hydr)oxide), and its reduced product (e.g., Mn(II)) is shown in red. These strategies can apply to other substrates, and the flow of electrons can also be reversed. From the top clockwise: protein that resides on the cell surface (blue) directly • Ainteracts with the extracellular substrate to transfer electrons from the cell to the substrate.

electron shuttle interacts with the substrate outside • Anthe cell; the substrate remains at a distance from the cell, with the shuttle catalyzing transfer of electrons between the cell and the substrate. The shuttle, in principle, could be reduced on the outside or inside of the cell. A cellular appendage such as an electrically conductive pilus forms a bridge between the cell and the substrate, catalyzing electron transfer. A chelator binds the substrate and transports the complex to the cell for reduction (either on the outside or on the inside); in the case of an electron shuttle (closed green oval, oxidized state; open green oval, reduced state).

• • 34

Se80

Introduction Microbial transformations including oxidation, reduction, methylation, and demethylation reactions affect the solubility, toxicity, sorption, volatility, and specific gravity of metals and metalloids. Bacteria are responsible for many transformations of selenium. Some of the genes and enzymes involved have been identified and characterized. However, at this point it is still not clear whether these reactions represent active responses to the presence of selenium or are more or less accidental. For example, methylated species are volatile and the presence of genes encoding selenium methylases makes cells more resistant. If these genes would be induced by the presence of Se, they would constitute an active resistance mechanism. However, this is not the case. Other selenium transformations have not been studied in great detail and the genes involved in selenium oxidation and demethylation have not been identified. In contrast, much has been learned about selenium incorporation into proteins via selenocysteine. Better tools to analyze

Reduction of Selenate Microorganisms are largely responsible for the reduction of soluble selenate (Se(VI), SeO2– 4 ) and selenite (Se(IV), SeO2– 3 ) to the insoluble mineral form Se(0) in most environments. The serABDC operon was shown to encode the genes responsible for dissimilatory selenium respiration in the anaerobic bacterium Thauera selenatis. The serABDC operon is closely related to operons involved in chlorate reduction, dimethyl sulfide (DMS) oxidation to DMSO, and possibly nitrate reduction. The homology serABDC to chrABDC is quite high and both SerA and ChrA are capable of chlorate and selenate reduction albeit with differing substrate affinities. In addition, they are probably both flanked by a transposase or other elements indicating mobility. No regulatory elements have been identified. The catalytic subunit SerA is predicted to be a periplasmic molybdenum-containing enzyme belonging to the DMSO reductase family. SerB is a Fe–S protein containing four cysteine residues that might bind 4[Fe–S] clusters. SerD is of unknown function but might be a molecular chaperone aiding protein maturation. Finally, SerC is thought to encode a b-type cytochrome located in the periplasm in analogy to DdhC from Rhodovulum sulfidophilum.

Methylation of Selenite (and Selenocysteine) to Dimethyl Selenide and Dimethyl Diselenide Dimethyl selenide and dimethyl diselenide are the prevalent volatiles of bacterial methylation of selenium. The enzymes involved can all methylate selenite and selenocysteine to dimethyl selenide and dimethyl diselenide but some such as the trimethyl purine methylase (Tmp) from Pseudomonas syringae can also methylate selenate. Two very different selenium methyltransferases could be isolated from a freshwater Pseudomonas strain. While the first is a bacterial thiopurine methyltransferase, the other, MmtA, is a homologue of calichaemicin methyltransferase with homologues in many bacterial species.

Acknowledgments This work was supported by United States Public Health Service Grants AI43428, GM52216, and GM55425 (to B.P.R.) and United States Public Health Service Grant GM079192 (to C.R.).

Environmental Microbiology and Ecology | Heavy Metals Cycle (Arsenic, Mercury, Selenium, others) See also: Heavy Metals, Bacterial Resistance; Heavy Metal Pollutants: Environmental and Biotechnological Aspects; Metal extraction and Biomining

Further Reading Bhattacharjee H and Rosen BP (2007) Arsenic metabolism in prokaryotic and eukaryotic microbes. In: Nies DH and Simon S (eds.) Molecular Microbiology of Heavy Metals, vol. 6, pp. 371–406. Heidelberg/New York: Springer-Verlag. Gralnick JA and Newman DK (2007) Extracellular respiration. Molecular Microbiology 65: 1–11. Messens J and Silver S (2006) Arsenate reduction: Thiol cascade chemistry with convergent evolution. Journal of Molecular Biology 362: 1–17. Mukhopadhyay R and Rosen BP (2002) Arsenate reductases in prokaryotes and eukaryotes. Environmental Health Perspectives 110(supplement 5): 745–748. Mukhopadhyay R, Rosen BP, Phung LT, and Silver S (2002) Microbial arsenic: From geocycles to genes and enzymes. FEMS Microbiology Reviews 26: 311–325. Rensing C and Grass G (2003) Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiology Reviews 27: 197–213.

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Shi L, Squier TC, Zachara JM, and Fredrickson JK (2007) Respiration of metal (hydr)oxides by Shewanella and Geobacter: A key role for multiheme c-type cytochromes. Molecular Microbiology 65: 12–20. Stolz JF, Basu P, Santini JM, and Oremland RS (2006) Arsenic and selenium in microbial metabolism. Annual Review of Microbiology 60: 107–130. Tebo BM, Bargar JR, Clement BG, et al. (2004) Biogenic manganese oxides: Properties and mechanisms of formation. Annual Review of Earth and Planetary Sciences 32: 287–328. Tebo BM, Johnson HA, McCarthy JK, and Templeton AS (2005) Geomicrobiology of manganese(II) oxidation. Trends in Microbiology 13: 421–428. Tottey S, Harvie DR, and Robinson NJ (2005) Understanding how cells allocate metals using metal sensors and metallochaperones. Accounts of Chemical Research 38: 775–783. Zumft WG and Kroneck PM (2007) Respiratory transformation of nitrous oxide (N2O) to dinitrogen by bacteria and archaea. Advances in Microbial Physiology 52: 107–227.

Relevant Website http://bicn.com – Bangladesh International Community News

Heavy Metals, Bacterial Resistance S Silver and L T Phung, University of Illinois, Chicago, IL, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Flavors of Arsenic and Its Resistance Mercury and Organomercurial Resistance Silver Resistance

Glossary antiporter A membrane pump that derives energy for pumping out one substrate from coupled uptake of another. arsenic resistance Microbial resistance by genes and proteins to inorganic and organoarsenicals. ATPase A membrane efflux pump that derives energy from hydrolysis of ATP. chromate resistance Microbial resistance to chromate oxyanion. efflux Pumping out of the cell via a membraneembedded protein.

Abbreviations CDF

mercury resistance Microbial resistance by genes and proteins to inorganic and organomercurials. organoarsenical An organic compound with an arsenic atom covalently linked to a carbon. organomercurial An organic compound with a mercury atom covalently linked to a carbon. regulatory gene A gene whose protein product binds to DNA in order to turn on or turn off mRNA synthesis. resistance The ability to grow on higher concentrations of toxic compounds than which is usual.

RND

Resistance, Nodulation, and Division

cation diffusion facilitator

Defining Statement Toxic heavy metals have occurred on Earth at high levels since before the beginning of life, nearly four billion years ago. Microbes exposed to these inorganic toxins early developed resistance mechanisms. Thus, the question of whether toxic metal cation (and oxyanions of some soft metals) resistance systems evolved in microbes in response to human pollution in the last few thousands of years is easily answered: they arose much earlier, almost since the origin of life. The arguments supporting such a broad hypothesis need to be addressed element by element. The primary basis is the widespread occurrence of such resistance systems, from bacterial type to type (and occasionally recognized in Archaea and fungi as well) and with frequencies ranging from a few percent in pristine environments to nearly 100% in heavily polluted environments. The amino acid (and DNA) sequences and structures of the genes and proteins concerned with arsenic resistance indicate an ancient origin,

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Cadmium Resistance Chromate Resistance Other Bacterial Toxic Metal Resistance Systems Further Reading

although one cannot conclude whether early bioavailable As occurred in As(III) or As(V). Genetic and mechanistic studies of toxic metal ion resistance systems have been reviewed frequently, and recent examples are provided in ‘Further Reading’, especially in the special issue of FEMS Microbiology Reviews edited by Brown and colleagues. Some toxic elements have been chosen for deeper consideration in this article, as they are better understood and provide models for general mechanisms. The most frequent mechanism of toxic divalent cation resistance is energy-dependent pumping out from the cytoplasm (Figure 1). The P-type ATPase (CadA for Cd2þ and SilP for Agþ are toxic metal efflux examples, but these are found in all cell types – prokaryote and eukaryote) is generally a single large polypeptide embedded in the membrane and has multiple protein domains (a membrane-embedded cation translocation pathway and a three-domain ATPase region; Figure 1). The single-polypeptide membrane potential-driven efflux

Environmental Microbiology and Ecology | Heavy Metals, Bacterial Resistance

221

Cd2+

Cd2+

H+

Cd2+

sm

Cd2+

H+

CzcCBA 1 NH2

ArsB

OM OM

la rip Pe

429 COOH

H+

IM IM

CadA

Figure 1 Membrane-associated efflux of metal ions. Families of carriers are represented by the single-polypeptide ArsB arsenite efflux protein, the three-component chemiosmotic antiporter system (CzcCBA), and the CadA P-type ATPase. IM, inner membrane; OM, outer membrane.

pump (ArsB for arsenite and ChrA for chromate are wellknown examples for toxic oxyanions) moves the substrate out of the cell without ATP energy (Figure 1). Similarly, the three-polypeptide RND (Resistance, Nodulation, and Division) complex (CzcCBA for Cd2þ, Zn2þ, and Co2þ is an example) of Gram-negative bacteria picks up the cation either from the cytoplasm or from the periplasm and moves it through a channel formed by the inner membrane protein (CzcA in Figure 1) and the outer membrane protein (CzcC in Figure 1). The third polypeptide (CzcB) links the inner and outer membrane proteins together and may be involved in energy transduction.

Flavors of Arsenic and Its Resistance Resistance to both arsenite (As(III), As(OH)3) and arsenate (As(V), AsO4 3 – ) is widely found among both Gramnegative and Gram-positive bacteria, and even in the known genomes of all Escherichia coli and related clinical bacteria. Usually, it is in the form of an ars operon with a minimum of three cotranscribed genes – arsR (determining the regulatory negatively acting repressor), arsB (determining the membrane transport pump), and arsC (the determinant of a small intracellular arsenate reductase enzyme). Indeed, the ars operon occurs more widely in newly sequenced bacterial genomes with over 1000 or 2000 genes than do the genes for tryptophan biosynthesis. It has been argued, partially because of the abovementioned fact, that arsenic resistance is very ancient, probably found in early cells.

Occasionally two additional genes are found in ars operons of Gram-negative bacteria, so the gene order is arsRDABC. ArsA is an intracellular ATPase protein that docks onto the ArsB membrane protein, converting its energy coupling from the membrane potential to ATP hydrolysis. The arsenite membrane efflux pump is unique in that it can function either chemisomotically (with ArsB alone) or as an ATPase (with the ArsAB complex). ArsD is thought to function as a polypeptide chaperone carrying the arsenite to the ArsA ATPase protein, functioning with two pairs of adjacent thiol residues, Cys12–Cys13 and Cys112–Cys113, which bind As(III). Sb(III) can bind to ArsD and ArsA as alternatives to As(III). The ArsB membrane efflux pump is specific for As(III), arsenite (and also for closely related Sb(III)), and therefore, As(V), arsenate, resistance requires conversion of As(V) to As(III). Thus, a small cytoplasmic enzyme, arsenite reductase, ArsC, is found. The reductase uses oxidized/reduced cysteine thiol cycling to reduce arsenate to arsenite. Three distinct and unrelated families of ArsC arsenate reductase proteins have apparently arisen by convergent evolution, analogous to the wings of birds and insects, which are unrelated but both enable flying. One class of arsenate reductase uses three cysteine thiols within the ArsC protein as an electron source and thioredoxin to regenerate reduced cysteines for the next cycle (Figures 2 and 3). This enzyme is paralogous in structure and in function to a class of cell division enzymes found in microbes and in animals, called low molecular weight protein tyrosine phosphates (Figure 2). The steps involved in the thioredoxin-linked arsenate reductase cycle are shown in

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Environmental Microbiology and Ecology | Heavy Metals, Bacterial Resistance

B. halodurans

Staphylococcus plasmids pN315B pI258 pSX267

B. subtilis insertion element

P. aeruginosa

Human PA1F A. ferrooxidans

0.1

B. subtilis YWLE

Figure 2 Phylogenetic tree showing evolutionary relationships among selected ArsC thioredoxin-coupled arsenate reductases and paralogous low molecular weight protein tyrosine phosphatase.

O– O–

As [V] = O (arsenate)

ArsC

Cys10

S–

Cys82

SH

Cys89

SH

O– ArsC 1

SH

S HS

HO

2H+

H2O

Cys10

S

Cys82

SH

Cys89

SH

S

4

OH

H+

OH

Trx

As [V] = O

2

(arsenite) As [III]

Trx HO

ArsC

Cys10

SH

Cys82

S

Cys89

3 S

ArsC disulfide cascade

OH Cys10

S

Cys82

S

Cys89

SH

Figure 3 The reaction cycle for thioredoxin-linked arsenate reductase with intermediates. Reproduced from Messens J and Silver S (2006) Arsenate reduction: Thiol cascade chemistry with convergent evolution. Journal of Molecular Biology 362: 1–17.

Figure 3, as an example of how these enzymes work. Initially, inorganic ionic arsenate forms a covalent As–S bond with a cysteine thiol, from Cys10 in the example shown. Next, arsenate is reduced to arsenite, concomitant with oxidation of two cysteines to cystine (Figure 3, step 2). The third cysteine displaces Cys10 in a cystine regenerating the reduced Cys10 (Figure 3, step 3), and finally, the small soluble cellular protein thioredoxin reduces arsenate reductase fully (Figure 3, step 4). For the other two classes of structurally and evolutionarily unrelated arsenate reductases, the tripeptide glutathione and the small protein glutaredoxin function as thiol electron intermediates, in a manner similar to Cys82 and

Cys89 of the thioredoxin-linked arsenate reductase. Although overall they are similar proteins, thioredoxin does not function with glutaredoxin-linked arsenate reductase and glutaredoxin does not function with thioredoxin-linked arsenate reductase. In recent years, additional and totally different enzymes involved in arsenic resistance and redox chemistry have been isolated and their genetic basis studied. These are the periplasmic respiratory arsenite oxidase (genes called aox or aso) and respiratory arsenate reductase (genes called arr). The arsenite oxidase is a resistance mechanism converting highly toxic high arsenite levels to relatively less toxic arsenate, while respiratory arsenate

Environmental Microbiology and Ecology | Heavy Metals, Bacterial Resistance

–O

Arsenite

–O

OH

OH As

As

–O

O arsenate

HO O Rieske Pt

[2Fe–2S]

[

S

OH VI

Mo

S

S S

] Pt

–O

OH As

–O

O

cys S N his

N his Fe

Fe S

S Fe S cys

Small subunit 14 kD

–2e–

S

Pt

[

S Fe

S Fe

S cys

223

S cys

S

S

OH IV

Mo

S

S

S

] Pt

+2e–

O cys S

HiPIP [3Fe–4S]

Pt

[

S

IV

Mo

S

S

S

] Pt

Large subunit [3Fe–4S] Mo-pterin 88 kD

Figure 4 The reaction mechanism of arsenite oxidase. Reproduced from Silver S and Phung LT (2005a) Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Applied and Environmental Microbiology 71: 599–608.

reductase functions as a terminal electron acceptor, allowing anaerobic growth in the absence of oxygen. The oxidase and reductase contain related molybdopterin centers and [Fe–S] cages (Figure 4), and both are coupled to inner membrane respiratory chains, with the oxidase as an initial electron donor and the reductase as the terminal electron acceptor in an anaerobic respiratory process. For the Aso respiratory arsenite oxidase (Figure 4), the oxyanion substrate is thought to enter a shallow conical pit on the enzyme surface, directly contacting the embedded Mo(VI). Concerted two-electron transfer occurs, arsenate is released, and molybdenum reduced to Mo(IV). Next, electrons are transferred to an [3Fe–4S] cage in the large molybdopterin subunit and from the [3Fe–4S] to an [2Fe–2S] cluster in the small subunit (Figure 4). From the small subunit, the electrons are transferred to the inner membrane respiratory chain, and eventually to oxygen, the terminal electron acceptor. For the functionally related Arr respiratory arsenate reductase, the electrons are transferred in the opposite direction, from the respiratory electron transport chain to the enzyme small subunit and finally from the Mo(IV)-pterin cofactor to the substrate arsenate. There is an additional aspect of microbial arsenic enzymatic transformations about which less is known. Microbes methylate and demethylate arsenic compounds, and inorganic arsenic is incorporated into small organic compounds, such as arsenobetaine, arsenolipids, and arsenosugars. The methylase and demethylation enzymes (and their genes) have recently been identified, so a microbial cycle occurs. Although microbial methylation of inorganic arsenic was recognized over a century ago

and attributed to fungi, when poisonous volatile arsenic compounds were released from ‘moldy wall paper’, methylation by prokaryotes and animal tissues has been identified only more recently. Major progress occurred with the isolation and sequencing of the arsenic methylase genes, first from mammals and more recently from prokaryotes. The methyl donor is S-adenosyl methionine and methylation requires arsenite as substrate. At each methylation step, from inorganic arsenite to monomethylAs, and subsequently to dimethyl-As and trimethyl-As, oxidation to As(V) occurs, and reduction to an As(III) organoarsenical is required before the next methylation step.

Mercury and Organomercurial Resistance Mercury resistance, together with arsenic resistance, is the best understood and most widely found of the toxic metal resistance systems. The same mechanism has been found for mercury resistance widely in all bacterial divisions where it has been sought (and also in some Archaea). This has not been found, however, in any eukaryote. Mercury resistance occurs widely in clinical and industrial isolates, as well as in environmental strains, supporting a wide and ancient occurrence. Mercury resistance genes are frequently found on plasmids and encoded by transposons. The first mercury-specific gene products for resistance encountered by Hg2þ approaching a Gram-negative bacterial cell are a small monomeric polypeptide in the periplasmic space, MerP, with a single cysteine pair for Hg2þ binding, and one of three inner membrane proteins,

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Environmental Microbiology and Ecology | Heavy Metals, Bacterial Resistance

and 4), where it is reduced by electron transfer from FADH cofactor (Figure 5, step 6) and Hg0 released. Monoatomic Hg0 is a membrane soluble gas and rapidly leaves the cell and under aerobic shaking or bubbling conditions is released into the atmosphere. Organomercurial lyase is a small monomeric enzyme that cleaves the Hg–C covalent bond releasing Hg2þ (the substrate of mercuric reductase) and reduced organic compounds, such as methane from methyl mercury (Figure 6) or benzene from phenyl mercury. The MerB primary sequence is unusual, having no paralogous enzymes with related sequences but different substrates. A wide range of MerB sequences have been identified (accessible in GenBank) and a wide range of organomercurial substrates are known, although at this time there is no understanding of how sequence differences determine substrate variation. The enzymatic reaction for organomercurial lyase was shown to be a concerted proton attack on the Hg–C bond by an SE2 reaction mechanism, after the organomercurial is initially bound to a cysteine thiol (Figure 6, step 1), either from a membrane protein, such as MerT, or directly from a small cellular thiol compound, such as glutathione (GSH in Figure 6). It is unclear whether a dicarboxylic acid residue, a tyrosine, or a second cysteine thiol is the source of a proton that

generally MerT, but sometimes its alternative forms MerC or MerF (all three of which have two sets of Hg2þ-binding cysteine pairs that are thought to function as a serial cascade of thiol Hg2þ-binding sites, in the absence of redox chemistry). Once at the inner surface of the inner membrane, Hg2þ is thought to be transferred in still another cysteine pair to cysteine pair exchange to the N-terminal cysteine pair of the homodimer enzyme mercuric reductase. Recent progress has occurred in the understanding of the structures and mechanisms of the two enzymes involved in mercury resistance, mercuric reductase (Figure 5) and organomercurial lyase (Figure 6). Most recognized MerA sequences (with the exception of that from Streptomyces) have this N-terminal domain that is homologous in sequence and considered to function as a thiol Hg2þ-binding site similarly to MerP. Hg2þ in the reductase enzyme is next transferred by another cysteine pair to cysteine pair exchange from the N-terminal domain to the C-terminal cysteine pair of the MerA subunit (C558 C559 in Tn21 MerA numbering; Figure 5, steps 1 and 2), forming an S–Hg–S ring structure. Hg2þ bound to mercuric reductase on the C-terminal Cys557 Cys558 of one subunit is then transferred by rapid thiol/thiol exchange to the Cys135 Cys140 thiol pair of the other monomer (Figure 5, steps 3

Mercury Hg2+ bound

Reduced MerA

Transfer to active site

NADPH

NADPH

NADPH

NADPH

FAD

FAD

FAD

FAD

S

S

S

S

C140

C140

Hg(SR)2

C135

SH

1

2

C135

C140 SH

3

C135

HS C557′

C557′

RSH

C558′

S Hg S

C558′

SH 7

C140

RSH SH

C135

HS

NADPH

Cyclic formation

S

Oxidized inactive enzyme

HgSR

S Hg S

C557′

C557′

C558′

C558′ SH

Hg Reduction

At active site

4

NADP+ FADH

FAD

FADH S

S C140 Hg

S C140 S

6

C135

C135

HS C557′ C558′ SH

Hg 0

5

S

HS

C140 Hg S C135 HS

C557′

C557′

C558′

C558′

SH

SH

Figure 5 The reaction cycle for mercuric reductase. Reproduced from Silver S and Phung LT (2005b) A bacterial view of the periodic table: Genes and proteins for toxic inorganic ions. Journal of Industrial Microbiology and Biotechnology 32: 587–605.

Environmental Microbiology and Ecology | Heavy Metals, Bacterial Resistance

C160 C159

C160

H3CHgSG

C159

SH

225

1

C117

S

C117 SH

HgCH3 SH

C96

C96 OH

Y93

O

OH

GSH

OH

Y93

D/E

OH

D/E

C558S C559S

(GS)2Hg or MerA

4

O

Hg

2

2GSH or MerA

C160 C159

C160 C159

S

C117

3

Hg S

Hg

CH3

S

C96

H

C96 OH

Y93

S

C117

O

O–

CH4

D/E

OH Y93

O

O

D/E

Figure 6 The reaction cycle for organomercurial lyase. Reproduced from Silver S and Phung LT (2005b) A bacterial view of the periodic table: Genes and proteins for toxic inorganic ions. Journal of Industrial Microbiology and Biotechnology 32: 587–605.

adds, for example, to methylmercury, forming methane. Results from recent mutagenesis and structural studies of organomercurial lyase have led to the suggestion that a cysteine thiol might be the source of the proton that attacks the Hg–C bond. In sum, the pathway in Figure 6 is explicit but still tentative. The four steps are shown in Figure 6, with methylmercury as the organomercurial are of greatest environmental and medical concern. However, experimental work has generally been done with less toxic mercurials. Initially, the organomercurial forms a thiol– mercury covalent bond with the conserved Cys159 (Figure 6, step 1). A second cysteine thiol, from Cys96, possibly forms a bond with the Hg (Figure 6, step 2) and the proton from Cys96, Tyr93, or from an aspartate or glutamate (Asp98 is conserved in organomercurial lyases) attacks the Hg–C bond releasing the methane (Figure 6, step 3). The inorganic Hg2þ bound to the organomercurial lyase by the two thiols is released in cell-free assays to added small thiols such as GSH, but more likely in intact cells, Hg2þ is directly transferred to the C-terminal vicinal cysteines of mercuric reductase (Figure 6, step 4).

Silver Resistance Silver compounds have come into wide use as antimicrobials over the last 40 years, following earlier use, for example, as antimicrobial rinses for the eyes of neonates. Current uses are primarily as burn ointments and on bandages intended for use on burns, trauma wounds, and

slow-healing diabetic ulcers. In addition, biocidal silver compounds are used for hygiene in dish and clothes washers, refrigerators, water purifiers, sports clothing, and a wide variety of other uses. It is perhaps not surprising that bacterial resistance to Ag compounds has been repeatedly reported. Silver ions are highly toxic to all microorganisms, probably due to poisoning of the respiratory electron transport chains and components of DNA replication. The genetic and physiological basis of bacterial Agþ resistance has been analyzed. These efforts are recent enough that one does not know if the same mechanism will be found broadly among bacteria. However, for enteric bacteria, nine genes contribute to Agþ resistance. These are found on large plasmids, and homologues of the central six genes contribute to low-level Agþ resistance governed by the E. coli chromosome. The nine silver resistance genes start with the silE gene that determines a small periplasmic metal-binding protein that is unrelated to other metal-binding polypeptide domains, except for the PcoE polypeptide of copper resistance. Upstream from silE, a gene pair, silRS, determining proteins involved in transcriptional gene regulation occurs. The membrane sensor kinase (SilS, with the conserved histidine residue that is predicted to be phosphorylated by ATP) and the regulatory responder protein (SilR, which is trans-phosphorylated on an aspartate residue from the SilS histidine) are homologous to other members of the large two-component family. The remaining six open reading frames in the Agþ resistance system occur in

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Environmental Microbiology and Ecology | Heavy Metals, Bacterial Resistance

the opposite orientation from silRSE with the silCBA genes determining a three-polypeptide RND membrane potential-driven cation/proton exchange complex, quite similar to the CzcCBA complex shown in Figure 1. This complex includes the SilA inner membrane proton/cation pump protein, the SilB membrane fusion protein that is predicted to physically contact SilA and SilC, and the SilC outer membrane protein. The product of the last gene of the silver resistance determinant, SilP, is predicted to be a P-type ATPase (similar to CadA shown in Figure 1) and represents an additional subclass with a monovalent cation substrate, differing in cation specificity from other members of the P-type efflux ATPases, most of which have divalent cation substrates. The full silver resistance determinant is unique among resistance systems in encoding both a periplasmic metal-binding protein and chemiosmotic and ATPase efflux pumps, rather than one or the other.

Cadmium Resistance Cadmium resistance is found widely in environmental and clinical bacteria, and the mechanism is generally a membrane efflux pump, either a P-type ATPase or a chemiosmotic pump (Figure 1). Occasionally binding by proteins or small anionic metabolites (phosphates or carbonates) has been reported. The best-studied Cd2þ efflux pumps are the CadA ATPase of Staphylococcus aureus and the CzcCBA complex of Cupriavidus metallidurans strain CH34 (the genus and species names of this strain have been changed five times over recent years). There are additional known P-type toxic cation efflux ATPases for Agþ, Cuþ (or Cu2þ), Zn2þ, Ni2þ, and Pb2þ and threepolypeptide RND (CBA) systems that pump out Cd2þ, Co2þ, Ni2þ, and Zn2þ. An additional single-polypeptide cation diffusion facilitator (CDF) chemiosmotic efflux system was first described with the CzcD Cd2þ and Zn2þ efflux system of C. metallidurans. Additional members of the CDF family include the Zn2þ efflux systems ZitB of E. coli and ZntA of S. aureus and the Fe2þ FieF efflux system of E. coli and C. metallidurans. CDF homologues are found encoded in many bacterial genomes and also in Archaea, yeast, plants, and animals. There are even seven CDF proteins encoded in the human genome. Thus, the CDF family is as widely occurring in different life forms as are P-type ATPases. The RND chemiosmotic proton/divalent cation exchangers are, however, limited to Gram-negative bacteria. Detailed protein structures of the Cd2þ P-type ATPase and CzcCBA chemiosmotic pump have been proposed by analogy to closely related structures that have been solved by X-ray diffraction or NMR analysis (Figure 1). The P-type efflux ATPase has four readily discerned protein domains and five movements between

domains and substrate-binding motifs during the reaction cycle, which could be diagrammed in steps similar to those for arsenate reductase as mentioned above. Within the membrane domain, CadA ATPase is thought to have eight predominantly alpha-helical transmembrane stretches. These move relative to one another during the reaction cycle. The cytoplasmic domains are the nucleotide-binding (N), phosphorylation (P), and activator/phosphatase domains. The CadA divalent cation ATPase has a Cys-Pro-Cys motif in helix no. 6, which is conserved in this group of P-type ATPases and is considered to be involved in cation translocation. The N domain includes a shared motif of GDGXNDXP toward the carboxyl-end of the domain sequence, while the P site TGTKD and the dephosphorylation motif TGES are shared by all P-type ATPases. When ATP binds to the N domain, it rotates above the P domain allowing close proximity to the gamma phosphate with the aspartate. ATP hydrolysis and ADP release result in rotation of the activator/phosphatase domain so that the TGES phosphatase motif is in contact with the phosphoaspartate, which becomes accessible by outward rotation of the N domain, with concomitant movement within the membrane domain responsible for energydependent cation efflux. Understanding of the CzcCBA RND family system is less advanced. There are structures of individual components from homologous systems, but there are no data available supporting polypeptide movements as for the P-type ATPases. It is proposed that the RND systems, such as CzcCBA, pick up cation substrates from either the periplasmic space or the cytoplasm (Figure 1) and release the cation through outer membrane porin proteins. CzcA contains four domains, two membrane-embedded domains each with six membrane-spanning alpha-helical regions (that are thought to form the cation and Hþ pathways) and two periplasmic domains of approximately equal size. The large periplasmic domains of CzcA may form the wall of a large central cavity (Figure 1) in contact at its top with the opening in the outer membrane protein CzcC (Figure 1). CzcC as a monomer may bridge halfway across the periplasmic space and dock with a CzcA trimer across the outer membrane to the cell surface (Figure 1). The pore through CzcC is bordered by alpha helices in the periplasmic space and a beta-barrel structure in the outer membrane region. CzcB is thought to be anchored by its N-terminus to the inner cell membrane and to make contact with the periplasmic domains of both CzcA and CzcC (Figure 1).

Chromate Resistance Two physiological functions are widely associated with bacterial chromate resistance: first, reduction from the highly soluble chromate (Cr(VI), CrO4 2 – ) oxyanion to

Environmental Microbiology and Ecology | Heavy Metals, Bacterial Resistance

the less soluble (Cr(III), Cr(OH)3), and second, efflux of chromate from the cells by the single chemiosmotic ChrA membrane protein. Although both membrane-associated and soluble cytoplasmic chromate reductases have been described, well-defined resistance is invariably associated with the ChrA efflux pump that is frequently genetically contiguous to other required functions. ChrA is very widely found in GenBank sequences and appears to occur in several subbranches, which as yet have not been assigned to specific functional differences. The most familiar ChrA products determined by Pseudomonas and by C. metallidurans strain CH34 have been shown to cross the cytoplasmic (inner) membrane 12 or 13 times.

Other Bacterial Toxic Metal Resistance Systems Additional bacterial toxic metal resistance systems for which genes have been sequenced and mechanisms proposed include those for Co2þ, Cu2þ (and Cuþ), lead (Pb2þ), Ni2þ, tellurite, and Zn2þ. Bacterial lead resistance is often reported from environmental studies. However, it is unclear whether a single mechanism of Pb2þ resistance commonly occurs or whether there are different mechanisms in different bacterial types. The DNA sequence of Pb2þ resistance for C. metallidurans strain CH34 contains six genes, including those for a positively acting (MerR-like) activator PbrR and genes that appear to encode the resistance mechanism. PbrD appears to be an intracellular Pb2þ-binding protein with a Cys-rich potential metal-binding motif (Cys-X7-Cys-Cys-X7-Cys-X7-His-X14-Cys). A resistance mechanism including intracellular sequestration may occur. However, the large PbrA P-type ATPase with the conserved CysProCys and other general sequence properties of soft metal cation P-type ATPases including CadA may function in Pb2þ efflux. PbrB is predicted to be a small outer membrane lipoprotein, which may be the pathway for removal of Pb2þ that was pumped by PbrA into the periplasmic compartment. In contrast to the Pbr system of C. metallidurans, intracellular lead phosphate precipitates have been associated with plasmid-governed Pb2þ resistance

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in Gram-positive bacteria. It also appears that intracellular and extracellular binding of Pb2þ may provide additional mechanisms for lead resistance.

See also: Heavy Metals Cycle (Arsenic, Mercury, Selenium, others)

Further Reading Barkay T, Miller SM, and Summers AO (2003) Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews 27: 355–384. Begley TP and Ealick SE (2004) Enzymatic reactions involving novel mechanisms of carbanion stabilization. Current Opinion in Chemical Biology 8: 508–515. Benison GC, Di Lello P, Shokes JE, et al. (2004) A stable mercurycontaining complex of the organomercurial lyase MerB: Catalysis, product release, and direct transfer to MerA. Biochemistry 43: 8333–8345. Brown NL, Morby AP, and Robinson NG (eds.) (2003) Interactions of bacteria with metals. FEMS Microbiology Reviews 27: 129–447. Cervantes C, Campos-Garcia J, Devars S, et al. (2001) Interactions of chromium with microorganisms and plants. FEMS Microbiology Reviews 25: 335–347. Messens J and Silver S (2006) Arsenate reduction: Thiol cascade chemistry with convergent evolution. Journal of Molecular Biology 362: 1–17. Muller D, Me´digue C, Koechler S, et al. (2007) A tale of two oxidation states: Bacterial colonization of arsenic-rich environments. PLoS Genetics 3(4): e53, 0518–0530. Schiering N, Kabsch W, Moore MJ, Distefano MD, Walsh CT, and Pai EF (1991) Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607. Nature 352: 168–172. Silver S (2003) Bacterial silver resistance: Molecular biology and uses and misuses of silver compounds. FEMS Microbiology Reviews 27: 341–354. Silver S and Phung LT (1996) Bacterial heavy metal resistance: New surprises. Annual Review of Microbiology 50: 753–789. Silver S and Phung LT (2005a) Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Applied and Environmental Microbiology 71: 599–608. Silver S and Phung LT (2005b) A bacterial view of the periodic table: Genes and proteins for toxic inorganic ions. Journal of Industrial Microbiology and Biotechnology 32: 587–605. Silver S, Phung LT, and Silver G (2006) Silver as biocides in burn and wound dressings and bacterial resistance to silver compounds. Journal of Industrial Microbiology and Biotechnology 33: 627–634. Taylor DE (1999) Bacterial tellurite resistance. Trends in Microbiology 7: 111–115.

High-Pressure Habitats A A Yayanos, University of California, San Diego, La Jolla, CA, USA ª 2009 Elsevier Inc. All rights reserved.

Introduction Historical and Current Background Instruments to Survey and Sample Microorganisms in High-Pressure Habitats High-Pressure Apparatus for Laboratory Studies of Microorganisms

Glossary barophile An organism growing or metabolizing faster at a pressure greater than the atmospheric pressure. This term is being replaced by the word piezophile. hyperpiezophile An organism whose maximum rate of growth over all possible growth temperatures occurs at a pressure greater than 50 MPa. To date, only hyperpiezopsychrophiles have been isolated. piezophile An organism whose maximum rate of growth over all possible growth temperatures occurs at a pressure greater than the atmospheric pressure and less than 50 MPa. To date, there are examples of piezopsychrophiles, piezothermophiles, and piezohyperthermophiles.

Abbreviations DSRV PTG

Deep Submergence Research Vessel pressurized temperature gradient

Introduction The influence of pressure on biological systems attracted the interest of scientists at Accademia del Cimento in Florence and of Robert Boyle in England during the seventeenth century. Science historian Stephen G. Brush credits Boyle for introducing ‘a new dimension – pressure – into physics’. Euler provided the first mathematical definition of pressure in the eighteenth century. By the end of the nineteenth century, the concept of pressure had been developed into its present-day meaning. Pressure and temperature are today fundamental parameters for physical–chemical theory, for the description of environments, in industrial chemistry and biotechnology, and in both laboratory and ecological investigations of organisms. An indispensable aspect to the analysis of

228

Distribution of Bacteria and Archaea in High-Pressure Habitats Properties of High-Pressure Inhabitants Further Reading

pour tube A test tube containing a nutrient medium that can be caused to gel by temperature change or following the addition of another component. Bacteria added to the pour tube become immobilized by the gelling. Each immobilized bacterium grows and divides to eventually form a visible colony of cells. Pour tubes are well suited for high-pressure microbiology. pressure The ratio of the force acting on a surface divided by the area of the surface on which the force acts. Atmospheric pressure at sea level is 0.101325 MPa. 1 Pa ¼ 1 N m2 (newton per square meter). PTk-diagram A graph of k, the exponential growth rate constant of a microorganism, versus temperature, T, and pressure, P.

PUFA ROV

polyunsaturated fatty acid remotely operated vehicle

organisms inhabiting high-pressure environments is that temperature and pressure are coordinate variables.

Historical and Current Background Seventeenth-century biological research at the Accademia del Cimento and by Boyle quantified and described the effects of decompression from atmospheric pressure. Hot air ballooning began in the late eighteenth century and provided another reason for studies in highaltitude physiology. Human problems similarly aroused interest in elevated pressures. Workers in diving bells, caissons, and deep mine shafts often became ill and experienced physiological difficulties. Bert’s classic treatise reviews many of these early studies. The interest in

Environmental Microbiology and Ecology | High-Pressure Habitats

what we now term high-pressure habitats, epitomized by the deep sea, began in earnest in the nineteenth century. Certes, Regnard, and Roger in France did pioneering research in experimental biology. The question of how high pressure influences deep-sea life was, indeed, an impetus for their work. Key references to nineteenth century and to early twentieth century high-pressure research are in the book of Johnson, Eyring, and Pollisar. Several oceanographic expeditions from the nineteenth century onward established the existence of life, both animal and microbial, in all of the deep sea, which is the largest high-pressure habitat on Earth. Scientific research on HMS Challenger during the first round-theworld oceanographic expedition from 1873 to 1876 showed the presence of animals throughout the seas to depths greater than 5000 m. Nearly 80 years later, participants on the Danish Galathea Expedition (1950–52) demonstrated the presence of life in the greatest ocean depths. ZoBell published in 1952 the results of his work done on the Danish research vessel Galathea. He found that bacteria from 10 000 m depths in the Philippine Trench grow at pressures as great as 100 MPa whereas bacteria from the upper ocean do not. This work, done with the most probable number method and microscopic examination of cultures incubated at high pressures, was with natural populations of microorganisms. Since 1979, pure cultures of deep-sea bacteria have been isolated by several investigators. Beginning in the late 1970s, expeditions utilizing Deep Submergence Research Vessel (DSRV) Alvin led to the discovery of hydrothermal vents at depths approaching 4000 m. At vents, high temperatures and high flow rates of seawater containing copious nutrients result in remarkably localized and productive communities of microorganisms and animals. The seawater effusing from a vent, moreover, may be carrying microorganisms from seafloor depths and locales distant from the vent. High-pressure habitats in addition to those in the oceans are found within the seafloor and beneath the surface of continents. These subterranean regions, also called subsurface environments, are inhabited by microorganisms. Geochemical analyses showed more than 30 years ago that microbes very likely inhabit the seafloor to depths of hundreds of meters. The continental subsurface has also been studied for many years, as reviewed by Amy and Haldeman in 1997. Microbiologists only recently began to sample a wide spectrum of subsurface environments. Although results unusual in terms of pressure have not been found, these are likely to appear as deeper parts of the subsurface environment are explored. Petroleum reservoirs in the seafloor and in subsurface continental locales have yielded thermophilic bacteria. Continental subsurface microbial inhabitants have been studied by scientists in Sweden to understand and quantify microbial processes that could influence plans to bury radioactive waste.

229

Finally, thermophilic bacteria were cultivated from samples collected at a depth of 3700 m in an African gold mine. The search for life buried in the seafloor has recently accelerated on a number of fronts. First, more scientists are attempting to cultivate microorganisms from cores collected under the auspices of the Deep Sea Drilling Program. Second, geochemists and molecular biologists are looking for patterns in chemical composition along depth profiles (i.e., along the length of a core) that would suggest microbial activity. Third, an examination of the morphology of inorganic surfaces on rocks in cores shows erosion features that provide compelling evidence for biological activity in buried environments. Fourth, the radiolysis of water by ionizing radiation from the decay of Earth’s naturally occurring radionuclides has been shown to provide enough hydrogen to fuel microbial metabolism in buried environments. Finally, several clever in situ experiments have revealed an abundance of microbial growth in the upper part of the seafloor. In summary, many high-pressure regions of the earth beneath our feet and the seafloor are inhabited. Definition of Pressure A stress, , is the ratio of the magnitude of a force,
F, to the area,
A, on which it is acting as
A ! 0. Force acting on an area can be further resolved into components parallel and perpendicular to the area. The parallel components are the shear stresses and the perpendicular ones are the normal stresses. These are summarized by the stress tensor, 3 11 12 13 7 6 7 ij ¼ 6 4 21 22 23 5 31 32 33 2

In fluids at rest and at a uniform temperature, shear stresses are absent or negligible (ij ¼ 0 for i 6¼ j ) whereas the three normal stresses are equal to each other (11 ¼ 22 ¼ 33). The normal stresses are called the hydrostatic pressure or simply the pressure, P, when dealing with fluids of uniform temperature and at rest. At any point in such a fluid, the pressure is the same in all directions. In solids, the pressure may or may not be hydrostatic. For example, some solids can have 11 6¼ 22 6¼ 33. The stress tensor is a comprehensive expression of stress distribution in a substance. The remainder of this article deals with hydrostatic pressure. High-Pressure Environments on Earth and Other Planets The pressure at the surface of the earth arises from the gravitational attraction between Earth and its atmosphere

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Environmental Microbiology and Ecology | High-Pressure Habitats

and is 0.101325 N m2 ¼ 0.101325  106 Pa ¼ 0.101325 MPa. Since water is considerably denser than air, the pressure increases rapidly with depth in the oceans to reach approximately 110 MPa in its deepest trenches. The pressure in the ocean increases with depth according to the equation dP ¼ gdz

where g is the gravitational ‘constant’,  is the density of the seawater, and z is the depth. On Earth at sea level, g ¼ 9.8 m s2. Although the values of g and  vary with latitude, longitude, and depth, these variations in the oceans are small for microbiological considerations so that the pressure in megapascal is approximately given by the depth in meters multiplied by 0.01013. At a depth of 10 000 m, the pressure in Earth’s ocean is close to 101.3 MPa. On Mars, g ¼ 3.71 m s2. Therefore, if Mars once had an ocean with the same maximum depth as the one on Earth, then the maximum pressure in the Mars Ocean would have been less than 38 MPa. Furthermore, if an ocean on Mars had been as cold as or warmer than the one on Earth, then pressure would not have been a limiting factor for Earth-like life. However, if the Mars Ocean had been substantially colder than that on Earth, then the effects of pressure would have been pronounced even at

38 MPa. Obviously, if life on Mars had been substantially different from that on Earth, then there is no way at present to surmise how pressure influenced it. New evidence suggests that there may be oceans on certain Jovian satellites. Europa, a Jovian satellite slightly smaller than our moon, has a g  1.23 m s2. Callisto has a g  1.3 m s2 and is about the size of the planet Mercury. The pressure in any of the oceans on these two moons of Jupiter can be determined by both depth and the local gravitational field. As shown in Table 1, the pressure at the bottom of a possible Europa Ocean that is 30 000 m deep would be approximately 40 MPa. A list of high-pressure environments is given in Table 1. There is no incontrovertible evidence that the Kola well at a depth of 8000 m or that hydrothermal vents at temperatures much greater than 115  C are inhabited. Figure 1 assists us in getting an idea of how temperature and pressure influence (1) where life is found; (2) where life is likely absent; and (3) where searches for life may lead to the discovery of new organisms. Figure 1 is a diagram of the PT-plane where P is the pressure and T is the temperature of a locus on Earth. Some of the environments listed in Table 1 are indicated in this figure as lines of constant pressure (isopiests) or constant temperature (isotherms) to show where life is found as well as where it may be found. Only a small portion of all possible

Table 1 High-pressure environments High-pressure environment

T ( C)

P (MPa)

Depth (m)

Earth’s oceans Weddell Basin Central South Pacific Central North Pacific Peru–Chile Trench Philippine Trench Tonga Trench Mariana Trench Celebes Sea Halmahera Basin Sulu Sea Mediterranean Sea Red Sea Hydrothermal vent

–0.5 1.2 1.5 1.9 2.48 1.8 2.46 3.26 7.54 9.84 13.5 44.6 2–380

45.6 50.7 50.7 60.8 101.3 96.3 110.4 63.0 20.7 56.5 50.7 22.3 25.3

4500 5000 5000 6000 10 000 9500 10 915 6300 2043 5576 5000 2200 2500

Freshwater bodies Lake Baikal Lake Vostok

3.5 >–5

16 ca. 35

>1600 3800 ice þ 500 water

Subsurface of earth’s continents Kola well in Russia

>155

88.3–205.9

8000

Subsurface of earth’s seafloor Nankai Trough, 30 m into sediments 6 km water depth, 1 km into seafloor

0 5 100

45.9 70.9

4530 7000

Planetary environment Venus (at its surface) Europa (conjectured values)

227 5

9 40

0 30 000

Environmental Microbiology and Ecology | High-Pressure Habitats 150 The deep earth

? Temperature (°C)

? 50

?

?

?

?

? Ice I

–50

?

A deep-sea hydrothermal vent The mediterranean sea The cold deep sea

Humans 5

?

?

100

0

Ice VI 100 200 Pressure (MPa)

300

Figure 1 Aspects of life in environments of different temperatures and pressures. (1) The heavy black line is an isotherm at 2  C that represents the cold deep sea inhabited to pressures greater than 110 MPa. The deep sea is populated with animals, archaea, and bacteria and is the most prominent highpressure habitat on earth. (2) The isotherm at 13.5  C, drawn as a dashed line, represents the habitats of the Mediterranean Sea and extends to 50 MPa. (3) The vertical dashed line is an isopiest (line of constant pressure) at 25 MPa. This represents the pressure–temperature environment around a hydrothermal vent at 2500 m depth where the maximum temperature observed, in excess of 350  C, undoubtedly exceeds the tolerance of organisms. The upper temperature limit for life has not been established. Many believe that it will be a temperature less than 150  C. (4) Two lines show how temperature and pressure might increase along a depth profile into the earth on continents. Similar lines could be drawn beginning, for example, at a pressure of 50 MPa and a temperature of 2  C to represent a temperature–pressure profile in the seafloor. (5) The two question marks in circles are to indicate that we do not yet know the upper pressure–temperature limits of life. (6) The question marks in squares represent extant temperature–pressure conditions on earth where life could plausibly exist. The three upper squares are with regard to conditions found deep in the seafloor. The square at negative pressures is at temperature–pressure conditions found in the xylem sap of tall trees. (7) The question marks in diamond symbols are positioned at temperature– pressure conditions that possibly do not exist on earth but in which life could exist. (8) The bottom line is approximately along the water–ice phase transition. Price (2007) reviews the possibilities for life and for the origin of life in icy places.

PT-habitats have been studied. Chief among these are the atmospheric pressure isobar, the cold deep-sea isotherm, the Mediterranean Sea isotherm, hydrothermal vent isopiests up to 40 MPa, and a few subsurface habitats. Potentially interesting habitats that have not been sampled enough are beneath the seafloor at water depths in excess of 5000 m. Figure 1 also serves to underscore the fact that the distribution of organisms is influenced and delimited by both temperature and pressure acting in concert. That is, the temperature range wherein life is found increases with pressure; and, quite possibly, the pressure range of life increases with temperature. There

231

is a line or transition zone of demarcation on the PTplane separating conditions compatible with life from abiotic ones. We know neither where to quite put this line or PT-envelope delimiting life processes nor the shape of the envelope. It may, for example, be a continuous concave inward curve. However, this is not yet known. At this time, a theoretical analysis to define a PT-envelope for life on Earth has not been made. No one can yet say, for example, that at 60 MPa life will be found at an upper temperature of X  C and never 1  C higher. Life exists in most of the high-pressure environments listed in Table 1. The world’s oceans contain an abundant and diverse group of animals, bacteria, and archaea. The deep Earth is a home for mainly bacteria and archaea. Environments on Mars and Europa could contain microorganisms similar to those on Earth. However, hard evidence must await further exploration. Venus has a surface temperature of 227  C that experiments and calculations suggest is far too high for Earth-like life processes. However, a final verdict on whether Venus is inhabited must await a determination of its subsurface temperatures. Table 1 and Figure 1 show that Earth’s oceans and seafloor have inhabited regions not easily reproduced on other planets and moons of the solar system. That is, almost all of the extraterrestrial conditions that are extreme and habitable in terms of pressure–temperature are represented somewhere on Earth in a marine setting. It is interesting how oceans provide this variety of pressure–temperature habitats. The temperature of the sea surface is warmest at tropical latitudes and coldest at polar regions. The deep sea is a nearly uniformly cold habitat. Seawater at the poles is less than 1.5  C. The cold surface waters acquire a density less than that of the underlying ocean waters. Enough cold surface water forms and sinks in polar oceans to initiate and sustain deep ocean circulation. Although sinking polar water masses compress in a mostly adiabatic fashion, they become only slightly warmer as they sink. The net result is a deep sea that is preponderantly a cold environment with temperatures close to 2  C. Only a few warm deep-sea environments exist and they arise in two distinct ways. One is through the presence of sills, elevated portions of the seafloor that completely separate the deep parts of adjacent basins. The sills block the entry of cold deep water derived from polar regions. Thereby, the abyssal regions of these seas are warm, as shown in Table 1. An example is the Sulu Sea that receives its water from the South China Sea over a sill depth of 400 m, reckoned from the sea surface to the top of the sill. The temperature of the deep sea in the Sulu Sea is 9.8  C. Another example is the Mediterranean Sea separated from the Atlantic Ocean by the Strait of Gibraltar sill at a depth of 320 m that

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Environmental Microbiology and Ecology | High-Pressure Habitats

blocks the entrance of cold deep Atlantic water. Furthermore, the Mediterranean Sea has strong vertical mixing driven by evaporation that forms a dense layer of warm water on the sea surface, which then sinks. Thus, horizontal mixing as well as vertical mixing plays a role in determining the temperature of about 13.5  C in the deep sea of the Mediterranean Sea. Figure 1 shows an isotherm at 13.5  C extending to a pressure of 50 MPa found at the greatest depth of the Mediterranean Sea off of the west coast of Peleponesus. The second cause for warm and hot habitats in the otherwise cold deep ocean is hydrothermal circulation, most notably at midocean ridges. The temperature in geothermal water can be over 370  C. Mixing with cold ocean water occurs rapidly. A vent environment, called a vent field, is a highly localized region of hundreds of square meters to over a square kilometer. The water depth at most midocean ridges is less than 4000 m. Vents are inhabited to temperatures somewhat above 110  C. The fluids emanating from vents derive from a sub-seafloor circulation. This implies that these fluids are inhabited for at least part of their journey through the seafloor. Noteworthy is evidence that the deep sea was not always as cold as it is today. Estimates suggest that the temperature of the deep sea has alternated several times between 15  C and its present value of 2  C over the past 800 million years. Thus, the comparative biology of organisms in the cold deep sea, the Sulu Sea, the Mediterranean Sea, and at hydrothermal vents is of value for paleoecology.

Other Parameters of High-Pressure Habitats In the previous section the case is made that temperature and pressure must be considered as twin parameters of habitats. An important part of the rationale for this is that the values of other essential parameters of habitats and of the intracellular milieu change along with changes in temperature and pressure. Chief among these temperature-dependent and pressure-dependent parameters are those of density, viscosity, dielectric constant, coefficient of thermal expansion, coefficient of compressibility, chemical equilibrium constants, phase transition and stability conditions, and chemical reaction rate constants. The dielectric constant, for example, increases with increasing pressure and decreases with increasing temperature. An understanding of how these essential parameters are affected by both temperature and pressure will ultimately offer two important explanations. One is to provide a basis for the existence of limits of temperature and pressure beyond which life cannot exist. The other is to explain (or, to predict) the temperature and pressure limits for the growth of a given organism.

Instruments to Survey and Sample Microorganisms in High-Pressure Habitats There are two distinct methods used to study life in highpressure habitats. One is the in situ approach whereby measurements, observations, and experiments are performed in the deep sea or in other high-pressure habitats. The second is to recover organisms from a high-pressure habitat and conduct research on them in the laboratory. Organisms during the sampling process may suffer decompression, temperature change, contamination, exposure to light, and a change in habitat chemistry. Specially designed sampling instruments minimize or eliminate these changes. Instruments used in marine microbiology include Niskin bottles, pressureretaining water samplers, pressure-retaining animal traps, pressure-retaining corers, and thermally insulated corers. Sediment microbiologists use cores collected mostly with decompression. Microbiologists sampling the terrestrial subsurface devise methods to minimize or detect contamination. Sometimes they flame the outer surface of a core to inactivate microbial contaminants. They also add to drilling fluids detectable particles whose presence in a core sample serves as a proxy for possible microbial contamination. DSRV Alvin and DSRV Shinkai 6500 are particularly effective in collecting samples both without the introduction of additional foreign microorganisms and from precisely identified locales. Scientists Kato, Li, Tamaoka, and Horikoshi of the Japanese Marine Science and Technology Center operated Kaiko, a remotely operated vehicle (ROV), using DSRV Shinkai 6500 to collect sediments from the deepest part of the Mariana Trench in 1997 without introducing contaminant organisms via the sampling procedure. Axenic sampling of seafloor sediments and subterranean habitats is difficult and likely impossible because these habitats are contaminated constantly with upper ocean organisms as a result of natural processes. It is, however, important to undertake efforts to minimize and assess any additional contamination due to sampling. If every experiment required samples strictly maintained at habitat conditions during sample collection, then research progress would be slow and expensive. Thus, development of alternate sampling strategies is important. Indeed, most deep-sea microbiology is conducted with decompressed samples collected with Niskin bottles and a variety of coring devices deployed from ships. An inescapable sampling requirement for microbiology of the cold deep sea is the avoidance of sample warming. The justification for this is seen in Figure 2 showing the rapid thermal inactivation of a deep-sea bacterium at temperatures encountered in the upper tropical and temperate

Environmental Microbiology and Ecology | High-Pressure Habitats

Surviving fraction of cells

10–0 10–1

20 °C

10–2 0 °C

10–3 10–4 27 °C

10–5 10–6

0

50

100

150

200

Minutes of exposure Figure 2 Thermal inactivation kinetics for two different bacterial isolates. The data in red are for bacterial isolate CNPT3, from a depth of 5782 m in the Pacific Ocean. One suspension of cells was placed at 27  C and another at 20  C. At selected times the cell suspensions were sampled and the pour tube method (Figure XX) was used to determine the fraction of cells that were still able to form colonies. This bacterium looses viability (colony-forming ability) at atmospheric pressure when kept at temperatures greater than 10  C. Sensitivity to warming has not been determined for very many bacterial strains from the cold deep sea. Nevertheless, high thermal sensitivity is likely one of their traits. One bacterial strain from the Mariana Trench, isolate MT41, looses viability at 0  C at atmospheric pressure. The dashed line shows the slow rate of death for isolate MT41. This slow death rate may be due to decompression per se. Reproduced from Yayanos AA (1995) Microbiology to 10 500 meters in the deep sea. Annual Reviews of Microbiology 49: 777–805.

ocean through which sampling gear must pass. Also shown in Figure 2 is the inactivation of a bacterium from the deepest part of the ocean while it is held at atmospheric pressure and 0  C. This shows that decompression per se, although lethal to this bacterium, does not instantly kill it. Death following decompression has so far been seen only in bacteria of the cold deep ocean. Most subsurface microbiology has been done with decompressed core samples from mesophilic and thermophilic habitats and with atmospheric pressure cultivation. Such samples have been adequate to establish enrichment cultures of mesophilic and thermophilic bacteria and archaea. Pressure retention on samples, however, remains an essential aim in work addressing questions on community composition, structure, and function and seeking to identify any variability in the manifestations of pressure adaptation. Research on samples from the Mid-Atlantic Ridge at a depth of 3550 m shows that certain hyperthermophilic microbes grew in enrichment cultures only if incubated at high pressure. One of the startling things about this work is that the organism Thermococcus barophilus thus isolated also grows at atmospheric pressure, raising the

233

question of why it was not isolated in atmospheric pressure enrichments. T. barophilus, moreover, is a hyperthermopiezophile since it exhibits its maximum growth rate at a high pressure when tested over all possible temperatures. This study by Marteinsson and colleagues shows clearly the value of imitating as much as possible the conditions of the sampled environment in enrichment cultures and also the necessity of experimentally determining the pressure–temperature growth responses of organisms under a variety of experimental conditions. Piezothermophiles survive exposure both to low temperatures and to low pressures whereas piezopsychrophiles do not survive low pressures and temperatures much above 20  C. This means that the dispersal path for the latter is through the cold deep ocean. In contrast, some piezothermophiles survive exposure to cold temperatures for at least 1 year. This gives piezothermophiles many pathways for dispersal from one vent to another. For example, they could be transported by cold deep-ocean currents from one vent field to another. Also, they could emerge from one vent and enter the seabed to become part of the subseafloor hydrothermal circulation and then emerge from another vent. Although speculative, these possibilities seem quite feasible.

High-Pressure Apparatus for Laboratory Studies of Microorganisms Essential tools of microbiologists are enrichment culture technique, plating for isolation of clones and assay of colony-forming ability (viability), replica plating for isolation of mutants, and determination of growth rates in liquid culture. Each of these methods needs to be modified for the study of microorganisms having a strong preference or absolute necessity for high pressure. Yayanos (2001) provides a more thorough treatment of the methods discussed in the remainder of this section. The key components, commercially available, of a high-pressure apparatus are a pressure gauge, a pump (sometimes called an intensifier), a pressure vessel, valves, and tubing connecting these components. Although pressure vessels can be purchased, the need to fabricate them with features otherwise unavailable often arises. The fluid used to compress a sample in a pressure vessel is usually water. Gases present problems as a hydraulic medium and find infrequent use. For example, they dissolve in biological phases to have effects other than those arising from compression. Also, the need for precautions such as barricades to ensure safety in work with compressed gases makes the work expensive. One of the technical difficulties in high-pressure microbiology is that cultures must be isolated from the hydraulic fluid. Although liquid cultures have been

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Environmental Microbiology and Ecology | High-Pressure Habitats

incubated with success in syringes, the possibility of contaminants entering the culture around the syringe plunger must always be kept in mind, especially in long-term incubations. A great boon to high-pressure microbiological research has been the advent of heat-sealed plastic containers such as bags and polyethylene transfer pipettes. These allow for pressure equilibration, isolation of the culture from microbes and chemicals in the hydraulic fluid, and a degree of control of gas composition in the culture. The latter is achieved through the selection of plastic bag materials with appropriate gas permeability allowing for the exchange of dissolved gases between the culture and the hydraulic fluid. Clones of bacteria can be obtained at high pressure with pour tubes. Figure 3 shows colonies of a marine bacterium grown at high pressure. Parafilm stretched tightly over the opening of a pour tube sealed it from contamination and served to transmit the pressure to the medium. The pour tube method can also be accomplished with heat-sealed polyethylene transfer pipettes rather than test tubes. The pour tube technique works for

Figure 3 Five pour tubes are shown. The one on the right was not inoculated. The other four, beginning with the tube on the left, were inoculated with a serial dilution of a culture of Micrococcus euryhalis, covered with parafilm and incubated at high pressure. After several days, visible colonies developed around each immobilized bacterial cell. Note that the colonies are larger at greater dilutions. This pour tube method is ideal for obtaining clones of bacteria in pressure vessels and for assay of cell viability following the exposure of cells to chemical or physical stress.

high-pressure microbiology as petri plates do for atmospheric pressure microbiology with one exception. That is, replica plating cannot be done with pour tubes. Mesophiles and thermopiles from habitats having a pressure of less than 50 MPa grow in enrichment cultures incubated at atmospheric pressure. For example, methanogens and sulfide oxidizers have been isolated through atmospheric pressure enrichment cultures that were inoculated with samples from deep-sea hydrothermal vent habitats. The enrichment culture method is also known as the ecological method because it is an attempt to grow an organism under the physical and chemical conditions of its natural habitat. Strict application of the ecological method would seem to require incubation of the enrichment cultures at the pressure of the hydrothermal vent habitat. If additional evidence, however, provides little doubt that an organism isolated at atmospheric pressure is an inhabitant of the sampled high-pressure habitat, then the need for conducting enrichments at high pressure seems diminished. Most of the cultures of mesophilic and thermophilic organisms from deep-sea hydrothermal vents and the terrestrial subsurface have so far been found amenable to cultivation and plating at atmospheric pressure. Perhaps, a requirement for strict high-pressure technique will be necessary for the successful isolation of mesophiles and thermophiles from warm and hot habitats having a pressure of 100 MPa or more. In contrast to mesophiles and thermophiles, the isolation and study of psychrophilic deep-sea bacteria is nearly impossible without the use of high-pressure laboratory methods, especially the pour tube technique modified for high pressures. An instrument called a pressurized temperature gradient (PTG) allows for the concurrent determination of growth parameters of bacteria incubated at different pressures and temperatures. It also allows for the isolation of clones of bacteria growing at different temperatures and pressures from a single inoculum. Scientists in Germany, Japan, and the United States have published three different designs of a PTG instrument. The fact that a wider use of such instruments has not been adopted suggests that further design improvements are needed.

Distribution of Bacteria and Archaea in High-Pressure Habitats Bacteria and archaea are ubiquitous in the ocean, including its greatest depths with a pressure of approximately 110 MPa. Since animals are found at all ocean depths, as shown in Figure 4, a highly plausible hypothesis is that animals, bacteria, and archaea could exist at pressures even higher than 110 MPa. It is further likely that microorganisms could live at pressures beyond those limiting

Environmental Microbiology and Ecology | High-Pressure Habitats

235

Properties of High-Pressure Inhabitants Before any research had been done on deep-sea bacteria, it was known that physiological and biochemical processes in bacterial and eukaryotic cells were influenced by pressure. Single-cell organisms from atmospheric pressure habitats typically show morphological and physiological aberrations when placed at 20–50 MPa. Against this background, the finding of pressure adaptation in deepsea bacteria is expected. Exactly how adaptation is achieved, however, remains an active area of research. Current biochemical and molecular biological research is primarily on pressure adaptation in Gram-negative heterotrophic bacteria isolated from the cold deep ocean and in archaea from both the cold ocean and hydrothermal vents. The most obvious manifestation of adaptation to high pressure was first seen in organisms of the cold deep sea. Only there, for example, have microorganisms that grow exclusively at high pressure been found so far. Such bacteria have been isolated from samples of ocean depths between 6000 and 10 000 m. Nevertheless, pressure adaptation, although less conspicuous, is present in cold deep-sea microorganisms inhabiting depths at least as shallow as 2000 m and in hyperthermophiles from submarine hydrothermal vents. The rest of this article describes some of the manifestations of pressure adaptation.

Figure 4 Photograph of amphipods swarming to food (dead fish) placed on the seafloor at a depth greater than 10 500 m. The dead fish were tied to one end of an expendable pole and the 35 mm camera in a pressure-resistant housing was mounted at the other end of the pole by means of a timed release mechanism. The pole and other ballast were left on the seafloor when the timed release mechanism was actuated to enable floats to raise the camera to the sea surface. The amphipods are Hirnodellea gigas. Pictures such as this one suggest that the animals inhabit this depth. But this does not prove that the animals reproduce at this depth. These animals were not able to survive decompression to even 34 MPa in the few experiments conducted to date.

animal life. Perhaps an environment having a pressure of 200 MPa would be habitable by bacteria. If such an environment existed and was as cold as or colder than the 2  C temperature of today’s deep sea, then metabolic processes probably would be very slow and confined within narrow ranges of temperature and pressure. Bacteria have been found beneath the surface of both continents and the seafloor. Among the principal factors bounding the distribution of life in subsurface environments are restricted space as judged by the size of pores in compacted sediments, the absence of cracks or fissures, and temperatures exceeding approximately 115  C.

Rates of Growth The kinetics of chemical transformations mediated by microbes and of microbial growth are of general interest because the associated rate constants are important parameters in models of food web dynamics and biogeochemical cycles. The question of whether bacteria at a given depth in the sea grow more slowly than do their relatives at a shallower depth is difficult to answer. This is because the growth rate of bacteria is a multivalued function of not only temperature and pressure but also other factors. These include pH of the medium, oxidation–reduction reactions, and nutrient types and levels. E. coli, for example, grows 20 times more rapidly in a complex nutrient medium than in a minimal medium with succinate as the sole carbon source. Experiments with a deep-sea bacterial isolate also show slower growth in a minimal medium. Remarkably, the growth rate of this bacterial isolate also exhibited a diminished response to pressure change when grown on glucose or glycerol minimal medium. To summarize, the pressure dependence of the growth rate is dependent on many factors. Nevertheless, a comparison of growth rates of different heterotrophic bacterial species grown in an identical nutrient-rich medium at their respective habitat temperatures and pressures shows a trend for deeper-living species to have the slowest growth rates. Thus, the fastest-growing

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Environmental Microbiology and Ecology | High-Pressure Habitats

deep-sea heterotrophic bacteria are usually from depths of less than 5 km. And, the slowest-growing ones are generally from the greatest ocean depths of about 10 km. Growth rates (at habitat temperatures and pressures) of bacteria isolated from depths of 2–11 km are in the range of 3–35 h, about as ZoBell reported nearly 50 years ago.

3584 m

k IN HR 0.1 0.2

–1

0.3

Strain PE36

15

In pure culture, the growth rate of microorganisms can be exponential,

3

PTk-Diagrams

IN

P

S*

R BA

6

10 T 5 IN 0 °C

–5

5782 m

Strain CNPT3 –1

k IN HR 0.2 0.1

dN =dt ¼ kN

–2

10

9

0.0

12

12 –2 0 *1 RS

9

0.0

BA

6

P

3

°C

0

10 T IN

IN

–5

7100 m

15 9

122 – 10

*

RS

6

5 °C

0

10 T IN

3

k IN HR 4 0.00 0.0

–1

0.08

Strain PT64

–5

P

IN

BA

8961 m

k IN HR 0.03

–1

0.06

Strain MT199

12 2 – 10

9

0.00

15

*

3

°C

0

IN

5

T

RS

6

10

–5

P

IN

BA

10 476 m

–1

k IN HR 0.06 0.00 0.03

Strain MT41

12 –2 0 *1 RS

9 6

10 T IN

0

°C

3

5

1. The pressure dependence of the growth rate is more pronounced in bacteria from the deepest habitats. Thus, strains MT199 and MT41 from deep habitats do not grow at atmospheric pressure whereas strain PE36 from a relatively shallow habitat of 3.5 km grows at atmospheric pressure. 2. The PTk-diagrams show that each bacterial species has a single pressure–temperature condition where its growth rate is a maximum. 3. The pressure where the growth rate has a maximum value is very nearly the pressure of the presumed habitat. This relationship between the pressure of maximum growth rate and the habitat pressure is not observed among heterotrophic bacteria from the warm deep-sea environments of the Sulu and Mediterranean seas. This fact shows very clearly that growth rate as an index of pressure adaptation depends greatly on the temperature of the high-pressure habitat. It is conceivable that physiological parameters other than growth rate will provide useful indices of pressure adaptation for inhabitants of high-temperature, high-pressure environments. 4. A particularly curious characteristic of deep-sea psychrophilic bacteria is that the temperature where the bacteria grow best is greater by 6–10  C than the constant habitat temperature of 2  C. This relationship, also evident with bacteria from the Sulu and Mediterranean seas, remains unexplained.

15

5

where N is the number of cells at time t and k is the exponential growth rate constant. The pressure dependence of the growth rate of an exponentially growing bacterial species is widely used as an index of pressure adaptation. Bacterial growth rates in media having high levels of nutrients provide the following view of cellular adaptation to pressure in the cold deep ocean. In Figure 5, growth rates of five heterotrophic bacterial strains, isolated from six different depths of the Pacific Ocean, are shown as a function of both temperature and pressure. These plots, called PTkdiagrams where P is the pressure, T the temperature, and k the specific growth rate constant, summarize a large number of growth rate determinations and reveal relationships otherwise difficult to see. Examples are as follows.

–5

P

IN

15

BA

Figure 5 PTk-diagrams of five deep-sea bacteria. This shows that pressure adaptation becomes more pronounced as the habitat depth of an isolate increases. Each bacterial isolate has a maximum growth rate close to the pressure of its habitat (indicated by the black dot). In this figure the unit of pressure is the bar. 1 bar ¼ 0.1 MPa and 1 atm ¼ 1.01325 bars.

Environmental Microbiology and Ecology | High-Pressure Habitats

The study of the properties of bacteria from the cold deep ocean and from warm deep seas with the aid of PTkdiagrams shows a progression in the properties of bacteria along the temperature and pressure gradients. That is, the growth properties of these microorganisms are in a sense a signature for the depth from which they came. Similar conclusions from the study of thermophiles cannot be made as yet. One possible reason for this is that thermophiles grow over large conditions of temperature and pressure. Another compounding effect is that the particular place where a sample was collected to isolate a thermophile may not be the principal habitat of that organism. That is, the process of hydrothermal circulation may be bringing the organism from a habitat of considerably different temperatures and pressures.

237

This binary categorization of microorganisms with respect to pressure was created before there were any studies with axenic cultures of deep-sea bacteria and of bacteria and archaea from high-pressure habitats having different temperatures. A newly suggested categorization of a given bacterial isolate is based on its PTk-diagram and is analogous to the scheme used to describe temperature adaptation. First, in this new scheme, non-pressure-adapted microorganisms are simply psychrophiles, mesophiles, thermophiles, and hyperthermophiles. They are all ‘barotolerant’ in the old terminology. Second, if the pressure, Pkmax, where the maximum growth rate occurs, as determined with a PTk-diagram, is 0:1 MPa < Pkmax < 50 MPa

then the isolate is a piezophile. Third, if

Physiological Classification of Bacteria and Archaea with Respect to Temperature and Pressure

Pkmax > 50 MPa

Microorganisms are called psychrophiles, mesophiles, thermophiles, or hyperthermophiles based on the temperature range wherein they grow. Although there is likely a continuum of adaptations along the temperature scale, this classification is useful. Even if these microorganisms are from atmospheric pressure habitats, they can grow at high pressures, usually less than 50 MPa. The conditions for maximum rate of growth, however, have always been found, so far, at atmospheric pressure. Since all organisms grow to some extent at high pressure, the older term ‘barotolerant’ to describe an organism that can tolerate high pressures has little significance. There are two ways microorganisms can be classified with respect to their growth as a function of pressure. The traditional classification is to call them ‘barophiles’ if they grow best at a pressure greater than the atmospheric pressure. This binary classification of the pressure-adaptive trait is thus different from the one used for classifying organisms based on their response to temperature. Furthermore, growth studies of bacterial isolates from high-pressure habitats of different temperatures make it apparent that an organism can be ‘barophilic’ at one temperature but not at another. Significantly, there are bacteria showing little or no ‘barophilic’ character at the temperature of their habitat while being distinctly ‘barophilic’ at a greater temperature.

then the isolate is hyperpiezophile. Table 2 shows the different possible types of microorganisms based on where the pressure and temperature of the maximum growth appears on a PTk-diagram. Inspection of Table 2 shows that this is a more useful classification than to simply state that an organism is a ‘barophile’. Also evident in Table 1 is that hyperpiezophiles have only been found in the cold deep ocean, that is, among the psychrophiles of a high-pressure habitat. As discussed in the context of Figure 1, the possibly inhabited environments having a pressure of approximately 100 MPa and a temperature in the range of 100  C may exist only in the seafloor beneath the deepest parts of the ocean. It is there that hyperpiezomesophiles, hyperpiezothermophiles, and hyperpiezohyperthermophiles will be found. Parenthetically, the prefix ‘piezo’ (having the meaning to press) rather than ‘baro’ (having the meaning of weight) is widely adopted in science, in terms such as piezochemistry and piezoelectric, to describe the role of pressure. To summarize, the scheme in Table 1 provides a succinct view of states of adaptation to both temperature and pressure. Molecular Biology and Biochemistry of Adaptation to High Pressures The structure of deep-sea bacteria viewed to date with light and electron microscopy is the same as that of

Table 2 Categorization of microorganisms P of kmax

P  0.1 MPa

0.1 MPa < P < 50 MPa

P > 50 MPa

T of kmax T < 15  C 15 < T < 45  C 45 < T < 80  C 80  C < T

Pychrophiles Mesophiles Thermophiles Hyperthermophiles

Piezopsychrophiles Piezomesophiles Piezothermophiles Piezohyperthermophiles

Hyperpiezopsychrophiles Hyperpiezomesophiles Hyperpiezothermophiles Hyperpiezohyperthermophiles

Representative bacteria or archaea in the categories shown in italics have not been found.

Environmental Microbiology and Ecology | High-Pressure Habitats

bacteria in general. The adaptations making possible their existence at high pressure can be presumed thereby to be at the molecular level. Several macromolecular structures and interactions as well as chemical reactions are altered by pressure change. These include microtubule assembly, ribosome integrity, helix-coil transitions in nucleic acids and proteins, protein conformation, protein–protein interactions, protein–nucleic acid interactions, enzyme activity, transport across membranes, and membrane fluidity. The list of pressure-affected processes is long. Bacterial membrane protein composition is also a function of pressure. The altered composition reflects pressure action on gene expression, on protein synthesis, and on membrane function. The changes observed are further dependent on the physiological state of the cell. For example, protein profiles not only change with pressure but also in a different way when cells grow on different carbon sources. Thus, the adaptation of marine microorganisms to nutritional states alters the manifestation of pressure adaptation. The final view of pressure and temperature adaptation will most likely comprise both an understanding of the pressure and temperature dependencies of many individual processes and a mathematical model of how the cell operates as a dynamical system. The latter is necessary because biological systems are quintessentially greater than the sum of their parts. The fatty acid composition of the membrane phospholipids of deep-sea heterotrophic bacteria is a function of the growth temperature and pressure. In some bacterial isolates, the trend in fatty acid composition change is consistent with the homeoviscous hypothesis. The crux of this hypothesis is that cells regulate their membrane composition to maintain the membrane in an appropriate physical state (fluidity, in particular). The regulation is done by altering membrane fatty acid composition and phospholipid molecular species. The regulation is activated in response to any physical or chemical factor that causes a change in membrane fluidity. The bacterial membrane lipid composition changes in response to temperature change have been long known. It is now well documented that the composition of the membranes of deep-sea bacteria varies with both the temperature and pressure of growth. Work remains to be done to show that the observed changes are along the lines of the homeoviscous hypothesis. Figure 6 shows the membrane fatty acid composition of a bacterial isolate from the Philippine Trench and its dependence on growth pressure. Deep-sea bacteria synthesize polyunsaturated fatty acids (PUFAs) for their membrane phospholipids. Figure 7 shows the chemical structure of the principal PUFAs found in many deep-sea bacteria. Although not demonstrated for deep-sea animals, it is generally believed that animals do not synthesize PUFAs and fulfill the requirement for them

(n – 3) bond

H3C

H2 C

H C

C H

(Δ – 4) bond

C H2

H C

C H

H2 C

C H

H C

C H2

H C

C H

H2 C

C H

H C

C H2

H C

C H

H2 C

O C H2

OH

22:6 (n – 3) Docosapentaenoic acid (n – 3) bond

H3C

H2 C

C H

H C

(Δ – 4) bond

C H2

H C

C H

H2 C

C H

H C

C H2

H C

C H

H2 C

C H

H C

C H2

H2 C

O C H2

OH

20:5 (n – 3) Eicosapentaenoic acid

Figure 6 The chemical structures of two polyunsaturated fatty acids (PUFAs) found in deep-sea bacteria. Prior to the discovery of these fatty acids in the membrane phospholipids of deep-sea bacteria, it was believed that few if any bacteria could synthesize these lipids. They now appear to be common among heterotrophic deep-sea bacteria.

27.5 MPa

55.2 MPa

82.7 MPa

40 % (w/w) of phospholipid fatty acids

238

30

20

10

0

13:0 14:1 14:0 15:0 16:1 16:0 18:1 18:0 20:5 Fatty acid

Figure 7 Fatty acid composition of membrane phospholipids of a deep-sea bacterium and how it changes with pressure. Plotted from data in DeLong and Yayanos (1985).

through their diet. It is conceivable that deep-sea bacteria contribute to this dietary need in the deep sea. The deep sea is also dark. The results in Figure 8 show that one deep-sea bacterial isolate has an extraordinary sensitivity to UV light. Similar results were obtained with four other deep-sea bacterial strains. However, it has not yet been determined if these bacteria have lost the genes to repair UV-damaged DNA. The sensitivity of deep-sea bacteria to ionizing radiation, moreover, is no different than that observed with shallow-water bacteria. This is in accord with the background of natural radioactivity being similar in both shallow water and the deep sea. Since the radiation

Environmental Microbiology and Ecology | High-Pressure Habitats

10–0

Surviving fraction

10–1 Upper ocean bacterium 10–2

10–3

10–4

Deep-sea bacterium

0

25

50 Dose in J m–2

75

100

Figure 8 The sensitivity of a deep-sea bacterium and of an upper ocean bacterium to UV light. Reproduced from Yayanos AA (1989) Physiological and biochemical adaptations to low temperatures, high pressures, and radiation in the deep sea. In: Hattori T et al. (eds.) Proceedings of the 5th International Symposium on Microbial Ecology, pp. 38–42. Tokyo: Japan Scientific Societies Press.

biology of deep-sea microorganisms is based on scant information, more work is needed before general statements can be made confidently. The genome sequences of several deep-sea bacteria have been completed recently. Studies of these genomes will certainly lead to hypotheses ultimately elucidating the nature of adaptation to the diverse high-pressure environments on Earth. As pointed out elsewhere in this article, the understanding of life in all of the extreme habitats on Earth will undoubtedly be of great value in finding life elsewhere in the solar system. See also: Deep Sub-Surface; Deep-Sea Hydrothermal Vents; Ecology, Microbial; Extremophiles: Hot Environments; Marine Habitats; Sediment Habitats, including Watery

Further Reading Amy PS and Haldeman DL (eds.) (1997) The Microbiology of the Terrestrial Deep Subsurface, 356 pp. Boca Raton, FL: CRC Lewis Publishers. Bartlett DH (2002) Pressure effects on in vivo microbial processes. Biochimica Biophysica Acta 1595: 367–381. Bert P (1943) Barometric Pressure. Researches in Experimental Physiology (Translated from the French by MA Hitchcock and FA Hitchcock). Columbus, OH: College Book. DeFlaun MF, Fredrickson JK, Dong H et al. (2007) Isolation and characterization of a Geobacillus thermoleovorans strain from an ultra-deep South African gold mine. Systematic and Applied Microbiology 30(2): 152–164.

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DeLong EF (1997) Marine microbial diversity: The tip of the iceberg. Trends in Biotechnology 15: 203–207. DeLong EF and Yayanos AA (1986) Biochemical function and ecological significance of novel bacterial lipids in deep-sea prokaryotes. Applied and Environmental Microbiology 51: 730–737. DeLong EF, Franks DG, and Yayanos AA (1997) Evolutionary relationships of cultivated psychrophilic and barophilic deep-sea bacteria. Applied and Environmental Microbiology 63: 2105–2108. Deming JW and Baross JA (1993) Deep-sea smokers: Windows to a subsurface biosphere. Geochimica et Comochimica Acta 57: 3219–3230. Furnes H and Staudigel H (1999) Biological mediation in ocean crust alteration: How deep is the deep biosphere. Earth and Planetary Science Letters 166(3–4): 97–103. Johnson FH, Eyring H, and Polissar MJ (1954) The Kinetic Basis of Molecular Biology, 874 pp. New York: John Wiley & Sons, Inc. Karl DM (ed.) (1995) The Microbiology of Deep-Sea Hydrothermal Vents, 299 p. Boca Raton, FL: CRC Press. Kato C and Bartlett DH (1997) The molecular biology of barophilic bacteria. Extremophiles 1: 111–116. Kato C, Li L, Tamaoka J, and Horikoshi K (1997) Molecular analyses of the sediment of the 11000-m deep Mariana Trench. Extremophiles 1: 117–123. Lauro FM and Bartlett DH (2008) Prokaryotic lifestyles in deep sea habitats. Extremophiles 12(1): 15. Marteinsson VT, Reysenbach AL, Birrien JL, and Prieur D (1999) A stress protein is induced in the deep-sea barophilic hyperthermophile Thermococcus barophilus when grown under atmospheric pressure. Extremophiles 3(4): 277–282. Middleton WEK (1971) The Experimenters: A Study of the Accademia Del Cimento, 415 pp. Baltimore: Johns Hopkins Press. Price PB (2007) Microbial life in glacial ice and implications for a cold origin of life. FEMS Microbiology Ecology 59(2): 217–231. Sakiyama T and Ohwada K (1997) Isolation and growth characteristics of deep-sea barophilic bacteria from the Japan Trench. Fisheries Science (Tokyo) 63: 228–232. Yayanos AA (1980) Measurement and instrument needs identified in a case history of deep-sea amphipod research. In: Diemer FD, Vernberg FJ, and Mirkes DZ (eds.) Advanced Concepts in Ocean Measurements for Marine Biology, pp. 307–318. Columbia, South Carolina: University of South Carolina Press. Yayanos AA (1986) Evolutional and ecological implications of the properties of deep-sea barophilic bacteria. Proceedings of the National Academy of Sciences USA 83: 9542–9546. Yayanos AA (1989) Physiological and biochemical adaptations to low temperatures, high pressures and radiation in the deep sea. In: Hattori T et al. (eds.) Proceedings of the 5th International Symposium on Microbial Ecology, pp. 38–42. Tokyo: Japan Scientific Societies Press. Yayanos AA (1995) Microbiology to 10,500 meters in the deep sea. Annual Reviews of Microbiology 49: 777–805. Yayanos AA (1998) Empirical and theoretical aspects of life at high pressures in the deep sea. In: Horikoshi K and Grant WD (eds.) Extremeophiles, pp. 47–92. New York: John Wiley & Sons. Yayanos AA (2001) Deep-sea piezophilic bacteria. Methods in Microbiology 30: 615–637. Yayanos AA and DeLong EF (1987) Deep-sea bacterial fitness to environmental temperatures and pressures. In: Jannasch HW, Marquis RE, and Zimmerman AM (eds.) Current Perspectives in High Pressure Biology, pp. 17–32. New York: Academic Press. Yayanos AA and Dietz AS (1982) Thermal inactivation of a deep-sea barophilic bacterium, isolate CNPT-3. Applied and Environmental Microbiology 43: 1481–1489. Yayanos AA, Dietz AS, and Van Boxtel R (1979) Isolation of a deep-sea barophilic bacterium and some of its growth characteristics. Science 205: 808–810. ZoBell CE (1952) Bacterial life at the bottom of the Philippine Trench. Science 115: 507–508.

Low-Nutrient Environments J S Poindexter, Barnard College, Columbia University, NY, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Oligotrophic Aquatic Habitats Extreme Habitats Oligotrophic Cultivation

Glossary allochthonous Nutrients that arrive in a habitat, having been produced or released from insolubility away from the habitat. autochthonous Nutrients that are produced or released from insolubility within the habitat. copiotrophic Nutritional class of microbes whose competitiveness in their natural habitat depends on their ability to exploit an abundance of nutrients. direct count/total count Microscopical technique for enumeration of bacteria-size particles. dissolved organic carbon (DOC) Organic material in solution, which may be labile (readily utilized by microbes) or refractory (not utilized or only slowly utilized by microbes). eutrophic Condition of the habitat characterized by abundant organic productivity and high density of microbial populations.

Abbreviations AODC Bchl a chl a CFU DAPI

acridine orange direct count bacteriochlorophyll a chlorophyll a colony-forming unit 49,6-diamidino-2-phenylindole

The Niche of Oligotrophic Bacteria in Low-Nutrient-Flux Aquatic Habitats Further Reading

heterotrophic Metabolism that utilizes organic carbon as its source of carbon for biosynthesis. limiting nutrient Nutrient required for metabolism present in the habitat in amounts that limit the yield of biomass produced. oligotrophic Of the habitat: condition of the habitat characterized by low organic productivity and low density of microbial populations. Of microbes: nutritional class of microbes whose competitiveness in their natural habitat depends on their ability to exploit a scarcity of nutrients. particulate organic matter (POM) Organic matter contained in microscopically visible particles, some animate and some inanimate. viable count Microscopical or cultivational enumeration of microbes able to reproduce in microculture or accumulate a discernible mass by reproduction in a laboratory culture.

DO DOC DOM NUCC POM

dissolved O2 dissolved organic carbon dissolved organic matter nucleoid-containing cell particulate organic matter

Defining Statement

Oligotrophic Aquatic Habitats

Oligotrophic aquatic habitats are defined as low-nutrientflux environments in which both primary production and microbial densities are distinctly lower than in eutrophic, heavily nourished environments. Progress in design of conditions for laboratory cultivation of oligotrophic bacteria is summarized, and characteristics and identities of some frequently cultivated oligotrophic bacteria are described.

The prokaryotic microbes, currently classified as the two domains Bacteria and Archaea, are the most ubiquitous as well as the oldest forms of life on earth. At present, it is safe to generalize regarding any locale that if it is wet, it is a microbial habitat; that the microbes present will be predominantly or exclusively prokaryotic; and that if the habitat is aerated, those prokaryotic microbes will be predominantly bacteria. Their presence may not be

240

Environmental Microbiology and Ecology | Low-Nutrient Environments

apparent to the casual observer – even to trained ecologists – because the prokaryotic populations of microbial habitats are often so sparse that detection of their presence requires laboratory-assisted techniques. Bacterial populations can be restricted to inconspicuous densities by one or the other of two major causes: either the environmental supply of nutrients is too low to support a dense community, or ambient abiotic conditions are beyond the extremes tolerated by most forms of life. Prokaryotic microbes that thrive in inhospitable, extreme habitats – especially archaea such as the extreme halophiles, thermophiles, and acidophiles – are known to possess unique properties with respect to their membranes, their metabolism, and even their genomes. In contrast, prokaryotic microbes known to occur particularly in communities that are sparse due to nutrient scarcity – oligotrophic microbes – appear to be similar to many other forms of life in their fundamental structural, metabolic, and genetic properties. Because bacteria constitute the great majority of oligotrophic prokaryotic microbes that have been studied, practically all references in this article will be to bacteria, on the understanding that the bacteriological work may or may not eventually prove a helpful guide to the elucidation of the physiology and ecology of oligotrophic archaea.

Parameters of Oligotrophic Waters With respect to environmental nutrient supply, microbial ecology borrows terms that originated for the classification of freshwater lakes and were promptly extended to marine environments. Extending these terms to soils requires some redefining that will not be done here because this article will emphasize aquatic (freshwater and marine) low nutrient supply environments. The widely used terms are oligotrophic and eutrophic for the scarce and abundant, respectively, ends of the spectrum of nutrient supply to watery habitats; ‘mesotrophic’ is used for the continuum of intermediate conditions. Generalized ranges for environmental parameters that define these classes are shown in Table 1. (The terms in Table 1 refer to nutritional conditions in the environment. See the glossary and Table 2 for uses of the terms ‘‘oligotrophic’’ and ‘‘copiotrophic’’ to refer to categories of microbes distinguished by competitiveness in lowand high-nutrient-flux environments, respectively.) The numbers given are averages of values that vary from one site to another within a body of water and may also vary with season of the year. The most important aspect of lake nutrition is nutrient flux (reflected in C flux, turnover time, and productivity in Table 1), not standing concentrations of nutrients. The concept of a low-nutrient environment should be

241

dynamic: it is the flux, the rate of delivery of utilizable nutrients, that defines a low-nutrient environment, and oligotrophic conditions should be described as low nutrient flux. A definition based on standing concentrations would not provide a useful or dependable distinction between oligotrophic and eutrophic waters because, except within the bodies of living organisms where constant concentrations and fluxes of soluble nutrients are maintained, microbial habitats generally contain very low concentrations of soluble organic materials. Any lively microbial population will consume available nutrients until the concentration of at least one nutrient (the limiting nutrient) is reduced to a level so low that the organisms cannot detect and transport it for use in metabolism. That nutrient, which may be organic or inorganic, will often also be undetectable by chemical analysis unless large volumes of water are concentrated or extracted for assay. Nevertheless, it is also evident in Table 1 that two standing concentrations can serve as convenient indicators of oligotrophic/low nutrient supply conditions: the relative abundance of dissolved O2 (DO) and the density of demonstrably living bacteria. These two parameters are inversely related to each other because wherever bacteria occur in abundance with adequate organic nutrients to support their respiratory metabolism, oxygen will be consumed to well below its saturation in water at the ambient temperature. With few exceptions, subsaturation levels of DO in natural waters – fresh and marine – are the result of microbial respiration, almost always using organic material as electron donors. Oligotrophy, as a nutritional state, is predictably an aerobic condition. To distinguish oligotrophic and eutrophic habitats from each other, it is also possible to perform simple organoleptic tests. Oligotrophic waters, whether fresh or marine, do not stink (in human estimation); are optically clear, not cloudy (see Secchi depth, Table 1); and have rocky or sandy, not muddy or mucky, bottoms. Oligotrophic waters are generally at least 15 m deep; on a sunny day appear blue, not green or brown; and harbor a diversity of deep-water animals, including fish, but few if any rooted plants. Microscopical examination of a sample of such water at 100 or higher will reveal very little evidence of life. In contrast, relatively shallow, cloudy, greenish, plant-inhabited, muddy-bottomed waters low in fish diversity are predictably sites supplied with an abundance of nutrients; they are recognizably eutrophic. Microscopical examination of samples of such water at 100 or higher will provide hours of informative observation of the forms and activities of diverse organisms, from bacteria to microfauna and the propagules of plants, animals, algae, and fungi; this (particularly the protozoa) is the entertaining pond water of biology classes.

Table 1 Characteristics of aquatic habitats

Habitat Freshwater lakes Oligotrophic Mesotrophic (LW 1971–79b) Eutrophic (LW 1963–66b) Eutrophic Marine regions Estuarine Coastal Oceanic  Surface  Deep a

C flux (mg C l1 day1)

Turnover time (Corg)

Productivity (mg C m2 y1)

Chl a (mg l1)

DOC,a (mg C l1)

Phosphate (mg P l1)

Total N (mg N l1)

Bacteria (CFU ml1)

O2, deep saturation

Turbidity (Secchi, m)

10–100

150–1200 h

6

65–950

>20

>500

106–109

3–40 2–40

3–900 6–450

1–20 3–25 0.5–100

14–200

102–106

0.2–2000 300–600

DOC is dissolved organic carbon. LW is Lake Washington, Seattle, WA, USA; 1963–66 was a period of eutrophication, and 1971–79 were years following recovery of mesotrophic conditions.

b

Bottom

0–10%

> mineralization cycle. Over the season from spring thaw to autumnal decrease in photosynthesis, the lake experiences its major annual surge of organic nutrients, and when primary production is high, the oligotrophic conditions of winter may alternate with meso- or eutrophic conditions in the summer. A second autumnal surge may occur when the biomass accumulated during the spring and summer begins to decline in activity, making it susceptible to disintegration and consumption by heterotrophic bacteria. As surface water (ice) warms, reversing the relative density of the liquid layers, a second period of vertical mixing occurs in dimictic lakes. Changes that occur according to season, such as fluctuations in temperature, illumination, and sequestering of inorganic nutrients, also accompany deep-ocean currents and disturbances such as storms or floods or exceptional tides, all of which translocate both organic and inorganic nutrients – and, of course, microbes. Consequently, these changes influence local primary production and account for the regular seasonal fluctuations in the chemical and biotic parameters of natural waters. Transport routes and vehicles

Most allochthonous nutrients are delivered to aquatic habitats in moving water (springs, streams, rivers, tides, sewage effluents, precipitation as rain or snow), although some arrive as dry precipitation (atmospheric dust) or solid masses (falling trees, animal carcasses, discarded shells, sinking ships, plastic trash, etc.). The quantity and identity of the nutrients borne by a river into a lake or estuary depend on natural productivity and population densities in the river itself, on biotic communities along the shore, and on the mineral composition of the local soil and the river bottom. The weather and the contour of the region determine the volume and velocity of the running water, which together determine the rate of delivery of the useable nutrients carried by the water. Dissolved organic carbon (DOC) is carried by rivers predominantly as small molecules: monomeric sugars and amino acids; small, soluble polymers of these compounds; low-molecular-weight by-products of photosynthesis such as glycolate; and smaller amounts of other nonnitrogenous carboxylic acids. In contrast to compounds such as alcohols, esters, long-chain fatty acids, and hydrocarbons, most of these organic nutrients are not volatile and are not borne by rain. They are readily consumed by heterotrophic microbes during their transport along the river’s

Environmental Microbiology and Ecology | Low-Nutrient Environments

course so that rivers do not carry substantial amounts of readily metabolizable (labile) organic carbon from lake to lake or from land to the ocean. Rivers do deliver significant amounts of some readily assimilated inorganic nutrients to lakes and estuaries. River-water addition to the seas is a major source of inorganic forms of silicates, sulfate, chloride, sodium, Mg, K, Ca, and bicarbonate. Rain naturally contributes sulfur in various oxidation states, and pollution increases delivery – in rain and/or in rivers – of phosphates, nitrate, ammonium, sulfate, chloride, sodium, and CO2. Respiration exhales CO2 into the atmosphere, where the amount of CO2 was stable for long periods in the past, prior to the Industrial Revolution. Even though atmospheric CO2 has probably never been the limiting nutrient for primary production, its recent increase (surge) – accelerated by the burning of fossil fuels – is having a significant impact on the ocean. Seawater is mildly alkaline, and its absorption of CO2 is causing measurable acidification of the open ocean and potential solubilization of the CaCO3 foundation of the shells of marine animals such as corals and of the skeletons of coralline algae. A river may be as deep as a lake at some points along its course, but mixing – both vertically and horizontally – will be greater and less seasonal than in a lake; lotic (running) waters are therefore less likely to have anoxic regions. However, heavy precipitation that causes a river to overflow its banks, reduction of the river’s flow due to withdrawal of large volumes of water for domestic use, or treefalls or dam construction that impedes a river’s flow may cause organic matter accumulation and O2 depletion. Unimpeded rivers are capable of self-cleansing through microbial activities and oxygenation so that assimilable inorganic nutrients added at one point along the river’s course are typically consumed within the river and built into biomass. Delivery of nitrate and phosphate to the ocean (or to lakes on the way to the ocean) is low because the salts are assimilated by microorganisms during the river’s flow, and most of the N and P that arrives at the seashore is in the form of suspended, relatively refractory POM. When the rivers are polluted, waters downstream from urban areas where human population density is high carry low (4000 m Benthic Realm (seabed) Supralittoral Littoral Sublittoral Bathyal Abyssal Hadal

Above high-tide mark Between the tides Between low tide and edge of continental shelves (200 m) Seaward of continental shelves to 4000 m 4000–6000 m >6000 m, including trenches

depending upon whether the habitat of interest is the overlying water column (fluid) or the seabed (solid), respectively (Figure 1; Table 1). Within each of these main categories, a number of additional subdivisions can be made depending, for example, on increasing water depth from the high tide mark. For the pelagic habitat, major subdivisions include neritic for waters overlying the continental shelves (200 m deep) and oceanic, for the vast open sea. The oceanic realm can also be further subdivided (Table 1). Benthic habitats include littoral (intertidal), sublittoral (from the low tide boundary to edge of continental shelf), bathyal, abyssal, and hadal. Other classification schemes use the topographic boundaries: continental shelf, continental slope, abyssal plain, and deep sea trenches (see Figure 1). Although these and other terms are routinely used, the depth ranges are not always identical, so they should be considered as guidelines rather than rules. Along the seawater depth gradient, whether in benthic or pelagic habitats, some physical and chemical characteristics systematically change (e.g., decreasing temperature and increasing pressure). In this regard, it is important to emphasize that the most common marine habitat is cold (50 bars). Consequently, many marine microbes are cold- and pressure-adapted, even obligately so; indeed, some abyssopelagic bacteria require high pressure (>400 bars) to grow. Other classification schemes based on the availability of sunlight (euphotic (light present) or aphotic (light absent)) or the relative rates of organic matter production (eutrophic (high), mesotrophic (medium), or oligotrophic (low)) have also been used.

Sharp horizontal gradients can also be observed in the surface of the ocean. For example, since the 1960s, oceanographers have used satellite-based remote sensing approaches to map various features of the global ocean, including sea surface temperature, winds, altimetry, and the distributions of photosynthetic microbes as inferred from observations of spectral radiance. The first satellitebased ocean color measurements were obtained using the Coastal Zone Color Scanner (CZCS) aboard the Nimbus7 satellite that was launched in October 1978; it provided useful data for nearly a decade. The CZCS sensor was eventually replaced with the Sea-viewing Wide Field-ofview Sensor (SeaWiFS), launched in September 1997, and still operational, followed by Moderate Resolution Imaging Spectroradiometer (MODIS) aboard the Terra and Aqua satellites (1999 and 2002 to present, respectively). And, although these instruments cannot provide information regarding spatial variability below 1 km resolution, they have provided unprecedented observations on the temporal variability or surface ocean macrohabitats (depth-integrated to one optical depth, 25 m in clear open ocean waters), as well as the mesoscale (10–100 s of km) and large-scale distributions of chlorophyll (chl). Daily synoptic global images can be pieced together to track the dynamics (days to decades) of photosynthetic microbial assemblages in the global ocean and their correlations with other environmental variables in ways that are not possible by any other means (Figure 2). Furthermore, systematic analyses of these ocean color datasets can be used to define spatial habitat structure in oceanic ecosystems, and the partitioning of the global ocean into a suite of ecological provinces or functional habitat units, leading to the novel subdiscipline of marine ecological geography. Unfortunately, there are no satellite-based sensors that can track non-chl-containing marine microbes, although several novel remote detection systems are under development for in situ application based on molecular/genetic probes and imaging-in-flow cytometry. There are distinct water types throughout the ocean that can be easily identified by measuring their temperature and salinity characteristics, which together determine their densities (T-S diagram; Figure 3). Using T-S diagrams as a fingerprinting tool, water types can be traced throughout the world ocean to specific regions of formation. A water mass results from the mixing of two or more water types, and is represented by a line between distinct water types on the T-S diagram (Figure 3). Water masses can also be tracked for great distances throughout the world ocean, and their microbial assemblages can also be sampled and characterized. The global circulation rate, as deduced by nonconservative chemical properties and radioisotopic tracers, has a time scale of hundreds to thousands of years. Consequently, ‘young’ and ‘old’ water masses can be identified based on the time that the water was last in contact with the atmosphere.

Environmental Microbiology and Ecology | Marine Habitats

263

2007.5 27

2007

26 2006 25

Year

2005.5 2005

24

2004.5 23

2004 2003.5

Sea surface temperature, °C

2006.5

22

2003 21 162

160

158

156

154 152 Longitude W

150

148

146

144

142

2007.5

0.12

2007

0.11

2006.5

0.1

2006

0.09

Year

2005.5

0.08

2005 0.07 2004.5

mg chl a m–3

164

0.06 2004 0.05 2003.5 0.04

2003

0.03 164

162

160

158

156

154 152 Longitude W

150

148

146

144

142

Figure 2 Satellite-derived temporal and longitudinal variability in sea surface temperature (Top) and chlorophyll (Bottom) for the region surrounding Station ALOHA (22.75 N, 158 W). The data, available from the NASA Ocean Color Time-Series Online Visualization and Analysis website (http://reason.gsfc.nasa.gov/), have been obtained through NASA’s Moderate Resolution Imaging Spectroradiomater (MODIS) sensor on board Aqua between July 2002 and June 2007 and correspond to the latitudinal average between 22.5 N and 23.5 N for the longitude band 142–164 W. The black line marks longitude 158 W where Station ALOHA is located.

Due to unique seafloor topography and interactions with the atmosphere, certain regions ‘short-circuit’ the mean circulation by serving as conduits for a more rapid ventilation of the deep ocean (bringing it into contact with the atmosphere) and the concomitant delivery of nutrient-rich deep water to the surface of the sea. These so-called upwelling regions occupy only 1% of the

surface ocean, but they are important areas of solar energy capture through enhanced photosynthesis and the selection of relatively large algae and short food chains; thereby they support some of the great fisheries of the world (Figure 4). In contrast, the more common condition (90% of the global ocean) is the oligotrophic habitat (low nutrient and low rates of organic

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Salinity (‰) Figure 3 Potential temperature versus salinity (T-S) plots are used to identify, trace, and compare distinct water types and water masses in the marine environment. (Top) T-S diagram for the Hawaii Ocean Time-series (HOT) Station ALOHA for the period 1988–2006. The inset shows the depth profiles of potential temperature and salinity. The ALOHA T-S fingerprint shows the presence of numerous water masses at specific depths. The contours show lines of constant density, or isopycnal surfaces, in density anomaly notation ((density in g cm3)1.000)1000). In addition to temperature and salinity (density) variations, these distinctive water masses also have distinctive chemical properties and may contain unique assemblages of microorganisms. The large variability of T and S at the top of the graph is a result of seasonal and interannual changes in near-surface water properties. (Bottom) Comparison of T-S fingerprints for a variety of oceanic time-series stations including: Ocean Station Papa (OSP; 50 N, 145 W), SouthEast Asia Time-Series (SEATS; 18 N, 116 E), Hawaii Ocean Time-series (HOT; 22.75 N, 158 W), and Bermuda Atlantic Time-series Study (BATS; 32 N, 64 W). The three North Pacific stations (OSP, SEATS, HOT) have a common deep water mass.

matter production) that selects for very small primary producers, long and complex microbial-based food webs, and relatively inefficient transfer of carbon and energy to higher trophic levels like fish. These

fundamental differences in physics result in marine habitats with diverse structures and dynamics that host dramatically different microbial assemblages, as discussed later in this article.

Environmental Microbiology and Ecology | Marine Habitats

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Figure 4 Importance of nutrient flux on the size distribution and efficiency of biomass and energy flow in marine habitats. The schematic on the left depicts a habitat where ‘new’ nutrient (as NO 3 ) flux is high (e.g., an upwelling region). This leads to a selection for large phytoplankton cells (PL) that are efficiently consumed by higher trophic levels (HTLs) including large zooplankton and fish. This results in a short and efficient food chain. In contrast to the upwelling regions, most open ocean habitats have low new nutrient (NO 3 ) fluxes and survive by local remineralization of required nutrients (‘recycled’). These conditions select for small phytoplankton cells (PS) that serve as the food source for long and complex microbial-based food webs (also called microbial loops; ML) that recycle mass and dissipate most of the solar energy that was initially captured. The great marine fisheries of the world are generally found in association with upwelling regions.

Marine Microbial Inhabitants and Their Growth Requirements The marine environment supports the growth of a diverse assemblage of microbes from all three domains of life: Bacteria, Archaea, and Eucarya. The term ‘microorganism’ is a catchall term to describe unicellular and multicellular organisms that are smaller than 100 150 mm. This grouping includes organisms with broadly distinct evolutionary histories, physiological capabilities, and ecological niches. The only common, shared features are their size and a high surface-to-biovolume ratio. A consequence of being small is a high rate of metabolism and shorter generation times than most larger organisms. Microorganisms, particularly bacteria and archaea, are found throughout the world ocean including marine sedimentary and subseabed habitats. There is probably no marine habitat that is devoid of microorganisms, with the possible exception of high-temperature (>100  C) zones. In addition to a physically favorable environment, the metabolism and proliferation of microorganisms also require a renewable supply of energy, electrons for energy generation,

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a A ‘mixotroph’ is an organism that uses more than one source of energy, electrons, or carbon.

carbon (and related bioelements including nitrogen, phosphorus, sulfur), and occasionally organic growth factors such as vitamins. Depending upon how these requirements are met, all living organisms can be classified into one of several metabolic categories (Table 2). For example, photolithoautotrophic microbes use light as an energy source, water as an electron source, and inorganic carbon, mineral nutrients, and trace metals to produce organic matter. At the other end of the metabolic spectrum, chemoorganoheterotrophic microbes use preformed organic matter for energy generation and as a source of electrons and carbon for cell growth. In a laboratory setting, only obligate photolithoautotrophs are self-sufficient; all other autotrophs and all heterotrophs rely upon the metabolic activities of other microorganisms. However in nature, even obligate photolithoautotrophs must tie their growth and survival to other, mostly deepsea, microbes that are vital in sustaining nutrient availability over evolutionary time scales. Most marine microorganisms probably use a variety of metabolic strategies, perhaps simultaneously, to survive in nature. Because needed nutrients in the ocean’s surface are often found in dissolved organic molecules, it seems highly improbable that sunlit marine habitats would select for obligate photolithoautotrophy as opposed to, for instance, mixotrophic growth. Across the full metabolic spectrum of possible modes of growth, some microbes are more self-sufficient than others. For example, while most microbes require a supply of chemically ‘fixed’ nitrogen, either in reduced (ammonia or dissolved organic nitrogen (DON)) or in oxidized (nitrate or nitrite) form to survive, a special group of N2-fixing microbes (diazotrophs) can use the nearly unlimited supply of dissolved N2 as their sole source of cell N. Additionally, some microbes can manufacture all their required building blocks (e.g., amino acids and nucleic acid bases) and growth cofactors (e.g., vitamins) from simple inorganic precursors, whereas others

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require that they be supplied from the environment; ‘auxotrophic’ microorganisms are, therefore, ultimately dependent upon the metabolic and biosynthetic activities of other microbes. These ‘incomplete’ microbes, probably the bulk of the total microbial assemblage in seawater, cannot grow unless the obligate growth factors are present in and resupplied to the local habitat. In this regard, most marine habitats provide the laboratory equivalent of a complex or complete medium containing low-molecularweight compounds (e.g., amino acids, simple sugars, nucleic acid bases, and vitamins), in addition to the mineral nutrients and trace metals. The active salvage and utilization of these biosynthetic precursors, in lieu of de novo synthesis, conserves energy, increases growth efficiency, and enhances survival. Over evolutionary time, some unused biosynthetic pathways in particular organisms appear to have been lost from the genome, perhaps, as a competitive strategy for survival in a mostly energy-limited environment. This process has been termed genome streamlining. Finally, growth and reproduction are often viewed as the most successful stages of existence for any microorganism. However, in many of the low nutrient concentration and low energy flux habitats that dominate the global seascape, the ability to survive for extended periods under conditions of starvation may also be of great selective advantage and ultimately may affect the stability and resilience of microbial ecosystems. The starvation-survival response in marine bacteria leads to fragmentation (i.e., cell division in the absence of net growth) and, ultimately, to the formation of multiple dwarf or miniaturized cells. Other physiological changes, including reduction in endogenous metabolism, decreases in intracellular adenosine triphosphate (ATP) concentrations, and enhanced rates of adhesion are also common consequences. These starved cells can respond rapidly to the addition of organic nutrients. This ‘feast and famine’ cycle has important implications for how we design in situ metabolic detection systems and model microbial growth in marine habitats.

Distribution, Abundance, and Biogeography of Marine Microbes The distribution and abundance of microbes is highly variable, but somewhat predictable, across globally distributed marine habitats. For example, phototrophic microbes are restricted to sunlit regions (0–200 m in the open sea) whereas chemotrophic microbes are found throughout the oceanic realm. However, because the abundance and productivity of marine microbes depend on the availability of nutrients and energy, there is often a decreasing gradient in total microbial biomass from the continents to the open ocean, and a decreasing gradient in total microbial biomass from the sunlit surface waters to the abyss. For the pelagic

zone, total microbial biomass in near-surface (0–100 m) waters ranges from 30 to 100 mg carbon m3 in neritic waters to 6–20 mg carbon m3 in oceanic waters. For open ocean habitats, this biomass decreases by approximately three orders of magnitude from euphotic zone to abyssal habitats, with values 99% 16S RNA identity) sampled from a temperate coastal marine habitat had at least 1000 distinct coexisting genotypes, and bacterial samples collected from the aphotic zone of the North Atlantic Ocean revealed an extremely diverse ‘rare biosphere’ consisting of thousands of low-abundance populations. The ecological implications of these independent reports of taxonomic diversity are profound; new ecological theory may even be required to build a conceptual framework for our knowledge of marine habitats and their microbial inhabitants.

Sunlight, Nutrients, Turbulence, and the Biological Pump Of all the environmental variables that collectively define the marine habitat, we single out three – namely, sunlight, nutrients, and turbulence – as perhaps the most critical for the survival of sea microbes. Together, these properties

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control the magnitude and efficiency of the ‘biological pump’, a complex series of trophic processes that result in a spatial separation between energy (sunlight) and mass (essential nutrients) throughout the marine environment. In the sunlit regions of most (but not all) marine habitats, nutrients are efficiently assimilated into organic matter, a portion of which is displaced downward in the water column, mostly through gravitational settling. As particles sink through the stratified water column, a portion of the organic matter is oxidized and the essential nutrients are recycled back into the surrounding water masses. Depending upon the depth of remineralization and replenishment to the surface waters by physical processes, these essential nutrients can be sequestered for relatively long periods (>100 yrs). The vertical nutrient profile, for example of nitrate, shows a relative depletion near the surface and enrichment at depth as a result of the biological pump (Figures 5(a) and 5(b)); regional variations in the depth profiles reflect the combination of changes in the strength and efficiency of the biological pump and the patterns of global ocean circulation (Figures 5(a) and 6). The highest nutrient concentrations in deep water can be found in the abyss of the North Pacific, the oldest water mass on Earth. The regeneration of inorganic nutrients requires the oxidation of reduced organic matter, so the concentrations of dissolved oxygen decrease with depth and with age of the water mass as a result of the cumulative effect of microbial metabolism (Figures 5(b) and 6). Turbulence in marine habitats derives from a variety of processes including wind stress on the ocean’s surface, ocean circulation, breaking internal waves, and other large-scale motions that can create instabilities, including eddies, in the mean density structure. Turbulence, or eddy diffusion, differs fundamentally from molecular diffusion in that all properties (e.g., heat, salt, nutrients, and dissolved gases) have the same eddy diffusion coefficient; a typical value for horizontal eddy diffusivity in the ocean is 500 m2 s1, a value that is 109 times greater than molecular diffusion. Vertical eddy diffusivity is much lower (0.6–1  104 m2 s1) suggesting that the upward flux of nutrients into the euphotic zone is a slower process than movement horizontally in the open ocean. Most near-surface dwelling microbes, particularly phototrophs that are also dependent upon solar energy and are effectively ‘trapped’ in the euphotic zone habitat, depend on turbulence to deliver deep water nutrients to the sunlit habitat. In addition to the eddy diffusion of nutrients from the mesopelagic zone, wind stress at the surface and other forces can mix the surface ocean from above. If the nearsurface density stratification is weak or if the mixing forces are strong, or both, then a large portion of the euphotic zone can be homogenized; in selected latitude regions the surface mixing layer can extend to 500 m or more, well below the maximum depth of the euphotic

zone. These well-mixed environments usually have sufficient nutrients but insufficient light to sustain photosynthesis because the phototrophs are also mixed to great depths, as in some polar habitats during winter months. Following these seasonal deep-mixing events, the ocean begins to stratify due to the absorption of solar radiation in excess of evaporative heat loss. As the wind forcing from winter storms subsides and the intensity of solar radiation increases, a density gradient develops in the upper water column. Phototrophic microorganisms in the euphotic zone gain a favorable niche with respect to both light energy and nutrient concentrations. Depending upon the presence or absence of grazers, this condition results in an increase in phototrophic microbial biomass, a condition referred to as the spring bloom. A comprehensive formulation of the ‘vernal blooming of phytoplankton’ presented by H. Sverdrup remains a valid representation of this important marine microbial phenomenon. However, in many portions of the world ocean, particularly in tropical ocean gyres, local forcing due to wind stress is too weak to break down the density stratification, so the nutrient delivery from below the euphotic zone through mixing is not possible. In these oligotrophic regions, the habitat is chronically nutrient-stressed and oftentimes nutrient-limited. Although surface mixed layers can be observed, they rarely penetrate deeper than 100 m. Even within the so-called mixed layer, gradients in chl, nutrients, dissolved gases, and microorganisms can be detected, suggesting that these regions are not always actively mixing. This subtle distinction between a mixing layer, where there is an active vertical transport of physical, chemical, and biological properties, and a mixed layer, which is defined operationally as a layer with weak or no density stratification, has important implications for microbial growth and survival, particularly for phototrophic microorganisms. Consequently, without additional information on mixing dynamics (e.g., a profile of turbulent kinetic energy), the commonly used term mixed layer can be misleading with regard to habitat conditions for microbial growth. The time required to change from a mixing layer to a mixed layer to a density-stratified surface habitat and back again will depend on the habitat of interest. One approach for distinguishing between a mixing layer and a mixed layer is to measure the near-surface concentrations and temporal dynamics of a short-lived photochemically produced tracer, for example, hydrogen peroxide (H2O2). The concentration versus depth profile of H2O2 in a mixing layer with a short mixing time scale (1 h) would be constant because the concentration of photochemically active DOM and average solar energy flux would also be relatively constant. On the other hand, the H2O2 concentration profile in a nonmixing (or slowly mixing, turnover >1 day) ‘mixed layer’ would

Environmental Microbiology and Ecology | Marine Habitats

approximate the shape to the flux of solar energy decreasing exponentially with depth nearly identical to a density-stratified habitat, assuming that the concentration of photosensitive DOM is in excess. It is also possible to use other photochemical reactions to obtain information on vertical mixing rates.

Time Variability of Marine Habitats and Climate Change Marine habitats vary in both time and space over more than nine orders of magnitude of scale in each dimension. Compared with terrestrial habitats, most marine ecosystems are out of ‘direct sight’, and, therefore, sparsely observed and grossly undersampled. The discovery and subsequent documentation of the oases of life surrounding hydrothermal vents in the deep sea in 1977 revealed how little we knew about benthic life at that time. Furthermore, because marine life is predominantly microscopic in nature, the temporal and spatial scales affecting microbial processes may be far removed from the scales that our senses are able to perceive. And, due to (a)

this physical and sensory remoteness of marine microbial habitats, even today unexpected discoveries about the ocean frontier continue to be made, many of these involving marine microbes. We have selected the North Pacific Subtropical Gyre (NPSG) for a more detailed presentation of relationships between and among habitat structure, microbial community function and climate. Our choice of the NPSG as an exemplar habitat is based on the existence of the Hawaii Ocean Time-series (HOT) study, a research program that seeks a fundamental understanding of the NPSG habitat. The emergent comprehensive physical, chemical, and biological data sets derived from the HOT benchmark Station ALOHA (A Long-term Oligotrophic Habitat Assessment) is one of the few spanning temporal scales that range from a few hours to almost two decades. More generally, we submit that the sampling and observational components of the HOT program at the deep water Station ALOHA are applicable to other locations that may be representative of key marine habitats. The NPSG is one of the largest and oldest habitats on our planet; its present boundaries have persisted since the

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Figure 5 (a) Nitrate versus depth profiles for the North Atlantic (Bermuda Atlantic Time-series Study; BATS) and the North Pacific (Hawaii Ocean Time-series; HOT) showing significant interocean differences including a steeper nitracline (i.e., a larger change in NO3 concentration per meter in the upper mesopelagic zone region) and higher deep water (>4000 m) NO3 concentrations for HOT. These differences in NO3 inventories and gradients, part of a systematic global pattern (see Figure 6), have significant implications for NO3 fluxes into the euphotic zone. Data available at the HOT and BATS program websites (http://hahana.soest.hawaii.edu; http:// www.bios.edu/). (b) Relationships between the vertical distributions of nitrate (NO3) and dissolved oxygen (O2) at Station ALOHA in the North Pacific Subtropical Gyre (NPSG). (Left) Graph of NO3 (mmol l1) versus depth (m) showing the characteristic ‘nutrient-like’ distribution of NO3 with regions of net NO3 uptake and DON cycling and particulate nitrogen (PN) export near the surface, and net NO3 remineralization at greater depths. The insert shows these main N-cycle processes, which are most intense in the upper 1000 m of the water column. (Right, top) NO3 and O2 concentration versus depth profiles of the 300–700 m region of the water column at Station ALOHA showing the effects of net remineralization of organic matter. (Right, bottom) A model 2 linear regression analysis of NO3 versus O2 suggests an average consumption of 80 mmol l 1 O2 for each 1 mmol of NO3 that is regenerated from particulate and DOM. Data available at the HOT program website (http://hahana.soest.hawaii.edu).

Pliocene nearly 107 years before present. The vertical water column at Station ALOHA can be partitioned into three major microbial habitats: euphotic zone, mesopelagic (twilight) zone, and aphotic zone (Table 3 and Figure 7). The main determinant in this classification scheme is the presence or absence of light. The euphotic zone is the region where most of the solar energy captured by phototrophic marine microbes is sufficient to support photosynthetic activity. In the twilight zone (200–1500 m), light is present at very low photon fluxes, below which photosynthesis can occur, but at sufficiently high levels to affect the distributions of mesozooplankton and nekton and, perhaps, microbes as well. At depths greater than 1500 m, light levels are less than 103 quanta

cm2 s1; the aphotic zone is, for all intents and purposes, dark. Each of these major habitats is characterized by specific physical and chemical gradients, with distinct temporal scales of variability, providing unique challenges to the microorganisms that live there, and resulting in a vertical segregation of taxonomic structure and the ecological function of the resident microbial assemblages. A recent report of microbial community genomics at Station ALOHA, from the ocean’s surface to the abyss, has revealed significant changes in metabolic potential, attachment and motility, gene mobility, and host–viral interactions. The NPSG is characterized by warm (>24  C) surface waters with relatively high light and relatively low

Environmental Microbiology and Ecology | Marine Habitats

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concentrations of inorganic nutrients and low microbial biomass (Figure 8). The euphotic zone has been described as a ‘two-layer’ habitat with an uppermost light-saturated, nutrient-limited layer (0–100 m) which supports high rates of primary productivity and respiration, and a lower (>100 m) light-limited, nutrient-sufficient layer. A region of elevated chl a, termed the Deep Chlorophyll Maximum Layer (DCML), defines the boundary between the two

layers (Figure 9). The DCML in the NPSG results from photoadaptation (increase in chl a per cell) rather than enhanced phototrophic biomass; this can also be seen in the near-surface ‘enrichment’ of chl in winter when light fluxes are at their seasonal minimum (Figure 9). Previously considered to be the oceanic analogue of a terrestrial desert, the NPSG is now recognized as a region of moderate primary productivity

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resulting from climate controls on habitat structure and function. At Station ALOHA the light-supported inorganic carbon assimilation extends to 175 m, a depth that is equivalent to the 0.05% surface light level (20 mmol quanta m2 day1). Most of the light-driven inorganic carbon assimilation (>50%) occurs in the upper 0–50 m of the water column (Figure 8), a region of excess light energy (>6 mol quanta m2 day1). In addition, chemoorganoheterotrophic microbial activities are also greatest in the upper 0–50 m. However, unlike photolithoautotrophic production (where light is required as the energy source and inorganic carbon is assimilated for growth), the metabolism of chemoorganoheterotrophs is not dependent on light energy so it continues, albeit at a reduced rate, well into the twilight zone and beyond. Recently, it has been observed that ‘heterotrophic production’ at Station ALOHA is enhanced by sunlight, suggesting the presence of microorganisms using light and both inorganic and organic substrate (photolithoheterotrophic) or light and organic substrates (photoorganoheterotrophic) to support their metabolism, or both. Several possible pathways for solar energy capture and carbon flux potentially exist in the euphotic zone at Station ALOHA, and we are just beginning to establish a

Table 3 Conditions for microbial existence in the three major habitats at Station ALOHA in the North Pacific Subtropical Gyre Depth range (m)

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high solar energy high DOM  low inorganic nutrients, trace elements, and organic growth factors  low solar energy  decrease in reduced organic matter with depth  increase in organic nutrients and trace elements with depth  no solar energy  low DOM  high inorganic nutrients and trace elements 

(150–200 g carbon m2 year1), despite chronic nutrient limitation. Furthermore, based on data from the HOT program it appears that the rates of primary production have increased by nearly 50% between the period 1989 and 2006 due in part to enhanced nutrient delivery

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Environmental Microbiology and Ecology | Marine Habitats

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comprehensive understanding of these processes, their roles and controls, and the diversity of microbes supporting them in the pelagic ecosystem. As described earlier, physical and chemical depth gradients in the water column affect the vertical distribution of microbial assemblages and their metabolic activities. Furthermore, at a macroscopic scale we can assess how each depth horizon is affected by different temporal patterns of variability, which, in turn, influence the microbial environment. For example, in the upper euphotic zone the variability in solar radiation due to cloud coverage and changes in day length associated with the seasonal solar cycle can affect the rates of photosynthesis. In this habitat, far removed from the upper nutricline (the depth at which nutrient concentrations start to increase), the dynamics of microbial processes will be controlled mainly by the rates of solar energy capture and recycling of nutrients through the food web. Furthermore, upper water column mixing rates also contribute significantly to the variability in the light environment. However, if the variability has a high frequency relative to the cell cycle, then microbes integrate the signal because the energy invested in acclimation may be greater than that gained

by maximizing photosynthetic and photoprotective processes along the variability (light) gradient. Between the base of the mixing layer and the top of the nutricline, the microbial assemblage resides in a wellstratified environment that is nevertheless still influenced by variability in light. Although mixing does not play a significant role in this habitat, unless a deep wind- or density-driven mixing event occurs, the vertical displacements of this stratified layer as the result of near-inertial period (31 h at the latitude corresponding to Station ALOHA) oscillation forces may introduce strong dayto-day variability in the light availability and photosynthetic rates (Figure 10); these vertical motions can affect the short-term balance between photosynthesis and respiration. The variability in solar irradiance described above propagates into the lower euphotic zone, penetrating into the upper nutricline. But the apparent presence of excess nutrients relative to the bioavailable energy that can be derived through photosynthesis in this region indicates that light is the limiting factor supporting microbial activity. For this reason, day-to-day variations, as well as the seasonal cycle of solar irradiance in this layer may trigger successional patterns in the microbial

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Figure 9 Vertical distributions of chloropigments (chlorophyll plus pheophytin) determined from in vivo fluorescence measurements and bottle calibrations. These graphs show average distributions at Station ALOHA for summer (June–Aug) versus winter (Dec–Feb) for 1999 showing and documenting changes in both total concentration of chloropigments at the surface and at the depth of the Deep Chlorophyll Maximum Layer (DCML). Both of these seasonal differences are caused primarily by changes in light intensity.

assemblage, and lead to pulses of organic matter export into the deeper regions of the ocean. At Station ALOHA, as well as in most oceanic regions, the gravitational flux of particles formed in the euphotic zone represents the major source of energy that links surface processes to the deep sea. In addition, these sinking organic particles represent energy- and nutrient-enriched microhabitats that can support the growth of novel microbial assemblages. The remineralization of particles with depth follows an exponential decay pattern indicating that most of the organic matter in these particles is respired in the upper layer below the euphotic zone. If the quality and quantity of organic rain was constant, we would expect to observe stable layers of microbial diversity and activity with depth. However, the long-term records of particle flux to abyssal depths at Station

ALOHA suggest that, during certain periods of the year, this flux increases significantly, representing potential inputs of organic matter into these deep layers driven by changes in upper water column microbial processes. Microscopic analysis of these organic matter pulses at Station ALOHA reveal that their composition is dominated by diatoms. These photolithoautotrophic microbes produce an external siliceous skeleton that can act as strong ballast when the cells become senescent. Several mesoscale physical processes have been observed that can modify the upper water column habitat at Station ALOHA, triggering an increase in the relative abundance of diatoms in surface waters and subsequent cascade of ecological processes. The passage of mesoscale features, such as eddies and Rossby waves, can shift the depth of nutrientrich water relative to the euphotic zone, leading to a possible influx of nutrients into the well-lit zone that can last from days to weeks. This sustained nutrient entrainment can alter the microbial size spectrum, in favor of rapidly growing, large phytoplankton cells (usually diatoms), resulting in a bloom. In addition, eddies can trap local water masses and transport microbial assemblages for long distances. A second mechanism triggering changes in the microbial community appears to occur during summer months at Station ALOHA, when the upper water column is warm and strongly stratified. Under these conditions, N2-fixing cyanobacteria, sometimes living in symbiosis with diatoms, aggregate in surface waters and provide an abundant supply of reduced nitrogen and organic matter to the microbial community. Although it is still not clear what triggers these summer blooms, in situ observations suggest that they significantly alter the structure and metabolic activity of the microbial assemblage. Finally, the mixing layer can periodically penetrate to a depth where it erodes the upper nutricline and delivers nutrients to the surface waters, while mixing surfacedwelling microbes into the upper nutricline. This deepening of the mixing/mixed layer can be driven by sudden events such as the development of a severe storm or the cooling of surface waters by the passage of a cold air mass. And, although each of these three mechanisms can lead to the entrainment of nutrients into the euphotic zone, they generate different microbial responses and interaction. For example, while the first two mechanisms do not involve a change in stratification, the third mixes the water column temporarily erasing the physical, chemical, and biological gradients that had existed before the event. Furthermore, while the passage of eddies and Rossby waves introduce nutrients into the base of the euphotic zone, affecting primarily the microbial populations inhabiting the upper nutricline, summer blooms have their strongest effect in the microbial assemblages residing in the upper few meters of the water column. Nevertheless, all these mechanisms appear to generate pulses of particulate organic matter rain that enhance the availability of

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Figure 10 Effect of isopycnal vertical displacements in accounting for day-to-day variability of Photosynthetically Available Radiation (PAR) at the DCML at Station ALOHA: (Top) Observed minimum and maximum depth range distribution of the DCML for each HOT cruise based on continuous fluorescence trace profiles obtained from 12 CTD casts deployed over a 36-h sampling period. (Center) Surface PAR measured at the HALE ALOHA mooring location during HOT-83 (5–9 May 1997). (Bottom) Estimated PAR at the DCML based on the vertical displacement of the DCML, surface PAR, and assuming kPAR ¼ 0.04 m1. Daily integrated PAR values (in mol quanta m2 day1) are displayed next to each light cycle in (Center) and (Bottom). These day-to-day variations in light caused by inertial period oscillations of the DCML and variations in surface PAR due to clouds are certain to have significant effects on rates of in situ photosynthesis. Reproduced from Karl DM, Bidigare RR, and Letelier RM (2002) Sustained and aperiodic variability in organic matter production and phototrophic microbial community structure in the North Pacific Subtropical Gyre. In: Williams PJ, le B, Thomas DR, and Reynolds CS (eds.) Phytoplankton Productivity and Carbon Assimilation in Marine and Freshwater Ecosystems, pp. 222–264. London: Blackwell Publishers.

ephemeral microenvironments, fuel the deeper microbial layers, and carry microbes to depth. In addition to mesoscale events and seasonal cycles that seem to support small transient changes in the microbial community structure and function, variability at longer time scales (interannual to decadal) may shift the taxonomic structure of the microbial community. For example, there is evidence suggesting that a significant shift in the

dominance of phototrophic taxa may have taken place in the NPSG as a result of changes in ocean circulation and wind forcing during the 1970s. More recently, changes in the stability of the upper water column since the 1997–98 El Nin˜o event may have also triggered long-term changes in the phototrophic community structure. Ultimately, these long-term habitat changes are the result of processes taking place over a broad range of

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scales propagating into the habitat experienced by a microbe. In this context, the advent of novel molecular tools such as metagenomic, proteomic, and transcriptomic analyses has provided an unprecedented opportunity to infer the diversity and biogeochemical relevance of microhabitats via the characterization of the genes being expressed in the environment. These new tools may help us better explore how physical and biological processes, by affecting the spatial and temporal distribution of these habitats, shape the microbial diversity and metabolism in the sea. However, understanding how microbial assemblages in different oceanic habitats may evolve over time in response to climate change will require not only a characterization of the microbes’ response to physical and chemical changes, but also the development of an understanding of how interactions among microbes contribute to the plasticity and resilience of the microbial ecosystem in the marine environment.

Summary and Prospectus All marine habitats support diverse microbial assemblages that interact through a variety of metabolic and ecological processes. The characteristics and dynamics of marine habitats determine the composition, structure, and function of their microbial inhabitants. Many microbial habitats (i.e., microhabitats) are cryptic, ephemeral, and difficult to observe and sample; the spatial and temporal domains of these environments are poorly resolved at present. The changing ocean will lead to different and, probably, novel marine habitats that will select for new microbial assemblages. Future ecological research should focus on the relationships among climate, habitat, microbes, and their individual and collective metabolic function. These comprehensive studies demand coordinated, transdisciplinary field programs that fully integrate physical and chemical oceanography with theoretical ecology into the wonderful world of marine microbes.

Acknowledgments We thank our many colleagues in the HOT and C-MORE programs for stimulating discussions, and the National Science Foundation, the National Aeronautics and Space Adminstration, the Gordon and Betty Moore Foundation, and the Agouron Institute for generous support of our research. See also: Algal Blooms; Aquaculture; Archaea (overview); Regulation of Carbon Assimilation in Bacteria; DNA Sequencing and Genomics; Deep-Sea Hydrothermal Vents; Ecology, Microbial; Food Webs, Microbial; High-

Pressure Habitats; Horizontal Transfer of Genes between Microorganisms; Low-Nutrient Environments; Metabolism, Central (Intermediary); Nitrogen Cycle; Phosphorus Cycle; Photosynthesis: Microbial; Picoeukaryotes; Regulation of Carbon Assimilation in Bacteria; Sediment Habitats, including Watery; Stable Isotopes in Microbial Ecology

Further Reading Cole JJ, Findlay S, and Pace ML (1988) Bacterial production in fresh and saltwater ecosystems: A cross-system overview. Marine Ecology Progress Series 43: 1–10. Cullen JJ, Franks PJS, Karl DM, and Longhurst A (2002) Physical influences on marine ecosystem dynamics. In: Robinson AR, McCarthy JJ, and Rothschild BJ (eds.) The Sea, vol. 12, pp. 297–336. New York: John Wiley & Sons, Inc. DeLong EF, Preston CM, Mincer T, et al. (2006) Community genomics among stratified microbial assemblages in the ocean’s interior. Science 311: 496–503. Fenchel T, King GM, and Blackburn TH (1998) Bacterial Biogeochemistry: The Ecophysiology of Mineral Cycling, 2nd edn. California: Academic Press. Giovannoni SJ, Tripp HJ, Givan S, et al. (2006) Genome streamlining in a cosmopolitan oceanic bacterium. Science 309: 1242–1245. Hedgpeth JW (ed.) (1957) Treatise on Marine Ecology and Paleoecology. Colorado: The Geological Society of America, Inc. Hunter-Cevera J, Karl D, and Buckley M (eds.) (2005) Marine Microbial Diversity: The Key to Earth’s Habitability. Washington, DC: American Academy of Microbiology. Johnson KS, Willason SW, Weisenburg DA, Lohrenz SE, and Arnone RA (1989) Hydrogen peroxide in the western Mediterranean Sea: A tracer for vertical advection. Deep-Sea Research 36: 241–254. Karl DM (1999) A sea of change: Biogeochemical variability in the North Pacific subtropical gyre. Ecosystems 2: 181–214. Karl DM (2007) Microbial oceanography: Paradigms, processes and promise. Nature Reviews Microbiology 5: 759–769. Karl DM, Bidigare RR, and Letelier RM (2002) Sustained and aperiodic variability in organic matter production and phototrophic microbial community structure in the North Pacific Subtropical Gyre. In: Williams PJ, le B, Thomas DR, and Reynolds CS (eds.) Phytoplankton Productivity and Carbon Assimilation in Marine and Freshwater Ecosystems, pp. 222–264. London: Blackwell Publishers. Karl DM and Dobbs FC (1998) Molecular approaches to microbial biomass estimation in the sea. In: Cooksey KE (ed.) Molecular Approaches to the Study of the Ocean, pp. 29–89. London: Chapman & Hall. Letelier RM, Karl DM, Abbott MR, and Bidigare RR (2004) Light driven seasonal patterns of chlorophyll and nitrate in the lower euphotic zone of the North Pacific Subtropical Gyre. Limnology and Oceanography 49: 508–519. Longhurst A (1998) Ecological Geography of the Sea. San Diego: Academic Press. Mann KH and Lazier JRN (1996) Dynamics of Marine Ecosystems: Biological-Physical Interactions in the Oceans, 2nd edn. Massachusetts: Blackwell Science. Martiny JBH, Bohannan BJM, Brown JH, et al. (2006) Microbial biogeography: Putting microorganisms on the map. Nature Reviews Microbiology 4: 102–112. Morita RY (1985) Starvation and miniaturization of heterotrophs, with special emphasis on maintenance of the starved viable state. In: Fletcher M and Floodgate GD (eds.) Bacteria in Their Natural Environments, pp. 111–130. Orlando: Academic Press. Platt T and Sathyendranath S (1999) Spatial structure of pelagic ecosystem processes in the global ocean. Ecosystems 2: 384–394. Purcell EM (1977) Life at low Reynolds number. American Journal of Physics 45: 3–11.

Environmental Microbiology and Ecology | Marine Habitats Rocap G, Larimer FW, Lamerdin J, et al. (2003) Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation. Nature 424: 1042–1047. Ryther JH (1969) Photosynthesis and fish production in the sea. The production of organic matter and its conversion to higher forms of life vary throughout the world ocean. Science 166: 72–76. Sverdrup HU (1953) On conditions for the vernal blooming of phytoplankton. Journal du Conseil International pour l9Exploration de la Mer 18: 287–295. Thompson JR, Pacocha S, Pharino C, et al. (2005) Genotypic diversity within a natural coastal bacterioplankton population. Science 307: 1311–1313. Yayanos AA (1995) Microbiology to 10,500 meters in the deep sea. Annual Review of Microbiology 49: 777–805.

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Relevant Websites http://www.agi.org – AGOURON Institute http://www.bios.edu/ – BIOS, Bermuda Institute of Oceanic Sciences http://hahana.soest.hawaii.edu – Microbial Oceanography, Hawaii http://reason.gsfc.nasa.gov/ – NASA, National Aeronautics and Space Administration http://woce.nodc.noaa.gov – NODC, National Oceanographic Data Center

Mats, Microbial R W Castenholz, University of Oregon, Eugene, OR, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Habitat Distribution Comprehensive Investigations

Glossary algae A general, nontaxonomic term that includes several unrelated or somewhat related phyla (or divisions), for example, the brown algae, chrysomonads, haptomonads, cryptomonads, green algae, euglenoids, charophytes, red algae, and some other groups with relatively few species. anoxygenic photosynthesis Photosynthesis having only a single photosystem and never evolving O2 (e.g., purple bacteria, green sulfur bacteria, green nonsulfur bacteria, and heliobacteria). bacteriochlorophyll a, b, c, d, e, g Types of chlorophylls that occur exclusively in anoxygenic bacteria. Many of these bacteria are obligate anaerobes; others can tolerate O2 or even use it in respiration. Although all bacteriochlorophylls have absorption maxima in the violet/blue or near ultraviolet (UV), they are distinguished by their absorption maxima in the near infrared (IR), for example, Bchl c (745– 760 nm), d (725–745 nm), and e (715–725 nm). Bchl a, on the other hand, has one or two absorption maxima between 800 and 900 nm. Only a few species of purple bacteria exclusively contain Bchl b, absorbing between 1015 and 1050 nm. Bchl g is a unique pigment of the heliobacteria, absorbing maximally at 780–790 nm. blue-green algae An earlier term for cyanobacteria used before genetic and cytological data proved that these were true bacteria, although with a green plant type of photosynthesis that produces O2. carotenoids A general term for C40H56-conjugated hydrocarbons such as the bicyclic -carotene. However, many are monocyclic or acyclic, with some having one or a few oxygen atoms as keto or hydroxyl groups, in which case they are referred to as xanthophylls. chemoautotrophy A process in which a reduced substance (usually inorganic) is oxidized (respired) biologically while reducing CO2 to organic matter. chloroflexi A recently created phylum (or kingdom) that includes the filamentous anoxygenic phototrophic bacteria Chloroflexus, Roseiflexus, and Heliothrix. chlorophyll a, b, c, and d Various chlorophylls that absorb in the blue, but have distinctive spectral maxima

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Case Studies of Selected Microbial Mat Ecosystems Stresses Experienced by Microbial Mats Concluding Remarks Further Reading

in the red (685 nm for chl a) or (715–717 nm for chl d). Chl a is the only chlorophyll in most of the cyanobacteria, although a few (e.g., Prochlorococcus) have chl a and b, and one genus has chl d. Only some eukaryotic chromophytes (e.g., diatoms, chrysomonads, haptomonads, and brown algae) have both chl a and chl c. combined nitrogen N that is combined with other atoms or molecules, such as NO–3 (nitrate), NHþ 4 (ammonium), or organic forms of N, unlike dinitrogen gas (N2). cyanobacteria Oxygenic photosynthetic bacteria with two photosystems that use H2O as reductant to convert CO2 to new cell material (as in green plants). They contain chlorophyll a and the blue pigment phycocyanin (some also with the red pigment phycoerythrin) and all contain allophycocyanin and carotenoids. diatom Eukaryotic alga characterized by a cell wall composed of silica (SiO2). diel A reference to the entire 24 h period rather than simply the daytime (¼ diurnal). ecotype A stable, genetically distinct variant of a species that is associated with a particular habitat or niche. The term has no official taxonomic status. The term was coined in 1922 by Swedish botanist Go¨te Turesson. fermentation A partial oxidation of one organic compound resulting in the reduction of another, providing ATP via substrate level phosphorylation. filamentous Refers to a chain of cells (a filament) with or without branching. GenBank (NCBI) National Center for Biotechnology Information. A database that includes nucleotide sequences of various nucleotides, proteins, and so on, including 16S rDNA, 18S rDNA, and the nucleotide sequences of many other DNA loci. gene expression The process by which the inheritable information in a gene (i.e., DNA sequence) is converted into a functional product such as an amino acid. Sometimes used to indicate the expression of an entire genetically controlled process. gliding motility A method of sliding along on a solid or semisolid substrate; no flagella are involved, and

Environmental Microbiology and Ecology | Mats, Microbial

the nature of the mechanism is still uncertain. Common in cyanobacteria, other filamentous bacteria, as well as unicellular bacteria such as myxobacteria. However, the mechanisms are not the same, even among different cyanobacteria. An entirely different mechanism is involved in gliding motility of the eukaryotic diatoms. heterocyst (or heterocyte) A specialized cell of some cyanobacteria that has differentiated from a normal photosynthetic cell and has lost the ability to produce O2 and because of other modifications has become anoxic internally, thus allowing N2 fixation to take place even in an external environment high in O2. meiofauna Small benthic invertebrates, such as small crustacea and nematodes, that would normally pass through a 1 mm mesh opening. methanogenesis Often regarded as a very specialized type of anaerobic respiration in which CO2 or an organic compound (e.g., acetate) is reduced to CH4. It takes place in various species of methanogenetic Archaea. morphotype An organism description based on the morphological or structural characters, rather than the genetic characteristics (genotype). oxidant The compound that becomes reduced in a chemical or biochemical reaction. In aerobic respiration the oxidant is O2 and it is reduced to H2O. oxygenic photosynthesis The type of photosynthesis used by green plants, algae, and cyanobacteria in which H2O serves as the reductant for Photosystem II from which O2 is evolved; electrons moving through a second light-activated photosystem (PS I) result in the reduction of CO2 to new cell material. PCR (polymerase chain reaction) Is a technique in molecular biology. One of its key components, a DNA polymerase (usually Taq polymerase), is used to replicate a piece of DNA by in vitro enzymatic replication. As the reaction progresses, the DNA generated is used as a template for further replication. This sets in motion a chain reaction in which the DNA template is amplified exponentially. parts per thousand (ppt) ppt or ‰ is used primarily for seawater salinity, equaling 10%. Thus, seawater generally is 30–34‰ or 3.0–3.4%. phototrophic A term often used synonymously with photosynthetic, but can also include microorganisms such as some purple bacteria that may use light simply for energy and not necessarily for reducing CO2 into cell material. phycocyanin and phycoerythrin Two pigments contained in cyanobacteria and the chloroplasts of red algae and cryptomonads. Phycocyanin is a water-

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soluble biliprotein that absorbs in the yellow and is blue when isolated. Different forms of phycoerythrin are generally reddish in color, and most absorb in the green region of the spectrum. phylogeny The evolutionary history of any group of organisms, currently mainly based on genetic relatedness, often arranged as a phylogenetic tree. precambrian The general geologic term for the beginning of the earth as a planet at about 4.7  109 bp to the Cambrian period, about 0.57  109 bp (before present). prokaryotes A general nontaxonomic term used almost universally by microbiologists to signify the Bacteria and Archaea – in other words, microorganisms that lack a defined membrane-bound nucleus and mitochondria and plastids, but might appear very similar to each other using microscopy. In one taxonomic ‘group’, the Planctomycetes, a membrane may surround the DNA molecule. proterozoic The age of stromatolites, from the latter part of the Precambrian from about 2.5  109 years ago to the Cambrian (0.57  109 years ago). purple bacteria A general term for bacteria in the Proteobacteria (a branch of the true bacteria) that contain bacteriochlorophyll a or b. Some are photoautotrophs using CO2 as the source of carbon (and usually a reduced sulfur compound or reduced iron as the reductant) or photoheterotrophs using organic carbon derived ultimately from other organisms but still using light as the source of energy. There are still others that are referred to as purple bacteria that use a small content of bacteriochlorophyll and light energy simply as a supplemental source of ATP, but are mainly chemoheterotrophs, using the aerobic respiration of organic compounds for energy and organic carbon for cell material. reductant Any compound that is capable of giving up electrons and therefore becoming oxidized. spring tide A tide that occurs in correlation with full moon and new moon phases, as opposed to a neap tide that alternates with spring tides. Generally, spring tides have greater amplitude (both high and low) than neap tides. sulfate reduction A form of anaerobic respiration in which SO2– 4 (or elemental S) replaces O2 as the oxidant in the oxidation of various organic compounds (or H2). trichome Literally means hair, but for the cyanobacteria and other filamentous bacteria this archaic term refers to the chain of cells minus a sheath. If a sheath is present, the term for trichome plus sheath is filament.

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Abbreviations DBL EPS ESSA ITS

diffusion boundary layer extracellular polysaccharides Exportadora de Sal internal transcribed spacers

Defining Statement Microbial mats are top accreting, cohesive microbial communities that are often laminated and are found growing at the sediment–water interface in shallow fresh or saline waters or on intertidal flats. They may be ephemeral (seasonal) and only a few millimeters thick or perennial (i.e., remaining for several years) and acquiring a thickness of several millimeters to more than a meter. A few become hardened with calcium carbonate, but most at the present time remain organic and relatively soft and gelatinous. They may be regarded as ecosystems in which an energy input (usually solar radiation) and various nutrient salts result in a cascading tier of biochemical transformations. Biofilms are very thin cohesive microbial communities that develop on many surfaces and in some cases may develop into ‘mats’. Although microbial mats are often referred to as modern or living stromatolites, the latter term technically refers only to ‘fossilized’ or lithified structures. Proterozoic stromatolites (those 100 m in a water column of a lake or the sea. Cyanobacteria may be of the unicellular type (e.g., Synechococcus). Various species or varieties of this genus may form biofilms or mats up to a temperature of 73  C in hot springs of western North America and eastern Asia (the Pacific Rim). However, hightemperature forms of Synechococcus are apparently missing from the rest of the globe. For example, Synechococcus species and other cyanobacteria of hot springs in Europe and New Zealand grow only up to temperatures of about 60–62  C. Thermophilic Synechococcus species are missing altogether in the numerous alkaline hot springs of Iceland. In all alkaline hot springs of the globe that issue at temperatures of 60–62  C or below, numerous genera and species of cyanobacteria occur and many of these are filamentous. Some species appear to be restricted to one geographic area (e.g., New Zealand), while another filamentous type (generally referred to as Mastigocladus laminosus) is present up to 57–58  C in neutral to alkaline hot springs almost everywhere, including Iceland (Figure 5). This cyanobacterium is thought to spread easily, since is quite capable of surviving desiccation, low temperature, and freezing. A few alkaline hot springs contain sulfide (i.e., HS– or H2S) in the source water of the spring and this compound excludes many species of

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Horm Het

Het

about 6.5 and this rises to 7.0 or over when the charge of CO2 is lost through effervescence. These springs deposit large quantities of CaCO3 (with some magnesium) often in the form of travertine terraces. The microbial mats are usually thinner, and sometimes become embedded within the travertine. Although precipitation of the travertine would and does occur without the cyanobacterial/microbial mat, the microorganisms influence the morphology or shape of the deposits to some degree. Mats of acid hot springs

Figure 5 Photomicrograph of Mastigocladus (¼Fischerella) showing branching, heterocysts (het) and a hormogonium (horm) that is a motile gliding stage. The width of the hormogonium is 3.5 mm.

cyanobacteria when the concentration in the bathing waters is high (i.e., >15–20 mM). Some cyanobacteria of alkaline hot springs, namely Mastigocladus, produce heterocysts when the waters are low in combined nitrogen (Figure 5). These specialized cells generate internal anoxia and thus are able to fix N2 in the daytime. Other cyanobacteria of hot springs, which do not produce heterocysts, are known to shift to N2 fixation as darkness settles, as in many saline or hypersaline mats. Below the cyanobacterial top layer, in the virtual absence of visible light, there is often a reddish-to-orange layer that consists primarily of phototrophic bacteria that lack the ability to produce oxygen, as in hypersaline mats. These are very prevalent in hot springs and are composed of one or more members of the Chloroflexi phylum, such as Chloroflexus and Roseiflexus. Their primary method of metabolism and growth is to use the IR radiation wasted by the cyanobacteria for their energy source, but usually use organic compounds for cell building and growth, compounds that are ultimately derived from the primary producing cyanobacteria. Below about 45  C in alkaline hot springs, eukaryotic algae may also occur. However, mat building is not common, because below this temperature, grazing animals such as ephydrid fly larvae (and adults) and small crustaceans such as ostracods and amphipods tend to decimate the various mat-producing microbiota. Most of the mats of alkaline hot springs described above are soft, but in some areas the deposition of silica (SiO2) is rapid and the mats become brittle and hardened (e.g., Upper Geyser Basin, Yellowstone National Park). There are also several locations globally where the spring waters are rich in calcium and bicarbonate rather than sodium, chloride, and silicates (e.g., Mammoth Hot Springs, Yellowstone). The pH at the source is usually

Although common only in regions of current or recent volcanic activity, pH levels below about 4.0 and down to 0.0 exclude all photosynthetic bacteria, including the cyanobacteria. However, there is an abundant group of unicellular eukaryotic algae (species of three or more genera of the red algal order Cyanidiales) that forms a dominant feature in warm to hot acidic streams and pools (Figure 6). These springs are acidic mainly because of the sulfuric acid produced through oxidation of sulfide. These streams are often a brilliant blue-green color (or more yellow-green under the high light of summer) due to the sheer numbers and great biomass of this one and only type of photosynthetic microorganism that lives in acid waters and in a temperature range of about 40–56  C. In some cases, particularly in Yellowstone National Park, stable microbial mats may accrete to thicknesses of over 0.5 cm. In these mats, however, the most prominent heterotrophic utilizer of the organic compounds produced by the photosynthesizer may be one or more types of fungi. Microbial Mats of Antarctic and Arctic Ponds and Lakes It was a surprising discovery that freshwater to saline melt ponds in the Antarctic and high Arctic also developed

Figure 6 View of Lemonade Creek, Yellowstone National Park at 45  C and pH 2. The green color of the mat is imparted by a member of the Cyanidiales, namely Galdieria sp.

Environmental Microbiology and Ecology | Mats, Microbial

Figure 7 View of a melt pond (Casten Pond) on the ablation moraine of the Ross Ice Shelf, Antarctica, a few hundred meters from Bratina Island. The submerged mats are seen in orangebrown color near the shores and particularly in the narrows between the two sections of the pond.

microbial mats dominated by cyanobacteria (Figure 7). In fact, without specific identification of the organisms, they mimic visually the mats of many hypersaline and hot spring habitats in temperate climates Cyanobacteria had previously been known as dominants primarily in warmer water. However, the cyanobacteria known from these cold waters that are ice-free for only 2–3 months of the year are very slow growing at typical temperatures both in culture and apparently in situ at the low temperatures experienced in these waters (4–10  C). It is clear that these slowly accreting and often perennial mats develop because of the absence of efficient grazers, and the inability of many potential eukaryotic algal competitors or herbivores to tolerate the winter conditions when most of these ponds freeze throughout the water column, not simply on the surface as in temperate climates. In many of these melt ponds that are slightly saline, a small amount of liquid bottom water remains after freeze-up, and this consists of a highly concentrated brine. Thus, the microbes located there must be halotolerant in addition to tolerating extreme cold as well as darkness for many months. In ponds at 78 S (e.g., McMurdo Ice Shelf) the only herbivores of note are rotifers, nematodes, and unicellular protists.

Terrestrial Microbial Mats and Crusts Surprisingly, extensive microbial mats or crusts, with many of the same attributes and microbial components of submerged mats, occur in hot and cold deserts of the world (Figure 8). For example, extensive mats occur on the Colorado Plateau, and on the Antarctic Peninsula. They are slow developers and therefore do not withstand the disturbance of humans or livestock. In undisturbed areas, they may form a ground cover between and around desert shrubs. However, if disturbance occurs, recovery

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Figure 8 Desert microbial crust, dominated by scytonemincontaining cyanobacteria, near Moab, Utah. The light-colored patches are areas from which the crust has been removed. Reproduced with permission from Whitton BA and Potts M (eds.) (2000) Ecology of Cyanobacteria: Their Diversity in Time and Space. Dordrecht: Kluwer Academy.

may take many years or never, even if the cause of disturbance is removed. Ephemeral Mats Many relatively thin microbial mats develop for only a short time, that is, seasonally. For example, major oil spills in the Persian Gulf have formed thickened tar-like coverings in the intertidal and have developed mats that are similar to mats in other saline habitats, with cyanobacteria forming the dominant top layer. The degradation of petroleum products takes place as a result of the activities of the entire community, not simply of the cyanobacteria. Other ephemeral mats, although thin, are well known, particularly in tropical sandy habitats, although they might best be regarded as ephemeral cyanobacterial biofilms. However, many mats that persist for many months do develop over gently sloping, extensive intertidal flats (e.g., sand flats of the North Sea). Some mats are able to persist, even for years (not very ephemeral), because of the multiple conditions that exclude grazers. These conditions include evaporative drying with increasing salinity during periods of neap tides or the persistence of high sulfide in some marine intertidal marshes that tend to exclude gastropods. Mats on Tropical Corals Black band disease is a disease of certain shallow subtidal corals that actually consists of a creeping microbial mat dominated by a dark phycoerythrin-rich oscillatorian cyanobacterium and other rather typical microbial components, including sulfate-reducing bacteria. The sulfide produced may be involved in the death of the coral underlying the mat.

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Comprehensive Investigations Rigorous studies of microbial mats have concentrated on mats located in relatively few areas. Only two are discussed here. Hypersaline mats have been extensively studied in the lagoons and salinas in the area of Guerrero Negro, Baja California Sur, Solar Lake, Sinai, Egypt, and the Camarque region of southern France. Although much is now known about the microorganisms of these and other mats, only recently has the common presence of phages (bacterial viruses) been documented. Most of the detailed studies of neutral to alkaline thermophilic mats have been in Yellowstone National Park, Wyoming, USA (e.g., Octopus and Mushroom Springs, and the travertine terrace-building springs of the Mammoth area). Acid spring microbial mats have also been extensively studied in Yellowstone Park, particularly in the Norris Geyser Basin. Microbial mats of Antarctica have been studied extensively in the ponds in the Bratina Island area of the Ross Ice Shelf, and in the region of the Antarctic Peninsula. Terrestrial mats and crusts of the Colorado Plateau have been studied intensively, as have the mats of the inselbergs (hammock-like limestone outcrops) of Venezuela and the ‘Guayanas’. Microbial mats are not static entities. Within various vertical zones or strata, various biochemical transformations are taking place in a diel manner or seasonally, and many cases of mutualism and dependence exist among the various individual types of microorganisms and also among guilds of organisms that more or less serve the same function. There is also movement, particularly vertical movement, that may be initiated by light intensity, the changing light regime, and the chemical gradients that exist or develop. Cyanobacteria and other photosynthetic microorganisms on the mat surfaces produce organic compounds that may be excreted, leaked, or released with cell lysis. These compounds (sugars, amino acids, glycollate, various organic polymers, etc.) may then be metabolized by fermentative bacteria in the anoxic undermat that largely excludes O2 at least during nighttime. The production of H2, CO2, and acetate by some of the fermenters promotes the growth of methane-producing archaea, although in marine or some hot spring mats the presence of high levels of sulfate (SO2– 4 ) favor the activity of sulfate-reducing bacteria that use most of the same organic compounds as methanogens or hydrogen gas, with the resultant production of H2S. The microenvironment within microbial mats has been accessed by various types of microelectrodes that can penetrate mechanically into mats at intervals of a few microns. The most common types of electrodes used today are those that individually measure O2, pH, CO2, sulfide, and several other ions. Thus, 24 h measurements of these conditions allow the researcher to know when

conditions are anoxic or oxic (Figure 2). Microprobes that measure light intensity at different depths within a mat have also been developed and used. For example, a complete spectrum of penetrating light can be completed within seconds using a recording spectroradiometer with a microprobe. This is not a trivial type of data collection, since the changes in various conditions and activities within a few microns depth in a mat may be equivalent to what is happening over many meters in depth in the water column of a lake or the sea.

Case Studies of Selected Microbial Mat Ecosystems Microbial Mats of Guererro Negro, Mexico The seawater evaporation ponds of Exportadora de Sal (ESSA) have been studied extensively by biologists and geochemists from many parts of the globe, but more often than not, they were under the guidance of Dr. David J. Des Marais of NASA. They are adjacent to the Laguna Ojo de Liebre at 28 N on the west side of Baja California. In summer the water temperatures generally range from 22 to 29  C, whereas winter temperatures are between 14 and 22  C. In ponds above about 65‰ total salinity, the invertebrate population is restricted to meiofauna that are not efficient or abundant enough to prevent the accretion of microbial mats dominated by cyanobacteria and diatoms on the surface. From about 65 to 100‰, the cyanobacteria are predominantly species of Microcoleus, Oscillatoria, and Spirulina. At higher salinities (>100 to about 200‰), unicellular types tend to predominate at the surface (e.g., Aphanothece, Cyanothece). In some ponds or lagoons where NaCl is at saturation and may be precipitating (>  265‰), the green alga, Dunaliella salina (which accumulates much -carotene), occurs along with the ever abundant orange-red, nonphotosynthetic haloarchaea (usually referred to as halobacteria), forming dense planktonic populations. However, these haloarchaea occur in abundance only in the highest salinity crystallizing ponds or lagoons where no microbial mats occur. These archaea contain the carotenoid (bacterioruberin) imparting the bright orange-red coloration. However, if O2 deficits occur, these organisms also synthesize bacteriorhodopsins and/or halorhodopsins in special membranes (purple membranes) that create a light-driven proton pump that produces ATP as a supplement to their normal chemoheterotrophic metabolism. However, these haloarchaea are planktonic (containing gas vesicles) and are an insignificant component of microbial mats. However, even above 125‰, embedded in the precipitated CaSO4 (gypsum), filamentous oscillatorian-type cyanobacteria are abundant. At the surface of the soft mats, there is a diffusion boundary layer (DBL) of a few millimeters through which flow is restricted, although

Environmental Microbiology and Ecology | Mats, Microbial

diffusion and exchange of nutrients takes place. The undermat at these ponds is highly anoxic a few to several millimeters below the surface in the daytime, depending on how translucent the mat is with respect to light penetration and how deep the O2 diffuses. However, at night anoxia essentially persists to the surface (see Figure 2). The production of sulfide (H2S, HS–, S2–) by anaerobic sulfate-reducing bacteria is extensive wherever anoxia occurs, and the sulfide diffuses to the surface during the night. In this general region, members of the Chloroflexi also occur. These are phototrophic filamentous bacteria that use dim light, especially in the IR region, and one at least (‘Chlorothrix’) uses sulfide as the photosynthetic reductant. Vertically migrating filamentous sulfideoxidizing, but nonphototrophic, bacteria (e.g., Beggiatoa) tend to follow the narrow, moving boundary of O2 and sulfide on a diel (24 h) basis, since most of these sulfide oxidizers require both sulfide and O2 for respiration. In the saline ponds of Guerrero Negro, much soluble organic matter is released from the mats, and the waters that flow slowly from pond to pond of increasing salinity foam easily with wind and wave action. Depending on light intensity, several of the filamentous cyanobacteria make a diel movement downward within the mat (in bright light and UV) and upward in low light or darkness (see ‘UV stress’). It should be pointed out that these microbial mats develop and accrete to nearly a meter in thickness in some ponds with only about 1–2 m of water above them. They do not precipitate calcium carbonate and thus remain soft throughout their existence. Accumulation of mat, however, is possible only because efficient grazers, such as gastropods, do not inhabit these saline waters, and higher aquatic plants and macroalgae do not compete because of their absence. However, in much earlier times (during the Precambrian and up to about 0.57  109 years bp), no efficient grazing animals were present or abundant enough to require microbial mats to ‘take refuge’ in hypersaline waters. Thus, it appears from the fairly extensive Precambrian record of stromatolites that mats such as these developed abundantly in shallow seas almost everywhere, whether saline or not. It appears that the actual growth rate of the mat (i.e., its accretion rate) is quite slow and that most of the time this compact mass of organisms in a gel-like matrix is simply maintaining itself and recycling its essential nutrients (e.g., N and P), with some loss of inorganic (i.e., CO2, HCO–3) and organic carbon to the water above. Along the widespread intertidal flats of the natural shoreline of Laguna Ojo de Liebre an almost complete coverage by a thin leathery and persistent mat of the cyanobacterium Lyngbya sp. occurs, a mat that is covered by tidal waters of about 35–50‰ salinity only during the twice monthly occurrences of spring tides (Figure 9). Bordering the upper edge of this mat is a corrugated

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Figure 9 Intertidal flats at Laguna Ojo de Liebre, Baja California Sur, Mexico. The dark area is covered by the scytonemincontaining cyanobacterium, Lyngbya sp. This area is covered by seawater of 35–50‰ salinity during days of spring tides (i.e., every 2 weeks).

mat of Calothrix sp., a cyanobacterium that is covered by water even less often than that of Lyngbya. However, when covered it fixes N2 in the daytime by use of its heterocysts, whereas Lyngbya fixes N2 at night during anoxic conditions, since heterocysts are not produced by this cyanobacterium. Microbial Mats of Octopus and Mushroom Springs, Yellowstone National Park Alkaline hot springs in the Lower Geyser Basin of Yellowstone National Park have been studied extensively by associates, students, and visitors of the lab of Dr. David M. Ward at Montana State University, as well as by other investigators from many parts of the globe. They are structurally quite similar to the submerged hypersaline mats of Guerrero Negro, although the species present are quite different, but with cyanobacteria again forming the top accreting layer (Figure 3). The description of hot spring microbial mats in the section titled ‘Hot spring mats of neutral to alkaline pH waters’ is based primarily on the situation in Octopus and nearby Mushroom Springs. However, more details are added here. Instead of basing species identification solely on morphology, pigments, and physiology of culture isolates, a large effort using molecular/genetic methods has been made. Most of the work has focused on the cyanobacterial genus Synechococcus (Figure 10). Not only are there a few species indicated by the sequence comparisons of the 16S rDNA gene, but slight differences in these sequences and greater differences in the sequences of the internal transcribed spacers (ITS) separating 16S and 23S rRNA genes have indicated that there are many more variants within a cluster of seemingly identical cyanobacteria, each of

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Environmental Microbiology and Ecology | Mats, Microbial

major portion of the undermat have been classified by molecular means in these springs. These include the phototrophic members of the genus Roseiflexus, Chloroflexus, and undescribed members of this phylum. Although sulfate-reducing bacteria operate in these springs, their role is minor compared to the methanogenic archaea that use H2, CO2, and/or acetate to produce methane (CH4) as a waste product.

Stresses Experienced by Microbial Mats

Figure 10 Photomicrograph of thermophilic Synechococcus sp. isolated in culture from an alkaline hot spring in Yellowstone National Park. A few of the cells may be seen dividing by fission across the midsection of the cell. The width of a typical cell is 1.2–1.5 mm.

which may be classified as a separate ecotype, meaning that certain small sequence differences may be correlated with differences related to dissimilarity in environment, such as temperature, light intensity, and chemistry optima and tolerances. One of the major contributions from the study of these springs has been the collection of environmental and genetic data over 24 h periods, showing that gene expression for certain physiological traits is not static. For example, when healthy high-temperature Synechococcus biofilms encounter normal high solar radiation in summer during late morning, inhibition of photosynthesis occurs (less so when UV radiation is filtered out), and recovery does not occur until the following morning, suggesting that repair and rehabilitation occurs mainly during the dark. Recent work from the Ward lab has shown that previously unknown N2 fixation genes of Synechococcus are turned on during the evening after anoxic conditions develop, that N2 fixation occurs then, and that genes for fermentation in Synechococcus are also switched on during the night period, while genes involved in photosynthesis and aerobic respiration are turned off as would be expected in a dark anoxic environment. Presumably, fermentation provides enough ATP required for N2 fixation, a process that requires much ATP. Previously, without the intensive 24 h molecular and physiological monitoring, the fact that Synechococcus was capable of N2 fixation was unknown. Additional work with these hot spring mats has shown that differences in ecotypes occurred with relatively small changes in the gradients of temperature and vertical light intensity within the mat, but all within the same species of Synechococcus. This type of work has just begun to reveal the complexity of mat interrelationships and functions. In addition to the photoautotrophic Synechococcus, anoxygenic members of the Chloroflexi that make up a

It is likely that most of the organisms within microbial mats are under some form of stress most of the time. Even if the mats occur in flowing systems only the uppermost cell layers have the benefit of continuously renewed nutrients, and at the same time these cells may be under the daytime stress of too much light and UV radiation. Most of the multitude of aerobic and anaerobic microorganisms within the crowded mat matrix may be nutrient starved, reductant starved, light starved, or high light inhibited depending on the circumstances. The combination of various factors normally results in a very slow growth rate or simply represents maintenance of life in the stationary phase with death of some cells being replaced by newly divided cells, and this over long periods. It has been shown, however, that an area recently denuded of mat (naturally by waves or hail storms or experimentally) will be colonized rapidly with a new biofilm and a relatively fast growth until the thickening materializes into a mat. High Light and UV Stress and Responses by Microorganisms Although most of the microorganisms inhabiting the uppermost layers of microbial mats are phototrophs (i.e., requiring light for energy), many biologists do not realize that too much light can be extremely detrimental. Since cyanobacteria generally constitute the uppermost layer of organisms in microbial mats, they have evolved various strategies to cope with the damaging effect of high solar radiation, which includes the most detrimental portion of this radiation, the UV component. Photons (expressed as quanta of energy) are absorbed by light-harvesting pigment molecules and then transferred from molecule to molecule to a reaction center where photochemistry occurs. However, the reaction center can become ‘saturated’ and the excess quanta can excite O2, for example, and produce the destructive singlet state (1O2). Carotenoid pigments in excess can quench such reactions and dissipate the energy as heat. Carotenoids are also important in preventing reactions that would ordinarily result in the production of the very destructive OH radical. The phenomenon of light intensity-dependent pigment regulation in cyanobacteria

Environmental Microbiology and Ecology | Mats, Microbial

and algae has been known for many decades. In general, the exposure to high light results in the active regulated degradation of pigments, especially of phycocyanin (the blue pigment of cyanobacteria), but also of chlorophyll. In some phototrophs, however (e.g., anoxygenic purple bacteria), the response to high light may be simply the cessation of pigment synthesis (e.g., of bacteriochlorophyll) and the consequent dilution of the pigment with each new cell division. In each case, the result is a lower pigment matrix for absorbing photons. As mentioned above, absorbance of photons in excess of what can be used photosynthetically usually results in the production of detrimental species of oxygen, causing photoinhibition or death. However, when light intensity is low, photons may become scarce, and this results in the synthesis of a greater content of light-harvesting pigments. Although the shorter wavelengths of UVR (UVC) 190–280 nm no longer impact the surface of the earth, the ozone/O2 shield in the early Precambrian was missing, and a damaging intensity of this germicidal wavelength would have reached the earth. At present, a small percentage of UVB (280–320 nm) does reach the earth’s surface and directly affects DNA, resulting in dimeric photoproducts between adjacent pyrimidines and other mutagenic damage, some of which can be repaired by photoreactivation or excision repair if the cells are metabolically active. Many other physiological processes and compounds are affected negatively by UVR. UVA (320–400 nm) has less direct effect on DNA, but is primarily associated with the production of reactive oxygen species that cause lipid peroxidation, chlorophyll bleaching, phycobilin degradation, and inhibition of growth and differentiation through a variety of targets. In metabolically active cyanobacteria, the process of repair and resynthesis of damaged compounds usually takes place at a rate that compensates for the damage done during periods of high solar radiation. However, several species of cyanobacteria that occupy exposed surfaces, such as microbial mats in shallow water, produce UV-shielding compounds that function even when the cyanobacterium is inactive metabolically, as during suboptimal temperature exposure, conditions of nutrient starvation, and during periods of desiccation or freezing. Filamentous, colonial, or unicellular cyanobacteria that produce an extracellular sheath or extracellular slime (both often simply referred to as EPS, i.e., extracellular polysaccharides) synthesize, with exposure to UVA radiation in particular, a yellowish-brown pigment called scytonemin that accumulates in the sheath and absorbs up to 97% of UVA radiation, thus preventing exposure within the cells. The content of scytonemin may be over 5% of the total cellular dry weight. It is also an effective absorber in the more dangerous UVB region. Scytonemin is a dimeric indole alkaloid with a molecular weight of 544. In many higher plants anthocyanins and flavones provide protection

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against UV radiation. Most of this information has come from the work of Dr. Ferran Garcia-Pichel in the lab of Richard W. Castenholz. A family of mycosporine-like amino acid derivatives (MAAs) represent another type of passive protective compound in cyanobacteria. These are a series of water-soluble low-molecular-weight condensation derivatives of a cyclohexonone ring and amino acid (or imino alcohol) residues. Some MAAs absorb maximally at wavelengths as low as 310 nm; others absorb maximally at longer wavelengths (e.g., 320–360 nm – UVA by definition). These compounds are widespread throughout the cyanobacteria, especially in hypersaline waters. These compounds also occur in eukaryotic algae, but in both groups of organisms they usually reside within the cytoplasm, and are thus less effective as screens. However, at least one species of the cyanobacterium, Nostoc, that often forms mats or is a part of terrestrial crusts accumulates MAA–oligosaccharide complexes in the extracellular sheath along with scytonemin, thus forming a twofold UV shield. Either MAAs or scytonemin or both may have evolved early in the Precambrian when the atmospheric penetrance of the UV solar flux was much greater than at present, a time when even the shorter wavelength UVC reached the earth’s surface. The vertical movements of motile cyanobacteria constitute another strategy to avoid the detrimental effects of high-light and UV radiation. Much of this work has come from the lab of Richard W. Castenholz. Although a few types of unicellular cyanobacteria are known to respond to changing light intensities by a slow upward or downward movement or by vertical or horizontal alignment, the most striking movements are by filamentous cyanobacteria in which the trichomes are able to move by gliding motility (i.e., a sliding movement in contact with a solid or semisolid substance; the mechanism is still uncertain) (Figure 11). In the bright light of midday,

Figure 11 Photomicrographs of Spirulina (helical trichome) and Oscillatoria (nontwisted trichome) from a hypersaline pond near Guerrero Negro, Mexico. These are the most prominent cyanobacteria that have been shown to perform diel vertical migrations. The width of the Oscillatoria trichome is 3.5 mm.

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the migrating cyanobacteria are situated a millimeter or more below the mat surface that may consist of a gravelly surface or of nonmotile cyanobacteria using another strategy. By late afternoon with declining light, the motile cyanobacteria gradually move to the surface where they remain throughout the night, only to descend again with the approach of high light the next morning. It has been shown in the hypersaline mats of the ponds at Guererro Negro, Baja California Sur, that the response cue is primarily to UV radiation. The cyanobacteria that migrate in this manner, when artificially subjected to the high light and UV of midday, are drastically inhibited photosynthetically, probably enough to cause death. The advantage of this type of escape strategy is that these filaments keep their high content of light-harvesting pigments that enables them to take advantage of the low light of morning and afternoon, and of overcast days. The migration system can easily be manipulated by covering portions of a midday mat with filters (e.g., neutral density). This results in the upward migration of the motile cyanobacteria within about 45–60 min. This response is much faster than the pigment regulation method of nonmotile species in preventing the damage caused by high radiation (see below). An almost identical response may be seen in some hot spring mats where a rapidly gliding oscillatorian cyanobacterium exhibits a similar diel upward and downward pattern. Other types of motile bacteria of microbial mats may also respond to the daily regime of light intensity changes. Beggiatoa, a nonpigmented but light-sensitive sulfideoxidizing chemoautotroph may often be seen at or near the surface of mats during darkness where they take advantage of the free sulfide and low O2 in the dark surface waters. In a few cases investigated, purple sulfur bacteria exhibit a somewhat similar response. These flagellated photosynthetic bacteria (e.g., Chromatium, Thermochromatium) occupy a protected position just below the surface in the day, and perform anoxygenic photosynthesis in which sulfide is used as the electron donor (reductant) rather than H2O. However, much of the sulfide is only partially oxidized, and consequently elemental sulfur is stored in the cells. At night, however, these cells may swarm to the surface water and respire the internal sulfur to sulfate for energy with O2 as the oxidant, thus acting as chemoautotrophs. Many of the migratory activities described here are known in hypersaline, marine, hot spring, and even terrestrial mats or crusts when the last named are moist with rain. Desiccation and Osmotic Stress Liquid water is a requirement for life on earth. Thus, the ability of some organisms to survive without water for extended periods of time is one of the many adaptations to life in extreme environments. Terrestrial mats have different types of cells from all three domains of life that

are able to withstand moderate to severe dehydration. When faced with desiccation, most microbial eukaryotes and many types of bacteria make a resting cell, such as a spore. However, there are some microbes in which the vegetative (photosynthetic) cells are able to withstand essentially complete dehydration for prolonged periods of time. Cyanobacteria are best known for this ability. Many occupy the top layer of terrestrial microbial mats. Desiccation-tolerant cyanobacteria can be found in environments ranging from hot or cold desert crusts to hypersaline intertidal mats. The effects of desiccation at the cellular level have been studied in some detail, especially by Malcolm Potts of Virginia Polytechnic University. The main consequence is the deformation of large molecules, such as proteins, nucleic acids, and lipids and their loss of function. These processes can occur during the loss of water and during rehydration. In addition, while in the desiccated state, cells are more susceptible to other stresses, particularly UV radiation, which may cause significant damage to DNA. The presence of water also helps maintain the fluidity of the cytoplasmic membrane. Thus, loss of water can lead to both hardening of the membrane and loss of structural integrity. Nevertheless, many prokaryotes, such as cyanobacteria, are able to recover from this stress. Temperature Stress The stress of high temperature becomes obvious in hot spring mats of North America and portions of the Pacific Rim where species composition changes, with fewer and fewer species present as the temperature rises to about 70–73  C, above which typical microbial mats are unknown. In other areas, such as New Zealand, Iceland, Italy, the upper limit of mat development is at about 60–63  C, simply because of the absence of a hightemperature form of the cyanobacterium, Synechococcus. As with hypersalinity, temperatures above about 45  C provide refuge for mat development in the absence of most grazers. Low temperature, on the other hand, is a less obvious stress for microbial mat formation. Freshwater and saline ponds in the Antarctic, for example, at 78 S, microbial mats, dominated primarily by cyanobacteria, accrete slowly during the short summer thaw. Although most of the cyanobacteria cultured from such ponds grow extremely slowly at their native temperatures of 4–10  C, grazing competitors are absent except for a few meiofauna. However, low temperature alone may not be responsible for the absence of herbivores, but the fact that most of these ponds freeze solidly (top to bottom) during the long winter, and if not, concentrate the remaining water at the bottom as a hypersaline brine (see ‘Microbial mats of Antarctic and Arctic ponds and lakes’). All of these conditions, plus possible problems in dissemination from other latitudes, tend to eliminate most

Environmental Microbiology and Ecology | Mats, Microbial

eukaryotic microorganisms, allowing the cyanobacteria to develop mats that structurally are very similar to those of hypersaline or hot spring mats in more temperate regions.

The Stress of Low pH, Metal, Metalloid, and Sulfide Toxicity Many habitats that are of low pH (i.e., 1, whereas those parameters that are unaffected or suppressed by roots would have R:S values equal to or less than 1, respectively. Typical R:S values based on microbial culturability vary among plant species, soil horizon, and microbial physiological groupings, as illustrated in Table 1. R:S ratios are typically higher in B horizon samples since its nonrhizosphere soil contains very low organic matter content capable of supporting microbial populations. High R:S ratios for protozoa reflect the increased abundance of their bacterial prey in this environment, and this higher bacteriovory activity accounts for a significant microbial loop that accelerates mineral nutrient cycling in the rhizosphere,

Environmental Microbiology and Ecology | Rhizosphere

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Table 1 Classical analysis data on measurements of the effect of various plant species on the major microbial groups and functional guilds in rhizosphere soila Microbial group or functional guild

Plant

Rhizosphere effect (R:S ratio)b

Bacteria Bacteria Bacteria Bacteria Bacteria Protozoa Filamentous fungi Microalgae Cellulose-degrading bacteria Nitrifying bacteria Ammonifying bacteria Denitrifying bacteria

Red clover Maize Yellow birch, A horizon Yellow birch, B horizon Mangles Mangles Wheat Wheat Wheat Wheat Wheat Wheat

24 3 15.1 57.8 120 3–23 6 11 5 1 50–125 90–1260

a

Data are based on viable plate counts and derived from studies by Katznelson and coworkers. The R:S is the metric that defines the magnitude of the rhizosphere effect on its associated microbial community and is typically computed as the value of the parameter measured (e.g., microbial abundance, diversity, or activity) in rhizosphere soil divided by its corresponding value in nearby nonrhizosphere control soil. Thus, root-associated enhancements would produce R:S values >1, whereas those parameters that are unaffected or suppressed by roots would have R:S values equal to or less than 1, respectively. b

thereby benefiting overall plant nutrition. A high R:S ratio of the denitrifier functional guild reflects this environment’s increased abundance of organic matter, lower pO2 due to the higher biological oxygen demand that accompanies degradation of organic matter and depletion of oxygen by microbial aerobic respiration, and the availability of NO 3 as the microbes’ second choice of alternate electron acceptor used to support ATP-yielding anaerobic respiration. Evidence for a lower in situ pO2 in the rhizosphere is supported by manometric measurements of total oxygen uptake in rhizosphere versus nonrhizosphere soil that produce R:S values ranging from 2 to 5, depending on the plant species. This increased indicator of aerobic respiration reflects the combined influence of readily oxidizable nutrient enrichments and the large community of metabolically active, O2-utilizing rhizosphere microorganisms.

Microbial Diversity in the Rhizosphere Other early studies characterized the diversity, morphology, and nutritional groupings of abundant culturable taxa in rhizosphere microbial communities. These studies indicated that the large diversity of culturable bacteria in the rhizosphere was typically dominated by Gramnegative, rod-shaped pseudomonads with nutritional requirements satisfied by one or more amino acids or organic acids. Populations of these distinctive rhizosphere bacteria increase rapidly and are most competitive when organic nutrient inputs are high, but decline dramatically when they become limiting. They are classified as zymogenous, copiotrophic R strategists. In contrast, the large diversity of culturable bacteria in nonrhizosphere soil are typically dominated by Gram-positive, Arthrobacter-like

pleomorphic forms with nutritional requirements satisfied by extractable factors in soil organic matter but not by simple sugars, amino acids, or organic acids. These nonrhizosphere bacteria are classified as autochthonous, oligotrophic K strategists that exhibit the opposite behavior of successful competition with low mortality in the absence of nutrient enrichments because of their efficient abilities in nutrient uptake, conversion of soil organic matter into microbial biomass, and synthesis/utilization of storage reserve polymers. Consistent with these early findings are more recent gene-based sequencing studies especially targeting the 16S ribosomal RNA gene of prokaryotes. These studies indicate that the -proteobacteria group (of which pseudomonads are representative) embody, on average, 35% of all bacterial cell clones isolated from rhizosphere samples (n ¼ 13 plant species), and their numerical dominance in the rhizosphere of some plant species extends up to 65% of all bacterial clones analyzed. Studies using mutant analysis of rhizobacterial pseudomonads indicate that their high competence in ability to colonize tomato rhizospheres involves several physiological systems, including chemotaxis, pili-mediated twitching motility, lipopolysaccharide O-antigen biosynthesis, anabolism of vitamins, nucleotides, and organic acids, cell autoaggregation, diverse nutrient uptake and utilization pathways, and protein secretion.

Nutrition and Cultivation Strategies of Typical Rhizosphere Microorganisms The zymogenous nature of typical rhizosphere microorganisms like Pseudomonas is also reflected in the significantly higher proportion of the resident community

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that can be cultured in the laboratory. Interestingly, 40–70% of the rhizosphere community can be isolated and cultured, whereas typically only 0.1–1.0% of the total bacterial community in nonrhizosphere bulk soil is culturable. Thus, this ‘plate count anomaly’ poses a more severe problem when attempting to culture the microbial community in bulk soil than in rhizosphere soil, and therefore the latter represents an accessible reservoir of culturable microorganisms with high phylogenetic and metabolic diversity. This significantly higher proportion of culturability for the rhizosphere community likely reflects microorganisms that are sampled while being in a more metabolically active physiological state, that is, when microorganisms have faster growth rates and less stringent nutritional and physiological growth requirements that can be satisfied by culture media formulations and cultivation conditions in the laboratory.

Root Exudation and Rhizodeposition in the Rhizosphere Studies by Rovira and colleagues analyzed root exudates of plants grown axenically (without associated microorganisms). These studies identified various low molecular weight organic compounds including numerous sugars, amino acids, vitamins, organic acids, nucleotides, enzymes, and miscellaneous phenolic compounds in root exudates. The diversity and abundance of the excreted plant metabolites varied among different species and ages of plants, and this information helped to solidify the concept that the rhizosphere effect is primarily a consequence of increased microbial metabolic/physiological activity made in response to nutrient enrichments through root exudation of carbon-rich and energy-yielding compounds in the soil environment surrounding roots. The high metabolic activity and productivity of the microbial community in the rhizosphere is mainly supported by carbon inputs from live roots, which are delivered into that environment in various forms, including water-soluble compounds in root exudates (see above), dead epidermal root cells, root cap cells, and mucilage polymers that slough off to lubricate the root tip during its penetration and geotropic growth through soil. All of the carbon lost from plant roots is termed ‘rhizodeposition’, and, in addition to the above sources, includes respiratory CO2 and other organic volatiles like ethylene. Much effort has been expended to measure rhizodeposition and identify factors that influence its magnitude. Values range widely in different experimental systems, reaching as high as 50% of net primary production in some ecosystems. For optimized lab studies, typically, 14 CO2 is provided to the photosynthetically active aerial plant system, and the amount of 14C-labeled photosynthates released from roots is measured. These studies

show that rhizodeposits of amino acids and carbohydrates are higher when roots are grown in some type of porous solid matrix (rather than in hydroponics), and the proportion of 14C-labeled rhizodeposits is significantly higher (18–25% vs. 4–10%) when plant roots are grown in unsterilized soil (with a rhizosphere microbial community) rather than in sterilized soil. Other studies show that extracellular metabolites of rhizosphere-competent microorganisms like Pseudomonas and Fusarium oxysporum can significantly increase carbon exudation from plant roots. Important implications of these rhizodeposition studies are that plants release significant amounts of newly fixed photosynthates through their roots without assimilating it into plant biomass, and that rhizosphere microorganisms can significantly increase – even double – the amount of fixed carbon lost from associated plants by root exudation. Such results have profound implications that lead to a provocative question: Who is in charge: the plant or its associated microbes? This rhetorical question generated a lively scientific debate when raised at a recent international congress on the rhizosphere, with approximately equal representation of conference delegates advocating the microbe or the plant component as the main driving force for the association. Various approaches have been used to locate the major sites of nutrient exudation on roots. These include ninhydrin staining of filter paper imprints of growing roots to locate concentrated sites of amino acid exudation, localized chemotropism of amino acid-requiring auxotrophs of Neurospora crassa fungi on roots, and localization of genetically engineered lacZ or Lux reporter strains of rhizobacteria inoculated on plants. All four methods revealed that the major sites of root exudation are just above the meristem in the elongation region of the root and at surrounding sites of lateral root emergence.

The Volume and Spatial Scale of the Rhizosphere The volume of the rhizosphere that extends from the root cylinder into surrounding soil is a dynamic function of the rate of plant rhizodeposition, the diffusion of nutrients within the soil matrix, and the extent of uptake and utilization of the rhizodeposits by the rhizosphere community. The radial distance of the rhizosphere has been examined by measuring the concentration gradient of carbon rhizodeposits and the density of colony-forming units and individual bacterial cells in soil sampled at various distances from the root surface. These studies indicate that the rhizosphere effect is typically most intense at the root surface (called the rhizoplane) and extends a few millimeters to little more than a centimeter out into the soil. Factors that influence this spatial scale include the species and growth stage of the plant, its root architecture and

Environmental Microbiology and Ecology | Rhizosphere Fibrous

Arable

Rhizosphere effect Figure 2 Schematic drawing illustrating how the root architecture influences the volume of the rhizosphere.

density, the light intensity and duration of the photoperiod, the texture, depth, fertility, and moisture status of the soil, and the types, abundance, and activities of the microbes, just to name a few. Figure 2 illustrates how root architecture influences the volume of different plant rhizospheres, and is much larger for root systems that are fibrous and highly branched in comparison with arable and nonfibrous systems. Rhizospheres of neighboring plants can occupy significant volumes and even overlap when their root densities are sufficiently high. For example, 30–40% of the top 10 cm of A horizon soil lies within 1 mm of a wheat root at its dough stage in development. Considering the high density of lawn turfgrass, all the soil to depths of their longest roots is rhizosphere.

Direct Microscopy of Microbial Colonization of Roots Early Microscopy Studies Starkey, Krasilnikov, Rovira, Bowen, and Foster conducted pioneering studies that utilized direct microscopy to examine the rhizosphere effect directly. A clever way to

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examine the rhizosphere effect in situ utilized a contact slide modification of the Rossi–Cholodny buried slide technique (Figure 3). Also, roots grown in soil were stained directly and examined by brightfield microscopy or processed for transmission and scanning electron microscopy to examine the rhizoplane microorganisms. These studies verified earlier cultivation methods that showed the increased abundance of microbes in soil surrounding roots, their growth into microcolonies, ultrastructural details of the rhizosphere soil fabric, and preliminary descriptions of the microbial distributions on the rhizoplane. Other key findings made by those early microscopy studies were the direct demonstration of a mucilage layer on the root surface that sometimes embeds microorganisms within it, bacteria surrounded by abundant fibrillar capsules whose periphery is covered with particulates of clay envelopes, the morphological diversity of the rhizoplane community, increased bacterial colonization in grooves between adjacent epidermal cells where exudation is predictably elevated, local sites of eroded epidermal walls allowing microbial penetration, and direct evidence of biological control via the mechanism of hyperparasitism, that is, beneficial microorganisms directly attacking and killing other microbial pathogens of plants. Examination of root cross sections of field-grown plants showed that their root cortex naturally supports large communities of microorganisms without the plant expressing symptoms of disease responses. This benign associated community of microorganisms was initially termed the ‘endorhizosphere’ but later the organisms were called ‘endophytes’ or ‘internal root colonists’. Figure 4 is a color fluorescence micrograph of the microbial community within rhizosphere soil surrounding a white clover seedling root. This image acquired by conventional epifluorescence microscopy is informative

(b) (a)

Planted seed

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Seedling root

2–3 days

Figure 3 (a) Schematic diagram illustrating the experimental design of the seedling contact slide method to examine the rhizosphere effect by direct microscopy. (b) Direct microscopy of the abundant, localized colonization of seedling root tissue by rhizosphere microorganisms on a seedling contact slide. The sample was stained with aniline blue and photographed using brightfield light microscopy.

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Environmental Microbiology and Ecology | Rhizosphere

Improvements Using CLSM

10 µm Figure 4 Color fluorescence micrograph of the microbial community within rhizosphere soil surrounding a white clover seedling root. The sample was stained with the nucleic acid stain acridine orange, then rinsed with a solution of sodium pyrophosphate and examined by conventional epifluorescence microscopy.

in showing the clustered distribution of the morphologically diverse unicellular and filamentous bacteria in close association with particulate, reddish-brown autofluorescent organic matter, but it suffers because a major portion of the foreground objects are outside the plane of focus.

Advancements in Rhizosphere Studies Using Modern Technologies Modern technologies provide deeper insights into the understanding of microbial community structures and their interactions in the rhizosphere of plants. (a)

The most significant advancement in the use of fluorescence microscopy to examine root-associated microbes was the development of confocal laser scanning microscopy (CLSM). The confocal imaging system has an optical design utilizing pinhole apertures at the laser light source and at the detection of the object’s image, thus eliminating the stray and out-of-focus light that interferes with the formation of the object’s image. This ability to use only signals from the focused plane eliminates a major limitation of the conventional fluorescence microscope when examining plant tissue, soil particles, and organic debris that emits a significant amount of objectionable background autofluorescence and/or absorbs the fluorescent dye itself. Because the light from outside of the plane of focus is not included when the image is formed, the 2-D (x, y) image becomes an accurate optodigital thin section with a thickness that can approach the theoretical 0.2 mm resolution of the light microscope. Also, by digitizing a sequential series of 2-D images while focusing through the specimen in the third (z) dimension (called the ‘Z series’), an all-inclusive flattened stack or a 3-D reconstructed image can be produced, rotated, and quantitatively analyzed. Dazzo and colleagues first reported the successful use of CLSM to visualize the rhizoplane microbial community stained with the fluorescent nucleic acid-staining dye, acridine orange. Examples are illustrated in Figure 5(a) and (b). By using this approach, one can map the distribution of the rhizoplane microbial community colonized on seedling roots grown in soil. Figure 6 shows a typical result, illustrating that the spatial distribution of microorganisms on the rhizoplane is discontinuous, being almost completely devoid of microbes at the root tip growing in soil, (b)

Figure 5 Use of confocal laser scanning microscopy (CLSM) to detect rhizoplane microorganisms colonized on roots grown in soil. White clover seedlings were grown in soil for 2 days, then cleared free of rhizosphere soil, using methods described in the book COST 631: Handbook of Methods in Rhizosphere Research, then stained with acridine orange, washed, and examined by epifluorescence CLSM. The image (a) is historically the first one illustrating this protocol, and the image(b) shows direct evidence of bacterial growth and formation of a microcolony biofilm (arrow) localized on the rhizoplane. Reproduced from Dazzo FB (2004) Applications of quantitative microscopy in studies of plant surface microbiology. In: Varma A, Abbott L, Werner D, and Hampp R (eds.) Plant Surface Microbiology, pp. 503–550. Germany: Springer-Verlag, with permission from Springer-Verlag.

Environmental Microbiology and Ecology | Rhizosphere 200 µm

Older

Younger

Figure 6 Binary montage image depicting the spatial distribution of microorganisms colonized on the rhizoplane of a white clover seedling after 2 days of growth in soil. Note the discontinuous density of colonization that is greater on older regions of the root.

individual cells just above the root tip, some of which eventually develop into microcolonies further up the root. Overall, the rhizoplane of actively growing roots is typically covered only up to 20% with microbes. In some cases, single microbial cells find the rhizoplane environment to be favorable for growth, resulting in localized microcolony biofilms with in situ population generation times as short as 2–3 h.

A Case Study: Rhizobium Colonization of Rice Roots Rhizobium is the well-known nitrogen-fixing root nodule bacterial symbiont of legume plants (see ‘Rhizobia’).

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Recent studies have shown that this soil microorganism also develops natural, intimate, and sometimes beneficial endophytic associations with various cereal crops like rice, wheat. This alternate ecological niche of Rhizobium can be exploited in sustainable agriculture in the form of efficient biofertilizers that are inoculated on the cereal plant host at planting and can significantly promote its growth, thereby reducing its need for chemical fertilizer applications to achieve maximum grain yield. Figures 7 and 8 illustrate aspects of the colonization and endophytic infection of rice roots by Rhizobium using CLSM and scanning electron microscopy, respectively. These show the colonization of the rhizoplane especially surrounding lateral root emergence and at junctions between epidermal cells, crack entry into void spaces created by lateral root emergence and fissures between epidermal cells, colonization of dead cortical cells within the root, and localized pit erosion mediated by Rhizobium cell-bound cellulases. Hartmann and colleagues demonstrated the value of CLSM combined with immunofluorescence microscopy (IFM) using fluorescent-labeled antibodies as specific molecular probes to examine colonization of a specific microorganism (Azospirillum sp.) on the wheat rhizoplane. In that study, a dual laser system was used to produce the

(a)

(b)

(c)

(d)

Figure 7 Epifluorescence confocal laser scanning microscopy showing colonization of the rhizoplane and internal root structures of rice by rhizobia. (a) and (b) Preferential colonization of a dense collar of rhizoplane rhizobia surrounding the base of emerged lateral roots. (c) and (d) Endophytic rhizobial colonization of underlying, lysed root cortical cells and the vascular cylinder of rice. Rhizobia are stained with acridine orange in (a) and (c) and are expressing the green fluorescent protein in (b) and (d). Images (a) and (c) reproduced from Reddy PM, Ladha JK, So R et al. (1997) Rhizobial communication with rice: Induction of phenotypic changes, mode of invasion and extent of colonization in roots. Plant and Soil 194: 81–98, with permission from (Plant and Soil). Images (b) and (d) reproduced from Chi F, Shen S-H, Cheng H-P, Jing Y-X, Yanni YG, and Dazzo FB (2005) Ascending migration of endophytic rhizobia from roots to leaves inside rice plants and assessment of their benefits to the growth physiology of rice. Applied and Environmental Microbiology 71: 7271–7278, with permission from the American Society for Microbiology.

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(a)

(b)

(c)

(d)

dm

CW2

uc

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ic

m b m

Figure 8 Scanning (a–c) and transmission (d) electron microscopy of the colonization and infection of rice by endophytic rhizobia. Images illustrate (a) colonization at epidermal root junctions (arrows); (b) localized eroded pits of the epidermal cell wall where rhizobia with cell-bound cellulases have been attached (arrows); (c) crack entry mode of infection of the rice root at fissures between epidermal cells (arrows); and (d) endophytic colonization of rhizobia within a dead cortical cell adjacent to an uninfected host cell with intact membranous organelles. Reproduced from Yanni YG, Rizk RY, Abd El-Fattah FK, et al. (2001) The beneficial plant growth-promoting association of Rhizobium leguminosarum bv. trifolii with rice roots. Australian Journal of Plant Physiology 28: 845–870, with permission from CSIRO.

green autofluorescence of the root background upon which the distinctive red immunofluorescent Azospirillum cells could be easily seen. The noninvasive optical sectioning ability of the confocal microscope was also used to locate the Azospirillum cells within the root mucigel layer. Use of CMEIAS Image Analysis Software to Analyze the Colonization of Rhizoplanes by Microbes In Situ Computer-assisted digital image analysis can significantly enhance the quantitative analysis of microbial colonization of roots by allowing one to extract, store, retrieve, and electronically transmit numerical information regarding selected and pertinent image features. Careful morphological analysis of bacterial cells can provide useful information on the diversity, microbial abundance, and 2-D spatial distribution of microbial community members. A computer-aided system has been developed by the Center for Microbial Ecology at Michigan State University to assist in such assessments. Center for Microbial Image Analysis System (CMEIAS) is a semiautomated analysis tool that uses digital image processing and pattern recognition techniques in conjunction with microscopy to gather size and shape measurements of digital images of microorganisms and classify them into

their appropriate morphotype, allowing culture-independent quantitative analysis of the morphological diversity and distribution of complex microbial communities. Also, quantitative studies of microbial biogeography are greatly facilitated using CMEIAS computer-assisted microscopy, especially for defining the appropriate in situ spatial scale of quorum sensing, niche overlap and competition, and microcolony biofilm colonization at spatial scales directly relevant to individual bacterial cells. To illustrate applications of CMEIAS for autecological biogeography studies of specific organisms colonized on root surfaces, digital image analysis was performed on the fluorescent antibody-labeled cells of Rhizobium leguminosarum bv. trifolii on the white clover rhizoplane shown in Figure 9. In approximately 2 s of computing time, CMEIAS extracted several measurement parameters from each individual, immunofluorescent bacterial cell, and reported the data summarized in Table 2 that defined their abundance and pattern of spatial distribution on the root surface. Of particular autecological biogeography interest are the image analysis data extracted by CMEIAS needed to perform plot-less distance-based and quadrat-based spatial point pattern analyses, and geostatistical analyses on spatial interactions among the bacteria. These attributes define spatial patterns of distribution indices of dispersion and mathematical modeling that predict bacterial

Environmental Microbiology and Ecology | Rhizosphere

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Figure 9 Epifluorescence micrograph of immunofluorescent cells of Rhizobium leguminosarum bv. trifolii 0403 colonized on the root surface (especially at junctions between epidermal cells) below the root hair region of a white clover seedling. The bar scale is 20.0 mm. Reproduced from Dazzo F, Schmid M, and Hartmann A (2007) Immunofluorescence microscopy and fluorescence in situ hybridization combined with CMEIAS and other image analysis tools for soil- and plant-associated microbial autecology. In: Hurst C, Crawford R, Garland J, Lipson D, Mills A, and Stetzenbach L (eds.) Manual of Environmental Microbiology, 3rd edn., pp. 595–792. Washington, DC: American Society for Microbiology Press, with permission from the American Society for Microbiology.

Table 2 CMEIAS image analysis of the abundance and spatial distribution of immunofluorescent bacteria on the white clover root epidermis shown in Figure 9 Parameter measured

Value obtained

Image area analyzed Cell count % Substratum area covered by microbes Total microbial biovolume Total microbial biomass carbon Total microbial biosurface area Spatial density Mean first nearest neighbor distance Mean second nearest neighbor distance Spatial randomness index Variance:mean ratio Morista’s dispersion index Negative binomial K index Lloyd’s mean crowding index Lloyd’s patchiness index

10 434 mm2 136 2.7% 259.0 mm3 51.8 pg 1 126.6 mm2 13 034 cells mm2 3.9  2.4 mm 5.7  2.9 mm 0.88 (clustered) 1.796 (clustered) 1.996 (clustered) 0.950 (clustered) 1.595 (clustered) 1.995 (clustered)

Reproduced from Dazzo F, Schmid M, and Hartmann A (2007) Immunofluorescence microscopy and fluorescence in situ hybridization combined with CMEIAS and other image analysis tools for soil- and plant-associated microbial autecology. In: Hurst C, Crawford R, Garland J, Lipson D, Mills A, and Stetzenbach L (eds.) Manual of Environmental Microbiology, 3rd edn., pp. 595–792. Washington, DC: American Society for Microbiology Press.

colonization behavior in situ. The spatial randomness index, computed from the image area, average and standard deviations of the first and second nearest neighbor distances (Table 2), provides quantitative data indicating that the bacteria in Figure 9 exhibit a clustered pattern of spatial distribution (values 10%) of the total sediment inventory. The two abovementioned examples demonstrate that the connection between cellular activity and bulk sediment activity is not well understood. One outstanding question is how many sedimentary microbes are undergoing exponential growth, and another is whether there are a few superconsumers or if all sedimentary microbes are tuned to respire within a certain range. It is certainly true that sedimentary microbial systems dominated by Corg respiration are much more complex than pure cultures examined during optimal growth conditions.

Can Microbial Populations Be Predicted from Chemical Layers? One of the key questions that arises from the study of sedimentary environments is that of whether or not it is possible to predict the nature of the microbial populations by the profiles that are present. We propose that it is to some extent possible to predict the general nature of the reactions that must have taken place to account for the observed profiles. However, that is probably as far as it goes. There is a tremendous redundancy in nearly every process – a redundancy that precludes the specification of which phylotypes should be expected to be present. Thus, one has to settle with knowing that oxygen utilizers, denitrifiers, manganese reducers, and so on are present, but other strategies must be employed to identify the organisms actually present and active. For example, as the geochemists would identify the ‘zone of manganese reduction’ by a profile of manganese as shown in Figure 2, microbiologists would call this the zone of manganese reducer activity. However, there are literally hundreds of different microbial species capable of manganese reduction, thus relegating one to specifying processes rather than species, or even genus, names.

The other part of the predictive process is that most of the processes being studied are in fact cyclical. The oxygen diffusing down into the sediments acts not only as the electron acceptor for heterotrophic metabolism, but as the oxidant of many inorganics, regenerating them for further oxidative activity in the zones of reduction. Thus, near every reductive interface, as described by the geochemists, is an oxidative one, in which the electron acceptors are regenerated, almost always via the microbial activity(ies). As with the reductive activities discussed earlier, these activities can be specified, but the microbes responsible for them can only be designated by metabolic group. For the microbial ecologist, then, the sedimentary environment remains a great challenge in terms of understanding the diversity and interactions that occur as one proceeds downward through the LMCs. Complications Introduced by Activities of the Macrofauna Sediments that are mixed by macrofaunal activities (bioturbation) show complexities in microbial distribution due to the introduction of oxygen (and other oxidants) at depth due to burrowing, pumping of water, and other activities of multicellular macrofauna of a variety of types (Figure 1). Despite these activities, however, which tend to mix the upper surface of the sediments, one sees that bacteria distribute themselves along geochemical gradients and, in fact, are often responsible for the maintenance of these gradients although sometimes pore water chemistry and microbial community structure are uncoupled by this process. Sediments that do not undergo physical mixing provide excellent systems for the calibration of bacterial community activity, gradient formation, and community evolution: Mixed sediments provide sites for understanding the same processes in a much more complex setting of 3-dimensional diffusion gradients, perhaps more reminiscent of soil environments. Intersection of Chemical Gradients A fascinating feature of sediment communities relates to the places in which chemical gradients of different types intersect with one another, such as can be seen the sulfate/methane horizon in deep-sea cores. At such interfaces abundant microbes are seen, and the process of anaerobic methane oxidation, which occurs seldom if at all elsewhere, is abundant. The advantage of living near a redox boundary is that the energy-yielding reaction space increases: For example, oxygen utilization in the respiration of Corg requires a microbial community capable of breaking Corg into oxidizable-sized compounds and it requires that the respiring microbes ‘find’ both the oxidizable compound and the oxygen simultaneously. The

Environmental Microbiology and Ecology | Sediment Habitats, Including Watery

presence of oxygen in contact with ammonia, diffusing up from deeper in the sediment column, provides a fixed location where both energy-yielding compounds coexist. In fact, the constancy of geochemical gradients and redox boundaries within the sediment column is evidence that sedimentary microbes adapt to and will aid in the perpetuation of these gradients and boundaries. Sometimes the net free energy yield of a coupled set of geochemical reactions is sufficient to support a sedimentary microbial community even when some of the ‘players’ are working in a free energy deficit. An example of this is where sulfate is used in the oxidation of methane, a process known to be catalyzed by a consortium of archael methanotrophs and SRB. It has been determined that while the SRB capture significant energy from this coupled reaction, using H2 as an intermediate, there must be something ‘in it’ for the archaea for their role in this coupled reaction costs them energy. The further one looks into sedimentary bacterial activities, the more one sees that a simple thermodynamic explanation (i.e., the free energy paradigm) is a rule that is commonly broken. Under eutrophic and low oxygen water columns lie sediments containing little or no oxygen. In these sediments live bacteria that can sequester nitrate from the overlying water column and transport that nitrate within the sediments to zones where sulfide is present in high quantities. Under diffusive conditions, nitrate would not penetrate to the depth of high sulfide, yet these bacteria, for example, Beggiatoa and Thioploca, are capable of bypassing the diffusive zone and injecting nitrate into sediments rich in sulfide, thereby oxidizing the sulfide to S0 or SO42. An unsolved enigma is that the greater free energy yield would be obtained if nitrate were to react with Corg compounds. Sedimentary bacteria find ways to utilize unusual combinations of reduced and oxidized compounds, not only those in association with free solutes but also those in the solid phase.

Nutrient-Poor Sedimentary Environments Recent studies of deep-sea sediments underlying low productivity oceanic zones have revealed that a major part of the deep sea (perhaps 20% or more) consists of sediments that are so organic poor that the numbers of microbes are far less than those usually encountered in near-shore and productive environments. For example, Steve D’Hondt, Bo Joergensen, and their colleagues, in studies of sediments in the South Pacific gyre area, have found surface sediments that typically harbor only 103–105 bacteria per gram of sediment – orders of magnitude below those found in other more nutrient-rich horizons. Predictably, the chemical gradients typical of more active zones are absent here. Life becomes ‘cryptic’ in terms of using geochemical profiles to define it. The

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nature of the microbes in such environments is curious – they appear to be alive, but nondividing or extremely slowly dividing, with estimates of doubling times extending to thousands of years. Subsurface Sedimentary Microbiology In a situation similar to that seen in nutrient-poor sediments, the deep subsurface of ocean sediments appears to harbor a low-density (105 per gram) population that is dividing at very low rates (again, perhaps thousands of years), This so-called starving majority as it has been called by Bo Joergensen constitutes one of the current great mysteries in microbial ecology, and it is tempting to speculate that it may be similar to environments of early Earth, when Corg may have been much less abundant, and organisms might have utilized mechanisms for survival not often needed on the nutrient and energy-rich surface of our planet. Paleontological Properties of Sediments: The Rock Record As noted earlier, diagenesis is the process of sediment alteration, a process that begins as soon as the sedimentary material arrives, and continues as long as energy is flowing through the system. Superimposed on this are the paleontological consequences of sedimentation. As diagenesis occurs, a number of biosignatures can be deposited as records left to be examined in ancient sediments – records of early metabolism, even in the absence of living biomass, or even cell structures. Biosignatures can consist of molecules indicative of certain taxa and the processes they catalyze (i.e., markers indicative of photosynthetic activity, particularly of oxygenic photosynthesis), as well as stable isotopes of carbon, sulfur, and/or nitrogen that can be fractionated by biological systems. Such molecular fossils are often the only records available in the ancient sediments, and even these are often altered beyond recognition by diagenesis and metamorphosis (cooking and alteration!) of the sediments. However, it is these ancient sediments and their geobiological records that provide hints as to the earliest metabolism(s) on Earth, and even to the first detectable signs of life on our planet.

Sediments in Shallow Water, and the Impact of Light Almost everything discussed in this article has dealt with the sedimentary environment in the deeper and dark zones of oceans and lakes. Of course, along the continental margin, and in many lakes, sunlight can penetrate the overlying water, adding new complexity to the sedimentary environment. Now the environment becomes even

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more distinctly layered, often with oxygen-producing cyanobacteria at the surface, and other layers of anoxygenic photosynthetic bacteria below. The oxygen penetration depth can change dramatically between day and night, with oxygen production by the cyanobacteria during the day often pushing the oxic zone to several centimeters, and oxygen consumption by the heterotrophs leading to anoxia, even at the surface at night. This being said, however, the general features of the environment still apply: It is a permanently wet, diffusion-controlled niche that is strongly impacted by physical (porosity, tortuosity, etc.) and chemical (salinity, anion content, etc.) factors.

7. Sediments in the photic zone take on new properties due to the daily cycles of photosynthesis and oxygen consumption, and are further complicated by the input of organic matter directly to the environment by photosynthesis. However, they are still permanently wet, diffusion-controlled systems, subject to the same rules as other sediments. 8. Sediments are in general excellent habitats for prokaryotes, with 109 per gram or more cells being common. The sedimentary niche may harbor up to 50% of the biomass on our planet, and interact strongly with the biogeochemical cycles of nearly every major biologically active element.

Conclusions

See also: Coccolithophores; Bacteriophage Ecology; Nitrogen Cycle; Sulfur Cycle; Marine Habitats; Mats, Microbial

In closing, it is appropriate to remind ourselves of the general properties of the sedimentary niche, which is perhaps the largest single niche available to life on the planet. 1. Sediments are not soils – they accumulate biomass by sedimentation from above, and are constantly water saturated. 2. Sediments are physically stabilized, and therefore solute distributions are primarily controlled by diffusion – nutrients must diffuse from above (or sometimes below), and this diffusion controls the overall character of the niche. 3. Sediments can (and often do) have zones of bioturbation, a type of mixing imposed by eukaryotic multicelled organisms that pump water and nutrients into the sediments, as well as physically mixing the environment. 4. Sediments typically become anoxic due to respiration of the microbial population removing the oxygen at a rate faster than it can be supplied by diffusion, thus leading to widespread anoxic sediment distribution. 5. Other oxidants (nitrate, manganese oxides, iron oxides, sulfate, and CO2) are then used up via respiration, leading to chemical layering of sediments, processes that result in LMC formation. 6. Pore water analyses reveal the existence and nature of the LMCs in sediments, and allow one to specify to some degree the nature of the processes occurring in a given sedimentary habitat.

Further Reading Aller RC (1990) Bioturbation and manganese cycling in hemipelagic sediments. Philosophical Transactions of the Royal Society of London Series A – Mathematical Physical and Engineering Sciences 331: 51–68. Berelson WM, McManus J, Coale KH, et al. (1996) Biogenic matter diagenesis on the sea floor: A comparison between two continental margin transects. Journal of Marine Research 54: 731–762. Berner R (1980) Early Diagenesis: A Theoretical Approach. Princeton, NJ: Princeton University Press. Brocks JJ and Summons RE (2004) Sedimentary hydrocarbons, biomarkers for early life. In: Holland HD and Turekian KK (eds.) Treatise on Geochemistry. Chichester, UK: John Wiley and Sons. Burdige DJ (2006) Geochemistry of Marine Sediments. Princeton, NJ: Princeton University Press. Jorgensen BB and D’Hondt S (2006) A starving majority deep beneath the seafloor. Science 314: 932–934. Madigan MT, Martinko JM, and Parker J (2000) Brock: Biology of Microorganisms, 9th edn. Upper Saddle River, NJ: Prentice Hall. Nealson KH (1997) Sediment bacteria: Who’s there, what are they doing, and what’s new? Annual Review of Earth and Planetary Sciences 25: 403–434. Nealson KH and Popa R (2005) Metabolic diversity in the microbial world: Relevance to exobiology. Society for General Microbiology Symposium 65: 1157–1171. Parkes RJ, Cragg BA, and Wellsbury P (2000) Recent studies on bacterial populations and processes in marine sediments: A review. Hydrogeology Reviews 8: 11–28. Whitman BW, Coleman DC, and Wiebe WJ (1998) Prokaryotes: The unseen majority. Proceedings of the National Academy of Science of the United States of America 95: 6578–6583. Ziebis W, Forster S, Huettel M, and Joergensen BB (1996) Complex burrows of the mud shrimp Callianassa truncata and their geochemical impact in the sea bed. Nature 382: 619–622.

Sulfur Cycle P Lens, Wageningen University, Wageningen, The Netherlands ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Planetary Sulfur Fluxes The Microbial Sulfur Cycle Sulfur Cycling within the Ecosystems

Glossary acidophiles Bacteria that prefer acidic conditions, that is, microbes with a low pH optimum, typically below 4. assimilatory reduction Reduction of a compound for the purpose of introducing its building elements into cellular material. dissimilatory reduction Reduction of a compound in an energy-yielding reaction. The element does not become incorporated into cellular material. sulfate reducing bacteria (SRB) Name of a group of bacteria belonging to a diversity of genera that gain their metabolic energy from the reduction of sulfate into sulfide. sulfide oxidizing bacteria Name of a group of bacteria belonging to a diversity of genera that gain their metabolic energy from the oxidation of sulfide into sulfate. Under certain conditions, the oxidation is incomplete and stops at elemental sulfur, thiosulfate, or sulfite. sulfur cycling A natural environmental process where sequential transformation reactions convert the sulfur atom in its different valence states ranging from –2 to þ6. The reactions of the sulfur cycle alter the chemical,

Abbreviations COS CS2

carbonyl sulfide carbonyl disulfide

Environmental Consequences and Technological Applications Further Reading

physical, and biological status of sulfur and its compounds so that sulfur cycling can occur. Many of the reactions of the sulfur cycle are mediated by microorganisms. The transformation reactions involved represent a continuous flow of sulfur-containing compounds among the various compartments (soil, water, air, and biomass) of the earth. Besides cycling of sulfur on a macroscale, internal sulfur cycles within one compartment exist. These internal cycles depend on the gradients of oxygen and sulfur compounds, which can occur on a large scale, for example, in stratified lakes or on a micrometer scale, for example, in laminated mats and wastewater treatment biofilms. Disrupture of the sulfur cycle can lead to several serious environmental problems. On the other hand, sulfur biotransformations are the basis of a whole set of environmental bioremediation technologies. sulfur reducing bacteria Name of a group of bacteria belonging to a diversity of genera that gain metabolic energy from the reduction of elemental sulfur into sulfide. sulfuretum Habitat with a complete sulfur cycle (plural: sulfureta).

DMS NMR SRB

dimethyl sulfide nuclear magnetic resonance sulfate reducing bacteria

Defining Statement

Planetary Sulfur Fluxes

The sulfur cycle plays an important role in global planetary geochemical cycles, in which microorganisms play a key role. The sulfur bioconversions are carried out by specialized, mainly Eubacterial genera. Unbalanced sulfur cycling in ecosystems can have devasting effects, whereas environmental biotechnology engineers sulfur cycle activities to benign processes.

Sulfur is the 8th most abundant element in the solar atmosphere and the 14th most abundant element in the earth’s crust. Sulfur is present in several of the large environmental compartments on earth (Table 1). The lithospheric compartment is the largest and contains roughly 95% of this element. The second largest compartment is the hydrosphere and the earth’s oceans

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containing reduced sulfur, for example, dimethyl sulfide (DMS), carbonyl sulfide (COS), and carbonyl disulfide (CS2), are volatile and can escape to the atmosphere. From there, sulfur compounds are redeposited into the litho-, hydro-, and pedosphere, either directly or after conversion to sulfate, via the gaseous intermediate SO2. Sulfide has high chemical reactivity with some metal cations, leading to the formation of poorly soluble metal sulfides. This results in an accumulation of solid-state reduced-sulfur stocks within the global planetary sulfur cycling, due to the formation of insoluble metal sulfides in most anaerobic environments, like marshes, wetlands, freshwater, and sea sediments all over the globe. The formation of solid-state reduced sulfur proceeded already from early history of the planetary biogeochemical cycling. Accumulation of sulfur in anaerobic, highly organic rich deposits of biomass resulted in the contamination of coal by metal sulfides as well as organic bond sulfur compounds (sulfur content ranging between 0.05 and 15.0%). Similarly, mineral oils and petroleum can contain substantial amounts of sulfur compounds (0.025–5%). Under certain conditions, large – commercially exploitable – quantities of elemental sulfur can accumulate in petroleum reservoirs (see ‘Sulfur cycling within the ecosystems’). Another stock of accumulated solid-state reduced sulfur are the sulfidic ores, for

Table 1 Amount of sulfur present in various components of the earth Component

Amount of sulfur (kg)

Atmosphere Lithosphere Hydrosphere Sea Freshwater Marine organisms Pedosphere Soil Soil organic matter Biosphere

4.8  109 2.4  1019 1.3  1018 3.0  1012 2.4  1011 2.6  1014 1.0  1013 8.0  1012

contain approximately 5% of the total sulfur. Sulfate is the second most abundant anion in seawater. The other compartments in which sulfur is found together comprise 2500 million years ago) – in units older than 2000 million years, the known record of preserved microbes becomes increasingly sparse and patchy, and the history of the various fossil lineages becomes increasingly difficult to decipher.

Stromatolites Stromatolites are laminated rock structures, most commonly composed of carbonate minerals (e.g., calcite, CaCO3) that are formed by the microbially mediated accretion of laminae, layer upon layer, from the surface of an ancient seafloor or lake bottom. Their layered structure reflects the photosynthetic metabolism of the mat-building microorganisms. The thin (millimeterthick) mats formed as such microbes multiplied and spread across surfaces were intermittently veneered by detrital or precipitated mineral grains that blocked sunlight. To maintain photosynthesis, mobile members of such communities, for example, gliding cyanobacterial oscillatoriaceans, moved upward through the accumulated mineral matter to establish a new overlying microbial mat. The repeated accretion and subsequent lithification of such mats, their microbial community commonly augmented by an influx of nonmobile microbes (such as chroococcacean, entophysalidacean, and pleurocapsacean colonial cyanobacteria), can result in the formation of stromatolitic structures that range from small millimetric columns and pustular mounds to large decimetric bioherms. During diagenesis, the series of changes that lead to the lithification of such

deposits, silica (SiO2) or, more rarely, phosphate-containing minerals, can replace the initially deposited carbonate grains. If replacement occurs early, before the mat-building microorganisms decay and disintegrate, cellularly intact microbes can be preserved. However, the great majority of stromatolites, unchanged by such replacement, are devoid of fossil microbes: during diagenesis, carbonate grain growth crushes and obliterates the stromatolite-forming microbes, leaving only an amorphous, thin coaly residuum of microbe-derived carbonaceous matter.

Cellular Fossils Two principal processes preserve cellular microbial fossils: compression and permineralization. Compressionpreserved microbes occur in fine-grained detrital sediments such as shales and siltstones, pressed and flattened along bedding planes as the sediment lithifies. Such fossils are rare in sediments of the Phanerozoic, evidently because of the bioturbation of such deposits by invertebrate metazoans, but are well known from the Precambrian. The microbial fossil record is best known from microorganisms preserved by permineralization. Of all modes of fossil preservation, this process (also known as ‘petrification’) provides the most faithful representation of original biologic morphology. Such preservation, common for plants and fungi as well as fossilized microbes, results from the pervasion of mineral-charged solutions into cells during the early

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stages of diagenesis, prior to their disintegration. The permeating fluids infill micellar, intercellular, and intracellular spaces – replacing the watery milieu of their biomolecular components – to produce a mineral-infused inorganic– organic mix that can preserve physically robust structures such as organic-rich cell walls. As a result, both the organismal morphology and cellular structure of such fossils can be preserved in microscopic detail. Although permineralized microbes have been reported from a variety of matrices (e.g., carbonate, phosphorite, and pyrite), the most common matrix is silica, the primary mineral that comprises the rock type known as chert or flint. Hundreds of microbepreserving cherts are known from the Precambrian when silica was abundant in the world’s oceans, well before the Phanerozoic appearance of silica-mineralized sponges, diatoms, and radiolarians that today mediate the oceanic silica budget. Such cherts can contain exquisitely preserved fossil microbes.

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Stromatolites: Lithified Microbial Mats Carbonate microbial stromatolites occur today (Figures 2(a), 2(b), and 2(d)) that in shape, size, environmental setting, and laminar structure are much like those known from the geological past (Figures 2(c), 2(e), and 2(f)). In Phanerozoic sediments, fossil stromatolites are rather uncommon, evidently because of the disruption of stromatolite-forming microbial mats by grazing and burrowing metazoans. But in shallow-water carbonate deposits throughout the Precambrian such structures are abundant and morphologically diverse, the earliest known being from rocks 3500 million years old (Figures 2(f), 3, and 4). Their distribution over time parallels that of the Precambrian rock record that has survived to the present: stromatolites are common in younger-aged Precambrian deposits but become more rare as the rock record peters out with increasing age (Figure 4). Such structures establish the presence of flourishing photosynthesis-based microbial communities, but because of their carbonate mineralogy they do not contain cellular fossils that might indicate whether the stromatolite-building photoautotrophs were oxygenic, like cyanobacteria, or anoxygenic, like photosynthetic bacteria.

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Figure 2 Modern and fossil stromatolites. (a) Modern stromatolites at Shark Bay (Hamelin Pool), Western Australia; (b) modern Shark Bay columnar and domical stromatolites for comparison with (c) fossil stromatolites from the 2300-Ma-old Transvaal Dolomite, Cape Province, South Africa; (d through f) modern and fossil vertically sliced columnar to domical stromatolites showing accretionary microbial lamination from Shark Bay (d), the 1300-Ma-old Belt Supergroup of Montana (e), and the 3350-Ma-old Fig Tree Group of the Eastern Transvaal, South Africa. Scale for (a) and (c) shown by the geologic hammers enclosed by red circles.

Taxonomy Paleontological taxa

Although current microbial classifications are based primarily on biomolecular data (e.g., rRNA, genome, and DNA base compositions), for many modern microbial taxa such assignments are consistent with traditional morphology-based groupings. For example, cyanobacteria, the

principal stromatolite formers over geologic time, have been assigned on the basis of their pattern of structure and development to five ‘sections’ (cf. morphology-defined taxonomic families) that for many genera mesh well with biochemically based classifications. Section I

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Carawine dolomite Ghaap plateau dolomite Malmani dolomite Chitradurga schist belt Schmidtsdrift subgrp. Cheshire fm. Snofield lake Black river volcanic complex Jeerinah fm. Black flag grp. Kwekwe area Ascot vale Manjeri fm. Zwankendaba grp. Deogiri fm. Joldhal fm. Dharwar spgrp. Bothaville fm. Rietgat fm. Klippan fm. Madina fm. Tumbiana fm. Helen iron-fm. Jurtel volcanic complex Lionora area Kylena fm. Muskrat dam greenstone belt Hardey fm. Mount roe basalt Vanivalas fm. Sebakwian grp. Steeprack grp. Keeyask metasediments Woman lake marble Mushandike fm. Bull assemblage Insuzi grp. Cattle well fm. Dixon island fm. Sheba fm. Buck reef chert Kromberg fm. Strelley pool chert Witkop fm. Panorama fm. Hooggenoeg fm. Mount ada fm. Dresser fm.

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Figure 3 Archean-age microbially laminated stromatolites. (a) domical, pseudocolumnar, and branching stromatolites, overlain by rippled sediments; (b) a domical stromatolite from the 2723-Ma-old Tumbiana Formation, Fortescue Group, Western Australia; (c) conical stromatolite; (d) stratiform and conical stromatolites from the 2985-Ma-old Insuzi Group, South Africa; (e through g) laterally linked conical stromatolites from the 3388-Ma-old Strelley Pool Chert of Western Australia.

(cf. Chroococcaceae) consists of predominantly spheroidal solitary and colonial unicellular cyanobacteria that reproduce by fission or by budding. Section II (cf. Pleurocapsaceae) consists of unicellular or pseudofilamentous forms that give rise to small daughter cells (baeocytes) by multiple fission. Section III (cf. Oscillatoriaceae) encompasses uniseriate cyanobacterial filaments that lack cellular differentiation (e.g., Oscillatoria and Spirulina). Section IV (cf. Nostocaceae) includes simple uniseriate filaments that exhibit cellular differentiation into akinetes and heterocysts. Section V (cf. Stigonemataceae) is composed of morphologically more complex filamentous cyanobacteria that exhibit true branching and heterocysts. Representatives of all of these groups are known from the fossil record: Sections I–IV dating from well into the Precambrian (Figure 1) whereas representatives of Section V are known earliest from Stigonemalike fossils of 400 million-year-old Rhynie Chert of Scotland. In general, Sections II, IV, and V are consistent with biochemically based phylogenies whereas cyanobacteria included in Sections I and III do not appear to comprise monophyletic lineages. Nevertheless, because the biochemical components of microbes, like those of all living systems, are geochemically labile – converted over geological time to coaly kerogen, a complex mix of interlinked polycyclic aromatic hydrocarbons – the classification of such

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fossils is necessarily based on their morphology, not on their original biochemistry.

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Identification of the major fossil types

As is shown in Figure 1, four major categories of ancient microbes are known from the fossil record: (1) cyanobacteria; (2) fossils of uncertain systematic position (bacteria incertae sedis); (3) sulfate-reducing bacteria; and (4) methaneproducing archaeans. Of these, the microbial fossils incertae sedis are all regarded as members of the bacterial rather than the archaeal domain, the uncertainty of their systematic position reflecting their morphological similarity both to cyanobacteria and to members of other bacterial groups. The sulfate reducers and the methane producers are known only from isotopic evidence, not from morphologically preserved microscopic fossils. Of the four categories, cyanobacteria have the best-documented fossil record, known from thousands of cellularly preserved specimens. Many such cyanobacterial fossils are essentially indistinguishable from their modern morphological counterparts in life cycle, behavior, and ecologic setting. In comparison with other microbes, whether bacterial or archaeal, most cyanobacteria have somewhat larger cells and more complex morphology. Because of their light-requiring oxygenic photosynthetic metabolism, cyanobacteria occupy the uppermost surface of microbial mats, rather than the lower regions of such biocoenoses where decay and cellular disintegration are prevalent. For this reason, cyanobacteria have a higher probability of becoming incorporated in the fossil record as cellularly intact specimens than do other prokaryotes, especially if they are preserved by permineralization during the early stages of sediment lithification. Of the various morphological components of cyanobacteria, extracellular sheaths and envelopes, initially composed largely of pentose sugar-containing carbohydrates and relatively resistant to degradation, are the most commonly preserved. Although physically robust and organic-rich, cell walls are somewhat less commonly preserved, and in the cells of fossilized filamentous forms the originally peptidoglycan-containing lateral walls are much more commonly preserved than their peptidoglycan-deficient transverse walls. The organic components of the aqueous cytosol of such cells are never preserved intact. Along with their watery milieu, such organics are usually leached out of the cells during preservation, but small aggregates of intracellular carbonaceous remnants occur in some cyanobacterial fossils. Cyanobacteria Coccoidal and ellipsoidal fossils (Chroococcaceae, Entophysalidaceae, and Pleurocapsaceae)

Figure 5 shows photomicrographs that illustrate the morphological similarities of extant coccoidal and ellipsoidal

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Figure 5 Modern and fossil coccoidal and ellipsoidal cyanobacteria; all fossils are shown in petrographic thin sections of stromatolitic chert. (a) Modern Gloeocapsa sp. (Chroococcaceae) for comparison with (b) Gloeodiniopsis uralicus from the 1500-Ma-old Satka Formation of Baskiria, Russia; (c) confocal laser scanning micrograph; (d) optical photomicrograph of Myxococcoides inornata, an ensheathed colonial chroococcacean from the 650-Ma-old Chichkan Formation of southern Kazakhstan; (e) modern Entophysalis sp. (Entophysalidaceae) for comparison with (f) Eoentophysalis belcherenisis from the 2100-Ma-old Kasegalik Formation, Belcher Group, of Northwest Territories, Canada; (g) Modern Pleurocapsa sp. (PCC 7327, Pleurocapsaceae) for comparison with (h) Paleopleurocapsa reniforma from the 650-Ma-old Chichkan Formation of southern Kazakhstan.

cyanobacteria (chroococcaceans, entophysalidaceans, and pleurocapsaceans; Figures 5(a), 5(e), and 5(g), respectively) to their fossilized counterparts (Figures 5(b), 5(f), and 5(h)). Figure 5(c) shows an image of a sheathenclosed chroococcacean colony within a thin slice of rock, obtained by the use of confocal laser scanning microcopy (CLSM), for comparison with an optical

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image of the same specimen (Figure 5(d)). Use of CLSM, recently introduced to such studies, provides high-resolution three-dimensional images of such rockembedded fossils that are unavailable from standard photomicrography. The morphological similarity of the fossil and modern cyanobacteria shown in Figure 5 is typical of chroococcaceans, entophysalidaceans, and pleurocapsaceans generally, groups that have established fossil records dating from more than 2500 million years ago (Figure 1). Figure 6 shows a fossil stalk-forming pleurocapsacean that provides evidence of its life cycle (inferred from specimens preserved at varying stages in its development), behavior (evidenced by the orientation of its elongate stalks), and ecologic setting (documented by paleoenvironmental facies relationships). Like its modern morphological and ecological counterpart, Cyanostylon of the Bahamian tidal banks, this ancient pleurocapsacean colonized surfaces by producing laterally extensive pincushion-like aggregates. The virtually identical morphology, life cycle, behavior, and ecologic setting of this fossil pleurocapsacean and its living counterpart are typical of the coccoidal and ellipsoidal cyanobacteria known from the geologic record.

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Filamentous taxa (Oscillatoriaceae, Nostocaceae)

Like coccoidal and ellipsoidal cyanobacteria, modern and fossil filamentous members of the group commonly exhibit essentially identical ecologic settings and organismal and cellular morphologies. Among the several extant families of filamentous cyanobacteria, stromatolitebuilding members of the Oscillatoriaceae have the most extensive fossil record, represented by diverse fossils in hundreds of ancient microbial communities (Figure 7). However, detailed studies of fossil oscillatoriaceans show that their optically discernible morphology (Figures 7(b), 7(d), 7(f), and 7(h)) does not always accurately reflect their true structure. In modern oscillatoriaceans, cells divide by the centripetal invagination of partial septations that fuse in the center of a cell to produce transverse cell walls (Figure 8). The lateral cell walls of such filaments are about twice the thickness of their transverse walls, and they contain rigidifying peptidoglycans that are absent from partial septations and transverse walls except at the cell periphery. Because of these differences, lateral cell walls tend to be relatively well preserved in fossil specimens whereas transverse walls, and their precursor partial septations, are preserved only in part. A fossil oscillatoriacean exhibiting such differential preservation is illustrated in Figure 9 in which an optical image of the specimen (Figure 9(c)) is compared with images obtained by use of CLSM (Figures 9(d)–9(h)) and three-dimensional Raman imagery (Figures 9(i)–9(k)). The Raman images show that the biopolymers originally comprising the cell walls have been converted to carbonaceous

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Figure 6 Specimens of the colonial stalk-forming pleurocapsacean cyanobacterium Polybessurus bipartitus in petrographic thin sections of stromatolitic chert from the 775Ma-old River Wakefield Subgroup of South Australia. (a through c) Vertically oriented and radiating, pincushion-like originally mucilaginous extracellular stalks shown in longitudinal (a) and transverse sections (b and c); (d) a single, mucilage-embedded, ellipsoidal cell at the upper surface of the colony; (e and f) fossilized asymmetrically laminated mucilaginous stalks; (f) capped by the stalk-forming cell; (g) interpretive drawings (based on tracings of photomicrographs) showing from (a) through (d) the ontogeny of stalk formation.

kerogen. Nevertheless, they and the CLSM images document the presence of thin incompletely preserved partial septations, showing that this ancient oscillatoriacean divided by the same processes, and in a living state evidently exhibited the same wall structure composed of the same biochemicals, as extant members of the family. Data such as these establish that the fossil record of the Oscillatoriaceae extends deep into geological time (Figure 1) and that such cyanobacteria have changed little or not at all over thousands of millions of years of evolutionary history.

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Figure 8 Schematic drawing, based on transmission electron micrographs, of a medial section of a cell of a modern filamentous oscillatoriacean cyanobacterium showing the symmetrically distributed primary and secondary partial septations that with ingrowth give rise to new medial cells. Modified from Schopf JW and Kudryavtsev AB (2005) Geobiology 3: 1–12.

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(h) 10 µm Figure 7 Modern and fossil filamentous (oscillatoriacean) cyanobacteria; fossils in (b) and (d) are shown in petrographic thin sections of stromatolitic chert whereas those in (f) and (h) have been separated from their siltstone matrices by acid maceration. (a) Modern Oscillatoria sp. for comparison with (b) Oscillatoriopsis breviconvexa from the 750-Ma-old Bitter Springs Formation of central Australia; (c) Modern Oscillatoria amoena for comparison with (d) Cephalophytarion grande from the 750-Ma-old Bitter Springs Formation of central Australia; (e) Modern Lyngbya sp. for comparison with (f) Paleolyngbya helva from the 950-Ma-old Lakhanda Formation, Siberia, Russia; (g) Modern Spirulina sp. for comparison with Heliconema turukhania from the 850-Ma-old Miroedikha Formation, Siberia, Russia.

In comparison with the fossil record of oscillatoriaceans, that of similarly filament-forming nostocaceans is poorly known. A prime characteristic of the Nostocaceae is the presence of intercalary heterocysts, thick-walled cells that protect the nitrogenase enzyme complex used to fix atmospheric nitrogen from oxidation. Such heterocysts develop when nostocaceans are

deprived of other sources of usable nitrogen (viz., nitrate and ammonia). In stromatolitic microbial biocoenoses, however, bacterially generated ammonia is plentiful, so the nostocaceans of such communities, both fossil and modern, are not heterocystous (such differentiated cells being earliest known from Stigonema-like Section V fossils of the nonstromatolitic Devonian Rhynie Chert). Nevertheless, based on organismal and cellular morphology, numerous ancient fossils have been assigned to the Nostocaceae. One such example is shown in Figure 10(b), compared with modern Nostoc (Figure 10(a)). Figures 10(c)–10(e) show images of a fossil akinete, a reproductive structure characteristic of nostocaceans that is known from geologic units dating to 2100 million years ago. The temporal distribution of the Nostocaceae shown by such fossils fits with other lines of evidence. Geochemical data indicate that a stable oxygenrich environment became established 2300 million years ago, before which the nitrogenase-protecting heterocysts of akinete-producing nostocaceans would have been of little selective advantage. Further, rRNA phylogenies indicate that the Nostocaceae, like other heterocystous cyanobacterial families, originated in a burst of evolution well after the appearance of coccoidal, ellipsoidal, and nonheterocystous filamentous oscillatoriaceans. Like other cyanobacteria, nostocaceans appear to have evolved little or not at all since their origination more than 2000 million years ago (Figure 1).

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Figure 9 Modern and fossil oscillatoriacean cyanobacteria showing partial septations; (a through c) show optical photomicrographs; (d through h) show confocal laser scanning micrographs; and (i through k) show three-dimensional Raman images (acquired in a spectral window centered in the kerogen ‘G’ band at 1605 cm 1 that establishes the carbonaceous composition of the fossil). (a and b) Two specimens of modern Oscillatoria sp. that exhibit well-defined partial septations (at arrows). (c) Optical image of the morphologically similar fossil Oscillatoriopsis media, in a petrographic thin section of stromatolitic chert from the 650-Ma-old Chichkan Formation of southern Kazakhstan, in which the red rectangles denote the areas of the trichome shown in (d), (e), and (f through k); (d) the trichome terminus, showing its rounded end cell and subtending disk-shaped medial cells. (e) A part of the trichome that exhibits well-defined partial septations (at arrows). (f through h) A second part of the trichome that exhibits partial septations (at arrows), in (f) showing the trichome as viewed from above its upper surface, in (g) with the image of the trichome tilted slightly to the right to show its interior, and in (h) with the image rotated to show the trichome as viewed from its side. (i) A Raman (chemical) image of the part of the specimen denoted by the red rectangle in (f), as viewed from above the trichome. (j) The part denoted in (g), titled slightly to the left; (k) the part denoted in (h), rotated to show the specimen from its side.

Bacteria Incertae Sedis

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Figure 10 Modern and fossil nostocacean cyanobacteria; (a through c) show optical photomicrographs; (d and e) show confocal laser scanning micrographs. (a) Modern Nostoc sp. (PCC 7936) for comparison with (b) Veteronostocale amoenum shown in a petrographic thin section of stromatolitic chert from the 750-Ma-old Bitter Springs Formation of central Australia; (c through e) Archaeoellipsoides longus, a reproductive akinete characteristic of nostocacean cyanobacteria, from the 650-Ma-old Chichkan Formation of southern Kazakhstan, in (c and d) shown as viewed from above the specimen, and (e) shown from its lower side.

Fossils regarded as bacteria incertae sedis – that is, fossil prokaryotes of the bacterial domain that cannot be firmly identified as members of any particular extant microbial group – are known throughout the geological record. Such remnants constitute the great majority of the fossils now known from Archean-age rocks. Because of geologic recycling, and the resulting decrease in the preserved rock record with increasing geological age, the record of Archean fossils is sparse, reported from only some 20 Archean units, 2500 to 3500 million years in age, and represented by only six fossil morphotypes. Of these units, 13 date from the interval between 3200 and 3500 million years ago, well documenting the existence of microbe-level life at least this early in Earth history. For most of these ancient microbes, the uncertainty in their classification stems from their morphological similarity both to cyanobacteria and to noncyanobacterial bacteria.

Representative fossils of uncertain systematic position

The specimens shown in Figures 11 and 12 are representative of the Archean microscopic fossils now known. Rod-shaped fossils (Figures 11(a)–11(f)), small-diameter coccoids (Figures 11(g)–11(r)), and narrow filaments (Figures 11(s)–11(y)) predominate, but larger-diameter uniseriate cellular microbes also

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Figure 11 Archean-age bacterial microfossils incertae sedis (of uncertain systematic position), shown in petrographic thin sections. (a through f) Solitary and paired rod-shaped; (g through l) coccoidal unicells from the 2600-Ma-old Monte Cristo Formation of the Eastern Transvaal, South Africa; (m through r) coccoidal unicells from the 3260-Ma-old Swartkoppie Formation of Swaziland, South Africa, in (n) through (r) ordered in a sequence inferred to represent stages of cell division; (Knoll AH and Barghoorn ES (1977) Science 198: 396–398); (s) narrow filamentous fossil from the 3320-Ma-old Kromberg Formation of Swaziland, South Africa; (t through y) narrow filaments (Archaeotrichion contortum) from chert units of the 3470-Ma-old Mount Ada Basalt of northwestern Western Australia.

occur (Figure 12). Some of the small-celled (Figures 11(a)–11(f)) and narrow filamentous forms (Figures 11(s)–11(y)) seem likely to be noncyanobacterial bacteria, but others (Figures 11(g)–11(r)) have morphologies consistent with their assignment either to relatively large-sized noncyanobacterial bacteria or to small-sized cyanobacteria. Similarly, the relatively large-diameter uniseriate trichomes shown in Figure 12 are morphologically similar to extant and fossil cyanobacteria (e.g., Figures 7(a)–7(f)), but are also comparable to large-celled sulfur-metabolizing bacteria (e.g., Beggiatoa). A firm taxonomic assignment of such fossils is not yet possible.

Carbon and sulfur isotopic evidence of ancient microbes

The carbon isotopic record of photosynthesis, based on thousands of analyses, shows that Earth’s ecology has been dominated by photoautotrophic primary producers since at least as early as 3500 million years ago. Such data are consistent with carbon fixation by Rubisco (ribulose bisphosphate carboxylase/oxygenase), the CO2-capturing enzyme of cyanobacteria (as well as

of photosynthetic protists and higher plants). But because of the mixing of carbonaceous matter from diverse sources that occurs during the deposition of sediments and the alteration of carbon isotopic compositions that can occur during geological metamorphism, these data do not indicate whether in early Earth history the dominant primary producers were cyanobacteria, anoxygenic photosynthetic bacteria, or anoxygenic chemosynthetic bacteria. Other carbon isotopic evidence, measured in carbonaceous matter so enriched in C12 as to be plausibly derived only from CH4-metabolizing methanotrophs, indicates that anaerobic methanogenic archaeans were present 3500 million years ago (Figure 1) and that such microbes were a major component of the ecosystem prior to the establishment of a stable oxygen-rich environment 2300 million years ago. Similarly, isotopic analyses of sedimentary pyrite (FeS2), enriched in S32 due to microbial activity, show that sulfate-reducing bacteria were also present in Earth’s earliest biota (Figure 1). The occurrence of C12-rich graphitic carbon in the oldest sedimentary rocks now known, from Akilia Island off southwestern Greenland, suggests that microbes may have existed as early as 3800 million years ago.

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identical morphologies, life cycles, behaviors, and ecologic settings of modern and fossil members of the group. Isotopic data suggest that such stasis may be characteristic of other microbial lineages as well – or of the enzyme systems involved in the production of the preserved isotopic signatures – but the evidence is not conclusive, since fossils morphologically identifiable as archaeal methanogens or bacterial sulfate reducers are yet to be reported. As for cyanobacteria, their early evolutionary success can be attributed to their photosynthetic production of gaseous oxygen, a toxin to the earlier-evolved anaerobic photosynthetic bacteria with which they initially competed for photosynthetic space. Once established, their evolutionary stasis (their hypobradytelic rate of evolution) was presumably a result in part of their asexual reproduction and the lack of genetic variability it provides. But the prime source of such stasis seems almost certainly to be a result of their exceptional ecologic tolerance. Over their exceedingly long evolutionary history, cyanobacteria adapted to the slowly changing global environment – from anoxic to oxygenic, UV-rich to UV-deficient, CO2-rich to CO2deficient, short to increasingly longer daylengths, and, perhaps, from relatively high ambient temperatures (60  C) to that of the present-day Earth (15  C). The genomes of cyanobacteria thus encode a history of adaptation virtually unparalleled by any other microbial group. Whether such marked evolutionary stasis is typical of other microbial lineages is yet to be determined. See also: Archaea (overview); Cell Structure, Organization, Bacteria and Archaea; Cyanobacteria; Ecology, Microbial; Methanotrophy/methane oxidation; Stable Isotopes in Microbial Ecology

Figure 12 Archean-age cyanobacterium-like microfossils incertae sedis (of uncertain systematic position), shown in petrographic thin sections. (a–d) Four specimens of Primaevifilum septatum from a chert unit of the 3465-Ma-old Apex Basalt of northwestern Western Australia; (e) a morphologically similar specimen of P. septatum from a chert unit of the 3470-Ma-old Mount Ada Basalt of northwestern Western Australia.

Fossil Evidence of Microbial History Evolutionary Stasis Numerous lines of evidence indicate that microbial communities composed of members of diverse microbial lineages have been extant since early in Earth history (Figure 1). Perhaps the most striking evidence is that showing the evolutionary stasis of cyanobacteria, the best documented of such lineages, over a vast segment of geological time. Such stasis may or may not be characteristic of microbial lineages generally, but for cyanobacteria it is established firmly by the essentially

Further Reading Giovannoni SJ, Turner S, Olsen GJ, Barnes S, Lane DJ, and Pace NR (1988) Evolutionary relationships among cyanobacteria and green chloroplast. Journal of Bacteriology 170: 3585–3592. Herdman M, Janvier M, Waterbury JB, Rippka R, Stanier RY, and Mandel M (1979) Deoxyribonucleic acid base composition of cyanobacteria. Journal of General Microbiology 111: 63–71. Knoll AH (2008) Cyanobacteria and Earth history. In: Herreo A and Flores E (eds.) The Cyanobacteria, pp. 1–19. Norfolk, UK: Caiser Academic Press. Rippka R, Deruelles J, Waterbury JB, and Stanier RY (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111: 1–16. Schopf JW (ed.) (1983) Earth’s Earliest Biosphere, Its Origin and Evolution. Princeton, NJ: Princeton University Press. Schopf JW (1995) Disparate rates, differing fates: The rules of evolution changed from the Precambrian to the Phanerozoic. In: Fitch WM and Ayala FJ (eds.) Tempo and Mode in Evolution, Genetics and Paleontology 50 Years after Simpson, pp. 41–61. Washington, DC: National Academy Press. Schopf JW (1996) Are the oldest fossils cyanobacteria? In: Roberts DM, Sharp P, Alderson G, and Collins M (eds.) Evolution of Microbial Life, Society for General Microbiology

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Symposium 54, pp. 23–61. Cambridge, UK: Cambridge University Press. Schopf JW (1999) Cradle of Life – The Discovery of Earth’s Earliest Fossils, Princeton, NJ: Princeton University Press. Schopf JW (2000) The paleobiologic record of cyanobacterial evolution. In: Brun YV and Shimkets LJ (eds.) Prokaryotic Development, pp. 105–129. Washington, DC: American Society for Microbiology Press. Schopf JW (2006) Fossil evidence of archaean life. Philosophical Transactions of the Royal Society of London B 361: 869–885.

Schopf JW and Klein C (eds.) (1992) The Proterozoic Biosphere: A Multidisciplinary Study, New York: Cambridge University Press. Schopf JW and Kudryavtsev AB (2005) Three-dimensional Raman imagery of Precambrian microscopic organisms. Geobiology 3: 1–12. Schopf JW, Kudryavtsev AB, Czaja AD, and Tripathi B (2007) Evidence of Archean life: Stromatolites and microfossils. Precambrian Research 158: 141–155. Schopf JW, Tripathi A, and Kudryavtsev AB (2006) Three-dimensional confocal optical microscopy of Precambrian microscopic organisms. Astrobiology 6: 1–16.

FUNGI Contents Aspergillus: A Multifaceted Genus Clavicipitaceae: Free-Living and Saprotrophs to Plant Endophytes Endophytic Microbes Entomogenous Fungi Fungi: Plant Pathogenic Yeasts

Aspergillus: A Multifaceted Genus C Scazzocchio, Institut de Ge´ne´tique et Microbiologie, Universite´ Paris-Sud, Orsay, France Department of Microbiology, Imperial College London, London, UK ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Note What is Aspergillus? Food Contamination by Aspergilli Aspergillus as Pathogens

Abbreviations APC HMG-CoA

anaphase-promoting complex 3-hydroxy-3-methylglutaryl-coenzyme A

Defining Statement In this article, different aspects of ongoing work in the genus Aspergillus are discussed, ranging from toxin production, pathogenicity to humans and animals, traditional and modern biotechnological uses, genomics, and the use of Aspergillus nidulans as a model organism to study fundamental problems of cell and molecular biology.

Note For Aspergillus genes and proteins, the standard nomenclature is followed, for example, the benA gene encodes the BenA protein, which is a -tubulin. When genes or

Useful Aspergilli A. (Emericella) nidulans as a Model Organism The Genus Aspergillus in the Genomic Era Further Reading

ORF ROS

open reading frame reactive oxygen species

proteins of other species are mentioned, the standard nomenclature for each species is used.

What is Aspergillus? An Aspergillum is an instrument used in the Roman Catholic mass to sprinkle holy water over the heads of the faithful. Aspergillus is a genus of the ascomycete fungi (see below). In 1729, Pietro Antonio Micheli, priest and botanist, described the asexual spore heads (conidiophores; see below) of a number of common molds. The heads of some of these molds showed rows of spores radiating from a globular central structure, which he thought resembled the Aspergillum he was familiar with. The morphology of the

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Figure 1 The left panel shows a scan of copper-engraving 91 from Micheli’s Nova plantarum genera, showing his drawings of Aspergillus conidiophores. The description in Micheli’s text suggest that Figure 1 of the engraving, called Aspergillus capitatus (muffa turchina, blue mold) by Micheli, may correspond to Aspergillus fumigatus or a close relative. The right panel shows on the top a scanning electron micrograph of the conidiophore of Aspergillus nidulans and at the bottom an epifluorescence micrograph. The preparation is stained with DAPI which reveals the nuclei of the conidia and of the subjacent structures of the conidiophore. Both pictures on the right panel have kindly provided by Reinhard Fischer. Reproduced with permission from Kues U and Fischer R (eds.) (2006) The Micota I, Growth Differentiation and Sexuality. Berlin: Springer-Verlag. Micheli describes in his text the condiophore as been formed by a stalk, and a head, which he called ‘placenta’ carrying the conidia. See legend to Figure 13 for the correspondence with the modern terminology.

conidiophore is still an essential taxonomic marker. Figure 1 compares the original Micheli’s drawing with modern observations of conidiophores. The classification of the kingdom fungi into major groups (Phyla, such as Ascomycota and Basidiomycota) is based on the morphology of the sexual reproductive structures. The Aspergilli should be placed among the ascomycetes (see below), those fungi that have the products of meiosis placed in a sac or ascus. However, most of the fungi we call Aspergilli (see below) do not have sexual reproduction, and thus no asci. To solve this problem, mycologists created the group fungi imperfecti or otherwise called deuteromycetes in which they placed all fungi without known sexual reproduction. This is a mixed bag without any phylogenetic significance. This provokes ridiculous situations, by which a fungus would change genus, and in fact phylum, every time sexual reproduction is detected. Thus the ‘imperfect’ fungus Aspergillus nidulans (see below) becomes the ‘perfect’ fungus Emericella nidulans, and it is placed in a different phylum from Aspergillus sydowii, in spite of the fact that morphological and molecular data show the two organisms to be close relatives. Names like Emericella, Eurotium, and Neosartorya design Aspergilli with a sexual cycle (also called teleomorphs). Thus, E. nidulans is the teleomorph (perfect form) of A. nidulans (anamorph, imperfect form). No one but a mycologist would know that we are talking about one and same organism. This situation exists for many other genera. The only solution to this conundrum is to completely abandon the

division ‘fungi imperfecti’ and choose in each case one and only one name for a given genus. As early as in 1926, Thom and Church, Thom and Raper (1945), and Raper and Fenell (1973) proposed that ‘‘the generic name Aspergillus should be applied to all these fungi whether or not an ascosporic (sexual) stage was produced’’. The main morphological characteristics of the genus, drastically abbreviated from Raper and Fenell (1973), are ‘‘vegetative mycelium consisting of septate branching hyphae . . . Conidial apparatus developed as condiophores . . . conidiophores . . . broadening into turbinate elliptical, hemispherical, or globose fertile vesicles . . . bearing fertile cell or sterigmata . . . conidia (asexual spores) . . . produced successively from the sterigmata. Ascocarps (asci, containing sexual spores) found in certain groups only, unknown in most species’’. Recent work shows that the Aspergilli are as a whole a monophyletic group, where loss of sexual reproduction has occurred many times independently. Not everyone agrees with the reasonable proposal of Raper and Fenell. A recent classification (2005) of the ascomycetes places the Aspergillus-related teleomorph genus names in the kingdom Fungi, subphylum Pezizomycotina, class Pezizomycetes, family Trichocomaceæ. The genera Aspergillus and the related Penicillium do not appear in this list as they are considered imperfect forms! Throughout this article, the generic name Aspergillus is used, as a genus comprising all the related ‘teleomorphs’ together with the forms where sexual reproduction is absent. The teleomorph name is also indicated when appropriate, as this is used in

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some important databases (as NCBI). The kiss of death to the concept of ‘fungi imperfecti’ was delivered by recent molecular data that show that the genomes of ‘imperfect’ Aspergilli, include the genes that determine mating types, these genes being clearly homologous to, and even placed in the same place in the chromosome as, the ones found in the ‘perfect forms’ (‘The genus Aspergillus in the genomic era’). Fungi of the genus Aspergillus, which includes about 200 species, are important in public health as toxin-producing food contaminants, as human and animal pathogens, as useful fungi in traditional and modern biotechnological processes, and finally one species has been used as a model to study a number of cellular processes. A recent development is the availability of eight complete genomes within the genus, a matter of obvious practical, taxonomical, and evolutionary importance.

Food Contamination by Aspergilli Many organisms, including bacteria, fungi, and plants, produce secondary metabolites. These are molecules that can be very complex and are not obviously necessary for the viability of the organism. In fungi, they are produced during the stationary phase and their synthesis is usually coordinated with asexual sporulation (see below). Some secondary metabolites are extremely toxic, and when fungi grow on stored foods, they secrete them, provoking food spoilage and eventually intoxications that may be fatal. Among the Aspergilli, the two main culprits are strains of Aspergillus flavus and Aspergillus parasiticus, which secrete aflatoxins, a group of highly substituted coumarins. Strains that are closely related may vary drastically in their ability to produce the toxin. These saprophytic fungi can grow on a variety of foodstuffs, or even on plants before harvesting. In fact, A. flavus can be considered a weak opportunistic, nonspecific plant pathogen. The aflatoxins were discovered in 1960 when thousands of turkeys died in an English hatchery. The contaminated food was a ground peanut meal. The most serious contamination is that of maize. While this contamination results in loss of hundreds of million dollars every year to farmers in developed countries, the impact on human health is extremely serious in developing countries. Of the related compounds called aflatoxins, Aflatoxin B1 is one of the most toxic and carcinogenic compounds known, as judged by tests on laboratory animals. Maize stored under warm and humid conditions becomes contaminated with aflatoxigenic Aspergilli, and when consumed by humans or animals, this can lead to liver failure and death. Periodic outbreaks of acute aflatoxin poisoning occurred in East Africa, the latest in 2004, leading to 125 deaths. It is more difficult to assess the damage caused by chronic aflatoxin poisoning and the correlation of the toxin in food with the frequency of liver cancer.

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Controls on aflatoxin levels are tight in developed countries; they are, however, impossible to be enforced in developing countries, where people would store grains in their homes and the stored grain may be the only available food. Human aflatoxicosis is a disease of poverty. The Aspergilli can contaminate food with other toxic molecules. Only the ochratoxins, produced by a number of Aspergilli and Penicillia will be discussed below. The ochratoxins comprise an isocumarin moiety and a phenylalanine ring joined by an amide bond. Ochratoxin contamination has been reported in many foodstuffs, including grapes, nuts, cacao, coffee beans, and spices. In poultry, and laboratory animals, ochratoxins provoke serious kidney lesions. It is difficult to assess damage to human health caused by chronic exposure to ochratoxins. They have been implicated as a cause of testicular cancer. The similarity of symptoms of porcine mycotoxin nephropathy with that of Balkan endemic nephropathy, a disease localized to regions of Bulgaria, Romania, and former Yugoslavia, has implicated ochratoxins as causal agents of the disease. A similar case can be made for chronic interstitial nephropathy of northern Africa. A case of acute renal failure, almost certainly due to the exposure to an ochratoxin, has revived the hypothesis that exposure to this mycotoxin is the cause of the ‘mummy curse’, which is alleged to have killed archaeologists who have braved the prohibition to open royal tombs.

Aspergillus as Pathogens The common fungus diseases are mild and superficial, while those that are deep-seated and endanger life are so rare that one man can seldom see enough cases to make any extensive study of them. (Henrici, Presidential Address to the Society of American Bacteriologists, 1939)

Aspergillus as Human Pathogens The emergence of species of the genus Aspergillus as, in many cases, intractable human pathogens, has gone hand in hand with the progress of medicine. All Aspergilli encountered as causal agents of human or animal diseases are opportunistic pathogens. The Aspergilli are all saprophytes, usually growing in decomposing vegetal material. The main pathogen, Aspergillus fumigatus, thrives on compost. Before the transplant era, Aspergillus infections were only encountered sporadically. Farmer’s lung is a general name for an allergic disease that could be due to different causal agents, bacteria or fungi, of which the Aspergilli are the main culprits. It is an occupational disease associated with high exposure of spores, in environments such as grain silos. In the nineteenth century, two exotic occupational diseases associated with A. fumigatus were described: the maladie de gaveurs de pigeons and the maladie de peigneurs de cheveux. These pulmonary diseases were associated

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with people who force-fed pigeons and with people who sorted hair for wigs, respectively. A perusal of the Pathogenesis chapter by Austwick, included in Raper and Fennell’s monograph of 1973, leaves the impression that a large number of Aspergillus species could be opportunistic pathogens, that pulmonary disease was basically an occupational hazard, that virtually every organ could be colonized by one or other Aspergillus species, and that once the fungus was established the prognosis was bleak. Henrici, compared invasive fungal diseases to autocatalytic processes, sluggish to start, but eventually becoming unstoppable. The comparison still holds today, except that immunodepression gives the fungus a head start. A. fumigatus, was then as now, the prevalent species, followed by A. flavus. Three types of respiratory pathologies are associated with the Aspergilli. Exposure to the fungus can result in allergic diseases, such as farmer’s lung and allergic broncopulmonary aspergillosis, encountered mainly in asthmatic and cystic fibrosis patients. Aspergillus spores can germinate in preexisting cavities such as the sinuses or those present in the lung as a result of tuberculosis. This leads to localized Aspergillomas in immunocompetent subjects, which can be treated surgically and/or with appropriate drugs. Finally, the most threatening form is the invasive Aspergillosis, associated, in most but not in every case, with a depression of the immune system. The ability to perform grafts of bone marrow cells in leukemic patients, of solid organs such as kidney, liver, and lung, has been accompanied by the emergence of invasive Aspergillosis. AIDS patients are also at risk, but Aspergillus spp. are encountered less frequently in these patients than Pneumocystis carinii, Candida spp., or Cryptococcus neoformans. Susceptible patients include those affected by neutropenia. Neutropenia can result from leukemia or from the chemotherapy used to control it, or be subsequent to treatment with immunodepressants used in bone marrow, stem cell, or organ transplants. Patients of systemic diseases treated with immunodepressing drugs, mainly corticosteroids, are also at danger. In all these patients, germination of Aspergillus spores leads to invasive aspergillosis, usually of the lung, which breaking through the blood vessels can infect other organs. There seems to be no organ in which the fungus cannot grow in the absence of an appropriate immune response. In almost all cases, spores enter through the respiratory tract and germinate in the parenchyma of the lung, leading to invasion of the bronchiolar walls and the adjacent blood vessels. Invasive Aspergillosis has been classified into angioinvasive and bronchio-invasive forms, but this classification is somewhat artificial, as invasion of both bronchioles and arterioles can be seen in the same patient. This leads eventually to respiratory failure and death. Figure 2 shows an Aspergillus mycelium grown in lung tissue. A very recent review estimates an eight-fold increase in Aspergillus-disseminated infections, from the 1970s to the

Figure 2 A neutropenic mouse lung tissue, experimentally infected with Aspergillus nidulans is shown at 20 h postinfection. The fixed sections were stained with Grocotts Methanamine Silver. The pictures show hyphae (stained brown) actively growing in the lung tissue (stained green). Photograph kindly provided by Elaine Bignell.

present day. Between 9 and 17% of all deaths in transplant recipients are due to Aspergillus infections according to recent data. The prognosis of invasive aspergillosis is grim; mortality in transplant patients infected with Aspergillus sp. is never lower than 60% for patients treated with antifungals and 100% in nontreated patients. The most common encountered species in all Aspergillusrelated pathologies is A. fumigatus; A. flavus, Aspergillus niger, A. nidulans, and Aspergillus ustus have also been recorded. One recent study of nosocomial infection reports that of 458 patients 154 were infected with A. fumigatus and 101 with A. flavus. The same and other studies establish a link between construction or renovation work in the vicinity of the hospital and frequency of invasive aspergillosis and conclude that even very low spore counts (1 spore per m3) are dangerous to immunocompromised patients. Genotyping has shown that there are no specific pathogenic strains and suggests that every A. fumigatus strain present in the environment is a potential risk for immunodepressed patients. Recently, an upsurge of Aspergillus terreus infections has been observed. This is particularly worrying, as the organism is resistant to Amphotericin B, the drug most widely used to treat invasive Aspergillosis. The prevalence of A. fumigatus infection has not been explained. We are all continuously exposed to fungal spores and two obvious factors can be considered to explain the prevalence of one or other species. The first is the spore density in specific environments. Unfortunately, many early studies simply report the density of ‘Aspergillus’ without any further species discrimination, let alone genotyping. It is generally accepted that the high frequency of A. fumigatus infections cannot be explained by a prevalence of the organism in the environment. The second parameter to be considered is spore size. The smaller the spores, the most

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likely they are to reach the alveolar tissue of the lung, as they will be less susceptible to removal by the mucociliary tissue of the respiratory tract. A. fumigatus spores are usually about 2–3 mm in diameter, at the lower end of the genus. Specific gravity of spores has, to my knowledge, never been measured. Another obvious parameter is thermotolerance, especially in relation to spore germination. However, it is unlikely that the combination of small spores and ability to germinate rapidly at 37  C be sufficient to explain the prevalence of A. fumigatus. Both characteristics are shared by A. fumigatus and A. nidulans, the latter being rarely encountered as an opportunistic pathogen. Another possibly interesting parameter is spore hydrophobicity. This is determined by a family of proteins called hydrophobins. Strains of A. fumigatus lacking a specific hydrophobin become more sensitive to macrophage killing. Sensitivity of different species to neutrophil and macrophage killing has been sporadically, but not systematically, assessed. It is important to distinguish putative specific virulence determinants from essential metabolic processes, even if the latter can be potential drug targets. Only those processes, that when blocked, by mutation or otherwise result in reduced virulence but do not affect the growth of the fungus outside infected tissues, can be considered proper virulence determinants. This is of course conditional to the media in which the fungus is tested, my feeling is that the more we know about the metabolism of the fungus in the wild, the less we will be inclined to call a specific metabolic step a ‘virulence determinant’. It is not surprising that engineered strains, deficient in essential biosynthetic pathways, or cell wall biosynthesis show reduced or no virulence. As an example, strains blocked in lysine biosynthesis show reduced virulence, but this tells nothing about virulence, it reveals that lysine is limiting in the alveolar environment. However, as some of these processes are fungal-specific, they are potential targets for antifungal drugs. Secondary metabolites and nonribosomal peptides vary considerably from one fungal species to the other, and thus they represent an interesting avenue of research bearing on virulence. These metabolites may have evolved in saprophytic organisms in response to the presence of competing organisms in a common environment. As such, they may be cytoxic and eventually involved in pathogenicity. One of these metabolites, gliotoxin, a substituted diketopiperazine, has received considerable attention. It been implicated in the suppression of the innate immune response, including inducing apoptosis of neutrophils. However, specific inability to synthesize gliotoxin does not affect the virulence of A. fumigatus in the neutropenic mouse model. However, deletion of laeA, a gene necessary for the transcription of a large number of genes encoding enzymes of secondary metabolite synthesis (see ‘Medically useful secondary metabolites’ and ‘Regulation of secondary metabolism’), including gliotoxin, does affect the virulence of A. fumigatus. Absence of

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LaeA leads to a pleiotropic phenotype, and the decreased virulence may result from a combination of factors. It seems that at present we simply do not know why A. fumigatus is the prevalent pathogen and why other Aspergilli are occasional pathogens. It is likely that a complex combination of characters is responsible for triggering the autocatalytic process proposed by Henrici. Opportunistic pathogens have not evolved as such, in a coevolutionary relationship with a host organism, it thus may be completely fortuitous that one or other of them be able to thrive in the tissues of immunocompromised patients. It is important to determine which are the barriers that prevent fungal infections in immunocompetent subjects. Alveolar macrophage would get rid of ungerminated conidia, while polymorphonuclear neutrophils destroy hyphae mainly through the action of reactive oxygen species (ROS). One proposed mechanism involves the recognition of fungal cell wall constituents, such as 1-3-glucans, by macrophage membrane receptors, leading to phagocytosis. Recent studies, however, imply a less clear-cut distribution of labor, with neutrophils also having an important role in preventing conidial germination, which correlates with the susceptibility of neutropenic patients. Dendritic cells are able to ingest Aspergillus spores, thus being able to present specific antigens to T cells, a role shared with macrophage. Both CD4þ T and CD8þ T cells respond to fugal antigens, CD4þ T cells produce cytokines, which further recruit neutrophils. The protective role of specific antibodies is subject to discussion, as they are found in infected patients, which they fail to protect. Recently, protective roles have been postulated for surfactants secreted by epithelial cells that interact with conidia and may facilitate phagocytosis. A crucial role in innate immunity to opportunistic fungi is carried out by PTX3, a protein belonging to the pentraxin family of secreted, soluble proteins. PTX3 is essential in conidial recognition by macrophage and dendritic cells, and homozygous knocked-out mice genes are highly susceptible to experimental infection. The study of the immune response to infection by Aspergillus spores has made considerable progress in recent years and may lead to treatments, which promote the recovery of the immune response of the patient as an alternative or in association with antifungal drugs. Fungi are eukaryotes, more closely related to metazoans than to plants, that is why ascomycetes such as Saccharomyces cerevisiae and A. nidulans are useful models in molecular and cell biology. Many cell processes are common to the fungal and the animal cell, and that, in order to find effective antifungal agents, is necessary to identify those processes that will inhibit growth of the fungal cell without damaging the host. Flucytosine (5-fluorocytosine) has been used as an antimycotic since 1968. In clinical practice, it is used mainly in candidiasis. It affects nucleic acid synthesis and thus can hardly be considered a specific antifungal agent. Four other classes of compounds are currently used to treat fungal infections in clinical practice. The polyenes, such as

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Amphotericin B, interact directly with ergosterol in the fungal cell membranes leading to leakage of potassium ions and cell death. Amphotericin B, one of the most used antifungals, interacts with animal cell membranes also, which can lead to acute kidney failure. The azoles, such as fluconazole or the newly developed voriconazole, inhibit specifically lanosterol demethylase, blocking the synthesis of ergosterol. They are less toxic than Amphotericin B, and a case has been made to use voriconazole as a first line, rather than a second line, drug for the treatment of invasive Aspergillosis. However, they are not free of secondary effects. The allylamines such as Terbinafine also result in ergosterol depletion by inhibiting squalene epoxidase. Finally, the echinocandins are really specific antifungal drugs, as they affect the fungal-specific process, the synthesis of the glucans of the fungal cell wall by inhibiting noncompetitively -1,3-glucan synthase. Better knowledge of fungal development and metabolism, the search for genes essential for the pathogen, but absent in, or not essential for the host should lead to the development of new specific antifungal drugs. A different and complementary approach is to reinforce the immunological response of the host. This includes the possible development of an antifungal vaccine. Besides the uncertainty as to whether protective antibodies can be produced, the large variety of fungi that can affect immunodepressed patients posits an additional difficulty. Recently, a whole roaster of new fungi appeared as opportunistic pathogens, such as Fusarium, black molds, and zygomycetes. Success against Candida has been followed by an increase of Aspergillus infections. Preventive treatment with voriconazole, effective against Aspergillus, has been followed by infections by a whole variety of zygomycetes. It has been proposed that a vaccine using -1,3-glucan as antigen, which is a universal component of fungal cell walls, may be worth exploring. An early diagnosis is essential in the successful treatment of invasive fungal infections. Immunological detection of cell wall components such as galactomannan and 1-3--D-glucan and detection of fungal DNA by PCR are being developed and evaluated. Aspergillus in Veterinary Medicine Aspergilli are encountered, even if uncommonly, in veterinary practice. Here, as in the human disease, A. fumigatus is the most frequently encountered pathogen, followed by A. flavus. In mammals, canine sinonasal Aspergillosis, guttural pouch mycosis of horses, and bovine mycotic abortion are the most common diseases, but infection of other species and pulmonary and generalized aspergillosis has also been described. The horse disease is correlated with the presence of an extension of the Eustachian tube, the guttural pouch, an organ of uncertain physiological significance exclusive of horses, other Equidæ, and rhinos and tapirs. This organ could provide temperature and humidity conditions suitable for the growth of Aspergillus. Bovine mycotic abortion is

correlated with confinement to sheds, which leads to exposure to high concentrations of spores. More surprising is the finding of pulmonary Aspergillosis in free-range dolphins. If the finding of Aspergillus infections in mammals is sporadic and infrequent, birds are at a much higher risk. The main pathogen is A. fumigatus and the route of entry is the respiratory tract. In a large postmortem study, 4% of more than 10 000 birds showed fungal infection of the respiratory tract, probably in most cases due to A. fumigatus. Aspergillosis affects both free-ranging and domestic birds. Turkeys, poultry, and waterfowl are commonly affected but fatal infections of penguins, ostriches, and rheas have also been reported. The susceptibility of birds to Aspergilli has been explained by both anatomical characteristics of the respiratory system and cellular differences related to innate immunity such as the absence of alveolar macrophage. A. sydowii, a Specific Pathogen for Gorgonian Corals An ecologically menacing new Aspergillosis affects Gorgonian (fan) corals, mainly but not exclusively Gorgonia ventalina (infections of Gorgonia flabellum and one outbreak affecting Pseudopterogorgia americana have been reported). Up to the present time, it has been recorded only in the Caribbean Sea, first identified in Saba in 1996 and studied intensively in the Florida Keys. In an epizootic starting in 1997, more than 50% of the sea fan corals were lost. A subsidence of the epizootic has been since reported. The organism responsible is exclusively A. sydowii. The restricted host–pathogen specificity contrasts with the situation described above for mammals and birds. This species is a common saprophyte, which can be isolated from a number of environments. Cultures isolated from diseased G. ventalina are infectious, while strains isolated from nonmarine environments are not. As only three and two strains respectively were analyzed, this experiment is not definitive. The pathogenic and nonpathogenic strains do not form separate clades when molecular markers are analyzed. As A. sydowii does not sporulate in seawater, aerial dissemination has been suspected. One hypothesis is that the spores are carried by dust storms, originating in the North Africa. While fungal spores are surely carried by dust storms, no genotyping work confirming this hypothesis has been reported. Warming and nutrient effluents, including nitrates, have also been blamed for the outbreak. Obviously, these possible causes are nonexclusive. It is possible that the decrease of the epizootic is due to selection for resistant strains of G. ventalina. Thus, sea fan infection by A. sydowii, besides being an ecological menace, provides an interesting opportunity to study a specific host–parasite interaction involving an Aspergillus, and the elucidation of the mechanism of resistance could lead to the discovery of new antifungal compounds, for which there is a crying need. An infected sea fan coral is shown in Figure 3.

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bacteria and yeasts. In the production of sake, the Japanese wine derived from rice, the steamed rice is inoculated with spores of A. oryzae, and the hydrolyzed product is used as the substrate for alcoholic fermentation by Saccharomyces sake. The Aspergillus strains used in the soy sauce production differ from those used for sake production, the former are selected for high protease, the latter for high amylase titers. Both A. oryzae and A. sojae belong to the A. flavus groups, and genomic analysis has confirmed the very close relationship between A. oryzae and A. flavus, while A. sojae is considered a domesticated strain of A. parasiticus. Through the centuries, the organisms have been in use, they have been selected for both high extracellular enzyme titers and nil toxin production, at least under fermentation conditions. Aspergillus fermented food products represent, according to a recent source, 2% of the gross national product of Japan. Extracellular Enzymes Produced by Aspergilli: Aspergilli as Hosts for Recombinant Proteins

Figure 3 A specimen of the fan coral Gorgonia ventalina infected with Aspergillus sydowii. The infected areas are deep purple. The purple gall-like growths may be a result of the infection. A necrotic area surrounded by a deep-purpled ring can be seen at the bottom left of the colony. Bar 5 cm. The photograph has been kindly provided by Kiho Kim.

Useful Aspergilli Aspergillus biotechnology ranges from the first steps of sake fermentation to the production of recombinant mammalian proteins. These processes are briefly summarized below. Oriental Food Uses of Aspergillus The use of Aspergilli in the food industry in the far East relies on the extracellular enzymes secreted by the fungus when grown on solid or semisolid substrates. These technologies originated in China more than 2000 years ago. An old review cites more than a hundred such different fungal fermentations. The main products are soyu (soy sauce), miso (fermented soybean paste), and sake (rice wine). The production of soy sauce involves the fermentation of a mixture of cooked soybeans and wheat. The mixture is inoculated in traditional production by ‘koji’, which derives from a previous fermentation, or in more modern procedures by a spore suspension of specific strains of Aspergillus oryzae or Aspergillus sojae. A second fermentation is carried out by lactic acid

The Aspergilli are major producers of enzymes such as carbohydrate hydrolases, lipases, and proteases, used in a variety of industries such as food, beverages, detergent, and animal food additives industries. The first microbial enzyme to be marketed (1894) was an amylase, ‘takadiastase’, produced from A. oryzae. At least 27 different enzymes are produced industrially by the Aspergilli. Different species, mainly but not exclusively, of the A. niger, A. oryzae, and A. sojae groups have been optimized for the production of specific enzymes. In some cases, increased production has been achieved through proprietary recombinant procedures, which allows an increase in the copy number of homologous and in a few cases heterologous enzyme genes. Chymosin (rennin) is an enzyme essential for cheese production, which prior to its heterologous production by A. niger var. awamori (and other microorganisms), had to be extracted from calf’s stomach. The stunning efficiency of some of the Aspergilli in the process of enzyme secretion (>20 g l1), the considerable experience of the fermentation industry, and the fact that many procedures involving Aspergilli are generally regarded as safe (GRAS) had suggested that the Aspergilli could be used as ‘cell factories’ for the production of heterologous proteins. This has been successful for some recombinant enzymes (chymosin, lipase, and phytase), but not for high valued, medically important mammalian proteins. Lactoferrin is produced in commercial quantities by recombinant strains of A. awamori. More research is needed to understand why some filamentous fungi are so efficient at secreting many fungal proteins but are inefficient as heterologous hosts. Tissue plasminogen activator and interleukin have been experimentally produced at a rate of 12–25 mg l1 in a protease-less mutant of A. niger. A number of bottlenecks, such as specificity of glycolsylation and the onset of the unfolded protein response by the translation of foreign proteins, are under active investigation.

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Aspergillus and Production of Organic Acids

Medically Useful Secondary Metabolites

Depending on culture conditions, strains of A. niger are able to excrete a number of organic acids such as oxalic (used in metal leaching), citric, and itaconic acids and are thus used in their industrial production. Citric acid, a tricarboxylic acid, is an intermediate of the Krebs cycle. It is used in the food, beverage, and pharmaceutical industries. The annual production of citric acid, quoted for 2001, was 1 million tons. The main producing organisms are strains of A. niger. Since the ability of the organism to divert its metabolism to the production of citric acid was detected, industrial strains, that can convert over 90% of the carbon source in the culture media (carbohydrates) into citric acid were selected. Industrial carbon sources are low-grade molasses (typically sugar beet), but in principle many other residues of industrial process could be used. Specific culture conditions such as high concentrations of carbon source, low pH, and limitation of ions such as manganese are essential. It is not clear how the metabolism of the organism is diverted to citric acid overproduction. The production of citric acid implies that there is a bottleneck in the Krebs cycle, so that much more citric acid is produced than that is utilized in the cycle. Citrate itself inhibits phosphofructokinase I, the enzyme that catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, a crucial step in glycolysis. It has been proposed that under the culture conditions used, this inhibition is counteracted by other metabolites, thus removing this feedback inhibition of citrate production. Alternatively or additionally, tricarboxylic acid mitochondrial transporters leak out citrate from the mitochondrion, thus depleting the cycle. Itaconic acid is a dicarboxylic acid, which is used in industry as a precursor of polymers used in plastics, adhesives, and coatings. New uses of itaconic acid-derived polymers are under active investigation. The production of itaconic acid for 2001 was quoted as 15 000 tons. There is a renewed interest in this chemical as industry searches for substitutes of petroleum-derived chemicals. Virtually all itaconic acid produced is by fermentation by specific strains of A. terreus. Itaconic acid production is a further perversion of the Krebs cycle, citrate is converted as normally into cis-aconitate, which for reasons unknown is, in some organisms, decarboxylated into itaconitate, which has no known metabolic role in the cell. The fact that different strains of Aspergillus and more generally of fungi can divert metabolic pathways to the overproduction and secretion of useful chemicals, coupled with the fact that these organisms can grow on residues of processes such as sugar and ethanol production, open the possibility of engineering pathways to produce high value chemicals through ‘green’, low polluting, waste-eliminating procedures.

Of the useful fungal secondary metabolites, the most wellknown are the -lactam antibiotics, penicillin and cephalosporin, and their derivatives. Some Aspergilli, such as A. nidulans, produce low titers of isopenicillin N. This has been useful in the elucidation of the genomic organization and regulation of the pathway. It has already been mentioned above (see ‘Aspergillus as human pathogens’) that the echinocandins are specific antifungal drugs. Anidulafungin is a semisynthetic derivative of echinocandin B0, produced by A. nidulans var. echinulatus. Anidulafungin has been recently introduced in clinical practice and it is specifically indicated to treat Candida infections of the digestive tract. The most widely used secondary metabolite produced by an Aspergillus is lovastatin, produced by A. terreus. This metabolite, as other statins, is used in medical practice to reduce cholesterol levels. The market for statins has been estimated at more than 12 billion US$ annually. Statins are specific inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which reduces HMG-CoA to mevalonate. Statins are built around a common polyketide skeleton, have a structure similar to HMG, and act as competitive inhibitors of the HMG-CoA reductase. As other secondary metabolites, statins are synthesized by complex sequential steps, involving polyketide synthases, the genes coding for the cognate enzymes map in a 64 kb gene cluster. Gene clustering and its possible role in the synthesis of secondary metabolites is discussed in ‘Regulation of secondary metabolism’.

A. (Emericella) nidulans as a Model Organism The A. nidulans Genetic System In 1953, Guido Pontecorvo published a 97-page review on the ‘Genetics of Aspergillus nidulans’. Why did Pontecorvo and coworkers spend a considerable amount of time and energy to develop a genetic system for what was then an exotic organism? One of the key problems of biology was, at the time, that of the nature of the gene. The classical image of the gene was that of a discrete element, and mutations were considered to be alternative states of this element. The gene was an abstract concept whose molecular nature was elusive. The image of the gene as an indivisible, discrete unit was based on the experimental fact that mutations that did not complement were not separable in recombination experiments. That is, crosses of individuals carrying different alleles of the same gene, wild-type progeny were never obtained. ‘Never’ was a few dozen progeny in mice, a few thousands in Drosophila melanogaster. The modern reader may not grasp how fundamental the problem was at the time. In the 1940s, there were already exceptions to the ‘nonrecombination’ rule. In D. melanogaster, a few

Fungi | Aspergillus: A Multifaceted Genus

A. nidulans entered the molecular era when relatively efficient transformation techniques were worked out in 1983 followed by the development of replicating plasmids. We are witnessing a second methodological revolution, with the development of techniques and modified strains that allow to inactivate genes, introduce point mutations, change promoters, or add tags in a very simple and rapid way, opening the possibility of a high throughput reverse genetics. This, together with the availability of a complete genome and microarrays is changing again the prospects for this model organism. Usually a technique is first worked out in A. nidulans, and then that is applied to the other Aspergilli, such as the pathogenic or industrially important organisms mentioned in previous sections. The life cycle of A. nidulans is shown in Figure 4. Diploid Heterokaryon As e

a xu

Parasexual cycle

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Haploidisation mitotic recombination

l cy

noncomplementing mutations could recombine in crosses to yield rare wild-type individuals. These mutations were called ‘pseudo-alleles’. Pontecorvo was looking for a system where hundreds of thousands progeny could be scored. A. nidulans happened to be such an organism. By early 1950s, it became clear in Pontecorvo’s laboratory, through the work of Alan Roper, followed by Bob Pritchard, that the gene was divisible. The paradigmatic work on the divisibility of the gene was carried out by Seymour Benzer using the bacteriophage T4. ,‘‘That is, that the gene as a working unit in physiological action is based on a chromosome segment larger than the unit of mutation or recombination’’ Roper (1953) and ‘‘The classical ‘gene’ which served at once as the unit of recombination, of mutation, and of function, is no longer adequate. These units require separate definition. A lucid discussion of this problem has been given by Pontecorvo’’ Benzer (1957). The phage system of Benzer was so powerful and elegant that A. nidulans, as a system to study the fine structure of the gene, seemed redundant. Nevertheless, a beautiful genetic system was there, ready for the taking. I dare say that in 1953 no system afforded the same degree of sophistication. This system allows conventional meiotic genetics, carried out by analyzing the progeny contained in a fruiting body (cleistothecium). The cleistothecium may contain as many as 100 000 thousand asci, each containing eight ascospores, the product of a single meiosis and an additional mitosis. In the standard genetic analysis, the products are analyzed ‘in bulk’, without isolating single asci, as those are small and difficult to dissect. However, tetrad analysis is possible and was employed in early work. The power of resolution of the ‘in bulk’ genetic analysis has permitted fine structure mapping to the extent that mutations separated by 11 nucleotides have been resolved by recombination. A. nidulans strains carrying different markers can form heterokaryons. Nuclei in heterokaryons can rarely fuse, giving origin to stable diploids, which allow another layer of genetic analysis, developed by Pontecorvo and Etta Ka¨ffer, the parasexual cycle. Diploids can revert to haploids in which all markers in one chromosome segregate as a unit, allowing the rapid assignment to any new mutation to one of the eight chromosomes of the organism. Mitotic recombination also occurs and can be selected for in diploids, permitting the mapping of markers in relation to the centromere. The discovery of the parasexual cycle led to two developments. The first one was the possibility to carry out genetic analysis in the Aspergilli and the related Penicillia where a sexual cycle was not described. It is not known why stable diploids, different from the transient diploids that occur during the sexual cycle, can be obtained in these organisms and not in other filamentous ascomycetes. The second was the analogous development of systems, initiated by Pontecorvo, and based on cell fusion to carry out somatic genetics in mammalian cells.

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Ascospore

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Asexual cycle

Cleistothecium

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Figure 4 Schematized life cycle of Aspergillus nidulans. Three cycles are shown, the asexual cycle, which has been described in the text (see A. nidulans as a model for cell biology and A. nidulans developmental pathways). In the sexual cycle, two nuclei divide synchronously as a dikaryon in a specialized structure. Eventually the nuclei fuse to give a diploid, which does not divide as such but undergo meiosis. No differentiated mating types exist; any given mycelium can generate fertile cleisthotecia. To cross two strains, nutritional and color markers are used. Classical genetics procedures are facilitated by the fact that one cleisthotecium derives from only one fertilization event. In the parasexual cycle nuclei fuse in mycelia, outside the specialized developmental structure to yield a diploid, which does not undergo meiosis (at variance with diploids produced during the sexual cycle) but divides as such. Breaking up of the diploid (haplodization) and mitotic recombination are additional genetic tools. For clarity we have supposed that the heterokaryon and diploid formation are carried out between green (yAþ) yellow strains (yA–) and nuclei are colored accordingly. Both diploids and heterokaryons can undergo the asexual cycle. The different structures are not shown to scale. Scheme kindly provided by Ste´phane Demais and modified by the author.

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Fungi | Aspergillus: A Multifaceted Genus

Work carried out with A. nidulans has served as a model mainly in three aspects of cell and molecular biology (but a few others will be briefly discussed below). Historically, each of these three aspects can be related to a specific scientific school. David Cove and John Pateman initiated the investigation on control of gene expression in A. nidulans. The cell biology work derives from an early article by Ron Morris (1976) where he isolated mutations blocked in the cell cycle and in nuclear migration. Bill Timberlake initiated an analysis of the development of the conidiophore, work which profited from the early genetic work of John Clutterbuck, himself a product of the Glasgow school of genetics. Work on secondary metabolism stems from a confluence of this work with work carried out in other species of Aspergilli producing noxious chemicals.

was published that the intron of the precursor of the ribosomal 26s gene of Tetrahymena termophila was self-splicing. It was noticed that pairings that were postulated by us and by Bernard Dujon and Franc¸ois Michel for mitochondrial group I introns were conserved in this self-splicing intron; thus the model for splicing of mitochondrial class I introns became a model of self-splicing, which was confirmed experimentally. Self-splicing of introns was essential to the concept of ribozyme and eventually to that of a primeval RNA world. Thus, the sequence of the mitochondrial DNA of A. nidulans contributed, albeit somewhat indirectly, to present ideas on the origin of life.

The Mitochondrial DNA of A. nidulans

The metabolic versatility of the Aspergilli led a group of Spanish scientists to use mutants blocked in amino acid degradation to identify the enzymes and the genes of human metabolic diseases, including those of aromatic and branched amino acid catabolism. As stated in a review article ‘‘The metabolic capacity of A. nidulans for amino acid degradation largely resembles that of human liver’’. Figure 5 shows the breakdown of phenylalanine and the

The possibility to construct heterokaryons allows the genetic study of mutations that occur in the mitochondrial genome, as these are cytoplasmically inherited. A few markers were characterized and a circular genetic map was established. In the late 1970s, two groups, led respectively by Hans Ku¨nzel and Wayne Davies, attempted to sequence the whole 33 000 bp mitochondrial DNA of A. nidulans. This was almost accomplished, except for a gap of around 200 bp. At the time, where the longest DNA sequenced was the 16 000 bp mitochondrial DNA of Homo sapiens, this was a less than trivial enterprise. A number of important results derived from this sequencing effort. It was possible to compare the whole DNA organization with that of the completely sequenced human mitochondrial DNA, and the ongoing sequence of the S. cerevisiae mDNA, an organism where sophisticated genetic and molecular studies were actively carried out. These comparisons were extended to other mitochondrial DNA sequencing projects such as those of Neurospora crassa, Podospora anserina, and Schizosaccharomyces pombe. A number of unidentified open reading frames (ORFs) were found in the mitochondrial DNA of A. nidulans and the human mitochondrial DNA, but not on that of S. cerevisiae or of S. pombe. These reading frames correspond to genes coding for complex I, the NADH dehydrogenase complex, which is absent in both model yeasts. The highlight of this work was the elucidation of the structure of class I introns. Metazoan mitochondrial genes have no introns, while introns of two different classes are present in the mitochondria of fungi and plants. Introns on the nuclear genome of the fungi are small, typically of >100 bp. Mitochondrial fungal introns are large, usually >1000 bp, and can contain ORFs. A comparison of the sequences of the mitochondrial introns of A. nidulans with those of wild-type and mutant strain of S. cerevisiae led to a model of the secondary structure of class I introns, including the proposal of a mechanism of intron splicing. Around the same time, it

A. nidulans as a Model for Genetic Metabolic Diseases

Phenylalanine

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Phenylketonuria

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4-hydroxyphenylpyruvate Tyrosinemia III

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Homogentisate Alkaptonuria

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Maleylacetoacetoacetate Fumarilacetoacetate Tyrosinemia I

fahA

Fumarate + acetoacetate Lactose + phenylalanine Figure 5 Aspergillus nidulans as a model for human metabolic diseases. To the left the degradation of phenylalanine is shown, to the right of the pathway, the steps blocked in the cognate human metabolic diseases. In italics the relevant corresponding genes of A. nidulans are shown. To the right the toxicity of fumaryl-acetoacetate and the suppression of the toxicity by the upstream hmgA null is shown together with the secretion of the purple oxidation product of homogentisic acid. Lactose is used as a poor, nonrepressing carbon source, as phenylalanine catabolism is subject to carbon catabolite repression (see ‘Nitrogen and Carbon utilization’). See text for details. Photographs of plates were kindly provided by Miguel Pen˜alva.

Fungi | Aspergillus: A Multifaceted Genus

cognate blocks in human diseases affecting this metabolism. The gene of A. nidulans coding for the fumaryl-acetoacetate hydrolase was cloned as a cDNA highly expressed in the presence of phenylacetic acid (fahA, Figure 5). Mutations in the human homologue result in the serious disease tyrosinemia I, and the A. nidulans ORF shows 47% identity with the human gene. Satisfactorily, the growth of A. nidulans is strongly inhibited by the accumulation of this metabolite in fahA-deleted strains. In a second step, suppressor mutations of this inhibited phenotype were isolated. These pinpointed the gene (hgmA) coding for homogentisate dioxygenase. Not only these mutations suppressed the toxicity of phenylalanine seen in fahA nulls, they also resulted in the accumulation of a purple pigment (see Figure 5). This is exactly the pigment that is accumulated in the urine of patients affected by a milder disease, alkaptonuria. The identification of the gene was straightforward and its sequence served to identify the hitherto unknown human gene and to identify the lossof-function mutations present in a number of patients. Alkaptonuria was identified by Garrod in 1902 as an ‘inborn error of metabolism’ and shown to be inherited as single Mendelian gene. The work of Beadle and Tatum in N. crassa, and the one gene-one enzyme proposal arising from it, can be seen as a completion of Garrod early proposals. The identification of the human gene through the cloning of the A. nidulans gene is of more than historical importance and underlines the necessity of choosing the correct model system for a particular problem. Control of Gene Expression Nitrogen and carbon utilization

The Aspergilli can utilize a large number of metabolites as nitrogen and/or carbon sources. Early work with A. nidulans has established some fundamental concepts pertaining to the control of gene expression and metabolic regulation. Some of the first eukaryotic pathway-specific regulatory genes (nirA, uaY; see below) were characterized in the 1960s. The genes encoding the key regulators for nitrogen (areA) and carbon catabolite repression (creA) were the first such genes to be described in any eukaryotic organism. Their mode of action was established by formal genetic analysis long before recombinant DNA technology came into existence. In general, the genes coding for the enzymes involved in the utilization of a specific metabolite are only transcribed in the presence of a specific inducer. Inducers act by activating specific transcription factors, which in turn elicit the transcription of specific catabolic genes. Thus, nitrate activates the NirA protein, necessary for the transcription of the genes encoding nitrate and nitrite reductase and the nitrate transporters, acetaldehyde activates AlcR, regulating ethanol utilization, uric acid activates UaY, regulating at least eight scattered genes encoding enzymes and transporters

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involved in purine utilization, proline activates PrnA, regulating all the other genes of the proline utilization gene cluster, while -alanine activates AmdR/IntA a protein that positively regulates the amdS gene (encoding acetamidase) and the gabA gene (encoding the -aminobutyrate transporter). Almost all the pathway-specific transcription factors belong to a group of proteins that bind DNA through a specific fungal motif, the Zn binuclear cluster (Cys6Zn2). Most bind DNA as dimers, including the paradigmatic S. cerevisiae protein GAL4. AlcR is an exception, which uniquely binds as a monomer. NirA and UaY are localized in the nucleus as a result of induction. Nitrate induces by breaking the association of NirA with KapK (the orthologue of the mammalian and S. cerevisiae exportins Crm1P and CRM1). PrnA and AlcR are always nuclear, PrnA necessitating induction to bind its cognate sequences in the promoter. The induction of genes involved in the utilization of nitrogen sources does not occur in the presence of preferred sources such as ammonium and glutamine, while the induction of genes involved in the utilization of carbon sources is strongly diminished in the presence of glucose. These processes, nitrogen metabolite repression and carbon catabolite repression, involve two additional regulators, AreA and CreA. AreA is a GATA factor, acting positively in synergy with the specific regulators (such as NirA or UaY). Ammonium and glutamine negate AreA function at a number of levels, including the stability of its cognate mRNA. The dependence on AreA is absolute for the niiA-niaD bidirectional promoter, driving the genes encoding nitrate and nitrite reductases, less marked for some of the genes of the purine utilization pathway. CreA acts as a genuine repressor in the presence of favored carbon sources, negating the activation by or competing with the binding of the pathway-specific factors such as AlcR. CreA is a Zn finger protein, with a Zn finger sequence extremely similar to Mig1p, the repressor mediating carbon catabolite repression in S. cerevisiae and related organisms. However, the similarity between CreA and Mig1p stops there. Little sequence conservation can be seen outside the DNA-binding domain. Neither the glucose signaling mechanism nor the downstream mechanism of transcriptional repression seems to be shared by Mig1p and CreA. Mig1 represses transcription by recruiting the Tup1/Ssn6p co-repressor complex, which is not the case in A. nidulans and most likely in the fungi where a CreA, rather than a Mig1p orthologue, is present. The Aspergilli can use a number of metabolites as both carbon and nitrogen sources, the mechanism of regulation having been elucidated for the prn gene cluster (comprising five genes involved in the utilization of proline) and the amdS gene. Repression occurs only when both repressing carbon (glucose) and nitrogen sources (ammonium or

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1

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Specific transcription factor

Specific transcription factor 2

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4 Figure 6 General scheme of the transcriptional regulation of genes involved in the utilization of nitrogen sources.

Figure 7 General scheme of the transcriptional regulation of genes involved in the utilization of carbon sources.

1. In the absence of a specific inducer (such as nitrate) and in the presence of a preferred, repressing nitrogen source (ammonium, glutamine), neither the specific transcription factor nor the broad-domain GATA factor AreA is activated. No or only basal transcription is seen. 2. In the absence of a specific inducer in the presence of a nonrepressive nitrogen source, only AreA is active. No or only basal transcription is seen. 3. In the presence of a specific inducer and in the absence of a repressing nitrogen source, both transcription factors are active, full transcription is seen. 4. In the presence of both a specific inducer and a repressing nitrogen source, the specific transcription factor is active, but the AreA factor is inactive and no transcription is seen. In the nitrate utilization pathway a further mechanism is in act, as AreA is necessary both indirectly through its regulation of transporters for the uptake of the specific inducer (nitrate) and for the binding of the specific transcription factor (NirA) to DNA. Thus, the situation will be identical to that seen in scheme 1.

1. In the presence of ‘neutral’ carbon source (such as glycerol) and the absence of an inducing carbon source, neither the specific positive-acting transcription factor nor the CreA repressor are bound to the promoter. No or only basal transcription is seen. 2. In the presence of an inducer carbon source, the specific transcription factor (such as AlcR in the ethanol utilization pathway) is bound to DNA and active, full transcription is seen. 3. In the presence of both inducing and repressing carbon sources, the specific transcription factor is active but the CreA repression partially or totally negates its effect. No or only basal transcription is seen. 4. In the presence of only a repressing carbon source, only the CreA repressor is bound to DNA, no or only basal transcription is seen.

glutamine) are present. This can be rationalized by thinking that if a repressing nitrogen source is present, it will be advantageous for the organism to use proline or acetamide as a carbon source, while if only a favored carbon source is present, it will still be advantageous to use proline or acetamide as a nitrogen source. Carbon metabolite repression requires the CreA repressor, while nitrogen metabolite repression operates through the inactivation of the AreA GATA factor. While, for example, in the nitrate assimilation pathway AreA is always essential for transcription to occur, for prn and amdS, it is only necessary when the CreA repressor is activated by a repressing carbon source. These regulatory patterns are conserved in the Aspergilli and more generally in the filamentous ascomycetes and are schematized in Figures 6, 7, and 8, while the nuclearcytoplasmic shuffling of NirA is illustrated in Figure 9. The gabA gene, encoding the -aminobutyrate transporter, is subject to an even more complex pattern of regulation. It is induced by !-amino acids and subject to concomitant repression by nitrogen, carbon, and alkaline pH.

Regulation of gene expression by external pH

Soil organisms, such as the Aspergilli, respond to a variety of environments and it is not surprising that a system that regulates gene expression as a function of external pH has evolved. External pH regulates genes coding for extracelullar enzymes or transporters or those encoding steps in the synthesis of exported metabolites. In neutropenic mice experimentally infected with A. nidulans, this process is necessary for virulence. Penicillin is synthesized by some Aspergilli but only at alkaline pH. The synthesis and uptake of siderophores is also regulated by pH. The elucidation of the mechanism of pH regulation is a superb scientific achievement of the groups of Herb Arst and Miguel Angel Pen˜alva. The signal transduction pathway described below is conserved throughout the ascomycetes. The key actor is PacC, a transcription factor of the classical Zn finger type. In its active form, PacC acts as a positive transcription factor of alkaline-expressed genes (such as alkaline phosphatase or isopenicillin synthase) and as a repressor of acid-expressed genes (such as acid phosphatase or the -aminobutyrate transporter). At acidic pH (pH usually tested 4.0), there is no activation signal, and the protein is in an inactive form. In the full-length PacC (PacC72), intramolecular interactions hold the protein in a folded inactive form, which is largely excluded from the

Fungi | Aspergillus: A Multifaceted Genus

1

Specific transcription factor

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3 Wide domain AreA transcription factor 4

Wide domain CreA repressor 5 Figure 8 General scheme of the transcriptional regulation of genes involved in the utilization of metabolites that can serve as both nitrogen and carbon sources. 1. In conditions where the inducer is not present the specific transcription factor (PrnA in the proline utilization gene cluster) is not bound to the promoter. In the scheme shown, under neutral conditions (e.g., urea as nitrogen source, lactose as carbon source) AreA would be bound, and CreA would not be bound. No or only basal transcription is seen. This applies to every other combination (not shown) where the specific inducer is absent. 2. Same conditions but in the presence of the inducer (proline in the example given in the text), both the specific transcription factor (such as PrnA) and AreA are bound, full transcription is seen. 3. In the presence of the inducer and a repressing nitrogen source (ammonium or glutamine) but no repressing carbon source. Only the specific transcription factor is bound. Full or almost full transcription 4. In the presence of the inducer and a repressing carbon source (glucose), but no repressing nitrogen source. The three regulatory proteins are bound; AreA negates the repressing action of CreA. Full or almost full transcription 5. In the presence of inducer (such as proline) and both carbon and nitrogen repressing metabolites (ammonium or glutamine and glucose). The specific transcription factor (such as PrnA) is bound and CreA negates its action. Efficient repression. No or only basal transcription is seen.

nucleus. At alkaline pH values (usually 8.0), the protein is activated by two proteolytic steps. The palA, palB, palC, palF, palH, and palI genes encode proteins involved in pH sensing and in signal transduction. Mutations in all these pal genes have an acidity-mimicking phenotype, while mutation in the transcription factor pacC can lead to acidity mimicking (loss-of-function mutations), alkalinity-mimicking or neutrality-mimicking phenotypes, where both ‘alkaline’ and ‘acidic’ genes are expressed. The pH sensor is probably PalH assisted by PalI, both of which are plasma membrane proteins. The C-terminus cytoplasmic tail of PalH interacts directly with PalF, a member of the arrestin family, which is,

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similarly to the mammalian arrestins, phosphorylated and ubiquinated. These modifications occur at alkaline pH and are dependent on the PalH and PalI proteins. Under alkaline pH conditions, PalA binds to motifs flanking a specific protease-sensitive sequence in the C-terminus of the fulllength PacC (PacC72). PalA interacts directly with the A. nidulans orthologue of Vps32, a protein involved in the formation of multivesicular endosomes. PacC/PalA interaction renders PacC sensitive to a specific cleavage, catalyzed by PalB, a protease of the calpain family. PacC72 is cleaved to PacC53. The cleaved form of PacC becomes susceptible to further processing by the proteasome, yielding PacC27. This is the active form of PacC, strictly localized in the nucleus, where it activates genes expressed at alkaline pH and represses genes expressed at acid pH. This account leaves open the mechanism of pH sensing and the connection between the arrestin-like PalF and PalA-PalB. The interactions of PalA with components of the mature endosome, and recent work on the related signal transduction pathway in S. cerevisiae, strongly suggests that endocytosis provides this connection. PalC, the unplaced actor of the process, has a functionally important Bro1 domain (also present in PalA), a domain of possible interaction with Vps32, strengthening the endosomal connection of the pH signaling pathway. The YPXL/I motif recognized by PalA is also recognized by its putative mammalian orthologue, AIP1/Alix, a protein involved in a variety of functions, including the budding of the human HIV virus from infected cells. The whole process has tantalizing similarities with the Hedgehog signaling pathway in metazoans, leading to the proteolytic activation of the Zn finger transcription factor cubitus interruptus/Gli, posing the question of whether these pathways are evolutionarily related. A simplified version of the pH signaling process is shown in Figure 10. Specific regulatory mechanisms acting at the level of transporters

The control of transporter synthesis and activity is a key step in metabolic regulation, as the activity of specific transporters modulates the entry of metabolites that serve as inducers or repressors of specific pathways. Work with A. nidulans has led to the identification of two new control processes affecting transporters, besides their tight specific transcriptional regulation. The transcription of a number of transporters is activated during the isotropic phase of conidial germination (see below). This is a developmental control, which bypasses other specific control systems. Recent transcriptomic work suggests that this mechanism occurs for many transporters and is general for the filamentous ascomycetes. It can be proposed that germinating fungal spores explore an unknown environment by expressing a whole range of transporters, to progress to specific induction once the spore has germinated. The second mechanism is posttranslational. In the presence of a favored nitrogen source such as ammonium, both purine and amino

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Fungi | Aspergillus: A Multifaceted Genus

NO3–

Arginine

NH4+

Figure 9 Cytoplasmic and nuclear localization of the NirA transcription factor. A construction where the whole NirA transcription factor is fused to green fluorescent protein (GFP) substitutes the NirA wild-type gene. This construction is competent to mediate induction by nitrate. NirA is localized in the cytoplasm in the presence of a noninducing, nonrepressing nitrogen source (arginine), of a repressing nitrogen source (ammonium) and localizes in the nucleus only when an inducing nitrogen source is present. See text for details. This figure illustrates also the technology of gene fusions and epiflourescence microscopy, which is been extended to all other Aspergilli and many filamentous fungi, including animal and plant pathogens. The original pictures were kindly provided by Joseph Strauss.

pH > 7 PalH

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Figure 10 Simplified scheme of the regulation of gene expression by external pH, redrawn from a number of articles of the groups of Arst and Pen˜alva. Alkaline pH is sensed by the PalH and PalI proteins, the signal is transduced to the PalF arrestin via the C-terminus of PalH. PalF is phosphorylated and ubiquinated and signals PalA, which has been shown to interact with the endosomal protein Vps32. The role of PalC is hypothetical. PalA leads to the opening of PacC72, which is cleaved by PalB at a specific site. PacC53 is further processed by the proteasome, leading to the active form PacC27. In this simplified scheme both proteolytic processing steps are shown in the cytoplasm, in fact the second step may occur in either or both the cytoplasm and the nucleus. In yellow, proteins of the pH signal transducing pathway, in blue cellular proteins or complexes nonspecific for the pathway. The ‘active’ portion of PacC is shown in green, the inhibitory, cleaved portions are shown in red.

Figure 11 Posttranslational regulation of transporters. The left panel shows the membrane localization of a fusion with the green fluorescent protein of the proline transporter (PrnB-GFP). The cognate gene prnB maps in the proline gene cluster, its transcriptional regulation is schematized in Figure 8 (see ‘Control of gene expression, Nitrogen and carbon utilization’). Conidiospores were grown for 16 h at 25  C in the presence of urea as nitrogen source, and induced with L-proline after 10 h. In the right-hand panel ammonium was added for the last 2 h. In the left panel, the PrnB-GFP fusion strongly stains also the basal septum. Localization in septa is a characteristic of all membrane proteins studied up to now. Confocal microscope images were kindly provided by Vicky Sophianopoulou.

Regulation of secondary metabolism

acid transporters are internalized to the vacuole, where they are possibly destroyed. This posttranslational mechanism is synergistic with but independent from the nitrogen metabolite repression mechanism (described above). Figure 11 illustrates this process.

Fungi produce an astonishing variety of secondary metabolites. Fungal toxins, the -lactam antibiotics and lovastatin, have already been mentioned. In the pregenomic days, conventional genetic analysis led to the identification, cloning, and sequencing of a number of

Fungi | Aspergillus: A Multifaceted Genus

genes encoding biosynthetic steps for a number of secondary metabolites, while many more metabolites were identified as secreted by a variety of Aspergilli. As many secondary metabolites involve nonribosomal peptide or polyketide synthases, putative fungal metabolite gene clusters have been identified in a number of fungal genomes. In A. fumigatus, the estimate is that of 22 secondary metabolite gene clusters. The best studied pathways are those leading to the biosynthesis of isopenicillin in A. nidulans, aflatoxin in A. flavus, and the aflatoxin precursor sterigmatocystin in A. nidulans. Secondary metabolism synthesis occurs late during mycelial growth and is generally correlated with conidiation and shares with this process some of its signaling pathway. Pathway-specific transcription factors have been characterized for the aflatoxin, sterigmatocystin, and gliotoxin pathways. The clusters of aflatoxin and sterigmatocystin biosynthesis include the regulatory gene aflR, necessary for the expression of the rest of the genes of the cluster. AflR belongs to the Cys6Zn2 family of specific fungal activators. It is not known whether the activation of AflR involves a specific metabolite or if it is only activated by the ‘fluffy’ signalling pathway to be described below (A. nidulans developmental pathways). No pathwayspecific activator has been described for isopenicillin biosynthesis, which is regulated by a number of environmental parameters, including extracellular pH. Clustering of genes is variable for genes involved in primary metabolism. In contrast, the genes of secondary metabolism biosynthesis are as a rule organized in large clusters. The 70 kb aflatoxin cluster comprises 25 coregulated genes. The gliotoxin gene cluster comprises 12 genes. Does the clustering of secondary metabolism genes have an evolutionary and/or functional significance? Possibly the two divergently transcribed genes responsible for isopenicillin-N synthesis have been horizontally transferred from a Streptomyces to an ancestor of the Aspergilli and Penicillia. There is no evidence for horizontal transfer for any other secondary metabolite gene cluster. Comparative genomics is providing some clues, even if not yet an answer, to the significance of secondary metabolite gene clustering. There is a significant bias toward the location of secondary metabolite clusters in subtelomeric regions. The fact that species of Aspergilli differ widely in the secondary metabolites they produce correlates with the mapping of the cognate genes in genomic regions where syntheny between species is broken. A fundamental advance in the understanding of the regulation of secondary metabolism arises from the discovery of the global regulator LaeA in the laboratory of Nancy Keller. LaeA is conserved in filamentous fungi, but not in yeasts. LaeA shows a domain typical of histone methyltransferases, the SAM domain, while lacking a second domain found in these enzymes, the SET domain. LaeA regulates positively the synthesis of isopenicillin, sterigmatocystin, gliotoxin, and lovastatin. The global role of LaeA has recently been investigated by transcriptomic studies

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with A. fumigatus. Of the 22 gene clusters, a deletion of laeA diminishes clearly the transcription of 13. Thus LaeA is a broad, but not a universal regulator of secondary metabolism. Recent work points to a role of LaeA in remodeling chromatin structure. In A. nidulans, deletion of a number of genes universally involved in gene silencing in heterochromatin result in premature secondary metabolite production. More strikingly, these deletions act as partial suppressors of a laeA deletion. Thus the exciting possibility arises that LaeA acts by reversing a heterochromatic state of the secondary metabolite gene clusters. Thus the study of the regulation of secondary metabolism may lead to an understanding of the role and genomic distribution of heterochromatin in filamentous ascomycetes.

A. nidulans as a Model for Cell Biology The life cycle of the Aspergilli includes a number of tightly regulated developmental pathways, from the germination of conidia or ascospores to the formation of complex structures involved in sexual (cleistothecia) or asexual (conidiophore) spore formation. The germination of conidiospores, but not that of ascospores, has been well studied. Conidiospores can stay dormant and viable for many years and contain (in A. nidulans) one nucleus arrested in the G1 phase. When plated on suitable media, they go through a phase of isotropic growth, where the conidium swells. The first mitosis may occur in this phase or after the emergence of the germ tube (Figure 12). Mitosis occurs synchronically, up to the eight nuclei stage when a perforated septum appears basally

Figure 12 Conidial germination. A group of germinating conidia from A. nidulans are shown. They are stained with the green fluorescent protein (GFP) fused to a strong nuclear localization signal, driven by a strong constitutive promoter. One white arrow indicates a conidia where the first mitosis has occurred before the production of the germinal tube, another mitosis occurring concomitantly with germination. Note that the signal is not lost during mitosis, which as in other fungi is closed. Photograph by Ana Pokorska in the laboratory of the author.

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Fungi | Aspergillus: A Multifaceted Genus

(Figure 11). Other septa are laid during hyphal growth out every —three to four nuclei. Only the nuclei comprised between the septum and hyphal tip are competent to divide and they do so synchronously. A second germ tube can arise from the conidiospore at 180 from the first one. Nuclei in nonapical compartments became again competent to divide when the conidiophore is developed (see below) and when branches arise from subapical compartments. Thus a highly coordinated process occurs, involving the regulation of mitosis, the establishment of a primary polar axis, the establishment of secondary polar axes in branches, nuclear migration, the laying down of septa and finally the appearance of another highly polarized structure, the conidiophore. Some processes, such as hyphal polar growth and the deposition of septa, are specific of fungi, while others are common to all eukaryotes, and the A. nidulans work matches and has added considerable information to the work carried out in S. pombe and S. cerevisiae. In both yeasts and most cells in higher eukaryotes, mitosis is followed by cytokinesis, where the two daughter cells separate. This is not exactly the case in filamentous fungi, where the whole mycelium is one syncytium, subdivided by perforated septa. It must be stressed that during condiogenesis the situation resembles budding, with proper cytokinesis, as metullae, phialides, and conidia are uninucleate cells, while ascospores are binucleate (see ‘A. nidulans developmental pathways’). Thus an understanding of Aspergillus cytokinesis involves understanding the generation of these different patterns. The determination of hyphal polarity and the related problem of the relationship of mitosis with septum formation are active fields of research at present, and recent work has shown that while some of the determinants of polarity and cytokinesis are common with the yeasts, some are entirely novel. In particular, a specific ceramide synthase is essential for polarity, probably by generating specific lipid rafts at the growing tip, which in turn would be involved in the localization of other polarity determinants such as formin, an actin nucleating protein. Particular to filamentous fungal growth is the Spitzenko¨rper, a subapical organelle that acts as a vesicle supply centre. The challenge for future research is to understand the coordination of signaling pathways, the polarization of the actin cytoskeleton, the formation of lipid rafts, and the activity of the Spitzenko¨rper to reach a complete understanding of polarity determination. Some of the highlights of the work relating to the cell cycle are indicated below, where A. nidulans has served as an eukaryotic model, while some specific aspects of Aspergillus development are summarized in ‘A. nidulans developmental pathways’. The judicious use of mutants resistant to the tubulin inhibitor benomyl led to the identification of the first - and -tubulin-encoding genes in any organism. It was then shown that the tubulins are involved in nuclear and chromosomal movement. The crowning of this work was the discovery of -tubulin by Berl and Liz Oakley.

A benA (encoding one of the isoforms of -tubulin) temperature-sensitive mutant, benA33, results in microtubules that are hyperstable (rather than nonfunctional) at the nonpermissive temperature. Three suppressors of benA33 mapped in a gene that when cloned and sequenced was shown to code for a new tubulin. This tubulin is critical for the nucleation of microtubules in all eukaryotes where it has been studied. The establishment of the function of -tubulin illustrates the use of A. nidulans as a model organism. While the inactivation of the cognate gene is lethal, the mutation could be maintained in a heterokaryon (see ‘The A. nidulans Genetic System’). As conidia are uninucleate, heterokaryons will produce two types of conidia, one of which carries the disrupted allele, where the phenotype caused by the mutation during conidial germination can be assessed microscopically. The disruption does not affect germination, but blocks nuclear division and to some extent nuclear migration. DNA is replicated, chromosomes condense, but spindles are not assembled. Thus, work that started with the isolation of tubulin inhibitor-resistant mutants led to the discovery of a new tubulin, which in all organisms is crucial for microtubule nucleation in centrosomes and in fungi (which have a closed mitosis) in spindle polar bodies. In the seminal Morris article of 1976, a large number of conditional mutants were characterized. These were temperature-sensitive mutants, which either failed to enter mitosis at the nonpermissive temperature (nim, never in mitosis), were blocked at different stages (bim, blocked in mitosis), or where the nuclei failed to migrate (nud, nuclear distribution), while sep mutants are defective in septum formation. Eventually, the cognate genes were cloned and sequenced, suppressors were isolated and identified, to give a growing picture of the genes involved in basic processes of cell biology. Cellular motors of the myosin class associate with actin filaments, while kinesis and dyneins move cargo (vesicles and organelles) along microtubules. nudA encodes the dynein heavy chain, nudG the dynein light chain, while other nud mutants defined hitherto undescribed regulatory proteins of the dynein complex. In particular, nudF encodes a close homologue of the human protein LIS1, which is mutated in Miller–Dicker lysencephaly, a human hereditary disease of the nervous system where neurons fail to migrate in the hemizygote. NudC, a protein that interacts with NudF is also conserved from fungi to mammals. It is likely that the primary effects of NudC/NudF in organisms with an open mitosis are in cytokinesis, a role obviously that can only be partially conserved in a syncytial organism with a closed mitosis such as A. nidulans. This pioneering work, which exploited both the A. nidulans genetic system and its specific morphology, has guided the work leading to the understanding of the function of the dynein complex in the nervous system.

Fungi | Aspergillus: A Multifaceted Genus

At variance with dyneins, kinesin genes are highly redundant and only one was identified through mutant screens. This is bimC, which defines a specific class of plus-end conserved kinesins. Mutants in this gene are defective in spindle pole separation and are thus blocked in nuclear division and provided the first direct evidence the kinesins are involved in mitosis. In the genetic screen, no mutants blocked in G1 were found, mutants blocked in the S-phase map at five loci, others blocked in the transition of G2 to mitosis map at six loci. Among the genes so defined, some are orthologues of genes previously known from S. pombe. nimX (not identified in the screen) encodes the orthologue of the cyclin-dependent S. pombe cdc2 kinase. The homologue of the cdc13 cyclin B is encoded by nimE, while the phosphatase activity necessary for the activation of NimXcdc2 is encoded by nimT. NimA, on the contrary, is a newly discovered serine/threonine kinase, which defines a whole class of proteins conserved throughout the eukaryotes. NimA functions downstream of NimXcdc2/cyclin B, which would then have two independent functions, one to promote spindle formation, through the activation of other kinases, the second to activate NimA, which in turn is necessary for chromosome condensation. NimA is necessary for entry into mitosis, mutants showing duplicated spindle polar bodies, while its destruction by proteolysis is necessary for exit from mitosis. There is a considerable evidence for similar roles in mitosis for NimA homologues in higher eukaryotes. A human protein, Pin1, interacting with NimA was identified in a two-hybrid screen. Pin1 mutants have a phenotype reciprocal to that of NimA mutants, suggesting that Pin1 (PinA in A. nidulans) is involved in the inactivation of NimA. Pin1 is a universally (in eukaryotes) conserved peptidyl-prolyl isomerase that catalyzes specifically the isomerization of prolyl bonds in a P-Ser/Thr-Pro dipeptide, increasing its rate by about 1000 times, thus allowing a drastic change in the peptide backbone conformation, NimA is only one of its substrates, another one being cdc2/cyclin B. It has recently been shown in HeLa cells that Pin1 is necessary for entry into mitosis, associates with mitotic chromosomes, and it strongly stimulates cdc2 phosphorylation. The discovery of Pin1 and its involvement in mitosis has led to flurry of activity, concerning its possible role in cancer, but more cogently in the onset of Alzheimer’s disease. Mice homozygously deleted for the Pin1 gene develop a neuronal degeneration with many of the histological characteristics of Alzheimer’s. Both tau, a microtubuleassociated protein, and APP (amyloid precursor protein) are phosphorylated at Ser/Thr-Pro motifs. These proteins are hyperphosphorylated and insoluble in Alzheimer’s. It had been proposed that the key regulator of the state of these proteins is actually Pin1, which would displace the equilibrium toward the nonphosphorylated, soluble forms. The anaphase-promoting complex (APC) is an ubiquitin ligase that targets key mitotic proteins such as cyclins and directs them to the proteosome. Mutants in its

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components will be expected to be blocked in metaphase and to show a bim phenotype. Two such components were first identified among the bim mutants. BimE was identified first as a negative regulator of mitosis. Biochemical work in Xenopus oocytes showed that a protein that copurified with APC (APC1) is the orthologue of BimE. bimA encodes the APC3 component. Once all chromosomes are attached to microtubules, APC activation results in degradation of securin. This releases and activates separase, a protease that cleaves cohesin. As cohesin keeps sister chromosomes together, this cleavage is the prerequisite for anaphase. bimB encodes separase. A component of cohesin, sudA, was identified as a suppressor of a bimD allele, which itself results in an anaphase block characterized for defective chromosome separation. Finally, mutations in bimG result in large, polyploid nuclei that fail to complete anaphase. Nuclei are clumped and conidia fail to germinate highlighting the link between the regulation of mitosis and the establishment of polarity. BimG is a phosphatase, showing striking identity with mammalian phosphatases of the PP1 class. BimG is localized to the spindle polar bodies, to the nucleolus, to the tip of the hypha, and transiently in the septum. There is no hint as to what are the substrates of BimG in the mitosis, septum formation, and polarity establishment.

A. nidulans Developmental Pathways In the sexually reproducing Aspergilli, the mycelial mat can follow two different developmental pathways. Meiosis and the formation of asci that contain ascospores, occurs in specialized structures, the cleistothecia. In A. nidulans, mature cleistothecia are globose, darkly pigmented structures of 100–200 mm in diameter. Ascospores have a characteristic bivalve morphology (about 4 mm  3.5 mm) showing two equatorial crests. Ascospore ornamentation is a valuable taxonomical character in the sexually reproducing Aspergilli. The protocleistothecium is generated from vegetative hyphae, which coil in a spherical structure developing into a cleistothecium, surrounded by specialized, modified hyphal cells called hu¨lle cells. A. nidulans is homothallic; two genetically identical nuclei can fuse to give diploids which, as in all other filamentous ascomycetes, are immediately committed to meiosis. In heterothallic Aspergilli, the sexual cycle occurs only when nuclei of opposite mating types meet in heterokaryons. Some nonsexual Aspergilli (A. flavus and A. parasiticus) form structures, sclerotia, which may be developmentally related to the cleistothecium. Conceptually, we can distinguish two processes in the development of the mature cleistothecium. One is the morphological process that leads to cleisthotecia, surrounded by hu¨lle cells. The second is the behavior of nuclei, which in the primordium of the cleisthotecium form dikaryons, in which two nuclei divide synchronously.

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Fungi | Aspergillus: A Multifaceted Genus

Dicaryotic nuclei fuse into transient diploids, which undergo immediate meiosis, followed by two mitoses leading to eight binucleate haploid ascospores per ascus. These processes can be experimentally separated, as it is possible to obtain morphologically perfect cleisthothecia that do not contain asci. Many genes, including transcription factors and G-coupled receptors, have been implicated in either or both processes. The availability of the genome and possibility of following tagged proteins through the developmental processes should lead to an understanding of the sexual maturation process, including the roles of mating types in homothallic and also heterothallic species. Very recent work has established that both  and HMR mating type genes (see ‘The genus Aspergillus in the genomic era’) are necessary for fertility but not for cleistothecial formation. At present, we cannot yet draw a scheme of the developmental pathway leading to the formation of sexually mature, fertile cleistothecia. The second developmental pathway is the formation of asexual conidia, which is present in all Aspergilli. These are formed from a specific structure, the conidiophore, which is the taxonomic marker of the genus. Conidiophores sizes range from 50 to 70 mm long in A. nidulans as much as 5 cm in Aspergillus giganteus. The structure of the conidiophore is shown in Figure 13. From the flat mycelial mat, a stalk grows from a foot compartment at a right angle from the mat. The stalk then swells into a multinucleate vesicle. From

the vesicle a first series of cells arise, the metulae or primary sterigmata. About 60 metulae are formed in each vesicle. Each metula buds at its tip to give two or three uninuclear phialides, also called secondary sterigmata. From the phialide, uninuclear conidia bud, only one nucleus enters each conidium. The process is repeated, in such a way that clonal rows of conidia are formed, the last conidium to be formed is adjacent to the metula, the first and oldest being the most distal one. This process is not identical in all Aspergilli; some species, called uniseriate (such as A. fumigatus), have only one series of sterigmata from where conidia arise directly, while in some Aspergilli, conidia contain more than one nucleus (such as A. oryzae). Two approaches were used to study this process. In the first one, mutants were isolated and blocked in different steps of conidiophore development; in the second, mycelia were synchronized, and by the technique of ‘cascade hybridization’, an early methodology to define a transcriptome, it was determined which genes were expressed at different stages of conidiophore development. John Clutterbuck published in 1969 the seminal article of the study of the conidiophore developmental pathway, while the first cascade experiment in any organism was published by the Timberlake Laboratory in 1980. Clutterbuck described a number of mutants blocked in different steps of conidiophore development. In bristle mutants (brlA), conidiophore stalks that fail to complete the developmental pathway

C

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P M C

brlA

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P V

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abaA V

S FC Figure 13 The condiophore of Aspergillus nidulans. In the left panel scanning electron microscopy images of condiophore of the wildtype and two mutant strains are shown, these carry loss-of-function mutations in the brlA (bristle) and abaA (abacus) gene respectively. The center panel illustrates the developmental process by showing the expression of a membrane protein (the UapA transporter fused to the green fluorescent protein (GFP)), which is specifically expressed in the metula stage, which is then diluted on in the phialides and conidia, the same transporter is then expressed again during conidial germination (see text). The right panel show a schematic representation of the conidiophore of A. nidulans, metulae and phialides are arbitrarily colored to facilitate identification. FC, foot compartment; S, conidiophore stalk; V, Vesicle; M, metula; P, phialide; C, conidia. Notice that for some metulae in the left and centre panels the two cognate phialides can be clearly seen. The pictures in the left hand panel has been kindly provided by Reinhrad Fischer. Reproduced with permission from Kues U and Fischer R (eds.) (2006) The Micota I, Growth Differentiation and Sexuality. Berlin: Springer-Verlag. One of the center panel by George Diallinas and Areti Pantazopoulou.

Fungi | Aspergillus: A Multifaceted Genus

originate from the mycelial mat. In abacus mutants (abaA), sterigmata continue to give row after row of additional sterigmata, without ever terminally differentiate phialidae or conidia (Figure 14). Stunted mutants (stuA) result in short conidiophores with conidia being made directly from the vesicle, while in medusa (medA), metulae do not immediately differentiate and produce series on metulae before giving origin to phialides. Finally, wet mutants (wetA) do not affect the development of the conidiophore, but result in defective conidia that autolyze. Once these genes were cloned, their function was analyzed by inactivating them, following their expression pattern and overexpressing them conditionally using the tightly regulated alcA promoter (see above). BrlA is a Zn finger transcription factor, which directly regulates the expression of abaA. AbaA is also a transcription factor, which regulates brlA in a feedback loop and wetA. WetA regulates lateexpressed and conidial-specific genes such as cell wall genes, and it is supposed to be a transcription factor. StuA has the characteristics of a transcription factor and limits in some unknown way the spatial distribution of the BrlA and AbaA proteins as well as being involved in conidiophore elongation and wall thickening, while MedA regulates the temporal expression of brlA along the developing conidiophore. Downstream of the three core regulators, BrlA,

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AbaA, and WetA, there are target genes, which are activated at different stages of the conidial developmental process. Some of these, as genes involved in spore pigmentation, were known from the earlier days of A. nidulans genetics, others were identified by the cascade hybridization methodology. These target genes show a variety of regulation patterns, some like wA (see Figure 14) being under the control of WetA, some like yA under the control of AbaA, some others requiring, in order to be expressed, various combinations of the three transcription factors. The number of downstream genes has been estimated 90% (A–T)-rich sequence that separates CDEI and CDEIII. CDEIII is a conserved 24 bp sequence. CDEI and CDEIII are essential for chromosome segregation, since deletion of CDEI increases chromosome loss tenfold and deletion or point mutations of CDEIII destroy centromere function. In contrast to the centromere sequence, centromerebinding proteins of S. cerevisiae show little similarity to analogous proteins in other yeasts and higher eukaryotes. However, in all cases, kinetochores are large protein assemblies. In S. cerevisiae, kinetochores comprise 60 different subunits arranged in layers as 14 discernable complexes. A four-protein inner complex, termed Cbf3, binds specifically to CDEIII and is key for initiating kinetochore assembly. After binding to CDEIII, one of the Cbf3 proteins, p58, is activated by phosphorylation and later degraded by ubiquitin-mediated proteolysis. By this mechanism, assembly of the kinetochore can be limited to one per chromatid and assembly can be restricted to a limited time during the cell cycle. The kinetochore links spindle microtubules to sister chromatids (Figure 5). The spindle microtubules emanate from the SPB, which is embedded in the nuclear membrane throughout the cell cycle. Duplication of the SPB takes place prior to the onset of S phase in S. cerevisiae. Surprisingly, the spindle (SPB with microtubules) first appears during S phase in budding yeast, suggesting that chromosome replication and spindle assembly comprise two separate regulatory pathways. Spindle elongation and subsequent chromatid separation requires kinesin-related proteins Kip1 and Cin8 and a dynein-related protein Dyn1 to provide both pushing and pulling forces. There is also evidence for mechanochemical motor activity associated with the kinetochore. In budding yeast, each kinetochore captures only one independent microtubule. A mechanism to detect improperly oriented attachments exists, and attempts to reattach are continued until the correct orientation is achieved. It is likely that only one of the sister chromatids initially becomes attached to a spindle microtubule. The attachment, however, causes the movement of both

chromatids, which oscillate as the microtubule grows and shrinks. This oscillation may prevent the remaining kinetochore from attaching to other microtubules from the same SPB. Only binding of the remaining kinetochore to a microtubule from the opposite SPB can produce the bidirectional force required to trigger dissolution of cohesions holding the chromatids together (Figure 5). Since the release of cohesion during the cell cycle is such a dramatic event, there are several checkpoint proteins complexed with the kinetochore to monitor the events in early mitosis and inhibit chromosome segregation until all chromatids are properly attached to microtubules. The loss of cohesion is synchronous and very tightly regulated. Scc1 is cleaved by a cysteine protease termed separase (Esp1), whose activity is held in check by a chaperone protein called securin. Securin is degraded by APC to release active separase only in anaphase (Figure 5). When Scc1 is cleaved, the linkage holding the sister chromatids together is broken, and the chromosomes are segregated by the motor forces in the microtubules and kinetochore. Origin Localization and Chromosome Segregation in Prokaryotes Little obvious structural organization within the cytoplasm of prokaryotes has been observed in E. coli or any other eubacteria, and there is certainly no structure equivalent to the mitotic spindle segregation system. However, bacterial cells must have a highly effective equipartitioning system to segregate sister chromosomes, since anucleate cells are rarely produced. This perplexing situation resulted in models that included the cell surface as part of the segregation machinery. The 1963 Replicon hypothesis proposed by Jacob, Brenner, and Cuzin suggested that the replication origin could act as centromere, and the sites at which the origin attached to the membrane could function as an SPB (centrosome) in E. coli. Although the physical force separating bacterial chromosomes has yet to be identified, it is clear that bacteria contain mechanisms to accurately relocate newly replicated chromosomal origins to specific intracellular sites followed by ordered, progressive segregation of duplicated loci in a way that maintains the orientation of segregated chromosomes into newly divided daughters (Figure 6). Most information on the intracellular position and movement of genomic loci has come from E. coli, Bacillus subtilis, and Caulobacter crescentus, using several elegant methods. One approach, termed fluorescent repressor operator system (FROS), involves inserting DNA cassettes containing multiple copies of the lac operator into the chromosome at the genetic location to be tracked. Time lapse microscopy then follows the intracellular location of green fluorescent protein (GFP) linked to Lac repressor, which binds to the cassettes. An alternate

Genetics, Genomics | Chromosome Replication and Segregation

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Ori C is at mid-point of the cell

Initation of DNA replication ori C is duplicated Ori C relocates to the one-fourth and three-fourth position Chromosome segregation begins Cells divide, ori C is again at mid-cell Figure 6 Localization of oriC during the cell cycle. Schematic diagram of the localization of oriC in E. coli or B. subtilis during the cell cycle. OriC is at mid-cell in newly divided cells, and remains there until DNA replication initiation. After duplication of oriC, the two origins are actively relocated to positions that are one-fourth and three-fourth the length of the cell. As the chromosome is replicated, the sister chromosomes are also separated into each half of the cell. After cell division, each daughter cell has a complete chromosome, and oriC is again located at mid-cell.

approach is fluorescence in situ hybridization (FISH), in which fluorescently labeled DNA probes for specific loci are hybridized to fixed cells on microscope slides. Partition proteins have also been localized during the cell cycle using fluorescent antibodies as in situ probes. Bacterial replication origins are found to be positioned at specific cellular locations prior to the onset of DNA synthesis (polar in C. crescentus and near mid-cell in E. coli and B. subtilis; see Figure 6). How the correct placement of oriC is achieved in most bacteria remains to be determined, but it is likely that the origin localization mechanisms will vary among bacteria depending on cell shape and life style. After DNA synthesis initiation, duplicated origins are actively separated to the one-fourth and three-fourth cell position in E. coli and B. subtilis (Figure 6) or to opposite poles in C. crescentus. There have been various proposals to explain how the force acting on replication origins is generated; it has been suggested to be derived from the replication process itself (see below), by a mechanism related to transcription, to the synthesis of membrane components between newly replicated origins, or by a dynamic cytoskeletal structure (see below). While none of these mechanisms can be ruled out and perhaps several will act cooperatively, origin translocation across cellular space is unusually rapid raising intriguing issues about the existence of kinetochorelike components that specifically act on origin regions to determine their position in cells and move them apart during the replication process. Several candidate components of a bacterial kinetochore have been described in the most widely studied bacterial systems. Two related systems were uncovered in sporulating B. subtilis. During sporulation, an originlocalizing protein, RacA, targets oriC to cell poles after binding preferentially to 25 regions spread over 612 kb across the origin portion of the chromosome. RacA has

the ability to condense chromosomal DNA. B. subtilis also contains chromosomally encoded proteins that are similar to members of the Par family, partition proteins encoded by plasmids. Most plasmid systems encode two interacting Par proteins (ParA, B) and carry a cis-acting centromere-like element (parS). In most plasmid systems, ParA is an ATPase, and ParB binds directly to parS. In B. subtilis, Spo0J is a homologue of the plasmid-partitioning protein ParB, binding to at least eight sites spread over 800 kb in the B. subtilis oriC proximal region. Loss of Spo0J leads to 100-fold increase in anucleate cells. Spo0J also plays a role in origin positioning in the forespore, suggesting redundancy in B. subtilis kinetochore systems. Par family proteins also exist in C. crescentus, with a role in preventing anucleate cells, but an actin-like protein called MreB is thought to direct origin movement, without affecting other chromosomal loci. MreB appears to associate directly with origin proximal DNA and forms a spiral corkscrew on the cell surface that could serve as a track for origin movement. MreB is also present in E. coli and B. subtilis where it has been implicated in determining cell shape as well as playing a role in chromosome segregation. However, it is not clear that MreB plays a role in oriC localization in cell types other than C. crescentus. In E. coli, a 25 bp locus termed migS was recently identified near oriC. Deletion of migS perturbs segregation, but proteins interacting with this locus remain to be found. It is not clear whether the forces responsible for separating origins are also responsible for segregating the bulk of chromosomal DNA. Recent studies are consistent with the idea that in B. subtilis and other bacteria, the replisome is a stationary factory located in the cell center and extruded DNA emanating from the factory is captured and directed to the appropriate cellular location rather than being pulled to the new position. In addition, during sporulation, a motor protein, SpoIIIE, pumps the bulk of

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Genetics, Genomics | Chromosome Replication and Segregation

chromosomal DNA into the prespore after asymmetric pole formation. This motor may also play a role in vegetative cells. Studies in B. subtilis suggest that chromosome condensation proteins are also required for proper segregation. Mutations in the smc gene of B. subtilis, a homologue of the yeast smc genes, produce less condensed nucleoids, 10% anucleate cells, and altered localization Spo0J. Although no clear SMC homologue exists in E. coli, at least 11 bacteria harbor this protein and it is likely to play an important role in bacterial chromosome biology. A search for partitioning mutants (anucleate cells) in E. coli identified the Muk family of proteins (encoded by mukB, E, and F). These mutant strains are defective in correct folding and condensation of daughter chromosomes suggesting Muk proteins are the best candidates to condense E. coli nucleoids in the absence of SMC proteins. MukB is a 177 kDa protein. In solution, it appears as an elongated molecule with globular domains at both N- and C-termini with a hinged rod connecting region, and it has structural features in common with myosin and kinesin motor proteins. MukB binds ATP and GTP, but does not hydrolyze these nucleotides. MukB also binds to DNA, but specific binding sites have not been identified. It remains to be determined whether MukB forms higher order structures similar to the spindle in eukaryotes or whether MukB is a motor protein that pulls or pushes DNA along a structure associated with the cell surface. Despite these uncertainties, MukBEF remains the best candidate for a motor protein component of the putative bacterial cytoskeleton. The kinetics of chromosome movement with respect to cell cycle timing has been the subject of some debate. The debate centers on whether segregation is a progressive process or whether sister chromosomes remain paired along their length during a significant portion of the cell cycle and then segregate as a unit. Although the presence of a bacterial cohesion mechanism, similar to the mechanism in eukaryotes that ensures proper partitioning during mitosis is attractive, most recent FROS studies support a progressive model for segregation of chromosomal loci. Irrespective of the kinetics of movement, all recent studies show clearly that the nucleoid is well organized and exhibits a highly regulated spatial location in cells during replication and partitioning of daughter chromosomes into newly dividing cells. Based on studies in C. crescentus and E. coli, replication divides the circular genome into two distinct and equal arms termed replicores. Replicores occupy separate halves of the cells, and

this spatial orientation is maintained generation after generation by the sequential layering of newly replicated replicore DNA to the inner and outer edges of developing nucleoids. It remains to be determined whether an additional mechanism is necessary to direct the placement of the chromosome arms. Although it is unlikely that the well-defined segregation structures seen in eukaryotic cells will be found in bacteria, an increasing body of evidence suggests that the segregation mechanism may be more similar between prokaryotes and eukaryotes than was believed previously. The notion of a complex bacterial ‘cytoskeleton’, which helps localization and movement of macromolecules inside the cell, now seems highly plausible. See also: Archaea (overview); Cell Cycles and Division, Bacterial; Chromosome, Bacterial; DNA Replication

Further Reading Barry ER and Bell SD (2006) DNA replication in the archaea. Microbiology and Molecular Biology Reviews 70: 876–887. Blow JJ and Tanaka TU (2005) The chromosome cycle: Coordinating replication and segregation. Second in the cycles review series. EMBO Reports 6: 1028–1034. Botchan M (2007) Cell biology: A switch for S phase. Nature 445: 272–274. Diffley JF (2004) Regulation of early events in chromosome replication. Current Biology 14: R778–R786. Ghosh SK, Hajra S, Paek A, and Jayaram M (2006) Mechanisms for chromosome and plasmid segregation. Annual Review of Biochemistry 75: 211–241. Kaguni JM (2006) DnaA: Controlling the initiation of bacterial DNA replication and more. Annual Review of Microbiology 60: 351–375. Kato J (2005) Regulatory network of the initiation of chromosomal replication in Escherichia coli. Critical Reviews in Biochemistry and Molecular Biology 40: 331–342. Leonard AC and Grimwade JE (2005) Building a bacterial orisome: Emergence of new regulatory features for replication origin unwinding. Molecular Microbiology 55: 978–985. Nasmyth K and Schleiffer A (2004) From a single double helix to paired double helices and back. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 359: 99–108. O’Donnell M and Jeruzalmi D (2006) Helical proteins initiate replication of DNA helices. Nature Structural & Molecular Biology 13: 665–667. Sawitzke J and Austin S (2001) An analysis of the factory model for chromosome replication and segregation in bacteria. Molecular Microbiology 40: 786–794. Sherratt DJ (2003) Bacterial chromosome dynamics. Science 301: 780–785. Stillman B (2005) Origin recognition and the chromosome cycle. FEBS Letters 579: 877–884. Thanbichler M, Viollier PH, and Shapiro L (2005) The structure and function of the bacterial chromosome. Current Opinion in Genetics & Development 15: 153–162. Watrin E and Legagneux V (2003) Introduction to chromosome dynamics in mitosis. Biologie Cellulaire 95: 507–513.

Chromosome, Bacterial K Drlica, University of Medicine and Dentistry of New Jersey, Newark, NJ, USA A J Bendich, University of Washington, Seattle, WA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Historical Introduction Chromosome Form and Number Gene Arrangements Recombination DNA Twisting, Folding, and Bending

Glossary DNA supercoiling A phenomenon occurring in constrained duplex DNA molecules when the number of helical turns differs from the number found in DNA molecules of the same length, but containing an unconstrained end that can rotate. Supercoiling creates strain in constrained DNA molecules. A deficiency of duplex turns generates negative supercoiling; a surplus generates positive supercoiling. Supercoils can be helical or plectonemic (similar to a twisted rubber band). DNA topoisomerases Enzymes that change DNA topology by breaking and rejoining DNA strands. Topoisomerases introduce and remove supercoils, tie and untie knots, and catenate and decatenate circular DNA molecules. genome The entire complement of genetic material in a bacterium or in the nucleus, mitochondrion, or chloroplast of a eukaryotic species.

Abbreviations FIS H-NS

factor for inversion stimulation histone-like nucleoid structuring protein

Chromosome Inactivation Chromosome Duplication and Segregation Chromosome Packaging Dynamics Concluding Remarks Further Reading

nucleoid A term for the bacterial chromosome when it is in a compact configuration, either inside a cell or as an isolated structure. origin of replication A location on the chromosome (oriC) where initiation of replication occurs. For E. coli, oriC is about 250 nt long and during initiation it specifically interacts with several proteins to form an initiation complex. Archaebacterial chromosomes may contain as many as three replication origins. recombination A process in which two DNA molecules are broken and rejoined in such a way that portions of the two molecules are exchanged. replication fork The point at which duplex DNA separates into two single strands during the process of DNA replication. Associated with replication forks are DNA helicases to separate the strands and DNA polymerases to synthesize new DNA strands.

IHF LRP

integration host factor leucine-responsive regulatory protein

Defining Statement

Historical Introduction

Advances in microscopy reveal intracellular locations and movements of specific chromosome regions. DNA conformations (supercoiling, folding, and looping) are also dynamic. Nucleotide sequence analysis reveals many historical additions, deletions, and rearrangements; strains within a species can display both common and diverse sequences.

Bacterial chromosomes were discovered much later than their eukaryotic counterparts, largely due to their small size. Moreover, bacterial chromosomes do not undergo the striking metaphase condensation that makes eukaryotic chromosomes so easy to see. Indeed, it was not until the early 1940s that bacteria were clearly shown to undergo spontaneous mutation and to have mutable

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genes. At about that time, Avery and associates discovered the chemical nature of genetic material: extracted DNA carried a character for polysaccharide synthesis from one strain of Pneumococcus to another. At first, the result was not universally accepted as evidence for genetic exchange, partly because the so-called ‘transforming principle’ exerted its effect after an unknown number of steps and partly because Avery lacked a molecular framework for explaining how DNA could function as genetic material. In 1952, Hershey and Chase announced that phage DNA, not protein, is injected into bacterial cells during infection, and a year later Watson and Crick provided the structural framework for DNA. At that point DNA became widely accepted as the carrier of hereditary information, and a search for bacterial chromosomes began. By 1956, nucleoids, as bacterial chromosomes are called, could be seen in living cells as discrete, compact structures (for recent example see Figure 1). Gentle extraction methods eventually yielded large, intact DNA molecules; by the early 1970s it became possible to isolate a compact form of the chromosome for biochemical study. DNA supercoiling had been discovered in the mid-1960s, and within a decade enzymes called DNA topoisomerases that introduce and remove supercoils were found. The existence of DNA topoisomerases gave credence to the idea that chromosomal DNA is under torsional tension inside cells. During the 1980s the dynamic, regulated nature of supercoiling emerged as a major structural feature that needed to be considered whenever the activities of the chromosome were discussed. The development of rapid methods for determining nucleotide sequences led to complete sequences for many bacterial genomes in the 1990s.

DNA sequence analyses led to the conclusion that all living organisms share a common ancestor, and inferences could be drawn about the nucleotide sequence history of chromosomes. An emerging theme is the dynamic nature of bacterial chromosomes. In terms of nucleotide sequence, massive gene shuffling has occurred over the course of evolution. With respect to three-dimensional structure, portions of the chromosome move to particular regions of the cell at specific times during the cell cycle, while the bulk of the DNA threads through replication forks. At the level of DNA conformation, changes can occur within minutes after alterations in cellular environment occur. These changes are influenced, and in some cases directed, by protein components of chromosomes. In the following sections we sketch major concepts concerning chromosome structure. We emphasize that a bacterial chromosome is not equivalent to a bacterial genome: a chromosome is a dynamic protein–RNA–DNA structure that can vary in conformation, size, DNA content, and form with growth conditions, whereas a genome is the genetic information content of the organism, its DNA sequence; a genome does not change with growth conditions.

Chromosome Form and Number Bacterial DNA has been found in both circular and linear forms. For Escherichia coli, chromosomal circularity is supported by three lines of evidence. First, circles were observed when radioactively labeled DNA was extracted from cells and then examined by autoradiography. Although almost all of the molecules in these experiments

(a)

(b)

(c)

(c)

Figure 1 Bacterial nucleoids. Nucleoids of Escherichia coli K-12 were visualized in a confocal scanning laser microscope as developed by GJ Brankenhoff (Nature 317: 748–749, 1985). Elongated cells were obtained by growth in broth. Then the nucleoids were stained with the DNA-specific fluorochrome DAPI (0.1 mg ml1) added to the growth medium. Under these conditions the stain had no effect on growth. The cells were observed either alive (a) or after fixation with 0.1% osmium tetroxide (b). Since the cell boundary is not easily visualized, it has been sketched in for reference (c). Multiple nucleoids were present because these fast-growing cells contain DNA in a state of multifork replication. In live cells the nucleoid has a cloud-like appearance and a smooth boundary with the cytoplasm (protuberances, if present, would be smaller than 200 nm). Magnification for panels (a) and (b) is 9000. Photo courtesy of Dr. Conrad Woldringh, Department of Molecular Cell Biology, University of Amsterdam, The Netherlands.

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were so tangled that their configurations were unclear, a few appeared as large circles more than a millimeter in length (cells are only 1 or 2 mm long). However, these circular molecules exhibited a size range of severalfold, which was not readily explained. Second, genetic mapping studies are most easily interpreted as the genes being arranged in a circle, although a linear interpretation is still possible (mapping can be ambiguous, since a large linear bacteriophage DNA is known to have a circular genetic map). Third, two bidirectional replication forks emerge from a single origin of replication, and DNA moves through a ‘replisome’ (or the replisome moves through DNA) such that the forks converge at a point located 180 opposite to the origin on the circular map. Recombination and decatenation events expected to be associated with large circular DNA then allow each daughter cell to inherit a chromosome. Conclusive evidence for circularity would be visualization of circular images for most of the DNA molecules present. In 1989, chromosomal DNA molecules of Borrelia burgdorferi were found to have a linear form. Linear chromosomes were subsequently observed in Streptomyces species, Rhodococcus fascians, and Agrobacterium tumefaciens. With Streptomyces, DNA ends contain repetitive sequences as well as terminal proteins that prime DNA synthesis complementary to the 39 end of the DNA. In B. burgdorferi, the ends are hairpins that facilitate complete replication. Thus bacteria have chromosomal ends that function much like the telomeres of linear chromosomes in eukaryotic cells. Many bacteria carry all of their genes in a single genetic linkage group, as if they have a single type of chromosome. However, there is a growing list of species in which useful or essential genes are found on two or more chromosomes. The number of large, circular-mapping DNA molecules is two for Vibrio cholerae, Leptospira interrogans, Rhodobacter sphaeroides, and Brucella species; three for Rhizobium meliloti; and 2–4 among the isolates of Burkholderia (Pseudomonas) capecia. Some Agrobacterium species contain one circular- and one linear-mapping chromosome. Thus the old idea that prokaryotes contain only one circular chromosome has been abandoned. Indeed, those with more than one chromosome (genetic linkage group) may constitute a sizable class, since the vast majority of bacterial species have yet to be examined. Distinguishing between chromosomes and plasmids can be difficult, since some plasmids are very large and contain genes essential for cell growth. Moreover, some large plasmids integrate into, and excise from, chromosomes. Thus chromosome number in some species may be variable. The existence of multiple copies of chromosomal regions, as well as entire chromosomes (multiploidy), is well known in eukaryotes. In addition, a eukaryotic cell can contain thousands of copies of mitochondrial and

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chloroplast genomes. Multicopy genomes are also common among bacteria. For example, the cells of E. coli growing rapidly in a rich medium (20-min doubling time) at low cell density are large and contain about ten genome equivalents of DNA per cell, whereas the number is between one and two in small cells during slow growth. Cells of Deinococcus radiodurans, contain ten genome equivalents during exponential growth and four during the stationary phase. Even slowly growing cells, such as Borrelia hermsii (minimum doubling time 8 h), can carry multiple genomes. This bacterium contains 8–11 genome copies when grown in vitro and up to 16 copies when grown in mice. Azotobacter vinlandii presents a dramatic example. Genome copy number in rich medium increases from 4 to 40 and then to greater than 100 as the culture progresses from early exponential through late exponential to stationary phase. DNA per cell then decreases at the start of a new growth cycle. The spectacular increase in genome copy number in Azotobacter is not observed with cells grown in minimal medium. High copy number may result from an ‘engorge now divide later’ reproductive strategy. When nutrients are abundant, it might be advantageous to carry many genomes in large cells to be diluted into smaller cells when nutrients become limited. An extreme practitioner of this strategy is the eubacterium Epulopiscium fishelsoni, which can fill its halfmillimeter-long cells with 100 000 genome-equivalents of DNA in times of plenty. When nutrients run out, the large cells subdivide into many smaller cells. Regardless of the reason for genome multiplicity, it is clearly not restricted to eukaryotic cells. Indeed, bacterial and eukaryotic chromosomes can no longer be considered different with respect to form (both types can be linear) and number of linkage groups (bacteria, which often have one, can have several; eukaryotes, which usually have many chromosomes, can have only one, as seen with the ant Myrmecia pilosula). What distinguishes bacterial and eukaryotic chromosomes is the coupling between replication and segregation: it is flexible in bacteria and tight among eukaryotes As a result, a nuclear chromosome never contains more than one genome equivalent of DNA as it segregates to daughter cells. In contrast, a bacterial chromosome may contain from one to many genome equivalents of DNA (depending on growth conditions) even during segregation.

Gene Arrangements Gene mapping in bacteria was originally based on the ability of an externally derived, genetically marked fragment of DNA to recombine with the homologous region of a recipient’s DNA. The frequency with which two nearby markers recombine is roughly proportional to the distance between them. As mutations were collected

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for a variety of purposes, characterization of a mutation usually included determining its map position on the genome. The resulting genetic maps revealed relationships among genes such as operon clusters, showed orientation preferences that might reflect chromosomal activities, and suggested that some chromosomal information may have been derived from plasmids and phage. The discovery of restriction endonucleases led to a quantum advance in genetic mapping, since these enzymes allowed the accurate construction of maps in terms of nucleotide distances. Practical nucleotide sequencing methods, which became available in the late 1970s, were expected to yield the complete nucleotide sequences for at least 1000 bacterial species by the year 2008. Data are being obtained at three levels: (1) the genetic map, with the genes and their map locations correlated with the role of the gene products in cell metabolism, structure, or regulation, (2) the physical map in terms of locations of restriction sites, and (3) the nucleotide and corresponding protein amino acid sequences. It is becoming clear that all living organisms probably arose from a common ancestor. Thus information on nucleotide sequence and gene function in one organism can be applied to many other organisms. The conservation of gene structure makes it possible to use nucleotide sequence information for comparison of genetic maps among bacterial species. One of the features revealed is clustering of related genes. For example, the genetic maps of Bacillus subtilis, E. coli, and Salmonella typhimurium show a grouping of many genes for biosynthetic and degradative pathways. Such grouping could be for purposes of coordinated regulation, since some adjacent genes produce polycistronic messages. A completely different view of the same data maintains that functionally related genes move horizontally (from one organism to another) as clusters, because the products of the genes work well together, increasing the probability of successful transfer. Both ideas are likely to be accurate. While it is clear that genomes are quite malleable, the time frame over which insertions and deletions occur can be large. Some perspective is provided by comparison of the maps of E. coli and S. typhimurium. One large genetic inversion occurred, and the maps have major differences at about 15 loci where pieces of DNA were either inserted into or deleted from one genome or the other. But the overall arrangement of gene order between the organisms is remarkably conserved. In contrast, the genomes of some other bacteria, such as Salmonella typhi, Helicobacter pylori, and strains of Pseudomonas aeruginosa, Fransciella tularensis, and Bartonella henselae, appear to have undergone substantial rearrangement. Interestingly, when S. typhi and S. typhimurium are grown in the laboratory, rearrangements are found for both, but when isolated from humans from all over the world, rearranged genomes are found for S. typhi but not S. typhimurium. The reason for

differences among organisms is not clear. Some rearrangements, such as insertions or deletions, may be more deleterious for certain bacteria. Alternatively, the opportunity for rearrangements, such as the occurrence of recombinational hot spots, may be greater in some organisms than in others. The latter explanation appears to be more likely for chloroplast and mitochondrial genomes, which in many ways resemble bacterial genomes. After some 300 million years of evolution, the order of chloroplast genes is highly conserved among most land plants, including mung bean. Yet chloroplast gene order is completely scrambled in pea, a plant closely related to beans. Massive rearrangement of genes is also evident when mitochondrial genome maps are compared among types of maize. Thus gene order in organelles appears to have little functional significance, and it can be subject to frequent recombination if the opportunity arises. Situations in which the opportunity is lacking clearly exist. As complete genomic nucleotide sequences become increasingly available, new questions arise. For example, what is the minimal number of genes required for independent life? Endosymbionts of sap-sucking insects currently hold the record: Carsonella ruddii DNA has only 160 000 bp arranged in 182 open reading frames. Nucleotide sequence analysis is also identifying genes involved in pathogenicity by comparison of virulent and avirulent strains of a pathogen. For example, such an approach has uncovered a ‘pathogenicity island’, a collection of virulence genes, in H. pylori. The island is bounded by 31 bp direct repeats, as if it had been transferred horizontally into an ancestor of H. pylori. In some bacteria, horizontal transfer may have been quite extensive. With E. coli as much as 15% of the genome, 700 kbp, may have been acquired from foreign sources such as integrative bacteriophage, transposons in plasmids, and conjugative transposons (genetic elements that cannot replicate independently but cause the occurrence of conjugation, a form of cell-to-cell DNA transfer).

Recombination Intracellular DNA experiences a variety of perturbations that must be repaired to maintain the integrity of the chromosome and to allow movement of replication forks. Cells have several ways to repair DNA damage, one of which involves recombination (Recombination is a process in which DNA molecules are broken and rejoined in such a way that portions of the two molecules are exchanged.) Damaged sequences in one molecule can be exchanged for undamaged ones in another. It is now thought that the raison d ’eˆtre for recombination is its role in DNA repair, a process that occurs thousands of times per cell generation.

Genetics, Genomics | Chromosome, Bacterial

Recombination is also involved in DNA rearrangements arising from the pairing of repeated sequences. When the repeats are in direct orientation, duplications and deletions arise; inversions arise when the repeats are inverted. In B. subtilis a cascade of sequential rearrangements has been identified in which large transpositions and inversions have been attributed to recombination at specific junction points in the genome. Several other examples are found for the rrn clusters, sets of similar assemblies of ribosomal and transfer RNA genes. Rearrangements at the three rrn clusters of Brucella are thought to be responsible for differences in chromosome size and number among species of this genus. Other repeated sequences that facilitate rearrangements are duplicate insertion sequences, the rhs loci (recombination hot spot), and experimentally introduced copies of the transposon Tn10. The third consequence of recombination is the insertion of genes from mobile elements into chromosomes. These elements, which include transposons, plasmids, bacteriophage, integrons, and pathogenicity islands, move from one cell to another and sometimes from one species to another. A spectacular example of gene transfer and its evolutionary effect across kingdoms is seen in the acquisition of a 500 kbp symbiosis island. This island of DNA converts a saprophytic Mesorhizobium into a symbiont of lotus plants that is capable of fixing nitrogen. Because they mediate such sweeping change, mobile genetic elements may represent the most important means for generating the genetic diversity on which selection operates. For mobility, and thus generation of genetic diversity, such genetic elements require recombination activities.

DNA Twisting, Folding, and Bending DNA Supercoiling Circular DNA molecules extracted from mesophilic bacteria have a deficiency of duplex turns relative to linear DNAs of the same length. This deficiency exerts strain on DNA, causing it to coil. The coiling is called negative supercoiling (an excess of duplex turns would give rise to positive supercoiling). Negative supercoils can assume either a helical or a plectonemic form (the latter is similar to a twisted rubber band). Superhelical strain is spontaneously relieved (relaxed) by nicks or breaks in the DNA that allow strand rotation; consequently, supercoiling is found only in DNA molecules that are circular or otherwise constrained so the strands cannot rotate. Since processes that separate DNA strands relieve negative superhelical strain, they tend to occur more readily in supercoiled than in relaxed DNA. Among these activities are the initiation of DNA replication and initiation of transcription. Negative supercoiling also makes DNA

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more flexible, facilitating DNA looping, wrapping of DNA around proteins, and the formation of cruciforms, left-handed Z-DNA, and other non-B-form structures. In a sense, negatively supercoiled DNA is energetically activated for most of the processes carried out by the chromosome. Negative supercoils are introduced into DNA by gyrase, one of the several DNA topoisomerases found in bacteria. DNA topoisomerases act through a DNA strand breaking and rejoining process that allows supercoils to be introduced or removed, DNA knots to be tied or untied, and separate circles of DNA to be linked or unlinked. The action of gyrase is countered by the relaxing activities of topoisomerase I and topoisomerase IV. Since gyrase is more active on a relaxed DNA substrate, while topoisomerases I and IV are more active on a negatively supercoiled one, the topoisomerases tend to reduce variation in supercoiling. Moreover, lowering negative supercoiling raises gyrase expression and lowers topoisomerase I expression. Thus negative supercoiling is a controlled feature of the chromosome. Supercoiling is influenced by the extracellular environment. For example, when bacteria such as E. coli are suddenly exposed to high temperature, negative supercoiling quickly drops (relaxes), and within a few minutes it recovers. The reciprocal response is observed during cold shock. Presumably these transient changes in DNA supercoiling facilitate timely induction of heat- and coldshock genes important for survival. Supercoiling is also affected by the environment through changes in cellular energetics. Gyrase hydrolyses ATP to ADP as a part of the supercoiling reaction, and ADP interferes with the supercoiling activity of gyrase while allowing a competing relaxing reaction to occur. Consequently, the ratio of [ATP] to [ADP] influences the level of supercoiling. Changes in oxygen tension and salt concentration provide examples in which cellular energetics and supercoiling change coordinately. Collectively, these observations indicate that chromosome structure changes globally in response to the environment. Supercoiling is influenced locally by transcription. During movement of transcription complexes relative to DNA, RNA polymerase does not readily rotate around DNA. Consequently, transcription generates positive supercoils ahead of the polymerase and negative supercoils behind it. Since topoisomerase I removes negative supercoils and gyrase positive ones, transcription and similar translocation processes have only transient effects on supercoiling. However, cases in which induction of very high levels of transcription results in abnormally high levels of negative supercoiling have been found. In such situations transcription-mediated changes in supercoiling provide a way for specific regions of a DNA molecule to have levels of supercoiling that differ greatly from average values.

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Problems can arise when negative supercoils build up behind a transcription complex and facilitate DNA strand separation, since nascent transcripts form long hybrids with the coding strand of DNA. Such hybrid structures, called R-loops, can interfere with gene expression. These hybrids are removed by ribonuclease H; consequently, a deficiency of topoisomerase I can be corrected by overexpression of ribonuclease H. Problems also arise from a buildup of positive supercoils ahead of a transcription complex, since helix tightening will slow transcription. That is probably why strong gyrase-binding sites are scattered throughout the chromosome immediately downstream from active genes.

DNA Looping In the early 1970s Worcel showed that multiple nicks are required to relax chromosomal supercoils, demonstrating that DNA must be constrained into topologically independent domains. Superhelical tension and topological domains were later detected in living cells, making it unlikely that the domains are artifacts of chromosome isolation. Dividing supercoiled DNA into independent domains keeps a few nicks or breaks from relaxing all the supercoils. Independent domains also allow supercoils to be introduced into the chromosome before a round of replication finishes – in the absence of domains, the gaps following replication forks would relax any supercoils that gyrase might introduce. While early studies estimated the number of domains at about 50–100, more recent work, some based on site-specific DNA breakage and supercoil-sensitive expression of particular genes, places the number in the hundreds, about one per 10 kb of DNA. A variety of processes probably contribute to domain structure. One may be coupled transcription–translation of proteins that transiently anchor the chromosome to the cell membrane. This process, called transertion, restricts strand rotation. Since domain barriers are present even when RNA synthesis is inhibited, additional factors, such as the MukBEK protein, are likely to be involved. MukBEK has two DNA-binding domains connected by a long flexible linker. Thus, it could restrict DNA rotation by binding to distant regions of DNA. Another type of constraint is seen after exhaustive deproteinization. Nearly every nucleoid in preparations from both exponential and stationary-phase E. coli appears by fluorescence microscopy as a rosette or loose network of 20–50 large loops. Such interactions involving only DNA may reflect the role of recombination in forming some of the domains. Still other factors may be the DNA-compacting proteins discussed below.

DNA-Compacting Proteins Five small, abundant, DNA-binding proteins have captured attention as possible elements of chromosome packaging. At one time these proteins were called histone-like proteins, but they have little resemblance to histones with respect to amino acid sequence. They have also been called nucleoid-associated proteins, but that term encompasses many more proteins. A unifying feature is the ability of these proteins to compact DNA. A variety of functions, including regulation of gene expression and in several cases participation in site-specific recombination, have evolved for these proteins. Most of our knowledge about the proteins comes from biochemical studies. The most abundant of the small compacting proteins is HU (60 000–100 000 monomers per cell). Each member of the dimer protein has a long arm; together the arms reach around DNA, using a proline at the tip of each arm to intercalate into DNA and either create or stabilize kinks in DNA. HU binding, which lacks nucleotide sequence specificity, shows a preference for supercoiled DNA. HU constrains negative supercoils, which led to the idea that the protein wraps DNA into nucleosome-like particles in vitro. Nucleosomes, which have long been recognized as a distinctive feature of eukaryotic nuclei, are ball-like structures in which about 200 bp of DNA is wrapped around histone proteins. Nucleosomes occur at regular intervals along DNA, giving nuclear chromatin a ‘‘beadson-a-string’’ appearance. True bacteria (eubacteria) do not have true histones or nucleosomes, although some archaebacteria do. Thus HU is more likely to be a bending rather than a wrapping protein. With some DNAs, HU introduces a 180 bend, although on average the bends are closer to 100 . The bending activity is especially clear when HU serves as an architectural protein, assisting in the formation of DNA–protein complexes that carry out site-specific recombination. HU also provides the DNA bending needed for certain repressors to bring distant regions of DNA together in loops that block initiation of transcription. Closely related to HU is a bending protein called IHF (integration host factor, 30 000–60 000 copies per cell). It bends DNA roughly 160 , but unlike HU, IHF recognizes specific nucleotide sequences (about 1000 specific IHFbinding sites are present in the E. coli genome). Many examples have been found in which IHF helps form a DNA loop between promoters and transcription activators located far upstream from promoters. IHF also participates as an architectural protein during the formation of site-specific DNA–protein complexes. The best known of these is the intasome generated by bacteriophage lambda during integration into and excision from the bacterial chromosome. Since cells contain many more copies of IHF than specific IHF-binding sites, the protein

Genetics, Genomics | Chromosome, Bacterial

may also have a nucleotide-sequence-independent mode of binding that contributes to DNA compaction. In vitro the protein can compact DNA by 30%, presumably through nonspecific binding. The third protein is called FIS (factor for inversion stimulation). FIS recognizes a weakly conserved 15 bp binding site that is present at almost 6000 copies per genome. FIS is a dimer of two identical subunits that appear to bind in adjacent major grooves of DNA. A bending angle of about 50 to 90 is generated as dimerization of the protein pulls on the two portions anchored to DNA. FIS also binds nonspecifically to plectonemic supercoiled DNA, clustering at DNA crossover points and at the apexes of DNA loops. Thus, FIS may stabilize DNA loops. The level of FIS expression is sharply elevated shortly after dilute bacterial cultures enter logarithmic growth, reaching a maximal copy number of about 60 000 per genome. In older stationary-phase cultures, the rate of FIS synthesis drops to almost zero. Some of the binding sites for FIS are so close to promoters that FIS acts as a repressor. In other cases, FIS acts as an upstream activator of transcription. It also serves as an architectural protein when it forms protein–DNA complexes for site-specific recombination. LRP (leucine-responsive regulatory protein; 3000 dimers per cell) is a small protein that responds to the nutrient status of the cell, particularly amino acid levels. It acts as a repressor for many genes involved in catabolic (breakdown) processes and as an activator for genes involved in metabolic synthesis. LRP appears to have two modes of action. At its own promoter it oligomerizes into an octomeric, nucleosome-like structure that wraps DNA. The second mode is much like the DNA bridging observed with H-NS (see below). As with HU, IHF, and FIS, LRP has architectural roles when it forms protein– DNA complexes for site-specific recombination. The fifth protein is called H-NS (histone-like nucleoid structuring protein, 20 000 molecules per cell). Unlike the four other small DNA-compacting proteins, H-NS does not actively bend or wrap DNA. Instead, it binds to DNA that is already bent, generally at AT-rich sequences; thus, H-NS stabilizes DNA bends. H-NS is composed of two domains connected by a flexible linker. The N-terminal domain dimerizes with another H-NS molecule, while the C-terminus binds DNA. Thus the dimer has two DNA-binding domains. Electron microscopy studies indicate that H-NS can form bridges between regions of double-stranded DNA, while biochemical work suggests that binding of H-NS stiffens DNA, perhaps in local patches. H-NS patches interacting with each other could be responsible for the DNA bridging effect, which could stabilize DNA loops. If an appropriate DNA bend is near the promoter of a gene, H-NS binding will repress the gene by preventing RNA

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polymerase binding. Binding within a gene can also act as a roadblock to transcription, and with some genes the bridging function of H-NS appears to trap RNA polymerase in a DNA loop. The expression of hundreds of genes is likely to be affected by H-NS. An interesting possibility is that H-NS binds to many regions of DNA when they enter a genome ‘horizontally’, thereby repressing large numbers of genes. Such gene silencing would allow cells to accept a new piece of DNA that might otherwise express deleterious genes. While the abundance and biochemical properties of DNA-compacting proteins make them good candidates for chromosome structural elements, the dynamic nature of their interactions with DNA and their multiple, sometimes redundant activities make it difficult to assign firm roles in DNA packaging. Nevertheless, combinations of compacting protein mutations are associated with reduced nucleoid compaction, as seen with HU/FIS double mutants. Conversely, overexpression of H-NS increases compaction.

Chromosome Inactivation In eukaryotic cells, large portions of genomes are rendered transcriptionally inactive by heterochromatinization, a local DNA compaction that is readily observed by light microscopy. Bacterial chromosomes are too small to see locally compacted regions; consequently, we can only guess about their existence. However, evidence is accumulating that bacteria have systems that condense entire chromosomes. Caulobacter crescentus serves as an example. In the life cycle of this organism, two cell types exist: swarmer cells and stalk cells. The latter are genetically active. When a swarmer cell differentiates into the stalked type, the nucleoid changes from a compact form into a more open structure, possibly reflecting transcriptional activation of the chromosome. In the second example, a histone H1-like protein in Chlamydia trachomatis appears to cause chromosomal condensation when the metabolically active reticulate body differentiates into an inactive, extracellular elementary body. Another example occurs during sporulation in Bacillus. In this case the chromosome of the spore is bound with new proteins as its transcriptional activity ceases. Still another case is seen when the archaebacterium Halobacterium salinarium progresses from early to late exponential phase of growth. The H. salinarium nucleoid, when obtained by gentle lysis, changes from a type containing naked DNA to one having the beads-on-astring appearance typical of nucleosomal DNA. This change, seen by electron microscopy, is also reflected in nucleoid sedimentation properties. Finally, fluorescence measurements of DNA and RNA within the enormous cells of E. fishelsoni, which, as pointed out above, are up to 500 mm long, suggest that decondensation and dispersion of

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the nucleoid is accompanied by increased transcriptional activity. Whether some of these diverse organisms share a common mechanism for chromosome activation–inactivation is unknown.

Chromosome Duplication and Segregation The major features of chromosome replication have been established for many years. Semiconservative replication was demonstrated by density-shift experiments in 1958, and a few years later the autoradiograms prepared by Cairns revealed a partially replicated circle containing a large replication ‘bubble’. In the early 1970s it became clear that bidirectional replication begins at a fixed origin (oriC) with a pair of forks pointing in opposite directions along the genetic map. With E. coli, about 40 min is required for the forks to reach a point located 180 from oriC on the genetic map, thereby completing the replication of the 4.6 megabase chromosome. The two daughter chromosomes then segregate. Under conditions of rapid growth, bacterial chromosomes can contain more than one pair of replication forks, allowing cells to inherit chromosomes containing more copies of genes near oriC than far from oriC. The location of highly expressed genes (such as those encoding rRNA and RNA polymerase) near oriC may be advantageous for bacteria capable of rapid growth, thereby providing a reason for segregation of branched DNA. In the nucleus of a eukaryotic cell, however, only unbranched DNA molecules that have completed replication can serve as chromosomes (segregating genetic units) during cell division. Initiation of replication has long been a focus of attention, since it is expected to regulate the cell cycle. Early in the study of initiation, heat-sensitive mutations were obtained in genes called dnaA and dnaC. These mutations made it possible to uncouple initiation from the elongation phase of replication. Then the origin was cloned by its ability to confer replication proficiency to a plasmid lacking an origin of replication. The availability of oriC on a small piece of DNA, plus purified initiation proteins, allowed Kornberg to develop an in vitro initiation system. From this system we learned that initiation involves the specific binding of DnaA to oriC and the wrapping of origin DNA around the protein. Local DNA strand separation then occurs at the origin, and single-stranded binding protein attaches to the separated strands. That helps stabilize what looks like a single-stranded bubble in duplex DNA. The DnaB helicase, helped by the DnaC protein, binds to the replication bubble and enlarges it. Then DNA polymerase binds to form two replication forks. The two forks point in opposite directions; thus, as DNA synthesis begins, the left half of the chromosome

moves through one fork and the right half through the other fork. Sensitive probes that bind to specific regions of the chromosome are being used to address major cytological questions such as whether DNA is drawn through stationary replication ‘factories’ or whether the replication machinery moves along the DNA. For a decade the former idea was favoured. Fluorescent labeling of DNA polymerase indicated that replication forks are located at the center of the cell, where they remained throughout most of the cell cycle. When multifork replication occurred, two additional replication centers, each probably containing a pair of forks, were seen situated between the midcell forks and the cell poles. Data from Sherratt recently argued for independent replication forks that follow the path of the DNA in E. coli cells. These opposing views have not been resolved. A second issue concerns the origin of replication (oriC), which can be located by fluorescent antibodies directed at proteins that bind to repeated nucleotide sequences placed near oriC (or any other specified region). In some studies of newly formed cells, oriC and the replication terminus are located at opposite poles of the nucleoid, implying that the dynamic nucleoid must have internal structure. At the beginning of replication, oriC moves briefly toward a midcell position, presumably to the replication apparatus. Later, two copies of oriC become visible at the same nucleoid pole, apparently having been drawn back to the pole after replication begins. Still later, one copy of oriC abruptly moves to the opposite edge of the nucleoid. Eventually the replication terminus is pulled to the replication apparatus at the midcell position, and late in the cell cycle two replication termini can be seen pulling apart. Then the septum that separates new daughter cells forms between the termini. The localization of oriC and its rapid movement, which is about 10 times faster than cell elongation, indicate that bacterial chromosomes undergo a form of mitosis. But unlike eukaryotic mitosis, the bacterial chromosome continues to be replicated and transcribed throughout segregation. While the details of bacterial ‘mitosis’ are still poorly understood and rapid movement of oriC is not always seen, several proteins exhibit properties expected of mitotic proteins. For example, in B. subtilis the ParB (SpoOJ) protein appears to participate in chromosome partitioning by binding to multiple sites on the chromosome near oriC. An attractive idea is that ParB holds the new and old copies of oriC near one pole of the nucleoid until the mitotic apparatus pulls one oriC copy to the opposite pole. Since mitosis is expected to be an essential activity, it is surprising that mutations in parB are not lethal. Clearly, there is much more to learn about the segregation of sister chromosomes to daughter cells. We expect DNA tangles to arise as replicated chromosomes pull apart. The double-strand passing activity of

Genetics, Genomics | Chromosome, Bacterial

gyrase and topoisomerase IV is well suited for resolving tangles, with the movement of daughter chromosomes to opposite cell poles providing the directionality needed by the topoisomerases to untangle loops. Consistent with this idea, both gyrase and topoisomerase IV are distributed around the E. coli chromosome, as judged by DNA cleavage induced by the quinolone inhibitors of the topoisomerases. Replication is also expected to leave daughter chromosomes catenated (interlinked). Plasmid studies indicate that unlinking may be a function of topoisomerase IV, although other topoisomerases are also able to perform the function. For example, gyrase shows decatenating activity in vitro, as do topoisomerases I and III if nicks or gaps are present in DNA. Some of these backup systems must function in Mycobacterium tuberculosis, Treponema pallidum, and H. pylori, since these bacteria lack topoisomerase IV.

Chromosome Packaging Dynamics Nucleoid compaction occurs at four levels. One is macromolecular crowding: cytoplasmic proteins and other large cytoplasmic molecules are present at such high concentration that they force DNA into a small volume. This packing level requires no specific DNA-compacting protein and therefore accommodates the apparent absence of nucleosome-like particles in eubacteria. A second level of packing is represented by DNA bends and loops stabilized by the small DNA-compacting proteins. Larger proteins, such as MukBEK, may also constrain loops. Some of these proteins are likely to be displaced when a segment of DNA encounters the replication apparatus or transcription complexes. DNA looping generated by plectonemic supercoils represents a third level of compaction. The fourth level is represented by macrodomains. These large (1000 kbp), contiguous regions appear by some assays to be independent units. E. coli contains four macrodomains (Ori, Ter, Left, Right) and two less-structured regions. The Ori and Ter macrodomains were initially recognized by colocalization of fluorescent probes binding at a variety of map positions near oriC or near the terminus of replication. However, years earlier it had been noticed that chromosomal DNA contains boundaries across which DNA inversion rarely occurs. These boundaries define the macrodomains genetically: a much lower frequency of site-specific recombination occurs between sites located in different macrodomains than within the same macrodomain. What causes regions of DNA within a macrodomain to interact more with each other than with other regions is unknown. We envision that chromosomal activities involving bulky protein complexes occur at the edges rather than in the center of the nucleoid. For example, the replication apparatus, which is likely to be attached to a multienzyme

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complex that supplies deoxyribonucleoside triphosphates, may be situated at the edge of the compacted portion of the chromosome, which would allow replication proteins to bind to the cell membrane. Likewise, transcription, which in bacteria is coupled to translation, also probably occurs on DNA emerging from the compacted mass of nucleoid DNA, because ribosomes are seen only outside the nucleoid (extrachromosomal localization is especially likely when transcription–translation complexes are bound to the cell membrane via nascent membrane proteins). Consistent with this idea, pulse-labeled nascent RNA is preferentially located at the nucleoid border, as is topoisomerase I (as pointed out above, topoisomerase I may serve as a cytological marker for transcription, since it is probably localized behind transcription complexes to prevent excess negative supercoils from accumulating). In special cases, such as transcription of ribosomal RNA during periods of rapid growth, transcription ‘factories’ pull together genes from different regions of the nucleoid, thereby creating local foci. If the replication and transcription–translation machineries are located on the surface of the nucleoid, DNA movement must occur to allow access to all nucleotide sequences. Such movement may not fully explain transcriptional access to the whole genome, since some genes can be induced when DNA replication is not occurring. Compacted DNA may be sufficiently fluid that genes frequently pass from interior to exterior without guidance from proteins. At any given moment, in some fraction of the cell population each gene may be at the surface of the nucleoid and available for transcription. Capture of a gene by the transcription–translation apparatus would hold that gene on the surface. During induction of transcription, the fraction of cells in which a particular gene is captured would increase until most cells express that gene. For the chromosome as a whole, many genes would be expressing protein during active growth, and many regions would be held outside the nucleoid core. Kellenberger suggested that such activity explains why the nucleoid appears more compact when protein synthesis is experimentally interrupted. Capture of the oriC region by the replication apparatus might be similar to gene capture for transcription. With the fluid chromosome hypothesis, replication proteins would assemble at oriC and move oriC from its polar position toward the midcell location of the replication apparatus. As oriC and nearby regions are passed through the replication forks and then replicated, new binding sites (parS) for the ParB chromosome partition protein would be created. Once these sites were filled, the two daughter oriC regions might pair through ParB–ParB interactions and return to the polar position. Other proteins would later disrupt ParB–ParB interactions, allowing the new ParB–oriC complex to move to the other pole of the nucleoid.

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Evidence for an oriC-pulling force has been obtained with V. cholerae. In this bacterium ParB interacts with a centromere-like site (parS) located near oriC. During segregation, parS pulls away from nearby chromosomal loci. Meanwhile, ParA, an ATPase capable of forming filamentous polymers in vitro, forms a band that extends from the distant cell pole to the segregating ParB/parS complex. The ParA band then appears to retract, pulling the ParB/ parS complex and the nearby oriC region. Whether a similar phenomenon occurs in other bacteria and whether conclusions derived from other bacteria apply to V. cholerae have yet to be established.

Acknowledgments

Concluding Remarks

Further Reading

Many of the features found in bacterial chromosomes are remarkably similar to those in eukaryotic chromosomes: one or more dissimilar chromosomes (the number can be as high as four among bacteria and as low as one in eukaryotes (2N ¼ 2)), high ploidy (copy number) levels, and, at least in some species, a mitotic-like apparatus used in cell division. Consequently, the prevalent belief that profound differences exist between prokaryotic and eukaryotic chromosomes is eroding. Even the distinction revolving around histones and their compaction of DNA into nucleosomes has exceptions. True bacteria lack histones and nucleosomes, and so DNA compaction must occur by other means. But some archaea have histones and stable nucleosomes, while some unicellular eukaryotes lack both. Nevertheless, the absence of a nuclear membrane and the segregation of actively replicating bacterial chromosomes are distinct. So is the idea of a pan-genome. Genomic sequencing using DNA from multiple strains of the same bacterial species reveals that with some species individual strains can share a core genome but the overall gene content differs from one strain to another. Thus the nucleotide sequence of the total genome of some species can be far greater than that found in any given strain. To our knowledge this phenomenon has not been reported with eukaryotic organisms. One of the next tasks will be to determine whether the core sequences are physically clustered on chromosomes.

Bendich AJ (2007) The size and form of chromosomes are constant in the nucleus, but highly variable in bacteria, mitochondria, and chloroplasts. BioEssays 29: 474–483. Boccard F, Esnault E, and Valens M (2005) Spatial arrangement and macrodomain organization of bacterial chromosomes. Molecular Microbiology 57: 9–16. Drolet M (2006) Growth inhibition mediated by excess negative supercoiling: The interplay between transcription elongation, R-loop formation and DNA topology. Molecular Microbiology 59: 723–730. Fogel M and Waldor M (2006) A dynamic, mitotic-like mechanism for bacterial chromosome segregation. Genes & Development 20: 3269–3282. Hendrickson H and Lawrence J (2007) Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. Molecular Microbiology 64: 42–56. Jin D and Cabrera J (2006) Coupling the distribution of RNA polymerase to global gene regulation and the dynamic structure of the bacterial nucleoid in Escherichia coli. Journal of Structural Biology 156: 284–291. Luijsterburg M, Noom M, Wuite G, and Dame R (2006) The architectural role of nucleoid-associated proteins in the organization of bacterial chromatin: A molecular perspective. Journal of Structural Biology 156: 262–272. Medini D, Donati D, Tettelin H, Masignani V, and Rappuoli R (2005) The microbial pan-genome. Current Opinion in Genetics & Development 15: 589–594. Nakabachi A, Yamashita A, Toh H, et al. (2007) The 160-kilobase genome of the bacterial endosymbiont Carsonella. Science 314: 267–268. Reyes-Lamothe R, Wang X, and Sherratt D (2008) Escherichia coli and its chromosome. Trends in Microbiology 18: 238–245. Thanbichler M, Wang SC, and Shapiro L (2005) The bacterial nucleoid: A highly organized and dynamic structure. Journal of Cellular Biochemistry 96: 506–521. Zimmerman SB (2006) Shape and compaction of Escherichia coli nucleoids. Journal of Structural Biology 156: 255–261.

We thank Marila Gennaro for critical comments on the manuscript. The authors’ work was supported by grants from the National Science Foundation, the American Cancer Society, and the National Institutes of Health.

See also: Cell Structure, Organization, Bacteria and Archaea; Chromosome Replication and Segregation; Intracellular Structures of Prokaryotes: Inclusions, Compartments and Assemblages; Quinolones

Conjugation, Bacterial L S Frost, University of Alberta, Edmonton, AB, Canada ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Conjugative Process Physiological Factors Conjugative Elements Gram-Negative Conjugation

Glossary coupling protein An ATPase responsible for the transport of DNA during conjugation. It is a hallmark of conjugative systems and homologues are widely distributed throughout nature. ICE (integrating conjugative element) Chromosomally encoded elements, similar to conjugative transposons, capable of excision, conjugation, and reestablishment in a new host via integration. The excision and integration operations are formally similar to those of integrative phages. plasmid An extrachromosomal DNA segment, usually circular, which is capable of autonomous replication via a segment of the plasmid called the replicon. relaxase The protein responsible for site-specific nicking at the origin of transfer (oriT) in the DNA as well

Abbreviations Cma Eex fi/Fin Hfr HFT HGT HSL ICEs

chromosome mobilization ability entry exclusion fertility inhibition high frequency of recombination high frequency of transfer horizontal gene transfer homoserine lactone-like integrating conjugative elements

Gram-Positive Conjugation Mobilization Transfer to Plants Evolutionary Relationships Conjugation in Natural Environments Further Reading

as recircularization after transfer. It covalently attaches to the 59 end of the nicked DNA via a tyrosine. It is a key component of the relaxosome. transconjugant A general term for a recipient cell that has successfully been converted to donor cell by conjugation. transposon A segment of DNA that is replicated as part of a chromosome or plasmid. It encodes a mechanism, called transposition, for moving from one location to another, leaving a copy at both sites. type IV secretion system (T4SS) A widely distributed mechanism for the secretion and uptake of protein and nucleic acids via secretion, conjugation, and transformation.

IHF Inc kb LPS Mpf Mps NLS T4SS Tc

integration host factor incompatibility groups kilobases lipopolysaccharide mating pair formation mating pair stabilization nuclear localization signals type IV secretion system tetracycline

Defining Statement

Introduction

Bacterial conjugation is a widespread mechanism for the transfer of DNA between cells in close contact with one another. This entry summarizes past findings and discusses the better-studied systems in Gram-negative and -positive bacteria as well as the phenomena of mobilization and tumorigenesis in plants, which are related processes.

Bacterial conjugation was first described by Lederberg and Tatum in 1946 as a phenomenon involving the exchange of markers between closely related strains of Escherichia coli. The agent responsible for this process was later found to be a site on the chromosome called the F (‘fertility’) factor. This finding was the basis of bacterial

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genetics in the 1940s and 1950s and was used extensively in mapping the E. coli chromosome, making it the preeminent prokaryotic organism at that time. It was also shown that F could excise out of the chromosome and exist as an extrachromosomal element or plasmid. It was capable of self-transfer to other bacteria and could cotransfer the chromosome, a serendipitous function of F, and integrate randomly into its host’s DNA. The F sex factor of E. coli also imparted sensitivity to bacteriophages that required the F pilus, which is encoded by the F transfer region, as an attachment site during infection. In the 1960s a number of other conjugative plasmids were isolated, many carrying multiple antibiotic resistance markers. These plasmids were termed R (‘resistance’) factors and were found in many instances to repress pilus expression and conjugation by F, a process termed fertility inhibition (fiþ). The number of conjugative plasmids discovered has grown tremendously in the last few decades and includes self-transmissible plasmids isolated from Gram-negative and -positive bacteria as well as mobilizable plasmids. Conjugative transposons or integrating conjugative elements (ICEs), which move between cells using a conjugative mechanism, excise and integrate into the host chromosome via a process reminiscent of lysogenic phages; an example of a conjugative phage has been described for Staphylococcus aureus. In general, the transfer and replication functions of these mobile elements are often physically linked and the type of transfer system is closely aligned with the nature of the replicon that is described by incompatibility groups (Inc). An excellent summary of the properties of many conjugative plasmids is given in Shapiro (1977). Bacterial conjugation is now realized to be one of the principal conduits for horizontal gene transfer (HGT) among microorganisms. The process is extremely widespread and can occur intra- and intergenerically as well as between kingdoms (bacteria to yeast or to plants). DNA sequence analysis has revealed that conjugation, and in some cases transformation, two of the main conduits for HGT, are effected by a transenvelope protein complex that belongs to the type IV secretion system (T4SS). The effect of this process on evolution has been immense with bacteria rapidly acquiring traits both good (hydrocarbon utilization) and bad (antibiotic resistance, toxins). Once again, bacterial conjugation is at the forefront of microbiology but this time the emphasis is on the process itself rather than its utility as a geneticist’s tool. Excellent reviews of the topic are provided in The Horizontal Gene Pool, Bacterial Plasmids and Gene Spread (C.M. Thomas, ed.) and Plasmid Biology (Phillips, G. and Funnell, B., eds.).

Conjugative Process Unlike other processes like transformation and transduction that contribute to HGT, conjugation can be distinguished by two important criteria. There must be close cell-to-cell contact between the donor and recipient cells and DNA transfer must begin from a specific point on the transferred DNA molecule, be it a plasmid, transposon, or chromosome (Figure 1). This point is encoded within the origin of transfer (oriT) called nic. The proteins that act on this site are encoded by tra (transfer) or mob (mobilization) regions although other designations such as vir are now common. In general, each conjugative element encodes an array of proteins for mating pair formation (Mpf) while another set of proteins are involved in processing and transferring the DNA (Dtr). The Mpf genes can further be classified into the genes for pilus formation or mating pair stabilization (Mps) in Gram-negative bacteria or aggregate formation in

7 1

6 F

2 8 5

RP4 9 4

3

* * *

* * * pCF10

Figure 1 Summary of the mating process for universal (plasmid F) and surface-preferred (plasmid RP4) conjugation systems in Gram-negative bacteria and the pheromoneactivated system of Enterococcus faecalis (plasmid pCF10). In universal systems, the pilus attaches to a receptor on the recipient cell surface (1) and retracts to form a stable mating pair or aggregate (2). DNA transfer is initiated (3), causing transport of a single strand in the 59!39 direction (4). Transfer is associated with synthesis of a replacement DNA strand in the donor cell and a complementary strand in the recipient (5). The process is terminated by disaggregation of the cells, each carrying a copy of the plasmid (6). The transfer systems of conjugative plasmids in Gram-negative bacteria can be repressed (7) or derepressed (constitutive; 8). Cells carrying RP4 and related plasmids express pili constitutively but the pili are not seen attached to the bacteria. Such cells form mating pairs by collision on a solid surface (8). In Gram-positive bacteria, such as the enterococci, the donor senses the presence of pheromone () released by the recipient cell, which triggers mating pair formation (Mpf) and DNA transfer (9). Donor cells are shown as oblongs (blue) and recipient (red) cells as ovals. Pili are blue.

Genetics, Genomics | Conjugation, Bacterial

Gram-positive cocci. A system to prevent close contact between equivalent donor cells is called surface exclusion. The gene products that process the DNA in preparation for transfer usually include a protein (relaxase) that cleaves the DNA in a sequence- and strand-specific manner at nic and remains covalently bound to the 59 end in all cases that have been examined. This nucleoprotein complex plus other auxiliary proteins bound to the oriT region is called the relaxosome whereas the complex formed between the relaxosome and the transport machinery is known as the transferosome. A hallmark of conjugative systems is the coupling protein, within the cytoplasmic membrane, that connects the relaxosome to the transferosome. A process that prevents the transfer of DNA into the recipient cell after Mpf has occurred is called entry exclusion (Eex). Previously, the terms surface exclusion and entry exclusion were used interchangeably; however, as the details of the process have been refined, it is important to make this distinction. In Gram-negative bacteria, the process of DNA transfer is triggered upon cell contact whereas in Enterococcus faecalis and T-DNA transport by Agrobacterium tumefaciens, among others, contact between cells induces a complex program of gene expression leading to DNA transport. Whereas the sequences for a number of conjugative elements have been completed and comparisons have revealed information on the evolution of conjugative elements, a study of the conjugative process has only been undertaken in some depth for IncF, IncI, IncP, IncW elements and the Ti plasmid of A. tumefaciens and other Gram-negative bacteria and for the pheromoneresponsive system found in some plasmids in Ec. faecalis, although studies on other systems such as pIP501 are ongoing. Information is now available on the integration and excision processes of conjugative transposons and ICEs as well as the role of the mob genes in mobilizable plasmids. In addition, conjugation in Streptomyces has been studied in some detail but is quite different than that described and may use a DNA transport mechanism related to the process of DNA partition during septation in Bacillus subtilis (see ‘Streptomyces’).

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circumstances. Factors affecting mating efficiency include temperature with very precise optimums usually being the rule. For instance, F and RP4 mate optimally at 37–42  C, and IncH plasmids and the Ti plasmid at about 20–30  C. Other factors include oxygen levels, nutrient availability, and growth phase. Silencing by host-encoded factors such as H-NS is an important phenomenon that is thought to provide control of gene expression by newly acquired DNA through HGT, a process now termed ‘xenogeneic silencing’. Fþ cells in late stationary phase are known as F phenocopies because they are able to accept incoming F DNA and are not subject to surface or entry exclusion. Available literature indicates conjugation to be maximal over a short temperature range, in nutrient-rich environments with good aeration for aerobic organisms. Liquid versus Solid Support The ability of some conjugative systems to mate equally well in liquid media or on a solid support is one of the hallmarks of conjugation. Whereas all conjugative elements can mate well on a solid support, usually a filter placed on the surface of a prewarmed nutrient agar plate, many transfer systems, including those of the IncF group and the pheromone-responsive plasmids of Enterococcus, mate very efficiently in liquid media. This difference can be attributed to the nature of the Mpf process as thick, flexible pili of Gram-negative bacteria are associated with systems that mate well in liquid media whereas rigid pili, not usually seen attached to the cells (e.g., IncP), require a solid support for efficient mating. The aggregation substance of Ec. faecalis allows high levels of transfer in liquid media but other Gram-positive systems and conjugative transposons mate at low levels and absolutely require a solid support. In general, it appears that mating systems requiring a solid support depend on collision between donor and recipient cells whereas systems that mate well on either medium have a mechanism for initiating contact between freely swimming cells (thick, flexible pili, and aggregation substance). The description of media requirements for many Gram-negative plasmid transfer systems is given in Bradley et al. (1980).

Physiological Factors Conjugative Elements The level of transfer efficiency varies dramatically among the various systems. For derepressed or constitutively expressed systems such as F (IncFI) or RP4 (IncP), maximal levels of mating (100% conversion to plasmidbearing status) are possible within 30 min. Plasmids undergoing fertility inhibition usually have a 100- to 1000-fold reduction in mating efficiency whereas other plasmids, especially the smaller plasmids of Grampositive bacteria and conjugative transposons, mate at barely detectable levels even under the best of

Naturally occurring conjugative elements including plasmids, conjugative transposons, or ICEs, which are incorporated into the host chromosome, can lead to chromosome mobilization ability (Cma), resulting in high frequency of recombination (Hfr). Free plasmids can be divided into self-transmissible (Mpf plus Dtr genes) or mobilizable (Dtr or Mob genes) plasmids and can vary in size from a few kilobases (kb) to large plasmids 100–500 kb in size.

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Plasmids In general, Gram-negative transfer systems are approximately 20–35 kb and reside on plasmids from 60 to 500 kb whereas mobilizable plasmids are under 15 kb. The transfer or mobilization regions often represent half or more of the coding capability of the plasmid. Table 1 contains a list of selected plasmids and their characteristics including their pilus type and mating medium preference. In nonfilamentous Gram-positive plasmids, the smaller plasmids ( G:G > A:C > C:C. Frame shift mismatches are also recognized and repaired. Since S. pneumoniae has no GATC methylation system, it would appear that the role of MutH is replaced, at least in DNA transformation, by the single-strand break that must appear as a part of single-strand displacement during the process of integration of the strand of DNA that has been taken up. Furthermore, the requirement for a DNA end, to avoid mutations in this and other organisms, could be satisfied by the ends on the leading and the lagging strands that must be present at the replication fork. The vsr system is a short-patch repair system that recognizes and cuts at the T in the G:T mismatches at the second C site in the sequence CC(A/T)GG and perhaps others. Both the removal of the T and resynthesis is carried out by DNA polymerase I and its exonuclease activity. The repair patches are presumably short and the activity of vsr is substantially reduced when mutL or mutS are disabled. A:G and G:G mismatches are repaired by the MutY system, first recognized as a system responsible for correcting the A of A:G mismatches in heteroduplex bacteriophage DNA and called MicA (mismatch induced correction). The identity of the genes MutY and MicA was recognized earlier as a function preventing mutations in growing bacteria and incorrectly interpreted to be a function that imposed increased discrimination during replication. The identity of the two functions has been demonstrated. More recently, its probable primary function has been identified as playing a role in the avoidance of mutations caused by 8-oxo-7, 9-dihydrodeoxyguanine lesions by functioning as a DNA glycosylase that removes A from a GO:A mismatch. There is also evidence for the same protein harboring an associated 39 apurinic lyase activity. The biochemical evidence for the activity of this purified protein is that it can remove an A from A:G or A:C mismatches. The nicking activity on A:C mismatches is, in vitro, substantially lower than it is on A:G. There is, in addition, evidence that the protein is also capable of removing the G of G:G and A:G mismatches. The latter observation provides a further argument against a role for

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A:G repair in mutation avoidance. The resynthesis of hydrolyzed nucleotides in this case is also carried out by DNA polymerase I and the patch length is short, between 9 and 27 nucleotides.

Some Roles of Mismatch Repair Systems In Vivo In E. coli, only the methyl-directed mismatch repair system is known to recognize heterologies in DNA sequence, that is, the presence of one or more nucleotides in one strand of the sequence with no complementing nucleotide(s) in the complementary strand. When the number of unpaired nucleotides is large, there is little or no processing of the heteroduplex. The heteroduplex is efficiently processed when the number of nucleotides is three or fewer; there is some processing when there are four nucleotides and little or no processing when there are five or more nucleotides that are unpaired. With one particular nonhomology, that in which there is a transposon on one strand and none in the other (constructed in vitro), there is efficient processing on transfection, with the efficient loss of the entire transposon sequence, by an unknown mechanism. It is possible that some feature of the replication fork could be responsible for the loss when such an unusual structure is encountered. Efforts to carry out recombination reactions either by transduction or by conjugation between E. coli and Salmonella typhimurium reveal a several-orders-of-magnitude reduction in the yield of recombinants when the divergence of homology has reached 10% or 20%. When MutS or MutL functions are disabled, most of this incompatibility is lost. The mechanism for the inhibition of recombination between diverged sequences is thought to be an inhibition of DNA strand transfer by the MutS product, prominently enhanced in the presence of the MutL product, when the DNA in as much as 3% diverged. The presence of these proteins has no effect in the in vitro system when the transferred DNA strand is homologous. In addition, it would seem that mismatch-stimulated events in gapcontaining molecules could provoke destruction of recombination intermediates by hydrolyzing one of the DNA strands of the heteroduplex that joins the two parental participants, as they do in products of transformation in Streptococcus pneumococcus. Such a processing would be independent of GATC methylation and therefore the MutH product. The long repair length of resynthesized DNA make this system a poor candidate for separating closely linked markers in recombination. In fact, the evidence suggests that the system eliminates substrates that could be converted into fine structure recombinants. It is the shortpatch mismatch repair processes that are major actors in the formation of such recombinants.

Fine Structure Recombination Investigation of bacteriophage lambda recombination has provided some evidence on the magnitude of the contribution of mismatch repair. In normal crosses, the length of heteroduplex DNA in primary products of recombination is about 4000 nucleotides. Crosses between phage with amber mutations P3 and P80, separated by 27 nucleotides in a gene whose function is required for DNA replication, would produce primary products of recombination that would, for the most part, harbor both mutant sites in the heterozgous heteroduplex region. In either of the two possible heteroduplex-containing molecules, the mismatch repair required to allow the message-producing strand to make a functional message, on an indicator host that cannot suppress the amber mutation, is A correction of an A:C mismatch. This must occur without correcting the wild-type nucleotide of the neighboring mismatch on the same strand. This is an activity that is part of the repertoire of MutY. Products of such a cross, in a host that is defective in MutL and in MutY, have been assayed on various nonsuppressing (Su) indicators. The number of recombinants evident on a MutL indicator is 3.7 times greater than on a wild-type indicator and 8 times greater on a MutL indicator harboring the plasmid pMutY. This observation shows that most of the recombinants that are capable of giving Pþ products are present in the progeny of the cross as nonrecombinant heterozygotes that are substrates for the formation of recombinants, not in the cross, but by MutY mismatch repair in the indicator used for the detection of recombinants. It would be reasonable to conclude that some fraction of fine structure recombinants in a normal cross are not made in the cross itself, but become recombinants on the indicator used to detect them. This is particularly true for markers, like those in genes O and P, whose presence prevents replication on the host used to detect recombinants. These observations confirm the evidence that has been reported for the presence of large numbers of fine structure recombinants that are derived by mismatch repair of unprocessed heteroduplex molecules in the host used to detect the recombinants.

Specificity of One Short-Patch Repair System Artificially constructed heteroduplex molecules of bacteriophage lambda have been packaged and the infectious particle used to assess the way in which the MutY system processes the mismatches. This assay system makes use of lambda plac5 phage, defective in P, with mutations in the lacZ gene. These lacZ mutants were constructed by C. Cupples and were introduced into lambda plac5 by

Genetics, Genomics | DNA Mismatch Repair, Bacterial

recombination. By isolating DNA from appropriately marked phages, mixing equal amounts pairwise, denaturing, annealing, and packaging, we can expect to find four products from each pair. For example, if one of the parents were plac5 cc101 Pam3 and the other plac5 Pam80, the packaged phage would include the following species in about equal abundance. The two parents - - - - - - -Zcc101- - - - - - - -/- - - - - -Pam3- - - -39(l) - - - - - - -Zcc101- - - - - - - -/- - - - - -Pam3- - - -5(r) and - - - - - - -Zþ- - - - - - -/- - - - - - - - -Pam80- - - - -(l) - - - - - - -Zþ- - - - - - -/- - - - - - - - -Pam80- - - - -(r) and the two heteroduplex types. (a) - - - - -Zcc101- - - -/- - - - - - - -Pam3- - - - - -39(l) - - - - - - -Zþ- - - - - -/- - - - - -Pam80- - - - - - - -59(r) and (b) - - - - -Zþ- - - - - /- - - - - - Pam80- - - - - - - -39(l) - - - - - - - Zcc101- – -/- - - - - - -Pam3- - - - - - - - - 59(r) The two later products harboring mismatches can be rewritten in terms of the nucleotides, which distinguish the phage parents, as - - - - - - -Am- - - - - - - - - -C- - - - - -Tm- - - - - - - - - 39 - - - - - - -G- - - - - - - - - - -Am— - - - G- - - - - - - - - -59 and - - - - - - - C- - - - - - - - - - Tm- - - - - -C- - - - - - - - - -39 - - - - - - -Tm- - - - - - - - - G- - - - - - -Am- - - - - - - - –59 These annealed preparations were packaged in an in vitro packaging mix and the packaged phage were adsorbed, at low multiplicity, to an E. coli host that was Su, unable to suppress the amber mutations in the P gene whose product is required for DNA replication, and mutL so that the bacteria are unable to carry out the long-patch mismatch repair. The reconstructed parental phages that harbor the same P mutation in both strands are lost because they cannot grow on the Su indicator. The phage with different P mutations in the two strands cannot grow either, unless the mutant nucleotide in the 59 transcribed strand is repaired to wild type, without changing the neighboring wild-type allele. During the time interval required for the appropriate Pam mutation to be corrected, the mismatch in the distant lacZ allele (20 kb) is subject to repair as an independent event. A single cycle of growth of a phage that has experienced the appropriate P correction will give rise to a burst of phage, all of which can grow on an Suþ host. They include the product of the transcribed strand that has been corrected to Pþ strand and the Pam phage products of the complementary strand, each harboring, for the most part, their appropriate lac genes. If the lac mutant A is corrected, then all of the phage in the burst are lacþ; if the G is corrected, all of the phage would be lac–; and if the lac mismatch escapes correction, the burst would contain a mixture of lacþ and lac– phages. These phenotypes can be readily

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detected on an Suþ host that is lacZ deleted, when the chromogenic substrate, Xgal (5-bromo-4-chloro-3-indolylb-D-galactopyranoside), and lactose are present. Phage with the P3 allele can be distinguished from the P80 phage by virtue of their suppression patterns, and so the entire allele composition of every burst can be determined and the composition of the parental heteroduplex can be ascertained.

Localized Repair of A:G and C:T Mismatches The Cupples mutants cc101 and cc104 are the result of transversions in adjacent nucleotides in the coding triplet for the amino acid that is the active center of the enzyme -galactosidase. In heteroduplex molecules, they create A:G and C:T mismatches at their respective sites in opposite strand orientations. This permits us to determine the extent to which repair is strand specific, sequence specific, and also let’s look at the effect of transcription. The bursts, after infection of Su mutL bacteria, under conditions of repression of the lac operon show that for cc101 with 94 bursts, 85% of the A:G mispairs are corrected and for 58 bursts of cc104 only 46% are corrected to C:G. These corrections include A correction to C:G and G correction to A:T. The A correction occurs about twice as frequently as the correction of G. These observations would suggest that the biochemical evidence for cutting only in the A-containing strand requires further inquiry. During transcription, when the lac operon is derepressed in the presence of the gratuitous inducer isopropylthiogalactoside (IPTG), the frequency of repair of A:G drops by a factor of three for both mismatches and the ratio of A repair to G repair remains two to one. The C:T mismatch is not repaired in either orientation. For each repair event the relative likelihood of repairing G or the A appears fixed. However, the likelihood of any repair event is influenced by the level of transcription. When transcription is turned on, the repair level is reduced. When the location of the mismatch is changed, the likelihood of repair is also changed but the direction of the repair is not influenced by transcription. The fact that the sequence location influences the likelihood of repair of the same mismatch in different locations in the P gene of lambda indicates that it is sequence location and not strand location that is influencing the likelihood of repair. To establish that MutY is essential for these repair functions, use was made of the O205 amber mutation of lambda. This is a mutation, near the transcription end of the O gene, that results in a display of weak activity. It does not make a burst on an Su host, but when a heteroduplex molecule is constructed with P3 in the

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complementary strand, the molecules with O205 in the transcribed strand give a delayed burst of a mixture of P3, and O205 phage, presumably by a single replication of the parental molecule and subsequent complementation. On the mutL, mutYþ host, 34% of these bursts that are mixtures of P3 and O205 phage display A:G correction in the lac gene. Of 121, 34 are correction to C:G and 10 to A:T. When these phages were used to infect a strain that is mutL, mutY, among the 268 bursts examined, none showed evidence of correction. These observations show that the repair of A:G mismatches is dependent on the mutY function. The repair can occur in either direction, to C:G or to A:T and the efficiency of repair depends on sequence location, and on whether the gene, in which the mismatch resides, is turned on or not. The T:C mismatches are not repaired.

Localized Repair of A:C and G:T Mismatches The Cupples mutant cc106, an A:T to G:C transition, allows the examination of the fate of A:C and G:T mismatches under these mutL conditions. Of 146 bursts of A:C-containing phage, occurring under repressed conditions, 13 were repaired to G:C. Among 105 that harbored T:G mismatches two were repaired to C:G. In 161 bursts, in the presence of IPTG, seven showed repair to G:C. Among 116 bursts under these conditions, none of the T:G mismatches displayed correction. The two T:G corrections, under conditions of repression, could be products of the inhibited level of vsr repair that occurs when the methyl-directed system is disabled. It is clear that A:C mismatches are repaired but we are not able to determine the effect of transcription.

Other Mismatches The Cupples lac mutation cc103, a C-to-G transversion, allows the construction of molecules with C:C and G:G mismatches. Of the 61 bursts of G:G-containing phage, under repressed conditions, seven were corrected to G:C and five to C:G. In the presence of inducer, no correction was detected in 38 bursts. For the C:C mismatch, no correction was detected in 36 bursts under repressed conditions and in 25 bursts under derepressed conditions. The conclusion is that G:G correction can occur, and here again there is evidence that transcription inhibits mismatch repair. The C:C mismatches do not appear to be processed. The final mismatches that were subject to a limited examination under conditions of repression were A:A and T:T. None of the 44 bursts with A:A mismatches showed evidence of repair and one of 35 bursts with T:T

mismatches was corrected to A:T. These mismatches are only minimally repaired if they are repaired at all. Some of the processes that play a role in the resolution of the products of recombination are also part of the process that accounts for the correction of errors in replication. The observations that we have described provide us with a glimpse of the process of error correction as part of the process of DNA replication.

See also: Chromosome Replication and Segregation; Chromosome, Bacterial; DNA Restriction and Modification; Genetics, Microbial (general)

Further Reading Chen JD and Lacks SA (1991) Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae. Journal of Bacteriology 173(1): 283–290. Claverys JP and Lacks SA (1986) Heteroduplex deoxyribonucleic acid base mismatch repair in bacteria. Microbiological Reviews 50(2): 133–165. Cupples CG and Miller JH (1989) A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proceedings of the National Academy of Sciences of the United States of America 86(14): 5345–5349. Eisen JA (1998) A phylogenomic study of the MutS family of proteins. Nucleic Acids Research 26(18): 4291–4300. Fox MS, Radicella JP, and Yamamoto K (1994) Some features of base pair mismatch repair and its role in the formation of genetic recombinants. Experientia 50(3): 253–260. Hennecke F, Kolmar H, Brundl K, and Fritz HJ (1991) The vsr gene product of E. Coli K-12 is a strand- and sequence-specific DNA mismatch endonuclease. Nature 353(6346): 776–778. Huisman O and Fox MS (1986) A genetic analysis of primary products of bacteriophage lambda recombination. Genetics 112(3): 409–420. Lieb M (1985) Recombination in the lambda repressor gene: Evidence that very short patch (VSP) mismatch correction restores a specific sequence. Molecular and General Genetics 199(3): 465–470. Lieb M (1987) Bacterial genes mutL, mutS, and dcm participate in repair of mismatches at 5-methylcytosine sites. Journal of Bacteriology 169(11): 5241–5246. Michaels ML, Cruz C, Grollman AP, and Miller JH (1992) Evidence that MutY and MutM combine to prevent mutations by an oxidatively damaged form of guanine in DNA. Proceedings of the National Academy of Sciences of the United States of America 89(15): 7022–7025. Modrich P (1991) Mechanisms and biological effects of mismatch repair. Annual Review of Genetics 25: 229–253. Nghiem Y, Cabrera M, Cupples CG, and Miller JH (1988) The mutY gene: A mutator locus in Escherichia coli that generates G.C – T.A Transversions. Proceedings of the National Academy of Sciences of the United States of America 85(8): 2709–2713. Parker BO and Marinus MG (1992) Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 89(5): 1730–1734. Radicella JP, Clark EA, and Fox MS (1988) Some mismatch repair activities in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 85(24): 9674–9678. Radicella JP, Clark EA, Chen S, and Fox MS (1993) Patch length of localized repair events: Role of DNA polymerase I in mutYdependent mismatch repair. Journal of Bacteriology 175(23): 7732–7736. Raposa S and Fox MS (1987) Some features of base pair mismatch and heterology repair in Escherichia coli. Genetics 117(3): 381–390.

Genetics, Genomics | DNA Mismatch Repair, Bacterial Tiraby JG and Fox MS (1973) Marker discrimination in transformation and mutation of pneumococcus. Proceedings of the National Academy of Sciences of the United States of America 70(12): 3541–3545. Tsai-Wu JJ, Radicella JP, and Lu AL (1991) Nucleotide sequence of the Escherichia coli micA gene required for A/G-specific mismatch repair: Identity of micA and mutY. Journal of Bacteriology 173(6): 1902–1910.

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Tsai-Wu JJ, Liu HF, and Lu AL (1992) Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs. Proceedings of the National Academy of Sciences of the Unites States of America 89(18): 8779–8783. Wagner R, Jr and Meselson M (1976) Repair tracts in mismatched DNA heteroduplexes. Proceedings of the National Academy of Sciences of the United States of America 73(11): 4135–4139.

DNA Restriction and Modification G W Blakely and N E Murray, University of Edinburgh, Institute of Cell Biology, Edinburgh, UK ª 2009 Elsevier Inc. All rights reserved.

Introduction Detection of Restriction Systems Nomenclature and Classification R-M Enzymes as Model Systems Control and Alleviation of Restriction

Glossary ATP and ATP hydrolysis Adenosine triphosphate (ATP) is a primary repository of energy that is released for other catalytic activities when ATP is hydrolyzed (split) to yield adenosine diphosphate (ADP). bacteriophage ( and T-even) Bacterial viruses. Phage lambda () is a temperate phage, and therefore on infection of a bacterial cell, one of two alternative pathways may result; either the lytic pathway in which the bacterium is sacrificed and progeny phage are produced, or the temperate (lysogenic) pathway in which the phage genome is repressed and, if it integrates into the host chromosome, will be stably maintained in the progeny of the surviving bacterium. Phage  was isolated from Escherichia coli K-12 in which it resided in its temperate (prophage) state. T-even phage (T2, T4, and T6) are virulent coliphage, that is, infection of a sensitive strain of E. coli leads to the production of phage at the inevitable expense of the host. T-even phage share the unusual characteristic that their DNA includes hydroxymethylcytosine rather than cytosine. DNA methyltransferases These enzymes (MTases) catalyze the transfer of a methyl group from the donor S-adenosylmethionine (AdoMet or SAM) to adenine or cytosine residues in the DNA. efficiency of plating (EOP) This usually refers to the ratio of the plaque count on a test strain relative to that obtained on a standard, or reference, strain. endonucleases Enzymes that can fragment polynucleotides by the hydrolysis of internal phosphodiester bonds. Escherichia coli strain K-12 The strain used by Lederberg and Tatum in their discovery of recombination in E. coli.

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Distribution, Diversity, and Evolution Biological Significance Applications and Commercial Relevance Further Reading

glucosylation of DNA The DNA of T-even phage in addition to the pentose sugar, deoxyribose, contains glucose attached to the hydroxymethyl group of hydroxymethylcytosine. Glucosylation of the DNA is mediated by phage-encoded enzymes, but the host provides the glucose donor. helicases Enzymes that separate paired strands of polynucleotides. recombination pathway The process by which new combinations of DNA sequences are generated. The general recombination process relies on enzymes that use DNA sequence homology for the recognition of the recombining partner. In the major pathway in E. coli the RecBCD enzyme, also recognized as exonuclease V, enters DNA via a double-strand break. It tracks along the DNA, promoting unwinding of the strands and DNA degradation. The activity of RecBCD changes when it encounters Chi, a special DNA sequence in the 39 to 59 orientation. Degradation at the 59 end is now favored and the single-stranded DNA with a 39 end becomes a substrate for RecA-mediated strand transfer into an homologous DNA duplex. SOS response DNA damage induces expression of a set of genes, the SOS genes, involved in the repair of DNA damage. Southern transfer The transfer of denatured DNA from a gel to a solid matrix, such as a nitrocellulose filter, within which the denatured DNA can be maintained and hybridized to labeled probes (single-stranded DNA or RNA molecules). Fragments previously separated by electrophoresis through a gel may be identified by hybridization to a specific probe. transformation The direct assimilation of DNA by a cell, as the result of which the recipient is changed genetically.

Genetics, Genomics | DNA Restriction and Modification

Abbreviations ADP ATP EOP HMC

adenosine diphosphate adenosine triphosphate efficiency of plating hydroxymethylcytosine

Introduction Awareness of the biological phenomenon of restriction and modification (R-M) grew from the observations of microbiologists that the host range of a bacterial virus (phage) was influenced by the bacterial strain in which the phage was last propagated. Although phage produced in one strain of Escherichia coli would readily infect a culture of the same strain, they might only rarely achieve the successful infection of cells from a different strain of E. coli. This finding implied that the phage carried an ‘imprint’ that identified their immediate provenance. Simple biological tests showed that the occasional successful infection of a different strain resulted in the production of phage that had lost their previous imprint and had acquired a new one, that is, they acquired a different host range. In the 1960s, elegant molecular experiments showed the ‘imprint’ to be a DNA modification that was lost when the phage DNA replicated within a different bacterial strain; those phage that conserved one of their original DNA strands retained the imprint, or modification, whereas phage containing two strands of newly synthesized DNA did not. The modification was shown to provide protection against an endonuclease, the barrier that prevented the replication of incoming phage genomes. The host-controlled barrier to successful infection by phage that lacked the correct modification was referred to as ‘restriction’ and the relevant endonucleases have acquired the colloquial name of restriction enzymes. The modification enzyme was shown to be a DNA methyltransferase that methylated specific bases within the target sequence, and in the absence of the specific methylation, the target sequence rendered the DNA sensitive to the restriction enzyme. When DNA lacking the appropriate modification imprint enters a restriction-proficient cell, it is recognized as foreign and cut by the endonuclease. Classically, a restriction enzyme is accompanied by its cognate modification enzyme and the two comprise an R-M system. Most restriction systems conform to this classical pattern. There are, however, some restriction endonucleases that attack DNA only when their target sequence is modified. A restriction system that responds to its target sequence only when it is identified by modified bases does not, therefore, coexist with a cognate modification enzyme.

PCR R-M SAM TRDs

539

polymerase chain reaction restriction and modification S-adenosylmethionine target recognition domains

Two early papers documented the phenomenon of restriction. In one, Bertani and Weigle, in 1953, using temperate phage ( and P2), identified the classical R-M systems characteristic of E. coli K-12 and E. coli B. In the other, Luria and Human, in 1952, identified a restriction system of a nonclassical kind. In the experiments of Luria and Human, T-even phage were used as test phage and after their growth in a mutant E. coli host they were found to be restricted by wild-type E. coli K-12, but not by Shigella dysenteriae. An understanding of the restriction phenomenon observed by Luria and Human requires knowledge of the special nature of the DNA of T-even phage. During replication of T-even phage, the unusual base 5-hydroxymethylcytosine (HMC) completely substitutes for cytosine in the T-phage DNA, and hydroxymethyl residues become substrates for glucosylation. In the mutant strain of E. coli used by Luria and Human as host for the T-even phage, glucosylation fails and, in its absence, the nonglucosylated phage DNA becomes sensitive to endonucleases present in E. coli K-12 but not in S. dysenteriae ; particular nucleotide sequences normally protected by glucosylation are recognized in E. coli when they include the modified base, HMC, rather than cytosine residues. These endonucleases, now accepted as restriction systems, were later discovered to attack DNA that includes methylated cytosine residues. Strains lacking these endonucleases enhanced the efficiency of cloning foreign DNA in E. coli. The classical R-M systems and the modificationdependent restriction enzymes share the potential to attack DNA derived from different strains and thereby ‘restrict’ DNA transfer. They differ in that in one case an associated modification enzyme is required to protect DNA from attack by the cognate restriction enzyme and in the other modification enzymes specified by different strains impart signals that provoke the destructive activity of restriction endonucleases.

Detection of Restriction Systems As a Barrier to Gene Transfer This is exemplified by the original detection of the R-M systems of E. coli K-12 and E. coli B by Bertani and Weigle in 1953. Phage  grown on E. coli strain C (.C), where

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E. coli C is a strain that apparently lacks an R-M system, forms plaques with poor efficiency (EOP of 2  104) on E. coli K-12 because the phage DNA is attacked by a restriction endonuclease (Figure 1). Phage  grown on E. coli K-12 (.K) forms plaques with equal efficiency on E. coli K-12 and E. coli C, since it has the modification required to protect against the restriction system of E. coli K-12 and E. coli C has no restriction system (Figure 1). In contrast, .K will form plaques with very low efficiency on a third strain, E. coli B, since E. coli B has an R-M system with different sequence specificity from that of E. coli K-12. Phage often provide a useful and sensitive test for the presence of R-M systems in laboratory strains of bacteria, but they are not a suitable vehicle for the general detection of barriers to gene transfer. Many bacterial strains, even within the same species and particularly when isolated from natural habitats, are unable to support the propagation of the available test phage and some phage (e.g., P1) have the means to antagonize at least some restriction systems (see ‘Antirestriction systems’). Gene transfer by conjugation can monitor restriction, although some natural plasmids, but probably not the F factor of E. coli, are equipped with antirestriction systems. The single-stranded DNA that enters a recipient cell by conjugation, or following infection by a phage such as M13, becomes sensitive to restriction only after the synthesis of its complementary second strand. In contrast, the single-stranded DNA that transforms naturally competent bacteria may not become a target for e.o.p. = 1

restriction because it forms heteroduplex DNA with resident (and therefore modified) DNA, and one modified strand is sufficient to endow protection. Transformation can be used to detect restriction systems when the target DNA is the double-stranded DNA of a plasmid. In Vitro Assays for DNA Fragmentation Endonuclease activities yielding discrete fragments of DNA are commonly detected in crude extracts of bacterial cells. More than one substrate may be used to increase the chance of providing DNA that includes appropriate target sequences. DNA fragments diagnostic of endonuclease activity are separated according to their size by electrophoresis through a matrix, usually an agarose gel, and are visualized by the use of autoradiography or a fluorescent dye, ethidium bromide, that intercalates between stacked base pairs. Extensive screening of many bacteria, often obscure species for which there is no genetic test, has produced a wealth of endonucleases with different target sequence specificities. These endonucleases are referred to as restriction enzymes, even in the absence of biological experiments to indicate their role as a barrier to the transfer of DNA. Many of these enzymes are among the commercially available endonucleases that serve molecular biologists in the analysis of DNA (Table 1; see ‘Applications and commercial relevance’). In vitro screens are applicable to all organisms, but to date R-M systems have not been found in eukaryotes, although some algal viruses encode them.

C

λ .K

Sequence-Specific Screens e.o.p. = 1 e.o.p. = 1

λ .C K–12 e.o.p. = 2 × 10–4 Figure 1 Host-controlled restriction of bacteriophage . Escherichia coli K-12 possesses, whereas E. coli C lacks, a Type I R-M system. Phage  propagated in E. coli C (.C) is not protected from restriction by EcoKI and thus forms plaques with reduced efficiency of plating (EOP) on E. coli K-12 as compared to E. coli C. Phage escaping restriction are modified by the EcoKI methyltransferase (.K), and consequently form plaques with the same efficiency on E. coli K-12 and C. Modified DNA is indicated by hatch marks. Reproduced from Barcus VA and Murray NE (1995) Barriers to recombination: Restriction. In: Baumberg S, Young JPW, Saunders SR, and Wellington EMH (eds.) Population Genetics of Bacteria Society for General Microbiology, Symposium No. 52, pp. 31–58. Cambridge, UK: Cambridge University Press.

The identification of new R-M genes via sequence similarities is sometimes possible. Only occasionally are gene sequences sufficiently conserved that the presence of related systems can be detected by probing Southern transfers of bacterial DNA. More generally, screening databases of predicted polypeptide sequences for relevant motifs has identified putative R-M systems in the rapidly growing list of bacteria for which the genomic sequence is available (see ‘Distribution’). Currently, this approach is more dependable for modification methyltransferases than restriction endonucleases, but the genes encoding the modification and restriction enzymes are usually adjacent. Many putative R-M systems have been identified in bacterial genomic sequences.

Nomenclature and Classification Nomenclature R-M systems are designated by a three-letter acronym derived from the name of the organism in which they

Genetics, Genomics | DNA Restriction and Modification

541

Table 1 Some Type II restriction endonucleases and their cleavage sitesa

Bacterial source

Enzyme abbreviation

Haemophilus influenzae Rd

HindII

HindIII

Haemophilus aegyptius

HaeIII

Staphylococcus aureus 3A

Sau3AI

Bacillus amyloliquefaciens H

BamHI

Escherichia coli RY13

EcoRI

Providencia stuartii

PstI

Sequences 59 ! 39 39 59

Noteb

GTPy#PuAC CAPu"PyTG # AAGCTT TTCGAA " GG#CC CC"GG

1, 5

#GATC CTAG" # GGATCC CCTAGG " # GAATTC CTTAAG " # CTGCAG GACGTC "

2, 3

2

1

2, 3

2

4

a

The cleavage site for each enzyme is shown by the arrows. 1, produces blunt ends; 2, produces cohesive ends with 59 single-stranded overhangs; 3, cohesive ends of Sau3AI and BamHI are identical; 4, produces cohesive ends with 39 single-stranded overhangs; 5, Pu is any purine (A or G), and Py is any pyrimidine (C or T).

b

occur. The first letter comes from the genus, and the second and third letters from the species. The strain designation, if any, follows the acronym. Different systems in the same organism are distinguished by Roman numerals. Thus HindII and HindIII are two enzymes from Haemophilus influenzae strain Rd. Restriction endonuclease and modification methyltransferases (ENases and MTases) are sometimes distinguished by the prefixes R.EcoRI and M.EcoRI, but the prefix is commonly omitted if the context is unambiguous. The current convention for ENase naming omits italicization.

Classification of R-M Systems R-M systems are classified according to the composition and cofactor requirements of the enzymes, the nature of the target sequence, and the position of the site of DNA cleavage with respect to the target sequence. Currently R-M systems are divided into three types (I, II, and III). In addition there are modification-dependent restriction systems, now referred to as Type IV. Early experiments identified Type I systems, but the Type II systems are the simplest and for this reason will be described first. A summary of the properties of different types of R-M systems is given in Figure 2.

Type II R-M Systems A classical Type II R-M system comprises two separate enzymes; one is the restriction ENase, the other the modification MTase. The nuclease activity requires Mg2þ, and DNA methylation requires S-adenosylmethionine (AdoMet or SAM) as methyl donor. The target sequence of both enzymes is the same; the modification enzyme ensures that a specific base within the target sequence, one on each strand of the duplex, is methylated and the restriction endonuclease cleaves unmodified substrates within, or close to, the target sequence. The target sequences are often rotationally symmetrical sequences of 4–8 bp; for example, a duplex of the sequence 59GAATTC is recognized by EcoRI. The modification enzyme methylates the adenine residue identified by the asterisk, but in the absence of methylated adenine residues on both strands of the target sequence the restriction endonuclease breaks the phosphodiester backbones of the DNA duplex to generate ends with 39 hydroxyl and 59 phosphate groups. Type II ENases cut within or close to their target sequences. The nature of the modification introduced by the MTase varies according to the system: N6-methyladenine (m6A) and N5- and N4methylcytosine (m5C and m4C). Irrespective of the target sequence, or the nature of the modification, ENases differ in that some cut the DNA to generate ends with 59 overhangs,

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Genetics, Genomics | DNA Restriction and Modification Type I

Type II

Type III

• Hetero-oligomeric enzymes • ENase and MTase • Require ATP hydrolysis for generally separate enzymes restriction • Cut DNA within or • Cut DNA at sites remote close to target from target sequence sequence • DEAD-box proteins • Do not require ATP e.g. EcoKI Genes

Subunits

hsdR

HsdR

e.g. EcoRI

Type IV

• Hetero-oligomeric ENase • Modification-dependent Enase • ATP required for restriction • Cut DNA close to target • Cut DNA outside the sequence target sequence • DEAD-box proteins • No cognate MTase e.g. EcoP1I

hsdM hsdS

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⎯⎯⎯⎯⎯⎯ ENase

Figure 2 The characteristics and organization of the genetic determinants and subunits of the different types of restriction systems. These systems are classified on the basis of their complexity, cofactor requirements and position of DNA cleavage with respect to their DNA target sequence. The Type I, II and III systems are the classical restriction and modification (R-M) systems. The restriction enzymes of the Type I and III systems contain motifs characteristic of ‘DEAD’-box proteins. These motifs are associated with ATP-dependent DNA translocation. The Type II restriction enzymes do not translocate DNA, and their properties are sufficiently variable for them to be allocated to subclasses. Some indication of this is given in the text. Modification-dependent endonucleases are now included as Type IV restriction systems. ENase, restriction endonuclease; MTase, methyltransferase (Adapted from King and Murray, 1995).

some generate 39 overhangs and others produce ends which are ‘blunt’ or ‘flush’ (see Table 1). Classical Type II restriction enzymes are generally active as symmetrically arranged homodimers, an association that facilitates the coordinated cleavage of both strands of the DNA. In contrast, Type II modification enzymes act as monomers, an organization consistent with their normal role in the methylation of newly replicated DNA in which one strand is already methylated. The genes encoding Type II R-M systems derive from the name of the system. The genes specifying R.BamHI and M.BamHI, for example, are designated bamHIR and bamHIM. Transfer of the gene encoding a restriction enzyme, in the absence of the transfer of the partner encoding the protective MTase, is likely to be lethal if the recipient cell does not provide the relevant protection. Experimental evidence supports the expectation that the genes encoding the two components of R-M systems are usually closely linked so that cotransfer will be efficient. The extensive characterization of restriction endonucleases during 40 years has led to significant broadening of the Type II class, and its subsequent division into many subclasses. Subclass IIP includes classical representatives such as EcoRI and HindIII, in which the endonucleases comprise symmetrically arranged homodimers that permit recognition of a symmetrical (palindromic) target sequence, and the cutting of each strand of DNA at fixed symmetrical locations, either within the target

sequence or immediately adjacent to it. DpnI, a similar dimeric endonuclease that cuts within a symmetrical target sequence, but only if methylated, is now accepted as a Type II system. It identifies subclass M (methylationdependent). Systems that recognize asymmetric target sequences are assigned to subclass IIA, but many of these, for example, FokI, cut the DNA at a precise, but short, distance from their recognition sequence, and therefore, they also meet the requirement for subclass IIS (where S refers to the shifted position of the cut). The endonucleases of members of the subclasses IIB, C, G, and H have hybrid structures that include both endonuclease and modification domains within a single polypeptide. The activity of members of subclasses IIE and IIF is mechanistically dependent on two target sequences. In subclass IIE one of the two targets only serves as an accessory site, while in subclass IIF both targets are substrates for coordinate cleavage. This brief survey of even the Type II subclasses illustrates enormous variation among sequence-specific endonucleases.

Type I R-M Systems Type I R-M systems are multifunctional enzymes comprising three subunits that catalyze both restriction and modification. In addition to Mg2þ, endonucleolytic activity requires both AdoMet and adenosine triphosphate (ATP). The restriction activity of Type I enzymes is associated

Genetics, Genomics | DNA Restriction and Modification

with the hydrolysis of ATP, an activity that correlates with the peculiar characteristic of these enzymes, that of cutting DNA at nonspecific nucleotide sequences considerable distances from their target sequences. The Type I R-M enzyme binds to its target sequence and its activity as an ENase or a MTase is determined by the methylation state of the target sequence. If the target sequence is unmodified, the enzyme, while bound to its target site, is believed to translocate DNA toward itself simultaneously in both directions in an ATP-dependent manner. This translocation process brings the bound enzymes closer to each other and experimental evidence suggests that DNA cleavage occurs when translocation is impeded, either by collision with another translocating complex or by the topology of the DNA substrate. The nucleotide sequences recognized by Type I enzymes are asymmetric and comprise two components, one of 3 or 4 bp and the other of 4 or 5 bp, separated by a nonspecific spacer of 6–8 bp. All known Type I enzymes methylate adenine residues, one on each strand of the target sequence. The three subunits of a Type I R-M enzyme are commonly encoded by three contiguous genes: hsdR, hsdM, and hsdS. The acronym hsd was chosen at a time when R-M systems were referred to as host specificity systems and hsd denotes host specificity of DNA. hsdM and hsdS are transcribed from the same promoter, but hsdR from a separate one. The two subunits encoded by hsdM and hsdS, sometimes referred to as M and S, are both necessary and sufficient for MTase activity. The third subunit (R) is essential only for restriction. The S subunit includes two target recognition domains (TRDs) that impart target sequence specificity to both the restriction and modification activities of the complex; the M subunits include the binding site for AdoMet and the active site for DNA methylation. Two complexes of Hsd subunits are functional in bacterial cells, one that comprises all three subunits (R2M2S1) and is an R-M system, and a second that lacks R (M2S1) and has only MTase activity. Type III R-M Systems Type III R-M systems are less complex than Type I, but nevertheless share some similarities with them. A single hetero-oligomeric complex catalyzes both the restriction and modification activities. Modification requires the cofactor AdoMet and is stimulated by Mg2þ and ATP. Restriction requires Mg2þ and ATP, and is stimulated by AdoMet. The recognition sequences of Type III modification enzymes are asymmetric sequences of 5–6 bp. Restriction requires two unmodified sequences in inverse orientation (Figure 3(a)). Recent evidence shows that Type III R-M enzymes, like Type I, can translocate DNA in a process dependent on ATP hydrolysis, but they hydrolyze less ATP than Type I systems, and probably only translocate DNA for a relatively short distance.

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Cleavage is stimulated by collision of the translocating complexes and occurs on the 39 side of the recognition sequence at a distance of approximately 25–27 bp: this contrasts with cleavage by Type I enzymes where cutting occurs at sites remote from the recognition sequence. Because only one strand of the recognition sequence of a Type III R-M system is a substrate for methylation, it might be anticipated that the immediate product of replication would be sensitive to restriction. In order to understand why this is not so, it is necessary to distinguish the target for modification from that needed for restriction. Restriction is only elicited when two unmethylated target sequences are in inverse orientation with respect to each other and, as shown in Figure 3(b), replication of modified DNA leaves all unmodified targets in the same orientation. The bifunctional R-M complex is made up of two subunits, the products of the mod and res genes. The Mod subunit is sufficient for modification, while the Res and Mod subunits together form a complex with both activities (see Figure 2). The Mod subunit is functionally equivalent to the MTase (M2S) of Type I systems and, as in Type I R-M systems, imparts sequence specificity to both activities. Type IV Restriction Systems These systems only cut modified DNA, but in contrast to Type IIM enzymes, not within a specific target sequence. They are variable in their complexity and requirements. E. coli K-12 encodes three distinct, sequence-specific, modification-dependent systems. Mrr is distinguished by its ability to recognize DNA containing either methylated adenine or 5-methylcytosine in the context of particular, but as yet undefined, sequences. McrA and McrBC both restrict DNA-containing modified cytosines (HMC or methylcytosine). The Mcr systems (modified cytosine restriction) are those first recognized by Luria and Human by their ability to restrict nonglucosylated T-even phage (originally called RglA and RglB; restricts glucose-less phage). McrBC is a complex enzyme with a requirement for GTP rather than ATP.

R-M Enzymes as Model Systems Sequence Recognition, Including Base Flipping Structures of the crystals of several Type II restriction ENases have been determined – some in both the presence and the absence of DNA. The symmetrically arranged dimers of the Type II enzymes bind to their specific target sequences by the combined effects of different types of interactions including hydrogen bonding and electrostatic interactions of amino acid residues with

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Genetics, Genomics | DNA Restriction and Modification (a)

CAGCAG GTCGTC

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Figure 3 (a) DNA substrates for a Type III R-M system (EcoP15I). The top strand of each duplex is written 59 to 39; the arrows identify the orientation of the target sequences. Solid lines indicate polynucleotide chains of undefined sequence. Only pairs of target sequences shown in inverse orientation (line 2) are substrates for restriction. A single site in any orientation is a target for modification; (b) replication of modified DNA leaves all unmodified target sites in the daughter molecules in the same orientation, and therefore insensitive to restriction.

the bases and the phosphate backbone of the DNA. No general structure, such as a helix-turn-helix or zinc finger (often found in proteins that interact with DNA), is characteristic of the protein–DNA interface, and amino acids that are widely separated in the primary sequence may be involved in interactions with the target nucleotide sequence. Comparisons of the active sites of EcoRV, EcoRI, and PvuII identify a conserved tripeptide sequence in close proximity to the target phosphodiester of the DNA backbone and a conserved acidic dipeptide that may represent the ligands for Mg2þ, the catalytic cofactor essential for ENase activity. The structure of a monomeric MTase interacting with its target sequence identified an important solution to the question of how enzymes that modify a base within a DNA molecule can reach their substrate. The cocrystal structure of M.HhaI bound to its substrate showed that the target cytidine rotates on its sugar–phosphate bonds such that it projects out of the DNA and fits into the catalytic pocket of the enzyme. Such base flipping was confirmed for a second enzyme, M.HaeIII, which also modifies cytosine, and circumstantial evidence supports the notion that this mechanism may be true for all MTases regardless of whether they methylate cytosine or adenine residues.

Comparative analyses of the amino acid sequences of many MTases identified a series of motifs, many of which are common to MTases irrespective of whether the target base is cytosine or adenine. These motifs enable structural predictions to be made about the catalytic site for DNA methylation in complex enzymes for which crystals are not available. DNA Translocation Specific interactions of large R-M enzymes with their DNA substrates are not readily amenable to structural analysis. The molecular weight of EcoKI is in excess of 400 000 and useful cocrystals with DNA have not yet been reported. Nevertheless, these complex enzymes have features of mechanistic interest. Much evidence now supports models in which DNA restriction involves the translocation of DNA in an ATP-dependent process prior to the cutting of the substrate. In the case of Type I R-M enzymes the breaks in the DNA may be many kb remote from the target sequence. Molineux and colleagues, in 1999, using assays with phage, have shown that EcoKI can transfer (translocate) the entire genome (39 kb) of phage T7 from its capsid to the bacterial cell.

Genetics, Genomics | DNA Restriction and Modification

For linear DNA, the evidence supports the idea that cutting by Type I R-M systems occurs preferentially midway between two target sequences. For Type III enzymes the breaks are close to the target sequence, but in both cases the endonuclease activity may be stimulated by the collision of two translocating protein complexes. The most conserved features of the polypeptide sequences of Type I and Type III R-M are the so-called DEAD-box motifs, which are also found in RNA and DNA helicases, and the motifs characteristic of adenine MTases. The DEAD-box motifs acquired their collective name because a common variant of one element is AspGlu-Ala-Asp, or DEAD when written in a single-letter code. The DEAD-box motifs, which include sequences diagnostic of ATP binding, are found in the subunit that is essential for restriction (HsdR or Res) but not for modification. It is not known how the ATP-dependent activity drives the translocation of DNA, although circumstantial evidence correlates ATPase activity with DNA translocation. Mutations in each DEAD-box motif have been shown to impair the ATPase, translocase and endonuclease activities of a Type I ENase.

Control and Alleviation of Restriction Control of Gene Expression The control of restriction activity is critical for survival of bacterial cells. This can be provided by the regulation of gene expression. It may be useful for protecting host DNA in restriction-proficient cells, but it is especially important when R-M genes enter a new host. Experiments show that many R-M genes are readily transferred from one laboratory strain to another. The protection of host DNA against the endonucleolytic activity of a newly acquired restriction system would be achieved if the functional cognate MTase is produced before the restriction enzyme. Transcriptional regulation of some of the genes encoding Type II systems has been demonstrated. Genes encoding regulatory proteins, referred to as C-proteins for controller proteins, have been identified in some instances. The C-proteins for a number of systems have been shown to activate efficient expression of the restriction gene. When the R-M genes are transferred to a new environment, in the absence of C-protein there is preferential expression of the modification gene, but following the production of the C-protein the consequent generation of restriction-proficient cells. Representatives of all three types of classical R-M systems have been shown to be equipped with promoters that could permit appropriate transcriptional regulation of the two activities. For complex R-M systems, despite the presence of two promoters, there is no evidence for transcriptional regulation of gene expression. The heterooligomeric nature of these systems offers opportunity

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for the regulation of the R-M activities by the intracellular concentrations of the subunits and the affinities with which different subunits bind to each other. Nevertheless, efficient transmission of the functional R-M genes of some families of Type I systems requires ClpX and ClpP in the recipient cell. Together these polypeptides comprise a protease. The ClpXP complex functions to degrade the HsdR subunit of an active R-M complex before the endonuclease activity has the opportunity to cleave unmodified chromosomal DNA. Restriction Alleviation The efficiency with which E. coli specifying a Type I R-M system restricts unmodified DNA is influenced by a number of stimuli, all of which share the ability to damage DNA. Induction of the SOS response leads to a decrease in restriction activity, and one consequence of this is a marked reduction of the efficiency with which the bacteria restrict incoming DNA. This alleviation of restriction is usually monitored by following the EOP of phage – unmodified in the case of classical systems or modified in the case of modification-dependent restriction systems. Alleviation of restriction is characteristic of complex systems and can be induced by ultraviolet light, nalidixic acid, 2-aminopurine and the absence of Dam-mediated methylation. The effect can be appreciable and host systems may contribute to more than one pathway of restriction alleviation. Recent experiments have shown that ClpXP is necessary for restriction alleviation of the EcoKI system; therefore there is a connection between the complex mechanisms by which restriction activity is normally controlled and its alleviation in response to DNA damage. Homologous recombination, required for DNA repair, can generate unmodified targets by synthesis of new DNA strands. A normal function of restriction alleviation is to protect the bacterial chromosome from restriction by resident Type I R-M systems when unmodified targets are generated. ClpXP is not relevant to all Type I R-M systems; therefore, alternative mechanisms of alleviation remain to be determined. Antirestriction Systems Many phage, and some conjugative plasmids, specify functions that antagonize restriction. An apparent bias of functions that inhibit restriction by Type I R-M systems may reflect the genotype of the classical laboratory strain E. coli K-12, a strain with a Type I but no Type II R-M system. The coliphage T3 and T7 include an ‘early’ gene, ocr or 0.3, the product of which binds Type I R-M enzymes and abolishes both restriction and modification activities. Ocr does not affect Type II systems. The ocr gene is expressed before targets in the phage genome are

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accessible to host restriction enzymes, so that ocrþ phage are protected from R-M by Type I systems. The crystal structure of T7 Ocr has shown that this protein mimics the shape and charge of the DNA substrate. Phage T3 Ocr has an additional activity; it hydrolyzes AdoMet, the cofactor essential for both restriction and modification by EcoKI and its relatives. Bacteriophage P1 also protects its DNA from Type I restriction, but the antirestriction function, Dar, does not interfere with modification. The Dar proteins are coinjected with encapsidated DNA, so that any DNA packaged in a P1 head is protected. This allows generalized transduction to occur between strains encoding different Type I R-M systems. Coliphage T5 has a well-documented system for protection against the Type II system EcoRI. As with the ocr systems of T3 and T7, the gene is expressed early when the first part of the phage genome enters the bacterium. This first segment lacks EcoRI targets, whereas the rest of the genome, which enters later, has targets that would be susceptible in the absence of the antirestriction protein. Some conjugative plasmids of E. coli, members of the incompatibility groups I and N, also encode antirestriction functions. They are specified by the ard genes located close to the origin of DNA transfer by conjugation, so that they are amongst the first genes to be expressed following DNA transfer. Like the ocr proteins of T3 and T7, the protein encoded by ard is active against Type I R-M systems. Bacteriophage  encodes a very specialized antirestriction function, Ral, which modulates the in vivo activity of some Type I R-M systems by enhancing modification and alleviating restriction. The systems influenced by Ral are those that have a modification enzyme with a strong preference for hemimethylated DNA. Unmodified ralþ  DNA is restricted on infection of a restriction-proficient bacterium, because ral is not normally expressed before the genome is attacked by the host R-M system, but Ral enhances the modification of those phage that escape restriction. Ral may act by changing the MTase activity of the R-M system to one that is efficient on unmethylated target sequences. Some phage are made resistant to many types of R-M systems by the presence of glucosylated HMC in their DNA, for example, the E. coli T-even phage and the Shigella phage DDVI. The glucosylation also identifies phage DNA and allows selective degradation of host DNA by endonucleases specified by the virulent phage. Nonglucosylated T-even phage are resistant to some classical R-M systems because their DNA contains the modified base HMC, but they are sensitive to modification-dependent systems, although T-even phage encode a protein (Arn) that protects superinfecting phage from McrBC restriction. It has been suggested that some phage have evolved to specify DNA that contains HMC, which counteracts classical R-M systems, and that

host-encoded modification-dependent endonucleases are a response to this phage adaptation. In this evolutionary story, the glucosylation of HMC would be the latest mechanism that renders T-even phage totally resistant to most R-M systems. In some cases, a phage genome can tolerate a few targets for certain restriction enzymes. The few EcoRII sites in T3 and T7 DNA are not sensitive to restriction, because this unusual enzyme requires at least two targets in close proximity and the targets in these genomes are not sufficiently close. For the Type III enzymes the orientation of the target sequences is also relevant. Since the target for restriction requires two inversely oriented recognition sequences, the T7 genome remains refractory to EcoP15I because all 36 recognition sequences are in the same orientation. The unidirectional orientation of the target sequences is consistent with selection for a genome that will avoid restriction. Considerable evidence supports the significance of counter-selection of target sequences in phage genomes, in some cases correlating the lack of target sequences for enzymes found in those hosts in which the phage can propagate.

Distribution, Diversity, and Evolution Distribution The technical importance of Type II endonucleases in biological sciences has extended their discovery to include enzymes with more than 250 different specificities, while the detection of Type I and Type III R-M systems continued to rely on in vivo experiments. More recently, the sequencing of genomes has revealed that R-M systems are almost ubiquitous in the Eubacteria and the Archaea, although identification of homologous sequences does not guarantee the activity of the predicted enzymes. From a survey of 496 genomes (http://rebase.neb.com/rebase/ rebase.html), only 35 lack homologues of known R-M systems. Eubacteria without R-M systems generally have small genomes (Sp1 >Sp2 >Sp3 >Sp4 >Sp5 >Sp6 >Sp7 >Sp8 >Sp9

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Phylogenomic tree Figure 3 Methods for phylogenomic inference based on primary sequences. Starting from genomic data, the alignment of orthologous genes is required. Once this crucial step is achieved, two alternative approaches can be used to infer phylogenetic trees. The supermatrix approach involves analyzing the concatenation of individual genes, and nonoverlapping taxa are coded as missing data. Alternatively, the supertree approach combines the optimal trees obtained from the analysis of individual genes, each of which contains data from only partially overlapping sets of taxa.

Because of the solid methodological background in sequence-based phylogenetic inference, these remain the methods of choice.

Supertree Approach This approach consists of combining the optimal trees obtained from the analysis of individual genes in a single ‘supertree’. Contrary to the classic consensus techniques (e.g., majority rule consensus tree used in bootstrap analysis), the source trees only need to have overlapping rather than identical taxon sets, giving much more flexibility and allowing incorporation of more data (e.g., a gene that has been lost in a single species can still be considered). Different methods for combining trees exist, but because of its intrinsic simplicity and its demonstrated accuracy, the matrix representation using parsimony (MRP) is most popular. In brief, starting with a set of trees, the presence of all the clades observed is coded as a binary character (missing species being represented by a question mark), and the obtained matrix is analyzed by parsimony to construct a supertree. This approach presents several major limitations, including the fact that the

resulting supertree is often biased toward large and/or unbalanced source trees. But, perhaps the major limitation is that single-gene phylogenies generally do not have enough discriminating power (stochastic error), and thus, by combining trees without considering their uncertainties, a too strong weight is given to potentially weak signals. Several variants have been developed to overcome this problem, for example, by weighting each column of the matrix according to the bootstrap proportions of the clade it represents. The supertree approach has several advantages. First, it can be used to combine trees that have been obtained from disparate sources such as molecular and morphological data; second, by calculating individual gene trees, one can separate trees that are relatively different from one another, for example, because of hidden paralogy or HGT; and, third, it can be easily parallelized and does not require as much memory resources as the supermatrix approach.

Supermatrix Approach The supermatrix approach follows the principle of total evidence, that is, combining all relevant available data,

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which in this case are the alignments of each individual gene. This ‘supermatrix’ will then be analyzed by the standard sequence-based phylogenetic inference methods described earlier (or by variants thereof). In this approach, the sequences of genes that cannot be used for some species, because they have been lost, horizontally transferred, or have not yet been sequenced, are coded as question marks. Using simulated and real data, several studies have shown that a certain degree of missing data (10–30%) does not seriously affect phylogenetic inference, provided that each taxon is sequenced for a sufficiently large number of positions (at least several thousands). This robustness makes the supermatrix approach powerful for phylogenetic reconstruction, as data sets can be assembled at low cost by mining existing databases or by the sequencing of multiple PCR-targeted loci or preferably randomly selected cDNA clones. This allows the incorporation of a large number of species, instead of being restricted to model organisms for which complete genome sequences are available. The supermatrix method has been criticized because combining genes with different histories will not produce a single rational phylogenetic reconstruction. Yet, in most studies, the possibility of incongruences is minimized by selecting genes having a priori the same evolutionary history (e.g., single-copy genes) and by checking a posteriori that single-gene trees are not incongruent with the supermatrix-based tree. But even if the genes combined support the same evolutionary relationships among species, they may have evolved in a different way, for example, faster or slower in each species. This additional heterogeneity is handled by partitioned models in which parameters, such as branch lengths, can be different for each gene. Because the supermatrix method has been intensively explored, tested, and validated, many of its weaknesses and strengths are known, and it is therefore widely used. The major limitations of the supermatrix approach are (1) the memory requirement and the computational load, (2) the reliability of heuristic searches, which often become trapped in local maxima separated by high barriers in the tree space, and (3) the increased effect of systematic errors. Several innovations have been used to address the two first points, including genetic algorithms, disk-covering methods, and parallelized computing. Systematic error is by far the major concern of phylogenomics but has been better characterized for supermatrix methods than for any other phylogenomic approach.

support), because the small amounts of phylogenetic signal contained in each gene should, in principle, add up and overwhelm stochastic errors. However, high statistical support does not necessarily mean that the obtained tree is correct, because of the systematic error. Moreover, when the strength of the systematic error is of similar magnitude as the genuine phylogenetic signal, this can lead to weak statistical support. Therefore, the study of potential systematic errors always deserves particular attention. Approaches to Detect Systematic Errors When using single genes for phylogenetic inference, the most straightforward way to detect systematic errors is to observe incongruences between different markers. This is obviously no longer possible when all the data are combined into a single supermatrix and therefore alternative approaches should be used to reveal incongruences and potential systematic errors: 1. Using different tree reconstruction methods. Because different methods are not sensitive in the same way and to the same extent to systematic errors, they will potentially produce slightly different topologies, identifying the most problematic parts of the tree. The congruence of all methods, although encouraging, is not a definitive proof of the absence of systematic errors, because all methods will have problems to correctly locate, for instance, a very fast-evolving, or rogue, lineage. 2. Partitioning the data set vertically or horizontally, that is, in subsets of genes or of species. For instance, one can compare trees based on supermatrices of informational and operational genes, or based on random subsamples of genes or positions (i.e., a jackknife test). Importantly, experience has demonstrated that varying taxon sampling is a very efficient way to reveal systematic errors because of the introduction or elimination of fast-evolving lineages, which are more likely to have accumulated homoplasies. Therefore, the targeted removal of divergent taxa is highly recommended. If sufficient computational resources are available, analyses of numerous random taxon subsamples could be illuminating. These approaches fundamentally test the coherence of a given approach (e.g., the supermatrix method). Even more conclusive evidence for the presence of systematic errors is the recovery of different topologies using phylogenomic approaches based on different character types (e.g., gene order data).

Supermatrices and Systematic Errors

Developing Better Models to Reduce Systematic Errors

The use of large data sets generally results in a global increase in the resolution of phylogenetic trees (increased statistical

The development of improved models of sequence evolution is a continuous quest in phylogenetics. Recently,

Genetics, Genomics | Phylogenomics

this field has experienced important advances, mainly because of (1) the availability of improved computational resources, (2) the introduction of fast algorithms such as the Monte Carlo Markov Chain, and (3) the use of large data sets allowing a more accurate estimation of a larger number of parameters (hence allowing more complex models). Among the most important recent improvements are models handling rate variation throughout time (heterotachy), nonstationarity of nucleotide or amino acid composition, site-specific amino acid propensity, and site-interdependent evolution due to protein tertiary structures. A major step in every phylogenetic analysis is to identify the model that fits the data best, because it should in theory result in the most reliable tree. However, an improved fit can be obtained by a better explanation of data characteristics that do not disturb phylogenetic inference; as a result, a less fitted model may sometimes lead to a more accurate tree. For this reason, the comparison of different suboptimal models is interesting, at least to potentially detect parts of the phylogeny affected by systematic errors. Ultimately, all of the known parameters shaping sequence evolution should be combined into the same model, but this would require a major increase in the computing resources needed, explaining why this integration is only beginning. Unfortunately, no model will ever capture all of the complexity of evolutionary patterns, and therefore alternative approaches are required to detect and overcome systematic errors. Alternative Approaches to Reduce Systematic Errors The theoretical basis of the alternative approaches to reduce systematic errors is that fast-evolving data (species or sites) are more prone to induce artifacts because they have accumulated a larger number of multiple substitutions. Accordingly, the most obvious approach is to simply remove the fast-evolving or aberrant taxa from the analysis. Unfortunately, this is only possible when all taxonomic groups under study are represented by at least one slow-evolving organism. The removal of fast-evolving genes is another possibility, but it is probably not a very efficient approach because genes are always a mixture of slow- and fast-evolving positions. In particular, a gene can appear slow because it contains many constant positions while its variable positions are extremely rapid. However, the specific removal of genes in which the problematic taxa are the fastest evolving has shown its efficiency in several cases. The removal of the fast-evolving positions is also promising. Several variations to estimate the evolutionary rate (e.g., compatibility-based, within predefined groups, or without the problematic taxa) have been proposed and successfully applied, although their relative performance is unstudied. The identification of fast-evolving species, genes, or sites

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requires, however, an a priori knowledge of the phylogeny to accurately estimate evolutionary rates. This circularity therefore constitutes the main limitation of this approach. An alternative method consists in discarding the fastest-evolving substitutions. For example, the RY coding for nucleotide data implies replacing the nucleotides by purines and pyrimidines in all the sequences, allowing elimination of transitions, which occur more often than transversions. A certain degree of compositional heterogeneity is also reduced by this approach because the frequency of purines and pyrimidines appears more stationary over time than that of individual nucleotides. A similar coding method has been proposed for amino acids in which they are grouped according to their biophysical properties. Finally, one should not forget that systematic errors are caused by the inability of algorithms to correctly detect multiple substitutions. To tackle this, the most obvious way would therefore be to increase taxon sampling to provide more information on the series of substitutions that occurred over time. In other words, breaking branches into small pieces on which multiple substitutions are unlikely, hence rendering the effect of homoplasy negligible. Although this increase in taxon sampling is not always feasible (e.g., Amborella or coelacanth) and is expensive, it should remain a priority.

Phylogenomic Inference Based on Whole Genome Features Methods for phylogenomic inference based on whole genome features have been introduced only recently, which limits their evaluation at this time point. In principle, these methods are promising because whole genome features such as gene content and order are more complex than primary sequence data and therefore less prone to homoplasy by convergence or reversal: changes in gene content and order have billions of possible character states compared to only 4 nucleotides and 20 amino acids. Other approaches, such as the DNA string approach, do not require the difficult step of homology assessment. The three methods explained below are illustrated in Figure 4. DNA String Approach This method relies on the fact that the frequency of short oligonucleotides (DNA strings) is relatively constant throughout a particular genome but variable across genomes. The comparison of the frequency of DNA strings between different organisms provides a measure of similarity that can be used for phylogenetic inference. In brief, for each genome, a DNA string vector is calculated as the ratio between observed and expected (from the nucleotide frequencies) oligonucleotide frequencies. The DNA

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Figure 4 Methods for phylogenomic inference based on whole genome features. Approaches based on gene content and gene order require previous orthology assignment. A character matrix can be constructed by scoring the absence/presence of genes or of pairs of genes per species, which will be analyzed by maximum parsimony or converted into distance matrix. A distance matrix can also be constructed by calculating break-point or rearrangement distances between pairs of sequences. Approaches based on DNA string frequencies do not require identification of orthologous genes and consist of calculating evolutionary distances among species from the differences in their oligonucleotide word usage, and reconstruct phylogenetic trees using standard distance-based algorithms.

string vector differences are calculated for each genome pair for all the species studied and transformed into a distance matrix. A standard distance-based method is then applied to generate a phylogenetic tree. The advantage of this approach is that, contrary to all other methods, it does not rely on homology or orthology and that it does not require any alignment. Although it seems possible to extract phylogenetic signals from oligonucleotide frequencies, this feature evolves fast and the phylogenetic signal in DNA strings saturates rapidly, which may limit the use of this method. Currently, DNA strings are transformed into evolutionary distances without any model of evolution, but improvements are easy to envision.

Gene Repertoire The analysis of gene repertoire is a straightforward way of comparing genomes and was the first published method used with complete genomes. This approach is based on the principle that closely related species will share a large proportion of genes and distantly related species will have differentially lost or gained a large proportion of genes with respect to their last common ancestor. The method consists of the construction of a data matrix that scores the

absence/presence of each gene. This matrix is then analyzed by parsimony or maximum likelihood. A distance matrix that represents the proportion of shared orthologues between each genome can also be derived from the data matrix and used by distance-based methods to construct a tree. The most important limitation of this approach is the definition of the gene repertoire itself. In most cases, orthologous genes are considered, but their identification is based on simple similarity searches, which are prone to error. In theory, phylogenetic analysis of each gene family would be required to accurately define gene repertoire, but this would not only imply a huge amount of computing time but the results would also be plagued by stochastic error. Alternatively, it has been proposed that only homologous genes be considered, but this drastically reduces the number of gene families. In both cases, it is often difficult to assess gene absence with certainty because an accelerated rate of evolution and/or a short sequence length can make a gene undetectable even when using sophisticated similarity search tools such as psi-blast. Another major problem of gene repertoire-based methods is the so-called small/big genome attraction artifact, which causes the grouping of unrelated species with small genomes: certain organisms, for example, intracellular parasites, tend to lose a similar

Genetics, Genomics | Phylogenomics

set of genes. This was in fact the first systematic error identified for non-sequence-based phylogenomic methods. Although probabilistic models of gene gains and losses are currently being developed, they do not seem to perform better than parsimony methods, and numerous improvements are still required.

Gene Order The gene order approach follows the same logic as gene repertoire-based methods, but requires the accurate recognition of orthologous relationships. The method starts with the construction of a character or a distance matrix. Scoring the presence/absence of gene pairs in each genome generates the character matrix. The distance matrix is generally based on break-point distances, that is, the number of adjacencies present in one genome but not in the other, or on rearrangement distances, that is, the number of rearrangements, inversions, transpositions, insertions, and deletions between genomes pairs. Among these three approaches (presence/absence of gene pairs, break-point distances, and rearrangement distances), only the latter implies the use of true evolutionary distances – the other two may severely underestimate the evolutionary distances between genomes. Because almost infinite combinations of gene order are possible and the probability of convergence is small, this approach is promising. However, although more trustworthy, rearrangement distances are extremely difficult to compute, even under the unrealistic assumption that only inversions have occurred in both genomes. Nevertheless, probabilistic methods for gene order evolution are currently under development. The computational burden presently makes the use of gene order for phylogenetic inference difficult, except for small genomes (mitochondria and plastids).

Rare Genomic Changes Finally, genomic data can also be analyzed using an approach similar to the one that has been used for morphology over the centuries: the identification, in complete genomes, of a few complex characters that are putatively homoplasy free because they evolve slowly (possibly only a single change). Various so-called rare genomic changes have been proposed, useful at different evolutionary depths: retroposon insertion of, for example, SINE and LINE, genetic code variation, gene fission and fusion, or presence/absence of introns. Because of their scarcity, these characters are well suited for ‘manual’ analyses, which is currently the rule. However, there are no reasons for not applying standard statistical methods to these types of characters, as is currently done for intron position evolution. More generally, any genomic characteristics

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that can be rigorously compared among organisms could be considered, and its usefulness should be evaluated. Limitations and Perspectives Like nucleotide and amino acid sequences, whole genome features are affected by homoplasy, and inferences can be misled by systematic errors. However, except for the small/big genome attraction mentioned earlier, systematic errors have not yet been characterized in detail, rendering evaluation of their impact uncertain. To deal with them, the same approaches as the ones described for the supermatrix method should be applied: modifying species sampling or removing fast-evolving data (e.g., regions in the genome with high recombination rates). Similarly, the use of improved models that better describe the evolution of these characters is the ultimate goal. Although a large amount of work is needed, obtaining reliable methods complementary to the supermatrix approach is of prime importance: because inferring ancient evolutionary events is extremely difficult, results corroborated by independent methods are the most trustworthy. This corroboration is the key to validate the inference of the Tree of Life.

Contribution of Phylogenomics to the Microbial Tree Because of large differences in genome size, data used in phylogenomics come mainly from complete genome sequences in the case of prokaryotes and from expressed sequence tag (EST) sequencing for eukaryotes (particularly in the case of animals). New sequencing technology will probably make available numerous complete eukaryotic genomes soon. Surprisingly, despite the fact that hundreds of complete bacterial genomes have been available for several years, very few bacterial phylogenies have been inferred with more than 100 and none with more than 200 species. Technical limitations, in particular userfriendly tools to handle this large amount of data, and computational burden explain this underuse of promising genomic data. Accordingly, phylogenomics has not yet produced a large number of new results, although the situation is currently changing. Eukaryotes Because of the availability of large amounts of sequence data from animals, fungi, and green plants, advances concerning the evolutionary relationships among these three groups represent the first and most spectacular achievements of phylogenomics. For instance, the sister group of vertebrates was shown to be tunicates, instead of cephalochordates as long assumed. Inferring the eukaryotic tree

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requires more than this, because much of the evolutionary diversity of the domain Eukaryota is contained outside these three kingdoms. Recently, large-scale genomic data have been generated for a range of poorly studied but important microbial eukaryotes commonly referred to as protists, and phylogenomic studies have been carried out to determine their evolutionary position in the eukaryotic tree. A number of these unicellular lineages have appeared to be related to animals (Choanoflagellata, Capsaspora, and Ichthyosporea), or fungi (Nucleariidae), but most of them have been regrouped in exclusively unicellular proposed superensembles – Amoebozoa, Excavata, Rhizaria, and Chromalveolata. Amoebozoa are a group of morphologically diverse amoebae, which includes slime molds (e.g., Dictyostelium), lobose amoeba (e.g., Amoeba), and anaerobic Archamoeba (e.g., Entamoeba). These organisms were thought to have evolved independently, but recent phylogenomic analyses based on hundreds of protein-coding nuclear genes have shown that they share a common origin. Excavata are an ensemble of organisms that do not possess a single common feature but that are united by a series of overlapping ultrastructural and molecular characters. Till now, there is no molecular phylogenetic evidence for the monophyly of this group, but recent phylogenomic analyses have found monophyly for a few subensembles such as the grouping of Jakobida, Euglenozoa, and Heterolobosea. Chromalveolates unite alveolates (ciliates, dinoflagellates, and apicomplexans), stramenopiles, haptophytes, and cryptophytes and the hypothesis of their monophyly is based on the assumption of a single secondary endosymbiosis with a red alga that is at the origin of vertically inherited chlorophyll-c-containing plastids. According to recent phylogenomic studies, haptophytes and cryptophytes could be sister groups, but there is no convincing evidence to cluster them with alveolates and stramenopiles. Indeed, recent multigene analyses associate the last superensemble, the Rhizaria, with alveolates and stramenopiles. In general, the progress concerning the eukaryotic phylogeny has not been as impressive as expected because taxon sampling remains sparse (often only one species to represent a large and diverse phylum, some phyla being completely absent), and because the relationships to infer are ancient (hence homoplasy is not negligible). With the increase of the available sequence data for many interesting unicellular eukaryotes and the development of better models of evolution, the situation is likely to improve. One of the most difficult questions is the position of the root of the eukaryotic tree. The Archaea, often used as outgroup, are too distantly related for a reliable inference. Although -proteobacteria constitute a much closer outgroup for the eukaryotic genes of mitochondrial origin, they have not yet been used at a genome scale to decipher the eukaryotic root. Although a few rare genomic changes (gene fusions and gene duplications) have been proposed

to address this issue, they are not fully congruent. They nevertheless suggest a root between unikonts (Amoebozoa and Opisthokonta) and bikonts (all other eukaryotes). Phylogenomic analyses using rich taxon sampling and improved models of sequence evolution are definitively needed to settle the root of the eukaryotic tree. Prokaryotes Despite the large number of complete prokaryotic genomes now available, the bacterial and archaeal phylogenies remain almost identical to the rRNA-based tree. The phylogeny of prokaryotes has been carefully inspected because of the supposed predominance of HGT, which would make the inference of the species phylogeny difficult or even impossible. For some researchers, HGT is considered to be so widespread that the phylogenetic signal is thought to have disappeared. Analyses of gene order, DNA strings, and other methods have shown that it is possible to robustly recover most relationships among prokaryotes, and several supermatrix and supertree approaches have shown that there is a core of genes that rarely undergo HGT and that these genes are therefore suitable for determining the phylogeny of prokaryotes. Relationships that were already supported by rRNA phylogenies, such as the monophyly of proteobacteria, cyanobacteria, spirochetes, and chlamydiales, have been confirmed by phylogenomic studies, but relationships between them are mostly unresolved. Based on orthologue distances, concatenated alignments, and supertree analyses at least three major new clades are strongly supported (albeit systematic error cannot be excluded, due to insufficient testing) – the sister group of chlamydiales and spirochetes, the sister group of aquificales and thermotogales, and the grouping of high GC Grampositive bacteria with cyanobacteria and deinococcales. In addition, several other major groupings were found by some but not by other approaches and should be considered tentative for the moment. The resolution of the bacterial radiation remains one of the major challenges, even in the age of phylogenomics. Within the Archaea, the separation between Euryarcheota and Crenarcheota was clear with rRNA phylogenies, but some uncultured taxa such as Korarchaeota were of uncertain position. Phylogenomics recently questioned the monophyly of Crenarcheota, but taxon sampling remains too sparse to draw any firm conclusions, many important lineages, such as Nanoarchaeota and Methanopyrales being represented by a single species. One of the most interesting debates concerning archaeal phylogeny is on whether the methanogens are monophyletic. Ribosomal RNA-based phylogenies did not support their monophyly, but suggest that Methanopyrus emerged early on. The nonmonophyly was confirmed by phylogenomic studies, but methanogens

Genetics, Genomics | Phylogenomics

appear to have emerged relatively late, although the position of Methanopyrus remains unsettled, possibly close to methanobacteriales and methanococcales. The sparse taxon sampling and the extreme evolutionary properties of archaeal genomes (in particular amino acid compositions) make phylogenomic inference quite difficult and further refined studies are needed.

Conclusion Phylogenomics is without doubt the most promising way to resolve the Tree of Life, as demonstrated in several cases. Improvements in species sampling and inference methods are still needed to avoid systematic errors and enhance phylogenetic signal. However, resolving power does not increase linearly with the number of characters considered, which implies that some closely spaced speciation events will most likely remain unresolved, albeit making such inference from lack of resolution premature. A serious drawback of phylogenomics is that the needed resources, particularly computational ones, increase dramatically with respect to single-gene phylogenetic inference, thus contributing to environmental degradation, that is, to the loss of biodiversity. Although it was already noticed that scientific observations often lead to the destruction of the object under study, this is particularly problematic in the case of the Tree of Life. See also: Genome Sequence Databases: Types of Data and Bioinformatic Tools; Horizontal Transfer of Genes between Microorganisms; Metagenomics

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Further Reading Beiko RG, Harlow TJ, and Ragan MA (2005) Highways of gene sharing in prokaryotes. Proceedings of the National Academy of Sciences of the United States of America 102: 14332–14337. Brochier-Armanet C, Boussau B, Gribaldo S, and Forterre P (2008) Mesophilic crenarchaeota: Proposal for a third archaeal phylum, the Thaumarchaeota. Nature Reviews Microbiology 6: 245–252. Burki F, Shalchian-Tabrizi K, Minge M, et al. (2007) Phylogenomics reshuffles the eukaryotic supergroups. PLoS ONE 2: e790. Daubin V, Gouy M, and Perrie`re G (2002) A phylogenomic approach to bacterial phylogeny: Evidence of a core of genes sharing a common history. Genome Research 12: 1080–1090. Delsuc F, Brinkmann H, and Philippe H (2005) Phylogenomics and the reconstruction of the Tree of Life. Nature Reviews Genetics 6: 361–375. Jeffroy O, Brinkmann H, Delsuc F, and Philippe H (2006) Phylogenomics: The beginning of incongruence? Trends in Genetics 22: 225–231. Lerat E, Daubin V, and Moran NA (2003) From gene trees to organismal phylogeny in prokaryotes: The case of the gamma-proteobacteria. PLoS Biology 1: e19. Moret BME, Tang J, and Warnow T (2005) Reconstructing phylogenies from gene-content and gene-order data. In: Gascuel O (ed.) Mathematics of Evolution and Phylogeny, pp. 321–352. Oxford, UK: Oxford University Press. Philippe H, Delsuc F, Brinkmann H, and Lartillot N (2005) Phylogenomics. Annual Reviews Ecology Evolution Systematics 36: 541–562. Rodrı´guez-Ezpeleta N, Brinkmann H, Burger G, et al. (2007) Toward resolving the eukaryotic tree: The phylogenetic positions of jakobids and cercozoans. Current Biology 17: 1420–1425. Rodrı´guez-Ezpeleta N, Brinkmann H, Roure B, Lartillot N, Lang BF, and Philippe H (2007) Detecting and overcoming systematic errors in genome-scale phylogenies. Systematic Biology 56: 389–399. Snel B, Martijn AH, and Dutilh BE (2005) Genome trees and the nature of genome evolution. Annual Reviews Microbiology 59: 191–209. Wolf YI, Rogozin IV, Grishin NV, and Koonin EV (2001) Genome trees and the tree of life. Trends in Genetics 18: 472–479.

Plasmids, Bacterial M Filutowicz, University of Wisconsin-Madison, Madison, WI, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Impact of Plasmid Research on Molecular Biology Plasmid Structures and DNA Synthesis

Glossary biofilm A spatially organized community of microorganisms associated with a surface. centromere The DNA segment of a replicon that is associated with spindle fibers and involved in DNA segregation. cis From Latin for ‘on this side of’. DNA replication Duplication of the genome to make two copies of it. helicase An enzyme that separates the DNA double helix into single strands. integron A gene-capture system found in plasmids, chromosomes, and transposons that requires a recombinase enzyme (integrase) and a proximal recombination attachment site for incorporation into the genome.

Abbreviations 2,4-D BHR dso HGT HR ICP IHF Inc Mpf ori

2,4-dichlorophenoxyacetic acid broad host range double-strand ori horizontal gene transfer homologous recombination interon-containing plasmid integration host factor incompatibility mating pair formation apparatus origin of replication

Defining Statement The focus of the article is on plasmids that establish themselves in bacteria. Strategies by which plasmids are reproduced, maintained, and transferred are described. The broader and interrelated issues of how plasmids generate a horizontal gene pool by recruiting genes from different environments, including genes for antibiotic resistance, are also discussed.

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Resolution and Distribution of Newly Replicated Plasmid DNA Horizontal Plasmid Transfer by Conjugation Further Reading

microcosm A small enclosed part of a habitat. origin of replication (ori ) The site at which DNA replication starts. polymerization The process of joining identical or similar subunits to make DNA, RNA, or proteins. relaxase A site-specific topoisomerase that removes superhelical twists from DNA. selfish DNA DNA whose only function is self-preservation (see also Further Reading). Single-strand binding proteins (SSB) Proteins that facilitate replication by coating single-strand DNA, thereby preventing complementary regions from pairing with each other. topoisomerase An enzyme that introduces or removes superhelical twists in the DNA. trans From Latin for ‘on the opposite side of’.

oriT PCD RCR RE Rep RM SSB ssi sso TIVSS

origin of (DNA) transfer programmed cell death rolling circle replication restriction enzymes replication proteins restriction–modification single-strand binding proteins single-strand initiation single-strand ori type IV secretion system

Impact of Plasmid Research on Molecular Biology Bacteria are the most abundant and diverse forms of life on Earth and they play host to a vast assortment of extrachromosomal genetic elements. Joshua Lederberg originally proposed the word ‘plasmid’ as a generic term for any extrachromosomal hereditary determinant, a broader definition than is commonly used today. However, past and

Genetics, Genomics | Plasmids, Bacterial

recent discoveries do point to common properties of certain bacteriophage and plasmids. Both can use very similar mechanisms for replicating, maintaining, and partitioning their genomes, and both can be evicted from a cell. From the outset, plasmid research profoundly contributed to the development of modern molecular biology as summarized in Figure 1. Bacterial plasmids are exemplary subjects for study, being conveniently dissected, reassembled, and introduced into various hosts. Their versatility and power make them eminently worthy of our attention. Unraveling the complexities of plasmids relied upon genetics to identify the genes (proteins), biochemistry to determine their function, and microscopy to observe the conformations of single DNA molecules in vitro and the behavior of plasmid communities in vivo. The monumental task of elucidating the various shapes of DNA and how they impact replication, transcription, and recombination benefited immeasurably from the ease with which plasmids can be isolated and genetically altered. Plasmids are selfish DNA that constitute a burden for the bacterial host cell. Their size can range from 2 to 500 kb and a given strain may contain several distinct plasmids. As a result, plasmids can commandeer a sizeable fraction of a cell’s resources, at times providing little to no demonstrable benefit. For example, very few detectable phenotypic differences were observed under laboratory conditions after a wild type Bacillus megatherium strain was cured of its seven plasmids, which comprised 11% of the cellular DNA. How is it, then, that plasmids are so

Recombination in bacteria

successful in colonizing bacterial communities and why are plasmid-free cell lines so infrequent among bacteria isolated from nature? The DNA of plasmids and their hosts have coevolved a complex network of control mechanisms assuring a highly effective symbiotic relationship, albeit a forced one.

Plasmid Structures and DNA Synthesis All circular and linear plasmids are double-strand, antiparallel DNA helices. When circular plasmids are isolated from bacterial cells, the entire double helix is ‘stressed’, which can lead to a change in the actual number of base pairs per helix turn. Alternately, this stress can cause regular spatial deformations of the helix axis. In either event, the axis of the double helix then forms a helix of a higher order. It is this deformation of the helix axis in closed circular DNA that gave rise to the interchangeably used terms ‘superhelicity’ and ‘supercoiling’. Supercoiling is a form of energy that can be stored in DNA molecules and used in various DNA transactions. As shown in Figure 2, supercoiled DNA is more compact in comparison to relaxed -nicked- circles. Several unusual DNA conformations can be stabilized by supercoiling and some wellcharacterized examples include cruciforms, R-loops, and open complexes, all of which have specific sequence requirements. Many proteins that modify superhelicity

Genome evolution

Mammalian cell transfection Gene expression

Conjugation and fertility

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Bacterial transposons Co-transformation

‘Operon’ PLASMIDS Restriction an modification enzymes

‘Replicon’

DNA replication

‘Antisense’ RNA DNA topology

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DNA cloning

Partitioning of DNA at cell division

Artificial chromosomes Reporter genes

Figure 1 Summary of some of the major contributions to bacterial genetics that have resulted from the study of plasmids. Reprinted from Elsevier, vol. 135, Cohen SN (1993) Bacterial plasmids: Their extraordinary contribution to molecular genetics. Gene 67–76. ª 1993, with permission from Elsevier and the author.

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(plasmid or chromosome), all bacterial DNA polymerases require a 39-OH group for initiating the synthesis of the leading DNA strand. This occurs via elongation that extends from one of three sources: an RNA primer (R-loop), a nick in one of the two strands of the double helix, or an amino acid of a protein covalently bound to the DNA. During synthesis of the lagging strand, many RNA primers are made. Eventually, the primer for the leading strand and the multiple RNA primers for the lagging strand are removed and the gaps are filled. DNA polymerases, multisubunit complexes by themselves, associate with many other proteins to form a molecular machine called the replisome. In some bacteria the replisome assembles at the cell midpoint or the predivision site. It was proposed that in Gram-positive bacteria with low GC content, two different polymerases might be used to replicate DNA (plasmids and chromosomes), one specializing in leading strand synthesis and a second that synthesizes lagging DNA strands. In Gram-negative bacteria, a single polymerase functions in both capacities. The replisome operates on single-strand template DNA created by the binding/activity of DNA helix-destabilizing proteins.

Figure 2 Electron microscope images of RI DNA. Predominant circular species appear as light images on dark background. The species in the top left and the bottom right corners represent superhelical plasmid DNA while two molecules in the center represent the relaxed form. Samples were prepared by a specialized technique that ‘thickens’ DNA and thereby amplifies the visual impression of DNA versus the background. Reprinted by permission from Macmillan Publishers Ltd: Nature. Cohen SN and Miller CA (1969) Multiple molecular species of circular R-factor DNA isolated from Escherichia coli. Nature 224: 1273–1277. ª 1969.

catalytically (e.g., topoisomerases) or by simply binding to DNA and constraining superhelicity (e.g., integration host factor (IHF) and HU) exist. The known topoisomerases (Topo) that alter supercoiling include Topo I, DNA gyrase, Topo III, and Topo IV. Topo I and Topo III break ‘one’ strand of the DNA duplex resulting in their classification as ‘type I’ enzymes. The type II enzymes DNA gyrase and Topo IV are related to one another and, as their classification suggests, they break both strands of the DNA simultaneously. In almost all cases, isolated plasmid DNA displays ‘negative superhelicity’ (DNA replication). Synthesizing the ends of linear plasmids (i.e., telomeres) bucks this trend, however, since it is stimulated by positive supercoiling. The process of DNA replication influences and responds to the superhelicity of the plasmid molecule. Initiation of plasmid DNA replication typically occurs asynchronously with respect to the bacterial cell cycle, unlike initiation at the chromosome’s oriC (Chromosome replication and segregation). Irrespective of their target

Unit of Replication: The Replicon Units of replication called replicons comprise unique DNA segments that are indispensable for the plasmids (and chromosomes) that contain them. The reason is that the ori sequence from which replication ‘originates’ is embedded within each replicon. Interestingly, an ori of a circular replicon has been converted into a linear replicon by adding telomeres. Conversely, the oris from linear plasmids have been similarly used to drive the replication of circular vectors. These results suggest that linear and circular replicons diverged from a common progenitor. Although a single replicon with a single ori is necessary and sufficient for the propagation of most plasmids, an additional level of complexity is found in a subset of plasmids that contain multiple oris. Studies of plasmid replicons have revealed that an ori must be distinguished from the rest of the DNA and it is the only site where the frequency of replication is regulated. Once replication begins, DNA is threaded through the machinery at the fairly steady rate of about 1000 bp/s. ‘Termination’ of DNA synthesis occurs either at the ori (for unidirectional replication) or at a site called ter (for bidirectional replication) where the replication fork is disassembled. In linear plasmids, a different strategy for terminating replication is employed at the telomeres, which is described later. To understand the components of the ori and the dynamics controlling its activity, an extraordinary effort has been made to isolate oris from a variety of naturally occurring plasmids. Typical analyses of plasmid replication functions employ constructs called ‘basic’ or

Genetics, Genomics | Plasmids, Bacterial

‘minimal’ replicons. These are defined as the minimal portion of a plasmid that replicates with a copy number characteristic of the parent replicon. Minimal replicons are cryptic plasmids; they encode no function other than replication. Hence, to assess an ori ’s ability to facilitate controlled replication, a selectable marker such as antibiotic resistance is often added (cloned) into these selfreplicating DNA molecules. The recombinant plasmids that are made by this process confer antibiotic resistance to their host without posing risks to the environment because their construction and use are strictly regulated and confined to research laboratories. In fact, genetic markers of various types and combinations can be added to a plasmid replicon as long as the aforementioned rules are adhered to. As might be expected from its complicated role, the ori coordinates multiple molecular interactions. Hence, it is somewhat surprising how simple some oris are, with hostencoded RNA polymerase being the sole machine responsible for producing a preprimer. More often, before the replisome is assembled and the replication fork is launched, various proteins move in and sometimes out of the ori in a controlled sequential pathway to provide for the regulated initiation of DNA replication. Typically, one or many copies of a plasmid-encoded ori-specific protein bind to the ori and change its structure in a supercoiling-dependent process. DNA sequences to which these replication proteins (Rep) bind tend to be reiterated, thus earning the name ‘iterons’, and plasmids that carry them are called iteron-containing plasmids (ICPs). The presence of Rep-binding iterons is a hallmark, not only of

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many prokaryotic plasmid oris, but of chromosomal, viral (phage), and eukaryotic oris as well. Several wellcharacterized Rep proteins are known to bend the ori DNA when they bind to target sequences, be they singular cognate binding sites or iterons. Moreover, besides containing Rep-binding sites, some oris contain multiple binding sites for a variety of proteins of which the most prominent are RNA polymerase, DnaA, IHF, HU, Fis, and H-NS. Each of these proteins is known to constrain supercoiling and some bend or even kink DNA upon binding. The distortions they produce lead to considerable changes in the DNA structure, and the resulting patterns of protein–protein interactions are needed to facilitate replication (Figure 3).

Examples of Replication oris and Mechanisms of Their Activation Circular plasmids are classified as belonging to one of three broad categories based on their mode of replication, which can be thought of in terms of their signature replication intermediates when visualized by electron microscopy. The terms that have been coined for two of these replication modes are particularly descriptive of these intermediates. Replicating theta-mode plasmids produce structures resembling the Greek letter theta, ‘’ (Figure 4). Rolling circle-mode plasmids, sometimes referred to as sigma mode for the Greek , appear as circles extruding linear product, giving the appearance of yarn rolling off a spinning wheel. The third replication

ori AT-rich region

Rep

Helicase

IHF

Polymerase

DnaA Primer

Primase DNA-synthesis

Figure 3 Replication steps – a model. The replication initiator protein (Rep) recognizes the origin of replication (ori) and induces a conformational change in the plasmid (e.g., DNA bending). Then, Rep protein, with or without host proteins engagement with their binding sites (IHF/HU, DnaA), triggers strand separation in an AT-rich segment of the DNA. This single-strand region is then targeted for the loading of DNA helicase and primase. DNA helicase will further unwind the DNA helix while primase will start synthesizing short RNA molecules that serve as primers for the initiation of DNA synthesis by ‘sliding’ DNA polymerase. Reproduced from Kru¨ger R, Rakowski SA, and Filutowicz M (2004) Participating elements in the replication of iteron-containing plasmids (ICPs). In Funnell BE and Phillips GJ (eds.) Plasmid Biology, ch. 2, pp. 25–45. Washington, DC: American Society for Microbiology. ª 2004, with permission from ASM Press.

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(a)

(c)

(e)

(b)

(d)

(f)

Figure 4 Electron micrograph of plasmid replication products synthesized in vitro: (a) Double-strand circular template DNA, (b) D-loop molecule, (c) and (d) Theta-type replicative intermediates containing two branch points and two doublestrand daughter segments. (e) and (f) Catenated daughter molecules. To enhance picture resolution, the template and replicative intermediates were prepared for microscopy by a technique that ‘relaxes’ DNA. If not treated in this way, all samples would resemble the supercoiled molecules shown in Figure 2. Reprinted from Elsevier, vol. 193, Levchenko I, Inman RB, and Filutowicz M (1997) Replication of the R6K origin in vitro: Dependence on wt  and hyperactive S87N protein variants. Gene 97–103. ª 1997, with permission from Elsevier.

mode, strand displacement, has perhaps a less catchy name but it still informs the imagination.

Plasmids replicating by the ‘theta’ mechanism

Theta-type plasmids are divided into several distinct subgroups and many of the previously mentioned ICPs fall, collectively, into a theta subgroup characterized by oris with AT-rich regions. For these plasmids, melting ori DNA during open complex formation typically requires the concerted action of both the plasmid-encoded Rep protein and the host-encoded initiator, DnaA. These proteins are believed to promote localized DNA melting at their binding sites, but there are insufficient data to explain how this would destabilize the spatially separated AT-rich segments. There is not even a consensus as to the type of nucleoprotein structure that DNA-bound Rep produces. In some cases, Rep proteins appear to form a discrete nucleoprotein complex into which none or only a short stretch of DNA is incorporated. In other ICPs, however, iteron-containing DNA seems to wrap around Rep, but only if another host protein (HU or IHF) is present, suggesting that interactions between the proteins

occur in association with ori DNA. In fact, IHF may also have a role in strand separation. Although the recognition of oris by Rep protein and auxiliary host factors is energy-independent, the DNA melting process requires energy. Given that DnaA possesses ATPase activity, one possible role for this host factor in initiation may be to generate energy for strand separation. Surprisingly, however, ATP hydrolysis is not obligatory for DnaA functioning in the melting of some oris despite the fact that data support crucial roles for DnaA–ATP and DnaA–ADP complexes in the regulation of chromosomal replication. Interestingly, in the presence of ATP, some Reps can change the conformation of plasmid DNA without any additional factors, suggesting that the Rep protein, itself, can somehow perceive the presence of ATP (Figure 5). Once melting is complete, the replication process proceeds to the synthesis of an RNA primer and the loading of DNA helicase. ATP is required as a cofactor as well as a substrate for RNA primers and there must be energy input for the process to progress. In a supercoiled DNA molecule, conformational energy stored after open complex formation can be tapped and, additionally, ATP hydrolysis occurs during the helicase movement and topoisomerase activities that take place ahead of the replication fork (gyrase, mentioned earlier). The mechanism employed for primer generation is an important distinction used to assign plasmids to subgroups of theta-mode replication. In the R-loop-type plasmids, RNA polymerase-driven transcription generates an RNA molecule that is complementary to the transcribed strand of the plasmid DNA. Recent studies of RNA polymerases show that the RNA transcript and the DNA exit through separate channels of the polymerase, but the displaced DNA strand is accessible for basepairing. When the 59 end of the RNA and the displaced DNA strand interact, a stable RNA–DNA heteroduplex (the R-loop) is formed at the ori. This is highly unusual. Typically, the product of transcription is messenger RNA, which is bound by ribosomes and translated into proteins. In the prototype plasmid ColE1, a primer precursor of approximately 550 bp, called RNAII, interacts with the displaced DNA strand as RNA polymerase transcribes the template DNA. For DNA polymerase (Pol I) to initiate replication, RNA degrading enzymes must process the nucleic acid heteroduplex to make an RNA–DNA junction that will be proficient in priming unidirectional DNA replication. This group of plasmids does not encode any protein that is essential for replication. Contrasting with this, ColE2-type plasmids encode a Rep that functions as a priming enzyme specific for the ColE2 ori. Such enzymes, called primases, are simply alternate forms of RNA polymerase that are dedicated to synthesize primers for DNA replication. In the case of ColE2, the Rep/primase generates a very short RNA

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Figure 5 RepE54-induced relaxation of mini-F plasmid. Supercoiled plasmid and RepE54 were incubated in the absence or presence of ATP (at optimal amounts of all three reactants). Atomic force microscopy images of (a) and (b) supercoiled mini-F replicon DNA in the absence of RepE54, (c) and (d) relaxed plasmids by RepE54 binding in the absence of ATP, and (e) and (f) relaxed plasmids by RepE54 binding in the presence of ATP are shown. The images in f represent the relaxed plasmids only from the sample longer in ‘apparent length’. Bound proteins are indicated with arrows. Image sizes are 1  1 mm (a, c, and e) and 300  300 nm (b, d, and f) (reproduced at 90% of original size). Reproduced from Yoshimura SH, Ohniwa RL, Sato MH, et al. (2000) DNA phase transition promoted by replication initiator. Biochemistry 39: 9139–9145. ª 2000, with permission from the American Chemical Society.

primer, a scant 3 bases in length, to prepare for the synthesis of the plasmid’s leading DNA strand. Rolling circle replication

For plasmids that employ rolling circle replication (RCR) such as the PT181 family, the initiation step involves the recognition of the double-strand ori (dso) by the plasmidand dso-specific Rep protein. Many dsos contain sequences that promote the formation of hairpin and cruciform structures in a supercoiling-dependent fashion. The binding of Rep to the dso is also known to enhance cruciform extrusion. Two enzymatic activities, nicking and strand

closing, accompany the Rep protein’s sequence-specific DNA-binding activity. In many RCR plasmids, a nic sequence is located in the loop of the hairpin and this sequence, in addition to the dso’s Rep-binding sequence, is required for plasmid replication. Reps are highly conserved among this family of plasmids, and sequence comparisons (of Reps and dsos) suggest that the hundreds of RCR plasmids may belong to only a few families. Rep proteins encoded by RCR plasmids remain covalently attached to the 59 end of the nick site via a conserved amino acid, tyrosine. Leading strand replication proceeds by extension from the free 39 end of the nicked DNA until a

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complete round of synthesis leaves the parental portion of the leading strand fully displaced. Cleavage and rejoining reactions at the regenerated nick site, catalyzed by the Rep protein (using another tyrosine), result in a covalently closed, circular, double-strand DNA that contains the newly synthesized leading strand. Perhaps the requirement for two tyrosines explains why many Rep proteins of RCR plasmids function as dimers in which each subunit performs a different role. A common feature of the RCR initiators is that they promote only one round of leading-strand replication, a consequence of the inactivation of the tyrosine that Rep needs for initiation (an oligonucleotide is attached). To complete the replication process, the displaced leading strand is subsequently converted to double-strand DNA by using a single-strand ori (sso) and, solely, host-encoded proteins (Figure 6). DNA replication during the process of conjugation

Conjugation, the self-controlled transfer of DNA, is an amazing property of some plasmids and a process of such significance that it warrants separate discussion later in this article (Conjugation, bacterial). The replication of DNA that occurs during conjugation is mechanistically very similar to RC plasmid replication, the major difference being that, in conjugation, the process commences in one cell but is completed in a different one. Conjugative plasmid replication begins with the relaxosome, which is an assemblage of proteins that processes the DNA at a site called the origin of (DNA) transfer (oriT). DNA relaxases are the key enzymes although they act, together, with

additional accessory proteins. In all systems of self-transmissible and mobilizable plasmids studied so far (except in actinomyces), DNA cleavage is the consequence of a strand transfer reaction that involves the formation of a covalent DNA–relaxase intermediate. During conjugation, a unique plasmid DNA strand called the transfer (T) strand undergoes 59-to-39 directional transmission; thus, the relaxase-bound end of the DNA enters the recipient first. The 39 end of this strand likely undergoes continuous extension by DNA polymerase in the donor cell, thereby generating a transfer intermediate that is longer than unit length and contains an internal nic site. The 59-bound relaxase recognizes the site after it enters the recipient and mediates the recircularization of the DNA molecule by a reverse strand transfer reaction. In other words, a cleavage–rejoining reaction is catalyzed between the free 39 OH end of the DNA and the covalently linked 59 terminus. The initiation and termination steps in a round of transfer require different sequence features at oriT, consistent with the model that initiation involves negatively supercoiled, double-strand DNA whereas the termination reaction acts on single-strand DNA. The T-strand that enters the recipient is ‘parental’ DNA. It is generally believed that replacement strand synthesis in the donor bacterium proceeds via a rolling-circle mechanism from the 39 hydroxyl group exposed at nic. In the recipient cell, conversion to double-strand DNA occurs using a sso and host-encoded proteins. Another intriguing aspect of certain plasmids is that conjugative DNA replication and RC replication for copy number maintenance must

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Figure 6 A model for plasmid RCR replication. See text for details. Reproduced from Khan SA (2004) Rolling-circle replication. In: Funnell BE and Phillips GJ (eds.) Plasmid Biology, ch. 4, pp. 63–78. Washington, DC: American Society for Microbiology. ª 2004, with permission from ASM Press.

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be strictly coordinated. After all, the initiation of DNA synthesis at oriT would lead to an immediate loss of superhelicity, thereby preventing cleavage at any other RCR type origin. Although the conjugative relaxases and Reps of RCR cleave DNA site- and strand-specifically, the relaxases have substantially higher affinity for the 39-terminal region of the substrate DNA. This property of relaxases allows the superhelical state of the cleaved DNA to be maintained, which in turn allows the plasmids to exist stably as ‘relaxosomes’ without being impaired in other plasmid functions. As a result, relaxosomes can be present throughout the entire replication cycle, awaiting the cues that will trigger the release of the 39 end. DNA cleavage by vegetative RCR initiators, in contrast, results in the spontaneous release of the 39 terminus, which subsequently becomes available for elongation by host-encoded DNA polymerases. Strand displacement: The IncQ family of replicons

The strand displacement mode of replication utilized by the RSF1010-like IncQ plasmids begins with a familiar scenario: Plasmid-encoded Rep proteins bind to iterons in the ori, which results in the melting of an adjacent AT-rich region. As a new twist, the opened DNA provides an entry point for a plasmid-encoded helicase whose activity exposes two single-strand initiation (ssi ) sites. These sites are located on opposite DNA strands and are recognized by a plasmid-encoded primase. DNA replication proceeds by continuous extension of RNA primers by the host polymerase originating from a primed ssi site, and this results in the displacement of the nontemplate DNA strand. IncQ plasmids are exceptionally broad in their host ranges and their features will be discussed in greater detail in the section describing strategies used by plasmids for transferring themselves to plasmidfree cells. Plasmid Replication: Regulation of Initiation Frequency Every replicon’s most basic function is to maintain itself at its characteristic, fixed, intracellular copy number. For some plasmids, the copy number can be as low as one per cell, whereas others can approach several dozen plasmid replicons per chromosomal equivalent. Ultimately, however, high copy and low copy plasmids adopt the survival strategy of controlling their rates of replication in accordance with the reproduction of their hosts. If the plasmid replicates ‘too slowly’ it will be lost from the bacterial population, but if it replicates ‘too fast’, cells will become ‘intoxicated’ by the excessive amplification of extrachromosomal DNA. Functional assays called incompatibility (Inc) tests have been used to identify elements that control the copy number of plasmids. The name of the assay derives

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from another use of this powerful genetic tool, determining the functional relatedness of plasmids. An Inc– phenotype often results from the inability of plasmids to coexist within the same cell over many generations because they share replication/copy number control elements; this eventually winds up excluding one of the plasmids from the cell. This assay led researchers to classify plasmids into several dozen Inc groups. By cloning fragments of minimal replicons into unrelated plasmids, Inc testing was adopted as a screen to identify factors and/or sequences that inhibit the replication of their plasmids of origin. This methodology demonstrated that, across the board, antisense RNA and iterons play a trans-acting regulatory role even though they encode no product; the RNA or DNA itself inhibits replication in a dosage-dependent fashion. The mechanism that senses the number of oris per cell (ori counting) is dependent on the binding of antisense RNA or iterons to their targets, another RNA, or Rep protein. These targets were identified by using another major genetic approach used to dissect plasmid replication, the isolation of ‘copy-up’ mutants with elevated plasmid copy numbers. Copy-up mutations typically fall within the ‘genes’ encoding antisense RNA or Rep. It is noteworthy that certain copy-up mutations (singularly or in combination) have been found to cause a plasmid–host relationship to become selfdestructive due to plasmid (over)replication. ICPs: Regulation of replication in Rep–iteron systems

As previously discussed, an understanding of the control of replication in iteron-containing plasmids relies on a familiarity with the interactions between Rep protein and iterons, the dominant players in DNA helix melting at the ori. Helix melting is an early step of open complex formation, which is considered to be a prime target for elements in plasmid replication control. Insight into one possible mechanism for modulating ori activity was gained by the observation in one system (plasmid R6K) that precise deletion of mutant iterons can sometimes restore the function of a defective replication ori. It was hypothesized that continuous alignment of iterons invites near-neighbor contacts between the Rep molecules bound to them. More recent experiments have demonstrated cooperative iteron binding by Rep protein, which presumably has a positive influence on filling the ori with initiator even when concentrations of active Rep might be suboptimal. Decades of research have demonstrated that Rep and iterons are not only necessary for initiating ICP replication, but under certain conditions they act as negative regulatory elements as well. Our advanced understanding of replication control in ICPs owes itself to the fact that many Rep proteins and iterons exhibit sequence homologies and a still larger group (including eukaryotic counterparts) appears to be related on a structural level. This has allowed insights made in one

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model system to be applied to others. It has become evident that Rep proteins are characterized by structural flexibility, allowing them to participate in a wide range of regulatory communications (Figure 7). Crystal structure analyses and biochemical data have revealed that Rep monomers are modular in nature with one subdomain dedicated to DNA binding while the other can alternately be used for DNA binding or dimerization (Figure 8). In iteron-regulated plasmids, copy-up Rep mutations typically destabilize protein dimers and overwhelming evidence indicates that Rep monomers are the initiators of replication, not the dimers that often seem to predominate. In one or two systems, dimers can compete with monomers for iteron binding; however, most Rep dimers seem to lack iteron-binding activity. Intriguingly, a number of experiments have demonstrated that dimers inhibit replication and some of the data come from systems where dimers do not bind to iterons. Whether or not dimeric versions of Rep bind iterons, they often play key regulatory roles in the autorepression of transcription and/or the inhibition of replication (Figure 7). One model for Rep-mediated inhibition of plasmid replication, known as handcuffing, has been proposed in variations that do and do not invoke direct DNA

interaction by Rep dimers. The handcuffing model centers on three main postulates: An individual ori with Rep monomers bound to its cognate iterons is replication proficient. Rep-mediated coupling of two oris by dimers blocks the initiation of replication for each of the participating plasmids. Finally, handcuffed structures fall apart and initiation potential is restored as cell volume increases, perhaps a result of dimers dissociating to yield two monomer-containing oris that are ready to recruit other replication components (Cyanobacterial toxins). There is ever-growing evidence to support handcuffing as a unifying mechanism of replication control among ICPs. Another model for negative replication control, called titration, also invokes Rep–iteron interactions but in an entirely different context. The underlying principle for titration rests on the notion that iterons could inhibit DNA replication by tying up Rep initiators in nucleoprotein complexes that are nonproductive for replication initiation. A straightforward prediction of the titration model is that increasing the concentration of the ratelimiting component should increase plasmid copy number. This prediction has been realized in ICP systems in which plasmid levels are proportional to Rep over a broad range of protein concentrations. Replication control by antisense RNAs

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Figure 7 Two essential components of a ‘minimal’ R6K replicon are the ori and its cognate Rep protein, , encoded by the pir gene.  monomers activate ori replication at low intracellular levels [1]. At elevated protein levels,  dimers form [2], autoregulating pir expression [3] and inhibiting replication.  dimers may use more than one mechanism to inhibit replication. Although handcuffing [4] is hypothesized to be a replication inhibitor in several plasmid systems, additional work established competitive protein–DNA binding as another viable mechanism for R6K (not shown). The monomeric initiator form of  and the dimeric replication inhibitor form bind iterons competitively and sequence specifically. Although  dimers are typically vastly more abundant, monomeric  has a couple of advantages, cooperativity and dual DNA binding domains.

Data from a wide assortment of systems controlled by antisense RNA have revealed the formation of highly structured molecules that act via sequence complementarity on targets called sense RNAs. Copy-up mutations that destabilize antisense RNA or alter its interaction with the cognate sense RNA are known. Antisense RNA-dependent regulation of replication is achieved by a variety of mechanisms: (1) inhibition of RNA primer utilization by DNA polymerase, (2) inhibition of the translation of Rep protein or the leader peptide needed for efficient Rep translation, (3) attenuation of transcription to limit the availability of Rep. In some instances, the antisense RNAs act alone. In other cases, antisense RNAs act in concert with regulatory proteins that are either transcriptional repressors or RNA-binding proteins. The use of antisense RNA as a regulatory control element is further elaborated on in RNAs, small etc.

Resolution and Distribution of Newly Replicated Plasmid DNA Resolution of the Products of Circular Plasmid Replication Multimerization of circular replicons is a persistent problem in all recombination-proficient bacterial species. The best studied case of multimer formation is the dimerization of circular chromosomes, which is lethal when unresolved. A host-encoded recombination system, Xer,

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Figure 8 Structures and functions of RepE. (a) Schematic representation of the functions of RepE initiator of plasmid F. (b) DNA sequence used for the cocrystallization of RepE with DNA. Arrows indicate the common 8 bp sequences shared by the ori iteron and the inverted half iterons present in the repE gene operator. (c) and (d) Two views (length and distance) of a RepE–operator DNA complex (c) and a RepE–iteron DNA complex. Each functional dimer is colored green (molecule A) or yellow (molecule B). The DNA models are omitted in the lower panels. The secondary structural elements of the RepE dimer are designated according to previously determined elements of the RepE54 structure. Reproduced from Nakamura A, Wada C, and Miki K (2007) Structural basis for regulation of bifunctional roles in replication initiator protein. Proceedings of the National Academy of Sciences USA 104: 18484–18489. ª 2007, with permission from National Academy of Sciences, USA.

is required to convert chromosomal dimers into monomers and its high level of conservation among bacteria and archaea (with circular genomes) reflects its crucial role in chromosome segregation. Not surprisingly, ongoing sequencing projects reveal that most circular and linear plasmids contain one, and often multiple, site-specific resolvase genes. Why is dimerization, and higher level multimerization, so problematic for a replicon? The formation of dimers affects replicon stability by lowering the number of segregation units at the time of cell division. As noted earlier, replication is controlled by ‘origin counting’, which means that a dimer counts as two plasmids for replication but only as a single unit for segregation. Imagine cell division advancing toward the production of two ‘daughter’ cells but with only one dimeric chromosome to be partitioned to them. Plasmids, even multicopy plasmids, are similarly illaffected by the formation of multimers, which increases the frequency of plasmid loss. In fact, although it may seem counterintuitive, dimer formation poses a greater risk to the high copy number plasmids. These plasmids follow a ‘random copy choice’ mode of ori activation and, as a result, dimers replicate at twice the frequency of monomers. The replicative advantage of the multimers causes their rapid accumulation in the progeny of the cells in which they appeared. This phenomenon, called the ‘dimer catastrophe’, is responsible for the greater fraction of segregation defects in plasmid-bearing cells because it

leads to the formation of a subpopulation that contains mostly multimers. Another serious disadvantage of multimer formation in circular, but not linear, plasmids is their high sensitivity to rearrangements caused by homologous recombination (HR) (Figure 9; Recombination, genetic). Certain recombination events among circular replicons result in the formation of a dimeric cointegrate molecule in which the two copies of the replicon are fused in a head-to-tail configuration. These events do not occur in a strain that is recombination deficient (e.g., recA) consistent with the view that the vast majority of plasmid dimers form by HR. In addition to multimerization, circular plasmids are confronted with another obstacle to plasmid segregation, ‘catenation’. The replication of both DNA strands of circular plasmids results in the formation of intercatenated structures in which the two sister double-strand DNA molecules remain interlinked (unseparable by pulling them apart). Sophisticated DNA-processing machines physically resolve catenanes and dimers (Figures 4 and 9). An enzyme mentioned earlier, type II topoisomerase, can promote decatenation by sequentially nicking and closing the two strands of the DNA backbone. Resolution of multimeric forms of circular plasmids (and chromosomes) is mediated by relatively simple molecular machines, termed site-specific recombinases or resolvases, that catalyze the essential DNA breakage and rejoining reactions. These enzymes are often plasmid-encoded with the recombinase

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HR

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Figure 9 Formation and resolution of circular replicon dimers. Homologous recombination (HR) occurring during or after replication of a circular plasmid or chromosome produces a dimeric DNA molecule in which the two copies of the replicon are fused in a head-to-tail configuration. The dimer is converted into monomers by site-specific recombination between the duplicated copies of the replicon resolution site (colored in black and gray). The core recombination sites where the recombinase catalyzes the strand-exchange reaction are represented by squares. The adjacent colored regions are regulatory sequences that are often associated with the recombination site to control the recombination reaction. Circles represent the plasmid or chromosome replication origin. Reproduced from Hallet B, Vanhooff V, and Cornet F (2004) DNA site-specific resolution systems. In: Funnell BE and Phillips GJ (eds.) Plasmid Biology, ch.7, pp. 145–180. Washington, DC: American Society for Microbiology. ª 2004, with permission from ASM Press.

gene and the target recombination site usually being associated side by side (i.e., linked loci). Resolvases fall into two major families of unrelated proteins that use different mechanisms to cleave and rejoin DNA molecules. These two groups of enzymes are now commonly referred to as the serine recombinase family and the tyrosine recombinase family according to the conserved residue that participates in the DNA cleavage–rejoining steps. Many plasmids, however, such as those of the ColE1 family, utilize the hostencoded Xer recombination system rather than encoding one of their own. Recombination between directly repeated sites on a circular DNA molecule will resolve the molecule into two separate rings (Figure 9). Multimer resolution activity is totally independent of other cellular processes such as replication, allowing site-specific recombination to take place at any stage of the cell cycle. This is crucial to ensure efficient resolution of multimeric forms of circular replicons.

Termination and Resolution of Replication by Machines Assembled on Linear Plasmids Linear plasmids and chromosomes have been identified in a number of widely divergent bacterial species and they generally retain the same features and mechanisms for replication initiation as their circular counterparts. In species that possess both linear and circular plasmids such as Borellia burgdorferi, a conserved mechanism for replication initiation appears to be the rule. Such

replicons contain internal oris from which replication proceeds bidirectionally toward the telomeres. In contrast to initiation, however, replication termination and the resolution of replicated plasmids are significantly different in linear and circular replicons. In circular plasmids replicating bidirectionally by the theta mechanism, the ter sequence is recognized by a contra-helicase called RTP that blocks the movement of the replicative helicase (DnaB), thereby promoting termination. The ter sequence signals the end point of replication for a molecule that has no physical ends. All linear replicons, whether eukaryotic, prokaryotic, or viral, are presented with a different challenge as replication nears completion: how to replicate the extreme 39 ends of the DNA? Various mechanisms have evolved to solve this problem and are discussed below. The ends of linear plasmids in bacteria fall into two main structural classes, telomeres with covalently closed hairpin ends and telomeres with unlinked DNA and protein-capped ends. Plasmids with hairpin ends can be described as continuous single strands of DNA that are self-complementary, so their structure is a double-strand linear molecule with direct linkages at both ends between the 59 end of one strand and the 39 end of the other. Due to the inherent stiffness of DNA, linear plasmids have loops composed of at least four unpaired nucleotides making the connection between the two strands, called a hairpin end. Plasmids containing hairpin telomeres versus free ends capped with terminal proteins require different termination mechanisms. For the linear plasmids with closed hairpin ends, replication initiates from an internal origin and continues around the hairpin telomeres, resulting in a circular dimer. Processing of the circular dimer into two linear plasmids is accomplished by the activity of telomere resolvases that recognize specific sites (i.e., replicated telomeres) in the circular dimer formed after replication. The enzymes cleave the joined telomeres of replication intermediates, subsequently religating them to generate two daughter plasmids with hairpin ends (Figure 10). An inverted repeat is presumed to be the only sequence feature required for telomere resolvase to recognize and cleave joined telomeres. Given that the replication of this type of linear plasmid produces head-to-head circular dimers, it is not surprising that organisms harboring such replicons contain enzymes related to the tyrosine recombinases described in the previous section. Evidence for an alternate method of processing the ends of linear plasmids can be seen in Streptomyces in which proteins are bound to the termini. As the bidirectional fork encounters the extreme 59 end of the newly synthesized DNA strand, with its last RNA primer removed, a terminal patching mechanism is required to fill the remaining gap. The new DNA chain is approximately 300 nt short at the 59 terminus, which leaves a single-strand 39 overhang (Figure 11) Two mechanisms

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and sequence analysis has revealed that the proteins are homologous. Initiation of replication from internal origin

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Figure 10 Model for replication of linear plasmids with covalently closed hairpin ends. Replication initiates from an internal origin and proceeds bidirectionally, producing a circular dimer intermediate with joined telomeric sequences producing inverted repeats (arrows). The replicated telomere sequence serves as a recognition site for the telomere resolvase ( ), which cleaves both DNA strands and then joins opposite strands together to create two linear plasmids with covalently closed hairpin telomeres. For further details, see Further Reading. Reproduced from Stewart P, Rosa PA, and Tilly K (2004) Linear plasmids in bacteria: Common origins, uncommon ends. In: Funnell BE and Phillips GJ (eds.) Plasmid Biology, ch. 13, pp. 291–301. Washington, DC: American Society for Microbiology. ª 2004, with permission from ASM Press.

have been proposed for filling in the missing nucleotides and each assigns roles for the terminal proteins, which are known to be essential although their exact roles in replication remain to be elucidated. In one model, the terminal protein functions as a protein primer to initiate gap filling. In a second model, the terminal protein nicks the template strand and attaches itself covalently to the 59 end with subsequent displacement and gap filling by DNA polymerase. Whatever functions terminal proteins possess, they are likely conserved among this category of linear plasmids. The genes encoding terminal proteins have been found to lie adjacent to the plasmids’ telomeres,

Plasmid Segregation Plasmid partitioning and plasmid replication are independent functions (Chromosome replication and segregation). This was perhaps best demonstrated by observations that several partitioning loci (coding and noncoding par ‘genes’) promote plasmid stability when cloned to different replicons striped of their own par systems. Partition machines also exert an Inc phenotype that is distinct from the replication-mediated Inc described earlier, the latter being the Inc that is traditionally used to classify replicons. As a result, two replicons that would be compatible based on their replication machinery will nonetheless be unable to stably coexist in the same cell if they are segregated by the same par system. This Inc mechanism derives from competition between identical partition systems on otherwise different plasmids and has been used to genetically dissect partition modules and their components. Elegant microscopic studies support the contention that Par machines prevent plasmid diffusion in the intracellular space by organizing DNA into communities called foci then actively distributing plasmids to each side of the cell division plane. By using fluorescence microscopy it has become possible to track segregating plasmid molecules. The components of par systems and the dynamics of segregating plasmid foci are reminiscent of the mitotic machinery of eukaryotic cells. Remarkably, plasmid molecules rely on just three essential components for their specific intracellular positioning and, thus, stable propagation. The first of these elements, the centromere, is required in cis for plasmid stability. Centromeres often contain one or more inverted repeats as recognition elements and they serve as the loading sites for the rest of the segregation machinery. In addition to the centromeres, two trans-acting Par proteins are required; one or both Par proteins usually autorepress their own expression. Adaptor proteins specifically recognize the centromeres and the energy-generating cytoskeletal ATPases (or GTPases) that move and attach plasmids to specific host locations. Plasmid partition systems are typically classified according to the nature of the cytoskeletal component they encode. ParM systems: Actin-like ATPases

Our understanding of partitioning systems is most advanced for the Escherichia coli plasmid RI that encodes ParM protein, an actin-like ATPase. As revealed by fluorescence microscopy, ParM assembles into transient and dynamic filaments that grow at similar rates at both ends and then depolymerize unidirectionally. Additional insights were gained when Par-mediated movement was reconstituted from purified components. By hydrolyzing

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Figure 11 Model for the replication of linear plasmids with protein-capped ends. Bidirectional replication from an internal origin results in a gap at the 59 end of the newly synthesized strand when the RNA primer is removed. Two general models for filling the gaps are depicted and are based on models for the replication of the linear chromosome of Streptomyces spp. Inverted repeat sequences of the single-stranded 39 overhang fold together to form stem-loop structures. (a) The terminal protein ($) recognizes the complex secondary structure of the 39 DNA strand and serves as a protein primer for DNA polymerase to initiate replication and fill the gaps. (b) The folded 39 terminus forms the double-stranded primer necessary for DNA polymerase to initiate replication and fill the gap. Subsequently, the terminal protein binds and nicks the DNA near the beginning of the inverted repeat regions. DNA polymerase then proceeds in a 59 to 39 direction from the original template strand and fills the remaining gap. For variations on these models, see Further Reading. Reproduced from Philip S, Rosa PA, and Tilly K (2004) Linear plasmids in bacteria: Common origins, uncommon ends. In: Funnell BE and Phillips GJ (eds.) Plasmid Biology, ch. 13, pp. 291–301. Washington, DC: American Society for Microbiology. ª 2004, with permission from ASM Press.

ATP, ParM polymerization was shown to facilitate the segregation of DNA complexes containing the adaptor protein (ParR) bound to centromeres (parC), thus providing strong evidence that these three elements of the par system are required and sufficient to mediate plasmid segregation. Researchers have been able to visualize the

segregation in vivo and measurements reveal that it is a speedy process. ParM filaments grow by the insertion of monomers at the filament–plasmid junction and, like actin, the protein assembles head-to-tail into a polarized filament with distinguishable plus and minus ends. Plasmid DNA invariably localizes to opposite ends of a

Genetics, Genomics | Plasmids, Bacterial

growing spindle, suggesting that polymerizing ParM actively pushes plasmids apart. How do plasmid molecules manage to interact with opposite filament ends at the same time? A clue was provided by crystal structure data showing that ParR dimerizes to form a DNA-binding structure that further assembles into a helical (or ring-shaped) array with DNAbinding domains presented on the multimer’s exterior. Additionally, electron microscopic analysis of ParR– parC complexes showed parC–DNA wrapped around the ParR protein scaffold. These findings suggest that the ParR–parC complex can encircle ParM filaments and slide along the polymer. As the ParR–parC complex has twofold symmetry, this interaction may occur in inverse orientations at opposite ends of the ParM filament, thus explaining the topological problem of how ParR–parC can interact with both ends of a polar filament. In addition to the long ParM filaments, shorter ones appear to emanate from a single plasmid, which suggests a model (shown in Figure 12) that might explain how the par spindle works. ParM filaments form continuously throughout the cytoplasm but rapidly decay in the absence of stabilizing interactions with plasmid molecules. However, if a filament at one end of the cell becomes stabilized, it will ‘search’ the cytoplasm and only after it ‘captures’ a second plasmid will the filament extend into a pole-to-pole spindle. This is similar to the way in which microtubules extend from the eukaryotic spindle pole body during mitotic pro-metaphase, searching for chromosomes. Although bipolar stabilization of ParM filaments is favored when two plasmid copies are in close proximity, plasmid pairing itself is not required. Other mechanisms for plasmid localization

The more widespread type I family of bacterial DNA segregation systems uses ATPases whose signature ATPbinding amino acid sequence is referred to as the Walker type. The cytoskeletal ParA proteins form filamentous structures that move through the cell in an oscillatory pattern. Like ParR of plasmid Rl, the DNA-binding adaptor proteins (ParB) of these systems serve as tethers between ParA and plasmid centromere sites (parS). Although the mechanism by which the ParA system functions is less well understood, it is equivalent to its ParM counterpart in stabilizing plasmid molecules and, in fact, manages to distribute multiple plasmids along the length of the cell. Yet another system has been recently discovered (called type III) that, quite unexpectedly, displays treadmilling behavior rather than dynamic instability. Despite the obvious functional similarities as intracellular transport machinery, no homology exists between the force-generating proteins or the DNA-binding adaptor proteins of the three types of partitioning systems. With few exceptions, the organization of par functions in linear plasmids is similar to those of circular replicons. The N15

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Figure 12 Molecular model of plasmid segregation by the RI par operon. (a) Nucleation of new filaments will happen throughout the cell. Filaments attached to one plasmid will search for a second plasmid. (b) Plasmids will diffuse around the cell until they get close enough to encounter each other. (c) When two plasmids come within close proximity, filaments will be bound at each end by a plasmid, forming a spindle. This will prevent the filaments from undergoing catastrophe. (d) As these stabilized filaments polymerize, the two plasmids will be forced to opposite poles. If the ends of a spindle run into the sides of the cell, it will be followed along the membrane to the ends of the cell. (e) After reaching a pole, pushing against both ends of the cell causes the filament to dissociate from the plasmid at one end and quickly depolymerize. Reproduced from The Journal of Cell Biology, 2007, 179: 1059–1066. ª 2007 The Rockefeller University Press.

linear replicon stands out as one of the odd balls. Its protein-encoding par loci are genetically unlinked from any centromere and it has palindromic sequences dispersed across the genome that function as centromeres. Overall, much remains to be elucidated concerning plasmid segregation mechanisms, but as more plasmids become sequenced and characterized, the plasmid segregation repertoire is likely to expand. This anticipated wealth of new data in combination with sophisticated fluorescence microscopy will lead to the advancement of this field. Until late 1990 it had been widely held that plasmid molecules are scattered throughout the cell. Thus, it came as a surprise when analyses of segregation kinetics indicated that the losses of some multicopy plasmids failed to conform to the expectations for a random distribution. Rather, the data were deemed to be consistent with the plasmid molecules being tethered inside the cell. These studies were conducted on ICPs paving the way for speculation that the aggregation of the plasmids might be a consequence of Rep-mediated handcuffing (described earlier). Evidence that implicates membrane association as being important for the in vivo functions of replisomes and Rep proteins is accumulating. In fact, some Rep proteins have amino acid signatures that are typical of transmembrane proteins. It would not be unreasonable, then, to suspect that these DNA-binding,

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membrane-binding proteins might act as effectors of intracellular plasmid localization. Even if true, however, the mechanisms that account for this type of plasmid localization are most likely independent of the classic partitioning systems, which stabilize plasmids without affecting their copy numbers. Addiction Modules Like all living organisms, bacteria die, and plasmids are known to facilitate this ultimate stage of life. Bacterial addiction to plasmids is a very basic, and at first glance counterintuitive, phenomenon, making its discovery exciting. One of the best studied forms of death in bacteria is mediated through specific genetic components called ‘addiction modules’ or toxin–antitoxin systems. Each consists of a pair of genes, a stable toxin and an unstable antitoxin that interferes with the toxin’s lethal action. The existence of these plasmid-encoded elements was inferred as a result of some interesting observations arising from studies (in E. coli) of low copy number plasmids such as Rts1, RI, and F. It was found that a mutant Rts1 plasmid with temperature-sensitive machinery for plasmid replication had the unexpectedly broader phenotype of making the growth of its bacterial host cells temperature sensitive as well. Dissecting the phenomenon revealed that at the nonpermissive temperature, the bacterial host cells lost all copies of Rtsl, and with it all copies of the antitoxin. Degradation of the antitoxin and its messenger RNA left the unneutralized toxin to linger in the cytoplasm and kill the plasmid’s former host. The Rts1 locus that was responsible for this effect that was later referred to as segregational killing, a term arising from studies of the analogous hok/sok locus of plasmid RI. Over the years, a variety of different genetic systems have been described that promote either bacteriostatic or bacteriocidal effects in bacteria. In all cases, a cell that liberates itself from the forced symbiosis with the plasmid DNA will most likely die or stop growing due to the activity of long-lived toxins. The highly unusual behavior of plasmid-bearing cells eventually led to a frontal attack on the chromosomally encoded toxin–antitoxin systems, some of which are homologous to the plasmid addiction modules. E. coli ’s mazEF, for example, is a stress-induced ‘suicide module’ that activates when a stressful condition interrupts the expression of MazE allowing the protein to exert its toxic effect and cause cell death. The presence of mazEF-like modules in the chromosomes of many bacteria suggests that cell death plays roles in bacterial physiology and/or evolution. Furthermore, there are observations suggesting an interplay between the plasmid- and chromosome-encoded addiction systems. For example, the toxic product (Doc) of the phd/doc addiction module of the plasmid prophage P1 requires the presence of the

cellular mazEF system to be bactericidal. Bacterial addiction modules are often classified as mechanisms of programmed cell death (PCD) or apoptosis, terms that are traditionally associated with eukaryotic multicellular organisms. Future studies of plasmid addiction and other PCD systems in bacteria will be important for revealing the death pathways involved and perhaps for designing new classes of antimicrobial agents (e.g., compounds that interfere with antitoxin expression or activity). Why would a genetic element that is potentially toxic to the genome ever be maintained? The far-reaching impact of the discovery of plasmid addiction is that it has fostered an important conceptual change in our understanding of bacteria. Death is clearly counterproductive for an individual bacterium; however, it might be advantageous for a whole cell population. Growing experimental evidence suggests that bacteria seldom behave as individual organisms. In fact, some species have evolved the ability to communicate with each other via quorum-sensing signal molecules, which allow coordinated responses to a variety of stimuli. As a result, bacteria can be induced to manifest multicellular-like behaviors and addiction may fall into this category. Plasmid-encoded and chromosomal toxin–antitoxin systems with their attendant killing of ‘afflicted’ bacteria can be viewed as examples of multicellular behaviors under stressful conditions. When challenged, the bacterial population seems to act like a closed society in which a subpopulation is excluded through forced suicide, thereby permitting the survival of the bacterial population, with its genome remaining intact.

Horizontal Plasmid Transfer by Conjugation Bacteria can acquire foreign DNA by various means including phage-mediated delivery (transduction) and the uptake of ‘naked’ DNA (transformation). In addition, certain plasmids are equipped to facilitate lateral gene transfer between bacteria through a process, mentioned earlier in this article. Conjugation is usually mediated by plasmids and transposons, and important details differ from system to system as a consequence of plasmid diversity (Conjugation, bacterial and transposable elements). Conjugative plasmids rely, at least in part, on plasmidborne gene products and specific DNA sequences to transfer themselves from hosts to recipient cells. One of the more simple conjugation systems, as judged by the number of participants it employs, can be found in mycelial streptomycetes. A single plasmid-encoded protein, the DNA translocator TraB, is sufficient to promote conjugal transfer of DNA in these organisms. Following primary transfer from the donor into the recipient, the plasmid is further distributed to the neighboring mycelial

Genetics, Genomics | Plasmids, Bacterial

compartments in a process that requires 5–6 host-encoded proteins. In contrast, plasmid-encoded factors play a much more prominent role in the conjugation processes of Gramnegative and Gram-positive bacteria, and they include DNA sequences such as oriT in addition to multiple conjugation-related proteins. Most conjugative plasmids depend on a relaxase to start the DNA-processing reactions, streptomyces being the remarkable exception to this rule. Coupling Mechanisms for Donor–Recipient Pairs Bacterial conjugative systems are grouped together into the type IV secretion system (TIVSS) and the proteins required for conjugal transfer fall into three groups. Mobilization proteins (Mob and nickase, frequently called the relaxosome) bind specifically to their cognate oriTs and produce a nick in the DNA from which the conjugal transfer begins, utilizing the RC-like pathway described earlier in the article. Transfer proteins (Tra) form a multiprotein complex called the mating pair formation apparatus (Mpf) that, among other transportrelated activities, is needed for pili formation and their extrusion to the cell surface. Tra proteins are not functionally confined to the oriT of the same plasmid. They can facilitate the transfer of plasmids that contain unrelated oriTs provided those plasmids also contain the cognate relaxosomes. Once plasmid DNA is prepared for transfer, it must be transported through the donor’s cytoplasmic membrane into the recipient. It is generally believed that DNA crosses the donor membrane with the aid of a coupling protein, so-called because it couples Tstrand DNA processing to a TIVSS. Free-living (planktonic) as well as surface-bound bacteria are capable of transferring plasmids, and one of the few diversifying elements in conjugation systems is the type of pili produced and used to facilitate conjugation; some pili are thick and flexible while others are long and rigid. Pili have the remarkable ability to retract, allowing them to promote intimate associations of cell surfaces over extended areas, which stabilize mating pairs against shearing forces. Plasmids signal their occupancy of the cell through a mechanism called surface exclusion that prompts bacterial cells to disengage before a redundant transmission of DNA occurs. Abundant membrane proteins called exclusion proteins are presented on the cell surface, mediating this process. Well-studied Gram-positive conjugation systems must employ a different strategy to bring plasmid donors in contact with potential recipients since no pili have been functionally linked to conjugation in this group of bacteria. Rather, small molecules called pheromones are known (e.g., pCF10) or suspected (e.g., pAW63) to facilitate cell-to-cell contact. Pheromones are peptides of seven or eight amino acids and are secreted in miniscule

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amounts, with as little as 1–10 molecules per donor needed to initiate the mating process. A given pheromone specifically activates the conjugative transfer system of a particular plasmid type. When a plasmid is acquired, secretion of the related pheromone is prevented, while unrelated pheromones continue to be produced to ‘seduce’ other potential donor cells. The capacity of several plasmids to produce a surface-exposed aggregation protein (in vitro and in vivo) in response to pheromones expressed during conjugation has been well established and, in the case of Enterococcus faecalis, functionally connected to the pathology of the plasmid-bearing organism. Factors Affecting Plasmid-Mediated Horizontal Gene Transfer In general, conjugation-mediated transfer of DNA appears to have few if any barriers. It can occur between Gramnegative and Gram-positive bacteria as well as fungi, actinomycetes, and the cells of higher eukaryotes. Some plasmids can be remarkably promiscuous as exemplified by two antibiotic-resistance plasmids: RK2 (also known as RP4), which was originally identified in a Gram-negative host, and pIP501, which was isolated from a Gram-positive strain. pIP501 has an extremely broad host range (BHR) for conjugative transfer that includes a variety of Grampositive bacteria, multicellular Streptomyces, and the Gramnegative E. coli. However, as impressive as this list is, RK2’s conjugative spectrum is even broader as it can be transferred to all tested Gram-negative bacteria, yeast, and even mammalian cells (Horizontal gene transfer: Uptake of extracellular DNA by bacteria). Conjugation efficiencies vary from one plasmid system to another and are affected by a plethora of factors. For many plasmids, conjugative functions are typically found to be in a repressed state and conjugation efficiency is very low. One out of million plasmid-free cells, or even less, may receive a conjugative plasmid even when the donor cells greatly outnumber the potential recipients. Interestingly, in repressed systems, transfer-proficient donors and potential recipients can initiate a cascade of conjugative transfer because the newly transferred plasmids are transiently derepressed in response to the initial lack of repressor proteins in the recipient. Mutations in repressor genes can also cause conjugation to become derepressed (drd mutants). In fact, in a couple of wellstudied plasmids, transfer functions are naturally derepressed by deletion or insertion mutations in the gene encoding the repressor. Remarkably, these derepressed plasmid systems are able to sustain conjugative DNA transfer under laboratory conditions with close to 100% efficiency (e.g., F and pCF10). Conjugation typically occurs within aggregates of multiple donor and recipient cells; in some cases twenty and in other cases thousands of aggregated and conjugating cells were observed.

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Consecutive DNA transfer events by the same donor are known to occur rapidly with estimated cycles of as little as 3–5 min for some pheromone-responsive and pilimediated machineries (e.g., pCF10 and F). The DNA transfer process occurs at the speed of replication previously noted, roughly 1000 bp/s. Interestingly, cells that acquired plasmid DNA (newly formed transconjugants) require much longer periods, up to about 60 min, to mature into proficient donors. This long maturation time is most likely required to synthesize and assemble the impressive protein machineries used in the multiple coordinated processes that contribute to conjugation: assembling and anchoring the relaxosome (into the cytoplasmic membrane) and the Mfp components (into the cell wall), then connecting both by a coupling protein. Once assembled, the DNA secretion machinery can be remarkably stable, and cells killed by several bactericidal treatments retain conjugation proficiency for several hours. In a related and unsettling finding, externally added antibiotics (e.g., tetracycline) can stimulate some conjugative systems up to 100-fold and this derepression may not be limited to laboratory settings. These observations may necessitate changes in the way we think about horizontal gene transfer (HGT) and its potential impact on microbial communities. We will return to this topic later in the article.

plasmids not only enhances the adhesive properties of bacteria but in some situations it can also dramatically restrict cellular motility, changes that are likely beneficial for surface-dwelling populations and of little to no value for planktonic cells. It would appear that biofilm formation and conjugation might be mutually reinforcing phenomena. Relevant to this, biofilms are sometimes regarded as ideal niches for conjugation although the relatively early stage of this work prevents general conclusions from being drawn. New mathematical approaches that model the spatial dynamics of plasmid transfer and persistence are increasingly being turned to, their purpose being to ascertain how three-dimensional structure affects the spread and loss of plasmids in surface-associated bacterial communities. Plasmid ecology is also studied in biofilm microcosms with the long-term objective of breaking down toxic compounds in wastewaters and other environments by disseminating degradative genes via conjugation. ‘Three-dimensional’ images of conjugating biofilm communities have been generated and transconjugants (tagged with green fluorescent protein) in different locations within the biofilm were observed. Scientists perform studies like these with hope of predicting how HGT might work in practical applications of conjugation in natural environmental contexts such as bioremediation and other emerging applications.

Environmental cues

Restriction enzymes

The acquisition of plasmids via conjugative transmission has been studied in numerous environments, providing ample evidence that both abiotic and biotic cues affect this process. Nonetheless, the focus of pioneering studies in this field needs to be narrower than ‘the environment’. Studies of the environmental factors that affect plasmid transfer rely on the use of discrete habitat subsamples called microcosms. Examples of microcosms include soils, plants, and water, all of which are being used to elucidate the effects of key ecological factors on the plasmid transfer process. Interested readers are advised to consult the Further Reading section for broader access to existing knowledge in this important area. Spatially structured microbial populations known as biofilms can form in the microcosms described as well as in clinical systems, and they appear to represent unifying experimental systems for studying plasmid transfer processes. To dissect the conjugation process in ‘natural’ and artificial (laboratory) microcosms, an experimental approach for the direct in situ monitoring of plasmid transfer in biofilms has been developed. Using plasmidencoded green fluorescent protein as a visual ‘reporter’, the intercellular movement of plasmids can be studied microscopically. Researchers have used this technique to demonstrate that conjugation has a dramatic stimulatory effect on the ability of transconjugant bacteria to participate in biofilm formation. The uptake of conjugative

One of the potential obstacles confronting the conjugal transfer of plasmid DNA is a group of host-encoded proteins, the restriction enzymes (RE), that are designed by nature to destroy DNA they recognize (DNA restriction and modification). Their resemblance to the addiction cassettes described earlier is evident in that they work with companion enzymes of opposing function, one enzyme cleaves DNA (analogous to toxin) and the other modifies the DNA recognition sequence thereby preventing DNA damage (analogous to antitoxin). Such restriction–modification (RM) systems protect cells from an invasion by foreign DNA. They are ubiquitous in bacteria and can be plasmid-encoded or reside on the bacterial chromosome. Although the DNA that is transferred during conjugation is single-strand and therefore not susceptible to restriction, there is a race to protect or restrict as the T-strand is converted into double-strand DNA. There is little doubt that RM systems affect the efficiency of plasmid spread. Conjugation and plasmid establishment are expected to occur more frequently between taxonomically related species in which plasmid DNA can evade restriction systems and replicate. It comes as no surprise, therefore, that the DNA of some BHR plasmids, which are capable of replicating in many hosts, contains fewer restriction sites when compared to the DNA of their narrow host range counterparts.

Genetics, Genomics | Plasmids, Bacterial

Additionally, many conjugative plasmids contain antirestriction loci (ard) as part of their so-called ‘leading region’ defined as the first portion of the plasmid to enter the recipient. The products of these genes act specifically to alleviate restriction by certain types of RE. Having an ardA locus present in cis allows an incoming, unmodified plasmid to evade restriction when transferred by conjugation but not by other processes that involve double-strand DNA (e.g., transformation or electroporation). Protection requires the expression of the incoming ard gene, which is enhanced by conjugative transport into the recipient cell. Replication Ranges of Conjugative Plasmids The conjugation ranges of plasmids and their replication ranges are related but distinct entities. The replication range refers to the variety of hosts that can maintain an extrachromosomal plasmid once it enters a cell; this range is typically narrower than the conjugation range. Despite the higher demands posed by plasmid maintenance, however, BHR plasmids are able to replicate in a diverse assortment of bacteria, employing various strategies to achieve their promiscuity. One strategy adopted by some BHR plasmids is to limit their reliance on host proteins by encoding their own helicase and primase. As a result, these plasmids have an advantage, but successful maintenance in any given strain is not assured. BHR replisome assemblies still require the expression of plasmid-encoded proteins in the bacterial host, productive interactions between the plasmid and accessory host proteins, and productive interactions between the host proteins and DNA-binding sites of the plasmid. Moreover, plasmid-encoded proteins must be expressed at an appropriate level, possibly even at a specific time in the cell cycle, and the proteins must be stable in different host backgrounds. Plasmids of the IncP group forgo encoding their own replisome proteins and employ two alternate strategies that enhance their replicative promiscuity. First, they produce a Rep that is versatile enough to recruit helicases of distantly related bacterial species. Indeed, in vitro and in vivo work have demonstrated that plasmids of this group use different pathways for helicase recruitment and activation. Structural differences in DnaB helicases from different species of bacteria are likely the basis for the diversity required to form a productive interaction. The second mechanism is more elaborate and reflects a unique interrelationship between DNA transfer and vegetative replication modes. Specifically, IncP conjugation systems have the unusual ability to transfer (unidirectionally) primases and single-strand DNA-binding proteins (SSB) into the recipient bacterium as nucleoprotein complexes. Transfer is abundant, amounting to several hundred molecules of primase. The enzyme contributes to transfer promiscuity by eliminating the requirement for

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the host enzymes of different bacteria to recognize the incoming DNA strand, which facilitates efficient secondstrand synthesis in different cellular backgrounds. SSB proteins, which are also transferred via the conjugation apparatus, are essential for DNA replication and repair. The traveling SSBs encoded by BHR plasmids presumably overcome deficiencies of host SSB in cells receiving single-strand DNA during conjugation, presumably leaving the metabolism of chromosomal DNA (replication and recombination) relatively undisturbed. Although naturally occurring plasmids with multiple oris have been isolated (e.g., R6K), none has been shown to specifically utilize different oris in different bacterial hosts. Nonetheless, engineered plasmids called ‘shuttle vectors’ contain two distinct replicons that are active in unrelated hosts and they prove that the presence of two narrow host range replicons on a single plasmid can extend its host range. Narrow host ranges can also be broadened by mutations in genes that encode an essential plasmid or host protein, the consequence of which is likely the strengthening of a required host–protein/plasmid–protein interaction (e.g., pPSlO and DnaA/RepA). In summary, the ability of a plasmid to transfer itself from one bacterium to another by conjugation or to be mobilized between hosts by conjugative functions provides the means by which a plasmid can pioneer new cellular landscapes. Once an immigrant plasmid is introduced, many pieces must come into play in a wellorchestrated manner for a plasmid to be able to survive. While restriction and replication processes are key, other factors contribute to plasmid promiscuity. Analyses of the partitioning and postsegregational killing systems clearly demonstrate the role of accessory functions in extending or limiting plasmids’ ability to be maintained. To be of use, a variety of contributing elements must be expressed and regulated in novel hosts. It is evident that we are just beginning to understand some of the key genetic and molecular factors involved in extending the host range of plasmids in all three kingdoms of life. Plasmid Evolution Evolutionary analysis relies upon the identification of unifying features. The last three decades have seen tremendous advances in the determination of the evolutionary relationships that connect all living organisms. The use of 16S ribosomal RNA genes to determine phylogenetic relationships has provided a unifying methodology for evolutionary analysis even as it resulted in recognizing a new branch in the tree of life. That tree is divided into the three domains (bacteria, archaea, and eukarya), and plasmids inhabit organisms belonging to all three domains but certainly are most prevalent in bacteria. Plasmids are not organisms in their own right; instead, they represent a horizontal gene pool, which is

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coevolving with their hosts. Not surprisingly, a signature DNA sequence such as 16S ribosomal RNA is lacking in plasmids primarily because the very nature of these elements is to not encode essential host information. Determining the evolutionary relatedness of replicons

Even the fundamental ability to replicate cannot be used to establish the common ancestor to all plasmids, as evidenced by the inherent diversity of replicons. It appears that plasmid replication functions likely originated more than once, independently of each other. However, the absence of a single identifiable reference sequence in all plasmid genomes has not impeded the construction of adequate phylogenies encompassing groups of plasmids. In fact, the debate about plasmid classification is

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reminiscent of the ongoing discussion regarding the concept of bacterial species. What level of DNA similarity makes it reasonable for researchers to contend that plasmids belong to the same plasmid group? Another issue is to decide what genes/sequences are best suited to generate plasmid phylogenies. The main contenders are the genes coding for the Rep (Figure 13) and Par proteins and the proteins facilitating DNA transfer. Homologies in these groups of proteins and their cognate DNA targets have been established and are emphasized on numerous occasions in the literature pertaining to this important topic. An example phylogeny based on the rep gene sequences of theta-type replicons is shown in Figure 13. However, as will be discussed, these phylogenies are valuable only in establishing the evolutionary relationship of plasmid ‘genes’; the task of establishing

pMJ101 pRSF1010

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Figure 13 Phylogenetic tree based on alignments of Rep proteins of various iteron-controlled plasmids or genomic sequences (strain names in italic). Alignments were made with Clustal W and the tree was constructed using SplitsTree 4.8. Protein sequences were obtained from GenBank either directly or by translating nucleotide sequences of putative genes located with Glimmer 3.02. Reprinted from Norman A, Hansen L H, She Q, and Sørensen (2008) Nucleotide sequence of pOLA52: A conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux. Plasmid 60: 59–74, with permission from Elsevier.

Genetics, Genomics | Plasmids, Bacterial

relationships between plasmids in their entirety is substantially more complex. Included in the Further Reading are references to the sources of plasmid DNA sequences. Resources such as these have been multiplying rapidly since the inception of research programs specifically designed for sequencing plasmids and annotating their genes (e.g., Wellcome Trust SANGER Institute). There is no doubt that plasmid classification systems based on DNA sequence similarities in the segments that make up the plasmid ‘backbone’ (replication, maintenance, and transfer regions) is gaining more and more importance. The increasing number of sequenced plasmids has prompted the use of DNA primers to amplify and determine the DNA sequence of plasmid backbonespecific segments, which in theory should help establish the relatedness of ‘new’ plasmids to ‘known’ ones. Attempts have been made to sample new plasmids from a variety of exotic and mundane environments using sequencing primers that recognize select, known plasmids from Enterobacteriaceae. However, these attempts have overwhelmingly failed to detect any relatedness in these isolates to the well-characterized oris. Clearly, much more remains to be discovered in our ongoing quest to sequence the mass of collective environmental DNA samples (i.e., metagenomes, discussed in Metagenomics). How many more signatures of new replicons will we find? How much will we learn about where they originated and for what ‘purpose’? So far, science has only touched the tip of an iceberg when it comes to uncovering the diversity of replication oris and their attendant genes for both chromosomal and plasmid origins. Plasmids as vehicles of genetic plasticity

Since the discovery of plasmids, we have learned that besides the machinery for their own maintenance and transfer, most also carry genes that confer a plethora of traits on their bacterial hosts. Frequently, these traits are ones that are useful intermittently or in certain environments, such as antibiotic resistance, virulence, or degradation of unusual substrates (some discussed below). The various functions, found on both circular or linear plasmids isolated from nature, can be as simple as a single gene (e.g., an antibiotic-resistance determinant) or may involve genes encoding whole metabolic pathways requiring hundreds of kilobases of DNA sequence (e.g., nitrogen fixation in rhizobia). Before we discuss some specific examples, it should be stressed that the factors that contribute to evolutionary change in plasmids are the same as those which are involved in evolution in general – single base pair substitutions, insertions, deletions, and genetic rearrangements such as inversions and translocations. The high adaptability of plasmid-bearing strains relies on various recombination events, which may occur at the borders of functional units with ‘recombinogenic ends’ designed for recombination (e.g., transposons

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and integrons; Transposable elements) or as a result of HR, often between parental and newly synthesized DNA (described earlier and Recombination, genetic). In fact, recent investigations have suggested that recombination between genes in plasmids may occur at a much higher frequency than chromosomal recombination. The mechanism accounting for this apparent difference remains to be determined. Hundreds of plasmid and bacterial genome sequences already available have revealed extensive HGT within and between these classes of replicons (DNA sequencing and Genomics, Genome Sequence Databases: Annotation, and Horizontal gene transfer: Uptake of extracellular DNA by bacteria). On an evolutionary scale, plasmid-mediated gene rearrangements appear to be particularly significant. Science continues to discover that genomes are full of mobile genetic elements and there is compelling evidence that many genes have joined bacterial genomes relatively recently from distantly related organisms, even from eukaryotes. In addition, comparisons of closely related genome sequences (e.g., E. coli K12 and E. coli O157:H7) suggest frequent rearrangements of DNA during their evolution. Perhaps related to this, most circular plasmids contain site-specific recombinase genes and the multiplicity of these genes found on large plasmids often correlates with their level of mosaicism. In other words, the greater the number of recombinases, the more likely it is that a plasmid will be regarded as a mishmash of sequences derived from multiple sources. Evidence exists that ‘illegitimate’ recombination mediated by plasmid and transposon-encoded resolvases (i.e., DNA inversion and intermolecular fusion reactions) has also contributed to plasmid evolution. Indeed, it has become apparent that plasmids are quite active in shuffling, recombining, and redistributing genes or sets of genes, and in so doing they facilitate not only their own evolution but also the evolution of microbial communities and individual strains. Antibiotic resistance: An example of plasmidenhanced bacterial adaptability

The classic principle of genetic selection of fitness is painfully illustrated by the fact that conventional antibiotic treatments are becoming increasingly ineffective due to the acquisition and dissemination of antibiotic-resistance genes by bacteria. In fact, the continuous manifestation of an antibiotic-resistance phenomenon is not new. The first observation of resistance to penicillin was described before the drug was even in clinical use. Furthermore, it was already evident in the 1950s, from the work of plasmid researchers in Japan, that antibiotic resistance was on the rise – and it has been increasing dramatically ever since. There are many examples of the astonishingly rapid acquisition of antibiotic resistance by bacteria. One particularly remarkable and disturbing example is found in the opportunistic pathogen Acinetobacter baumannii, which has

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acquired close to 50 resistance genes in just 40 years! Integrons and associated gene cassettes (Figure 14) have been shown to be of major importance in the acquisition of antibiotic-resistance genes by this and other species. Several factors have played a pivotal role in the remarkable speed with which bacteria have adapted to our chemical arsenal. First of all, antibiotics do not specifically target bacteria that cause infections; they are indiscriminate killers of susceptible bacteria – be they harmful, benign, or beneficial organisms. This becomes problematic on the recognition that resistance genes are rarely fixed in the chromosome of a bacterial cell, rarely restricted in their transmission to only that cell’s progeny. Instead, such genes are typically found on transmissible plasmids and transposons. The transmission problem becomes further exacerbated in specific cases where

antibiotics can stimulate transposition such as those that occur in the human commensal organism, Bacteroides. Another possible factor in the rapid dissemination of resistance, noted earlier in the article, is the ability of bacteria harboring plasmids to continue transferring the DNA to other cells long after the donating bacterium has been killed. Finally, cells under various forms of stress have higher mutation rates. As a result, antibiotics that cause DNA damage, such as mitomycin C, can directly elevate the frequency of mutation. Remarkably, antibiotics that affect translation fidelity also boost the mutation rate in bacteria. All the factors just described along with others that are probably yet to be discovered allow reservoirs of resistance to emerge and rapidly spread within diverse microbial communities (Figure 14). Although niches

Antibiotic resistance gene pool Antibiotic-producing strains Antibiotic-resistant strains Resistance-encoding DNA

Dissemination of resistance genes through intra- and interspecific transfer

Uptake of resistanceencoding DNA by bacteria

R plasmids and conjugative transposons

Resistance genes in bacterial cytoplasm

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Figure 14 A scheme showing the route by which antibiotic-resistance genes are acquired by bacteria in response to the selection pressure of antibiotic use. The resistance gene pool represents all potential sources of DNA encoding antibiotic-resistance determinants in the environment; this includes hospitals, farms, or other microenvironments where antibiotics are used to control bacterial development. After uptake of single- or double-stranded DNA by the bacterial host, the incorporation of the resistance genes into stable replicons (DNA elements capable of autonomous replication) may take place by different pathways, which have not yet been identified. The involvement of integrons, as shown here, has been demonstrated for a large class of transposable elements in the Enterobacteriaceae. The resulting resistance plasmids could exist in linear or circular form in bacterial hosts. The final step in the cycle, dissemination, is brought about by one or more gene transfer mechanisms discussed in the text. From Davies J (1994) Inactivation of antibiotics and the dissemination of resistance genes. Science 264: 375–382. Reprinted with permission from AAAS.

Genetics, Genomics | Plasmids, Bacterial

such as the human or animal gut, manure, sewage, soils, plant surfaces, and water systems are often thought of as being distinct, they are actually microbiologically connected. Favorable plasmid-borne genes can be found to circulate between different microenvironments and selective pressure by antibiotics might exacerbate this genetic exchange. For instance, there is evidence identifying very similar replicons with very similar streptomycin- and tetracycline- resistance genes in diverse hosts in two distinct habitats, clinical hospitals and agricultural fruit orchards. The fact that both the ‘habitats’ have been subjected to streptomycin and tetracycline selective pressure for decades is unlikely to be a coincidence. From the foregoing discussion it is evident that a change in our understanding of microbial evolution is necessary to fully appreciate why antibiotics and other antimicrobial agents are destined to eventually have their utility undermined by resistance acquisition.

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important mechanism for expanding metabolic pathways. The aforementioned examples of plasmid-mediated host adaptation illuminate a principle of broad significance at the interface of plasmid biology and microbial sciences: the survival of plasmids appears to be heightened by genes that provide selective advantages to their host organisms. In that light, perhaps ‘selfish’ is a little harsh as a descriptor of these versatile and diverse conduits of bacterial evolution. But admittedly, ‘benevolently selfinterested DNA’ really does not have the same cachet. See also: Antibiotic Resistance; Chromosome Replication and Segregation; Conjugation, Bacterial; Cyanobacterial Toxins; DNA Replication; DNA Restriction and Modification; DNA Sequencing and Genomics; Genome Sequence Databases: Annotation; Horizontal Gene Transfer: Uptake of Extracellular DNA by Bacteria; Metagenomics; RNAs, Small etc.; Transposable Elements

Broader contributions to microbial evolution

Another way of ascertaining plasmid–host coevolution is by monitoring novel metabolic capacities harbored by plasmid-bearing bacteria. Of particular interest, bacterial responses to toxic compounds (e.g., xenobiotics) in the natural environment have provided an opportunity to study the evolution and acquisition of new catabolic processes. Such toxins are sometimes organic in composition and include pesticides, herbicides, refrigerants, and solvents – one of which, the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) – has been in use for over 50 years. Genes that break down 2,4-D are carried on a conjugal plasmid called pJP4. This plasmid has provided a model for studies of the evolution and spread of catabolic pathways in bacterial communities, a process that is mechanistically more demanding than the acquisition and spread of antibiotic-resistance genes described above. Abundant data suggest that there has been extensive interspecies transfer of pJP4. Moreover, there is evidence to suggest that the genes in the 2,4-D degradative pathway may have evolved elsewhere, for other purposes, and then recent recombinations and modifications were selected in response to 2,4-D in the environment. The adaptive transfer and reorganization of genetic modules is also well illustrated by the analysis of bacteria from polluted environments; these organisms sometimes acquire the ability to degrade chemicals that would otherwise persist for long periods of time. In this case too, the incorporation of new genetic material has been the most

Further Reading Casjens S (1999) Evolution of the linear DNA replicons of the Borrelia spirochetes. Current Opinion in Microbiology 2: 529–534. Chaconas G and Chen CW (2005) Replication of linear bacterial chromosomes: No longer going around in circles. In: Patrick Higgins N (ed.) The Bacterial Chromosome. Washington, DC: ASM Press. Clewel DB (1993) Bacterial Conjugation. New York and London: Plenum Press. Cohen SN (1993) Bacterial plasmids: Their extraordinary contribution to molecular genetics. Gene 135: 67–76. Dawkins R (1976) The Selfish Gene. Oxford: Oxford University Press. Doolittle WF and Sapienza C (1980) Selfish genes, the phenotype paradigm and genome evolution. Nature 284: 601–603. Funnell BE and Philips GJ (2004) Plasmid Biology. USA: ASM Press. Giraldo R (2003) Common domains in the initiators of DNA replication in Bacteria, Archaea and Eukarya: Combined structural, functional and phylogenetic perspectives. FEMS Microbiology Reviews 26: 533–554. Lipps G (2008) Plasmids: Current Research and Future Trends. Germany: Caister Academic Press. Orgel LE and Crick FHC (1980) Selfish DNA. Nature 285: 645–646. Summers DK (1996) The Biology of Plasmids. Oxford: Blackwell Science Ltd. Thomas CM (2000) The Horizontal Gene Pool – Bacterial Plasmids and Gene Spread. Amsterdam: Harwood Academic Publishers.

Relevant Websites http://www.embl-ebi.ac.uk – European Bioinformatics Institute http://www.sanger.ac.uk – The Wellcome Trust Sanger Institute http://www.essex.ac.uk – University of Essex

Transduction: Host DNA Transfer by Bacteriophages P C Fineran, N K Petty, and G P C Salmond, University of Cambridge, Cambridge, UK ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Specialized Transduction Generalized Transduction Variations on Transduction

Glossary abortive transductant A bacterium that has acquired transduced DNA, which has not been degraded or stably integrated into the bacterial DNA. Abortive transductant DNA can be expressed, but cannot be replicated. gene transfer agent A prophage-like element that promotes generalized transduction of bacterial DNA but cannot replicate to form infective phage particles. generalized transduction The phage-mediated transfer of any region of bacterial DNA from one bacterium to another. Generalized transduction can be mediated by temperate or virulent phage. lysogen A bacterium that harbors a prophage. prophage A phage in lysogeny. Prophage are replicated as part of the bacterial chromosome or as a plasmid-like element. specialized transduction The phage-mediated transfer of regions of bacterial DNA, located adjacent to

Abbreviations cos sites GTA HFT

cohesive end site gene transfer agent high-frequency transducing

Transduction as a Genetic Tool Transduction in the Environment Conclusion Further Reading

the site of prophage insertion, from one bacterium to another. Specialized transduction is mediated by temperate phage upon the incorrect excision of the chromosomal prophage. temperate phage A phage that is capable of entering either the lytic or lysogenic life cycles. transducing particle A phage capsid that has packaged bacterial DNA and hence is proficient for transduction. transducing phage A phage capable of mediating transduction. transductant A recipient bacterium that has stably acquired the transduced DNA. transduction The phage-mediated transfer of bacterial DNA from one bacterium to another. virulent phage A phage that is able to replicate only via the lytic cycle.

HGT HT TEM

horizontal gene transfer High-transducing transmission electron microscopy

Defining Statement

Introduction

Transduction is the bacteriophage-mediated transfer of host DNA between bacterial cells. Transduction occurs in the natural environment, where phage are numerous, and is predicted to be a significant driver of bacterial evolution. Model phage–host studies have revealed transduction mechanisms and have led to the development of sophisticated genetic methods based on transducing phage.

Bacteria can acquire genes by vertical gene transmission to daughter cells and by horizontal gene transfer (HGT). The three main classes of HGT are transformation, conjugation, and transduction. This article will focus on transduction, with transformation and conjugation being dealt with in other chapters. Transduction is the bacteriophage (phage)-mediated transfer of bacterial DNA from a donor bacterium to a recipient bacterium. It was first

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observed by Zinder and Lederberg in 1952 whilst studying genetic recombination in Salmonella enterica serovar Typhimurium. The recombination observed did not require bacterial cell–cell contact and was not susceptible to DNase treatment, suggesting a genetic transfer mechanism distinct from conjugation and transformation. The temperate phage P22 was the agent responsible, and these authors coined the term transduction to describe this new form of gene transfer. Transduction can be divided into two major types called specialized (or restricted) and generalized, which will be detailed in sections ‘Specialized transduction’ and ‘Generalized transduction’, respectively. Briefly, specialized transduction occurs when the prophage of a temperate phage incorrectly excises from the chromosome of the lysogen taking with it genes flanking the prophage insertion site. The recipient bacterium will acquire the ‘new’ genes when it is lysogenized by the specialized transducing phage or following recombination between homologous sequences in the transducing phage DNA and the recipient chromosome. In generalized transduction, which can occur during the lytic mode of phage growth of both virulent and temperate phage, segments of bacterial DNA roughly equal to the genome size of the transducing phage are accidentally packaged into capsids. The resulting transducing particles can still adsorb to, and inject DNA into, recipient bacteria and the transduced DNA may be incorporated into the recipient chromosome by homologous recombination, resulting in stable bacterial transductants. Variations on specialized and generalized transduction, including the effects of nonreplicative prophage-like gene transfer agents (GTAs), will be addressed in section ‘Variations on transduction’. Transduction, especially generalized transduction, has become a powerful tool in bacterial genetics. Phage have been used for gene mapping, construction of strains, and localized mutagenesis, etc. In the section titled ‘Transduction as a genetic tool’, we will discuss techniques that have utilized transduction and how transducing phage can be used as ‘molecular reagents’. Transduction is a useful tool under laboratory conditions and also occurs in the natural environment. The formation of transducing particles is often considered an accidental process. However, evidence from bioinformatics and experiments on transduction in the environment suggest that transduction can benefit the bacterial hosts, and, therefore, their molecular parasites – the phage (see ‘Transduction in the environment’).

Specialized Transduction Definition and Discovery In 1956, a few years after the discovery of generalized transduction, the process of specialized transduction was

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first observed using Escherichia coli and phage lambda (). Since then, many other temperate phage that infect various bacteria have been shown to be proficient at specialized transduction. The term ‘specialized’ was derived from the ability of  to mediate the transfer of only a limited number of E. coli genes. The main difference between specialized and generalized transduction lies in what DNA is packaged into the phage capsid. Whereas generalized transducers can package bacterial DNA without phage DNA, specialized transducers can package host DNA along with some, or all, of their own genome. Briefly, a specialized transducing particle arises from the incorrect excision of a prophage from the host chromosome and the resultant packaging of bacterial and phage DNA. Bacteriophage  is one of the most thoroughly studied and understood biological entities on the planet and our understanding of many of the known gene regulation mechanisms derives from ingenious experiments using this model phage. Not surprisingly, the most complete picture of the steps involved in specialized transduction has been elucidated using . Therefore,  will be discussed to illustrate the general mechanism of specialized transduction. Replication of Phage  To understand fully how specialized transduction occurs, it is necessary to discuss the process of  replication. Lambda is the classic example of a temperate phage, being able to undergo two alternative life cycles: lytic and lysogenic. As mentioned earlier, specialized transduction relies on temperate phage that are able to form prophage during the lysogenic cycle. Therefore, the life cycle of , with the emphasis on the lysogenic process, will be outlined here and is depicted in Figure 1. Phage  recognizes host cells via the LamB outer membrane protein and, upon binding to this receptor, injects its linear double-stranded DNA (dsDNA) into the cell. The linear dsDNA then cyclizes via the pairing of cos sites (cohesive end site). The cos sites contain 12 nucleotide single-stranded overhangs that self-anneal via complementary base pairing and are covalently linked by ligation. Based on a well-characterized regulatory mechanism, the circular  genome now ‘decides’ which life cycle to enter. Replication via the lytic cycle is promoted by the Cro regulatory protein, which results in the assembly of new phage particles and their release from the cell following bacterial lysis. Alternatively, during lysogeny,  can integrate into the host chromosome and thus replicate as a prophage. The initial insertion into the bacterial genome requires the phage attachment site (attP) on  and the bacterial attachment site (attB) located between the galactose (gal) and biotin (bio) operons in E. coli (Figure 1). This insertion

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DNA replication and cell division of these  lysogens. However, factors such as cellular stress can trigger the regulatory switch from lysogeny to the lytic pathway, which will ultimately generate mature phage. The first step in this process requires the excision of the prophage to regenerate the circular form. Since the attP and attB sites are now hybrid sequences (Figure 1), they are not recognized by Int alone, and in addition require a phage excisionase called Xis. The combined action of Int and Xis catalyzes the site-specific recombination of the hybrid att sites and enables excision of the  prophage, leading to the formation of the circular  genome. Lambda then begins replication to generate long concatemers of  DNA interspersed with cos sites. Once phage heads are assembled, the DNA is packaged and cleaved into genome length units in a single-stranded staggered manner, regenerating each of the cos sites at the ends of the linear dsDNA. This process of packaging involves numerous phage and host proteins. Phage tail proteins are then added to the heads and the completed phage particles are released from the host following lysis and can reinitiate another infective cycle. Incorrect Prophage Excision Creates Specialized Transducing Particles

λ prophage

attPB

Figure 1 Normal phage  lysogenic and lytic life cycles. The linear dsDNA genome of  is injected into Escherichia coli following binding of the phage to the LamB receptor. The genome then cyclizes via the complementary single-stranded regions of the cos sites and is covalently closed by ligation. Lambda may then enter the lytic pathway with replication, packaging, and lysis of new phage particles from the cell. Alternatively, site-specific recombination between the  attP sites and the E. coli attB site is promoted by Int, resulting in establishment of the  prophage in the E. coli genome. Induction of the prophage by stresses (e.g., UV light) causes the excision of the  prophage catalyzed by the Int and Xis proteins that recombine the hybrid attBP and attPB sites. The recyclized genome is then replicated, packaged, and released from the cell as mature  phage particles. E. coli DNA is depicted in blue and phage  DNA in black. Transmission electron microscopy (TEM) image of phage  stained with phosphotungstic acid.

is catalyzed by the phage Int protein, a site-specific recombinase, that recognizes and promotes recombination between these short, relatively dissimilar, sequences. Int is a member of the tyrosine family of recombinases that have an active site tyrosine residue, which forms a covalent link to the 39 phosphate after cleavage. Because  is circular and integration occurs at attP and not the cos site, site-specific recombination into the bacterial chromosome results in a different linear gene order in the prophage than in the mature capsids. Once in the chromosome, the prophage state is maintained by the CI repressor and can be stably propagated upon bacterial

Occasionally, the  prophage does not excise using the Int/Xis system, but instead recombines in a nonspecific manner (illegitimate recombination) as shown in Figure 2. These excision events are rare (approximately 10 6 compared with normal excision) and result in circular phage DNA molecules that contain phage and bacterial genes. If the site of illegitimate recombination maintains the approximate size of the  genome and still contains the cos sites, following rolling circle replication, these specialized transducing phage can be packaged into capsids. Due to the location of the  prophage in the E. coli chromosome, the first genes shown to be transduced were the gal and bio operons that flank the insertion site. A number of methods have been developed to use  to transduce genes that are not located adjacent to attB. Initially, strains that contained large deletions or rearrangements were utilized because they positioned genes closer to attB and enabled packaging by . In another strategy, the attB site was deleted and  lysogens were isolated where the prophage had inserted with reduced specificity into the genome at alternative (secondary) sites. This allowed the transduction of genes located near these integration points. Furthermore, these experiments provided information about the sequence specificity and efficiency of the Int protein. Finally, it is possible to engineer  to contain a transposon and then use the same transposon to mutagenize the bacterial host. The prophage is then able to insert into these sites in a process requiring homologous recombination. Therefore,

Genetics, Genomics | Transduction: Host DNA Transfer by Bacteriophages E. coli λ lysogen gal

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Aberrant excision cos

λ genome concatemers etc

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gal

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Figure 2 Aberrant excision of a  prophage creates specialized transducing particles. At a low frequency, illegitimate recombination events cause incorrect excision of the  prophage. This can result in the cyclization of phage genomes that lack some genes but, that now also carry E. coli chromosomal segments that were flanking the prophage. In the figure, the generation of a gal transducing particle is depicted. Replication of the genome produces a long concatemer that is packaged and cleaved at the cos sites resulting, in this example, in gal transducing phage particles. E. coli DNA is depicted in blue and phage  DNA in black. Transmission electron microscopy (TEM) image of phage  stained with phosphotungstic acid.

using this technique it is theoretically possible to insert  in any nonessential gene in the E. coli genome and thereby transduce flanking host genes. Once the specialized transducing particles have been packaged they are capable of initiating infection of a new recipient bacterium.

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phage head and tail genes and, therefore, cannot undergo fully productive lytic growth, but can lysogenize the host. Alternatively, a bio transducer, pbio (the ‘p’ means plaque forming), lacks the int and xis genes and subsequently is unable to become a prophage, but can replicate lytically. To form a transductant, the horizontally acquired DNA must be maintained stably in the recipient cell via one of a number of possible routes. Homologous recombination between sequences common to both the transducer and host genomes can provide stable transductants that have incorporated the transducing phage genome (Figure 3(a)). These lineages will have two copies of the transduced DNA segment (merodiploid) and can be useful for complementation analyses. However, if multiple recombination events occur, the host locus may be exchanged for the equivalent transduced DNA (Figure 3(a)). This process of forming a haploid transductant is less frequent and is essentially how transductants are obtained by generalized transduction. Alternatively, introduction of the transduced DNA can proceed via the normal  integration route, with the aid of a helper phage that provides the missing functions in trans or in cis via recombination (Figure 3(b)). First, coinfection with a wild-type  and the transducing particle can result in recombination at sites shared by these phage, which can then integrate at attB via the attP and Int functions supplied by the helper phage. The resulting lysogens harbor the genomes of both the transducer and helper phage and hence are called double lysogens (or dilysogens). These dilysogens are extremely useful for the generation of high-frequency transducing (HFT) lysates upon prophage induction. A single excision of both prophage yields a hybrid circular DNA that is replicated into concatemers and packaged according to the cos sites, giving rise to alternate transducing and wild-type  particles. The final mode of stable transductant formation can occur when  is able to replicate as a plasmid in the recipient. Mutation of the  N gene or the host nusA gene causes reduced expression of the  O and P genes, which are required for replication and, at low levels, allows maintenance of  essentially as a plasmid. Alternatively, acquisition of a plasmid origin of replication also provides  DNA with stable replication and maintenance. Therefore, any one of these mechanisms can produce heritable transductants.

Formation of Transductants

Other Specialized Transducing Phage

The dsDNA of the infecting transducing particle is injected into the new host and cyclized as described above. Because specialized transducing particles usually have lost some phage genes, this can have deleterious effects on  function, especially in terms of integration and generation of new particles. For example, a gal transducing phage, dgal (the ‘d’ means defective), has lost the

The  phage has provided an excellent system for unraveling the mechanisms of specialized transduction. This model predicts that aberrant excision of any prophage that packages its DNA based on phage-specific sequences (analogous to cos sites) could lead to production of specialized transducing particles. Indeed, many specialized transducers have been identified, including Pseudomonas

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(a)

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cos

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bi o attBP

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Figure 3 Formation of transductants and the creation of high-frequency transducing lysates. (a) Formation of diploid and haploid transductants via Rec-dependent recombination with the chromosome of an Escherichia coli gal mutant. In this example, the dgal phage is used for illustrative purposes. A single recombination event between the shared E. coli sequences can result in the merodiploid lysogen shown, or one where the mutant gal allele is present in the prophage (not shown). A second recombination event that can lead to the replacement of the mutant gal allele with the wild-type gal sequence is possible. (b) Production of dilysogens occurs when an E. coli gal mutant is coinfected with wild-type  and the specialized transducing particle (dgal). The phage genomes cyclize and can then recombine together at shared sequences and integrate into the host chromosome in an Int-dependent manner giving the dilysogen shown. Induction of the prophage leads to the excision of either the wild-type  or the entire dilysogen, which are then capable of replication, packaging, and release from the cell. E. coli DNA is depicted in blue and phage  DNA in black.

aeruginosa phage D3 and Bacillus subtilis phage SP . Comparative genomics may provide further numerical data on the frequency of specialized transduction since it is also theoretically possible that some of the apparently degenerate prophage present in the many sequenced bacterial genomes may have been the result of past specialized transduction events. Alternatively, there is evidence that mutational events leading to inactivation of prophage can occur following prophage acquisition by the host bacterium.

Generalized Transduction Generalized transduction is the process whereby any section of the bacterial DNA can be transferred from one bacterium to another via a phage virion. This phenomenon was first identified by Zinder and Lederberg in 1952 in Salmonella, where the temperate phage P22

transferred chromosomal DNA from one strain of Salmonella to another. A few years later, in 1955, Lennox identified phage P1 as a generalized transducing phage of E. coli, and the knowledge about transducing phage and the mechanisms of generalized transduction has been gleaned from investigations using these two ‘model’ phage. Early studies of P22 transduction in Salmonella and P1 transduction in E. coli have been extensively reviewed elsewhere (see ‘Further reading’). The findings from many of these studies, together with studies of other transducing phage, have contributed to current knowledge on generalized transduction, which is summarized below.

Properties of Generalized Transducing Phage The known, naturally occurring generalized transducing phage are dsDNA tailed phage that utilize a sequential headful DNA packaging mechanism. Generalized

Genetics, Genomics | Transduction: Host DNA Transfer by Bacteriophages

transducing phage can be either virulent or temperate and occasionally, bacterial DNA, instead of phage DNA, is packaged into the head of an otherwise unaltered virion, resulting in a transducing particle instead of a fully functional phage. In order for this to occur, a generalized transducing phage must not degrade the host DNA completely upon infection. The life cycle of a generalized transducing phage is summarized in Figure 4. Generalized transducing particles arise through a mistake in DNA packaging so that the host chromosomal or plasmid DNA is taken up into the phage head in place of the phage DNA. The frequency of this mispackaging, and therefore the frequency of transducing particle formation, is dependent upon the mechanism of DNA packaging employed by the phage. During the phage lytic cycle, the structural proteins are made and the phage capsid proteins make up the virion head or prohead with the tails assembled separately. The DNA is then replicated and forms concatemers of phage genomes, arranged head to tail in tandem, usually around four genomes long, depending on

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the phage. This DNA must then be packaged into the proheads. In both P1 and P22, headful packaging is initiated by recognition of a specific sequence present in the phage DNA called the pac site by the phage packaging apparatus. The phage terminase recognizes the pac site and the DNA is cleaved at or near this site. The cleaved pac end is bound to the large terminase subunit, attached to the prohead, which initiates the packaging of the linear DNA concatemer into the prohead. The size of the head determines how much DNA can be packaged; hence the term ‘headful packaging’. This is usually a little more than the size of the phage genome. For example, the genome of P1 is 95 kb in length, but up to 115 kb of DNA is usually packaged into the phage head. Therefore, in addition to a single copy of the phage genome, there is also some extra phage DNA packaged, which is terminally redundant. Once the prohead is full, the DNA is cleaved and the cut end of the remaining concatemer is recognized by the packaging apparatus and used to initiate packaging of the next empty prohead. Several proheads are filled sequentially in this manner from the remaining DNA,

Phage adsorption to donor Phage DNA injection

Phage Lytic Cycle

Host lysis

Phage DNA replication

Virion assembly

Transducing particle adsorption to recipient

Donor DNA injection

Homologous recombination with recipient DNA

Transductant

Figure 4 Generalized transducing phage life cycle. During the lytic cycle a phage adsorbs to a host bacterium, injects and replicates its DNA, makes virion heads and tails, packages its DNA into the heads, attaches the tails and lyses the host, releasing the phage to infect new bacteria. Upon infection by a generalized transducing phage, phage-encoded enzymes can cleave the bacterial DNA into large sections, and occasionally these lengths of bacterial DNA can be mispackaged into a phage head. A small number of virions made will contain host DNA in place of phage DNA, producing a transducing particle instead of a functional phage. Transducing particles can adsorb to, and inject the bacterial DNA into, susceptible hosts, where this donor DNA can undergo homologous recombination with the recipient genome, causing a transfer of any genetic markers encoded. Phage DNA is shown in blue, donor DNA in red, and recipient DNA in green.

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with 3–4 headfuls produced on average from one concatemer. The phage tails are then attached to the filled proheads to complete the virion and the host bacterium lysed. The released phage, on adsorbing to a susceptible host, injects its DNA into the cell. The DNA undergoes circular permutation by recombination of the terminally redundant ends, to protect it from nucleases. Next, the DNA will either insert into the bacterial chromosome or remain as a plasmid as in the case of P1 if entering the lysogenic cycle or initiate the construction of new phage if entering the lytic cycle.

required for pac-site recognition and initiation of subsequent DNA encapsidation. Therefore, mutations in packaging specificity determinants can improve and enhance transducing phage and support the model for P22 packaging DNA at sites related to pac. The only difference between a phage and a transducing particle is the origin of the DNA in its head so that once the host DNA is packaged, the remainder of the lytic cycle occurs as for the phage, up to the point of DNA injection into a susceptible host. Fate of Transduced DNA

DNA Packaging in Transducing Particles The random mispackaging of bacterial DNA that occurs in a generalized transducing phage happens infrequently. Estimates of the number of transducing particles in a P22 lysate from density transfer experiments show that they account for about 2% of all the particles, so that approximately 1 out of every 50 phage produced is a generalized transducing particle. Generalized transducing particles are thought to arise by one of two possible ways. First, the phage packaging apparatus recognizes pac-like sequences on the bacterial DNA, which are sufficiently similar to the phage pac site, and packages host, instead of phage, DNA. This is believed to be the stimulus for transducing particle formation in P22. However, there is little evidence to support this hypothesis for P1, and it seems most likely that, for this phage, the bacterial DNA is mispackaged from nicks or ends in the host DNA. Whichever the method of host DNA recognition, generalized transducing particles are filled in the same manner: by the headful, cleaving the DNA, and filling the next head from lengths of the bacterial chromosome or plasmid DNA. Again, the amount of host DNA that can be packaged and therefore transduced is dependent on the size of the phage head. P22 can transduce approximately 44 kb (around 1% of the host genome) in one transducing particle, whereas P1 is larger and can transduce around 115 kb (around 2% of the host genome), and the B. subtilis phage PBS1 is capable of packaging up to 300 kb (around 8% of its host genome). Any region of the host DNA can be packaged into a virion in this way although the frequency of transduction of different regions of the genome can vary. This is particularly noticeable if generalized transduction occurs through mispackaging from pac-like sites as the frequency of transduction of a particular marker depends on its location in the bacterial genome relative to a pac-like site. High-transducing (HT) mutants of P22 display an increased efficiency of transduction and this appears to be due to a reduced specificity in pac-site recognition and packaging. The resulting P22 HT particles transduce different regions of the chromosome with a similar frequency. Indeed, the mutations in P22 HT phage map to gene 3, which encodes the terminase

The fate of the transduced bacterial DNA differs from that of phage DNA once it has been injected into a new bacterium. These possible fates are listed below and summarized in Figure 5. Abortive transduction

Following DNA injection, the majority of the DNA (90%) remains extrachromosomal within the recipient bacterium. In this case, the linear DNA is injected into the cell, along with a phage-encoded protein found in the prohead. The protein binds to the ends of the transduced DNA and circularizes it, protecting the DNA ends from host nucleases and preventing recombination with the recipient bacterial chromosome. The DNA can remain stable in this way for several generations and can even transcribe genes that are present on the DNA. However, abortively transduced DNA is unable to replicate, and is therefore inherited by only one daughter cell following division. So, any phenotype encoded in this region will be apparent in only a very small minority of the offspring, giving rise to minute colonies. Abortive transductant DNA is only rarely able to recombine with the recipient chromosome. However, recombination may be stimulated if the DNA has been damaged. Nicks that have formed in the circularized DNA can promote the action of the host DNA repair system, resulting in recombination. For example, it has been shown that UV irradiation of generalized transducing phage lysates achieves a higher rate of stable transductants, due to the rendering of abortive transductants as recombinogenic. Recycling of nucleotides

A small percentage of the DNA injected by transducing particles is left unprotected by the phage prohead proteins found in abortive transduction, and if this DNA is unable to undergo homologous recombination with the recipient bacterium, it is recycled by host degradation into component nucleotides that are incorporated into the bacterial genome during DNA repair. It has been estimated that not more than 15% of the transduced

Genetics, Genomics | Transduction: Host DNA Transfer by Bacteriophages

10%

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Figure 5 Fate of transferred DNA in generalized transduction. Once the donor DNA is injected into the cell from a transducing particle, there are alternative fates that it can undergo. The majority of the injected DNA will be protected by phage proteins, which bind to the ends of the transduced DNA and circularize it, protecting it from nucleases and recombination. This process, known as abortive transduction, accounts for the fate of 90% of the transduced DNA. Two percent of the unprotected DNA is able to undergo homologous recombination into the recipient genome in stretches of at least 500 bp, with the remaining 8% degraded to its constituent nucleotides and incorporated into the recipient DNA. Phage proteins are shown in blue, donor DNA in red, and recipient DNA in green.

DNA is liable to this degradation, and that usually only around 8% of total transduced DNA undergoes this process. Chromosomal recombination

If the recipient bacterium is sufficiently related to the donor bacterium at the DNA level, stable insertion of the transduced DNA into the recipient chromosome can occur via homologous recombination. This process usually occurs within 1 hour of transducing particle infection in E. coli and Salmonella. After this time, successful recombination is unlikely to happen and any remaining transduced DNA is usually degraded and recycled. Chromosomal recombination and subsequent stable transductant formation requires RecA-dependent replacement of the equivalent DNA on the recipient chromosome by the donor DNA, leading to the expression of the corresponding encoded phenotype carried by the transduced DNA. Stable recombination into the chromosome is only a rare event; it has been shown for P1 and P22 that only about 2% of the bacterial DNA injected by a transducing particle normally undergoes homologous recombination into the recipient genome in continuous stretches of at least 500 bp. Plasmid inheritance

Some, but not all, generalized transducing phage are able to transduce plasmids from one host to another. In this

case, the plasmid DNA is injected by the transducing particle and recircularizes into a stable plasmid that replicates along with the new host and is inherited by daughter cells. Broad host range phage, which may not be able to successfully transduce chromosomal DNA into bacteria lacking sufficient genetic identity, may still be able to transduce plasmids between genetically diverse bacteria, as recombination and therefore genetic identity with the recipient bacterium, is not required. Development of Other Generalized Transduction Systems Virulent phage that use the headful packaging method are obvious targets for development into generalized transducing phage, and some such phage have been manipulated to make them generalized transducers. For example, the well-characterized E. coli phage T4, which packages its DNA by the headful mechanism but is not capable of transduction in its wild-type state, has been modified for use as a generalized transducing phage. Mutation of T4 genes encoding endonucleases prevents the degradation of host DNA upon T4 infection, thus enabling transduction. This simply means that the bacterial DNA is left sufficiently intact to allow mispackaging to occur, and as T4 does not require pac sites in order to package its DNA, it is able to package both bacterial and

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phage DNA equally well, making it an extremely efficient generalized transducer. The model temperate phage , described earlier in this article as a specialized transducing phage, can also, with adaptation, make generalized transducing particles. Lambda does not use the headful packaging mechanism of most known transducers, but utilizes an alternative mechanism whereby the DNA concatemers are cleaved at a specific cos site found at either end of the phage genome. Whereas only one pac site is needed to initiate headful packaging and DNA cleavage, two cos sites, one at either end of the DNA, are required for  packaging. Packaging is initiated by recognition of the first cos site. The second cos site signals the end of the DNA to be packaged and that the DNA should be cleaved. As with pac, mistakes in DNA packaging can occur if DNA sequences resembling cos sites are found on the bacterial chromosome. However, the chances of two cos sites being found on the host DNA, exactly the required length apart, are minimal. Therefore, in the absence of a ‘signal’ sequence to cleave the DNA, when  does package bacterial DNA into its proheads by mistake, there is usually a protrusion of a length of DNA and the phage tails are unable to bind to make a functional transducing particle. Simple in vitro DNase treatment of the lysate containing such partially formed particles cleaves the excess DNA and the phage tails are then able to attach to the proheads to complete the generalized transducing particle. However, even with other manipulations,  is only a poor generalized transducer and its preferred use as a laboratory tool is that of a specialized transducer. Another highly studied phage, Mu, can also operate as a generalized transducing phage although it is perhaps better known for its qualities as a transposable element. This phage packages its DNA via a headful mechanism, and a pac site is required to initiate the packaging of its DNA into the prohead. The genome of phage Mu when it is packaged is found flanked by host DNA of variable sequence; a short region of up to 150 bp at the left-hand end and a larger region of up to 3 kb on the right-hand end. Mu-transducing particles are believed to arise primarily due to the mispackaging of host DNA as for other headful packagers. However, it cannot be ruled out that at least some transduction events observed for Mu are derived from recombination with host DNA present at the right-hand end. Generalized transduction is also possible with miniMu, a Mu derivative with the central region of the genome deleted, leaving only the ends intact plus the transposase A gene. In the presence of a helper Mu, mini-Mu can be induced and will be packaged into a prohead together with the adjacent bacterial chromosome to a total length of 39 kb of DNA. Ninety percent of transductants that arise following infection of a susceptible host with this mini-Mu/donor DNA

transducing particle, result from RecA-dependent homologous recombination with the recipient DNA. The remaining 10% of transductants result from mini-Muduction, whereby the donor DNA has a copy of mini-Mu attached at each end, which can insert anywhere into the recipient DNA in the same way as for random transposon insertion. Homology between the donor and recipient DNA is not required for mini-Muduction, therefore allowing transduction of DNA between any species of bacteria that Mu is able to infect. Mu has a relatively broad host range, and is able to infect and replicate in many different bacteria, making it a very useful genetic tool, particularly for bacterial strains without an existing identified generalized transducing phage. Clearly, Mu can be considered to display properties of both generalized and specialized transducing phage, in addition to transposable elements. A combination of experimental and bioinformatic approaches may be utilized in the search for generalized transducers for a particular bacterial strain. When trying to isolate generalized transducers, headful packaging phage can be enriched by using their reduced sensitivity to chelating agents. Indeed, sodium pyrophosphate has been successfully used in this way to isolate transducing phage for Streptomyces venezuelae. With advances in the number of phage genomes sequenced, searches for generalized transducers for certain bacteria could also be undertaken at the genome level. Terminases of many headful packagers can be recognized and classified into functional groups using comparative genomics, which may narrow the search for generalized transducers. As previously mentioned, the terminase gene product is involved in recognizing sequences in phage DNA and initiating the series of packaging events that result in mature phage particles. Therefore, generalized transducing phage terminases that have reduced specificity are better transducers, a prediction borne out with P22 HT gene 3 terminase mutant phage compared with parental P22. It may be worthwhile in the future to develop rational and randomized mutagenesis approaches on sequenced phage with predicted headful packaging strategies, with the aim of developing generalized transducers, by mutation of terminases (e.g., P22) or genes involved in host DNA degradation (e.g., T4). For phage that are poor transducers, or when using markers that are only transduced at low frequencies, methods have been devised to increase the frequency of transduction. As already mentioned, the frequency of generalized transduction can be greatly enhanced by UV irradiation of the donor lysate, whereby damage is introduced into the transduced DNA to stimulate recombination with the recipient’s chromosome. Also, insertion of a phage pac site into the bacterial DNA will greatly increase the efficiency of transduction of that region. This

Genetics, Genomics | Transduction: Host DNA Transfer by Bacteriophages

is particularly useful when transducing plasmids, as these are often transduced at lower frequencies than chromosomal markers.

Variations on Transduction Gene Transfer Agents GTAs are a class of prophage-like elements that package short random segments of bacterial DNA. Following release from the donor cell, GTAs infect neighbors, thereby enabling the transduction of bacterial genes that are incorporated via homologous recombination. These agents are reported to promote ‘constitutive transduction’ or ‘capsduction’ and this generalized transduction phenomenon was first discovered in 1974 by Marrs while studying genetic recombination in Rhodobacter capsulatus. Since the original observation, GTAs have also been found in diverse bacteria, including Brachyspira hyodysenteriae, Methanococcus voltae, Desulfovibrio desulfuricans, and Bartonella spp. A common feature of sequenced GTAs is that the genes encoding the phage particles are present in the bacterial genome; they encode all of the products required for assembly of the phage (structural genes), but lack early genes responsible for the replication of phage DNA. Upon assembly, these unusual phage-like particles typically package between 4.5 and 14 kb of random host DNA, depending on the particular GTA. However, the prophage-like gene clusters encoding the R. capsulatus and the B. hyodysenteriae GTAs are 14.1 and 16.3 kb, respectively, which is considerably larger than the DNA these particles can package (4.5 and 7.5 kb, respectively). In contrast, tailed dsDNA phage typically package at least 40 kb of DNA. Most of the GTAs characterized also have a small head morphology compared to tailed phage, ranging from 30 to 80 nm in diameter. Therefore, due to the reduced capsid size, even when these GTAs randomly package their own DNA at a frequency as low as any other chromosomal genes it is clear that they are unable to package their entire genome. The inability to package their entire genome and the lack of early replication genes result in phage-like particles that are nonreplicative and, hence, do not form plaques on any host tested. As such, the genes encoding these phage-like elements are inherited in a predominantly vertical fashion from parent to daughter cells. Rare cases of GTA horizontal transfer have been inferred in phylogenetic studies using the R. capsulatus GTA, but the mechanisms are unknown. Once the GTA transducing particles are formed, it is unclear how they are released from donor cells because detection of these particles does not usually correlate with lysis of the host bacterium. Indeed, the sequence of the R. capsulatus GTA is not predicted to encode homologues of phage lysin or holin genes.

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Conversely, the B. hyodysenteriae GTA possesses copies of lysin and holin genes and purified lysin was shown to disrupt cell walls by degrading peptidoglycan. The theory that these ‘defective’ prophage-like elements are actually host-adapted gene transfer modules is supported by regulation studies. Work on R. capsulatus has demonstrated that its GTA is controlled by the CckA/ CtrA two-component phosphorelay regulatory system that also controls motility. In addition, expression of the GTA structural genes is activated by a LuxIR-type quorum sensing system that enhances gene transfer in a cell-density-dependent manner in response to the N-hexadecanoyl-homoserine lactone signal. Therefore, gene transfer is promoted when there is a large number of signal-related recipients nearby, which would increase the chance of a successful DNA transfer event. Finally, the presence of R. capsulatus-like GTA sequences in many -proteobacteria indicates functional selection for their maintenance in diverse genomes. The limited functional studies and sequence data for most GTA elements highlight a paucity of information on this unusual mode of ‘generalized transduction’. Further studies on GTAs could provide more information on DNA packaging mechanisms and transduction. Indeed, phylogenetic studies have demonstrated that homologues of the R. capsulatus GTA terminase product cluster in a single group, which presumably represent enzymes with reduced sequence specificity proficient in packaging random host DNA. If phage that contain GTA-like terminases are identified, it might indicate the potential of these phage to function in generalized transduction. Areas requiring further analysis include the mechanisms of relaxed packaging specificity, particle release, and adsorption to bacteria. It is likely that many more diverse GTAs exist in other bacteria, which await discovery through both bioinformatics and functional studies. Are ‘Cargo’ Genes a Special Case of Transduction? The recent genome sequencing efforts of both bacteria (including their prophage) and phage have revealed that many phage and prophage genomes contain nonessential genes of putative bacterial origin. Prophage-associated genes often impart an advantage to bacterial lysogens via the expression of toxins or virulence factors that aid bacterial pathogenesis. This phenomenon is called lysogenic conversion due to the ‘conversion’ of the bacterial host upon lysogeny. A familiar example is the prophageencoded shiga toxin produced by certain pathogenic E. coli lysogens. Alternatively, phage-encoded gene products, of bacterial origin, can aid in phage lytic infection. One example is the production of photosynthetic proteins by certain cyanophage upon infection of cyanobacteria. The phage-encoded photosystem genes (e.g., psbA and

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psbD) are thought to increase energy production during phage infection of the cyanobacterial host, ultimately assisting in phage replication. Phylogenetic and host range studies demonstrate that these photosystem genes were acquired by phage from bacteria, have been shuffled among the phage (probably via coinfection), and in some cases have been recombined back into some cyanobacteria. The generation of a bacterial recombinant following infection with a virulent phage must only arise when the infection is nonproductive. Therefore, it is theoretically possible to define both virulent and temperate phage that carry these ‘cargo’ genes (or ‘morons’) as a form of potential transducing phage, since these genes may be acquired and maintained in the infected bacteria. Obviously, this sort of genetic promiscuity or modularity in phage genomes represents the dynamic evolution of these biological entities and it is interesting to consider how these ‘cargo’ genes might have been acquired (e.g., by illegitimate recombination and/or incorrect prophage excision). Clearly, phage are a constant source of genetic mixing that often blurs the boundaries between what are considered phage and bacterial genes. When reassessed in this broader context of general phage evolution, many more phage might be considered as transducers, albeit at a very low frequency compared to the well-characterized classes of specialized and generalized transducing phage.

Transduction as a Genetic Tool Since the discovery of phage, researchers have rapidly harnessed their knowledge of phage biology for the development of new tools and applications. Examples include phage (and their products) as antibacterial agents, as DNA delivery vehicles for expression of particular genes in a desired host (e.g., for transposon delivery or luciferase reporter expression), and as components of DNA cloning, integration, and expression systems. These cases represent just a handful of phage uses, with those related to their transducing (especially generalized) properties being covered here. Isolation and Characterization of Generalized Transducing Phage Obviously, the first requirement to enable the use of a generalized transducing phage is to isolate one for the bacterium of interest. There is now extensive evidence of the ubiquitous distribution of phage in the natural environment. In fact, it is estimated that there are approximately 10 phage for each bacterium on Earth. Therefore, a good starting point for phage isolation is the native environment from which the bacterium was originally cultured. For enteric bacteria, a good source of phage is raw and treated sewage. Using a variety of

sources, with the target bacteria as an indicator, phage have been isolated for many bacterial genera. Phage are then screened for transducing ability using, for example, donors with transposon insertions in defined genes with screenable phenotypes (e.g., auxotrophy). Transduction is performed in a wild-type recipient, selecting for the transposon antibiotic resistance marker. Putative transductants are then screened for cotransduction of the phenotype (e.g., auxotrophy on minimal media). In addition, transduction of plasmids can be tested. Transduction of multiple loci is required to confirm isolation of a generalized transducing phage. Using strategies similar to those described above, generalized transducing phage have been isolated for strains of many bacterial genera, including Bacillus, Caulobacter, Citrobacter, Erwinia, Myxococcus, Pseudomonas, Salmonella, Serratia, Staphylococcus, Streptococcus, and Streptomyces. In these cases, and others, generalized transducing phage have enhanced the genetic tractability of their host organisms. Below, the most common uses of generalized transducing phage are discussed. Constructing Strains The ability to transduce loci with selectable or screenable phenotypes has greatly facilitated bacterial genetic manipulations and the power of generalized transduction has been aided particularly by its use in conjunction with transposon mutagenesis. Typically, following transposon mutagenesis, it is desirable to confirm the presence of a single transposon insertion, which can be checked by Southern blot analysis. However, it is also possible that other secondary (e.g., point) mutations may have arisen in the mutant strain. In order to move the transposon insertion of interest into a background likely to be free from either secondary transposons or other mutations, generalized transduction is the facile method of choice. Cotransduction of the marker and the phenotype studied confirms that the appropriate transposon copy has been selected. Furthermore, where selectable markers are available, generalized transduction makes the construction of double and multiple mutant strains quick and easy compared with alternative marker (allelic) exchange procedures. Currently, in most molecular microbiology laboratories in a postgenomics era, this is the major use of generalized transducing phage. Localized Mutagenesis To examine gene function in detail, it is necessary to analyze the effects of mutations. There is often value in studying more subtle mutations than those generated by knockout or transposon disruption. In the current era of molecular biology, localized mutagenesis can be performed by error-prone polymerase chain reaction (PCR)

Genetics, Genomics | Transduction: Host DNA Transfer by Bacteriophages

followed by a strategy to recombine mutated sequences in a single copy into the bacterial chromosome. Although this is a powerful and ‘clean’ technique, it relies on suitable delivery (e.g., transformation and/or conjugation) systems for the organism studied. An alternative strategy is to subject the bacterium to UV or chemical mutagenesis and screen for mutants. However, any interesting mutants may contain other mutations elsewhere in the genome, complicating further analysis. If the gene of interest is located in the vicinity of a selectable marker (e.g., a transposon), it can be transduced from the mutated strain into an unmutagenized background, selecting for the marker and, by linkage, any nearby mutations in the gene of interest. Alternatively, a transducing lysate, prepared on the transposon-tagged strain, can itself be exposed to mutagenic agents such as hydroxylamine or nitrous acid. Therefore, only the DNA packaged within the phage is mutated and upon transduction into a recipient will be linked to the selectable marker. Due to the relatively short stretches of DNA transferred by transduction, mutations linked to the selected marker can be identified and characterized. Genetic Mapping Transductional mapping was used in E. coli and Salmonella to determine the fine genetic structure of closely linked genes and mutations within genes. With the huge expansion and ease of genome sequencing, there is now little need for these traditional genetic mapping experiments. The basic principle of transductional mapping relies on measuring the genetic linkage of loci that depends on the efficiency of cotransduction and recombination into particular recipients. Simply, if two mutations are linked (within the size of DNA packaged in the phage capsid), they will be transduced together at a particular frequency. Calculation of linkage depends on the distance of the loci from each other and the number of recombination events required to isolate a transductant. Although historically interesting and extremely powerful in early bacterial genetics, these techniques are no longer widely pursued as their utility has to a large extent been overtaken by advances in molecular biology methods. Plasmid Transduction For many genetic manipulations in bacteria, it is necessary to introduce plasmids into recipient cells. Commonly, conjugation and transformation are the systems of choice for plasmid transfer. However, phagemediated plasmid transduction can be a useful method for bacterial strains with poor transformation efficiency or a paucity of suitable conjugal plasmids. Transduction of plasmids has been demonstrated with many generalized transducing phage. However, the efficiency can vary

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greatly depending on the phage and the size and sequence of the transduced plasmid. The currently accepted model is that multimeric double-stranded plasmid DNA, generated during rolling circle replication, is accidentally packaged by the phage. After injection of the plasmid DNA, the recipient bacterium regenerates the plasmid in a process requiring homologous recombination and RecA. For efficient transduction of pBR322 by wild-type P22, it is necessary to introduce a pac sequence. Furthermore, a P22 HT derivative, with reduced packaging specificity, can transduce pBR322 without any cloned DNA fragments. The plasmid transducing mechanism has also been analyzed for a modified T4 that is proficient in generalized transduction (see ‘Generalized transduction’). Transduction of pBR322 by this modified T4 involves packaging of the equivalent of 38 monomers of the plasmid arranged as multimers and establishment in the recipient requires homologous recombination. Phage P1 can package plasmids that contain a P1 inc site and, due to the broad host range of P1 (see below), transduce these into a variety of strains. In the absence of pac sequences, pBR322 requires cloned P22 DNA fragments for efficient plasmid transfer by P22. The method of transduction involves an initial homologous recombination step between P22 and the plasmid in the donor prior to transduction into the recipient. This dramatic increase in transduction efficiency, when a portion of phage DNA is cloned into plasmids, has also been observed for multiple bacteria, including species of Bacillus, Lactobacillus, Staphylococcus, and Streptomyces. Intergeneric Gene Transfer Phage–host receptor interactions are often highly specific with a phage only recognizing a single strain of a given species. In other cases, phage can have a very broad host range, with P1 and Mu providing classic examples. The obvious benefit of isolating a broad host range transducing phage is that it is possible to use it for multiple bacterial isolates. Phage P1 has an invertible region that can switch the expression between two alternative tail fiber products, a major determinant in phage–host interactions. Depending on what tail fiber form is expressed, P1 can adsorb to, and replicate in, Citrobacter, Enterobacter, E. coli, Erwinia, Klebsiella, Pseudomonas, and Salmonella. P1 can also inject its DNA into strains of Agrobacterium, Flavobacterium, Myxococcus, and Vibrio but cannot produce phage progeny on these. In this manner, plasmids have been introduced into Myxococcus by P1, where they are unable to replicate. This is the basis of a suicide vector delivery system that is used for transposon mutagenesis and targeted gene disruptions in these bacteria. Intergeneric gene transfers between E. coli and Salmonella have been performed using P1. Some

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researchers have used the transduction efficiency as a rough indicator of DNA homology between donor and recipient. However, these transductions are often unsuccessful due to the large genomic differences between these genera and the requirement of long stretches of nearidentical sequence for efficient homologous recombination. Analysis of the bacterial transductants shows that recombination has frequently occurred between highly conserved regions, such as the ribosomal (rrn) loci, which can result in large genome alterations. It is now understood that the recipient’s methyl-directed mismatch repair system is responsible for some of the recombinational stringency and, as such, mismatch repair mutants are more efficient recipients of donor DNA from different genera. To date, such intergeneric gene transfer experiments have not been widely utilized outside of E. coli and Salmonella. Lambda as a DNA Delivery Vehicle Lambda has been developed as an efficient transduction tool for the introduction of DNA into host cells. This includes the packaging and transduction of cosmids and transposons to target cells. Cosmids are plasmid vectors that contain  cos sites, hence their hybrid name. Genomic libraries can be generated by ligation of large chromosomal DNA fragments (up to 47 kb) into cosmids. Packaging of the cosmids is performed in vitro with  capsids via the recognition and cleavage at consecutive cos sites. Phage tails are attached and the completed particles can then transduce the cosmid into a suitable recipient, where it is replicated by virtue of a plasmid origin of replication. These cosmids can be further packaged in vivo following infection with . In a similar mechanism,  derivatives containing transposons (e.g., Tn5, Tn10, TnphoA, and TnblaM) can be transduced into a target bacterium. Transposition (mutagenesis) events can be selected and mutants subsequently characterized. Lambda replication is inhibited by amber mutations in the phage morphogenesis genes, whereas in some other genera (see below) wildtype  is unable to replicate. Compared with plasmid-based (conjugation) mutagenesis procedures, -based systems are faster and have no requirement for counterselection of E. coli donors. The requirement of recipient bacteria for the LamB phage  receptor originally restricted the host range use of . However, a wider range of Gram-negative hosts for  adsorption and DNA injection have been generated by introducing the lamB gene on a suitable plasmid. Bacteria capable of expressing and transporting LamB to the outer membrane may then be infected by phage . Plasmids containing lamB have enabled  infection of species of various genera, including Agrobacterium, Erwinia, Klebsiella, Mesorhizobium, Pseudomonas, Salmonella, Serratia, and Vibrio, making delivery of cosmids and transposons relatively

straightforward in these systems. In an interesting extension of the  host range, virons can be taken up by certain eukaryotic cells in culture and also by antigen presenting cells in vivo. This mechanism enables the delivery of cosmids and other vectors into eukaryotic cells for expression studies and vaccine delivery.

Transduction in the Environment With increasing numbers of sequenced bacterial genomes and advances in comparative genomics, it is now clear that HGT accounts for a large degree of the genetic diversity found within bacterial species. Phage are believed to be the most abundant biological entities, with estimates of 1031 phage on the planet. The number of transduction events per year has been estimated to be 1014 in the Tampa Bay Estuary and 1013 in the Mediterranean Sea. This demonstrates just how important the role of phage-mediated transduction might be for HGT in the natural environment. Indeed, interrogation of the genomes of nearly all sequenced bacteria reveals the presence of numerous prophage and prophage-like elements, and these regions are often associated with adjacent horizontally acquired regions and ‘cargo’ genes of bacterial origin. Possible explanations for these ‘cargo’ genes is that specialized transduction or illegitimate recombination has taken place. Some of these phagetransferred regions encode bacterial virulence factors that can convert the host into a pathogenic strain. For example, it is known that the cholera toxin is encoded on a phage, CTX, and infection of a nonpathogenic Vibrio cholerae strain with this phage will render it pathogenic. More recently, generalized transducing phage have been shown to transduce both the VPI pathogenicity island, which encodes the receptor for CTX, allowing adsorption and infection of strains that are normally resistant to this phage, and the genes encoding CTX itself. Unfortunately, since transduction frequently relies on homologous recombination between highly related sequences, in these instances, its impact on bacterial genomes is not readily detected using current bioinformatic sequence tools. Therefore, the importance of transduction in bacterial evolution has been inferred from a combination of laboratory experiments, an understanding of the global abundance of phage, and the detection of transduction in the natural environment. Several studies have shown transduction of both chromosomal and plasmid DNA to occur in a variety of natural environments. Transduction of both plasmid and chromosomal markers between strains of Pseudomonas aeruginosa has been demonstrated in freshwater environments, as well as between bacteria on leaf surfaces, even when the donor and recipient were originally on different plants. Broad host range phage have been

Genetics, Genomics | Transduction: Host DNA Transfer by Bacteriophages

shown to transduce plasmids among a diverse range of bacteria in natural populations of both fresh and marine water environments. It has also been reported that chromosomal markers have been transferred from virus-like particles, spontaneously released from five strains of marine bacteria, to convert different auxotrophic mutants of E. coli to prototrophy. This suggests generalized transduction between bacteria of different families, although these conclusions were not reinforced by verification of the corresponding prototrophic gene acquisition. Similar studies on virus-like particles isolated from thermal vents and hot spring bacteria have also been undertaken, and the same phenomenon observed. If verified, these findings would indicate that generalized transduction can occur between a broad range of bacterial species in the environment. Generalized transducing phage have also been detected containing various bacterial 16S rRNA genes, which are bacterial species-signature-specific sequences. This could imply a role for generalized transduction in the horizontal transfer of 16S rRNA gene sequences between bacteria of different genera. Many phage are very stable and resistant to degradation in the environment, particularly in comparison to naked DNA, and transducing particles thereby represent a comparatively stable repository for bacteria DNA. Given these observations, together with the global abundance of phage, it seems possible that phage-mediated transduction events in the environment are a major force driving HGT and adaptive evolution of bacteria.

Conclusion The phage-mediated transfer of bacterial DNA between donor and recipient cells is a form of HGT called transduction. The current understanding of the molecular mechanisms of transduction has been fueled by studies of a small number of ‘model’ phage–host systems. With advances in modern molecular biological techniques (many of which are themselves derived from the products of phage research), it is important not to forget the power of simple genetic techniques that exploit phage. Indeed, development of a generalized transducer is still an extremely useful tool for genetic analysis of any bacterial strain. A recent stimulation in phage research and the ‘-omics era’ has provided major advances in knowledge of the genomes, evolution, ecology, and diversity of phage. For example, the host-adapted function of GTAs is a salient reminder that some sequences that may appear like ‘defective’ prophage in bacterial genomes can in fact be highly effective HGT mechanisms proficient in

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generalized transduction. It is anticipated that the recent rejuvenation in phage biology research will continue to further our appreciation and understanding of transduction, both as a tool for bacterial geneticists and as an important evolutionary force in the adaptation of phage and their bacterial hosts. See also: Bacteriophage Ecology; Bacteriophage (overview); Horizontal Transfer of Genes between Microorganisms; Bacteriophage Therapy: Past and Present

Further Reading Calender R (ed.) (2006) The Bacteriophages, 2nd edn. New York: Oxford University Press. Lang AS and Beatty JT (2001) The gene transfer agent of Rhodobacter capsulatus and ‘‘constitutive transduction’’ in prokaryotes. Archives of Microbiology 175: 241–249. Margolin P (1987) Generalized transduction. In: Ingraham JL and Neidhardt FC (eds.) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 1st edn., pp. 1154–1168. Washington, DC: American Society for Microbiology. Masters M (1996) Generalized transduction. In: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikopp WS, Riley M, Schaechter M, and Umbarger HE (eds.) Escherichia coli and Salmonella typhimurium: Cellular & Molecular Biology, 2nd edn., pp. 2421–2441. Washington, DC: American Society for Microbiology. Miller RV (2001) Environmental bacteriophage–host interactions: factors contribution to natural transduction. Antonie Van Leeuwenhoek 79: 141–147. Morse ML, Lederberg EM, and Lederberg J (1956) Transduction in Escherichia coli K12. Genetics 41: 142–156. Mulholland V and Salmond GPC (1995) Use of coliphage  and other bacteriophages for molecular genetic analysis of Erwinia and related Gram-negative bacteria. In: Adolph KW (ed.) Microbial Gene Techniques, pp. 439–454. London: Academic Press. Paul JH (1999) Microbial gene transfer: and ecological perspective. Journal of Molecular Microbiology and Biotechnology 1: 45–50. Smith MCM and Rees CED (1999) Exploitation of bacteriophages and their components. In: Smith MCM and Sockett RE (eds.) Methods in Microbiology, vol. 29, pp. 97–132. London: Academic Press. Sternberg NL and Maurer R (1991) Bacteriophage-mediated generalized transduction in Escherichia coli and Salmonella typhimurium. Methods in Enzymology 204: 18–43. Toussaint A (1985) Bacteriophage Mu and its use as a genetic tool. In: Scaife J, Leach D, and Galizzi A (eds.) Genetics of Bacteria, pp. 197–215. London: Academic Press. Weinbauer MG (2004) Ecology of prokaryotic viruses. FEMS Microbiology Reviews 28: 127–181. Weisberg RA (1987) Specialized transduction. In: Ingraham JL and Neidhardt FC (eds.) Escherichia coli and Salmonella typhimurium: Cellular & Molecular Biology, 1st edn., pp. 1169–1176. Washington, DC: American Society for Microbiology. Weisberg RA (1996) Specialized transduction. In: Neidhardt FC, Curtiss III R, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikopp WS, Riley M, Schaechter M, and Umbarger HE (eds.) Escherichia coli and Salmonella typhimurium: Cellular & Molecular Biology, 2nd edn., pp. 2442–2448. Washington, DC: American Society for Microbiology. Zinder ND and Lederberg J (1952) Genetic exchange in Salmonella. Journal of Bacteriology 64: 679–699.

Transposable Elements W S Reznikoff, Marine Biological Laboratory, Woods Hole, MA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction The Advent of In Vitro Transposition Systems Genome-Wide Knockout Analyses

Glossary bacteriophage A virus that infects bacteria. CFP Cyan fluorescent protein. donor DNA The DNA on either side of the transposon from which the transposon moves during transposition. recognition end sequences The short DNA sequences that define the two ends of a DNA transposon. Recognition end sequences are recognized by the transposon-specific transposase protein. synapsis The formation of a transposase–transposon DNA dimeric complex, t is a required intermediate in transposition.

Abbreviations CFP

cyan fluorescent protein

Defining Statement Transposons are powerful molecular genetic tools for performing both genome-wide genetic analyses and studies targeted to particular genes or gene regions. In addition to being amenable to standard genetic analyses, transposon technologies are important adjuncts to modern high-throughput techniques.

Introduction The first use of transposable elements (to be called transposons for the remainder of this article) as tools for microbial genetics dates from the discovery of bacteriophage Mu by AL Taylor over 40 years ago. Taylor’s initial report on Mu described how one could generate a wide variety of auxotrophic mutations merely by creating Mu lysogens. The frequency and variety of these mutations within a lysogen population suggested that all lysogens were mutants in which the phage genome had inserted into one of a wide variety of bacterial genomic

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Transposable Element-Based Deletion Studies Targeting Individual Genes Conclusion Further Reading

target DNA The DNA into which the transposon DNA is inserted following transposition. Tn5 Transposon 5, a particular DNA transposon. transposase The protein that catalyzes DNA transposition. transposition The process by which a defined DNA sequence moves from one site in the genome to a second site. transposon The defined DNA sequence that is moved as a consequence of transposition. YFP Yellow fluorescent protein.

ER end recognition YFP yellow fluorescent protein

sites. In other words, Taylor had discovered a biologically based mutagen that was incredibly efficient, apparently random, and very easy to use. It was this observation by Taylor, bolstered by discussions of Mu’s properties at Cold Spring Harbor Phage Meetings, that led various investigators to adopt Mu as a genome-scanning, knockout, mutagen. My own adoption of this tool, soon after I arrived at the University of Wisconsin–Madison as a new faculty member, was initiated by my attempt to use Mu to identify positive regulatory genes for the Escherichia coli tryptophan operon. I used a trp-lac fusion strain and looked for Lac Mu lysogens on Lactose-MacConkey agar. As it frequently happens in genetics, the selection/screen worked to yield interesting mutations (in the gene encoding phosphoglucose isomerase), but not in the mythical gene that I had imagined to exist. Nonetheless, the power of transposons as genetic tools was not lost on me and many other investigators. The widespread acceptance of transposons as genetic tools in the E. coli and Salmonella typhimurium research communities followed from the discovery of two other types of transposons. An analysis of spontaneous knockout

Genetics, Genomics | Transposable Elements

mutations in E. coli indicated that many of them were caused by the insertion of identical DNA sequences. These became to be known as IS elements, each type encoding its own transposase and containing specific terminal sequences that were recognized by the cognate transposase at the initiation of transposition. A second related class of mobile genetic elements was discovered in the mid-1970s. These elements were antibiotic resistance-encoding transposons that could move from one replicon (for instance, an antibiotic resistanceencoding plasmid called an R factor) to a second replicon (such as bacteriophage ). I directly benefited from these discoveries as Jim Shapiro (one of the discoverers of IS elements) and I were postdoctoral fellows together shortly after his discovery of IS-associated mutations, and the next step in my research career led me to a laboratory adjacent to that of Julian Davies, who shortly thereafter discovered the antibiotic resistance-encoding transposon Tn5. The discoveries of IS elements and antibiotic resistanceencoding transposons were coupled with the then ‘new’ recombinant DNA techniques to enable the structural dissection, modification, and genetic analysis of several transposons. A generic view of transposon structure is presented in Figure 1. An important result of these studies was the detailed determination of the key steps in the transposition process for several transposons (Figure 1 also presents a schematic of transposition through one well-studied mechanism, ‘cut and paste’ transposition).

Molecular Participants in Transposition The key molecular participants in ‘cut and paste’ transposition (see Figure 1) are quite simple. As described below, these participants include transposon-specific transposase, transposon DNA defined by recognition end sequences, target DNA, and Mg2þ. 1. Transposase For each type of transposon, there exists an element-specific protein called a transposase, which catalyzes the biochemical steps in transposition. For many frequently used transposons, the transposase is the only required protein although in some cases host proteins or additional transposon-encoded proteins are required. 2. Recognition end sequences (defining the transposon structure) Each transposon end is defined by a specific DNA recognition end sequence to which the transposase specifically binds to initiate the transposition process. For some transposons, these ends are as short as 18 bp. If the transposase is encoded elsewhere, the content and length of the DNA found between the two recognition end sequences can be anything, provided it is not too short (a 256-bp Tn5 derivative has been

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successfully used) or too long as to prevent the formation of transposition intermediates (see Figure 1). 3. Target DNA The final step in transposition involves the integration of the transposon DNA into the target DNA sequence on the same or on a different DNA molecule (Figure 1). Many transposons are rather random in their target sequence choice with the exact target primarily chosen based on the random collision between the excised element and the target. However, there are some sequence biases that have been determined for those transposons that have been carefully studied. 4. Mg2þ (or Mn2þ) The divalent cation Mg2þ plays an absolutely key role in the catalytic steps for transposition.

Transposon Content The realization that transposon DNA is flexible in content and the use of recombinant DNA technology to modify the content of transposons have been important steps in developing transposons as true tools in molecular genetic analyses, making them far more than mere knockout mutagenesis agents. In essence, anything that the investigator can conceive of with regard to a desired DNA sequence can be incorporated between the two recognition end sequences. General categories of interesting DNA sequences to consider include the following. 1. Selectable functions The fundamental genetic information that is needed within a transposon for almost all applications is a selectable marker. In the original Mu work of Taylor, the selectable marker was immunity to Mu superinfection. Transposons in use today typically encode resistance to one or more antibiotics. The architects of these antibiotic-resistant transposons have merely borrowed from or copied the transposons that were discovered in the 1970s. 2. Reporter functions A variety of reporter functions have been included in the body of transposons typically with the reporter gene abutting one recognition end sequence. In operon fusion systems, the reporter gene is complete with its own translation initiation signals, but lacks a transcription initiation signal between the reporter gene and the upstream end of the element. With this type of construct, the reporter gene expression will be driven by transcription that reads from the DNA adjacent to the target site and provides a qualitative measure of the level of the resulting fusion mRNA synthesis. Gene fusion reporter systems fuse an N-terminal truncated protein encoded by the transposon to a protein encoded by the target sequence. Thus, the fusion can be used to tag the target protein for determining its subcellular localization and to evaluate the level of transcription and translation of the

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Genetics, Genomics | Transposable Elements Recognition end sequence Transposon

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Figure 1 Transposon structure and ‘cut and paste’ DNA transposition. DNA transposons are DNA sequences that are defined by specific end sequences (represented by two open triangles). Typically, a natural transposon contains a gene that encodes the specific transposase (represented by an open circle) that catalyzes transposition after binding to the specific recognition end sequences. However, if the transposase is supplied from somewhere else, the DNA between the specific end sequences can contain a wide variety of other genetic information. The figure additionally presents a simplified schematic of ‘cut and paste’ DNA transposition. The transposase binds to the recognition end sequences to form a synaptic complex. In the presences of Mg2þ, the transposase in the synaptic complex catalyzes cleavage of the DNA between the recognition end sequences and the donor DNA, thus releasing the transposition complexes. The transposition complexes then bind to target DNA and the transposase then catalyzes strand transfer, thus inserting the transposon into the target. Further details on transposon structure and the ‘cut and paste’ transposition mechanism can be found in Nancy Craig and Williams Reznikoff.

targeted gene. Typical reporter genes include those that encode -galactosidase, alkaline phosphatase, and various fluorescent proteins. A specific example of the use of fluorescent protein fusions will be given later. 3. Landmarks Transposons by their very nature represent landmarks. That is, their integration inserts a recognizable DNA sequence within the target DNA and their locations and orientations can be easily mapped against other known genome locations. In addition,

transposons have been designed to carry specialized landmarks. An obvious class of landmarks carried by all transposons is a primer-binding site that can be used for sequencing adjacent target DNAs. It is for this reason that transposons are frequently used as powerful DNA sequencing tools. Transposons have been designed to carry sequences that are targets for sitespecific recombination systems (such as a P1 lox site, a  att site, or an FRT site) so that additional genetic information can be subsequently incorporated or

Genetics, Genomics | Transposable Elements

unwanted transposon sequences can be removed. Rare cleavage sites can be included for physical mapping of insert locations or as counterselectable markers against the presence of the transposon or to allow the removal of unwanted transposon-encoded sequences (see below for an example of such a sequence removal process). Finally, transposons that carry multiple copies of lacO or tetO can be located through fluorescence microscopy within a cell or on long DNAs using cognate repressors fused to GFP derivatives (for instance, LacI-CFP and TetR-YFP). An interesting class of landmarks, which can be derived from inserted transposons, are sequences that encode specific amino acid residues in the interrupted target gene product. For instance, the encoded sequence may involve the insertion of a unique protease-sensitive site or a particular epitope. In general, these types of insertion products are examples of linker-scanning mutations. Typical transposons that are used to generate these insertions include a translation-fusion reporter function so that only insertions in the desired orientation and in the correct reading frame are chosen for subsequent analysis. They also have modified

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recognition end sequences so that they can encode the desired peptide-encoding sequence and there are sitespecific recombination sites or rare restriction sites that allow the simple excision of the bulk of the transposon. Following excision, an insert containing N  3 nucleotides encoding the desired sequence is left behind within the target-encoded protein (Figure 2). In some cases, the modification is even more detailed, for instance, the resulting protein can have a single amino acid insertion, substitution, or deletion. Further examples will be presented subsequently. 4. Controlling elements Controlling elements are cis active DNA (or RNA) sequences through which the inserted transposon can direct adjoining DNA (or RNA) activities. Examples of controlling elements include the following: a. Promoter sequences for directing the transcription of adjacent DNAs. b. Transcription termination sequences to insure that the inserted transposon shall have a polar knockout effect on downstream genes. c. Origins of replication that will allow the inserted transposon to act (in conjunction with restriction

Cleavage sites Reporter-selector

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Figure 2 Transposons and the generation of linker-scanning mutations. Transposons can be used to generate linker-scanning mutations. The transposon showed contains genes for a protein fusion reporter gene and an antibiotic resistance (selector). Inserts are selected with the selector and those inserts in the correct orientation and reading frame are identified with the reporter. The bulk of the transposon is excised using a specific cleavage site and ligation leaving the linker sequence in various random locations. See Williams Reznikoff for a modified version of this figure.

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cleavage beyond the boundaries of the transposon followed by ligation) as a cloning tool for adjacent DNAs. d. Origins of DNA transfer. e. End recognition sites for an alternative transposase, thus constructing a composite transposon (a use for including alternative transposon end recognition sites will be described subsequently).

The Advent of In Vitro Transposition Systems The initial applications of transposon technology were performed with in vivo transposition reactions. Typically, the investigator would construct a transposon within a socalled suicide vector. Suicide vectors cannot replicate within the target host under defined conditions. In many cases, the gene encoding the transposase would be located outside of the boundaries of the transposon. The first consequence of the suicide vector design is that the only way in which the transposon-encoded selectable marker could be inherited by the target host would be as a consequence of the transposon transposing off of the suicide vector into the genome of the host. The second consequence would be that the transposase gene would be lost from the host cell. The latter result is important because it would prevent subsequent confounding transposition events. The above systems were typically tailor-made for work with E. coli and S. typhimurium. Moreover, they often required genetic manipulations that may not be familiar to today’s molecular biologists. In addition, these approaches do not lend themselves to transposition procedures that are targeted to defined DNA regions. These limitations have been addressed as a result of the development of in vitro methodologies for studying transposition. Although in most cases, in vitro transposition studies were directed at achieving a basic science molecular understanding of DNA transposition mechanisms, the obvious fruit of these studies was the development of molecular genetic tools based on these in vitro technologies in whole or in part. The key transposition systems that were developed into in vitro molecular genetic tools include Ty1, Tn7, Tn5, Mariner, Mu, and Tn552. For original references and descriptions of these systems see Further Reading. The most obvious application of the in vitro technologies is performing the transposition events on target DNA in the test tube and then introducing the mutagenized DNA into the target cells. The main advantage of this technology is that the target can be restricted to one specific DNA sequence; in some cases an individual gene or gene segment, and in other cases a bacterial artificial chromosome or virus genome. One use of the latter is in the application of transposition technology for

high-throughput DNA sequencing (the transposon contains two divergent mobile primer binding sites). A second important derivative of the in vitro studies is the ability to perform a partial in vitro reaction (constructing transposase–transposon DNA complexes in vitro) followed by electroporating or micro-injecting the transposase–transposon complexes into living cells after which the transposase integrates the transposon DNA into the cells’ genomes. This combined in vitro–in vivo methodology has only been published for the Tn5 and Mu systems. The specific applications of both of the above techniques are mentioned in detail below.

Genome-Wide Knockout Analyses An important approach to genome functional analysis is to generate knockout mutations in as many genes as possible. Obviously, transposons can be the mutagen of choice. The mutagen is highly efficient and in most cases the resulting mutation is an absolute knockout. The mutagen can be delivered in such a way that the great majority of survivors only have single unique mutations, the distribution of mutation sites is relatively random, and the location of individual mutation sites is easy to determine. There are two general approaches that use this methodology. First, one generates a large library of viable insert mutations and then screens or selects for the mutants with the desired phenotype. That is exactly the approach that I used as a new independent investigator when I accidentally isolated a phosphoglucose isomerase-defective mutant. With the advent of genome sequencing information and techniques such as microarray analyses, one can now identify and analyze several different mutants that have similar phenotypes at the same time. For instance, a number of research groups have described techniques in which a large collection of inserts is interrogated in bulk for their members by generating runoff transcripts that include both ends of the transposable element and adjacent DNA and then hybridizing the RNAs to microarrays, thus identifying which inserts are present in the collection (Figure 3). By presenting the collection with particular growth challenges and repeating the microarray analyses, one can determine which inserts in which genes cause growth impairment. Thus, this procedure identifies the phenotypes (nutritional requirements) for a class of inserts. The second approach is designed to identify putative essential genes. In this technology, a large insert library (hopefully a saturated collection with all genes suffering inserts within the collection) is generated. The experimentalist then determines which genes fail to have any inserts represented in the collection. One particular execution of this essential gene hunt was described by Svetlana Gerdes and colleagues in 2002. The pool of transposable element inserts is interrogated by polymerase chain reaction (PCR) analysis using a transposon-based primer and one of the

Genetics, Genomics | Transposable Elements T7 promoter Transposon T7 promoter

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The assumption from these studies is that if no inserts for a particular gene are found in the collection, the gene must encode an essential function. However, there are alternative trivial explanations for not finding inserts in a specific gene, such as bad luck or target sequence biases. Therefore, the investigator needs to confirm the identification of a particular gene being essential by other means, such as deletion analysis or individual targeted gene studies that are described below.

Select for viable colonies Transposon library construction Competitive library outgrowth CONTROL

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Isolate chromosomal DNA, label and hybridize samples to oligonucleotide microarrays (see Figure 2 for details)

Microarray data analysis Identify transposon insertion locations (CONTROL)

Identify transposon insertion locations (TEST)

Compare data sets to identify mutants lost during selective outgrowth Figure 3 Genome-wide microarray screening of transposon insertions. The identification, localization, and tracking of multiple transposon inserts can be accomplished by using microarray hybridization. In one version of this technology, the transposon is constructed to have outward-facing T7 promoters. The collection of inserts is harvested, the DNAs extracted, and probes of the DNA sequences adjacent to the inserts are generated using T7 RNA polymerase. The RNA is labeled and hybridized to appropriate microarrays. Reproduced from Winterberg KM and Reznikoff WS (2007) Screening transposon mutant libraries using full-genome oligonucleotide microarrays. In: Kelly T Hughes and Stanley R Maloy (eds.) Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering, Methods in Enzymology Series, Vol. 421, pp. 110–125. San Diego, CA: Elsevier Inc.

several strategically located genome primers. All of the viable inserts are represented by PCR products of predicted sizes, whereas essential genes are tentatively identified by the absence of blocks of PCR products.

Transposable Element-Based Deletion Studies Transposable elements of the composite transposon class have the capacity to generate adjacent deletions. In this section, I describe the use of this property to study the essentiality of genes (or groups of genes). In a subsequent section, I describe how composite transposon deletion generation can be used to generate nested families of protein deletions. Composite transposons (such as Tn5 or Tn10) can be thought of as being composed of four different types of transposable elements depending on the precise recognition end sequences that are chosen by the transposase for synaptic complex formation (Figure 4). Using the nomenclature presented in Figure 4, one can see that Tn5 transposition will involve OLER–ORER synapsis. But it is also possible to have OLER–ILER or IRER– ORER synapsis, in which case one would have IS50L or IS50R transposition, respectively. Of interest to adjacent DNA deletion formation is the possibility of having IRER– ILER synapsis, in which case a new transposable element has been formed; the IS50 elements and the donor DNA now compose the transposable element (Figure 4). IRER–ILER intramolecular transposition results in one of the two types of DNA rearrangements for the DNA between these two ends, either a deletion or an inversion (Figure 5). The IRER–ILER intramolecular deletion formation potential has been developed into a practical chromosome deletion tool by utilizing a transposase that is selective for I-ER sequences (as apposed to O-ER sequences) and by genetically marking the components of the new composite transposon so that only deletions are isolated. The resulting system has been used to delete random sections of the E. coli chromosome and thereby can be used to define which genes are not essential (they can be deleted and still yield a viable organism).

Targeting Individual Genes The most important consequence resulting from the development of in vitro transposition systems is the ability

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generate a nested family of deletions of the target gene. These deletions can be used to map epitopes or specific domains of interest to the investigator.

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IS50L–donor DNA–IS50R Figure 4 Composite transposons can give rise to four types of transposons. Composite transposons such as Tn5 contain two identical or nearly identical insertion sequences (IS50L and IS50R in the case of Tn5) that bracket additional genes. Depending on which end recognition (ER) sequences are chosen by the transposase during synapsis, four different transposons can be mobilized: Tn5 mobilization involves OLER–ORER synapsis, IS50L mobilization involves OLER–ILER synapsis, IS50R mobilization involves IRER–ORER synapsis, and IS50L–donor DNA–IS50R mobilization involves ILER–IRER synapsis.

to target transposition to specified DNA sequences such as individual genes. From this general technology a number of specific applications are derived. An obvious use of targeted in vitro transposition is to generate gene-specific knockouts and then attempt to introduce the knockouts as substitutions for the intact gene in the host organism. If the knockout organism can be isolated and propagated, the disrupted gene is not essential. If the experimenter is unable to isolate cells that contain the insertion, the negative result is prima facie evidence that the gene is essential. As described below, other applications of targeted in vitro transposition lend themselves to a variety of techniques that allow the analysis of protein structurefunction.

Protein Structure-Function Studies: Generating Random Nested Deletions As mentioned above, intramolecular transposition can be used to generate deletions. A straightforward adaptation of the intramolecular transposition/deletion technology to generate nested deletions in a protein-encoding gene first requires the construction of a transposon containing the target gene through recombinant DNA techniques. Once it is constructed, intramolecular transposition will

Protein Structure-Function Studies: Generating In-Frame Microinsertions, Deletions, and Substitutions Transposons have become widely used (and commercially available) tools for generating in-frame linker insertions. The principle, as outlined in Figure 2, typically follows the general steps mentioned below. At first, one uses translational fusion technology (inserting a reporter gene lacking transcription and translation initiation signals into the target gene of choice) to capture inserts with the correct orientation and reading frame. Then one excises the bulk of the transposable element either using rare restriction enzyme digestion followed by ligation or a site-specific recombination system leaving an in-frame insertion, whose sequence is dictated by the residual transposon sequence. Of course, a precisely constructed transposon needs to be used for this technology. The small inserts can be used to map structural domains of the protein (functional proteins typically result only from insertions in unstructured regions) or to insert an epitope or protease target sequence. An exciting extension of this insertion technology has been developed in the laboratory of Dafydd Jones. In this technique, a specialized version of a mini-Mu transposon is used for the initial mutagenesis, and the off-set cleavage activity of a type IIS restriction enzyme is used to perform the excision of the bulk of the transposon. Depending on precisely how the procedure is applied, and whether an intermediate cloning is performed or not, the technique can be utilized to generate precise 3-bp (base pairs) deletions or additions, or precise 3-bp substitutions at the site of transposon insertion. The latter is particularly powerful. Imagine generating a random collection of single insertions on the target gene of choice and then generating known sequence substitutions at each of the sites. Protein Structure-Function Studies: Generating Random Protein Fusions In all the above protocols, a constant sequence (the transposon) is juxtaposed against a random sequence (the target). By utilizing a specifically designed composite transposon and transposases that were specific for the two types of recognition end sequences (I-ER and O-ER), we developed techniques that fused two genes in a random fashion with a sequence composed of an I-ER and O-ER between the two fused gene sequences (Figure 6). We applied this technology only once. The results were that active product fusion proteins were generated only when the resulting fusion partners were in the

Genetics, Genomics | Transposable Elements

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Figure 5 Intramolecular transposition and adjacent gene deletion formation. Intramolecular transposition that utilizes ILER–IRER synapsis as shown in Figure 4 has been used to generate adjacent chromosomal deletions as a means to define nonessential genes and to reduce the size of the Escherichia coli chromosome. A transposase specific for the open triangle end recognition sequences forms synaptic complexes, cleaves the DNA free of the Kan-Tnp-EK/LP-encoding donor DNA, and catalyzes intramolecular transposition, which can generate deletion formation to give the ‘restored chromosome’ shown. Reproduced from Goryshin IY, Naumann TA, Apodaca J, and Reznikoff WS (2003) Chromosomal deletion formation system based on Tn5 double transposition: Use for making minimal genomes and essential gene analysis. Genome Research 13: 644–653.

same reading frame and orientation and when the proteins were fused utilizing unstructured regions between secondary structure domains. There is no obvious reason why this technology cannot be applied in generating a variety of functional fusion proteins. Protein Structure-Function Studies: Generating Random Reporter Gene Fusions The ability to generate reporter fusions to a target protein is very useful. An example is suggested above in

the ‘Landmarks’ in which a LacI-CFP (cyan fluorescent protein) and TetR-YFP (yellow fluorescent protein) were used to locate Mariner constructs that contained (lac O)n or (tet O)n, respectively. Typically, tagged fusion proteins are constructed using recombinant DNA technology to generate N- or C-terminal fusions. This construction method makes the assumption that one or the other type of fusion will maintain optimal protein function and probably eliminates the possibility of using the reporter as a probe for changes in protein conformation.

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Figure 6 Two sequential transposition events used to generate random gene fusions. The gene fusion technology utilizes a transposase that recognizes closed triangle end recognition sequences to first insert the transposon into target gene A and then a transposase that recognizes open triangle end recognition sequences to insert the newly formed transposon carrying the two halves of gene A into gene B. Some of the resulting products encode random fusions of genes A and B. Reproduced from Naumann TA, Goryshin IY, and Reznikoff WS (2002) Production of combinatorial libraries of fused genes by sequential transposition reactions. Nucleic Acids Research 30: e119, with permission from Oxford University Press. See also Williams Reznikoff.

Genetics, Genomics | Transposable Elements Tn5 RS

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GCCCGGGCAGATGTGTATAAGAGACAG AlaArgAlaAspValTyrLysArgGln

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Figure 7 Transposon for generating random yellow fluorescent protein (YFP) (or cyan fluorescent protein, CFP) fusions. The transposon shown will generate KanR, YFP fusion as a result of insertions in the correct orientation and reading frame into a target gene. Digestion with Sr f I and ligation will excise the bulk of the transposon, generating a fusion of both the N- and C-terminal portions of the target gene sequence to YFP. Alternatively, digestion with AscI followed by ligation will generate a fusion to CFP. Reproduced from Reznikoff WS (2006) Tn5 transposition: a molecular tool for studying protein structure-function. Biochemical Society Transactions 34(part 2): 320–323 and Sheridan DL and Hughes TE (2004) A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins. BMC Biotechnology 4: 17–25.

One can also make CFP or YFP fusions using transposon technology. By using the construct described in Figure 7, one can search a wide variety of fusions for the ones that have optimal properties. The concept is to initially generate random YFPþ fusions (in the correct orientation and in frame) to the gene of interest. Following the fusion generation, an in-frame portion of the transposon (including the translation termination signal) is removed using Sr f I cleavage and ligation. The resulting product encodes the N-terminal target-YFP (active)-C-terminal target. These fusion constructs are then examined to find those that have maintained maximal target protein activity and/or those that make YFP emission sensitive to target protein environment or function. The construct is designed also to allow the generation of CFP fusions from the same inserts.

Conclusion Transposons are powerful tools in the whole genome structure studies and in the analysis of protein (and RNA) structure-function. Although there is an energy barrier to their adoption in some laboratories, once accepted they combine ease of use, great flexibility, and straightforward interdigitation with other technologies. See also: Conjugation, Bacterial; Genetically Modified Organisms: Guidelines and Regulations for Research; Horizontal Transfer of Genes between Microorganisms; Genetics, Microbial (general); Plasmids, Bacterial; Transduction: Host DNA Transfer by Bacteriophages; DNA Sequencing and Genomics; Genome Sequence Databases: Genomic, Construction of Libraries; Genome Sequence Databases: Sequencing and Assembly; Recombinant DNA, Basic Procedures

Further Reading Berg CM and Berg DE (1996) Transposable element tools for microbial genetics. In: Neidhardt FC, Curtiss R III, Ingraham JL, et al. (eds.) Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn., pp. 2588–2612. Washington, DC: ASM Press. Berg DE, Davies J, Allet B, and Rochaix JD (1975) Transposition of R factor genes to bacteriophage. Proceedings of the National Academy of Sciences of the United States of America 72: 3628–3632. Boeke JD (2002) Putting mobile DNA to work: The tool box. In: Craig NL, Craigie R, Gellert M, and Lambowitz AM (eds.) Mobile DNA II, pp. 24–37. Washington, DC: ASM Press. Craig NL, Craigie R, Gellert M, and Lambowitz AM (2002) Mobile DNA II. Washington, DC: ASM Press. Gerdes SY, Scholle MD, D’Souza M, et al. (2002) From genetic footprinting to antimicrobial drug targets: Examples in cofactor biosynthetic pathways. Journal of Bacteriology 184: 4555–4572. Goryshin IY, Naumann TA, Apodaca J, and Reznikoff WS (2003) Chromosomal deletion formation system based on Tn5 double transposition: Use for making minimal genomes and essential gene analysis. Genome Research 13: 644–653. Hughes KT and Maloy ST (2007) Methods in Enzymology, Vol. 421. Advanced Bacterial Genetics: Use of Transposons and Phage for Genetic Engineering. San Diego, CA: Academic Press. Jones DD (2005) Triplet nucleotide removal at random locations in a target gene: The tolerance of TEM-1 -lactamase to an amino acid deletion. Nucleic Acids Research 33: e80. Reznikoff WS (2002) Tn5 transposition. In: Craig NL, Craigie R, Gellert M, and Lambowitz AM (eds.) Mobile DNA II, pp.403–422. Washington, DC: ASM Press. Reznikoff WS (2006) Tn5 transposition: A molecular tool for studying protein structure-function. Biochemical Society Transactions. 34(part 2): 320–323. Shapiro JA (1969) Mutations caused by the insertion of genetic material into the galactose operon of E. coli. Journal of Molecular Biology 40: 93–105. Sheridan DL and Hughes TE (2004) A faster way to make GFP-based biosensors: Two new transposons for creating multicolored libraries of fluorescent fusion proteins. BMC Biotechnology 4: 17–25. Taylor AL (1963) Bacteriophage-induced mutation in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 50: 1043–1051. Vinopal RT, Hillman JD, Schulman H, Reznikoff WS, and Fraenkel DG (1975) New phosphoglucose isomerase mutants of Escherichia coli. Journal of Bacteriology 122: 1172–1174. Viollier PH, Thanbichler M, McGrath PT, et al. (2004) Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. Proceedings of the National Academy of Sciences of the United States of America 101: 9257–9262.

HISTORY AND CULTURE, (AND BIOGRAPHIES) Contents AIDS, Historical Biographies Cholera, Historical History of Microbiology Methods, Philosophy of Plague, Historical Smallpox, Historical Spontaneous Generation Syphilis, Historical Typhoid, Historical Typhus Fevers and Other Rickettsial Diseases, Historical

AIDS, Historical D S Jones, Massachusetts Institute of Technology, Cambridge, MA, USA A M Brandt, Harvard Medical School, Harvard University, Boston, MA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Pathophysiology of HIV and AIDS Origins and Spread of HIV Emergence and Recognition of AIDS

Glossary ART (anti-retroviral therapy) Broadly defined, these include any medications active against HIV. In practice, ART usually refers to a combination of a protease inhibitor and two reverse transcriptase inhibitors that have powerful inhibitor effects against HIV replication. Also known as highly active anti-retroviral therapy (HAART). HIV (human immunodeficiency virus) An RNA retrovirus, related to a series of simian immunodeficiency viruses, which is the cause of AIDS. opportunistic infections Infections that do not cause significant disease in nonimmunocompromised hosts,

AIDS Before Effective Treatment: Fear and Blame The Development of Effective Treatments The Challenge of Providing Care Further Reading

but can cause severe disease in people with AIDS; for example, Kaposi’s sarcoma (KS) and pneumocystis carinii pneumonia (PCP). stigmatization The process by which the stigma attached to certain behaviors, notably homosexuality and intravenous drug use, is transferred to a disease, such as AIDS, having a substantial impact on theories of, and responses to, the disease. viral load A measure of the concentration of HIV RNA in a person’s blood. It can be used to screen patients for HIV and to monitor the progress of therapy.

1

2 History and Culture, (and Biographies) | AIDS, Historical

Abbreviations 3TC ACT UP AIDS ART AZT CDC CMV ELISA HPA-23 HAART

Lamivudine AIDS Coalition to Unleash Power Acquired immune deficiency syndrome anti-retroviral therapy azidothymidine Centers for Disease Control and Prevention cytomegalovirus enzyme-linked immunosorbent assay Heteropolyanion-23 highly active anti-retroviral therapy

HIV HTLV-III IDUs KS LAV PCP PCR PEPFAR SIV STDs

human immunodeficiency virus human T-lymphotropic virus type III intravenous drug users Kaposi’s sarcoma lymphadenopathy virus pneumocystis carinii pneumonia polymerase chain reaction President’s Emergency Plan for AIDS Relief simian immunodeficiency virus sexually transmitted diseases

Defining Statement

Pathophysiology of HIV and AIDS

The appearance and rapid globalization of the AIDS pandemic demonstrate the social and economic processes that shape the distribution of diseases at present. These same processes also determine access to life-saving treatment which, as of yet, has reached only a minority of people who would benefit from it.

HIV is an RNA retrovirus with fewer than 10 000 bp. These code for three structural genes and six regulatory genes that are, in turn, transcribed and spliced in a variety of ways to produce a total of 15 proteins. The viral particles, or virions, have an icosahedral structure. Two identical strands of RNA and the virus’s crucial proteins – protease, integrase, and reverse transcriptase – are enclosed within a bullet-shaped core, composed of the capsid protein (p24). This, in turn, is surrounded by the viral envelope, composed of an inner layer, made up of the matrix protein (p17), and an outer lipid layer. Seventy-two spikes, made up of proteins gp120 and gp41, protrude from this envelope. The virus can attack any cell that expresses the CD4 molecule and a coreceptor, often the chemokine receptors CCR5 or CXCR4. The virus’s gp120 proteins bind to CD4, allowing the viral envelope to fuse with the cell membrane, releasing the core into the cell’s cytoplasm. Once there, viral reverse transcriptase translates the viral RNA into DNA. This viral DNA is then inserted, by viral integrase, into the genome of the infected cell. The cell’s own machinery then transcribes and translates the viral DNA, producing a single long protein. This is cleaved, by the viral protease, into the 15 proteins. New virions form and bud from the infected cell. A chronically infected person can produce more than 10 000 000 000 virions each day. HIV primarily targets the CD4þ subset of T lymphocytes, known as the helper T cells, which regulate crucial aspects of the normal immune response. But any cell that expresses CD4 can be infected. Infection has been reported in macrophage, epithelial cells in the gastrointestinal and urinary tracts, astrocytes, microglial cells, cardiac myocytes, and a variety of other cell types. Infected individuals can transmit infection through

Introduction Acquired immune deficiency syndrome (AIDS), first identified in 1981 is an infectious disease caused by the human immunodeficiency virus (HIV). The virus attacks the host’s immune system, causing its eventual failure. This failure leaves affected individuals vulnerable to many infections and cancers, leading inexorably to severe morbidity and high mortality. HIV emerged in the middle of the twentieth century, following the infection of humans with simian immunodeficiency viruses (SIV). Spread sexually and through blood, it penetrated populations in Africa, Europe, and the United States in the 1970s. AIDS appeared in the 1980s caused considerable fear and provoked dramatic social responses. Despite rapid progress in scientific understanding and medical treatment of the disease, and despite the existence of valuable preventive technologies, HIV continued to spread rapidly throughout the world in the 1980s and 1990s. Decisive treatments appeared in the late 1990s, triggering debates about whether or not these could be implemented on a global scale. Although dramatic progress has been made, disparities in risk of infection and in access to treatment continue to expose critical inequities in the distribution of social and medical resources in developed and developing countries.

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blood, semen, and other genital secretions. Heterosexual transmission is responsible for most of the infections worldwide, possibly as many as 85%. However, substantial transmission has also occurred through maternal–fetal infection (during pregnancy, birth, or breast-feeding), homosexual contact, needle-sharing by intravenous drug users (IDUs), and contaminated blood products. Transmission has also been reported through organ donation, renal dialysis, artificial insemination, and acupuncture. HIV cannot be transmitted by insect bites, and it is almost never transmitted through casual contacts at home or in public. The predominant mode of transmission varies substantially from country to country. In sub-Saharan Africa, most cases have occurred from heterosexual contact. In the United States, in contrast, roughly 45% of the cases have occurred among men who have sex with men, 25% among IDUs, and just over 10% among heterosexuals. Meanwhile, in India, transmission through drug use predominates in the northeastern states, while heterosexual transmission is most common in the south. Since modes of transmission vary so widely from place to place, public health campaigns to prevent transmission must be targeted to reflect local circumstances. When HIV enters a body, initial infection and replication produce a surge in viral load in the victim’s blood (viremia). The viremia triggers a vigorous immune response: antibodies are generated, especially against p24 and gp41, and the infection is initially contained, largely within the body’s lymphoid tissues. This first stage of infection often goes unnoticed, though some individuals do experience a flulike prodromal syndrome characterized by fever, rash, and malaise. After this initial stage, an individual may remain free of symptoms for many years. During this time, the virus continues to attack the immune system, causing rapid turnover of CD4þ lymphocytes. In most people, the virus eventually gains the upper hand: the CD4þ count slowly but steadily declines and immune functions become increasingly compromised. This latent period, in which infected individuals are generally healthy, typically lasts for 10 years. Other outcomes are also possible. In roughly 10% of people, for instance those with mutations to CCR5, the immune system contains the virus and there is no progressive loss of immune function. These people have remained disease free for as long as 20 years after infection. In other individuals, especially in the setting of malnutrition and tuberculosis, the progression from infection to AIDS to death occurs within 1 year. In the majority of cases, HIV eventually cripples the host’s immune system, leaving the infected individual vulnerable to other disease-causing agents in the environment. Clinical signs of infection generally begin to appear when the person’s CD4þ count, normally around 1000 cells ml1, drops below 500. As the immune system deteriorates further, a series of opportunistic infections

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begin to appear, including thrush, tuberculosis, pneumocystis carinii, histoplasmosis, toxoplasmosis, herpes, cytomegalovirus (CMV), and Mycobacterium avium complex. According to the current criteria, established in 1993, the diagnosis of AIDS is given to patients who are HIVþ when either their CD4þ count drops below 200 or they develop opportunistic infections or cancers (especially Kaposi’s sarcoma (KS)), AIDS encephalitis, or AIDS wasting syndrome. Clinical presentations of AIDS have varied over the course of the epidemic and in different countries. In the United States, the first cases presented with pneumocystis carinii pneumonia (PCP) or KS. In sub-Saharan African countries, a syndrome of chronic diarrhea and severe weight loss (‘Slim disease’) was common; many people also presented with fulminant tuberculosis. Now that anti-retroviral therapy (ART) controls many HIV infections in the United States, patients often have greater problems with medication side effects than with opportunistic infections. HIV is accepted as the causative agent of AIDS by an overwhelming majority of physicians, scientists, and public health experts. Despite this, opposition to the HIV explanation appeared early in the epidemic and has persisted in a vocal minority. Citing the existence of clinical syndromes that resembled AIDS in HIV individuals (known as idiopathic CD4þ lymphocytopenia), some observers have argued that HIV is an insufficient explanation for AIDS. One theory argued that HIV and other factors together trigger an autoimmune response that produces immunodeficiency. Another replaced the infection model with a pollution model: recreational drug use, overuse of antibiotics, multiple sexually transmitted infections, exposure to impure blood products, and malnutrition somehow converged to produce AIDS. The most prominent skeptic of HIV has been South African President Thabo Mbeki, who long refused to acknowledge HIV as the cause of AIDS. His advocacy of alternative theories proved to be a major obstacle to the implementation of ART in South Africa in the early 2000s.

Origins and Spread of HIV HIV was first isolated in January 1983 by Francis Barre´Sinoussi and Luc Montagnier at the Pasteur Institute in Paris. They named it lymphadenopathy virus (LAV) and suspected that it might be the cause of AIDS. In April 1984 Robert Gallo and his team at the National Institutes of Health announced the discovery of another virus that they believed to be the cause of AIDS. Recognizing the virus as a retrovirus, related to two retroviruses that Gallo had previously shown to cause lymphoma, they named it human T-lymphotropic virus type III (HTLV-III). A third group, led by Jay Levy at the University of

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California at San Francisco, independently isolated the virus as well. After a bitter priority dispute between Montagnier and Gallo, researchers realized that LAV and HTLV-III were the same virus. In 1986 an international commission officially assigned the name HIV. Subsequent study revealed that many different strains of HIV circulate in human populations. In 1985 a second virus, now named HIV-2, was identified in some cases of AIDS in West Africa. In 1994 two subgroups of HIV-1 were described: HIV-1 group M (main) and HIV-1 group O (outlier). In 1998 a third subgroup was described, HIV-1 group N (non-M/non-O, or new) in two individuals in Cameroon. HIV-1 M counts for the vast majority – over 90% – of all cases of AIDS. Many subtypes of HIV-1 M now exist, which vary in their geographic distribution. While subtype B dominates in the Americas and western Europe, A and B can be found in eastern Europe, B and C in North Africa and East Asia, and A, D, F, G, C, H, J, K, and others in sub-Saharan Africa. Analysis of these subtypes has shown that a single individual can become infected with multiple subtypes and that these can recombine to produce novel forms of the virus. Careful analysis of viral genetics and host phylogeny has revealed the origins of the different subgroups of HIV. The family of retroviruses has two main divisions. The first division, the oncoviruses, had been described in animals early in the twentieth century; Gallo described the first human oncogenic retrovirus in 1980, HTLV-1. HIV belongs to the second division, the lentiviruses, which are cytopathic. They have been found in a variety of animal species, including horses, goats, humans, and other primates. The various strains of SIV are the closest relatives of HIV. The first SIV was identified in 1985 in a rhesus monkey, in captivity, which suffered from an AIDS-like illness. Subsequent study has found over 20 species-specific strains of SIV. Recombination between these lineages obscures the details of their phylogeny. However, genetic analysis of the strains suggests a common origin, in Africa, in the Pleistocene epoch. Each species-specific strain is harmless in its natural host, a fact consistent with the ancient association between the strains and their hosts. But when a strain infects an individual from another species, an AIDS-like illness results. For instance, the rhesus money from which SIV was first identified was infected with a strain from a sooty mangabey. HIV reflects a similar phenomenon. At some point in the past, a single subspecies of chimpanzee, Pan troglodytes troglodytes, was infected with two different strains of SIV, one from red capped mangabeys, and one from spot nosed monkeys. These two strains recombined in chimpanzees to form a novel strain, SIVcpz. This strain then jumped to humans and evolved into HIV-1 M. When and where did this jump take place? Chimpanzees infected with SIVcpz have been found in Cameroon, Gabon, Congo, Central

African Republic, and Equatorial Guinea, an area that corresponds well with the epicenter of the human epidemic. Transmission from chimpanzees to humans likely occurred repeatedly, wherever humans hunted chimpanzees for food. The three different subgroups of HIV-1 (M, N, O), for instance, represent infections of humans with three different strains of SIVcpz. HIV-2, in contrast, came from strain of SIV found in sooty mangabeys in West Africa, where the monkeys are hunted and kept as household pets. Some researchers argue that humans were infected hundreds or thousands of years ago. Most, however, believe that HIV first emerged in the 1930s or 1940s. Many theories have been offered to explain why significant spread of HIV among humans began at this time. During the final decades of French colonial rule in Africa, policies of resettlement, forced labor, and urbanization destabilized African populations. Malnourished and overworked, some people turned to chimpanzees as a food source. This increasingly brought SIV into contact with human populations who had reduced resistance to infection. This foothold may have facilitated the evolution of SIV into HIV. Once human-to-human transmission became possible, HIV spread during the turbulent social changes that followed decolonization after World War II. Civil wars produced migrations of refugees. Urbanization disrupted traditional social practices, marginalized women, and promoted prostitution. Governments increased spending on military programs and sacrificed social services and education. Poorly funded health care programs had to reuse needles, something that accelerated the spread of HIV. Major vaccination campaigns in the 1950s and 1960s might have played a crucial role. Highway construction and improving transportation allowed the virus to spread from its central African origins throughout subSaharan Africa, and then throughout the world. International aid workers and tourists who indulged in commercial sex industries became infected and brought the virus back to their home countries. Once HIV gained access to developed countries, it found many routes of spread. Needle sharing spread HIV among IDUs in New York City in the 1970s. The 1970s also saw the emergence of gay identity, especially in San Francisco where, for some gay men, promiscuous anonymous sex became an important part of their identity. Blood sharing technologies, particularly transfusion and fractionated blood products, had also become more widespread. These varied factors transformed a local practice – hunting of chimpanzees for food – into a global pandemic. This rapid evolution of HIV over several decades may be a harbinger of further changes yet to come. Researchers have scrutinized medical records to detect HIV or make retrospective diagnoses of AIDS to reconstruct a detailed prehistory of the epidemic. Collections of old blood samples have been tested to find direct evidence of the presence of HIV in human populations. The oldest

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match comes from a 1959 sample from Leopoldville (Kinshasa), Congo. This strain is the common ancestor of three of the current subtypes of HIV-1 M. The earliest definitive clinical evidence of AIDS comes, surprisingly, from a Norwegian family. In 1961 a Norwegian teenager worked on a merchant ship that sailed down the west coast of Africa, stopping in many ports from Senegal to Cameroon. During this trip, he was treated for gonorrhea. He subsequently returned to Norway and began to show symptoms of AIDS in 1966. His wife fell ill in 1967 and their daughter, born that year, became sick in 1969. All three died in 1976. Subsequent testing of blood samples taken from them between 1971 and 1973 have tested positive for HIV-1 O, a subgroup found most often in Cameroon. The earliest case in the United States has been identified in a 15-year old from St. Louis. Diagnosed with a severe chlamydial infection in 1968, he died in 1969. Autopsy revealed extensive, aggressive KS. Tests on surviving blood samples, performed in 1987, confirmed the diagnosis of HIV. Dozens of other likely cases of AIDS, that lack confirmatory blood tests, have been found in Africa, Europe, and the United States in the 1970s. How prevalent was the infection? Screening of surviving blood samples has revealed its insidious spread. Samples from sub-Saharan Africa show a prevalence of 0.1% in 1959, 0.3% in 1970, and 3% in 1980. Data from clinics for IDUs in New York City show that HIV had entered the city by 1976. Infection had reached 9% of IDUs by 1978, 39% by 1980, and over 50% by 1984. These data suggest that HIV existed, but was rare, in human populations in the 1950s. By the 1970s, however, parallel epidemics had begun in sub-Saharan Africa, the United States, and Europe.

Emergence and Recognition of AIDS In the late 1970s, physicians in New York and California noted the increasing occurrence of KS, PCP, and other rare infections among previously healthy young men. Because of the unusual nature of these diseases, which are typically associated with a failure of the immune system, epidemiologists began to search for characteristics that might link the cases. In the summer of 1981, Michael Gottlieb described an outbreak of PCP among five homosexual men in Los Angeles. By the end of the year, similar cases, as well as unusual cases of KS, had been described among homosexuals, IDUs, and female sexual partners of IDUs, in Los Angeles, San Francisco, New York City, and Europe. Researchers traced these opportunistic infections to a specific deficit in CD4þ cells. Although several risk groups had been identified, etiological hypotheses focused on particular aspects of gay culture which might explain the outbreak of the disease, including use of amyl nitrite (‘poppers’) and steroid skin creams, antigen

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overload, and pathogenic sperm. Some researchers suggested a link to CMV. Other risk groups were identified in 1982. Researchers traced cases of immunodeficiency and opportunistic infections to contaminated blood products, especially the clotting factors used to treat hemophilia. Although the risk posed by clotting factor concentrates was soon recognized, many people with hemophilia, tragically, had no choice but to continue taking them. Meanwhile, during the summer of 1982, deaths from opportunistic infections, notably toxoplasmosis, were described among Haitians at detention camps in Florida. The occurrence of cases in diverse risk groups suggested infection with a bloodborne agent. In September 1982, the Centers for Disease Control and Prevention (CDC) replaced the range of early labels (gay plague, gay cancer, and gay-related immune deficiency) with a more neutral term: AIDS. By the end of the year, over 1000 cases had been described. By early 1983, the CDC had defined AIDS as the result of infection with an unknown transmissible agent, characterized its mode of transmission, and defined four risk groups, the ‘4H Club’: homosexuals, heroin addicts, hemophiliacs, and Haitians. Recognizing that many European cases had links to Africa, researchers speculated that Africa might be the source of the virus. This suspicion was strengthened by reports of slim disease and KS beginning to emerge from Uganda, Zaire, and Zambia. The identification of HIV by Barre´-Sinoussi, Montagnier, and Gallo enabled intensive scientific scrutiny of the epidemic. The fortuitous emergence of HIV at exactly the moment when scientists had developed the techniques needed to study it allowed rapid progress in understanding the biology and pathophysiology of the virus. The problems posed by the virus – biologically, clinically, and epidemiologically – quickly moved virology and molecular immunology onto the center stage of scientific research. The first crucial innovation came in March 1985 with the licensing of the first blood screening tests. The enzyme-linked immunosorbent assay (ELISA) and the Western Blot did not detect the virus itself. Instead, both identified the high levels of antibody produced by most infected individuals within several months of infection. Although the tests could miss people who had recently been infected, they allowed clinicians to test patients, epidemiologists to survey populations, and officials to screen donated blood to protect the blood supply from HIV. With both a clinical definition of AIDS and a screening test for HIV, officials could map the spread of the epidemic more accurately. Through the 1980s the United States had the greatest number of cases of AIDS, with 100 000 by 1989 and 200 000 by 1991. By the mid-1980s, however, concern had begun to shift to Africa. The first cases of AIDS in Africa had been described in Uganda in 1982. Reports from Zaire,

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Zambia, and Rwanda of patients with slim disease, KS, thrush (oral candidiasis), and meningitis emerged in 1983 and 1984. By 1985, with additional cases in Kenya and Tanzania, the WHO began to fear a vast epidemic in Africa. These fears have been realized: fully two-thirds of all cases of HIV have been in sub-Saharan Africa. The first cases of AIDS in Asia were reported in Thailand in 1984. Reported cases of AIDS, however, remained rare for several years. Rapid spread began in 1988 with prevalence among IDUs and commercial sex workers quickly approaching 50%. By 1991 HIV had spread into the general Thai population, with 10–15% of military conscripts testing positive for the virus. In contrast to the rapid spread in these areas, many regions were largely spared during the 1980s. There were few cases of AIDS in Japan in the 1980s and those were largely limited to transmission from blood products, which were often imported from the United States. The Soviet Union and eastern European countries reported few cases before the breakup of the Soviet empire, and most of the acknowledged cases came from iatrogenic transmission. For instance, in 1990, Romania reported HIV among 2000 children who had been abandoned by their parents. According to folk practice, many newborns received small blood transfusions to increase their strength; this practice exposed the infants to unsterilized needles and a contaminated blood supply. However, even countries that were spared during the 1980s experienced accelerating epidemics in the 1990s. China, for instance, reported very few cases in the 1980s, mostly among foreigners or people who had traveled abroad. However, in the mid-1990s, the virus began to spread among IDUs and commercial blood donors. India similarly saw few cases in the 1980s, but by the mid-1990s, HIV was spreading through drug use, the blood supply, and heterosexual contact.

AIDS Before Effective Treatment: Fear and Blame As scientists struggled to characterize AIDS and HIV, the epidemic caused considerable suffering and generated a worldwide health crisis. The epidemic began at a moment of relative complacency, especially in the developed world, concerning epidemic infectious disease. Not since the influenza epidemic of 1918–20 had an epidemic with such devastating potential struck. Developed countries had experienced a health transition from the predominance of infectious to chronic disease and had come to focus their resources and attention on systemic, noninfectious diseases. In this respect, AIDS appeared at a historical moment in which there was little social or political experience in confronting a public health crisis

of its dimension. The epidemic fractured a widely held belief in medical security. Not surprisingly, early sociopolitical responses were characterized by blame and discrimination. AIDS first appeared among homosexuals and IDUs, some of the most marginalized groups in society. Echoing earlier assessments of other sexually transmitted diseases (STDs), observers divided victims of HIV into categories: the ‘innocent victims’, who acquired their infections through transfusions or perinatal exposure, and the ‘guilty perpetrators’, who engaged in high-risk, morally condemnable behaviors. Since the disease was associated with ‘voluntary’ behaviors considered to be immoral, illegal, or both, individuals were typically blamed for their disease (Figure 1). Some religious groups in the United States, for example, saw the epidemic as an occasion to reiterate particular moral views about sexual behavior, drug use, sin, and disease. AIDS was viewed as ‘proof’ of a certain moral order. Hostile commentators called for a series of regressive, even punitive, measures to control the epidemic, including universal testing and quarantine. While such measures were never implemented in the United States, victims did suffer from discriminatory behavior, including loss of jobs, housing, and insurance. Homophobia became more visible as violence against gays and lesbians increased. The Food and Drug Administration (FDA) barred blood donations by members of the risk groups, including all Haitians. As the global extent of HIV became clear in the mid-1980s, the nature of blaming and bias also globalized. Early commentators condemned African behaviors that had supposedly spread the disease from monkeys to humans, such as the reported use of monkey blood as an aphrodisiac. Others blamed ecological disruption, particularly the destruction of tropical rain forests. In 1985 Soviet and Indian media reported that HIV had been created by biological warfare experts in the United States and released in Zaire in 1978. This theory received extensive international attention until it became clear that it had its origins in rumors actively spread by the KGB starting in 1983.

Figure 1 In the early years of the acquired immune deficiency syndrome (AIDS) epidemic in the United States, AIDS was associated with a series of stigmatized behaviors, as in this poster indicating the risk of shared needles among intravenous drug users. Such stigmatization blamed victims for their disease and hindered early public health efforts to contain the spread of HIV.

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This early period of the epidemic in the United States also saw considerable fear of casual transmission despite reassurances that HIV could only spread through blood or sexual contact. A 1985 poll showed that 47% of Americans felt that infection could be transmitted through shared drinking glasses and 28% feared infection through toilet seats. The link between AIDS and receiving blood transfusions became transformed into belief that one could be infected by donating blood. Realtors in California were instructed to warn customers if a house had previously been owned by someone with AIDS. In some communities, parents protested when HIV-infected school children were permitted to attend school. In one instance, a family with an HIV infected child – an ‘innocent victim’ – was driven from a Florida town when their home was burned down. As the disease, deadly and poorly understood, continued to spread, governments and victims’ advocacy groups struggled to make difficult decisions. The debates about bath houses are particularly revealing. In the 1970s an outbreak of hepatitis B revealed that these bath houses, and the sexual behaviors they facilitated, had become dangerous sites of disease transmission. In the early 1980s they were recognized as a dominant site of transmission of HIV. Many public health officials demanded that they be closed. The gay community was torn between those who saw bath houses as an essential expression of gay identity and those who feared the growing impact of AIDS. San Francisco shut down its bath houses in 1984 but similar efforts failed in Los Angeles. The United States government initially showed little interest in AIDS. The defined risk groups had little political clout, and even less political appeal to the conservative administration of President Ronald Reagan. Failing to display leadership crucially needed in the early years of the epidemic, his administration repeatedly denied the need to make special appropriations for HIV and AIDS. However, as the potential ramifications of the epidemic became evident, national institutions began to mobilize. Congressional appropriations for research and education rose steeply. The National Academy of Sciences and a Presidential Commission both issued prominent reports. Meanwhile, international programs proliferated. Medical researchers organized a series of International AIDS Conferences, beginning in 1985. The World Health Organization established a Global Program on AIDS in 1986 to coordinate international efforts at epidemiologic surveillance, education, prevention, and research. The WHO also tried to guide responses to the epidemic, lobbying against the use of coercive measures, such as Cuba’s experiment with mandatory isolation of HIV-infected individuals. Instead, it advocated for the use of less restrictive measures to contain the epidemic. Circumstances changed dramatically in 1985 with the development and wide implementation of screening tests.

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Just as the CDC definition of four risk groups shaped the early years of the epidemic, the ELISA and Western Blot generated a new range of responses to the epidemic. The tests quickly became a crucial method of reassurance. For the first time, people could know whether or not they were infected, without the uncertainty of the long latency that preceded the symptoms of AIDS. The tests also allowed public health officials to reestablish the safety of the blood supply. Prior to this, blood banks could only reduce risk by restricting donations from defined risk groups and testing for various surrogates, such as hepatitis B, a virus that shared many of the epidemiological characteristics of HIV. But despite these methods, over 10 000 people were infected by blood products in the United States by 1985. However, with the implementation of universal screening, the safety of the blood supply quickly improved. Since then, there have been very few transfusion-related cases of AIDS in the United States. The advent of screening, however, raised difficult issues about the balance of individual rights and public health. The ability to screen for HIV made it likely that people who were infected, but still healthy, would suffer the full burden of discrimination faced by people with AIDS. Many questions were debated. Should public health officials require mandatory reporting and case tracing of HIV? Should states demand premarital testing, as they did for syphilis? Should employers be able to screen prospective employees? Should health insurers be able to screen prospective customers? Should hospitals be able to test all patients, and should patients be able to demand testing of their doctors? The Department of Defense, citing the risks posed by its vaccination programs and by potential battlefield transfusions, began screening all recruits in 1985. Critics of this policy saw it as a thinly veiled effort to enforce the military’s ban on homosexuals. Heated controversy also arose in 1987 when Congress forced the CDC to add HIV to the list of ‘dangerous contagious diseases’ which would be used to ban immigrants from the United States, despite evidence that HIV was not easily communicable. For the most part, the rights of privacy won out in the United States. Few states required reporting of HIV at a time when reporting of other diseases, from tuberculosis to syphilis, was routine. In other countries the situation was quite different. The Soviet Union, for instance, implemented a massive compulsory testing program. Travelers, homosexuals, soldiers, pregnant women, prisoners, and those thought to engage in casual sex were all tested, 142 000 000 altogether. Infected foreigners were banned from entering the country. The advent of screening also set the stage for a series of tragic scandals. In the United States, public and private blood banks implemented screening relatively quickly. In other countries, notably France and Japan, the tests were not immediately implemented. Thousands of patients, particularly hemophiliacs, were infected. Subsequent investigations have ended in both civil and

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criminal convictions, with prison sentences for negligent officials and substantial payments to infected victims. Although the ability to screen for HIV reduced many of the fears associated with the epidemic, many new fears quickly emerged. By 1986, two factors fueled increasing fear in developed countries that heterosexual spread would bring HIV into the general population. First, public health officials began to shift emphasis from the stigmatized ‘risk groups’ to more precise ‘risk behaviors’, stressing that dangerous behaviors such as needle sharing and anal intercourse could be found among people not identified as IDUs or homosexuals (Figure 2). Second, epidemiologists realized that heterosexual transmission was the dominant mode of spread in Africa and Haiti. Since these epidemics were believed to have begun earlier than in Europe and the United States, they were perceived as glimpses into the future of AIDS in Europe and the United States. The fears were exacerbated by slow progress in developing treatments for HIV and AIDS. Many treatments were hailed initially, including interleukin-2, alphainterferon, bone marrow transplants, heteropolyanion-23 (HPA-23), and various immune stimulants. However, subsequent work inevitably showed them to be both toxic and of little value. Vaccines proved similarly elusive. When Gallo announced the discovery of his virus in 1984, federal officials optimistically predicted that a vaccine would be available within 2 years. By the end of the decade, however, researchers had little to show for their efforts other than skepticism and pessimism. In the absence of treatment, physicians and patients had little to offer people with HIV. Patients tried diets and alternative medicines to delay the progression to AIDS. Doctors could only work to prevent, and then treat, the devastating opportunistic infections.

Figure 2 During the late 1980s, public health officials increasingly recognized the threat of spread of HIV through heterosexual contact. Campaigns such as this one, moving outside of the traditional risk groups, encouraged women to protect themselves from incurable infection with HIV.

Although there was little that could initially be done to help people infected with HIV, public health officials knew everything that they needed to know to prevent the spread of the epidemic. Once the two primary modes of transmission had been identified – sexual intercourse and needlesharing by IDUs – two modes of prevention became obvious: widespread distribution of condoms and sterile needles. These techniques, combined with screening programs, could have slowed the epidemic. Some countries, notably the Netherlands, England, and Canada, did implement such programs. However, it quickly became clear that a diverse range of obstacles prevented effective deployment of public health programs. In the United States, for instance, the conservative federal government criticized condom and needle distribution programs as condoning morally and legally sanctioned behaviors. In 1988 Congress criminalized needle distribution programs. In the absence of federal support, grassroots needle exchange programs were begun in Boston, New Haven, New York, and San Francisco. Some programs received support from local governments, while others faced opposition and arrests. Such marginalization seriously impaired efforts to slow the spread of HIV. By the end of the decade, the panic created by the emergence of AIDS had settled into a new equilibrium. Incidence rates began to level off in Europe and the United States. Public faith in the safety of the blood supply had been restored. Education efforts curtailed the spread of HIV among homosexuals. Fears that HIV would spread into the general population diminished. New cases remained confined to marginalized groups, notably urban minority populations. AIDS was no longer seen as a wildfire epidemic that would sweep the globe and threaten civilization. Once compared to the great epidemics of the past – plague, cholera, and influenza – AIDS now evoked comparisons to chronic diseases, particularly cancer. It had become routinized. Some observers feared that this new complacency would have adverse consequences. Public health officials in the United States, for instance, feared that increased public apathy would lead to decreased vigilance and allow a resurgence of the epidemic (Figure 3). Meanwhile, the view in developing countries was much bleaker. HIV continued to spread quickly in sub-Saharan Africa. Health officials faced difficult choices. Should they recommend that HIVþ mothers breast feed their children, which risked transmitting HIV, or should they encourage use of infant formulas, which exposed the children to contaminated water supplies? Seeing no good solution, the WHO supported breast feeding in the late 1990s. HIV also began to spread in countries, such as Thailand and India, and then China, that had initially been spared. Despite this, developed countries, no longer fearing a global pandemic, had become less supportive: international assistance for AIDS

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Figure 3 Belief that HIV would not spread widely beyond traditional risk groups, or beyond marginalized urban populations, generated considerable apathy among many people. Public health officials tried to overcome this apathy by highlighting the general threat that would be posed if HIV was not acknowledged and managed.

actually dropped between 1991 and 1992. Although some progress had been made, the epidemic continued.

The Development of Effective Treatments As soon as HIV was identified, pharmaceutical companies began screening drug compounds for antiviral activity. In 1985 researchers realized that azidothymidine (AZT), which had been developed as an anticancer drug in 1964, blocked reverse transcriptase, an enzyme essential to the viral life cycle. On the basis of promising results from clinical trials, Burroughs Wellcome received authorization to market AZT in 1987. However, initial enthusiasm that AZT might be a magic bullet for HIV quickly gave way to frustration. The drug had many toxic side effects. Long-term studies showed that AZT conferred no survival benefit, in part because patients quickly developed resistance to the drug. Meanwhile, the research process became highly politicized. AIDS activists protested cumbersome FDA regulations that slowed the approval of new drugs. AIDS Coalition to

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Unleash Power (ACT UP), founded in 1987 by playwright Larry Kramer, issued scathing critiques, sought increased funding for drug research, and demanded more rapid access to promising treatments. In October 1988, ACT UP took over the FDA offices in Washington, DC, to bring attention to the plight of patients who died while the FDA worked to decide whether or not to approve new drugs. ACT UP also organized drug sharing, buyers clubs, and basement laboratories to provide underground access to potentially valuable treatments. Such advocacy eventually led to major reforms in FDA policy, facilitating the testing and licensing of new treatments for HIV and AIDS. Another problem with AZT quickly rose to the forefront. When the drug was approved by the FDA in 1987, it costed roughly $10 000 per patient per year of treatment. While this might have been within reach of people with HIV in Europe and the United States, international health activists realized that few developing countries had the resources to provide such an expensive treatment. At the Fourth International AIDS Conference in Stockholm in 1988, officials debated ways of getting treatment to the poor worldwide. Realizing that AZT would not solve the challenges posed by HIV, researchers and drug companies continued to seek new and better treatments. By 1991 several new reverse transcriptase inhibitors became available (ddI, ddC, Lamivudine (3TC)). Clinicians hoped that combination therapy would be more powerful than AZT alone. While this proved to be true, even a combination of these drugs did not stop the infection. Resistance continued to emerge quickly. Moreover, clinical experience showed that even when patients had an excellent clinical response to therapy, the virus could survive, dormant, in various parts of the body (e.g., the testes). There seemed to be no way to completely eradicate the infection and cure patients. New hope came in 1994 when a major research trial (ACTG 076) showed that AZT could reduce the risk of transmission of HIV from pregnant women to their children by over 60%, from 20 to 8%. This gave patients greater hope that they would not transmit the infection to their children, and it provided a method for preventing a significant burden of new cases of HIV. Efforts to implement campaigns against maternal-to-child transmission, however, also became mired in controversy. The 6-month course of AZT used in the research study cost $800 per patient, a sum beyond the reach of most patients in developing countries. Researchers wanted to know whether or not a shorter course, that cost only $50, would be as effective. The quickest way to learn this would be to compare a short course of AZT to a placebo control group. A 1997 trial of a such shorter, cheaper regimen became embroiled in fierce controversy over what ethical guidelines should regulate international health research. Angry confrontations among researchers united by their desire to reduce the suffering created by

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History and Culture, (and Biographies) | AIDS, Historical

HIV demonstrated the complicated scientific, social, and moral problems raised by the epidemic. While health officials attempted to optimize the implementation of reverse transcriptase inhibitors, researchers continued to study the biology of HIV intensively. By 1991 they hoped that they would soon be able to block the HIV protease, crippling the activity of HIV. The first such protease inhibitor, Saquinavir, was approved by the FDA in December 1995. To maximize its efficacy and to minimize the emergence of resistance, clinicians combined the protease inhibitor with two reverse transcriptase inhibitors. The flexibility of such regimens increased in 1996 when researchers developed a third class of drugs, the non-nucleotide reverse transcriptase inhibitors (e.g., nevirapine). Combination ‘triple therapy’ (initially called HAART, for highly active antiretroviral therapy, but now generally referred to as ART proved to be a powerful weapon against HIV. Patients who had been near death from AIDS were miraculously revived: not cured, but restored to health, an effect that quickly became known as the Lazarus syndrome. AIDS mortality dropped quickly in western Europe, the United States, Canada, Australia, and New Zealand (Figure 4). AIDS, which had been the leading cause of death in young men in the United States in 1995, fell to fifth place. The incidence of HIV began to decrease as well. Physicians hoped that protease inhibitors might succeed where earlier drugs had failed and actually eliminate HIV from patients, curing them of their disease. At the 11th

International AIDS Conference in Vancouver in 1996, protease inhibitors were celebrated as a ‘quantum leap’ in the treatment of HIV. Another major advance also appeared in 1996: viral load testing. The initial blood tests for HIV relied on detection of antibodies produced by the body in response to infection. While both sensitive and specific, these tests often did not become positive until several months after infection. The viral load test, in contrast, used polymerase chain reaction (PCR) technology to detect directly viral RNA in the patient’s blood. This had two advantages. First, it could detect the virus much earlier in the infection. Second, it could be quantified to measure the activity of the infection. Changes in viral load could then be used to gauge prognosis and response to treatment. Motivated by the optimism that followed the development of protease inhibitors, the United Nations created a new program devoted to AIDS. However, as had happened with the advent of AZT, a series of obstacles quickly emerged that threatened to undermine the efficacy of combination ART. First, the initial ART regimens were complex, requiring up to 16 pills taken at various times during the day. Outcomes in clinical practice were inferior to those attained in clinical trials, presumably because patients struggled to adhere to the complex regimens. Physicians feared that imperfect adherence to drug regimens would facilitate the emergence of resistant strains. Second, it became clear that the virus could become resistant to each class of

Trends in Annual Age-Adjusted* Rate of death due to HIV Disease, United States, 1987–2004 18

Deaths (per 100 000 population)

16 14 12 10 8 6 4 2 0 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

Note: For comparison with data for 1999 and later years, data for 1987–98 were modified to account for ICD-10 rules instead of ICD-9 rules. *Standard: age distribution of 2000 US population

Figure 4 Rate of death from human immunodeficiency virus (HIV). Death rates from HIV in the United States climbed steadily through the 1980s and early 1990s, peaking in 1995. The introduction of protease inhibitors and combined anti-retroviral treatment led to a dramatic reduction in HIV death rates. Bringing this treatment to all populations in the United States, and worldwide, has proven exceedingly difficult. Reproduced from CDC, Divisions of HIV/AIDS Prevention, Slide Series L285, Slide #4.

History and Culture, (and Biographies) | AIDS, Historical

medications, even when they were used carefully in combination. Third, patients developed a series of side effects in response to protease inhibitors, particularly lipodystrophy, a disruption of fat metabolism that could increase the risk of diabetes and heart disease. Fourth, researchers increasingly realized that the ability of HIV to insert its genetic material into the genome of host cells made eradication of HIV from an individual nearly impossible. Reservoirs of infected cells persisted, even when viral load became undetectable in patients’ blood. If treatment was ever stopped, these internal reservoirs of HIV could become seeds of reinfection. Finally, the regimens were extremely expensive, initially costing $10 000–$20 000 per patient per year, a cost beyond the means of the developing countries where most infections occurred. By the late 1990s a familiar pattern had been established. Public health officials knew that powerful methods could prevent the spread of HIV, but these were rarely utilized to their full potential. Physicians had developed promising new treatments, from AZT to ART, but these faced difficult obstacles, including drug resistance, side effects, and cost. While better treatments could always be developed, the greatest challenge became making good use of existing technology.

The Challenge of Providing Care Throughout the history of AIDS, the nature of the epidemic had depended on where it took place. Modes of transmission varied significantly from country to country, as did the most prevalent opportunistic infections. These geographic variations persisted over the first three decades of AIDS. By 2000, however, a new disparity had emerged, one in many ways more troubling. For the first time a technology existed that could control, though not cure HIV. Patients in developing countries had good prospects for accessing ART and experiencing substantial gains in life expectancy after infection. Meanwhile, ART initially remained out of reach in developing countries, creating enormous disparities in the experience of infection. The advent of ART led to substantial changes in the nature of the AIDS epidemic in developed countries. Since 1981, the United States had seen over one million cases of AIDS with over 500 000 deaths, the eighth highest total for a country worldwide. Mortality rates had climbed steeply from the 1980s to the 1990s, reaching over 16/100 000 in 1995. With ART, however, the United States witnessed an 80% reduction in mortality, with the rate falling to under 6/100 000. With mortality declining, a diagnosis of HIV no longer seemed to be the life-ending event it had been in the 1980s. Testing became much more widespread. In 1996 the FDA

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licensed home test kits for HIV. Although testing services were regulated to ensure that patients who received positive test results by telephone also received adequate counseling, HIV testing became nearly as simple as home pregnancy tests. Testing technology continued to improve, allowing clinics to provide results within 30 min. Treatment, even though expensive, was well funded by both insurers and government programs. As a result, everyone with HIV in the United States had access, in theory, to ART. Pharmaceutical companies also produced complex pill formulations, allowing a one-pill-a-day regimen for people infected with nonresistant strains. Widespread access to treatment led to a dramatic increase in life expectancy for people with AIDS: over 13 years by 2006, a number expected to grow with continuing use of the powerful medications. Decreased mortality, easier testing, and better access to treatment all contributed to the routinization of HIV in the United States. Health officials, physicians, and patients became more willing to accept control measures that, during the height of AIDS fear, would have been dismissed as unacceptable infringements of civil liberties. By 1998, 30 states (but representing only 30% of all cases) had established some form of mandatory reporting. The CDC worked hard to increase the coverage of its HIV surveillance efforts. In 1999 and again in 2005, the CDC pushed all states to report all cases of HIV to the CDC. The CDC argued that this was already done routinely for many communicable diseases and that there was no longer any reason to treat HIV differently. Eventually, tying mandatory reporting to over $1 400 000 000 in federal aid, the CDC achieved this goal: by the end of 2007, the United States finally had a system of nationwide mandatory reporting in place, something that had taken 30 years to implement. Working in parallel, physicians pushed to make HIV testing a routine part of annual physical exams and prenatal care. Officials heralded the end of ‘AIDS exceptionalism’: while special measures to protect privacy had been appropriate during the early years of AIDS, such protections were increasingly seen as missed opportunities for controlling the epidemic. However, some old problems persisted. Strong moral judgments about patients and modes of infection continued to disrupt efforts to prevent and contain HIV. Condom and clean needle distribution programs remained mired in political debate. In 1998 President William Clinton refused to lift the ban on federal funding for needle exchange programs. Political opposition to needle exchange programs continued under President George W. Bush, despite evidence that they were the most effective means of preventing HIV among IDUs, and that they might actually decrease the prevalence of drug use, presumably because the site of needle exchange programs became a valuable site for educational programs. Public health officials also feared that declining

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mortality and routinization would lead to complacency and carelessness about prevention. Although mortality dropped, the incidence of HIV remained stable, at roughly 40 000 new cases each year since 1992. Most men (58%) continued to be infected through homosexual contact; incidence even began to increase among young homosexual men. Among women, however, most cases (71%) came through heterosexual transmission. Even more worrisome was the accelerating spread of HIV among the most marginalized communities in the United States. By the mid-2000s, African Americans made up 50% of all cases, even though they represented only 12% of the population. The incidence among African Americans compared to whites was 7 times higher for men, and 21 times higher for women. Women also made up an increasing share of the cases, especially among younger age groups. Among HIVþ teens, for instance, nearly two-thirds were women, mostly African Americans, infected through heterosexual contact. Enormous disparities existed not just for incidence of HIV, but also for outcomes after infection. Treatment failure was especially prevalent in minority populations, with 50% of AIDS deaths occurring in these populations. The AIDS mortality rate in Boston and New York City, for instance, was roughly 15 times higher in black women than in white women. Many obstacles prevented successful implementation of ART in these populations. One study found that only 37% of patients in Baltimore had undetectable viral loads after 1 year of treatment. Such data suggested that in the United States, where adequate resources existed to control HIV and AIDS, failures of social and political will disrupted responses to the epidemic. The situation outside of the United States was even more complex. Some developing countries, notably Brazil, successfully implemented ART to reduce mortality from AIDS substantially. Many more countries, however, were devastated by HIV, a testimony to the global failure to contain the epidemic. By the end of 2006, an estimated 33 200 000 people lived with HIV, nearly 0.8% of the world’s population. Despite the ability, in theory, to prevent infection through abstinence, condom use, and clean needle distribution, over 2 500 000 people were newly infected in 2006. Despite the ability of ART to extend life expectancy dramatically, nearly 2 100 000 died of HIV in 2006. Much of the burden of the epidemic remained in sub-Saharan Africa. Over 60% of people living with HIV, and over 70% of deaths from AIDS, were in sub-Saharan Africa, where overall prevalence of HIV among adults reached 5.9%. Many countries in southern Africa had adult prevalence rates over 20%. Swaziland, suffering the most intense epidemic in 2006, had an estimated prevalence rate of 33.4%. South Africa continued to have the highest number of cases, 5 500 000, without evidence that spread of infection had slowed.

Most of the people living with HIV continued to be unaware of their infection. In contrast to southern Africa, many other regions of Africa experienced much less severe epidemics. In eastern and western Africa, many countries had prevalence rates below 5%, and some remained below 1%. Outside of Africa, rates also remained low, but with concerning evidence of rapid increases in the late 1990s and early 2000s. Rates of HIV increased by 70% in eastern Europe and central Asia between 2004 and 2006, with 90% of this increase confined to Ukraine and Russia, where most spread occurs among IDUs. Rates have also increased in East Asia, south Asia, the Middle East, and north Africa. Although China reported only 650 000 cases overall, nearly half among IDU, prevalence in that group rose in the early 2000s, reaching over 50% in some Chinese provinces. Increased rates have also been seen among pregnant women. India, poised to displace South Africa as the country with the highest number of cases, experienced a highly varied epidemic, with most cases confined to industrial centers in the south, west, and northeast. Preliminary data suggest that prevention programs had begun to slow transmission. A few countries, notably Cuba, have been largely spared from the epidemic, with prevalence less than 0.1%. The HIV epidemic has had striking demographic impacts. AIDS has orphaned 15 000 000 children, of whom 80% live in sub-Saharan Africa. Many subSaharan Africans have also seen marked decreases in overall life expectancy. Although life expectancy had reached 65 years in Botswana by the late 1980s, it fell to less than 36 years because of HIV. Similar drops occurred in Zimbabwe and Swaziland. Throughout southern Africa, AIDS became a major barrier to economic development and political stability. Tragically, in the absence of development and stability, AIDS will remain difficult to control. How has this happened, 20 years after the development of successful preventive strategies and 10 years after the development of effective treatment? When ART became available in the late 1990s, many leading health officials believed that it could not be implemented in developing countries. In 1998, for instance, many prominent officials argued that ART was inappropriate for poor countries. The treatment was too expensive, far exceeding the annual per capita health expenditures of every country. It was too difficult to implement, requiring sophisticated testing services and health infrastructure. If implemented imperfectly, it would facilitate the emergence of resistant strains, which would then threaten populations worldwide. Instead of treatment, health officials emphasized prevention. One influential comparison of treatment and prevention concluded that prevention was 28 times as cost effective as ART and argued that funding ART would, overall, lead to a greater loss of life. The focus on prevention achieved

History and Culture, (and Biographies) | AIDS, Historical

a powerful consensus. At the 13th International AIDS Conference, held in South Africa in 2000, over 5000 attendees signed the ‘Durban Declaration’: while ongoing research might lead to cheaper treatments, even a vaccine, AIDS campaigns needed to focus on prevention of sexual transmission. Proponents of prevention had evidence that such approaches could be successful. Even amidst the many calamities of the HIV pandemic, some countries achieved remarkable success at slowing the spread of HIV through preventive programs including health education, drug treatment, employment, and distribution of condoms and clean needles. In Uganda, for instance, education programs emphasized abstinence and condom use among sexually active teens. Such programs decreased prevalence, which peaked in the late 1980s and fell through the 1990s. Knowing that the presence of other STDs substantially increased the risk of transmitting HIV, officials in Tanzania implemented an STD treatment program that led to a 42% reduction in the incidence of HIV. Prevalence rates have also declined in Zambia, Kenya, Ghana, Rwanda, and Burkina Faso. Countries in southern Africa generally had less success, with Zimbabwe standing out as an exception. Despite substantial poverty and civil unrest, the prevalence of HIV among pregnant women fell from over 30% in 2000 to 24% in 2004. There have been successes outside of Africa as well. Aided by WHO, the Thai government began a major campaign for public education and condom use by prostitutes. As a result, prevalence among commercial sex workers declined significantly over the 1990s. Overall incidence in Thailand has decreased from 143 000 in 1991 to only 18 000 in 2005. Critics, however, argued that strict emphasis on prevention consigned the 30 000 000 people living with HIV to death. They insisted that large-scale treatment programs must be implemented, both because of belief in equity and social justice and because treatment could potentially be a highly effective form of prevention: if reducing viral loads to undetectable levels decreases the infectiousness of HIVþ individuals, then comprehensive treatment would substantially slow the spread of HIV. Arguing that no developed country would tolerate withholding treatment from a large sector of its own population, treatment activists called on developed countries to make resources available for global treatment programs. Noting that the roughly $8 000 000 000 needed each year for global AIDS control represented only 0.044% of the GDP of the 22 wealthiest countries, they argued that resources for AIDS treatment clearly existed. The challenge was simply one of priorities. Treatment advocates were especially critical of the use of costeffectiveness analysis to support prevention at the expense of treatment, arguing it would be better to mobilize the resources needed for both. When Lee Jong-Wook took over as director general of the WHO in 2003, he

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pushed aggressively for treatment. With only 2% of HIVþ people having access to ART, he argued that a worldwide health emergency existed. Although such advocacy of treatment was initially seen as naı¨ve and unrealistic, remarkable progress was made quickly. Success came from several directions. First, a series of reforms substantially reduced the cost of HIV treatment. In the late 1990s, pharmaceutical companies in Thailand, Brazil, and India began producing generic versions of HIV medications, in violation of international patent law. Brazilian companies, for instance, reduced the cost of ART in 1996 from $15 000 per person per year to only $4000. In 1998 South Africa attempted to import generic ART from these companies. Fearing that this would serve as a precedent for violations of patent rights worldwide, 39 pharmaceutical companies sued South Africa, initially with the support of the United States government. This lawsuit proved to be immensely embarrassing for the United States, which was seen as protecting the interests of profitable corporations while millions of people died from AIDS. As a result, in 2000, President Clinton issued an executive order forcing the companies not to enforce their patents in South Africa. Nearly simultaneously, those companies agreed to slash prices for their drugs in developing countries by over 90%. This created a two-tiered system in which developed countries paid full price while developing countries received steep discounts. Such arrangements were formalized in 2001 by the World Trade Organization’s agreement on Trade-Related Aspects of Intellectual Property Rights. Known as the Doha Declaration, this agreement gave governments the right to take certain measures to protect public health. In the midst of a health crisis, governments may impose compulsory licensing and allow local companies to produce medications in violation of international patents. Seen as a recognition of the primacy of public health over private property, the Doha Declaration enabled further reductions in the cost of ART. The nongovernmental organization Me´decins Sans Frontie`res worked with Cipla, an Indian pharmaceutical company, to produce combined ART at a price of only $350 per person per year. Following this lead, the Clinton Foundation negotiated further price reductions in 2003, to only $140 per person per year. The marked reduction in the price of ART motivated the second major development, a dramatic increase in funding for global AIDS control. When ART became available, the United States provided limited funding for international AIDS programs, only $121 000 000 in 1998. Activists asserted that much greater resources existed and that corporations had the knowledge needed to deploy treatment effectively worldwide. As prices for ART fell by 99% in the early 2000s, resistance to treatment programs dissipated and donor countries began to develop funding mechanisms. In 2002 the Global Fund to Fight

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History and Culture, (and Biographies) | AIDS, Historical

AIDS, Tuberculosis and Malaria was created as a partnership between governments, communities, and industry to direct resources to areas suffering from those diseases. Although it received only a fraction of the funding it needed, the Global Fund made progress in many areas. In 2003 President Bush increased the commitment of the United States, promising $15 000 000 000 in AIDS assistance over 5 years; in 2007 he called for another $30 000 000 000 to extend this 5 years further. This initiative, which became the President’s Emergency Plan for AIDS Relief (PEPFAR), significantly increased ART treatment worldwide (Figure 5). Private donors, notably Bill Gates and Warren Buffett, also made substantial contributions. All told, funding available for global AIDS treatment increased from only $292 000 000 in 1996 to over $8 000 000 000 in 2005. Decreased cost of ART and increased funding for global AIDS made both prevention and treatment possible on a previously unprecedented scale. Actual implementation proved difficult. One crucial challenge was ensuring that treatment, once started, was never interrupted, something that would facilitate the emergence of resistant strains. This required careful attention to planning and logistics. Experience also showed that simply making treatment available, through voluntary counseling and testing, did not get enough patients into treatment. The most successful HIV control programs instead tied HIV services more broadly to the provision of primary health care services, especially STD prevention and treatment, as well as maternal and child health programs. Wraparound services, including nutrition

Figure 5 The PEFPAR testing center in Migori, Kenya. In the early to mid-2000s, a variety of programs made anti-retroviral therapy (ART) available to people in developing countries on an unprecedented scale. The most effective programs combined voluntary counseling and testing with access to ART and a broad range of primary care services. Reproduced from The Power of Partnerships: Third Annual Report to Congress on PEPFAR (2007), p. 103 (http://www.pepfar.gov/press/ c21604.htm).

assistance, substance use treatment, and social support, made it possible to achieve high rates of treatment compliance even in the world’s poorest regions. However, as happened in the United States, a variety of political factors conspired to undermine optimal implementation of HIV prevention and AIDS treatment. President Thabo Mbeki’s opposition to ART delayed its widespread introduction into South Africa until 2004. Meanwhile, critics argue that domestic social politics in the United States interfered with AIDS programs funded by the United States. Specifically, the Bush administration mandated that one-third of the funding it gave to prevention programs be used for advocacy of abstinence and monogamy, efforts that were not always well suited to local regions. As has happened since the 1980s, ideology and pragmatism remained in tension, disrupting HIV prevention. One of the most dramatic efforts to increase AIDS treatment worldwide came in 2003 when the WHO launched its 3 by 5 campaign, an effort to get 3 000 000 people in developing countries on ART by 2005. This program met fierce resistance from critics and skeptics. Some complained that the target of 3 000 000 represented only half of the people who needed treatment. Others worried that infrastructure did not exist in enough countries and that the WHO did not control enough funding to make this possible. The outcome of 3 by 5 was a mixed success. By the end of 2005, only 1 300 000 people were on ART in developing countries. Few of the target countries had reached the goal of getting 50% of eligible patients on treatment. However, this still reflected a threefold increase over 2 years. Moreover, some countries, notably Botswana, made enormous progress, bringing ART to 85% of the people in need. Such successes demonstrated the feasibility of extensive treatment in developing countries and fueled growing enthusiasm for ART. Countries acting on their own, such as Brazil, or in collaboration with either PEPFAR or WHO brought ART to millions of patients. By the end of 2006, the Global Fund reported that it had 770 000 on ART, PEPFAR 822 000, and WHO 1 800 000. South Africa alone claimed over 1 000 000 people, a tenfold increase since 2003. Despite such progress, a majority of people who would benefit from ART – over 70% – still did not have access. World leaders repeatedly pledged to do better. In 2006, for instance, the General Assembly of the United Nations committed member nations to provide universal access to prevention and treatment by 2010. While most global health leaders supported this goal, no one produced a clear road map for how to make it possible amidst daunting obstacles. Health officials believe that vaccines will ultimately provide the best solution for the long-term control and prevention of HIV in developing countries. An ideal vaccine would be safe, cheap, and stable enough for mass vaccination campaigns; it would provide long-lived

History and Culture, (and Biographies) | AIDS, Historical

protection; and it would be active against the diverse subtypes of HIV. Such a vaccine remains the Holy Grail of AIDS research. Despite more than 25 years of research, there has been little success. The first clinical trial of an AIDS vaccine began in 1998, but nothing promising enough for widespread use has been developed. Researchers predict that a vaccine will not be available for many years. Some even confess that the task might be impossible because of the ways in which HIV sabotages the immune system. But research continues with the hope that a partial success would still be valuable. Even a ‘therapeutic vaccine’, one that did not prevent infection but instead strengthened an individual’s response to infection, would help. Efforts to develop other measures to prevent infection have also been disappointing. Researchers have long tried to develop an anti-HIV microbicide that women would insert into their vaginas to protect themselves against infection by HIVþ partners. A series of clinical trials, however, have shown that each proposed microbicide actually increased rates of infection, presumably because of irritation of the vaginal mucosa. New hope came from an unexpected direction. Epidemiologists had recognized by the 1990s that rates of HIV transmission were lower in countries where male circumcision was widely practiced than in countries where it was not. This led to the suggestion that circumcision somehow protected against HIV transmission. Several clinical trials were begun to test prospectively whether circumcision of adult men could be an effective means of reducing the spread of HIV. In 2006 researchers stopped two randomized trials prematurely because the outcomes were so dramatic: circumcision reduced transmission rates by over 50%. Since even a 50% reduction could have a substantial impact on the epidemic, circumcision could work as a powerful, if imperfect, vaccine. However, as with condoms and needle distribution, circumcision is tied to social beliefs and practices that might limit its popularity as a means of preventing HIV. As AIDS approaches the end of its third decade, it has attained a complicated status, characterized by a series of striking disparities. Medical research and public health campaigns have produced some dramatic successes. Condom and clean needle distribution, as well as male circumcision, can substantially reduce the transmission of HIV. Such measures have been implemented in some countries. Furthermore, people who have become infected with HIV can control their illness with ART, increasing their life expectancy dramatically. However, a variety of social, economic, and political obstacles have disrupted successful implementation of these powerful technologies. Although AIDS mortality has decreased significantly in developed countries, marginalized minority populations experience marked disparities in both rates of infection and mortality. Although some

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developing countries have successfully deployed both prevention and treatment, AIDS continues to devastate sub-Saharan Africa, where the millions of people now infected with HIV will likely develop and die from AIDS. The impact on other areas, notably Russia, China, and India remains unclear. These countries have a limited window for containing the epidemic before beginning to suffer the decrease in overall life expectancy seen in southern Africa. At every level, AIDS has been an epidemic created by, and starkly reflecting, inequalities within and between countries. Poverty creates dangerous vulnerabilities to HIV, and AIDS exacerbates poverty. The same inequalities that create the epidemic also hinder responses to it. While patients in wealthy countries have access to powerful treatments, the vast majority of victims do not yet. As a result, scientific success to date has served to expose economic inequality. In the mid-2000s, a strong consensus emerged that called for bringing access to effective prevention and treatment to all countries worldwide. Will it be possible to make universal access a reality? If such efforts contain the epidemic, will it be possible to maintain support for lifelong treatment for everyone now infected with HIV? Will a vaccine emerge that will someday replace ART as the cornerstone of global HIV campaigns? Unprecedented efforts will be needed, over decades, to implement the necessary medical, public health, and social reforms. Such efforts could have an enormous reward, preserving the health of populations worldwide and restoring health to the many areas already devastated by HIV and AIDS. See also: Antiviral Agents; Emerging Infections; Epidemiological Concepts and Historical Examples; Evolution, Viral; Global Burden of Infectious Diseases; HIV/AIDS; History of Microbiology; Phylogenomics; Sexually Transmitted Diseases; Syphilis, Historical

Further Reading AVERTing HIV and AIDS. Available at http://www.avert.org. Bailes E, Gao F, Bibollet-Ruche F, et al. (2003) Hybrid origins of SIV in chimpanzees. Science 300: 1713. Bayer R and Fairchild AL (2006) Changing the paradigm for HIV testing – the end of exceptionalism. The New England Journal of Medicine 355: 647–650. Bayer R and Oppenheimer GM (2000) AIDS Doctors: Voices from the Epidemic. Oxford: Oxford University Press. Epstein S (1996) Impure Science AIDS, Activism, and the Politics of Knowledge. Berkeley: University of California Press. Farmer P (2006) AIDS and Accusation: Haiti and the Geography of Blame, 2nd edn. Berkeley: University of California Press. Fee E and Fox DM (eds.) (1988) AIDS: The Burdens of History. Berkeley: University of California Press. Fee E and Fox DM (eds.) (1992) AIDS: The Making of a Chronic Disease. Berkeley: University of California Press. Gao F, Bailes E, Robertson DL, et al. (1999) Origin of HIV-1 in the chimpanzee Pan troglodytes trogdolyes. Nature 397: 436–441.

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Garrett L (1994) Hatari: Vinidogodogo (Danger: A very little thing): The origins of AIDS. In: The Coming Plague: Newly Emerging Diseases in a World Out of Balance, pp. 281–389. New York: Penguin Books. Marseille E, Hofmann PB, and Kahn JG (2002) HIV prevention before HAART in sub-Saharan Africa. Lancet 359: 1851–1856. Rothman DJ (2000) The shame of medical research. The New York Review of Books 30 November: 60–64. Shilts R (1987) And the Band Played On: Politics, People, and the AIDS Epidemic. New York: St. Martin’s Press. Simon V, Ho DD, and Karim QA (2006) HIV/AIDS epidemiology, pathogenesis, prevention, and treatment. Lancet 368: 489–504.

‘t Hoen E (2002) TRIPS, pharmaceutical patents, and access to essential medicines: A long way from Seattle to Doha. Chicago Journal of International Law 3: 27–46. Treichler P (1987) AIDS, homophobia, and biomedical discourse: An epidemic of signification. October 43: 31–70. UNAIDS. AIDS Epidemic Update. World Health Organization, December 2006. Walensky RP, Paltiel AD, Losina E, et al. (2006) The survival benefit of AIDS treatment in the United States. Journal of Infectious Diseases 194: 11–19. Walton DA, Farmer PE, Lambert W, Le´andre F, Koenig SP, and Mukherjee JS (2004) Integrated HIV prevention and care strengthens primary health care: Lessons from rural Haiti. Journal of Public Health Policy 25: 137–158.

Biographies W C Summers, Yale University School of Medicine, New Haven, CT, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Genres of Scientific Biography

Glossary bio-bibliography A list bibliography or a list of sources that provides biographical information, such as biographical dictionaries, obituaries, memoirs, biographies, and autobiographies. hagiography Originally biographical accounts of the Christian saints, but now a term referring to ‘worshipful’ and uncritical biographical praise.

Abbreviations DSB

Standard Biographical Sources Conclusion Further Reading

prosopography A form of collected biographies that seeks to embed the various life stories of the subjects in the context of institutional and cultural networks to provide a broad explanation of the social roles of the subjects.

NLM

National Library of Medicine

Dictionary of Scientific Biography

Defining Statement The general nature of biography as a literary form is discussed with special reference to scientific biography. Well-known and widely available standard sources as well as newer electronic databases are described.

the use of direct quotes from the subject, along with personal details and motivations as opposed to a simple account of accomplishments, and was a vivid attempt to provide a portrait of ‘the complete man’. This work is often said to be the ‘greatest biography ever written’ and probably accounts for the continuing fame of Dr. Johnson.

Introduction

Genres of Scientific Biography

Since the time of Plutarch (c. 46–120 CE), biography has been esteemed as both history and moral instruction. The Parallel Lives, paired biographies of eminent Greeks and Romans, provided the personal character and motives for individual actions and ethical evaluations of the great men of Plutarch’s time. This emphasis on the story of a life as moral exemplar was propagated in the genre of hagiography, or lives of the saints, a popular form in the Middle Ages. It was Giorgio Vasari in his Lives of the Artists (1550) whose landmark collected biography focused on secular lives rather than holy lives. Vasari created heroic figures, his subjects became celebrities, and his book was widely read, even down to the present. Of course, the modern author whose name is synonymous with the genre is James Boswell, whose biographical study of his friend and hero Dr. Samuel Johnson was both revolutionary and successful. His Life of Johnson (1791) introduced

Scientific biography is a genre that is less well defined than some other forms of writing but has a substantial history. On the one hand, so-called scientific biographies deal with the ideas, scientific work, and impact of the subject, while more personal biographies can emphasize an exemplary life, perhaps one held up as an inspiration to the young scientist. On the other hand, scientists, as often as others, are motivated to write memoirs, personal reflections on life and its meaning. Memoir, and its more self-critical cousin, autobiography, often suffers from the normal human need for justification. Scientific biographies can be so focused on the work of the subject that the personal qualities recede into the background. In Lavosier – The Crucial Year: The Background and Origin of His First Experiments on Combustion in 1772, Henry Guerlac famously describes the life of Lavosier in terms of his thought and reformulation on how chemistry should be

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History and Culture, (and Biographies) | Biographies

done and Lavosier the man seems hard to fathom. Harvey Cushing’s Life of Osler (in two volumes), on the other hand, seems to leave nothing out and the reader is almost swamped in detail. The memoir, when well written and perceptive, has its own charms. Examples that come to mind include Advice to a Young Scientist by the famous neurophysiologist Ramo´n y Cajal, and As I Remember Him by the microbiologist and poet Hans Zinsser. Zinsser, with his usual brio, wrote his autobiography in the third person, a conceit that was, of course, completely transparent. Both authors used the memoir to illuminate the people, places, and problems they encountered in their scientific and personal lives rather than to simply recount a self-centered view of their individual universes. Memoir and autobiography are both exercises in controlled remembering, and, as such, they have the advantage and disadvantage of the personal and temporal perspectives. Memory is faulty and often reconstructs events in ways that they did not occur. The best writings exhibit an awareness of these limitations and a critical attitude demanding caution or corroboration by contemporary documentation. Maclyn McCarty in his memoir The Transforming Principle: Discovering that Genes Are Made of DNA is remarkably candid about his, and others’, dim recollections. He takes particular pains to flag secondhand recollections, stories for which he can find no support in his laboratory notebooks or personal diaries, and the like. On occasion, two participants in the same events differ in their recollection of what took place. One has only to see Michael Frayn’s play Copenhagen and read the controversies it entailed about the role of Werner Heisenberg in Germany’s atomic bomb project in World War II to realize that conflicts in memory may be irreconcilable. In a similar vein, the memories of the key participants in the famous Meselson–Stahl experiment on semiconservative replication of DNA do not accord with data from their own laboratory notebooks and centrifuge records as shown by F. L. Holmes in Meselson, Stahl, and the Replication of DNA. Biographers face a dual challenge: access to their subject by personal interviews with the subject and/or the subjects’ relatives and colleagues or by examination of personal papers and documents on the one hand and by the maintenance of a critical stance in evaluating the evidence from these sources on the other hand. Subjects, being only human, are accustomed to project only a carefully crafted ‘public face’ while the biographer is trying for a more complete and full analysis of a scientist’s life. Oral histories, while fashionable, are fraught with such problems, and must be used with care. Interviews frequently elicit ‘canned stories’ that have been rehearsed many times in the past. The skilled interviewer can prevent such obstacles by diligent preparation and intimate knowledge of the subject’s life and work and by asking

the unexpected question or by following up a canonical account with further probing questions. Scientists frequently say that they don’t trust nonscientific biographers to ‘get it right’. This is another barrier for the biographer, to establish trust. Access to personal papers, sometimes in public repositories but sometimes still in the possession of the subject, can be difficult. Such archives may contain sensitive personal information or scientifically damaging material. The case of Newton is famous: he sorted his papers near the end of his life to create the precise historical impression he was constructing for his subsequent biographers. Likewise, the famous physicist Robert Millikan worked hard to recover both sides of all his correspondence from the time he was about 17 years old, because he just ‘knew’ he would be famous. He even kept copies of love letters written as a young college student. Again, the savvy biographer will be on guard against such potential obfuscation. Biographies may be hampered by incomplete sources because of records being withheld for long periods at the request of the subject. Louis Pasteur was so upset at the treatment of Claude Bernard’s life and work by his biographers that Pasteur instructed his family to never show anyone his private laboratory notebooks. His family honored that request for nearly a century, and only in 1971, 76 years after his death, did his personal correspondence and notebooks become available to scholars. They provided a highly revisionistic account of Pasteur and his work. Another genre of scientific biography is the collection of biographies that provides not only information about the lives of individuals, but also something of the networks in which they lived and worked. Pioneer Microbiologists of America, by Paul F. Clark, goes beyond a list of subjects and their biographical entries and, instead, it locates its subjects in geographic and intellectual traditions while giving a broad history of the development of microbiology in North America. As such it represents a form of biography sometimes termed prosopography, a biographical form that uses the stories of many individuals to present an account of the relationships, the contexts, and the social and institutional embeddedness of these individuals. Albert Delaunay’s L’Institut Pasteur des origines a´ aujourd’hui is a similar prosopographical study of many of the scientists who have been associated with the Pasteur Institute in Paris. Hagiography, or uncritical, reverential praise, abounds in scientific biography. Scientists are cultural heroes, worthy of emulation and admiration. Biographers, who are often admiring students, prote´ge´s, or scientific followers, are only too happy to sing the praises of their favorite icon. One of the two ‘fathers’ of microbiology, Louis Pasteur has been, without much doubt, the subject of the greatest number of hagiographic scientific biographies, starting with The Life of Pasteur, written by Rene´

History and Culture, (and Biographies) | Biographies

Valery Radot, a no less interested author than Pasteur’s son-in-law. This was followed by a series of articles, A l’ombre de Pasteur, by his former laboratory assistant Adrien Loir, Paul deKruif’s breathless account in Microbe Hunters, and even Rene´ Dubos’ Louis Pasteur, Free-lance of Science. When a biography appeared, based on the newly available Pasteur notebooks, which was less than entirely laudatory (Geison, The Private Science of Louis Pasteur), it was christened ‘the Geison Affair’ and reported in the New York Times, eliciting outcries of rage from many quarters in defense of the purity and sanctity of Pasteur. The last form of biography of note is the obituary. Often bordering on hagiography, but frequently more restrained, obituaries may be the only source of detailed information about many scientists. For an individual not famous enough to command the attention of a biographer and not egotistical enough to have written an autobiography or memoir, the obituary is often the primary and sole source of biographical information. Obituaries in newspapers are frequently indexed so that they can be found with relative ease. If well written, they can give clues as to where to search for more information: surviving family members, academic and professional affiliations, and, rarely, depositories of personal papers. In the past, many professional journals regularly published obituaries of noted individuals in the field. This practice, unfortunately, is rapidly becoming obsolete.

Standard Biographical Sources Biographical dictionaries are a useful and widely available source of biographical data, both for scientists and for nonscientists. A particularly useful collection is the Dictionary of Scientific Biography (DSB), a work in 18 volumes started in 1970 and last updated in 1990. It contains articles on the lives and works of selected scientists in all fields and throughout history with a critical analysis of their lives, major works, and key bibliographies. Other less extensive sources include American Men and Women of Science and the various versions of Who’s Who and Who’s Who in America. Such biographical sources exist for other national groups of scientists throughout the world. Specialized biographical dictionaries abound in various forms; some are devoted to certain periods or fields and others, such as Fruton’s A Biobibliography for the History of the Biochemical Sciences since 1800, are lists of collected sources, such as biographies, memoirs, and obituaries, for further reference. Bulloch’s History of Microbiology has a useful appendix of short biographies of microbiologists working prior to the 1930s. Society memoirs are a standard source of biographies for a select set of scientists who were members of the National Academy of Sciences of the United States of America or of the Royal Society. The memoirs of these two societies are obituaries prepared by other members

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who were both personally and professionally acquainted with the subject, often a friend, student, or colleague. The Bulletin of the Pasteur Institute, an example of an institutionally based journal, is a source of memoirs about deceased Pastorians. Similarly, the Nobel Institute in Stockholm maintains online information, including autobiographical statements, about its prizewinners. The development of electronic databases, search engines, and online access has revolutionized the ease of access to biographical information. Not only do many libraries with important collections of manuscripts and archives provide online finding aids and indexes to their collections, but there are centralized databases that facilitate access to existing sources, now in electronic form. The National Library of Medicine (NLM), part of the US National Institutes of Health (NIH), has made some of its biographical holdings available online in its Profiles in Science project (http://profiles.nlm.nih.gov/), digitized material such as correspondence, photographs, and scientific papers of key scientists of international stature or who have been closely associated with the NIH. In addition to the NLM, the Wellcome Library in London (http://library.wellcome.ac.uk/) is another major source of online information based on its extensive holdings in history of medicine. The Biography Resource Center (http://www.gale.cengage.com) is a commercial, comprehensive database of biographical information throughout history, around the world, and across all disciplines and subject areas. Many larger public and academic libraries subscribe to this service. Table 1 includes a list of generally available booklength biographies or autobiographies of well-known microbiologists. The list, which is certainly not exhaustive, was selected based on recognized contributions to microbiology (the Nobel Prize; landmark advances in the field, or national leadership in microbiology). Most works are available at the Library of Congress/NLM if not from other scholarly collections. Some, however, are rare and are included because the subject is noteworthy but without much biographical attention.

Conclusion Scientific biography is more than simply genealogy or unqualified praise; at its best it describes the life and work of a scientist in the context of his or her times, interactions with family, colleagues, and institutions. It situates ideas and influences of the scientist in the period and place of that scientist. In the best tradition of biography, it can serve as moral instruction on how science is done well or poorly, and how human thought develops and progresses in ways contingent on circumstance. Sources of biographical information are varied, from the unique resources of personal papers found in archives,

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History and Culture, (and Biographies) | Biographies

Table 1 Representative book-length biographies of some noted microbiologists Subject

Title

Author

Publisher

Date

Avery, Oswald

The Professor, the Institute, and DNA

Rene´ J. Dubos

1976

Baltimore, David

Ahead of the Curve: David Baltimore’s Life in Science George Beadle: An Uncommon Farmer, the Emergence of Genetics in the 20th Century Emil von Behring: Infectious Disease, Immunology, Serum Therapy

Shane Crotty

Rockefeller University Press University of California Press Cold Spring Harbor Laboratory Press

2005

Beijerinck, Martinus

Martinus Willem Beijerinck, His Life and His Work

Benzer, Seymour

Time, Love, Memory: A Great Biologist and His Quest for the Origins of Behavior Reconceiving the Gene: Seymour Benzer’s Adventures in Phage Genetics How to Win the Nobel Prize: An Unexpected Life in Science Hepatitis B: The Hunt for a Killer Virus

G. van Iterson, Jr., L. E. den Dooren de Jong, and A. J. Kluyver; foreword by C. B. van Neil preface by Thomas D. Brock Jonathan Weiner

American Philosophical Society Science Technical Publications

Knopf

1999

Frederic L. Holmes edited by William C. Summers

Yale University Press

2006

J. Michael Bishop

Harvard University Press Princeton University Press Heinemann

2003

Oxford University Press Oxford University Press Methuen

1999

Fundac¸a˜o Oswaldo Cruz Weidenfeld and Nicolson J.U. Kern’s Verlag

1993

Science History Publications

1976

Relume Dumara: Rio Arte Harcourt, Brace & World Norton

1996

1999

Ekkehard Grundmann

Yale University Press LIT Verlag

2005

Thomas Hager

Harmony Books

2006

Carol L. Moberg

ASM Press

2005

Beadle, George

Behring, Emil

Bishop, J. Michael Blumberg, Baruch Burnet, F. M.

Paul Berg and Maxine Singer

Derek S. Linton

Baruch S. Blumberg

Changing Patterns; an Atypical Autobiography Burnet: A Life

Macfarlane Burnet

Calmette, A.

The B. C. G. Vaccine

K. Neville Irvine

Chadwick, Edwin

The Life and Times of Sir Edwin Chadwick Meu pai

???

The Life of Ernst Chain: Penicillin and Beyond Ferdinand Cohn: Blatter der Erinnerung

Roland W. Clark

Chagas, Carlos Chain, Ernest Cohn, Ferdinand

Cruz, Oswaldo

De Kruif, Paul Delbru¨ck, Max d’Herelle, Fe´lix Domagk, Gerhard

Dubos, Rene´

Beginnings of Brazilian Science: Oswaldo Cruz, Medical Research and Policy, 1890–1920 Oswaldo Cruz: entre micro´bios e barricadas The Sweeping Wind, a Memoir Thinking about Science: Max Delbru¨ck and the Origins of Molecular Biology Fe´lix d’Herelle and the Origins of Molecular Biology Gerhard Domagk: The First Man to Triumph over Infectious Disease The Demon under the Microscope: From Battlefield Hospitals to Nazi Labs, One Doctor’s Heroic Search for the World’s First Miracle Drug Rene´ Dubos, Friend of The Good Earth: Microbiologist, Medical Scientist, Environmentalist

Christopher Sexton

Carlos Chagas Filho

Ferdinand Cohn. Assembled by his wife Pauline Cohn; with contributions of F. Rosen Nancy Stepan

Moacyr Scliar Paul De Kruif Ernst Peter Fischer and Carol Lipson William C. Summers

2001 2003

1983

2002 1968

1934 1952

1985 1901

1962 1988

(Continued )

History and Culture, (and Biographies) | Biographies

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Table 1 (Continued) Subject

Title

Author

Publisher

Date

Dulbecco, Renato Ehrenberg, Christian

Scienza, vita e Avventura Christian Gottfried Ehrenberg. Ein Tagwerk auf dem Felde der Naturforschung des 19 Jahrhunderts Paul Ehrlich, Scientist for Life

Renato Dulbecco J. Hanstein

Sperling & Kupfer Adolph Marcus

1989 1877

Ernst Ba¨umler translated by Grant Edwards Arthur M. Silverstein intro by Sir Gustav Nossal Virginia Law Burns

Holmes & Meier

1984

Academic Press

2002

Enterprise Press

1993

Carlos E. Finlay edited by Morton C. Kahn Andre´ Maurois Translated by Gerard Hopkins Intro by Robert Cruickshank Kevin Brown

Oxford University Press Jonathan Cape

1940

Sutton

2004

Gwyn Macfarlane

Oxford University Press University of Wisconsin Press Johns Hopkins University Press

1979

Ehrlich, Paul

Evans, Alice C. Finlay, Carlos Fleming, Alexander

Florey, Howard Fred, Edwin B. Gajdusek, D. Carlton Haffkine, Waldemar Hershey, Alfred D. Jacob, Francois Jenner, Edward Jerne, Niels Kitasato, Shibasburo Kluyver, Albert

Paul Ehrlich’s Receptor Immunology: The Magnificent Obsession Gentle Hunter: A Biography of Alice Evans, Bacteriologist Carlos Finlay and Yellow Fever The Life of Sir Alexander Fleming, Discoverer of Penicillin Penicillin Man: Alexander Fleming and The Antibiotic Revolution Howard Florey, The Making of a Great Scientist Edwin Broun Fred: Scientist, Administrator, Gentleman The Collectors of Lost Souls: Kuru, Moral Peril, and The Creation of Value in Science The Brilliant and Tragic Life of W.M.W. Haffkine, Bacteriologist We Can Sleep Later: Alfred D. Hershey and The Origins of Molecular Biology The Statue within: An Autobiography Edward Jenner, 1749–1823 Science as Autobiography: The Troubled Life of Niels Jerne Kitazato Shibasaburo

Diane Johnson Warwick Anderson

Selman A. Waksman F. W. Stahl (ed.) Francois Jacob translated by Franklin Philip Richard B. Fisher Thomas Soderqvist translated by David Mel Paul Daizo Nagaki

1959

1974 2008

Rutgers University Press Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press Andre Deutsch Yale University Press Keio Tsushin

1964 2000 1995 1991 2003 1986

Albert Jan Kluyver, His Life and Work Biographical Memoranda, Selected Papers, Bibliography and Addenda Robert Koch, A Life in Medicine and Bacteriology Alphonse Laveran, sa vie, son oeuvre Antony van Leeuwenhoek and His ‘‘Little Animals’’ Lord Lister

A. F. Kamp, J. W. M. La Riviere and W. Verhoeven (eds.)

North-Holland

1959

Thomas D. Brock

Springer-Verlag

1988

Marie Phisalix Clifford Dobell

Masson et cie Russell & Russell

1923 1958

R. J. Godlee

2005

A Slot Machine, a Broken Test Tube: An Autobiography Jeux et Combats Patrick Manson, the Father of Tropical Medicine Imperial Medicine: Patrick Manson and the Conquest of Tropical Disease

S. E. Luria

The Classics of Medicine Library (reprint) Harper & Row

1984

Andre´ Lwoff Philip Manson-Bahr

Fayard T. Nelson

1981 1962

Douglas M. Haynes

2001

Metchnikoff, Ilya

Life of Elie Metchnikoff, 1845–1916

Oxford University Press

1991

Monod, Jacques

Metchnikoff and the Origins of Immunology: From Metaphor to Theory Jacques Monod

Olga Metchnikoff preface by Sir Ray Lankester Alfred I. Tauber and Leon Chernyak

University of Pennsylvania Press Houghton Mifflin

Flammarion

1996

Koch, Robert Laveran, Alphonse Leeuwenhoek, Antony Lister, Joseph

Luria, Salvador Lwoff, Andre´ Manson, Patrick

Patrice Debre´

1921

(Continued )

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History and Culture, (and Biographies) | Biographies

Table 1 (Continued) Subject

Title

Author

Publisher

Date

Nicolle, Charles

Charles Nicolle Pasteur’s Imperial Missionary: Typhus and Tunisia Dr. Noguchi’s Journey: A Life of Medical Search and Discovery The Man Who Lived for Tomorrow; A Biography of William Hallock Park, M. D. Louis Pasteur

Kim Pelis

University of Rochester Press Kodansha International E. P. Dutton

2006

Johns Hopkins University Press Princeton University Press S. Hirzel

1998

University Press of Virginia [?] Mexico M.I.T. Press

1982

Muller

1950

Macmillan

1997

Rockefeller University Press G.P. Putnam’s Sons

1971

Peter G. Hesse and Joachim S. Hohmann

Lang

1995

E. O. Jordan, G. C. Whipple, and C.-E. A. Winslow, intro. by Mary K. Sedgwick K. Codell Carter and Barbara R. Carter Sherwin B. Nuland

Yale University Press

1924

Greenwood Press

1994

Norton

2003

Kiyoshi Shiga Claude E. Dolman and Richard J. Wolfe

Nihon Tosho Senta Harvard University Press

1997 2003

Angela N. H. Creager

University of Chicago Press

2002

Geoffrey M. Cooper, Rayla Greenberg Temin, and Bill Sugden Selman Waksman Selman Waksman

ASM Press

1995

Simon & Schuster Rutgers University Press

1954 1953

Wu Liande

W. Heffer

1959

Henri H. Mollaret and Jacqueline Brossollet Hans Zinsser

Fayard

1985

Little, Brown

1940

Noguchi, Hideo Park, William H.

Pasteur, Louis

The Private Science of Louis Pasteur Pettenkofer, Max v. Reed, Walter Ricketts, Howard T. Rivers, Thomas M.

Rogers, Leonard Ross, Ronald

Harald Breyer

Dr. Howard Taylor Ricketts Tom Rivers: Reflections on a Life in Medicine and Science; an Oral History Memoir Happy Toil; Fifty-Five Years of Tropical Medicine Ronald Ross: Malariologist and Polymath: A Biography

Ruben Saucedo Fuentes Saul Benison

A Notable Career in Finding Out

Salk, Jonas

Splendid Solution: Jonas Salk and the Conquest of Polio Friedrich Schaudinn, 1871–1906: Sein Leben und Wirken als Mikrobiologe: Eine Biographie A Pioneer of Public Health, William Thompson Sedgwick

Sedgwick, Wm T.

Semmelweis, Ignaz

Shiga, Kiyoshi Smith, Theobald

Stanley, Wendell

Temin, Howard

Waksman, Selman Winogradsky, Sergei Wu, Lien teh Yersin, Alexandre Zinsser, Hans

Patrice Debre´ translated by Elborg Forster Gerald L. Geison

Max von Pettenkofer: Arzt im Vorfeld der Krankheit Walter Reed: A Biography

Rous, Peyton

Schaudinn, Friedrich

Atsushi Kita translated by Peter Durfee Wade W. Olive

Childbed Fever: A Scientific Biography of Ignaz Semmelweis The Doctors’ Plague: Germs, Childbed Fever, and The Strange Story of Ignac Semmelweis Aru saikin gakusha no kaiso Suppressing the Diseases of Animals and Man: Theobald Smith, Microbiologist The Life of a Virus: Tobacco Mosaic Virus as an Experimental Model, 1930–1965 The DNA Provirus: Howard Temin’s Scientific Legacy My Life with the Microbes Sergei N. Winogradsky: His Life and Work; the Story of a Great Bacteriologist Plague Fighter; The Autobiography of a Modern. Chinese Physician Alexandre Yersin, le vainqueur de la peste As I Remember Him; The Biography of R. S.

William B. Bean

Leonard Rogers foreword by Sir John W. D. Megaw E. R. Nye and M. E. Gibson; foreword by Brigadier H. S. Langstaff Peyton Rous Jeffrey Kluger

2005 1941

1995 1981

1953 1967

2004

History and Culture, (and Biographies) | Biographies

through personal interviews, and through electronic databases available to anyone with Internet access. It is the responsibility of the user of these resources to apply the critical skills of the biographer’s craft in their analysis. See also: AIDS, Historical; Cholera, Historical; History of Microbiology; Plague, Historical; Spontaneous Generation; Syphilis, Historical; Typhoid, Historical

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Fruton JS (1992) A Bio-bibliography for the History of the Biochemical Sciences Since 1800, 2nd edn. Philadelphia, PA: American Philosophical Society. Geison GL (1995) The Private Science of Louis Pasteur. Princeton, NJ: Princeton University Press. Gillispie CC (ed.) (1970–1990) Dictionary of Scientific Biography. New York: Scribner. Vallery-Radot R (1902) The Life of Pasteur. Westminster, UK: A. Constable & Co.

Relevant Websites Further Reading Bulloch W (1938) The History of Bacteriology. London: Oxford University Press. Clark PF (1961) Pioneer Microbiologists of America. Madison, WI: University of Wisconsin Press. Delaunay A (1962) L’institut pasteur des origines a´ aujourd’hui. Paris: E´ditions France-Empire.

http://www.gale.cengage.com/BiographyRC – GALE, CENAGE Learning http://profiles.nlm.nih.gov/ – Profiles in Science, National Library of Medicine http://library.wellcome.ac.uk/ – Wellcome Library, Wellcome Collection

Cholera, Historical C D Meehan and H Markel, University of Michigan Medical School, Ann Arbor, MI, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Etymological Considerations Pathogenesis, Signs, and Symptoms Etiology

Glossary anticontagionism The belief that disease originates not from the transmission of germs from person to person but rather from interaction with an unsuitable environment, contact with rotting or deteriorating organic material, inhalation of polluted or contaminated air, or immoral behaviors. contagionism The belief that disease is transmitted by direct or indirect contact between living organisms, especially humans, in the form of microscopic living entities (e.g., germs). miasma Literally, foul or bad air arising from decaying animal or vegetable matter, thought by anticontagionists to cause disease.

The Pandemics Control, Prevention, and Treatment Cholera and Society Further Reading

quarantine Originates from the Italian words quarantina and quaranta giorno, referring to the 40-day period ships entering the port of Venice were required to remain in isolation before any disembarkment. Defined by public health authorities in the nineteenth century as the process of inspecting all ships, cargos, and passengers for evidence of contagious diseases. sanitarians Public health officials who worked to eradicate disease by disinfecting the environment and imposing sanitary restrictions on citizens.

Defining Statement

Etymological Considerations

Cholera is an acute diarrheal disease in which Vibrio cholerae, serogroup 1, found only in humans, are present in large numbers in the small intestine. Marked by severe abdominal pains and rapid dehydration, cholera has brought horrific and untimely death to millions as it swept the world in as many as seven pandemics in the past two centuries. Cholera’s modern history as a worldwide pandemic disease begins in 1817. Long endemic in India, cholera at this time began to make its way out of the subcontinent and spread as far as the United States by 1832. Cholera, described by one historian as ‘the classic epidemic disease of the nineteenth century’, has played a major role in medical history, particularly due to its widespread physical and emotional effects on individuals, societies, and cultures. The disease manifests with little warning, induces frightfully painful symptoms in its victims, and kills between 30 and 80% of those infected. Moreover, the cholera pandemics spanned generations, crossed political and geographic boundaries, and fueled fears of disease during a time of dramatic growth in the life sciences, public health, and international migration.

The Greek word kholera, meaning ‘bile’ or ‘to flow’, is found in sources as early as the Hippocratic Corpus. Classical writers such as Celsus (first century AD) used the term ‘cholera’ to describe illnesses marked by sporadic diarrhea, vomiting, and gripping abdominal pain. Although this term has been in use for millennia, it was applied in various ways to describe a variety of conditions. For example, the term ‘choleric’, meaning ‘easily moved to anger’, is also derived from kholera, but was a concept employed by humoralists to describe the condition resulting from vitiated humors. It was not until 1669 that Thomas Sydenham first used the term ‘cholera morbus’ to distinguish sporadic and endemic diarrhea from the choleric concept. By the early nineteenth century, when cholera reached global epidemic proportions, the term ‘cholera nostras’ had come to designate diseases with symptoms similar to sporadic and endemic cholera (i.e., profuse diarrhea, dehydration, and cramps), whereas cholera morbus described the more virulent occurrences of epidemic cholera. Other terms synonymous with epidemic cholera are cholera asiatica, malignant cholera, cholera asphyxia, and cholera spasmodica.

This article is reproduced from the 2nd edition, volume 1, pp. 801–807, Ó 2000; Elsevier Inc.

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History and Culture, (and Biographies) | Cholera, Historical

Pathogenesis, Signs, and Symptoms The disease called cholera today occurs when the etiological organism Vibrio cholerae is ingested and passes through the stomach into the small bowel, where it colonizes and multiplies. The V. cholerae is an acid-sensitive bacterium and a human must ingest a relatively large inoculum for sufficient numbers to evade the acidic environment of the stomach and reach the small intestine. The organism then releases a toxin that enters the epithelial cells, disrupting the absorption of electrolytes, and results in copious, watery discharges relatively free of fecal material accompanied by shredded mucous and epithelial cells giving it a characteristic ‘rice water’ appearance. The signs and symptoms of cholera are the result of a rapid loss of fluid and electrolytes. Symptoms begin with the victim’s sense of generally not feeling well. This condition lasts only a few hours and is then followed by a violent bout of vomiting and diarrhea. The victim experiences painful spasms of the abdominal muscles as a result of forceful propulsions of the gut, and matters only worsen as hypovolemic shock ensues. The effects of dehydration are painfully apparent on the face of the victim; the skin becomes blue and tight, the eyes are sunken deeply in their sockets, and the lips and mucus membranes of the mouth are dry and cracked. As diarrhea and vomiting continue, patients become increasingly more disoriented and eventually lapse into convulsion and/or coma. From 30 to 80% of all cholera cases end in death.

Etiology From antiquity until well into the nineteenth century, the medical theory of the cause of cholera was relatively static. For the most part, illness was perceived as an imbalance in the bodily humors due to poor diet, change in weather or seasons, geographic environment, or exposure to poisoned air or miasmas, such as the foul air caused by rotting organic material or animal and human waste. Although humoral theory waned during the nineteenth century, miasmatic theory and environmental influences continued to serve as explanations for disease. In a world still influenced largely by religious morality, people also often invoked metaphysical conceptions to explain cholera and other diseases. In America during the 1830s, for example, many ministers, moralists, and physicians agreed that those suffering from cholera had engaged in sinful activities and thereby predisposed themselves to sickness. When the second pandemic reached Europe and the United States (1832), delegations of scientists and physicians were sent out to study the spread of cholera, and

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questions about its contagious nature slowly began to surface. Germ theory was far from well established at this time, but burgeoning interests in contagionism and increasing knowledge of microorganisms stirred the imagination of medical scientists to search beyond the traditional metaphysical and environmental explanations of disease. Perhaps one of the greatest challenges to miasmatic theories on the cause of cholera came from John Snow, a British physician and epidemiologist. In the summer of 1849 he published a pamphlet ‘‘On the Mode of Communication of Cholera’’, in which he proposed that cholera was a waterborne disease. In 1854, his theory would be put to the test. He mapped incidences of cholera that occurred near Golden Square in London and noted that many cases were clustered around a particular water pump on Broad Street. After careful analysis, his cholera pamphlet was expanded and reprinted in 1855, reporting findings that sewage had seeped into the well that the recent victims of cholera used and concluding that contaminated water was the ‘predisposing cause’ of cholera. Although Snow was unaware of the causative agent, he posited that materis morbi (poisonous matter) must have caused the infection. Although many papers and pamphlets were published on studies similar to Snow’s (such as the work of William Budd) and some measures were taken to avoid contaminated water, many medical and scientific elites who subscribed to anticontagionist theories, such as the German public health expert Max Von Pettenkofer, remained resolute in their belief that cholera was caused by imbalances in the air, soil, and environment or exposure to human and animal filth in overcrowded cities. Shortly after Snow announced his findings, Italian microscopist Filippo Pacini reported his discovery of a microorganism with a terminal flagellum, isolated from the excreta of a cholera victim. This report, however, did not attract much interest among the medical community. Moralist ideas and claims about miasmatic theory were slow to die. Anticontagionism, however, steadily lost scientific credibility as the nineteenth century progressed. For example, in 1868, the French chemist Louis Pasteur first articulated the germ theory when he elucidated the microbial cause of silkworm diseases. Fifteen years later, the famed microbiologist Robert Koch isolated the V. cholerae during the cholera epidemics in Calcutta and Alexandria. Indeed, Koch’s studies confirmed that cholera is transmitted through drinking water and food contaminated with fecal matter from those infected with cholera. The acceptance of germ theory and rapid growth in the science of bacteriology yielded more advances in the field of cholera research at the turn of the century. Recent investigation has elucidated the existence of 60 or more serogroups of V. cholerae, of which only a single group, serogroup 1, is responsible for the epidemic cholera; others are responsible for sporadic diarrhea and

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History and Culture, (and Biographies) | Cholera, Historical

symptoms similar to epidemic cholera. Serogroup 1 of the V. cholerae exists as two serotypes, Ogawa and Inaba, each characteristic of different somatic antigen structures in the organism. V. cholerae also occur as two major biotypes: the classic (first described by Koch) and El Tor. The El Tor vibrio was discovered in 1905, during the sixth pandemic, in the El Tor quarantine camp in Egypt. Six unusual strains of vibrios were isolated from the dead bodies of pilgrims returned from Mecca. Gotschlich, who isolated the strains, performed tests that revealed the reactions of classic V. cholerae, though the victims did not show any signs of the cholera disease post mortem or while they were alive. Upon reexamination of the strains, Kraus and Pribram found that the El Tor vibrios produced a hemotoxin found to be lethal in experimental animals. They hypothesized that variability in the biotypes could be responsible for individual cholera epidemics. This was clearly demonstrated in the 1960s when El Tor was found to be the cause of many cholera outbreaks throughout the world. The previous hypothesis was further supported in early 1993 when a V. cholerae strain, with an antigenic structure differing from that of El Tor, was found responsible for a large outbreak in India. The existence of the newly identified strain, named O139 Bengal, demonstrates that epidemic potential is not exclusive to the classic and El Tor vibrios bearing the O1 antigen. The existence of O139 Bengal is indicative of an emerging level of complexity in the relationship of genetic and epidemiological factors facing today’s researchers and public health officials.

The Pandemics First Pandemic (1817–1824) In 1817, northeastern India experienced a particularly heavy rainfall that brought deluge to the state of Bengal and other regions between the Ganges and Brahmaputra Rivers. Floods resulted in failed crops and destroyed villages, and following the devastation cholera appeared with extraordinary virulence. In July of that year, cholera broke out in several districts in Bengal. Within a month, 25 000 people in Calcutta were being treated for the disease, 4000 of whom perished. This episode marks the beginning of cholera as a worldwide pandemic disease. Only 3 months later the province of Bengal was consumed by the disease, and by 1818 cholera extended in all directions to Nepal, Burma, and further into India: Delhi, Bombay, and as far as Ceylon. Although cholera affected these regions in the past, the rapidity with which it spread and the numbers mortally endangered in 1817 cast a shadow over any previous record of the disease. By 1820, the disease had spread by sea and land routes to Southeast Asia, moving from Burma to Siam’s (Thailand)

capital city of Bangkok and to Indonesia, where as many as 100 000 people succumbed to the cholera menace. By 1822, incidences of cholera began to subside in India, although the disease was widely established outside the subcontinent. Ships traveling from Southeast Asia carried the epidemic to China, where it had been previously introduced by land travelers as early as 1817. Soon after, the British army landed infected troops from Bombay into Oman, Arabia, from where the disease spread and was established in Muscat by 1821. In the same year, 15 000–18 000 people were killed along the Persian Gulf in port cities such as Basra. From this region cholera continued on, moving across the Mediterranean region, carried by caravans into countries such as Syria, where it raged until the end of 1823. The spread of cholera was greatly facilitated by Persian troops victoriously returning home to present-day Iran after warring with Turks; they served as vectors for the deadly vibrio, disseminating cholera in areas between the Tigris and Euphrates Rivers. After raging across the Far and Middle East for as many as three or four seasons, cholera subsided but remained active in Lower Bengal, India. Some have posited that control measures may have been responsible for cholera’s retreat in the early 1820s, although others have ascribed its decline to a severe winter during 1823 and 1824. Second Pandemic (1829–37) By 1824 cholera settled back in its home, the endemic area of the Ganges Delta in Bengal, India. There the disease remained active until 1827, when it was reported to have moved west to the Punjab. Cholera continued to exist in isolated regions outside of India during the interpandemic period; therefore, when the disease spread to the north and west of the Caspian Sea, into Russia and neighboring countries, it was thought that the pandemic’s origin came from Astrakhan, Azerbaijan, where cholera had persisted throughout the 1820s. However, cholera did not disperse from Astrakhan until 1830, although it was observed to be a problem in Orenburg, Russia, as early as 1829. Reports from that period in China demonstrated that the second pandemic made its way back into areas of China, particularly in and around Peking, via India, and from there gradually extended into Russia. Similarly, cholera was creeping across Persia from Afghanistan into Russia by way of caravans. Although these scenarios might not explain cholera’s appearance in Orenburg, they demonstrate the vast space through which cholera steadily advanced early in the second pandemic. Well established in Russia by 1830 in cities such as Moscow and Kharkov, cholera was then introduced into Poland by the Russian army. A ship transported the disease from Riga to the Prussian city of Danzig, resulting in

History and Culture, (and Biographies) | Cholera, Historical

a wave of infection that soon spread to Berlin, Vienna, and Hamburg by late 1831. At this time, some of the first cases of cholera were appearing in England in the port city of Sunderland, which had shipping connections with Germany. Cholera effectively made its way through England in 1832, infecting as many as 15 000 people and leaving more than one-third of them dead. The same year cholera visited Ireland and France. All of Paris’ districts were reportedly overcome with the disease and as many as 7000 people died in 18 days. By early 1832, much of Europe was suffering from the devastating effects of cholera. Having traveled from Asia through the Near East and into Europe, it crossed the Atlantic and released its fury in North America. On 8 June, a ship from Dublin carried 173 passengers to Quebec City and Montreal, of which 42 died before reaching port. On 23 June, cholera appeared in New York, and 2 weeks later it surfaced in Philadelphia. From the north the epidemic spread to Mexico, Latin America, and the Caribbean. It was not until 1837 that the second cholera pandemic finally subsided. Third Pandemic (1846–55) Scholars have long debated the dates of the second and third pandemics. Authorities such as Pollitzer considered the second pandemic to have run until 1851, marking the commencement of the third in 1852. Others, however, noting the significant decrease in cases of cholera and its withdrawal in some areas during the period of the mid1830s to the mid-1840s, have concluded that the resurgence of the disease in 1846 marks the beginning of a third pandemic. In 1846 cholera again erupted in and around the endemic regions of South Asia. Having already extended into Persia and central Asia during 1844 and 1845, Europe was again invaded by cholera by 1848 in much the same fashion as the disease progressed during the second pandemic. In the same year, New York and New Orleans were miserably reacquainted with cholera. From these cities cholera spread rapidly across North America, reaching as far as California in 1850. Present throughout Europe since 1849, cholera continued its course, eventually surging in 1854, making this year one of the worst on record. Fourth Pandemic (1863–79) In the fourth pandemic, cholera flourished in the same regions as it had in past pandemics, but it infiltrated Europe via new routes. Instead of moving from Asia, across Persia and the Caspian Sea, and into the heart of Europe, cholera traveled across Arabia. In Mecca, for example, 30 000 of 90 000 pilgrims died in 1865. The scourge progressed throughout the Mediterranean

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regions from Alexandria, Egypt, and this time entered Europe through Italy and southern France. From Egypt, Africa’s countries along the eastern coast were ravaged as far south as Mozambique. When cholera traveled north in 1866, some of the worst episodes occurred in Germany; where up to 115 000 deaths were reported. In Berlin, approximately 5500 deaths occurred out of 8000 cases. It has been suggested that Austria’s wars with Germany and Italy may have contributed to cholera’s dissemination. In 1866, the United States was just recovering from the Civil War when cholera returned for a third American epidemic. New Orleans was hit by several waves of cholera through 1868 and the disease spread through the South and the Midwest via the extension of the railway system. However, the outbreaks were mild in comparison with previous epidemics. In New York, measures such as the cleaning of city streets and the isolation of the ill were successful in preventing the further transmission of disease. Cholera resurfaced again in New York and New Orleans in 1873–75, though casualties were minimal. Russia, Persia, Arabia, Africa, India, and much of East Asia, however, suffered repeated serious attacks of cholera during this period. Central Europe also had its share of cases, but epidemics were less widespread due to the increasing awareness of public health. Fifth Pandemic (1881–96) Many of the early epidemics of this period occurred in Asia, particularly China, and around the Mediterranean. However, in 1892 Hamburg suffered a huge outbreak of cholera due to the contamination of unfiltered water from the Elbe River; approximately 7500 deaths occurred. In North America, potential epidemics were again prevented due to advanced notice and modern sanitary measures, although isolated cases were recorded, particularly in New York City. Sixth Pandemic (1899–1923) Cholera recrudesced in India in 1899, marking the start of the sixth pandemic. Cholera never disappeared completely from China and other regions of Asia after the previous pandemic, as was also the case for Egypt. From these regions, cholera spread to Persia, Arabia, and Afghanistan. During this period, America and most of Europe were largely unaffected, although sporadic cases occurred in southern Europe and Hungary. Seventh Pandemic (1961–Present) Many of the epidemics occurring in the present era have been traced to the El Tor biotype. El Tor was isolated in Indonesia and China in 1961 and in 1962 the World

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Health Organization recommended the inclusion of diseases caused by El Tor to appear in the definition of cholera. Both the classic and El Tor biotypes were detected in Afghanistan, from where the El Tor spread to neighboring Iran. An upsurge in cholera infection was also documented to have spread into Europe and Africa in 1970 and 1971. Since 1972, the number of countries affected in any 1 year has declined. However, in South America, where nearly a century had passed since the last reported cholera cases, the disease broke out violently in Peru in early 1991. El Tor was found responsible for this episode and for the recurrent Latin American epidemics that followed in the early 1990s. In late 1992, cholera-like disease appeared in Madras, India. The outbreak spread north into Bangladesh, where 100 000 cases were reported in the next 6 months. The new strain of V. cholerae, O139 Bengal, was the source of this epidemic that subsequently spread to Pakistan, Nepal, and regions of Southeast Asia. Cholera remains endemic in areas of Asia, Africa, and many countries of South and Central America, and rarely isolated cases of cholera have been imported to America and Europe. One relatively recent occurrence was reported in Los Angeles in 1993 after a woman returned from a 6-week visit to Hyderabad, India. Geographical Considerations Cholera owes much of its geographical distribution to pilgrimage, the industrial revolution, international shipping, traveling armies, and immigration. For example, large religious festivals in India convene near sacred sites, usually along the banks of sacred rivers. Upon people’s return home from such festivals, where thousands camped and used the same water supplies, cholera often followed. A similar process has been observed during the Islamic pilgrimages to Mecca. As transportation, such as with the steamship, became more efficient and continental and intercontinental railways linked city and village, the dissemination of the disease accelerated. The need for large workforces during the industrial revolution and the desire of many to emigrate because of either political or religious persecution contributed to the crowding of cities and the contamination of water supplies.

Control, Prevention, and Treatment Some of the most significant topics explored in the history of medicine, such as the modern-day understanding of contagious diseases, the rise of sanitation and public health institutions, and developments in medical therapy, find context in the history of cholera. For much of the nineteenth century, doctors found that they could do very little to prevent or cure cholera. Revolutions in industry and transportation brought about the rapid growth of urban

populations. It was in this setting, including the living conditions of crowded and dirty cities, contaminated water supplies, and large-scale migration of peoples, that cholera proliferated before traveling into bucolic areas. Before the 1830s, city officials had little interest in public health. With news of cholera moving from East or West, however, both European and American newspapers and popular magazines reported the spread of the disease, raising the attention of civic leaders and the public. Physicians and public health officials published pamphlets and articles and posted signs encouraging personal hygiene. Anticontagionists such as Von Pettenkofer warned of bad air and soil and encouraged proper nourishment and sanitary measures. John Snow, on the contrary, encouraged attention to maintaining clean water and Koch’s discovery of the etiology of cholera, V. cholerae, led to the development of modern public health policies and sanitation procedures and techniques that continue to maintain health in much of the industrialized world. One of the initial responses to cholera’s appearance or impending crossing of borders was the use of quarantine. For example, soon after it was reported that cholera broke out in Canada in 1832, both Canadian and US ports issued quarantine measures on all shipping and goods. In 1892, an epidemic of cholera was effectively prevented by the quarantining of ships in New York Harbor, where infected passengers were found. Entire vessels were quarantined for days until it was certain that no cholera would be introduced to the population on land. As a result of bacteriologists’ increasing abilities to recognize organisms and epidemiologist’s facility to track disease, scientists, physicians, and public health officials have been successful in controlling the spread of cholera. Treatment of cholera before and during the early nineteenth century employed the use of emetics, purgatives, and even bloodletting. Such practices probably only worsened the conditions of the ill. As early as 1830 research in fluid balance led physicians to attempt injecting water into terminally ill cholera patients. Many of these patients demonstrated the return of a strong pulse, but soon died. Injections of saltwater proved slightly more helpful, although determining a proper and effective solution was not always possible. Hence, such a treatment did not become successful until the early decades of the twentieth century when physicians had a clearer understanding of water and electrolyte balance. After the discovery of antibiotics in the 1940s, their use and a carefully planned regimen of fluid replacement greatly improved the therapeutic resources of physicians.

Cholera and Society Epidemics are commonly viewed as crises and engender massive reactions by physicians, public health workers,

History and Culture, (and Biographies) | Cholera, Historical

and laypeople. One unfortunate theme in the history of cholera (as with other epidemic diseases) is the scapegoating of those people and social groups perceived to be the vectors of the epidemic in question. The history of immigration illustrates such examples, where massive movements of peoples resulted in the transmission of disease across borders. In large cities that attracted great numbers of immigrants, such as New York City, the meeting of various ethnic groups and cultures set the stage for dramatic shifts in civic and social organization. When dealing with the cholera menace, societies, governments, and groups such as public health organizations at times did not hesitate to point the finger at marginalized peoples, labeling them as the bearers of disease. Immigrants were perceived to be predisposed to disease based on the association with the relatively few immigrants who did arrive in US ports ill with cholera and hence stigmatized. In reality, cholera was a social leveler that attacked people of all social classes and backgrounds. Nevertheless, the fear of cholera often inspired certain majority social groups to scapegoat and blame minority groups, such as immigrants and the urban poor. On the contrary, the cholera pandemics in the nineteenth century helped solidify ideas and sentiment toward concerns for public health in a time of scientific, economic, and societal growth. During this period, the pandemics also inspired the formation of the first international health agency (the International Sanitary

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Conferences), demonstrating that epidemics also have the power to bring nations together. Hence, the history of cholera illustrates that epidemic disease is an intimate part of social organization and change. See also: Plague, Historical; Typhoid, Historical

Further Reading Bilson G (1980) A Darkened House: Cholera in Nineteenth-Century Canada. Toronto, ON: University of Toronto Press. Delaporte F (1986) Disease and Civilization: The Cholera in Paris, 1832. Cambridge, MA: MIT Press. Evans RJ (1987) Death in Hamburg: Society and Politics in the Cholera Years, 1830–1910. Oxford: Oxford University Press. Kudlick CJ (1996) Cholera in Post-Revolutionary Paris: A Cultural History. Berkeley, CA: University of California Press. Longmate N (1966) King Cholera: The Biography of a Disease. London: Hamish, Hamilton. Markel H (1997) Quarantine! East European Jewish Immigrants and the New York City Epidemics of 1892. Baltimore, MD: Johns Hopkins University Press. McGrew RE (1965) Russia and the Cholera (1823–1832). Madison, WI: University of Wisconsin Press. Morris RJ (1976) Cholera, 1832: The Social Response to an Epidemic. New York: Holmes & Meier. Pollitzer R (1959) Cholera. Geneva: World Health Organization. Rosenburg CE (1962) The Cholera Years, the United States in 1832, 1849, 1866. Chicago: University of Chicago. Snow J (1936) Snow on Cholera: Being a Reprint of Two Papers. Richardson BW (ed.) London: Milford. Snowden FM (1995) Naples in the Time of Cholera, 1884–1911. Cambridge, UK: Cambridge University Press.

History of Microbiology W C Summers, Yale University School of Medicine, New Haven, CT, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Microscopy in the Seventeenth Century Spontaneous Generation and Microbes Classification of Microbes Contagion Germ Theories Applications of the Germ Theories Antimicrobial Therapies

Glossary adaptation Change in the ability of a microbe to utilize a specific nutrient after exposure to that nutrient. animalcules Term given to microscopic organisms first described by Leeuwenhoek. antisepsis Practice of using chemical agents to kill microbes during surgical or other procedures. asepsis Practice of surgical or other procedures carried out in the absence of microbes. attenuation Reduction in the virulence of a microbe by certain growth conditions or chemical or physical treatments. chemotherapy Treatment for disease with specific drugs of known composition, usually a natural or synthetic organic compound, sometimes including the toxic, antimicrobial natural products of other organisms, termed antibiotics. contagion The belief that an illness can pass from one individual to another by contact or by indirect transfer of germs. cyclogeny A theory of life cycles of bacteria, usually in terms of an entire population of microbes. dissociation The phenomenon by which bacteria with different properties, usually related to virulence, arise from a homogeneous population of bacteria. Now attributed to gene mutations. episome An extrachromosomal genetic element in cells, which can sometimes become associated with the chromosome. fermentation Classically, the process of conversion of sugar into alcohol. infusion A suspension resulting from soaking or heating organic material in water, for example, tea. inoculation Deliberate transfer of material from one source, such as a sick animal or a laboratory culture, into another.

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Public Health Clinical Microbiology Agricultural Microbiology Virology Microbial Physiology Microbial Genetics Bacterial Transformation and DNA Physiological Genetics Further Reading

miasma Concept of contagion by some atmospheric influences, ‘bad airs’, or emanations, not perceivable by the senses. microbe Generic term for living organisms too small to be seen by the unaided eye. monomorphism Nineteenth century doctrine that bacteria are of a fixed form or morphology and that the different forms indicate distinct bacterial species. mutation An abrupt change in a bacterial character, which is stably heritable. Now known to be the result of a change in the genetic code of an organism. operon A concept applied to a group of genes for functions that are regulated coordinately by means of control of the transcription, or messenger RNA synthesis, of those genes. plasmid An extrachromosomal, self-replicating genetic element, which may or may not become associated with the chromosome. More general term than episome. pleomorphism Nineteenth-century doctrine that bacteria are of variable form or morphology and that the different forms are not indicative of distinct bacterial species, in contrast to the concept of monomorphism. poisson distribution A statistical function that predicts the probability of occurrence of all-or-none rare events, such as mutations, under the assumption that their occurrence is random. putrefaction The process of breaking down, decomposition, or rotting of organic matter, similar to, but often contrasted with, fermentation. septicemia A bacterial infection carried in the blood of the affected organism. spontaneous generation The process whereby living organisms arise from nonliving matter. The matter may be inorganic (abiogenesis) or organic matter from previously living organisms (heterogenesis). transformation A change from one form of bacteria to another, usually in a ‘single property’, mediated by

History and Culture, (and Biographies) | History of Microbiology

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exposure to some material, now known to be DNA, from bacteria exhibiting the second property. vaccine A preparation (made in various ways) from an infectious organism that induces immunity, but not a full infection, when inoculated into a susceptible animal.

virulence The capacity of a specific organism to cause disease when inoculated into a susceptible host. vitalism The concept that all living organisms possess a ‘life force’ or ‘vital principle’ which accounts for the distinct properties of life, beyond the chemical and physical organization of the organism.

Abbreviations

PPLO RTF TMV

HIV MALT

human immunodeficiency virus mucosa-associated lymphoid tissue

Defining Statement This article surveys the key landmarks in both the conceptual and the practical history of microbiology.

Introduction The field of microbiology is defined, more or less, by the physical scale of the living objects of its study. In this sense, it began with the invention of instrumentation to visualize objects below the limits of the human eye, that is, the microscope. In another sense, however, microbiology also includes the study of properties of these objects which can be observed macroscopically or indirectly, for example, the metabolic consequences, the diseases, and the products of microbial activity. In this latter sense, the history of microbiology includes fermentations and other processing of foods, preservation of materials from microbial decay, and concepts of disease and contagion, to mention just a few examples. The term ‘microbe’ is a broad and somewhat general one, meant to embrace the biological organisms that are characterized principally by their small size. It was first suggested by Maximilien-Paul-E´mile Littre´ (1801–81), the great French lexicographer and linguist who was also trained in medicine, and proposed to the Acade´mie impe´riale de Me´decine by the French surgeon Charles Se´dillot (1804–83) and quickly adopted by the French school. ‘Microbe’ allows an agnostic stance until one is sure of the precise biological nature of the organism under discussion, a particular virtue in the early days of microbiology. Bacteria, molds, viruses, protozoans, and sometimes even small multicellular parasites and mammalian cells in culture are subsumed under this designation. For historic reasons, no doubt, the singlecell algae seem to have remained the province of the botanists and are rarely considered as microbes. Also, although resistance and responses to microbial infections are of great

pleuropneumonia-like organisms resistance transfer factors tobacco mosaic virus

importance in microbiology, many of these phenomena have become the domain of immunology and will not be included here. This article will focus on the history of microbiology since the invention of the microscope for two reasons: first, the new mechanistic philosophy of the seventeenth century provided a context in which to understand microbes, and second, the ability to observe microbes, albeit indirectly with an optical instrument, was essential to the further development of the subject. Since any brief survey must be selective, this article will emphasize those events and discoveries that are considered common to all microbiological investigations. Some areas have been omitted or treated with less attention that they deserve (e.g., parasitology and mycology), and the history of some newer topics (such as microbial evolution) have been left to the text articles on those subjects. The bibliography includes standard monographs of historical interest, key textbooks that give historical accounts of various topics, and a few references to the literature of the history of biological science and medicine. Articles on the history of microbiology are included in such searchable databases as Medline and History of Science and Technology (Research Libraries Group), both accessible on the Internet.

Microscopy in the Seventeenth Century The invention of the telescope at the beginning of the seventeenth century quickly led Galileo to his revolutionary astronomical discoveries, and shortly thereafter experimenters in optics produced the first microscope, reportedly demonstrated for the first time in Paris in 1620. These first microscopes were ‘simple’ in that they had a single lens of high curvature, often a glass bead, and were first exhibited as curiosities. By the mid-seventeenth century, several serious investigations were underway, which employed the microscope to examine the invisible structures, if any, of

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matter. This interest in the structure of matter was stimulated by the broad seventeenth-century philosophical debates on the mechanistic concept of the universe, which included speculation that matter, animate and inanimate, is made of small particles, some of which function as tiny machines to impart function to the whole. Notable among the seventeenth-century microscopists were Robert Hooke (1635–1703), Nehemiah Grew (1641–1712), Marcello Malpighi (1628–94), Jan Swammerdam (1637–80), and Antony van Leeuwenhoek (1632–1723). It was Leeuwenhoek who is most remembered today for his voluminous investigations and for the superior quality of his self-made instruments, and who was most concerned with observations on suspensions that contained objects we now consider as the subjects of microbiology. Leeuwenhoek was a local cloth merchant in Delft, Holland, who had little formal education but who developed a passion for lens-making and microscopic observations and descriptions. Through the Delft anatomist Reinier de Graaf, Leeuwenhoek came to the attention of Henry Oldenburg, the secretary of the newly formed Royal Society of London for the Improvement of Natural Knowledge (‘The Royal Society’), who began publishing Leeuwenhoek’s descriptive studies in 1673. Leeuwenhoek’s ‘Letters’ to the Royal Society span over 50 years of microscopic observations on infusions, body fluids, and a wide range of other materials. His skill as a lens-maker became widely recognized, and his reports were appreciated for his careful descriptions as well as for his technical virtuosity. His studies on human semen and description of spermatozoa (his own) contributed to the seventeenth-century debates on animal generation and the demise of various preformation theories of reproduction. In his own time, Leeuwenhoek was best known for these contributions, and his observations on microorganisms, now celebrated as the beginning of microbiology, were of relatively little interest and significance in his own time. Robert Hooke was the other microscopist most credited as a founder of the field of microbiology. Hooke’s famous book, Micrographia, published in 1665, astounded people with the details of the invisible world revealed by the new microscopes, some of which were devised by Hooke himself. This book contains for the first time illustrations drawn by Hooke of his microscopic observations. The most famous of these, showing the microscopic structure of cork, has been taken as the beginning of the cell theory of life, later developed by Schleiden and Schwann in the nineteenth century. Hooke was able to confirm and extend Leeuwenhoek’s results in the presence of members of the Royal Society, thus establishing the credibility of the rather reclusive Dutch cloth merchant. While Leeuwenhoek’s reports on what he called ‘animalcules’ (little animals) in rainwater and in various infusions indicate that he had indeed seen various

common protozoa, yeasts, and some bacteria, he did not extend these observations very much beyond his careful descriptions. Throughout the eighteenth century, dedicated microscopists pursued the study of these objects and refined their descriptions, but all were limited by the optics of the simple microscope: chromatic and spherical aberration, and relatively low magnification. Specimen preparation methods, such as sectioning and staining, were primitive. For example, prior to the introduction of the mechanical microtome in 1770, thin sections had to be cut freehand. Contemporary ideas about life, matter, and disease did not allow seventeenth- and eighteenth-century microscopic observations to be easily interpreted. Even in the late eighteenth century, Linnaeus was puzzled as to how to treat these little animals in his grand classification schemes. He assigned many of them to the genus and species: Chaos infusoria, perhaps indicative of the ambiguous status of microbes at that time.

Spontaneous Generation and Microbes In parallel with microscopic studies, complementary debates on the origin of living things were intense during the seventeenth and eighteenth centuries. Although these two topics started from quite different points, they would become intimately connected in the nineteenth century, so much that we now think of them as part and parcel of one tradition. Common observations of the apparently spontaneous appearance of insects, worms, and other small creatures (even mice) suggested to many observers that these living beings could arise from nonliving sources such as mud or putrefying vegetable or animal matter. Even the famous chemist and philosopher Jan Baptist van Helmont (1577–1644) believed that mice could arise from grain stored in granaries. In 1668, Francesco Redi (1626–97) published his investigations to test the belief that maggots and flies arose spontaneously from meat. Redi was a courtier and natural philosopher under the patronage of the Medici Grand Duke in Tuscany and, as such, was expected to present ‘observations’ and ‘demonstrations’ for the enlightenment and amusement of the court. At one point, he conducted a series of observations on rotting animal flesh, some portions of which had been protected from the air or from other sources of infection. The interpretation of these ‘experiments’ was that the maggots, flies, and ova seen in the exposed samples but rarely in the protected samples, did not have their origin from the meat itself, but rather the rotting flesh only served as suitable nest for the growth and nourishment of the eggs of the animals that were deposited there. While Redi is often cited in textbooks as having used or even invented

History and Culture, (and Biographies) | History of Microbiology

the ‘controlled experiment’, when his work is viewed in the context of his own time, it is clear that his view of ‘experiment’ was quite different from the modern interpretation. For Redi, experimentation meant visual demonstration of already ‘known’ truths. Despite work such as that of Redi and others, the idea that living beings were continuously being created from more basic material persisted. Georges-Louis Leclerc, Compte de Buffon (1707–88), a famous French naturalist, in his monumental Histoire naturelle (1749 et seq.), described his beliefs that all living things contain an indestructible vital principle. Buffon incorporated newer ideas about the chemistry of ‘molecules’ (small globules or particles) into his theories and regarded spontaneous generation as the result of the intrinsic properties of these organic ‘molecules’. The noted British naturalist, John Turberville Needham (1713–81) employed microscopic studies of various infusions, both heated and unheated, both open and closed to the air, and consistently observed the development of many of Leewenhoek’s little animals. He interpreted such results in terms of Buffon’s hypotheses and was a strong supporter of the notion of spontaneous generation. When Needham’s work was repeated and extended by the Italian naturalist Lazzaro Spallanzani (1729–99), Spallanzani found that heating was quite effective in preventing the appearance of the little animals, and furthermore, that Buffon’s ideas about the vitality of the material present in the infusions, thought to be the precursor of the little animals, were untenable. Spallanzani did many careful experiments with heated sealed and unsealed flasks from which he concluded that exposure to air was the source of the organisms that appeared in the infusions after heating. Neither Needham nor Spallanzani had the microscopical techniques that might have allowed resolution of their disputes. Furthermore, the biological status of the ‘little animals’ remained unclear, with the result that well into the nineteenth century, the questions of spontaneous generation and ‘vital principles’ remained unresolved. As discussed below, motivated by practical concerns with fermentations and disease, Pasteur and others pursued the questions already framed by their eighteenth-century predecessors with newly developed techniques, concepts, and instruments and by the end of the nineteenth century, serious discussion of spontaneous generation waned. The belief in Buffon’s vitalism, however, has been more durable, and even the most current textbook writers feel a need to exorcise the ghosts of this eighteenth- and nineteenthcentury doctrine by proforma reference to Wohler’s famous synthesis of the ‘organic’ compound urea, from an ‘inorganic’ cyanate (a reductionistic view that claimed that only living organisms, because of the vital principle, could produce ‘organic’ compounds). Even in twenty-first century popular culture one can find strains of these eighteenthcentury ideas.

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Classification of Microbes The biological nature of the animalcules began to attract the attention of naturalists in mid-eighteenth century with the classification of many of these forms by the Danish naturalist Otto Friderich Mu¨ller (1730–84), who devised some of the terminology still in use today. He grouped the animalcules into two major groups: those without visible external organs and those with such structures. The first group was divided further into two groups, one of which included five genera, two of which (Monas and Vibrio) contained bacterial forms. One of Mu¨ller’s species, Monas termo, would become the subject of most nineteenth-century attention, much as the colon bacillus, Escherichia coli, has been the paradigmatic species in the late twentieth century. The science of optics was sufficiently developed by the 1820s to allow construction of compound microscopes with achromatically corrected lenses and some correction for spherical aberrations. Better microscopy resulted in more detailed observations of the microbial world. In some sense, modern classification in microbiology started in 1838 with the work of Carl Gustav Ehrenberg (1795–1876), who published Die Infusionsthierchen als vollkommene Organismen, a massive folio of 574 pages with an atlas of 64 hand-colored plates. Many of his drawings reflect his belief that ‘Infusoria’ (a category that included bacteria, protozoa, rotifers, and diatoms) were small animals and were organized on the same principles as larger animals; thus, he believed all these little organisms had tiny stomachs and were perfect and complete (vollkommene) animals. Three of Ehrenberg’s 22 families of Infusioria include organisms that are recognizable as bacteria: Monadina, Cryptomonadina, and Vibronia. Vibrionia was composed of five genera: Bacterium, Vibrio, Spriochaeta, Sprillium, and Spirodiscus. These groups were defined by gross morphology: for example, Bacterium included filaments or threads of rod-like forms, which showed transverse divisions. Although Ehrenberg gave detailed descriptions and drawings of many of his observations, it is difficult, if not impossible, to determine with certainty how these organisms would be classified today. Subsequently, many classifications of microorganisms were proposed, based predominantly on morphologic characteristics with some attempts to incorporate growth characteristics such as growth by transverse division or budding. After Ehrenberg, the next important classification scheme was that of Ferdinand Cohn (1828–98). In 1854, Cohn published an important work in which he suggested that Ehrenberg’s Vibroinia should be regarded as belonging to the vegetable kingdom rather than to the animal kingdom, and suggested that bacteria were very similar to the well-known microscopic algae. He suggested that bacteria be classified as Mycophyceae or

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History and Culture, (and Biographies) | History of Microbiology

‘Wasserpilze’ (water fungus). Cohn’s major work in bacteriology, Untersuchungen u¨ber Bacterien (1872, 1875, 1876), was highly influential and established the outlines of modern bacteriological thought. While Cohn was arguing for the establishment of a taxonomy for bacteria that positioned them within the known biological realm, that is the kingdom of plants, another German biologist, Carl von Na¨geli (1817–91), maintained that microscopic fungi may arise spontaneously. Na¨geli was an important figure in nineteenth-century biology, and his views gave strong support to an alternative view of bacterial classification, namely the idea that there would be little or no constancy of form in bacteria, which were produced spontaneously from animal or plant precursors. This belief in the multiplicity of forms became known as pleomorphism, and was supported by observations that many fungi seemed to change form depending on conditions and stage in its growth cycle. The late nineteenth century saw intense investigation and debate about these changes or alteration of forms among the fungi and bacteria. In contrast to the pleomorphic hypothesis was the view of Cohn and his followers that each kind of organism had a definite and fixed form. This view became known as ‘monomorphism’. Needless to say, the outcome of the monomorphism– pleomorphism controversy was crucial to the future of bacterial classification and taxonomy. An example of this confusion is observed in the work of Wilhelm Zopf (1846–1909), who sided with Na¨geli in supporting the pleomorphic doctrine, but implicitly followed the monomorphists in his widely influential classification scheme published in 1885 (Die Spaltpilze). Toward the end of the nineteenth century the classification of microbes was influenced by three major developments: improved morphologic methods (both better instruments and staining methods), pure culture techniques, and better knowledge of the functional activities of bacteria (e.g., pathological actions and fermentations). By the turn of the century, classifications such as that proposed by Migula and Orla-Jensen included properties of bacteria beyond morphology, such as pathogenicity, growth requirements, and staining properties. The filamentous fungi as well as the protozoa were also included in these early classification schemes, and their distinctive growth patterns, natural histories, and pathologies were recognized. The spore formation by fungi, the colonial growth forms, and the life cycles were characteristics that allowed classification of these organisms to proceed more surely than that of the smaller bacteria. Many of the animalcules of Leeuwenhoek were first called infusoria. The first genus for these infusoria (Paramecium) was introduced in 1752 and in 1817 the generic term protozoa was employed by Goldfuss. While classification methods, stemming from the work of Ehrenberg and culminating in the early work of David

H. Bergey (1860–1937) and the Society of American Bacteriologists (Bergey’s Manual of Determinative Bacteriology), have been of great pragmatic use in categorizing and recognizing bacterial isolates, another goal of classification and taxonomy is to delineate evolutionary relationships. At the light microscopic level, the observable structures of bacteria are relatively uninformative, however, and there has been little confidence that simple size and shape determinations carry much evolutionary weight. Only with the more detailed investigations and classification of bacteria using biochemical and genetic analyses (genomics) has it been possible to devise classifications that go significantly beyond those of the beginning of the twentieth century. Direct comparisons of the DNA of different organisms became possible with the discovery of nucleic acid ‘hybridization’ as a method for testing the relatedness of DNA sequences. Prior to the invention of DNA sequencing methods, the extent and rate of DNA helix formation from complementary DNA strands from different microbial species was used, first by Schildkraut, Marmur, and Doty (1961) to compare the genomes of bacterial isolates. Substantial improvements in gene-based classifications were achieved when Woese (1977) showed that the comparison of the sequences of RNA in the smaller (16S) ribosomal subunit gave meaningful classificatory information. Since the aim of most systems of classification is to reflect evolutionary relationships, such gene-based schemes were highly satisfying. The comparisons of 16S RNA sequences (RNA sequencing was technically more advanced than DNA sequencing methodologies in the 1970s) allowed Woese to propose a novel group of evolutionarily ancient microbes, which he termed Archaea. He proposed that all life be classified into six kingdoms (Eubacteria, Archaebacteria, Protista, Fungi, Plantae, and Animalia). With the development of rapid methods for the determination of entire genomic DNA sequences since the late 1980s, the comparison and classification of microorganisms has become an industry, based almost entirely on gene relatedness. By 1990, Woese had revised his classification to three ‘Domains’ rather than ‘Kingdoms’ (Bacteria, Archaea, and Eukarya). Viruses, however, still resist attempts at rational classification into a single, consistent system.

Contagion The periodic and widespread occurrence of disease in populations has been noted by historians for several millennia. Both popular and medical accounts abound with descriptions, explanations, and prophylactic advice about epidemic diseases. Many of these accounts suggest that disease is transmissible from an afflicted person to a healthy person, that is, the disease is contagious.

History and Culture, (and Biographies) | History of Microbiology

Although ‘contagious’, in modern thinking, almost always implies ‘infectious’, this is not at all the way the notion of contagion should be read in these older accounts. Without the knowledge of microbes and their relation to disease, the concept of ‘infection’ is especially problematic. Instead, contagion sometimes implied active maliciousness such as the ‘evil-eye’, hex-casting, or other such magical practice. Alternatively, the contagion could reside in local conditions such as ‘foul’ air, or on the clothing or other belongings of the afflicted person, or emanate from decaying material or earth. Thus, the notion of contagion was broad and flexible. While much has been written on the history of epidemics from the Plague of Athens in 430–425 BCE (Thucydides) to AIDS and SARS, and many writers have tried their hands at retrospective diagnosis and search for precursors to modern ideas of infectious disease, the small book usually called De Contagione by Girolamo Fracastoro (1478–1553) is usually taken as the first, more or less clear, recognition that a contagious disease might be transmissible by some sort of ‘infectious’ agent. Fracastoro treats three types of contagion: by contact alone, by fomites (things ‘which are not corrupted themselves, but are able to preserve the original germs and give rise to their transference to others’), and by contagion at a distance. In spite of Fracastoro’s rather clear exposition, however, his ideas seem to have lain fallow for several centuries. Contagionist doctrines, while held by many scientists, did not develop in the direction of infectionist beliefs until a plausible mechanism, that is microbes, was known.

Germ Theories While many of the early microscopists speculated that their little animals might be related to disease, fermentations, and putrefaction, such ideas did not take hold partly because the existing concepts of these phenomena had no way to incorporate microbes into their explanatory schemes. While the diligent historian can find many examples of writings that appear to be precursors to modern germ theories, it is quite clear that they represent a rather silent or ignored tradition in medicine and biology in the seventeenth and eighteenth century. As the biological understanding of bacteria developed in the nineteenth century, and as scientists turned more and more to physiology and ‘animal chemistry’ for understanding of biology and medicine, the newer findings about bacteria found their applications. No longer content with explanations of diseases based on miasmas, supernatural retribution for sinful behaviors, and simple environmental conditions, late eighteenth- and early nineteenth-century thinkers looked for causes of disease based within the body itself, in its function, normal, and deranged. These explanations often incorporated specific

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aspects to account for the contagiousness of certain diseases. The process of fermentation was so important and widely studied that it formed the model by which other biological processes were conceived. Thus, digestion was seen as analogous to fermentation. Likewise, the processes of putrefaction, and by extension, tissue degradations in abscesses, for example, were thought to be related to fermentation. The great German chemist, Justus von Liebig (1803–73), conceived of disease as a putrefaction of tissue produced by nonliving (yet ‘vital’) substances (which we now recognize as enzymes, ferments in French) with the production of toxins. These toxins, when transferred to another individual, could induce further putrefaction ‘by contact’. Such a mechanism could explain both contagion and physiological effects without the need for living, infectious agents. In Liebig’s theory, fermentations, too, could be brought about by introduction of the nonliving ferments to suitable circumstances, for example, in wine-making. Debate on the theories of contagion thus evolved into investigation of the biological processes of both fermentation and digestion in the mid-nineteenth century. As might be inferred, too, these debates were intimately related to the controversies over spontaneous generation and vitalism mentioned above. Did fermentation (and by extension, disease) require the presence of living organisms? Was fermentation a strictly chemical process, or did it require some essentially ‘vital’ component? If fermentations always required living organisms (yeast), where did the yeast come from in the apparently spontaneous fermentations seen in wine- and beer-making? From his studies on the fermentation of beer and wine, Louis Pasteur (1822–95) concluded that the agent that is responsible for alcoholic fermentation is a living organism, yeast, which must be introduced either accidentally or intentionally. Occasional accidents of fermentation (‘diseases’ of wine and beer) occur when the wrong organism is present. This pathway of experimentation led Pasteur to strongly attack the belief in spontaneous generation. His vigorous experimentation and even more vigorous rhetorical activities led to his fame as the slayer of the doctrine of spontaneous generation. The English physicist, John Tyndall (1820–93), provided strong support for Pasteur’s ideas by his investigation of dust and small particles in the air as a source of the contaminations in experiments, which claimed to show the existence of spontaneous generation. Along with studies of ‘diseases’ of fermentations, Pasteur undertook the study of a disease of silkworms that was causing severe economic difficulties for French silk producers, and in 1866 found the ‘cause’ of this disease to be a specific microbe. By the mid-1870s, Pasteur had come to believe that all contagious diseases were caused by specific microbes, a grand generalization that had many versions, which now go under the rubric of ‘germ theories of disease’.

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History and Culture, (and Biographies) | History of Microbiology

Pasteur devised methods for preparation of pure liquid cultures of bacteria by serial transfers at high dilution, and exploited such pure culture techniques to investigate the role of bacteria in disease and later to devise protective vaccines. A related problem also engaged the attention of nineteenth-century physicians: the nature and cause of diseases variously called putrid intoxication, septicemia, surgical fever, and pyemia. Experiments based on early belief in toxins showed that these diseases were often transmissible by inoculation of blood from one animal to another. Pus from a human wound could induce disease in animals, for example. The precise relationships between pus globules, white blood cells, and other components of blood were unclear. Casimir-Joseph Davaine (1812–82) examined the blood of animals with anthrax and septicemia and reported that bacteria were only present in the blood of diseased animals. Much work of this sort from 1850 onward supported the association between the presence of bacteria and the occurrence of disease. Davaine extended this work to deliberate inoculation experiments with graded injections of material from sick animals and showed that the transmissible agent (called the ‘virus’ in nineteenth-century terminology) could be titrated and that different animal species varied in their susceptibility to the virulent agent. Davaine’s work attracted great interest in France and elsewhere, yet some investigators interpreted this work to show that the bacteria were considered a consequence of the disease rather than a cause. Theodor Albrecht Klebs (1834–1913), working in military hospitals in Karlsruhe, studied septic deaths from gunshot wounds and found bacteria of different forms present in almost every case he examined. He added much to the knowledge of wound infections and devised several approaches to the study of wound infection, which were soon adopted by Koch. Robert Koch (1843–1910) was a medical practitioner in the small town of Wollstein in Pozen, where he undertook the study of wound infections to determine if they were of parasitic (bacterial) origin. He conducted animal experiments and self-consciously considered just what kind of experiments he must do to prove, as conclusively as possible, that the wound infections that he produced in animals by injection of pus were, indeed, caused by the bacteria found in the pus. Koch was aided in his work by newly available aniline dyes for staining samples, by the newly available Abbe´ microscope illuminator, and by the fine oil-immersion lenses made in Jena by Carl Zeiss. In his publication, Untersuchungen u¨ber die Aetiologie der Wundinfectionskrankheiten, he clearly raised the standards for the study and description of the relationship between bacteria and disease. In spite of the minor stir that this publication caused, many physicians remained unconvinced. Similar investigations were carried out by many

other workers, including Alexander Ogston (1844–1929) in Scotland and Daniel E. Salmon (1850–1914) in America. Koch undertook the study of the life history of the anthrax bacillus in which he noted the formation of spores, which were especially heat-resistant. He immediately realized the relevance of the spore form both from an experimental point of view as well as from the epidemiological standpoint. Koch communicated this work to Ferdinand Cohn, a leader in the German academic world, who recognized its importance and arranged for its publication. From this work on anthrax, Koch became widely recognized as a masterful experimentalist and creative thinker. Early in his research, Koch recognized the need for better cultivation and identification methods in bacteriology and he worked hard to develop the tools he saw as essential. Both his new staining methods and his improvements of the methods of solid surface cultures led to major new advances in his research work. He devised fixation methods, which preserved the morphology of the bacteria and thus was able to study the organisms in a nonmotile form. He tested a wide variety of fixatives and stains (much of this work based on the early studies of Paul Erhlich) and was able to obtain preparations far superior to any previously available. Koch also pioneered the use of photomicrography, which had an important role in helping spread his theories as well as convince his critics. As early as 1865, there had been prior attempts to obtain pure cultures of bacteria by growth of individual ‘colonies’ on solid medium but these were generally unsatisfactory. In 1881, Koch reported that he could grow individual colonies of bacteria on the sterilized cut surface of a potato slice and soon he followed up on prior work by Oscar Brefeld, who laid the theoretical and practical foundation for work with pure cultures. Koch realized that what was needed was a medium that was sterile, transparent, and solid. Initially, he found gelatin to be very useful and later employed the more stable carbohydrate, agar–agar, as the gelling agent in the medium. At first the medium was simply poured on a glass plate and allowed to gel, but soon Koch’s assistant Richard Julius Petri (1852–1921) made slight modifications and introduced the flat dish with an overhanging lid (Petri dish), still in use today. The solid surface culture of bacteria was a revolutionary advance in technique that had major consequences. With this method for pure culture isolation, many of the ambiguities in the monomorphism–pleomorphism debate were resolved. Bacterial identification and classification became easier and more certain. Most importantly, Koch was finally able to affirm the identity of a specific bacterial type with the virulent agent in the blood, pus, or tissue extracts in his animal inoculation experiments. Koch and several other thoughtful advocates of germ theory doctrines of disease were concerned about the

History and Culture, (and Biographies) | History of Microbiology

knowledge claims they were making: what experimental evidence was needed to prove that a specific bacterium was the cause of a specific disease? Over a period of several years (1876–82), Koch evolved a set of criteria for such evidence. In his 1878 paper on wound infections, he gave a set of three rather weak criteria, but in 1882 in his landmark paper on the etiology of tuberculosis, he stated: ‘‘To prove that tuberculosis is a parasitic disease, that it is caused by the invasion of bacilli and that it is conditioned primarily by the growth and multiplication of the bacilli, it was necessary to isolate them [free] from any disease-product of the animal organism which might adhere to them; and, by administering the isolated bacilli to animals, to reproduce the same morbid condition which, as known, is obtained by inoculation with spontaneously developed tuberculous material.’’ Later versions by Koch and textbook authors have combined this statement with some of Koch’s other writing to synthesize three criteria which have come to be known as ‘Koch’s postulates’. (Apparently, however, he never referred to these criteria as ‘postulates’.) In America, Daniel Salmon was investigating the cause of hog cholera and had developed his own set of criteria for disease causation by bacteria. In addition to the criteria that Koch proposed, Salmon believed that the causal connection required the added demonstration that the killing of the bacteria was curative of the disease. In the final decades of the nineteenth century, with the new methods available and the conceptual framework of Koch, Pasteur, Davaine and others, advocates of germ theories of disease were hard at work ‘hunting microbes’. With each success of associating another disease with a specific pathogenic organism, the rush to find bacterial causes for all disease increased. In addition to bacterial causes of cancer, there even were reports of bacterial causes for mental disorders.

Applications of the Germ Theories With a clearer understanding of the causes of contagious diseases, there was a strong impetus to use this knowledge in ways to treat or prevent the disease. These efforts took several forms: sanitation and public health, asepsis and antisepsis, and preventive vaccinations. Specific chemotherapy came later. Germ theories of disease fit well with the program of the nineteenth-century public health movement with its emphasis on ‘filth’ as the cause of disease. From the mid-century work of the lawyer-sanitarian Edwin Chadwick (1800–90) in England, it was believed that mortality rates, and perhaps health in general, could be affected by sanitary reform. Bacteriologists noted that the very places and conditions considered ‘filthy’ were generally the places and conditions where bacteria were

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likely to flourish. It is no surprise, then, that many of the leading nineteenth-century bacteriologists were interested in water supply sanitation, food quality control, and sewage and waste treatment and disposal. In America, William T. Sedgwick (1855–1921) was the most accomplished sanitary scientist and water bacteriologist of his day, and as chair of the Harvard – MIT School for Public Health Officers, he educated a generation of public healthoriented bacteriologists. In England, Joseph Lister (1827–1912) was the most active advocate for the application of Pasteur’s germ theories to the practice of surgery. In 1868, he reported on his use of antisepsis (not asepsis) during surgery to prevent the occurrence of surgical wound infections. He employed phenol (carbolic acid) in an oil suspension. His results and his approaches to surgical cleanliness initiated a new era in surgical practice and led to a dramatic decline in postsurgical septic mortality. Lister’s work led others to study antiseptics in detail, and Koch soon was able to make the important distinction between agents that simply arrest bacterial growth without killing (bacteriostatic agents) and those that are able to kill bacteria (bacteriocidal agents). Germ theories did not find application only in medicine. Crop diseases, especially those involving fungi, were recognized as infectious in origin. As early as 1767, Giovanni Targioni-Tozetti (1712–83) proposed that rusts of cereals might be caused by infection with microscopic fungi and by 1807, Isaac Be´ne´dic Pre´vost (1755–1819) had demonstrated experimental smut infections and showed that copper sulfate solutions could be used to disinfect plant seeds. The biological study of fungi led to better understanding of their life cycles and of the alternation of hosts required by some organisms. For example, by 1865 Anton de Bary (1831–88) explained the role of barberry plants as an intermediate host in wheat rust, and recommended that rust epidemics could be controlled by elimination of the practice of using barberry hedges near wheat fields. Ergot of rye was another important pathogen, which led to widespread human illness in certain years when the infection was prevalent. Several fungi (mildews) were recognized as pathogenic to grape vines by the mid-nineteenth century and direct treatment of vineyards with agents known to kill the organisms were developed. Copper sulfate and lime suspended in water were widely employed and became known as Bordeaux mixture. Human diseases caused by fungi and protozoa were also recognized in the nineteenth century. Skin diseases (dermatomycoses) such as favus (Scho¨nlein, 1839) and thrush (Langenback, 1839) were shown to be related to specific fungi (Achorion schonleinii and Candida albicans, respectively), and the monograph Histoire Naturelle de Ve´ge´taux Parasites (1853) by Charles-Phillippe Robin (1821–85) was a landmark summary of early medical

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History and Culture, (and Biographies) | History of Microbiology

mycology. In 1910 when Sabouraud devised a medium on which pathogenic fungi could be easily cultivated, the development of medical mycology greatly accelerated. The role of protozoa in disease was suggested early by observations such as Alphonse Laveran’s (1845–1922) discovery of a specific organism in the blood of malaria patients in 1880. Later Giovanni Battista Grassi (1854–1925) and Ronald Ross (1857–1932) were able to discover the role of the mosquito in the transmission of malaria (1890s). Ross and his collaboration with Patrick Manson (1844–1922) exemplify the way the field of tropical medicine developed from the need to better understand newly encountered diseases during nineteenth-century European colonial expansion. Other forms of microorganisms were found to be the cause of disease as well. For example, in the early twentieth century, small, bacteria-like, obligate intracellular forms now known as Rickettsia were found to be the infectious agent in diseases such as louse-born epidemic typhus (Charles Nicolle, Howard Ricketts, and Stanislaus Prowazek) and Rocky Mountain spotted fever (Ricketts). Chlamydiae, another group of obligate intracellular pathogenic forms, thought to be distantly related to Gram-negative bacteria, were recognized in 1952 as distinct from large viruses. Mycoplasmas, the smallest known free-living microbes, are a pleomorphic and widespread group of organisms. Since they often pass through conventional filters, they were originally classified as filterable viruses. The first known mycoplasma disease was pleuropneumonia of cattle, which was studied by such luminaries as Pasteur (1883), Nocard (1898), and Bordet (1910). From this example, mycoplasmas came to be called ‘pleuropneumonialike organisms’ or PPLO until recently. One isolate from primary atypical pneumonia in humans was propagated in chicken embryo tissue and was known eponymously as the Eaton agent (1944). Perhaps the most dramatic advances that followed the new understanding of germ theories of disease were those involving preventive vaccination. The general phenomenon of resistance and immunity had been recognized for a long time, but beyond general knowledge that a prior smallpox attack protected the individual in a subsequent epidemic, and that some diseases seemed to be speciesspecific, there seemed to be no way to understand or manipulate these phenomena except in the unusual case of smallpox. Smallpox had long been deliberately transmitted by contact from a sick person to a healthy person with the intent of inducing a (hoped for) mild case of the disease, which would then result in lifelong immunity. This practice was called ‘variolation’, and was introduced to England and America in the early eighteenth century apparently from the Middle East, although this practice was widespread in India and East Asia before that. In 1878, Edward Jenner (1749–1823) started his studies on

the role of cowpox infection in humans as a protective experience against smallpox. He was following an apparently well-known local folk belief, but through his careful work and clear exposition in his report of 1798 he gave wide attention to the effectiveness of this procedure, called ‘vaccination’ (Latin: vacca, cow), in protection against smallpox. Pasteur seemed to have been influenced by notions of such cross-immunity resulting from similar but slightly different diseases, and when he was called upon to study chicken cholera in France, he saw an opportunity to apply this notion to another disease. While studying the bacteria associated with chicken cholera, Pasteur transferred the cultures serially and noted that the virulence of the cultures for killing inoculated chickens decreased with the number of laboratory culture transfers. He termed this decrease ‘attenuation’. Apparently, he sought to ‘challenge’ some birds that had recovered from an inoculation of an attenuated culture with a highly virulent form of the bacterium and discovered that the attenuated inoculum had produced strong immunity to the lethal form of the disease. From these observations, Pasteur went on to formulate rather elaborate and complex theories of immunity and attenuation. These theories are no longer accepted, but they stimulated much important research on vaccines, immunity, and the nature of bacterial virulence. Out of this work came Pasteur’s famous rabies vaccine as well as his famous work on anthrax vaccine, which resulted in highly publicized trials of his vaccine on a herd of sheep, some cows, and a goat at Pouilly-le-Fort in 1881. Vaccine development has proceeded from this time partly along the empirical approach used by Pasteur and his colleagues, and partly along the more theory-based approaches developed by Paul Ehrlich (1854–1915), Emil von Behring (1854–1917), and later immunologists. In trying to explain the mechanisms by which vaccines induce immunity, early research focused on the interaction of the bacterium and the blood. Richard Pfeiffer (1858–1945) noted that the serum of immune animals was able to cause lysis of the specific bacterium against which the immunity was directed, and later Jules Bordet (1870–1961) discovered that multiple serum components (complement) are involved in the immune cytolysis (cell lysis) phenomenon. The antibody molecules induced in response to vaccine treatment were studied intensely, and Ehrlich explained the specificity of the antibody-antigen interaction in terms of the structural features of each of these components. The science of immunology took new directions with the landmark studies of Karl Landsteiner (1868–1943) on the specificity of serological reactions. While germ theories of disease gradually gained adherents in the last two decades of the nineteenth century, and hunts were underway for microbes in every conceivable situation, doubts remained. For example,

History and Culture, (and Biographies) | History of Microbiology

the discovery of the healthy carrier state in cholera by Koch and his colleagues provided a serious challenge to germ theories. When Max Von Pettenkofer (1818–1901), a major critic of the germ theories, drank a pure culture of cholera vibrios and remained healthy, Koch registered his worry that the germ theory had suffered a serious setback. Vaccination campaigns were not always readily accepted by the public and the validity of vaccination for a variety of diseases was doubted not only by many layperson, but also by many physicians as well. The new science of microbiology did not simply march triumphantly into the clinics and up to the bedsides to take over medicine by force of reason, superior science, and undeniable successes.

Antimicrobial Therapies Following on the work of Lister on antiseptics, many microbiologists undertook searches for agents, which could be used to kill bacteria in vivo. Various attempts to use chloroform, iodine, thymol, and many other disinfectants were reported. Paul Ehrlich reasoned that just as there were dyes as well as antibodies that are specific for certain bacteria, there must be other kinds of chemicals that can bind to, and inactivate, specific microbes. His search for such compounds led to the discovery of several drugs useful in the treatment of protozoal and spirochetal diseases between 1905 and 1915. The arsenical compound, arsphenamine (salvarsan, compound 606), was used to treat syphilis until the advent of penicillin. In a continuation of Ehrlich’s approach, another product of the dye industry, prontosil, was introduced as an antibacterial agent by Gerhard Domagk (1895–1964) in 1935. This compound was metabolized to sulfanilamide, a fact which when recognized, led to the development of many new sulfa drugs. While investigating lysozyme, a bacteriolytic enzyme present in some body fluids and abundant in egg white, in 1929 Alexander Fleming (1881–1955) noted that cultures of the mold Penicillium produced a substance (penicillin) that inhibited the growth of Staphylococcus on culture plates. Although Fleming was unable to purify and more fully characterize penicillin, an accomplishment of Howard Flory and Ernst Chain in 1940, his finding suggested to others that saprophytic fungi might be a useful source of antimicrobial agents. Rene´ J. Dubos (1901–82) discovered one such product, tyrothricin, which has the peptide gramacidin as its active component. This success marked the beginning of a search for microbial products that can be used as antimicrobials. The discovery of streptomycin by Selman A. Waksman (1888–1973) and Albert I. Schatz (1920–2005) in 1944 was the beginning of a cornucopia of useful antibiotics. Potent microbial toxins, such as the highly carcinogenic aflatoxin from Aspergillus

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flavus, cholera toxin and botulinus toxin, among many others, have been discovered.

Public Health The origins of public health microbiology are rooted in the earlier concerns of public health with sanitation and with control of epidemic diseases. The linkage of epidemiology and microbiology was a natural outgrowth of germ theories of disease and the general understanding of the distribution of microbes in the environment. W. T. Sedgewick wrote a key text in 1902, Principles of Sanitary Science and Public Health, which described the examination of drinking water for microscopic forms (bacteria, diatoms, algae, and infusoria), the use of coliform counts to assess the effectiveness of water filtration and the processing of sewage involving microbial actions. Major problems in public health included food microbiology, such as testing milk cows for tuberculosis (1889), epidemics, exemplified in Sources and Modes of Infection published in 1910 by Charles V. Chapin (1856–1941), and sanitation testing through the establishment of government microbiological laboratories. Partly in response to the cholera epidemics in the United States in the nineteenth century, several major cities established permanent, active boards of health by the end of the century. The US Public Health Service established the Laboratory of Hygiene in a Marine Hospital on Staten Island in 1887, to serve as a cholera study unit. This laboratory evolved into the National Institute of Health in 1930. Governmental laboratories, both municipal and state, served as diagnostic centers for physicians and public health officers to carry out microbiological isolations, diagnostic identification, and community surveillance. In New York, for example, the Public Health Laboratory under Herman Biggs, Haven Emerson, and William H. Park provided diagnostic services, bacteriological screening of food handlers and of school children, and later supervised immunization programs. These public health laboratories combined diagnostic microbiology, epidemiology, field work, quarantine, and research to define the current scope of public health microbiology.

Clinical Microbiology Microbiology in medicine often was in the professional domain of pathologists, because in the period before effective antimicrobial therapies, many infections were studied in the autopsy room after the demise of the patient. Within the hospital, then, microbiology grew up along with pathology, and until the middle of the

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twentieth century these two disciplines were frequently practiced in the same department, usually called ‘pathology and bacteriology’ recognizing the seniority and dominance of pathology. Starting in the early twentieth century, however, bacteriology began to claim its independence from pathology. Educational programs in bacteriology were separated from pathology, laboratory practices differentiated bacteriology from anatomic pathology, and professional boundaries began to develop. The birth of medical microbiology in association with pathology probably made it relatively easy for microbiology to take on service roles in examination of clinical specimens as had pathology. Many of the early microbiologists had been trained as physicians and they could relate well to their clinical colleagues and argue for the importance of increased bacteriological study of patients prior to death. Especially with the discovery of antimicrobial therapies, starting with serums and vaccines, and later with bacteriophage and then sulfas and antibiotics, the role of the bacteriological laboratory in clinical medicine became central to the practice of medicine. With the advent of specific antimicrobial therapy, it became crucial to know the identity and antibiotic sensitivities of the infectious agent isolated from the patient. This demand for accurate and rapid microbiological analysis of clinical samples continues to the present, with great emphasis put on technological innovation and the rapid processing of massive numbers of samples.

Agricultural Microbiology Microbiology developed from an early stage around both the practices and the products of agriculture. The study of the microbes in the soil, the microbiology of animal health, and the processes of food production all were important from the very beginning of the field. While soil microbiology has developed into its own disciple, it originated in questions relating to the chemistry and fertility of soil and broad questions about the role of soils in chemical processes involving nitrogen. The sources of ammonia and nitrates in the soil puzzled chemists such as Pierre-Euge`ne-Marcelin Berthelot (1827–1907) who soon surmised that it was the microbes in the soil which were responsible for the ‘fixation’ of nitrogen into ammonia and nitrates. Other nineteenth century microbiologists found that the root nodules in legumes were symbiotic colonies of bacteria with the special capacity to reduce atmospheric nitrogen to ammonia and other forms that were then available to the plants for assimilation. Sergei Winogradsky (1856–1953), the Great Russian microbiologist, focused on soil microbiology and through his work and that of his research school, developed our current understanding of microbial nitrogen fixation, a process essential for life on earth. The Dutch school of general

and comparative microbiology, founded by Martinus W. Beijerinck (1851–1931) and his protege, Albert Jan Kluyver (1888–1956), grew from this same interest in soil and environmental microbiology. Both the diseases and special biology of animals and plants provided important opportunities for crucial advances in microbiology. Pasteur refined his concepts of the role of microbes both in fermentation and in disease by his studies on the ‘diseases’ of beer and wine, and in his study of diseases of silkworms, beehives, and chicken flocks, not to mention the famous case of anthrax. Such diseases afforded chances to carry out investigations on animals that would not have been possible in analogous human diseases. Animals also provided novel biological opportunities because of their specialized physiology. The symbiotic relationship between bacteria and digestion in ruminants was an important advance in the understanding of animal husbandry. The production of silage by microbial action furthered the knowledge of both animal nutrition as well as the science of fermentation. The role of microbes in the processing and preservation of foods provided another path of investigation in microbiology. Both the use and the avoidance of microbes in food technology has been crucial to improving food yields, reducing spoilage, and avoiding illnesses associated with food contamination. Food preservation by microbial action is ancient, familiar examples being wine, beer, and cheesemaking. The use of vinegar, a product of bacterial action, and salt to inhibit contamination in pickling is also an ancient practice. Smoking and drying fish and meat are traditional practices to limit bacterial growth.

Virology One of the basic methods for ‘microbe hunting’ from the very early period of microbiology was filtration. Very fine filters were designed to remove small particles such as microbes to sterilize fluids and to characterize the particulate nature of infectious materials. Two widely used filters were the Chamberland filter and the Berkfeld filter. The former, named for Charles Chamberland (1851–1908) of the Pasteur Institute, is a tube of unglazed porcelain (a ‘candle’ filter) through which liquids can be passed under pressure. The latter is a column of diatomaceous earth (Kieselguhr), named for the owner of the mine near Hannover, which produced the material. If an infectious agent was not retained by such a filter, it was termed ‘filterable’ (sometimes spelled filtrable) or ‘filter-passing’. Of course, both particulate and soluble substances could be ‘filterable’. The term ‘virus’ was initially used to designate the component of an inoculum, which was the causative agent of the disease (Latin: virus: poison, venom). The

History and Culture, (and Biographies) | History of Microbiology

term was generically applied to bacterial agents as well as other organisms. One of the main goals of the early germ theorists was to isolate and identify the virus that was present in blood, tissue extract, sputum, or stool sample, which could transmit disease when inoculated into susceptible hosts. Soon it was found that some of these viruses of disease could pass through the filters, which were known to retain bacteria. These agents of disease came to be known as ‘filterable viruses’ to distinguish them from all the other viruses. Since the nature of the filterable viruses was obscure in the early part of the twentieth century, the term ‘filterable virus’ persisted in the literature until the 1930s; for example, the classic 1928 text, edited by Thomas M. Rivers (1888–1962), was entitled Filterable Viruses. Shortly thereafter, common usage came to drop the qualifier ‘filterable’ in favor of simply ‘virus’ to designate the filter-passing forms of infectious agents. The first disease that was recognized as being caused by a filter-passing agent was tobacco mosaic disease. This disease was economically devastating to Dutch tobacco growers and its cause was actively studied in Holland starting with the work of Aldof Mayer (1843–1942) in 1879 who was able to transmit the infection but failed to find the causative virus, believed to be a bacterium. Martinus W. Beijerinck worked on tobacco mosaic disease off and on for over a decade and by 1898 he found that the virus was ‘filterable’, that it would diffuse through agar, and that it was serially transmissible. For the virus of tobacco mosaic disease, Beijerinck proposed a new category of agent, living and nonparticulate, a contagium vivum fluidum. A few years earlier, Dimitri Ivanovski (1864–1920) had shown the filterabilty of the agent of tobacco mosaic, but because he was able to transfer the disease via bacterial colonies (probably contaminated with residual filterable virus), Ivanovski believed that tobacco mosaic was a bacterial disease. The nature of filterable viruses as represented by the tobacco mosaic virus (TMV) was controversial. Some thought of the agent as chemical, perhaps a cellular component, while others thought of it as an ‘ultramicrobe’, an organized living being, too small to be seen in the microscope. Subsequent attempts to study TMV were hampered by the inability to grow the agent outside of the infected plant, and by difficulties in its detection and quantification. Some of these difficulties were overcome by the preparation of large amounts of infected plant material by Carl G. Vinson (1927) and an improved quantitative assay by Francis O. Holmes (1928). In 1935 Wendell Stanley (1904–71) reported that he had crystallized TMV, a startling observation that forced reconsideration of the concept of ‘living’, ‘infectious’, and ‘microbe’. Stanley and his coworkers, based on their chemical analyses at the time, believed that TMV was composed only of protein. Since crystallization was the standard criterion for purity

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of organic compounds and by extension, of proteins, this work was interpreted to show that filterable viruses such as TMV were self-replicating, infectious proteins. Indirectly, of course, this conclusion strengthened the belief that genes, too, were protein molecules. The RNA component of TMV (about 7%, by weight) was soon found by Frederick Bawden (1908–72) and N. W. Pirie (1907–97) in England. Still, the controversy over viruses remained. How did they replicate? Where did they originate? Were they autonomous or part of the cell? Should they be conceived as macromolecules or as microbes? From the beginning of the twentieth century, filterable viruses were found as the causes of many contagious diseases that had resisted bacteriological etiologies: Herpes labialis (fever blisters), influenza, and poliomyelitis, to name a few human diseases; hog cholera, rabies, and cowpox among the mammalian diseases; and leukemia and sarcomas in birds. It was eventually realized that one of the defining, although ‘negative’, characteristics of all these viruses is that they are obligate intracellular agents, and they cannot be grown independently of their host or at least host cells. This realization finally led to searches for better ways to grow and assay viruses apart from animal or plant inoculations. The tissues of the embryo in the chicken egg became a convenient and standardized growth and assay medium for many viruses and are still in use today for some purposes. Egg cultures of many viruses were studied by Ernest Goodpasture (1886–1960), who perfected this method in the 1930s. By the 1950s the ability to grow explanted mammalian cells in various culture media (‘tissue culture’ or ‘cell culture’) provided a new and improved way to grow, assay and study many viruses. In particular, the growth of poliovirus in monkey kidney cells in culture in 1949 by John Enders and colleagues led to better understanding of this virus and its ultimate control through preventative immunizations first with a killed preparation of poliovirus (the Salk vaccine) and later a live, attenuated orally effective vaccine (Sabin). A special class of filterable agents was discovered in the first decade of the twentieth century, which deserve special mention: the cancer-causing viruses. This group of viruses, not related by structure, pathogenesis, or genealogy, has provided important insights into the interplay between genes, viruses, and cells. In 1911, Peyton Rous (1879– 1970) observed that a cell-free filtrate prepared from a chicken sarcoma could induce similar sarcomas when inoculated into other chickens. Likewise, in 1908 Ellerman and Bang suggested that leukemia in fowl could be caused by inoculation with a filterable agent. The notion that cancer might be an infectious disease was so potentially frightening to the public that Rous tried to avoid any unnecessary publicity of his work, and eventually abandoned it. In searches for the causes of cancer, there were many reports of bacteria associated with various malignancies, but only the filterable viruses seemed to emerge as

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possible causal agents for animal cancers. (One possible exception is the mucosa-associated lymphoid tissue (MALT) lymphoma, associated with Helicobacter pylori infection of the stomach. In plants, the causative role of Agrobacterium tumefaciens is causing tumor-like proliferation in well established.) By the mid-1950s several classes of viruses had been identified, which could induce cancers in experimental animals. However, it was not until much later that candidate human tumor viruses were isolated. Most of these tumor viruses have RNA genomes, which can be copied into DNA and integrated into the cell genome to reside there in symbiosis while causing a neoplastic transformation in many cases. Interestingly, the organism associated with AIDS, human immunodeficiency virus (HIV), first identified in the 1980s, turned out to be in this same retrovirus group of viruses, although HIV does not cause tumors. Bacteriophage are now recognized as viruses that infect bacteria; however, originally they were not believed to be similar to the filterable viruses. In 1915 F. W. Twort (1877–1950) in England reported ‘glassy transformation’ of micrococci, which contaminated his attempts to grow vaccinia virus on cell-free culture media. This phenomenon of glassy transformation was serially transmissible and killed the bacteria. Twort’s interpretation of this phenomenon was unclear and ambiguous. In 1917, Fe´lix d’Herelle (1873–1949), a French Canadian working in Paris, independently observed lysis of dysentery cultures, and noted that the lytic principle was filterable, and that something in the lysed culture could produce clear spots (plaques) on confluent bacterial cultures. This lytic principle was serially transmissible and by d’Herelle’s interpretation, particulate. He conceived of this agent as a microbe, which parasitizes the bacteria, that is a virus of bacteria. Not only did he devise the quantitative plaque counting method, but he also worked out the basic life cycle of this agent, which d’Herelle termed bacteriophage (although long known by the noncommittal term ‘Twort-d’Herelle Phenomenon’). The biological nature of bacteriophage was hotly debated for about 20 years, with the majority view in opposition to d’Herelle’s ultravirus hypothesis and in favor of some sort of endogenous autocatalytic process. Only when uniform virus particles were observed by electron microscopy in 1940 did the particulate view of bacteriophage become widely accepted. D’Herelle and Twort engaged in a 10-year polemic over the issue of priority of discovery of bacteriophage which tarnished the reputation of both scientists. Twort did not pursue phage research, and d’Herelle focused mostly on the application of phage as therapy and prophylaxis for infectious diseases in the era before antibiotics. While this application seemed to offer promise and is now being reexamined, it was eclipsed by the marvels of the new antibiotics in the early 1940s.

The study of phage from the biological point of view has been central to the development of molecular biology. Viruses in general, and phage in particular, were recognized as very useful ‘probes’ for cellular processes which they exploit during their life cycles. The problem of gene duplication, especially, seemed amenable to study with phage. Both in the United States and in France, phage research since the 1940s was directed at understanding what happens during the half hour or so that it takes for one infecting phage to produce a hundred progeny in an infected bacterium. This was the basic research program that Max Delbru¨ck (1906–81) set for the American Phage Group. The work of this loosely defined school of research has been instrumental in deciphering much about the nature of the gene, mutagenesis, recombination, and gene expression and regulation. In virology the electron microscope has had a major impact. Prior to about 1940, viruses were defined by their small size, by filtration and diffusion properties, and their invisibility in the light microscope. The electron microscope was invented in Germany in 1931 by Max Knoll and Ernst Ruska, and by the late 1930s had sufficient resolving power to demonstrate the particulate nature of several viruses, including bacteriophage. The RCA company designed and constructed the first electron microscopes in the United States, and in 1942 Thomas Anderson (1911–91) and Salvador Luria (1912–91) used such an instrument to examine phage in detail. The ability to visualize viruses at last (albeit indirectly) brought some long-awaited unity to virology, and bacteriophage were finally accepted as viruses of bacteria.

Microbial Physiology With the recognition of the role of microbes in ancient processes such as fermentations and other types of food processing (cheese, bread, soy sauce, and silage), a better understanding of these uses of microbes and their by-products was possible. Antibiotics represent just one aspect of this exploitation of microbial metabolism. Several groups of researchers, including Sergei Winogradsky and Martinus Beijerinck, emphasized the diversity of microbial forms and metabolism and pioneered the fundamental study of the physiology of bacteria. These studies led directly to the use of microbes to produce useful ‘secondary metabolites’, compounds such as glycerol, lactic acid, pigments, and other intermediates that are products of the metabolism in specific organisms. Soil and dairy microbiology developed in colleges of agriculture and in the agriculture experiment stations established in the United States, in the late nineteenth century. This work promoted the understanding of microbial processes used in cheesemaking, such as the use of specific molds to achieve the best products, in spoilage of foods, and in the use of manure, legumes, and

History and Culture, (and Biographies) | History of Microbiology

composts to fertilize the soil. By the 1930s this work on bacterial metabolism was central to the new science of biochemistry, representing, for example, a major focus in Gowland Hopkins’ department in Cambridge under the leadership of Marjory Stephenson (1885–1948). Interest in animal and plant nutrition, especially work on growth factors and vitamins, was extended to microbes in the 1920s and 1930s. In Paris, Andre´ Lwoff (1902–94), for example, investigated the growth requirements of protozoa, while in England, Stephenson, B. C. J. G. Knight (1904–81) and Paul Fildes (1882–1971) studied bacterial growth requirements, and in America, Edward Tatum (1909–75) turned to fungal biochemistry. These kinds of investigations led to the concepts of essential nutrients and specific metabolic reactions in microbes.

Microbial Genetics When George Beadle (1903–89) and Tatum were investigating the genes for eye color in the fruit fly (Drosophila melanogaster), they saw an opportunity to examine the biochemistry of gene action in a more tractable system, the biosynthesis of vitamins in fungi. In 1941 they produced pyridoxine-requiring mutants of Neurospora crassa by X-ray mutagenesis, and showed that these mutants behaved in a Mendelian fashion in mating experiments. This result suggested to Beadle and Tatum that genes were more or less directly involved in the control of metabolic steps, that is of enzymes. While the exact chemical nature of the gene was unclear at the time (many scientists thought that the genes were the enzymes themselves), they formulated a theory of the gene that came to be known as the ‘one gene:one enzyme hypothesis’, a basic principle of modern molecular genetics (notwithstanding the additional complexities introduced later by discovery of messenger RNA, splicing, operons, and oligomeric enzymes). This approach, employing both genetic and biochemical studies, was remarkably fruitful and in a few years the notion that genes controlled all the biochemical pathways in cells was widely (although not universally) accepted. While some of the literature on cyclogeny (a theory based on bacterial growth cycles) suggested that mating, with the formation of zygotic pairs, might occur in bacteria, and some data were presented to support this concept, none of these studies led to new understanding and further progress in the genetics of bacteria. However, after the successful discovery of nutritional mutants in Neurospora, C. H. Gray and Tatum in 1944 were able to produce nutritional mutants in bacteria, and in 1947, Joshua Lederberg (1925–) and Tatum carried out experiments with two nutritionally defective mutants of the K-12 strain of E. coli (a clinical isolate that had been used in the student laboratory at Stanford University

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where Tatum had recently taught), and they found that they could obtain recombinant forms of E. coli K-12 in a clear demonstration of sexual mating in bacteria. By means of bacterial recombination, a genetic map of E. coli K-12 was constructed and by 1952 William Hayes (1913–94) clarified this process of conjugation by his explanation of unidirectional transfer of genetic material. Lederberg and Cavalli formulated bacterial mating as a rudimentary form of sexuality with the donor cell called Fþ (fertility plus, ‘male’), the recipient called F (fertility minus, ‘female’), and the recipient cell with the donated genes called the zygote. The transfer of the genetic material from donor cell to recipient cell could be interrupted by mechanical agitation of the culture, and Franc¸ois Jacob (1920–) and E´lie Wollman (1917–) were able to investigate the nature and sequence of the gene transfer by this ‘interrupted mating’ (‘coitus interruptus’) experiment. Since some of the genes they studied did not seem to be associated with the bulk of the cell genes, they hypothesized that some genes exist on extrachromosomal elements, not always present in all cells, which they termed ‘episomes’ (1958). While the understanding of microbial genetic processes was increasing during the 1940s and 1950s, the understanding of the nature of the gene itself and of the processes involved in gene stability and change, that is mutation, were also maturing, but from a somewhat different tradition. By the mid-1920s, two strands of investigation converged to direct attention to the problem of heredity in microbes. First, many studies on the virulence of bacterial isolates suggested, even from the time of Pasteur’s work on attenuation of virulence, that supposedly pure strains of bacteria could give rise to variants with altered virulence. Second, many bacteria with recognizable colony morphologies, for example, smooth or rough colonies, pigmented or nonpigmented, were noted to throw off variants of the other type now and then. This phenomenon in which a ‘pure’ culture ‘dissociated’ into a mixture of two types was called ‘bacterial dissociation’ and was widely studied. Interestingly, explanations of dissociation first centered on the ideas that cultures had ‘life cycles’ and that the different forms of bacteria represented stages in the life cycle of the culture. Contemporary work on sporulation, protozoal development, ‘phase variation’ in some bacteria such as Salmonella, and fungal growth variations all supported this concept of bacterial ‘cyclogeny’ as it was termed. The theory of cyclogeny led to a resurgence of the pleomorphism concept of the nineteenth century and was a rather widely held belief in the period between World War I and World War II. The more recent success in the alternative explanation, genetic variation, has all but obliterated memory of the heyday of cyclogeny. While some bacteriologists, such as Paul Henry DeKruif (1890–1971), better known as a science writer

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History and Culture, (and Biographies) | History of Microbiology

and the author of Microbe Hunters, advocated genetic mutation of individual cells as the explanation for bacterial dissociation, the continued focus on the entire culture rather than the individual cell obscured the genetic basis for this phenomenon. Indeed, the genetic status of bacteria and some other microorganisms was unclear. The science of genetics arose from breeding experiments, both in plants and in animals, and its focus was on reassortment and sexual transmission of characters from parent to offspring. Without easily visible chromosomes and without a sexual phase of reproduction, bacteria did not fit into the existing genetic paradigms. As late as 1942, the eminent British biologist Julian Huxley (1887–1975) wrote about bacterial heredity: ‘‘One guess may be hazarded: that the specificity of their composition is maintained by a purely chemical equilibrium, without any of the mechanical control supposed by the mitotic (and meiotic) arrangements of higher forms.’’ This view was no doubt strongly influenced by Huxley’s contact with Cyril Hinshelwood (1897–1967) and his school which saw bacterial physiology and genetics in terms of chemical kinetics, and which minimized the centrality of the gene in the life of the bacterium. Perhaps because of the well-known phenomena of adaptation and ‘training’ of bacteria to different growth conditions, much of the discussion of mutation in bacteria during the 1930s and early 1940s has a strong neoLamarckian quality: the exposure of the organism to the agent somehow provoked the observed changes. Exposure of bacteria to bacteriophage led to the emergence of cultures resistant to the phage and often displaying new properties such as altered virulence, changed colony morphology, and different antigenic types. The ‘adaptation’ of cultures upon exposure to antiseptics, drugs, extremes of pH, and so on, were often seen as purposeful and directed responses. A minority view in the 1930s was, however, that the dissociation phenomenon was happening independently of the exposure to the agent used to detect the change, that is the phage, drug, or chemical. The major problem in this work was one of experimental design: how to observe a rare event that happened in a huge population prior to the selection for the outcome of that event? In a particularly clear and convincing work, Isaac M. Lewis (1873–1943) examined the mutational change from the inability to ferment lactose to the ability to use this sugar source in the E. coli strain mutabile (in 1907 Rudolf Massini (1880–1955) reported on a variant of Bacterium coli (E. coli), which had lost its ability to utilize lactose but which frequently regained this ability and called it B. coli mutabile). Lewis spread the parental (lactose-negative) bacteria on glucose-containing plates and also on lactose-containing plates. About one in a million of the bacteria grew on the lactose-containing plates and when retested, all the cells in these lactose-utilizing colonies bred true and

could use lactose. By laboriously picking colonies from the glucose plates and testing them for their ability to utilize lactose, he estimated the frequency of lactoseusing bacteria in the culture in the absence of exposure to lactose. This frequency was similar to that determined by plating of the mass culture on lactose medium, so he concluded that the mutations to lactose utilization occurred prior to the selection, not as a consequence of exposure to the selective conditions. This elegant and clear approach, however, did not change many minds, and it was not until the 1940s that two related experimental approaches gave results that comprise the canonical account of bacterial mutation. In their work on the reproduction of bacteriophage, Max Delbru¨ck and Salvador Luria were aware that bacteria often developed resistance to phage. If a bacterial culture was infected with phage and lysed, sometime later a ‘secondary’ growth of bacteria would often appear, and these bacteria were resistant to infection with the original phage. While the occurrence of resistant bacteria was a drawback to the use of phage as an antibacterial therapeutic agent, it afforded Luria and Delbru¨ck (1943) an opportunity to study the change in another hereditary property of bacteria. They were also aware of the problem in the interpretation of bacterial mutation experiments. No doubt because of their routine use of statistical models in their work on the inactivation of bacteria and phage by radiation, as well as their backgrounds in atomic physics, they devised a statistical approach to show that phage-resistant mutants exist in the bacterial population prior to exposure to the lethal effects of phage. Their method (‘fluctuation test’) was based on the properties of the Poisson distribution and the arrangement of the experiment to analyze the frequency of occurrence of mutants in numerous replicate cultures of bacteria. If the occurrence of the mutants took place prior to the analysis (i.e., prior to the application of selective conditions), the Poisson distribution predicted that the frequencies of mutants would fluctuate widely among the replicate cultures. Conversely, if all the mutations occurred during the analysis (i.e., induced by the selective conditions), the frequencies of mutants would be very similar among the replicate cultures. The frequencies fluctuate as predicted by the Poisson distribution; however, Luria and Delbru¨ck showed that this fluctuation could be used to estimate the mutation rate, that is, probability of a given type of mutation per cell division. The method of Luria and Delbru¨ck and a related procedure devised in 1949 by Howard B. Newcome were indirect, mathematical, and subject to suspicion. However, in 1952 Joshua Lederberg and Esther Lederberg (1922–2006) devised a simple and direct method to demonstrate that mutations were occurring in a random way, independent

History and Culture, (and Biographies) | History of Microbiology

of the selection procedures. They reasoned that the approach of Lewis was logically correct, but that the methods for its execution had to be improved. They devised a method for transferring very large numbers of colonies from one plate to another by the use of velvet cloth as a transfer tool. The tiny fibers that stand out from the velvet acted as small inoculating needles and picked up some bacteria when pressed against the surface of a culture plate studded with colonies. When the velvet was pressed on a fresh, sterile plate, this plate was inoculated with bacteria in exactly the same pattern as the original plate. Thus, this ‘replica plate’ could be used to test colonies in great numbers for mutant properties. Lederberg and Lederberg (1952) applied this technique to the study of phage resistance as well as to the streptomycin resistance, and in both cases it was clear that the mutants had appeared before the application of the selective agent. Although by mid-century it was generally recognized that bacteria have some sort of genetic apparatus, since they do not have cytologically visible nuclei and chromosomes, the exact organization of the genes was unclear. Perhaps influenced by the widespread interest in cytoplasmic inheritance in higher organisms, there was much discussion about the possibility that bacterial heredity was carried on plasmagenes or that bacteriophage were ‘raw genes’ from bacteria. Joshua Lederberg (1952) introduced the term ‘plasmid’ to explain and encompass the wide variety of genetic determinants, which had been observed or hypothesized to exist in microbes. Thus, examples of cytoplasmic inheritance in protozoa, lysogeny in phage, and sometime later, bacterial fertility factors, came under this rubric. This concept, in a variant form described by Jacob and Wollman (1959) under the name of ‘episome’ nicely explained the genetic studies with fertility factors, specialized transfer of one or a few markers (e.g., the lactose-utilization genes) in some mating experiments, and later on, the ‘infectious’ nature of antibiotic resistance (resistance transfer factors, RTF) and the variation in virulence and toxin production in many pathogens. Today, the term plasmid has come to signify a circular, extrachromosomal DNA molecule, which autonomously replicates in the cell, and is not essential for that species (although culture conditions, such as the presence of certain nutrients or antibiotics, can make the plasmid essential under specific conditions). The plasmid concept has facilitated the understanding, too, of the ‘gene flow’ between the chromosome proper and exogenous agents such as viruses. The ability of some plasmids to undergo genetic recombination with the chromosome explained the mechanisms by which genes can move between species in nature and also established some of the key steps in the process of lysogeny of bacteria by certain bacteriophage. As such, the plasmid concept has been important in understanding the genetics of cancer, as well as certain schemes for evolution of

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microbes. Of course, the role of plasmids as vectors for gene transfer in current biotechnology is too well known to need elaboration here.

Bacterial Transformation and DNA With the beginning of the understanding of the process of bacterial mutation as well as the discovery of ways of manipulation of bacterial genes by conjugation, the field of microbial genetics entered the modern period. Two disparate strands of research led to the current understanding of the nature of genes in bacteria. One strand comes from work on bacterial virulence and pathogenesis in pneumonia, and led to the identification of DNA and the chemical stuff of which genes are composed. The other strand comes from the study of bacteriophage replication. In 1928, Frederick Griffith (1877–1941) was investigating the virulence of the organism that is responsible for pneumonia (Streptococcus pneumoniae or ‘pneumococcus’), and he found that heat-killed bacteria of one form of pneumococcus could somehow convert live bacteria of another form to exhibit some of the antigenic and virulence properties of the heat-killed form. While these results were of no apparent interest or relevance to the few bacteriologists interested in microbial heredity, they were of real importance to the pathologists interested in pneumonia. Oswald T. Avery (1877–1955), working at the Rockefeller Institute, was one of several scientists who confirmed and followed up Griffith’s work with a research program on what he called ‘transformation’ of antigenic types. Work in his laboratory, first by Martin Dawson followed by James L. Alloway, and then by Colin MacLeod (1909–72) and Maclyn McCarty (1911–2005), eventually led to characterization of the transforming material from the heat-killed bacteria as DNA itself. The final characterization, published in 1944, relied on the use of the newly purified and characterized enzymes, DNase and RNase, as well as several well-known proteolytic digestive enzymes. Still, appreciation of DNA was not universal: at the mid-century meeting of the Genetics Society of America, DNA was hardly mentioned. Because proteins exhibited the diversity expected of genes and the chemistry of DNA as it was then understood suggested its ‘information content’ was too low to have genetic potential, DNA was not taken seriously as the genetic material. Two major works soon challenged that belief: using new analytical methods resulting from wartime research, Rollin D. Hotchkiss (1911–2004) showed that the base compositions of DNAs from different organism were not identical and Erwin Chargaff (1905–2002) noted certain regularities in the analyses of all DNAs: the number of purines always equal the number of pyrimidines, and the ratios of adenine to thymine and guanine to cytosine were always very near to one.

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History and Culture, (and Biographies) | History of Microbiology

While the chemistry of the ‘transforming principle’ in pneumococcus was being established, a second line of work was going on to understand the process of gene duplication. Max Delbru¨ck saw bacterial viruses (bacteriophage) as a simple model for the process of gene replication and he recruited a group of like-minded associates to attack this problem. In the early stages of their work, the American Phage Group (as Gunther Stent has named Delbru¨ck’s school) treated the host bacterium more or less as a ‘black box’ and studied phage replication as a simple input–output process. In a widely cited experiment, in 1952 Alfred Hershey (1908–97) and Martha Chase (1927–2003) labeled bacteriophage with two isotopes, 32P in the DNA, and 35S in the protein, and in an attempt to follow the fate of the two major components of the phage through one life cycle, they noted that most of the 32P label entered the cell and a significant fraction ended up in progeny phage, while very little of the 35S label entered the cell and even less ended up in the progeny phage. While this result is often described in texts and reviews as if the results were ‘all or none’, the experimental results given in the original paper, while certainly supportive of the ‘only DNA’ hypothesis, are far from conclusive. Another member of the American Phage Group was James D. Watson (1928–), a student of Luria in Indiana University. Watson’s thesis was on the radiobiology of bacteriophage and he firmly believed that DNA was the chemical substance of the gene. Watson and Francis Crick (1916–2004), working at the Cavendish Laboratory in Cambridge England, devised a plausible model for the three-dimensional structure of DNA, which finally provided the much-needed explanatory framework for the genetic role of DNA (1952). Accounts of this landmark research abound, and do not need repetition here.

Physiological Genetics While the understanding of the role of the gene in the hereditary transmission of properties from parent to progeny organisms and in the explanation of the mutational process derived from research in diverse areas of microbiology as pneumonia research, microbial growth factors, and bacteriophage reproduction, the role of the gene in the growth, development, and physiology of microorganisms was studied in different contexts. Although as early as Pasteur microbiologists investigated microbial physiology, this field developed rapidly in the 1930s. In parallel with the general advances in biochemical understanding, microbial physiology was important in the discovery of growth factors, in understanding the complexities of nutrition in animals with symbiotic flora (e.g., ruminants), and in appreciating the

role of bacteria in conversion of atmospheric nitrogen to organic forms of nitrogen in the process of ‘nitrogen fixation’ that occurs in the bacteria-packed root nodules of leguminous plants. A particularly interesting problem in bacterial physiology, one that represented a general phenomenon in microbes, was that of ‘enzymatic adaptation’. It was widely observed that cultures growing on one substrate (e.g., glucose) and then shifted to another substrate (e.g., lactose) exhibited a short lag in growth and then ‘adapted’ to the new substrate by the appearance of an enzyme (or several enzymes), which function to metabolize the new substrate (e.g., lactose-splitting enzymes). The mechanism by which the adaptation occurred was subject of intense discussion and debate up to the mid-1950s. By that time, the work of Jacques Monod and colleagues at the Pasteur Institute had focused on the specific case of the response of E. coli to the shifts between glucose and lactose as the sole carbon source for growth. Their early approach was classically physiological, that is, they studied the rates of growth, the kinetics of the adaptations, the levels of the relevant enzymes, and the concentration of substrates involved. A key question arose: when the enzyme appeared in the adaptation (termed ‘enzyme induction’), was it made de novo, perhaps upon the ‘instruction’ of the inducer (lactose), or did the enzyme exist in a preformed state, which was somehow stabilized or activated by the inducer? These basically physiological questions were brilliantly reformulated by the group at the Pasteur Institute into genetic terms by the reconceptualization of the process as involving an intermediate system for synthesis of the enzyme, which was distinct from the enzyme itself, and by exploiting mutations affecting the induction phenomenon to identify the components in this hypothetical enzyme-forming system. This work resulted in the operon concept of gene function as first described in 1960 by Jacob, Perrin, Sanchez, and Monod and fully elaborated by Jacob and Monod in 1961. This synthesis of physiology and genetics provided a broad explanation of the biology of a bacterium, E. coli, yet it unified as well much of the rest of biological thought. It ended the long estrangement between transmission genetics (Morgan and the ‘nuclear monopoly’) and the Continental embryologists, who viewed genes primarily as the agents directing growth and development, and rejuvenated interest in what is now known as cell biology.

See also: AIDS, Historical; Biographies; Cholera, Historical; Methods, Philosophy of; Plague, Historical; Smallpox, Historical; Spontaneous Generation; Syphilis, Historical; Typhoid, Historical; Typhus Fevers and Other Rickettsial Diseases, Historical

History and Culture, (and Biographies) | History of Microbiology

Further Reading Brock T (1988) Robert Koch. A Life in Medicine and Bacteriology. Madison, WI: Science Tech Publishers. Brock T (1990) The Emergence of Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Bulloch W (1938) The History of Bacteriology. London: Oxford University Press. Cairns J, Stent GS, and Watson JD (eds.) (1966) Phage and the Origins of Molecular Biology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory for Quantitative Biology. Clark PF (1961) Pioneer Microbiologists of America. Madison, WI: University of Wisconsin Press. Dobell C (1955) Antony van Leeuwenhoek and His ‘Little Animals’. New York: Russell & Russell. Dubos RJ and Hirsch JG (eds.) (1965) Bacterial and Mycotic Infections of Man, 4th edn. Philadelphia, PA: Lippincott. Duclaux E (1920) Pasteur: The History of a Mind (trans. Smith EF and Hedges F). Philadelphia, PA: Saunders. Evans AS (1993) Causation and Disease. New York: Plenum. Geison GL (1995) The Private Science of Louis Pasteur. Princeton, NJ: Princeton University Press.

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Helvoort T van (1991) What is a virus? The case of tobacco mosaic disease. Studies in History and Philosophy of Science 22: 557–588. Lechavalier HA and Slotorovsky M (1965) Three Centuries of Microbiology. New York: McGraw-Hill. Maurois A (1959) The Life of Sir Alexander Fleming. London: Jonathan Cape. McCarty M (1985) The Transforming Principle. New York: Norton. Monod J and Borek E (eds.) (1971) Of Microbes and Life [Les microbes et la vie]. New York: Columbia University Press. Olby R (1994) The Path to the Double Helix. The Discovery of DNA. New York: Dover. Topley WWC and Wilson GS (1929) The Principles of Bacteriology and Immunity, vol. 2. New York: William Wood. Wilson C (1995) The Invisible World: Early Modern Philosophy and the Invention of the Microscope. Princeton, NJ: Princeton University Press. Woese CR, Kandler O, and Wheelis ML (1990) Towards a natural system of organisms: Proposal for the domains archaea, bacteria, and eucarya. Proceedings of the National Academy of Sciences of the United States of America 87: 4576–4579.

Methods, Philosophy of K F Schaffner, George Washington University, Washington, DC, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Scientific Method in General Scientific Method in Biology; Objective Truth and Scientific Progress Specific but Philosophically General Methods of Experimental Inquiry

Glossary cause A condition that is necessary in the circumstances for bringing about an event. model An idealized, usually causal, mechanism or collection of mechanisms that account for a biological outcome.

Abbreviations BSE CJD IPTG ONPF

bovine spongiform encephalopathy Creutzfeldt-Jakob disease isopropylthiogalactoside o-nitrophenyl-fucoside

Defining Statement Philosophy of method is a branch of the philosophy of science that investigates scientific methodology. Though ‘scientific method’ is largely implicit in microbiology, microbiologists do implement a number of methods of scientific inquiry that have been analyzed by philosophers, such as the methods of difference and comparative experimentation. In addition, these methods are put into practice against a backdrop of more general philosophical assumptions concerning scientific progress, scientific realism, scientific inference, and the nature of empirical evidence. The philosophy of method is best described using particular microbiological examples illustrating the application of specific methods of experimental inquiry.

Scientific Method in General The search for a method that would articulate and codify the principles of scientific inquiry has a long history. This article is reproduced from the 2nd edition, volume 3, pp. 227–239, Ó 2000; Elsevier Inc.

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Microbiology: Its Scope and Subject Areas; Representative Illustrations of the Philosophy of Method Further Reading

scientific method A collection of general principles of scientific inquiry, test, and evaluation. strong inference A systematic attempt to devise mutually exhaustive alternative hypotheses and subject them to crucial experimental tests. theory Generally, a collection of related principles that explain a domain.

PrP PrPc PrPsc TSE

prion protein cellular PrP scrapie PrP transmissible spongiform encephalopathy

Philosophers, in particular, have proposed a number of different approaches to scientific method, beginning with Aristotle. In more modern times, Descartes and Francis Bacon wrote on the subject, but the study of scientific method received its most systematic treatments in the work of the nineteenth-century philosophers and scientists William Whewell, Stanley Jevons, and John Stuart Mill. It was the last who forcefully re-presented the methods of agreement, difference, concomitant variation, and other methods that continue to have an influence among contemporary philosophers. Later in this article, some of the more current positions of such philosophers as Karl Popper and Thomas Kuhn will also be cited as contributing in important ways to this subject. Though some philosophers studying scientific method, such as Francis Bacon, hoped to furnish a method of ‘discovery’, the majority of philosophical thinking in this area has tended to follow Popper’s view that states The initial stage, the act of conceiving or inventing a theory, seems to me neither to call for logical analysis or be susceptible of it. The question how it happens that a new idea occurs to a man – whether it is a musical theme,

History and Culture, (and Biographies) | Methods, Philosophy of 49 a dramatic conflict, or a scientific theory – may be of great interest to empirical psychology; but it is irrelevant to the logical analysis of scientific knowledge. The latter is concerned not with questions of fact but only with questions of justification or validity.

This view of Popper’s has come under attack by a number of recent philosophers and artificial intelligence theorists. These proponents of a logic of scientific discovery have been able to develop discovery programs in a number of domains, including organic chemistry and mathematics, and active research in this area is continuing. Advances in this area are quite technical and typically domain specific, and, thus, it will not be possible in the context of the present article to discuss in any detail these various attempts to develop a logic of discovery; this selection will primarily be concerned with the logic of testing and of proof. It should be noted that ‘scientific method’ has been largely ‘implicit’ in the writings of microbiologists. There are some occasional reflections on methodological issues, for example, Koch’s development of his ‘postulates’ for assessing the causation of disease by a bacteriological agent and Peter Duesberg’s recent appeals to methodological issues in connection with the cause(s) of HIV disease. Another interesting methodological debate is the recent controversy about the causative agent – putatively a prion – of the spongiform encephalopathies, such as ‘mad cow disease’. Though these explicit debates are the exception, microbiologists do put into practice various philosophies of method, and it is the function of the present article to make explicit what figures in a largely silent way in microbiology.

Scientific Method in Biology; Objective Truth and Scientific Progress Theory and Experiment in Physics Compared and Contrasted with Biology Biology in general, and microbiology in particular, shares a number of common methodological assumptions with all the natural sciences, including the physical sciences. The life sciences, however, also posses some special methodological features that will be useful to distinguish between them. Because each of the natural sciences seeks reliable general knowledge, the methods mentioned briefly in ‘Scientific method in general’, such as the methods of agreement and difference, are widely employed in the life and nonlife sciences. These methods can be thought of as attempting to discern the causal structure of the world, and, in their application, scientists endeavor to identify possible confounding factors that can lead to spurious inferences about causes and effects. Thus, all

natural scientists attempt to control for interfering and extraneous factors, frequently by setting up a control comparison or a control group. Such controls are a direct implementation of what Mill termed the method of difference and Claude Bernard the method of comparative experimentation, which will be reviewed in some detail in ‘Specific but philosophically general methods of experimental inquiry’. In the biological sciences, including microbiology, some added complexity is frequently encountered due to biological diversity and the number of systems that strongly interact in living organisms. It is the backdrop of evolutionary theory that allows one to understand why there can be both extensive and subtle variation in organisms and mechanisms, as well as why there may be narrowly defined precise mechanisms that are (nearly) biologically universal, such as the genetic code. Variations due to meiosis, mutation, and genetic drift, for example, predict that extensive variation should occur most frequently in evolving populations where strong selection pressures toward precise and universal mechanisms are not present. Alternatively, where variations would almost certainly be lethal, there exist strong pressures toward the fixation of (nearly) universal mechanisms. Thus, evolutionary theory, at a very general level, explains some of the specific and general features of other theories and models in microbiology. Because broad and subtle variations may be encountered in microbiology, special attention frequently needs to be given to ensuring the (near) identity of the organisms under investigation, except for those differences that are the focus of the scientist’s inquiry. Accordingly, the development of special strains of organisms and the identification and classification of populations (and subpopulations) assume an urgency that can be frequently ignored in the physical sciences, where, for example, all electrons are identical. It is in connection with the satisfaction of these urgent needs that the development of the techniques of pure culture becomes so vitally important. Organism and subsystem variation do not only influence the experimental arena, but also figure in what constitutes the biological analogue of ‘theory’. Genetic and environmentally produced variation frequently results in the need of biologists (and microbiologists) to focus on ‘model’ organisms and on prototypical systems, which are highlighted against a backdrop of similar but different organisms and mechanisms. Biologists, thus, often find themselves practicing what a 1985 National Academy of Sciences report called ‘many–many modeling’ in a complex ‘biomatrix’. The report, which re-presented the results of a series of workshops directed by H. Morowitz, introduced a notion of the ‘matrix’ of biomedical knowledge: ‘‘The workshops demonstrated that the results of biomedical research can be viewed as contribution to a complex body, or matrix, of interrelated biological knowledge built from studies of

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many kinds of organisms, biological preparations, and biological processes at various levels.’’ From within this multidimensional matrix, many–many modeling occurs, in which analogous features at various levels of aggregation are related to each other across various taxa. The report notes: ‘‘An investigator considers some problem of interest – a disease process, some normal physiological function, or any other aspect of biology or medicine. The problem is analyzed into its component parts, and for each part and at each level, the matrix of biological knowledge is searched for analogous phenomena. . . . Although it is possible to view the processes involved in interpreting data in the language of one-to-one modeling, the investigator is actually modeling back and forth onto the matrix of biological knowledge.’’ Method and Scientific Progress P. Collard, in his historical monograph on the development of microbiology, distinguished four historical eras. The period of ‘speculation’ comprised the epoch from about 5000 BC until the work of the microscopist Leeuwenhoek around 1675 ushered in the era of ‘observation’. Beginning in the mid-nineteenth century, the era of ‘cultivation’ began with Pasteur’s studies of fermentation and Koch’s development of pure culture methods employing solid media. Collard suggests that the modern physiological era, which is dominated by the elucidation of bacteriological and biochemical mechanisms, commenced about 1900. Implicit in Collard’s chronology is the inference that the more recent methodologies are more scientifically sound and objective, in contrast to the earlier, more speculative inquiries in microbiology. It may initially appear odd to scientifically trained microbiologists to even raise the question of whether there is any ‘objective truth’ in science. Over the past 25 years or so, however, serious questions have been raised about the nature of scientific ‘truth’ and ‘progress’ by Kuhn in his influential work, some philosophers of science (e.g., Feyerabend), and several sociologists of science (e.g., Latour and Woolgar), and a brief discussion of these issues may be helpful. Truth and Progress in Science Throughout this century, philosophers of science have engaged in vigorous disputes about the nature of scientific truth. An examination of the history of science in general, and microbiology in particular, would lead one to the conclusion that there have existed many ‘good’ scientific theories that have not survived to the present day. Kuhn’s characterization of scientific revolutions provides a superb (if ultimately misleading) introduction to examples of these discarded theories. Such theories have gone through the stages of discovery, development, acceptance,

rejection, and extinction. Further examination of extinct theories, however, would show that they possessed a number of beneficial consequences for science. Incorrect and literally falsified theories have several explanatory functions, and have systematized data, stimulated further inquiry, and led to a number of important practical consequences. For example, the false Ptolemaic theory of astronomy was extraordinarily useful in predicting celestial phenomena and served as the basis for oceanic navigation for hundreds of years. Newtonian mechanics and gravitational theory, which is incorrect from an Einsteinian and quantum mechanical perspective, similarly served both to intelligibilize the world and to guide its industrialization. In the biological sciences, the false evolutionary theory of Lamarck systematized and explained significant amounts of species data, and in microbiology, Pasteur’s false nutrient depletion theory of the immune response, nonetheless, served as the background for the development of the anthrax vaccine. Such examples lead one toward what has been termed an ‘instrumentalistic’ analysis of scientific theories (or hypotheses). The basic idea behind such a position is to view theories and hypotheses as ‘tools’ and not as purportedly true descriptions of the world. For a thoroughgoing instrumentalist, the primary function of scientific generalizations is to systematize known data, to predict new observational phenomena, and to stimulate further experimental inquiry. Such an approach bears strong analogies to the ‘constructivist’ program of several sociologists of science, such as Latour and Woolgar, who conceive of many biomedical entities (e.g., neuroendocrine releasing factors) as being ‘constructed’ rather than as ‘discovered’. To constructivists, such putatively ‘real’ microbiological substances are actually ‘constructed socially’ (as conceptualized entities), as part of a complex give-and-take among laboratory machine readings and discourse among laboratory scientists. Though such positions as Kuhnian relativism, instrumentalism, and social constructivism are prima facie attractive, they are inconsistent with other features of scientific inquiry. The relation of scientific theory to ‘observations’ in the laboratory is exceedingly complex. In spite of the weblike connections between theoretical postulation and laboratory experiments, however, multiple empirical constraints on theoretical speculations generate a stability and ‘stubbornness’ of both data and theory, to use Galison’s terms. For example, scientists view the distinction between what they term ‘direct’ and ‘indirect’ evidence as important. Even though, as shall be seen in the following, the distinction is relative, it is, nonetheless, significant to note that scientists ‘behave’ as if the distinction is important, and that ‘direct evidence’ would seem to support a more ‘realistic’ analysis of scientific theories (or hypotheses). A realistic type of alternative to the instrumentalist position would characterize scientific theories as ‘candidates’ for ‘true’

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descriptions of the world. Though not denying the importance of theories’ more instrumentalistic functions, such as prediction and fertility, the realist views these features as partial indications of a theory’s ‘truth’. The history of recent philosophy of science and the more general discipline of ‘science studies’ has seen an oscillation between these realist and instrumentalist positions, as well as the development of some interesting variants of these positions. The relativist reviews have been strongly criticized, especially by realismdefending scientists, the give-and-take of which has been described as the 1990s ‘science wars’. This, however, is a subject that cannot be pursued within the limitations of this article. Suffice it to say that, in spite of the variation in positions that scientists and philosophers of science have taken about the nature of ultimate scientific truth, these varying positions do not disagree about the need to report accurately and faithfully what the scientist has observed or reasoned to in his or her investigations.

Specific but Philosophically General Methods of Experimental Inquiry This section provides a more systematic and in-depth review of the classical methods of experimental inquiry that were very briefly introduced in ‘Scientific method in general’. It begins with John Stuart Mill’s characterization of these experimental methods, since Mill’s analysis is ‘provisionally’ adequate for our purposes and is also well known.

‘Mill’s’ Methods Mill’s first experimental method is known as the ‘method of agreement’. This was as follows: If two or more instances of the phenomenon under investigation have only one circumstance in common, the circumstance in which alone all the instances agree, is the cause (or effect) of the given phenomenon.

It should immediately be pointed out that this method is most difficult to satisfy in microbiological inquiry because of the complexity of organisms. To vary every relevant character save one is largely unrealizable, though it may occasionally be done in very narrowly circumscribed investigations in molecular biology, for example, in genetic codon analysis. The applicability of the method of agreement is also suspect in complex biological organisms for two other general reasons, originally pointed out by Mill in his System of Logic. These general reasons were referred to by Mill as the ‘plurality of causes’ and the ‘intermixture of

effects’. In the former, Mill noted that in those complex cases where the same consequence could be the result of different jointly sufficient antecedents, the method of agreement yielded ‘uncertain’ conclusions. In complex and adaptable biological organisms, such a situation is often likely. The problem of the ‘intermixture of effects’ produced even greater difficulties for the method of agreement. This occurred in cases where effects were ‘intermixed’, that is, either they were not separable into clearly defined components, or the result was emergent and incalculable on the basis of the causal components. As examples of these two species of the intermixture of effects, Mill cited the vector addition of forces in mechanics and the production of water from oxygen and hydrogen. Mill also believed that biological properties were emergent with respect to the physicochemical, with attendant difficulties. Suffice it for now to note that the method of agreement possesses serious imperfections in its application to complex circumstances, as are found in biological organisms. Mill’s methods also include, besides the better known method of agreement and the method of difference, the method of residues and the method of concomitant variations. The latter, according to Mill, may be stated in canonical form as follows: Whatever phenomenon varies in any manner whenever another phenomenon varies in some particular manner, is either a cause or an effect of that phenomenon, or is connected with it through some fact of causation.

This method has important uses in biology and medicine, and an illustration of it will be considered in the following section on specific implementation of the methods. Again, as in the case of the method of agreement, the complexity of biological organisms makes a simple and straightforward application of the method of concomitant variations uncertain. When attempting to determine a major causal role which, say, an entity plays in a system, a simple recording of concomitant variations is almost always insufficient. The method of residues is likewise suspect in its simple application to biological and microbiological causation. This method, in which one ‘‘subduct[s] from any phenomenon such part as is known by previous inductions to be the effect of certain antecedents’’, and considers ‘‘the residue of the phenomenon . . . [to be] the effect of the remaining antecedents’’, is similarly difficult to apply in complex systems in which the interfering residues are likely to be very large, both in number and in their interactions. The method of difference supplemented in a number of cases with a statistical interpretation, which bears certain analogies to Mill’s methods of concomitant variation, is often the most appropriate method of experimental

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inquiry to establish empirically and directly scientific claims. The method of difference was stated by Mill in the following manner: If an instance in which the phenomenon under investigation occurs, and an instance in which it does not occur, have every circumstance in common save one, that one occurring only in the former, the circumstance in which alone the two instances differ is the effect or the cause, or an indispensable part of the cause of the phenomenon.

The application of the method of difference, like the other methods, is not automatic in any experimental situation and, as has been observed by a number of thinkers, ‘presumes’ an analysis of the situation into all relevant factors that can be examined one at a time. This rather demanding requirement can be ameliorated in a way that is reasonably faithful to Mill’s approach by following some suggestions of the great nineteenth-century physiologist and philosopher of method, Claude Bernard. In his influential monograph An Introduction to the Study of Experimental Medicine, Bernard noted that these presumptions of an antecedent analysis of the experimental situation and the ability to examine factors one at a time were not necessarily valid in experimental medicine, and he urged that the method of difference be further distinguished into (1) the method of ‘counterproof ’ and (2) the method of ‘comparative experimentation’. According to Bernard, in the method of counterproof, one assumes that a complete analysis of an experimental situation has been made, that is, all complicating and interfering factors have been identified and controlled. Subsequently, one eliminates the suspected cause and determines if the effect in which one is interested persists. Like Karl Popper in the twentieth century, Bernard believed that experimental medical investigators avoided counterproof as a method, since they feared attempts to disprove their own favored hypotheses. Bernard defended a strong contrary position and maintained that counterproof was essential to avoid elevating coincidences into confirmed hypotheses. He argued, however, that those entities that fell into the province of biology and medicine were so complex that any attempt to specify all of the causal antecedents of an effect was completely unrealistic. As a remedy for this problem, he urged the consideration of ‘comparative experimentation:’ Physiological phenomena are so complex that we could never experiment at all rigorously on living animals if we necessarily had to define all the other changes we might cause in the organism on which we were operating. But fortunately it is enough for us completely to isolate the one phenomenon on which our studies are brought to bear, separating it by means of comparative experimentation from all surrounding complications. Comparative experimentation reaches this goal by adding to a similar organism,

used for comparison, all our experimental changes save one, the very one which we intend to disengage.

Bernard referred to comparative experimentation as ‘the true foundation of experimental medicine’. Strong Inference The use of counterproof, crucial experiments, and comparative experimentation finds unified application in what J. R. Platt termed ‘strong inference’. Platt contended that rapid progress could be made in the biological sciences if well-formulated, alternative hypotheses were subjected to crucial experiments, designed to eliminate most of the alternatives. He characterized this approach as involving the following steps: 1. devising alternative hypotheses; 2. devising a crucial experiment (or several of them) with alternative possible outcomes, each of which will, as nearly as possible, exclude one or more of the hypotheses; 3. carrying out the experiment so as to get a clean result; 1.9 repeating the procedure, making subhypotheses or sequential hypotheses to refine the possibilities that remain; and so on. In the next section, some examples of how this ‘strong inference’ approach is coupled with the methods of experimental inquiry in several recent examples in microbiology are discussed.

Microbiology: Its Scope and Subject Areas; Representative Illustrations of the Philosophy of Method The scope of microbiology is astonishingly broad. The most recent edition of Zinsser’s well-known textbook comments on this subject, stated as follows: With each passing year the term microbiology becomes a less satisfactory umbrella for the many disciplines that it attempts to cover. Bacteriology, immunology, virology, mycology, and parasitology have each long since become separate and independent disciplines.

Zinsser provides a continuing rationale for the subject area, however, noting that these numerous specialities ‘‘are treated together . . . simply because they deal with the agents that cause infectious diseases and with the mechanisms by which hosts defend against them’’. Be that as it may, any analysis of the philosophy of method of a domain that includes infection, bacteria, spontaneous generation and origin of life, the germ theory of disease, viruses, immunity, and chemotherapy must, of necessity, be selective. In the following paragraphs, several examples are discussed that are representative of methodology in

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microbiology. These illustrations will be used to provide a specific and accessible approach to the somewhat abstract topic of the philosophy of method in microbiology. The Example of Koch’s Postulates and AIDS Virology As noted in the discussion earlier, self-conscious, explicit methodology is rare in the writings of microbiologists. One prominent and influential exception to this general rule are the Koch’s postulates – more accurately termed the Henle–Koch postulates – developed to assess bacteriological causation of disease. About 10 years ago, these postulates returned to prominence at the center of a potentially momentous controversy in AIDS virology. The postulates can also be used as a framework for investigating another controversial example of disease causation – prions – as well as to point toward the limits of ‘direct evidence’ in microbiology. A brief account of the postulates’ history and the philosophical presuppositions will facilitate an understanding of their role in the contemporary debate to be discussed later in this article. The postulates have their proto-origin in Henle’s work in 1840 and were then further developed by his pupil, the great microbiologist Robert Koch, in lectures in 1884 and 1890. There are several slightly different formulations of the postulates (see Evans, who follows Rivers as well as Zinsser), but the essence can be summarized as follows: 1. The infectious agent occurs in every case of the disease in question and under circumstances that can account for the pathological changes and the clinical course of the disease. 2. It must be possible to isolate the agent from all cases of the disease and to grow the agent in pure culture. 3. After being fully isolated from the diseased animals (or humans) and repeatedly grown in pure culture, it can induce the disease anew by inoculation into a suitable host. In addition, some formulations add a fourth postulate: 4. The infectious agent occurs in no other disease as a fortuitous and nonpathogenic agent. These are stringent conditions, and even at the time that Koch enunciated them, a number of etiological agents were known that did not fully meet the criteria, including bacteria isolated from typhoid fever and cholera. In the ensuing 100 years since the articulation of the postulates, several types of hitherto unrecognized forms of disease, including chronic diseases, cancer, and a number of viral diseases, have required the modification and extension of these postulates. In 1976, Evans attempted to bring together a number of these different themes into a ‘unified’ epidemiological conception of disease causation. It is, however, the more traditional and stringent form of the postulates that was cited over the course of the

past decade in an attempt by molecular virologist Peter Duesberg to question the causal relationship between HIV and AIDS. The theses of Duesberg’s work were that (1) HIV (the AIDS virus in either its HIV-1 or HIV-2 form) is neither necessary nor sufficient for AIDS, though HIV is a good ‘marker’ for American AIDS, and (2) AIDS is actually an autoimmune disease caused by as yet unknown pathogens, probably acting in concert with a variety of environmental insults, including hard psychoactive as well as medical drugs (AZT) and blood transfusion (particularly in the case of hemophiliacs). In the eyes of most scientists, Duesberg’s principal arguments have been effectively answered, though Duesberg has continued to criticize the standard view in several recent books. One of Duesberg’s major arguments is that HIV fails to satisfy Koch’s postulates. He wrote concerning the first of Koch’s postulates that ‘‘there is no free virus in most – and very little in some – persons with AIDS, or in asymptomatic carriers’’. Though he admits that various viral elements (and, by definition of the disease, antibody to HIV) can be found in most or all of AIDS patients, he contended that HIV is not present in ways that can account for the loss of T cells or for the clinical course of the disease, which lags 8 or more years behind infection. Further, with respect to Koch’s second postulate, Duesberg contends that there is an ‘‘often over 20% failure rate in isolation of HIV from AIDS patients’’. Finally, citing Koch’s third postulate, Duesberg notes that HIV does not produce AIDS in any animal model (chimpanzees had been inoculated unsuccessfully), and the data from accidental inoculations of humans (through donor semen, laboratory accidents involving HIV researchers, and blood transfusions) are not consistent with the role of HIV as the sole cause of AIDS. The response to Duesberg’s arguments has been threefold. First, a number of commentators have pointed out that Koch’s postulates listed earlier fail to be satisfied in a number of other diseases, primarily of the viral and immunological type encountered in AIDS, and that the postulates need to be taken as ‘guidelines’ and supplemented by broader epidemiological evidence. Second, when a broader, more epidemiological conception of causation is implemented using epidemiological data, that the evidence for HIV as the cause of AIDS is overwhelming. Finally, molecular investigations of HIV pathophysiology have deepened in recent years. New coreceptors for the virus have been identified that explain some resistance to HIV and may also help explain its oddly delayed pathogenesis. These receptors have also permitted better animal models of HIV/AIDS to be created. Duesberg has rejoined to this criticism, arguing that appeals to epidemiological considerations do not make the case that the proponents of HIV think they do and continues to aggressively defend his own theory.

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It is not the purpose of this article to delve further into the details of the ongoing controversy regarding HIV and AIDS, but several points need to be highlighted that illustrate the role of the different philosophies of method involved. First, problems with the failure of HIV to satisfy Koch’s first postulate have led to further speculations about the mechanism(s) of T-cell depletion. These speculations include syncytia formation, ‘co-factor’ pathogens, HIV as the trigger for an autoimmune disease, and, most recently, the proposal of a ‘super-antigen’ theory. Investigators generally believe that the evidence for these hypotheses are ‘indirect’, at best, and that more ‘definitive or direct’ proof is needed. Thus, the Henle–Koch postulates provide a framework and the failure to satisfy them in any simple way should provide the motivation for additional investigation, which may move the subject more in the direction of ‘direct’ evidence for explanatory models of AIDS pathogenesis. In the next illustration of the philosophy of method, another example of how ambiguous results that do not fully meet the requirements of Koch’s postulates is examined, which raises the question of whether fully conclusive evidence is ever forthcoming in microbiology. The last example of just how ‘direct’ evidence can be obtained will partially answer this question affirmatively, with the continuing caveat, however, that in science, even direct proof continues to remain somewhat conditional. Prions and the Question of the Causative Agents of the Transmissible Spongiform Encephalopathies Modifications needed to extend Koch’s postulates to take into account later microbiological developments are also a useful framework for a consideration of the cause of the transmissible spongiform encephalopathies (TSEs). These diseases include Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE), or ‘mad cow’ disease, in cattle. A number of investigators have attempted to determine the etiological agents and the details of the pathogenesis of the TSEs. At present, TSEs are generally thought to be caused by a novel infectious agent, a ‘prion’ – a term coined by S. Prusiner, to represent a ‘proteinaceous infectious particle’. Prusiner received a Nobel Prize in 1997 for his accomplishments in this area and was cited for his ‘discovery of ‘Prions – a new biological principle of infection’y.’ That prions are the fundamental cause of TSEs is still somewhat controversial, and an alternative viral causation hypothesis continues to have its defenders. If one followed Koch’s postulates, as outlined previously, in the TSE area, one would attempt to (1) show the (various) infectious agent(s) occurs in every case of the diseases and accounts for the pathogenesis, (2) isolate the agent and purify it, and (3) use the purified agent(s) to

induce the disease(s) in new hosts, In addition, one might want to (4) show the agent does not occur in any other disease as a nonpathological agent. Though Prusiner does not explicitly use Koch’s postulates in his various writings on prions and TSEs, the postulates can function as a framework to approach his various arguments. Prusiner’s early work was directed at the sheep TSE, scrapie. Following work by Alper that suggested the infectious agent was a protein that did not include nucleic acid, Prusiner was able to show that scrapie prions contained a protein he called PrP, short for ‘prion protein’. These proteins are also unusually resistant to degradation by protease enzymes. Working with Leroy Hood, the protein was then partially sequenced, and the sequence used to construct a probe that could identify the genetic source of the protein. Remarkably, it turned out that the PrP gene can be found in hamsters, mice, and humans, but, most of the time, cells that make PrP are not pathological. An unorthodox, unprecedented, but possible, explanations was that PrP could exist in two different tertiary forms with the same amino acid sequence: one normal, called ‘cellular PrP’ (PrPc), and the other associated with scrapie, termed ‘scrapie PrP’ (PrPsc). This heretical idea was subsequently confirmed and considerable work has been done that examines various conformational changes in PrP and their pathological effects. Prusiner and his group initially attempted (implicitly) to conform to Koch’s second postulate by using the PrP gene to generate pure copies of PrP. They thought they could then implement Koch’s third postulate and, as they wrote, ‘‘inject the protein molecules into animals, secure in the knowledge that no elusive virus was clinging to them. If the infections caused scrapie, we would have shown that protein molecules could, as we had proposed, transmit the disease’’. But as Prusiner adds, ‘‘by 1986, we knew the plan would not work’’. It was difficult to induce the gene to make the high levels of PrP needed, and, in addition, the PrP that was made was the benign ‘cellular’ type. An alternative strategy was then devised that looked to inherited, rather than transmissible, forms of the SEs. Gestmann – Straussler–Scheineker disease is a very rare familial human SE that Prusiner and other investigators were able to link with a point mutation in the PrP gene. This served as the backdrop for the creation of genetically altered mice carrying a mutated PrP gene. One could first examine whether the mutated gene in the transgenic mice produced scrapie and then whether brain tissue from these mice could cause scrapie in ‘healthy’ mice, albeit only healthy transgenic mice expressing a low level of mutant PrP. These events do, in fact, occur and are claimed by Prusiner in 1995 to be ‘‘solid evidence that the protein encoded by the mutant gene had been solely responsible for the transfer of the disease’’. Step back and

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examine this claim more carefully and also consider a critic’s view of this ‘solid’ or ‘persuasive’ evidence. Prima facie, the experiment seems to satisfy Koch’s first postulate, since all of the transgenic mice making high levels of PrP did develop mouse scrapie. Transplanted infected brain tissue, however, caused only many (not all) of the recipients to develop the disease. A proponent of the competing viral hypothesis, B. Chesebro, finds the transgenic mouse experiment ‘inconclusive’. He notes that the transgenic mice differ from all known TSE models, because no abnormal protease-resistant PrP – a hallmark of scrapie PrP – is detectable in the diseased brain tissue. Also, disease occurs only when the mutant gene is overexpressed, not when it is found in its normal site as a single copy. Finally, transmission by infected brain tissue is only successful in transgenic mice – the low-level PrP producers – and not in nontransgenic animals. Chesebro suspects that ‘other molecules’ from the diseased brains could be causative of the disease, and, further, doubts the utility of the transgenic model, because none of the transmitted materials has been demonstrated to have typical properties of TSE agents, such as resistance to inactivation by heat. Other investigators also believe that additional TSE experimental findings point toward something more than prions as the cause of these diseases, perhaps a virus. The prion hypothesis and the competing viral hypothesis are, thus, not unambiguously and definitively tested by this transgenic model. But this is only one of a number of lines of evidence supporting the prion hypothesis, and this article cannot delve into the multiple arguments and counterarguments in support of and against the prion view, which continue to evolve (see, e.g., the very recent report by J. Safar and colleagues in Nature Medicine, that provides data that different protein conformations are responsible for variations in prions’ ‘strains’). This fragment of the story, however, does suggest that Koch’s postulates can be a useful framework within which to analyze possible etiological agents of disease, albeit one that needs extensions. Such extensions include both new understandings of molecular biological processes never dreamed of by Koch, as well as powerful new experimental techniques, such as the use of transgenic and knockout animals. But the story also indicates how difficult it is to obtain conclusive or ‘direct’ evidence of a causal hypothesis in microbiology, particularly in the still comparatively early stages of an investigation of a novel model of infection, even with this new powerful molecular knowledge and associated techniques. The Example of the Isolation of the Repressor The third example illustrating the application of methods of experimental inquiry is drawn from molecular genetics and involves progress in clarifying the nature of the

mechanism of genetic control in bacteria, more specifically, the development and testing of the operon model of genetic regulation. The model, which is principally due to the work of Jacob and Monod, developed over a number of years. The initial form of the mechanism dates from 1960 to 1961 and has had a most important impact on both experiment and theory construction in molecular biology (and in embryology). It has been extraordinarily well corroborated by a variety of experiments. It was further developed in the 1960s, 1970s, and 1980s. The model proposed the existence of a new class of genes, termed regulator genes, which were responsible for the synthesis of a substance, later determined to be a protein of about 160 000 molecular weight, which was termed a repressor. The repressor, in inducible systems, such as the lac region of Escherichia coli, binds specifically with a DNA region termed the operator locus. This operator has adjacent to it several structural genes, which are under the operator’s control. When the repressor is bound to the operator, the associated structural genes are not transcribed into messenger RNA, and, accordingly, no proteins or enzymes associated with those structural genes are synthesized. An inducer can specifically interact with the repressor, altering its three-dimensional structure, and thus render it incapable of binding to the operator. The enzyme RNA polymerase, which attaches to a DNA sequence called a promoter in the presence of a protein called CAP and which can transcribe the structural genes, is then no longer prevented from initiating transcription of the structural genes adjacent to the operator, and mRNA synthesis and then protein synthesis commence. Repressible systems employ a similar regulatory logic, only, in that case, the initially ineffective repressor is aided in its operator-binding capacity by an exogenously added corepressor. It has been often noted by biologists that such a regulatory system is most useful to the organism since it allows unnecessary genes to be turned off when the proteinaceous enzyme products of those genes are not required for a metabolic process. This results in energy saving and is conceived of as evolutionarily useful to the organism. In spite of the breadth of evidence for the operon model when it was first proposed in 1961, it can be said that the evidence, at that time, was primarily of an indirect genetic type. Monod in his Nobel lecture suggested, for example, that in the early 1960s the repressor – the crucial regulatory entity – seemed ‘‘as inaccessible as the matter of the stars’’. The difficulty of being able to provide more direct biochemical evidence for the existence of the repressor and its postulated properties stimulated at least one alternative theory of genetic regulation, involving a mechanism different from that of the operon model. In 1966, however, W. Gilbert and B. Mu¨ller-Hill reported that they had been able to isolate the elusive

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repressor, and, in 1967, the same authors were able to demonstrate that the repressor bound directly to the operator DNA. These experiments were most important for the field of regulatory genetics, and they were confirmed by Mark Ptashne’s essentially simultaneous isolation of the repressor in the phage  system and the discovery that it also bound to DNA. These experiments, and the analyses of the experimental results, illustrate both the application of Mill’s methods and Platt’s ‘strong inference’. Gilbert and Mu¨ller-Hill noted in their article reporting their results that though they felt the Jacob and Monod model to be the simplest, other models would also ‘‘fit the data’’ available. ‘‘Repressors’’, they continued, ‘‘could have almost any target that would serve as a block to any of the initiation processes required to make a protein. A molecular understanding of the control process has waited on the isolation of one or more repressors’’. Isolating the repressor was most difficult because of the hypothesized very low concentration of repressor in the E. coli cells. Gilbert and Mu¨ller-Hill solved this problem by looking for and finding a mutant form of the bacteria (it or tightbinding), which produced a repressor with about a ten fold increased affinity for the inducer. As an inducer, the chemical IPTG (isopropylthiogalactoside) was used, which induces the lac operon but which, in contrast to lactose, is not metabolized by the induced enzyme. The repressor was detected by placing an extract of the it mutant in a dialysis sac in a solution of radioactive IPTG (labeled to facilitate detection of very low amounts of it), and looking for an increased concentration of IPTG inside the sac after about 30 min (at 4  C). Using the increased concentration as a sign of the presence of repressor, Gilbert and Mu¨ller-Hill purified the extract and analyzed the binding component. They discovered that the enzymes which would attack DNA and RNA would not destroy the inducer binding ability of the presumed repressor, but that an enzymes which attacked proteins would. Heating (above 50  C) also destroyed the ability to bind inducer. This was rather direct evidence (though still conditional on the hypotheses that (1) the enzymes possessed anti-DNA/anti-RNA capacity and (2) heat denatures proteins) that the repressor was a protein. The repressor (labeled with the radioactive inducer) was centrifuged and sedimented on a glycerol gradient. The ‘profile’ of the sedimented repressor indicated that it had a molecular weight of about 150 000– 200 000. Gilbert and Mu¨ller-Hill, in their article, cited what they termed ‘negative and positive controls’ on their supposition that they had isolated the repressor product of the i gene. The most important ‘negative controls’ involved examining the extracts from i s (the superrepressed) and i  (constitutive) mutants. The former type (i s) presumably synthesizes a repressor that has lost the ability to bind

inducer, and the latter (i ) had been hypothesized to produce an (significantly) incomplete repressor. For both of these mutants, no binding of IPTG was observed. (Gilbert and Mu¨ller-Hill noted that ‘‘the various mutant forms of the i gene were put into identical genetic backgrounds, so that unknown variations from strain to strain could not confuse the issue’’.) Gilbert and Mu¨ller-Hill plotted graphs representing the binding constants of the wild type and i t mutant to demonstrate the essential identity of the repressors, which were used as positive controls. An examination of the binding ability of the repressor for various other substances, including galactose and glucose (and yielding a decreasing sequence of affinities), ‘‘gave further support for the [thesis that the isolated] material. . . [was] the repressor’’. In 1967, Gilbert and Mu¨ller-Hill used this partially purified radioactive repressor to test for in vitro binding to the operator of lac DNA. In this experiment, they used strains of bacteriophage that carried different forms of the lac operon in their DNA. By sedimenting mixtures of the various strains of the phage DNA with the radioactive repressor, Gilbert and Mu¨ller-Hill found the following: 1. In the absence of inducer, the radioactive repressor binds to the DNA. 2. In the presence of IPTG, which releases the repressor from the DNA, no binding is observed. 3. The effect of the IPTG is specific, for substitution by a chemical ONPF (o-nitrophenyl-fucoside; a substance that binds to the DNA but does not induce) has no effect. 4. Use of two mutant phage oc strains, in which the affinity of the operator for the repressor is expected to be weaker by a factor of 10 and 200, yielded sedimentation profiles in rough quantitative conformity with these binding expectations. These experiments, which appear to utilize the methods of comparative experimentation or difference (the ‘negative control’) and concomitant variation (the ‘positive controls’), have been characterized as offering ‘direct’ evidence and ‘proving’ the hypotheses of the Jacob– Monod model. Clearly, they do so, however, with the aid of auxiliary hypotheses involving, among others (1) the effects of growth in radioactive media so as to specifically label the ‘observed’ entities and (2) the analysis of centrifugation and sedimentation techniques in density gradients. The experiments require a chain of reasoning. There is, in addition, (3), a very general negative auxiliary assumption that what is observed is not an artifact. It must be further emphasized that the direct proof of the Jacob–Monod operon model is contingent on the assumption that other, and perhaps more important and overriding, factors are not involved, that is, some

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competing, but as yet unarticulated, hypothesis that could account for all these results. The proposal of a novel mechanism of control that would account for the genetic data as well as Gilbert and Mu¨ller-Hill’s findings (perhaps by showing some of them to be artifacts), but which would disagree with basic tenets of the operon model cannot be logically excluded. The existence of Gilbert and Mu¨llerHill’s experiments are thus a ‘conditional’ direct proof, the conditions depending on (1) the truth of auxiliary hypotheses and (2) the nonexistence of an inconsistent competing theory or model that would account for the same data as the operon model. Generalizing from this example, it is obvious that the ‘directness’ and force of the evidence is obtained by choosing one or more ‘central’ (roughly equivalent to ‘essential’) property(ies) of the hypotheses in the theory or model to be tested. In the operon model’s case, these properties were initially suggested in Monod’s work, namely, the repressor’s ‘recognition’ of the inducer and the operator. In addition, plausible competitor theories and models are scanned to determine if the test condition(s), if realized, would support the model under test while falsifying the competitors. Recall that ‘genetic’ evidence was insufficient to do this, whereas the Gilbert–Mu¨ller-Hill experiments (as well as Ptashne’s) were. This exhibits Platt’s ‘strong inference’ in specific ways. The evidence was considerably strengthened by providing both positive and negative controls. These controls are essentially equivalent to the well-known Mill’s methods of difference and concomitant variation, as discussed previously. These three particular examples, the use of the Henle– Koch postulates to assess the causal role of HIV and the TSEs, and the employment of a strategy of strong inference implementing methods of difference and concomitant variation to identify and characterize the repressor, illustrate the application of several methodological themes found in microbiology. Each of these examples also indicates that the application of such methods requires creative insight coupled with detailed knowledge of the subject domain, as well as exemplary technique, in the case of laboratory investigations. In addition, the examples illustrate that the path from speculation and indirect evidence for scientific hypotheses to direct proof is difficult and frequently depends on background assumptions that themselves could, at some time, be called into question. Such a process of continued questioning of received assumptions, coupled with the

proposal of new, precisely formulated hypotheses subject to experimental test and evaluation by rigorously applied scientific method, is the essence of excellent science, and though this process is typically implicit in microbiology, it is no less an important constituent of scientific progress in the subject. See also: AIDS, Historical; History of Microbiology; Prions

Further Reading Chesebro B (1998) BSE and prions: Uncertainties about the agent. Science 279: 42–43. Collard P (1976) The Development of Microbiology. Cambridge: Cambridge University Press. Collins HM and Pinch T (1994) The Golem: What Everyone Should Know About Science. New York: Cambridge University Press. Duesberg PH (1991) AIDS epidemiology: Inconsistencies with human immunodeficiency virus and with infectious disease. The Proceedings of the National Academy of Sciences Online (US) 88: 1575–1579. Duesberg PH (1996) Inventing the AIDS Virus. Washington, DC: Regenery Publishing Co. Evans AS (1989) Does HIV cause AIDS? An historical perspective. Journal of Acquired Immune Deficiency Syndromes 2: 107–113. Gilbert W and Mu¨ller-Hill B (1966, 1967) Isolation of the Lac repressor: The Lac operator is DNA. The Proceedings of the National Academy of Sciences Online (US) 56: 1891–1898; 58: 2415–2521. Gross PR and Levitt N (1994) Higher Superstition: The Academic Left and Its Quarrels With Science. Baltimore, MD: Johns Hopkins University Press. Holmes FL (1974) Claude Bernard and Animal Chemistry: The Emergence of a Scientist. Cambridge, MA: Harvard University Press. Keyes M (1999) The prion challenge to the ‘Central Dogma’ of molecular biology. 1965–1991. Part I: Prelude to prions; Part II: The problem with prions. Studies in History and Philosophy of Biological and Biomedical Sciences. 30C/1: 1–19; 30C/2: 181–218. Kuhn TS (1970) The Structure of Scientific Revolutions., 2nd edn. Chicago: University of Chicago Press. Laudan LL (1981) Science and Hypothesis: Historical Essays on Scientific Methodology. Boston, MA: Kluwer Inc. Morowitz H (1985) Models for Biomedical Research: A New Perspective. Washington, DC: National Academy of Sciences Press. Platt JR (1964) Strong inference. Science 146: 347–353. Popper KR (1959) The Logic of Scientific Discovery. New York: The Free Press. Prusiner S (1995) The prion diseases. Scientific American. 272(1): 48–57. Safar J, Wille H, Itri V, et al. (1998) Eight prion strains have PrP(Sc) molecules with different conformations. Nature Medicine. 4(10): 1157–1165. Schaffner K (ed.) (1985) Logic of Discovery and Diagnosis in Medicine. Berkeley, CA: University of California Press. Schaffner K (1986) Exemplar reasoning about biological models and diseases: A relation between the philosophy of medicine and philosophy of science. Journal of Medicine and Philosophy 11: 63–80. Schaffner K (1998) Model organisms and behavioral genetics: A rejoinder. Philosophy of Science 65: 276–288. Wolfgang K, Joklik HP, Willett D, and Amos B (eds.) (1992) Zinsser Microbiology, 20th edn. Norwalk, CT: Appleton and Lange.

Plague, Historical A G Carmichael, Indiana University, Bloomington, IN, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Recent Debates about Historical Plagues Plague in Late Antiquity Plague in Europe, 1347–c. 1550

Glossary BCE, CE Refer to calendar dating before and after the current era, by convention assigned from the year one of the Gregorian calendar of 1582. There is no year zero, and this dating system only applies to historical

Defining Statement It is difficult to align historical evidence with modern questions about the biological causes of past epidemics that were called ‘plague’. New archeological and molecular biological approaches confirm a traditional biomedical view: that great human plagues recurred in late antiquity, in late medieval and early modern Europe, and globally over the last 120 years. Recurrent plague experience led to persisting literary and religious themes, and led to many modern public health practices in epidemic surveillance and control.

Evolution of Plague Containment and Surveillance Practices, 1400–1900 Modern Plague, 1894 to the Present Further Reading

(document-based) time. Archeologists also use BP to refer to time before the present. Black Death The epidemic in Europe that began in 1347 and persisted until 1353. The term was not used until the 1700s, and in popular treatments is not always used with historical precision.

whether anything like this level of mortality could occur again, while others pose scenarios of an unmanageable future pandemic. The possibility of renewed world conflict, ecological degradation within great periurban slums, the emergence of extreme antibiotic resistance, global environmental and ecological changes consequent to global warming, and the instructive experience of the 1918 influenza pandemic are all related to recent interest in a better understanding of historical plagues as models of human and ecological catastrophe.

Recent Debates about Historical Plagues Introduction This article accepts a century-long consensus within the biomedical community that Yersinia pestis best accounts for some of the most dramatic plagues of the past 1500 years. Earlier plagues illustrate the bacterium’s destructive potential and extraordinary virulence. Plagues in the past were, nonetheless, general intervals of crisis mortality, and not best understood exclusively as epidemics of bubonic plague. In Western tradition the word plague came from the Latin plaga, meaning a strike or a blow; medici plagarum were wound doctors, attending the blow that resulted in a wound. Thus even the word lacked specificity. Past plagues could result in levels of mortality nearly unimaginable today 30, 40, and 50% of a population dying within a 2-year interval. Some question

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In 1951, French investigator R Devignat, working in the Belgian Congo (now Democratic Republic of the Congo), advanced an elegant hypothesis about historical plagues. He aligned three great waves of plagues in history with three known biovars of Y. pestis. The biovars were defined by variance in the biochemical ability to ferment glycerol and the ability to reduce nitrate to nitrite. One strain was called antiqua and had two ancient epicenters – North Asia and Central Africa. The plagues of the medieval and early modern centuries were biovar mediaevalis, with an origin in the Eurasian steppes. Finally the plagues erupting in South China during the 1890s were collectively biovar orientalis. Devignat knew that the three biovars were all capable of causing epizootics and epidemics, and research of the 1920s through 1940s had shown that the variant strains could be introduced to a new region

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independently of one another, as in Japan during the 1920s, when both mediaevalis and orientalis appeared during different epidemics. Historical studies informed by Devignat’s hypothesis began with an implicit assumption that the three great pandemic waves would have general similarities but not necessarily the same impacts on human communities. Microbiologists during the mid-twentieth century also assumed that the virulence of a mammalian pathogen tended to weaken over time, and therefore that the plagues of the late nineteenth and twentieth centuries were naturally less virulent. The most important historical use of these hypotheses about bubonic plagues was made by French physician-historian, Jean-Noel Biraben during the 1970s. Biraben explained that comparatively avirulent Yersinia pseudotuberculosis and Yersinia enterocolitica were recently evolved because they were less virulent. Human plagues in the modern world then became less virulent because these other two Yersinia species provided some cross-immunity to affected populations. Since the late 1980s, two different lines of investigation have unraveled this microbiology/history synthesis in plague studies, one from within historical communities, and the other from molecular and evolutionary microbiology. Some historians since the 1980s, especially medievalists, have expressed strong opposition to the notion that premodern plagues could be understood through modern scientific interpretations of written evidence. Because concepts of plague occupied a very different range of ideas and images in the past, historians generally favor the study of historical records within historical contexts. While the ranks of professional historians do include many who find modern scientific studies helpful in analyzing the past, most would prefer that we understand the past within its own terms and frameworks. Cohn and others have recently challenged the microbiology/history plague synthesis, specifically claiming that recurring plagues of the 1340–1700 period in Europe could not have been caused by Y. pestis, in other words that Y. pestis is not a good fit with that historical evidence. Y. pestis could not have been responsible for the Black Death of 1347–53, they argue, because the medieval plague spread extraordinarily fast and caused catastrophic losses in all seasons of the year. A few historians further claimed that there exists little written evidence for the presence of Rattus rattus, the ideal rat host for Y. pestis, in ancient and medieval Europe, but recent archeological investigations have excluded that particular objection. Rats were well established in western Europe by Roman times. Meanwhile, recent study of molecular and genetic microevolution of Yersinia species has shown that historical genomic changes to Y. pestis did not evolve from three different geographical points of origin. All Y. pestis variants descend from Y. pseudotuberculosis, and the genetic additions and subtractions within Y. pestis species reflect eight different populations, not just three biovar-related groups.

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Recently, Achtman and others have shown that Y. pestis evolved at some point during the last 1500–20 000 years and that the emergence of particular variations cannot be pinpointed in time. Overall, Y. pestis species are unusually monomorphic and all evolved recently enough to have caused any of the historical pestilences. Virulence – and Y. pestis remains an exceptionally virulent pathogen for humans – requires the presence of a plasmid common to all three pathogenic Yersiniae. Work has only just begun on the mechanisms responsible for the emergence of the particularly lethal capabilities of Y. pestis. The central problem for historical studies of plague ultimately stems from the fact that humans are not part of the primary ecology and microbiology of Y. pestis. Human plague epidemics are rare, even in regions where susceptible rodent hosts have succumbed to epizootic die-off, even when competent flea vectors exist in great numbers. The history of plague thus begins with events that occur atypically in nature. The catastrophic event of a human plague epidemic also produces emotional and confusing accounts. Historical evidence before around 1900 rarely reveals anything of the much broader ecological collapse that led to an epidemic. Human historical records are human-oriented, and even when aggressively analyzed yield little detail about the world where Y. pestis does most of its damage. Furthermore, historical plague evidence comes only from areas where literate survivors existed who were inclined to record any details of an epidemic – city dwellers for the most part. This means that historical accounts of plague come from epidemiological circumstances very unlike those people and places where deaths from Y. pestis have been documented in the late 1900s. Several teams of investigators have recently combined the efforts of historians, microbiologists, and archeologists to recover evidence of Y. pestis from known plague burial sites, and in human remains specifically from dental pulp where decay of the microbial DNA might not have been completed. Evidence from a southern French medieval site and a seventh-century Bavarian site confirm the presence of unique Y. pestis DNA sequences. English investigators, on the other hand, examined medieval plague pits in Britain and Scandinavia, but found no evidence of Yersinia. Today we find and suppress epidemics of Y. pestis long before urban rodents are affected, and we have effective insecticides to contain any transmission of the pathogen to humans once an epizootic has occurred. Even in the 1890s, Western-trained physicians who handled cases of plague believed that attention to sanitation and personal hygiene substantially protected people from plague, and they assumed that modern experience of Y. pestis epidemics would not replicate past circumstances. Yet in the three great multicentury waves of plague pandemics described here, no other known pathogen fits so closely to the physical illness that eyewitnesses and survivors reported. Hence, in many ways we are at the beginning of a new era in

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understanding historical plagues from a microbiological perspective. Review of traditional historical evidence is thus quite useful, even with its many limitations.

Plague in Late Antiquity The earliest human plague for which we have reasonably suggestive accounts of a Y. pestis outbreak come from the 540s of the current era. Two famous earlier pestilences, the Plague of Athens described by Thucydides (430s BCE) and the ‘Antonine plague’ in the 160s CE at the time of Roman emperor Marcus Aurelius, are not generally considered to be epidemics of bubonic plague. Two detailed survivor accounts of the epidemic that reached Constantinople in 542 CE report events and effects that strongly resemble a bubonic plague epidemic. Additionally, there are less full notices of the epidemic elsewhere in the Mediterranean littoral, and salient nonliterary evidence. The wide geographical dissemination of this initial epidemic wave was impressive; however, the real damage and demographic collapse of late ancient urban societies seem to have proceeded from recurrences in port cities and their trade hinterlands. Plague also spread along the Eurasian Silk Road, and caused repeated destruction in the 600s and 700s CE (Tang dynasty, China), while plague likely spared desert peoples such as the Arabs. The spiritual vacuum left in plagues’ wake may have facilitated the rapid rise and spread of Islam. Plague has also been linked to the successful missionary spread of Roman Catholic Christianity over western Europe in the early Middle Ages. Both of these great religious traditions have ever since been of enormous importance to world history, and both contributed a strong apocalyptical cast to the ways that great plagues and pestilences were subsequently understood and reported. The Plague of Justinian, 542 CE From the city of Pelusium, a major center for grain markets in the ancient Mediterranean near today’s Port Said, plague spread during summer of 541 CE, moving northeast into Gaza by August, and west to Alexandria by September. Grain imports were crucial to Constantinople, a city of over 300 000, and Alexandria was the port that shipped grain to the capital city. Thus Constantinople was not infected until the spring of 542, while Palestine and Syria were already in the throes of plague. From Constantinople the plague diffused inland, and by ship to Italy and all the major trade centers of the western Mediterranean, reaching Spain and Southern France by 543. Britain and Ireland were not stricken until 549, if written sources can be trusted on this point. Two great, often-cited literary accounts of the plague exist. Procopius, in a leadership role while Emperor

Justinian was struggling to recover, provides one of them, and Syrian bishop John of Ephesus, who saw plague’s sudden appearance as a clear indication of the apocalyptical last days, wrote the other. Both Procopius and John of Ephesus emphasize the overwhelming mortality, the stench of unburied bodies, the desperate attempts to maintain any sort of order and decorum over burials, routine nutrition, and provision of care to the ill, and in general the climate of acute fear that challenged all military, religious, and civic authorities. All of these issues became standard themes in western European plague literature. John of Ephesus provided the longer of the two accounts. His testimonial is extraordinary because his apostolic mission from Constantinople to Alexandria coincided with the outbreak of plague in the latter city. Fearing the worst, he headed home to Syria, describing in vivid detail burial scenes and the collapse of agricultural activities. He became overwhelmed with the extent of the disaster, and later wrote of the events as part of God’s plan for the end of time, a persistent plague theme in Western literature. He also emphasized contemporary famines and earthquakes, a cluster that became an integral part of many other narratives of the plague. Although Procopius did not leave Constantinople, his account is more famous because it is part of his larger body of work on Byzantine politics and history, and because Procopius detailed the administrative and psychological effects of a great plague on a large city. Therefore, his narrative speaks of issues that have long interested historians of all great plagues and pestilences, especially the gradual, week-by-week descent into terror, the inability to attend the most basic civilities of cosmopolitan life – bodies were packed into empty towers, caverns, and houses, or hauled to the wharfs and plopped into the sea. Procopius claims that some administrative order was maintained. He asserted that the dead were counted up to 230 000 cadavers, surely an exaggeration. The ability to maintain order reflected well on Justinian’s administration, himself included, and underscored the necessity of some of the draconian taxes that Justinian would impose after the plague. The full picture that Procopius provided of plague’s victims bears a strong resemblance to modern bubonic plague infection. He offers a very clear account of the prominent buboes appearing on the bodies of plague victims, and in the regions of the body where bubonic plague is classically seen – the inguinal/groin region, the axilla, and in cervical nodes, typically behind the ears. Some victims simply dropped dead on the streets, some were delirious in fever, and some manifested lentil-sized pustules as well, all of which have been amply described in epidemics of Y. pestis since the 1890s. Recurrent Plague in Late Antiquity 550–750 CE The Byzantine Empire depended on the economic health of Constantinople. Nearly half its population died or fled

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in 542–543 CE, but afterward in-migration substantially repaired these losses. Justinian (born 482/483) himself lived a long life, to 565 CE, and focused his efforts on maintaining military strength sufficient to repulse Persian expansion in Anatolia (modern Turkey). The fiscal bases of the Empire’s well-being nevertheless began to weaken considerably. Recurrent plague, first in the late 550s, then in the 570s, and next most famously in the 590s, when plague hit Rome especially hard, undermined the financial and demographic health of the Empire. Grain shipments essential to the ancient city could not be maintained; trade and commerce contracted; throughout Mediterranean towns came to rely on local markets. By the 700s Constantinople’s population was only around 40 000. The gradual economic retreat profoundly impacted the western Mediterranean, ushering in a very different historical era: the rural world once called ‘the Dark Ages’. Because plague in late antiquity relates to the histories of globally important religious traditions today, plaguerelated study of both early medieval Christianity and early Islam emphasizes the important role spiritual authorities had for people confronting sudden and catastrophic pestilences. The plague of 590 in Rome quickly claimed the current Pope’s life, and a monk living outside the city triumphantly assumed the post, becoming Gregory I, later ‘Gregory the Great’. Hagiography attributes his accession to cessation of the plague, including an apparition of the Archangel Gabriel over the ancient mausoleum of Roman Emperor Hadrian. Renamed the Castel Sant’Angelo for the angel’s sign that plague would end, the structure is now part of the Vatican. Pope Gregory I (born 540 CE–604) became the first of several plague-tested saints available to the faithful in times of epidemic. The early military successes of followers of the Arabian prophet Muhammad (c. 570 CE–632) may have been facilitated by recurrent plague losses in the Middle East. Population decline created significant power vacuums in the buffer zones separating Persia from the Byzantine Empire, eroding secular and religious authority. Possibly related to the speed of conversions, deep rifts within early Muslim communities appeared soon after the prophet’s death in the 600s, divides that persist unmended to this day. But the overall eastward and westward expansion of Islam spread almost as fast as the plague had spread when the Mediterranean was still united at the peak of the late Roman and Byzantine Empires. The disassembly of trade networks and grain provisioning to great cities over the centuries of recurrent plague effectively ended Greco-Roman antiquity. Long distance Eurasian commerce also suffered. Trade across Eurasia had extended along the Silk Road, and plague epidemics in the 600s and 700s occurred at both eastern and western ends of the caravan routes. Along this ancient route enzootic plague persists today, and it is interesting

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that both Christians and Muslims regarded the region as a land of darkness, where the forces of Gog and Magog threatened to break through and usher in the end of the world. From today’s Ukraine, through the former Soviet state of Azerbaijan to Turkmenistan and Kazakhstan, across eastern Russia to Mongolia and Manchuria, Y. pestis foci always threatened to break the security of trade and travel through these regions. Ancient and early medieval Islam grew wealthy by consolidating southern-centered trade routes, developing maritime networks that linked northeastern Africa to the Indian subcontinent, to Indonesia and southeast Asia, and eventually, through Javanese and Fujian mariners to China. The lands of Islam thus recovered from early plague losses far faster than did Christian Byzantium or western Europe, and by the 800s Muslim regions were entering a golden age. In contrast, the last recorded plagues in western Europe occurred in the mid-eighth century. The last plagues of Tang China occurred in western provinces during the mid-ninth century, but for the most part of China, by turning inward, also had entered a period of extraordinary growth and development. Plague epidemics reflect patterns of trade in human history, and plague appears to have died out in the late ancient world when the links between Central Eurasia and the western Mediterranean were fully broken. It took Europe longer to recover. The historical evidence is quite thin concerning the precise nature of many of the recurrent plagues of late antiquity. Testimonies from some of the epidemics, including the plague of Rome in 590, mention the characteristic buboes of bubonic plague. But for other epidemics no discrete descriptive or demographic evidence survives, and the presumption of plague comes from the larger geographical pattern of reported crises. Plague disappeared from western Europe by the mid700s, contemporary with the last reports of epidemics in Constantinople. We do not know whether plague reached populous centers within the lands consolidated under Islam, but if it did the losses were not sufficient to interrupt the spectacular growth and extension of a great civilization. The return of plague in the fourteenth century, however, did cripple the economic vitality of the Muslim heartlands.

Plague in Europe, 1347–c. 1550 All sizeable epidemics among medieval Christians were generically called peste, pestilentia, or simply mortalitas, and were understood to be a collective punishment for sin. Medieval physicians nevertheless accepted naturalistic explanations for the appearance of particular acute epidemics, citing the poisoning or corruption of air, water, or earth as underlying explanations for a large mortality. By

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eating, drinking, or breathing corrupted elements, the body’s humoral balance was compromised. If the poisoning was particularly strong, it could overwhelm any of the natural processes through which nature healed. Before the Black Death, however, epidemics were characteristically local or regional, and so both religious and scientific authorities needed to explain the catastrophe that unfolded in 1348. By autumn that year, reports reached Paris of the sheer magnitude and widespread effects of pestilence in Italy and Spain. The king of France asked his elite academic physicians to explain how a truly great pestilence could occur. They reasoned that celestial events had created a ripple effect through all the elements (earth, air, fire, and water), leading to a massive mortality. The ‘Black Death’ was initially assumed to be an epidemic like others known from traditional authorities, and did not lead to a rejection of the mechanisms by which disease and epidemics were explained. It seems to have taken Europeans a couple of years to understand just how disastrous this plague was. The economic aftershocks of the mortality were felt acutely in the disruption to wages and prices, as laborers everywhere in Europe began to realize that they were in a better bargaining position. Furthermore, recent study of surviving wills and testaments during and after the Black Death illustrates that ordinary survivors may not have realized how widespread the catastrophe was until 1350. Ordinary Christians making a pilgrimage to Rome to receive forgiveness for their sins that ‘Jubilee Year’ probably first learned how devastating the plague had been all over Europe. Recurring plague epidemics hit western Europe and the Middle East over the next 400 years, but no subsequent epidemic had the vast geographical sweep of the initial wave. At the local and regional level, later plague epidemics could be as brutal as the initial Black Death had been, and there were intervals during which much of Europe and eastern Asia were affected in one larger pandemic. But no wave of plague ever reached both the ubiquity and the magnitude of the first one. Eyewitnesses to the later plagues frequently, but not always, distinguished plague from other pestilences by the appearance of buboes in the groin, axilla, or cervical regions. More importantly, the more discrete, less universal nature of recurrent plagues after the Black Death led European observers to practices and strategies, which they hoped would contain or mitigate the costs and losses in plague years. Thus, the greatest biomedical legacy of recurrent plagues in Europe is the piecemeal elaboration of barrier technologies and supportive ideas describing how and why plague could be contained. There are two phases to this legacy, each about two centuries long.

The Black Death Pandemic and Late Medieval Plagues Most medieval physicians were quite certain that the pestilence of 1348 could be causally explained by natural events, although a few of them thought those natural causes were well poisoners who knew how to isolate and spread plague poison. Physicians’ ideas of natural causes could be seamlessly fused to such ideas, as well as to the pervading religious frameworks. Some graphic chronicles of the great pestilence reported with complete credence that these remote lands were subjected to raining frogs and serpents, and that all manner of natural disasters accompanied an unheard-of dying. Reinforcing apocalyptical expectations of the coming end days, a great earthquake in the Carinthian mountains of southeastern Austria occurred in January 1348. The tremors were felt over a 300-mile radius and survivors close to the epicenter fled in all directions with stories of unparalleled destruction. This quake was one of the two greatest earthquakes in European history over the last 1000 years. A different set of ideas about the coming of pestilence operated in many popular rumors: plague came by ship from the Crimea, carried in the bodies and goods of sailors and merchants. Such stories from nonexpert but literate survivors did not fit neatly into medical or religious systems that explained how a universal pestilence could occur. The most famous of these stories pointed to an episode that is often called an early attempt at biowarfare: the Genoese colony at Kaffa (now Feodosiya, in the Ukraine) became a source of plague after non-Christian warriors hurled the bodies of their dead over the walls of the town, supposedly causing plague to break out inside it. The Genoese were unable to rid the city of cadavers, trying first to empty their own dead into the Black Sea. Then they set sail for home, stopping at multiple points along the way, carrying plague to each stop. The saga thus featured vivid details, such as desperate plague-infected men seeding plague in western ports all consequent to contaminated goods and people. Neither the events at Kaffa nor the natural disasters further east were witnessed by the individuals who recorded the stories, in contrast to the testimonials of the great earthquake. Many Western accounts of the Black Death thus emphasize patterns and means of plague’s spread that logically suggested interventions were possible. Two particular Black Death survivors offered other reflections that empowered their readers to define and understand ways of mitigating plague. Francesco Petrarca (1304–74) and Giovanni Boccaccio (1313–75), remembered as architects of the Western humanistic tradition, enlarged the plague literature by focusing on patterns of human behavioral responses and the difficulty of surviving the death of so many friends. Neither man had much interest in settling scores, in chronicling events in a descriptive

History and Culture, (and Biographies) | Plague, Historical

fashion, nor in making the connections to Biblical prophecies of the apocalypse. Neither was much concerned with understanding plague in a medical–scientific, or in a religious–cosmological framework. Petrarca focused his attention on personal losses and the pain of grieving: specializing in the genre of consolation, believing that words of comfort could help more than any medicine to relieve the deep sadness of losing loved ones. Thus his approach is generally regarded as secular because it acknowledges that grief is not completely allayed by knowing that the dead have gone to a better life. Boccaccio meanwhile escaped the summer of plague in Florence by fleeing to a villa in the surrounding hillsides. Writing sometime in 1349, Boccaccio described the plague in a prologue to a collection of 100 edifying stories, the Decameron. Boccaccio used the plague as a literary device to explain the escape of his seven young storytellers. They would amuse and instruct each other on the subject of love in order to take their minds off the terrible sights and smells of the unfolding epidemic. Boccaccio categorized typical human responses to a great epidemic: living modestly and piously in the hope of escaping divine wrath; eating, drinking, and otherwise enjoying themselves because survival seemed unlikely; or choosing a moral, middle ground. Nowhere did Boccaccio discuss altruistic behaviors or the actions of priests and physicians serving the ill. Instead he admitted flight was the most basic plague advice, as it would be for the coming centuries. Cito, longe, tarde was the dictum: flee quickly, go far away, and return slowly. Over time, those who could flee a plague tended to survive: plague’s dangers accompanied particular places. Collective responses to the Black Death included both characteristic and extraordinary examples of medieval. Traditional religious responses to great epidemics included efforts of spiritual authorities to organize frightened communities in great processions, in order to seek God’s intervention. During and after the Black Death, pious processions seeking God’s healing intercession were common. Typically they called upon local saints, still connected to the living through their relics, to help make the case for God’s merciful suspension of the pestilence. By the 1500s, many believed that such gatherings might present greater danger for propagating plague, and thus religious responses to plague began to pit church authorities against local secular governors. Flagellant processions, on the other hand, were less common but far more dramatic. A feature of transalpine Europe, flagellant bands emerged in 1348–49 along the Danube river valley, moving west and north along the Rhine river. Most accounts of the flagellants during the Black Death show that fear of the end of days propelled the movement, and the fullest of the descriptions of the movement explicitly links the flagellants to the earthquake and apocalyptical expectations.

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It is difficult to categorize the wave of scapegoat murders as a component of traditional religiosity. There was no express textual rationale or justification for the elimination of Jewish communities, and the Pope expressly condemned Christians for such actions. Explosive antiJewish riots first occurred in the spring of 1348, in southern France, Navarre, and the eastern Rhone valley. Jews had been expelled from much of France, Italy, and all of England, during the century before the Black Death, so most of the subsequent murders occurred in towns north of the Alps. Independent of the unprecedented human mortality from the Black Death, the pandemic led to the near annihilation of many Jewish communities. During the following two centuries, Jewish refugees moved eastward into Germany and Poland, where their labor was actively recruited by feudal lords. Permission and forgiveness for genocide was also effectively dispensed by the Pope’s plenary indulgence after the epidemic, obviating the need for communities to prosecute criminals, or the need for individuals to seek forgiveness for their sins during the plague through a personal and specific penance. The epidemic’s huge territorial range, claiming victims across at least Egypt, Syria, Byzantium, Anatolia, Mediterranean Europe, transalpine continental Europe, and Britain and Scandinavia, needs to be considered. Plague was not universal, as many survivors who wrote about it assumed. Plague did not strike the entirety of Eurasia. Recent research by Chinese historians shows that Yuan China, holding the area north of the Yangtze, was not devastated by this particular pandemic. Sub-Saharan Africa was not affected, and the Indian subcontinent south of the Ganges likely escaped as well. To account for a wide latitudinal propagation of plague, French plague research over the last century has emphasized the importance of the human flea, Pulex irritans. M. Baltazard at the Pasteur Institute in Paris noted the importance of human fleas in North African plague outbreaks of the 1950s: clouds of fleas surrounded recent victims of plague, abandoning a cooling body in order to find new human hosts. The feeding apparatus of the human flea does not become blocked with Y. pestis organisms as does the rat flea, and thus cannot establish an enzootic focus for plague in Europe. Systematic study of the sanitary and hygienic conditions of both rural and urban environments in the 1300s is not possible with surviving historical evidence, and so this model for a Y. pestis pandemic remains speculation. Those historians who doubt the Black Death was caused by bubonic plague point to survivors’ lack of consensus that buboes were its most characteristic feature. In Europe it was the second wave of plague, in the 1360s, that first led chroniclers to the conclusion that the pestilence of 1348 was a distinctive and specific disease. Survivors in Mediterranean Europe who witnessed both

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epidemics noted the specific similarity the second one bore to the previous one. The most famous among them was a papal physician, Guy de Chauliac, himself a plague victim who recovered. Guy described an 8-month course of the epidemic in Avignon, on the Rhone River, beginning late winter, more virulent in spring and early summer, gradually losing its strength in September and October. He attributed his own survival to becoming infected rather late in the epidemic’s duration, even though he was quite ill for 6 weeks and recovered only after the bubonic abscess drained. Plague and Demographic Change, 1350–1500 Where recent, painstaking historical investigations have been made to estimate the extent of mortality during and immediately after the Black Death, the overall losses have been revised upward. This first great pandemic caused between 30 and 45% mortality across Europe and in Egypt, higher than any other pestilence known in history. Extensive study of the demographic effects of the Black Death is fuller for England and Italy than for the rest of Europe, partly due to the nature of surviving sources. English court and manorial records are in some localities complete enough that historical demographers have been able to reconstruct the age structure and size of the population before and after the epidemic. Italian and southern French records, in contrast, are typically based on urban tax records, wills and testaments, and on survivals within large families or clerical/monastic communities. English studies show that the Black Death took more lives among children and those over age 40. There were geographically very few localities that were not affected at all, but a few do exist, and interest in the localities that the epidemic by-passed has become an important venue of current research. Despite these extraordinary losses, population and economic recovery were under way in the 1350s. The precipitate decline of European population instead began with the recurrences of pestilence in 1360 and afterward. In England, 1362–63 was recalled as the ‘children’s plague’, because deaths in the baby boom generation following the Black Death were more visible, although on the continent victims of the pandemic did not seem to be confined to the generation born after the Black Death. As with the Justinianic plagues, the second wave seemed the demographic tipping point, after which replacement rates collapsed. By the mid-1400s, the overall population of Europe was 40% of its total in 1300, and most regions of western Europe carried populations less than 50% of their levels in 1300. Although we do not have comparable statistics for much of North Africa and the Middle East, we know that in Egypt the collapse was even more profound because agrarian production was the economic mainstay

of the region, and plague losses left a labor force insufficient to maintain irrigation ditches. The population losses also led to quite different demographic responses across Europe. In England and northwestern Europe (the Low Countries and northern France), the late medieval era is the point at which a distinctive pattern of late marriage and lower overall fertility emerges. The overall shape of European demographic change over two centuries, between 1300 and 1500, presents some curiosities for economic historians. Agrarian western Europe had supported very large numbers of people in 1300, and at levels not reached again until the 1800s. Many were living at or below subsistence levels. Malnutrition and misery increased from 1300 to 1348, such that the Black Death hit at a time when a great many people were nutritionally and socially vulnerable. If a vast undernourished population increased mortality rates during the Black Death, the loss of some 40% should have relieved population pressure. This seems to have happened initially, but demographic decline accelerated after the second medieval plague wave of the 1360s. Population decline in the later Middle Ages thus had little to do with absolute scarcities of resources, and the punishing, recurring plague epidemics are generally cited as an exogenous mediator of population change in this era. Many were more prosperous than their grandparents had been, and by the fifteenth century the standard of living of ordinary farmers in England was rising. General economic recovery thus occurred despite demographic depression, because the 1400s and 1500s saw a turn to urbanization and market integration. People living in successful towns and cities were richer and trade supplying these centers was both more diversified and linked directly to more distant suppliers. The century of demographic recession was a time of prosperity for many, in part because of the transformation of European markets. The era of recurrent plagues in late medieval Europe thus is a striking contrast to the profound decline in the late ancient plague era. Larger scale shipping and transport began to link the North Sea and Baltic ports directly to the Mediterranean, and maritime fishing in the north Atlantic dramatically increased. Overland routes through German-speaking regions and in eastern Europe multiplied. New patterns in both maritime and overland transport in turn influenced the geographical diffusion of early modern plagues, which could occasionally, but only locally or regionally, become as brutal as the Black Death was. Even minor plagues of the late medieval period were quite costly in human lives, typically culling over 10% of town or city population. Any plague epidemic thus exceeded the mortality level of a great famine. Plagues could be further distinguished from subsistence crises demographically. The latter claimed greater mortality rates among the very young and among older adults,

History and Culture, (and Biographies) | Plague, Historical

whereas plagues included sizeable losses among teens and young adults. And plague always provoked flight of the wealthy, who increasingly demanded more reliable information concerning the whereabouts of plague.

Evolution of Plague Containment and Surveillance Practices, 1400–1900 In the mid-twentieth century, plague was often described as ‘the great teacher’ – that is, the historical disease that led Western Europeans to the discovery of the fundamentals of modern epidemic control. The historical reality is far more complex, and the lessons that can be drawn even from recent past plagues reveal much that is not progressive. To simplify centuries of ambivalent lessons, this section will omit much of historical interest in order to emphasize evolving ideas of epidemiology and public health that endured, and will divide the subject into two temporal intervals. First, the late medieval and Renaissance period, roughly 1350–1600 CE, created a rudimentary set of ideas and practices that not only persist today, but also likely altered Europeans’ actual experiences with recurrent plague. Second, the period between 1600 and the late-nineteenth century developed some important practical implications of plague-related ideas, and initiated long-lasting approaches to international health surveillance. Late Medieval Plague Control Facing recurrent plagues, Europeans – mostly Italians – of the late medieval period elaborated two strategies for the management of epidemic crises: information-gathering and barrier technologies. Together, these administrative and technical strategies are cornerstones of modern epidemic control. During 1348, some Mediterranean cities created temporary committees of leading citizens to orchestrate street cleaning, but urban sanitation was not again a significant component of epidemic control until the 1700s. Instead, surveillance, by ascertaining where plague was, where one could safely flee, when plague was nearby, became the principal way to translate fear into a strategy for survival. Recurrent plagues by the 1400s proved that plague moved through and across geographical regions slowly enough that information gathering could provide some time to prepare. Flight from an area where plague appeared was thus a generally useful tactic, but one that was realistically open only to the privileged. Surveillance thus became the mechanism underpinning the advice to flee plague. Abandoning cities and towns to those without means to flee or access to information created other problems for elites, and gradually imposing civic order became important topics of plague management in the early modern centuries. By

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the mid-1500s, information gathering in the wake of plague also served the purposes of medical and administrative debriefing: what had and what had not worked in the control of a plague. Managing plague risks through the creation of many different kinds of barriers between the well and the ill was the other large area of public health innovation. Among the barrier strategies invented in this time period are quarantine, pest houses, official passes declaring that a person had not contacted plague, public health bureaucracies, disinfection of persons and property that had touched plague, and identification of causes of death. Plague could so overwhelm administrators left in charge of urban properties that frequently hit urban regions in southern Europe not only invented mechanisms to protect property and economic interests of the governing elite, but also needed means to insure their own personal safety if forced to remain within a plague-stricken region. Most of these barriers were crafted piecemeal and in many different locations. Only by the later 1400s do we begin to see coordination and explicit rationales provided for separation of the well from the ill, the effectiveness of hospitals specifically for the plague-stricken, and the need to cleanse or destroy all items touched by plague. Initially, quarantine specifically referred to the passive isolation of individuals who were not ill, but were deemed likely to become ill. This fundamental control strategy was invented in 1374 by Venetian governors of Ragusa, a Dalmatian port town now known as Dubrovnik, in Croatia. Because Ragusa was then easily accessible only by sea, governors reasoned that plague entered through the port. Venetian rulers of the port town mandated a 30-day waiting-and-watching period, holding incoming persons and merchandize on an island until it could be demonstrated that they did not also bring plague. Naturally such costly measures were difficult to enforce rigorously. When plague could not be kept at bay, the desperate months of massive epidemic mortality required different actions and types of surveillance. As early as the mid1400s the first dedicated hospitals for the plague-stricken emerged, in part because Church-controlled hospitals typically refused to shelter victims of plague. The earliest permanent hospitals for the confinement of the plaguestricken (the lazzaretto, or pest house) opened in the early 1500s, and by 1600 many Italian cities had added separate facilities for isolating those who recovered and those who had contacted, but not yet manifested plague symptoms. The first permanent public health agencies also took origin during this era, especially within those commercial and maritime cities that saw cases of plague frequently. Appointing permanent committees mandated to monitor plague’s whereabouts was an important step: plague was thereby judged an ongoing threat to the health and security of the state. The bureaucracies created to protect the

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state became semiautonomous, surviving sweeping military, political, and religious changes. Operational costs of supporting urban populations through a plague were sustainable only in the wealthiest commercial areas of Europe at this time, and were thus rarely implemented in rural areas such as the Germanspeaking lands and England. Much was left up to the individuals to protect themselves. By 1500, the risk of dying during a plague was strongly associated with urban poverty and the inability to flee to some secure location. Privileged observers thus increasingly viewed the poor as dangerous plague carriers. Such perceptions had significant consequences to the management of epidemics. Could the poor be allowed to flee a plague-ridden city, too? Such debates about the legality and morality of flight emerged 150 years after the Black Death. The rationale and the ethics of flight provoked more general religious and secular debates by the early 1500s. The Church undertook charitable interventions for those who could not flee a plague-stricken area. From 1510 to 1550 legal treatises first appeared to delineate who did and did not have the right to flee a plague. Religious reformers in Italy undertook care of the urban poor in times of plague; for his Protestant followers, Martin Luther addressed the theological dimensions of fleeing plague. Discussions of the mechanisms for plague’s spread multiplied in university discussions, and by 1546, Girolamo Fracastoro (1478–1553) published his On Contagion and Contagious Diseases, helping to focus ongoing medical debates about plague as a contagious disease. In the sixteenth century, academic medical discussions about plague finally confronted the conceptual dissonance between practices that assumed plague could be spread person to person (thus isolation) and medical theory that held all disease as an imbalance of the body’s four humors. In records of the late fifteenth and early sixteenth centuries, plague also became increasingly better distinguished from other epidemic infections. The printing press facilitated distribution of pamphlets and testimonials related to plague experience. Because printing offered greater access to information about antiplague strategies and concerns outside one’s own locality, convergence and consensus about the ways to recognize and respond to plague is a hallmark of the early 1500s. Even more importantly medical and governing authorities began to distinguish among differently caused epidemics. Some epidemics were seen as altogether novel – such as petechial fever (likely epidemic typhus fever) and the ‘great pox’ (likely syphilis), both of which produced rashes unlike the buboes and blackened skin typically associated with plague cases. Monitoring the length of time that individuals were ill was a bureaucratic proxy for plague; adults ill less than 7 days before death became plague suspects. This habit, too, had a large pay-off in

plague control. Epidemics of petechial fevers had a 2-week course of illness, and the great pox allowed its victims to linger for months or years. Epidemics were otherwise distinguished from plague by being associated with discrete and identifiable subpopulations – for example, diphtheria and smallpox among children, dysenteries among the homeless. Fracastoro offered the first clear theoretical framework of a mechanism that could explain how a particular disease could be transmitted from one person or place to another. He imagined the existence of ‘seedlets of contagion’, or seminaria contagionis, which acted like poison within the body and could be dispersed through air or by direct contact. Fracastoro admitted only a short list of ‘contagious diseases’ that were propagated in one of three paths: by touch directly, by fomites (including clothing and other items touched by the ill), and by a glance. The last of these paths included both conjunctivitis and the evil eye as contagions, reminding us now that Fracastoro did not think of the disease seeds as living germs, and did not view plague and his other contagious diseases (smallpox, measles, leprosy, phthisis, and petechial fever) as we do today. The single most influential and comprehensive adaptation of Fracastoro’s views of contagion in the 1500s was subsequently made by a physician in Palermo, Sicily, working as the chief physician, or Protomedico, of the Spanish state. Giovanni Filippo Ingrassia (1510–80) detailed the proper concerns of a public health office, investigating reports and rumors of sudden deaths, verifying cases of plague through postmortem inspection, and sometimes autopsy, then establishing secure locations where the ill and those associated with the ill could be separately housed and cared for. Differentiating the obligations of individual physicians from the obligations of public health physicians, Ingrassia reviewed the rationale and mechanisms for disinfecting houses where plague cases had occurred, assembling findings and observations from treatises published over the previous century. His is the first study of a significant urban plague analyzed through the personal experience of a medically trained public health officer, including knowledge of current theories of plague spread, and review of the literature on plague management. After Ingrassia, individual public health and medical authorities typically published their own personal reports in similar vein, allowing observations about plague epidemics a general comparative format. Thus Christian Europeans began to assume that either Church or State, or both together, had an obligation to commit funding and an infrastructure for mitigating human misery. Times of epidemic were moments of great physical and spiritual need. Reinforcing this broad cultural change were new themes within artistic, religious, and literary production specifically linked to

History and Culture, (and Biographies) | Plague, Historical

plague. Such issues are of greater interest to historians, and thus will not be considered here. Refinement and Internationalization of Plague Control in Europe, 1600–1894 Plagues recurred in continental western Europe until the great plague of Marseilles, 1720–22, and the plague of Messina in 1743. During the previous 200 years, spectacular epidemics periodically affected large cities, with mortality rates that could still reach 40–60%. Equally high demographic costs of plague occurred in rural areas where no centralized government authority undertook surveillance and isolation of early plague cases. Overall, as Eckert has shown with detailed geographical and epidemiological analysis of plagues in Central Europe, 1560–1720, plague gradually receded, largely through the imposition of large-scale regional barriers. The cordon sanitaire, blocking communication with an entire region affected by plague, braked commerce and migration across regions, and was likely the single most important reason why plague’s hold in Europe gradually eased, although many other causes of plague’s recession played some role. For example, scholars have speculated that the widespread appearance of white arsenic (AsO3) in European markets of the late seventeenth century made available a powerful rat poison, thus culling the rodent population in interepidemic intervals. Similarly, geographical changes in routine European commerce may have contributed, as trade moved from the Mediterraneandominated exchanges of the late medieval world to the Atlantic, North Sea, and Baltic commerce of early modern centuries. New trade patterns likely reduced the routine maritime transmission of plague-carrying rodents and fleas. Here, too, Eckert is able to show that commerce from southeastern Europe to the Baltic and North Sea ports was a principal route through which the Netherlands and Britain were infected in the seventeenth century, and that plague spread along eastern overland and riverine routes in the sixteenth through eighteenth centuries. Nevertheless, the intensification of plague control methods invented in the previous centuries had measurable effects on the mitigation of plague mortality among Europeans, both at home and abroad. From the seventeenth to the mid-nineteenth centuries, wherever commerce was Mediterranean-based and traditions of quarantine and lazaretto confinement were longstanding, cities developed aggressive bureaucratic management of plague outbreaks. Small armies of assistants performed all the duties which anxious administrators believed necessary: guards, house fumigators, street cleaners, inventory takers, informants, perfumers, launderers, clerks, porters, boatmen, gravediggers, cart drivers, and lamp carriers, bedside attendants, and those who disrobed,

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inspected, and disinfected all who were deemed suspect. In many places special hospitals were dedicated to the plague victims, other facilities to the recovering, still other structures for the suspect – those not yet determined to have plague. Publishing reflections on the best practices among these minutiae of plague barrier technologies became widely available knowledge, and these publications bolstered medical and bureaucratic careers. Prosperous citizens preferred to be confined to their own homes if isolation were mandated, so that the municipality or parish church carried the duty and expense of bringing food and guarding to prevent escapes. Priests set up altars in the streets, so that those confined to their houses could celebrate the Mass from windows and balconies. In France, national controls dictated that infected houses be marked with a large white cross, and that all physicians and priests dealing with the plague-stricken carry a white handkerchief signifying infection. Plague controls were staggeringly expensive for small communities, and likely did little to control plague once it had breached environmental thresholds for its propagation. Most administrators assumed that plague could only be eradicated by fire, and thus destroyed houses and belongings of victims. In some cases the conflagrations were mandated by remote authorities, who could be arbitrary and sweeping in their demands. Descriptions of plague in seventeenth century villages emphasize the sudden silence and empty streets once plague took hold, in part because the full costs and uncertainties of life after plague were in limbo. The most-often cited refinement of barrier technologies invented during this period is a visual metaphor for plague: the bird-beaked, robed physician, although it need not be a physician beneath the garment. A royal physician to King Louis XIII of France claimed the credit for inventing this protective gear during 1621, when a minor plague reached Paris. Charles DeLorme’s (1584–1678) costume was not affordable by any but the wealthiest healers, but the idea of an oiled or waxed robe diffused quickly across northern and Central Italy, and was more used there than in France. Priests also wore the robe. DeLorme’s headgear was to be made of fine Moroccan leather, the beak packed with herbs and perfuming blossoms; the waxed robe tucked into pants and high boots. More modest costumes were made of linen or even flax; the wax or oil was impregnated with aromatic herbs and applied to the robe, head covering, gloves, and shoes that were humble wooden clogs. Occasionally even the carriages that conveyed doctors to the stricken were covered in oiled cloth. Refinements to the costume were debated throughout the eighteenth century. Confidence in the rigorous application of cleansing and disinfection measures increasingly preoccupied individuals in charge of pest houses. Some physicians had bowls of vinegar carried before them, and dipped their hands into

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the vinegar before touching the patient’s pulse. Vinegarinfused herbs were scattered around the hospital space; smoking braziers or even gunpowder was used to purify air. Elaborate procedures were devised for disinfecting mail, airing luggage and cargoes, and were aggressively maintained along the Austrian–Hungarian boundary between Christian Europe and Ottoman (Muslim) Turkey. Lazarettos did not work reliably well against individual cases of plague, but were useful as a regional or national strategy, so long as the costs to trade were not great. Creating impediments to the flow of goods – particularly grain and other foodstuffs that rats followed – seems to have squeezed plague back toward the Balkans and Ukraine by the later seventeenth century. Implementation at a large scale depended on the centrally directed administration of cordons sanitaires, blocking commerce and migration at a regional or national level, and thus came to be recognized as a state prerogative. In the 1780s, John Howard (1726–90), an eccentric and wealthy Englishman interested in reforming prisons, visited many Mediterranean lazarettos, interviewed the administrators and the physicians, noted their procedures and regulations, drew floor plans of the confinement areas, and then presented this information in a 1789 volume. Howard noted the rituals of disinfection and quarantine, and was surprised that even with roughly similar protocols in place everywhere, the physicians and surgeons who staffed the lazarettos did not agree on whether the plague was contagious or a miasmatic environmental change. Howard ultimately used lazarettos as a model for the modern penitentiary, a confinement space where recovery and rehabilitation of those dangerous to the public was managed. Elaborate protocols provided a brake on communication between western Europe and the Ottoman Empire. As plague lingered in the Balkans, the Middle East, and the eastern Ukraine and Crimea, two conclusions emerged. First, plague was increasingly viewed as an affliction foreign to Europe and Europeans, a disease of alien origin. Europeans had replaced the perception of plague as a punishment from God with punishment for crossing some social boundary or failing to maintain proper vigilance. Second, European travelers of the late eighteenth and early nineteenth centuries attributed the continuing presence of plague in these regions to the reluctance of Muslim nations to use quarantines aggressively. While Europeans urged, and would later demand, that non-Europeans apply traditional containment strategies in times of epidemic disease, during the same time at home they were dismantling the principle of quarantine as the principal disease-containment strategy. European merchants and travelers in the Middle East believed that they could only escape recurrent plague by completely isolating themselves from the native population. Especially in Egypt and Turkey, Europeans of the late

eighteenth and nineteenth centuries sealed themselves in their homes during plague, hired trusted porters and servants to fumigate all correspondence, dip money in vinegar, destroy noncaged animals on their premises, rigorously wash all food brought in, and seal the openings found around walls, doors, and windows. If anyone had to puncture this isolation bubble, he typically wore the garb that the Marseilles plague made famous. Should cases of plague occur, any items associated with a victim were either burned or aired on the rooftops for weeks. Meanwhile they berated non-Europeans as fatalistic and ignorant, and occupied themselves with cleanliness during times of plague. While a few modernizing Muslim leaders of the early nineteenth century became convinced that quarantine was a useful personal strategy, the vast majority instead feared isolation, forcible removal to a pest house, and violation of customs honoring a deceased family member. Thus the imposition of quarantine in the 1830s’ epidemics – both cholera and plague appeared in succession – provoked widespread popular resistance to European-style epidemic controls. Beginning in the 1830s, for 60 years Europeans focused on cholera as the new plague, and with its appearances refashioned plague controls. Cholera first appeared in Europe as a new and extremely frightening disease in 1830–32, and the epidemics geographically replicated the east-to-west spread of medieval plague. Quarantines were applied in Russia and eastern Europe – often brutally and with military resources, but nowhere did such measures work well or even staunch cholera’s progress. Thus British and French authorities were in a position to doubt quarantine’s utility against cholera. During the 1830s and 1840s, medical and political authorities in these two nations debated the utility of quarantine as a domestic strategy to contain disease spread and the empirical evidence for contagion. Attention instead to environmental sanitation took shape in both Paris and London of the 1840s, with demonstrably better morbidity and mortality rates. A second pandemic of cholera between 1848 and 1855 led to the first International Sanitary Conference of 1851, held in Paris, and for the next 40 years such conferences would focus on cholera and the role environment played in epidemics. International conventions and standardization were broad objectives of western nations during the later nineteenth century. There were 58 times more international events in the second half of the century, compared to the first half. The 12 International Sanitary Conferences that met between 1851 and the outbreak of the First World War occurred in the same decades as the International Statistical Conferences, (gradually standardizing weights, measures, time, as well as leading to the creation of standard causes of death by 1892). International Medical Congresses met in these years, international world fairs, and numerous other ventures. The International Agency

History and Culture, (and Biographies) | Plague, Historical

of Notification was created in 1874, taking advantage of recent communication successes in laying undersea cables. Focused almost exclusively around cholera, the first eight of these conferences (to 1894) ultimately strove for protection of Europeans’ commercial interests by considering the problem of global infectious diseases as the threat poor, non-Western nations posed to ‘civilized’ nations. Over these decades, quarantine and cordons sanitaire were increasingly depicted as anachronistic and unworkable means of impeding the spread of feared diseases from one region of the world to another. The speed of transportation, the numbers of individuals traveling, and the mounting costs of interrupting regular trade led political representatives of the International Sanitary Conferences to reject the kinds of mechanisms devised in the plague centuries. Most of the medical internationalism of these decades was also suffused with strong optimism about the ability of technology and good governance to effect progress, even if very little was actually achieved at the international level. One of the great ironies of the cholera conferences lay in the extent to which medical and diplomatic discussion of cholera – its causes, origins, and best means of control – led delegates to reject both plague-style controls and scientific evidence that cholera was a discrete waterborne pathogen. Reversing centuries of faith in personal barrier technologies, European commentaries on news of plague in ‘Mesopotamia’ and southeastern Russia reflected lack of attention to environmental sanitation, now their hallmark of civilization. Plague took hold where the fodder of filth supported it, and not otherwise. By the 1890s, increasing scientific consensus about the germ theory of disease made such scientific and diplomatic equivocation about the cause and transmission of cholera untenable, and therefore permitted the first ratification of an international sanitary convention. The seventh and eighth International Sanitary Conferences of 1892 and 1894 were able to reach such accord only because these conferences excluded consideration of pilgrims to Mecca, on whom many European delegates wanted to apply the full weight of disinfection and quarantine controls. They approved a weak sanitary convention regarding the inspection and disinfection of ships, and even then the most powerful nation, Great Britain, demanded many exceptions to its compliance with the convention. When the participant nations finally ratified the convention in 1897, plague was back and on the agenda. Plague reached colonial enclaves in southeast Asia, 1894–98; suddenly, discussion of quarantine and the need for international barriers became more focused and resolute, and the perceived need for oldstyle controls no longer appeared outmoded. European whitewash brigades doused plague-ridden sectors of Hong Kong and Mumbai (then Bombay) with sulfuric acid, hydrochloric acid, and lime, razed houses and flooded streets, but plague spread easily. In desperation the British,

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in both these cities, returned to past models, evicting the native population from whole sectors of the cities. With enzootic foci in both India and South China, plague next spread to most of the main port cities of the southern hemisphere.

Modern Plague, 1894 to the Present Over the 1800s, plague threatened most of the southern borderlands of the vast Russian empire. From the Balkans to Central Asia, plague occasionally produced dramatic epidemics countered with militarized cordons. During the nineteenth century, plague also moved steadily from rural enzootic foci in the Himalayan foothills to South China ports. By the late nineteenth century, however, steamship and rail travel linked all the continents, permitting plague’s global spread to many port cities within a just few years. In the spring of 1894, plague had reached Guangdong (then called Canton) through commerce and migration along the Pearl River, exploding into a massive epidemic. From Guangdong it spread to Hong Kong, an English colony established during the previous half-century. By the 1890s, Europeans had learned the benefits of government spending on basic public sanitation and had streamlined and focused maritime quarantine protocols, diminishing the costs plague had once imposed on routine commerce among privileged nations. The combined approach was largely able to contain plague spread before it could move to inland cities, but the application of epidemic controls had a vicious imperialist cast. Equally important in the 1890s was the authority that laboratory science had acquired in understanding the microbial causes of feared infections. A century of intensive research in pathological anatomy prepared physicians to differentiate diseases postmortem, although pioneers in the first generation of germ theory proponents made the crucial difference. The modern era in plague history begins with Alexandre Yersin (1863–1943) and Shibasaburo Kitasato (1852–1931) in Hong Kong. Within a matter of weeks these two investigators separately identified a bacterial cause of plague, although Kitasato likely did not isolate the bubonic plague bacillus. During the late 1880s, Kitasato had studied with Robert Koch (1843–1910) in Berlin. Meanwhile Swiss-born Yersin, a young physician who had joined Louis Pasteur’s (1822–95) new institute in Paris in the mid-1880s, had been instrumental in bringing Koch’s bacteriological techniques to the Pasteur Institute in Paris. During the early 1890s both men moved to East Asia: Yersin with the French merchant marine went to Indochina, Kitasato returned home to Japan. Thus both were within a week’s travel when plague reached Guangdong and Hong Kong. Kitasato and his highly

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capable clutch of assistants arrived on 12 June 1894. Yersin, working without hospital infrastructure or local governmental support, reached Hong Kong 3 days later, accompanied by an Annamite servant or ‘boy’ and two hired Chinese ‘coolies’. Despite opposition to his efforts, Yersin was nevertheless able to identify the microorganism before Kitasato and his team of investigators, largely because Yersin cultured pulp from victims’ buboes. Kitasato’s team tried to culture the organism from blood and from internal organs. Yersin also noticed dead rats in the streets of Hong Kong where greater numbers of human victims had occurred; he noted buboes on rats and guinea pigs that he used as experimental animals; and he observed that birds seemed immune. Within 2 weeks Yersin was able to satisfy Koch’s postulates, proving that the Gramnegative bacillus with clubbed ends – mon microbe, he called it – was the cause of plague. His first published claims did not appear until September 1894, several weeks after Kitasato’s first report in the Lancet. A priority dispute raged for decades, resolved only in 1925, when Kitasato graciously acknowledged Yersin’s priority. Meanwhile Hong Kong was a British-controlled Treaty Port, with a population of around 200 000 when the plague arrived. The vast majority of its population were from Guangzhou province, and were highly skeptical of Western medical approaches to plague. The crisis situation quickly overwhelmed any attempts at diplomacy between the colonial government and their subject population. Local British authorities controlled plague with draconian containment and sanitary procedures, desperate to avoid suspension of international trade. Efforts to control the epidemic in April and May had led the colonials to hire cheap Chinese labor, who were ordered to convey the sick to emergency hospitals and hospital ships, and remove garbage and human waste, all without effect on the epidemic. In the crowded, filth-glutted district of Taipinshan, coffins piled up and rumors of plague intensified tensions among the colonial population living in the hills above; Chinese residents meanwhile stepped up their resistance to European-style controls. Escalating mortality and door-to-door surveillance fueled even harsher colonial cleansing and disinfection efforts. A Scottish regiment now fought the sanitary war against Chinese ‘backwardness’, and Indian Sikhs were imported to serve them. Each morning the soldiers stripped naked, bathed in disinfectants, donned work clothes, fanned out to empty laborers’ hovels, removed paper from the walls, burned rubbish in the streets, groped through dark and airless shanties over dead and dying bodies, and marched back to the disinfection rooms after 4 h surges. At the hospital that served as their staging ground, they gargled a carbolic acid solution mixed with cologne, camphor and water, then took a dose of quinine, changed clothes, had lunch. Afternoon gangs made the next assault. All these

military men were rewarded with alcohol, monetary compensation, the best obtainable Cuban cigars, and afterward were awarded handsome medals commemorating their hazardous service. They were very similar race- and class-based public sanitation campaigns in every city that plague reached – in Australia, the United States and Hawaiian Islands, British India, Indian Ocean islands, South and West Africa, Portugal, Brazil, Argentina, Uruguay, and even Scotland. Seemingly oblivious to this parallel application of aggressive colonial sanitation and quarantine controls over non-Western populations, a new generation of bacteriologists pursued prospects related to the morphology and biochemistry of the plague microbe, and experimented with vaccines. Waldemar Haffkine (1860–1930), working in Bombay in 1896–97, is typically given the credit for the development of a killed-bacteria plague vaccine, though Yersin’s biographers always note his work in Paris during the summer of 1895, trying to culture an attenuated plague strain for use as a vaccine. Convinced that a reliable vaccine was not possible, Yersin next developed a serum therapy, inoculating horses with his bacillus, then harvesting serum for later use. The tenth International Sanitary Conference was the only one to deal exclusively with plague. Meeting in Venice in 1897, delegates quickly agreed that plague was caused by Yersin’s bacillus, and that rats were involved in its spread. Then delegates from Germany, Austria-Hungary, Russia, Italy, Ceylon (now Sri Lanka), and Egypt all dispatched national biomedical commissions to investigate plague in Bombay, none fully trusting the two British commissions to manage plague containment impartially. This plague-centered conference seems to have been the tipping point in focusing the long-term objectives of subsequent conferences. A few of the older participants, still tied to the pre-germ-theory era, insisted that plague surveillance include rigorous attention to water sources. But by the next conference, in 1903, when plans for a permanent Office d’Hygie`ne International were finalized, thinking about plague and the purposes and mechanisms of quarantine converged toward a new biomedical model. International sanitary conventions were redrawn. Between the tenth and the eleventh conferences, research on malaria and yellow fever, in addition to cholera and plague, further defined the range of needed control strategies. Nineteenthcentury approaches were now outmoded. Control of water supplies was important to cholera, but not involved in plague transmission. Between 1897 and 1903, Paul-Louis Simond (1858–1947) proved that the rat was the primary host for plague (Yersin had not followed up his initial hunch), and then proved the flea was the likely vector between rodent and human. Simond caught a still-living rat in a plague victim’s house, bottled the rat with some fleas harvested from a cat.

History and Culture, (and Biographies) | Plague, Historical

(Entymological precision was not greatly important to plague research until the 1950s.) Simond waited for the rat’s death throes and then placed it in a two-compartment cage with a healthy uninfected rat on the other side of a grille mesh. The sick rat died, and autopsy showed blood and organs with ‘Yersin’s bacillus’. Six days later the second rat died, externally showing both inguinal and axillary buboes, as well as identical autopsy findings to those of the first rat. ‘That day, 2 June 1898, I felt an emotion that was inexpressible in the face of the thought that I had uncovered a secret that had tortured man since the appearance of plague in the world’. In 1897, Japanese investigator Masanori Ogata of the Hygiene Institute in Tokyo, working in Taiwan (then Formosa), had similarly postulated the role of fleas, but did not publish his work until 1905. After World War I, with its massive experimentation in the use of airborne chemicals to kill both humans and insects, international plague control focused on the routine use of first sulfur dioxide and/or hydrogen cyanide to exterminate rats and fleas in ships’ holds. Here again, Western nations created diplomatic mechanisms allowing domestic and regional shipping concerns to escape onerous and merchandize-damaging procedures: the Deratization Exemption Certificates. For the first time, also, Americans were moving into technical leadership by being the first to develop the chemical protocols. Plague not only spread to great port cities of the late nineteenth century. Two great epicenters of Eurasian plague foci flared. Tsarist Russian microbiologists, trained in Koch’s bacteriological methods, revised approaches to plague prevention. Epidemics in the steppe region of the lower Volga, beginning in 1878, still led to the reenactment of older militarized plague suppression, and also to the creation of the Central Microbiological Institute in Saratov. Outbreaks in the Russian Transbaikal District became a second important training ground for scientific microbiologists. At this juncture, global political and economic events helped to create another plague drama. Between 1898 and 1903, Russia completed the Trans-Siberian railroad, during the same interval that Western European nations formed military coalitions to suppress a Chinese nationalist movement, the Boxer Rebellion. Boxers hoped to evict ‘foreign devils’ from China, so Western nations sent in troops to rescue diplomats, merchants, and missionaries, hoping to open China to Western capitalism in the process. Warfare, development, dire famines, and a politically moribund Qing dynasty, unable to exert economic and military control over Manchuria’s natural resources, all helped to stage the great Manchurian outbreak of 1910–11, the most severe pneumonic plague ever recorded. Harbin lay at the intersection of the Russian, Chinese, and Japanese railway lines and within the orbit of the newly American-controlled Philippines. Although only China had legal jurisdiction, Russian interests in plague initially prevailed, with Russian police forcing victims and their contacts into railroad wagons. The tsar’s Imperial

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Institute of Experimental Medicine in St Petersburg dispatched Danilo Zabolotny (1866–1929), a plague-seasoned Ukrainian microbiologist who had also been present at the Bombay epidemic. The Qing emperor chose Wu Liande (also written Wu Lien-Teh, 1879–1960), a 29-year-old graduate of Cambridge University teaching Westernstyle medicine at the Peking Union Medical College. Wu arrived on 24 December 1910 in Harbin, quite able to take over both laboratory and epidemiological investigation of the epidemic, as well as work amicably with the Russian team. Within 2 months Wu identified the source of human pneumonic plague, transmitted by fleas in the fur of Siberian marmots (or, tarbagans). Marmot fur had recently become a highly desired substitute for sable among Western furriers, and thousands of seasonal Chinese migrants came to Manchuria and Mongolia to capture and kill the animals. As winter set in and migrants returning home for the new year clumped at flophouses near railway stations, pneumonic plague dramatically worsened. Over 60 000 people died between late October and mid-February (1911), when Wu brought the epidemic under control. An International Plague Conference held in Mukden, April 1911, assembled teams from 11 nations to report various aspects of plague research conducted during the late winter. Wu was in charge of the meeting, much to the annoyance of many more senior individuals. American Richard Pearson Strong, who had been developing his own plague vaccine in Manila the previous decade, seized the opportunity to publish the proceedings in 1912. In many different respects the International Plague Conference marked an important divide in plague research. This was the first international infectious disease conference not run and ordered by Western Europeans and North Americans, although the scientific triumph it represented was decidedly Western. Wu himself was clearly aware of the significance of his own chairmanship over the proceedings, as well as of the inauguration of a new age in plague management. Wu and the plague of Manchuria made famous the importance of using masks for anyone attending persons with an airborne infectious disease. Finally, after the Manchurian plague the focus of forward-looking plague management shifted from urban, rat-borne plagues to careful surveillance of enzootic plague foci. French Pasteurians would make the shift in perspective by the mid-twentieth century, greatly expanding knowledge of the broader ecological variation in Y. pestis transmission. The Russians, and Wu himself with his Manchurian Plague Prevention Service, first turned the focus of plague research away from humans, toward the vast remote, rural, spaces where Y. pestis spread among rodents, squirrels, gerbils, and lagomorphs.

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The single greatest scientific legacy of Yersin’s Hong Kong success over the twentieth century was the global leadership role of French plague investigators and the worldwide placement of Pasteur Institutes. The Parisbased Office d’Hygie`ne Internationale Publique served as the communication hub for international health surveillance until the creation of the World Health Organization in 1947. Russian physicians and scientists were in good position to share dominance with French investigators, but during the Cold War turned instead to the study of plague as a potential biowarfare agent. Most of the historical research on Soviet antiplague institutes and the extensive biowarfare investigations is yet to be done. A recent series of articles in Critical Reviews in Microbiology (2006) outlines the gradual process by which Russian governors and investigators before the Soviet period came to understand the plague as an indigenous, enzootic threat rather than an imported, human-carried infection. Until quite recently the full extent of a biowarfare development program in the post-Stalin era was mostly known only from the often-contradictory and unverifiable claims of participant scientists who sought asylum in the West. The earlier Soviet Union had a vast territorial system of AntiPlague Institutes outside Russia, with around 100 field stations and laboratories. These posts were all controlled by a Central Ministry of Health; they in turn served both military and epidemiological monitoring functions, including weapons test sites and notorious closed cities. True historical perspective on Y. pestis and biowarfare research is not yet possible, because secrecy, denial, and destruction of records attend research in this arena since World War II. Biowarfare, however, is a long-standing plague topic. Yet like a mantra, histories of biowarfare recite a cluster of plague history moments to stress a continuity in yet another plague theme: the (unproven) incident at Kaffa in the 1340s, when Tatar forces supposedly launched plague-ridden cadavers into the Genoese fortress; the sadly ironic Japanese use of Wu’s Mukden facilities to conduct biological warfare studies during World War II; the accusations of North Koreans against both China and the United States during the early 1950s; and these latest still-unfinished saga of Soviet manipulation of Yersin’s bacillus for supposedly defensive

purposes. The weight of plague’s historical past carries considerable influence, making re-searching, and thus re-thinking, difficult to do. Y. pestis’s unique and frightening capabilities include legacies and patterns of human behavior that can serve as both warnings and sources of encouragement. See also: Cholera, Historical; Epidemiological Concepts and Historical Examples; History of Microbiology

Further Reading Achtman M, Morelli G, Zhu P, et al. (2004) Microevolution and history of the plague bacillus, yersinia pestis. Proceedings of the National Academy of Sciences of the United States of America 101(51): 17387–17842. Benedict C (1996) Bubonic Plague in Nineteenth-Century China. Stanford: Stanford University Press. Biraben J-N (1975) Les Hommes et la peste, 2 volumes. Paris: Mouton. Cipolla C (1981) Fighting the Plague in Seventeenth-Century Italy. Madison: University of Wisconsin Press. Cohn SK, Jr. (2002) The Black Death Transformed: Disease and Culture in Early Renaissance Europe. London: Edward Arnold. Crawford EA (1996) Paul-Louis Simond and his work on plague. Perspectives in Biology and Medicine 39: 446–458. Devignat R (1951) Varie´te´s de l’espe`ce Pasturella pestis: Nouvelle hypothe`se. Bulletin of the World Health Organization 4: 247–263. Echenberg M (2006) Plague Ports: The Global Urban Impact of Bubonic Plague, 1894–1901. New York: New York University Press. Eckert EA (2000) The retreat of plague from Central Europe, 1640–1720: A geomedical approach. Bulletin of the History of Medicine 74: 1–28. Horrox R (ed. and trans.) (1994) The Black Death. Manchester: Manchester University Press. Howard-Jones N (1975) The Scientific Background of the International Sanitary Conferences. Geneva: WHO. Huber V (2006) The unification of the globe by disease? The International Sanitary Conferences on Cholera, 1851–1894. The Historical Journal 49:2: 453–476. Little LK (ed.) (2006) Plague and the End of Antiquity: The Pandemic of 541–750. Cambridge and New York: Cambridge University Press. Mollaret HH and Brossollet J (1985) Alexandre Yersin, la vainqueur de la peste. Paris: Fayard. Naphy W and Spicer A (2004) Plague: Black Death and Pestilence in Europe, 3rd edn. Stroud, Gloucestershire: Tempus Publishing, Ltd. Nutton V (1990) The reception of Fracastoro’s theory of contagion: The seed that fell among thorns? Osiris Series 2, 6: 196–234. Ouagrham-Gormley S, Melikisvili A, and Zilinskas R (2006) The Soviet anti-plague system: An introduction. Critical Reviews in Microbiology 32: 15–64. Stathakopoulos D (2004) Famine and Pestilence in the Late Roman and Early Byzantine Empire: A Systematic Survey of Subsistence Crises and Epidemics. London: Ashgate.

Smallpox, Historical D A Henderson, University of Pittsburgh Medical Center, Baltimore, MD, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction History Smallpox, the Disease

Glossary case-fatality rate The number resulting from dividing the total number of deaths caused by a disease by the total number of cases of the disease. inoculation (sometimes called ‘variolation’) The introduction of pustular or scab material containing variola virus, the causative agent of smallpox, into the skin. This usually induced a localized infection that protected against natural smallpox but sometimes proved fatal. poxviruses Members of a large family of viruses, each of which tends to be specific to a particular species

Abbreviations HI

The Global Eradication Campaign Posteradication Further Reading

(e.g., canarypox, mousepox, and raccoonpox). Only four are known to infect humans: variola, vaccinia, cowpox, and monkeypox. vaccination The introduction of pustular or scab material containing vaccinia or cowpox virus into the skin. variola The word is used almost interchangeably with ‘smallpox’. It is derived from the Latin word ‘pock’ or ‘pustule’. The term ‘great pox’ was used to describe syphilis, the lesions of which are much larger.

WHO

World Health Organization

hemagglutinin-inhibiting

Defining Statement The history and nature of smallpox is summarized beginning in earliest historic times. A devastating disease, it remained uncontrolled until the discovery of a vaccine in the eighteenth century. Disease control was superseded by a global program of eradication which resulted in the last cases occurring in 1977.

Introduction Throughout history, until its eradication in 1980, smallpox was the most serious and feared of all the pestilential diseases with a history dating back to the time of the Pharaohs. It was a disease caused by the variola virus, which infected only humans. All countries with no regard to climate once experienced the disease. The severe form of smallpox, variola major, killed some 30% of its victims, but among the unusually susceptible Amerindian populations, case-fatality rates as great as 60–80% were recorded. There was no treatment for this disease.

Smallpox was transmitted directly from person to person. Those who recovered from the disease were immune from acquiring a second infection. The disease was marked by high fever and the development of a characteristic pustular rash. As the disease progressed, scabs formed over the pustules and eventually separated leaving permanent, deeply pitted scars, which were most prevalent over the face. Some victims were permanently blinded. Of all the infectious diseases, none had the capacity both to spread so widely and to inflict such a high mortality as did smallpox. In the eighteenth century England, it was widely recognized that milkmaids, in particular, seldom acquired smallpox and some attributed this to their having experienced a pustular infection of their hands resulting from contact with pustules on cows. As we now know, these infections were caused by the cowpox virus, which is closely related to variola. In 1796, Edward Jenner, a physician, demonstrated that this pustular material could be deliberately transferred from person to person by inoculation into the skin. He showed that individuals so treated were protected from acquiring smallpox when

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History and Culture, (and Biographies) | Smallpox, Historical

they were later challenged. This was the world’s first vaccine and its discovery is acknowledged as one of the most important in the history of medicine. As vaccine use increased and more potent and heatstable vaccines became available, smallpox steadily receded until, in 1967, with only 34 countries still infected, the World Health Organization (WHO) launched a global campaign to eradicate the disease. This proved to be a success. The last naturally occurring case was a Somali resident who became ill on 26 October 1977. In 1980, smallpox was declared to have been eradicated and vaccination ceased everywhere.

History Smallpox was an ancient disease and affected specifically humans. Those who became ill could transmit infection only during the 2- to 3-week period of acute illness; second attacks of smallpox were rare. For the virus to survive, it was necessary for it to infect one person after another in a continuing chain of transmission. Thus, to sustain the chain of infection, a moderately large population in reasonably close contact with each other was required. For these reasons, it is assumed that the disease must have arisen less than 10 000 years ago, a time when agricultural settlements were initially developing. Three Egyptian mummies from the 18th to 20th dynasties (1570–1100 BC), apparent victims of the disease, all show the characteristic pustules of acute smallpox. The subsequent spread of smallpox is difficult to trace because of the paucity of historical records that describe disease symptoms and epidemics in sufficient detail to identify them. Accounts of the disease in India suggest its existence there for almost as long a period as in Egypt. From India, it appears to have spread across China (about 250 BC) and to other parts of Asia. Periodic outbreaks occurred in Europe but not until the eighth century did smallpox became firmly established throughout the continent. By the seventeenth century, smallpox killed more than 400 000 people in Europe every year. The saga of smallpox in the Americas represents one of the most catastrophic, yet little known chapters in the history of mankind’s struggle against disease. In 1507, soon after contact with the New World, the Spaniards brought smallpox to Hispaniola. By 1541, the island’s population, estimated to have originally been between 300 000 and 1 000 000 persons, numbered only 500. Several diseases contributed to this disaster but smallpox was the main offender. In 1520, Cortes, with an army of 500, was sent to Mexico to capture additional slaves. Smallpox traveled with him and soon spread throughout Mexico and Middle America and eventually into the vast Incan Empire of the Andean Region. By the end of the sixteenth century, populations throughout Central and

South America were estimated to have decreased by 80–90%. North American Indians fared no better. In all parts of the world, smallpox was known and feared. Special deities were found among populations in many parts of Asia and Africa. However, in India, sometime early in the Christian era, an unusual practice was discovered which served to protect against fatal smallpox. It was called inoculation. Pustular or scab material was taken from a smallpox lesion and inoculated into the skin of a susceptible person. Smallpox infection induced in this manner rather than by the usual route of inhalation, usually resulted in a much less serious disease. Customarily, the recipient would experience fever and malaise and after 5–7 days would develop a pustular lesion at the inoculation site. Most persons recovered uneventfully and were thereafter immune to acquiring smallpox. However, the inoculated individual could transmit infection to others and, in some, a more severe illness occurred which was sometimes fatal. The practice of inoculation spread to China and Southwest Asia although it is uncertain as to what proportion were so treated. In 1721, the practice was introduced into England and later into other parts of Europe as well as North America. Although it was accepted in some areas, the practice was not enthusiastically embraced everywhere, in part because of the sometimes fatal outcomes and in part because of the occurrence of new smallpox outbreaks developing as a result of spread of the virus from inoculees. The concept of inoculation as a protective measure effectively established this technique as a common practice. Thus, Edward Jenner utilized a form of inoculation in his now famous experiment in which he took pustular material from a cowpox lesion on the hand of a dairymaid and inserted it into the arm of a young boy, James Phipps. The use of cowpox was suggested by local lore, which held that dairymaids did not acquire smallpox because of having had a cowpox infection. Jenner attempted to inoculate Phipps after some weeks with pustular material from a smallpox lesion and demonstrated that he could not be infected. He published this and other observations in 1798 and soon thereafter the cowpox vaccine (an orthopoxvirus, like smallpox) was shipped to countries around the world. Jenner’s discovery was acclaimed at the time and is recognized even now as one of the most important of all medical discoveries. However successful vaccination, its widespread application proved difficult. Until late in the nineteenth century, vaccination was conducted by the arm-to-arm transfer of the vaccine material. When a vaccination was unsuccessful and no other material was available, fresh material had to be obtained. Cowpox, however, was found only in Europe and occurred only sporadically. The reason for this, as we now know, is that the natural reservoir of cowpox is actually small rodents. Thus, although the virus was continuing to circulate, cows themselves only

History and Culture, (and Biographies) | Smallpox, Historical

occasionally acquired infection from the rodents but this was not known at the time. When efforts were made to ship new material, it often would deteriorate en route. Eventually, it was discovered that syphilis and hepatitis B could also be transferred by inoculation. Incidents occurred, in fact, in which cutaneous lesions of syphilis were thought to be vaccinial pustules and large-scale syphilis outbreaks resulted when the pustular material was transferred. Late in the nineteenth century, cowpox began to be grown on the scarified flank of a calf or sheep, thus assuring a more reliable and ample supply of vaccine. In the 1920s, French and Dutch scientists showed that a dried preparation could be produced which was stable at ambient tropical temperatures. However, methods for drying the vaccine frequently resulted in a final product that was heavily contaminated with bacteria. Finally, a commercial process for freeze-drying the vaccine, perfected in England in the early 1950s, opened the way for widespread vaccination throughout the world and eventually for the global eradication program.

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The causative agent of smallpox, variola virus, belongs to the genus Orthopoxvirus, subfamily Chordopoxvirinae, family Poxviridae. Three other members of this genus can also infect humans: monkeypox, cowpox, and vaccinia. However, none of the three are readily transmitted from person to person. The poxviruses are the largest and the most complex of all viruses. The virion is a brickshaped structure with a diameter of about 200 nm. Its lipoprotein outer membrane (envelope) encloses a single linear, double-stranded DNA. Replication of the poxviruses occurs in the cytoplasm. Unlike other DNA viruses, the poxviruses encode the dozens of enzymes required for transcription and replication of the viral genome. The viral core is released into the cytoplasm after fusion of the virion with the plasma membrane. Restriction endonuclease maps of the genome definitively identify the species of the Orthopoxvirus genus.

minor. This form of smallpox may have been present in some areas of the world before this time but few records are available which set forth numbers both of cases and deaths along with a reasonable description of the disease. Variola minor appeared in the United States at the beginning of the twentieth century and spread rapidly across the country eventually displacing variola major. From the United States, variola minor spread to Latin America and to Britain. Throughout Asia, variola major was the only type of smallpox seen, whereas in Africa, other variants of variola major occurred resulting in case-fatality rates of between 10 and 15%. Although epidemics of variola minor with the very low case-fatality rates were found in Ethiopia beginning in the mid-1970s, historical reports suggest that before this time, case-fatality rates more closely resembled those found elsewhere in Africa. Natural infection occurred by the implantation of variola virus on respiratory mucosa. After migration to and multiplication in regional lymph nodes, viremia developed followed by multiplication of virus in the spleen, bone marrow, and lymph nodes. A secondary viremia with fever and toxemia began about the 8th day. The virus, contained in leukocytes, then localized in small blood vessels of the dermis and mucosa. This process resulted in formation of the characteristic vesicular and pustular lesions. Hemagglutinin-inhibiting (HI) and neutralizing antibodies could be detected beginning about the 6th day of illness. Neutralizing antibodies were long lasting, whereas HI antibodies declined to low levels within 5 years. Little is known about the development of cell-mediated immunity. Vaccinia-induced antibody responses developed more rapidly, accounting for the fact that complete or partial protection of individuals was possible even among those vaccinated several days after exposure. Secondary bacterial infection was not common, and death, when it occurred, probably resulted from the toxemia associated with circulating immune complexes and soluble viral antigens. As the patient recovered, the scabs separated and the characteristic pitted scarring gradually developed. The scars were most evident over the face and resulted from the destruction of sebaceous glands followed by shrinking of granulation tissue and fibrosis.

Pathogenesis

Symptoms

Two distinct forms of smallpox have been observed over the years: variola major with a case-fatality rate of 20–30% and variola minor with a case-fatality rate of 1% or less. Variola major appears to have predominated throughout the world at least through the nineteenth century. A much less virulent form of the disease, usually called variola minor or alastrim, was first definitively identified in South Africa toward the end of the nineteenth century. The rash of variola major is indistinguishable from that of variola

After an incubation period of 7–12 days, the patient experienced a 2- to 5-day period of high fever, malaise, and prostration with headache and backache. Those with variola major were usually sufficiently ill to want to stay in bed. A maculopapular rash then began to develop at which time the patient first became contagious to others. The rash appeared first on the face and forearms and on the mucosa of the mouth and pharynx. It then spread to the trunk and legs. Within 1–2 days, the rash became vesicular and then

Smallpox, the Disease Etiology

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pustular. The pustules were characteristically rounded, tense, and deeply embedded in the dermis. Crusts began to form about the 8th or 9th day. When they separated, they left pigment-free skin and eventually scars. The eruption was characteristically more extensive on the face and distal parts of the arms and legs, and lesions were occasionally found on the palms and soles. Although the case-fatality rate among unvaccinated victims of variola major was usually about 30%, a more severe form accompanied by extensive hemorrhage occurred in about 1 case in 20 and was invariably fatal. Persons with variola minor usually experienced a less extensive rash and less prostration than with infection caused by variola minor but the rash was indistinguishable from that of variola major. A milder form of disease was also seen among those who had previously been vaccinated, the rash in such persons tended to be more scant and atypical and the evolution of lesions more rapid. Supportive therapy, maintenance of fluids and nutrition, and good nursing care were the best that could be offered to most patients. On the infrequent occasions when bacterial infections supervened, antibiotics were appropriate. Antiviral drugs, most notably methisazone and the arabinosides, were reported in some early studies to be effective in early treatment or prophylaxis, but confirmatory studies showed otherwise. More recently, cidofovir, an antiviral agent, has been shown to be effective in monkeys experimentally infected with monkeypox virus, but only if given within 24 h after infection and well before clinical symptoms develop. Given that vaccination performed even as late as 2–3 days after infection of the patient provides protection against development of clinical disease and because of the fact that cidofovir is highly toxic to the kidneys, the use of cidofovir, even on an experimental basis, is not advised. Laboratory Diagnosis Diagnosis of a poxvirus infection is confirmed rapidly by electron microscopic identification of virus particles in vesicular or pustular fluid or scabs. All orthopoxvirus virions have the same appearance. The four orthopoxviruses, which infect humans, grow and produce a cytoplasmic effect in cultured cells derived from many species but they cannot usually be differentiated from each other in most preparations. For diagnostic purposes, PCR techniques are now widely available which readily identify the different orthopoxvirus species, each of which has a distinctive DNA map.

The Global Eradication Campaign A global campaign for the eradication of smallpox was proposed by the Soviet Union in 1958 and agreed by the

World Health Assembly. However, over the succeeding 8 years, little progress was made. At the time, WHO and many of the smallpox-endemic countries were fully preoccupied with a global malaria eradication program which had begun in 1955 and which consumed a substantial proportion of the WHO’s budget, personnel, and time. As the years passed, many began to express skepticism that malaria or, for that matter, any disease could actually be eradicated. Soviet Union delegates, joined by others, expressed increasing displeasure that the WHO did not assign a higher priority to the smallpox effort, and demanded finally that steps be taken to work out a definitive plan and cost estimates for an intensified program. Meanwhile, in recognition of International Cooperation Year, the United States, in November 1965, announced its intention to provide support to 18 countries of Western and Central Africa in an effort to eradicate smallpox and control measles. At the 1966 World Health Assembly, the United States joined the Soviet Union in arguing for the development of a greatly intensified global eradication campaign. Some countries, as well as the Director General of WHO, opposed the concept of smallpox eradication as being technically impossible; others objected to increasing the WHO budget by $2.5 million per year to provide a portion of the needed financial support for the effort. Eventually, by the margin of only two votes, the decision was taken to intensify the eradication effort. Assembly delegates proposed a 10-year target. In 1967, the year the program began, 46 countries reported smallpox of which 33 were considered to have endemic disease while 13 recorded imported cases. Estimates later showed that some 10–15 million cases and 2 million deaths occurred the same year. The basic strategy for the program was twofold: population-wide vaccination and a surveillance-containment program. These were to be initiated simultaneously at the outset of each program. The vaccination program was intended to reach 80% of the population in each of the endemic countries and in neighboring countries at risk of imported cases. Those directing the program reasoned that a substantial level of vaccination immunity would sufficiently diminish the rapidity of virus spread so as to permit the surveillance-containment program to be fully effective. The latter program required that a reporting system be developed such that each hospital and health center would report each week as to the number of smallpox cases diagnosed. When cases were reported, a surveillance team would go to the field, seek to find other cases, and vaccinate household and village contacts in hopes of stopping the chain of transmission of infection. The strategy of surveillance and containment was soon discovered to be far more effective than was expected based on the conventional wisdom of the time which held that smallpox spread rapidly and widely. Through

History and Culture, (and Biographies) | Smallpox, Historical

field epidemiology, it soon became clear that a given patient seldom infected more than two to five others, usually household members or close friends and they normally did not develop illness until 7–12 days after exposure. Thus, smallpox usually spread comparatively slowly and cases tended to cluster within cities and within geographic areas. This greatly facilitated the containment activities. Many observations of importance gradually shaped the program. It was found that with good planning and involvement of community resources, vaccinators could regularly average 500 or more vaccinations per day and vaccination coverage of more than 90% was to be expected. In contrast, vaccination coverage that relied solely on clinics and hospitals for dispensing vaccine seldom exceeded 60%. Quality control was essential. To assure performance in vaccination, independent assessment teams checked a sample of villages vaccinated by the mass vaccination teams to assure that both coverage and vaccine efficacy standards were met. Where they were not, the entire area was revaccinated. The supply of vaccine was a special problem. As the program began, it was discovered that not more than 10% of the vaccine then in routine use met accepted international standards. Smallpox vaccine was then being produced in more than 40 different laboratories scattered throughout the world. Few undertook to perform routine quality controls of their product. Given the fact that the WHO allocation for smallpox eradication was too meager to purchase required vaccine from established laboratories, the decision was made to foster improvement in vaccine quality in the existing production facilities. Two laboratories, one in the Netherlands and the other in Canada, undertook to perform independent testing for all vaccine to be used in the program. WHO convened a panel of expert consultants from the major production laboratories to develop a standard manual of procedures. Many consultants then traveled to the various laboratories for purposes of training and evaluation. Remarkably, within 4 years, all vaccine met international requirements and more than 80% of the vaccine was able to be produced in the endemic countries themselves. Research made important contributions. A new method for vaccination resulted in more successful vaccinations utilizing much less vaccine. In 1967, a new vaccination needle (the so-called bifurcated needle) was in the late experimental stages at Wyeth Laboratories in the United States. With Wyeth approval, WHO undertook additional studies with the needle, altered the technique for its use so that it was given by multiple punctures, and introduced its use worldwide. Using the needle, only one-fourth as much vaccine was required as was needed using the standard techniques. Training of vaccinators could be done within a matter of hours and

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the result was a higher proportion of successful takes. The needles were fabricated from stainless steel and could be boiled or flamed and reused many times. A second important finding derived from field epidemiological studies, which revealed that the protection afforded by primary vaccination was not the 3–5 years that most textbooks asserted but, rather, showed a 90% efficacy rate at 20 years. With this discovery, it was possible to focus efforts on assuring that every person had a vaccination scar rather than assuring that everyone had been vaccinated within the preceding few years. Rapid progress was made in the campaign so that by 1973, smallpox was largely confined to four countries of South Asia and, in Africa, to Ethiopia. However, in Asia, efforts such as had been successful in the Americas and in Africa proved far less efficacious. The problem was that with a more extensive infrastructure for road and rail travel in Asia, people traveled frequently and over considerable distances, often returning as a family to a home village when one of the families became ill. The result was that smallpox spread more rapidly than in other countries and over longer distances. In the summer of 1973, it was decided with Indian Government authorities to assign large numbers of health personnel to undertake a 10-day search throughout all the villages, towns, and cities of the country. It was hoped that this would permit cases to be found much earlier so that surveillance teams could move rapidly to vaccinate household and village contacts and so prevent further spread of the disease. The first search found tens of thousands of previously unreported cases, many more than had been expected, but early containment was then possible. Every few months the searches were repeated with increasing precision, eventually reaching every household in every village in India. Less than 2 years elapsed before India and all of Asia became smallpox-free. As the numbers of cases decreased, it became the policy to offer a small reward to anyone reporting a case that proved to be smallpox. As the cases became fewer, the reward was increased so that by the end of the program, a reward of $1000 was being offered for the report of a case that could be confirmed as smallpox. This technique proved to be exceedingly valuable in the closing phases of the program in which each country had to demonstrate to an independent commission that it had a surveillance network that had been functioning effectively for at least 2 years after an endemic area was free of smallpox and that surveillance was sufficiently sensitive to detect cases if present. The last naturally occurring case of smallpox occurred on 26 October 1977 in Somalia, albeit two further cases occurred, both in Birmingham, England, in 1978, as the result of a laboratory accident. As smallpox incidence declined, an independent international commission was created to review the work of the individual national

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commissions and to define additional studies and data that it required to assure itself that eradication had been achieved. The Committee met in December 1979 and, at that time, pronounced that it was satisfied that eradication had been achieved. The Chairman of the Committee reported its findings to the Director General and the World Health Assembly in May 1980. The Assembly, in a specially convened session, passed a resolution indicating that eradication had been achieved and recommended that all countries cease vaccination.

replace smallpox as a dangerous contagious disease. The reports in 1997 of large numbers of human monkeypox cases were widely publicized and caused alarm. However, as investigations progressed, it became clear that a large proportion of the cases were chickenpox and that there was little evidence to suggest that the virus had changed its character. However, field studies could not be pursued because of civil strife until very recently. As of 2007, intensive surveillance for cases throughout a large area in the Democratic Republic of the Congo have been resumed but evidence of more frequent human-to-human spread has not been detected.

Posteradication A Possible Natural Reservoir of Variola Virus From the inception of the program, eradication program staff had been concerned about the possibility, however remote it may have seemed, that there might be an unsuspected natural reservoir of smallpox. Not to be forgotten was the fact that a yellow fever eradication program had to be abandoned in 1932 when it was discovered that there was an unsuspected jungle reservoir. Thus, special efforts were made throughout the course of the eradication program to identify the source of all smallpox cases that were reported from countries that were believed to be free of the disease. It was believed that, if there were a natural reservoir of some sort or if it were possible for people to be infected from scabs or other material still remaining in the environment, smallpox cases would be found for whom the source of infection was not apparent. No cases of this type were found. Monkeypox, a Disease in Man Similar to Smallpox Monkeypox is caused by a virus that is a member of the Orthopoxvirus genus and so is closely related to smallpox. In man, it resembles smallpox clinically and is associated with a case-fatality rate of 10–15%, similar to that of smallpox in central African countries. Initially, there was concern that it might adapt to spread in humans. Human monkeypox cases were first detected in 1970 and studied intensively by WHO teams throughout the 1980s. They documented the fact that the natural monkeypox virus reservoir was actually ground squirrels and that man only infrequently became infected. However, the teams discovered that the virus could be passed from person to person although it was much less contagious than was smallpox. Moreover, the studies indicated that it was highly unlikely that the virus could continue to spread even in populations that had never been vaccinated. During 1997–98, reports of large outbreaks of human monkeypox cases in the Democratic Republic of the Congo raised questions as to whether monkeypox might be spreading so readily from person to person as to

Destruction of Remaining Smallpox Virus Stocks Following the 1980 declaration of smallpox eradication, a WHO expert committee was constituted to consider additional steps which might be taken to assure confidence in the fact of eradication and to advise regarding the continuation of a vaccine stockpile among other matters. One important question was to decide whether the known remaining stocks of variola virus should be destroyed. As a result of WHO recommendations, 74 of 76 laboratories known to have stocks of variola in 1980, had destroyed their stocks or sent them to one of two WHO reference laboratories, located, respectively, in Moscow, Russia, and in Atlanta, Georgia. At meetings in 1986 and 1990, the expert committee affirmed the desirability of destroying the variola stocks but advised that this be deferred until virus strains could be appropriately mapped, cloned, and sequenced. By 1994, this work had been completed and endorsements supporting the destruction of the virus strains had been obtained from five prominent scientific bodies including the International Union of Microbiological Societies, the Russian Academy of Medical Sciences, and the board of scientific counselors of the US National Center for Infectious Diseases. At its 1996 meeting, the World Health Assembly voted to accept the recommendations of the expert committee and proposed that the virus strains at the two laboratories be destroyed in June 1999. However, at subsequent meetings of the World Health Assembly, the last in 2006, several countries argued that it was important to retain the virus so as to permit studies to be undertaken to develop a more attenuated vaccine than that which had been routinely used and to develop antiviral drugs that would be suitable for treating smallpox cases should cases recur. On each occasion that a postponement in destruction has been agreed upon by the Assembly, it has added the provision that research studies using smallpox virus must be approved in advance by a designated WHO Committee and reported to the Assembly and that each

History and Culture, (and Biographies) | Smallpox, Historical

of the two laboratories be visited by WHO-appointed experts to provide independent reassurance to all member countries that adequate measures were being taken to preclude smallpox virus from being released. The rational for retaining the virus has continued to be viewed with skepticism by the majority of countries and many scientists who believe that the argument for virus destruction is a compelling one as it would make it less likely that the virus would be released either by accident or for malicious purposes.

Smallpox as a Terrorist Weapon There is increasing concern that biological weapons might be deployed by terrorists either acting as a dissident group or under state sponsorship. Until the early 1990s, biological weapons had not been considered to be a threat. The primary reason was that all countries had agreed in 1975 to abide by a Biological Weapons Convention which required all countries to cease work on organisms for use as biological weapons and to destroy such stocks as they possessed. Smallpox, in any case, was considered an unlikely agent because of the high level of population immunity, the availability of a vaccine, and the knowledge that vaccination of contacts can rapidly and effectively control epidemics. Thus, vaccination programs ceased in 1980, all smallpox vaccine production stopped, and the facilities for producing vaccine were converted into other uses. During the 1990s, it became known that the Soviet Union, ever since the Biological Weapons Convention had come into force, had been engaged in a massive research effort to develop biological weapons, to develop new mechanisms for their dispersal, and to produce them in large quantity. Such information was provided by a highranking defector from the program. President Yeltsin later confirmed publicly that the program had indeed existed. He took measures to close the civilian component of the program but the status of the military component is unknown. In all, more than 50 000 persons were involved in more than 50 of the most advanced biological laboratories. Smallpox virus was considered to be among the most effective of the possible biological weapons and, of the smallpox strains, one

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that had been recovered in India in 1966 was considered to be the most pathogenic. Special studies were undertaken to determine the possibility of developing recombinants of smallpox with Venezuelan equine encephalitis virus and with Ebola virus, and a major smallpox virus production unit was built that was capable of producing tons of smallpox virus. Subsequent to the break-up of the Soviet Union, many of the former biological weapons laboratories have had to sharply reduce the sizes of their staffs. Some of those previously engaged have emigrated to Western countries and some are believed to have consulted and worked in laboratories in countries that are involved in fostering terrorism. It is unknown whether some or any have carried their knowledge or strains of smallpox or other agents elsewhere. Because of these factors, smallpox vaccine production has now commenced in at least four laboratories; a number of countries now have stockpiles of vaccine; a WHO reserve stockpile is in process of being augmented; new diagnostic tests for smallpox have been developed and distributed; and special teams have been trained for immediate deployment should an outbreak be suspected. See also: Biological Warfare; Epidemiological Concepts and Historical Examples; Evolution, Viral; Smallpox, Historical; Surveillance of Infectious Diseases

Further Reading Alibek K and Handelman S (1999) Biohazard. New York: Random House. Breman JG and Henderson DA (1998) Poxvirus dilemmas – monkeypox, smallpox, and biologic terrorism. The New England Journal of Medicine 339: 556–559. Fenner F, Henderson DA, Arita I, Jezek Z, and Ladnyi ID (1988) Smallpox and its Eradication. Geneva: WHO. Henderson DA and Borio LL (2007) Smallpox and vaccinia. In: Plotkin SA and Orenstein WA (eds.) Vaccines. Philadelphia: Saunders. Hopkins DR (2002) The Greatest Killer: Smallpox in History. Chicago: University of Chicago Press. Jezek Z and Fenner F (1988) Human monkeypox. . In: Melnick JL (ed.) Monographs in Virology, vol. 17. Basel, Switzerland: Karger. Tucker JB (2001) Scourge; the Once and Future Threat of Smallpox. New York: Atlantic Monthly Press.

Spontaneous Generation J Strick, Franklin and Marshall College, Lancaster, PA, USA ª 2009 Elsevier Inc. All rights reserved.

Introduction Early History The Needham/Spallanzani Controversy Worms, Molecules, and Evolution The Role of Louis Pasteur

Glossary abiogenesis Term used in 1870 by Huxley to mean life arising from a combination of inorganic starting materials. Since that time, has come to be used more widely to indicate origin of life from any nonliving matter. archebiosis Term coined by Bastian in 1870 to mean life arising from strictly inorganic starting materials. biogenesis Term used in 1870 by Huxley to mean life arising only from other living things, the opposite of which he termed abiogenesis. The term seems to have been first coined by Bastian earlier that year, but to mean exactly the opposite: spontaneous generation. It is Huxley’s usage that became famous and remains the definition of this term today. heterogenesis Nineteenth century term used to mean the origin of living things from organic materials, for example, from infusions of plant or animal matter, or from decaying tissue in a diseased or dying organism. Thought to be the source of tumors, parasitic worms, microorganisms from putrefaction, as these organized themselves from the smallest microscopically visible particles (molecules) of organic tissues. Note that many supporters of heterogenesis might not support the more extreme position of archebiosis/abiogenesis from inorganic matter.

Introduction The idea that living things can originate from nonliving materials, spontaneous generation, has a long history, inseparably intertwined until about 1880 with the development of microbiology as a science. No less an authority than Aristotle claimed that cases of spontaneous generation could be observed in nature, and his support was important in establishing the idea for many centuries. Beginning around the time of the Scientific Revolution of the seventeenth century, however, the doctrine of

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The British Debate of the 1870s: Darwin, Spontaneous Generation, and the Germ Theory of Disease Twentieth Century Ideas Further Reading

molecules Term used from the writings of Buffon (c.1750) onward to indicate the tiniest (bacteria-sized) microscopically visible particles of organic matter in blood, tissue, infusions, and so on. This term was used by histologists, pathologists, and cytologists until the 1870s, obviously in a way quite different from the chemists’ use of the term that developed through the same time period. Important examples include the ‘active molecules’ of Robert Brown (1828–29), the cellforming cytoblastema ‘molecules’ of Theodor Schwann (1839), and the histological ‘molecules’ of John Hughes Bennett (1840–75). Many thought clumping together of these units was the basis for heterogenesis. pleomorphism The doctrine that microorganisms did not come in distinct, stable Linnean species but rather were highly variable in shape and metabolic capabilities in response to changes in their environment. Many believed this to be true of the bacteria, especially in Britain and Germany through the 1880s. More extreme versions held that bacteria, yeasts, molds, and algae were all interconvertible stages in the life cycle of a single organism. Heterogenesis can be seen as the next logical step, including the transition from nonliving organic matter to the simplest bacterial forms as but one more stage in that fluid transformability.

spontaneous generation was increasingly challenged and became the subject of numerous episodes of controversy. As combatants tried to answer one another’s criticisms, the new breakthroughs in technique and in experimental design that were developed served as some of the most important foundations upon which a science of microbiology could be built. As just one example, the development of a sterile technique as well as procedures to sterilize glassware and growth media all grew directly out of experiments trying to prove or disprove the possibility of spontaneous generation of microorganisms.

History and Culture, (and Biographies) | Spontaneous Generation

In seeking to answer a question as basic as how life originates, theory played a role as important or more so than technique or experiment. From the first, the doctrine of spontaneous generation was seen to be fraught with religious implications. If life could originate spontaneously from lifeless matter, the position of philosophical materialism, then a Creator God was irrelevant. If spontaneous generation occurred in present times, this was at odds with a single original Creation as told in the Bible. However, for those interested in a naturalistic worldview, such as supporters of Darwinian evolution, there was also a potential conflict. The doctrine of evolution was based on a profound philosophical assumption of continuity in nature, that is, that there were no sudden unbridgeable gaps between similar living forms, which would require supernatural intervention. Furthermore, Darwin’s theory implied that the vast diversity of living things had come from one or at most a few original ancestral organisms, and these must have originated somehow. For many, then, to believe in evolution and a completely naturalistic worldview required the belief that no unbridgeable gap occurred between living matter and nonliving matter and that living organisms must have been capable of arising from nonlife, at least once on the early earth. Those with this view, for example, Henry Charlton Bastian, challenged hypocrisy on the part of those who supported Darwin but were unwilling to believe in the necessity of spontaneous generation. Still another important source of disagreement, even among scientists who claimed not to care about religious implications, was fundamental epistemological assumptions about the nature of life. Some believed very deeply that all living things must reproduce by ‘seeds’ or ‘germs’, by analogy with the large number of organisms for which this process had been observed. So fundamental was this belief that, even in cases where microscopic life appeared in tubes of fluid boiled for hours, those scientists concluded that either the tubes must have leaked after boiling or there must be some kind of structures produced by microorganisms that were capable of withstanding previously unheard of temperatures, even though nobody had ever seen such structures. Their opponents, as late as the 1870s, believed that the most clearly observed and reliably established fact known about living things was that they were totally unable to survive the boiling temperature of water for more than a few minutes. From this equally tenable premise, they concluded that spontaneous generation was a less strained explanation of organisms in boiled infusions than the ad hoc invoking of ‘spores’ that nobody had ever experimentally demonstrated with totally unprecedented heat-resisting abilities. Thus, before ever carrying out an experiment, both sides in the spontaneous generation controversies were often begging the question at issue. Clearly, these philosophical issues were crucial points showing how experimenters

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could disagree because they were talking past one another, more than because of differences in the experiments themselves. The doctrine of spontaneous generation rose and fell in popularity repeatedly at different times in different countries in the past several centuries, and to tell the story of spontaneous generation controversies only as a series of battles about ‘dueling experiments’ would be to misunderstand much or even most of what the controversies were really about.

Early History From antiquity it had been maintained that frogs, eels, mice, and numerous worms and insects, especially parasitic worms living inside animals, could arise by spontaneous generation, mostly heterogenesis. With Leeuwenhoek’s discovery of microorganisms, many naturalists assumed that these too could arise without parents. Indeed, as microbes seemed exceedingly simple, and bacteria simplest of all, it was believed that they were the most likely to be organizable from nonliving materials. Francesco Redi, natural philosopher to the same Tuscan court that was patron to Galileo, carried out some famous experiments in 1668 that investigated specifically the origin of insects. Redi’s experiments showing that maggots come from fly eggs, not from rotting meat, usually led off histories of spontaneous generation debates. It was commonly believed until that time that the appearance of maggots in rotting meat was a clear example of spontaneous generation. Redi placed samples of many different types of meat and fish in glass jars. One set of jars was open to the air and soon swarmed with maggots. The other set was covered with fine muslin cloth. Redi saw that, while maggots never appeared in the meat of those jars, flies crawled about on the cloth and sometimes laid eggs there. Those eggs were seen to hatch into maggots, disproving spontaneous generation as their origin. Though Redi himself continued to believe that some insects, such as gall flies, did arise by spontaneous generation, many naturalists assumed from this time onward that spontaneous generation, if it occurred, only did so among parasitic worms and microorganisms. Of course, working at the time of the Scientific Revolution, Redi did participate at a time when experiment came to the fore as an activity ever afterward central in natural philosophy. Spontaneous generation debates, like all other natural philosophic questions, featured experiments more and more prominently from the late seventeenth century onward. Just as ‘natural history’ or ‘natural philosophy’ historically did not mean what is now called ‘science’ until fairly recently, neither did ‘experiment’ always mean what it means now. Many natural philosophers in the seventeenth century were interested in the power of the experimental method, but were just as

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interested in public demonstrations of ‘experiments’ as a way of convincing audiences, for example, prospective patrons, that their enterprise was qualitatively different from the book-dominated natural philosophy of the past, and from the subjective and often bloody religious disputes that had traumatized European life for over a century. Recent studies have looked closely at Redi’s career, especially his relationship with the Medici Grand Dukes, his principal patrons. One such study concludes that Redi’s public demonstration of experiments was a form of court entertainment in which the final arbiter of the meaning and success of the outcome was the Grand Duke. Thus there was a world of difference between Redi’s procedures and what would by the nineteenth century be called ‘controlled experimentation by a biologist’. He set high value on repeated observation and demonstration, saying, ‘‘I do not put much faith in matters not made clear to me by experiment.’’ Paradoxically, however, he suggested elsewhere that he experimented ‘‘in order to make myself more certain of that of which I am already most certain.’’ For Redi, then, especially in his primary role as an early modern courtier, ‘experiment’ meant something quite different from what we understand by that term today, particularly with regard to the role of ‘preconceived expectations’. The paradox results only if we assume in a naively ahistorical way that what Redi called ‘experiment’ should be assumed to have the same meaning that term came to have after ‘natural philosophy’ had been transformed into ‘science’. Once we gain this richer sense of what experiment meant in the historical context in which Redi lived and worked, we may begin to question the validity of an ahistorical narrative that uncritically compares his work with experiments performed by Pasteur, Tyndall, and their antagonists two centuries later, merely because of superficial similarity in ‘the use of controls’. Perhaps there are more similarities in the meaning of ‘experiment’ in such different historical settings, but this is an open question that can only be answered by looking at the full context in which each investigator had to operate, including such questions as the existing system of patronage upon which each depended to support work on scientific pursuits. Such points of comparison will be developed further when discussing Pasteur’s work.

The Needham/Spallanzani Controversy Eighty years after Redi, in 1748, another series of experiments by the Irish priest John Turberville Needham was published, and this time was widely believed to support the spontaneous generation of microorganisms. Needham soon collaborated with the French aristocrat Comte de Buffon. And over the next several decades the work of

Buffon and Needham was opposed by many, including Charles Bonnet and Lazzaro Spallanzani. Needham’s first experiments found that, when mutton gravy was stoppered and sealed with mastic in glass tubes and heated to boiling for a time, the fluid afterward teemed with microorganisms (protists). Buffon and Needham later reported seeing tiny particles (in the size range of bacteria) that they called ‘organic molecules’, which came from disintegrating organic material and moved very actively. These, they said, clumped together to form the larger animalcules (protists). They also claimed that sealing and boiling proved that the molecules did not originate from ‘‘insects or eggs floating in the atmosphere.’’ Most naturalists considered this, if true, to be a case of spontaneous generation. Spallanzani, an Italian cleric, carried out experiments challenging these claims and first published his results in 1765. His opposition was based on two main arguments. First, he said that with his own microscope he never saw anything like the ‘organic molecules’ and therefore charged that Buffon and Needham were only seeing what they wanted to see, because Buffon’s philosophy of nature had predicted that such particles must exist. This claim was widely repeated. Second, he said that when he boiled infusions similar to Needham’s, he did not find any living microbes in them, as long as he sealed the tubes by melting the glass shut in a flame before boiling and continued the boiling for at least an hour. Thus, Spallanzani claimed that the microorganisms in Needham’s infusions must indeed have gotten in, despite Needham’s precautions, from the atmosphere after boiling. Needham responded that boiling for as much as an hour would clearly be so severe a treatment as to deprive the air in the tubes of its power to generate or support life. Later experiments showing that indeed such treatment decreased or consumed the oxygen content in the tubes seemed to support Needham’s claim. Thus the experimental outcome was underdetermined by the experiments themselves, so the controversy was able to continue through the 1780s. A popular notion of experiment has it that ‘proper science’ can only occur when the scientist approaches his experiment with no ‘preconceived ideas’ about the outcome, and intends to ‘‘let the chips fall where they may.’’ In many histories of the spontaneous generation debates, the ‘losers’ are often accused of having been biased beforehand by their belief in a ‘vital force’ or some similar overarching philosophy, while Redi, Spallanzani, Pasteur, and Tyndall epitomize the openminded investigator. A closely related claim has been that Spallanzani, Pasteur, and others were often able to defeat their foes by virtue of possessing better instruments, especially superior-quality microscopes. Thus, as the story has been told since the early nineteenth century,

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the Comte de Buffon and his collaborator Needham in the 1740s were portrayed as ‘armchair philosophers’ who cooked up a doctrine of ‘organic molecules’, a vital ‘plastic force’, and spontaneous generation; and they had such an inferior microscope that they were able to interpret whatever fuzzy images they saw as supporting the fuzzy ideas they wanted the data to confirm. Victorian biologist T. H. Huxley’s version established one of his most famous cliche´s by describing Buffon and Needham’s work as an object lesson for generations of young scientists: Such as it was, I think it [Buffon and Needham’s doctrine] will appear . . . to be a most ingenious and suggestive speculation. But the great tragedy of Science – the slaying of a beautiful hypothesis by an ugly fact – which is so constantly being enacted under the eyes of philosophers, was played almost immediately, for the benefit of Buffon and Needham. Huxley (1870) Biogenesis and Abiogenesis. In: Discourses Biological and Geological, Essays New York: Appleton, 1925, pp. 232–274, 239

Huxley argued, based perhaps on Pasteur’s similar claim, that Buffon and Needham had a poor microscope and their opponent Spallanzani had a much more objective outlook and understanding of the method of controlled experiment. Though seldom emphasized, it is very important to note that spontaneous generation was seen to be directly supportive of two other broader doctrines: epigenesis and materialism. Epigenesis is the doctrine that all the parts of an embryo are assembled gradually, having at first been nonexistent. Epigenesis was opposed in the seventeenth and eighteenth centuries by the doctrine of preformation, which claimed that all embryonic organs existed inside the sperm or the egg in miniature from even before fertilization and only needed to grow, unfold, and expand as gestation proceeded. This implied logically that all the generations of, for example, humanity must have been contained, each within the previous one like Russian dolls, all the way back to the eggs in Eve’s ovaries or the sperm of Adam. (The overwhelming majority of naturalists writing during this period were Christian.) Since Buffon and Needham’s description of the origin of protist microbes suggested their organization from originally homogeneous ‘molecules’, this seemed a blow for preformationists. Buffon indeed ridiculed preformation theory, insisting that the microscope showed no trace of these successively enclosed generations within the sperm or eggs of animals. Spallanzani eventually came down on the side of preformation and rejected Needham’s theory. We would probably admit today that, since his microscope could not have shown him little preformed homunculi inside eggs or sperm cells, Spallanzani, too, must have been willing to go some way in believing in

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things he could not verify and was thus also operating in an atmosphere of ‘philosophizing’. More than that, if Buffon and Needham’s observations were correct, they showed that matter contained within itself all the properties necessary to organize into life. This was the basic tenet of philosophical materialism, a doctrine profoundly at odds with Christianity or any kind of Deism. Voltaire, among others, feared that Needham’s claims would support atheism and materialism. It was thought by many that this theory implied that life could originate by a chance, random combination of substances. This was so contrary to mainstream religious beliefs, and to a natural philosophy still very much in the service of demonstrating the existence of a beneficent Creator, that it generated heated opposition. Further, this led to spontaneous generation becoming strongly associated, beginning around 1750–70, with atheism, materialism, and political radicalism. (Since this chance combination of chemicals has become such a crucial element of modern views of the origin of life, such as the famous 1953 Urey–Miller experiment, it is interesting that Buffon and Needham are not celebrated as thinkers far ahead of the religious biases of their time. Voltaire seems to have been one of those who most actively spread the opinion that Needham and Buffon were poor scientists, though this was based on seriously misreading Needham.) Only recently for the first time has close study been applied to the actual technical evidence on what kind of microscopes were being used by Spallanzani and by Needham. This work has shown that Buffon and Needham’s results cannot be successfully explained by assuming that they worked with poor instruments, nor that their work was sloppy or biased in advance by a priori theorizing, despite the fact that these explanations are ubiquitous among previous accounts of the controversy. Indeed, a close review of the evidence on the actual experiments suggests that the experiments of Needham and Buffon were careful, high-quality work with quite a bit of sophisticated hypothesis formation and testing. Many of the contemporary critics of Buffon and Needham, including Spallanzani in the 1760s, had originated this claim of a priori bias, at least partly because they assumed the pair to have worked with a British Cuff compound microscope, common at that time (Figure 1). This type of device had a maximum magnification of only about 100, and even at that low power the image it produced suffered from severe chromatic and spherical aberration, as indeed was a problem with all compound microscopes prior to the achromatic lenses not perfected until 60 years later. By careful analysis of the original publications, however, it has been shown that the instrument used by Needham was actually a high-quality single lens microscope of the

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Figure 1 Plate from Buffon’s Histoire naturelle depicting an eighteenth century scientist using the compound Cuff microscope. Perhaps because of the illustration, many assumed that Buffon and Needham had used this instrument to make their observations, when in fact they had used the much superior Wilson screw-barrel design (see Figure 2). Compound microscopes of that era suffered from severe chromatic and spherical aberration, and the Cuff microscope had a maximum magnification of only about 100. Courtesy of Beinecke Rare Book and Manuscripts Library, Yale University.

Wilson screw-barrel design, capable of at least 400 magnification with outstanding resolution, that is, not plagued by the aberrations of compound scopes (Figure 2). This was similar to the instrument with which Robert Brown discovered the cell nucleus in plant cells and Brownian movement in pollen particles in the 1820s and 1830s. In fact, Buffon and Needham’s observations anticipate those that led to the discovery of Brownian movement, their ‘organic molecules’ being what Brown called ‘active molecules’. The simple microscope employed in the famous experiments by Spallanzani was a variant of the Lyonnet aquatic microscope. Spallanzani’s instrument was incapable of the short focal length, high-resolution work permitted by the Wilson screw-barrel design, so that he could not

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Figure 2 Single-lens Wilson screw-barrel microscope used by Buffon to make his observations in support of his interpretation of the theory of spontaneous generation. In this theory, smallbacterium-sized particles, which Buffon called ‘organic molecules’, clumped together to form living ‘animalcules’. Courtesy of Beinecke Rare Book and Manuscripts Library, Yale University.

even see the bacteria-sized ‘organic molecules’ under dispute. Thus the truth of this controversy appears to be that Spallanzani was the technically handicapped participant, though exactly the opposite story has become universal. Buffon and Needham’s discovery of ‘organic molecules’ was not the work of imagination but an actual discovery of active molecules and of Brownian movement, some 80 years before Brown. This explains why Buffon and Needham refused to back down from their original observations despite accusations of atheism and decades of controversy. Furthermore, both parties seem to have had plenty of a priori philosophical commitments at stake, and nobody approached as explosive a subject as materialism with a completely open mind.

History and Culture, (and Biographies) | Spontaneous Generation

Worms, Molecules, and Evolution The appearance of parasitic worms within animals was long seen, until perhaps the 1840s, as the strongest single piece of evidence supporting spontaneous generation claims. The complex life cycles of most of these parasites involved intermediate life cycle stages that had to live in other host animals. Thus, egg-feeding experiments would not have been able to show eggs from one animal directly producing worms in another. Only in the 1840s and 1850s, with the working out of the complex life cycles and intermediate hosts, did it finally become unambiguously clear that the worms reproduced via eggs. Despite the denunciation of Buffon and Needham, a great many pathologists, histologists, and, later, cytologists continued to observe bacteria-sized, actively moving particles in blood, tissues, and infusions. These particles continued to be called ‘molecules’ by life scientists. They were using the term, obviously, in a way quite different from the chemists’ use of the term that developed through the same time period. Important examples include the ‘active molecules’ of Robert Brown (1828–29), the cellforming cytoblastema ‘molecules’ of Theodor Schwann (1839), and the histological ‘molecules’ of John Hughes Bennett (1840–75). Many thought clumping together of these units was the basis for heterogenesis, and that this furthermore supported epigenesis as the correct explanation of embryonic development. Especially vocal in this belief were Lorenz Oken, Karl Burdach, and other German biologists of the Naturphilosophie school. (It should be emphasized that neither Brown nor Schwann themselves interpreted their ‘molecules’ in this way, and Schwann did some significant experiments early in his career that seemed to disprove certain cases of spontaneous generation.) In Britain, both Charles Darwin and Richard Owen worked in private on the molecule theory during the 1830s, as did William Addison in the 1840s. More importantly, the British histologist Hughes Bennett was one of the first professors to teach a physiology course in a university and medical school, and he taught from 1840 until almost 1870. His theory of ‘molecules’ as the essential units from which cells formed, explicitly embracing heterogenesis by the late 1860s, was thus widely influential among British scientific and medical circles for over a generation. J. B. Lamarck, the early French evolutionist, had made spontaneous generation of microorganisms an integral part of his evolution theory from its beginning in 1800. Lamarckian supporters were numerous and widespread, though often associated with radical politics, as selforganization and development of matter was seen as a crucial scientific underpinning for democratic, bottom-up organizing political theories. A few such Lamarckians did rise to university teaching posts, however, and notable

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among them was the outspoken British comparative anatomy professor Robert Grant. Though considered not quite respectable in Victorian intellectual circles, his teaching of evolution and spontaneous generation continued from the 1820s through his death in 1874, and thus gave those ideas a presence in British thought decades before On the Origin of Species appeared. Charles Darwin, for instance, was one of Grant’s earliest students and prote´ge´s (though Darwin later felt he had to avoid any contact with the less-than-respectable Grant when he was developing his own evolution theory in secret for 20 years). Grant and other Lamarckians had established in the public mind a strong link between evolutionary theories and spontaneous generation.

The Role of Louis Pasteur As Louis Pasteur’s experiments on spontaneous generation, particularly his ‘swan-necked flasks’, are among the most famous of all, the Pasteur–Pouchet debate of the 1860s in France deserves close attention. It is also worth noting that the Historical Introduction to Pasteur’s famous 1862 Memoir on the subject has served as the model upon which almost all subsequent histories of the controversy were based for over a 100 years . This has led to the casting of the controversy as a series of what I call ‘dueling experiments’, and has often stripped it of its crucial philosophical context. Pasteur also chose to leave out an entire chapter of the story: the argument that parasitic worms must have arisen by spontaneous generation. So dominant has Pasteur’s master narrative been that it is only with historical detective work in the early 1970s that this omission of Pasteur’s was finally restored to its place of importance in the story. Between 1860 and 1862 Pasteur carried out a lengthy series of experiments focusing exclusively on the development of microbes in previously boiled infusions, in response to the experiments in favor of heterogenesis published by Felix Pouchet. With great ingenuity, Pasteur designed numerous experiments that would allow the boiled infusions to come in contact with air that itself had not been altered by the heat, to answer Needham’s objection of a century before. Air was led through dense gun cotton filters, for example, before coming in contact with the infusions. The filters were then dissolved and the solid particles trapped from the air were examined microscopically. Pasteur saw some spherical bodies that he assumed were the ‘germs’ of microbes, since in those infusions microbial life rarely developed. Perhaps most famous of Pasteur’s experiments was a series of flasks which, after being filled with infusion, had their necks heated in a flame then drawn out into long, curved shapes, for example, like a ‘swan-neck’ (Figure 3). Air could enter through the long neck, but

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Figure 3 Pasteur’s illustrations of his ‘swan-necked flasks’. Courtesy of the American Society for Microbiology Press.

dust particles settled in the dip and could not come in contact with the flask’s contents. Again, if these flasks were boiled then cooled, rarely did any microbial growth ever appear in them. However, if the neck was broken off and dust allowed to enter the flask, microbial growth in the infusion soon followed. Pasteur concluded that this proved microbes or their germs were carried on dust particles, so the exclusion of dust followed by no growth meant the disproof of spontaneous generation. As the swan-necked flasks have in our time become textbook icons for exemplary experimental practice, it is difficult for us to reconstruct the historical period when, even after Pasteur’s famous public lectures on his experiments, a significant number of scientists remained unconvinced. In particular Pasteur claimed to have sweepingly proven that germs must be the source of growth in previously boiled infusions, but his opponents pointed out that what he had shown could be read to be only that dust was a necessary ingredient for spontaneous generation with the yeast/sugar water infusions that he used. Recent historians have also pointed out, as did Pouchet and other supporters of spontaneous generation at the time, that Pasteur never replicated some of Pouchet’s most convincing experiments: those involving boiled hay infusions. (It was only recognized a decade

later that the hay bacillus, Bacillus subtilis, produces heatresistant endospores.) If Pasteur had tried his famous swan-necked flask method with a hay infusion, it has been suggested that the debate might have ended quite differently on this point alone. Since even in this most famous of textbook cases, it still seems the experimental evidence at the time was not as finally conclusive as Pasteur declared it to be, why did the French Academy of Sciences award the victory to Pasteur and declare the spontaneous generation controversy settled once and for all? Spontaneous generation was still just as politically and religiously charged a subject in the French Second Empire of Louis Napoleon as it had been 100 years earlier in Buffon and Needham’s time. Furthermore, while spontaneous generation had in the past tended to be associated with the doctrine of transmutation of species, the latter doctrine was currently undergoing a particularly heated wave of notoriety in the wake of the recent publication of Darwin’s On the Origin of Species. The first French translation, by Cle´mence Royer, of the Origin had just appeared in 1862 and was prefaced by a long diatribe by Royer against the Catholic Church. The Church was, however, on closer terms than ever with the conservative government, since Louis Napoleon’s coup of 1851. In this environment, since the Academy of Sciences was a state-supported institution, Darwinian evolution was regarded in France as a politico-theological doctrine allied with forces that threatened the Church and State. In addition, spontaneous generation was seen as directly undermining belief in a providential Creator, so that the outcome of the Pasteur– Pouchet debate carried implications of enormous importance to the power structure of the Second Empire. Even foreign savants observing the controversy at the time, including Britain’s premier anatomist Richard Owen and Germany’s Friedrich Lange, noted that opposition to spontaneous generation seemed intimately tied to support for the forces of conservatism and the established Church. The force of Pasteur’s experimental genius has been surpassed historically only by his rhetorical skill. For, while he broached all these controversial subjects in a famous 1864 Sorbonne lecture on spontaneous generation, yet he succeeded in portraying himself (and science done at its best) as able to remain entirely aloof from such matters, saying that he had approached the question in a totally unbiased manner, equally ready to accept or reject the existence of spontaneous generation, based solely on the outcome of the experiments. While Pasteur was truly an experimental genius compared to Pouchet, the crucial problem with this ‘‘let the chips fall where they may’’ portrayal of himself is that it is not true. By this time Pasteur had been dominated for some years already by his own preconceived notions (derived from his work on crystalline asymmetry) of life as dependent upon a Cosmic Asymmetric Force. He had done numerous

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experiments in private, trying to produce living conditions in the laboratory by application of light, electricity, and magnetism to experimental setups, and he was convinced that Pouchet was wrong, not so much because spontaneous generation was somehow impossible, but because Pouchet was not aware of the importance of asymmetric forces to the problem of life, and was not approaching the experiments by that route. Furthermore, Pasteur was fully aware that all of his important scientific patrons were entrenched in the conservative regime, and he was constantly trying to cultivate those contacts, particularly with the Emperor and Empress personally. Thus it is no surprise that during all the years of the Second Empire he kept silent about his own attempts to simulate or create life in the laboratory and concentrated in public on refuting the work of Pouchet, which he knew to be very unpalatable to the Academy and the State.

The British Debate of the 1870s: Darwin, Spontaneous Generation, and the Germ Theory of Disease Charles Darwin himself had remarkably ambivalent feelings about spontaneous generation. In the first edition of On the Origin of Species he appears to have carefully avoided the topic through almost the entire book. Then in a single throwaway line near the very end, he upset many of his supporters by adopting this language: Therefore I should infer from analogy that probably all the organic beings which have ever lived on this earth have descended from some one primordial form, into which life was first breathed. Darwin, On the Origin of Species London: John Murray, 1859, p. 484, italics mine

In the second edition, released just 7 weeks later on 31 December 1859, Darwin made it ‘‘. . . breathed by the Creator.’’ Darwin received much criticism, from both supporters and opponents of evolution, about dissembling in this passage. In the 1862 third edition he simply dropped the entire phrase after ‘‘one primordial form’’ and left it that way through all remaining editions of the Origin in his lifetime, essentially taking a public position of ‘no comment’ on the implications of his theory for the origin of life on Earth. In private, to close confidantes, he said, ‘‘I have long regretted that I truckled to public opinion, and used the Pentateuchal term of creation, by which I really meant ‘appeared’ by some wholly unknown process. It is mere rubbish, thinking at present of the origin of life; one might as well think of the origin of matter.’’ He thought Pasteur’s experiments much more convincing than Pouchet’s. But to others Darwin said it

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would be a great thing if spontaneous generation could be proven, as it would undergird his theory in an important way. His reluctance to discuss the matter in 1859 seems to have become genuine ambivalence on the topic. On the British scientific scene in the 1860s and 1870s the most entrenched advocates of spontaneous generation and those most resistant to the germ theory of disease were the medical community. At that time, despite Pasteur’s suggestive research on silkworm diseases, British doctors strongly favored a theory of contagious disease as analogous to fermentation via chemical catalysis, a theory first promulgated by Justus Liebig in the early 1840s. This was in part due to a brief but highly publicized theory of 1849 that cholera was caused by a fungus. When the supposed cholera fungus was soon discovered to be a common mold contaminant, many British medical men who might have had leanings toward a germ theory felt that it had been discredited. Thus, when Pasteur’s claims of the mid-1860s reached Britain, most British doctors were skeptical that living microbes were the sources of contagion, and they continued to favor a chemical poison, viewing the microbes as some kind of product or concomitant of the disease process rather than its cause. Thus, when the physicist John Tyndall gave a famous lecture on ‘‘Dust and Disease’’ in January 1870, arguing that doctors must accept the germ theory of Pasteur in order to have any really scientific approach to disease, many British doctors took offence. Tyndall was known for his brash public persona and as a general spokesman for science in London society circles. In addition to his quite haughty tone, he was trained as a physicist and certainly had no clinical experience of disease or of the role of patients’ constitutions in causing their susceptibility to be so highly varied. So it was not surprising that doctors accused Tyndall of being an interloper in biology and medicine, an area where his opinions had no weight. They thought he was totally unaware of the cholera fungus hoax of 1849 and of other important technical reasons for medical skepticism about an oversimplified germ theory. Tyndall’s version of the germ theory denied any role to the ‘constitution’: it compared patients to so many identical tubes of infusion. If germladen dust particles fell into the patient, he would get sick, Tyndall said. Those in hospitals or towns with epidemics who did not get sick were those who such particles, or ‘germ clouds’, passed by. Chief among the medical professionals who opposed Tyndall was Henry Charlton Bastian, professor of pathological anatomy at London’s University College Medical School. Bastian was an avowed supporter of Darwin’s and Spencer’s writings on evolution, and did much experimental work to try to show that microorganisms could arise by spontaneous generation, or biogenesis as he at first called it. Bastian, like Alfred Russel Wallace and many others interested in natural selection, thought at

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that time that Darwin’s theory required spontaneous generation to explain in a nonmiraculous way where the original common ancestor of all species came from. Wallace wrote excitedly to Darwin and in a review in Nature in August 1872 claimed that Bastian’s findings could help Darwinian evolution out of the time crunch presented by William Thomson’s (later Lord Kelvin) challenge: that the age of the earth – a few tens of millions of years at most, by his physics calculations – was utterly inadequate to allow the hundreds of millions of years of evolution. Darwin’s gradualist theory required to get from single-celled creatures to complex vertebrates by a single, unbroken chain of descent. Darwin was tempted but still ambivalent. By this time he wrote to T. H. Huxley and J. D. Hooker that he certainly believed that life at first emerged from nonlife by natural processes in ‘some warm little pond’. However, Bastian’s idea of spontaneous generation going on all the time, everywhere, up to the present meant many different possible lines of descent with new ones beginning constantly, a model similar to Lamarck’s. Darwin was still deeply committed intellectually to a single branching tree of descent, which would require only one (or a small handful) progenitor(s) created by an original emergence of life entirely confined to the very earliest period of life on Earth. He followed closely the debates over Bastian’s experiments, as did all Darwinians who had come to know Bastian as one of their own, indeed one of their brightest rising young stars in experimental science. Bastian also thought that bacteria in diseased patients resulted from spontaneous generation, as by-products of the disease process. Between 1870 and 1875 he had published the results of hundreds of experiments in which he showed that bacteria could be found in tubes of various infusions boiled for periods varying from a few minutes up to an hour. Attempting to refute Bastian’s work from 1875 to 1877, Tyndall devised an ingenious dust-free cabinet in which to carry out the experiments (Figure 4). Tyndall’s close friend T. H. Huxley was at first interested in Bastian’s experiments, but soon concluded that the pathologist must be mistaken in his conclusions, especially given Huxley’s belief at this time in pleomorphism. He declared that Bastian ‘‘had gotten out of his tubes exactly what he put into them,’’ that is, organisms must have gotten into the tubes as contaminants. Both Huxley and Tyndall were also evolutionists, but found it much easier to believe in living things able to survive boiling somehow (or, even more believable, that Bastian was a sloppy experimenter) than to believe that organisms as complex as protozoa could come from anything other than the ‘seeds’ or ‘germs’ of other like organisms. They lobbied widely among scientific colleagues to convince others that Bastian was at best a poor experimenter and at worst a fraud and a cheat. As mentioned above, Bastian, for his part, found it much more difficult to believe that life could

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Figure 4 Tyndall’s dust-proof chamber for carrying out spontaneous generation experiments. Courtesy of the Royal Institution, London.

survive the boiling temperature for more than a minute or two, than to believe (especially since he saw it as a logical necessity of continuity in nature and of evolutionary science) that the transition in stages from nonliving to living matter should be possible. A noted physiologist, John Burdon Sanderson, observed Bastian’s technique carefully and reported that it was up to high scientific standards. Huxley, in a famous address to the British Association for Advancement of Science meeting at Liverpool in September 1870, moved to gain the rhetorical upper hand in the debate by defining the terms. He defined ‘biogenesis’ to mean life only from other life. The opposite belief he termed ‘abiogenesis’ and began to argue that it very well might be possible, even probable, but only in the conditions of the primitive earth. This convenient dualistic terminology was rapidly picked up by contemporaries and propagated in textbooks. Given this, it is more than a little ironic that Huxley had hijacked the term ‘biogenesis’ from Bastian, who was using it up until that time to mean exactly the opposite, that is, spontaneous generation (After this, Bastian substituted the term archebiosis, but it never caught on as Huxley’s terms did.). Also important, it is from this time that all discussion of the origin of life question began to be pushed more and more into the distant past. Only

History and Culture, (and Biographies) | Spontaneous Generation

after Huxley’s talk, for example, did Darwin advance the argument that if spontaneous generation had occurred in the earth’s distant past, it would no longer be possible after the evolution of the first heterotrophs, since they would consume any organic molecules that formed before those could assemble into a new organism. Those opposed to both evolution and spontaneous generation had a simpler solution. The physicist William Thomson, Lord Kelvin, for example, responded to both Huxley and Bastian in his own Presidential Address to the BAAS in 1871. He suggested that the germs of life might have originally been brought to earth from another world via a meteorite. To those demanding a completely naturalistic origin of life, of course, this merely pushed the question back a step to some other planet. Beginning in the autumn of 1876, Tyndall began to have difficulty with the experiments carried out in his dust-free cabinet. Infusions that had been sterilized by only 5 minutes’ boiling a year earlier now could not be sterilized even after hours of boiling. In 1876, the discovery by the German botanist Ferdinand Cohn that certain species of Bacillus, especially common in hay and in cheese (and in infusions made from these that showed microbial growth after extended boiling), were capable of producing heat-resistant endospores was taken to explain why Bastian’s infusions were full of microbial growth after boiling, and upon being handed a copy of Cohn’s article Tyndall immediately adopted this stance to explain his own recent difficulties. Tyndall struggled for several months and finally discovered that sterility could be achieved by repeated short boilings, followed by a period of allowing the remaining spores to germinate. This process came to be known as ‘fractional sterilization’ or ‘Tyndallization’. This was largely superseded with the development of the autoclave in the 1880s. Equally interesting from a historical point of view is that though this rapid about-face also showed that Tyndall and Huxley had been wrong that Bastian must be a sloppy, incompetent experimenter, that is precisely the version of Bastian (and most spontaneous generation advocates) that has usually gone into the textbooks until quite recently. The sole exception was Pasteur’s student Emile Duclaux, the only contemporary writer among the winners who credited Bastian’s perseverance in sticking to his experimental results with the value that it truly had, that is, that without the goad of Bastian and his popularity, Pasteur, Cohn, and Tyndall might never have discovered that they were wrong about the existence of bacteria in boiled hay infusions, and thus been led to realize that heat-resistant spores exist and require autoclaving or fractional sterilization to guarantee their being killed. Pasteur was a devout Catholic and never believed, to his dying day, the viewpoint favored by many modern origin of life hypotheses, that random or chance organization of materials could have formed life, even the very first living organisms.

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Twentieth Century Ideas The origin of life has continued to be a subject of active interest in the twentieth century, and, despite avoidance of the term ‘spontaneous generation’ since 1880, some modern concepts have resembled older ideas more than superficially. Beginning with Bastian (who revived experiments on spontaneous generation from 1900 to 1915), a number of investigators came to believe that the basic chemistry specific to the living was the chemistry of colloids. A burst of research in colloid chemistry from 1910 until the 1930s included many workers who believed that this avenue would lead to understanding the simplest combination of materials to cross the boundary from nonlife to living matter. Among these were biochemists Benjamin Moore and Albert Mary, bacteriologist Arthur I. Kendall, the Mexican biologist Alfonso Herrera, and medical doctor and researcher George Crile. Geneticist H. J. Muller suggested that the first chemical assembly of a gene should be considered the origin of life, though this still suggested a sudden origin of life from nonlife, perhaps the most central idea implicit in the doctrine of spontaneous generation. The Russian biochemist A. I. Oparin proposed instead a gradual chemical evolution of life, probably proceeding through the intermediate stage of coacervates. Research into life’s origin had lost none of its larger associations: the issue still attracted researchers with anticlerical leanings (Herrera was one) as well as affiliations with radical, even Communist political beliefs. J. B. S. Haldane and J. D. Bernal did research in the field and were avowed Communists, as was Muller until the late 1930s. Furthermore, Oparin and Wilhelm Reich explicitly credited the philosophy of dialectical materialism as having been crucial to their origin of life research programs. Research on viruses, particularly Wendell Stanley’s crystallization of tobacco mosaic virus in 1935, lent credence for a time to the idea that a virus might be the simplest ‘living molecule’. In addition, after the rise of molecular biology and the discovery of the Watson–Crick DNA structure, many considered Muller’s idea of ‘‘the gene as the origin of life’’ more likely. The Urey–Miller experiment, published just 3 weeks after Watson and Crick’s paper on DNA, lent further support to the notion that the building blocks of life could have assembled very rapidly. In that experiment, an electrical discharge passed through a mixture of steam, hydrogen, methane, and ammonia in a sealed container produced amino acids and other organic compounds after only a few days. Throughout the 1950s and 1960s, such views were in tension with those of Oparin and his students in the Soviet Union, that the process of chemical evolution was a more gradual one, with no single decisive ‘living molecule’. Cold War and anti-Lysenkoist hostilities were

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involved, as any scientific theory openly lauding dialectical materialism was seen as suspect in the West in the aftermath of Lysenko’s takeover of Soviet genetics. The involvement of NASA as a major patron for origin of life research, beginning in 1960, led to the funding of a wide variety of approaches, including gradual evolution models as well as sudden, gene-based models; ‘protein-first’ or ‘metabolism-first’ as well as ‘nucleic acid-first’ or ‘information-first’ models. This work took place under the rubric of ‘exobiology’ (broadened and renamed ‘astrobiology’ in the late 1990s). Thus, ‘spontaneous generation’ has been considered a dead-end, disproven belief since 1880 or so. But, despite the dropping of the older term, the conceptual continuities between modern and older origin of life ideas, particularly for abiogenesis in the earth’s distant past, are in some ways as interesting as the discontinuities. The continued association of these ideas with larger philosophical and political concerns is also a striking feature of their history well into the twentieth century.

See also: History of Microbiology; Methods, Philosophy of; Plague, Historical

Further Reading Bulloch W (1938) A History of Bacteriology. Oxford: Oxford University Press.

Dick SJ and Strick JE (2004) The Living Universe: NASA and the Development of Astrobiology. New Brunswick, NJ: Rutgers University Press. Farley J (1977) The Spontaneous Generation Controversy from Descartes to Oparin. Baltimore: Johns Hopkins University Press. Farley J (1978) The political and religious background to the work of Louis Pasteur. Annual Review of Microbiology 32: 143–154. Findlen P (1993) Controlling the experiment: Rhetoric, court patronage and the experimental method of Francesco Redi. History of Science 31: 35–64. Fry I (2000) The Emergence of Life on Earth. New Brunswick, NJ: Rutgers University Press. Geison GL (1995) The Private Science of Louis Pasteur. Princeton, NJ: Princeton University Press. Graham LR (1987) Science, Philosophy and Human Behavior in the Soviet Union. New York: Columbia University Press. See Chapter 3, ‘‘Origin of Life’’. Lazcano A (1992) Origins of life: The historical development of recent theories. In: Margulis L and Olendzenski L (eds.) Environmental Evolution: The Effects of the Origin and Evolution of Life on Planet Earth, pp. 57–69. Cambridge, Mass: MIT Press. Pelling M (1978) Cholera, Fever and English Medicine, 1825–1865. Oxford: Oxford University Press. Roe S (1983) John Turberville Needham and the generation of living organisms. Isis 74: 159–184. Roll-Hansen N (2007) Louis Pasteur. In: The New Dictionary of Scientific Biography. New York: Charles Scribners. Sloan PR (1992) Organic molecules revisited. In: Roger J (ed.) Buffon’88, pp. 415–438. Paris and Lyon: J. Vrin. Strick JE (1999) Darwinism and the origin of life: The role of H.C. Bastian in the British Spontaneous Generation Debates, 1868–73. Journal of the History of Biology 32: 51–92. Strick JE (2000) Sparks of Life: Darwinism and the Victorian Debates over Spontaneous Generation. Cambridge, MA: Harvard University Press. Strick JE (2001) Evolution and the Spontaneous Generation Debate, 6 vols. Bristol, UK: Thoemmes Press. Strick JE (2004) The Origin of Life Debate: Molecules, Cells, and Generation, 6 vols. Bristol, UK: Thoemmes Press.

Syphilis, Historical D S Jones, Massachusetts Institute of Technology, Cambridge, MA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction The Origins of Syphilis The History of a Disease

Glossary magic bullet The name given to Paul Ehrlich’s Salvarsan, the first specific antibiotic, representing the hope that use of a specific drug to kill the specific bacterial cause of syphilis would control the disease. morbus gallicus (the French disease) The most common name given to a new disease which appeared

Managing Syphilis The Limits of Biomedicine Further Reading

in Europe in the late fifteenth century; many physicians and historians identify morbus gallicus as syphilis. paleopathology The technique of examining ancient skeletal remains to identify the diseases suffered by historical populations. Treponema pallidum The bacteria that causes syphilis. Other subspecies, all extremely similar to each other, cause endemic syphilis (bejel), yaws, and pinta.

Abbreviation CDC

Centers for Disease Control and Prevention

Defining Statement

The Origins of Syphilis

Despite centuries of study by historians and scientists, using the latest techniques of molecular biology, details of the origins and spread of syphilis remain unclear. Moreover, despite the existence of decisive treatment, syphilis has not been eradicated and instead thrives wherever social and political conditions allow.

The recorded history of syphilis began in the late fifteenth century with the appearance of a new disease, widely called ‘morbus gallicus’, the French Disease. The earliest cases were described in 1495 as the mercenary army of French King Charles VIII retreated from its siege of Naples. Victims suffered from fevers, open sores, disfiguring scars, and disabling pains; many were consumed by the disease and met gruesome deaths. As the French army disbanded, infected soldiers carried the disease throughout Europe, to Germany in 1495, and to Holland, England, and Greece by 1496. The voyage of Vasco da Gama took it to India in 1498. By 1505 it had reached Japan. Witnesses described the disease with horror. Joseph Gru¨nbeck (1473–1532) left a typical account: ‘In recent times I have seen scourges, horrible sicknesses and many infirmities affect mankind from all corners of the earth. Amongst them has crept in, from the western shores of Gaul, a disease which is so cruel, so distressing, so appalling that until now nothing so horrifying, nothing more terrible or disgusting, has even been known on this earth’. This dramatic appearance has puzzled observers for centuries. The 1490s witnessed great transformations in Europe: Columbus encountered the Americas; France invaded Italy; the Spanish government expelled Jews and Moors from

Introduction The history of syphilis has long been one of the most popular topics in the history of medicine. Despite 500 years of research and debate by historians, physicians, and anthropologists, many questions about the origins of the disease remain unresolved. The evolution of syphilis since the sixteenth century demonstrates remarkable changes in both its medical symptoms and the cultural meanings of those symptoms. The history of efforts to control syphilis reveals the inevitable moral judgments about syphilis and other sexually transmitted infections, the balance between disease control and individual rights, and the limited ability of even powerful medical remedies to control the disease. These lessons take on new relevance as fears of HIV/AIDS motivate efforts to eradicate syphilis.

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Spain; the Pope abolished leper hospitals. Each of these factors might have contributed to the emergence of a new disease. Spanish witnesses traced the disease to Columbus’s voyages to the Americas: they heard from natives that the disease had long been endemic in Hispaniola, and they witnessed unchaste Spaniards acquiring the infection while there. Details of the timing of the early Spanish voyages suggest that syphilis could have been carried back from the New World to Spain in time to appear in Italy by 1495. This would make syphilis the counterpart to the many diseases, notably smallpox, which the Spaniards carried from Europe to America. However, definitive evidence does not exist. The traditional alternative to this theory asserts that syphilis always existed in Europe and Asia, but was not recognized before the 1490s. Detailed analyses of Egyptian papyri, the Hebrew Bible, Hippocratic writings, and medical texts from India and China contain abundant evidence of the prevalence of sexually transmitted infections. However, these records do not provide clear evidence of the specific presence of syphilis. Ambiguous medieval descriptions of leprosy, which observers believed could be transmitted sexually, have caused particular confusion. Several novel explanations have been suggested in recent decades. Four distinct diseases – venereal syphilis, endemic syphilis (bejel), yaws, and pinta – are all caused by the same species of bacteria, Treponema pallidum. Scientists have long been unable to find histological or immunological differences between the subspecies that cause these different diseases. This suggested that the four diseases might simply be different manifestations of infection with the same microbe, with the results of infection depending on social and environmental conditions. Yaws and endemic syphilis, long present in Europe, transformed into venereal syphilis in the fifteenth century when changes in sanitation, personal hygiene, and sexual behavior produced new conditions favorable for the venereal transmission and manifestations of the disease. A competing theory argued that differences did exist and that venereal syphilis emerged as a result of new and virulent mutations in existing subspecies of T. pallidum. This theory has won support from recent genetic analyses. In 1998 scientists sequenced the 1 138 006 bp of the treponema genome. Subtle variation, at only 1000 bp, distinguishes the subspecies that cause venereal syphilis, endemic syphilis, and yaws. When and where did these subspecies emerge? Immunologists looking for the origins of syphilis have sought treponemal antigens in mummies and skeletal remains, finding the oldest in a Pleistocene bear from Indiana. Paleopathological evidence, from thousands of bones from pre-Columbian America, Europe, Africa, and Asia, confirms the presence of treponemal infections in each location. Although interpretation has been controversial, researchers are now confident in their ability to distinguish the bony lesions caused by the different subspecies. Such analyses suggest that yaws has long existed in Africa, Europe, Asia, and America. Syphilis, in contrast, first appeared in the

Americas 2000 years ago, returned to Europe with Columbus’s crews, thrived in its new environs, and produced the outbreak of morbus gallicus. This story, however, is not fully supported by genetic analyses, which confirm neither the status of the yaws subspecies as the ancestral form nor the status of the syphilis subspecies as a recent arrival. Recognition of the existence of lateral gene transfers between the subspecies further confuses these analyses. As a result, the hopes that state-of-the-art science would finally resolve this historical question remain unfulfilled.

The History of a Disease The new disease had many dramatic impacts. Observers were horrified by its emergence and manifestations. Everyone blamed someone else. The Italians called it the ‘morbus gallicus’ or the ‘mal francese’. The French named it the ‘mal de Naples’. Others labeled it ‘scabies hispanicus’, the ‘American disease’, or – in Japan and the East Indies – the ‘Portuguese disease’. Some names reflected its manifestations: the ‘Great pox’, ‘fire-piss’, ‘gangrene grossa’, the ‘Neapolitan itch’, and ‘plum-blossom sores’. ‘Syphilis’ first appeared in the 1530 Latin poem of humanist and physician Girolamo Fracastoro (1483–1553), but this name was not widely used until the eighteenth century. Early observers traced syphilis to astrological origins, noting the ominous conjunction in 1484 of Mars, Jupiter, and Saturn in Scorpio, the constellation most closely associated with genital affairs. By the 1520s, the disease had been connected to sexual transmission and given a new name: ‘lues venerea’, the venereal disease. Many people saw the disease as a punishment from God for sexual debauchery (Figure 1). Public bath houses closed, distrust divided friends and lovers, and Platonic love emerged as a vibrant social cult. Physicians struggled to understand the disease for centuries. Many perceived the varied symptoms, including urethral discharges, penile chancres, and skin rashes as a single phenomena, all caused by a single poison. However, over the eighteenth century, the concept of a single disease became increasingly contested and physicians increasingly argued for the existence of many different ‘morbi venerei’. This debate drove English physician John Hunter (1728–93) to his famous 1767 experiment. He reportedly inoculated his own penis with pus from a patient with gonorrhea; when he developed a characteristic syphilitic chancre, he concluded that the existence of a single lues venerea had been proven. Careful work over the nineteenth century settled the controversy. In 1838, Phillipe Record (1799–1889) reported the results of over 2500 experimental inoculations performed at a Paris hospital. He demonstrated that the primary, secondary, and tertiary stages of syphilis all represented a single disease, distinct from other venereal infections. Rudolf Virchow (1821–1902) and Alfred Fournier (1832–1913)

History and Culture, (and Biographies) | Syphilis, Historical

Figure 1 Illustration from Joseph Grunpeck’s ‘Tractatus de pestilentiali scorra, sive mala de Franzos’ (1496). This image is traditionally interpreted as showing rays of divine wrath striking sinners with syphilitic lesions. It illustrates theological interpretations of the appearance of the new epidemic.

extended this work, describing the characteristic pathological lesions of syphilis and the consequences of congenital infection. In 1879, Albert Neisser (1855–1916) settled lingering doubts by isolating the gonococcus, the causative agent of gonorrhea. The characterization of syphilis, in its modern form, was completed in the early twentieth century. In 1905, protozoologist Fritz Schaudinn (1871–1906) and syphilologist Erich Hoffman (1869–1959) described the slender, spiral bacteria, Spriochaeta pallidum, later renamed T. pallidum. In August 1906, Wassermann (1866–1925), working with Neisser and Carl Bruck (1879–1944), developed the Wasserman test, a complement fixation reaction which became the definitive serological test for syphilis. The disease had been defined, the agent identified, and an objective diagnostic test developed.

Managing Syphilis When morbus gallicus first appeared, physicians treated its victims by purging their bodies of its poison. They

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recommend hot houses and extreme exercise (ball games, running, boar-hunting, or farming) to induce sweating. They practiced blood-letting and prescribed purgatives. Mercury, given topically or orally to induce sweating and salivation, became popular. Its severe side effects – loss of teeth, gum ulcerations, skeletal deterioration, and gastrointestinal problems – matched the severity of the disease. Such punitive treatment seemed appropriate for a disease attributed to venery. However, patients and physicians soon became confused about which symptoms came from the disease, and which from the treatment. In the 1510s, guaiac, a resin extracted from a West Indian tree, arrived in Europe. Although expensive, and not explained by medical theories, guaiac quickly became popular as rumors credited it with miraculous cures. It satisfied a popular theory that treatments for a disease ought to come from the same place (the Americas) as the disease itself. In addition, its side effects were much less severe than those of mercury. Desperation led physicians to attempt many other treatments: massage, tobacco smoking, abstinence from pork and peas, and decoctions of vulture broth with sarsaparilla. Hospitals and hospices appeared throughout Europe. Most were established as hospitals for the incurable, but as the effects of the Great Pox moderated over the century, they increasingly became places for cure and care. The Catholic Church even developed a special mass, ‘Missa contra morbum gallicum’. Mercury and guaiac dominated syphilis treatment for centuries. Surgeons developed ways to excise or cauterize the sores. Medical entrepreneurs produced and marketed secret remedies. But by the late nineteenth century, some physicians had begun to suspect that their treatments provided no benefit. This suspicion was confirmed by the famous Oslo Study, in which 2000 patients, between 1890 and 1910, received no treatment, and fared just as well (or poorly) as those receiving mercurial ointments and other treatments. The infamous Tuskegee Syphilis Experiment sought to extend the results of this study by describing the ‘natural history’ of syphilis among AfricanAmerican men in the rural south. Public Health Service officers deceptively promised treatment, but then provided none, from 1932 until 1972. But in contrast to the Oslo Study, these physicians withheld treatments – including penicillin – which they believed to be effective. As physicians and scientists struggled to develop effective treatments of syphilis, its prevalence steadily increased. In the nineteenth century, syphilis fueled widespread fear of cultural decay and the breakdown of social values. It was identified as a family poison which spread from profligate men to their innocent wives and children. Fears of casual transmission – through pens, pencils, drinking fountains, toilet seats, and doorknobs – proliferated. In the United States, immigrants were stigmatized as a source of infection. These fears, which reveal deep cultural anxieties about disease and sexuality,

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motivated many attempts at social engineering. Shocked to learn that as many as one-third of its troops were infected with syphilis, the British government passed the Contagious Disease Acts of 1864, 1866, and 1869. The laws acknowledged that sexual continence was an unrealistic goal. Syphilis could only be contained by minimizing the consequences of the inevitable transgressions: prostitutes would be inspected and, if infected, detained until cured. These laws produced an outcry from purity reformers, who argued that prostitution needed to be criminalized and repressed, not acknowledged and regulated. The laws were repealed in 1886. Similar calls for social hygiene dominated syphilis control in the United States from the 1890s through the 1920s. Education and moral encouragement beseeched men to be disciplined and restrained, faithful to their wives and families. During World War I, the US military, which saw syphilis as a substantial threat to its effectiveness, conducted extensive campaigns against the disease. Just as they drained swamps to prevent malaria, public health officials shut down red light districts and detained 20 000 suspected prostitutes. The Training Camp Commission distracted soldiers with organized sports and educated them with unambiguous messages: ‘A German bullet is cleaner than a whore’. Faced with the French system of regulated prostitution, the Army refused to distribute condoms. Instead, officials established prophylaxis stations to provide treatment if exposure occurred. They hoped that the painful urethral irrigations would be a disincentive to sex. The Navy contributed to the campaign by removing doorknobs from battleships to prevent transmission of syphilis among sailors. Despite all of this work, syphilis rates remained high. After the war, interest in venereal disease control diminished. Prudish censorship blocked public education in magazines or on the radio. Surgeon General Thomas Parran (1892–1968) led a campaign to reverse this. He decried the ‘conspiracy of silence’ that surrounded venereal diseases. He argued that sexual continence remained an impossible ideal and that victims of syphilis were victims of a disease and not criminals. His efforts led to the 1938 National Venereal Disease Control Act, which provided federal funding for diagnosis and treatment. States began requiring premarital serological tests (Figure 2). This new pragmatic attitude dominated efforts during World War II. Moralistic campaigns against venereal diseases did continue (Figure 3). But instead of repressing sexual behavior, officers of the Social Protection Division sought to modify it: instead of providing moral education, they distributed condoms. These efforts substantially reduced the incidence of syphilis among military personnel. The contrast between the efforts during World War I and World War II illustrates the two extreme options of social management of syphilis. Social hygienists asserted a

Figure 2 ‘Happiness Ahead – for the Healthy but not for the Diseased’. During the 1930s, many states began to require premarital screening for venereal diseases. In this poster from a public health campaign, syphilis was portrayed as poison that would destroy marriages and families.

moral ideal and believed that abstinence was the only way to prevent infection. Pragmatists, in contrast, acknowledged that pre- and extramarital intercourse were inevitable; they sought to prevent infection by encouraging safer sexual practices and by providing unstigmatized treatment.

The Limits of Biomedicine While public health officials swung between these two extremes of social management, medical researchers gradually produced powerful methods of medical management. In 1909, Paul Ehrlich (1854–1915) announced that his new drug, Salvarsan, had specific activity against syphilis. It was the first time anyone had found a specific drug that killed a specific microbe. Ehrlich called it a ‘magic bullet’. With this drug the promise of modern medicine seemed fulfilled: medical scientists had established the specific cause of syphilis, they had developed a specific diagnostic test, and they had a specific treatment. The control of syphilis seemed at hand. Hopes for easy control went unrealized. Salvarsan was toxic and difficult to administer, with some patients requiring two years of

History and Culture, (and Biographies) | Syphilis, Historical

Figure 3 ‘V.D.: Worst of the Three’. During World War II, the United States military waged a campaign against syphilis that combined clearly moral messages, as seen in this poster, with condom distribution campaigns. Note that in this image, venereal disease is personified as an evil, dangerous woman, and not as a promiscuous soldier.

treatment; only 25% of patients received the full series of infections. Hope was restored in 1943 when John F. Mahoney (1889–1957) demonstrated that syphilis could be cured with a single injection of penicillin. The miracle drug had been found. Incidence fell rapidly, from 94 957 cases in 1946 to only 6392 cases 10 years later. Syphilis seemed vanquished. But again, syphilis defied expectations. Despite the power of penicillin, syphilis began a slow recovery during the 1960s. Many factors have been implicated: the promiscuity of the sexual revolution, the availability of oral contraceptives, and decreased funding for education, case tracing, and other disease control programs. Some physicians even refused to prescribe penicillin, fearing that easy treatment only encouraged illicit sexual activity. Whatever the cause, syphilis rates began to rebound. Rising and falling in 10-year cycles, with each peak higher than the last, by 1990 the incidence of syphilis had risen to 50 578, the highest number of cases seen since 1948, with most of the burden falling on impoverished, minority populations. How could syphilis continue to spread despite the existence of penicillin, an affordable and decisive treatment? ‘Magic bullets’, even those as powerful as penicillin, are never panaceas. The history of

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syphilis shows the range of scientific, social, political, and cultural factors which contribute to the prevalence of the disease. A single intervention cannot treat the many different problems which become embodied as syphilis. Successful management requires concerted efforts against all of the causes of the disease. Interest in the history of syphilis took on new urgency in the 1980s with the appearance of HIV and AIDS. Syphilis became both a significant facilitator of the transmission of HIV and a dangerous infection in people with AIDS. In addition, public health officials and policy makers turned to the history of syphilis to guide their efforts against HIV. Many lessons were suggested. Just as penicillin had not solved syphilis, effective treatments for HIV would not end the epidemic. Educational programs, whether advocating abstinence or safe sex, would not cause people to avoid high risk sexual behaviors. Compulsory measures, whether quarantine or serological screening, would bring few results, at high costs to civil liberties. Fear, blame, and stigmatization would shape the development of public health policy. This new urgency, combined with growing awareness of the risks of sex, fueled more aggressive public health efforts that, combined with periodic ebbing of the prevalence of disease, reversed the slow rise of syphilis. By 2000 the Centers for Disease Control and Prevention (CDC) reported only 5979 new cases, the lowest number reported since it began keeping data in 1941. This reversal triggered optimism that syphilis could be contained. The CDC launched a National Plan to Eliminate Syphilis, which initially made substantial progress. Cases fell most dramatically among African Americans, women, and newborns; the black: white disparity in incidence dropped from 28.6:1 to 5.6:1. However, despite this progress, the absolute number of cases has increased since 2000, reaching 8724 by 2005. Most of the growth has been among men, but rates have also begun to increase among women. Proponents of elimination have known that the most important barriers to eradication would not be scientific, but rather the considerable social and economic obstacles that exist. Syphilis continues to be seen as a moral problem, reducing the willingness of victims to seek treatment. It flourishes in communities which lack basic financial and social resources, which perceive more serious threats from education, unemployment, crime, and other health issues. The shadow of the Tuskegee study has also left many afflicted communities suspicious of public health campaigns. The situation is much worse in developing countries, with roughly 12 000 000 cases worldwide each year. This represents an enormous toll of preventable morbidity, especially in light of the synergy of syphilis and HIV. The history of syphilis, therefore, demonstrates many important characteristics of diseases. Syphilis is not simply a biological phenomena. Instead, its incidence reflects political and economic structures and cultural beliefs and practices. Its manifestations and meanings change over

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time, shaping efforts to control the disease. Isolated medical remedies, even those as powerful as penicillin, cannot be the only solution. Successful control requires comprehensive programs, which acknowledge the cultural and social dynamics of syphilis.

See also: AIDS, Historical; History of Microbiology; Horizontal Transfer of Genes between Microorganisms; Paleontology, Microbial; Phylogenomics; Sexually Transmitted Diseases; Spirochetes

Further Reading Arrizabalaga J, Henderson J, and French R (1997) The Great Pox: The French Disease in Renaissance Europe. New Haven, CT: Yale University Press. Baker BJ and Armelagos GJ (1988) The origin and antiquity of syphilis: Paleopathological diagnosis and interpretation. Current Anthropology 29: 703–737. Brandt AM (1987) No Magic Bullet: A Social History of Venereal Disease in the United States Since 1880. New York, NY: Oxford University Press. Centers for Disease Control and Prevention (2006) The National Plan to Eliminate Syphilis From the United States. Atlanta, GA: Department of Health and Human Services.

Crosby AW (1972) The Columbian Exchange: Biological and Cultural Consequences of 1492. Greenwood, CT: Greenwood Publishing Company. Fleck L (1935, 1979) Genesis and Development of a Scientific Fact, Bradley F and Trenn TJ (trans.). Chicago, IL: University of Chicago Press. Fraser CM, Norris SJ, Steinstock GM, et al. (1998) Complete genome sequences of Treponema pallidum, the syphilis spirochete. Science 281: 375–387. Garnett GP and Brunham RC (1999) Magic bullets need accurate guns – Syphilis, eradication, elimination, and control. Microbes and Infection 1: 395–404. Gray RR, Mulligan CJ, Sun ES, et al. (2006) Molecular evolution of the tprC, D, I, K, G, and J genes in the pathogenic genus Treponema. Molecular Biology and Evolution 23: 2220–2233. Hudson EH (1965) Treponematosis and man’s social evolution. American Anthropologist 67: 885–901. LaFond RE and Lukehart SA (2006) Biological basis for syphilis. Clinical Microbiology Reviews 19: 29–49. Morison SE (1942) Admiral of the Ocean Sea: A Life of Christopher Columbus. Boston, MA: Little, Brown and Company. Que´tel C (1990) History of Syphilis, Braddock J and Pike B (trans.). Baltimore, MD: Johns Hopkins University Press. Reverby SM (ed.) (2000) Tuskegee’s Truths: Rethinking the Tuskegee Syphilis Study. Chapel Hill, NC: University of North Carolina Press. Rothschild BM (2005) History of syphilis. Clinical Infectious Disease 40: 1454–1463. Rothschild BM and Rothschild C (1996) Treponemal disease in the new world. Current Anthropology 37: 555–561. St Louis ME and Wasserheit JN (1998) Elimination of syphilis in the United States. Science 281: 353–354.

Typhoid, Historical W C Summers, Yale University School of Medicine, New Haven, CT, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Background and History Taxonomy and Classification

Glossary carrier An individual who harbors and may excrete virulent bacteria while remaining free of symptoms of the disease. cyclogeny A theory, widely held in the 1920s and 1930s, that asserted that bacteria in cultures go through stages of a life cycle in which the bacterial cells may undergo changes in shape, size, staining, and biochemical properties. endotoxin Molecules contained within the bacterial cell, but which when released by lysis of the cell can

Defining Statement This chapter focuses on the historical aspects of typhoid fever in its clinical manifestations, epidemiology and etiology.

Background and History Typhoid is the classic ‘enteric fever’ which begins with malaise, anorexia, and headache, followed by high fever and abdominal tenderness and distention. Rose spots appear on the skin. Patients may become delirious, the ‘typhoid-state’ for with the disease is named (typhus/ ujo&: stupor), have shock from intestinal hemorrhage, and die. It often occurs in epidemics, especially under conditions of poor sanitation such as in military camps and after widespread natural disasters. Its symptoms and epidemiology overlap with that of typhus (camp fever), so typhoid had been confused with this rickettsial disease for much of its history. The name, typhoid, implies a ‘typhuslike’ condition, and these two diseases have been clearly delineated only since the mid-nineteenth century. The typhoid organism infects only humans and chimpanzees, entering the body through the alimentary tract. In the sick individual the organisms are usually found in the spleen, bone marrow, lymphoid tissue associated with the

Prevention and Treatment Epidemiology Further Reading

exert toxic effects on the host cells by a variety of biological mechanisms. flagellum A flexible fiber-like structure on the surface of some bacteria. Flagellar movement is responsible for bacterial motility. The flagellum is driven by an internal cellular structure, the ‘motor’, which is powered by a proton gradient across the cell membrane. Peyer’s patches Localized lymphoid tissue in the small intestine of mammals.

gut (Peyer’s patches), and almost always in the gall bladder. Much of the pathology and the symptoms of typhoid are the result of endotoxin which is released upon lysis of the bacteria. The typical incubation period is 7–14 days. Mortality in untreated cases is about 10%; 75% of these have intestinal hemorrhage or perforation. About 3% of clinically recovered patients still excrete the typhoid organism in the feces after 1 year and are designated as carriers. In England, Huxham (1739) in his ‘Essay on Fevers’ described the Plymouth epidemic of 1737, and he distinguished between putrid typhus (febris putrida) and slow nervous fever (febris nervosa lenta) that is now recognized as typhoid. As disease nosology evolved, and the concept of specific disease entities developed, by the nineteenth century typhoid and typhus were being recognized as diseases with distinct clinical features. The American physician and medical educator, Nathan Smith, provided the classic description of typhoid in 1824 in his essay ‘A Practical Essay on Typhous Fever’, in which he recounted his observations of what is clearly typhoid in the Connecticut River Valley in New England. The great Parisian clinician Pierre-Charles-Alexandre´ Louis named the condition fie`vre typho¨ıde in his major work on the disease in 1829. Scho¨nlein (1839) in Germany distinguished Typhus exanthematicus (typhus) and Typhus abdominalis (typhoid), terms which became established in the German literature.

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The English physician William Budd (1811–73), who studied under Louis at the La Pitie´ hospital in the 1830s, saw outbreaks of typhoid fever in his rural general practice and by careful observations on the spread of the disease concluded that typhoid was spread from person to person. This put him in the camp of the ‘contagionists’ who were vigorously opposed by the ‘anticontagionists’, physicians who favored the miasma theory of disease for diseases such as typhoid. Budd published his first detailed ‘natural history’ of typhoid (what would now be called an epidemiological study) in 1856. He described similar outbreaks in later publications, and even before the rise of bacteriology, Budd concluded that typhoid was spread by fecal contamination of water and milk. He also proposed that a convalescent patient could still be a source of contagion. Budd was an enthusiastic advocate of disinfection, both of drains and of privies as well as water supplies. This work on typhoid and its waterborne dissemination was contemporaneous and apparently independent of parallel work by John Snow on cholera in London. Carl Joseph Eberth described the typhoid organism in tissues of patients in 1880, and Georg Gaffky isolated and grew this organism in 1884. Since the typhoid bacterium is relatively easy to grow in culture, soon many different bacterial isolates were found in typhoid-like cases, both in humans and in other species. For example, in 1888 Gaerntner isolated what is now designated Salmonella enteritidis from a patient who had eaten contaminated meat, and shortly thereafter Durham and de Noeble isolated Salmonella typhimurium from gastroenteritis patients who also had eaten contaminated meat. The classification of these early isolates was based on the disease entity (Salmonella cholerasuis, i.e., hog cholera), the geographic place of isolation (e.g., Salmonella montivideo, Salmonella newport), or the name of the investigator (Salmonella schottmu¨lleri). In 1896 Gru¨nbaum and Widal independently observed that serum from infected or recovered patients could agglutinate dead typhoid bacteria, and this test (Widal reaction) became the basis for a serological diagnosis of infection as well as a tool to study Salmonella pathogenesis.

Taxonomy and Classification The early nomenclature of the typhoid bacillus is even more confusing and inconsistent as that of most bacteria. The typhoid organism was variously called Bacillus typhosa, Bacterium typhosum, Eberthella typhosa, Salmonella typhosa, and most recently Salmonella typhi. The genus is named in honor of Daniel Elmer Salmon (1850–1914), the first director of the Bureau of Animal Industry of the US Department of Agriculture and one of the founders of bacteriology in America. Salmon investigated hog cholera and, along with his prote´ge´ Theobald Smith, identified

the hog cholera bacillus (S. cholerasuis), long believed to be the cause of hog cholera (later found, however, to be caused by a filterable virus). When it was found that individual isolates could be distinguished serologically from one another, classification of the typhoid bacillus and its relatives was revised and based on the antigenic reactivities of a particular isolate. Some strains grew in more diffuse colonial form on surface cultures, and this growth form had come to be described as similar to the appearance of ‘breath’ (perhaps the water condensate) on the agar surface. These strains shared antigenic properties termed H-antigens (Hauch : breath). Later it was recognized that this diffuse colony morphology results from the motility of the individual organisms and that the H-antigens are associated with the flagella of these strains. The antigens of the strains without H-antigens were designated as O-antigens (Ohne : without), and are now recognized as ‘somatic’ antigens, belonging to the ‘body’ of the bacterium. A third category of serological reactions was believed to be related to virulence of the organism in experimental infections, and these antigens were designated as the Vi or virulence antigens. The H-antigens were observed to vary with the culture conditions, and this two-state variation was interpreted in terms of the cyclogenic theories of the interwar period as characteristic of different growth phases of the culture, hence the concept of ‘phase variation.’ Once the H-antigens were understood in terms of the flagellar antigens and structure, phase variation was reinterpreted as a problem in flagellar biosynthesis and regulation. Fritz Kauffmann (1937) and Philip B. White (1926) studied the antigenic structures of the typhoid and typhoid-like bacteria in detail, and the Kauffmann– White system of classification became standard. This system of classification of the genus Salmonella employs the H, O, and Vi antigenic specificities to identify a particular isolate. In 2005, the International Commission on the Nomenclature of Prokaryotes designated the ‘type’ culture for the genus Salmonella as Salmonella enterica. The species includes over 2300 serotypes, many of which were formerly designated as different species, for example, S. cholerasuis, S. typhi, Salmonella paratyphi A, and S. typhimurium. The variants are termed serovars, for example, S. enterica serovar Typhimurium.

Prevention and Treatment Although vaccines for typhoid have been in use for a long time, it was only in the 1960s that definitive evidence of their effectiveness was obtained through studies on human volunteers. The best vaccine preparations give over 90% protection for at least 3 years against challenges with doses of typhoid similar to that expected in contaminated water supplies. Higher challenge doses,

History and Culture, (and Biographies) | Typhoid, Historical

however, can overcome the vaccine-induced immunity and cause disease. Chloramphenicol (discovered in 1948) was the first effective drug to be used in typhoid, and it was followed by the newer penicillin derivatives as treatment for typhoid and other related Salmonella infections. In addition to vaccines and antibiotics, however, the most effective means to prevent and control typhoid have been sanitary treatment of sewage and establishment of clean drinking water supplies.

Epidemiology The epidemiology of typhoid, as exemplified by the work of William Budd, has been of great interest, both for medical and for sociological reasons. Based on epidemiological evidence, Robert Koch suggested that new cases of typhoid could come from convalescents long after the disease disappeared, and in support of this assertion, Drigalski (1904) isolated typhoid bacteria from individuals with no signs of illness. Observations such as these led to the concept of the healthy carrier of disease. Soon it was recognized that the gall bladder was a common site in which to find bacteria in these carriers, and ways were sought to eradicate the asymptomatic infections in order to eliminate the carrier state. Gall bladder removal (cholecystectomy) was a favored treatment. Less drastic approaches such as chemotherapy and various disinfectants were tried without success. Food contamination, usually by flies in summer, but sometimes by food handlers, led to the notion of the carrier as a danger to society. Quarantine and sometimes long-term detention of suspected or proven carriers were justified in the cause of protecting the public from contamination. Probably the most notorious and egregious case was that of Mary Mallon, an Irish immigrant domestic worker in New York, who seemed resistant to treatment and who was deemed uncooperative by the New York City Public Health authorities. As ‘Typhoid Mary’ she was de facto incarcerated in Riverside Hospital on North Brother

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Island in the East River by Manhattan for many years. Indeed, the term ‘Typhoid Mary’ has become generic for a carrier of a dangerous disease who is uncooperative in preventing its spread. Typhoid and its agent played another role in the American legal system when the city of Chicago constructed a drainage canal to help channel Chicago sewage from the Chicago river into the Mississippi River watershed. Downstream, the city of Saint Louis, Missouri was the potential recipient of the diverted sewage. Worried about typhoid outbreaks from Chicago, St. Louis went to court to block the Chicago plan. This case was the first in US history in which the new science of bacteriology was accepted as expert testimony in court. In circumstances of social disruption, such as wars, civil unrest, famine, or natural disasters which affect water and sewage systems, typhoid is still a major health problem. However, immunization, antibiotics, and sanitary measures have reduced the US typhoid mortality from 26 per 100 000 in the period 1906–10, to about 500 in the entire US population in 1967, to a few sporadic cases at present. See also: Enteropathogenic Infections; Epidemiological Concepts and Historical Examples; Gastrointestinal Microbiology in the Normal Host; Global Burden of Infectious Diseases

Further Reading Garrison FH (1929) An Introduction to the History of Medicine, 4th edn. Philadelphia: Saunders. Leavitt JW (1996) Typhoid Mary: Captive to the Public’s Health. Boston: Beacon Press. Moorhead R (2002) William Budd and typhoid fever. Journal of the Royal Society of Medicine 95: 561–564. Morgan HR (1965) The enteric bacteria. In: Dubos RJ and Hirsch JG (eds.) Bacterial and Mycotic Infections of Man, 4th edn., pp. 610–648. Philadelphia: Lippincott. Topley WWC and Wilson GS (1929) The Principles of Bacteriology and Immunity. New York: William Wood.

Typhus Fevers and Other Rickettsial Diseases, Historical C Socolovschi and D Raoult, Universite de la Mediterranee, Marseille, France ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Rickettsia and Human Rickettsioses Epidemic Typhus Murine Typhus Rocky Mountain Spotted Fever

Abbreviations AFR ATBF DDT DEBONEL ISFR ITTR MLST MSF

Astrachan spotted fever rickettsia African tick bite fever Dichloro diphe´nyl trichloroe´thane Dermacentor-borne necrosis–erythema– lymphadenopathy Israeli spotted fever rickettsia Indian tick typhus rickettsia multilocus sequence typing Mediterranean spotted fever

Defining Statement This article focuses on the historical aspects of rickettsial diseases, the causative agents and their arthropod vectors, and the diagnosis of epidemic and murine typhus, Rocky Mountain spotted fever (RMSF), Mediterranean spotted fever (MSF), African tick bite fever (ATBF), and other rickettsioses.

Introduction Rickettsial diseases existed since antiquity and have had great effects on the history of human populations. Massive population movements, poor hygiene, and famine favored the development of epidemics, and many wars and invasions were associated with or followed by outbreaks that included typhus. Typhus may cause more deaths than weapons during wartime. The Napoleonic wars were examples of the terrible consequences of a combination of war and diseases, and thousands of soldiers succumbed to typhus, as proven recently by molecular biology. Rickettsioses occurred not only in the history of the military, it has been a continuing threat to public health too. The serious danger of rickettsial diseases stimulated remarkable medical research and clinical observations, which clarified their specific etiology, mode of transmission, pathogenesis, therapy, and means of control. Because

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Mediterranean Spotted Fever African Tick Bite Fever Other Rickettsioses Conclusion Further Reading

MST ORF PABA PCR POB RMSF SFG TG TIBOLA

multi-spacer typing open reading frame p-aminobenzoic acid polymerase chain reaction p-hydroxybenzoic acid Rocky Mountain spotted fever spotted fever group typhus group Tick-borne lymphadenopathy

of their unique biological characteristics, such as small size, ranging to the limits of microscopic vision and beyond, ability to pass through a filter, inability to grow on bacteriologic media, and their obligate intracellular habitat in vertebrate and arthropod hosts, researchers had serious difficulties in identifying of rickettsial agents. Moreover, the rickettsial agents were treated as dangerous organisms due to their aerosol transmission, low infectious dose, and high risk-associated morbidity and mortality. Several microbiologists in the laboratory and physicians, particularly in the preantibiotic era, died of rickettsial infection like Howard Ricketts and Stanislav von Prowazek (Table 1). A remarkable progress was made in 1904 when Wilson and Chownin proposed related hypothesis on the transmission of rickettsia by tick bite. In 1909, Nicolle showed the louse transmission of typhus. In 1938, Cox demonstrated that rickettsiae could be cultivated in the yolk sacs of chick embryos. In 1948, it was demonstrated that broad-spectrum antibiotics were shown to be specifically and therapeutically effective. Tetracycline and chloramphenicol are the currently used antibiotics of choice in treating rickettsial diseases and have not been challenged since their discovery. The rickettsial scientific field has undergone significant evolution at the epidemiological, microbiological, and molecular levels in the past 20 years. Currently, the

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Table 1 The great scientists who died of typhus fever 1744–1814 1784–1850 1871–1910 1875–1915 1870–1915 1858–1915 1874–1915 1870–1916 1876–1916 1890–1921 1866–1922 1879–1922

Johann Bartholoma¨ us (Barthel) von Siebold, the ancestor of the ‘‘Wurzburg family’’ of physicians and scientists Zachary Taylor, president of the United States Howard Taylor Ricketts, researcher Stanislaus von Prowazek, zoologist and parasitologist Hugo Lu¨thje, scientist Georg Cornet, researcher, scholars of Robert Koch Georg Jochmann, physician of the Department of Infectious Diseases, Berlin Artur Pappenheim, Doctor Habilitatus, professor haematologist Paul Ro¨mer, the professor of Hygiene at the University of Halle Wolfgang Ga¨rtner, lecturer at the University of Kiel Arthur William Bacot, entomologist Edmund Weil, researcher

Rickettsia genus contains 24 recognized species, in contrast to three human pathogens and five endosymbionts as mentioned in the 1922 Brumpt list. In this article, we review the milestones of historical aspects of rickettsial diseases, discovery of the infectious agents, their vectors and mechanism of transmission and reservoir, and the major technological developments that have facilitated the study, control, and treatment of rickettsiae and rickettsial diseases.

Rickettsia and Human Rickettsioses The term ‘rickettsia’ applies only to arthropod-borne bacteria, belonging to the genus Rickettsia within the family Rickettsiaceae of the order Rickettsiales. Rickettsiae are small Gram-negative microorganisms that grow in association with eukaryotic cells. These bacteria are described as obligate intracellular organisms that retain basic fuschin when stained by the method of Gimenez. Rickettsial cells divide by binary fission for multiplication and can be cultivated in cell cultures, embryonnated eggs, or susceptible animals. Members of the Rickettsia genus are closely related phylogenetically and have a high degree of 16S rDNA sequence homology to each other. Historically, cross reactions of patient’s sera with somatic antigens of strains of Proteus OX19, OX2, and OXK were used to classify rickettsial isolates into three groups, namely, the spotted fever group (SFG), the typhus group (TG), and the scrub TG. Subsequently, the scrub typhus species Rickettsia tsutsugamushi was reclassified within the genus Orientia. Other phenotypic criteria that have been used to describe Rickettsia species include the geographical distribution of the strain, the arthropod vector, pathogenicity in humans, mice, and guinea pigs, optimal culture temperature, time for plaque formation, size of plaques, growth in embryonated chicken eggs, and hemolytic activity. Recently, guidelines were proposed to classify rickettsial species and the diseases they cause.

Presently, the genus Rickettsia, with 24 currently validated species, is divided into two main groups: the TG with Rickettsia prowazekii and Rickettsia typhi and the SFG that accounts for most tick-borne rickettsiosis and also Rickettsia felis, which is the agent of flea-borne spotted fever and Rickettsia akari is the agent of Rickettsialpox transmitted by mite (Table 2). Rickettsial organisms are mainly transmitted to humans by bites from infected arthropods. However, infections from aerosols of infected insects, feces, and blood transfusions have also been described. Besides the known pathogens, many other rickettsial strains have been found in arthropods, in particular ticks, but their roles as human pathogens have yet to be determined. New developments in the epidemiologic profile of rickettsioses include the emergence of spotted fever rickettsioses due to Rickettsia rickettsii in Argentina, Brazil, Columbia, and the United States; identification of other Rickettsia species in ticks in South America and Mexico; and outbreaks of louse-borne typhus in Burundi, Russia, Peru, and Algeria. To date, 16 known rickettsioses are recognized and represent a global problem. Rickettsioses are seasonal outbreaks and the arthropod host determines their epidemiology and geographic distribution. The clinical presentation of rickettsial diseases can vary from mild to very severe, with the case fatality for highly virulent rickettsiae ranging from 2 to >30% (Table 3). The main clinical signs include fever, headache, rashes that are maculopapular or sometimes vesicular, inoculation eschars at the site of the arthropod bite, and localized lymphadenopathy. The severity of the diseases varies with the pathogen virulence and the host factors. The main pathologic mechanism in rickettsioses is vasculitis following infection of the endothelial cells. Rickettsiae represented a paradigm of reductive evolution. The reconstruction of ancestral genomes, using seven Rickettsia genomes that inferred the origin and fate of the genes found in today’s species, indicates that their last common ancestor contained more genes, but already possessed most traits associated with cellular parasitism.

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Table 2 Historical data on diseases caused by Rickettsial species Disease and etiological agent

Event

Researcher

Siberian tick typhus R. sibirica

The first account of disease (1930) Description of disease, ‘Primary tick borne disease’ or ‘far Eastern tick-borne typhus’ (1936–37) Discovery of tick vector of the disease and the mammalian reservoir (1938) Isolation of R. sibirica in guinea pig (1938) Naming of the disease: Siberian tick typhus (1944)

Mil Mil, Antonov, and Naishat Unknown

Rickettsial pox R. acari

Description of Rickettsial pox and isolation of R. acari (1946) Discovery of the role of rodents and mites (1947)

Huebner Pomerantz

Queensland tick typhus R. australis

Isolation of R. australis from human (1946) Description of diseases (1946) Naming of the rickettsia (1950)

Andrew et al. Plotz and Smadel Philip CB

Tick-borne lymphadenopathy (TIBOLA) R. slovaca

Isolation of R. slovaca from Dermacentor marginatus ticks (1968) First documentation of a human case of infection (1997) Naming the clinical syndrome: TIBOLA (1999) DEBONEL: Dermacentor-borne necrosis–erythema– lymphadenopathy (2004) Determining the genome sequence (2007)

Brezina et al. Raoult et al. Lakos A Oteo et al.

Japanese spotted fever R. japonica

Description of diseases (1984) First documentation of human cases (1985) Isolation of R. japonica (1985) Naming the rickettsia (1992) First identification in ticks (1996)

Mahara Mahara Uchida et al. Uchida et al. Mahara

Far east spotted fever R. heilongjiangensis

Isolation of rickettsia and first identification in ticks (1982) Description of rickettsia (2003) Description of diseases (2004)

Lov Fournier et al. Mediannikov et al.

Flinders island spotted fever R. honei

Description of diseases (1991) Isolation of R. honei from human samples (1992) First identification in the ticks (1993) Naming the rickettsia: R. honei ep.nov (1998)

Stewart Baird Graves et al. Stenos et al.

Lymphangitis-associated rickettsioses R. sibirica mongolitimonae

First identification in Hyalomma asiaticum ticks (1991) Culture and identification of rickettsiae (1993) First clinical description (1996) Naming the rickettsia (2005)

Yu et al. Yu and Raoult Raoult et al. Fournier et al.

Flea-borne spotted fever R. felis

First account, detection in Ctenocephalides felis (1918) Detection in cat fleas (1990) First case of flea-borne spotted fever, the name ‘Elb agent’ (1994) The name of rickettsia (1996) Culture of R. felis (2000) Genome sequence (2005)

Sikora H Adams et al. Schriefer and Azad Higgins et al. Raoult et al. Ogata et al.

R. aeschlimannii

Isolated from H. marginatum marginatum (1992) The name: R. aeschlimannii (1997) First case of infection (2002)

Beati et al. Beati and Raoult Raoult et al.

R. helvetica

Isolation of R. helvetica from Ixodes ricinus (1979) The name of rickettsia (1993) First case of acute infection (1997)

Burgdorfer and Peter Beati et al. Fournier et al.

R. parkeri

Isolation of rickettsia from Amblyomma maculatum ticks (1939) The name: R. parkeri (1965) First case (2004)

Parker

Isolation of R. massiliae from Rh. sanguineus tick (1992) The name: R. massiliae (1993) First recognition of infection (2005) Genome sequence (2007)

Beati et al.

R. massiliae

R. raoultii

First detection in Ixodid ticks (1999) Isolation of Rickettsia sp. ‘DnS14’ (2001) The first human case (2007) The name of rickettsia (2008)

Korshunova et al. Pavlovsky

Raoult D

Lackman et al. Paddock et al.

Beati et al. Vitale et al. Blanc et al. Rydkina et al. Oteo et al. Fournier et al.

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Table 3 Effect of specific antibiotics in the course of major rickettsiosses Disease

Rocky Mountain spotted fever Epidemic typhus Murine typhus Scrub typhus

Untreated Average duration of the fever (days) 16 14 12 14

Mortality (%)

Treated Average duration of the fever after treatment (days)

Mortality (%)

21 30–40 2–5 15

3 2 2 1

0 0 0 0

The obligate endocellular microorganisms tend to evolve faster as a consequence of reduced effectiveness of selection, and suggest a major role of enhanced background mutation rates on the fast protein divergence in the obligate intracellular -proteobacteria.

10%), and other cases reported in Rwanda, Russia, Peru, the United States, and Algeria are reminder that epidemic typhus can reemerge as a result of a catastrophic breakdown of social conditions. The clinical onset of epidemic typhus is usually abrupt after an incubation period of about 14 days. Classical epidemic typhus is the most severe rickettsial disease, with symptoms including a high fever, headache, chills, myalgias, conjunctival injection, and rales. Erythematous macules (2–6 mm) appear most often on the trunk on day 5 and later on the extremities; the face, palms, and soles are usually spared. The characteristic rash of typhus is also the origin of more descriptive names, such as spotted fever in English, Fleckfieber in German, and thyphus exanthe´matique in French. Myocarditis, pulmonary involvement, severe neurologic complication, and gangrene of the distal extremities may occur in severe cases. Without the availability of antibiotics, the disease is fatal in 13–60% cases (Table 3). Flying squirrel-associated typhus (sylvatic form) has been less severe (fever, headache, maculopapular rash, confusion, and myalgia), with no fatalities records. Once infected with R. prowazekii, convalescent patients maintain the infection for all their life, and with weakening immunity recrudescent typhus, Brill–Zinsser disease may occur. The symptoms of recrudescent typhus are less pronounced, and the associated mortality rate is

Typhus, the ‘spotted fever’ of the sixteenth century in England, the ‘gaol fever’ of the eighteenth, and the ‘Irish fever’ of the mid-nineteenth centuries, has a long and distinguished history intimately associated with the social upheavals caused by war and famine. Greek, etymology of typhus, meaning smoky or hazy, was originally applied by Hippocrates to the confused states of mind frequently associated with high fevers. Some historians such as Haeser and Hecker contend that the disease was described by Thucydides during the Plague of Athens (430–425 BC), but recently typhoid fever was reported as the causing agent. The first account of typhus was recorded during the civil wars of Grenada 1489–90, when typhus was introduced in Spain by soldiers from the island of Cyprus (Table 4). The distribution of typhus from Spain to Italy, France, and then northward continued in an almost uninterrupted succession of small outbreaks. The disease was described by several physicians such as Girolamo Fracastorius in 1546 in De Contagione et Contagiosis Morbis, referring to the Italian epidemics of 1505 and 1528 and distinguished it from plague; Girolamo Cardano in his book De Malo recentiorum medicorum Ursu Libellus in 1536; and by Von Zavorziz in 1676 in The Infection of Military Camps. Villalba suggests that typhus was transported from Spain to the New World during the first half of the sixteenth century, but historical evidence suggests that typhus fever existed among South American natives in pre-Columbian days and recognizable epidemics occurred in Mexico before the arrival of Cortez at Vera Cruz in 1519. Moreover, there is a legend, credited by Bernal Diaz and by Nicolas Le´on, that typhus destroyed the Toltec city of Tollan in 1116 AD. The description of typhus fever in Mexico was done by missionaries in 1545. Toward the end of the sixteenth century, typhus killed

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Table 4 Milestones of epidemic typhus Discovery of the epidemic typhus Rickettsia prowazekii

Author

1489 The first account of typhus 1739 First distinctions between typhus and typhoid 1760 Description of exantematic typhus 1810 Description of diseases 1837 Histological distinction between typhus and typhoid 1909 Role of body lice in typhus and transmission to chimpanzee and monkeys 1910 Description of recrudescent form 1910 Serology test based on Proteus for typhus fever 1911 Isolation of R. prowazekii in guinea pig 1916 Description of bacteria and named R. prowazekii, the genus name after H. Ricketts and the species after the Polish researcher Stanislas von Prowazek 1916 Weil–Felix test for typhus fever 1922 Human histopatology 1930 Typhus vaccine 1934 Tissue cultures 1937 Cell culture (mouse and chick embryos in Koll flasks) 1938 Role of feces of the louse 1938 Culture in yolk sacs of embryonated chicken eggs 1942 The antirickettsial action of p-aminobenzoic acid 1948 Treatment by tetracycline and chloramphenicol 1952 The base ratio of DNA 1956 Vaccine Madrid E attenuated 1975 Reservoir: Flying squirrels (Glaucomys volants) 1998 Genome sequence

more than 2 million native Indians in the Mexico highlands. It is tempting to speculate that typhus was born in the chance meeting of an American rickettsia and a Spanish louse. A century after the Thirty Years’ War (1618–48), when the epidemic typhus spread in Europe (Germany lost three-fourth to one half of her total population), Huxham, in 1739, made the first distinctions between typhus and typhoid (Table 5). Boissier de Sauvages confirmed this in the eighteenth-century in France and called it exantematic typhus. In 1836, William Gerhard of Philadelphia clearly distinguished between typhus and typhoid fevers based on postmortem findings of six patients revealing the remarkable absence of ulceration of Peyer’s patches, contrary to observation of typhoid. By the 1860s, the physicians distinguished typhus from another louseborne disease, relapsing fever (Borrelia recurentis), while problems of diagnosis remained. Later, Bacot established that trench fever and typhus were different diseases; the former was caused by Bartonella quintana and the latter by R. prowazekii. This was tragically confirmed by a typhus infection acquired by him in 1922. In 1909, Nicolle showed the transmission of the disease through the blood of a typhus patient to a chimpanzee and from the chimpanzee to a macacus monkey. The disease was then transmitted from macacus to macacus via the louse. In 1911, the typhus patient transmitted the infection to the guinea pig. Although fever was the only sign of the disease, the guinea pig remained for many decades the

Huxham Boissier de Sauvage Johann V. von Hildebrand William Wood Gerhard Charles Nicolle (Nobel prize) Nathan Brill Wilson Charles Nicolle Henrique da Rocha Lima Weil and Felix Wolbach Rudolph Weigl Kligler and Aschner Zinsser and Schoenbach Starzyk Cox Snyder Woodward Wyatt and Cohen Clavero and Gallardo Fox Bozeman et al. Andersson et al.

most useful experimental animal. Later, Wolbach detailed the clinical observations and the human histopathology, and the effect of R. prowazekii infection on guinea pigs. During 1896–1910, Nathan Brill, in New York, studied 221 febrile patients whose illness reminded him of typhus rather than typhoid fever, in spite of protestations of his colleagues. Negative blood cultures and Widal reactions led him to call it an illness of unknown cause; it was a mild form of typhus fever, later named as Brill’s disease. In 1933, Zinsser and Castenada isolated R. prowazekii from these cases. Through careful analysis of epidemiological data, Zinsser concluded that it is a recrudescent typhus case and it served to maintain endemic prevalence of R. prowazekii by bridging breaks in the chain of man– louse–man propagation. Zinsser’s hypothesis was confirmed by Murray in 1950.

Infectious Agent: Rickettsia prowazekii In 1910, Ricketts, while working on the etiology of typhus fever in Mexico, speculated that the agent of typhus had the same morphology of bubonic plague and Rocky Mountain spotted fever (RMSF) organism. He described bacillary or pleomorphic bodies, with bipolar dark staining and intervening pale areas, in the blood of patients suffering from typhus and in the dejecta of lice that had fed on these patients. However, these descriptions do not fit with our current knowledge and represent something else. In 1911, Charles Nicolle isolated these bacteria from guinea pig. In

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Table 5 Milestones of suspected outbreaks of epidemic typhus Years

Country

Number of deaths

1489–90 1528 1542 1545 1552 1557–59 1618–49

Civil wars of Grenada, Spain Naples, Italy Hungary Mexico Metz, France

17 000 Spanish soldiers 21 000 French soldiers 30 000 German and Italian soldiers 2 million native Indians 10 000 soldiers 10% of the English population One half of total population of Germany 60 000 deaths in Lyon and 25 000 Limoges Thousands???? 550 000 French soldiers 61 964 Russian soldiers 2 million people throughout Europe 250 000 people in Germany 700 000 people 20 000 Irish immigrants 134 000 soldiers 150 000 Serbs and 30 000–60 000 Austrian prisoners 3 million Russian people (30 million cases) 150 000 Serbs and 60 000 Australian prisoners 23 000 cases 77 000 cases 17 000 29 cases 43 345 cases

1813–14

The Thirty Years War, France (1628) The Seven Years War, Europe Napoleon’s invasion of Russia (confirmed by PCR assay) Europe

1816–19 1847 1854–56 1914 1917–25

Ireland Canada Crimean war Australian invasion in Serbia World War I and Russian Revolution

1942

Egypt French North Africa World War II Russia Burundi

1756–63 1812

1945 1989 1997

1913, Hegler and von Prowazek described appearances of neutrophilic leucocytes in 51 typhus cases and in a smear from louse, which they believed to be microorganism. In 1914, in Istanbul, and in 1915, in eastern Germany, Rocha Lima and Stanislaus von Prowazek confirmed the observations of previous workers, and together they described R. prowazekii (Figure 1). In 1916, Rocha Lima grouped the microorganisms in the order Rickettsiales and named the agent of typhus R. prowazekii after Ricketts and his deceased friend von Prowazek. In the same year,

Edmund Weil and his English associate Arthur Felix showed that a strain of Proteus bacillus was agglutinated by the sera of typhus patients. Some European bacteriologists declared that the Proteus bacillus was the ‘exciting organism’ of typhus, while others argued that Proteus and the typhus virus were simply variants of the same organism. The debate about infectious agent was resolved in 1937 by Herarld Cox with the development of the tissue culture technique. Later, cross adsorptions showed that there was a common cross-reacting epitope among Legionella bozemanii Wiga, Rickettsia TG, and Proteus OX19, due to a common lipopolysaccharide antigen. In 1998, the complete genome sequence (1.1 Mb) of R. prowazekii showed 834 protein coding genes, contains the highest proportion of noncoding DNA (24%) detected so far in a microbial genome and the phylogenetic analysis proved new evidence of an evolutionary relationship between rickettsiae and intracellular mitochondria. Disease Vector: The Human Body Louse (Pediculus humanus corporis)

Figure 1 Rickettsia prowazekii in the L929 cell culture (day 6) by Gimenez staining.

In 1740, James Lind, a physician observed that typhus was carried on the bodies of men, on clothes; but only in 1909, Charles Nicolle discovered the role of lice in transmission of typhus, later receiving the Nobel Prize (Figure 2). By 1903, Tunis was full of typhus patients; Nicolle observed that patients infected others on the street and their clothing was infectious. After the patients had a hot bath and

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History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical

Figure 2 The human body louse, Pediculus humanus humanus (P. h. corporis).

were dressed in hospital clothing, they ceased to be infectious. Later, he fed uninfected lice on an experimentally infected bonnet monkeys (Macacus sinicus) and then transferred the lice to uninfected monkeys that later developed typhus. This accomplishment became a powerful impetus for the establishment of delousing procedures. In the same year, Ricketts and Wilder confirmed these results in Mexico City and N. F. Gamaleia in Russia. In 1916, Da Rocha Lima discovered that the R. prowazekii multiplied within the epithelial cells of the louse’s stomach, which was confirmed by others researchers, such as No¨ller; To¨pfer, and Schu¨ssler. In 1922, Wolbach observed that R. prowazekii escapes from alimentary tract through feces and they supposed that it may be introduced by scratching or by the mouth parts of the louse after becoming soiled with feces. They did not see rickettsia in the salivary glands or in the esophagus of louse. At that time, it was difficult to investigating lice as many were infected by B. quintana (Rickettsia pediculi). However, only in 1938, Starzyk demonstrated that patients are infected by the faces and not by the bite of the louse, and R. prowazekii could be transmitted to man by inhalation or in a conjunctive way. The role of lice as a vector of epidemic typhus has been studied in many countries, and in 2002, by using modern tools, an experimental model was established,which reproduces the natural infection of body louse. The question of survival for R. prowazekii during interepidemic periods has led to several rather controversial hypotheses about reservoir. As suggested by Zinsser, after his study of 538 Brill’s disease cases, the main reservoir appears to be in humans, because lice die of the infection and humans who contract typhus retain some rickettsiae for the rest of their life. This finding was confirmed by Nicolle (1934), Giroud (1948), Mooser (1946), Willsch (1952), Gear (1952), and Murray (1951). In contrast, Reiss Gutfreund in 1956 found, in Ethiopia, R. prowazekii in domestic animals and their ticks.

R. prowazekii was isolated from adult Amblyomma variegatum and Hyalomma marginatum rufipes and Hyalomma truncatum in Ethiopia. Transmission from experimentally infected larvae to nymphs took place in 50% of A. variegatum and Amblyomma lepidum. R. prowazekii was maintained for several years in experimentally infected Ornithodoros moubata and in Ornithodoros papillipes; however, adverse effect on Dermacentor marginatus and Discoglossus pictus ticks has been reported. Recently, R. prowazekii was detected by molecular detection and isolated by shell vial culture in Amblyomma ticks collected in Mexico. Later, Dyer and Mooser found R. prowazekii in the fleas, and several experiments to infect fleas with R. prowazekii were reported. The head louse Pediculus humanus capitis is capable of maintaining R. prowazekii experimentally; its role in the transmission of this rickettsiosis is not well established. An extensive work by Ormsbee and McDade failed to support the hypothesis that an extrahuman reservoir of infection plays an important role in the ecology of epidemic typhus. The search was futher stimulated by the isolation of Rickettsia canadensis from a pool of ticks taken from rabbit. While investigating the ecological niche of R. canadensis, Bozeman unexpectedly recovered several strains of R. prowazekii from flying squirrels (Glaucomys volans volans) that had been captured in Virginia and Florida. Later, it was found in their ectoparasites. Thus, the hypothesis of a widespread extrahuman reservoir of epidemic typhus was confirmed. Diagnosis and Treatment of Epidemic Typhus: Historical Aspects Serology: During the first two decades of the twentieth century, numerous attempts were made to cultivate the bacteria from the blood, urine, and stools of typhus patients. In 1910, Wilson reported the isolation of a coliform bacterium, designated Bacillus U, from stools and rarely from urine, and not from blood. His work demonstrated that the bacterium was agglutinated by sera of typhus patients to a titer 3–10 times greater than by sera from normal individuals or patients with other diseases. Comparable results were obtained in 1916, in Berlin, by Weil and Felix with a Proteus isolate from patients’ urine. Other agglutination tests were suggested like agglutination with Vibrio cholerae by Cantacuze`ne (1919), with Pseudomonas aeruginosa, V. cholerae infection, Bacillus prodigiosus, and Bacillus subtilis by Violle (1920). Epstein and Morawitz named it ‘the Weil–Felix test’ in honor of the researchers, and it remained for many decades the chief tool for the serological diagnosis of the typhus fevers. Staining rickettsiae: Rickettsiae stained poorly with the Gram stains, most of early observations were made on smear and sections stained with Giemsa. In 1930, Castenada substituted, to some advantage, the azur II and eosin of Giemsa’s with methylene blue and safranin.

History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical

More successful was the procedure introduced in 1937 by Macchiavello using basic fuchsin (rickettsiae retain the red strain), following decolorization with citric acid and counterstaining with methylene blue. In 1964, Gimenez changed the methylene blue with malachite green. This procedure has gained wide acceptance until now (Figure 1). Culture: In 1937, Kligler and Aschner proposed the same technique for cultivating R. prowazekii such as the one presented earlier by Nigg and Landsteiner for murine typhus using Maitland cultures in tissue cultures. Zinsser found that greater yields of rickettsiae could be obtained on agar cultures with the same constituents. In later experiments, he used Kolle flasks for the mass cultivation of rickettsiae from minced tissues obtained from mouse and chick embryos. Herald R. Cox’s discovery of using the yolk sacs of chick embryos for cultivating rickettsiae in 1938 had an enormous impact on rickettsial technology, paved the way for a new generation of vaccines and diagnostic reagents, and facilitated studies of rickettsial susceptibility to chemotherapeutic agents and rickettsial physiology. In the following years, R. prowazekii was cultivated in chick fibroblast cell culture, in cotton rat macrophages, in guinea pig macrophages, L929 cell line mouse, vero cells (kidney epithelial cells extracted from African green monkey), and NIAS-AeAl-2 insect cells. Recently, R. prowazekii was isolated from a blood sample from a patient by the shell vial cell culture technique using human embryonic lung fibroblasts. Control and eradication: The first prevention method used for the patients with epidemic typhus was double delousing process, more complete than that practiced on admission to the hospital using kerosene and ‘lightwood oil’. This method was used since eighteenth century by James Lind in America and later by all physicians after Nicolle discovery. Dichloro diphe´nyl trichloroe´thane (DDT), a poisonous powder for insects, was used in Mexico, Algeria, Italy, and Egypt for delousing. In 1988, Darby proposed a protocol of powder dusting of the entire clothing with 10% DDT, 1% malathion, or 1% permethrin dust, but a resistance of lice to DDT was detected in Korea and Japan. A standard treatment protocol with 1% permethrin has been created by WHO in 1993. Because treatment should be repeated every week, of 3–5 tons of powder is required to treat 100 000 people on one occasion. In 1987, Strong and Brown sowed the therapeutic efficacy of ivermectin to eradicate lice. Treatment: In 1919, Danielopolu recommended to use strophantin or chlorine injection for typhus treatment. Some investigators employed large doses of the serum of convalescent. Digitalis or strychnia was given for possible supportive effect in most cases, and in case of a collapse camphor or caffeine. Preantibiotic chemotherapy of rickettsial diseases was dominated by p-aminobenzoic acid (PABA), discovered in 1942 by Snyder, and

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separately in 1944 by Greiff. In 1951, Snyder and Davis showed that the growth of rickettsiae is inhibited by PABA and requires p-hydroxybenzoic acid (POB); later, Weiss isolated PABA-resistant mutants of R. prowazekii, suggesting a role of POB in rickettsial biosynthesis. The newly discovered antibiotic compounds, namely, the tetracyclines family and chloramphenicol changed the natural history of epidemic typhus. Initial clinical trials, carried out by Payne and Smadel, demonstrated the effect of chloramphenicol on patients ill with epidemic typhus. Chloramphenicol treatment in recrudescent typhus fever (Brill’s disease) produced defervescence in approximately 4 days. Now, recommended treatments for epidemic typhus are doxycycline and chloramphenicol. Therapy for a minimum of 5 days and at least 2–4 days after defeverscence should be provided to preclude relapses, especially with early treatment. In case of an outbreak, a single 200-mg oral dose of doxycycline is extremely efficient. Vaccination and prevention: One of the earliest protective measures for typhus was consuming human lice. The value of this procedure is undocumented. Based on the protocol of RMSF vaccines, Rudolph Weigl developed, in 1930, a similar vaccine against epidemic typhus from the intestine of experimentally infected lice. Initially, more than 100 infected lice were needed to produce a single dose of vaccine; later 90 lice provided three doses of the vaccine. This vaccine was produced on a large scale in Weigl’s laboratory before World War II in Poland and was used in China (1936, 1943), Ethiopia (1939), and other countries. After discovery of cultivation of R. prowazekii in embryonated chicken eggs, it was possible to produce very large quantities of live Rickettsiae, inactivated by 1.5% solution of phenol. A total of 5–6 millions of individuals were vaccinated against typhus during German occupation in the eastern zone of war operations. Two additional attempts of inactivated vaccine production deserve to be mentioned, one in the guinea pigs on a vitamin-deficient diet or by X-ray radiation made by Zinsser and Castenada in 1932 and the E strain vaccine reported by Fox in 1956, as they provide information on the biology of rickettsiae. The isolation of vaccine strain E, originated from a 1941 typhus case in Madrid, was passed routinely into eggs and reduced virulence for guinea pigs after the 11th passage. Extensive field trials were conducted in Peru and Burundi with strains passed into eggs at least 256 times.

Murine Typhus Murine typhus, caused by R. typhi, was found throughout the world, and found to be prevalent in tropical and subtropical seaboard regions, where the most important rat reservoirs (Rattus spp.) and flea vectors (Xenopsylla cheopis) (Figure 3) are found, during warms months of year. It is difficult to

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History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical Table 6 Milestones of murine (endemic) typhus Discovery on murine (endemic) typhus: Rickettsia typhi 1913 First clinical description 1920 The first name: Dermacentroxenus typhi 1921 Identification of R. typhi (guinea pig) 1924 The first study on typhus 1926 Isolation of R. typhi from the blood 1926 The first hypothesis of reservoir and vector of diseases 1930 Role of fleas and rat 1930 Tissue cultures (guinea pig tunica tissue) 1931 Mechanism of transmission 1940 Distinction of scrub typhus 2004 Genome sequence

Authors Paullin Wolbach and Todd Mooser Stuart Maxcy Maxcy Dyer and Mooser Nigg and Landsteiner Ceder and Dyer Lewthwaite and Savoor McLeod MP et al.

Figure 3 Xenopsylla cheopis femelle, the flea vector of endemic typhus.

establish the true incidence of the disease, and the number of reported cases does not reflect the true prevalence, because the patients usually mark a rather mild course and present with nonspecific symptoms. During the 1-month attack of Khmers in Thailand, the rate of murine typhus infection was 172 in 100 000 adults. Murine typhus may occur in travelers returning from endemic regions. After an incubation period ranging from 6 to 14 days, the clinical manifestations were high fever, severe headache, chills, myalgia, weakness, and nausea. The rash described as macular (49%), maculopapular (29%), papular (14%), petechial (6%), and morbiliform (3%) is usually centrally distributed on the trunk, but also found on the extremities. Childhood murine typhus is often mild, with fever (100%) and rash (57%), lymphadenopathy (usually cervical (37%)), and severe headache (29%). Splenomegaly or hepatomegaly was less frequent findings (24 and 10%, respectively). Physicians practicing in or near R. typhi endemic areas need to consider murine typhus in the differential diagnosis with a febrile illness without a clear source of infection. Occasionally, patients develop some complications: central nervous system abnormalities (9.6%), renal insufficiency, hepatic insufficiency, respiratory failure are seen in older patients. There was one death in Maxcy’s series of 114 cases, no deaths in a series of 180 cases by Stuart and Pullen, and 13 of 345 cases reported by Dumler. Historical Aspects of Murine Typhus Disease From the time of Brill’s report, a sporadic typhus-like illness was designated throughout the world as ‘endemic typhus’ to distinguish it from the more serious classic louse typhus. Ricketts and several of the early researchers wondered if Mexican and European forms of typhus were

the same diseases. Paullin made the first clinical description of a ‘milder form of typhus’ without mortality in 1913 in Atlanta (Table 6). In 1917, Neil noted that male guinea pigs inoculated with the blood of typhus patients in south Texas often developed a scrotal swelling and inflammation, along with hemorrhage beneath the tunica, similar to the lesions elicited by R. rickettsii. His results, confirmed in 1928 by Mooser, showed that the American and the European variety could be clearly differentiated by their reactions in the guinea pigs. These studies contributed in distinguishing the endemic from the classic typhus. It is difficult to differentiate when the workers were dealing with flea-borne typhus and when with epidemic typhus; both forms were present and both were referred to as Mexican typhus. For several years the murine typhus was misdiagnosed, the cases observed in 1920 in Rome by Carducci would be, according to Pe´cori, of the cases of murine typhus. In 1929 in Sao Paulo, during an outbreak of tabardillo, researchers were able to isolate the bacteria not only from ticks (Amblyomma spp.), but also from fleas, urban rats, and small mammals. In 1923, in the Annual Reports of the Department of Health of Palestine, there was a reference to a mild course of typhus fever, similar to Brill’s disease and different from the classic form of typhus. Stuart, in 1924, made the first comprehensive study on typhus in Palestine, and Fletcher and Lesslar in Malaya. In 1925, an outbreak of 200 cases of endemic typhus occurred in Australia. Plazy, Marcandier, and Pirot observed the first Mediterranean cases of murine typhus in sailors on the warships of Toulon (France), Blanc in Morocco, and Nicolle in Tunis (Tunisia). During 1926 and 1932, 135 cases of murine typhus on the warships of Toulon were noted. Le´pine in Greece and in Liban observed identical

History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical

cases, and showed the presence of the microorganism in the brains of infected rats. In 1935–36, Suarez, Palacios, Chavez in Chili, and Sesnic and Giroud in Tunisia identified asymptomatic cases of murine typhus. In the period 1931–46 42 000 cases were reported in the United States. Late reports established the worldwide distribution of endemic typhus. In 1940, Lewthwaite and Savoor reiterated the important distinction between the X. cheopis-borne murine typhus (‘shop typhus’) and the chigger-borne scrub typhus (‘tsutsugmushi’ or ‘rural typhus’). The designation ‘murine’ typhus was adopted since the infectious agent was well entrenched in the rat population and its flea throughout the world. About the origin of murine typhus, Traub suggested that the R. typhi has always associated with the genus or subgenus Rattus, and that, in its ancestral homeland, which was probably in southeast Asia, ectoparasites other than X. cheopis were the original intramurid vectors. In contrast, murine typhus existed in a sylvan cycle involving other small mammals (theraphions) and ectoparasites, but spilled other into Rattus, which proved to be unusually well adapted to maintaining it and disseminating it over the world and bringing it in contact with man. The nature of such an aboriginal cycle and its geographic location are yet unrecognized, but the data to date indicate that northern Africa, which was believed to be the origin of X. cheopis, is unlikely to be involved.

The Infectious Agent: Rickettsia typhi In the late 1920s and early 1930s, through the efforts of many investigators, the agent of endemic typhus was considered more virulent for the rat than R. prowazekii. There was considerable discussion of whether they were variants of the same species or two different species. In 1920, Wolbach and Todd proposed the designation Dermacentroxenus typhi for the Mexican variety of typhus. With time D. typhi was changed to R. typhi, although some rickettsiologists still prefer the designation R. mooseri in honor of Herman Mooser. In 1926, Maxcy isolated rickettsia from the blood and noted their antigenic similarity and differences with R. prowazekii and R. rickettsii. In 1930, Dyer isolated the suggested new ‘virus’ from fleas collected in Baltimore during an outbreak of three cases of typhus. Mooser recovered the microorganism from brains of rats in a prison of Mexico City, where several immates had presented typhus fever. The studies of Lepine, Lorandos, Lemiere, Combiesco (1934), and Combiesco (1935) led to the isolation of bacteria from cats and dogs. R. typhi is an obligate intracellular bacterium that infects endothelial cells in mammalian hosts and midgut epithelial cells in the flea host. In 2004, complete genome (1 111 496 bp) analysis of R. typhi revealed 877 genes, 40 pseudogenes, and 838 proteins.

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Murine Typhus Vectors In 1926, Kenneth Maxcy, a wise epidemiologist with a clinical background, observed patients and the environments related to their illness, particularly in Alabama, Georgia, North Carolina, and Virginia. He concluded ‘‘that the reservoir is rodents, probably rats or mice, from which the disease is occasionally transmitted to man. The parasitic intermediaries which are first suspected are fleas, mites, or possible ticks’’. Hone in 1927 postulated the same theory, and the vector other than the louse for endemic typhus was reported in Palestine. Maxcy’s theory was validated by two different groups. In 1930, a physician-pharmacist, Dr. J. Lipsky presented fever, headaches, and a rash. His physicians diagnosed typhoid fever, but blood cultures and the Widal reaction were negative, and the Proteus OX19 reaction reached a titer of 1:320. Endemic typhus fever was the discharge diagnosis. On the day of discharge, Dr. Lipsky’s female clerk was hospitalized and placed in the same room with the same illness, as was the male clerk 3 weeks ago. Dyer and his associates, Rumreich and Badger, successfully transmit the rickettsial agent to guinea pigs from rats’ brains, and fleas, trapped in the basement of Dr. Lipsky’s drugstore, Baltimore. They identified the murine reservoir of the rickettsia and incriminated the flea as a vector, showing that the organisms persisted in rat fleas for at least 9 days and were present in feces of infected fleas. The authors found that the flea X. cheopis became highly infectious on the fifth or sixth day, remained infectious for 40 days without evidence of ill effect with an inoculum representing 1/128 000 of a flea. In the same year, Mooser recovered the microorganism from the brains of rats in a prison in Mexico City, where several inmates had presented typhus fever. The attempt of these two groups elucidates the epidemiological cycle of the disease by revealing the reservoir (rat) and the vector (fleas). Later, Ceder and Dyer noted that the disease was transmitted not by flea’s bite, but by inoculation into the flea-biten part or by skin abrasions with the flea feces or crushed fleas. Mooser and Castenada followed, by cytologic methods, the development and multiplication of the rickettsia of murine typhus in tissues of several species of fleas in epithelial cells of the stomach and in cells of the malpighian tubules. Rickettsiae were not observed in the salivary glands and not in the genitals, thus the fleas were considered inefficient vectors of typhus. Lewthwaite in Malaysia confirmed the findings about transmission via contact with crushed fleas or flea feces, which remains infectious for 8 or 9 years (potential for aerosol transmission) and the absence of transovarian infection. In 1931, Dove and Shelmire raised a serious doubt as to the exclusive role of flea transmission of endemic typhus, following their studies with the tropical rat mite,

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History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical

Ornithonyssus bacoti. They demonstrated the transovarial transmission and the passage of disease from guinea pig to guinea pig and to rat via infected mites. Later, Pang noted natural infection with O. bacoti in China. The Polyplax, lice of rat, was found infected with murine typhus and was able to transmit the disease experimentally. However, this louse did not bite humans and therefore would not be a direct cause of human infection, but may be important in maintaining enzootic infection. On the contrary, R. typhi was isolated from the human lice P. humanus from patients in India, Mexico, and Ethiopia. Experimentally infected lice did not transmit R. typhi to their progeny. In 1930, Castaneda and Zinsser reported that by experimental model the bedbug Cimex lectularius can acquire the rickettsiae of ‘Mexican typhus fever’, but failed to transmit it to another animal. R. typhi was isolated from a pool of Boophilus ticks collected from bushes in India and from Hyalomma ticks collected from cattle in Ethiopia. Some ecological and epidemiological studies indicate that the ticks and trombiculidae mites (chiggers) may be of some importance as vectors but they do not challenge seriously the theory that fleas are the principal carriers to man (Table 7). Transmission by aerosols and contamination with urine or feces of infected rats or cats that contaminated foodstuffs might account for some cases. Also, the transmission of the disease by mouth by animal experiments and the possibility of transmitting the disease from human to rabbits and

Table 7 Arthropods implicated as actual or potential vectors of murine typhus Arthropod Fleas Xenopsylla cheopis (rat flea) Xenopsylla astia (rat flea) Xenopsylla brasiliensis Xenopsylla bantorum Ctenocephalide felis (cat flea) Echidnophaga gallinacea (sticktight flea) Leptopsylla segnis (mouse flea) Monopsyllusa nisus (murine flea) Nosopsylla fasciatus (ceratophyllid flea) Pulex irritans (human flea)

Distribution

Almost worldwide Southern Asia and coastal East Africa Africa, South America, India Africa (Ethiopian region) Cosmopolitan Cosmopolitan Cosmopolitan Northern Asia Cosmopolitan Cosmopolitan

Lice Hoplopleura oenomydis Pediculus humanus Polyplax spinulosa

Cosmopolitan Almost worldwide Cosmopolitan

Mites Echinolelaps echidninus Ascoschoengastiain dica Ornithonyssus bacoti

Almost worldwide Asia Almost worldwide

to guinea pigs was proved. Some native wild rodents were susceptible to infection with R. typhi: the woodchuck, Marmota monax monax; the meadow mouse, Microtus pennsylvanicus pennsylvanicus; the white footed mouse, Peromyscus leucopus neveborascensis; the house mouse, Mus musculus; and the spermophiles, genus Citillus. But, the most important components of the murine typhus life cycle are commensal rats of the subgenus Rattus, such as R. rattus and R. norvegicus, and their fleas, in particular the oriental rat flea X. cheopis.

Diagnosis and Treatment: Historical Aspects Diagnosis: The first test for diagnosis of murine typhus was the Weil–Felix test. Weigl reported a more sensitive and specific serological test using suspensions of rickettsia of European typhus and murine typhus grown in the intestine of lice. Today, indirect immunofluorescence assay is the reference method for serodiagnosis of rickettsia in most laboratories, and Western blotting assay associated with the cross-adsorption technique to eliminate the false-positive results through cross reactions between bacteria sharing common antigens, such as Legionella and Proteus OX-19. A diagnosis of murine typhus can be confirmed by immunohostological demontration of R. typhi in tissues, by polymerase chain reaction (PCR) amplification from blood and by cell culture system. Treatment: Since 1950, chloramphenicol is the treatment for murine typhus. In the first study, the authors noted that the temperature reached normal levels within 3 days after beginning the therapy. The current recommendation is administration of 100 mg of doxycycline orally twice daily for 2 to 3 days after deffervescence, when tetracycline is contraindicated, chloramphenicol is an alternative treatment. Vaccine: A vaccine for typhus more unique than Weigl’s was developed by Blanc and Baltazar using ‘attenuated’ R. typhi-infected fleas’ feces with ox bile/saline diluent, in the Institut Pasteur, Casablanca. A later study of this vaccine in volunteers showed that protection against epidemic typhus occurred only when the flea feces in the vaccine was viable and caused a mild murine typhus infection with serological conversion after vaccination. No effective vaccine is available for murine typhus. Prevention: Prevention is directed primarily toward the control of flea vectors and potential flea hosts. In 1952, a decline in cases of murine typhus in the United States was reported after DDT dusting campaigns. The rate of infection remained low and no human cases occurred in the area for at least 3 years after cessation of the dusting operations. The recent recommendations are rat trapping and application of rodenticides and dusting harborages with carbaryl or permethrin for flea control.

History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical

Rocky Mountain Spotted Fever RMSF caused by R. rickettsii is transmitted by the bite of infected ticks. RMSF has not occurred in epidemic proportions, but causes the most severe clinical manifestations of any rickettsioses in early studies with the virulence to kill before the introduction of effective therapies, 10% of children and 30% of adults (Table 3). Currently, it is not so deadly and has a death rate comparable to that of Mediterranean spotted fever (MSF). The average annual RMSF incidence in the United States during 1997–2002 was 2.2 cases per million persons. The disease also occurs in Central and South America, where it is currently largely unrecognized and possibly misdiagnosed as dengue or other febrile exanthems; hence RMSF has been described as a ‘wolf in sheep’s clothing’ and ‘the great imitator’. Around 1 week following the bite of an infected tick, symptoms of illness begins with fever, chills, myalgia, and headache. During the next few days, these symptoms continue and may be accompanied by anorexia, nausea, vomiting, abdominal pain, diarrhea, photophobia, and cough. The fever is typically high and associated with a severe frontal headache. Rash, considered as the hallmark feature of RMSF, appears on day 3 of fever as small, pink, blanching macules, typically on the wrists, ankles, and forearms, which evolve into maculopapules. Within 24 h, it spreads centrally to involve the legs, buttocks, arms, axilae, trunk, neck, and face. The entire body may be involved, including the mucous membranes of the palate and pharynx. Characteristic of the rash is petechial lesions, the distribution that includes the palms and soles and occurs in only 36–82% of patients. In some severe cases, petechiae may coalesce to form large ecchimoses. Severe manifestations may include pulmonary edema and hemorrhage, cerebral edema, myocarditis, renal failure, disseminated intravascular coagulopathy, and gangrene. Three factors were independent predictors of failure by the physician to initiate therapy the first time a patient was seen: absence of a rash, presentation between 1 August and 30 April, and presentation within the first 3 days of illness. In untreated patients who survive their illness, the natural course of the fever terminates after 2–3 weeks.

Historical Aspects of Rocky Mountain Spotted Fever Disease It is not known how long R. rickettsii has been indigenous to the Western Hemisphere, a reflection of the 500 years or so since the first explorers, slaves, and immigrants inhabited what is now the United States, Canada, Mexico, and Central and South America. According to the US Public Health Service, the earliest account of RMSF dates to 1873 (Table 8). Major W. W. Wood, who collected

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Table 8 Milestones of Rocky Mountain spotted fever Discovery of the Rocky Mountain spotted fever: Rickettsia rickettsii 1873 The earliest records of RMSF 1896 Description of RMSF 1899 The first clinical account 1904 Association: tick disease (not proved) 1905 Transmission from tick to man 1906 Isolation of bacteria in guinea pigs 1906 Tick role in RMSF 1919 Description of the etiologic agent and proposed the name as Dermacentroxenus rickettsii 1922 The name of R. rickettsii 1924 Vaccine 1954 Natural reservoir: the meadow vole ‘Microtus pennsylvanicus’ 2004 Genome sequence (unpublished) 2007 R. rickettsii str. Sheila Smith genome sequence

Author US Public Health Wood WW Maxey Wilson and Chowning McCalla Ricketts Ricketts and King Wolbach

Brumpt Spencer and Parker Gould and Miesse

Madan et al. Eremeeva et al.

descriptions of cases from eight Idaho physicians, apparently first recorded the disease in 1896. Edward Maxey published the first report in the medical literature of a case in the Snake River Valley of Idaho in 1899 and he gave a vivid description of the disease known locally as the ‘blight on the Bitterroot’ or ‘black measles’. Its severe dark rash and folk wisdom of the day suggested that infection occurred from drinking the melted snow water that gushed out of Lo Lo Canyon during spring runoff, the notorious site of the infection after Michie and Persons. In 1902, Wilson and Chowning, both from the University of Minessota, began a significant epidemiological study. Their valid observations included the first description of severe RMSF, findings from eight autopsies, and geographic distribution on the western side of the Bitterroot River. They correctly concluded that ‘spotted fever’ was a disease of the capillary circulation caused by a noncultivable infectious agent with a wild reservoir with no evidence of transmission of the disease from person to person or by means of a common water or food supply. With Ricketts’ findings of 1906, RMSF became a true disease, not a syndrome. From 1904 to 1913, 96 (63%) of 153 patients died of RMSF in Montana. Clinically and epidemiologically compatible cases of RMSF were described from New York State as early as 1912; however, the first clinically recognized, laboratoryconfirmed case, east of the Rocky Mountains, was

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documented in an Indiana child in 1925. In 1931, Badger described cases in Delaware, Maryland, North Carolina, Pennsylvania, Virginia, and Washington, establishing the widespread distribution of RMSF in the United States. In 1929 in Sao Paulo, the physicians diagnosed several patients with a febrile exanthematous disease, named ‘Sao Paulo exantematic typhus’ or ‘Brazilian spotted fever’ with a high fatality rate (70%), identifying in ticks (Amblyomma spp.) and small mammals a rickettsia similar to the RMSF agent. Recently, contemporary diagnostic methods have permitted the retrospective confirmation of fatal case of spotted fever in a Maryland patient that occurred in 1901. During the next several decades, RMSF was confirmed in Columbia (Toba petechial fever), Mexico (Fiebre de Coix and Fiebre manchada), Canada, Panama, Costa Rica, and Argentina. The Infectious Agent: Rickettsia rickettsii At the beginning of 1900, research of infectious agent was very difficult because the techniques of visualization of bacteria were imperfect. Sometimes, certain works were false tracks like those of Wilson and Chowning (1904) of Fricks, and Noguchi later (1916), which attribute to the parasite Pyroplasma hominis identified in the hypochromic and sledged red cells of an afflicted patient and in Columbian ground squirrels an etiological role of RMSF. In 1904, Stiles and Craig intensively studied the blood and tissues of spotted fever patients and both showed the absence of protozoan parasites. The first experimental transmission of infection from the blood of patients to guinea pigs and monkeys was carried out by Rickets in 1906. The maintenance of the agent via serial animal passages alternating between monkeys and guinea pigs was suggested in his laboratory. Ricketts found that the infectious agent was retained by a small Berkefeld filter, was unable to grow in bacteriologic media, and the guinea pigs could also be infected with serum or washed cells. Later, he described ‘‘small spherical, ovoid and diplococcoid forms’’ in the blood of guinea pigs and monkeys infected with RMSF that seemed to be bacteria and ‘‘no so frequently in the blood of men’’. By using Giemsa stain, he saw ‘‘two somewhat lanceolate chromatin staining bodies, separated by a slight amount of eosin-staining substance’’. He wrote, ‘‘I have devised no formal name for the organism discussed, but it may be referred to tentatively as the bacillus of Rocky Mountain spotted fever’’. It is difficult to recognize rickettsiae based on this description. In his carefully prepared laboratory notes, Ricketts made drawings of his parasite. Wolbach was the first to demonstrate R. rickettsii in human tissues by using a modified Giemsa stain. He felt that the bodies, when present in the circulating blood, were only within phagocytic cells. In this point of identification of the parasite, Wolbach compared one of Ricketts’ original

preparations with those of his own obtained from eggs of infected ticks. He felt that ‘‘while Ricketts may have encountered the true parasite of the disease in ticks, he was led hopelessly astray by the occurrence of bacteria in his infected as well as noninfected ticks’’. In the middle of 1916, Wolbach described the morphology of an intracellular bacteria that he called ‘‘a wholly new kind of micro-organism’’, a Gram-negative bacterium measuring from 0.2 to 0.5 mm, and has the capacity to infect and replicate in the cytosol and occasionally in the nucleus of vertebrate and invertebrate cells. This was probably the first description of Rickettsia. Later, he reported the experimental infection of primates and the post-mortem findings of five cases of RMSF. His meticulous and detailed descriptions of rickettsiae in the tissues of patients with fatal RMSF established the foundation for the current understanding of the pathogenesis of this disease. The similarity of the agent of typhus and tsutsugamushi fever was remarkable as noted by Wolbach. In 1933, Harris showed rickettsiae in the skin of a patient who contracted RMSF in Tenessee. Wolbach named it Demacentoxenus rickettsii, the genus name after its vector ticks and the species name after Howard Ricketts. In 1922, Brumpt proposed the name R. rickettsii and Bengston in Bergey’s ‘Manual of Determinative Bacteriology’ calls it ‘Rickettsia rickettsii (Wolbach)’. The complete genome sequence has not been fully characterized, but contains 1 257 710 bp with an estimated 1365–2849 open reading frames (ORFs) and contains considerable inactivated genetic material in the process of spontaneous degeneration. Tick Vector The most brilliant discoveries of the century, quite on par with the great findings of Robert Koch, were the first demonstration of arthropod biological transmission of any disease. The possibility that wood ticks (genus Dermacentor) were responsible for transmitting the infection was suggested by an Idaho physician, when a tick was found in the genitals of one of the fever victims. Wilson and Chowning made a crucial association between the discovery of the agent of Texas Fever (Babesia bigemina) carried by the cattle tick Boophilus annulatus and RMSF. Their acclaimed hypothesis of transmission by tick bite was later rejected when the acknowledged experts – Stiles, Ashburn, and Graig – did not find any protozoa in the patient’s blood. In 1905, McCalla, an Idaho physician, in association with Brereton, placed a tick obtained from the chest of a man who was very ill with spotted fever on the arm of another patient, with the full consent of the participants. The tick remained on the second patient for 48 h and then placed on the leg of a woman, where it remained for at least 10 h. The incubation periods were 9 and 3 days, respectively. McCalla did not publish his finding. Ricketts, during his investigation of RMSF in western Montana, demonstrated that Dermacentor andersoni was the principal vector and showed the

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Figure 5 Rhipicephalus sanguineus (the brown dog tick), female (left), male (2nd of left), nymphs, larve, and egg, the main vector of MSF and sometimes vector of RMSF.

Figure 4 Dermacentor variabilis adults (the American dog tick), male (left) and female (right), nonengorged, implicated in the transmission of Rickettsia rickettsii collected in Lawrence country, OH, USA.

transovarial transmission, confirmed independently by Walter W. King. Microscopically, Ricketts found the peculiar microorganisms in salivary glands, alimentary sac, and ovaries of infected females as well as in eggs. In collaboration with King, Ricketts showed the ability of a female tick to acquire the infection by feeding on experimentally infected guinea pigs and to subsequently transmit the illness by feeding on another guinea pig. In 1907, Ricketts had shown that Columbian ground squirrels, chipmunks, and woodchucks were susceptible to R. rickettsii. Later, Wolbach concluded that there was no cellular reaction in the tick’s tissues for the presence of the parasites, ‘even when presented in enormous numbers’. Other rarely recognized routes of transmission of R. rickettsii include blood transfusion and inoculation of rickettsiae through mucous membranes. Later, R. rickettsii was isolated from other ticks like Dermacentor variabilis (Figure 4), Rhipicephalus sanguineus (Figure 5) and Amblyomma cajenennse. Burgdorfer (1963) showed 100% transovarial development of rickettsiae through four additional tick generations and filial infection rates in the seventh generation, but the low proportion of infected ticks in nature was explained by negative effect. Transstadial and transovarial transmission of rickettsiae in tick hosts is central to the maintenance of R. rickettsii in nature, the same for the horizontal transmission among tick-feeding animals and vertebrates host. Over many years, extensive research was carried out to find the reservoir of this disease. R. rickettsii was first isolated from small mammals such as a meadow vole (M. pennsylvanicus) trapped near Alexandria in 1954 by Gould and Miesse. A decade later, it was isolated from eight other mammalian species, including a pine vole (Pitymys pinetorum), white-footed mouse (P. leucopus), a cotton rat (Sigmodon hispidus), cottontail rabbits

(Sylvilagus floridanus), an opossum (Didelphis marsupialis virginiana), chipmunks (Eutamias amoenus), a snowshoe hare (Lepus americanus), and golden-mantled ground squirrels (Spermophilus lateralis tescorum). The significance of birds as reservoir hosts for R. rickettsii remains unproven. The rather limited list of animals from which R. rickettsii has been isolated, compared to the extensive list of seropositive animals, is not really surprising when one considers the transient level of rickettsemia in animals.

Diagnosis and Treatment: Historical Aspects Diagnosis: For the first time a definitive, objective laboratory method for the diagnosis of RMSF was established after Rickett’s discovery in 1907, named ‘infection test’. The macroscopic changes in infected guinea pigs yielded a distinctive criterion for the diagnosis of the infection. Later, Ricketts developed an agglutination test using infected ticks, but the Weil–Felix test resides as the most widely used confirmatory procedure. In 1948, the test was completed by the complement fixation test, a specific reaction for the spotted fever strain of rickettsiae. Actually, a diagnosis by serology, by PCR assays, immunohistochemistry, or by the isolation of rickettsiae in cell culture was performed. Prevention: In 1913, Fricks implemented a program for the Public health Service focused on controlling the tick vector in the Bitterroot Valley. In 1946, Smith showed that spraying an area with DDT or other insecticides was ineffective because of the huge territory and the difficulty in getting under low vegetations. Control of infection in nature is not feasible. The best protection to an individual lies in preventing the attachment of a tick to the skin or in removing it before the rickettsias have become ‘activated’. Vaccination: In 1924, Spencer and Parker developed a vaccine for RMSF from infected tick’s intestine. In 1938, they showed that during the period of several years in western Montana there were 38 deaths in 50

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nonvaccinated adults (76% fatality rate) and only three deaths in 59 vaccinated persons (9% fatality rate). This vaccine not only gives effective protection, but also produces many local and occasionally severe systemic reactions. It is not certain that vaccines against RMSF developed in 1973 by DuPont and in 1983 by Clement do better. At present, no licensed vaccine exists for RMSF. Serotherapy: In 1908, Ricketts demonstrated passive serum prophylaxis of experimental disease in guinea pigs, but of nine human patients treated with hyper immune horse serum, six died. Later in 1943, the therapeutic use of an immune rabbit’s serum as therapeutic doses administered before the third day of the appearance of the rash diminished mortality around 19%; the initiation after the third day of the rash did not alter the ultimate prognosis, it only reduced the toxemic symptoms. The development of antibiotics relegated serum therapy to use only in extremely toxemic patients or in those who have contraindication to chemotherapeutic agents. Therapy: The goal of chemotherapy was to develop a rickettsiocidal drug. In 1949, Harrel suggested treatment with ‘heavy metals’ (arsenic and mercury) and with a solution of neoarsphenamine. After discovery of contraindication of sufonamides, the researcher proposed the use of PABA as it showed an inhibitory effect on the growth of rickettsiae in vivo, but in practice a very large amount of the drug was required. After chloramphenicol discovery, a series of 37 patients with RMSF were successfully treated showing an average on the sixth day of illness, without fatalities. In certain patients with profound toxic syndrome, chloramphenicol with steroids suggested a significant antitoxemic effect. The isolation of viable rickettsiae several years after active infection from RMSF confirmed that the mode of action of this antibiotic is suppressive rather than killing. In 1949, Harrel reported the first results of aureomycin treatment, highly effective in chick embryos and in guinea pigs, but having a rickettsiostatic effect. After the introduction of broad-spectrum antibiotics in 1948, the incidence of RMSF in the United States dropped to about 250 cases per year (from 500), with only about 24 deaths. In 2007, the recommended therapy for RMSF is doxycycline in a doseage of 100 mg twice daily for adults for 7 days and is continued for 2 days after the patient has become afebrile. Glucocorticoids are sometimes given to severely ill patients, but no documentation of efficacy has occurred. Moreover, they are not recommended, although in experimentally infected dogs treated simultaneously with doxycycline, no detrimental effects of prednisole were noted.

Mediterranean Spotted Fever Ricketsia conorii, the causative agent of MSF is transmitted to humans by tick bite, particularly by Rh. sanguineus ticks.

MSF is endemic in the Mediterranean area, including northern Africa and southern Europe, but new cases were reported sporadically in central Europe, Central and South Africa, and India. The role of MSF in history is unknown because the disease has a moderate severity, but it plays an important role as a public health treat. In 2002, in Italy, the national incidence rate is 1.6 cases per 100 000 persons (but, in Sicily 10 cases per 100 000 persons); in Portugal between 1989 and 2003, the annual incidence was 8.9/100 000 inhabitans; and in Spain as well as in Marseilles between 1983 an 1985, the estimated incidence was 23–45 cases per 100 000 persons. The incubation period from the time of infection to the onset is 6 days. Often, patients present abrupt fever, flu-like symptoms (headache, chills, arthromyalgias), and a tache noire at the tick-bitten site. The tache noire, the hallmark of the disease, is an inflamed red papule, the center of which becomes necrotic and black, indolent, and usually localized on the trunk and the legs and arms; in infants it is often on the scalp. It is present in the majority of cases; however, in some patients it cannot be found unlike clinics, in 14–28% cases. On the seventh day of the fever, a generalized maculopapular rash erupts first on the extremities and then on the trunk, often involves the palms and soles, and to a lesser extent the face. Other common clinical manifestations were myalgia (73%), headache (69%), conjunctivitis (32%), hepatomegaly (44%), and splenomegaly (19%). Severe forms of the disease have been reported in 6% of patients, especially in adults with one of the following conditions: diabetes, cardiac disease, chronic alcoholism, glucose-6-phosphate dehydrogenase deficiency, end-stage kidney disease. The mortality rate may reach 2.5% among diagnosed cases. In 1997, in Beja, a southern Portuguese district, the case fatality rate of MSF was 32.3% in hospitalized patients, explicated by the authors by the differences in virulence strains. Historical Aspects of Mediterranean Spotted Fever Disease It is evidentl the first cases of MSF were observed before the first description by Conor and Brush of the Pasteur Institute in Tunis in 1910. The authors described seven cases of spotted fever; they differentiated the disease from epidemic typhus and other eruptive fevers and compared with RMSF (Table 9). Advised by Charles Nicolle, they called the Tunisian spotted fever ‘‘fie`vre boutonneuse de Tunisie’’. They made the first experimental infection to chimpanzee (cured of typhus since 45 days). After 10 days of fever, the chimpanzee died. In the same year, Conor and Hayat described the clinical features of other four clinical cases (abrupt onset, high fever, headache, chills, arthromyalgias, and cunjunctivitis). The exanthema was papular rather than macular; they named it ‘bouton’ and it often turned red-purple in color, involving the palms and

History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical Table 9 Milestones of Mediterranean spotted fever Discovery: Mediterranean spotted fever: Rickettsia conorii 1910 First case reported of MSF 1914 Tick role in MSF: the brown dog tick Rh. sanguineus 1925 Description of ‘tache noire’ 1932 Isolation of R. conorii conorii 1946 First cases of Israeli spotted fever rickettsia 1950 Isolation of R. conorii indica from tick collected in India 1972 The first case of a febrile exantema in Astrakhan 1974 Culture of R. conorii israelensis 1991 Culture and identification of R. conorii caspia 1991 Description of Astrakhan fever disease 2001 Description of Indian tick typhus rickettsia 2001 Genome sequence R. conorii strain 7 2005 Subspecies of R. conorii based on MLST

Author Conor and Bruch Wilson Pieri Brumpt Valero Philip et al.

Goldwasser Tarasevitch and Raoult

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group with the typhus. He wrote about several form of typhus: severe winter typhus (epidemic typhus) and benign summer typhus, which consisted of Brill’s disease and Marseilles’ fever. Several histological studies of MSF between 1932 and 1935 on the skin biopsies showed that R. conorii caused vasculitis and was responsible for the clinical anomalies. The spotted fever disease was clinically described in India by Megaw (1921), who named it Indian tick typhus rickettsia (ITTR); in (1946) Israeli spotted fever rickettsia (ISFR); and in Astrachan, a region of Russia located on the shores of the Caspia Sea (1972), Astrachan spotted fever rickettsia (AFR). Later subspecies of the etiological agent R. conorii was isolated from human samples of these diseases.

Tarasevich

The Infectious Agent: Rickettsia conorii

Murali et al.

In 1927, Netter supposed that the agent of spotted fever of Marseille was the same organism associated with Brill’s disease, only that it lost some virulence factors by the phenomenon of mutation. In 1932, Blanc and Caminopetros characterized some small bacteria, which seemed probably Rickettsiae on the ticks’ organ smears using Giemsa staining, and showed that the infectious agent crossed through the filters Chamberland L2 and L3 and remained infectious after centrifugation for humans. P. Durand, Kuszyncki, and Hohenadel showed the same results from the skin biopsies of MSF patients. Brumpt described the infectious agent in tick samples and gave the name R. conorii in honor of the work of Conor (Figure 6). R. conorii is transmitted by biting, through tick’s feces, or by projection of the liquid contents of the ticks on the mucous membrane conjunctivae. The transmission by ingestion as well as the possibility of infection through respiration of environmental pollutants containing Rickettsiae from ticks feces was already noted. In 1978, Philip by using mouse serotyping demonstrated that Malish, Moroccan, and Kenyan R. conorii isolates were closely related antigenically to ITTR and that they belonged to a single serotype. Later, it was demonstrated that AFR differed from both ISFR and R. conorii in terms of SDS-PAGE mobility, PFGE profiles, and PCR RFLP, but using a combination of genotypic criteria, the differences were too faint to classify as new species. Among the 39 isolates or tick amplicons studied recently, four multilocus sequence typing (MLST) genotypes were identified; furthermore, they were also classified into four types using multi-spacer typing (MST) genotyping as well as mouse serotyping. Then, four subspecies were reported: R. conorii subspecies conorii (type strain Malish, ATCC VR-613, formerly MSF), R. conorii subspecies indica (type strain ATCC VR-597, formerly ITTR), R. conorii subspecies caspia (type strain A-167, formerly, Astrakhan fever rickettsia), and R. conorii subspecies israelensis (type strain ISTT CDC1,

Ogata et al. Zhu et al.

soles. The duration of illness was 12–15 days, without fatality cases. During the same time, several cases with atypical generalized rash, called ‘Marseilles’ fever’, was reported in the South France. Later, not only sporadic cases, but small endemic cases of spotted fever in the warm months were mentioned in this region. In 1920, in Italy, Carducci reported 16 cases of spotted fever collected during the last 10 years and distinguished two different diseases by clinical and epidemiological criteria: remittent fever and spotted fever, named after ‘‘Febbre eruttiva, forma speciale descritta dal Pr. Carducci’’. In 1925, Jean Pie`ri and Brugeas described a small lesion ‘tache noire’, an eschar of inoculation, the hallmark of diseases. In 1928, Roche, a physician of Sorgues, South of France reported, ‘‘During 31 years of my professional work, I carried each year, some clinical cases of one mysterious disease to which I was embarrassed to give a name’’. The physician named it ‘‘exanthe`me infectieux e´pide´mique, maladies exanthe´matique, fie`vre exanthe´matique du littoral, and exantheme infectieux me´diterrane´en’’. In 1925–27, on the Academy of Medicine, Olmer gave an excellent clinical description of the disease and projected new experimental researches on the guinea pigs and monkey for differentiation of epidemic, endemic typhus, and Brill’s disease. Boinet and Pieri reported an epidemiological study of disease and insisted that the disease was not very contagious to human, benign, and very different of typhus. Two opposite opinions were projected: Jean Olmer classified this disease in the same

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Figure 6 Rickettsia conorii detected in hemolymph of infected Rhipicephalus sanguineus adult ticks by using Gimenez staining.

formerly ISFR). Complete genome sequence determined the 1 268 755 nt of R. conorii conorii, containing 1374 ORFs. Vectors History Several years after the description of the first case by Conor, the researchers tried to identify the vector of disease. Netter and Olmer proposed a red Japanese mite, Thrombidium akamushi. After the discovery of ‘tache noire’, an eschar of inoculation of an infectious agent, all researchers believed in the existence of a vector for the disease. Olmer (1928) thought that the vector of the disease was the brown dog tick, a hypothesis confirmed by P. Durand and E. Conseil, who inoculated to humans the crushed infected Rh. sanguineus ticks; the patients contracted MSF (Figure 7). In 1932, it was shown that the different stages of ticks and over winter unfed males and females could act as vectors of MSF. It was also shown that when eggs or

larvae obtained from infected Rh. sanguineus females were crushed and used to inoculate humans, the patients contracted MSF. Later, the transtadial passage and transovarial transfer to at least some eggs in the next generation were demonstrated. Ranque supposed that the Rh. sanguineus retained the R. conorii for at least five or six generations. The reproduction and maintenance of R. conorii in tick populations may be related largely to the environment of the tick than of the host. However, the transovarial transmission rate, or the filial infection rate, was not proved yet. Later, a negative effect of R. conorii on Rh. sanguineus ticks was shown in experimentally infected ticks, which may explain the low prevalence of infected ticks in nature. No animal reservoir of R. conorii has been definitely identified, but A. Raybaud (1929) accused the rabbits, hares, weasels, and fox; Godlewski thought that the herds transhumant that return from the Alps could import in the suburbs of Avignon their ticks and spotted fever; Brumpt suspected the dog, but also various wild animals, cats, rabbits, the rodents, or insectivorous; and Plazy, Marcon, and Carboni suspected the rats. In 1930, Durand infected two 6-week-old puppies with an emulsion of Rh. sanguineus ticks collected in Tunis, and no clinical symptoms were observed. The administration of blood collected from these puppies (10 days postinfection) caused typical symptoms of MSF in human subjects. Laboratory experiments found that the dog, rabbit, guinea pig, and rat were not susceptible to infection. Swiss mice, Hartley guinea pigs, spermophile (Citillus citillus), and the chimpanzee are susceptible. In 1932, the European rabbit Oryctolagus cuniculus was considered a possible reservoir. In 1940, Violle and Joyeux isolated R. conori by inoculation of wild rabbit’s blood, brain suspension, and crashed ticks into guinea pigs. Furthermore, in 1952, myxomatosis destroyed almost all the rabbits in southern France. Immediately thereafter, there was a spectacular drop in the incidence of MSF, and the reappearance of wild rabbits several years later was followed by an increased incidence of MSF. The role of small rodents such as Pitymys duodecimcostatus, living in rabbit’s burrows, was suspected to be involved in the life cycle of R. conorii; however, this was never confirmed. Later, R. conorii was isolated from rats and mice by inoculation of spleen and brain material into guinea pigs. Each of these rodents is a characteristic host of immature Haemaphysalis leachi and Rhipicephalus simus ticks in Kenya. Recently, some ticks collected on a hedgehog were tested positive for R. conorii, but their role in transmission is not proved yet. Diagnosis and Treatment: Historical Aspects

Figure 7 Amblyomma variegatum (female left) and male (right) adults, nonengorged implicated in the transmission of Rickettsia africae.

Diagnosis: The first keys for diagnosis were the clinical presentation with generalized maculopapular rash that often involves the palms and soles and the black eschar ‘tache noire’. The diagnosis may be established by

History and Culture, (and Biographies) | Typhus Fevers and Other Rickettsial Diseases, Historical

immunofluorescent detection, latex agglutination, enzyme immunoassay, Western blot, compliment fixation, immunohistologic demonstration of R. conorii in skin biopsy, isolation in shell vial cell culture, and PCR can be applied to blood or skin biopsy. Recently, diagnostic criteria have` been proposed to help physicians in diagnostic MSF and other rickettsial diseases. Treatment: In 1949, Janbon and in 1950, Fouquet and Morin investigated chloramphenicol as treatment for MSF. Actually, the treatment of choice for MSF is 200 mg of doxycycline, a single dose, although for severe forms, it should be administered until the patient becomes afebrile.

African Tick Bite Fever African tick bite fever (ATBF) is due to Rickettsia africae transmitted by Amblyomma ticks in rural sub-Saharan Africa and the French West Indies. Some reports indicate that ATBF pose a significant problem to local populations. During Zimbabwean war of independence in the late 1970s, army medical authorities reported several thousand cases of tick typhus. Both European and African soldiers were affected, although the infection rate seemed to be greater among the former. In Zimbabwe, the incidence rates of ATBF was 60–80 cases per 100 000 patients each year in areas where Amblyomma tick was endemic. Whereas reports on ATBF in indigenous populations are scarce, the number of reported cases has recently increased in travelers from Europe and elsewhere. The clinical course typically comprises an abrupt onset of fever, nausea, headache, and neck myalgia beginning 5–10 days after a tick bite. Most patients develop an inoculation eschar at the site of the tick bite and up to 54% of patients have multiple eschars, a pathognomonical clinical sign. A painful regional lymphangitis is common and may be seen in the absence of a franc eschar. A generalized cutaneous rash, sometimes vesicular and usually best seen close to the eschar, is present in 15–46% of the patients. Less frequent clinical signs of ATBF include aphtous stomatitis and arthralgia. Complications are rarely seen, but it was reported in one patient presenting long lasting fever, reactive arthritis, subacute cranial or peripheral neuropathy, chronic fatigue, neuropsychiatric symptoms, myocarditis, and there are no other known fatal cases. ATBF should be considered along with malaria and other tropical fevers in the differential diagnosis of all febriles returning from the tropics. Historical Aspects of African Tick Bite Fever Disease ATBF is probably a very old disease in sub-Saharan Africa, which for several decades has been confused with the MSF. The first human case was described in 1911 by

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Table 10 Milestones of African tick bite fever Discovery: African tick-bite fever: Rickettsia africae 1911 The first human cases 1930 Isolation of R. africae and description of the disease 1990 Isolation of R. africae 1992 The name of diseases and of bacteria 1996 Official nomenclatures 2007 Genome sequence

Author McNaught, Sant’Anna JF Pijper Kelly Kelly and Raoult Kelly and Raoult Raoult D

McNaught in Mozambique and South Africa, and by Sant’Anna and Nutall in Angola and Kenya (Table 10). They named this disease as tick bite fever because it was transmitted by ticks. In 1929, Be´ros and Belozet suggested that this disease was the same as MSF. During the 1930s, Pijper, a pathologist working in Pretoria, described the distinct epidemiology and the clinical features of ATBF, isolated a causative organism from ticks, and concluded that ATBF was a separate disease from MSF (which at the time was known to be caused by R. conorii). Pijper noted that the vector of the disease was cattle ticks; the clinical manifestation was milder, less illness period with few complications, rare cutaneous rash, and no fatality case. However, subsequent experiments by Gear failed to confirm these findings and he reported the infection of R. conorii. For the next 52 years, ATBF were erroneously considered as MSF, which was also present in the same geographic areas, and the observed differences in clinical presentation and epidemiology were attributed to different host factors, such as age and risk behavior. The final advancement came several decades later; in 1992, a 36-year-old woman presented a history of tick bite behind the right ear, high temperature, and a severe headache. The skin at the bite was erythematous and she had regional lymphadenopathy. The bacterium was isolated and molecular tools confirm that it was distinct of MSF’s agent. The authors proposed the name ATBF for the disease. Since then, there have been several reports describing the clinical and epidemiologic features of a number of patients with laboratory-confirmed ATBF.

The Infectious Agent: Rickettsia africae In 1930, Pijper isolated for the first time the causative agent of ATBF in guinea pigs and showed the difference with R. conorii and R. rickettsii by using cross-protection studies. He described the clinical signs of experimentally infected guinea pigs and the difference with other rickettsial infection. Unfortunately, his isolate was lost. Several years later, different strains from South Africa, Kenya, and Northwest Africa were tested by the toxin-neutralization test in mice,

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serological and cross-protection tests, but have shown no significant differences with R. conorii. Extensive experiences with rickettsial agents and their clinical manifestations have led American and Russian specialists to agree in considering R. conorii as a single African rickettsial species and MSF as a single clinical entity. The French school still regards this subject differently. SFG rickettsiae other than R. conorii from Africa were reported from Amblyomma ticks in Ethiopia and from Amblyomma hebraeum in Zimbabwe. These strains were indistinguishable through serological tools, but distinct from R. conorii. Finally, in 1992, one rickettsial agent was isolated from a woman presenting a typical ATBF, and shown to be distinct from R. conorii but identical with rickettsial strains isolated in Amblyomma ticks collected in Etiopia and Zimbabwe. The authors proposed the name R. africae, nomenclatures that were made official in 1996. The genome of R. africae consists of a circular chromosome of 1.2 Mb.

Tick Vector Sant’Anna and Nutall described, in 1911, one new disease transmitted by A. hebraeum ticks, especially in places frequented by cattle in Mozambique. Later in South Africa, Pijper described tick bite fever as a rural disease occurring in people having contact with cattle tick. He showed that A. hebraeum, Rhipicephalus appendiculatus, and Boofilus decoloratus ticks are able to transmit the infectious agent. In the following years, Rickettsial agent was reported as R. conorii from Amblyomma ticks. In the light of actual data, these results are amazing; it would be interesting to test by genotyping the old samples for the presence of R. conorii or R. africae. In 1990, Kelly isolated rickettsial strains from A. hebraeum ticks collected in Zimbabwe and demonstrated the transovarial and transovarial transmission of R. africae in this tick. Thus, A. hebraeum in southern Africa and A. variegatum in West, Central, and East Africa and in the Caribbean are the vectors of ATBF and in the same time the reservoir of R. africae.

Diagnosis and Treatment Diagnosis: In addition to the archaic and poorly specific Weil–Felix test, which continues to be used as a first-line test in many countries, immunofluorescence assay is the most widely used microbiological test. More specific serological tests for ATBF include multiple antigen immunofluorescence assay, Western blotting, and crossadsorption assay. Isolation of R. africae from cell cultures and antigen detection by immunohistochemistry or PCR are available to such specialized laboratories, and should be attempted in unusual or complicated cases.

Treatment: The treatment described by Sant’Anna in 1911 for a new tick bite disease with a little effect was the aspirine and alkalies given internally, besides purgatives, and an ointment of potassium iodide and iodine applied externally. Currently, then, doxycycline 100 mg twice daily for 7 days or until 48 h after defervescence can be recommended in severe cases of ATBF. Fluoroquinolones may be also beneficial. Patients with mild symptoms may not require any treatment at all.

Other Rickettsioses A bacterial species reflects the sum of the biological information on a group of closely related strains isolated in pure culture. It is currently considered that strains with more than 3% divergence belong to different species. Specific rules for recognizing, naming, and classifying species have been established, which avoid redundant descriptions and the use of the same name for more than one species. Several prerequisites are necessary before a new bacterial species is validated. The bacteria should be isolated in pure culture and the description should be based on a minimum of five isolates for environmental bacteria. The new species should exhibit both genomic and phenotypic discriminative characteristics. A type strain should be identified for each new species and available to the scientific community through two independent official culture collections. Finally, the new bacterial name should appear in the approved lists of bacterial names. With the advent of molecular classification, many of the currently accepted Rickettsia species were identified genetically before they were cultured in the laboratory. As is the case with other bacteria, Rickettsia could be given Candidatus status if they fulfill the genomic criteria but have to be cultured. A subspecies is the lowest taxonomic rank that is recognized officially. It contains closely related strains that have distinct phenotypic traits, but do not meet the genomic criteria required for classification as a distinct species. The official validation of a subspecies is comparable to that of species. The rickettsiae designated as human pathogens have been isolated in cell culture and/or have been detected by molecular methods from blood or tissues of patients presenting illnesses clinically compatible with spotted fever rickettsioses and seroconversion using reference laboratory methods. We have briefly described, in Table 2, the first clinical description, rickettsia isolation in cell culture, vector discovery, and other historical aspects of human pathogens R. sibirica sibirica (Siberian tick typhus), R. acari (Rickettsialpox), R. australis (Queensland tick typhus), R. slovaca (Tick-borne lymphadenopathy (TIBOLA), Dermacentor-borne necrosis– erythema–lymphadenopathy (DEBONEL)), R. japonica (Japanese or oriental spotted fever), R. heilongjiangensis

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Table 11 Rickettsial species of possible or undetermined pathogenicity in humans R. asiatica R. bellii

R. canadensis

R. montanensis R. peacockii

R. rhipicephali R. tamurae

Candidatus Rickettsia amblyommii

Candidatus Rickettsia andeanae

Candidatus Rickettsia tarasevichiae

1993 First isolation from Ixodes ovatus ticks in Japan 2006 The name: R. asiatica 1966 Isolation in embryonated chicken eggs from D. variabilis 1983 The name: R. bellii 2006 Genome sequence 1967 Isolation of bacteria from the Haemaphysali leporispalustris tick and naming of rickettsia 1970 Serological evidence 2007 Genome sequence 1963 First isolation from D. variabilis and D. andersoni 1925 Rickettsia-like organism in D. andersoni tick smears 1997 The name of rickettsia after molecular identification in D. andersoni ticks 2001 Isolation from D. andersoni tick 1975 Isolation from Rh. sanguineus ticks 1978 The name of rickettsia 1993 Isolation from A. testudinarium ticks 2006 The name of rickettsia 2006 Serological evidence 1974 First isolation of R. ambliomii but lost 2000 Serological evidence 2007 Isolation in cell culture 2002 Detection by molecular tools in A. maculatum and I. boliviensis 2005 Phylogenetic analysis 2002 Detection by molecular tools in I. persulcatus 2005 Isolation from ticks

(Chinese spotted fever or Far-Eastern tick-borne rickettsiosis), R. honei (Flinders Island spotted fever), R. sibirica mongolitimonae (Lymphangitis-associated rickettsiosis), R. felis (Flea-borne spotted fever), R. aeschlimannii, R. massiliae, R. parkeri, R. raoultii, Candidatus Rickettsia marmionii, Candidatus Rickettsia kellyi, and Candidatus Rickettsia monacensis. Many other rickettsiae, not rewarding the pathogenicity criteria, have been indentifed in ticks and other biological niches. The list of rickettsial species of possible or undetermined pathogenicity in humans is provided in Table 11 with validated species: R. asiatica, R. canadensis, R. montanensis, R. peacockii, R. rhipicephali, R. tamurae, and the rickettsiae classified as Candidatus species.

Fujita et al. Unknown Philip et al. Ogata et al. McKiel et al. Bozeman et al. Eremeeva et al. Bell et al. Parker and Spencer Niebylski et al. Simser et al. Burgdorfer et al. Burgdorfer et al. Fujita et al. Fournier et al. Phongmany et al. Pretzman et al. Dasch et al. Labruna et al. Blair et al. Jiang et al. Shpynov et al. Shpynov et al.

for rickettsiologists and rickettsiology. High levels of awareness, including careful history taking and through physical and laboratory examinations by primary physicians along with the use of contemporary methods based on molecular biology techniques, have facilitated the discovery and description of all emerging human rickettsioses and will undoubtedly lead to the discovery of new tick-borne rickettsial diseases around the world.

See also: Emerging Infections; Endosymbionts and Intracellular Parasites; History of Microbiology

Further Reading Conclusion During more than a century of microbiological studies of rickettsial infections, our knowledge has fabulously progressed through description of clinical features, discovery of infectious agents and their arthropod vectors, and understanding mechanism of transmission, several pathogenic mechanisms of tissue injury, the modern diagnostic tools and a optimal treatment. These arthropod-associated intracellular bacteria are now being recognized in all parts of world. Rickettsial diseases retain several of its mysteries, and continue to provide countless exciting opportunities

Blanc G, Ogata H, Robert C, et al. (2007) Reductive genome evolution from the mother of rickettsia. PLoS Genetics 3(1): e14. Harden VA (1987) Koch’s postulates and the etiology of rickettsial diseases. Journal of the History of Medicine and Allied Sciences 42: 277–295. Jensenius M, Fournier PE, Kelly P, et al. (2003) African tick bite fever. The Lancet Infectious Diseases 3(9): 557–564. Parola P, Paddock CD, and Raoult D (2005) Tick borne rickettsioses around the world: Emerging diseases challenging old concepts. Clinical Microbiology Reviews 18(4): 719–756. Quintal D (1996) Historical aspects of the rickettsioses. Clinics in Dermatology 14(3): 237–242. Raoult D (2005) Rickettsioses and ehrlichioses. In: Mandell GL, Bennet JE, and Dolin R (eds.) Principles and Practice of Infectious Diseases, 6th edn. Philadelphia, PA: Elsevier, Churchill Livingstone.

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Raoult D, Fournier PE, Eremeeva M, et al. (2005) Naming of Rickettsiae and rickettsial diseases. Annals of the New York Academy of Sciences 1063: 1–12. Raoult D and Parola P (2007) Rickettsial Diseases. NY: Informa Healthcare USA, Inc. Raoult D and Roux V (1997) Rickettsioses as paradigms of new or emerging infectious diseases. Clinical Microbiology Reviews 10(4): 694–719. Raoult D, Woodward T, and Dumler JS (2004) The history of epidemic typhus. Infectious Disease Clinics of North America 18(1): 127–140.

Traub R, Wisseman CL, and Farhang-Azad A (1978) The ecology of murine typhus-a critical review. Tropical Diseases Bulletin 75(4): 237–317. Walker DH (1988) Biology of Rickettsial Diseases. Boca Raton, FL: CRC Press, Inc. Woodward TE (1981) Rickettsial disease: Certain unsettled problems in their historical perspective. In: Burgdorfer W and Anacker R (eds.) Rickettsiae and Rickettsial Diseases, pp. 17–40. New York: Academic Press. Zinsser H (1935) Rats, Lice, and History. London: Broadway House.

MUTUALISM AND COMMENSALISM Contents Behavior Modification of Host by Microbes Endosymbionts and Intracellular Parasites Lichens Mycorrhizae Rumen

Behavior Modification of Host by Microbes M Lyte, Texas Tech University Health Sciences Center, Lubbock, TX, USA R P A Gaykema and L E Goehler, University of Virginia, Charlottesville, VA, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Diversity of Systems Immune versus Non-immune-Mediated Behavioral Alterations Nervous System Substrates

Glossary central nervous system The part of the nervous system represented by the brain and spinal cord. c-Fos An immediate-early gene product used as an activation marker for many cells, including neurons. circumventricular organs Regions of the brain with access to substances in the general blood circulation that provide access for microbes to invade or influence the brain. enteric nervous system An interconnected network of neurons in visceral organs, most notably the gastrointestinal tract, that comprises sensory, motor, and intrinsic neurons that function to control functions such as digestion. peripheral nervous system The part of the nervous system consisting of nerves and cell bodies outside the

Methods for Studying Microbial Effects on Behavior Methodological Issues Conclusion Further Reading

brain and spinal cord that sense conditions within and control or modulate the functions of bodily tissues. sickness syndrome A constellation of behavioral symptoms associated with infection that include inhibition of social, ingestive, and sexual behavior and arousal, as well as increased somnolence (sleepiness) and inhibited motor behavior. vagus nerve A nerve that communicates between the brain and internal tissues including the heart, lungs, liver, and gastrointestinal tract. This nerve carries signals from these tissues to the brain and from the brain to the tissues to modulate their functioning. viscerosensory A term used to refer to nerves and functions associated with signaling states within internal tissues.

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Abbreviations CNS CSF

central nervous system cerebrospinal fluid

Defining Statement The intersection of the fields of microbiology and neurobiology represents the emerging interdisciplinary field of behavioral microbiology that seeks to understand the mechanisms by which microbes, both in health and in disease, can interact with the host to influence behavior.

Diversity of Systems Even a cursory survey of the literature reveals the diversity of etiological agents and complexity of behavioral assessments that have been employed to assess the ability of microbes to influence behavior. This includes bacteria, viruses, and parasites in hosts ranging from mammals to insects. Behavioral assessments have spanned from measurements of anxiety in mice to changes in the aggressive behavior of worker honeybees. The following sections will examine behavior and its measurement in depth. While providing an exhaustive review of the literature is beyond the scope of the present article, it is nonetheless apparent that in nature the influence of microbes on behavior is widespread and pervasive. In insects, the alpha-proteobacterium Wolbachia has been shown to influence the fitness of their hosts in order to facilitate their own spread within the host population. For example, Wolbachia-infected females display increased sexual activity as compared to noninfected controls. Infection of the European honeybee Apis mellifera L. with the picorna-like Kakugo virus has been shown to result in highly aggressive behavior that causes normal worker bees to become attackers. Ants, which normally inhabit the forest floor and do not climb surrounding vegetation such as trees and stalks, dramatically change their behavior to begin climbing such structures upon infection with ascomycetes of the genus Cordyceps. It has been recognized for decades that the challenge of animals with infectious microorganisms could lead to behavioral alterations. In rodents, infection with the parasite Toxoplasma gondii has been shown to reduce anxiety to such a level that infected animals no longer show fear of feline predators. Such infection-induced alteration of behavior, which has been described as a suicidal attraction to predation, has been postulated by a number of groups to provide a means by which T. gondii can complete its life cycle within its feline definitive host. On the other hand, infection of mice

ENS IBD

enteric nervous system irritable bowel disease

with the nematode Schistosoma mansoni resulted in reduced levels of exploration and grooming. Such changes in behavior indicate heightened levels of anxiety. Intracerebral infection of mice with the choriomeningitis virus resulted in long-term behavioral modifications, such as alteration in response to a sudden acoustic stimulus, known as the startle response, as well as general or locomotor activity. Infection can even produce behavioral alterations in animals of the same species that are not infected. For example, noninfected mice avoid approaching infected littermates. In the case of humans, there is extensive literature documenting behavioral alterations accompanying microbial infection or alterations in normal microbial flora. Two of the most prevalent health care problems, AIDS and irritable bowel disease (IBD), have behavioral components, which are due, in large part, to a microbial etiology. Infection of macrophages and microglia in the central nervous system (CNS) by HIV results in cognitive impairment in a significant percentage of infected individuals, which may in time progress to dementia. IBD represents a group of disorders characterized by intestinal inflammation that has been correlated with changes in gastrointestinal flora. Numerous studies have demonstrated that IBD patients have poorer emotional functions (anxiety or depression) than observed in the general population. And infection with T. gondii, which was noted above to cause behavioral alteration in rodents, not surprisingly also causes aberrant behavior in the human population. Individuals acutely infected with T. gondii may experience psychotic episodes that are similar to schizophrenia.

Immune versus Non-immune-Mediated Behavioral Alterations The predominant mechanism that has been proposed to account for infection-induced alterations in behavior is one involving the activation of specific immunologic responses. Infection-induced activation of the immune system can result in profound behavioral changes. Specific immune responses such as those that involve the elaboration of pro-inflammatory cytokines can exert effects on neural pathways and ultimately behavior through multiple mechanisms, although many facets of the neural–immune interaction have yet to be fully elucidated (Figure 1). Much less is known, however, about the ability to influence behavior in infections that do not result in immune activation. Early evidence supporting behavioral alterations

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Mood and affective changes Sickness symptoms Increased anxiety

General circulation

Vagal sensory nerves

Internal organs

Spinal visceral Sensory nerves Cytokines

Microbial pathogens

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Little is known regarding the ability of the >1014 microbes that inhabit the gut to influence behavior. While these microbes are generally regarded as commensals, and thus harmless, they are in close proximity to the 100 million neurons that comprise the enteric nervous system (ENS) as well as to nerve fibers associated with the autonomic nervous system. As discussed in the following sections, the ENS may provide a conduit, via the vagus nerve, to the brain to effect alterations in behavior that may be driven, in part, by changes in the gastrointestinal flora. Such changes may occur in the absence of any detectable immune response (except possibly at the local microenvironmental level) and suggests that changes in the composition of the gut flora may play a hitherto unrecognized role in behavior. Evidence for this can be found in children treated with oral antibiotics such as amoxicillin that disturb the normal gut flora, who often display emotional disturbances that dissipate upon cessation of oral therapy.

Immune cells

Figure 1 Microbes, in particular infectious pathogenic ones, can influence the brain by triggering the immune system, which mediates an inflammatory response, including the release of cytokines (e.g., interleukins and interferons, indicated with black arrows). The brain senses immune-derived signals either via viscerosensory nerves of the vagus and spinal cord, in the event of a local inflammatory response, or by way of general circulation. The sensory information propagated into the higher brain centers can then lead to changes in physiological, neuroendocrine, and behavioral sequelae in the animal’s coordinated adaptive response to infection (black arrows). Alternatively, microbes or their secreted products can influence sensory nerves directly (indicated by light gray arrows) to ultimately induce changes in behavior.

in the absence of a detectable immune response was first derived from a wound infection model in rats. The seeding of otherwise sterile wounds in the muscle and cranium with low numbers of bacteria resulted in behavioral alterations such as the extent of movement on an open platform (known as open-field activity) as well as an increase in freezing behavior, suggesting an enhanced anxiety-like state. Importantly, these behavioral alterations were shown to occur not only in the apparent absence of immune activation but also with no signs of sickness. This work led to the employment of strict sterile techniques in animal facilities in which surgically manipulated animals were to be later used in behavioral tests. More recent work has shown that oral challenge of mice with Campylobacter jejuni resulted in the display of anxiety-like behavior and in the activation of neurons within specific brain regions associated with such behavior. These studies suggest that microbes themselves, or their products, may directly interact with neurons to influence behavior in the relative absence of an immune response.

Nervous System Substrates An understanding of the ability of microbes to influence behavior must encompass an understanding of the neural mechanisms that govern behavior. Behavior is mediated by the nervous system. Indeed, the principal role of the nervous system is coordinating complex functions, including behavior, required of multicellular organisms. Behavior supports survival by enabling the acquisition of nutrients and water, mating partners, and the establishment and defense of territory. In addition, behavioral responses are critical for coping with external and internal challenges (e.g., predators or pathogens). Thus, any influence of microbes on behavior has central implications for host functioning. Design Features of Nervous Systems For the nervous system to perform regulatory functions, it must possess mechanisms for detecting conditions within the external environment (ambient conditions, presence of food or predators, etc.) as well as in internal tissues (metabolic needs, presence of pathogens, etc.). Detection of potential challenges must then be followed by central neural processing that integrates this sensory information with ongoing behavior and initiates appropriate behavioral (or physiological) responses. The vertebrate nervous system is divided into ‘peripheral’ and ‘central’ components. The peripheral components include nerves that directly provide sensory information to the central component (brain and spinal cord) as well as nerves that carry motor output signals to control muscles and glands. In addition, intrinsic (enteric) nerve cells reside in internal organs, where they detect local conditions

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within internal organs and coordinate visceral functions, including secretomotor functions of the gut and regulation of heart contractions. This information is available to the CNS via sensory nerves. Integration of sensory information is carried out primarily in the brain, in overlapping systems that process internal and external signals, and ongoing behavioral, mood, and cognitive (e.g., learning and memory) states. Mechanisms for Microbial Effects on Behavior Microbes can affect behavior by a number of different mechanisms. Microbes can behave as sensory stimuli by producing molecules that activate immune and/or sensory neurons to signal the brain; they interact with endocrine tissues or directly infiltrate the brain itself. Thus, identification of tissues harboring microbes can provide clues to the effects on behavior and neural functioning. This includes developmental stages such as insect larvae. Interaction of Microbes with Innervated Organs The major pathways for microbial access to internal tissues are ingestion or inhalation. Thus, in vertebrates, the gastrointestinal tract and lungs are most often the initial sites of colonization. These tissues are innervated by viscerosensory nerves associated with the enteric system (intrinsic neurons) as well as extrinsic nerves of the vagus (a cranial nerve derived from the caudal brainstem) and the spinal visceral nerves that run with the sympathetic system. Recent findings have indicated that these two extrinsic neural pathways contribute to different effects on behavior. The vagus nerve contributes to classical sickness behavior and anxiety, and spinal viscerosensory nerves are involved in hypersensitivity and enhanced pain states. To date, studies on microbial effects on behavior have focused on mechanisms by which immune responses influence these peripheral nervous pathways via circulating mediators including cytokines. However, some microbes, including bacteria in the gastrointestinal tract, influence behavior in the absence of immune-derived mediators, suggesting that there may be direct interactions between bacteria and nerves. Such interactions have yet to be identified, but could follow from association of bacterial polysaccharides with receptors on peripheral nerves or via toxins. There is good documentation indicating that the cholera toxin gains access to neurons and influences them directly. Following initial contact with internal tissues, some microbes enter the systemic circulation to eventually occupy other tissues, including endocrine organs and the brain. Within endocrine organs, for example, the gonads, microbes may influence the secretion of hormones such as testosterone, thereby influencing behavior dependent on

these hormones, such as aggression. Similarly, roundworm larvae gain access to the brain, where they induce behavioral changes indicative of anxiety, classic sickness, or neurological dysfunction. We do not know how these microbes gain access to the brain, as the brain has protective measures, such as the blood–brain barrier, that limit access to substances in the blood. The blood–cerebrospinal fluid (CSF) barrier limits access to the CSF. Interestingly, circumstances associated with stress (as well as during immune activation) make these barriers more permeable, implying that ongoing conditions within the host may regulate the pathogenicity of microbes influencing the brain.

Signal Pathways to the Brain The effects of microbes on the brain and on behavior are either through specific brain pathways that mediate or modulate behavior and/or physiology or through less specific stimuli that induce immune responses within the brain, thereby disrupting normal functioning. Microbe-induced signals carried by viscerosensory nerves (such as the vagus) activate neural pathways within the brain that modulate arousal states and maintain optimal states within the body, such as feeding, regulation of the endocrine system, and behavioral defense responses. These signals enter the brain in the brainstem and propagate to forebrain regions including components of the reticular activating system, the hypothalamus, and basal forebrain. Thus, signals induced by microbes can specifically modulate behavioral arousal and cognition, as well as behavioral, motivational, and mood states. Furthermore, via influences on the hypothalamus, such signals can also influence neuroendocrine regulation of stress responses and reproductive behavior. Microbes within the Brain Microbes that invade the brain enter via the vasculature, meninges (brain coverings), or circumventricular organs. Circumventricular organs are small brain regions located along the ventricles (the fluid-filled canal-like system that extends throughout the CNS) where the blood–brain barrier is weak. Here, substances normally excluded from the brain can gain access. Microbes infecting circumventricular organs can thus influence brain functions associated with these organs, including eating behavior, body fluid regulation, and fever. Microbes that migrate into the brain can also influence brain regions around the circumventricular organs, including the septal area, the hypothalamus, and the midbrain periaqueductal gray, that are implicated in anxiety-like and defensive behavior. The hippocampus, a region in mammals involved in memory, cognition, and modulation of stress responses, is also adjacent to the ventricular system, and therefore a potential target of microbial influences on behavior.

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Microbes that cross into the brain from the vasculature can be expected to influence functions associated with the invaded brain area, or may produce nonspecific neurological deficits associated with the brain’s responses (i.e., cerebral innate immunity) to the microbe. Because the brain itself maintains a relatively anti-inflammatory environment, such effects may be small and localized. If, however, the microbe has induced a systemic immune response, peripheral immune cells will follow the microbe into the brain and initiate inflammatory responses that can lead to marked dementia and/or other neurological deficits. Thus, microbial effects on the brain can vary widely, and depend upon a number of considerations, notably the mechanisms and pathways by which they interface with the nervous system.

Methods for Studying Microbial Effects on Behavior One of the remarkable features of microbial effects on behavior concerns the array of behavioral effects that can be induced and observed. Most experimental observations have been made in rodents, in which pathogen load can be controlled, and behavior can be carefully monitored with well-validated experimental paradigms. However, such approaches can be complemented with studies of animals in natural environments as well when feasible. Typical approaches toward identifying behavioral effects of microbes are briefly described below. The ‘classic’ sickness behavior syndrome was initially characterized in animals treated with bacterial lipopolysaccharides. This syndrome overlaps with symptoms of behavioral depression, including anergy, psychomotor retardation, anhedonia (the reduced experience of reward or pleasure), social withdrawal, suppressed appetitive behavior (eating, drinking, sex), increased somnolence, and can also involve memory impairment. Sickness behavior is assessed using tests directed toward specific symptoms. For instance, sick animals show reduced interest in their environment and show reduced interaction with novel individuals of their own species. This effect is typically assessed by introducing a juvenile animal into the sick animal’s home cage, and recording the instances of interaction, usually anogenital sniffing and following behavior. In addition, sick animals eat and drink less, even when the food or drink is highly palatable. This effect is easily quantified by measuring intake of the food or drink over a specified time and comparing to baseline values and those of control animals. Finally, depression is sometimes likened to ‘learned helplessness’ in which animals fail to exhibit escape behavior from noxious environments. Examples of tests for this behavior include the tail suspension test and the forced swim test.

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Anxiety-like behavior is usually expressed as a preference to remain in a ‘safe’ environment and is often characterized by an increase in the display of risk assessment behavior, as well as reduced exploration of novel or exposed environments. Thus, tests used to assess anxietylike behavior use ambiguous or potentially dangerous environments, such as the elevated plus maze or open field, to determine the extent to which an animal is willing to explore. Anxiety-like behavior can also be evinced as an increased priming of reflexes, such as startle. A commonly used test to assess such reflexes is the startle box, which contains bars on the floor, on which the rodent sits or stands, that allow measurement of the amplitude of a jump (‘startle’) made in response to a loud noise. Aggressive behavior is characterized by increased territoriality or irritability and a tendency to attack novel members of their own species. A convenient way to assess aggressive behavior in the laboratory is the residentintruder test. For this test, an animal is maintained in its home cage, without changing the bedding, a novel conspecific of the same gender is introduced, and the number of attacks on the intruder is recorded. Increased aggressive behavior of animals in the wild is sometimes quantified by counting wounds on the body. However, this technique is compromised by the lack of information regarding the nature of aggressive episodes. For instance, does an animal have more wounds because it engages in more fights or because it has been attacked and is not an effective fighter, perhaps thus less aggressive? Tests for cognitive deficits can be simple, for instance the use of a T- or Y-shaped maze that the animals explore (spontaneous alternation) or the bait of palatable food that the animal learns is present in or on the arms, which require spatial memory. Tests assessing learning, however, can involve rigorous training protocols that can reveal relatively subtle effects on cognitive tasks such as attention and vigilance or the ability to make new learned associations. Although descriptive analyses of microbial effects on behavior are important, it is of interest to also characterize the specific neural substrates in the brain that are influenced. A convenient way to assess this issue involves the use of neuronal activation markers. These markers are proteins that are expressed in brain cells under conditions of activation. This technique is advantageous because it provides cell-specific information regarding activation patterns throughout the brain and can be used with other markers that provide information regarding the neurotransmitters the activated cells use as well as regarding connectivity with other brain regions; that is, this technique can be used in conjunction with neuronal tract-tracing techniques. The most widely used activation marker, and the only one yet used in studies of microbial infection, is c-Fos. c-Fos protein is induced in neurons within about 45 min of activation (Figure 2). The use of this marker has already demonstrated the role of the

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Caveats Important for Behavior Assessment

100 µm Figure 2 Gastrointestinal infection with the bacterial pathogen Campylobacter jejuni in the mouse leads to activation of parts of the brain that receives and processes viscerosensory input carried by the vagus nerve (the nucleus of the solitary tract is shown in the photomicrograph, with the black box in the brain diagram inset indicating the anatomical location). Neuronal activation is visualized with immunohistochemical staining for the expression of c-Fos protein in the nerve cell nuclei, a marker for functional activation (arrows point at black bead-like staining).

vagus nerve in signaling bacteria in the gastrointestinal tract as well as in the brain regions subsequently influenced. This technique holds great promise for elucidating the effects of other microbes on the brain as well.

Methodological Issues There are a number of variables that may contribute to the finding of microbe-induced behavioral alterations. It is critically important to distinguish whether the microbe being examined is administered to the test host in an ecologically relevant manner. Many studies that have demonstrated infection-induced alterations in behavior have used doses and routes of administration that may not reflect actual real-world exposure. For example, while intracranial administration of a gastrointestinal pathogen may produce behavioral alterations, the relevance of such results and identified pathways is open to debate. Further, while challenge with microbes (especially in large doses not encountered in the clinical setting) that results in overt disease may produce robust immune activation and subsequent behavioral changes, often referred to as ‘sicknessinduced behavior’, this may only describe one part of a much larger picture and may not accurately reflect the development of behavioral alterations observed in a specific patient population. Thus, design of translational research experiments employing animal models should endeavor to as closely parallel the microbial (e.g., route of infection and numbers of microbes) as well as the behavioral (including their temporal association to the timing of the infectious challenge) aspects that are observed in the clinical setting.

Like all behavioral studies, assessments of microbial effects must take into account a number of issues that can confound interpretation of these studies. Among the most important caveats for behavioral assessments are interpretational issues related to the specificity of the behavioral response observed. Importantly, signals induced by microbes can induce a panoply of symptoms that can confound attempts to evaluate them. One major confound when evaluating behavior on, for example, an elevated plus maze to assess anxiety can be the lack of arousal that can be associated with sickness. This lack of arousal is typically associated with psychomotor retardation and even somnolence. Thus an animal may neglect to explore a maze, not because it is anxious but because it is sleepy or fatigued. This underlines an important point: assessments of behavior cannot rely solely on computerized assessment. Rather, experimental sessions must be recorded and assessed by an experimenter capable of discriminating between behavior states such as sickness and anxiety and other states that can be defined by similar motor behavior. It is also important to provide control experiments that differentiate between cognitive and mood or motivational effects on behavior and the ability for an animal to perform tasks demanded by the behavioral test. For instance, if a microbe influences the functioning of motor systems necessary for running a maze, or for responding to a threatening stimulus, it can be difficult to attribute the behavioral response to cognitive or emotional states such as ‘fear’ or ‘anxiety’. Further, behavioral tests themselves can induce states such as anxiety, thus complicating the delineation of differences in emotional states between infected and control animals. Thus experiments assessing the effects of microbes on behavior must be carefully conceptualized and implemented, and the results must be interpreted with caution. Nonetheless, characterizing microbial effects on the brain and behavior can provide important information regarding many aspects of host–pathogen interaction.

Conclusion Microbes can provoke marked changes in behavior, which can have profound effects on an animal’s functioning. These effects can include memory dysfunction, behavioral depression, anxiety, and increases or decreases in aggression. In many cases, the host’s responses are probably adaptive. For instance, the suppression of ingestive and social behavior associated with microbe-induced behavioral depression can prevent further ingestion of microbes, promote recuperation, and inhibit spread of microbes to conspecifics. Anxiety-like behavior, especially early on during an infection, can inhibit risky behavior, including foraging in unknown or exposed territory that may leave an animal vulnerable to predators if

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infection induces ‘classic’ sickness (e.g., behavioral depression, psychomotor retardation, and somnolence) later. The emergence of the field of behavioral microbiology is one that offers the microbiologist a theoretical and conceptual framework by which to pursue interdisciplinary research that seeks to go beyond the traditional constructs of microbiology. The realization that the immune system could interact with the nervous system led to the creation of the fields of neuroimmunology and psychoneuroimmunology, and helped to gain a better understanding of the role of the immune system not only in the pathogenesis of disease, but in normal homeostasis as well. In much the same way, the examination of the ability of microbes to affect behavior may lead to a fuller understanding of the myriad ways in which microbes govern both health and disease. See also: Immunity; Toxoplasmosis

Further Reading Acheson DW and Luccioli S (2004) Microbial-gut interactions in health and disease. Mucosal immune responses. Best Practice & Research. Clinical Gastroenterology 18: 387–404. Berdoy M, Webster JP, and Macdonald DW (2000) Fatal attraction in rats infected with Toxoplasma gondii. Proceedings of the Royal Society of London Series B-Biological Sciences 267: 1591–1594.

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Bradfield JF, Schachtman TR, McLaughlin RM, and Steffen EK (1992) Behavioral and physiologic effects of inapparent wound infection in rats. Laboratory Animal Science 42: 572–578. Crawley JN (2000) What’s Wrong with My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. New York: WileyLiss. Dantzer R (2006) Cytokine, sickness behavior, and depression. Neurologic Clinics 24: 441–460. Dean MD (2006) A Wolbachia-associated fitness benefit depends on genetic background in Drosophila simulans. Proceedings of the Royal Society of Biological Sciences 273: 1415–1420. File SE (2001) Factors controlling measures of anxiety and responses to novelty in the mouse. Behavioural Brain Research 125: 151–157. Gaykema RP, Goehler LE, and Lyte M (2004) Brain response to cecal infection with Campylobacter jejuni: Analysis with Fos immunohistochemistry. Brain Behavior & Immunity 18: 238–245. Goehler LE, Gaykema RPA, Opitz N, Reddaway R, Badr NA, and Lyte M (2005) Activation in vagal afferents and central autonomic pathways: Early responses to intestinal infection with Campylobacter jejuni. Brain, Behavior, and Immunity 19: 334–344. Hanstock TL, Clayton EH, Li KM, and Mallett PE (2004) Anxiety and aggression associated with the fermentation of carbohydrates in the hindgut of rats. Physiology & Behavior 82: 357–368. Kamiya T, Wang L, Forsythe P, et al. (2006) Inhibitory effects of Lactobacillus reuteri on visceral pain induced by colorectal distension in Sprague–Dawley rats. Gut 55: 191–196. Lyte M, Wang L, Opitz N, Gaykema RPA and Goehler LE (2006) Anxietylike behavior during initial stage of infection with agent of colonic hyperplasia Citrobacter rodentium. Physiology & Behavior 89: 350–357. Morales J, Larralde C, Arteaga M, Govezensky T, Romano MC, and Morali G (1996) Inhibition of sexual behavior in male mice infected with Taenia crassiceps cysticerci. Journal of Parasitology 82: 689–693.

Endosymbionts and Intracellular Parasites A E Douglas, University of York, York, UK ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Chronic Microbial Infections of Eukaryotes Functional Significance of Endosymbionts

Glossary chronic infection Persistent infection by microorganisms. horizontal transmission Acquisition of microorganisms from the environment or a host other than the parent, with the consequence that the microorganisms in parent and offspring hosts are not necessarily related.

Transmission of Microbial Symbionts and Parasites Persistence of Associations Further Reading

symbiosis The intimate association between phylogenetically different organisms, often restricted to relationships from which all organisms derive benefit. symbiosomal membrane Membrane of host origin bounding an intracellular symbiotic microorganism. vertical transmission The transmission of microorganisms from a parent to offspring host.

Abbreviation ROS

reactive oxygen species

Defining Statement Eukaryotes bear persistent (i.e., chronic) microbial infections. Most microorganisms are not deleterious to the eukaryotic host, and some are beneficial or even required by the host. Many mutualistic microorganisms expand the metabolic repertoire of their host or are beneficial through effects that cannot be attributed to defined microbial traits.

Chronic Microbial Infections of Eukaryotes Eukaryotes (‘hosts’) provide multiple habitats for proliferating populations of microorganisms, including representatives of the eubacteria, archaea, protists, and fungi. In other words, eukaryotes bear chronic (i.e., persistent) infections with microorganisms that generally are not deleterious and, in some cases, are beneficial or even required by the host. To illustrate, more than 90% of the cells and an estimated 99% of all genes in a healthy human are microbial. Only a minority of microorganisms associated with most eukaryotes are pathogens. The relationships are most conveniently classified by two complementary criteria: their impact on the eukaryotic host and their location.

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Impact on the Eukaryotic Host By convention, the relationships between microorganisms and their eukaryotic hosts are assigned to three categories: mutualism, where the host benefits; commensalism, where the microorganism has no observable effect on host fitness; and parasitism, where the microorganism is deleterious to the host. Despite its widespread use, this classification is recognized to be artificial. The effect of a microorganism on eukaryotes can vary with host species, genotype, developmental age and condition, and with environmental circumstance. For example, opportunistic pathogens have no detectable impact on healthy hosts but cause disease in immunocompromised or otherwise debilitated hosts. Similarly, mycorrhizal fungi associated with plant roots are generally beneficial to plants by enhancing plant mineral nutrition, but their demand for plant-derived photosynthetic carbon can be deleterious to young seedlings with limited photosynthetic capacity. A further term, symbiosis, is used widely to describe associations between microorganisms and their hosts. Some authorities regard symbiosis as synonymous with mutualism, while others subscribe to the original definition of symbiosis as ‘any association between differently named organisms’. Similarly, the term ‘symbiont’ is interpreted by some to describe mutualistic microorganisms (as distinct

Mutualism and Commensalism | Endosymbionts and Intracellular Parasites

from commensal and parasitic microorganisms) and by others to mean any microorganism in a eukaryotic host. The more general definition of symbiosis and symbiont is used extensively in the symbiosis literature but very rarely in the parasitology literature, and overt parasites are rarely considered as symbiotic. For this reason, this article refers to symbionts as microorganisms, which are generally advantageous to their host. Immediately relevant to the definition of symbiont are symbiont-derived organelles in eukaryotes. As the term implies, these organelles have evolved from intracellular symbionts. A symbiont-derived organelle is characterized by two linked traits: first, genes ancestrally in the symbiont have been transferred to the host nucleus; second, the cognate proteins are targeted back to the organelle. In this way, the symbiont-derived organelle is inextricably dependent on the host lineage in which it evolved. There is overwhelming evidence for a symbiotic origin of mitochondria and plastids, both of which retain small numbers of genes. Location in Eukaryotic Host Microorganisms living within their hosts are termed endosymbionts (and endoparasites), as distinct from ectosymbionts (and ectoparasites), which are located on the surface of the host. Endosymbionts are classified as either intracellular (within cells) or extracellular (external to cells). Within multicellular hosts, the extracellular symbionts may reside in cavities or other spaces, for example, the body cavity and gut of animals, or between closely apposed host cells, for example, endophytic and mycorrhizal fungi in plant shoots and roots, respectively, and the latter are sometimes referred to as ‘intercellular’. Most intracellular microorganisms are restricted to the cytoplasm and separated from the cytoplasmic contents by a membrane of host origin, variously known as the symbiosomal membrane or parasitiphorous vacuole. Exceptionally, some intracellular symbionts and parasites lie free in the host cell cytoplasm, for example, the

-proteobacterium Wigglesworthia symbiont in tsetse fly, or are localized to specific organelles, for example, rickettsias in mitochondria of the tick Ixodes, various bacteria in the nucleus of ciliates. Although a broad phylogenetic range of microorganisms adopt the intracellular lifestyle, the diversity of intracellular microorganisms in any one host is low, generally one to several taxa. A far higher diversity of microorganisms colonize extracellular habitats in eukaryotic hosts. In particular, the guts of many animals bear very diverse microbial communities, comprising hundreds of taxa and including both ‘resident’ species that persist for substantial periods up to the full lifespan of the host and ‘transients’ that pass through the digestive tract with food. Among vertebrate animals, the only known intracellular microorganisms are parasites. The reasons

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for the apparent absence of intracellular symbionts are unknown, but may be related to the adaptive immune system of these animals.

Functional Significance of Endosymbionts Symbioses as a Source of Metabolic Capabilities Many endosymbioses are founded on the fact that microorganisms have a wider metabolic repertoire than their hosts. In particular, the lineage giving rise to the eukaryotes lacked the key metabolic capabilities of aerobic respiration, photosynthesis, and nitrogen fixation. Various eukaryotic groups also lack additional capabilities; for example, some protists and all animals cannot synthesize 9 of the 20 amino acids that contribute to protein (the ‘essential’ amino acids) and some coenzymes required for functioning of key enzymes (vitamins); the insects additionally cannot synthesize sterols de novo, and vertebrates cannot degrade cellulose. Repeatedly, eukaryotes have gained access to these metabolic capabilities by forming symbioses with microorganisms. Eight key metabolic capabilities of eukaryotes have a symbiotic basis, and an overview of these is provided in Table 1 and the following text. Aerobic respiration

Eukaryotes have acquired aerobic respiration from just one lineage of microorganisms, an -proteobacterium allied to rickettsias, which evolved into mitochondria. The evidence is that all mitochondria are autonomous organelles (i.e., a cell line from which mitochondria have been eliminated cannot resynthesize these organelles) and possess coding DNA with unambiguous sequence similarity to rickettsias. Various genes derived from the mitochondrial ancestor have been transferred to the nucleus of the eukaryotic host and are present in the nucleus of taxa lacking mitochondria (e.g., trichomonads), leading to the current consensus that the ancestor of all modern eukaryotes was mitochondriate. It is disputed whether the acquisition of mitochondria predated (and was perhaps instrumental in) the evolutionary origin of eukaryotes, or occurred later, in a host bearing the key eukaryotic trait of a membranebound nucleus. Photosynthesis

Oxygenic photosynthesis evolved once, in the ancestor of the cyanobacteria, and has been acquired multiply by eukaryotes. By far, the most widespread and important photosynthetic symbiont is the cyanobacterial lineage that evolved into plastids. Phylogenetic analyses point firmly to a single evolutionary origin of plastids, acquired by the protist ancestor of chlorophytes (which gave rise to the land plants), rhodophytes (red

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Table 1 An overview of endosymbioses that enhance the metabolic repertoire of eukaryotes Metabolic capability

Examples of endosymbioses

Aerobic respiration Oxygenic photosynthesis

Mitochondria (evolved from -proteobacteria) in most eukaryotes Plastids (evolved from cyanobacteria) in algae and plants. Cyanobacteria (e.g., Nostoc) and algae (e.g., Trebouxia) in lichenized fungi. Various algae (e.g., freshwater Chlorella and marine Symbiodinium in protists and animals (e.g., corals) Various bacteria in marine animals, e.g., Pogonophora (e.g., Riftia) and bivalves Rhizobia in legumes, Frankia (actinomycete) in various dicot plants,a cyanobacteria in some lichenized fungi, plants (e.g., Gunnera, Azolla, cycads) and diatoms (e.g., Rhizosolenia). Various bacteria in termites, teredinid mollusks Various bacteria and fungi in protists and animals, especially insects feeding on vertebrate blood, plant sap, and wood Various bacteria (e.g., Ruminococcus) in herbivorous vertebrates, for example, ruminants; protists in lower termites Methanogenic bacteria associated with anaerobic ciliates Bacteria (Vibrio, Photobacterium) in marine teleost fish and squid Bacteria in various animals, endophytic fungi in grasses

Chemoautotrophy Nitrogen fixation

Essential nutrient provision (e.g., essential amino acids, vitamins, and sterols) Cellulose degradation Methanogenesis Bioluminescence Secondary metabolites as protective toxins a

Members of eight families: Betulaceae, Casuarinaceae, Coriariaceae, Dastiscaceae, Eleaginaceae, Myricaceae, Rhamnaceae, and Rosaceae.

algae), and a small third group of algae, the glaucophytes. Representatives of chlorophytes and rhodophytes have been acquired by other protists, giving rise to additional algal groups bearing complex plastids bounded by multiple membranes (Figure 1). Some host lineages have subsequently become nonphotosynthetic but retained their erstwhile plastids, which perform different but essential functions. For example, the apicomplexan protists bear an organelle, the apicoplast, required for its capacity to synthesize essential terpenoids, and the genome of which is unambiguously allied to the genome of chromophyte plastids. Various photosynthetic cyanobacteria and algae enter into symbioses with nonphotosynthetic hosts. Of particular importance are the lichens, associations of fungi with algae or cyanobacteria, which dominate large areas of tundra and are abundant in temperate and tropical forests. An estimated 14 000 fungal species, including nearly half of all described ascomycetes, are lichenized, and the symbionts include 30–40 genera of algae, mostly chlorophytes, and at least 12 genera of cyanobacteria. In the marine environment, the symbiosis between corals and dinoflagellate algae of the genus Symbiodinium is the architectural foundation of shallow-water coral reefs, which are highly productive and diverse ecosystems of immense ecological and socioeconomic importance. Chemoautotrophy

Chemoautotrophic bacteria fix carbon dioxide using the energy and the reductant derived from the oxidation of reduced (usually inorganic) compounds, generally with molecular oxygen as the electron acceptor. Various chemoautotrophs form symbioses with animals living at the interface between oxic and anoxic environments,

representing a source of oxygen and reduced substrate, respectively. Habitats include the zone in marine sediments where oxygen-rich seawater percolating downward meets anoxic sediment water, deep-sea hydrothermal vents, natural gas and methane seeps, and the immediate environment around sewage outfalls. Most symbiotic chemoautotrophs are sulfur oxidizers or methane oxidizers. Their animal hosts include bivalves, pogonophoran worms, nematodes, and annelids, and these symbioses are particularly conspicuous at hydrothermal vents, where the hosts include vestimentiferan tube worms, such as Riftia, up to 2 m in length, and very large bivalves, such as Calyptogena species. A recent metagenomic analysis of the symbiosis in the interstitial oligochaete annelid Olavius algarvensis has revealed microbial complexity. This symbiosis involves four bacterial taxa located internal to the host body wall cuticle: 1-, 3-, 1-, and 4-proteobacteria. Shotgun sequencing, gene identification, and metabolic reconstruction in silico indicated that the -proteobacteria are sulfide-oxidizing chemoautotrophs and that the -proteobacteria are sulfate-reducing bacteria that can also fix carbon dioxide by the reductive acetyl coenzyme A pathway and the TCA cycle. The complementary pathways of sulfate reduction and sulfide oxidation provide for a symbiotic sulfur cycle, ensuring that the

-proteobacteria are provided with sulfide, even though the external habitat has no detectable sulfide. The animal host lacks any gut, mouth, or anus and is believed to gain most of its nourishment by engulfing and digesting the subcuticular bacteria. The bacteria also have the genetic capacity to consume host waste ammonia, and this may explain the reduced nephridial excretory system in this species.

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Figure 1 The phylogenetic distribution of plastids in eukaryotes, mapped onto an unrooted phylogenetic tree of eukaryotes based on a combination of molecular phylogenetic and ultrastructural data. According to this evolutionary scenario, the diversity of plastids can be explained by three evolutionary events. (1) The cyanobacterial ancestor of all plastids was acquired by the ancestor of the archaeplastida, giving rise to three groups – the rhodophytes (red algae) and the glaucophytes containing chlorophyll a and the chlorophytes (green algae) and allies, including the land plants, containing chlorophyll a and b. (2) A rhodophyte alga was acquired by the ancestor of the chromalveolates, generating complex plastids that have chlorophyll a and c. (3) A chlorophyte was acquired by the ancestor of the euglenids and the chlorarachniophytes. Among the complex plastids generated through steps (2) and (3), the nucleus of the primary host has been retained as a nucleomorph in the chlorarachniophytes and one group of chromalveolates, the cryptophytes. Reproduced from Baldauf S. L. (2003). The deep roots of eukaryotes. Science 300: 1703–1706.

Nitrogen fixation

The phylogenetic distribution of nitrogen fixation is broad but irregular, indicative of multiple evolutionary acquisitions and losses. The lateral transfer of nitrogen fixation genes has apparently been widespread among bacteria, but eukaryotes have gained this capability exclusively by symbiosis with bacteria, none of which (to our knowledge) have evolved into organelles. The nitrogen-fixing endosymbionts are best known in plants. Of particular importance are the nitrogen-fixing bacteria in the root nodules of legumes (comprising two separate lineages of -proteobacteria, one including Bradyrhizobium and Azorhizobium and the other including Rhizobium, Sinorhizobium, and Mesorhizobium, and -proteobacteria of the genera Burkholderia and Ralstonia) and the actinomycete Frankia in various dicot plants. An estimated 150 species of vascular plants, including many cycads, the water fern Azolla, and the

dicot Gunnera, associate with cyanobacterial symbionts of the family Nostocaceae that comprise filaments of vegetative cells (capable of oxygenic photosynthesis) and heterocysts (which fix nitrogen), and the chief advantage to the plant host is access to fixed nitrogen derived from the heterocysts. In addition, an estimated 550 species of lichenized fungi associate with both photosynthetic algae and cyanobacteria restricted to specialized structures called cephalodia, in which they fix nitrogen at high rates. These cyanobacterial symbionts are the only known nitrogen-fixing symbioses in fungal hosts. Nitrogen-fixing bacteria are present in the gut microbiota of many animals, but they generally are at low abundance and of no nutritional significance to the animal host. Some animals feeding on nitrogen-poor wood (e.g., some termites, Tenebrio shipworms) gain nitrogen from nitrogen-fixing bacterial endosymbionts. There is a general expectation that nitrogen-fixing symbioses are less

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significant in animals than in plants because animals have a limited capacity to utilize the primary nitrogen fixation product, ammonia. Indeed, ammonia is a potentially toxic waste product of metabolism for most animals. Provision of essential nutrients

Most research on microorganisms that provide specific classes of primary nutrients, such as amino acids and vitamins, has focused on intracellular endosymbionts of insects that are restricted to specific organs, variously known as mycetomes or bacteriomes (Table 2). Although these symbioses have evolved independently multiple times between diverse groups of insects and microorganisms, the associations have three common traits. (1) The microorganisms are restricted to specific insect cells, the sole function of which appears to be to house and maintain the microorganisms; these cells are known as bacteriocytes or mycetocytes (the terms are synonymous), forming organs known as bacteriomes or mycetomes. (2) The microorganisms are obligately vertically transmitted, usually by insertion from the maternal bacteriocytes directly into the eggs in the female ovary. (3) The association is required by both insect and microorganisms. The anatomical location and structural organization of the bacteriome vary widely; the bacteriome may be associated with the animal gut (e.g., tsetse flies), fat body

(e.g., cockroach), Malpighian tubules (e.g., book lice), or lie free in the body cavity (e.g., aphids). The microorganisms are restricted to the cytoplasm. They are generally unculturable and molecular methods have revealed their diversity, including -, -, and -proteobacteria, flavobacteria, and fungi, some of which are not closely related to any formally described taxa. Where similar or identical sequences are identified in multiple host species of one order or family, the endosymbiont has been assigned a novel candidate generic name (Table 2). The nature of the symbiont–host interactions has been inferred from the distribution of these relationships, which are particularly prevalent in insects feeding on nutrient-poor diets, such as plant sap (phloem or xylem) deficient in essential amino acids and vertebrate blood deficient in B vitamins (Table 2). Direct experimental evidence for the translocation of essential amino acids from bacteria to insects has been obtained for cockroaches and aphids, and the nutritional role of other symbiotic microorganisms is inferred from their complement of metabolic genes, identified either by PCR amplification or from completely sequenced genomes. Microsopical studies have revealed various other animals, usually invertebrates including earthworms, leeches, tunicates, and nematodes, which universally bear dense aggregations of microorganisms, in specific anatomical

Table 2 Distribution of intracellular microbial endosymbioses in insects Insect (a) plant sap feeders Heteroptera Plataspids (stinkbugs) Alydids (broad-headed bugs) Homoptera Auchenorrhyncha (including leafhoppers, planthoppers, cicadas) Aphids Whitefly Psyllid jumping lice Scale insects & mealy bugs) (b) vertebrate blood Heteroptera Cimicid (bedbugs) Triatome bugs Anoplura (sucking lice) Diptera Pupiparia (c) general feeders Blattidae (cockroaches) Mallophaga (biting lice) Psocoptera (book lice) Beetles, e.g. Weevils Anobiid timber beetles Hymenoptera Camponoti (carpenter ants)

Microorganisms

Ishikawella ( -proteobacteria) Burkolderia ( -proteobacteria) Baumannia cicadellinicola ( -proteobacteria) and Sulcia muelleri (Bacteroidetes); Pyrenomycete fungi in some planthoppers Buchnera ( -proteobacteria) or pyrenomycete fungi Portiera aleyrodidarum ( -proteobacteria) Carsonella ruddii ( -proteobacteria) Tremblaya princes ( -proteobacteria)

-proteobacterium allied to Serratia Arsenophonus triatominarum ( -proteobacteria) Riesia pediculicola (( -proteobacteria) in human head louse & body louse Wigglesworthia in Glossina Arsenophonus in streblids and hippoboscids Blattabacterium (flavibacteria) Not known Rickettsia sp. Various -proteobacteria Symbiotaphrina (yeasts) Blochmannia ( -proteobacteria)

Mutualism and Commensalism | Endosymbionts and Intracellular Parasites

Animal nitrogenous waste compounds

Nitrogenous compounds synthesized by the microorganisms Animal

Microorganisms

Figure 2 Nitrogen recycling by endosymbiotic microorganisms. The microorganisms (collectively displayed here as a circle) transform nitrogenous waste products of the animal (ammonia, urea, etc.) into nitrogenous compounds valuable to animal metabolism, and these compounds are translocated back to the animal tissues.

locations. Similarly, some protist species consistently bear intracellular bacteria. The identity and function of the microorganisms have received little study, but a nutritional role is often invoked. Genome sequence analysis is assisting with the construction of specific hypotheses. For example, the bacteria Wolbachia in filarial nematodes have been implicated in the provision of nucleotides and heme to their host on the basis of the predicted gene complement of the complete genome sequence. Immediately related to the nutrient provisioning is the role of microorganisms in recycling, especially of nitrogen. Nitrogen recycling refers to the microbial consumption of animal waste nitrogenous compounds (e.g., ammonia and urea) to synthesize ‘high-value’ nitrogenous compounds (e.g., essential amino acids), which are released back to the host (Figure 2). Nitrogen recycling has been implicated from radiotracer and 15N-tracer studies of several symbioses, including cockroach– flavobacteria and the relationship between corals and their dinoflagellate algal symbionts Symbiodinium. Cellulose degradation

Vertebrate animals lack the capacity to digest cellulose and other plant cell wall polysaccharides, such as hemicellulose, and many herbivores can exploit these compounds only by association with cellulolytic microorganisms. Typically, the microorganisms are restricted to an anoxic portion of the gut, the ‘fermentation chamber’ where they degrade the plant polymers to support their own growth, releasing short-chain fatty acids as the waste products of anaerobic respiration. These compounds diffuse into the host bloodstream and are used as substrates for aerobic respiration by the animal host. These symbioses are known in the hindgut (colon or cecum) of virtually all herbivorous mammals (the giant panda is reputedly an exception) and various herbivorous birds and lizards. They are called postgastric symbioses because the fermentation chamber is distal to the

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enzymatic region of the gastrointestinal tract. Some mammals, for example, ruminants, such as cattle, sheep, deer, kangaroos, and colobine monkeys, and at least one bird, the leaf-eating hoatzin, additionally have pregastric fermentation chambers, that is, proximal to the digestive region. Cellulolytic symbioses are apparently rare among invertebrate animals, probably because many invertebrates have intrinsic cellulases and because, for small animals, the costs of maintaining an anoxic fermentation chamber would be unduly high. However, a minority of wood-feeding termites exploit cellulolytic microorganisms in an enlarged hindgut, known as the paunch. Unlike the bacterial cellulose-degrading symbionts of vertebrates, the cellulolytic symbionts in termites are obligately anaerobic flagellate protists of the orders Hypermastigida, Trichomonadida, and Oxymanidida. At least 400 species have been reported, most unknown from any other location, and several, including Trichomitopsis termopsidus and Trichomympha sphaerica, have been brought into culture. Related protists occur in wood-eating roaches of the genus Cryptocercus. The cellulolytic microorganisms represent one of many functional groups of microorganisms in the digestive tracts of many animals (see below). For the majority of associations that are postgastric, only diffusible microbial products (e.g., short-chain fatty acids) are available to the host. Other microbial products (proteins, vitamins, etc.) are lost from the system via the feces and are recovered only in host species that display coprophagy, that is, consumption of feces. Animals with pregastric fermentation chambers, by contrast, can gain nutrients from their microbiota by the digestion of microbial cells or other products that pass from the fermentation chamber into the gastric stomach. Methanogenesis

Methanogenic bacteria generate ATP by synthesizing methane under strictly anoxic conditions, most commonly by the reduction of carbon dioxide with hydrogen. All known methanogens are euryarchaeote Archaea. The consumption of hydrogen by methanogens is advantageous to anaerobic eukaryotes because the rate of oxidative reactions, such as glycolysis, can otherwise be depressed by high levels of hydrogen. In other words, methanogens can act as an electron sink for anaerobic hosts. Methanogenic symbioses are prevalent in anaerobic ciliate protists. Phylogenetic analyses suggest that the symbiotic habit has evolved multiple times among methanogens and that most symbionts are closely related to free-living taxa. The symbiotic methanogens may be ectosymbiotic, for example, on the surface of ciliates of the family Entodiniomorphida living in the rumen of cattle, or intracellular, as for Methanobacterium formicicum in Plagiopyla and Metopus species in rice paddies and landfill sites, and Methanobrevibacter species in cellulolytic protists in termite guts.

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Bioluminescence

Luminescence is the generation of light by the oxidation of a substrate, generically known as luciferin, catalyzed by the enzyme luciferase. Bioluminescence has evolved at least 35 in eukaryotes, involving at least 5 different biochemical reactions, and is rare in bacteria, being restricted to four genera, Vibrio, Photobacterium, Alteromonas, and Xenorhabdus. Linked to this, most instances of luminescence in eukaryotes are intrinsic; the only known luminescent symbioses involve Vibrio/Photobacterium in a minority of marine teleost fish and squid, and Xenorhabdus in some terrestrial entomopathogenic nematodes (see ‘Synthesis of secondary compounds’). Marine animals use light for communication in shoaling and courtship (e.g., flashlight fish), as a startle response to distract potential predators in deep waters, as a lure (e.g., angler fish) and as camouflage (by counterillumination to obscure the silhouette of the animal otherwise evident against downwelling light to predators lower in the water column). The luminescent symbioses in marine fish and squid are housed in light organs. The symbiotic bacteria are restricted to many narrow tubules, with access to the nutrients and oxygen required for sustained light production. They are maintained at high densities; this is essential for light production, which is regulated by quorum sensing via an autoinducer that, on reaching a certain concentration, induces the expression of the lux genes coding for the luciferase. Bacterial luminescence is generally less intense than intrinsic sources, requiring mirrors and lenses to maximize emission, and, unlike intrinsic luminescence, it is continuous (it cannot be turned off), requiring shutters, chromatophores, and so on to control the timing of its emission. Essentially, the greater anatomical complexity of symbiotic light organs than intrinsic light organs relates to the limitations of bacteria as a light source.

Synthesis of secondary compounds

Various eukaryotic groups have exploited the capacity of microorganisms to synthesize bioactive secondary compounds, principally as mediators of antagonistic interactions with natural enemies or prey. One of the best-studied relationships is between grasses, particularly agronomically important species, and endophytic fungi of the form genus Neotyphodium allied to the genus Epichloe¨. Hyphae of these fungi, which ramify through the shoot of the grass, contain alkaloids that are toxic to animals. Animals that feed on fungal-infected grasses ingest these alkaloids and become debilitated and may die. For example, ergot and indole diterpene-type alkaloids present in endophytes of tall fescue and perennial ryegrass cause neurological disorders known as ‘the staggers’ in cattle and sheep and also confer resistance to various insect and nematode pests.

The incidence of animals that derive protection from secondary metabolites synthesized by symbiotic microorganisms is uncertain, and only a few examples have been explored in detail. These include the production of compounds known as ‘bryostatins’ by the bacterium Endobugula sertula in the marine bryozoan Bugula neritina; polyketides, such as pederin, by uncultured pseudomonads both in beetles of the genus Pederus and in sponges, including Theonella swinhoei; and a variety of cyclic patellamide peptides by cyanobacteria of the genus Prochloron in marine ascidians (sea squirts). All these compounds have been suggested to have a protective role against predators, and this has been demonstrated for the Pederus beetles. Furthermore, the genes for pederin synthesis are borne on a putative pathogenicity island in the pseudomonad genome, suggesting that this capability is horizontally transmissible among bacteria. A further instance of secondary product biosynthesis by microbial symbionts is provided by the luminescent bacterium Xenorhabdus luminescens, the obligate symbiont of soil-borne heterorhaditid nematodes that parasitize insects. When the nematodes infect a susceptible insect, the bacteria are released into the insect hemolymph (‘blood’) and proliferate rapidly. In this location, they synthesize compounds, including antibiotics, thereby preventing invasion by other bacteria. This ensures that the insect habitat is sustained for the 2 weeks required for the reproduction of the nematodes. The bacteria also produce a red anthraquinone pigment and light, believed to attract new insect hosts by day or night toward the young infective nematodes as they emerge from the insect host. Some authorities, however, doubt the significance of the luminescence in this context because the light intensity is very low. An alternative possible role of the luciferase is as a terminal oxidase with greater affinity for oxygen than the cytochrome pathway, promoting aerobic metabolism at low oxygen tensions in the insect cadaver. A remarkable instance of evolutionary change in function is provided by the protein GroEL synthesized by the symbiotic bacteria Enterobacter aerogenes in antlions (insects of the family Myrmeleontidae, a group of lacewings). GroEL is a bacterial chaperone that mediates protein folding, but the GroEL homologue in the symbiotic E. aerogenes is a potent toxin, released in the insect saliva that paralyzes prey. Symbiont-Mediated Modification of Host Physiology and Vigor Comparisons of the traits of various hosts bearing and experimentally deprived of their symbiotic microorganisms have revealed differences that cannot be attributed readily to defined traits of the microorganisms. For example, plants infected with mycorrhizal fungi are commonly more drought-tolerant than uninfected congeners, the gut

Mutualism and Commensalism | Endosymbionts and Intracellular Parasites

microbiota of animals can confer resistance to gut pathogens, the tolerance of high temperatures by some insects is promoted by specific microbial symbionts, and calcification and skeletal growth of many corals is promoted by the presence of symbiotic dinoflagellate algae. In most instances, the underlying processes are obscure or uncertain, but microbial impacts on the host metabolism or hormonal and immune systems have been invoked. It should also be recognized that, although generally beneficial, these interactions can be deleterious to the host in certain circumstances. For example, the gut microbiota can promote obesity in mice and humans, and the commensal gut bacteria in insects are instrumental in the mortality of insects that ingest the -endotoxin of Bacillus thuringiensis (the toxin-mediated modification of the insect gut wall allows commensal gut microorganisms to gain entry into the insect body cavity, where they proliferate causing lethal septicemia). Microbial-mediated modification of host traits has been studied in detail for one group of insects, the aphids. Many aphids bear one to several ‘secondary symbionts’ in addition to the obligate intracellular bacterium Buchnera, which has a required nutritional role. The secondary symbionts have a wider tissue distribution than Buchnera and can be acquired horizontally both by the oral route and sexually. The secondary symbionts are not required by the aphids and are absent from many individual aphids in natural populations. They are all bacteria and include a rickettsia, a Serratia species, recently assigned the candidate name S. symbiotica, and two further -proteobacteria with the candidate names Regiella insecticola and Hamiltonella defensa that are not closely related to any formally described taxa. S. symbiotica can ameliorate the negative impact of heat shock on aphid fecundity and survivorship, while R. insecticola promotes aphid resistance to entomopathogenic fungi and H. defensa can protect aphids from parasitoids. In the pea aphid, the prevalence of the secondary bacteria differs with the plant from which the aphids are isolated. For example, aphids on clover species generally bear R. insecticola, while those on alfalfa tend to have H. defensa. The contribution of the bacteria and aphid genotype to these patterns is uncertain because experiments on the impact of manipulating the secondary symbiont complement on the capacity of aphids to use plant species have yielded contradictory results. Intracellular Parasites The intracellular habitat has several plausible advantages for microorganisms: evasion of extracellular defenses (e.g., antimicrobial peptides, complement and circulating antigens in the blood and tissue fluids), access to an alternative source of nutrients, and as a route to colonize new tissues in multicellular hosts. It is

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exploited by various parasitic microorganisms: facultative intracellular parasites, which can also thrive in the extracellular condition (e.g., in blood or the tissue fluids of animals), and obligate intracellular parasites, which can proliferate only within eukaryotic cells. Facultative parasites include Shigella species, which proliferate and spread among epithelial cells of the colon, and Mycobacterium tuberculosis, which invades the alveolar macrophages of the lungs. Rickettsias, mycoplasmas, and Chlamydia species are all obligate intracellular parasites. Most intracellular parasites are deleterious by causing disease, either through direct effects, for example, toxins, or as a consequence of the host immune response to their presence. One exceptional type of intracellular parasite is the reproductive parasites. These parasites are transmitted vertically via females and specifically target male hosts, for example, by killing or feminizing them or by inducing parthenogenesis in females (see below). Reproductive distortion by microorganisms is apparently restricted to arthropods and is mediated by very few microorganisms. Most known instances involve the proteobacterium Wolbachia, reportedly present in at least 20% of all insects, but Cardinium (Bacteroidetes), microsporidia, flavobacteria, and spiroplasmas have also been implicated.

Transmission of Microbial Symbionts and Parasites Two modes of transmission are recognized. Vertical transmission is the transfer of microorganisms from parent to offspring, usually via the mother in sexually reproducing hosts. Horizontal transmission is the acquisition of microorganisms from the environment or a host other than the parent, with the consequence that the microorganisms in parent and offspring hosts are not necessarily related.

Vertical Transmission Vertical transmission has important evolutionary consequences for microorganisms. The offspring of the host represent habitats for the progeny of vertically transmitted microorganisms, with the consequence that the microorganisms have a selective interest in the reproductive output of their host. Most vertically transmitted symbionts are, consequently, beneficial to their hosts, and vertical transmission can be considered as a route by which hosts ‘impose’ mutualistic traits on their microbial partners. Furthermore, where vertical transmission is obligate, the phylogenies of the host and microorganism are congruent.

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The advantage to the host of vertical transmission is that its offspring are assured of gaining a compatible symbiont – or, at least, a symbiont compatible with its parent. Vertical transmission is particularly important where the symbionts are rare in the free-living environment. Vertical transmission may involve highly regulated, coordinated processes in host and microorganism. This is illustrated by the transmission of the bacteria Riesia pediculicola in the human body louse Pediculus humanus. The mycetocytes in this insect are aggregated together as a coherent organ, the stomach disc, just ventral to the stomach. During the final molt of the female insect, the bacteria are expelled from the mycetome and they migrate to the reproductive tract, such that in the adult female louse, all the bacteria are associated with the reproductive organs. In the male, they are retained in the stomach disc. The bacteria in the adult females are initially associated with the lateral oviducts. They subsequently penetrate the oviduct wall and gain access to the insect cells lining the oviduct. From here, they are transferred to the insect cells in the pedicel and then to the cytoplasm of each egg as it matures (Figure 3). Although, as considered above, vertical transmission promotes overlap in the selective interests of the host and its complement of microorganisms, it does not eliminate conflict between the partners. In sexual hosts with exclusively maternal transmission, the source of conflict is the sex ratio of the host offspring, with a 1:1 female to male ratio generally optimal for the host, and an excess of females optimal for the microorganism; all microbial cells transmitted to a male host will die with that host.

Stomach disc Ovary

Reproductive distortion is widespread in arthropods, mediated by Wolbachia and other taxa in several different ways as follows: 1. Parthenogenesis, to give twice the number of female offspring relative to uninfected hosts, appears to be restricted to host taxa with haplodiploid sex determination (i.e., fertilized (diploid) eggs develop as females and unfertilized (haploid) eggs develop as males in uninfected hosts) and most examples of microbialmediated parthenogenesis are in insects of the order Hymenoptera, especially wasps. 2. Male hosts are feminized, thereby doubling the number of female offspring, the same consequence as microbial-mediated parthenogenesis (described above). The principal taxa susceptible to feminization by microorganisms are Crustacea, specifically isopods (woodlice) and amphipods, but feminization of insects (e.g., the moth Eurema hecabe) has also been reported. 3. Uninfected eggs are killed by a factor associated with the sperm from infected hosts, but crosses between infected males and females, and between uninfected males and infected females, are fertile. (Table 3) This mode of host reproductive manipulation is usually known as cytoplasmic incompatibility. By killing uninfected eggs, the frequency of infected females in the population is increased, often to very high levels or fixation, at which point the microorganism has a very small, or no, impact on the reproductive output of the host population. Although the microorganisms causing cytoplasmic incompatibility occur in the testes of the male insect, the mature sperms are uninfected. 4. Preferential killing of male hosts can result in increased fitness of the female hosts (and therefore of the vertically transmitted microorganism) where the females benefit from the death of their brothers through reduced chance of inbreeding or depressed intersib competition. Most sexual eukaryotes with vertically transmitted microorganisms have an unbiased sex ratio, and this suggests that the sex determination mechanisms in most eukaryotes are difficult to distort. However, the possibility cannot be excluded that reproductive parasitism is

Bacterial inoculum in basal egg Pedicel 1h

Figure 3 Vertical transmission of endosymbionts of insects. Symbiotic bacteria Riesia sp. in the human body louse Pediculus humanus. (a) Transmission of bacteria from stomach disc to ovaries of female insect. (b) Transfer of bacteria from pedicel at the base of each ovary to basal egg in one ovariole. See text for full description. Redrawn from Ries, E. (1931). Die symbiose der lau¨se und federlinge. Zeitschrift fur Morphologie undO¨kologie der tiere 20: 233–367.

Table 3 Disruption of host reproduction by cytoplasmic compatibility caused by infection with the bacterium Wolbachia: p compatible ( ) and incompatible (X) crosses between hosts Male host Female host Uninfected Infected

Uninfected

Infected

p p

X p

Mutualism and Commensalism | Endosymbionts and Intracellular Parasites

one factor limiting the incidence of vertical transmission. Two further factors may also be important. The first is the cost of housing and nourishing symbiotic microorganisms. This may be significant at developmental stages where the microorganisms confer little or no benefit, such as during early development of the host. For example, the mycetocyte symbionts of insects occupy up to 10% of the egg volume and often proliferate rapidly after transfer to the egg, presumably utilizing the egg’s nutritional reserves. The second factor limiting the incidence of vertical transmission is anatomical barriers in the host, which restrict microbial access to the gametes of the host. For example, the root symbionts of plants, for example, rhizobia and mycorrhizal fungi, are invariably horizontally transmitted, while shoot-borne microorganisms, such as bacteria in the leaf nodules of Ardisia species and cyanobacteria in leaflets of the water fern Azolla, are vertically transmitted. In animals, the gut wall is a crucial barrier to microbial colonization of the body cavity and, ultimately, the gonads; gut-borne microorganisms are generally horizontally transmitted, while microorganisms in the body cavity or internal organs (e.g., mycetocyte symbionts) are vertically transmitted. The anatomical barrier of the gut wall can, however, be bypassed by animal behavior. For example, vertical transmission of the -proteobacterium Ishikawella in the gut of stinkbugs is achieved by the maternal deposition of fecal pellets bearing these bacteria, which are consumed by the larvae immediately on hatching from the egg. Horizontal Transmission Chemical signaling between the microorganisms and the host plays a critical role in the establishment of symbioses with horizontally transmitted symbionts. This is particularly well understood in the relationship between legumes and nitrogen-fixing rhizobial bacteria because the symbiosis is amenable to molecular dissection, including the analysis of symbiosis formation by panels of mutants and the use of reporter genes to identify when and where individual genes are expressed as the symbiosis is established. It is now apparent that the so-called flavonoidNod factor-kinase signaling cascade (Figure 4) plays a crucial role in symbiosis formation. The first step in this cascade is the detection of a compatible plant host by rhizobial cells in the rhizosphere. The host signal is a specific flavonoid in the root exudates that diffuses into the rhizobial cell. The rhizobium responds to this chemical signal by synthesizing a responding signal, specifically an acylated oligosaccharide called a Nod factor. The Nod factor is the signal to the plant to allow the rhizobia to colonize the root and to initiate the development of the nodule that, in due course, houses the rhizobia. It is also an important determinant of the specificity of the symbiosis. Nod factors vary in the

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Plant flavonoid

Nod factor of rhizobium Nodule formation

NFR/1 DMI1 DM12

NSP2 Ca2+ spiking

DMI3

Figure 4 Signal transduction between symbiosis of alfalfa roots with rhizobia. The DMI2 protein is a receptor kinase with leucine-rich repeats, similar to plant kinases involved in defense against pathogens; DMI3 is a calcium-calmodulin kinase; and NSP2 has a GRAS domain characteristic of transcriptional regulators.

structure of the acyl chain and number and position of acetyl and sulfate groups, and each legume species associates only with rhizobia that produce Nod factors of certain chemical structures. The molecular basis of the plant response to the rhizobial Nod factor includes a protein, specifically a serine/threonine receptor kinase with one or more LysM motifs that is predicted to bind the acetyl-glucosamine backbone of the Nod factor. The result of this molecular interaction is a signaling cascade in the plant cell, as outlined in Figure 4. The relationship between the molecular signalingmediated flavonoid-Nod factor and the route by which rhizobia infect the root and the development of the nodule is understood in broad outline. The initial interaction occurs at the zone of contact between the rhizobial cell and a root hair, which responds to the Nod factor by curling at the site of contact. The cellular machinery for growth in the root hair is then reorganized to create an invaginated tube, known as the infection thread, which extends through the cell toward the root cortex, with its dividing population of rhizobial cells. At this early stage in infection, the region of the cytoplasm surrounding the nucleus undergoes regular oscillations in Ca2þ concentration, a process known as calcium spiking. This is required for the activation of the calcium-dependent calmodulin kinase DMI3 and the transfer of the putative transcription factor NSP1/2 to the nucleus. The first sign of the developing nodule is a wave of cell division near the center of the root. It is apparent about 12 h after rhizobial contact with the root, while the infection threads and the rhizobia are extending down through the epidermal and outer cortical cells, and it results in a mass of cells known as the nodule primordium. As the infection thread extends into this region, rhizobia are endocytosed from the infection thread into cells, where they differentiate into nitrogen-fixing bacteroids. Some plant cells remain uninfected and these form the nodule meristem.

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Gaining Entry into Host Cells For intracellular microorganisms, especially horizontally transmitted taxa, access to the host cell contents is an essential feature of transmission. This topic has been studied extensively for facultatively intracellular bacterial parasites of mammalian cells. These microorganisms gain entry into the host cell by phagocytosis: some, such as M. tuberculosis, are phagocytosed by macrophages (specialized phagocytic cells of the immune system), but many others induce phagocytosis by cells that are not normally phagocytically active. Two broad mechanisms by which bacteria invade nonphagocytic cells have been identified: the trigger mechanism and the zipper mechanism. The bacterial proteins mediating uptake of various pathogens have been identified, and they are key virulence factors. In the trigger mechanism adopted, for example, by Salmonella and Shigella, the effector proteins are secreted from the bacterial cell via the type III secretion system into the host cell, resulting in membrane ruffling. Actinrich protrusions are thrown up and fold over, engulfing the bacterium. The underlying mechanisms include the activation of Rho family GTPases, such as Rac and Cdc42, and direct binding to F-actin, resulting in reorganization of the actin microfilament network. These responses of the host cell are reminiscent of their response to growth factors, suggesting that the same intracellular signaling cascades may be involved. The zipper mechanism displayed, for example, by Listeria monocytogenes and Yersinia pseudotuberculosis to induce phagocytosis is mediated by adhesin proteins on the bacteria surface that bind to host cell surface molecules, which normally mediate adherence to other cells or the extracellular matrix. The key protein in Y. pseudotuberculosis is known as invasin and it binds to a subset of 1-integrins (receptors for fibronectin), and the internalin protein of L. monocytogenes binds to E-cadherin. The resultant cytoskeletal rearrangements in the host cell lead to phagocytosis of the bacteria. Intracellular symbionts also gain entry into host cells by phagocytosis, but the underlying mechanisms have not been studied extensively. Some authorities predict that internalization is mediated by specific surface molecules of the symbiont, analogous to the virulence factors of pathogens. However, studies of the uptake of symbiotic Chlorella by hydra suggest that the relatively nonspecific trait of surface charge may be the key discriminant of an acceptable symbiont.

Persistence of Associations Host Controls Over Microbial Infections Endosymbioses persist, meaning that the population of microorganisms is retained within the host for extended

periods, potentially for the full lifespan of the host and, in vertically transmitted associations, through multiple host generations. Furthermore, the density and proliferation rate of the microorganisms are tightly regulated such that the microbial population increases in parallel with the host, neither overgrowing nor being diluted out by host growth. Generally, this requires suppression of microbial growth rates. For example, the doubling time of the dinoflagellate alga Symbiodinium is 99%) involve ascomycetes. It is estimated that nearly 50% of all known ascomycetes are lichenized. All of these are so-called inoperculate ascomycetes, a group that includes virulent plant and insect pathogens. Approximately 90% of lichens involve an algal photobiont. Some 25–30 genera of algae, which may represent hundreds of species, are involved in lichen associations. In addition, more than a dozen genera of cyanobacteria participate in lichen symbioses. However, information on photobiont taxonomy is incomplete because in most lichens neither the algal nor cyanobacterial photobionts have been identified to the level of species. Most lichens involve two partners, a fungus and either an alga or cyanobacterium, but many species include both an algal and a cyanobacterial partner. Generally, the cyanobacterial partner in a tripartite relationship is considered as a secondary partner and is sometimes housed separately on the surface of the thallus. The cyanobacterium in a tripartite symbiosis is most significant for its input of biologically useable nitrogen into the thallus system. Lichens with cyanobacterial partners are significant nitrogen sources for many ecosystems, and net nitrification of soils under lichens without cyanobacterial partners has been documented as well. Biological soil crusts with significant lichen populations contribute to potential nitrogen fixation, reduce nitrogen leaching, and moderate soil microclimate. Moisture and temperature fluctuations beneath lichen thalli are slowed, and certain potentially toxic metals, for example aluminum, are absorbed by lichen thalli. Lichens ameliorate and improve soil conditions, encourage the establishment of microbial organisms, and produce an environment that supports a diversity of invertebrates. They can be considered as important contributors to pedogenesis (soilbuilding) in many ecosystems. Lichen mycobionts are similar to other fungi that associate in a long-term, stable, cell-to-cell collaborative interface with other species. But in other interspecies symbioses that include fungi, for example, mycorrhizal relationships, which affect approximately 90% of plant species, nutrient exchange occurs in both directions

between the mycobiont and the photobiont. With all of the nutrients in lichens flowing from the photobiont to the mycobiont, the relationship has been considered as a controlled parasitism, in which the fungal partner maintains a population of carbohydrate-producing photobionts. Anatomically, lichen fungi communicate with photobionts in simple cell-to-cell interfaces or through the extension of haustoria or appressoria, specialized outgrowths of the fungal cells that produce invaginations in the photobiont (Figure 17). In most cases, the relationship is nonpathogenic. Photobiont cell membranes are not penetrated and photobiont cells are not regularly destroyed in a stable lichen thallus. Mutual sustained recognition between bionts is essential for thallus success, and some control is apparently exerted by the photobiont. Some authors note that an extended photobiont cell cycle, such as that observed in many lichens, would reduce the number of times the symbiosis must be reestablished on a cell-to-cell basis, minimizing opportunities for pathogenic encounters. The rate at which photobiont cells undergo the cell cycle is apparently under regulation, but that whether and to what extent that regulation is influenced by the fungal partner is unknown. Recognition between the mycobiont and photobiont may be mediated by polysaccharides that are found in both, especially on membrane surfaces. Lectins, which are produced by the mycobiont and are present on the mycobiont cell wall, have been demonstrated to bind to specific ligands on the cell wall of algal photobionts, suggesting a role in communication between the partners. Studies of reestablishment of lichens from axenic culture suggest that certain nonself recognition and defense signals are weakened during the early stages of thallus development. The genomic implications of this mutual ‘quieting’ have not been fully explored.

Light

Moisture

Mycobiont Photobiont Communication Control (?) Haustoria

Carbohydrates

Appressoria

Figure 17 Summary of nutrient exchange and mutual control between mycobiont and photobiont in the lichen thallus.

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Most lichens experience profound drying for pronounced periods. During these periods, respiration rates are extremely low and photosynthesis is not in operation. Lichen thalli hydrate at different rates when exposed to moisture, but most require sustained exposure to moisture before physiologically significant hydration occurs. It is estimated that within minutes of effective hydration, photobionts release a very high proportion of stored photosynthate (up to c. 90% of the cell’s reserves), which is absorbed by the fungus. Simple carbohydrates like glucose are then converted to the fungal sugar mannitol, which is stored in fungal cells or converted for use in cellular respiration in the mycobiont. Cell wall building and other cellular functions of both partners occur in the presence of available nutrients and when adequate moisture is available for cellular activity. Hence, thallus growth is limited by the frequency and duration of drying events. These events may be diurnal, for example, in desert lichens that obtain their moisture from morning dew, or in foliicolous (leaf-inhabiting) lichens in rainforests that experience daily rainfall. Drying events may be less frequent but are longer and lasting for several weeks or even months. Frozen water is generally unavailable for photosynthesis and other cellular activities, and lichens may experience many months of inactivity under snow or ice. Many lichens are able to take advantage of microscopic amounts of liquid water that may occur under snow or ice in very cold temperatures. These species can apparently maintain marginal physiological activities in extremely cold conditions. Lichens are notoriously slow growers and generally this has been attributed to the wetting-drying patterns that they experience. However, fungal growth may also be limited by controls exerted by the photobiont host that have yet to be elucidated. The lichen thallus has evolved in response to potentially very high levels of thallus moisture fluctuation. When dry, the lichen surface may be any shade of opaque white, yellow, brown, orange, greenish, or even purple-black, but when moistened, most lichens appear translucent. Lichens recover relatively rapidly from desiccation, but studies have shown that water saturation can limit photosynthesis and effective gas exchange. The role of hydrophobins, a class of proteins produced in the thallus, have been intensively studied in relation to this problem. Hydrophobins have been implicated in several adaptive strategies, such as allowing water translocation, maintaining water-free interhyphal spaces (for optimal oxygen diffusion), and defining strata within the thallus that slow water loss. Light response curves of net photosynthesis in lichens are highest at relatively low light levels, perhaps as an adaptive response to desiccation that occurs at higher light levels. This lends support to the hypothesis that some photobionts, which reach maximal photosynthetic levels in attenuated light, achieve greater fitness in lichen thalli than in free-living conditions.

Photobiont algae, and to an extent cyanobacterial partners, undergo changes in morphology in the lichen thallus. These changes have been intensively studied. Less is known about physiolgical and morphological changes that the fungus experiences. Lichen fungi grown axenically often appear mold-like, with branching hyphae growing more or less concentrically outward from the point of establishment. This is a profound difference from the appearance of the fungi in lichen thalli. Within thalli, fungal cells that are in contact with photobionts may be thin-walled, presumably as a mechanism for obtaining nutrients from photobionts. This is especially the case with haustoria and appressoria, which are cellular outgrowths of the fungus. In thallus regions where there is no photobiont contact, fungal cells may become quite thick-walled, agglutinating into sclerotiumlike bodies that appear hard and sometimes carbonized. Fungal cells organize into pseudo-tissues within the thallus, stratifying, partitioning, and otherwise organizing its internal space. Agglutinated fungal cells may provide adherence to rocks, tree trunks, and other substrata. They may also participate in water relations of the thallus. The fungal partner has been implicated in directing the overall growth of the thallus. Mersitem-like regions of very tightly packed fungal cells with no direct photobiont content have been demonstrated to undergo coordinated growth that leads to macrophenomena such as thallus branching (Figures 18 and 19). Although it is still a matter of some controversy, it appears that secondary metabolite production is accomplished mostly if not exclusively by the fungal cells. It is generally assumed that production of these metabolites is undertaken in association with (or at least through contact with) photobionts. However, the production of secondary metabolites has been observed in many lichen fungi when grown axenically. The presence of photobionts has also been implicated in inhibiting the production of certain

Figure 18 Meristem-like fungal regions at growing tip of Cladina mitis thallus. Fungal bundles guide branching events as well as subtle torsion of the thallus. Scale ¼ c.100 mm.

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Figure 19 Meristem-like fungal regions at very young growing tip of Notocladonia sp. Development of these regions, including enlargement, splitting, and torsion is synchronized and is reflected in later thallus development. Scale ¼ c. 50 mm.

metabolites, perhaps through the secretion of urease inhibitors by algal partners. Generally, secondary metabolites are more varied and abundant in species with algal photobionts than in species with cyanobacterial partners.

Discussion Lichens are problematic. The thallus, a highly integrated functioning body, is morphologically simple and in many species, indistinct. Its developmental biology and functional anatomy are only obliquely understood. How morphogenetic roles within the thallus are partitioned is not understood. Likewise, the modes of production of secondary metabolites, the partitioning and control over nutrients, and the role of secondary partners such as cyanobacteria are incompletely known. The mycobiont– photobiont relationship provides a model for studying other symbioses. Yet, the state of our knowledge about mutual control between symbionts in lichens is in its very early stages. What is the extent of control? What is the agency of control? Is there a suite of interrelated chemical signals that maintain control? Are associated proteins activated from signals apprehended at the membrane level? What are the feedback mechanisms that modulate immune recognition between partners? How are signals between (or among) symbionts mediated? What causes the profound morphological and physiological changes that both symbionts undergo in the lichen thallus? The nature of the symbiosis requires further consideration. From one perspective, lichens may be considered as a sort of commensalism. In this framework, the photobiont and mycobiont have coevolved such that the photosynthetic host suffers no damage and the mycobiont is resistant to the host’s immune system. However, the loss

of sexuality in the photobiont is a significant loss in terms of fitness, and the fact that most myco- and photobionts require the symbiotic thallus to live suggests that in some respects the fitness of both the partners is compromised. Mutualism, a model in which both partners benefit, may better describe lichens. However, the unidirectional flow of nutrients into the fungal partner complicates this model and suggests that parasitism describes the lichen symbiosis more accurately. Some authors have accepted the classification of mutualism on the basis of the increased ecological amplitude of both symbionts in the lichen and the fact that lichen relationships are relatively long-lived while parasitic symbioses are generally shorter in duration. However, the parasitic nature of the fungus on the photobiont cannot be overlooked. It seems that Ahmadjian’s (1993) term ‘controlled parasitism’ is the most likely descriptor for lichens at present. It is ironic that while lichens may be the best known symbiotic relationship, they defy easy classification. Terms such as commensalism, mutualism, parasitism, and others that have been mustered to describe other symbiotic phenomena do not adequately describe the lichen symbiosis. Lichens make a significant contribution to the health of the biosphere. Most studies that describe lichen activities within ecosystems point to the beneficial effect of lichens as ground cover, forest epiphytes, soil stabilizers, and soil builders. Lichens are an important food for some ungulates and probably they provide nutrition for other organisms as well. Yet lichens do not appear in most macro-ecological analyses of the biosphere. A great deal more ecological work is needed in order to model the role of lichens in natural ecosystems. Lichens are widely distributed, but many species are highly sensitive to their environment and reflect a narrow and specialized geographic distribution. While all lichen species tolerate a great range of environmental conditions, especially as regards water relations, they are threatened by numerous anthropogenic changes in their environment. There is much to learn about the physiological mechanisms by which lichens function as photosynthetic units in challenging environments. Lichens have always been difficult to identify on the basis of their variability and human limitations in describing lichen form. Generally, the group has suffered because too few scientists undertake the study of lichens. Much more floristic work is required in order to better document lichen populations worldwide. As lichen habitats are destroyed by human interference, the species will inevitably be lost. Further resources are required, not only to identify and record lichen species but also to explore the numerous physiological phenomena that characterize the symbiosis. Lichens are subject to evolution like every organism on the planet. Their evolutionary history suggests an ancient lineage that has weathered innumerable changes

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in the environment. That evolutionary history is complicated by the presence of two or more symbionts in every lichen species. Further research will elucidate not only the evolutionary relationships of lichens but also the nature of their symbiosis. See also: Ecology, Microbial; Mycorrhizae; Photosynthesis: Microbial

Further Reading Ahmadjian V (1993) The Lichen Symbiosis. New York: John Wiley & Sons. Ahmadjian V and Paracer S (1986) Symbiosis: An Introduction to Biological Associations. Worcester, Massachusetts: University Press of New England. Elix JA (1996) Biochemistry and secondary metabolism. In: Nash TH III (ed.) Lichen Biology, pp. 154–180. New York: Cambridge University Press. Galun M (ed.) (2000) CRC Handbook of Lichenology (2 vols). Boca Raton, FL: CRC Press.

Green TGA, Lange OL, and Cowan IR (1994) Ecophysiology of lichen photosynthesis: The role of water status and thallus diffusion resistances. Cryptogamic Botany 4: 166–178. Hammer S (2001) Lateral growth patterns in the Cladoniaceae. American Journal of Botany 88: 788–796. Hawksworth DL and Pirozynski KA (eds.) (1988) Coevolution of Fungi with Plants and Animals. London: Academic Press. Hill DJ (2004) The cell cycle of the photobiont of the lichen Parmelia sulcata (Lecanorales, Ascomycotina) during the development of thallus lobes. Cryptogamic Botany 4: 270–273. Honneger R (1993) Developmental biology of lichens. The New Phytologist 125: 659–677. Kershaw KA (1985) Physiological Ecology of Lichens. Cambridge: Cambridge University Press. Lawrey JD, Torzilli AP, and Chandhoke V (1999) Destruction of lichen chemical defenses by a fungal pathogen. American Journal of Botany 86: 184–189. Nash TH III (ed.) (1996) Lichen Biology. New York: Cambridge University Press. Piercey-Normore MD (2006) The lichen-forming ascomycete Evernia mesomorpha associates with multipe genotypes of Trebouxia jamesii. The New Phytologist 169: 331–344. Schwenender S (1869) Die algentypen der flechtengonidien. Programm fu¨r die Rectorsfeier der Universita¨t Basel 4: 1–42. Taylor T, Hass H, Remy W, and Kerp H (1995) The oldest fossil lichen. Nature 378: 244.

Mycorrhizae J Dighton, Rutgers University Pinelands Field Station, New Lisbon, NJ, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Types of Mycorrhizae Mycorrhizal Function

Glossary collembola A wingless hexapod invertebrate characterized by the possession of a furculum or springtail, usually soil dwelling and often mycophagous. fruitbody A general term for a spore-bearing organ of both microfungi and macrofungi. mycorrhiza (plural mycorrhizas or mycorrhizae) ‘Fungus root’ a symbiotic, nonpathogenic, association of a fungus and the roots of a plant. mycorrhizal helper bacteria (MHB) Rhizospheric bacteria, usually fluorescent pseudomonads, that assist in the recognition process between mycorrhizal fungi and their host plant root to initiate the development of the specific structures associated with that mycorrhizal type.

Ecology of Mycorrhizae Mycorrhizae and Plant Production Mycorrhizae and Pollution Further Reading

nematode A small unsegmented roundworm, dwelling in soil and aquatic ecosystems. Members may feed on bacteria, fungi, other animals, or plant roots for each of which their mouthparts are specifically adapted. rhizomorph A root-like aggregation of hyphae having a well-defined apical meristem and frequently differentiated into an outer cortex and inner cells or lumen for conduction of nutrients or carbon. soil aggregate A combination of soil mineral particles (frequently clays), organic matter, and bacteria and fungi. The development and size of these aggregates are important for soil structure and stability. The possession of ‘protected organic matter’, organic matter that is relatively unavailable for decomposition by the soil community, within aggregate is important for carbon sequestration.

Abbreviation MHB

mycorrhizal helper bacteria

Defining Statement Mycorrhizal symbiosis come in a variety of types that influence plant growth and community composition by changing nutrient and water uptake and providing defense from root grazers and pathogens. Mycorrhizal fungi are important food sources for animals. Their interactions with pollutants make them potentially useful for restoration and remediation.

Introduction It would appear that terrestrial fungi emerged at about the same time as land plants. In addition to their role as saprotrophs, some fungi became intimately associated with roots of plants, enhancing their abilities to sequester nutrient elements. This became a symbiotic association

known as mycorrhizae, which has evolved in a number of directions, forming contrasting morphological changes to root structure and providing different ecological services to different groups of plants. Fossil records show these associations as possibly primitive endomycorrhizae in the Rhynie cherts (410–360 mya) and as ectomycorrhizae of pines in the Princeton cherts (50 mya). The term mycorrhiza is derived from the combination of two Greek words – ‘mykos’ meaning fungus and ‘rhizos’ meaning roots. Thus the ‘fungus roots’ have been distinguished as a specialized adaptation of plant roots, occurring in some 85% of all plants species on this planet. Recent estimates suggest that some 3617 plant species of 263 families have a mycorrhizal association. Thus, it is regarded that the mycorrhizal condition is the most prevalent symbiotic condition on earth. The evolution of the mycorrhizal state is believed to have arisen as a means of enhancing inorganic nutrient

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uptake by plants, with the fungal hyphae emanating from the root surface being able to explore a larger volume of soil than roots and root hairs alone could do. To make this a true symbiosis, the benefit for the fungus is a supply of carbohydrates from the photoassimilates of the host plant to support fungal growth. There is also evidence to suggest that the function of mycorrhizae goes beyond that of pure nutrient acquisition, to include access of less readily labile nutrients (organic nutrient sources), enhanced root access to water, and protection of roots from pathogenic bacteria and fungi and from grazing of soil invertebrates. An additional attribute of mycorrhizae is their ability to provide protection to plants from heavy metals by limiting translocation of metals into aboveground plant parts. Due to the nutrient uptake-enhancing properties of mycorrhizae, the ability of mycorrhizae to stimulate plant growth has been used in agriculture and forestry. Particularly in nursery conditions, the addition of mycorrhizal fungi at an early stage of plant growth has been shown to yield larger plants in comparison with uninoculated plants. The findings that mycorrhizal inoculation of plants in polluted soils often enhances plant survival and growth have led to the commercial production of mycorrhizal inocula for use in horticulture, agriculture, forestry, and restoration.

Types of Mycorrhizae The mycorrhizal association is made between fungal hyphae and the plant root. Fungal hyphae and fungal spores in soil can act as inocula for roots, and there is evidence to show that the juxtaposition of a root to a spore stimulates spore germination in response to root exudates. Current research is revealing the signaling systems that are involved in host/fungal recognition, whereby the plant and fungi recognize their compatibility and, given appropriate environmental factors, will result in the fungal hyphae associating with the root to form the morphological structures associated with that specific symbiosis. In arbuscular mycorrhizae, plant-exuded chemical signals, such as flavonoids, strigolactones, and surface or thigmotropic signals, are recognized by receptor proteins on the fungal plasma membrane. One of these receptors may be the Gin1 gene, which interacts with ATPase to initiate an internal fungal signaling process allowing the fungus to enter a symbiotic mode. In ectomycorrhizae, it appears that a combination of endogenous rhythms of growth flushes in both shoot and root (carbohydrate supply) and the expression of fungal-stimulated genes that are not present in the root alone stimulates the initiation of the symbiosis. The ability of fungi and plants to form mycorrhizae may be assisted by the presence of, particularly, fluorescent pseudomonad bacteria. These bacteria help to elicit mycorrhizal formation by

production of cell wall softening enzymes, possible chelating agents, and chemicals to elicit the plant/fungus recognition system. These bacteria have been termed mycorrhizal helper bacteria (MHB). There are varying degrees in specificity of plant– fungal association in mycorrhizae and dependency of the plant on mycorrhizal associations. A number of plant species and families will only associate with a limited number of fungal species, leading to specific mycorrhizal types, such as ericoid, arbutoid, orchid, and monotropid mycorrhizal associations being highly specific to limited plant families. A large number of grasses, herbs, and trees form associations with a relatively restricted fungal flora to form arbuscular mycorrhizal associations, whereas a more limited set of plant species (mainly trees) associate with a vast diversity of fungal species in the ectomycorrhizal state. Even within these broad categories of specificity, there is specificity within plant and fungal families. Some plants are heavily or entirely dependent upon mycorrhizal associations for their survival (obligate mycosymbionts), whereas others may only associate with mycorrhizal fungi under times of need (facultative mycosymbionts). Certain fungal species may only associate with one plant species (e.g., the European larch will only associate with Suillus grevillei), whereas others may have broad host specificity. The factors determining these degrees of specificity are not clearly understood, but it is likely that a combination of genetics, evolution, and environmental factors are involved.

Endomycorrhizae As the name suggests, endomycorrhiza is a general term for all mycorrhizal associations where the fungal component is predominantly internal to the root structure, with fungal penetration into host cortical cell walls. Two major groups are the arbuscular and ericoid mycorrhizae, but others, such as the dark septate endophytes, may also be included in this category. In all of these mycorrhizal forms, there is hyphal penetration into the host cells. Arbuscular mycorrhizae

This group of mycorrhizae are formed between a limited number of fungal species (approximately 150) of the phylum Glomeromycota with a very large number of vascular plant species, including grasses, herbs, and tress – particularly tropical tree species. Fungal hyphae penetrate the epidermal cells by a combination of enzyme activity and hydrostatic force, leaving an appressorium as a hyphal swelling on the root surface where pressure builds up. The hyphae then penetrate through cortical cell walls, pushing aside the plasma membrane and branch into characteristic

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arbuscules, tree-like structures, to maximize the area of contact between the fungus and host cell contents. This large surface area facilitates nutrient and carbon exchange between the fungal and plant component of the symbiosis. There are classically two types of arbuscule development, the Arum and Paris type. The Arum type develops multibranched hyphal structures that form tree-like arbuscules, whereas the Paris type consists of extensive hyphal coils in the host cells to form their arbuscules. As a result of colonization of root tissue by the mycorrhizal fungus, root hair development is suppressed as extraradical hyphae (hyphae running from the root surface into soil) effectively take over the role of root hairs to increase the absorptive surface area. In some fungal genera, excluding Gigaspora and Scutellospora, vesicles may be formed within the root tissue. These are fungal structures that completely fill the host cell and are stained by lipid stains. The presence of vesicles gave rise to the older name of vesicular-arbuscular mycorrhizae. These vesicles are terminal hyphal swellings that contain many nuclei and lipid bodies. These are thought to be involved with material storage. Asexual spores may be produced either externally or within the root. Spores of different fungal species are of contrasting size and have characteristic ornamentation and layering of chitin filaments in the spore wall that allow fungal species identification. These spores are dispersed in the air, by water, or by grazing soil animals. Ericoid mycorrhizae

Ericoid mycorrhizae are a restricted group of fungi associated with a restricted diversity of plant species in the Ericaceae, Epacridaceae, and Empetraceae. Hymenoscyphus (Pezizella) ericae was the first fungal species identified as an ericaceous endosymbiont. However, more recently a number of other fungal genera (Oidiodendron, Myxotrichium, and Gymnascella) have been identified as forming mycorrhizal associations with ericoid plants. In a similar manner to arbuscular mycorrhizae, the fungal hyphae invade cortical cells, usually of very fine roots, in which they form hyphal coils, rather than arbuscules. These fine roots of ericoid plants consist of a vascular bundle and one outer layer of cortical/ epidermal cells.

Ectomycorrhizae As their name suggests, ectomycorrhizae have a significant proportion of their fungal partners biomass external to the root. This comprises two parts – the sheath or mantle of fungal hyphae that wrap around the outside of the root and the extraradical hyphae and hyphal structures that extend into the surrounding soil. The sheath can be of variable complexity, from a loose weft of hyphae to a thick and structured multilayer of cells, which have

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the appearance of plant parenchyma tissue; termed pseudoparenchymatous. Internal to the root structure, but external to the cortical cells is a layer of hyphal penetration between cortical cells inward to the endodermis. This is termed the Hartig net and is the region of juxtaposition of fungal and plant symbionts where nutrient and carbon exchange occurs. The colonization of root tips by ectomycorrhizal fungi characteristically suppresses root hair development and changes root branching by the induction of altered levels of cytokinins, resulting in increased branching. Branching patterns are varied and can consist of simple bifurcations, through pinnate and pyramidal, with ultimate branching to such an extent that multiple (50 or so) root tips may be enveloped by a continuous hyphal sheath, a condition called coralloid or tuberculate. The fungal hyphae also impart their characteristic color to the mycorrhizal root surface. The degree of branching, surface color, degree of complexity of the sheath, and character of emanating structures (hyphae, rhizomorphs, cystidia, etc.) serve as morphological characteristics by which mycorrhiza may be identified. More recently, molecular analysis of mycorrhizae, together with morphological characteristics, are being used to create a more comprehensive database of mycorrhizal identification. In the same way as extraradical hyphae increase the surface area of arbuscular mycorrhizal roots, this is taken to a greater degree by many ectomycorrhizae, where extraradical hyphae can extend considerable distances from the root surface. This is particularly the case where congregations of hyphae, rhizomorphs, which are structured entities for rapid, long-distance translocation of nutrients, and water may extend meters from the root surface into soil. In some species and habitats, the hyphal extension is extensive, forming fungal mats, which can alter the soil physical conditions, particularly making them hydrophobic. The ectomycorrhizal association may be formed by a range of Basidiomycotina, Ascomycotina, and some Zygomycotina, in which about 5500 species have been identified as mycosymbionts. Many of the fungal associates are common forest and woodland mushrooms (e.g., Russula, Hebeloma, Cortinarius, Lactarius, Laccaria, Amanita, and Lycoperdon) and truffles (e.g., Tuber). These fungi associate with a limited number of tree species in all biomes. Tree genera include most coniferous trees, larch, birch, beech, oak, and eucalypts. In addition to pure ectomycorrhizae, pines and larches can produce a mycorrhizal type having characteristics of both ectomycorrhizae and arbuscular mycorrhizae, a condition known as ectendomycorrhiza. This association has been attributed to what is called E-strain fungi and is likely to be due to members of the Pezizales (Wilcoxina spp. and Sphaerosporella brunnea).

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Orchid Mycorrhiza Orchid mycorrhizae may be considered the epitome of plant fungal interdependency in the mycorrhizal world. Some 17 000 species of orchids exist and depend upon their basidiomycete fungal partners for acquisition of nutrients. In some cases, the dependency of the plant on its fungal partner has become so extreme that fungal propagules are carrier within the seed of the orchid in order to be present at seed germination and enhancing nutrient uptake at an early stage of plant development. This is important where seed size is so small as to limit the nutrient reserve that can be carried in the absence of an endosperm. Initial protocorm development after seed germination is dependent on the symbiotic fungus also for carbohydrate supply, until photosynthetic capacity can be developed. This may take up to a year in some species, and in achlorophyllous orchid species, it never develops. The fungi develop highly coiled arbuscules (peletons) within the cortical cells of the host plant. These fungal coils have a finite lifespan and upon death, deposit cellulose and pectin within the host cell. These cells may be subsequently ‘invaded’ by new hyphae that can access these carbohydrate and nutrient supplies.

Other Mycorrhizae Arbutoid

This specific mycorrhizal association occurs between two specific members of the Ericaceae, Arbutus and Arctostaphylos, and several genera in the Pyrolaceae. A number of ectomycorrhizal fungal species have been established to form these arbutoid relationships, which consist of a very thin fungal sheath surrounding the outside of the root, a paraepidermal Hartig net consisting of fungal penetration between the epidermis, and outer layer of cortical cells into which the hyphae invade to form hyphal coils (intracellular hyphal complexes). It is suggested that there is exchange of nutrients and photosynthates between arbutoid plants and adjacent tree species. Monotropoid

These mycorrhizal types are restricted to the Monotropoideae in the family Ericaceae, which have largely lost their photosynthetic capacity and live as achlorophyllous plants on the forest floor. At first thought to be parasitic on forest tree species, it is now known that they share mycorrhizal symbionts that are common with their neighboring trees. In this way, these plants obtain carbohydrates from adjacent trees through mycorrhizal bridges between their root systems. Fungal species identified forming these associations include Tricholoma, Russula, and Rhizopogon. On their monotropoid hosts, these fungi form fungal sheaths and Hartig nets in a

similar manner to the ectomycorrhizal condition on trees. However, they also produce hyphal pegs from the inner sheath layer of hyphae into the tangential wall of host cortical cells, with one peg per cortical cell. The abundance of peg formations appears to be related to growth and development of the host plant, increasing up to flowering and subsequently declining. The classic work of Bjo¨rkman in the 1960s, using radiotracers, established that there was movement of photosynthates from trees into Monotropa. Subsequent work has shown that this occurs via mycorrhizal bridges between the shared fungal hyphae forming mycorrhizae with both the Monotropa plant and the adjacent trees. The nutrient acquisition by monotropoid mycorrhizae has been less well studied, but it may be assumed that it is similar to that of the ectomycorrhizal condition. Dark septate endophytes

These root endophytes have been recognized in a number of plant species from almost all plant families, particularly those growing in cool, nutritionally poor environments. Many of the fungal species have not been identified, but members of the genera Chloridium, Leptodontidium, Phialocephala, and Phialophora have been identified from roots. These fungi enter the root via root hairs or cortical cells and establish runner hyphae between cortical cells from which dense, multibranched structures, called microsclerotia, develop within the host cell. Initially hyaline hyphae frequently deposit melanin in their cell walls resulting in the dark color that gives these fungi their name. Although these mycorrhizal types are frequently found, there is relatively little information on their function. However, given their greater abundance in oligotrophic and climatically limited environments, it is likely that they have the enzymatic competence to access nutrients for inorganic sources (see ‘Influence of Mycorrhizae on Plant Communities’).

Mycorrhizal Function The traditional concept of mycorrhizal function is that they enhance inorganic nutrient uptake from soil into the host plant. Indeed this is true, but may be an oversimplification of their function as there is evidence of multiple host benefits of mycorrhizal association. However, most of these functions have been shown under contrived experimental conditions and the magnitude of these effects in natural ecosystems has recently been questioned. Nutrient Acquisition Numerous studies have shown that plants grown in the presence of mycorrhizae grow larger than those grown in the absence of mycorrhizae. In an elegant

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study on arbuscular mycorrhizae, Nye and Tinker used radioactively labeled phosphate in soil and autoradiography to demonstrate that the phosphate uptake capability of mycorrhizal roots was larger than nonmycorrhizal roots. They demonstrated that the nutrient depletion zone developing around a mycorrhizal root system was greater than that for a root with root hairs due to the greater exploratory capacity of multiple, small diameter hyphae taking up phosphorus from the soil pore water. It has been suggested that the energetics of producing extensive and far-reaching hyphae in place of root hairs provides a larger absorptive surface for nutrient acquisition at less expense to the plant. However, the energetic cost–benefit hypothesis has recently been challenged. Carbon is made available to the fungus by way of photosynthates. In some ectomycorrhizal associations, this may account for some one-third of the plant’s photoassimilates. Both the plant and the fungus increase their hexose importer gene function to accomplish this carbohydrate exchange. Sugar-dependent gene expression has been shown to provide a number of physiological functions of ectomycorrhizae, such as defense against faunal grazing and to pathogenic bacterial and fungal attack. In arbuscular mycorrhizae, phosphate acquisition is achieved through membrane integral proteins, including PHT phosphate transporter and the P-type HþATPase. Following uptake, transport within fungal tissues of both arbuscular and ectomycorrhizae is mainly in the form of polyphosphates, which may also be deposited in cortical cells as nutrient reserves in polyphosphate granules. Within arbuscular mycorrhizae, specific MtPT4 protein production is closely associated with arbuscule formation, and thus with phosphorus uptake. There is similar genetic regulation of nitrogen uptake. In ectomycorrhizal association with Hebeloma crustuliniforme, three ammonium transporters and one nitrate transporter have been identified for inorganic nitrogen uptake. Both amino acid and polypeptide transporter genes along with protease and subtilase genes have been identified for acquisition of organic nitrogen. Enhanced nutrient uptake by mycorrhizae has led to the assumption that the growth benefit of mycorrhization was entirely nutrient driven. Indeed, there is plenty of evidence from experimentation, agricultural plant production, and forest nurseries to show the nutritional benefits of mycorrhizae for improving plant growth (height and stem diameter), foliar nutrient content and mass, and nutrient content of plant products (peanuts, grain, etc.). In addition, it has been shown in a number of cases that the presence of mycorrhizae improves plant survival, especially at the establishment phase. As a result of these growth benefits, mycorrhizae are being used commercially for enhancing growth of agricultural crops, forest trees in nurseries, and in horticulture. This has resulted in a recent increase in the number

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of companies producing and selling mycorrhizal inoculum for arbuscular mycorrhizae, ectomycorrhizae, and, more recently, ericoid mycorrhizae. However, there are a number of non-nutritional benefits of mycorrhizal associations. Water Acquisition The ability of mycorrhizal extraradical hyphae and, in particular, rhizomorphs of ectomycorrhizae to translocate nutrients from soil to plant also enables them to translocate water. This has also been found to be of great importance for plant growth in dry environments. Water uptake and the interaction between water and nutrient acquisition is plant species-dependent. For example, the presence of arbuscular mycorrhizae on Acacia did not improve plant growth under drought stress in both the absence and the presence of addition of P fertilizer. However, in another tropical tree Leucaena, the presence of mycorrhizae significantly improved plant growth under drought conditions at both levels of P supply. The finding of mycorrhizal associations of Acacia roots at depths of 30 m in Senegal is likely to be more associated with water acquisition than that of nutrients. Plant Defense The presence of mycorrhizae within the root provides some degree of protection from both grazing soil fauna and plant pathogens. This defense appears to take two forms. One form of defense is the production of a physical barrier to root tissue created by an ectomycorrhizal sheath. The other is a biochemical defense mechanism in which secondary metabolites of fungi defend the mycorrhizal root against pathogenic fungi and bacteria. For example, the presence of Glomus mosseae arbuscular mycorrhizae on peanuts significantly improved plant growth (28%), pod production (22%), and seed weight (12%). In the presence of two fungal pathogens, Fusarium solani and Rhizoctonia solani, the presence of mycorrhizae negated the growth suppression of the pathogens for all parameters and increased production over the pathogen alone by 26% (plant weight), 35% (pods per plant), and 39% (seed weight). In seedling studies, the ectomycorrhizal fungi Laccaria laccata, Paxillus involutus, H. crustuliniforme, and Hebeloma sinapizans significantly reduced the effect of the root pathogen Phytophthora cinnimomi on the growth of chestnut tree seedlings. These disease prevention attributes of mycorrhizal fungi are important in both the agricultural context and tree nurseries, where both the density of plants and the intensive management practices are favorable for plant pathogen development. The role of mycorrhizae in disease prevention in more natural systems has not been adequately quantified. In a meta-analysis of published

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data on the interactions between mycorrhizae and fungal pathogens and nematodes, Borowicz, in 2001, showed that mycorrhizal fungi inhibited the pathogen and nematode effects on plants. However, the defense against nematode attack was heavily biased by the large numbers of references to a sedentary nematode, Heterodera, suggesting that mycorrhizal defense against other nematode species may be less than reported.

Ecology of Mycorrhizae Global Distribution of Mycorrhizal Types and Soil Nutrients Naturally, the distribution of mycorrhizal types follows closely the global distribution of appropriate plant host species. However, Read suggested an underlying factor that linked both plant and mycorrhizal symbionts with soil factors. Using the analogy of an altitudinal cline up a mountain to the variability in soil characteristics from poles to the equator, Read suggested that the dominant mycorrhizal types (ericoid mycorrhizae, ectomycorrhizae, and arbuscular mycorrhizae) were closely related to the nutrient sources and availability in soil and the enzymatic competence of the mycorrhizal community. Organic matter in soil, derived from dead plant and animal remains, provides soluble mineral nutrients in soil as a result of decomposition and mineralization processes carried out by saprotrophic bacteria and fungi, with the help of soil animals. In colder latitudes and higher altitudes, organic matter accumulates. Here, in colder and wetter environments, litter quality is reduced by the presence of complex chemistry and toxic secondary plant metabolites and where the climatic window of opportunity for decomposition is restricted. In these conditions, soil nutrient capital can be high, but availability as soluble nutrients is low and plants are frequently ericaceous with ericoid mycorrhizae. These mycorrhizae benefit their host plant not only by enhancing soluble nutrient uptake, but also by possessing protease enzymes, which allow them to directly access organic forms of nitrogen. In more mesic environments supporting coniferous and mixed coniferous and deciduous forest, the ability of the predominantly ectomycorrhizal fungal community allows access to organic forms of both nitrogen and phosphorus by the production of protease and acid phosphatase enzymes. In most of the arbuscular mycorrhizaldominated plant communities of deciduous trees and grasslands and at lower altitudes and in the tropics, nutrient mineralization is more rapid, the pool of inorganic nutrients is larger, and the mycorrhizal community has limited enzymatic capabilities. Thus, the link between host plant and mycorrhizal type appears to be a mixture of fungal–host compatibility and environmental limitations.

The interaction between soil conditions and mycorrhizae can be seen during forest growth where changes in dominant ectomycorrhizal fungal species in the community change with tree age. As the forest floor accumulates more tree-derived and recalcitrant woody material, there is a shift from fungal species that efficiently scavenge for inorganic nutrients in soil to those with enzymatic capabilities to decompose organic complexes to access the mineral nutrients within. This short-circuiting of the saprotrophic community mineralizing nutrients has been named the ‘direct nutrient cycling hypothesis’. There is still debate in the literature regarding the interactions between mycorrhizal and saprotrophic fungi in this role, where some literature support the idea that mycorrhizal fungi compete against saprotrophs in low inorganic, high organic nutrient conditions and others suggest they work in harmony. Influence of Mycorrhizae on Plant Communities Plant establishment and growth is enhanced by the ability of the plant to form mycorrhizal associations. Within a plant community, not all plants have the same capacity to form mycorrhizae, nor the same dependency on mycorrhizae for their survival. Hence, the outcome of competition between plant species in a community is a complex series of interactions with the environment and availability and effectiveness of mycorrhizal fungal symbionts. These interactions have been explored by Bever and Schultz, in 2005, who show that a range of scenarios of plant–mycorrhizal strategy can influence the growth and competitive ability of different species in a community, which results in niche partitioning and the maintenance of diversity within the plant community. The relative dependence of a plant species on mycorrhizal fungi for nutrient uptake may also influence plant community composition depending on availability of nutrient resources, plant competition, and other environmental factors influencing mycorrhizal and plant community assembly rules. Indeed, diversity of mycorrhizal community often increases correspondingly to that of plant species diversity. Following the classic research of Bjo¨rkman who used radioactive carbon to trace carbohydrate flow from trees to the forest floor achlorophyllous plant, Monotropa, via mycorrhizal linkages between the two plants, interest has arisen about the potential for exchange of information between plants via mycorrhizae. The ability of like species of plants to share arbuscular mycorrhizal bridges between their root systems has been shown to benefit the status of that species in the community by the ability to move photosynthates and nutrients from dying plants to live members of the population. The discovery that a similar sharing of information may occur between plants of different species, in both the arbuscular and

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ectomycorrhizal plant community, has connotations that change our view on the rules for establishing plant community composition. Given that a ‘nurse effect’ between plant species can be established by sharing resources via mycorrhizal bridges and ‘cheater’ effects where plants effectively steal resources from others via mycorrhizal bridges invokes the concept of both competition and synergism being involved in regulating plant communities. This is in contrast to the classical plant community theory of competition alone as being the main driving force influencing plant community assembly rules. Mycorrhizae as Food Fungi are rich in important nutrients, particularly nitrogen, phosphorus, minerals, and vitamins, and have been reported to be as good as beef as a food resource. Thus the fruitbodies of ectomycorrhizal fungi basidiomycete mushrooms are grazed upon by a variety of invertebrates including mollusks and fly larvae. Many fly larval species are even tolerant of the toxic component of Amanatia spp., -amanitin. Additionally, many mushrooms and, particularly, hypogeous fruitbodies (e.g, truffles) are an important food source for small mammals. In Australia, 37 species of native and 4 species of feral mammals exhibit extensive mycophagy, where fungi may comprise more than 25% (by volume) of the diet of brush-tailed potoroo (Potorus longipes) at all times of the year. In old growth Douglas fir forests in western Oregon, 11 052–16 753 (some 2.3–5.4 kg ha1 dry mass) fruiting bodies of hypogeous mycorrhizal fungi can be produced per hectare per year. As these fungi have a higher content of nitrogen, phosphorus, potassium, and micronutrients than epigeous fungi (fungi fruiting aboveground), it makes them a high quality food resource for mammals. Indeed, it has been shown that small mammals foraging in adjacent intact forest for ectomycorrhizal fungi carried spores and propagules into refugia in the ash fields of Mount St. Helens to significantly enhance revegetation by trees following the volcanic eruption. Belowground, the mycelia of both ectomycorrhizal and arbuscular mycorrhizal fungi are important food sources for a variety of soil animals. Populations of nematodes and Collembola (springtails) are maintained by fungal consumption. Not all fungi are, however, equally palatable or provide adequate nutrition, resulting in hierarchies of feeding preference. There continues to be some controversies regarding the influence of soil animal grazing on mycorrhizal effectiveness with suggestions that severing hyphal connections between the root and surrounding soil reduces nutrient influx. It is likely that natural densities of grazing animals have minimal effects. An indirect effect of arbuscular mycorrhizae on animals can be seen by mycorrhizal enhancement of host plant nutritional status and grazing herbivores. The

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presence of Glomus spp. mycorrhizae on Lotus corniculatus significantly reduces larval mortality and increases growth rates of the leaf-grazing larvae of the butterfly Polyommatus icarus.

Mycorrhizae and Plant Production Agriculture Mycorrhizal growth-enhancing properties have been explored in agricultural settings. However, due to the application of fertilizer in most farming practices, the impact of the fungal inoculum is variable due also to the fact that there is frequently an abundance of native fungal propagules in the soil. The beneficial effects of enhanced plant nutrition often go hand in hand with protective benefits of mycorrhization to protect crops from root-feeding nematodes and bacterial and fungal pathogens. During the process of these trials, research was conducted on the methods of mycorrhizal delivery and a number of systems were developed for commercial production. These included incorporating arbuscular mycorrhizal spores into pellets or producing large numbers of spores in a loosely packed matrix (rockwool, sand, clay-brick granules). Due to demand for mycorrhizal inoculum for both arbuscular and ectomycorrhizal plants, a number of companies have developed, selling their mycorrhizal inoculum to gardeners, horticulturalists, and forest nurseries (e.g., Horticultural Alliance Inc.; Soil Moist; Mycorrhizal Products). Commercial Forestry The fact that mycorrhizal inoculation of tree seedlings enhances growth under experimental conditions has stimulated an interest in using ectomycorrhizal and arbuscular mycorrhizal inoculum in commercial forestry. The fungi (especially ectomycorrhizae) have to be the species that are readily culturable and easy to grow in bulk. These may be made commercially available in perlite–peat mixture or in alginate-entrapped mycelial cultures. The growth benefit provided by commercial inocula in the nursery has been demonstrated on many occasions, and the survival and enhanced growth of seedlings planted into harsh environments has been shown. For example, the presence of the ectomycorrhizal fungus Pisolithus tinctorius on tree seedlings allows improved survival and growth on heavy metal-contaminated mine spoils. However, generally outplanting tree seedlings that have been inoculated with mycorrhizal fungi often do not provide a significant growth enhancement – a fact that varies by tree species and locality. Part of this problem may lie with the fact that in a nursery setting, nutrients and water are rarely limiting. Hence, plants growing in rich soil conditions with poorly competitive,

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ruderal, mycorrhizal fungal associates are outplanted into poor soils. It would appear that in many instances, there is a replacement of the nursery-applied mycorrhizae with a native flora of the site of planting. During the time that this mycorrhizal competition occurs, any benefit of mycorrhizal colonization is lost and tree performance is reduced until a new mycorrhizal community has been established, which has the physiological adaptations to the site. A comprehensive review of the literature suggests that the benefits of nursery application of mycorrhizal inoculum to tree seedlings may not be as advantageous as may be thought. Restoration The ability of mycorrhizae to promote enhanced growth and establishment of plants in disturbed habitats suggests that mycorrhiza can be important for restoration projects. As we see below, many mycorrhizae have the ability to protect the host plant from pollutants, but in the context of restoration, the ability of mycorrhizal association to improve soil structure may be of equal importance. First, mycorrhizal fungi assist the establishment of vegetation on, usually, poor soils in a restoration site. This allows the return of plant parts to soil, establishing the organic component of a more healthy soil. Second, the ability of fungi to bind soil mineral particles together either by physical means or by the production of the sticky glycoprotein, glomalin, by some arbuscular mycorrhizal fungal species increases soil stability and prevents soil erosion. The combination of both the factors increases soil aggregation and the storage of carbon as protected organic matter within soil aggregates. Mycorrhizal fungal assisted long-term carbon sequestration in soil aggregates is a potentially valuable resource in a world of increasing atmospheric CO2. In some agricultural contexts, particularly in tropical areas, frequent crop irrigation leads to an accumulation of salt and the development of highly saline soils. In these conditions, arbuscular mycorrhizae have been shown to have significant benefit for crop production enabling plants to grow more effectively under these stressed conditions. This allows crop production to continue in areas that would otherwise be abandoned.

Mycorrhizae and Pollution Acidifying Pollutants It is in the late 1970s when soil ecologists became involved in the research on acid rain (sulfuric acid from dissolved SO2 in rain) in relation to the ‘Waldsturben’ effect of the forest die-back in Bavarian forests, where the observations of Ulrich, Huttermann, and Blaschke alerted researchers to the fact that acid rain was affecting root

growth and the ectomycorrhizal status of trees. Based on the idea that mycorrhizal formation was affected by both carbohydrate supply and nutrient levels in soil, which in turn influence hormone levels in roots, a two-directional impact model of acidifying pollutants on the development of mycorrhizae was developed. In this model, reduction of mycorrhizal associations of the plant roots occurred (1) via a reduction in photosynthesis in the tree canopy, reducing the energy supply to roots and (2) via acidinduced increase in the availability of toxic metal ions (aluminum, manganese, and magnesium) in soil, resulting in root damage and loss or changes in community composition of ectomycorrhizal fungal symbionts. This model has confirmation from a number of studies showing that ectomycorrhizal community composition changes with increasing acid rain loading and evidence that the photosynthesis is significantly reduced by acid rain. Using historical mushroom foray records, Arnolds detected changes in the ratio of ectomycorrhizal to saprotrophic basidiomycete fruitbody abundance in The Netherlands over recent years. He attributed this to enhanced nitrogen deposition that acted both as a fertilizer (reducing the effectiveness of mycorrhizae for nitrogen uptake into host plants) and as an acidifying pollutant. Both experimental and observational studies have shown that community shifts in ectomycorrhizae occur with increasing nitrogen loading to forest ecosystems, where the shift from nitrophobic to nitrophilic species could be used as an indicator of nitrogen pollution. Heavy Metal Pollutants Heavy metals are known to be toxic to many living organisms. Fungi are no exception to this; however, fungi have a degree of tolerance to heavy metals, which is especially apparent in ectomycorrhizae. In the late 1970s, Don Marx observed enhanced survival and growth of pine trees in mine spoil soils with trees inoculated with ectomycorrhizal fungi. Of particular interest was the fungus P. tinctorius, which appeared to be more frequent in these polluted sites than in other habitats. The effect of inoculation with P. tinctorius resulted in tree volumes 250% greater than those trees assuming natural inoculum from the site or inoculation with Thelephora terrestris. These trees also had higher foliar phosphate levels, but reduced levels of Ca, S, Fe, Mn, Zn, Cu, and Al, suggesting that the effect of this mycorrhizal fungus may reduce the uptake of heavy metals into the host tree. Similar prevention of heavy metal toxicity to host plants afforded by mycorrhization has also been seen in ericaceous plants of the genera Calluna, Vaccinium, and Rhododendron. The mechanism of plant protection in the ectomycorrhizal system was elucidated by Denny and Wilkins. Using electron microscopy, coupled with X-ray

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diffraction (EDAX), they identified adsorption of heavy metals onto fungal hyphae in the extraradical hyphal network, the fungal sheath, and Hartig net, preventing translocation of the metal into the host cortex and, particularly, preventing movement into the vascular tissue. Some of this binding capacity is related to extracellular slime formed by the hyphae of the ectomycorrhizal fungus P. tinctorius and some to Cu and Zn complexing with polyphosphate granules, which metabolically inactivate the heavy metals within the fungal hyphae. Similar protection has been seen in arbuscular mycorrhizae, where the rate of uptake of heavy metal was increased in the presence of mycorrhizae, but the transfer of metals to the host plant was reduced, effectively locking metals up in the fungal component of the mycorrhizal association. Protection in arbuscular mycorrhizae has been attributed to oxidative stress alleviation, where the production of zinc transporter genes, metallothionien, a 90 kDa heat shock protein, and glutathione have been shown to be induced in extraradical hyphae of Glomus intraraices in the presence of heavy metals.

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retain high levels of heavy metals and radionuclides, there are suggestions that they may be beneficial in the restoration and remediation of polluted environments. In particular, the high accumulation into basidiomycete fruiting structures in ectomycorrhizal fungi could be used as a harvestable product that could be exported from the site, resulting in a net export of the pollutant chemical. Organic Pollutants In a screening of 21 mycorrhizal fungi for PCB decomposition, 14 species were found to be able to decompose some of the PCBs by at least 20%. The ectomycorrhizal fungi Radiigera atrogleba and Hysterangium gardneri were able to degrade 80% of 2,29-dichlorobiphenyl and two ericoid mycorrhizae, Hymenoscyphus ericae and Oidiodendron griseum had decomposer abilities, but they were less effective than the ectomycorrhizal species. The interaction between ectomycorrhizal fungi, rhizospheric bacteria, and the decomposition of organic pollutants is a new area of study.

Radionuclide Pollutants

Climate Change

In the same way that fungi can accumulate heavy metals, it seems that they have the capacity to accumulate radionuclides. Accumulation into fruitbodies of basidiomycetes is often higher than surrounding soils. For example, facultative mycorrhizal fungal fruitbodies had ten times the concentration of radiocesium than leaf litter or organic soil horizons in Sweden following the Chernobyl accident, whereas saprotrophic fungal fruitbodies had half that level of accumulation. Many of these hyperaccumulators are ectomycorrhizal species, for example, members of the Cortinariaceae. The analysis of the isotope ratio of radiocesium from Chernobyl (137Cs:134Cs) in fruitbodies of ectomycorrhizal basidomycete fungi indicates that a large proportion (25–92%) of 137Cs was accumulated that originated from sources occurring prior to the accident at Chernobyl. This suggests long-term accumulation of radionuclides by these fungi. Research suggests that this is achieved by internal translocation within the mycelium and directional transport to fruiting bodies, along with other nutrients. In both ericoid and arbuscular mycorrhizal host plants, there appears to be a mycorrhizal effect of enhancing radionuclide uptake by the plant, but a significant change in the internal allocation of those radionuclides. Radionuclides appear to be prevented from being translocated into the shoots of plants by these mycorrhizae – a possible defense mechanism. Given the fact that mycorrhizal fungi, in association with their host plants, have the capacity to absorb and

Climate change can encompass a number of changes in our environment, including increase in atmospheric CO2 levels, increased temperature, and changes in rainfall amount and distribution pattern (both spatial and temporal). The impacts of these factors on mycorrhizae are in their infancy, but it is clear that changes in CO2 concentrations in the atmosphere influence the C:nutrient ratio of plant material that enters the decomposition pathways. As a result, the change in nutrient availability causes changes in both the composition of mycorrhizal communities as nutrient content is reduced. The response of mycorrhizae to rainfall, ozone, UV light, as well as CO2 appears to be unclear from the few studies carried out to date. Fungal Conservation Long-term fungal foray records are a potential source of information about log-term trends in abundance of fungal fruitbodies. Using such records, Arnolds discovered that the fungal flora of The Netherlands had changed from being dominated by mushrooms of ectomycorrhizal fungal species to that of saprotrophs. By overlaying information about levels of acidifying pollutants (particularly nitrogen deposition), combined with the knowledge of the effects of acidifying pollutants on mycorrhizae, he produced evidence to suggest that pollution was the cause of this change. As a result, he was the first to construct a red data list of fungal species that he thought were in danger of extinction. Subsequently, fungal conservation

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has developed both in the United Kingdom and North America, where the discussion relates to the maintenance of habitat for declining fungal species, many of which are ectomycorrhizal. In addition, there is concern in a number of localities about the effects of mushroom harvesting, where research is starting to evaluate if there is a relationship between harvesting rates and fungal species decline. Additionally, the way in which we manage our ecosystems, particularly forest systems, may have a profound effect on the ectomycorrhizal fungal flora.

See also: Ecology, Microbial; Food Webs, Microbial; Heavy Metal Pollutants: Environmental and Biotechnological Aspects; Heavy Metals Cycle (Arsenic, Mercury, Selenium, others); Nitrogen Cycle; Phosphorus Cycle; Rhizosphere

Further Reading Agerer R (1987–2006) Colour Atlas of Ectomycorrhizae. Munich: Einhorn-Verlag. Bever JD and Schultz PA (2005) Mechanisms of arbuscular mycorrhizal mediation of plant-plant interactions. In: Dighton J, White JF, and Oudemans P (eds.) The Fungal Community: Its Organization and Role in the Ecosystem, 3rd Edn., pp. 443–459. Baton Rouge, USA: Taylor & Francis.

Castellano MA (1996) Outplanting performance of mycorrhizal inoculated seedlings. In: Mukerji KG (ed.) Concepts in Mycorrhizal Research, pp. 223–301. The Netherlands: Kluwer. Dighton J (2003) Fungi in Ecosystem Processes. New York, USA: Marcel Dekker. Durall DM, Jones MD, and Lewis KJ (2005) Effects of forest management on fungal communities. In: Dighton J, White JF, and Oudemans P (eds.) The Fungal Community: Its Organization and Role in the Ecosystem, 3rd Edn., pp. 833–855. Baton Rouge, USA: Taylor & Francis. Koide RT and Mosse B (2004) A history of research on arbuscular mycorrhiza. Mycorrhiza 14: 145–163. Peterson RL, Massicotte HB, and Melville LH (2004) Mycorrhizas: Anatomy and Cell Biology. Wallingford, UK: CABI. Read DJ, Lewis DH, Fitter AH, and Alexander IA (eds.) (1992) Mycorrhizas in Ecosystems. Wallingford, UK: CABI. Smith SE and Read DJ (1997) Mycorrhizal Symbiosis, 2nd edn. San Diego, CA, USA: Academic Press. van der Heijden MCA and Sanders IR (eds.) (2003) Mycorrhizal Ecology. Berlin: Germany: Springer. Wang B and Qiu YL (2006) Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza 16: 299–363. Watling R (2005) Fungal conservation: Some impressions – A personal view. In: Dighton J, White JF, and Oudemans P (eds.) The fungal Community: Its Organization and Role in the Ecosystem, 3rd Edn., pp. 881–896. Baton Rouge, USA: Taylor & Francis.

Relevant Websites http://www.hortsorb.com – Horticultural Alliance Inc http://www.mycorrhizalproducts.com/ – Mycorrhizal Products http://www.soilmoist.com – Soil Moist

Rumen J B Russell, Robert C Holley Research Center, Ithaca, NY, USA Published by Elsevier Inc.

Defining Statement Introduction Recognition of Ruminal Fermentation Rumen as a Microbial Habitat Polymer Degradation Ruminal Acidosis Effect of pH on Ruminal Microorganisms Transport and Phosphorylation Crossfeeding among Ruminal Microorganisms Ruminal Fermentation Schemes Growth, Maintenance, and Energy Spilling

Glossary abomasum The gastric stomach of the ruminant. energy spilling The process microorganisms use to dissipate excess energy. eructation The process that lets fermentation gases escape from the rumen. hemo-concentration The ability of the body to absorb water from blood. hemo-dilution The ability of the body to add water to blood. ionophore A class of antibiotics that alters ruminal fermentation end products. maintenance Energy used to maintain ion gradients and turnover macromolecules.

Abbreviations CLA CMC CSIRO EMP FDP

conjugated linoleic acid Carboxymethylcellulose Commonwealth Scientific and Industrial Research Organisation Embden–Meyerhof–Parnas fructose-1,6-diphosphate

Toxic Compounds Effects of Ionophores on Ruminal Microorganisms Genetic Engineering Rumen Microbial Ecology Ruminal Bacteria and Nonculturable Bacteria Genomics Ruminal Protozoa Ruminal Fungi Models of Ruminal Fermentation Further Reading

omasum A chamber that traps large feed particles as digesta passes from the rumen to the abomasum. reticulum A small pouch that extends from the anterior of the rumen. It collects either large feed particles for rumination or small particles for passage to the lower gut. rumen The large pregastric stomach of a ruminant that acts as a fermentation chamber. rumination The process by which ruminants force large feed particles from the rumen up the esophagus to the mouth to be chewed again. VFA Volatile fatty acids are the primary end products of ruminal fermentation, primarily acetic, propionic, and butyric.

GDH GOGAT PEM Pi PTS USDA VFA

glutamate dehydrogenase glutamine oxoglutarate aminotransferase polioencephalomalacia inorganic phosphate phosphotransferase United States Department of Agriculture volatile fatty acids

Defining Statement

Introduction

Approximately 180 species of animals (ruminants) have a specialized pregastric structure called the rumen, which is inhibited by a highly diverse population of strictly anaerobic microorganisms (bacteria, protozoa, and fungi). These microorganisms enable ruminants to feed on fibrous materials that would otherwise not be efficiently digested.

Ruminal fermentation is an exergonic process that converts feedstuffs into short-chain volatile fatty acids (VFA), CO2, CH4, NH3, and heat. Some of the free energy is trapped as ATP, and this energy is used to drive the growth of anaerobic ruminal microorganisms. The ruminants absorb VFAs and digest the microbial protein to

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obtain energy and amino acids. CH4 and NH3 represent losses of energy and nitrogen to the animal. Some ruminal microorganisms can degrade cellulose, and this attribute gives ruminants the ability to digest fibrous materials. The rumen is inhabited by a highly diverse population of bacteria, protozoa, and fungi.

Rumen Omasum

Reticulum

Abomasum

Small intestine

Recognition of Ruminal Fermentation Cecum

Mankind and domestic ruminants share a long and mutualistic history, and this relationship has allowed man to maintain stable food supplies, harvest the photosynthetic potential of the grasslands, expand his geographical range, and develop stable cultures. The Bible recognizes the value of ruminants as livestock, and the Old Testament states, ‘‘Whatsoever parteth the hoof, and is clovenfooted, and cheweth the cud among the beasts, that shall ye eat.’’ The evolutionary path of ruminants did not entail an inherent capacity for fiber (cellulose and hemicellulose) digestion, but ruminants developed a symbiotic relationship with ruminal microorganisms. The animal provides a habitat for microbial growth, and the microbes, in turn, provide the animal with nutrients that would otherwise be unavailable. Zoologists have described more than 180 species of ruminants, but man’s efforts in ruminant domestication have largely been devoted to cattle (bovines), sheep (ovines), and goats (caprines). Camels have a slightly different digestive anatomy than ‘true ruminants’, but their pregastric fermentation is remarkably similar. Some nonruminant species (e.g., horses, zebras, rabbits, and rodents) have a larger fermentative capacity, but fermentation occurs postgastrically and the microbial protein can only be harvested by coprophagy. The Webster’s dictionary describes ruminants as ‘‘any of a group of four-footed, hoofed, even toed, and cud-chewing mammals which have a stomach consisting of four divisions or chambers, the rumen, reticulum, omasum, and abomasum; the grass etc. that they eat is swallowed unchewed and passes into the rumen or reticulum from which it is regurgitated, chewed, and mixed with saliva, again swallowed, and then passed through the reticulum and omasum into the abomasum where it is acted on by gastric juice.’’ This definition is anatomically correct, but it lacks any mention of microorganisms or fermentation.

Rumen as a Microbial Habitat The rumen is an ideal habitat for the growth of anaerobic microorganisms. Salivary bicarbonate buffers the ruminal fluid, and VFAs, arising from fermentation, are absorbed across the rumen wall. Some O2 enters the

Colon VFA & pH

Figure 1 The digestive tract of a ruminant and its various compartments. Reproduced from Russell JB and Bruckner GE (1991) Microbial ecology of the normal animal-intestinal tract. In: Woolcock JB (ed.) World Animal Science: Microbiology of Animals and Animal Products, vol. 7, pp. 1–14. Amsterdam, Netherlands: Elsevier Scientific Publishing Co.

rumen with the feed, but the fermentation produces CO2 and CH4 that displace O2 from the rumen. The remaining O2 is consumed by a small population of facultative anaerobes. The rumen accounts for 1/7th to 1/10th of the animal’s body weight and has a volume of 80 l (Figure 1). The ruminal contents are forced back and forth over two pillars, and these mixing movements inoculate the ingested feed with microorganisms and transfer VFAs to the mucosal surface where they can be absorbed. The reticulum is a much smaller pouch that collects large feed particles so they can be forced back up the esophagus to be chewed again (ruminated). Gases exit the rumen through the esophagus via a process known as eructation. Feed from the rumen passes to the omasum. Liquid and small feed particles pass through the laminae of the omasum, but large feed particles are trapped and back-washed into the rumen. Feed then enters the abomasum (the gastric stomach), and from this point, digestion is analogous to the process observed in simple-stomached animals. Because feed enters and leaves the rumen at regular intervals, the rumen operates as a continuous culture device, but selective retention of feed creates at least two major dilution rates. Large feed particles turn over at a slower rate (2–4 times) than do the liquid and small particle fractions. Many ruminal microorganisms prolong their residence time in the rumen by attaching to feed particles that have a slow dilution rate.

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Polymer Degradation Feedstuffs are primarily composed of large, and sometimes complex, polymers (cellulose, hemicelluloses, pectin, starch, proteins, etc.). These polymers must be degraded by extracellular enzymes before they can be utilized by ruminal microorganisms. Some enzymes are secreted into cell-free ruminal fluid, but most hydrolases remain cell-associated. By sequestering enzymes to their outside surfaces, ruminal microorganisms increase their chance of receiving the end products. A variety of hydrolases have been purified and cloned, but it is still not clear if all of the important enzymes have been identified. Carboxymethylcellulose (CMC) degradation has been used as an assay for cellulases, but CMCases lack cellulose-binding domains and are usually unable to degrade intact insoluble cellulose. Cellulase activity seems to be constitutive, but some enzymes (e.g., amylase) are repressed by glucose or other sugars. Because hydrolases can only come in contact with feed via water interactions, solubility can be a key factor regulating feed degradation. When proteins are heated, hydrophobic groups previously buried in the molecule come to the surface, solubility decreases, the protein becomes more resistant to ruminal degradation, and more amino nitrogen passes to the lower gut. Since World War II, American cattle have often been fed grain as a supplement to dramatically increase animal growth or milk production. The starch granules of cereal grains are encased in protein. Ruminal bacteria can quickly bore through the protein coat of some cereal grains (e.g., barley), but the starch granules of corn are coated by zein, a slowly degrading protein. However, because heat ruptures the zein, the degradation rate of corn grain can be enhanced, and heat treatments are a common practice of the feed industry. However, if the rate of carbohydrate fermentation in the rumen is very rapid, the buffering capacity of the rumen can be exceeded.

Ruminal Acidosis The rumen is well buffered by the bicarbonate of saliva, but pH can decline. Acute ruminal acidosis is usually caused by lactate accumulation, but chronic acidosis can be caused by a simple increase in total VFA. When animals are fed grain or other nonfibrous feeds, rumination and mixing movements decrease, and VFA absorption from the rumen is impaired. Ruminal acidosis can be prevented by feeding cattle sodium bicarbonate, but this practice has little impact on the buffering capacity of the ruminal fluid per se. Because the rumen is one of the largest compartments in the body, its osmotic pressure must be carefully regulated to prevent hemo-concentration or hemo-dilution. The propensity of water flux to or from

the rumen to stabilize ionic concentrations prevents significant changes in bicarbonate or ruminal buffering. When ruminants are fed salts like sodium bicarbonate, they drink more water, ruminal fluid dilution rate increases, VFAs are washed to the abomasum (the gastric stomach), a greater fraction of the VFAs are undissociated, and the absorption rate is much faster. When grain is substituted for fiber in the diet, graindependent increases in ruminal VFA concentration are aggravated by decreased mixing motions. Grain-fed cattle ruminate for shorter periods of time than those consuming forage, and rumination is coordinated with ruminal mixing motions. The impact of ruminal mixing on VFA absorption has not been studied in a systematic fashion, but it should be noted that the diffusion rate of substances through liquids is inversely proportional to the diffusion distance (Fick’s first law of diffusion). Because the rumen is a very large compartment, the mixing motions bring the VFAs in contact with the rumen wall and enhance VFA absorption. If saliva flow is severely depressed, ruminal viscosity increases, and effective mixing and VFA absorption are further impaired. Fiber is the key factor stimulating rumination and saliva production, but another potential effect of lack of fiber on ruminal pH is the ability of fiber itself to act as a ruminal buffer. Fiber has much less buffering capacity that bicarbonate, but the effective pKa of bicarbonate is approximately 6.7. If ruminal pH is one unit less than this value, the ability of bicarbonate to buffer the rumen is dramatically decreased, and the buffering capacity of fiber becomes more important. Because the rumen wall is not protected by the mucous, ruminal acidosis can have a very negative impact on ruminants. If the ruminal pH is chronically depressed, the rumen wall becomes inflamed and ulcerated. Potentially harmful bacteria such as Fusobacterium necrophorum migrate through the ulcers into portal blood and eventually to the liver, where they cause abscesses. A decrease in ruminal and blood pH can cause inflammation of the tissues above the hoof (founder). In severe cases, water rushes out of the blood into the rumen to equilibrate the increase in VFA concentrations, and the animal dies from hemoconcentration.

Effect of pH on Ruminal Microorganisms Ruminal bacteria differ greatly in their sensitivity to pH. Cellulolytic bacteria and methanogenic archaea are particularly sensitive to declines in pH, and even mild cases of ruminal acidosis can decrease cellulose digestion and CH4 production. Starch-fermenting ruminal bacteria are generally pH-resistant, and low ruminal pH does not seem to decrease starch fermentation. The negative effect of pH on sensitive ruminal bacteria can be explained by

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the transmembrane pH gradient. The pH-sensitive ruminal bacteria often try to maintain a near-neutral intracellular pH, and the pH gradient causes a logarithmic accumulation of VFA anions. Acid-tolerant ruminal bacteria have adapted to tolerate a low intracellular pH; therefore, the low pH gradient does not drive as large an accumulation of VFA anions.

Transport and Phosphorylation Ruminal microorganisms must compete for extracellular degradation products, and most ruminal bacteria have high-affinity transport systems and efficient mechanisms of phosphorylation. Some ruminal bacteria use phosphotransferase (PTS) systems to take up mono- and disaccharides, and these systems phosphorylate sugars as they pass across the cell membrane. Because at least some of the sugar is already phosphorylated, the ATP demand of a glucokinase reaction is decreased. Ruminal bacteria lacking PTS systems have an alternative strategy to conserve ATP. When disaccharides are translocated into the cell via active transport (ion- or ATP-driven), the disaccharides can be phosphorylated by phosphorylases. Phosphorylases conserve the free energy of the hydrolytic bond and phosphorylate one of the sugar residues. The rumen is a sodium-rich environment, and many active transport systems are sodium-dependent.

Crossfeeding among Ruminal Microorganisms Cellulolytic bacteria are always outnumbered by noncellulolytic species in vivo, and cellulolytic and noncellulolytic species can be cocultured in vitro on cellulose. Wolin and his colleagues hypothesized that the noncellulolytic species live on the extracellular products of cellulose digestion, but at least some cellulolytics have a reversible-cellodextrin phosphorylase. Cellodextrins that are excreted by the cellulolytic bacteria can be ‘stolen’ by noncellulolytics, but the interaction with other ruminal bacteria is even more complicated. Cellulolytic species require branched-chain VFA, but noncellulolytic, amino acid-fermenting bacteria produce these essential nutrients. Another interaction between ruminal bacteria is observed in protein degradation. The most active amino acid-fermenting bacteria are not proteolytic and must in turn depend on other proteolytic species. Crossfeeding is also illustrated by the observation that pure cultures of ruminal bacteria can form products in vitro that are not detected in the ruminal fluid. Formate and succinate are converted to CH4 and propionate, respectively, by methanogenic- and succinate-decarboxylating species. Some species produce lactate. However, this acid does not accumulate in the ruminal fluid

until the pH declines, and its utilization by Megasphaera elsdenii or Selenomonas ruminantium is impaired.

Ruminal Fermentation Schemes Virtually all of the ruminal hexose carbon is metabolized by the Embden–Meyerhof–Parnas (EMP) pathway, but pentose carbon can be metabolized by either the pentose pathway or a scheme involving phosphoketolase (Figure 2). Pyruvate arising from carbohydrate catabolism can be converted to lactate, but this method of fermentation provides only a modest amount of ATP. Acetate is the dominant end product of ruminal fermentation, and the ATP yield can be twice as much as the one obtained from lactate. When pyruvate is converted to acetate, the NADH of the EMP must be oxidized by other methods. NADH oxidation via H2 production is thermodynamically unfavorable, but methanogenic archaea, by scavenging H2, can keep the partial pressure of H2 low enough, and so even this reaction is possible. Interspecies hydrogen transfer promotes acetate production and increases ATP production. Not all ruminal bacteria can produce large amounts of H2, and some species have developed other schemes of reducing equivalent disposal. Butyrate-producing bacteria also convert pyruvate to acetyl CoA, but two acetyl CoA can be condensed and reduced by butyryl and butyryl CoA dehydrogenases. Propionate can be produced by either the randomizing (through succinate) or the acrylate pathway. These pathways have dehydrogenase reactions, malate dehydrogenase, and fumarate reductase or two acrylyl CoA dehydrogenase steps. The fumarate reductase of succinate or propionate production is a cytochrome-linked, ATP-producing reaction, but schemes that use other methods of reducing equivalent disposal (alcohol, lactate, butyryl CoA, and -hydroybutyryl CoA dehydrogenases) produce less ATP than acetate production. In this regard, rumen bacteria often have to counterbalance reducing equivalent disposal with opportunities for ATP production. Ruminal CH4 production is a process that involves the uptake of H2 and the stepwise reduction of CO2, and it is a dominant mechanism of reducing equivalent disposal in the rumen. The energetics of CH4 production were until recently not well understood. The free energy change of the overall process is very negative (8 kJ), but initial steps of CO2 formation are low or even positive. The terminal step appears to create a protonmotive force that then drives ATP synthesis. Ruminal acetogens can also utilize H2 and CO2, but these bacteria have a lower affinity for H2 than the methanogens and prefer utilizing sugar. Some ruminal bacteria ferment amino acids, but the ATP yield of amino acid fermentation is very low. It initially seemed that carbohydrate-utilizing ruminal bacteria were responsible for all of the deamination, but their

Mutualism and Commensalism | Rumen 167 Glucose ATP or PEP

ADP or Pyruvate G6P

ATP

ADP FDP

Triose Phosphate NADH ATP

NAD ADP

CO2

PEP

OAA

ADP

ATP

NAD Pyruvate

Lactate

Malate Formate or CO2 and XH

NADH Lactyl CoA

[Acetyl CoA]

Acetyl CoA

NAD

NADH Acrylyl CoA X

Acetyl P XH

Propionate

ADP

NADH

Butyryl CoA ATP

Fumarate

NADH ADP

NAD ATP Succinate CO2

Butyryl P

Acetate

NAD

Propionate ATP

ADP Butyrate

Figure 2 Fermentation schemes of various ruminal bacteria. Reproduced from Russell JB (2000) Rumen Fermentation. Encyclopedia of Microbiology, vol. 4, pp. 185–194.

rates of amino acid fermentation could not explain all of the ruminal ammonia. The rumen also has a small population of obligate amino acid-fermenting bacteria, and these highly specialized bacteria have exceedingly high rates of amino acid and peptide uptake. Some anaerobic habitats have bacteria that can oxidize volatile and even long-chain fatty acids, but these bacteria grow so slowly that they cannot persist in the rumen. Ruminal bacteria can, however, saturate the double bonds of polyunsaturated fatty acids, and biohydrogenation is yet another mechanism of reducing equivalent disposal. Biohydrogenation is normally complete, but a recent work indicates that low ruminal pH can cause the accumulation of partially hydrogenated fatty acids (conjugated linoleic acid, CLA). Some CLA can depress the milk fat of lactating cattle, but it appears to have beneficial dietary effects for humans.

Growth, Maintenance, and Energy Spilling In its simplest sense, bacterial growth is a process that assembles simple monomers (either preformed or

synthesized) into the macromolecules. Protein is usually the most abundant polymer in bacteria, and this macromolecule is relatively expensive to synthesize. Peptide bonds have less than 1 kJ of free energy per mole, but protein synthesis is a four-step process that consumes approximately 4 ATP equivalents or approximately 7 kJ of free energy per mole of amino acid polymerized. As much as 2/3rds of the ATP consumed can be used to polymerize amino acids. Polysaccharides (e.g., glycogen) and nucleic acids account for a smaller fraction of the bacterial mass, and the polymerization of sugars and nucleic acids requires less energy. Most ruminal bacteria can utilize ammonia as the sole source of nitrogen, and this gives ruminants the capacity to be reared on diets virtually devoid of protein, so long as ammonia or some other form of nonprotein nitrogen (e.g., urea) that can be converted to ammonia is available. For many years, it was believed that ruminal bacteria only use the enzyme glutamate dehydrogenase (GDH) to utilize (assimilate) ammonia. However, GDH has a relatively high affinity constant (Km) for ammonia, and this enzyme could not explain the growth of bacteria that

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grow in environments where ammonia concentration is often very low. It is now apparent that at least some ruminal bacteria use an alternative method of ammonia assimilation as demonstrated in many other bacteria. This scheme involves two enzymes, and operates in a cycle. The previously unrecognized enzyme, glutamine oxoglutarate aminotransferase (GOGAT) or glutamate synthase, is actually involved in the second step. The first step involves a common enzyme, glutamine synthetase (glutamate þ NH3 þ ATP ! glutamine þ ADP þ Pi þ H2O). Glutamine is then converted to glutamate by GOGAT (glutamine þ oxoglutarate þ NADPH ! 2 glutamates þ NADPH). The only significant difference between the GOGAT cycle and GDH is ATP. Bacteria utilizing the GOGAT cycle have to expend energy to fix ammonia. The GOGAT cycle has been clearly demonstrated in the ruminal bacterium S. ruminantium, but the significance of GOGAT in the assimilation of ammonia in the rumen is probably important only in extreme conditions of nitrogen derivation. Because the rumen is typically an energylimited rather than a nitrogen-limited ecosystem, GDH is probably the dominant means of nitrogen assimilation by ruminal bacteria. Many ruminal bacteria (the only major exception being the cellulolytics) prefer to utilize amino nitrogen (e.g., amino acids and peptides), and they typically grow more efficiently when amino nitrogen sources are available. However, this increase in growth cannot efficiently be explained by either carbon sparing or the cost of amino acid biosynthesis per se (see energy spilling below). Bacterial growth is driven by the ATP of catabolic schemes (YATP), but observed yields are often much lower than predicted by ATP availability alone. Bacterial cells must expend some of their ATP on maintenance functions. When cells grow, protein is synthesized, but the degradation and resynthesis of protein creates an additional expenditure. Growing cells take up ions and concentrate them, but ions can also leak back across the cell membrane, and their reuptake entails an additional expenditure. Motility is also nongrowth energy expenditure. When bacteria have large amounts of all essential nutrients and the growth rate is rapid, maintenance expenditures are ‘insignificant’, but it should be realized that maintenance expenditures are, in reality, a ‘fixed overhead’. When energy is restricted, and the growth rate decreases, maintenance become a dominant avenue of ATP utilization, and rumen bacteria expend a significant portion of their ATP on maintenance functions (10–30% of their ATP). As mentioned earlier, the rumen generally operates as an energy-limited system, but other nutrients can something be limiting. When other nutrients are limiting (e.g., amino nitrogen or branched VFA), the excess energy is

spilled. The utility of energy spilling to ruminal bacteria is illustrated by the observation that bacteria lacking energy-spilling mechanism can be killed by excess carbohydrate. Energy spilling and maintenance are both nongrowth energy expenditures, but their physiology is distinctly different. All bacteria must be ‘maintained’, but cells ‘spill energy’ only when it is in excess and some other factor is limiting. Streptococcus bovis is a ruminal bacterium that has very high rates of catabolism, and it has been used as a model of energy spilling. S. bovis regulates energy spilling via changes in the concentration of intracellular fructose1,6-diphosphate (FDP). When the glycolytic rate is fast, intracellular FDP increases, which causes a decrease in intracellular inorganic phosphate (Pi). Decrease in Pi causes an increase in the free energy of ATP hydrolysis and an increase in the protonmotive force. When the protonmotive force increases, the resistance of the cell membrane to protons decreases and protons enter the cell. The inward flux of protons is counteracted by the membrane-bound ATPase, and the excess ATP is dissipated. Energy spilling appears to explain the effect of amino nitrogen on the growth efficiency of ruminal bacteria previously mentioned, but it is not known if all ruminal bacteria have the same futile cycle as the one described for S. bovis. When ruminal bacteria have amino acids, they grow faster and can better match their anabolic and catabolic rates. Because there is less ‘excess’ ATP, energy spilling declines and overall growth efficiency (YATP) increases. It is worth mentioning that energy spilling is not restricted to ruminal bacteria. A wide variety of bacteria (even archaea such as methanogens), yeast, and the mitochondria of mammals have the mechanisms of energy spilling. A recent work indicates that at cancer, cells have very high rates of energy spilling, and leading authorities have speculated that energy spilling is a requirement of cancer transformation.

Toxic Compounds Ruminal bacteria can degrade toxic plant materials, but these specialized bacteria may initially be present at very low numbers in the rumen. In some cases, ruminal bacteria can produce toxic compounds from relatively innocuous feed ingredients. A dramatic case involves the degradation of the toxic amino acid mimosine. Tropical trees and leguminous shrubs belonging to the genus Leucaena originated in Central America, but Leucaena leucocephala is now widely distributed in the tropical and subtropical areas. Leucaena grows rapidly, and its leaves are rich in protein. However, the leaves and seeds contain mimosine. Mimosine is converted to 3-hydroxy-4-(1H)-pyridone (3,4 DHP) by the enzymes present in both leucaena and ruminal bacteria.

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DHP causes low weight gain, hair loss, and goiter and esophageal ulceration in cattle. In the late 1970s, Ray Jones of the Commonwealth Scientific and Industrial Research Organisation (CSIRO; Australia) noted that cattle and sheep in Hawaii could consume large amounts of leucaena without showing signs of toxicity. Australian cattle inoculated with ruminal fluid from a leucaena-adapted, Indonesian goat recovered. Milton Allison of the United States Department of Agriculture (USDA) isolated the DHP-degrading bacterium and named it Synergistes jonesii. S. jonesii is a Gram-negative, strictly anaerobic rod that does not use carbohydrates, but it utilizes histidine and arginine. S. jonesii is widely distributed in animals at regions where leucaena is a native plant, but this bacterium could not be detected in cattle in Florida. Inoculation of cattle in Florida with S. jonesii protected them from DHP toxicity. Because DHP-degrading ruminal bacteria are geographically isolated, there has been a great interest in cross-inoculation. Another toxic compound common in the western part of the United States is the organic acid oxalate. The plant Halogeton glomeratus accumulates as much as 25% oxalate, and this poisonous compound causes gastroenteritis and renal damage. In the 1970s, Allison and his colleagues isolated an oxalate-degrading bacterium from the rumen. Oxalobacter formigenes takes up oxalate and converts it to formate and carbon dioxide. If cattle are adapted gradually, O. formigenes numbers increase, and oxalate is degraded. O. formigenes is widely distributed in herbivores, but some humans are not colonized. The absence of these bacteria in humans (due to diarrhea or antibiotic treatments) causes an increased absorption of oxalate, and increases the risk of kidney stones. Plants such as Oxylobium parviflorum accumulate fluoroacetate, and so it has been used as a ‘pesticide’ to combat rabbits and wild dogs (dingo) in Australia. The toxicity of fluoroacetate is due to its conversion to fluorocitrate by mammalian enzymes. Fluorocitrate inhibits the citric acid cycle enzyme, aconitase. Fluorocitrate-degrading bacteria could not be enriched in cattle. However, Keith Gregg of the CSIRO used recombinant engineering to transfer a ‘dehalogenating gene’ to Butyrivibrio fibrisolvens. However, concerns over genetically engineered bacteria (see below) prevented its practical application. Cattle and sheep that graze lush grass in the spring may suffer from hypomagnesemia, or ‘grass tetany’. Grasses and plants causing grass tetany often contain high concentrations of trans-aconitate, an acid with three carboxyl groups. When plants grow rapidly in the spring, plants use transaconitate as an anion to counteract increases in potassium. It was initially hypothesized that trans-aconitate binds magnesium in the gut and decreases its availability, but later work demonstrated that trans-aconitate is fermented rapidly by ruminal bacteria. Some of the trans-aconitate is

fermented to acetate, but much of it is converted to another tricarboxylic acid, tricarballylate. Tricarballylate is absorbed but not metabolized by the animal. Studies with rats indicated that tricarballylate could chelate divalent cations (magnesium, calcium, and zinc) in blood and promote urinary excretion. S. ruminantium is the most important producer of tricarballylate, and it uses this reaction to reduce equivalent disposal. Because S. ruminantium has a high affinity for sucrose and rapidly growing plants have this sugar in abundance, S. ruminantium numbers in the rumen are already high. Grass tetany is a disease that sets in suddenly. Acidaminacoccus fermentans can convert trans-aconitate to acetate, but this bacterium is normally present in low numbers in the rumen. In the 1970s, J. R. Carlson of Washington State University and his colleagues noted that beef cattle taken off range and allowed to graze lush-irrigated pastures developed acute pulmonary emphysema. Similar symptoms have been noted when they are fed rape, kale, turnip tops, small grains, alfalfa, and a variety of grasses. Emphysema is caused by the ruminal conversion of tryptophan to a lung toxin, 3-methylindole (also called skatole). Tryptophan is first deaminated and decarboxylated by the normally occurring microorganisms. Indoleacetate is then converted to 3- methylindole by the lactobacilli. Because lactobacilli are Gram-positive bacteria that are inhibited by monensin, a common feed additive (see ionophores below), the problem has largely been alleviated. Nitrates contaminate the drinking water through nitrate fertilizers, and some plants accumulate nitrate. Nitrate itself is not highly toxic, but it is rapidly converted to nitrite by the ruminal bacteria. Once absorbed, nitrite oxidizes the ferrous iron of hemoglobin. Because this form of hemoglobin does not transport oxygen, nitrite causes metabolic anoxia, rapid breathing, trembling, and in extreme cases, death. Denitrifying bacteria convert nitrite to nitric oxide, nitrous oxide, and ultimately to nitrogen gas, but this process is slower than the conversion of nitrate to nitrite. Nitrite can be converted to ammonia by a process known as assimilatory nitrate reduction, but this pathway is repressed by ammonia in the rumen. However, if animals are slowly adapted to nitratecontaining diets, nitrite does not accumulate. When large amounts of sulfate are present in the ration (or drinking water), animals can die from hydrogen sulfide poisoning. Sulfide is not rapidly absorbed from the rumen, but it is found in ruminal gases that are passed to the lungs during eructation. Absorbed sulfide is a potent inhibitor of mitochondrial electron transport (cytochrome a to cytochrome a3), and animals can die from metabolic anoxia. Animals that consume large amounts of sulfate may also develop polioencephalomalacia (PEM) and brain lesions virtually identical to viral PEM or thiamine deficiency. Desulfovibrio desulfuricans, a bacterium that

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utilizes lactate, has been detected in the rumen in sufficient numbers (>108 cells per milliliter) to account for sulfide production in vivo. Many legumes including the species of Astragulus (e.g., crown vetch), Lotus, Cornilla, and Indigofera have nitrotoxins (organic compounds containing one or more NO2 groups), which cause increased heart and respiration rates, frothy salivation, and a lack of coordination. Nitrotoxins are present as glycosides, and their hydrolysis releases 3nitro-1-propionate, 3-nitropropanol, or nitroethane. H2, formate, and lactate serve as reductants in the conversion of nitro groups to their corresponding amines. Thus, 3nitro-1-propionate is converted to -alanine, and 3-nitropropanol is converted to 3-amino-propanol, but the pathways have not been defined. Cattle given nitroethane, the least toxic analogue, adapted so they could metabolize 3-nitropropanol at a faster rate. Bacteria capable of detoxifying nitrotoxins have been isolated from the rumen and placed in a new genus and species, Denitrobacterium detoxificans. Selenium is an essential mineral for mammals, but large amounts can be toxic. Some plants (e.g., Astragulus) accumulate selenium, and drinking water, too, can have high concentrations of selenium. Selenite is watersoluble, is more readily absorbed, and is more toxic than selenate. The ruminal conversion of selenite to selenate is a detoxification mechanism. S. ruminantium, Wolinella succinogenes, and Prevotella ruminicola are able to use selenite, and this reaction is the reverse of nitrate metabolism. The feed additive monensin (see below) inhibits selenite conversion to selenate in vitro, but it is not clear if this occurs in vivo.

Effects of Ionophores on Ruminal Microorganisms Beef cattle, and more recently dairy cattle, in the United States are routinely fed a class of antibiotics known as ionophores, and these compounds decrease H2 and CH4 production and increase propionate and energy retention. Obligate amino acid-fermenting ruminal bacteria are also sensitive to ionophores, and this inhibition decreases NH3 production and conserves amino acids. Some lactic acidproducing bacteria are inhibited by ionophores, and this activity may modulate ruminal pH. Ionophores translocate ions across cell membranes. When ion gradients (e.g., potassium, sodium, and protons) are dissipated, the bacteria must expend energy to reestablish the gradients, and thus their growth is impaired. Because Gram-negative bacteria are generally more resistant than the Gram-positive species, it initially appeared that the outer membrane was acting as a protective barrier to exclude ionophores from the cell membrane. However, ionophore resistance now appears to be a more complicated

phenomenon. Some Gram-positive ruminal bacteria are more sensitive to ionophores than the Gram-negative species; however, both Gram-positive and Gram-negative bacteria can adapt. Ionophore resistance now appears to be mediated by extracellular polysaccharides (glycocalyx) that excludes hydrophobic ionophore molecules from the cell membrane. In 2006, the European Union banned the use of antibiotics, including ionophores, in animal feed as growth promotants. Some questions then arise. How safe are ionophores? Do ionophores increase resistance to therapeutic antibiotics? Should they be banned in the United States as well? This is a very controversial subject, but some facts can be cited: (1) ionophores have and never will be used in human medicine due to toxicity; (2) cattle not receiving ionophores always have large populations of ionophore-resistant bacteria; (3) the increase in ionophore-mediated resistant bacteria is quickly reversed as soon as the ionophore is removed from the diet; (4) ionophore resistance appears to be a physiological selection that involves an increase in extracellular polysaccharide rather than a mutation- or plasmid-mediated event; (5) the adaptation and the development of ionophore resistance in a ruminal bacterium initially sensitive to ionophore did not cause an increase in resistance to 20 therapeutic antibiotics; and (6) ionophores have been very widely used for more than 20 years in the United States, and there has been little change in their effect on the feed efficiency of cattle.

Genetic Engineering In the 1980s, microbiologists perfected the use of restriction endonucleases and so genes could be transferred from one bacterium to another in a methodical fashion. This success lead to the question, could rumen microbiologists use recombinant DNA to improve ruminal fermentation? At least four projects were proposed: (1) improvement of fiber digestion by enhancing cellulase production, (2) addition of genes to Fibrobacter succinogenes so that it could use xylans, (3) transfer of fluoroacetate degradation genes to B. fibrisolvens, and (4) creation of an acid-resistant ruminal bacterium that could digest cellulose at low pH by reconstructing the -glucanase of Prevotella bryantii so that it could have a cellulose-binding domain. These efforts were thwarted by the fear that the release of genetically engineered organisms into the environment might be dangerous, and there is now little interest in pursuing any of these goals. Another feature that complicated the genetic engineering of ruminal bacteria was the observation that Escherichia coli clones carrying ruminal genes sometimes produced highly truncated proteins. This point is illustrated by -glucanase of P. bryantii. The -glucanase

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gene of P. bryantii encodes a protein that has a molecular weight of 88 kDa, but the first E. coli clones produced a protein that was only 42 kDa. Subsequent work indicated that this truncated protein had just as much activity against -glucan as the native protein from P. bryantii, but it lacked the N-terminal domain that tethered the protein to the cell surface of P. bryantii. This observation indicated that E. coli had at least one promoter that recognized a site in the -glucanase gene, but this promoter was not recognized by P. bryantii. The entire -glucanase gene of P. bryantii was eventually cloned into E. coli, and subsequent work indicated that the addition of a cellulose-binding domain from a nonruminal bacterium, Thermomonospora fusca, resulted in a protein that had tenfold more capacity to degrade insoluble native cellulose. However, restriction barriers and the failure to identify a suitable promoter prevented all attempts to return the reconstructed gene to P. bryantii and create an acid-resistant cellulolytic bacterium. Abigail Salyers at the University of Illinois and her colleagues developed a conjugation procedure involving the transfer of genes first from E. coli to Bacteroides uniformis and then to P. bryantii. However, the efficiency of this two-step conjugation procedure was extremely low.

Rumen Microbial Ecology Robert Hungate, the father of rumen microbiology, explained the complexity of ruminal microorganisms in three ways. His first and second principles were based on the idea that feedstuffs are relatively complex materials and that a single kind of cell cannot contain ‘‘the diversity of conditions and enzymes needed for all the individual reactions in the combination yielding maximum cell growth.’’ Hungate’s third hypothesis of ruminal microbial diversity was based on the supposition that even the ‘most-fitted’ species, by its proliferation, would eventually change the habitat and create additional niches for yet another bacterium. Rumen microbial diversity can also be explained by generation time. Most microorganisms in natural habitats generally grow very slowly, but ruminal microorganisms have been allowed to grow continuously at a relatively rapid rate (average doubling time of approximately 7 h) for over 70 million years. Because ruminal bacteria have relatively short generation times, there has been a much greater opportunity for adaptation, selection, and diversity.

Ruminal Bacteria and Nonculturable Bacteria In 1966, Hungate listed approximately 20 species of ruminal bacteria that achieved numbers of at least 107 cells

per gram of rumen contents (Table 1). These species were classified by physiological and morphological traits typical of Bergey’s Manual of Systematic Bacteriology, but some were renamed in the 1970s and 1980s. Strains within a given species sometimes had DNA homologies that were as low as 20%, and 16S rRNA sequencing indicated that many strains and some species should be reclassified. Relatively few 16S rRNA genes of ruminal bacteria have been sequenced, but it is clear that the diversity of ruminal bacteria is very great. An ecological analysis of any habitat is dependent on the quantitative measurements of activity, but the activities of pure cultures and mixed culture populations have not always been compared. For example, in the 1960s, it was generally assumed that carbohydrate-fermenting ruminal bacteria were the most important NH3-producing species, but later work indicated that these species had NH3 production rates 50% lower than mixed ruminal bacteria. This comparison led to the isolation of obligate amino acid-fermenting bacteria with 20-fold greater activity. Many ruminal bacteria are difficult to culture in the laboratory, and a direct microscopic count is usually tenfold greater than the culturable count. With the advent of molecular techniques, it has become apparent that many habitats have large populations of nonculturable bacteria. Very recently Stevenson and Weimer used real-time PCR of 16S rDNA to examine rumen bacterial ecology, and they concluded that ‘‘aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus’’ (Prevotella). So far, the latter bacteria have not been isolated and cultured, and further work will be needed to see if this interesting finding is reproducible and not diet-dependent.

Genomics Ruminal cellulose digestion is very rapid (as fast as 10% per hour), but attempts to isolate and characterize the cellulase of ruminal bacteria have been disappointing. In the 1960s, Halliwell and Bryant at the University of Illinois attempted to extract ‘true’ cellulases from three most active ruminal species. Cell-free extracts of the ruminococci solubilized some native cellulose, but extracts from F. succinogenes, the most active strain, had virtually no activity. Microscopic examination indicated that ruminal fungi and Ruminococcus albus had cellulases that were organized in structures (cellulosomes) similar to those found in Clostridium thermocellum. However, such structures are not evident in F. succinogenes, and all of its cloned enzymes turned out to be -glucanases or xylanases rather than true cellulases that could degrade insoluble cellulose. To address this perplexity, the US Department of Agriculture provided a grant to the North American Consortium for Genomics of Fibrolytic Ruminal

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Table 1 Taxonomy and physiological traits of predominant ruminal bacteria Taxonomy C. 1966 Bacteria Bacteroides succinogenes Ruminococcus albus Ruminococcus flavefaciens Butyrivibrio fibrisolvens Bacteroides amylophilus Selenomonas ruminantium Bacteroides ruminicola

Succinimonas amylolytica Succinivibrio dextrinosolvens Streptococcus bovis Eubacterium ruminantium Peptostreptococcus elsdenii Lachnospira multipara

Vibrio succinogenes Anaerovibrio lipolytica Methanobacterium ruminantium Protoza Holotrichs Entodinomorphs Fungi

Now

Fermentation productsa

Primary niches

Fibrobacter succinogenes Ruminococcus albus Ruminococcus flavefaciens

S, F, A A, F, E S, F, A, H2, E

Cellulose Cellulose Cellulose

Butyrivibrio fibrisolvens

B, F, L, A, H2

Ruminobacter amylophilus Selenomonas ruminantium Prevotella ruminicola

S, F, A, L, E L, A, P, B, H2, F S, A, F, P

Prevotella albensis

S, A, F

Prevotella brevis

S, A, F

Prevotella bryantii

S, A, F

Succinimonas amylolytica Succinivibrio dextrinosolvens

S, A, P S, A, F, L

Cellulose, hemicellulose, starch, pectin, sugars Starch Sugars, starch, lactate Starch, hemicellulose, pectin, -glucans, proteins Starch, hemicellulose, pectin, -glucans, proteins Starch, hemicellulose, pectin, -glucans, proteins Starch, hemicellulose, pectin, -glucans, proteins Starch Maltodextrins

Streptococcus bovis Eubacterium ruminantium Megasphaera elsdenii

L, A, F, E A, F, B, L P, A, B, Br

Strach, sugars Maltodextrins, sugars Lactate, maltodextrins, amino acids

Lachnospira multipara Peptostreptococcus anaerobius Clostridium aminophilum Clostridium sticklandii Wolinella succinogenes Anaerovibrio lipolytica Methanobacterium ruminantium

L, A, F, E, H2 Br, A A, B A, Br, B, P S A, S, P CH4

Pectin, sugars Peptides, amino acids Amino acids, peptides Peptides, amino acids Malate, fumarate Glycerol, lactate H2, CO2, formate

Isotricha, Dasytricha Entodinium, Diplodinium, Epidinium, Orphryoscolex Neoxallimastix, Caecomyces, Piromyces, Oprinomyces, Aneromyces

A, B, L A, B, H2

Soluble sugars Starch grains

A, L, H2, F

Cellulose

a

A, acetate; P, propionate; B, butyrate; F, formate; L, lactate; E, ethanol; Br, branched-chain VFA.

Bacteria, and genomes of predominant cellulolytic bacteria are now available (http://www.tigr.org). A computer search of the F. succinogenes genome supported the idea that this bacterium has enzymes that lack dockerin and cohesin sequences, commonly called cellulose-binding domains. However, the genome did have a single family 45 -glucanase gene and signal sequence. This gene did not encode a cellulose-binding domain, but some of the enzymes from this family have good activity against crystalline cellulose. Based on this information, primers were designed to clone this gene and express it in E. coli. The cloned protein was then purified and characterized, but once again little activity against native, insoluble cellulose could be demonstrated. This

observation supports the notion that F. succinogenes has a novel mechanism of cellulose degradation. The inability of researchers to locate and purify enzymes important in ruminal cellulose digestion may be related to feedback inhibition of the cellulases. This tentative hypothesis is supported by two observations. First, thiocellobiose, a nonmetabolizable form of cellobiose, is a very potent inhibitor of cellulose digestion. Second, when administered to nitrogen-limited F. succinogenes cultures, cellobiose is very toxic and causes a dramatic decease in intracellular ATP, protonmotive force, and viability. If the cellulases are, indeed, strongly inhibited by any significant accumulation of the product, it would be difficult, if not impossible, to assay them in vitro.

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Ruminal Protozoa Ruminants can be reared without protozoa or be defaunated with chemicals, but ruminal protozoa can account for as much as half of the microbial mass in the rumen. These eukaryotic, and sometimes even sexual organisms, are 20–100 times larger than bacteria, and can engulf bacteria, feed particles, and even other protozoa. The rumen has two major classes of protozoa – holotrichs that have cilia over their bodies, and entodinomorphs that have cilia around the oral cavity. Holotrichs use soluble sugars as the energy source, but entodinomorphs prefer utilizing starch grains. Entodinomorphs also seem to digest the cellulose, but our understanding of protozoal metabolism in the rumen has been stymied by the observation that most ruminal protozoa have an obligate requirement for bacteria. The impact of protozoa on fermentation can be both positive and negative. Protozoal engulfment of starch grains modulates the ruminal pH and prevents acidosis. Protozoal predation of bacteria and protozoal lysis increase ruminal NH3 and decrease amino acid availability. Large protozoa can take up or cannibalize the smaller ones. In the 1960s, Margaret Eadie of the Rowett Research Institute (Scotland) and her colleagues noted that sheep tended to have one or the other of two stable populations that the authors designated as ‘A’ and ‘B’. Population A was dominated by large protozoa, while population B was dominated by the smaller protozoa. After the ruminal fluid carrying population A was inoculated into a sheep having population B, the population B in the sheep disappeared and only population A types were observed. When Polyplastron, an A type protozoan, was introduced into sheep, the Eudiplodia increased in size, presumably in an attempt to evade predation. Another antagonism occurs between Entodium bursa and Entodinium caudatum. E. bursa is the larger of the two, and it can engulf E. caudatum. However, if E. bursa is present, E. caudatum develops spines and resists uptake.

observations lead microbiologists to believe that fungi are better able to digest cellulose than bacteria, but there has been little direct demonstration to show that this hypothesis is true. The ruminal fungi grow slower than bacteria, have very low growth yields, and rarely make up more than 6% of the microbial mass in the rumen.

Models of Ruminal Fermentation Computer models have been used to simulate complex systems for more than 40 years, and some groups have developed models to describe ruminal fermentation. To date, ruminal fermentation models have not provided a detailed description of the ecology, and have primarily emphasized the kinetics of feed digestion and its removal from the rumen. In 1980s, workers at Cornell University developed a model that had equations describing the basic principles of microbial growth (effects of maintenance energy, pH, and amino acid availability), substrate availability (fermentation vs. passage rates) as well as different microbial pools. The microbial ‘ecology’ was, however, still highly simplified. Ruminal bacteria were divided into only two groups (structural and nonstructural carbohydrate-fermenting types). Protozoa were simply a factor that decreased the theoretical maximum growth yield of the bacteria, and the fungi were completely ignored. Nevertheless, the impact of the Cornell model in the cattle industry was very positive. Feed savings as great as 17% were reported, which eventually provided a basis of the 1996 National Recommendations for Beef Cattle. See also: Ecology, Microbial; Energy Transduction Processes; Evolution, Theory and Experiments with Microorganisms; Fermentation; Food Webs, Microbial; Gastrointestinal Microbiology in the Normal Host; Methanogenesis

Further Reading Ruminal Fungi Early workers noted large flagellated microorganisms in the rumen, which were mistakenly classified as protozoa. Most of these flagellates were actually fungal zoospores. The ruminal fungi have a complex life cycle similar to the phycomycetes. Zoospores give rise to a mycelium that covers the feed particles, and sporangia release zoospores that can colonize fresh feed. Ruminal fungi have been classified under chytridomycetes, the most primitive group of fungi. Ruminal fungi are found in greatest numbers in animals that consume poor-quality forage, and the fungi seem to have very active cellulases. These

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White BA (eds.) Gastrointestinal Microbiology, pp. 319–379. New York, NY: Chapman and Hall. Gregg K and Sharpe H (1991) Enhancement of rumen microbial detoxification by gene transfer. In: Tsuda T, Sasaki Y, and Kawashima R (eds.) Physiological Aspects of Digestion and Metabolism in Ruminants, pp. 140–195. New York, NY: Academic Press. Hungate RE (1966) The Rumen and Its Microbes. New York, NY: Academic Press. Morrison M, Nelson KE, Antonopoulos D, et al. (2004) New and emerging approaches to improve herbivore nutrition: Rumen microbiology in the genomics era. In: t’Mannetje L, Ramirez-Aviles L, Sandoval-Castro C, and Ku-Vera JC (eds.) Proceedings of the Sixth International Symposium on the Nutrition of Herbivores. Mexico: Universidad Autonoma de Yucatan. Nagaraja TG and Chengappa MM (1998). Journal Animal Science 76: 287–298. Owens FN, Secrist DS, Hill WJ, and Gill DR (1998) Journal of Animal Science 76: 275–286. Russell JB and Houlihan A (2002) FEMS Microbiology Reviews 27: 65–74.

Russell JB and Wallace RJ (1997) Energy yielding and consuming reactions. In: Hobson PN and Stewart CS (eds.) The Rumen Microbial Ecosystem, pp. 246–282. New York, NY: Chapman and Hall. Sauvant D (1997) Rumen mathematical modeling. In: Hobson PN and Stewart CS (eds.) The Rumen Microbial Ecosystem, pp. 685–708. New York, NY: Chapman and Hall. Stewart CS, Flint HJ, and Bryant MP (1997) The rumen bacteria. In: Hobson PN and Stewart CS (eds.) The Rumen Microbial Ecosystem, pp. 10–72. New York, NY: Chapman and Hall. Weimer PJ (1992) Critical Reviews in Biotechnology 12: 189–222. Wolin MJ (1975) Interactions between the bacterial species in the rumen. In: MacDonald IW and Warner ACI (eds.) Digestion and Metabolism in the Ruminant, pp. 134–148. Armidale, Australia: The University of New England Publishing Unit.

Relevant Website http://www.tigr.org – J. Craig Venter Institute

PATHOGENESIS Contents Airborne Infectious Microorganisms Aminoglycosides, Bioactive Bacterial Metabolites Antibiotic Resistance Antifungal Agents Antiviral Agents Bacteriophage Therapy: Past and Present Bacteriophage Therapy: Potential and Problems -Lactam Antibiotics Cyanobacterial Toxins Diagnostic Microbiology Emerging Infections Enteropathogenic Infections Epidemiological Concepts and Historical Examples Exotoxins Food and Waterborne Illnesses Fungal Infections, Cutaneous Fungal Infections, Systemic Fungicides and other Chemical Approaches for use in Plant Disease Control Gastrointestinal Microbiology in the Normal Host Global Burden of Infectious Diseases Glycopeptides, Antimicrobial Immune Suppression Immunity Infectious Waste Management Lipopolysaccharides (Endotoxins) Macrolides Mycotoxins Oral Microbiology Plant Disease Resistance: Natural, Non-Host Innate or Inducible Plant Disease Resistance: Breeding and Transgenic Approaches Plant Pathogens and Disease: General Introduction Plant Pathogens and Disease: Newly Emerging Diseases Plant Pathogens, Bacterial Plant Pathogens, Minor (Phytoplasmas) Prions Quinolones Sexually Transmitted Diseases Skin Microbiology Subversion of Host Defences by Microbes

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Surveillance of Infectious Diseases Vaccine Development: The Development of Avian Influenza Vaccines for Human Use Vaccines, Viral Viral Pathogens of Domestic Animals and Their Impact on Biology, Medicine and Agriculture Zoonoses

Airborne Infectious Microorganisms L D Stetzenbach, University of Nevada, Las Vegas NV, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Bioaerosols Infectious Airborne Viruses

Glossary aspergilloma Pulmonary fungus ball consisting of a solid mass of hyphae growing in a lung cavity. aspergillosis Disease caused by the inhalation of some species of the genus Aspergillus resulting in growth of the fungus in the lungs. bioaerosol An airborne suspension of biological particles and microbial by-products. blastomycosis A symptomatic infection caused by the inhalation of the fungus Blastomyces dermatitidis. chickenpox Disease caused by the virus varicellazoster. coccidioidomycosis Disease caused by the inhalation of Coccidioides immitis arthroconidia (also called San Joaquin Valley fever). cryptococcosis Disease caused by the fungus Cryptococcus neoformans. endotoxin A lipopolysaccharide component of the Gram-negative bacteria cell released during active cellular growth and after cell lysis that can cause respiratory distress when inhaled. fomite An inanimate object that transmits infectious agents to a new host.

Abbreviations HPS HVAC NLVs

Hantavirus pulmonary syndrome Heating, ventilation, and air conditioning Norwalk-like viruses

Infectious Airborne Bacteria Infectious Airborne Fungi Airborne Bioterrorism Further Reading

H5N1, H7N2, H9N2, and H7N3 Strains of avian influenza A viruses that have been linked to respiratory disease in humans. histoplasmosis Disease caused by the infectious fungus Histoplasma capsulatum that can manifest as mild flu-like symptoms to chronic lung disease that resembles tuberculosis. HPS Hantavirus pulmonary syndrome. Legionnaires’ disease A severe respiratory disease resulting from the inhalation of Legionella pneumophila. measles Term for the disease caused by the virus of the same name. Mycobacterium tuberculosis The causative agent of human tuberculosis. NLVs Norwalk-like viruses. Pontiac fever A mild flu-like syndrome following inhalation of nonviable Legionella pneumophila. rubeola Another name for the disease measles. SARS Severe acute respiratory syndrome. SARS-CoV Severe acute respiratory syndrome associated coronavirus. Sin Nombre virus Causative agent of the majority of hantavirus pulmonary syndrome cases in the United States.

SARS SARS-CoV WMD

Severe acute respiratory syndrome SARS-associated coronavirus weapons of mass destruction

Pathogenesis | Airborne Infectious Microorganisms

Defining Statement Inhalation of airborne pathogenic microorganisms can elicit adverse human health effects including infection, allergic reaction, inflammation, and respiratory disease. This chapter discusses viruses, bacteria, endotoxin, and fungi that are dispersed through the air and can result in infection following inhalation.

Introduction Inhalation exposes the upper and lower respiratory tracts of humans to a variety of airborne particles and vapors. Airborne transmission of pathogenic microorganisms to humans from the environment, animals, or other humans can result in disease. The lung is more susceptible to infection than the gastrointestinal tract as ingested microorganisms must past through the acidic environment of the stomach before they can colonize tissue, while inhaled microorganisms are deposited directly on the moist surfaces of the respiratory tract. Inhalation of microbial aerosols can elicit adverse human health effects including infection, allergic reaction, inflammation, and respiratory disease.

Bioaerosols A bioaerosol is an airborne collection of biological material. Bioaerosols can be comprised of bacterial cells and cellular fragments, fungal spores and fungal hyphae, viruses, and by-products of microbial metabolism. Pollen grains and other biological material can also be airborne as a bioaerosol. Microbial aerosols are generated in outdoor and indoor environments as a result of a variety of natural and anthroprogenic activities. Wind, rain and wave splash, spray irrigation, wastewater treatment activity, cooling towers and air handling water spray systems, and agricultural processes such as harvesting and tilling are examples of activities that generate bioaerosols outdoors. Indoors bioaerosols are generated and dispersed mechanical and human activity. Industrial and manufacturing practices and biofermentation procedures can generate high concentrations of microbial aerosols. Heating, ventilation, and air conditioning (HVAC) systems, water spray devices (e.g., showerheads and humidifiers), and cleaning (e.g., dusting, sweeping, vacuuming, and mopping) result in the transport of microbial materials in the air. Talking and coughing generate bioaerosols from individuals, some of which may be infectious. Facilities with medical, dental, or animal care practices can generate infectious microbial aerosols.

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The individual particle size of particulate material in bioaerosols is generally 0.3–100 mm in diameter; larger particles tend to settle rapidly and are not readily transported in the air. Virus particles are nanometer in size, bacterial cells are approximately 1 mm in diameter, and fungal spores are >1 mm. These microorganisms can be dispersed in the air as single units, but are often present as aggregate formations. The larger aggregates have different aerodynamic properties than single-cell units; therefore their dispersal may be different than singleunit particles. Aggregates of biological material also afford protection from environmental stresses such as desiccation, and exposure to ultraviolet radiation ozone and other pollutants in the atmosphere. Often bacterial cells and virus particles are associated with skin cells, dust, and other organic or inorganic material. During agricultural practices (e.g., during harvesting, and tilling), fungus spores are released from plant surfaces and the soil and raft on other particulate matter. This ‘rafting’ affects the aerodynamic characteristics and the survival of the cells in the bioaerosol. When biological material is dispersed from water sources (e.g., splash, rainfall, or cooling towers and fountains), it is generally surrounded by a thin layer of water. This moisture layer also changes the aerodynamic properties and aids in the survivability of the microorganisms while airborne. Airborne particulate will remain airborne until settling occurs or they are inhaled. Following inhalation, large airborne particles are lodged in the upper respiratory tract (i.e., nose and nasopharynx). Particles 1000 mm3). As mentioned above, HIV is a complex retrovirus with six additional genes (tat, rev, nef, vpr, vif, and vpu). The CD4 receptor for HIV is present on T-helper lymphocytes, macrophages, and dendritic cells, so these are the cells that can be infected in vivo. Infection of T-helper lymphocytes results in cell killing, whereas infected macrophages and dendritic cells are not killed. During the initial stages of infection, there is typically substantial loss of T-helper lymphocytes. The initial infection may be accompanied by flu-like symptoms, or it may be asymptomatic. Development of cell-mediated and humoral immune responses limits the initial infection, after which levels of circulating virus (viral loads) substantially decrease and T-helper (CD4) counts recover. The infected individual then enters an asymptomatic period (typically 810 years) where there are relatively low viral loads and normal T-helper counts. However the infection has not been eliminated, as low levels of virus infection persist, and individuals continually produce virus-specific antibodies (the common diagnostic test for infection). Ultimately, the immune system is unable to control the viral infection, and rising viral loads result in progressive loss of T-helper lymphocytes. Because T-helper lymphocytes are essential for function of both cell-mediated immunity and humoral immunity, loss of Thelper lymphocytes leads to failure of both arms of the

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adaptive immune system. This results in the appearance of the symptomatic opportunistic infections and cancers. One of the reasons that infected individuals cannot eliminate HIV infection is that the virus can establish latency – in latently infected cells the viral genetic information is present but the virus is not produced. Latently infected cells can evade the immune system because they do not express viral proteins. Latently infected cells include macrophages and T-lymphocytes and they persist in infected individuals for many years. At later times they may be reactivated to produce virus that can spread. Another mechanism by which HIV evades host defenses is rapid and progressive mutation in infected individuals, particularly in the env gene. This mutation is largely driven by viral evasion of host immune responses (antibodies and cytotoxic T-lymphocytes) to the virus. In individuals being treated with antiviral drugs, the high virus mutation rate also leads to development of drug-resistant virus. Temporal variation of HIV is also seen in infected individuals. The initial virus transmitted generally is one that uses the CCR5 coreceptor. With time, the virus will mutate so that it is more cyopathic (killing T-helper lymphocytes efficiently), which will often be accompanied by a switch to usage of the CXCR4 coreceptor. Antiviral drugs targeted at HIV have substantially improved the clinical outlook for infected individuals. So far, antivirals targeted at RT and PR have been developed, and one drug targeted at IN has been approved. RT inhibitors are divided into two classes: nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). NRTIs (typified by AZT) are chain-terminating nucleoside analogues that are incorporated by RT into viral DNA during reverse transcription; once incorporated, an NRTI cannot be further extended, which kills the growing DNA molecule. The antiviral selectivity of NRTIs is because they are relatively inefficiently incorporated by host cell DNA polymerases. NNRTIs (e.g., nevirapine) inhibit reverse transcription by binding to RT and blocking its enzymatic activity. Additional drugs that block the initial entry events for HIV (fusion and coreceptor binding) have been developed or are nearing approval. Before the development of different classes of inhibitors, treatment with single HIV antivirals (NRTIs) proved to be of limited benefit. Monotherapy of infected individuals resulted in initial suppression of viral loads (with improvements in immune system function), but the effects were transient. Emergence of drug-resistant virus occurred quite quickly, with resulting decline in immune function. The development of additional classes of antivirals (notably PR inhibitors) paved the way for combination therapies. Combination therapies with several antiviral drugs are highly effective at reducing viral loads and improving the immune systems of individuals with AIDS. There are multiple combinations that are effective, and they always include inhibitors of more than one class. The reduction in viral loads can be prolonged,

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resulting in clinical improvement or even recovery in individuals with AIDS. However despite these very positive effects, combination therapies have limitations. The therapies must be taken continually because the virus has not been eliminated. Development of drug resistance is a major limiting factor, even for combination therapies. Clinical management of HIV-infected individuals includes monitoring for appearance of drug-resistant virus and developing new combinations of drugs that control it. A second limitation is that many of the HIV antiviral drugs have side effects; this is particularly important because they must be taken continually to control the virus. Side effects include anemia, neuropathies, pancreatitis, diabetes, cardiac disease, and lipodystrophy. The antiviral drugs are also expensive – beyond the means of many infected individuals in the developing world. For these reasons there is tremendous interest and effort in developing an effective preventative HIV vaccine. However, efforts so far have not been successful. In addition to HIV, other retroviruses also induce immunodeficiency in animals and they have been studied for insights into HIV pathogenesis. The most frequently studied animal retroviruses that cause immunodeficiency are simian lentiviruses (SIVs) infected into nonnative primate species (e.g., SIVmac into rhesus macaques). Simian retroviruses of the betaretrovirus class (simian retrovirus Type-D) also induce immunodeficiency in rhesus macaques. In addition, a murine retrovirus complex also induces murine AIDS (MAIDS). The MAIDS complex consists of an MuLV as well as a replicationdefective MuLV derivative. The replication-defective component is responsible for the development of MAIDS. The mechanism of immunodeficiency is distinct from that associated with HIV infection, with the virus actually inducing proliferation of B-lymphocytes.

Neurologic disease Several retroviruses also induce neurologic symptoms. HIV-infected individuals frequently develop such symptoms in the central nervous system (dementias) and in peripheral nerves (neuropathies). The dementias are associated with loss of neurons in the brain (spongiform encephalopathies) but the neurons themselves do not appear to be targets for HIV infection. Infection is observed in supporting cells, most notably microglial cells. Thus the mechanism of neuropathogenesis by HIV appears to be indirect. Some strains of MuLV also induce neurologic symptoms such as hind limb paralysis (in particular those derived from wild mouse ecotropic MuLV). These neuropathic strains of MuLV also induce spongiform encephalopathies of the brain or spinal cord. Similar to the situation with HIV, neuropathic MuLVs do not appear to infect neurons, so the pathogenesis likely results indirectly from infection of other cells.

The human retrovirus HTLV-I, which induces T-cell leukemia (see below), also induces a demyelinating disease known as HTLV-associated myelopathy or tropical spastic paraparesis (HAM/TSP). The symptoms resemble multiple sclerosis, and the disease may be immune-mediated.

Retroviruses of Humans While early research on retroviruses involved studies of animal retroviruses, several retroviruses of humans have been discovered, some of which cause disease. The first human retrovirus discovered was HTLV-I, which causes adult T-cell leukemia (ATL) and HAM/TSP. This virus is a member of the deltaretrovirus family, and it encodes several additional proteins from different reading frames in the X region. These include the transcriptional transactivator Tax and the Rex regulatory protein. Leukemogenesis by HTLV-I is relatively inefficient, with decades elapsing between infection and development of the leukemia. The Tax protein is likely involved in the process, because it can immortalize primary T-lymphocytes and transform fibroblasts in culture. Tax can transactivate cellular promoters in addition to the viral LTR, including the promoters for interleukin-2 (IL-2, T-cell growth factor) and the IL-2 receptor; the activation of these cellular genes may be involved in tumorigenesis. Simultaneous expression of IL-2 and IL-2R in a lymphocyte can lead to an autocrine loop in which there is continual signaling for cell division. On the other hand, many HTLV-induced ATL tumor cells actually do not express Tax; therefore Tax may be involved in establishment but not maintenance of the tumor. A recently discovered protein is encoded by the opposite strand of HTLV-I, HBZ. This is a DNA-binding protein with a B-zip motif that can affect transcription of both HTLV and cellular genes negatively or positively. Moreover it is expressed in all ATL cells, which suggests that it is required for maintenance of HTLV-induced tumors. A virus related to HTLV-I, HTLV-II has also been isolated. HTLV-II has not been causally linked with any neoplasms, although it was first isolated from hairy cell leukemia cells (a form of T-cell leukemia). HTLV-II infection is present in indigenous populations in North and South America, whereas HTLV-I infection is predominantly in Africa, the Caribbean, and Japan. The most important human retroviruses in terms of disease are HIV (HIV-1 and HIV-2). These viruses cause human AIDS, and as described above they represent relatively recent movement of infection from African primates into humans. Recently, another human retrovirus has been detected in tumor tissues of patients with a familial form of prostate cancer. These individuals carry a variant form (R462Q) of the RNase L gene, which was reported in some studies to

Viruses | Retroviruses

elevate the risk of prostate cancer twofold. RNase L is an effector molecule in the interferon-mediated innate antiviral response; when RNase L is induced by interferon, it degrades viral and cellular mRNAs. The R462Q form of RNase L has reduced activity, which led investigators to test the hypothesis that the elevated prostate cancer susceptibility reflects increased infection by an oncogenic virus. cDNA array experiments indicated that a significant fraction (40%) of the tumors from these individuals are infected at low level with a novel retrovirus closely related to an endogenous xenotropic MuLV, XMRV-1. Tumor tissues from sporadic prostate cancer patients showed a much lower frequency of XMRV-1 infection. The XMRV-1-infected cells in the familial prostate cancer tissues are not the tumor cells themselves, and the frequency of infected cells is low. Although it is still unclear whether XMRV-1 is involved in prostate oncogenesis, molecular experiments confirmed that this is an infectious virus of humans. Given the close phylogenetic relationship with xenotropic MuLV, it is possible that XMRV-1 represents a zoonotic infection of humans. Several reports have suggested that a virus closely related to MMTV is present in human breast cancer or tissues from biliary cirrhosis patients. These reports are controversial, because other investigators have not confirmed them.

Endogenous Retroviruses Most eukaroytic organisms carry retroviral DNA in their genomes as stably inherited elements. These elements are referred to as endogenous retroviruses (ERVs) and they represent retroviral infection of germ cells during evolution of a species. Once a germ cell is infected by a retrovirus, all progeny will inherit the proviral DNA. The ‘endogenization’ of retroviruses has occurred throughout evolution, with some ERVs entering the genomes relatively early during speciation (e.g., entry of some human ERVs (HERVs) in common ancestors to humans and great apes) whereas others have entered the germ lines relatively recently (e.g., entry of other HERVs after separation of humans from chimpanzees). Indeed, endogenization of retroviruses is presently occurring in some species (e.g., at high levels in the koala). In addition to the ERVs, there are also related genetic elements that resemble retroviruses except they lack coding sequences for an Env protein. These elements, LTR retrotransposons, assemble particles and reinsert reverse-transcribed DNA into the genome without an extracellular phase (retrotransposition). Over evolutionary time scales, multiple copies of ERVs or LTR retrotransposons have accumulated in the genomes of most eukaryotes; once inserted, there is no molecular mechanism for removal. Indeed, it is estimated that 8% of the

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human genome is made up of ERVs and LTR retrotransposons. This includes ‘solo LTRs’ that result from excision of the coding sequences of an endogenous provirus by homologous recombination between the two LTRs. Insertion of retroviral proviruses into the host genome can be detrimental to the host organisms if the provirus insertion disrupts a gene or leads to activation of a proto-oncogene. Thus there has been genetic selection against replicationcompetent ERVs that could continually reinfect the host; most ERV genomes carry mutations and/or deletions. However, for other species there are ERV genomes that can encode full-length proteins, and some species carry replication-competent ERV proviruses (e.g., mice). There are no replication-competent HERVs in the human genome. Because of the potential deleterious effects of ERVs and infection by exogenous retroviruses, many species have evolved restriction systems that limit exogenous retrovirus infection or transposition by retrotransposons. The initial infection step can be blocked by expression of an endogenous envelope protein that binds to the receptor, blocking infection by an incoming retrovirus (viral interference). Certain chicken and mouse strains express such envelope proteins that block incoming retroviral infection. There are also host restriction systems that block viral infection intracellularly. The first intracellular restriction identified was the Fv-1 system for MuLVs in mice. There are two alleles for Fv-1, Fv-1n, and Fv-1b; likewise, MuLVs can be classified as N-tropic or B-tropic. Fv-1n/n mice (or cells derived from them) are permissive for replication by N-tropic but not B-tropic MuLVs, whereas B-tropic but not N-tropic MuLVs can replicate in Fv-1b/b mice. Resistance is dominant, that is, Fv-1n/b mice are resistant to both N- and B-tropic MuLVs. Fv-1 restriction occurs at an early postentry step in viral replication – between reverse transcription and integration of viral DNA. The Fv-1 protein appears to be distantly related to a retroviral gag protein and it may interfere with viral replication by interacting with or mimicking MuLV Gag protein. Another early postentry restriction system is the TRIM proteins – proteins containing a tripartite motif consisting of a RING domain, a B Box-2, and a coiled-coil domain. This restriction was first identified in studies of HIV-1 replication. Cells of Old World primates (e.g., rhesus macaques) are poorly permissive for HIV-1 infection, and it was found that they express a species-specific restriction factor, TRIM5 – a TRIM protein containing a C-terminal extension with a B30.2 domain. The B30.2 domain binds viral CA protein; the species specificity of different TRIM5s is determined by this binding. Rhesus TRIM5 restricts HIV-1 replication at an early step, similar to the stage for FV-1 restriction. Restriction may result from TRIM5mediated degradation of viral cores, through a ubiquitin ligase activity on the protein. Species-specific restriction by TRIM proteins affect more retroviruses than HIV-1.

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For instance, human TRIM5 restricts intracellular replication of N-tropic MuLV, and TRIM5 proteins from African green monkeys and cattle restrict HIV-1, HIV-2, SIVmac, and N-tropic MuLV. Like Fv-1, TRIM5 restriction can be abrogated by high levels of viral Gag protein. Another interesting retroviral restriction system is the APOBEC3 proteins. APOBEC proteins are cytidine deaminases that deaminate cytosine residues to uracils in DNA or RNA. If incorporated into retroviral particles, certain APOBEC proteins can induce cytidine deamination in the first strand of reverse-transcribed DNA, leading to G ! A mutations in the viral genome. This restriction was first discovered by studies of HIV-1 and its vif gene. In vif-negative HIV-1, one APOBEC protein (APOBEC3G) is incorporated into virions as particles are assembled. Upon infection into the next cell, cytidine deamination of viral DNA during reverse transcription leads to extensive G ! A mutations and blockage of viral replication. The Vif protein counteracts this restriction by binding to APOBEC3G protein in the initially infected cell and triggering proteosomal degradation by recruitment of a ubiquitin ligase. Thus virus particles produced from a Vif-expressing infected cell are free of APOBEC3G. The APOBEC restriction also affects other viruses. For instance, human APOBEC3G also restricts MuLV infection in human cells, and murine APOBEC3 restricts replication of HIV-1 in mouse cells. It is interesting that multiple APOBEC genes are present in many species; for instance, mice have one APOBEC3 gene, whereas humans have seven (APOBEC3A – G). One hypothesis is that this resulted from evolutionary selection for resistance to infection by retroviruses and other viruses, as well as retrotransposition of endogenous retroelements. In vitro assays indicate that human APOBEC3A and -B reduce retrotransposition of IAP retroelements in human cells, and murine APOBEC3 reduces retrotransposition of MusD elements in mouse cells. Moreover, all primate APOBEC proteins restrict retrotransposition of ubiquitous non-LTR retroelements (e.g., LINE and ALU elements). ERVs have several biological effects on their host species. In some cases, ERVs can prevent infection by related exogenous retroviruses. A common mechanism is expression by an ERV envelope protein (see above). This has been observed for restriction of alpharetroviruses in chickens. For instance, C/A chickens express an endogenous envelope protein for subgroup A retroviruses; these cells are resistant to infection by exogenous subgroup A viruses that use the same receptor. In mice, strains carrying the Fv4 gene are expressing the Env protein of an endogenous MuLV that renders them resistant to infection. ERVs can also interact genetically with related exogenous retroviruses when they infect the cell. For instance, when exogenous MuLVs infect mice, recombinant MuLVs containing a new env gene appear, known as MCF

recombinants; MCFs result from recombination with an endogenous MuLV. MCF recombinants have an expanded host range in that they can infect cells of both mouse and nonmouse origin (polytropic); this reflects the host range of the Env protein encoded by the endogenous MuLV donors (polytropic or modified polytropic ERVs). MCF recombinants have been implicated in the development of MuLVinduced leukemia. It is noteworthy that the polytropic and modified polytropic ERV proviruses are themselves replication-defective, even though they can donate a functional env gene by recombination. Finally in some cases, ERVs have affected expression of a cellular gene by insertion into it. For instance, the dilute coat color mutation in mice results from insertion of an MuLV-type ERV. The most dramatic biological effects of ERVs is the role of ERV envelope proteins in normal embryonic development. During early human development, extrafetal embryonic cells fuse to make syncitial trophoblasts. The fusion is mediated by the proteins sincytin-1 and -2. Cloning and sequencing of these genes revealed that they are actually endogenous retroviral envelope proteins encoded by HERV-W class proviruses. It is striking that in mice and sheep ERV envelope proteins also have been coopted by the host to mediate syncitial trophoblast fusion. However different ERVs have been used for each of these different species. Thus in at least three independent cases the host has taken advantage of the membrane-fusing capacity of endogenous retroviral envelope proteins and used them to optimize fusion of host cells during embryogenesis. Presumably a less efficient mechanism independent of ERV Env proteins is the default mechanism for syncitial trophoblast fusion in all mammals, but enlisting fusion by ERV Env proteins facilitates this process.

Retroviral Vectors Retroviruses have been developed as gene therapy vectors. Retroviral vectors take advantage of several aspects of the retroviral lifecycle, most notably that integration of proviral DNA into the host genome is a step in the replication process. As a result, these vectors offer the prospects of prolonged high-level expression of the target gene in the vectored cell. Retroviral vectors based on gammaretroviruses (e.g., MuLVs), alpharetroviruses (e.g., spleen necrosis virus), and lentiviruses (e.g., HIV-1) have been developed. A typical retroviral vector DNA plasmid is generated by inserting the target gene into a retroviral provirus by molecular cloning. While some retroviral sequences may be eliminated during the cloning (e.g., those encoding the viral structural proteins), the essential retroviral sequences maintained in the vector plasmid are the LTRs and the RNA-packaging signal ( ). This vector DNA is then cotransfected into cells

Viruses | Retroviruses

along with helper expression plasmids for the viral structural proteins; the mRNAs from the latter plasmids lack the sequences so they themselves cannot be incorporated into retroviral particles. Alternatively, the vector DNA may be transfected into a retroviral vector ‘packaging cell’ that permanently expresses viral structural proteins from -negative mRNAs. In either case, the transfected cells will produce retroviral particles that contain only RNAs encoded by the vector plasmid (‘helper-free’). The resulting vector particles will infect susceptible cells, reverse transcribe the vector DNA, and integrate it into the chromosomal DNA where it can be stably expressed. However, because the retroviral vectors do not express the viral structural proteins, they will not assemble infectious particles that spread the vector infection. Introduction of genes into cells by way of retroviral vector infection is termed transduction. Variations of the basic retroviral vector design have been developed that address practical and theoretical concerns. Some retroviral vectors rely on the viral LTR as the promoter for the target gene, in which case the transcriptional specificity is dictated by that of the viral LTR. To increase expression in other kinds of cells, substitutions by LTRs with different transcriptional specificities have been used. Alternatively, an internal cellular or viral promoter is inserted within the vector to drive target gene expression independent of the LTR. Expression of the target gene protein (or expression of two target genes from the same promoter) can be enhanced by insertion of an internal ribosome entry site (IRES) upstream of the target gene-coding sequences. Another challenge has been that integrated retroviral vector DNAs tend to undergo transcriptional silencing over time (e.g., through DNA methylation). Approaches to this problem have been to include cellular transcriptional insulator elements in the vectors, or to use lentiviral vectors where there may be less silencing. The original retroviral vectors used retroviral envelope proteins on the vector particles for cell entry. Thus the host and tissue specificities of the vectors were determined by the Env proteins; moreover, retroviral envelope proteins are dissociated from viral particles relatively easily during purification or concentration. It is possible to use the heterologous G-protein from vesicular stomatis virus (VSV, a rhabdovirus) to form pseudotypes in which the vector particles contain retroviral Gag and Pol proteins, but with VSV-G protein in the envelope. These pseudotypes have the advantages that VSV-G has a broad host range and it is tightly anchored into the lipid bilayer of the viral envelope. As a result, infectious vector particles can be efficiently concentrated by ultracentrifugation. In contrast, ultracentrifugation strips many retroviral Env proteins from the particles.

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Other aspects of retroviral vector design address safety concerns. One concern is that recombination between retroviral vector and helper plasmid DNAs or RNAs could result in recovery of RCRs. To reduce the likelihood of RCR formation, modern vector systems generally use at least three different plasmids to express the vector genome and viral structural proteins. Thus formation of an RCR would require three or more recombination events. In practice, in clinical experiments all retroviral vector stocks are tested for the absence of RCRs by sensitive biological assays before use. The second safety concern is that introduction of retroviral vectors into patients could lead to induction of cancers by insertional activation of protooncogenes (see above). Recent clinical trials have indicated that this is a real concern in at least one setting (see below). One solution is self-inactivatin (SIN) vectors. In these vectors, the vector plasmid has a complete upstream LTR, but deletion of the promoter and enhancer sequences in the U3 region of downstream LTR; target gene expression is driven by an internal heterologous promoter (Figure 5). In the transfected vector-producing cell, the vector genome transcription is driven by the wildtype upstream LTR. However, in the transduced cell, reverse transcription will result in the integrated vector provirus containing U3-deleted (and inactive) LTRs in both upstream and downstream positions. The transgene will be expressed from the internal promoter, but the likelihood of activation of adjacent proto-oncogenes will be reduced because no active LTRs are present. Retroviral vectors have been tested in human clinical trials for several medical applications. One application is to use retroviral vectors to restore genes to individuals with single-gene defects such as hemophilia, adenosine deaminase deficiency (SCID), and common gamma chain deficiency (X-linked SCID). In some cases, target cells (e.g., hematopoietic progenitors) are isolated from the patient and subjected to vector transduction ex vivo. Alternatively, infectious vectors are infected directly into target tissues. In some cases, positive clinical results have been obtained, most notably correction of the severe combined immunodeficiencies (SCIDs) associated with inherited adenosine deaminase and common gamma chain deficiency. However, in the treated X-linked SCID patients (treated with a first-generation MuLVbased vector), a substantial percentage developed T-cell leukemia resulting from insertional activation of a protooncogene (LMO2). Although this had been a theoretical possibility, the actual occurrence of leukemias in at least one clinical setting demonstrated the importance of this concern. Another application of retroviral vectors has been to use them to express antigens or immunostimulatory molecules in cancer cells with the goal of stimulating a host immune response to the tumors – cancer vaccines.

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Viruses | Retroviruses Retroviral vector

Ψ

LTR

Target gene

LTR Vector plasmid

Ψ

gag

( )

pol

Helper plasmids

Ψ (

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)

(

(

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Self-inactivating (SIN) retroviral vector

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P

Target gene Vector plasmid

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Vector particle Producer cell

Transduced cell Figure 5 Retroviral vectors. Components to generate a typical retroviral vector are shown at the top. They include a plasmid encoding the retroviral vector as well as helper plasmids encoding the viral structural proteins from mRNAs that cannot be packaged since they lack the RNA packaging signal ( ). Vector particles are generated by transient co-transfection of vector and helper plasmids, or by transfection of vector plasmid into cells that stably express the helper plasmids (packaging cells). A self-inactivating (SIN) vector is shown at the bottom, in which the U3 promoter/enhancer sequences are deleted from the downstream LTR of the vector plasmid. Transient transfection into a producer cell yields vector particles with deleted U3 regions in their RNA; after infection into a target cell (transduced cell), the resulting provirus will contain deletions in both LTRs. Transcription of the target gene will be driven by an internal promoter.

See also: Transposable Elements; AIDS, Historical; Oncogenic Viruses; Virus Infection; HIV/AIDS; Evolution, Viral; Recombinant DNA, Basic Procedures

Fields BN, Knipe DM, and Howley PM (2007) Fields Virology, 5th ed., pp.1999–2262. Philadelphia: Lippincott Williams & Watkins.

Relevant Websites Further Reading Coffin JM, Hughes SH, and Varmus HE (1997) Retroviruses. New York: Cold Spring Harbor Laboratory Press.

www.ictvonline.org – International Committee on the Taxonomy of Viruses (ICTV) database http://rtcgd.abcc.ncifcrf.gov – Mouse retrovirus tagged cancer gene database

Viroids/Virusoids B Ding and X Zhong, Ohio State University, Columbus, OH, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Viroid Classification and Structure Host Range of Viroids Viroid Infection

Glossary agroinoculation Use of engineered Agrobacterium tumefaciens that infect certain groups of plants as a vehicle to transfer experimental DNA into plant cell nuclei for transient gene expression. biolistic bombardment A technique in which gold particles coated with experimental DNA or RNA are delivered under certain amount of pressure into cells for expression. BY2 cell A cell line derived from Nicotiana tabacum cv Bright Yellow 2. It can be propagated easily in an appropriate culture medium. The cells are fast-growing and nearly translucent. They have been extremely useful for studying viral and viroid replication and for studying basic plant cell biology and gene expression. phloem A type of vascular tissue consisting of several types of living cells that is responsible for the long-distance transport of nutrients and signaling molecules in plants. It is also used by viroids and many viruses to spread infection within a plant. plasmodesmata Cytoplasmic channels between plant cells that allow cell-to-cell diffusion of small molecules and selective trafficking of RNAs, proteins, viruses, and viroids.

Abbreviations ASBVd CCR CChMVd CEVd CsPP2 HPII

Avocado sunblotch viroid central conserved region Chrysanthemum chlorotic mottle viroid Citrus exocortis viroid PP2 from cucumber hairpin II

Viroid Pathogenecity Virusoids Conclusion Further Reading

protoplast A plant cell with its cell wall removed by treatment with cellulase and pectinase, enzymes that remove the major cell wall components cellulose and pectin. Protoplasts are prepared fresh for each experiment from plant materials or cultured cells and have been extremely useful for studying viral and viroid replication and for studying basic plant cell biology and gene expression. RNA silencing A recently discovered mechanism of gene regulation in many organisms. It is mediated by 20–26 nt small RNAs produced from various RNA and DNA sources and functions in regulating RNA stability and translation as well as chromatin modification underlying numerous developmental processes. It also plays a significant role in microbe–host interactions. viroid A noncoding and nonencapsidated circular RNA that replicates autonomously without helper viruses in a plant. virusoid A specific group of satellite RNAs, associated with sobemoviruses, that are circular and assuming similar secondary structures with viroids. They have no protein-coding capacity. They replicate by utilizing helper viral-encoded factors and are encapsidated by helper viral coat proteins.

HSVd LTSV PLMVd pol II PSTVd RYMV

Hop stunt viroid Lucerne transient streak virus Peach latent mosaic viroid polymerase II Potato spindle tuber viroid Rice yellow mottle virus

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Defining Statement

Viroid Classification and Structure

Viroids and virusoids are the smallest pathogens that infect plants. They are single-stranded, circular RNAs and do not encode any proteins. They present simple models to study how an infectious RNA replicates in a host cell and spreads systemically to cause diseases. They are also excellent models to investigate the basic structure–function relationships of RNAs.

There are over 30 species of viroids in the current database (http://subviral.med.uottawa.ca/cgi-bin/home.cgi). They belong to two families, Pospiviroidae and Avsunviroidae, with their type members being PSTVd and Avocado sunblotch viroid (ASBVd), respectively. Many species have sequence variants. All viroids are listed in Table 1, under their respective families and genera. Their sizes and number of sequence variants are also listed. The distinguishing features of the two families of viroids are summarized in Table 2. The members of Avsunviroidae have a highly branched secondary structure (Figure 1(a)). There is limited sequence or secondary structural conservation among the different species. They all replicate in the chloroplast and have ribozyme activities. The members of Pospiviroidae generally have a rod-shaped secondary structure (Figure 1(b)) and conserved sequences among some species, replicate in the nucleus, and are generally considered to lack ribozyme activities. Five broad structural domains are defined in the secondary structures of some viroids in Pospiviroidae. These include the left-terminal domain, pathogenicity domain, central domain that contains a central conserved region (CCR), variable domain, and right-terminal domain (Figure 1(b)).

Introduction Theodor O. Diener was credited with the discovery of the first viroid, Potato spindle tuber viroid (PSTVd), in 1971. This viroid is the causal agent of potato spindle tuber disease first described in the 1920s. Extensive research over the past three decades has established viroids as the simplest form of RNA-based infectious agents. All viroids are single-stranded, circular RNAs with sizes ranging from 250 to 400 nucleotides (nt). Differing from viruses, these RNAs do not have protein-coding capacity and are not encapsidated in a protein or membrane shell. They do not require the presence of a helper virus to establish infection. Thus, the viroid genomes and/or their derivatives contain all of the genetic information for direct replication in single cells and systemic trafficking throughout a plant to establish infection. Depending on viroid–host combinations, an infected plant may or may not develop disease symptoms. Common viroid disease symptoms include growth stunting, leaf epinasty and deformation, fruit distortion, stem and leaf necrosis, and plant death. Since viroids do not encode proteins, viroid diseases must result from direct interactions between viroid genomic RNAs or their derivatives and specific cellular components. Virusoids are also circular RNAs that are similar to viroids in size and secondary structure. They do not encode any proteins. However, they rely on protein factors encoded by their helper viruses for replication and encapsidation. Virusoids are a special group of satellite RNAs associated with plant viruses. Recent research has contributed significant insights into the sequence/structural elements in viroids that are critical for various aspects of replication, systemic spread, and disease formation in an infected plant. Knowledge of potential host proteins that assist various stages of viroid infection is also emerging. Much less is known about the biological functions of virusoids. It is proposed that continuing studies on these subviral pathogens should yield valuable insights into the simplest mechanisms of infection in eukaryotic cells and help uncover the basic principles of RNA structure–function relationships.

Host Range of Viroids Unlike many viruses, viroids have relatively narrow host ranges, each infecting one or a few plant species in the field. Avsunviroidae mostly infect woody species whereas Pospiviroidae mostly infect herbaceous species. There is evidence for the recent expansion of host ranges for many viroids. For instance, PSTVd was long known to infect potato only in the field. It has recently been reported to infect avocado and tomato in the field. Under experimental conditions, some viroids can infect more species. For example, PSTVd can infect Nicotiana benthamiana and some of its variants also infect N. tabacum. The infected plants usually do not exhibit noticeable symptoms. Recent studies tested whether weedy plant species characteristic for potato and hop fields, which are not natural hosts for PSTVd and Hop stunt viroid (HSVd), can be potential hosts for transmitting and spreading infection of these viroids, respectively. Indeed, when the leaves of 12 weedy species from the potato field and 14 from the hop field were inoculated with viroid RNAs or cDNAs through biolistic bombardment, many species supported replication of these two viroids. Sequencing revealed the presence of many variants of these viroids in different infected species. Therefore, there is always the potential for viroids to invade new species if conditions permit.

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Table 1 Current viroid species Family Genus Pospiviroidae Apscaviroid

Pospiviroid

Cocadviroid

Hostuviroid Coleviroid

Avsunviroidae Pelamoviroid Avsunviroid Elaviroid

Species name

Size (nt)

Number of variants

Pear blister canker viroid (PBCVd) Grapevine yellow speckle viroid-2 (GYSVd-2) Grapevine yellow speckle viroid-1 (GYSVd-1) Citrus viroid-III (CVd-III) Citrus bent leaf viroid (CBLVd) Australian grapevine viroid (AGVd) Apple dimple fruit viroid (ADFVd) Apple scar skin viroid (ASSVd) Apple fruit crinkle viroid (AFCVd) Citrus viroid-I-LSS (CVd-LSS) Citrus viroid-OS (CVd-OS) Japanese citrus viroid 1 (JCVd) Tomato planta macho viroid (TPMVd) Tomato apical stunt viroid (TASVd) Potato spindle tuber viroid (PSTVd) Mexican papita viroid (MPVd) Iresine viroid 1 (IrVd) Columnea latent viroid (CLVd) Citrus exocortis viroid (CEVd) Chrysanthemum stunt viroid (CSVd) Tomato chlorotic dwarf viroid (TCDVd) Hop latent viroid (HLVd) Coconut tinangaja viroid (CtiVd) Coconut cadang-cadang viroid (CCCVd) Citrus viroid IV (CVd-IV) Hop stunt viroid (HSVd) Coleus blumei viroid 3 (CbVd-3) Coleus blumei viroid 2 (CbVd-2) Coleus blumei viroid 1 (CbVd-1) Coleus blumei viroid (CbVd)

314–316 361–363 187–368 291–297 315–329 369 306–307 329–333 368–372 325–330 329–331 331 360 360–363 341–364 359–360 370 359–456 197–475 348–356 359–360 255–256 254 246–301 284–286 267–368 361–364 295–301 248–251 295

22 6 65 53 21 9 10 8 29 5 4 1 1 8 133 7 3 25 151 26 3 10 2 11 6 206 3 2 9 1

Chrysanthemum chlorotic mottle viroid (CChMVd) Peach latent mosaic viroid (PLMVd) Avocado sunblotch viroid (ASBVd) Eggplant latent viroid (ELVd)

397–401 335–351 120–251 332–335

23 297 83 9

The number of sequence variants are obtained by removing duplicate sequences in the database. Data are obtained from Subviral Database (http://subviral.med.uottawa.ca/cgi-bin/home.cgi).

Table 2 Distinct features of Pospiviroidae and Avsunviroidae Family Features

Pospiviroidae

Avsunviroidae

Secondary structure Replicate site Rolling circle Ribozyme activity Hosts

Rod-shaped Nucleus Asymmetric Uncertain Mostly herbaceous species

Branched for most members Chloroplast Symmetric Yes for all current members Mostly woody species

Reproduced from Ding B and Itaya A (2007) Viroid: A useful model for studying the basic principles of infection and RNA biology. Molecular Plant-Microbe Interactions 20: 7–20.

The reasons for the narrow host range of a viroid are not clear. Recent studies suggest a possibility that low level of replication and/or inability to traffic between cells contributes to the limited host range for some, if not all, viroids. For instance, none of the known viroids

infect Arabidopsis thaliana when inoculated onto this plant. In transgenic A. thaliana plants expressing the dimeric (þ)-RNAs of Citrus exocortis viroid (CEVd) and HSVd, species of Pospiviroidae, replication took place. Agroinoculation of A. thaliana with CEVd, HSVd, and

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(a)

Transcription initiation (U121)

Cleavage site (C55-U56)

(+) ASBVd

(–) ASBVd

Cleavage site (C90-G91)

Transcription initiation (U119)

(b)

(+) PSTVd HPI

GC box

HPII

HPI

Loop E

GC box HPII

Transcription initiation (C1 or U359)

TL

Pathogenicity

C

Variable

328

(c) 87 G – C102

80

G–C A–U C–G U–G U–A C–G G–C

HPI

109

TR

227

G–C G–C G–C A–U G – C HPII C–G G–C G–C G–C 319 G – C 236

Figure 1 Secondary structures of ASBVd (a) and PSTVd (b), type members of avsunviroidae and pospiviroidae, respectively. Reproduced, with modifications, from Ding B and Itaya A (2007) Viroid: A useful model for studying the basic principles of infection and RNA biology. Molecular Plant-Microbe Interactions 20: 7–20. For PSTVd, the five structural domains are indicated. TL, left-terminal domain; c, central domain; TR, right-terminal domain. Arrows indicate the transcription initiation sites and cleavage sites on the viroid genomic RNAs. HPI and HPII in (b) indicate the positions of nucleotide sequences in PSTVd for the formation of metastable structures HPI and HPII, which are shown in (c). Reproduced, with modifications, from Zhong X, Archual AJ, Amin AA, and Ding B (2008) A genomic map of viroid RNA motifs critical for replication and systemic trafficking. Plant Cell 20: 35–47; www.plantcell.org. Copyright American Society of Plant Biologists.

Coleus blumei viroid 1 dimeric cDNAs showed that these viroids did not traffic from the inoculated leaves to distal parts of a plant. For some viroids, their host range may be underestimated if disease symptom is used as the main detection method, because their infection of certain plants will not necessarily produce visible symptoms.

Viroid Infection Viroid Transmission Like viral infection in many cases, viroid infection easily spreads between individual plants through wounding of

plants caused by farming tools, human contact, or plant– plant contact. Some viroids can also be transmitted via infected seeds or pollens. Vegetative propagation by grafts or tubers also readily transmits viroids. For instance, PSTVd can be transmitted by infected potato tubers and infected tomato seeds. ASBVd can be seedtransmitted in avocado. In contrast to the common transmission of viruses, insect transmission of viroids is not common in the field. There is a report of infrequent transmission of PSTVd by the potato aphid Macrosiphum euphorbiae. Quarantine and elimination of infected plant/ seed stocks are the most effective means currently available to control the spread of viroid infection.

Viruses | Viroids/Virusoids

General Scheme of Systemic Infection in a Plant The establishment of systemic infection by both families of viroids involves the following mechanistic steps (Figure 2): (1) import into specific subcellular organelles (the nucleus for Pospiviroidae and the chloroplast for Avsunviroidae), (2) replication, (3) export out of the organelles, (4) cell-to-cell trafficking, (5) entry into the vascular tissue, (6) long-distance trafficking within the vascular tissue, (7) exit from the vascular tissue and subsequent invasion of nonvascular cells to repeat the cycle. As discussed further below, some steps have been well studied whereas others remain completely unknown.

Intracellular Localization and Replication Pospiviroidae

In order for replication to take place, members of the family Pospiviroidae must first enter the nucleus. How this is achieved is still poorly understood. The first study to address this question examined nuclear import of fluorescent-labeled in vitro transcripts of PSTVd in protoplasts of tobacco BY2 cells. The protoplasts were prepared by the removal of cell walls via digestion with enzymes such as cellulase and pectinase. The protoplasts were further treated with a detergent such as Triton-X 100 to permeabilize the plasma membrane. When the fluorescentlabeled transcripts were incubated with such protoplasts, they entered the cells through the permeabilized plasma membrane and then accumulated in the nucleus within 15–20 min, which was visualized under a fluorescence microscope. When fluorescent transcripts were mixed with a 10 molar excess of nonlabeled transcripts, nuclear import of the former was inhibited. This suggests that PSTVd import is a specific and regulated process, presumably mediated by a protein carrier that remains to be

539

identified. These findings were confirmed with an independent approach, in which PSTVd could function in cis to mediate nuclear import of a large fusion RNA in N. benthamiana leaves. Using the latter approach, a recent study showed that the conserved sequence in the upper strand of the PSTVd secondary structure was able to mediate nuclear import of a fusion RNA. The biological significance of this for viroid infection can now be tested. The cellular factor(s) that recognizes and imports the viroid RNA is not known. How the viroid RNAs exit the nucleus remains to be investigated. Within the nucleus, the viroids replicate via an asymmetric rolling circle mechanism (Figure 3(a)). Briefly, the circular (þ)-RNA is first transcribed into concatemeric linear (–)-strand RNA in the nucleoplasm. This long RNA then acts as the replication intermediate for the synthesis of concatemeric, linear (þ)-strand RNA. In one possible mechanism, the latter is transported into the nucleolus, where it is cleaved into unit-length monomers. Subsequent intramolecular end-to-end ligation of each monomer yields the mature, circular progeny viroid RNA. Alternatively, the cleavage and ligation occur in the nucleoplasm, and the mature viroid RNA is transported into the nucleolus for storage. Several lines of data suggest that the DNA-dependent RNA polymerase II (pol II) is involved in transcription. The purified tomato pol II can transcribe a PSTVd template in vitro. The CEVd RNA is associated with the largest subunit of pol II in vivo. Treatment of cells or nuclear extracts with pol II inhibitor -amanitin inhibits replication of CEVd and PSTVd in vivo or transcription in vitro. In the past few years, a new DNA-dependent RNA polymerase IV and several RNA-dependent RNA polymerases have been discovered in plants. It remains to be tested whether any of these enzymes are involved in viroid transcription.

ASBVd (7) Vascular exit and invasion of new cells

(2) Replication

Chloroplast (1) Organellar import

(3) Organellar export

(4)Cell-to-cell trafficking

(5) Vascular entry

Nucleus (6) Long-distance trafficking PSTVd Figure 2 Distinct steps of systemic infection of ASBVd and PSTVd. Reproduced from Ding B and Itaya A (2007) Viroid: A useful model for studying the basic principles of infection and RNA biology. Molecular Plant–Microbe Interactions 20: 7–20.

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(a)

PSTVd

(b)

ASBVd Chloroplast

Nucleoplasm

– +

+ –

Transcription Transcription Self-cleavage

Transcription

Ligation Nucleolus Cleavage Ligation Transcription

Self-cleavage Ligation

Figure 3 Rolling circle replication mechanisms of PSTVd (a) and ASBVd (b). The secondary structure sketches of the genomic RNAs illustrate the approximate transcription initiation sites. Reproduced, with modifications, from Ding B and Itaya A (2007) Viroid: A useful model for studying the basic principles of infection and RNA biology. Molecular Plant–Microbe Interactions 20: 7–20.

by the observation that enlargement of this loop by mutagenesis inhibited replication in protoplasts or infection in a plant in N. benthamiana. Furthermore, disruption of each of three consecutive loops from the left-terminal end (loops 2, 3, and 4 in Figure 4), respectively, also inhibited replication in protoplasts. The transcription initiation site on the (–)-strand PSTVd template is yet to be identified. There are two GC boxes in the PSTVd secondary structure (Figure 1(b)). Mutational studies suggest that they may play a role in transcription. Further studies are

Where transcription initiates in a viroid RNA of the family Pospiviroidae remains to be understood. Recent studies examining the de novo synthesis of the (–)-strand PSTVd RNAs in potato nuclear extracts mapped the transcription initiation site on the circular (þ)-RNA to U359/ C1 of the left-terminal loop (Figure 1(b)). This can be further tested by loss-of-function genetic experiments in combination with biochemical analysis of where on the viroid RNA the transcription complex binds. The importance of the left-terminal loop for replication was supported

T

T

1

1

2

3

4

359

R

T

5

6

7

8

9

10

314

R R

11 12

13

T T

14

15

16

17

R

T

T

T

148

18

19 20 21 22 23

240

R

Pathogenicity

T 122

286

R

TL

T

T 73

46

179

24

25

26 27 180

212

R

Central

Variable

TR

Figure 4 A genomic map of PSTVd loop motifs critical for replication (R) in single cells or for systemic trafficking (T) in a whole plant. Reproduced, with modifications, from Zhong X, Archual AJ, Amin AA, and Ding B (2008) A genomic map of viroid RNA motifs critical for replication and systemic trafficking. Plant Cell 20: 35–47; www.plantcell.org. Copyright American Society of Plant Biologists.

Viruses | Viroids/Virusoids

needed to determine their functions. There is evidence that loop E located in the CCR of PSTVd (see Figure 1(b)) is critical for replication. Recent studies provided evidence that the PSTVd loop E motif exists in vivo, has a defined tertiary structure, and disruption of this structure leads to loss of function in replication. A more recent study using whole genome mutational analysis has identified several additional PSTVd loops as motifs critical for replication in single cells. Some loops are located within the central region (loops 13 and 14 in Figure 4) and others are located in the left-terminal domain (loops 2, 3, and 4 in Figure 4). The specific roles of these loops in RNA stability, nuclear transport, transcription, cleavage, and ligation need to be determined. A thermodynamically metastable hairpin II (HPII) structure is predicted to form through base interactions involving nucleotide sequences 227–236 and 319–328 (Figures 1(b) and 1(c)) during thermal denaturation of the PSTVd secondary structure. The HPII structure has been detected in vitro and in vivo, suggesting its importance in viroid infection. It is also suggested that another metastable structure, HPI (Figures 1(b) and 1(c)), is important for infection of tomato. However, recent mutational analyses showed that neither HPII nor HPI is critical for PSTVd replication in N. benthamiana. Whether these metastable structures function in PSTVd infection in some plant species but not in others is an important question in further studies. The sequence and structural conservation of the CCR of several members of Pospiviroidae suggests its potential importance in viroid processing. In vitro studies mapped the cleavage and ligation site to between G95 and G96 of CCR. In recent work with transgenic A. thaliana plants that express dimeric (þ)-RNAs of CEVd, HSVd, and Apple scar skin viroid, the in vivo processing site for these viroids was mapped at equivalent positions of a putative HPI/double-stranded structure formed by the upper strand and flanking nucleotides of the CCR. More specifically, the substrate for in vivo cleavage is the proposed conserved double-stranded structure, with HPI potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. It is generally thought that a cellular RNase catalyzes the cleavage of concatemeric RNAs. It has long been known that wheat germ extract and Chlamydomonas reinhardtii contain ligase activities that circularize PSTVd linear RNAs. The biochemical identities of any enzymes and associated factors that are responsible for cleavage and ligation of viroids in the family Pospiviroidae remain unknown. Avsunviroidae

Viroids of the family Avsunviroidae replicate in the chloroplast. How they enter and exit the chloroplast is

541

not known. Within the chloroplast, these viroids replicate via a symmetric rolling circle mechanism (Figure 3(b)). The circular genomic (þ)-RNA is first transcribed into a linear, concatemeric (–)-strand RNA. This RNA is cleaved into unit-length molecules and circularized to serve as the template to generate linear, concatemeric (þ)-strand RNA. This RNA is subsequently cleaved into unit-length monomers and circularized. In vitro studies showed that the Escherichia coli RNA polymerase can transcribe Peach latent mosaic viroid (PLMVd) RNA templates in vitro, suggesting that the plastid-encoded bacterial-like multiunit RNA polymerase may be involved in transcription in vivo. However, sensitivity of ASBVd replication to treatment with targetoxin suggests that the nuclear-encoded and phage-like single-unit polymerase is involved in replication in vivo. Further studies are necessary to determine which polymerase is responsible for replication during infection. The transcription initiation sites have been determined for ASBVd and PLMVd. For ASBVd, in vitro capping and RNase protection assays mapped U121 as the initiation site on the (þ)-RNA and U119 as the site on the (–)-RNA. Both sites are located in the AU-rich terminal loops of the RNA secondary structures (Figure 1). For PLMVd, studies on a wide repertoire of PLMVd variants revealed A50/C51 and A284/A286 as the universal transcription initiation sites for the (þ)- and (–)-strand RNAs, respectively. Furthermore, a highly conserved CAGACG sequence appears to be important for defining these sites. The possibility that some variants can start transcription in other sites cannot be formally ruled out. The viroids in Avsunviroidae form hammerhead ribozymes in both the (þ)- and (–)-strands of RNAs to catalyze self-cleavage in vitro. The general thought is that these viroids self-cleave during infection in vivo. However, there is evidence that cellular factors may enhance this cleavage. UV-crosslinking of viroid RNA–protein complex in infected tissues in conjunction with biochemical analyses identified a chloroplast protein, PARBP33, that interacts with ASBVd in vivo. This protein has an RNA-binding motif and accelerates selfcleavage of concatemeric ASBVd RNAs in vitro. The in vivo role of this factor for viroid replication and general RNA processing remains to be further studied. Although self-cleavage of both the linear concatemeric (þ)- and (–)-RNAs is well demonstrated for members of the family Avsunviroidae, little is known about how monomeric molecules are ligated into circles. Nonenzymatic intra- and intermolecular ligation has been demonstrated for PLMVd in vitro. The self-ligation produces a 29,59phosphodiester bond in vitro. Recent studies demonstrated the presence of the 29,59-phosphodiester bond at the ligation site of the circular PLMVd RNAs isolated from infected peach plants. It will be important to determine

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whether this mode of ligation functions during PLMVd replication. A recent technical advance may enable genetic investigation of the cellular factors involved in cleavage and ligation. When the dimeric cDNAs of three species of Avsunviroidae, including ASBVd, Chrysanthemum chlorotic mottle viroid (CChMVd), and Eggplant latent viroid, are expressed in the transformed chloroplasts of C. reinhardtii, the dimeric (þ)- or (–)-RNA transcripts are correctly cleaved into unit length molecules and circularized. There is no evidence for replication to have taken place. Given the complete genome sequence information and well-established genetic and molecular approaches in C. reinhardtii, this model system may prove to be of great utility to identify the protein factors important for viroid RNA processing. Viroid Cell-to-Cell and Long-Distance Trafficking To establish a systemic infection, viroid RNAs must traffic from initially infected cells into neighboring cells and distant organs. Studies on PSTVd indicate that cellto-cell trafficking occurs through cytoplasmic channels plasmodesmata and long-distance trafficking occurs through the vascular tissue phloem. Without encoding proteins, one can assume that viroids either diffuse between cells and through the phloem or they have sequence/structural motifs to mediate the trafficking process. The first clue for motif-mediated trafficking came from studies showing that two mutations in the rightterminal domain of PSTVd do not appear to affect replication in tomato roots, but affect systemic infection. Further studies with microinjection showed that PSTVd could function in cis to potentiate cell-to-cell trafficking of a heterologous RNA, suggesting that the viroid RNA has a motif that mediates trafficking. Furthermore, in an infected flower PSTVd traffics into sepals but not the other floral organs, suggesting that the phloem has a mechanism to recognize and traffic PSTVd RNAs into selective sink organs. Mutational studies on two PSTVd strains, PSTVdNT and PSTVdNB, which differ by 5 nt, identified a bipartite motif that is required for trafficking from bundle sheath to mesophyll, but not required for trafficking in the reverse direction. Importantly, this motif is required for trafficking in young leaves, but not in mature leaves. Thus, plant development is also a major factor for the fine-tuning of trafficking controls. Whether the bipartite motif interacts with separate cellular factors or they form a particular tertiary structural motif via conformational changes to interact with a cellular factor for trafficking remains an outstanding question. A tertiary structural motif, which consists of at least U43/C318 (loop 7 in Figure 4) that interacts with

cis-Watson–Crick base pairing with water insertion, has been shown to be required for PSTVd to traffic from the bundle sheath into the phloem to initiate long-distance transport in N. benthamiana. Mechanistically, water insertion distorts the structure of the local helix. This distortion is necessary for trafficking. This motif recurs in many other RNAs. In rRNAs, this motif is a binding site for a ribosomal protein. These studies imply the existence of multiple PSTVd structural motifs to mediate trafficking across various cellular boundaries in an infected plant. Indeed, a recent study has identified many additional loops in the PSTVd secondary structure that are critical for systemic trafficking in N. benthamiana (Figure 4). Closing of these loops by nucleotide substitutions/deletions to create Watson– Crick base pairing abolishes systemic infection while allowing replication. The tertiary structure of each loop, whether each of these loops function individually or in some combinations to mediate trafficking across specific cellular boundaries, and what host proteins interact with each of these loops for function are outstanding issues for future investigations. A current hypothesis is that certain cellular proteins recognize specific viroid RNA motifs to potentiate trafficking between cells and among organs. These proteins have not yet been identified. Several promising candidates have been reported but their functions have to be conclusively tested. These include the mobile phloem lectin PP2 from cucumber (CsPP2) that binds HSVd in vitro and in vivo as well as two phloem proteins that bind ASBVd. A tomato protein, VIRP1, interacts in vitro with the right-terminal region of PSTVd and HSVd. Recent work showed that VIRP1 appears to be important for infection. When its expression is repressed by antisense method in transgenic N. benthamiana, the protoplasts prepared from this transgenic plant fail to support PSTVd replication.

Viroid Pathogenecity Without encoding proteins, viroid diseases must result from interactions between the viroid genome, or genomederived RNAs, and cellular factors. Such interactions disturb the normal course of plant development leading to disease formation. Viroid diseases show great variations, depending on viroid–host combinations. They range from nearly symptomless to host lethality. One of the most devastating diseases is the cadang-cadang disease that killed over 30 million coconut palms, caused by infection of Coconut cadang-cadang viroid. Environmental conditions affect symptom expression. In particular, high temperatures enhance disease symptoms. No natural resistance to viroid infection has been reported.

Viruses | Viroids/Virusoids

Mild

KF440-2

543

RG1

MildU257A

RG1U257A

KFU257A

2 cm

1 cm

1 cm

Figure 5 Mild to lethal disease symptoms caused by infection of several PSTVd variants in Rutgers tomato plants. Reproduced from Qi Y and Ding B (2003) Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA. Plant Cell 15: 1360–1374. www.plantcell.org. Copyright American Society of Plant Biologists.

In many cases, small sequence or structural variations in a viroid genome can cause symptoms of different degrees of severity (Figure 5). The viroid RNA structure and disease relationships have been studied most extensively for members of the family Pospiviroidae. Early studies with PSTVd and CEVd showed that many nucleotide changes in association with different degrees of symptom severity occur in the so-called pathogenicity domain (Figure 1(b) for PSTVd). More recent studies indicate that all five structural domains play a role in pathogenicity. Sequence comparisons among ASBVd clones isolated from diseased and healthy tissues of infected avocado suggest that a ‘U’ insertion between nt 115 and 118 in different variants is responsible for the symptoms. Studies on symptomatic and nonsymptomatic variants of CChMVd identified tetraloop UUUC (nt 82–85) as a major pathogenicity determinant. Conversion of this tetraloop to GAAA in natural variants or by mutagenesis renders the viroid nonsymptomatic. Other than correlations between viroid sequences and symptom severity, little is known about the mechanisms of pathogenicity. In general, viroid replication levels and tissue localizations are not major factors for the varying degrees of symptoms. This suggests that specific molecular interactions between viroid sequences/structures with host factors are prevailing disease mechanisms. The cellular factors that interact with specific viroid sequences/structures for disease development are not known. Infection of tomato by mild and severe PSTVd strains induced or suppressed expression of common and unique sets of host genes. These include genes involved in general defense/ stress responses, cell wall structure and metabolism, chloroplast functions, and so on. Similar alteration of host gene expression has also been reported in Etrog citron leaves infected by Citrus viroid III. How the altered expression of any host genes contributes to disease formation is not known. PSTVd infection also causes phosphorylation of a protein kinase that is immunologically related to the mammalian interferon-induced, double-stranded RNAactivated protein kinase. Further studies showed differential in vitro activation of the mammalian protein kinase P68 by PSTVd strains of different pathogenicity. The

biological significance of this activation for viroid symptom expression remains to be understood. Recent studies on PSTVd and PLMVd have started to shed light on the molecular mechanisms underlying pathogenicity. A U257A change in the CCR converted several strains of PSTVd into lethal strains that caused severe growth stunting and premature death of infected plants (see Figure 5). The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism of PSTVd. The stunted growth of infected tomato plants resulted from restricted cell growth, but not cell division or differentiation. This is correlated positively with downregulated expression of an expansin gene, LeExp2, that is known to play an important role in cell expansion in young growing organs. The peach calico symptom, characterized by extreme chlorosis of infected tissues, is associated with the insertion of an extra 12–13 nt sequence that folds into a hairpin in the left-terminal loop of PLMVd. Intriguingly, the insertion occurs sporadically de novo and can be acquired or lost during infection. Recent work shows that presence of this hairpin impairs processing and accumulation of chloroplast rRNAs. This eventually affects the structure and function of the chloroplast translation machinery. Chloroplast development is severely disturbed. Still, the underdeveloped chloroplasts retain the capacity to import proteins encoded by nuclear genes, which includes a chloroplast RNA polymerase, and support PLMVd replication. Altogether, these findings support the view that specific viroid sequence/structural elements can interact with yet to be identified host factors in a highly specific manner to alter host gene expression and developmental processes. An emerging model for viroid pathogenicity is that small RNAs of 20–24 nt derived from viroid RNA sequences during infection can guide RNA silencing of host genes, thereby leading to development of disease symptoms. Consistent with this hypothesis, there is a positive correlation between the levels of small RNAs and symptom severity for PSTVd and ASBVd. Moreover, symptom development is correlated with production of small RNAs in some transgenic tomato lines expressing nonreplicating, hairpin PSTVd RNAs.

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Viruses | Viroids/Virusoids

However, the correlation between viroid small RNA accumulation and symptom expression is not universal. It also remains to be tested whether viroid small RNAs can indeed target host genes for silencing and whether such silencing is crucial for disease development.

Virusoids Virusoid, a term used less frequently today, refers to a group of circular satellite RNAs associated with viruses in the genus Sobemovirus, the members of which have nonenveloped icosahedral virions containing one molecule of linear, single-stranded and positive-sense RNA. They bear structural similarity with viroids but share biological properties with satellites. Satellite RNAs are found to be mostly associated with plant viruses. All satellite RNAs must coinfect with a helper virus in order to replicate. The satellite RNAs have little sequence similarity with their helper viruses. They replicate on their own templates by utilizing the replicating enzymes encoded by the helper viruses. The virusoid RNA genomes are 220–388 nt long. They have a single-stranded, circular genome assuming rod-shaped secondary structure due to intramolecular base pairing. A virusoid genome does not code for any proteins. In these aspects, virusoids are similar to viroids. However, their thermostability is distinct from that of viroids, showing no cooperativity but rather random base sequences. Furthermore, like satellite RNAs, virusoids are replicated by the helper virus RNA-dependent RNA polymerases in the cytoplasm and are encapsidated by the coat proteins encoded by the helper viruses. They are encapsidated separately from the helper viral RNAs. Five virusoids are currently known (Table 3). All helper viruses are members of the Sobemovirus family. These include Rice yellow mottle virus (RYMV), Lucerne transient streak virus (LTSV), Subterranean clover mottle virus, Velvet tobacco mottle virus, and Solanum nodiflorum

mottle virus. By convention, the encapsidated and infectious form of the RNA is designated as the (þ)-strand. It accumulates to higher levels than the (–)-strand that is produced during RNA–RNA transcription. Virusoids replicate via rolling circle mechanisms similar to viroids. Both the (þ)- and (–)-strands of virusoid vLTSV contain hammerhead ribozyme activity in vitro, which catalyzes self-cleavage during replication. There is little understanding of the biology of virusoids, in terms of their interactions with helper viruses, how they initiate replication, how they move between cells and through a plant, and how they influence viral disease symptoms. A recent study revealed no involvement of virusoid vRYMV in symptom modulation or ability to break host–plant resistance to the viral disease.

Conclusion Viroids and virusoids are RNAs that are small in size, simple in structure, and yet complicated in biological functions. Their replication and systemic trafficking raise the fascinating questions of what RNA structural motifs within the RNA direct all of the biological functions and what host factors are employed by these motifs to accomplish each function necessary to establish a systemic infection. The viroid disease can be considered as an example of RNA-regulated expression of host genes. Further studies on viroid–host interactions and virusoid–host–helper virus interactions are expected to contribute new and exciting knowledge about the evolution of RNA-based pathogens and about the basic mechanisms of noncoding RNA functions. As compared to viroids, the biology of virusoids has been greatly understudied. It can be anticipated that with appropriate experimental tools developed, virusoids can serve as another powerful set of simple RNA models, like viroids, for fundamental discoveries in biology.

Table 3 Virusoids

Virusoid

Helper virus

Genome size (nt)

Accession #

vRYMV vLSTV vSCMoV

RYMV LSTV SCMoV

vVTMoV vSNMV

VTMoV SNMV

220 324 332 388 366 377

AF039909 X01984 M33000 M33001 J02439 J02388

(þ)-strand ribozyme

()-strand ribozyme

HH HH HH

HH

HH HH

RYMV, Rice yellow mottle virus; LSTV, Lucerne transient streak virus; SCMoV, subterranean clover mottle virus; VTMoV, Velvet tobacco mottle virus, SNMV, Solanum nodiflorum mottle virus; HH, Hammerhead.

Viruses | Viroids/Virusoids See also: Plant Disease Resistance: Natural, Non-Host Innate or Inducible; Plant Pathogens and Disease: Newly Emerging Diseases; Prions; Plant Pathogens: RNA viruses; RNAs, Small etc.; Virus Infection

Further Reading Diener TO (2003) Discovering viroids – a personal perspective. Nature Reviews in Microbiology 1: 75–80. Ding B and Itaya A (2007) Viroid: A useful model for studying the basic principles of infection and RNA biology. Molecular Plant–Microbe Interactions 20: 7–20. Flores R, Hernandez C, Martı´nez de Alba AE, Daro`s JA, and Di Serio F (2005) Viroids and viroid–host interactions. Annual Reviews of Phytopathology 43: 117–139. Gas ME, Herna´ndez C, Flores R, and Daro`s JA (2007) Processing of nuclear viroids in vivo: An interplay between RNA conformations. PLoS Pathogens 3: e182. Go´ra-Sochacka A (2004) Viroids: Unusual small pathogenic RNAs. Acta Biochimica Polonica 51: 587–607. Hadidi A, Flores R, Randles JW, and Semancik JS (eds.) (2003) Viroids. Australia: CSIRO: Collingwood. Hammond RW and Owens R Viroids: New and Continuing Risks for Horticultural and Agricultural Crops. http://www.apsnet.org/online/ feature/viroids. Matousek J, Orctova´ L, Pta´cek J, et al. (2007) Experimental transmission of pospiviroid populations to weed species

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characteristic of potato and hop fields. Journal of Virology 81: 11891–11899. Owens R (2007) Potato spindle tuber viroid: The simplicity paradox resolved? Molecular Plant Pathology 8: 549–560. Qi Y and Ding B (2003) Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA. Plant Cell 15: 1360–1374. Qi Y, Pe´lissier T, Itaya A, Hunt E, Wassenegger M, and Ding B (2004) Direct role of a viroid RNA motif in mediating directional RNA trafficking across a specific cellular boundary. Plant Cell 16: 1741–1752. Rodio ME, Delgado S, De Stradis A, Go´mez MD, and Floresand Di Serio RF (2007) A viroid RNA with a specific structural motif inhibits chloroplast development. Plant Cell 19: 3610–3626. Tabler M and Tsagris M (2004) Viroids: Petite RNA pathogens with distinguished talents. Trends in Plant Science 9: 339–348. Zhong X, Archual AJ, Amin AA, and Ding B (2008) A genomic map of viroid RNA motifs critical for replication and systemic trafficking. Plant Cell 20: 35–47. Zhong X, Tao X, Stombaugh J, Leontis N, and Ding B (2007) Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafficking. The EMBO Journal 26: 3836–3846.

Relevant Website http://subviral.med.uottawa.ca/cgi-bin/home.cgi RNA Database

– Subviral

Virus Infection W C Summers, Yale University School of Medicine, New Haven, CT, USA ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Virus Life Cycles Modes of Entry and Transmission of Viruses Responses to Virus Infections

Glossary attenuation The reduction in virulence or ability to cause disease or other consequences of infection by a microbe while it still remains viable. burst The abrupt release of many progeny virus upon the disintegration of the infected cell in which the virus has been growing. eclipse The period from the entry of a virus into a cell when it loses it infectivity as an independent particle to the appearance inside of the cell of fully infectious progeny virus particles. envelope The biological lipid–protein membrane that surrounds the protein and nucleic acid core of some viruses. interferon Specific cellular proteins that are synthesized in response to virus infection and are secreted outside the infected cell, rendering the neighboring uninfected cells resistant to virus infection. latent A virus that has entered into a nonreplicative mode of existence in a cell but is propagated along with the cell by certain mechanisms that allow limited viral genome replication without full expression of viral

Abbreviations AZT ddC

azidothymidine dideoxycytidine

Defining Statement Viruses are obligate intracellular parasites that can exist as potentially active but inert entities outside of cells. While there are viruses that infect many animal, plant, and protist cells and result in effects on the host that range from inapparent infection to lethality, all virus infections have some features in common. These include an entry phase, an intracellular phase consisting of multiplication, integration, or latency formation, a virus release phase, and usually some sort of host responses to the presence of

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Outcomes of Virus Infections Viral Virulence Antiviral Therapy and Prophylaxis Further Reading

functions and production of mature infectious virus particles. lysogenic A mode of virus interaction with cells in which the virus enters the latent state and can be induced to enter the full replicative cycle with subsequent disruption (lysis) of the cell and production of a full burst of progeny virus. lytic A mode of virus interaction with cells in which the virus undergoes a complete replicative cycle with the production of many progeny virus particles and the subsequent release of these virus upon disruption (lysis) of the cell. persistence A mode of virus interaction with a population of cells in which a few cells are always in a lytic mode of infection but the majority of cells are uninfected but potentially susceptible to lytic virus infection. tropism The specificity of a virus for infection of a specific cell type or specific species. viremia The presence of virus in the bloodstream. virulence The ability of a virus to cause more or less severe disease symptoms of a specific type.

HIV HPV

human immunodeficiency virus human papillomavirus

the virus infection. It is often these host responses that appear as the most prominent signs and symptoms of virus infection.

Virus Life Cycles Viruses are small and relatively simple microbes that cannot grow outside of living cells, that is, they are obligate intracellular parasites (Figure 1). At the structural level, all viruses have some general features in common: a

Viruses | Virus Infection

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(b) (c) (d)

(a)

(g) (e)

(h)

(f)

Figure 1 Electron microscope images of representative viruses. (a) poliovirus (30 nm diameter); (b) adenovirus (90–100 nm diameter); (c) influenza A virus (80–120 nm diameter); (d) herpes simplex type 1 (180 nm diameter); (e) rabies virus (180  75 nm); (f) Norwalk virus (27–38 nm diameter); (g) SARS coronavirus (120–160 nm diameter); (h) Ebola virus (80  1000 nm). Reproduced from the Public Health Image Library, Center for Disease Control and Prevention, in the public domain.

virus has a core of nucleic acid (either RNA or DNA) that acts as the genome of the virus and encodes some of the biological functions of the particular virus. Also, at the functional level, all viruses share some common processes of interactions with their host cells. While virus classification schemes are still debated among virologists, the nature of the genome and the mode of mRNA synthesis of a given virus are generally useful and widely employed bases for classification. David Baltimore has proposed a scheme based on mRNA metabolism (Figure 2). The chemical structure of the viral genome (DNA vs. RNA, single vs. double strandedness) is also a widely used basis for viral taxonomy (Table 1). At the cellular level a virus must first have some way of entry into the cell, often by adsorption or attachment to some structure or specific molecule on the surface of the target host cell. Often, the virus attachment site can be a molecule or group of molecules that the cell uses for other purposes, for example, a protein in the maltose transport system is used by bacteriophage lambda for attachment to Escherichia coli, and one of the lymphocyte cell recognition molecules is used by the human immunodeficiency virus (HIV) as its cell surface attachment site. In all virus infections the genome of the virus enters the host cell;

in some cases only the viral nucleic acid enters the cell, leaving the protein coat of the virus outside of the cell; in other cases, the entire virus is taken into the cell and the genome is exposed after a process of intracellular ‘uncoating’. In some instances, the viral nucleic acid enters the host cell with one or a very few genome-associated proteins while the bulk of the virus structural proteins remains on the outside of the host cell. Upon entering the cell, or soon thereafter, the infectious virus particles are disrupted, and even if the cell is artificially broken open, no infectious viruses are found. This period between the loss of infectivity and the appearance of fully infectious progeny virus is called the ‘eclipse’ phase of the virus life cycle. In the cases where some viral proteins enter along with the genome, such proteins play a necessary role in helping to express the viral genes or in the replication of the viral genome. In some instances, some of the imported viral proteins function to suppress the host gene expression so as to help the virus in effectively shutting down host functions as the virus subverts the cellular processes to its own program. After the viral genome enters the cell, some or all of its genetic information is expressed. In the case of

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Viruses | Virus Infection

Vl

Vll dsDNA

(+) ssRNA

l

ll

dsDNA

ssDNA

DNA/RNA hybrid ssRNA dsDNA dsDNA

V (–) ssRNA

lll mRNA

dsRNA

(–) ssRNA The Baltimore classification

lV (+) ssRNA

Figure 2 The Baltimore classification of viruses based on the method of viral mRNA synthesis. Reproduced from NIH, in the public domain.

Table 1 Virus classification based on genetic structure and replication strategy of virus Group

Genome

Typical representative

I II III IV V VI VII

DNA (double-stranded) DNA (single-stranded, þ sense) RNA (double-stranded) RNA (single-stranded, þ sense) RNA (single-stranded,  sense) RNA (single-stranded, þ sense, DNA intermediate) DNA (double-stranded, DNA intermediate)

Herpesviruses (fever blisters) Parvoviruses (fifth disease) Reoviruses (rotavirus) Picornaviruses (polio) Orthomyxoviruses (influenza) Retroviruses (HIV) Hepadnaviruses (hepatitis B)

viruses that have evolved to be ‘virulent’, that is, virus that will replicate and kill the host cell, some of the genes of the virus are expressed immediately and their translation into proteins results in the beginning of the intracellular replication phase of the virus. The usual genetic program of such viruses (e.g., bacteriophage T4, herpes simplex virus) is to direct the synthesis of viral DNA and when there are many copies of the viral genome, then to express the genes for the structural components of the virus, for example, the capsid (coat) proteins, and the envelope proteins, in the case of enveloped viruses. Once a large number of viral genomes have been produced and once a sufficiently large pool of virus structural proteins has accumulated, virus assembly is possible. When a large number of mature virus particles have accumulated, the cell often bursts because of disrupted metabolism or is lysed from within by specific lysis enzymes. This process releases the progeny virus in a ‘burst’ of

hundreds to thousands of new infectious virus particles able to initiate another round of infection. Figure 3 shows in diagrammatic outline the various steps in the infectious cycle of a specific virus, the influenza A virus. Some viruses, however, do not undergo this ‘lytic cycle’ but instead have evolved to enter into a symbiotic relationship with the host cell by promoting the integration of the viral genome into the host cell chromosome in a ‘repressed’ or latent state. Because these latent viral genomes can usually be reactivated by some conditions to full virus replication and gene expression with consequent virus production, cell lysis, and bursts of new virus particles, they are often called ‘lysogenic’ viruses (this terminology is most commonly used for bacterial viruses). The processes by which the infecting viral genome is integrated into the host chromosome is quite complex and differs for RNA- and DNA-containing viruses.

Viruses | Virus Infection

549

Virion

1

Nucleus 3a 4

Translation

Transcription, splicing 2

mRNAs Ribosomes

5a 3b

Golgi apparatus

RNA replication 5b 6

Cell

7

Figure 3 Diagrammatic representation of influenza virus replication cycle. Virus entry, uncoating, transcription, RNA replication, mRNA translation, protein processing in the Golgi apparatus, insertion of viral proteins into the infected cell membrane, and final virus assembly and budding are illustrated. Reproduced from NCBI, in the public domain.

Modes of Entry and Transmission of Viruses At the organismal level, virus infection is related to the physiology of a particular organism. In plants, for example, virus entry and release is often promoted by cellular injury and the virus is carried through the vascular system of the plant. In animals, there are many routes of entry, each exploited by different viruses. Common routes of infection are through the respiratory tract, the gastrointestinal tract, directly into the bloodstream, and by venereal contact. Airborne viruses, such as the common cold virus (rhinoviruses), measles virus, and influenza virus, enter the body through small droplets (aerosols) and the virus attaches to and penetrates the cells lining the surface of the respiratory tract. These viruses often replicate in the cells of the respiratory tract and cause these cells to initiate a local inflammatory response that results in many of the symptoms of these viral diseases. Viruses present in the respiratory secretions can be subsequently

transmitted by coughing, sneezing, and other similar modes of spread to other susceptible individuals. Some viruses spread from these localized infections to the bloodstream (viremia – virus in the blood), which allows for dissemination throughout the body. Other viruses, such as polioviruses, enter the body through ingested material (contaminated food and water), and because of their structural features they are able to survive the digestive actions of the stomach and intestines and then to infect cells of the intestinal tract. These viruses may cause local inflammation, such as various enteric fevers (various diarrheas, e.g.), or they may replicate and then be shed into the bloodstream for dissemination to other parts of the body. Poliovirus, for example, initially replicates in the gut with few symptoms but is borne by the blood to the central nervous system, where it infects specific cells to cause devastating effects. The viruses that replicate in the intestinal cells are often shed into the feces and passed on to others by the fecal– oral route of transmission.

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Some viruses are efficiently transmitted by direct inoculation into the bloodstream. In nature these viruses often require insect vectors to effect this transmission. Well-known examples include the yellow fever virus, dengue fever virus, and the encephalitis viruses, all transmitted by blood-feeding arthropods such as mosquitoes and ticks. Many viruses, for example, hepatitis B virus and HIV, although not transmitted by direct inoculation into the bloodstream in nature, can be transmitted by blood inoculation through medical procedures (transfusions, injections) or trauma. The venereal route of transmission is also utilized by some viruses. Well-known examples include the herpes simplex virus type 2, certain human papillomavirus (HPV) strains, and HIV. After the local infection of susceptible cells with an initial round of viral multiplication, the initial viremia (primary viremia) serves to transport the virus to specific target cells or tissues in the body where the virus may replicate further, giving rise to additional virus in the blood (secondary viremia). Often, the immunological responses of the individual are provoked only by massive secondary viremia because the primary viremia may be inadequate in duration or intensity to do so. Certain unusual modes of virus transmission have been observed, for example, in rabies, where the virus enters the tissues by trauma, often an animal bite, whereupon it enters the peripheral nerve cells and the virus migrates along the nerves to the central nervous system where it then replicates and causes damage. The virus can find its way, perhaps by the bloodstream or by the nerves, to the salivary glands where it can be excreted through the saliva and thereby transmitted to another susceptible host. Table 2 summarizes the modes of transmission and average incubation periods of some common human pathogenic viruses.

Responses to Virus Infections Most virus infections are asymptomatic or, at most, cause such common and inconsequential symptoms that the infection passes unnoticed. Analysis of the antiviral antibodies in normal human serum shows that we have many antibodies, which indicates a history of prior encounters with viruses of which we have been unaware. For example, approximately 85% of adults in the United States harbor latent Epstein–Barr virus (a herpesvirus) in their lymphocytes. Likewise, many individuals who cannot recall having fever blisters carry herpes simplex virus type 1 in a latent form in their bodies. Infection with poliovirus in infancy often provokes only a mild, selflimited febrile illness in contrast to the devastating infections of the central nervous system seen upon primary infection in older children and adults. The first cellular response to infection by many viruses seems to be the induction of interferon-specific proteins that are secreted by the infected cells and function to render neighboring cells more resistant to virus replication. The interferon response aims at producing local resistance to virus infection so as to limit the spread of the virus. This response is immediate and occurs within hours to days of the initial infection. Some side effects of the production of interferon include fever as well as the general malaise associated with many virus infections. The viremic phase of virus infection allows the cells of the immune system to detect and respond to the presence of virus. If the virus is sufficiently immunogenic (recognized as foreign to the body), the immune system produces a primary antibody response in about a week. This primary immune response results in the production of long-lasting memory-B-lymphocytes, which can be

Table 2 Routes of infection and incubation periods of some common human viral infections Virus

Routes of infection

Average incubation period

Epstein–Barr Virus Hepatitis A virus Hepatitis C virus Hepatitis B virus Herpes simplex virus Human papilloma virus Human immunodeficiency virus Influenza virus Measles virus Mumps virus Norwalk virus Parvovirus B19 (fifth disease) Poliovirus Rabies virus Rhinovirus (common cold) SARS coronavirus West Nile virus Yellow fever virus

Saliva, respiratory Orofecal, some body fluids Some body fluids Some body fluids Skin contact Skin contact Blood, some other body fluids Respiratory Respiratory Respiratory, direct contact Orofecal Respiratory Orofecal Animal bite Respiratory Respiratory, direct contact Mosquito bite, blood Mosquito bite

30–50 days 15–45 days Years 1–6 months 1–2 weeks Months to years 0–10 years 2–3 days 8–12 days 12–24 days 4–48 h 4–20 days 5–35 days 3–7 weeks 3–7 days 2–10 days Variable 3–6 days

Viruses | Virus Infection

activated later by subsequent exposure to the same virus to provide a more rapid and more intense secondary immune response. This immunological memory is the primary reason that we usually are more or less immune for life once we have survived a particular virus infection. The specific antibodies produced by the primary immune response can combine with the virus in the blood and result in circulating immune complexes that facilitate the destruction and clearance of the virus from the body. Such circulating immune complexes, however, also result in activation of some other processes such as the production of fever. Some viruses, such as the herpes simplex virus, cause local immunological reactions of such intensity that much of the inflammation and pain at the site of the infection is the result of the action of the immune cells rather than the destruction of the infected cells by the virus alone. Another unusual immune reaction is observed in the case of infection by Epstein–Barr virus. This virus enters certain cells of the B-lymphocyte lineage and results in a growth transformation of these cells, providing them with the potential for unlimited cell division (‘immortalization’, the first step in the formation of a B-cell malignancy). The normal immune surveillance mechanism that involves the T-lymphocyte system is activated to respond to these transformed B-cells and kill them. The process of T-cell activation gives rise to a large population of unusual T-cells, and it was this population of activated T-cells (large cells with a large nucleus, initially thought to be unusual monocytes) that gave Epstein–Barr virus infection its name: infectious mononucleosis, or ‘Mono’. That is, it was named for the cells that reacted to the virus infection rather than for the virus-infected cells themselves. Some viruses that enter into a latent or symbiotic state within the host cell can provoke the cell to behave in abnormal ways. Many such viruses carry extra genes that regulate cell division and can result in the malignant transformation of the cell to produce a cancer. These cancer-causing viruses (oncogenic viruses) are a special group of viruses that are of great current interest for both their special biology and their practical importance.

Outcomes of Virus Infections The usual outcome of a virus infection is recovery of the organism with long-lasting immunity. After the initial local multiplication, viremic phase, and immunological responses, the virus is eliminated from the body and the memory cells of the immune system stand ready to guard against another infection. It is this sort of immunity that is produced by successful iatrogenic virus infection called vaccination. If, however, the immune system is compromised, the virus replication overwhelms the immune system, or the virus manages to get into cells or tissues

551

that are hidden from the immune system, the virus may destroy critical tissues or organs and result in serious illness or death. Some viruses, after the primary infection, may enter into a latent form and be asymptomatic until periodic reactivation at later times. The herpes group of viruses are especially prone to such latent infections. Initial infection, such as with the chicken pox virus, gives rise to the viremia and generalized skin rash. The virus then enters into a latent infection of the dorsal root ganglia of the spinal cord and later, at times of lowered immunity, the virus may replicate and cause lesions in the skin along the local distribution of a particular spinal nerve, giving rise to the condition called ‘shingles’. Both chicken pox and shingles are disease manifestations of the same virus, the varicella zoster virus. A few viruses are known that may be present in the body and replicate at such a low level and be relatively benign yet escape the immune system and thereby establish a true persistent infection. The early phase of HIV infection seems to be an example of this mode of virus– host interaction. Varying degrees of cell proliferation may result from latent virus infections. These outcomes can result in local, limited growths such as viral warts and the small skin lesions caused by the virus of molluscum contagiosum, or can lead, in steps not yet fully understood, to malignant diseases such as Burkitt’s lymphoma, nasopharyngeal carcinoma, Kaposi’s sarcoma, and some types of cervical cancer.

Viral Virulence Viruses vary in their ability to infect and cause changes in their host cells. Even the specific cell type (tropism) may vary. These variations may be because of heritable properties (genetic mutations) or because of properties acquired from the most recent host, for example, viral envelope structures of cellular origin (pseudotype variations). Viruses are said to be virulent if they have a high propensity to cause disease or other evidence of infection in the specific test organism. Thus, a virus stock may be virulent for one species and avirulent for another. Repeated selection for virulence in one species may select for mutations that render the virus less virulent (attenuated) in another. This principle has been widely exploited to produce vaccine strains of virus. Some virulence may be related to the interaction of essential viral functions with related cellular functions. Other aspects of virulence may be simply a matter of virus interactions with the specific cell receptors for the virus. In certain cases, the genes of the virus that are known to be required for certain functions can be deleted or modified to make avirulent variants. Thus, nononcogenic

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Viruses | Virus Infection

forms of some retroviruses can be constructed by deletion of their specific viral oncogene. Virulence is a concept reserved for the capacity of a virus to produce an effect, not for the ability of the virus to survive inactivation. Some viruses are especially sensitive to drying, for example, and others are sensitive to organic solvents. These viruses may be virulent, even though they are, in some sense, very fragile and easily killed.

Antiviral Therapy and Prophylaxis Because virus infections rely on many pathways and processes of the host cell, there are very few unique virus-specific steps in the infectious process that provide vulnerable points of attack for antiviral drugs or treatments. Viruses with RNA genomes, however, differ significantly from the host cell with regard to genome replication and, thus, antiviral agents have been designed to target this unique step in virus infection. Chemical analogues of specific RNA precursors can inhibit the RNA replication process. These nucleoside analogues, such as azidothymidine (AZT) and dideoxycytidine (ddC), have been effective in treating HIV infections. Other drugs such as nevirapine also target the RNA replication step by direct inhibition of the replication enzyme, reverse transcriptase, of HIV. The prototype antiviral drugs have been analogues of DNA precursors such as the halogenated pyrimidines (iodo-deoxyuridine and bromo-deoxyuridine) and the sugar ring-opened purines (acycloguanosine and ganci-

clovir) used against viruses in the herpes group (herpes simplex, cytomegalovirus). These viruses encode a specific enzyme, thymidine kinase, which can activate these drugs to their toxic form. Uninfected cells lack this virusspecific kinase and hence are not harmed by the inactive prodrug form of these compounds. The best approach to controlling virus infection, however, is by active immunization. The first such success with this method was the well-known discovery of inoculation, and later, vaccination for small pox. Many vaccines have been developed that are highly effective in preventing or mitigating viral diseases, for example, rabies, measles, yellow fever, chicken pox, rubella, and influenza to name a few. This immunological approach has recently been extended to prevention of infection by human papilloma viruses with the consequent prevention of one of the long-term effects of such virus infection, cervical cancer. For malignancies that are caused or initiated by virus infection, this approach is highly promising. See also: Antiviral Agents; Bacteriophage (overview); Evolution, Viral; Plant Pathogens: DNA viruses; Plant Pathogens: RNA viruses; Vaccines, Viral; Viroids/ Virusoids; Viruses, Environmental

Further Reading Dimmock NJ and Primrose SB (1994) Introduction to Modern Virology, 4th edn. Oxford: Blackwell. Fields BN, Knipe DM, and Howley PN (eds.) (1996) Fields’ Virology, 3rd edn. Philadelphia: Lippincott-Raven. Levy JA, Fraenkel-Conrat H, and Owens RA (eds.) (1994) Virology, 4th edn. Englewood Cliffs, NJ: Prentice Hall.

Viruses, Environmental R -A Sandaa, University of Bergen, Bergen, Norway ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Introduction Viruses in the Environment Ecological Importance

Glossary autotrophic Describes an organism that is able to use CO2 as its sole source of carbon. bacteriophage (phage) Viruses that infect prokaryotes (domain Bacteria). burst size Number of virus particles originating from one host cell during a lytic cycle. culture-independent molecular techniques Methods that make it possible to study microbes without the need to culture them. Most of the microbes in the environment are not known to be culturable. cyanophage Viruses that infect cyanobacteria. heterotrophic Describes an organism that uses organic compounds for both energy metabolism and growth. lysogenic virus A virus that integrates its DNA into the host genome, whereby the phage genome becomes a prophage that is transmitted to daughter cells. A later event (such as exposure to UV radiation) can release the prophage, causing proliferation of new phage via the lytic cycle. Lysogeny also occurs in eukaryotes, although the process is not yet fully understood. lytic virus A virus reproducing through replication inside the host cell, resulting in lysis (disruption) of the host cell and release of the newly synthesized viral particles. metagenomics The study of DNA sequences recovered directly from environmental samples. metagenome clone libraries Collections of cloned DNA recovered directly from environmental samples. phylotype A unique DNA sequence presumed to correspond to a unique organism.

Abbreviations DGGE DMS DMSP DOC dsDNA

denaturing gradient gel electrophoresis dimethylsulfide dimethylsulfoniopropionate dissolved organic carbon double-stranded DNA

Methods for Studying Environmental Viruses Conclusions Further Reading

polymerase chain reaction (PCR) amplification Enzymatic amplification of a DNA fragment. Particularly useful when the targeted sequence is present in single or low copy number. PCR can be modified extensively to perform a wide array of genetic manipulations. pyrosequencing An inexpensive high-throughput method for sequencing DNA. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand alongside it. Each time a nucleotide is incorporated into the growing chain, a cascade of enzymatic reactions that causes a light signal to be emitted is triggered. 16S rDNA and 18S rDNA The main RNA component of the small ribosomal subunits of prokaryotes and eukaryotes, respectively. The most commonly used macromolecules for phylogenetic classification. shotgun libraries Clone libraries with long randomly selected DNA inserts. temperate viruses Viruses that, upon infection of a host, do not necessarily cause lysis but may become integrated into the host genome. virus A submicroscopic infectious agent that is unable to grow or reproduce outside a host cell. Outside the cell, viruses exist as inert free particles called virions, which are typically RNA or DNA genomes surrounded by a protein shell called a capsid. The term denotes viruses infecting both eukaryotic and prokaryotic cells. virus-like particles (VLPs) Term used to describe dots that are visualized by direct counting of viruses in an environment because it is not absolutely certain that these dots represent viruses.

dsRNA EFM EhV FCM LGV MCP

double-stranded RNA epifluorescent microscopy Emiliania huxleyi virus flow cytometry large genome viruses major capsid protein

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MGV MPN PCR PFGE PFU SGV

middle genome viruses most probable number polymerase chain reaction Pulsed-field gel electrophoresis plaque-forming unit small genome viruses

Defining Statement Viruses are the most numerous biological entities in the environment. They may also be the most diverse. Viruses have been found everywhere there is cellular life and are regarded as important players in various ecological processes, such as nutrient cycling, gene transfer, and biodiversity.

Introduction Viruses are the most numerous and probably most diverse biological entities in the environment. There are an estimated 1031 viruses on Earth, and most of them infect bacteria. Although the presence of viruses in the sea has been known since the 1950s, this knowledge was not initially appreciated because the vast pool of aquatic microbes had not yet been discovered. At that time, environmental microbiology consisted mainly of culturing microbes from different environments, and it was believed that most environments contained relatively few microbes. Thus, it was thought that these microbes had only minor importance in ecological processes. In 1989, Bergh and colleagues showed that viruses are the most abundant biological entities in the ocean, with their numbers often exceeding the host (microbe) abundance by several orders of magnitude. Almost at the same time, as new molecular techniques facilitated the study of the total microbial communities without the need to culture the organisms, it was discovered that the number and diversity of microbes in the environment is also enormous. These findings resulted in a major shift in thinking regarding the function of microbes in the marine environment. As viruses are infectious particles that are dependent on a host for replication, it was speculated that viruses may also be important players in various ecological processes such as nutrient cycling, gene transfer, and generation of biodiversity.

Viruses in the Environment Viruses have been found in every environment in which there is cellular life, from polar ice caps to hot springs. As viruses depend on their hosts for replication, the relative

ssDNA ssRNA TEM VBR VLPs

single-stranded DNA single-stranded RNA transmission electron microscopy virus-to-bacterium ratio virus-like particles

abundance of specific virus types roughly parallels that of the organisms they infect. Viral hosts span the three domains of life, Eukarya, Archaea, and Bacteria; however, most of the hosts that have been identified in the environment are bacteria. Consequently, bacteriophage are by far the most abundant viruses in the environment. The global population of environmental phage has been estimated to be of the order of 1031 particles. If these phage were laid end to end, they would span approximately 10 million light years, or the distance between the Earth and the Sun 1013 times. Numbers like these point to an important role for these biological entities in the environment.

Aquatic Viruses Microscopic observation indicates the presence of 3  107 virus-like particles (VLPs) in 1 ml of marine or fresh water. This gives a virus-to-bacterium ratio (VBR) in aquatic environments of between 10 and 15. This ratio is important because it demonstrates that the probability (likelihood) of a virus meeting a prospective host in order to reproduce itself before it disintegrates is high. This is critical because the half-life of a viral particle in the marine environment is approximately 2–4 days. This means that a virus population must be sufficiently large to ensure that at least one of its members meets up with an appropriate host during that time period. Current estimates indicate that viruses constitute 90% of the nucleic acid-containing particles in the ocean, while prokaryotes (Bacteria and Archaea) and protists (unicellular heterotrophic and autotrophic eukaryotes) only constitute 10 and 1%, respectively. However, because of their small sizes, viruses constitute only 5% of the total microbial biomass in the ocean, while the prokaryotes constitute 90%. If we convert the biomass of viruses into an equivalent amount of carbon, the total amount of viruses in the sea would contain more carbon than 75 million blue whales. This makes viruses the second largest component of biomass in the ocean after prokaryotes. Most information on viral ecology in aquatic systems is from the marine environment; however, those studies available from freshwater and

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estuarine environments have shown similar numbers and dynamics to those seen in marine environments.

Viruses that infect prokaryotes: Bacteriophage

On the basis of studies of cultured marine bacteriophage, it is assumed that most marine phage are double-stranded DNA (dsDNA) viruses. A high proportion of these viruses, at least in the surface ocean, belong to three phage groups, the Podoviridae, Siphoviridae, and Myoviridae (Figure 1). These are phage that infect members of the phyla Proteobacteria, Firmicutes, and Cyanobacteria and so far there is no evidence that they infect Archaea. The Podoviridae are phage with a very short tail and an icosahedral head with a diameter of 60 nm. The Siphoviridae have long flexible noncontractile tails, and also have an icosahedral head of diameter similar to podoviruses. Viruses classified as Myoviridae have a contractile tail; the head is isometric to prolate in shape and has a diameter of 50–110 nm. All three types are nonenveloped viruses with linear dsDNA genomes. The size of the genomes seems to be roughly distinct:

(a)

podoviruses and siphoviruses tend to have smaller genomes (80  C) infect a broad spectrum of the extremely thermophilic Archaea, the Crenarchaeaota. These viruses infect representatives of the genera Sulfolobus, Thermoproteus, Acidianus, and Pyrobaculum. Typically, these viruses show no clear similarity in their morphology or at the genomic level to either bacterial or euryarchaeal viruses. These crenarchaeote viruses have been classified into seven new families, of which four are approved by the International Committee of Taxonomy of Viruses. These families are lemon-shaped Fuselloviridae, filamentous Lipothrixvidae, stiff rod-shaped Rudiviridae, droplet-shaped Guttaviridae, spherical Globuloviridae, two-tailed spindle-shaped Bicaudaviridae, and bottle-shaped Ampullaviridae. The rod-shaped virions of the Rudiviridae and Lipothrixvidae show some similarity with the tobamoviruses and closteroviruses of vascular plants, respectively, while those of the Globuloviridae resemble viruses of the Paramyxoviridae, which infect vertebrates. All these hyperthermophilic viruses contain dsDNA genomes that are either linear or circular and their genomic sizes range from 15 to 75 kb.

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The deep-sea vent areas are among the most extreme habitats on Earth, characterized by high pressure, wide temperature ranges (10–400  C), and the presence of a variable composition of fluids and gases, which are often incompatible with life for most eukaryotes, and indeed most prokaryotes as well. Viruses in these systems can reach abundances of between 105 and 107 VLPs/ml. The morphology of these viruses is diverse, with morphotypes resembling typical archaeal viruses, including lemonshaped viruses and pleomorphic types such as spoonshaped and spindle particles with bipolar expansions, but also some morphotypes that are known to infect bacteria. These morphotypes are filamentous and rodshaped viruses. The only virus that has been isolated from this environment is named PAV1, and it is lemonshaped measuring 120  80 nm, with a short tail terminated by fibers. This virus infects the hyperthermophilic euryarchaeote Pyrococcus abyssi. The genome consists of 18 kb of circular dsDNA. Genomic comparison with other viral sequences has resulted in no significant similarity. Viruses in deserts

Deserts are extremely dry, and are exposed to extremes of UV light irradiation and temperature variations. Nonetheless, both eukaryotic and prokaryotic microbes have adapted to these extreme conditions. VLPs have been found in surface sands from different dessert locations in North Africa. These particles resemble morphotypes representing the three major bacteriophage families: Myoviridae, Siphoviridae, and Podoviridae. They also have dsDNA genomes in the same size range as these viral families, from 45 to 270 kb. Viruses in polar environments

Microbial life has also been detected in extremely cold environments such as high-latitude glaciers, polar permafrost, and the Dry Valleys of Antarctica, as well as in sea ice. In Arctic and Antarctic sea ice, microbes belonging to the prokaryotes and microeukaryotes, especially microalgae, have been detected together with VLPs. The viral abundance in sea ice is 10–100 times higher (106–108/ml) than in the surrounding water. This correlates with the bacterial abundance, which is also higher in the sea ice than in the adjacent water column. Viral proliferation appears to be enhanced in the sea ice relative to the open water, and the VBRs are among the highest reported in natural environments. In contrast, the number of VLPs in Arctic glaciers is 10–100 times lower than the average for marine and freshwater ecosystems in temperate regions. The VBR in this environment is on average >10, and there is a strong positive correlation between viral and bacterial abundance. Three Arctic viral isolates have been cultured and they all infect psychrophilic bacteria whose closest relatives are Shewanella frigidimarina, Flavobacterium hibernum, and Colwellia psycherythrae. All three

phage are lytic, have dsDNA genomes, and are morphologically similar to the families Siphoviridae and Myoviridae. They are adapted to a cold environment, and phage development has a lower maximum temperature than the maximum growth temperature of the host bacterium.

Ecological Importance Viral ecology is the study of the interaction of viruses with other organisms and the environment. Viruses are agents of mortality in both prokaryotes and eukaryotes, and thus they play important roles in the ecology of these organisms. As mentioned earlier, the global population of environmental phage contains 1031 particles. This viral pool turns over every few days, from which it can be calculated that every second somewhere on Earth 1023 microbes are infected by a phage. When the host organisms are lysed, nutrients are released to the surrounding environment; in this way viruses are important players in the cycling of carbon and nutrients. Viruses also directly affect the abundance and diversity of host cell communities and contribute to microbial gene exchange, which is important for the evolution of the host community. To date, virtually all information on the interaction between environmental viruses and their hosts has been gathered from aquatic systems, while little information is available on the importance of viruses in soil or in extreme environments. Consequently, most of the following discussion of viral ecology will focus on aquatic viruses. Viral Effect on Nutrient Recycling As the main drivers of oceanic biogeochemical cycles, microorganisms play a significant role in regulating the ecosphere. Marine viruses also play an important role in the microbial food web as catalysts that accelerate the transformation of nutrients from the particulate to the dissolved state. From this the nutrients can then be incorporated by microbial communities. The marine microbial food web consists of a consortium of heterotrophic and autotrophic prokaryotes, as well as their predators (viruses and protozoa). The structure of the food web determines the transfer of energy and nutrients (nitrogen and phosphate) to higher trophic levels and greatly influences global carbon and nutrient cycles (Figure 2). Viruses are a crucial and ubiquitous component of this microbial food web and play their part by lysing their host organisms. It is estimated that phage kill between 4 and 50% of the bacteria produced every day and that 2–10% of phytoplankton primary production is channeled through ‘the viral shunt’ in the microbial food web. Cell lysis implies that organic material is lost from the grazing food chain and

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Fish

Mineral nutrients

Heterotrophic flagellates

Ciliates

Zooplankton

Virus

Bacteria

Small algae

Large algae and diatoms

CO2 DOC

Figure 2 Microbial food web and virus-mediated carbon flow. The red arrows represent virus-mediated pathways. The black arrows represent transport of dissolved organic carbon (DOC) in the microbial food web. The black dotted arrows show the contribution to the CO2 cycle and the blue lines the transport of mineral nutrients in the microbial food web. DOC is the largest biogenic pool of carbon in the ocean. The dynamics of DOC may have an indirect impact on global carbon cycling, contributing to the control of atmospheric CO2. DOC is only accessible to microbes, primarily heterotrophic bacteria. When viruses lyse the host, carbon is transformed from the particulate form (POC) into the dissolved form (DOC). Figure: Professor Gunnar Bratbak, Department of Biology, University of Bergen.

becomes available to bacteria, which thrive on dissolved organic material and nutrients. The net effect of this is to increase nutrient recycling and respiratory loss of organic carbon in the lower part of the food chain. In other words, viruses short-circuit the flow of carbon and nutrients from phytoplankton and bacteria to higher trophic levels. This means that viral activity has a direct effect on the carbon budget of the ocean, and hence on the global climate. Viruses can also have an indirect effect on the climate by influencing the formation of dimethylsulfoniopropionate (DMSP), which is the precursor of dimethylsulfide (DMS). Viral infections in algae have been found to cause increased production of DMSP. In the ocean DMSP is degraded to DMS by the activity of bacteria and phytoplankton. A portion of this DMS diffuses from the ocean to the atmosphere, where it is involved in cloud formation and also causes acid rain. Clouds affect the radiation balance of the Earth and thereby strongly influence its temperature and climate. Virus–Host Interaction and its Effect on Microbial Diversity Viruses can influence the genetic diversity of their hosts in various ways. Simple models have been developed that

assume that prokaryotic diversity is controlled by a combination of predation, viral lysis, and substrate limitation. If substrate (nitrogen or phosphate) is limiting (bottom-up control), it can control which species are present because different species are specialized to use different substrates. This leads to a system where all species are limited by different substrates, and organismal diversity reflects the diversity of substrates. The host community can also be regulated by specific viral lysis and/or grazing by protozoa (top-down control). The so-called ‘killing the winner’ model explains the role of viruses in the maintenance of high biodiversity in marine systems, where coexisting hosts compete for the same resource. Because viruses control the most abundant or fast-growing host populations, less competitive or slower growing populations can coexist with the dominant fast-growing hosts (Figure 3). Population reduction (lysis) of the most dominant host populations creates open niches, which enable new hosts to become abundant. This provides an opportunity for less competitive host populations to exist in the presence of more competitive hosts, thus enabling maintenance of high diversity in the marine environment. Empirical evidence concerning the intensity or the outcome of the regulation of viral activity on the host community structure is still scarce, and contradictory

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A A-D: Phage-hostsystems Phage or bacteria (ml–1)

8

Bacteria populations Phage populasjons

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107 106 105 104

A

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Figure 3 Model of Lotka–Volterra oscillations in virus–host interactions. For each phage–host system, a selective factor stimulates growth of a specific host. An epidemic phage infection begins at a critical host cell density and the abundance of a specific phage increases. Phage lysis causes the abundance of host cells to decline to baseline levels and thus prevents dominance of a single host species. At the end of the epidemic, the number of infective phage declines to a baseline level at a decay rate specific to each phage.

minimal set of essential functions encoded in the viral genome, and no or few genes dedicated to coping with the host defense system. These viruses usually have small genomes, and the reproductive strategies of these viruses are optimized to outrun the host by rapid replication. For example, viruses belonging to the Podoviridae family may be regarded as r-strategists as the viruses are abundant and virulent, and have a rapid replication rate, a high burst size, and small genomes. In contrast, complex viruses with large genomes have evolved considerable autonomy of their replication and expression systems, in addition to having accrued elaborate gene ensembles to overcome host defenses. The mimivirus and some temperate phage, which exhibit a lysogenic lifestyle, may be examples of the latter. These viruses may be regarded as K-strategists, because they have low burst size, large genomes, and a slow decay time, which is the time it takes for the virus to be degraded in the environment. Viral Effect on Gene Transfer and Evolution of the Host

results have been reported. The picture is quite complicated because prokaryotic hosts can be susceptible to multiple phage infections and because sensitivity toward phage infection is strain-specific and exists as a continuum from highly sensitive to highly resistant. Nevertheless, the results of some studies clearly demonstrate that there is a link between viral and host biodiversity and that coexistence of nutrient-competing bacterial hosts might indeed be controlled by viral lysis. The results of other studies, however, indicate limited effects of viral activity on the bacterial community structure; these studies suggest that viral infections affect only a few abundant phylotypes, while other phylotypes may be resistant to infection. Alternatively, it has been suggested that the most dominant hosts are dominant because they are less susceptible to viral lysis. This means that rare marine bacterial groups might be the most susceptible and therefore the losers in the competition over resources. If, however, there is a trade-off between competitive ability among the hosts in terms of nutrient uptake and defensive ability (resistance) against viral infection, fast-growing competition specialists (r-strategists) would presumably be the least abundant, while their associated viral populations will have the highest abundance. Furthermore, the hosts that are defense specialists (Kstrategists), being more or less immune to viral attack, will grow more slowly but will be more abundant and have an associated viral population of low abundance. A complementary view of the dichotomy between simple and complex viruses regards simplicity and complexity as two fundamentally different strategies of virus–host interaction. Simple viruses rely almost entirely on the host for their replication and expression, with a

Viruses affect the microbial community through the introduction of new genetic traits via horizontal gene transfer. The most well-known cases of horizontal gene transfer by phage are in the context of pathogenesis, where phage can carry toxin genes and other virulence factors. In an environmental context, horizontal gene transfer is an essential factor in evolution. Phage are major conduits of genetic exchange, and transduce an estimated 1025–1028 bp of DNA per year in the world’s oceans. Gene transfer can occur during the viral replication cycle when some of the host genes are by ‘mistake’ packed inside the viral particle. During infection, these genes may be transferred to a new host. Viruses have several different life or replication cycles. The two most dominant are the lytic and the lysogenic (latent) cycles. Lytic (or virulent) viruses infect a cell, replicate, and are released following lysis of the host cell. Temperate viruses infect the host, but the viral DNA stays within the host cell, often physically integrated into the host genome. The viral genome then replicates along with the host genome, until external factors, such as DNA damage, induce the lytic cycle. For prokaryotes in the ocean, it has been suggested that in nutrient-rich waters, which are characterized by a high abundance of hosts, lytic phage dominate. It has also been speculated that viruses might use the lysogenic cycle as a survival strategy in harsh or low host abundance environments. This might be particularly important for the survival of viruses in extreme environments. Lysogeny has been suggested to be a beneficial lifestyle for viruses in Arctic environments. There are arguments both for and against such a theory, because all the phage

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that have been isolated from Arctic freshwater and glaciers so far are lytic. In the Arctic marine environment, however, a high percentage of marine bacterial genomes harbor integrated viral genomes (prophage). This finding demonstrates that viruses with lysogenic lifecycles are common in the Arctic marine environment. In fact, metagenome studies of the Arctic viral community have shown that all the five most abundant recognizable phage sequences represented prophage. This is in stark contrast to sequences obtained from temperate regions, which are dominated by genes from lytic phage. In other environments, such as salt lakes and hot springs, temperate phage have been isolated together with a few lytic viruses. However, the majority of these viruses exhibit a virus–host relationship that is different from those observed in less extreme environments. In this relationship, the infected hosts are not lysed, but virus particles are continuously produced. This is consistent with an equilibrium state being established between viral replication and host cell multiplication. Moreover, the viruses persist in host cells in a stable state and are not lost during continuous growth of the infected cultures. It has been suggested that such a survival strategy may help the virus population to avoid direct, and possibly prolonged, exposure to the harsh conditions of the host habitat. Nevertheless, large numbers of viruses have been reported in some hot springs, at densities of 106/ml. Hot-spring viruses have been shown to be resistant to shifts in temperature: 75% of such viruses were still intact after incubation on ice, showing that they are able to withstand extreme environmental conditions. The dynamic nature of the oceanic gene pool has resulted in genetic diversification of both donor and recipient genomes and has also probably guided their functional diversification. As an example, genes involved in photosynthesis have recently been detected in cyanophage. These genes (psbA and psbD) code for two proteins, D1 and D2, which form the reaction center of photosystem II (PSII). The D1 protein is common to all oxygenic phototrophs and has a high turnover rate as a result of photodamage. Both the psbA and psbD genes have been reported in cyanophage infecting Synechococcus and Prochlorococcus, and have been identified in BAC clones (metagenomes) and polymerase chain reaction (PCR) amplicons from environmental samples. These viral photosynthetic genes may be expressed during infection of the host, which suggests that they probably have a functional role during the infection cycle. The genetic transfer of photosynthetic genes between cyanobacteria and phage might thus have significant implications for the evolution of both hosts and phage (coevolution). Host genes retained in a particular phage could reflect key selective forces in the host environment. Indeed, phosphate sensing and acquisition genes have been found in phage that infect organisms in low phosphate

environments. Likewise, the most abundant viralencoded enzymes found through extensive sequencing of marine viral communities appear to be involved in scavenging host nucleotides (e.g., riboreductases) and supporting host metabolism throughout the infection cycle (e.g., carboxylases and transferases). Thus, it might be speculated that phage genes are important players in different metabolic processes in their hosts, increasing the fitness of both the host and the phage, and that the functionality of the phage may be coupled to the physiological state of the host.

Methods for Studying Environmental Viruses Documentation of both community diversity (richness of species) and composition (evenness of species distribution) are fundamental elements of ecological research. Consequently, information about abundance, diversity, and community structure is of crucial importance to understanding the ecological roles of viruses in the environment. For this purpose, a number of both qualitative and quantitative methods are available for describing their diversity and dynamics in the analysis of viral communities. Several methods are also available for determining the abundance of these particles in environmental samples. Enumeration and Measurement of the Concentration of Viruses There are essentially two different ways to enumerate viruses: the indirect viable count and the direct total count. Viable count techniques are based on the lysis of a cultivable host, while direct counts involve counting the viruses in the environmental sample without the need for a cultivable host. The methods available for performing viable counts are the plaque-forming unit (PFU) and most probable number (MPN) techniques. Direct counts may be performed using TEM, epifluorescent microscopy (EFM), or flow cytometry (FCM). PFU and MPN techniques are both used to quantify the number of particles released from a lysed host. This means that the host must be cultivable, which is not the case for most of the microbes in the environment. The PFU method is used to determine the number of viruses that cause lysis of bacteria, cyanobacteria, or algae that can grow on a solid medium. Initially, the host has to be grown in a liquid medium, and then it is mixed with a sample that contains the virus. The mixture of virus and host is then combined with molten agar (soft agar) and poured on to a plate in which the agar content is high and contains a medium that the host can utilize. After

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incubation, the plaques, which are areas in which cells have been killed (or transformed), can be counted and the number of infective virus particles in the original suspension estimated. The MPN technique is used to quantify the number of infectious viral units for hosts unable to grow on solid media. Serial dilutions of the virus are made in replicates. The last dilution in which the host is lysed (no growth) is considered to contain one infectious viral particle. The number of infectious particles can then be calculated based on the number of replicates in each dilution where lysis has occurred. The PFU and MPN methods can also be used to capture new environmental viral isolates and to purify viral isolates in culture in order to obtain a clonal virus. Direct counts

Direct counts of viruses from the environment provide the total number of VLPs without the need for culturing, and often give estimates that are 100–1000 times higher than PFU counts. For TEM, viruses are harvested directly on to electron microscopy grids by centrifugation or are concentrated by ultrafiltration (see below) and then transferred to grids. Thereafter, the viruses are stained with an electron-dense material, for example, uracil acetate (Figure 4). This method can also be used to describe the VLPs according to their morphology, a trait used in viral taxonomy. In this way, TEM can be used to give information that enables the tentative taxonomic affiliation of the viruses to be established. In addition, TEM can be used to visualize infected cells and thus to estimate the burst size of individual cells. For EFM, the viruses are concentrated on membrane filters and then stained with a fluorescent dye that binds specifically to the nucleic acids of the viral particles (Figure 5). FCM is the newest method that has been used to enumerate aquatic viruses (Figure 6). It is a technique for counting, examining, and sorting microscopic particles that are suspended in a stream of fluid. FCM allows populations to be analyzed based on the physical and/or chemical characteristics of single cells. This accurate high-throughput method also allows the quantification of subpopulations of fluorescent-stained viruses that differ in their characteristics of fluorescence and light scattering. The major advantages of FCM are its ability to analyze a large number of cells rapidly and the provision of data suitable for statistical analysis.

Figure 4 Transmission electron micrograph of a lysed bacterium and phage particles in a seawater sample. Scale bar ¼ 500 nm. Photo: Senior Scientist, Mikal Heldal, Department of Biology, University of Bergen.

Figure 5 Epifluorescence micrograph of prokaryotes and viruses in a seawater sample stained with a fluorescent dye, SYBR Green I. This dye specifically stains doubled-stranded DNA (dsDNA). The smallest dots are viruses and larger ones are prokaryotes (bacteria or archaea). The prokaryotes are approximately 0.5 mm in diameter. Photo: Prof. Gunnar Bratbak, Department of Biology, University of Bergen.

Concentration

For most viral analyses, it is necessary to concentrate viruses from the environmental sample prior to performing the specific analysis. Concentration is necessary for counting and isolation of virus particles, but also for methods used to study the viral community structure.

The most commonly used methods for concentrating viral particles from environmental samples are ultrafiltration and ultracentrifugation. During ultracentrifugation, the viral particles are pelleted by high-speed centrifugation, whereas ultrafiltration is based on filters with very

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(DNA stained with SYBR green I)

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Side scatter Figure 6 Biparametric flow cytometry plot showing characteristic viruses (pink, blue, and green dots) in a seawater sample stained with a fluorescent dye, SYBR Green I. The particles are discriminated using side scatter versus green fluorescence signals. The green-colored dots represent Emiliania huxleyi virus (EhV) infecting the microalga Emiliania huxleyi. The beads are 1 mm in diameter. Figure: Dr. Aud Larsen, Department of Biology, University of Bergen.

small pore size that retain the viral particles in the sample. The latter method involves a prefiltration step in which either 0.2 or 0.45 mm pore size filters are used to remove microbes from the sample. Thereafter, the viruses are concentrated in the sample by using filters with either a 30 kDa or a 100 kDa cutoff. The viruses in a sample of 200 l of seawater can be concentrated into a volume of 50 ml, and thus be concentrated by a factor of 4000, using the ultrafiltration system. Viral Diversity Culture-dependent methods

Culture-based studies for estimating environmental viral diversity are based on hosts that are able to grow either in cultures or on solid media. Thus, the information available on diversity is based only on a small fraction of the total viral community. Nevertheless, such information is of crucial importance in increasing our knowledge of the ecology of environmental viruses. Studies of virus–host interactions under cultivation conditions may provide valuable information about factors that influence such relationships. In addition, sequencing of whole viral genomes is important to provide information that might give us new insight into the ecological functions of these viral particles. One method that is used for such a purpose is the plaque assay, which makes it possible to obtain and purify the viral isolates infecting one specific host.

Another method for isolating viruses is to add concentrated viruses from an environmental sample to different potential hosts and monitor these for cell lysis. To ensure that the lysis has occurred due to virus propagation, the host cells are investigated using TEM to confirm the presence of VLPs inside the host cells. Culture-independent methods

The introduction of culture-independent molecular techniques combined with the unraveling of phylogenetic information harbored in genes that are common to all microbes, such as 16S rDNA and 18S rDNA, has enabled the study of the total diversity and dynamics of environmental samples. The results of these studies have shown that most microbes in the environment belong to as yet uncultured groups. The fact that most hosts have not yet been cultured has severely limited studies of viral diversity. In addition, there is no single universal gene, analogous to 16S rDNA and 18S rDNA in prokaryotes and eukaryotes, which is present in all (or even most) viruses. This makes it difficult to study the total viral diversity and dynamics of natural phage communities. However, whole genome comparisons have shown that conserved structural and functional genes, which are shared among all members within certain viral taxonomic groups, do exist and can be used for studies of viral diversity (Table 2) Thus, it is possible to study the diversity within certain taxonomic viral groups using

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PCR amplification and sequencing on viral concentrates. The dynamics of different viral groups can also be studied by the use of denaturing gradient gel electrophoresis (DGGE) and subsequent sequence analysis. The DGGE technique is a molecular fingerprinting method that is used to separate PCR-generated DNA products on the basis of sequence differences leading to different melting behavior of the DNA. Studies that are based on conserved viral genes are not able to investigate the total viral community. To access such information, it is necessary either to produce and sequence metagenome clone libraries of the total environmental viral community, or to sequence viral DNA isolated from the environment directly. Both these approaches are based on concentration of the environmental viruses by ultrafiltration. For metagenome cloning, the DNA is cut into short fragments either by enzymes or through random shearing of the DNA. These fragments are then cloned into vectors, such that each vector contains DNA from one viral particle. This sorting by cloning is not necessary with direct sequencing (now by pyrosequencing), in which individual DNA molecules are sorted on beads. A new high-throughput technique, 454 pyrosequencing, generates shorter fragments than the conventional techniques; however, this limitation is compensated for by the very large number of sequences that can be generated quickly and at low cost. Both techniques rely on extensive sequencing, and advances in bioinformatics, refinement of DNA amplification, and increased computational power have greatly aided the analysis of DNA sequences recovered from environmental samples. Another method that is available for study of the viral community structure without the need to employ sequencing and analysis of conserved genes is PFGE. This method exploits one characteristic of viruses that varies over a wide range and can be easily determined, namely, the size of the genome. The PFGE technique provides separation over the full size range of intact viral genomes. Environmental dsDNA viral genomes are reported to range from a few to several hundred thousand kilobases in size. The variation in genome size for RNA and ssDNA viruses is not as dramatic and these genome types are smaller, rarely exceeding 20 kb of total nucleic acid. Thus, genome size is a phenotypic characteristic of sufficient variability and universality to characterize the most dominant communities of dsDNA viruses. The method was first used in this way to analyze the bacteriophage community in the rumen of sheep, and it was demonstrated that one PFGE band consisted of DNA from one single phage genotype. This approach has been used recently in several studies that have explored the dynamics of communities of dsDNA viruses in the marine environment. The method allows investigation of the nonculturable fraction of the viral community and, when applied together

with PCR and specific primers, it is possible to characterize dominant unculturable viral populations.

Conclusions Viruses are found in every environment in which there is microbial life. They occur in high numbers and exhibit extraordinary diversity. As a result, viruses influence the composition of their host communities and are a major force behind biochemical cycles. They also affect the evolution of both host and viral assemblages through viral-mediated gene transfer. However, our understanding of these processes is far from complete, and the story of environmental viruses is still emerging as methodology improves. The area of viral research is quickly advancing and new discoveries seem to be forcing a paradigm shift in the thinking on viral ecology.

See also: Bacteriophage Ecology; Bacteriophage (overview); Ecology, Microbial; Food Webs, Microbial; Metagenomics; Transduction: Host DNA Transfer by Bacteriophages

Further Reading Breitbart M, Miyake JH, and Rohwer F (2004) Global distribution of nearly identical phage-encoded DNA sequences. FEMS Microbiology Letters 236: 249–256. Breitbart M and Rohwer F (2005) Here a virus, there a virus, everywhere the same virus? Trends in Microbiology 13: 278–284. Brussaard CPD (2004) Viral control of phytoplankton populations – a review. The Journal of Eukaryotic Microbiology 51: 125–138. Edwards RA and Rohwer F (2005) Viral metagenomics. Nature Reviews Microbiology 3: 504–510. Filee J, Tetart F, Suttle CA, and Krisch HM (2005) Marine T4-type bacteriophages, a ubiquitous component of the dark matter of the biosphere. Proceedings of the National Academy of Sciences 102: 12471–12476. Hambly E, Tetart F, Desplats C, Wilson WH, Krisch HM, and Mann NH (2001) A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2. Proceedings of the National Academy of Sciences 98: 11411–11416. Millard A, Clokie MR, Shub DA, and Mann NH (2004) Genetic organization of the psbAD region in phages infecting marine Synechococcus strains. Proceedings of the National Academy of Sciences 101: 11007–11012. Paul JH, Williamson SJ, Long A, et al. (2005) Complete genome sequence of phi HSIC, a pseudotemperate marine phage of Listonella pelagia. Applied and Environmental Microbiology 71: 3311–3320. Raoult D, Audic S, Robert C, et al. (2004) The 1.2-megabase genome sequence of Mimivirus. Science 306: 1344–1350. Romancer ML, Gaillard M, Geslin C, and Prieur D (2007) Viruses in extreme environments. Reviews in Environmental Science and Biotechnology 6: 17–31. Short SM and Suttle CA (2002) Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature. Applied and Environmental Microbiology 68: 1290–1296.

Viruses | Viruses, Environmental 567 Sullivan MB, Coleman M, Weigele P, Rohwer F, and Chisholm SW (2005) Three Prochlorococcus cyanophage genomes: Signature features and ecological interpretations. PLoS Biology 3: e144. Suttle CA (2005) Viruses in the sea. Nature 437: 356–361. Suttle CA (2007) Marine viruses – major players in the global ecosystem. Nature Reviews Microbiology 5: 801–812.

Weinbauer MG (2004) Ecology of prokaryotic viruses. FEMS Microbiology Reviews 28: 127–181. Weinbauer MG and Rassoulzadegan F (2004) Are viruses driving microbial diversification and diversity? Environmental Microbiology 6: 1–11.