Physiological and Biotechnological Aspects of Extremophiles [1 ed.] 0128183225, 9780128183229

Table of contents :
Physiological and Biotechnological Aspects of Extremophiles
Copyright
Contents
List of contributors
About the editors
Preface
Acknowledgments
1 Overview of extremophiles
1.1 Introduction
1.2 Eukaryotic extremophiles
1.3 Prokaryotic extremophiles in diverse habitats
1.4 Biotechnological potential of extremophiles
1.5 Molecular approaches like metagenomics and whole genome sequencing (WGS) of extremophiles
1.6 Conclusion
Acknowledgments
References
Further reading
2 Physiology of extremophiles
2.1 Introduction
2.2 Taxonomy of extremophiles
2.3 Diversity of extremophiles
2.4 Physiological adaptations of extremophiles
2.4.1 Psychrophiles
2.4.2 Thermophiles
2.4.3 Alkaliphiles
2.4.4 Acidophiles
2.4.5 Halophiles
2.4.6 Peizophiles
2.5 Genomics and evolution
2.6 Chemotaxis in extremophiles
2.7 Conclusions and future directions
Acknowledgments
References
Further reading
3 Mechanism of resistance focusing on copper, mercury and arsenic in extremophilic organisms, how acidophiles and thermophi...
3.1 Introduction
3.2 Mechanism 1—cellular sequestration by thiol systems
3.2.1 Low molecular weight (LMW) thiols
3.2.2 Protein thiols
3.3 Mechanism 2—none thiol, extracellular and intracellular complexation
3.3.1 Nanoparticles
3.3.2 Inorganic polyphosphates
3.4 Mechanism 3—enzymatic detoxification
3.4.1 Copper (Cu)
3.4.2 Mercury
3.4.3 Arsenic
3.5 Mechanism 4—efflux pumps and transporter
3.5.1 Copper
3.5.2 Mercury
3.5.3 Arsenic
3.6 Conclusions and future perspectives
References
4 Halotolerant microbes and their applications in sustainable agriculture
4.1 Introduction
4.2 Halotolerant biota
4.3 Rhizospheric bacteria and plant growth promotion
4.4 Stress alleviation through halotolerant rhizospheric bacteria
4.5 Beneficial attributes of halotolerant PGPR
4.6 Conclusions and future perspective
Acknowledgments
References
5 Halophilic microorganisms: Interesting group of extremophiles with important applications in biotechnology and environment
5.1 Introduction
5.2 Habitats of halophilic microorganisms
5.3 Classification
5.4 Mechanisms of salt adaptation
5.5 Structural characteristics of halophilic proteins
5.6 Current and potential applications of halophiles
5.6.1 Food fermentation
5.6.2 Production of stable enzyme
5.6.3 Production of organic osmotic solutes
5.6.4 Production of biosurfactants and exopolysaccharides
5.6.5 Liposomes production
5.6.6 Processing of halogenated products
5.6.7 Production of alternative energy
5.6.8 Production of polyhydroxyalkanoates (PHA)
5.6.9 Transfer of the halo-tolerance
5.6.10 Production of bacteriorhodopsin with original roles
5.6.11 Important role in the bioremediation
5.7 New molecular and genomic approaches
5.7.1 Development of new genetic tools for halophiles
5.7.2 Genomic and metagenomic sequencing
5.8 Conclusion
References
6 Overview of extremophiles and their food and medical applications
6.1 Introduction: what are extremophiles?
6.2 Adaptations of extremophiles at a molecular level
6.3 Thermophiles: life at high temperature
6.3.1 Habitats and diversity
6.3.2 Physiology and adaptation to high temperature
6.3.3 Thermophiles in medicine and food
6.3.4 Thermophilic enzymes and their applications
6.4 Psychrophiles: life at low temperature
6.4.1 Habitats and diversity
6.4.2 Physiological adaptation to low temperature
6.4.3 Applications of enzymes and metabolites from psychrophiles
6.5 Halophiles
6.5.1 Habitats and diversity
6.5.2 Physiological adaptations to high salt concentration
6.5.3 Medical applications of molecules from halophiles
6.5.4 Halophiles and food products
6.6 Acidophiles
6.6.1 Habitats and diversity
6.6.2 Physiological adaptation to low pH
6.6.3 Food and medicinal relevance of acidophiles
6.7 Alkaliphiles
6.7.1 Habitats and diversity
6.7.2 Physiological adaptation to high pH
6.7.3 Applications of alkaliphile enzymes
6.8 Piezophiles
6.8.1 Habitats and diversity
6.8.2 Physiological adaptation to high pressure
6.9 Radioresistant microorganisms
6.9.1 Diversity and survival strategy
6.9.2 Defense against ultraviolet radiation: sunscreen molecules and their applications
6.10 Xerophiles: life with little or no water
6.11 Metallophiles
6.12 Conclusions
References
7 Applications of extremophiles in astrobiology
7.1 Introduction and historical background
7.2 Study of extremophiles in astrobiology
7.3 Planetary field analogue sites in India and its extremophilic microbial diversity
7.3.1 Lonar lake
7.3.2 Extremophiles from rocks, seawater and intertidal sea zones in arabian sea
7.3.3 Salt deposits and saline systems in Rajasthan, Gujarat and Maharashtra
7.3.4 Mud volcanoes of Andaman
7.3.5 Geothermal hotsprings, cold deserts and glaciers in Leh Ladakh, Himalayas
7.4 Extremophiles from planetary field analogue sites in Europe: astrobiological implications
7.4.1 Rio Tinto, Spain
7.4.2 Ny-Ålesund in Svalbard archipelago of Norway
7.4.3 The Ibn battuta center near Marrakech, Morocco
7.4.4 The Kamchatka Peninsula, Russia
7.4.5 Tirez Lake, Spain
7.5 Micro-organisms in earth’s upper atmosphere and outer space: applications in astrobiology missions
7.6 Extremophiles from space craft assembly room: applications in planetary protection
7.7 Conclusion and future outlook
Acknowledgments
References
Further reading
8 High-pressure adaptation of extremophiles and biotechnological applications
8.1 Introduction
8.2 Effects of pressure on macromolecules and cells
8.2.1 Nucleic acids
8.2.2 Proteins
8.2.3 Phospholipids
8.2.4 Cells
8.3 Pressure adaptation in piezophiles
8.3.1 Genomes
8.3.2 Proteins
8.3.3 Membrane lipids
8.4 Pressure biotechnological applications
8.4.1 Food industry
8.4.1.1 Food preservation
8.4.1.2 Pre-treatment
8.4.2 Allergenicity and digestibility
8.4.3 Medical applications
8.4.3.1 Antiviral vaccines
8.4.3.2 Bacterial ghosts
8.4.3.3 Vaccine preservation
8.4.3.4 Cryopreservation
8.4.4 Biotechnological applications
8.4.4.1 Bio-purification
8.4.4.2 Modulation of cell activity
8.5 Biotechnological applications of piezophiles
8.6 Conclusion and future perspectives
References
9 Fructanogenic halophiles: a new perspective on extremophiles
9.1 Introduction
9.2 Fructans
9.3 Microbial fructan synthesis mechanism
9.4 Fructanogenic halophiles
9.5 Putative GH68 family enzymes of haloarchaea
9.6 Levan and levansucrase from Halomonas smyrnensis AAD6
9.7 Conclusions and future directions
References
10 Applications of sulfur oxidizing bacteria
10.1 Introduction
10.2 Oxidation behavior of sulfur oxidizing bacteria
10.3 Photoautotrophic oxidation
10.4 Chemolithotrophic sulfide oxidation
10.5 Enzyme responsible for sulfur oxidation
10.6 Applications
10.6.1 Sulfur oxidizing bacteria in biogeochemical cycling
10.6.2 Bioleaching
10.6.3 Bioremediation
10.6.4 Biofilteration
10.6.5 Biofertilizers
10.6.6 Bio controlling agent
10.6.7 Deodorization
10.6.8 Rubber recycling
10.6.9 Biosensor
10.7 Conclusions
References
Further reading
11 Physiological and genomic perspective of halophiles among different salt concentrations
11.1 Introduction
11.2 Classification and evolutionary relationships among halophiles
11.3 Mechanism of salt adaptation in halophiles
11.3.1 ‘‘High-salt-in’’ strategy
11.3.2 ‘‘low-salt-in’’ strategy
11.4 Extracellular hydrolytic enzymes from haloarchaea
11.5 Genomic insights into halophilic prokaryotes
11.5.1 Case study of square archaeon Haloquadratumwalsbyi
11.5.2 Case study of Halobacterium sp. NRC-1
11.6 Concluding remarks
References
Further reading
12 CRISPR/Cas system of prokaryotic extremophiles and its applications
12.1 Introduction
12.2 Organization of CRISPR/Cas in bacteria
12.3 Classification of CRISPR cas system
12.3.1 Type I CRISPR system
12.3.2 Type II CRISPR or gRNA-Cas9 complex system
12.3.3 Type III CRISPR system
12.4 CRISPR-Cas system in extremophiles
12.5 CRISPR/Cas system of halophilic archaea
12.6 Delivery methods
12.7 Applications
12.8 Conclusion and future directions
Acknowledgments
References
Further reading
13 Lipases/esterases from extremophiles: main features and potential biotechnological applications
13.1 Introduction
13.2 Structural features and classification of esterases/lipases
13.3 Thermophilic esterases/lipases
13.4 Psychrophilic esterases/lipases
13.5 Other extremophilic esterases/lipases
13.5.1 Halophiles
13.5.2 Alkalophiles/acidophiles
13.6 Running and potential applications for extremophilic esterases/lipases
13.6.1 Detergent
13.6.2 Food
13.6.3 Biodiesel
13.6.4 Drug
13.6.5 Oleochemical
13.6.6 Dairy
13.7 Conclusion and future
References
14 Thermostable Thermoanaerobacter alcohol dehydrogenases and their use in organic synthesis
14.1 Introduction
14.2 Thermoanaerobacter ADHs and their role in physiology
14.3 Structure and thermostability
14.3.1 Structure and binding pocket specificity
14.3.2 Thermal stability of TADHs
14.4 Biocatalysis using thermostable TADHs
14.5 Enzyme improvement
14.5.1 Altering cofactor preference
14.5.2 Altering stereoselectivity
14.5.3 Altering substrate specificity
14.6 Conclusions and future directions
References
15 Biotechnological platforms of the moderate thermophiles, Geobacillus species: notable properties and genetic tools
15.1 Introduction
15.2 Overview of the genus Geobacillus
15.2.1 History
15.2.2 Species placed under the genus Geobacillus
15.2.3 Diverse habitats and their implications
15.2.4 Cellular characterization
15.2.5 Genomic features
15.3 Genetic tools for Geobacillus spp.
15.3.1 Plasmid replicons
15.3.2 Antibiotic resistance markers
15.3.3 Counterselection markers
15.3.4 Recombinant plasmids
15.3.5 Protoplast transformation
15.3.6 Electroporation
15.3.7 Conjugative plasmid transfer
15.3.8 Strategic circumvention of restriction-modification (RM) systems
15.3.9 Genetic elements to control gene expression
15.3.10 Reporter proteins
15.3.11 Protein secretion
15.4 Geobacillus spp. that have potential in whole-cell applications
15.4.1 G. caldoxylosilyticus T20
15.4.2 G. kaustophilus HTA426
15.4.3 G. stearothermophilus ATCC 12978
15.4.4 G. stearothermophilus NUB3621
15.4.5 G. thermocatenulatus 11
15.4.6 G. thermodenitrificans OS27
15.4.7 G. thermodenitrificans T12
15.4.8 G. thermoglucosidasius DSM 2542
15.4.9 G. thermoglucosidasius M10EXG
15.4.10 G. thermoglucosidasius NCIMB 11955
15.4.11 G. thermoglucosidasius NY05
15.4.12 G. thermoglucosidasius PB94A
15.4.13 Geobacillus sp. LC300
15.4.14 Geobacillus sp. XT15
15.5 Conclusion and perspective
Acknowledgments
References
16 Thermophiles and thermophilic hydrolases
16.1 Introduction
16.2 Discovery and diversity of thermophiles
16.3 Thermophilic adaptations
16.3.1 Membrane level adaptations
16.3.2 Genome level adaptations
16.3.3 Proteome level adaptations
16.3.3.1 Amino acid composition
16.3.3.2 Surface and core distribution of amino acids
16.3.3.3 Ion pair networks, hydrogen bonds and aromatic interactions
16.3.3.4 Hydrophobic interactions and disulfide bonds
16.3.3.5 Secondary structures
16.3.3.6 Protein packing and folding
16.4 Thermophilic enzymes
16.4.1 Amylases
16.4.2 Proteases
16.4.3 Cellulases
16.4.4 Xylanases
16.4.5 Lipases
16.5 Conclusion
References
17 Effects of single nucleotide mutations in the genome of multi-drug resistant biofilm producing Pseudomonas aeruginosa
17.1 Introduction
17.2 β-Lactam resistance
17.3 Fluoroquinolone resistance
17.4 Aminoglycoside resistance
17.5 Target efflux pumps (before jumping to each system give 2-3 lines details about this)
17.5.1 MexAB-OprM
17.5.2 MexXY-OprM
17.5.3 MexCD-OprJ
17.5.4 MexEF-OprN
17.6 Antibiotic resistance and bacterial phenotype in biofilm formation
17.7 Conclusion
References
18 Understanding the structural basis of adaptation in enzymes from psychrophiles
18.1 Introduction
18.2 Cold adapted enzymes
18.3 Structure-function relationship of cold adapted enzymes
18.4 Conclusion and future directions
References
19 Molecular and functional characterization of major compatible solute in Deep Sea halophilic actinobacteria of active vol...
19.1 Introduction
19.2 Ectoine – a major compatible solute in halophilic eubacteria
19.3 Physicochemical properties of ectoine
19.4 Osmolytic properties of ectoine
19.5 Biosynthesis of ectoine
19.6 Transport of ectoine
19.7 Industrial production of ectoine
19.8 Biotechnological applications of ectoine
19.8.1 Chemical chaperones for protein folding
19.8.2 Enhancing PCR
19.8.3 Cryo-protection of microorganisms
19.8.4 Use in cosmeceuticals and pharmaceuticals
19.8.5 Generation of stress-resistant transgenic organisms
19.8.6 Ectoine based products in market
19.9 Molecular and functional characterization of ectoine in deep sea halophilic actinobacteria, nocardiopsis alba
19.10 PCR amplification, cloning and sequencing of ectoine biosynthesis genes
19.11 Molecular characterization of ectoine biosynthesis genes
19.12 Sequence analysis of ectA, B and C genes
19.13 Phylogenetic tree construction and analysis of ectoine biosynthesis genes
19.14 Concluding remarks
Acknowledgments
References
20 Antarctic microorganisms as sources of biotechnological products
20.1 Introduction
20.2 Bioprospection of microbial derived bioactive compounds in Antarctica
20.2.1 Enzymes
20.2.1.1 Discovery and purification
20.2.1.2 Activity retention
20.2.1.3 Enzymes for biorefinery and biodiesel production
20.2.1.4 Enzymes for pharmaceuticals and cosmetics production
20.2.1.5 Enzymes for agriculture and brewing
20.2.1.6 Immobilization of Antarctic-derived enzymes
20.2.2 Drug discovery
20.2.2.1 Antimicrobial drug discovery
20.2.2.2 Anticancer drug discovery
20.2.3 Ice-binding proteins
20.3 Nanoparticles
20.3.1 Cadmium nanoparticles
20.3.2 Iron-oxide nanoparticles
20.4 Conclusion and future directions
References
21 The secretomes of extremophiles
21.1 Introduction
21.2 The Sec pathway
21.3 The Tat pathway
21.4 The signal sequence
21.5 Secretomes of archaea
21.6 Conclusion and future directions
References
22 Carbonic anhydrase from extremophiles and their potential use in biotechnological applications
22.1 Extremophiles
22.2 Bacterial carbonic anhydrases
22.3 Carbonic anhydrases in extremophilic bacteria
22.4 Potential use of extreme carbonic anhydrases in biotechnological applications
22.4.1 Biosensors
22.4.2 Artificial lungs
22.4.3 Post-combustion carbon dioxide capture
22.5 SspCA immobilization
22.5.1 Polyurethane foam
22.5.2 Ionic liquid membranes (supported ionic liquid membranes)
22.5.3 Magnetic particles
22.5.4 In vivo immobilization
22.6 Conclusion
References
23 Understanding the protein sequence and structural adaptation in extremophilic organisms through machine learning techniques
23.1 Introduction
23.2 Databases
23.3 Machine learning
23.3.1 Machine learning platforms
23.3.2 Feature extraction and representation
23.3.3 Feature selection
23.3.4 Model performance validation
23.3.4.1 Types of validation method for testing the performance of trained machine learning models
23.3.5 Model performance evaluation metrics
23.4 Statistical analysis for inferring the molecular basis of extremophilic adaptation
23.5 Inferences from preceding methods
23.6 Conclusion
References
24 Exploration of extremophiles genomes through gene study for hidden biotechnological and future potential
24.1 Introduction
24.2 Types and characteristics of extremophiles
24.2.1 Temperature adaptation
24.2.2 pH adaptation
24.2.3 Salt adaptation
24.2.4 Pressure adaptation
24.3 Survival strategy to combat cold stress
24.3.1 Membrane fluidity
24.3.2 Protein synthesis and cold-accustomed protein
24.3.3 Structural adaptation of cold-active enzyme
24.3.4 Mutational study
24.4 Bioactive natural products by extremophiles
24.4.1 Gene study
24.4.2 Bioactive natural products
24.5 Biotechnological use of extremophiles
24.5.1 Polymerase chain reaction
24.5.2 Biomining
24.5.3 Biofuel production
24.5.4 Industrial use
24.5.5 Medicinal aspects
24.6 Conclusion
References
25 The ecophysiology, genetics, adaptive significance, and biotechnology of nickel hyperaccumulation in plants
25.1 Introduction
25.2 Physiology: mechanisms of Ni uptake, translocation, chelation, and storage
25.2.1 Uptake
25.2.2 Chelation
25.2.3 Transport
25.2.4 Localization and storage
25.3 Why hyperaccumulate nickel?
25.3.1 Elemental defense
25.3.2 Nutritional demand
25.3.3 Elemental allelopathy
25.3.4 Drought tolerance
25.4 Genetics of nickel accumulation
25.4.1 Identification of target genes involved in Ni hyperaccumulation by transporters
25.4.2 Identification of target genes involved in Ni hyperaccumulation by chelators
25.5 Phytoremediation and agromining
25.6 Conclusion
Acknowledgments
References
Further Reading
Index

Citation preview

Physiological and Biotechnological Aspects of Extremophiles

Physiological and Biotechnological Aspects of Extremophiles

Edited by Richa Salwan Assistant Professor (Microbiology), Department of Basic Sciences, College of Horticulture and Forestry, (Dr. YSP- University of Horticulture and Forestry), Neri, Hamirpur, Himachal Pradesh, India

Vivek Sharma University Centre For Research and Development, Chandigarh University Punjab, SAS Nagar, India

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www. elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-818322-9 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre G. Wolff Acquisitions Editor: Linda Versteeg-Buschman Editorial Project Manager: Sam W. Young Production Project Manager: Maria Bernard Cover Designer: Miles Hitchen Typeset by MPS Limited, Chennai, India

Contents List of contributors About the editors Preface Acknowledgments

xiii xv xvii xix

Part I Physiological aspects

1

1. Overview of extremophiles

3

Richa Salwan and Vivek Sharma 1.1 1.2 1.3 1.4 1.5

Introduction Eukaryotic extremophiles Prokaryotic extremophiles in diverse habitats Biotechnological potential of extremophiles Molecular approaches like metagenomics and whole genome sequencing (WGS) of extremophiles 1.6 Conclusion Acknowledgments References Further reading

2. Physiology of extremophiles

3 3 4 5

6 7 7 7 11

13

Richa Salwan and Vivek Sharma 2.1 2.2 2.3 2.4

Introduction Taxonomy of extremophiles Diversity of extremophiles Physiological adaptations of extremophiles 2.4.1 Psychrophiles 2.4.2 Thermophiles 2.4.3 Alkaliphiles 2.4.4 Acidophiles 2.4.5 Halophiles 2.4.6 Peizophiles 2.5 Genomics and evolution 2.6 Chemotaxis in extremophiles 2.7 Conclusions and future directions Acknowledgments References Further reading

13 13 14 15 15 17 17 17 17 18 18 18 19 19 19 22

3. Mechanism of resistance focusing on copper, mercury and arsenic in extremophilic organisms, how acidophiles and thermophiles cope with these metals 23 Javiera Norambuena 3.1 Introduction 3.2 Mechanism 1—cellular sequestration by thiol systems 3.2.1 Low molecular weight (LMW) thiols 3.2.2 Protein thiols 3.3 Mechanism 2—none thiol, extracellular and intracellular complexation 3.3.1 Nanoparticles 3.3.2 Inorganic polyphosphates 3.4 Mechanism 3—enzymatic detoxification 3.4.1 Copper (Cu) 3.4.2 Mercury 3.4.3 Arsenic 3.5 Mechanism 4—efflux pumps and transporter 3.5.1 Copper 3.5.2 Mercury 3.5.3 Arsenic 3.6 Conclusions and future perspectives References

4. Halotolerant microbes and their applications in sustainable agriculture

23 24 24 25 25 25 26 26 26 27 27 29 29 30 30 31 31

39

Jayant Kulkarni, Sandeep Sharma, Ashish K. Srivastava and Suprasanna Penna 4.1 Introduction 4.2 Halotolerant biota 4.3 Rhizospheric bacteria and plant growth promotion 4.4 Stress alleviation through halotolerant rhizospheric bacteria

39 40 41 42 v

vi

Contents

4.5 Beneficial attributes of halotolerant PGPR 4.6 Conclusions and future perspective Acknowledgments References

43 45 46 46

5. Halophilic microorganisms: Interesting group of extremophiles with important applications in biotechnology and environment 51 Lobna Daoud and Mamdouh Ben Ali 5.1 5.2 5.3 5.4 5.5

Introduction 51 Habitats of halophilic microorganisms 51 Classification 52 Mechanisms of salt adaptation 53 Structural characteristics of halophilic proteins 54 5.6 Current and potential applications of halophiles 55 5.6.1 Food fermentation 56 5.6.2 Production of stable enzyme 56 5.6.3 Production of organic osmotic solutes 56 5.6.4 Production of biosurfactants and exopolysaccharides 57 5.6.5 Liposomes production 57 5.6.6 Processing of halogenated products 57 5.6.7 Production of alternative energy 57 5.6.8 Production of polyhydroxyalkanoates (PHA) 57 5.6.9 Transfer of the halo-tolerance 58 5.6.10 Production of bacteriorhodopsin with original roles 58 5.6.11 Important role in the bioremediation 59 5.7 New molecular and genomic approaches 60 5.7.1 Development of new genetic tools for halophiles 60 5.7.2 Genomic and metagenomic sequencing 60 5.8 Conclusion 61 References 62

6. Overview of extremophiles and their food and medical applications 65 Jane A. Irwin 6.1 Introduction: what are extremophiles? 6.2 Adaptations of extremophiles at a molecular level 6.3 Thermophiles: life at high temperature 6.3.1 Habitats and diversity

65 66 67 67

6.3.2 Physiology and adaptation to high temperature 6.3.3 Thermophiles in medicine and food 6.3.4 Thermophilic enzymes and their applications 6.4 Psychrophiles: life at low temperature 6.4.1 Habitats and diversity 6.4.2 Physiological adaptation to low temperature 6.4.3 Applications of enzymes and metabolites from psychrophiles 6.5 Halophiles 6.5.1 Habitats and diversity 6.5.2 Physiological adaptations to high salt concentration 6.5.3 Medical applications of molecules from halophiles 6.5.4 Halophiles and food products 6.6 Acidophiles 6.6.1 Habitats and diversity 6.6.2 Physiological adaptation to low pH 6.6.3 Food and medicinal relevance of acidophiles 6.7 Alkaliphiles 6.7.1 Habitats and diversity 6.7.2 Physiological adaptation to high pH 6.7.3 Applications of alkaliphile enzymes 6.8 Piezophiles 6.8.1 Habitats and diversity 6.8.2 Physiological adaptation to high pressure 6.9 Radioresistant microorganisms 6.9.1 Diversity and survival strategy 6.9.2 Defense against ultraviolet radiation: sunscreen molecules and their applications 6.10 Xerophiles: life with little or no water 6.11 Metallophiles 6.12 Conclusions References

7. Applications of extremophiles in astrobiology

68 68 69 69 69 69 70 71 71 71 72 72 73 73 73 74 74 74 74 75 75 75 76 76 76

77 78 78 78 79

89

Rebecca S. Thombre, Parag A. Vaishampayan and Felipe Gomez 7.1 Introduction and historical background 7.2 Study of extremophiles in astrobiology 7.3 Planetary field analogue sites in India and its extremophilic microbial diversity 7.3.1 Lonar lake 7.3.2 Extremophiles from rocks, seawater and intertidal sea zones in arabian sea

89 90 91 91 91

Contents

7.3.3 Salt deposits and saline systems in Rajasthan, Gujarat and Maharashtra 7.3.4 Mud volcanoes of Andaman 7.3.5 Geothermal hotsprings, cold deserts and glaciers in Leh Ladakh, Himalayas 7.4 Extremophiles from planetary field analogue sites in Europe: astrobiological implications 7.4.1 Rio Tinto, Spain ˚ lesund in Svalbard 7.4.2 Ny-A archipelago of Norway 7.4.3 The Ibn battuta center near Marrakech, Morocco 7.4.4 The Kamchatka Peninsula, Russia 7.4.5 Tirez Lake, Spain 7.5 Micro-organisms in earth’s upper atmosphere and outer space: applications in astrobiology missions 7.6 Extremophiles from space craft assembly room: applications in planetary protection 7.7 Conclusion and future outlook Acknowledgments References Further reading

94 95

95

96 96 96 97 97 97

98

123

Gu¨lbahar Abaramak, Onur Kırtel and ¨ ner Ebru Toksoy O 9.1 9.2 9.3 9.4 9.5

Introduction Fructans Microbial fructan synthesis mechanism Fructanogenic halophiles Putative GH68 family enzymes of haloarchaea 9.6 Levan and levansucrase from Halomonas smyrnensis AAD6 9.7 Conclusions and future directions References

10. Applications of sulfur oxidizing bacteria

123 123 124 125 125 127 128 128

131

Kavita Rana, Neerja Rana and Birbal Singh 99 99 100 100 104

8. High-pressure adaptation of extremophiles and biotechnological applications 105 M. Salvador-Castell, P. Oger and J. Peters 8.1 Introduction 8.2 Effects of pressure on macromolecules and cells 8.2.1 Nucleic acids 8.2.2 Proteins 8.2.3 Phospholipids 8.2.4 Cells 8.3 Pressure adaptation in piezophiles 8.3.1 Genomes 8.3.2 Proteins 8.3.3 Membrane lipids 8.4 Pressure biotechnological applications 8.4.1 Food industry 8.4.2 Allergenicity and digestibility 8.4.3 Medical applications 8.4.4 Biotechnological applications 8.5 Biotechnological applications of piezophiles 8.6 Conclusion and future perspectives References

9. Fructanogenic halophiles: a new perspective on extremophiles

vii

105 105 106 107 107 108 109 109 109 111 113 113 113 114 114 115 115 116

10.1 Introduction 10.2 Oxidation behavior of sulfur oxidizing bacteria 10.3 Photoautotrophic oxidation 10.4 Chemolithotrophic sulfide oxidation 10.5 Enzyme responsible for sulfur oxidation 10.6 Applications 10.6.1 Sulfur oxidizing bacteria in biogeochemical cycling 10.6.2 Bioleaching 10.6.3 Bioremediation 10.6.4 Biofilteration 10.6.5 Biofertilizers 10.6.6 Bio controlling agent 10.6.7 Deodorization 10.6.8 Rubber recycling 10.6.9 Biosensor 10.7 Conclusions References Further reading

11. Physiological and genomic perspective of halophiles among different salt concentrations

131 131 132 132 133 133 133 133 133 134 134 134 134 135 135 135 135 136

137

Ashish Verma, Sachin Kumar and Preeti Mehta 11.1 Introduction 11.2 Classification and evolutionary relationships among halophiles

137 137

viii

Contents

11.3 Mechanism of salt adaptation in halophiles 11.3.1 ‘‘High-salt-in’’ strategy 11.3.2 ‘‘low-salt-in’’ strategy 11.4 Extracellular hydrolytic enzymes from haloarchaea 11.5 Genomic insights into halophilic prokaryotes 11.5.1 Case study of square archaeon Haloquadratumwalsbyi 11.5.2 Case study of Halobacterium sp. NRC-1 11.6 Concluding remarks References Further reading

Part II Biotechnological aspects 12. CRISPR/Cas system of prokaryotic extremophiles and its applications

139 140 141 143 143 143 145 146 146 151

176 176 176 176 176 177 178 178 178 178 178 179

14. Thermostable Thermoanaerobacter alcohol dehydrogenases and their use in organic synthesis 183

153 155

Richa Salwan, Anu Sharma and Vivek Sharma 12.1 Introduction 155 12.2 Organization of CRISPR/Cas in bacteria 157 12.3 Classification of CRISPR cas system 158 12.3.1 Type I CRISPR system 159 12.3.2 Type II CRISPR or gRNA-Cas9 complex system 160 12.3.3 Type III CRISPR system 160 12.4 CRISPR-Cas system in extremophiles 160 12.5 CRISPR/Cas system of halophilic archaea 161 12.6 Delivery methods 161 12.7 Applications 161 12.8 Conclusion and future directions 162 Acknowledgments 163 References 163 Further reading 168

13. Lipases/esterases from extremophiles: main features and potential biotechnological applications 169 Valentina De Luca and Luigi Mandrich 13.1 Introduction 13.2 Structural features and classification of esterases/lipases 13.3 Thermophilic esterases/lipases 13.4 Psychrophilic esterases/lipases

13.5 Other extremophilic esterases/lipases 13.5.1 Halophiles 13.5.2 Alkalophiles/acidophiles 13.6 Running and potential applications for extremophilic esterases/lipases 13.6.1 Detergent 13.6.2 Food 13.6.3 Biodiesel 13.6.4 Drug 13.6.5 Oleochemical 13.6.6 Dairy 13.7 Conclusion and future References

169 170 173 175

¨ rlygsson Sean M. Scully and Jo´hann O 14.1 Introduction 14.2 Thermoanaerobacter ADHs and their role in physiology 14.3 Structure and thermostability 14.3.1 Structure and binding pocket specificity 14.3.2 Thermal stability of TADHs 14.4 Biocatalysis using thermostable TADHs 14.5 Enzyme improvement 14.5.1 Altering cofactor preference 14.5.2 Altering stereoselectivity 14.5.3 Altering substrate specificity 14.6 Conclusions and future directions References

183 183 185 185 186 187 188 189 189 190 190 191

15. Biotechnological platforms of the moderate thermophiles, Geobacillus species: notable properties and genetic tools 195 Keisuke Wada and Hirokazu Suzuki 15.1 Introduction 15.2 Overview of the genus Geobacillus 15.2.1 History 15.2.2 Species placed under the genus Geobacillus 15.2.3 Diverse habitats and their implications 15.2.4 Cellular characterization 15.2.5 Genomic features 15.3 Genetic tools for Geobacillus spp. 15.3.1 Plasmid replicons

195 196 196 196 197 200 200 200 201

Contents

15.3.2 15.3.3 15.3.4 15.3.5 15.3.6 15.3.7 15.3.8

Antibiotic resistance markers Counterselection markers Recombinant plasmids Protoplast transformation Electroporation Conjugative plasmid transfer Strategic circumvention of restriction-modification (RM) systems 15.3.9 Genetic elements to control gene expression 15.3.10 Reporter proteins 15.3.11 Protein secretion 15.4 Geobacillus spp. that have potential in whole-cell applications 15.4.1 G. caldoxylosilyticus T20 15.4.2 G. kaustophilus HTA426 15.4.3 G. stearothermophilus ATCC 12978 15.4.4 G. stearothermophilus NUB3621 15.4.5 G. thermocatenulatus 11 15.4.6 G. thermodenitrificans OS27 15.4.7 G. thermodenitrificans T12 15.4.8 G. thermoglucosidasius DSM 2542 15.4.9 G. thermoglucosidasius M10EXG 15.4.10 G. thermoglucosidasius NCIMB 11955 15.4.11 G. thermoglucosidasius NY05 15.4.12 G. thermoglucosidasius PB94A 15.4.13 Geobacillus sp. LC300 15.4.14 Geobacillus sp. XT15 15.5 Conclusion and perspective Acknowledgments References

16. Thermophiles and thermophilic hydrolases

201 202 202 204 204 204

205 206 206 207 207 208 208 208 208 209 209 209 209 210 210 210 210 211 211 211 211 211

219

Shilpi Ghosh, Khusboo Lepcha, Arijita Basak and Ayan Kumar Mahanty 16.1 Introduction 16.2 Discovery and diversity of thermophiles 16.3 Thermophilic adaptations 16.3.1 Membrane level adaptations 16.3.2 Genome level adaptations 16.3.3 Proteome level adaptations 16.4 Thermophilic enzymes 16.4.1 Amylases 16.4.2 Proteases 16.4.3 Cellulases 16.4.4 Xylanases 16.4.5 Lipases 16.5 Conclusion References

219 219 220 220 220 223 225 225 226 227 227 228 229 229

ix

17. Effects of single nucleotide mutations in the genome of multi-drug resistant biofilm producing Pseudomonas aeruginosa 237 Sanjay Gunabalan, Chew Jactty and Babu Ramanathan 17.1 17.2 17.3 17.4 17.5

Introduction β-Lactam resistance Fluoroquinolone resistance Aminoglycoside resistance Target efflux pumps (before jumping to each system give 2-3 lines details about this) 17.5.1 MexAB-OprM 17.5.2 MexXY-OprM 17.5.3 MexCD-OprJ 17.5.4 MexEF-OprN 17.6 Antibiotic resistance and bacterial phenotype in biofilm formation 17.7 Conclusion References

237 239 239 239

239 240 240 240 240 240 241 241

18. Understanding the structural basis of adaptation in enzymes from psychrophiles 245 Mahejibin Khan 18.1 Introduction 18.2 Cold adapted enzymes 18.3 Structure-function relationship of cold adapted enzymes 18.4 Conclusion and future directions References

245 245 246 249 249

19. Molecular and functional characterization of major compatible solute in Deep Sea halophilic actinobacteria of active volcanic Barren Island, Andaman and Nicobar Islands, India 253 Balakrishnan Meena, Lawrance Anburajan, Nambali Valsalan Vinithkumar, Ramalingam Kirubagaran and Gopal Dharani 19.1 Introduction 19.2 Ectoine a major compatible solute in halophilic eubacteria 19.3 Physicochemical properties of ectoine 19.4 Osmolytic properties of ectoine 19.5 Biosynthesis of ectoine 19.6 Transport of ectoine 19.7 Industrial production of ectoine

253 255 256 256 256 258 258

x

Contents

19.8 Biotechnological applications of ectoine 259 19.8.1 Chemical chaperones for protein folding 259 19.8.2 Enhancing PCR 259 19.8.3 Cryo-protection of microorganisms 259 19.8.4 Use in cosmeceuticals and pharmaceuticals 260 19.8.5 Generation of stress-resistant transgenic organisms 260 19.8.6 Ectoine based products in market 260 19.9 Molecular and functional characterization of ectoine in deep sea halophilic actinobacteria, nocardiopsis alba 261 19.10 PCR amplification, cloning and sequencing of ectoine biosynthesis genes 262 19.11 Molecular characterization of ectoine biosynthesis genes 262 19.12 Sequence analysis of ectA, B and C genes 263 19.13 Phylogenetic tree construction and analysis of ectoine biosynthesis genes 263 19.14 Concluding remarks 264 Acknowledgments 264 References 264

20. Antarctic microorganisms as sources of biotechnological products

269

Tarcı´sio Correa and Fernanda Abreu 20.1 Introduction 20.2 Bioprospection of microbial derived bioactive compounds in Antarctica 20.2.1 Enzymes 20.2.2 Drug discovery 20.2.3 Ice-binding proteins 20.3 Nanoparticles 20.3.1 Cadmium nanoparticles 20.3.2 Iron-oxide nanoparticles 20.4 Conclusion and future directions References

269 269 270 275 279 280 280 280 281 281

21. The secretomes of extremophiles

285

Eyad Kinkar and Mazen Saleh 21.1 Introduction 21.2 The Sec pathway 21.3 The Tat pathway

285 285 287

21.4 The signal sequence 21.5 Secretomes of archaea 21.6 Conclusion and future directions References

287 288 292 293

22. Carbonic anhydrase from extremophiles and their potential use in biotechnological applications 295 Claudiu T. Supuran and Clemente Capasso 22.1 Extremophiles 295 22.2 Bacterial carbonic anhydrases 295 22.3 Carbonic anhydrases in extremophilic bacteria 296 22.4 Potential use of extreme carbonic anhydrases in biotechnological applications 298 22.4.1 Biosensors 298 22.4.2 Artificial lungs 298 22.4.3 Post-combustion carbon dioxide capture 298 22.5 SspCA immobilization 299 22.5.1 Polyurethane foam 299 22.5.2 Ionic liquid membranes (supported ionic liquid membranes) 300 22.5.3 Magnetic particles 300 22.5.4 In vivo immobilization 300 22.6 Conclusion 303 References 304

23. Understanding the protein sequence and structural adaptation in extremophilic organisms through machine learning techniques 307 Abhigyan Nath and S. Karthikeyan 23.1 Introduction 23.2 Databases 23.3 Machine learning 23.3.1 Machine learning platforms 23.3.2 Feature extraction and representation 23.3.3 Feature selection 23.3.4 Model performance validation 23.3.5 Model performance evaluation metrics 23.4 Statistical analysis for inferring the molecular basis of extremophilic adaptation 23.5 Inferences from preceding methods 23.6 Conclusion References

307 307 308 308 308 309 309 310

310 311 312 313

Contents

24. Exploration of extremophiles genomes through gene study for hidden biotechnological and future potential 315 Pijush Basak, Arpita Biswas and Maitree Bhattacharyya 24.1 Introduction 315 24.2 Types and characteristics of extremophiles 316 24.2.1 Temperature adaptation 316 24.2.2 pH adaptation 316 24.2.3 Salt adaptation 317 24.2.4 Pressure adaptation 317 24.3 Survival strategy to combat cold stress 317 24.3.1 Membrane fluidity 317 24.3.2 Protein synthesis and cold-accustomed protein 318 24.3.3 Structural adaptation of cold-active enzyme 318 24.3.4 Mutational study 318 24.4 Bioactive natural products by extremophiles 318 24.4.1 Gene study 319 24.4.2 Bioactive natural products 319 24.5 Biotechnological use of extremophiles 320 24.5.1 Polymerase chain reaction 320 24.5.2 Biomining 320 24.5.3 Biofuel production 321 24.5.4 Industrial use 321 24.5.5 Medicinal aspects 322 24.6 Conclusion 322 References 323

xi

25. The ecophysiology, genetics, adaptive significance, and biotechnology of nickel hyperaccumulation in plants 327 Anthony L. Ferrero, Peter R. Walsh and Nishanta Rajakaruna 25.1 Introduction 327 25.2 Physiology: mechanisms of Ni uptake, translocation, chelation, and storage 328 25.2.1 Uptake 328 25.2.2 Chelation 328 25.2.3 Transport 329 25.2.4 Localization and storage 331 25.3 Why hyperaccumulate nickel? 332 25.3.1 Elemental defense 332 25.3.2 Nutritional demand 333 25.3.3 Elemental allelopathy 333 25.3.4 Drought tolerance 334 25.4 Genetics of nickel accumulation 334 25.4.1 Identification of target genes involved in Ni hyperaccumulation by transporters 335 25.4.2 Identification of target genes involved in Ni hyperaccumulation by chelators 336 25.5 Phytoremediation and agromining 336 25.6 Conclusion 339 Acknowledgments 340 References 340 Further reading 347 Index

349

List of contributors Gu¨lbahar Abaramak Bioengineering Department, IBSB—Industrial Biotechnology and Systems Biology Research Group, Marmara University, Istanbul, Turkey

Anthony L. Ferrero Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA, United States

Fernanda Abreu Paulo de Go´es Microbiology Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Shilpi Ghosh Department of Biotechnology, University of North Bengal, Raja Rammohunpur, Siliguri, India

Lawrance Anburajan Atal Centre for Ocean Science and Technology for Islands, National Institute of Ocean Technology, Port Blair, India

Felipe Gomez Instituto Aeroespacial (INTA) (CAB), Madrid, Spain

Arijita Basak Department of Biotechnology, University of North Bengal, Raja Rammohunpur, Siliguri, India

Sanjay Gunabalan Department of Biological Sciences, School of Science and Technology, Sunway University, Kuala Lumpur, Malaysia

Pijush Basak Jagadis Bose National Science Talent Search, Kolkata, India Mamdouh Ben Ali Laboratory of Microbial Biotechnology and Enzyme Engineering (LBMIE), Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia; Astrum Biotech, Business incubator, Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia Maitree Bhattacharyya Jagadis Bose National Science Talent Search, Kolkata, India; Department of Biochemistry, University of Calcutta, Kolkata, India Arpita Biswas Department of Biochemistry, University of Calcutta, Kolkata, India Clemente Capasso Istituto di Bioscienze e Biorisorse, CNR, Napoli, Italy Tarcı´sio Correa Paulo de Go´es Microbiology Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Lobna Daoud Laboratory of Microbial Biotechnology and Enzyme Engineering (LBMIE), Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia; Astrum Biotech, Business incubator, Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia

Nacional de Te´cnica Centro de Astrobiologı´a

Jane A. Irwin Veterinary Sciences Centre, School of Veterinary Medicine, University College Dublin, Dublin, Ireland Chew Jactty Department of Biological Sciences, School of Science and Technology, Sunway University, Kuala Lumpur, Malaysia S.

Karthikeyan Department of Computer Science, Institute of Science, Banaras Hindu University, Varanasi, India

Mahejibin Khan CSIR-Central Food Technological Research Institute-Resource Centre, Lucknow, India Eyad Kinkar Department of Biology, University, Sudbury, ON, Canada

Laurentian

Onur Kırtel Bioengineering Department, IBSB— Industrial Biotechnology and Systems Biology Research Group, Marmara University, Istanbul, Turkey Ramalingam Kirubagaran Marine Biotechnology Division, Ocean Science and Technology for Islands Group, Ministry of Earth Sciences, Government of India, Chennai, India

Valentina De Luca Institute of Protein Biochemistry, National Research Council, Naples, Italy

Jayant Kulkarni Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India; Department of Botany, Savitribai Phule Pune University, Pune, India

Gopal Dharani Marine Biotechnology Division, Ocean Science and Technology for Islands Group, Ministry of Earth Sciences, Government of India, Chennai, India

Sachin Kumar Microbial Type Culture Collection & Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh, India xiii

xiv

List of contributors

Khusboo Lepcha Department of Biotechnology, University of North Bengal, Raja Rammohunpur, Siliguri, India; Department of Microbiology, University of North Bengal, Raja Rammohunpur, Siliguri, India Ayan Kumar Mahanty Department of Biotechnology, University of North Bengal, Raja Rammohunpur, Siliguri, India

Sean M. Scully Department of Natural Resource Sciences, University of Akureyri, Akureyri, Iceland Anu Sharma University Centre for Research and Development, Chandigarh University, Chandigarh, India

Luigi Mandrich Research Institute on Terrestrial Ecosystem, National Research Council, Naples, Italy

Sandeep Sharma CSIR-Central Salt & Marine Chemicals Research Institute, Bhavnagar, India; Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India

Balakrishnan Meena Atal Centre for Ocean Science and Technology for Islands, National Institute of Ocean Technology, Port Blair, India

Vivek Sharma University Centre for Research and Development, Chandigarh University, Chandigarh, India

Preeti Mehta DBT-IOC Centre for Advanced Bioenergy Research, Research & Development Centre, Indian Oil Corporation Limited, Sector-13, Faridabad, India

Birbal Singh Indian Veterinary Research InstituteRegional Station, Palampur, India

Abhigyan Nath Department of Biochemistry, Pt. Jawahar Lal Nehru Memorial Medical College, Raipur, India Javiera Norambuena Rutgers Brunswick, NJ, United States

University,

New

P. Oger Universite´ de Lyon, CNRS, UMR, Villeurbanne, France ¨ rlygsson Department of Natural Resource Jo´hann O Sciences, University of Akureyri, Akureyri, Iceland Suprasanna Penna Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India; Homi Bhabha National Institute, Mumbai, India J. Peters Universite´ Grenoble Alpes, LiPhy, Grenoble, France; Institut Laue Langevin, Grenoble, France Nishanta Rajakaruna Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA, United States; Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa Babu Ramanathan Department of Biological Sciences, School of Science and Technology, Sunway University, Kuala Lumpur, Malaysia Kavita Rana University of Horticulture and Forestry, Nauni, India Neerja Rana University of Horticulture and Forestry, Nauni, India Mazen Saleh Department of Biology, University, Sudbury, ON, Canada

Laurentian

Ashish K. Srivastava Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India; Homi Bhabha National Institute, Mumbai, India Claudiu T. Supuran Sezione di Scienze Farmaceutiche, Dipartimento Neurofarba, Universita` degli Studi di Firenze, Florence, Italy Hirokazu Suzuki Faculty of Engineering, Tottori University, Tottori, Japan; Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan Rebecca S. Thombre Department of Biotechnology, Modern College of Arts, Science and Commerce, Pune, India; School of Physical Sciences, University of Kent, Canterbury, United Kingdom ¨ ner Bioengineering Department, IBSB— Ebru Toksoy O Industrial Biotechnology and Systems Biology Research Group, Marmara University, Istanbul, Turkey Parag A. Vaishampayan Biotechnology and Planetary Protection Group, NASA-Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, United States Ashish Verma Microbial Type Culture Collection & Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh, India Nambali Valsalan Vinithkumar Atal Centre for Ocean Science and Technology for Islands, National Institute of Ocean Technology, Port Blair, India

M. Salvador-Castell Universite´ de Lyon, CNRS, UMR, Villeurbanne, France

Keisuke Wada Research Institute for Sustainable Chemistry, Department of Materials and Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Hiroshima, Japan

Richa Salwan Department of Basic Sciences, College of Horticulture and Forestry (Dr. YSP - University of Horticulture and Forestry), Neri, Hamirpur (HP), India

Peter R. Walsh Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA, United States

About the editors Dr. Salwan is presently working as Assistant Professor (Microbiology) at College of Horticulture and Forestry (Dr. YS Parmar-University of Horticulture and Forestry), Neri, Hamirpur. She has also worked as DST-Young Scientist under SYST scheme of DST. She has made tangible contribution in exploring actinobacteria with promising plant growth promoting attributes as well as molecular mechanisms underlying biocontrol attributes of Trichoderma. She is able to publish several research articles on the same aspect. Moreover, she has been awarded National-Post Doctoral Fellowship by SERB-DST in 2015 and 2017. Dr. Salwan has completed her PhD in Biological Sciences from Academy of Scientific & Innovative Research (AcSIR) at CSIR-Institute of Himalayan Bioresource Technology, Palampur, India. She has made her contribution on diversity of psychrotrophic bacteria from Western Himalayas and their utilization for industrial applications. Her research work on the exploration of psychrotrophic protease producing microbes from the cold deserts of Lahaul and Spiti in the Indian Western Himalayas has revealed the prevalence of industrially important microbes which are still under explored. She has published many research articles in international journals of repute. She has also published book chapters and presented her research in international conferences. She is working on the exploration of extremophiles for agriculturally and industrially important attributes. Dr. Sharma is working on molecular aspects of plant beneficial microbes. He has published several research papers in journals including International Journal of Biological macromolecules, Frontiers in Microbiology, European Journal of Plant Pathology, Current Microbiology, Pesticide Biochemistry and Physiology. He is in editorial board of journals like PLoS One, Frontiers in Bioengineering and Biotechnology and Chemical and Biological Technologies in Agriculture. Besides this, he is a recognized reviewer in dozens of international journals such as MDPI Pathogens, Molecular Biotechnology, Folia Microbiology, Physiological and Molecular Plant Pathology and Archives of Microbiology. Dr. Vivek Sharma did his PhD from CSIR-Institute of Himalayan Bioresource Technology, Palampur, India. Dr. Sharma has qualified CSIR-UGC Junior Research Fellowship and awarded DST-Young Scientist under Fast Track Scheme of DST. Dr. Sharma has also been selected for ARO Post Doctoral Fellowship at Israel in 2017 18. He is presently working as Assistant Professor in University Centre for Research and Development at Chandigarh University, Punjab. He is having research experience of more than 12 years in exploring molecular attributes of Trichoderma involved in different plant benefits.

Richa Salwan1 and Vivek Sharma2 1

Department of Basic Sciences, College of Horticulture and Forestry, Dr. YSP- University of Horticulture and Forestry, Neri, Hamirpur, India, 2

University Centre for Research and Development, Chandigarh University, Mohali, India

xv

Preface The present book on “Physiological and Biotechnological Aspects of Extremophiles” describes the role and importance of extremophiles under extreme conditions. Extremophiles can be found in all domains including Bacteria, Archaea and Eukarya and adapt themselves to survive in extremities which are important for evolutionary differences across different ecological areas. Extremophilic organisms are blessed with ability to tolerate and survive extremes of pH, salt, pressure, heavy metals, organic solvents, and growth in presence of toxic wastes and other habitats which are harsh for normal survival. Extremophiles are differentiated into a variety of groups including psychrophiles, psychrotrophs and thermophiles, alkaliphiles and acidophiles, peizophiles, metal and radiation tolerant. Different extremophiles are source of inspiration and often explored for getting a deep insight into the physiological adaptations. Further, the genomic cockpit of these microbes is known to encode information which helps these microbes to survive under harsh conditions. Extremophiles have vital importance in industrial applications as they are known for the production of enzymes and metabolites produced under extremes of environmental conditions. These enzymes and metabolites are being applied in detergent, food, leather, paper and pulp, pharmaceutical, textile and agricultural industries. These metabolic products offer properties such as high stability and catalytic efficiency, high salt and alkalinity, oxidant and bleach stability, low water activity and shelf life. The higher catalytic efficiency of their encoding gene products offers industrial advantages over their contemporaries under normal environmental conditions. The thrust for these microbes offers vast potential in present climatic scenario for developing efficient processes. The recent advancements in technology like whole genome sequencing and gene/ genome editing coupled to bioinformatic tools have enhanced the pace of mining microbial diversity of extremophiles and their genome plasticity for human welfare. The book comprises of 25 chapters that covers both physiological and biotechnological aspects of extremophiles. Chapters on physiological aspects like mechanisms and adaptations of metal tolerance, halotolerant, peizophiles, marine and Antarctic microbes are included. On the other hand, biotechnological aspects cover role of extremophiles in the production of enzymes such as lipases, carbonic anhydrases and thermophilic hydrolases as well as advances in molecular tools such as CRISPR-Cas, metagenomics, SNPs in Pseudomonas and adaptations in plants including Nickel Hyperaccumulation. Written with the cooperation of leading international experts with already published research and a strong background in relevant field from academia, government institutions or industries, it will contribute towards interdisciplinary knowledge and a common resource platform on extremophiles at global level. Overall, this book volume seeks to spur the role of extremophiles in bioremediation, industries and ecosystems. The book will be beneficial to the scientific community including students pursuing their doctoral studies, researchers and scientists working in the area of Microbiology in various research institutions and academic Universities.

xvii

Acknowledgments This book is the first of its kind to offer a comprehensive and up-to-date discussion on several aspects of extremphiles. It describes the adaptations underlying survival mechanisms under extreme conditions, diversity of extremophiles and their applications in various industrial processes. This is the first book edited by the authors and we are as dependent as ever on the wisdom of others, begins one, and another, plucked at random from a Barnes & Noble new-arrival shelf: The creation of this book has removed any notion I may have had of it being a solo endeavor. I express my thanks to God for providing me his Holy Spirit and covering all my sins. Dr. Richa extends her heart-felt gratitude to her soul mate for continuous motivation to come forward and bringing this book as an execution. This book was not possible without his valuable inputs, suggestions and kind support. He always encouraged independent and critical thinking, and shared his valuable research and professional insight to inculcate in me the attributes of an independent researcher. Working with him has been a great experience in learning. We acknowledge all the authors residing in India and Abroad who have contributed wonderful chapters for the successful publication of this book. The chapters contributed by the authors residing in countries including Brazil, Canada, France, Iceland, Ireland, Italy, Japan, Tunisia, Turkey and United States are highly acknowledged for timely submission. We are highly thankful to Elsevier Editorial Team members for their generous and constant support in finalizing this book and other content. Special thanks are due to Samuel Young and Swapna Praveen for timely intimation, suggestions and planning of tasks. My sincere thanks are due to Mr. Samuel Young for spending his time for repeated plagiarism check for every chapter. The Editors of this book Dr. Richa Salwan and Dr. Vivek Sharma do acknowledge Science for Equity, Empowerment and Development (SEED) Division, Department of Science and Technology, India for the financial support provided under the project SP/YO/125/2017.

xix

Chapter 1

Overview of extremophiles Richa Salwan1 and Vivek Sharma2 1

Department of Basic Sciences, College of Horticulture and Forestry (Dr. YSP - University of Horticulture and Forestry), Neri, Hamirpur (HP),

India, 2University Centre for Research and Development, Chandigarh University, Chandigarh, India

1.1

Introduction

Extremophiles are living organisms that have the ability to grow under conditions where normal organisms are not able to survive. These extremophilic organisms are always attracted towards conditions like extremely high and low temperature, extreme acidic or basic pH, high exposure to radiations, high salinity, low and high pressure, growth in presence of toxic wastes, organic solvents, heavy metals and other habitats which are harsh for normal survival. According to the growth conditions, extremophiles are categorized into extremophilic and extremotolerant organisms. The extremophilic category includes organisms which have the ability to grow under one or more extreme environmental conditions whereas extremotolerant includes organisms that normally grow under optimal conditions but can also survive on exposure to extreme environmental conditions [1]. The extremotolerant organisms are also known as extremotrophs [2]. Besides this, there are organisms which can tolerate more than one extreme condition like extreme temperature and pH, radiations, metals etc are known as polyextremophiles. Extremophiles include prokaryotic bacteria and archaea as well as eukaryotic organisms. Among prokaryotes, most archaebacteria are extremophilic because of their high versatility and adaptive behavior towards extreme conditions. These archaea are salt loving, high temperature and acid tolerant, and strictly anaerobic. Archaea such as Pyrolobus fumarii are also known as hyperthermophiles as they can tolerate temperature up to 121
C whereas bacteria Geothermobacterium ferrireducens can tolerate up to 95
C [3,4]. Archaebacteria identified as Methanopyrus kandleri and Picrophilus torridus grow at high temperature 122
C and 0.06 pH, respectively. Similarly, cyanobacteria are highly adaptive in combating extreme environmental conditions by forming mats with other organisms. These cyanobacteria can tolerate extremes of salt and metal concentrations, alkalinity and less water in dry areas but can’t tolerate low pH conditions [1]. Gloeocapsa spp. is an extremotolerant which can withstand extreme conditions in space such as temperature shifts, radiation and vacuum exposure. Similarly, spiral shaped Helicobacter pylori can survive extreme acidic environment of stomach. Previously, extremophile term was used to include organisms which are unicellular and prokaryotic but studies have reported that all extremophiles are not unicellular organisms [5].

1.2

Eukaryotic extremophiles

Eukaryotic multicellular organisms including fungi have well adapted physiology to survive in extreme conditions. Various microorganisms such as Chlamydomonas, Dunaliella, Euglena and Ochromonas, zooplankton, fungi and protists can grow and tolerate acidic and metal-rich conditions [611]. The fungal species can also thrive in acidic and alkaline environments, salt and metal tolerant but they cannot tolerate extremely high temperatures as they do not grow above 60
C [12]. Species of Exophiala and Cladophialophora have the capacity to metabolize hydrocarbons to obtain energy and survive in polluted environments [13]. Micro-algae can also withstand extremophilic conditions as they are resistant towards light, high temperature, acidic or alkaline pH, CO2 and metal concentration [14]. In similar studies, a red algae Cyanidioschyzon merolae can adapt to extreme environmental conditions by regulating the expression of 35% genes in blue and red light [15]. Moreover, lichens such as Usnea antarctica and Umbilicaria cylindrica representing algal and fungal association can also tolerate extremes of low temperatures [16]. These lichens have the ability to do photosynthesis at subzero temperature and protects photosystems from damage [17]. Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00001-0 © 2020 Elsevier Inc. All rights reserved.

3

4

PART | I Physiological aspects

Eukaryotic diversity has also been reported in acid mine drainage and certain aquatic environments [8,1821]. A microscopic invertebrate Tardigrade also known as polyextremophilic organism can tolerate 2272
C temperature, dry and dehydrated conditions, high pressure and radiation exposure. Tardigrades undergo a process known as cryptobiosis to survive in extreme environmental conditions by suspending their metabolism. Tardigrades can survive in such extreme conditions for several years and become active during the onset of favorable environmental conditions [22]. Similarly, Artemia salina also known as Sea Monkey can survive in extreme of salt concentrations. Poecilia mexicana, a viviparous teleost can grow in environment when there are low oxygen availability and high hydrogen sulfide concentrations [23].

1.3

Prokaryotic extremophiles in diverse habitats

Microorganisms constitute the major component of the earth’s biodiversity. The species biodiversity under extreme conditions such as hot and springs, saline and alkaline lakes, hot and cold deserts, and ocean beds is mainly limited to microbes as these extremes are harsh for the existence of life. Even in space where harsh environment like extreme radiation, extreme temperatures, altered gravity, extreme salinity and nutrients restrict the growth of other organisms but do allow the growth of these microbes. Nearly 70% of the earth’s biosphere like Arctic, Antarctic, and moderately cold regions are having temperature below 5 oC [2427]. Such cold environments are occupied by microorganisms cat% Psychrophiles are known to show optimum growth at or below 15
C egorized into psychrophiles and psychrotrophs. but are able to show growth maximum and minimum growth within 020
C. Psychrotrophs show optimum growth at or above 20
C but even tolerate a temperature below 5
C [2830]. Psychrophilic microorganisms inhabit environments such as deep seas mountains and Polar Regions which are permanently cold whereas psychrotrophs inhabit environments where temperature fluctuates [29,3134]. Similarly, thermophilic microorganisms are found in hydrothermal vents, hot springs and heated mud flats. Extremely thermophilic Thermus thermophilus, Thermoanaerobacter tengcongensis and Thermotoga maritima have wide biotechnological potential and importance in studying structural biology [3537]. Similarly, bacteria such as Pyrodictium abyssi can survive in hot boiling water [38] and Desulforudis audaxviator which lives in groundwater below the Earth’s surface. These microbes can survive without oxygen, light and can resist heat. Besides hot and cold areas, there are certain environments which have high salinity, high pressure, low water content, high radiations exposure and metals ions which are also considered as extreme environments (Fig. 1.1). Extremophilic microbes that can tolerate high metal concentrations such as arsenic, cadmium, copper and zinc are generally adapted to acidic environments, hot springs or from bio-oxidation/bioleaching processes where metal concentration is high.

Xerophiles Radioresistant Oligotrophs Endoliths & hypoliths

Peizophiles (70 to 120 MPa)

Acidophiles (at or below 3–4) Alkaliphiles (above 10)

Metal tolerant (copper, cadmium, arsenic, and zinc)

Psychrophiles (0 to 20 °C) Extremophiles Thermophiles (above 40 to 122 °C)

Health-related applications such as bio-pharmaceuticals

Red

Gray

Halophiles (0.5 to 5 M NaCl)

White

Environmental applications such as bioremediation

Enzymes with application in industrial processes

FIGURE 1.1 Categories of microorganisms including psychrophiles, thermophiles, halophiles, alkaliphiles, acidophiles, peizophiles, metal tolerance and others based on temperature, pH, salt, pressure, metal and other type of stress conditions. The metabolic products of these extremophiles find applications in red, white and gray biotechnology.

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Extremophiles possess many systems that are common to all microbes likewise Acidithiobacillus ferrooxidans showed resistance towards heavy metals cadmium and copper, Thermus thermophilus towards mercury, Ferrimicrobium acidiphilum towards iron and zinc, and Geobacillus stearothermophilus towards cadmium [3942]. Various organisms such as Thiobacillus and Thiobacillus thioparus have capacity to oxidize reduced sulfur to harmless state and hence play important role in the biogeochemical cycling of sulfur. Sulfur oxidizing bacteria also finds applications in bioleaching, bioremediation, biofertilizer, biofilters, biosensors and biodeodourizers and rubber recycling [4349]. Halophilic microorganisms another category of extremophiles thrive in saline environments. These organisms have the capacity to tolerate high sodium chloride concentrations ranging from 0.5 to 5 M [50]. Diverse prokaryotic organisms have been reported from Dead Sea, Salterns, Great Salt Lake and solar lakes in USA, Europe and Africa [51]. Halophilic organisms Haloarcula marismortui, Halofrex volcanii, Halorubrum lacusprofundi , Natronomonas pharaonis and square archaeon Haloquadratum walsbyi have been reported from Dead Sea, Antarctic lake and Soda Lake [5255]. Halotolerance has been reported in several yeasts such as Hortaea werneckii [5658] and plants Chenopodium quinoa. Both of these are adapted to osmotic stress and excessive salinity [59]. There are some environmental areas where pressure remains very high. These areas are occupied by the microorganisms that can tolerate ambient to high pressure raging from 70 to 120 MPa and are designated as strict or obligate piezophiles. Piezophiles excel in sustaining pressure conditions beyond the usual limits for humans. Piezophilic bacteria Shewanella benthica, Colwellia marinimaniae, Pyrococcus yayanossi, and Photobacterium profundum have well adapted proteins, lipids and genes for tolerating stress due to high pressure [6063]. Moreover, microorganisms also survive in environments with limited nutrients like carbon, iron, nitrate, and phosphate source which plays important role in biogeochemical cycles for biomass production and nutrient cycling. These microorganisms are known as oligotrophs. Several organisms including Deinococcus peraridilitoris survive under extreme desert conditions Besides this, various organisms can live inside rocks known as endoliths and some lives inside rocks in cold deserts called as hypoliths. Some organisms such as Dienococcus radiodurans can resist high levels of ionizing radiation called as radioresistant [64] and organisms able to withstand damaging agents including organic solvents called as toxitolerant. Some organisms are capable of tolerating desiccation known as xerophiles.

1.4

Biotechnological potential of extremophiles

The presence of extremophiles in extremes of environments has evolved biotechnologically suitable additives, enzymes, proteins and other metabolites. The capacity of extremophiles to perform better in harsh environment such as salinity, alkalinity and/or low water activity has opened exciting opportunities in industrial processes as compared to mesophilic counterparts. In today’s world, emphasis is given on biological products such as biofuels, bioplastics and biosurfactants to overcome the high production costs and hazardous aspects of chemically produced products. All types of extremophiles including thermophiles, alkaliphiles, halophiles and psychrophiles offer applications in white, gray and red biotechnology. Extremophiles mostly offer applications which are enzymes based but biomolecules such as antifreeze proteins, lipids, and other molecules also find applications in industrial processes. For examples, various proteases have been reported from Acinetobacter sp., Bacillus cereus, Colwellia sp., Curtobacterium luteum , Exiguobacterium undae Su-1 and Stenotrophomonas sp. for detergent industry [65]. Metabolic products known as extremozymes including enzymes such as amylases, cellulases, esterases, keratinases, lipases, pectinases, peroxidases, proteases and xylanases finds applications in agriculture, beverages, detergent, food and feed, pharmaceuticals, textiles, leather, pulp and paper industries. These extremozymes have characteristic properties like high stability and catalytic efficiency under varied temperature and pH conditions, salinity, low water activity, low oxygen and more shelf life [66,67]. Enzymes such as Taq DNA polymerase obtained from thermophilic Thermus aquaticus is widely used and finds applications in molecular biology [68]. Similarly, ligases, alkaline phosphatases, restriction enzymes and other thermostable polymerases have been reported from various extremophilic organisms [69]. The enzymes responsible for cellulose degradation including cellobiohydrolase, endoglucanase and β-glucosidase have been reported from thermophilic Thermotoga maritima, T. neapolitana and Pyrococcus furiosus [68]. Genencor International first commercialized cellulase from alkaliphile with applications in textiles and detergents [69]. Besides the production of extremozymes, extremophiles also produce organic compounds known as extremolytes under stressed conditions. These extremolytes include polyols, carbohydrates like trehalose, mannose and their derivatives like mannosylglycerate and mannosylglyceramide, glucosylglycerate (GG), glucosylglucosylglycerate, and amino acids [70]. Other derivatives including phosphodiesters di-myoinositol-1,10 -phosphate, cyclic 2,3-diphosphoglycerate and α-diglycerol phosphate and trianionic pyrophosphate are produced by archaeabacteria [71,72]. other compounds including bacterioruberin, ectoines, melanin, scytonemin and mycosporin-like amino-acids (MAAs) have been reported

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from UV-resistant extremophilic bacteria [7375]. These extremolytes find applications in pharmaceutical sector like cosmetics, therapeutics for developing pharmacophore with antiproliferative and anti-inflammatory activity and chemopreventive agents [76,77]. For example, Pseudomonas has been reported for the production of pyochelin, an iron binding compound with antifungal activity against Candida and Aspergillus spp [78]. Extremophiles are also known for the production of metabolites like exopolysaccharides biosurfactants, biopolymers and peptides with diverse industrial potential [79]. Biosurfactants are mostly employed as adjuvants for herbicides, pesticides formulations, bioremediation processes and biocontrol agents [70]. In different studies, halophiles are explored for the production of polysaccharides made of fructans with applications in food, medical, pharmaceutical, chemical and cosmeceutical industries [8082]. Several halophiles are reported for poly-hydroxyalkanoate as biodegradable plastic, exopolysaccharides as emulsifiers, osmotic solutes as stabilizers and bacteriorhodopsin in energy conversion [83]. Additionally, extremophilic organisms such as Bacillus licheniformis are efficient in degrading complex materials produced by industrial wastes and effluents, sewage and petroleum hydrocarbons [84]. Planococcus is a halophile having the capacity to tolerate up to 25% NaCl can degrade BTEX in oil-contaminated soil. Similarly, studies have reported that halophilic archaea degrade phenol, pyrene and naphthalene and produce biosurfactants [85]. Similarly, β-carotene from halophilic microalgae Dunaliella is used as supplements in food products, pharmaceutical industries for colorant and antioxidant properties [86].

1.5 Molecular approaches like metagenomics and whole genome sequencing (WGS) of extremophiles In the present scenario, extremophiles are being considered as attractive candidates for studying their adaptations with respect to physiological, biochemical and other fundamental cellular processes. As extremophiles are being adapted to extreme environmental conditions, it becomes difficult to cultivate such organisms in laboratory conditions. Therefore, molecular techniques like recombinant DNA technology for cloning and heterologous expression in a suitable host either bacteria or cell lines using vectors provide easy way for genetic manipulation and successful commercialization of gene products. Molecular approaches like ligation-independent cloning [87] and hybridization cloning [88] have also been used to obtain recombinant proteins for studying structure-function relationship of proteins and other enzymes. Omic based approaches such as comparative genomics, proteomics, transcriptomics, metallomics and secretomics are preferred for understanding mechanisms underlying physiological, biochemical and structural adaptations of extremophiles [8994]. To obtain the adaptations of extremophilic microorganisms, it is imperative to obtain the complete genome sequence of a particular microbe. Studies have reported biomass degrading genes in Bacillus cellulosilyticus [95], Cellulomonas spp. [96], Dictyoglomus turgidum [97] and Fibrobacter succinogenes [98,99] by WGS. Similarly, the genomes of halophilic strains belonging to Halomicrobium, Chromohalobacter, Haloferax, Haloarcula, Halorubrum, Natronomonas and Haloquadratum have been reported for various metabolites profiling such as membrane lipids, cell wall components and bacteriorhodopsin related to high salt concentrations from various habitats. The whole genomes of these halophiles have been explored to identify the possible role of genes DNA polymerase, thioredoxin reductase, cytochrome oxidase, multiple TATA binding proteins (TBP), transcription factors involved in adaptation to hyper saline environments [100]. Various studies on peizophilic bacteria Shewanella, Photobacterium profundum, Moritella profunda and Saccharomyces cerevisiae have properties that can withstand high pressure. DNA binding protein such as RecD and other proteins such as Hsp60, Hsp70, OmpH, RecA, F1F0 ATPases, Cct, Tat2 and Ypr153w have important role in adaptations for tolerating high pressure [101]. Moreover, dihydrofolate reductase from peizophilic Moritella profunda is involved in tolerance towards high pressure upto 50 MPa [63,102104]. Psychrophilic organisms Exiguobacterium antarcticum, Pseudoalteromonas arctica, Pseudoalteromonas haloplanktis and Aquaspillium arcticum contain enzymes such as β-glucosidase, protease, malate dehydrogenase, DNA repairing enzymes which are adapted to stress conditions such as low temperature [105109]. By obtaining the knowledge of gene sequences in whole genomes, engineering of proteins and genes with precision for desired property can be done. Similarly, microbial community’s analysis has been studied to evaluate their potential to produce different metabolites and their interaction among extreme environments using metagenomic approaches. Molecular phylogenetic analysis such as metagenomics provides athe total genetic pool and arrangement of genes of microbes in a culture-independent manner among particular environments. Studies have revealed most predominating phyla across the extreme environments and prediction of the role of genes responsible for various functional aspects. Metagenomic library from alkaline hot spring revealed predominance of bacterial phyla Acidobacteria, Aquificae, Chloroflexi, Deinococcus-Thermus, Firmicutes and Proteobacteria and presence of genes encoding for enzymes such as galactosidases, lipases proteases and xylanases with biotechnological potential [110]. In similar studies, metagenomics has been reported for new and novel biocatalysts from hypersalted biotopes, cold environments, hot springs and deep thermal vents with potential attributes for industries.

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Various enzymes like halophilic cellulases, proteases, xylanases and lipases have been reported from soil microbial consortia with suitability in food, detergent and textile industrial processes [111]. Besides omic technologies, genome wide editing tools such as Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-CAS) are becoming more significant as it includes an array of short palindromic repeats (CRISPR) and CRISPR associated genes (Cas) initially discovered for its role in bacterial immunity acts together to protect against foreign attacking agents [112]. The CRISPR-CAS uses a combination Cas systems and CRISPR to edit genome/gene (s). This technique includes improve or modify the properties of target gene products which can be more efficiently applied to biotechnological, industrial and agricultural applications.

1.6

Conclusion

Microorganisms are ubiquitously distributed in extremes of environment with respect to high or low temperature, acidic or alkaline pH, high or low pressure and radiation and metals. Various studies have revealed abundance of microbes residing in these extremes. But these extremophilic microbes are hidden repository and their metabolic potential is still under explored. In the post-genomics era, the advances in genomics, transcriptomics and metagenomics tools have enabled us to explore and characterize the microbial diversity and metabolic potential of microbial diversity residing in extreme environmental environments. Presently, a large number of whole genomes are available in the database for prediction and annotation but to completely understand the adaptations underlying survival of extremophiles both protein structure and biochemical properties need to be studied. In this book, different chapters have covered the physiological, biochemical and molecular aspects of different classes including, halophiles, thermophiles, psychrophiles and peizophiles.

Acknowledgments The corresponding author is thankful to SEED Division, DST for providing financial support under the project SP/YO/ 125/2017. The authors also acknowledge College of Horticulture & Forestry, Neri and Chandigarh University, Gharuan for providing necessary infrastructure for the successful accomplishment of this work.

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Levan and levansucrases: polymer, enzyme, microorganisms and biomedical applications. Biocatal Biotransformation 2017. Available from: https://doi.org/10.1080/10242422.2017.1314467. [82] Li W, Yu S, Zhang T, Jiang B, Mu W. Recent novel applications of levansucrases. Appl Microbiol Biotechnol 2015;99(17):695969. Available from: https://doi.org/10.1007/s00253-015-6797-5. [83] Oren A. Industrial and environmental applications of halophilic microorganisms. Environ Technol. 2010;31(89):82534. Available from: https://doi.org/10.1080/09593330903370026. [84] Li H, Li X, Yu T, Wang F, Qu C. Study on extreme microbial degradation of petroleum hydrocarbons. Mater Sci Eng 2019;484 (2019):012040. Available from: https://doi.org/10.1088/1757-899X/484/1/012040 IOP Publishing. [85] Khemili-Talbi S, Kebbouche-Gana S, Akmoussi-Toumi S, et al. Isolation of an extremely halophilic arhaeon Natrialba sp. C21 able to degrade aromatic compounds and to produce stable biosurfactant at high salinity. Extremophiles Life Extreme Cond. 2015;19(6):110920. [86] Shariati M, Hadi M.R. Microalgal Biotechnology and Bioenergy in Dunaliella, 2011. Available from: https://doi.org/10.5772/19046. [87] Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U. Phosphorothioate-based ligase-independent gene cloning (PLICing): an enzyme-free and sequence-independent cloning method. Anal Biochem 2010;406:1416. Available from: https://doi.org/10.1016/j. ab.2010.07.011. [88] Van den Ent F, Lo¨we J. RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochem Biophys Methods 2006;67:6774. Available from: https://doi.org/10.1016/j.jbbm.2005.12.008. [89] Yoon SH, Reiss DJ, Bare JC, Tenenbaum D, Pan M, Slagel J, et al. Parallel evolution of transcriptome architecture during genome reorganization. Genome Reorganization Genome Res 2011;21:1892904. Available from: https://doi.org/10.1101/gr.122218.111. [90] Cvetkovic A, Menon AL, Thorgersen MP, Scott JW, Poole II FL, Jenney F E Jr, et al. Microbial metalloproteomes are largely uncharacterized. Nature 2010;466:77982. Available from: https://doi.org/10.1038/nature09265. [91] Adams MW, Dailey HA, DeLucas LJ, Luo M, Prestegard JH, Rose JP, et al. The southeast collaboratory for structural genomics: a highthroughput gene to structure factory. Acc Chem Res 2003;36:1918. Available from: https://doi.org/10.1021/ar0101382. [92] Joachimiak A. High-throughput crystallography for structural genomics. Curr Opin Struct Biol 2009;19:57384. Available from: https://doi. org/10.1016/j.sbi.2009.08.002. [93] Lecompte O, Ripp R, Puzos-Barbe V, Duprat S, Heilig R, Dietrich J, et al. Genome evolution at the genus level: comparison of three complete genomes of hyperthermophilic archaea. Genome Res 2001;11:98193. Available from: https://doi.org/10.1101/gr.GR1653R. [94] Menon AL, Poole II FL, Cvetkovic A, Trauger SA, Kalisiak E, et al. Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome. Mol Cell Proteomics 2009;8:73551. Available from: https:// doi.org/10.1074/mcp.M800246-MCP200. [95] Mead D, Drinkwater C, Brumm PJ. Genomic and enzymatic results show Bacillus cellulosilyticus uses a novel set of LPXTA carbohydrases to hydrolyze polysaccharides. PLoS One 2013;8:e61131. Available from: https://doi.org/10.1371/journal.pone.0061131. [96] Christopherson MR, Suen G, Bramhacharya S, Jewell KA, Aylward FO, Mead D, et al. The genome sequences of Cellulomonas fimi and Cellvibrio gilvus reveal the cellulolytic strategies of two facultative anaerobes, transfer of Cellvibrio gilvus to the genus Cellulomonas, and proposal of Cellulomonas gilvus sp. nov. PLoS One 2013;8:e53954. Available from: https://doi.org/10.1371/journal.pone.0053954. [97] Brumm P, Hermanson S, Hochstein B, Boyum J, Hermersmann N, Gowda K, et al. Mining Dictyoglomus turgidum for enzymatically active carbohydrases. Appl Biochem Biotechnol 2011;163:20514. Available from: https://doi.org/10.1007/s12010-010-9029-6. [98] Brumm P, Mead D, Boyum J, Drinkwater C, Gowda K, Stevenson D, et al. Functional annotation of Fibrobacter succinogenes S85 carbohydrate active enzymes. Appl Biochem Biotechnol 2011;163:64957. Available from: https://doi.org/10.1007/s12010-010-9070-5. [99] Suen G, Weimer PJ, Stevenson DM, Aylward FO, Boyum J, Deneke J, et al. The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist. PLoS One 2011;6:e18814. Available from: https://doi.org/10.1371/journal.pone.0018814. [100] DasSarma S, Berquist BR, Coker JA, DasSarma P, Muller JA. Post-genomics of the model haloarchaeon Halobacterium sp. NRC-1. Saline Systems. 2006;2:3. Available from: https://doi.org/10.1186/1746-1448-2-3. [101] Bartlett DH, Kato C, Horikoshi K. High pressure influences on gene and protein expression. Res Microbiol 1995;146:697706. Available from: https://doi.org/10.1016/0923-2508(96)81066-7. [102] Feller G. Psychrophilic enzymes: from folding to function and biotechnology. Scientifica (Cairo) 2013;2013:128. Available from: https://doi. org/10.1155/2013/512840. [103] Ohmae E, Murakami C, Tate SI, Gekko K, Hata K, Akasaka K, et al. Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli. Biochim Biophys Acta - Proteins Proteom 2012;1824:51119. Available from: https://doi.org/10.1016/j.bbapap.2012.01.001. [104] Bidle KA, Bartlett DH. RecD function is required for high-pressure growth of a deep-sea bacterium. J Bacteriol 1999;181:23307.

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[105] Zanphorlin LM, de Giuseppe PO, Honorato RV, Tonoli CCC, Fattori J, Crespim E, et al. Oligomerization as a strategy for cold adaptation: structure and dynamics of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. Sci Rep [Internet] 2016;6:23776. Available from: https://doi.org/10.1038/srep23776. [106] Kim SY, Hwang KY, Kim SH, Sung HC, Han YS, Cho YJ. Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum. J Biol Chem 1999;274:117617. [107] Olufsen M, Smala˚s A, Moe E, Brandsdal B. Increased flexibility as a strategy for cold adaptation  a comparative molecular dynamics study of cold- and warm-active uracil DNA glycosylase. J Biol Chem 2005;180428. [108] Park HJ, Lee CW, Kim D, Do H, Han SJ, Kim JE, et al. Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacte˚ resolution: structural adaptations to cold and functional analysis of a laundry deterrium, Pseudoalteromonas arctica PAMC 21717, at 1.4 A gent enzyme. PLoS One 2018;13(2):e0191740. [109] Siddiqui K, Poljak A, Guilhaus M, De Francisci D, Curmi P, Feller G, et al. Role of lysine versus arginine in enzyme cold-adaptation: modifying lysine to homo-arginine stabilizes the cold-adapted α-amylase from Pseudoalteramonas haloplanktis. Proteins: Structure, Function, Bioinforma 2006;64(2):486501. [110] Lo´pez-Lo´pez O, Knapik K, Cerda´n M-E, Gonza´lez-Siso M-I. Metagenomics of an alkaline hot spring in galicia (spain): microbial diversity analysis and screening for novel lipolytic enzymes. Front Microbiol 2015;6:1291. Available from: https://doi.org/10.3389/fmicb.2015.01291. [111] Yin J, Chen JC, Wu Q, Chen GQ. Halophiles, coming stars for industrial biotechnology. Biotechnol Adv 2015;33:143342. [112] Maier LK, Fischer S, Stoll B, et al. The immune system of halophilic archaea. Mob Genet Elements. 2012;2(5):22832. Available from: https://doi.org/10.4161/mge.22530.

Further reading Bolhuis H, Poele EM, Rodriguez-Valera F. Isolation and cultivation of Walsby’s square archaeon. Environ Microbiol 2004;6(12):128791. Kamekura M, Kates M. Structural diversity of membrane lipids in members of Halobacteriaceae. Biosci Biotechnol, Biochem 1999;63(6):96972. Li H, Liu YH, Luo N, et al. Biodegradation of benzene and its derivatives by a psychrotolerant and moderately haloalkaliphilic Planococcus sp. strain ZD22. Res Microbiol 2006;157(7):62936. Madern D, Ebel C, Zaccai G. Halophilic adaptation of enzymes. Extremophiles 2000;4(2):918. Mescher MF, Strominger JL. Structural (shape-maintaining) role of the cell surface glycoprotein of Halobacterium salinarium. Proc Natl Acad Sci 1976;73(8):268791. Oesterhelt D, Stoeckenius W. Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nat New Biol 1971;233(39):149.

Chapter 2

Physiology of extremophiles Richa Salwan1 and Vivek Sharma2 1

Department of Basic Sciences, College of Horticulture and Forestry (Dr. YSP University of Horticulture and Forestry), Neri, Hamirpur (HP), India,

2

University Centre for Research and Development, Chandigarh University, Chandigarh, India

2.1

Introduction

Microorganisms constitute the considerable part of the earth’s biodiversity. The species biodiversity under extreme habitats such as hot springs, acidic springs, saline and alkaline lakes, hot and cold deserts, and ocean beds is mainly limited to microbes as these extremes are harsh for the existence of life. The microorganisms surviving in these extreme environments have adopted several ecological processes to thrive. These microorganisms play important role in cycling of available nutrients and production of biomass. Sometimes, these ecological processes are performed by chemotaxis and motility but have remained unexplored in case of extreme environments such as cold habitats [1]. Extremophiles reported from all three domains including bacteria, archaea and eukarya are important candidates for studying evolutionary relationship as the ancient living organisms on earth [2]. These microorganisms represent an ideal source of industrially important enzymes, biomolecules, biomaterials and metabolites [3,4]. Archaea includes members which are less versatile but are the promising individuals for attaining adaptations to extreme environment. Archaea includes halophiles, alkali and acidophiles and hyperthermophiles including Methanopyrus kandleri and Picrophilus torridus, etc. Among bacteria, a remarkable diversity of Arthrobacter, Flavobacterium, Idiomarina, Micrococcus, Pseudomonas, Rheinheimera, Sphingobacterium and Vibrio have been reported from the cold areas and high altitude alkaline lakes [5 10]. The ability of cyanobacteria to form microbial mats in areas such as cold deserts, hot springs, acidic mines, hypersaline lakes help them to survive under water stress. Similarly, eukaryotes including algae, fungi and lichens can tolerate all types of extreme environments except extremes of high temperature [11]. Extremophiles have long history in evolution because archaea were reported as first organisms from extreme environment.

2.2

Taxonomy of extremophiles

Microbial taxonomy based on chemotaxonomic, phenotypic and phylogenetic methods assign organisms, different groups or taxa based on their close relatedness. Chemotaxonomic methods include fatty acids and lipid profiling, quinones, proteins whereas the phenotypic methods are based on morphological characters. The phylogenetic method includes use of molecular markers such as 16S rDNA, RFLP, RAPD, DNA probes as well as DNA-DNA hybridization and total G 1 C content which assigns the bacteria to particular taxa [12]. For example, when DNA-DNA hybridization value are less than 70% and 16S rDNA similarity less than 97% with representative strain then the strain is presumed to differ and belong to another group or species [13]. In general, 16S rDNA is used as a gold standard in assigning organisms to new or higher taxa [14]. Microbial communities thriving at low temperature has been analyzed using molecular methods such as denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism (T-RFLP) and metagenomics [15]. As described earlier, extremophiles are found in three domains which occupy different niches across the globe. The microbes confer adaptive strategies for their survival to extreme habitats which is also responsible for evolutionary differences and speciation across different ecological areas. On the basis of their tolerance to extreme temperature, pH, salt, metal ions and radiations, extremophiles have been classified. For example, majority of earth’s surface is having temperature below 15  C [16]. Various bacteria including Colwellia, Deinococcus, Desulfuromonas, Flavobacterium, Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00002-2 © 2020 Elsevier Inc. All rights reserved.

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Glaciecola, Moritella, Psychromonas, Psychroflexus and Shewanella have been reported from cold environments including Arctic and Antarctic, deep-sea, polar areas, glaciers and alpine regions based on 16S rDNA. The cold tolerant microbes are known as psychrophiles [17]. On the other hand, high temperature tolerant, known as thermotolerant are also identified based on 16S rDNA and confined to different genera such as Bacillus thermophilus, Thermus aquaticus, Thermoplasma acidophilum, Thermodesulfobacter. It is reported that thermophilic representatives are found among majority of the bacterial phyla. Among all, Sulfolobus and Thermus were the first extremophile isolated and characterized based on chemotaxonomic and phenotypic characters [18,19]. Themophilic groups comprised of Aquificae, Thermotogae, Thermales and the genus Thermodesulfobacter have deep lineage in the evolutionary history and are represented as the ancient life forms on Earth [20]. Another class representing halophiles is also widely distributed in bacteria, archaea and eukarya such as algae. Bacteria and archaea reside in saline environments such as natural lakes and deep-sea basins having 25 33% salinity. The grouping of halophiles which includes both archaea and bacteria has been done based on second edition of Bergey’s Manual of Systematic Bacteriology [12,21]. Various species of bacterial genera including Rhodospirillum, Rhodovibrio, Methylarcula, Dichotomicrobium, Halothiobacillus, Nitrosomonas and archaea including Halobacterium, Haloarcula, Halococcus, Haloferax and Natronomonas are interested expectants for applications in biotechnological sector [22]. Acidophiles, another category of extremophiles are also found in all domains. These organisms have the ability to resist acidic environments that evolved during evolution. Acidophiles can survive in environment having pH ,3 and are the source of acidity in that environment. Bacteria including members of class α-proteobacteria, β-proteobacteria and γ-proteobacteria such as Acidithiobacillus, Acidimicrobium, Ferrimicrobium, Ferrovum, Hydrogenobacter, Leptospirillum and archaea such as Acidianus, Ferroplasma, Picrophilus, Thermoplasma and Sulfobacillus of phylum Euryarchaeota and Crenarchaeota [23,24]. On the other hand, eukarya including fungi, algae ciliates, diatoms and flagellates are also known which can survive in acidic environments. Alkaliphiles as the most important group of extremophiles survive in extreme of alkaline pH as found in soda lakes, alkaline Lonar lake, Sambhar Lake, Mono Lake, Wadi Natrun Lakes and Saline Qinghai Lake distributed all over the World. A diversity of microorganisms such as aerobic, anaerobic, methanogenic, chemolithotrophic and phototrophic are considered as alkaliphiles and their evolutionary relatedness proved the association among Gram negative, Gram positive with both high and low G 1 C content. Alkaliphiles are known since the discovery of indigo reported in Japan. The first report on alkaliphilic protease was recorded in 1971 [25]. Alkaliphiles can be aerobic, anaerobic, haloalkaliphile, sulfur-oxidizing and sulfate reducing, methanogens and methanotrophic as well as cyanobacteria based on their physiological activities [26]. High pressure is also considered as extreme environmental conditions. The effect of extreme pressure along with temperature alters the physiological parameters by altering the structure and metabolic reactions. Piezophiles adapt to extreme pressure by possessing lipids contatining unsaturated fatty acids which is crucial requirement for their growth [27]. Peizophiles produce DNA-binding proteins and restriction endonucleases at high pressure under osmolarity conditions. These piezophiles can be a source of novel restriction endonucleases and DNA-binding proteins [28]. Besides these groups, organisms based on their physiological functions are distributed or grow in areas such as toxic waste, heavy metals, organic solvents, low nutrient content, methane, sulfur and iron rich niches and exposed to high pressure. The microbes from such environments can be promising candidates for their role in industrial applications where harsh and tough conditions are required and normal microbes are unable to resist those harsh environments.

2.3

Diversity of extremophiles

Exploration of microbial diversity under extreme habitat is of para mount importance. Both culture dependent and culture independent approaches are being employed for identification of diverse microbial community residing in the extreme environments. Halophilic bacteria such as Salinibacter, Halomonas, Salicola and various haloarchaea commonly reside in hypersaline environments [29]. Similarly, haloalkaliphiles have adapted to both salt and alkaline environments [30]. Salar de Uyuni is the largest salt flat situated in the Southwest of the Bolivian Altiplano and harbors a unique microbial community of halophiles, radiotolerant, metal tolerants and alkaline and acidic pH tolerants. The halophiles can grow in 10% brine solution and also thrive as salt entrapped cell entities for many years [31 33]. Studies from Salar de Uyuni have also reported halophiles with special tolerance towards acidic pH. Moreover, halophilic organisms with tolerance pH range from 2.4 to 4 6 are also in existence [34]. Similarly, in India, a remarkable diversity of halophiles, halotolerant and haloalkaliphiles has been reported from Gujarat, Himachal Pradesh, Maharashtra, Goa and Rajasthan [35 38]. Besides the alkaline and saline environments, microorganisms are also prevalent in

Physiology of extremophiles Chapter | 2

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terrestrial and submarine heated soils, deep sea sediments, hydrothermal vents, hot springs and volcanoes. The microorganisms inhabiting hot environments are considered as thermophiles and are considered as closed relatives of early life forms on earth. But thermophiles have also been reported from cold environments, temperate areas and petroleum reservoirs. Studies have reported the existence of thermophilic methanogens from cold lakes but also have the capacity to tolerate low temperature of 15 30  C [39,40]. These organisms maintain themselves in inactive state or may slow their growth. Most common thermophiles belong to the genus Bacillus sp., Geobacillus, Pyrobaculum calidifontis and Sulfolobus acidocaldarius [41 43]. Another type of microorganisms includes piezophiles, can withstand high hydrostatic pressure. These peizophiles are found in environments such as deep sea, sub-seafloor and continental subsurface. Peizophiles are also difficult to explore because of the difficulty in sampling and experiments to be run at high pressure. Extremophilic microorganisms are difficult to grow in laboratory conditions due to the limitations of culture dependent approaches. So far only less than 1% of microbes have been cultivated under lab conditions. The exploration of the adaptive behavior of extremophiles has been made easy in omics era. Extremophiles play important roles in industrial processes, pathogenesis and antimicriobial resistance and biogeochemical cycling of minerals. Advances in omic technologies such as transcriptomics, proteomics, metabolomics and metagenomics have facilitated and enhanced our access to harness microbial community and microbial interactions within the environment [44]. Prokaryotic communities have been studied using metagenomic tools from glacier ice, hot springs, cold water lake Sayram, Borax lake with extremes of arsenic and NaCl and Alvord Desert [45 47]. Besides the diversity of prokaryotes, eukaryotic algae, lichens, fungi and viruses are also known [11]. Microbial diversity is of elementary importance in conserving and maintaining global genetic resources. Marine microorganisms play important role in maintaining ecosystem and being employed as suitable candidates in conserving energy. Cyanobacteria and microalgae use sunlight to fix CO2 and produce biomass and O2. Most algae are aquatic and reside in fresh water. These water bodies are polluted by natural causes like acidic environment which is caused by pyrite oxidation. Many species have adapted themselves and withstand high acidic conditions in lakes, ponds and rivers that receive mine water discharge. High acidic pH causes metals like Fe S, Cu, Cd and Zn to solublize in water leading to water pollution. Therefore, studies focused on identification of acid mine drainage tolerant species finds potential role in bioremediation. Various fungi, protists and algae including Ochromonas, Euglena, Dunaliella and Chlamydomonas [48 51] are prevalent in acidic and metal rich conditions with limited studies on zooplanktons [52]. Viral communities can also grow in extreme environments such as high salinity, high temperature, hydrostatic pressure, anoxic conditions and heavy metals which creates chances of exploring such extremophiles. Studies on these unusual biotypes have reported viral communities belonging to Caudovirales, Phycodnaviridae and Iridoviridae. Moreover, reports from deep sea brines revealed viruses in both upper and lower interface. The upper interface contains viruses which use marine bacteria as their host while lower interface conatins halophages and haloviruses. Still, the full exploration of viruses from these extreme environments is underexplored. Besides, bacteria, algae and protists, flamentous fungi are also considered as extremophiles as they can grow at low temperature environments such as Antartic environments. Different species of fungi have been reported as psychrophilic, psychrotolerant, and mesophilic-psychrotolerant [53,54]. But only a few species of fungi are also known which can tolerate high temperature and are called as thermophilic fungi.

2.4

Physiological adaptations of extremophiles

Exremophilic microbes have adapted to extreme environmental conditions by keeping their cellular metabolites stable under harsh conditions. Diverse groups of extremophiles have adaptations to survive under conditions such as temperature, salt, pH, organic solvents and metal ions (Fig. 2.1). Some extremophiles have capacity to tolerate more than one extreme conditions as they have special adaptation system which makes them more valuable in understanding not only the fundamental aspects but for their biotechnological applications [30]. These organisms have role in the origin of life and provide clues stability of the macromolecules and for the survival of microbes under extreme conditions. Therefore, their studies would provide important clues for adaptation under extreme environmental conditions.

2.4.1 Psychrophiles The psychrophiles can tolerate temperature range of 0 20  C but their optimal temperature for growth is 5  C. Psychrophiles are well adapted to low temperature as they possess several unique features such as presence of unsaturated fatty acids in cell membranes which remains as liquid even at low temperature to facilitate solute transport across

Peizophiles

Thermophiles • Permeability barrier for inward and outward flow of nutrients • High G+C contents containing more charged amino acids on the surface for intramolecular salt bridges

• Pressure responsive genes involved in chemotaxis pathway, hydrogenases and formate metabolism for energy, translation, and palindromic sequences associated with cellular apoptosis susceptibility proteins

14

Psychrophiles • Unsaturated fatty acids in cell membranes for decreasing membrane fluidity, • Reduced levels of transcription andtranslation and structure of ribosomes to changes in cellular machinery at low temperature • Accumulation of compatible solutes like betaine, glycine, mannitol and sucrose synthesis of antifreeze proteins for preventing ice crystal growth • Production of exopolysac charides 0

1

2

3

4

13 12 11 10 9 8 7 5

6

Mildly acid or alkaline-tolerant

Acidophiles • Maintaining the pH homeostasis and metal/metalloid resistance • Membrane impermeability which controls the proton influx inside the cell • Tetraether lipids in their cell membrane • Reduction in the size of membrane pores, proton efflux systems such as antiporters, symporters and H+ATPases and accumulation of buffering components such as arginine, histidine and lysine which help in the proton sequestration

Alkaliphiles • Cytoplasmic pH homeostasis and uptake of H+ by using electrogenic, secondary cation/proton antiporters • Protective layer of acidic substances including acidic amino acids, teichuronopeptide and teichuronic acid outside the cell to avoid environmental alkalinity • Phosphoserine aminotransferase for increases hydrophobic interactions and negatively charged amino acids at the interface for enhancing stability in alkaline conditions

Halophiles • Synthesis of osmoprotectants such as compatible organic solutes • Creating equilibriumin cellular and environmental salt concentrations • Presence of more negatively charged amino acids to compete with ions for water molecules and helps in protein solubilization

FIGURE 2.1 Physiological adaptations posed by different groups of extremophiles for their survival in extremes of low and high temperature, acidic and alkaline pH, salt and pressure.

Physiology of extremophiles Chapter | 2

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the membrane, cold shock proteins and cryoprotectants for synthesizing cold adapted enzymes. Several changes like membrane fluidity, reduced levels of transcription and translation and structure of ribosomes contribute to changes in cellular machinery at low temperature. Psychrophiles protect themselves from freezing by accumulating compatible solutes like glycine betaine, mannitol and sucrose or by synthesizing antifreeze proteins which prevents ice crystal growth. Moreover, psychrophiles also induces production of exopolysaccharides as a mechanism against cryoprotection [55]. These microbes also offer complex metabolic adaptations such as altered mechanisms of nutrients transport, cold-denaturation of proteins, intracellular ice formation and protein folding [5,56 58].

2.4.2 Thermophiles Thermophilic organisms are known for their growth at high temperature because of the stability acquired by their membrane lipids, permeability barrier which controls the inward and outward flow of nutrients, rRNA and tRNA molecules. These organisms are characterized by higher G 1 C contents, more charged amino acids on the surface and intramolecular salt bridges which contribute for their adaptations at high temperature [59 61].

2.4.3 Alkaliphiles Alkaliphiles are organisms that grow at high pH values. They adapt themselves by maintaining cytoplasmic pH homeostasis and uptake of H1 using electrogenic, secondary cation/proton antiporters. These microbes also develop a protective layer of acidic substances including acidic amino acids, teichuronopeptide and teichuronic acid outside the cell to avoid environmental alkalinity. Some alkaliphiles produce cytochrome C on the outer membrane which regulates transfer of protons on membrane. Members of alkaliphiles are known to synthesize phosphoserine aminotransferase which increases hydrophobic interactions and negatively charged amino acids at the interface for enhancing stability in alkaline conditions [62].

2.4.4 Acidophiles Acidophiles thrive under highly acidic conditions such as marine volcanic vents, and acidic sulfur springs, acid rock drainage (ARD) and acid mine drainage. These microorganisms have adapted themselves by maintaining their cellular pH neutral and also acquire resistance towards metals [24,63,64]. Members of the genus Acidobacterium, Leptospirillum, Picrophilus and Ferroplasma are found in acidic sites. These microorganisms generally require intracellular neutral pH but the mechanism of homeostasis in acidophiles is not well understood. The major factor that contributes to intracellular pH is membrane impermeability which controls the proton influx inside the cell. Archaea have been adapted to withstand low pH because tetraether lipids in their cell membrane have low permeability for proton influx. Similarly, Leptospirillum ferriphilum genome possesses genes for the biosynthesis of cell membrane and cell wall structural components for tolerance towards acidic pH [65]. Moreover, adaptations like reduction in the size of membrane pores, proton efflux systems such as antiporters, symporters and H1 ATPases and accumulation of buffering components such as arginine, histidine and lysine help in the proton sequestration. Besides this, other adaptation strategies include degradation of organic acids for proton dissociation, synthesis of chaperones that protect proteins and DNA at low pH from damage.

2.4.5 Halophiles The organisms which can grow in high concentrations of salt are called as halophiles. These organisms can grow upto 15% or 2.5 M concentration of salt [66]. These microbes survive in high salt concentration by opting specialized adaptations related to cellular and enzymatic machinery for maintaining osmotic balance. Cellular adaptations include strategies such as synthesis of osmoprotectants including compatible organic solutes or by creating equilibrium in cellular and environmental salt concentrations. These strategies are popularly known as low salt, organic solute-in and high salt-in strategy for surviving in saline environment. Moreover, halophiles have well adapted their enzymes by attaining more negatively charged amino acids which compete for exchange of ions and helps in protein solubilization.

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PART | I Physiological aspects

2.4.6 Peizophiles The organisms capable of growing under high hydrostatic pressure are known as peizophiles. These peizophiles adapt to resist high pressure by expressing genes operating under high pressure as reported in S. violaceae, or by regulating the expression of transcription, translation or by structural modifications in proteome for more stability under high pressure [67]. A case study of peizophilic Pyrococcus yayanosii CH1 has revealed pressure responsive genes involved in chemotaxis pathway, formate and hydrogenases metabolism for energy, translation, and palindromic sequences involve in apoptosis of susceptible proteins [68].

2.5

Genomics and evolution

Extremophiles are known for their remarkable versatility and complexity which are being explored using molecular tools such as metagenomics and next generation sequencing. The studies have explored the genomes of thermophiles, alkaliphiles, psychrophiles and psychrotolerants for functional applications. The Geological Survey of India has reported approximately 340 hot springs situated in India which are characterized by their orogenic activities [69]. Considering the size and diversity, the metagenomic studies from India are still in the infancy stage. Efforts are required to explore the untapped functional microbial diversity located in environmental sites associated with particular stress. Metagenomics have provided an easy way of exploring the microbial diversity and their genes associated with particular environmental stress [70]. Targeted metagenomics is one of the important tools for studying targeted gene associated with ecological processes such as biogeochemical cycling among microbial diversity and their historical relationship [71]. Metagenomic study of alkaline hot springs has revealed variations in the microbial community structure such as methanogens Methanocella and Methanomassiliicoccus and cyanobacteria and Chloroflexi at different temperature [72]. Similar studies have reported taxa Fischerella, Leptolyngbya, Geitlerinema, Stenotrophomonas, and Aquaspirillum from different hot springs [73 75]. Additionally, studies from acidic hot springs have also reported Acidithiobacillus spp., Bacillus megaterium, Bacillus sporothermodurans , Cellovibrio mixtus, Clostridium bifermentans, Clostridium lituseburense , Hydrogenobacter sp., Opitutus terrae, Rhodococcus erythropolis, Thermus thermophilus, Thermus brockianus and Verrucomicrobia [76 80]. The advances in next generation sequencing has provided 83 complete or permanent draft genome sequences of psychrophiles corresponding to 78 bacterial, 4 archaea and 1 eukaryote and 102 genomes are still in process [55]. These psychrophiles have been reported from marine environments, Antractic continent, Pacific and Southern oceans. Similarly, not only complete genome sequences and functional annotation of alkaliphiles but the relationship of microbial taxa residing in alkaline environments has been obtained using NGS technologies. A total of 288 genomes have been reported from microbes residing in alkaline environment till now [81]. Genomic studies have also revealed diverse acidic microbiome from environments involving sulfuric acid accumulation, volcanic and geothermal areas etc. These acidic microbiomes have diverse metabolic potential and ecological networks and applications in biotechnological sector. Microbial communities reported from acid mine drainage have been studied which play important role in generation of acid and exhibiting adaptations to the environment. Acidophiles including Acidithiobacillus, Leptospirillum and Acidiphilium, Thermoplasma and Ferroplasma have been reported for expressing genes involved in low-pH, carbon, nitrogen and phosphate assimilation, environmental stress resistance and energy generation [82].

2.6

Chemotaxis in extremophiles

Extremophiles are known for their remarkable adaptive behavior which provides flexibility to their survival and also a producer of natural compounds of vital importance [83]. Quorum sensing (QS) is essential part of cell to cell communication in a population where bacteria invade, defend, develop resistance against stress and build populations through coordinated gene expression. QS also regulates cellular processes such as motility, cell competency, bioluminescence, expression of genes, virulence and production of antibiotics, exopolysaccharides, enzymes and secondary metabolites [84]. The QS also attribute in tolerating extreme environmental conditions but needs in depth study in case of extremophiles whereas the phenomenon is well characterized in bacteria such as Allivibrio fischeri, Bacillus pseudofirmus., Escherichia coli, Oceanobacillus iheyensis and Pseudomonas aeruginosa [85]. The first report on quorum sensing was reported in Vibrio harveyi and A. fischeri for luminescence when cells reached a specified optical density [84]. The bacteria possess LuxI-LuxR, Autoinducer-1 (AI-1), and AI-2 universal systems for quorum sensing [86,87]. In case of extremophiles, quorum sensing has been reported in halophiles including Halomonas eurihalina andHalomonas

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anticariensis by producing molecules such as acylated homoserine lactones (AHL) while Halobacillus halophilus is reported for Lux S operon which codes for AI-2 signaling molecules. Lux S system has been reported as first system that is chloride dependent. Ferroplasma acidarmanus is also known for motility and biofilm formation and contain genes similar to LuxR or LuxS [88]. Similarly, Acidithiobacillus ferrooxidans produce AHLs like compounds which are stable under acidic conditions. Leptospirillum ferrooxidans is a closed relative of Acidithiobacillus ferrooxidans and is reported for production of genes which are a component of QS system homologous to LuxI-LuxR [84,89]. Thermus sp. produce AHLs like compounds during cold shock and are involved in biofilm formation. Pyrococcus furiosus and Thermotoga maritime have also been reported for biofilm formation and exopolysaccharide production but no genes related to luxI-luxR and luxS has been reported. Studies have reported that co-culture of Pyrococcus furiosus and Thermotoga maritime produce AI-2 type signaling molecules playing role in heat shock response [90]. Other organisms including Deinococcus radiodurans, Pseudoaltermonas haloplanktis, Photobacterium profundum and Shewanella spp. produce AI-2 type signaling molecules and a luxS homologue is present. Besides bacteria, archaea are also known for production of AHL molecules involved in quorum sensing. AHL based quorum sensing has been reported in Methanothrix, harundinacea, Natrialba magadii and Natronococcus occultus [84,91,92]. Similarly, chemotaxis has been reported in several alkaliphilic Bacillus subtilis, B. halodurans, B. clausii which show motility by making use of electrochemical gradients such as sodium-motive force opposite to that of H1 proton motive force used by neutralophiles [85].

2.7

Conclusions and future directions

The continuous exploration of extreme environments has led to the increase in the richness of microbial diversity. Extreme environments have tremendous biodiversity of microbes that can be explored as suitable candidates for various industrial and biotechnological applications. Extremophiles have adapted themselves by opting several mechanisms such as antiporters’ efflux pumps, membrane fluidity and cytoplasmic pH homeostasis. Genomic analysis provides an excellent way for targeting their enzymes and other metabolites suitable for industrial processes. Additionally, comparative genomics played vital role in exploring physiological mechanisms underlying the adaptive behavior of extremophiles and genome dynamics in studying evolution of microbial genomes. Metagenomics approaches are being studied to assess microbial community and their suitability for the production different metabolites. Moreover, chemotaxis and motility in extremophiles also provide an ideal way of their survival in extremities but the detailed study and mechanism underlying their metabolic adaptations are still in infancy stage. Recent advances in omics technologies can provide considerable knowledge and functional analysis of extremophiles to completely exploit their potential for industrial applications. Further studies including transcriptomic and proteomic based approaches will facilitate the role and importance of these rare organisms in the community.

Acknowledgments The corresponding author is thankful to SEED Division, DST for providing financial support under the project SP/YO/ 125/2017. The authors also acknowledge College of Horticulture & Forestry (Dr YSP-UHF), Neri and Chandigarh University, Gharuan for providing necessary infrastructure for the successful submission of this work.

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Further reading Terry J, Mcgenity TJ, Gemmell RH, Stan-Lotter WG. Origins of halophilic microorganisms in ancient salt deposits. Environ Microbiol 2000;2 (3):243 50. Yayanos AA. Microbiology to 10,500 meters in the deep sea. Ann Rev Microbiol 1995;49:777 805.

Chapter 3

Mechanism of resistance focusing on copper, mercury and arsenic in extremophilic organisms, how acidophiles and thermophiles cope with these metals Javiera Norambuena Rutgers University, New Brunswick, NJ, United States

3.1

Introduction

Most metals are essential for life as we know it, but even essential metals can become toxic. Cells have developed different mechanisms to avoid metal toxicity (Fig. 3.1): cellular sequestration by (1) thiol molecules or (2) by none-thiol complexation, (3) efflux pumps, (4) enzymatic conversion, (5) exclusion and (6) reduced sensitivity of cellular targets [1,2]. The first line of defense that cells possess to avoid any type of stress are low molecular weight thiols, these molecules can buffer metals, whereby preventing cell damage. Cellular sequestration may occur extracellularly or intracellularly. Intracellularly, the main buffering systems are small thiol molecules such as glutathione (GSH) or bacillithiol (BSH), which also act as redox buffers [1]; there are some specialized proteins bind metals, e.g., metallothioneins (MT), which are rich in cysteine (Cys) residues [1,3]. These metal buffers can prevent cellular damage, while they can FIGURE 3.1 Resistance mechanisms in prokaryotes. The first intracellular line of defense against metals (black circles) is cellular sequestration (pink), represented by glutathione (GSH) and proteins like thioredoxins (Trx). Metals can also be complexed by polyphosphates (PolyP, in purple) or like nanoparticles (NP, black and purple/white circles). Another resistance mechanisms are efflux pumps (various orange colors), enzymatic conversion (blue), exclusion (light green), and reduce sensitivity of cellular targets (dark green). Adapted from Nies DH. Microbial heavymetal resistance. Appl Microbiol Biotechnol 1999;51(6):730 50; Dopson M. Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic micro-organisms. Microbiology 2003;149(8):1959.

Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00003-4 © 2020 Elsevier Inc. All rights reserved.

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be detoxified or pumped out of the cell. Mobility of metals can also be changed by extracellular or intracellular complexation (none thiol based) of metals (mechanism 2), like by formation of nanoparticles or phosphate complexes. Metals can be pumped out of cells; the three main types of efflux pumps (mechanism 3) are (a) A- or P-type ATPases, use ATP to pump metals out, but P-type ATPases can also uptake metals; (b) RND (root, nodulation, cell division) superfamily uses proton gradient to pump metals out; and (c) CDF (cation-diffusion facilitator) family, which efflux can be driven by concentration gradient, Δψ, pH, or chemiosmotic gradient [4]. Metals cannot be metabolized or transformed like organic toxicants; to be detoxified their redox state need to be changed, which can decrease their environmental mobility, this process can be performed by enzymes (mechanism 4). For this, the redox potential of the metal has to be close to that of the reducing agents present in the cytoplasm. An efficient way that microbes have developed to decrease metal toxicity is by exclusion, for example in acidophiles the phosphate transporter gene has a mutation that decreases import of arsenite and favors phosphate [2]. It has also been described that metal targets can be modified, so the metal does not interfere with the cellular processes. [2], like specific cytochrome c-oxidase that renders the cell more tolerance to Hg(II) [5]. The last two mechanisms will not be covered in this chapter, but they are reviewed elsewhere [2]. This chapter will focus on describing defense mechanisms on thermophiles and acidophiles against on copper, mercury and arsenic.

3.2

Mechanism 1—cellular sequestration by thiol systems

There are two main thiol systems that maintain intracellular redox state in Bacteria, a low molecular weight (LMW) thiol/redox buffer system and a protein thiol system(s). The composition of these systems varies among organisms. As in neutrophils, some studies have shown the importance of these systems in metal resistance.

3.2.1 Low molecular weight (LMW) thiols LMW thiol systems differ between different organisms, but they all possess a free cysteine, reach millimolar concentrations and act as the main intracellular redox buffer. LMW thiols have a general role of maintaining the intracellular redox state and protect the cell from different toxicants [1,6,7]. Organisms typically have one main LWM thiol, like glutathione (GSH) [8] or bacillithiol (BSH) [9], that is found at higher concentrations than other accessory LWM thiol (s) [10,11]. Some organisms have similar concentrations of different LWM thiols [11]. The main LMW weight thiols found in extremophiles are GSH, BSH, coenzyme A (CoA) or modified versions of these thiols. In the genera Leptospirillum, the main LMW buffer remains unknown. GSH predominates in Gram negative bacteria [12] and its biosynthesis consists in 2-steps [13,14]; some extremely halophilic bacteria appear to have lost (or did not acquire) the second gene of GSH biosynthesis and contain approximately millimolar concentrations of L-γ-glutamyl-L-cysteine (produced by the first enzyme) and thiosulfate as a secondary buffer [15]. In neutrophiles, GSH has been linked to mercury (Hg), arsenite, copper (Cu), tellurite, cadmium and zinc metal resistance [16 23]. There are three GSH variations found in microbes: glutathione amide, glutathionylspermidine and trypanothione which have not been well studied [24]. Due to the interaction of GSH with different enzymes/proteins it is involved in several cellular processes [25 28]. Some extremophilic Gram negative bacteria like Acidithiobacillus and thermotolerant Pseudomonas [29] possess GSH as the main LWM thiol [30 32]. Exposure of Acidithiobacillus ferrooxidans to Cu(II) or cadmium causes an upregulation of the glutathione biosynthetic genes [33,34], Cu(II) also increases protein levels of enzymes involved in cysteine and glutathione biosynthesis [35]. These results correlate with an increase of intracellular levels of GSH and cysteine in cells exposed to cadmium stress [34,36]. Some Gram positives (Firmicutes) and the taxon Deinococcus/Thermus have BSH as the main redox buffer [10,11], which has been suggested complex metals more efficiently when compared to GSH and other thiols [11]. The biosynthesis of BSH is carried in three steps by three enzymes [9]. In neutrophils, BSH has been proposed to carry Fe-S clusters carrier [37], involved in Cu trafficking [38], zinc buffering [39,40], and confer resistance to Zn, Cu, Hg, cadmium, arsenic and dichromate [10,39,41,42]. Not much research has been performed in extremophiles that produce BSH and resistance to metals, in Thermus thermophilus, BSH has been linked to Hg resistance by buffering it and quenching the reactive oxygen species triggered by Hg(II)-stress [10,43]. In Actinomycetes, the most abundant LMW thiol is mycothiol (MSH) [30], and its biosynthesis involves five steps and is similar to BSH [24,44]. In Corynebacterium, MSH has been involved in arsenate detoxification [45] and resistance to resistance to chromium(VI), zinc, cadmium, cobalt and manganese, also in Cu(II) resistance, but only when other Cu-resistance genes were mutated [46]. The acidophilic Gram-positive Acidimicrobium ferrooxidans and Ferrimicrobium acidiphilum [47] and the thermophilic bacteria Defluviitalea phaphyphila [48] should produce MSH as

Mechanism of resistance focusing on copper, mercury and arsenic Chapter | 3

25

the main buffer, but no studies have revealed the role of this LMW thiol in metal resistance for these microbes. Interestingly, Acidi(thio)microbium spp. encode for a putative CoA disulfide reductase [32] which suggest that CoA also plays a role as a LMW thiol. In some neutrophilic Actinomycetes CoA and MSH are found in similar intracellular concentrations [30], so for these extremophilic microbes it remains to be determine the LMW thiol composition, concentrations and roles in metal toxicity. Other organisms produced less characterized thiols like ergothioneine, the aforementioned CoA, coenzyme M, coenzyme B and lower levels of cysteine. Ergothioneine is an antioxidant produced in non-yeast fungi, some Actinobacteria and Mycobacteria [24], but has only been shown to interact with metals in vitro [49]. CoA is produced in lower concentrations in Firmicutes and is the main thiol in some thermophilic Archaea [24] and it might play an important role in acidophiles [32]. CoA is the main thiol in thermophilic Archaea: Pyrococcus furiosus, Thermococcus litoralis , and Sulfolobus solfataricus [50]. Cysteine is found as a secondary buffer in many organisms and has been shown to respond to metal toxicity in several extremophiles. In the Firmicute, Bacillus stearothermophilus, cysteine synthase and cysteine desulfurase have been associated with tellurite resistance [51,52]. Heterologous expression of Geobacillus stearothermophilus V cysteine desulfurase gene (iscS) confers tellurite resistance to Escherichia coli [51]. Overexpression of genes involved in cysteine biosynthesis was observed in At. ferrooxidans when exposed to Cu(II) [35], same results were found for protein levels involved in cysteine biosynthesis in Metallosphaera sedula [53]. In At. thiooxidans Licanantay a genomic island (GI) encodes an extra cysteine biosynthetic pathway cysJIHDNG [54]. In T. thermophilus homocysteine and cysteine biosynthetic genes are upregulated in response to Hg(II); furthermore, one gene involved in the biosynthesis of homocysteine is part of this organism mer operon and co-expressed with other mer genes [10]. As well as, the mer operons of several Alphaproteobacteria, which encode for GSH related genes [10].

3.2.2 Protein thiols Protein thiols also help in the maintenance of a reduced intracellular environment. E. coli has two main protein thiol systems, the glutaredoxin and thioredoxin systems [8]. The thioredoxin system, present in all Bacteria, is composed in E. coli by two thioredoxins (Trx) and the NADPH dependent flavoenzyme thioredoxin reductase (TR) [8]. The glutaredoxin system predominates in Gram negative bacteria and is associated with the presence of the LMW thiol GSH. This system is composed of glutaredoxins, GSH S-transferases and a glutathione reductase (GR) [8,27]. Homologues systems are present in bacteria with BSH (bacilliredoxins, BSH S-transferases), MSH (MSH S-transferases, MSH reductase) and CoA (CoA disulfide reductase) have also been described [27,55 57]. These protein thiol systems have been shown to play a role in metal responses in extremophiles. Exposure of L. ferriphilum to iron (III) causes an increase in thioredoxins and TR expression and activities [10]; TR was also expressed in response to arsenite in Ferroplasma acidarmanus [58]. In At. ferrooxidans GR gene was upregulated upon growth on Cu(II) [33], as well as an increase in protein levels of GSH S-transferase and glutaredoxins [35], GR gene expression and activity was also increased in response to cadmium (II) [34]. Finally, it has been proposed that the high resistance of acidophiles to metals in their natural environment is due to complexation metals with environmental sulfates [59]. Either way, sulfur compounds play a fundamental role in metal tolerance and are the first response that cells have to “decrease” intracellular metal concentrations, while other more specific defense mechanism can be synthesized.

3.3

Mechanism 2—none thiol, extracellular and intracellular complexation

Cells have developed different strategies to decrease bioavailability of metals that are not thiol-based, these mechanisms involved the complexation of these metals with polyphosphates or formation of nanoparticles.

3.3.1 Nanoparticles For reviews on this topic check [60 62]. Nanoparticles (NP) are composed of any material that in one of their dimensions the size ranges from 1 to 100 nm [63]. They display unique physicochemical properties that are often unrelated to the characteristics of their bulk material. Nanoparticles have many technological applications, such as antimicrobials [64,65], conductors [66], catalysts [67], drug delivery systems [68] and they are thought to be a natural defense mechanism against metal toxicity. Bacterial nanoparticle biosynthesis has been widely studied in model organisms for silver, gold and oxides (either copper or zinc) [6,69,70]. Synthesis of high-quality elemental copper (CuNP) and silver (AgNp) nanoparticles has been described for the genera Morganella [71,72]. The cold-tolerant, M. psychrotolerans has higher

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PART | I Physiological aspects

levels of copper and silver resistance than the mesophilic counterpart, supporting the suggested link between nanoparticle synthesis and metal resistance [72 74]. At. thiooxidans is able to form gold NPs inside the cells [75], the production of extracellular gold NP has been reported for the thermophilic actinomycetes Thermomonospora sp. [76]. The thermophilic bacteria, Thermoanaerobacter, is able to synthesize magnetite and substituted magnetite with cobalt, chromate or nickel [77]. The synthesis of fluorescent semiconductor nanoparticles (quantum dots, QDs), has been reported in the Acidithiobacillus genus and is influenced by the presence of cysteine and glutathione [5]. Addition of inorganic phosphate also favored the biosynthesis of CdS QDs; it has been suggested that is due to its role in cadmium uptake and tolerance [78]. Cadmium and tellurite resistant Antarctic bacteria are also able to synthesize CdTe and CdS QDs [79]. But, the CdS QDs produced by psychrotolerant Pseudomonas spp. isolated from Antarctic was not related to thiol content as for Acidithiobacillus [80]. The mechanism by which these NPs are produced is not fully understood, further research is needed to fully comprehend the molecular mechanism. Furthermore, are the same mechanisms employed for NP biosynthesis used by neutrophilic and extremophiles? The most likely answer is no, given that different organism produce different shapes and size nanoparticles [81].

3.3.2 Inorganic polyphosphates For reviews check [82 84]. Inorganic polyphosphates (polyP) are a linear polymers of phosphate residues linked by a phosphoanhydride bonds, that is synthesized by a polyphosphate kinase (PPK) and degraded by a polyphosphatase (PPX), polyP have been described as metal chelators in neutrophilic organisms [82]. Enzymes necessary for polyP biosynthesis have been reported in different Acidithiobacillus strains [84 86] and the genomes of the acidophiles L. ferriphilum and Acidimicrobium ferrooxidans suggest that these organism could produce polyP [87], but there is no experimental data to corroborate their presence. At. ferrooxidans accumulates high amounts of polyP granules, which decrease in presence of Cu(II). It has been proposed that metal-phosphate complexes are formed when the polyP granules are dissolved, and these complexes are transported out of the cell as a tolerance mechanism to Cu(II) [88]. In a different strain of Acidithiobacillus biosynthesis of CdS QDs was favored by the degradation of intracellular polyP, suggesting a link between these two processes [78]. The thermophilic green-sulfur genera Chlorobium has the ability to sorb several metal ions (manganese, iron, nickel, copper, lead, zinc and cadmium) [89], these phenomenon might be partly due to the presence of two exopolyphosphatases, these Cb. tepidum phosphatases have different catalytic specificities for substrates and chain lengths [90]. So far no experimental data corroborates this. In Archaea, polyP have been reported in Methanosarcinae [91], Sulfolobus [92 94] and M. sedula [95], but high amounts of polyP (granules) are only accumulated in S. metallicus [92]. The ability of S. metallicus to accumulate high concentration of polyP had been linked to its ability to grow at high copper concentrations, as compared with low levels of polyP and low tolerance to copper of S. solfataricus [92]. As in At. ferrooxidans, when S. metallicus was grown in presence of copper the polyP levels decreased and Pi export increased [92]. Furthermore, a S. solfataricus mutant deficient in polyP had an increased sensitivity to Cu(II); interestingly, the strain lacking polyP had a significant upregulation of the Cu efflux pump gene copA, suggesting complementary role of these systems in Cu-resistance [96]. Homologous genes to the PPK and exopolyphosphatase of Sulfolobus were found in the genome of M. sedula [95], the exopolyphosphatase gene was upregulated in presence on copper and as other acidophiles, the polyP granules decrease when cells were grown in Cu(II) [97].

3.4

Mechanism 3—enzymatic detoxification

There are several reviews that cover these mechanisms of detoxification [1,2]. For a cell to be able to detoxify a metal by reduction, the redox potential of the metal has to be close to the intracellular redox potential, 2421 mV and 1808 mV, this means that Hg(II), Cu(II) and arsenate can be reduced by microbes [1]. This section will cover the enzymes that are present in different extremophilic microbes able to reduce these metals.

3.4.1 Copper (Cu) In E. coli the a periplasmic multicopper oxidase, CueO, mediates copper resistance by oxidizing Cu(I) to Cu(II), which is less toxic [98 100]. In some strains of E. coli a second copper oxidase, PcoA, is present in the periplasm. PcoA interacts with PcoC to convert Cu(I) to Cu(II); as for CueO, strains lacking PcoA are more sensitive to Cu than the WT strain [101,102]. Another type of multicopper oxidase is present in Rhodobacter capsulatus, cutO [103]. In At. thiooxidans ATCC 19377, cueO was upregulated in response to Cu and a ΔcueO mutant was more sensitive to copper than the WT. cueO is only present in the genomes of At. caldus ATCC 51756 and SM-1 and maybe Alicyclobacillus

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27

acidocaldarius LAA1; but in At. ferrooxidans, Leptospirillum, Sulfolobus, Ferroplasma and Acidimicrobium no cueO homologues are present [104]. In Leptospirillum, a multicopper oxidases are present in Leptospirillum group IV UBA BS species and L. ferriphilumT, the latter has an homologue to cutO [105], but in group IV the authors do not clarify the closest relative of this protein [106]. For thermophiles, the multicopper oxidase of T. thermophilus has been characterized biochemically [107,108], but no physiological studies have been performed.

3.4.2 Mercury The main enzymatic detoxification mechanism consist of mercury reductase (MerA), this flavoprotein reduces Hg(II) to Hg(0) by oxidation of NADP(H), Hg(0) is volatile and can leave the cell [109]. MerB is an organomercuric lyase that removes mercury from organomercurials [109], this enzyme will not be covered in this section. A metagenomic analysis revealed that MerA homologues are mostly present in acidic springs, these hot spring contained archeal and bacterial genomes [110]. MerA appears to be evolved from Aquificae being the deepest-branching lineage in the phylogenetic reconstructions. The Aquifecae thermophiles, Hydrogenobaculum sp. and Hydrogenivirga sp., were resistance to high levels of Hg(II) and the genomes encode for mer operons (merA, merT and merP) [111]. These organisms were also able to reduce Hg(II) to Hg(0) and crude cell extracts had MerA activity; interestingly the merA gene and MerA activity was not induced by Hg(II), which correlates with the lack of a MerR (regulator of the mer operon) homologue in these genomes [111]. The lack of a MerR-type regulator has been shown in other systems [109]. On the other hand, the MerA from the deepest branching bacterial lineage, T. thermophilus, had an optimal temperature between 70 and 80  C and its activity as well as merA transcripts were induced when cells were exposed to Hg(II), indicating that in this system the operon is regulated by Hg(II), not like in Aquificae [112]. A T. thermophilus mutant strain lacking the merA gene was very sensitive to Hg(II) and cell extracts lacked MerA activity [112]. At. ferrooxidans TFI 29 MerA, has been biochemically characterized and in strain BA-4 MerA activity was measured [113,114]. Strain BA-4 volatilized mercury and cell extracts also possessed MerA activity, the latter was optimal at neutral pH [113], this is not the only strain that has been shown to volatilize Hg(II) [115]. In At. ferrooxidans E15, there are 2 mer operons a merCAR and the other cluster includes a transposon gene along with a merRCA, both merA genes appeared to be truncated from their nucleotide sequence and no enzymatic activity was detected when expressed in E. coli [116]. Acidithiobacillus T3.2 has merRTPA operon, which is induced by Hg(II) in E. coli cells that heterologously expressed it [117]. Now, with genome sequencing and metagenomics it has been shown that some strains of Acidithiobacillus have an extra genomic island (GI) that carries extra genes for metal detoxification. The GI of At. ferrooxidans strain ATCC 53993 encodes for mercury detoxification (merACR) and Cu-resistance genes (covered in the next section), strain ATCC 23270 lacks this GI [87,118,119]. The genome of a biomining isolate of At. thiooxidans also possesses a GI encoding a mer operon (merRBC) [54]. Finally, in At. ferrooxidans SUG 2-2, a none MerA-Hg(II) reduction was proposed, in this organism a membrane a cytochrome-c oxidase was able to reduce Hg(II) in presence of Fe(II) [115,120,121]. Strain MON-1 was isolated from a SUG 2-2 culture and presented higher Hg(II)-resistance than the SUG 2-2 strain; even though both strains had similar levels of NADPH-dependent mercury reductase activity in cell extracts, strain MON-1 had higher levels of cytochrome-c oxidase, corroborating its role in Hg(II) reduction [122]. Leptospirillum groups II and III genomes encode for a merA, but in some members of group II merA is frame shifted (insertions/deletions in multiple reads) suggesting that this group cannot reduce Hg (II) [123]. In Leptospirillum group IV UBA BS species and two members of group II, L. ferriphilumT and L. ferriphilum ML-04, encode for a mer operon (merRAC) [105,106], this operon was not expressed in L. ferriphilumT grown in laboratory conditions with only trace metals [105], but it was expressed in biofilm samples of Leptospirillum group IV UBA BS, this results clearly show that this operon is expressed in their natural environment [106]. So far no physiological tests have been performed in the genera Leptospirillum. Another bioleaching genome, S. thermosulfidooxidans, encodes a merA [124]. So far no physiological tests have been performed in the genera Leptospirillum or Sulfobacillus. In the Archaea S. solfataricus, merA is responsive to Hg(II), and a merA mutant was more sensitive to Hg(II) than the WT strain and was not able to produce Hg(0) [125]. In Metallosphaera sedula, MerA is a thermostable enzyme that can remain active after extended incubation at 97  C, which its crystal structure has been obtained [126]. No physiological studies or information is available in acidophilic Archaea.

3.4.3 Arsenic On the other hand, some reduced products are more toxic to the cell than the original form of the metal, like arsenic. For reviews check references [1,127]. Arsenic exists as arsenate, As(V) usually found AsO42 3 , or arsenite, As(III)

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PART | I Physiological aspects

usually found AsO2 2 or As(OH)3, the latter being the most toxic form of arsenic [1,2]. Due to the structural similarities with phosphate, ars operons encode for arsenate reductase (ArsC) that reduces As(V) to As(III) which then is pumped out of the cell by either ArsA/B (covered in Section 3.5) and it can contain two regulators ArsR and ArsD (not covered in this chapter) [1,2]. ArsC can use as electron donors glutathione, glutaredoxin or thioredoxin, depending on the presence of a GSH as main LMW thiol [128 130]. Some Thermus species can respire arsenate (Thermus sp. HR13) and while others, like T. aquaticus and T. thermophilus can oxidize arsenite [131]. T. thermophilus is able to oxidize and reduce arsenic, its genome encodes for an arsC, but the ars genes are not clustered in a single operon like other organisms [132,133], same genetic organization was found in trans HB27 and HB8 [131]. arsC expression was induced in strain HB27 by arsenate and ArsC is a thioredoxin-coupled arsenate reductase [131]. In Acidiphilium multivorum AIU 301, the ars system is composed of arsR, arsD, arsA, arsB, arsC genes and are present in a plasmid with Hg(II) resistance genes; when heterologously expressed in E. coli, this operon conferred resistance to arsenate and arsenite [134,135]. In strain ATCC 33020 of At. ferrooxidans, the ars operon is comped by arsBCHR and when a Δars E. coli strain was complemented with arsCB from At. ferrooxidans resulted in resistance to arsenite, arsenate, and antimony; but thioredoxin was required for arsenate resistance in E. coli [136]. In At. caldus KU, there is also a chromosomal ars operon (arsR and arsC genes transcribed in one direction and in the opposite direction arsB) that is induced by arsenate, arsenite and antimony; like the At. ferrooxidans ars operon, when Δars E. coli was complemented with the operon, the strain was able to reduce arsenate to arsenite [137,138]. In a highly arsenic-resistant strain of Acidithiobacillus there is an extra set of ars genes, which appeared to be located in a Tn21-like transposable element, TnAtcArs, this operon has one arsR, one arsC, two arsD, two arsA and one arsB [139]. An E. coli strain expressing TnAtcArs was more resistant to arsenate than the strain harboring the chromosomal ars genes; same results were observed in when an At. caldus strain lacking TnAtcArs was complemented with TnAtcArs genes [138,139]. As mentioned, strain At. ferrooxidans ATCC 53993 has a GI island, which also has a cluster with five-gene potentially encoding different ars and heavy metal P-type ATPases [87,118,119], but in the GI of At. thiooxidans no arsC is present [54]. In strain At. thiooxidans A01 the ars operon encodes for an arsC (arsRC and arsB divergenlyt) that was upregulated in response to As(III) [140]. In Leptospirillum group III the ars operon (arsABCDR) is present and ArsC was detected in a proteomic analysis of acid mine drainage sample [123]. Leptospirillum group IV UBA BS species does not have an arsC and some members of Leptospirillum group II also lack this gene [106,123]. L. ferriphilum strains that possess ars operons are: L. ferriphilumT, L. ferriphilum YSK, ATCC 49881, Warwick, Fairview and ML-04 encode for one arsRBC operon [105,140 142]. This operon was named LfArs and was poorly expressed in L. ferriphilum strains Fairview and E. coli; furthermore, it conferred low resistance to E.coli when heterologously expressed [142]. Co-culture experiments of strain YSK and At. thiooxidans A01 revealed that At. thiooxidans A01 strain dominates the culture when arsenite is present; this is not the case when the co-culture is done in absence of arsenite, then YSK dominates the culture [140]. The loss of dominance in presence of arsenite of YSK might be explained by the low resistance to arsenic that homologous operon (Fairview strain) conferred to E. coli, [142], anyhow this operon was upregulated in strain YSK when exposed to arsenite [140]. Strains Fairview and ML-04 have an extra set of arsenic resistance genes (arsRCDAB), these are in a transposable element related to TnAtcArs from At. caldus [141,142]. As in At. caldus, the transposon of L. ferriphilum Fairview conferred resistance to arsenite and arsenate when expressed in E. coli and it was transpositionally active [142]. In L. ferriphilum ML-04, this element was slightly different it contain seven genes arsRCDA(CBS)B and ORF7, CBS and ORF7 are not present in the Fairview strain [141]. S. thermosulfidooxidans strain ST has two arsC genes, one of them is closely related to the homologue in Thermaerobacter subterraneus and the other one to Alicyclobacillus acidocaldarius, this gene has not been previously described in S. thermosulfidooxidans [124]. The Archaea, F. acidarmanus is extremely resistant to arsenic, but cannot reduce arsenate in vivo which correlates with the lack of an arsC in the genome [143,144]. S. metallicus strain BC has a membrane-bound As(III) oxidase that was induced by As(III) [145], similar arrangement of was observed in Thermoplasma acidophilum [2]. Some organisms are able to respire arsenic with a membrane arsenate oxidase, this allows them to “dump” electrons to arsenate [146]. Some aerobic organisms have arsenite reductase by which they can obtain energy from arsenite. In A. faecalis asoA and asoB encode the 2 subunits from the arsenite oxidase and are located in a GI with over 20 genes related to arsenite resistance, similar genes were found in Centibacterium arsenoxidans, but these genes were named aox [146]. Some extremophiles, like Thiomonas sp. and Chloroflexus aurantiacus, contain some sequences that encode for arsenite oxidase. In Archaeal genomes of Aeropyrum pernix and Sulfolobus tokodaii, asoA and asoB are present [146].

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3.5

29

Mechanism 4—efflux pumps and transporter

Efflux pumps function to decrease intracellular concentrations of metals. In general, genomes of acicidophilic organisms have a high number of genes for transport proteins; for example, approximately 12% of the genome of Picrophilus torridus (the most acidophilic organism known) encodes for transport proteins [147]. It is important to keep in mind that most of these pumps are not specific for only one metal; they can respond and pump more than one metal.

3.5.1 Copper Cu can be moved across the membrane by different types of transporters: (i) P-type ATPases located in the internal membrane can move Cu between the cytoplasm and the periplasm (CopA and CopB) and (ii) RND pumps which can pump Cu directly out of the cell (Cus system). Both play fundamental roles in Cu homeostasis. P-type ATPases play a role in moving Cu(I) and Cu(II) across the cell, for reviews [1,4,86,104,148,149]. CopA in some microbes acts as an exporter of Cu(I) from the cytoplasm to the periplasm to a periplasmic protein, CusF or CopZ [150 152]; copA expression is induced by Cu [150 152]. In other organisms, CopA is an importer of Cu(I) used for biogenesis of Cucontaining proteins, in this organism CopB is a Cu(I) and silver exporter [153 155]. CusA is a member of the RND superfamily of proteins. CusA, CusB and CusC form a multi-unit transporter complex which pump Cu from the cytoplasm or periplasm (in which CusF appears to be involved) directly to the outside of the cell, this system con also pump silver [156 158]. In T. thermophilus (HB8 and HB27) copA and copB are present. Purified CopA was more active with Cu(I) and CopB with Cu(II). Overexpression of CopA or CopB in T. thermophilus increased the Cu(II) tolerance, compared to the wild type; similarly, mutant strains lacking wither of the cop genes were more sensitive to Cu (II) [159]. At. ferrooxidans strains have different resistances to metals, as mentioned due to the presence of GIs. The genome of At. ferrooxidans ATCC 53993 (highly resistant to copper) and ATCC 23270 encode for Cus and Cop systems [87]. The first Cop protein associated with copper transport in At. ferrooxidans was afe_0454 (suggested to be copD), whose transcript levels were shown to be Cu dependent [160], as well as for the gene afe_1073 (that bioinformatically encodes for a Cu(I)-exporter transmembrane P-ATPase), it was induced in two At. ferrooxidans strains exposed to Cu(II) [161]. This correlates with another study that compared different At. ferrooxidans strains, when exposed to Cu(II) 4 genes related with Cu(II) transport were upregulated [162]. In strain ATCC 23270 there are 10 genes related to copper resistance (Cop and Cus systems) and most of them are upregulated when cells were exposed to Cu(II); furthermore, when E. coli mutants (ΔcopA or ΔcusCFBA ΔcueO) were complemented with their respective homologue gene from At. ferrooxidans, the complemented strains were more resistant to Cu(II) than the mutant strain [163]. This correlates with the up-regulation in response to Cu(II) of RND-type Cus systems and efflux pumps described in a proteomic analysis [35,164]. The high Cu(II) resistance in strain ATCC 53993 has been proposed to be due to a GI that has six ORFs involved in Cu resistance [119]. Most of these genes were upregulated in presence of Cu(II) and they complemented a copper-sensitive E. coli strain [119], recently a proteomic analysis showed overexpression of this GI in response to Cu, RND efflux systems and CusF chaperone were upregulated [104]. In At. ferrivorans SS3 four cus clusters (three cusABCF and one cusABC) are predicted in metagenomic islands, two of those clusters have a divergent a copA [85]; At. thiooxidans strain isolated from a biomining operation also has a GI encoding extra putative Cu(II) exporting ATPase [54]. The genome of At. thiooxidans has two cusABC clusters [104]; the genomes of At. caldus strains ATCC 51756 and SM-1 encode for one cus system (cusABC) and two cop operons (copBZ), and a putative periplasmic copper chaperone (cusF), respectively [104]. In the genera Leptospirillum no physiological studies have been performed in response to copper, but the genome of L. ferriphilum ML-04 two cus operons are present (cusABCF) and one homologous to CopA was found; on the other hand, in L. ferrooxidans only one cus operon is present (cusABCF), as well as one possible copA [104]. The cusABCF operon is also present in L. ferriphilumT genome, but this strain also encodes for a Cop-type ATPase [105]. In Leptospirillum group II UBA a copper -transporting ATPase is present in a genomic island, but Leptospirillum group III genome does not possess any transporter [123]. The genome of the Gram positive Acidimicrobium ferrooxidans encodes for one Cop system (copAZ), the same is true for S. thermosulfidooxidans genome, which also encodes for four possible copAB proteins and one of them is found contiguous to a putative copZ [104]. In the Archaea S. solfataricus the cop operon encodes for a metallochaperone (copM), a P-type copper-exporting ATPase (copA) and a novel transcriptional regulator specific for archaea (copT), copMA transcript responds to high Cu(II) concentrations, whereas the regulator appears to be constitutive expressed; CopT binds to the copMA promoter at multiple sites and this affinity is modulated by Cu [165]. On the other hand, S. solfataricus 98/2 has two

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copper-transporting proteins, CopA and CopB. copA andcopT are in an operon (in this case, CopT is a small copper binding protein, not the regulator, which is named copR) and this operon is induced by Cu(II) [166]; meanwhile copB is located in a different loci. Both Cop transporters are involved in export of Cu(I) and silver, but copA mutants were extremely sensitive to Cu and copB appears to play a role only at high Cu concentration [167]. S. metallicus genome encodes for two cop clusters composed of a metallochaperone (copM), a Cu-exporting ATPase (copA), and a transcriptional regulator (copT). As S. sulfataricus, copM and copA, in both gene clusters, were cotranscribed when cells were exposed to Cu [168]. In F. acidarmanus the cop loci has a putative transcriptional regulator (copY), a putative metalbinding chaperone (copZ) and a putative Cu-transporting P-type ATPase (copB). copZ and copB are co-transcribed and induced by exposure to high levels of Cu(II) [58]. Interestingly, these proteins were not upregulated in 2D-PAGE analysis, in response to Cu; authors suggested that is due to this technique, because hydrophobic membrane proteins, like CopB, and very small proteins, like CopZ, are difficult to detect [58]. The hyperthermophilic archaea, Archeoglobus fulgidus also possesses two Cop transporters, CopA transports Cu(I) and CopB transports Cu(II) and can partially transport and partially by Cu(I) and Ag(I) [169 171]. Same is the case for Metallosphaera sedula, a CopA and a CopB are present, the role of copRTA operon was demonstrated by a copR mutant of S. solfataricus that was copper sensitive, crossspecies complemented S. solfataricus strain was able to grow in presence of Cu corroborating the role of this operon in Cu resistance [172]. Furthermore, a copA mutant strain of M. sedula was more sensitive to copper than the WT strain and in a copR mutant, copB transcripts are highly induced on the mutant strain [172]. Proteomics of this microbe also revealed the upregulation in response to Cu(II) of a P-type ATPase (Msed_0490) associated with the Cop system (6-fold), this protein was also induced in response to Zn(II) [53]. The genomes of Thermoplasma acidophilum, Thermoplasma volcanium, P. torridus and Acidiplasma cupricumulans also encode for a CopA [104].

3.5.2 Mercury Hg(II) can get inside the cell without a transport system, but some mer operons encode for Hg(II) transport systems and the occurrence vary among organisms. Gram negatives can have the inner membrane MerT, MerC, MerF and MerE transportes, but Gram positives usually have MerT-like transporter [109]. MerT is the main Hg(II) transporter in Bacteria, MerC is thought to be needed at very high Hg(II) concentrations, the other two transporters are less studied, for review check [109]. As mentioned different mercury resistance operons have been characterized from a variety of At. ferrooxidans strains. The first strain to be characterized had only one merC as transporter (AfMerC), when expressed in E. coli, the Hg(II) uptake by AfMerC was lower than the MerT-MerP system from pDU1358, but AfMerC was not sensitive to lead as the plasmidial system was [173]. In At. ferrooxidans E15, there are two merC genes and one of them is present in a transposable element [116]. On the other hand, mer operons of Acidihiobacillus T3.2 and At. ferrooxidans Tn5037 encode for a merT [117,174]. Some strains of L. ferriphilum, L. ferrooxidans, L. rubarum carry a merT [106,123]; L. ferriphilumT and ML-04 encode for a merC gene located in the mer operon, merT is located in a different loci, but Leptospirillum group IV UBA only encodes for a merC [105,106]. S. solfataricus mer locus encodes for merHAI and merR in the opposite direction, but lacks orthologs for transport genes [125,175]. This may suggest that such transport proteins are unique to this organism or perhaps to archaea in general.

3.5.3 Arsenic E. coli has two phosphate transporters, the regulated high phosphate affinity-low velocity Pst (phosphate specific transport) and the constitutively expressed low affinity-high velocity Pit system (Pi transport), both systems can import arsenate [176 178]. The transport of arsenate by phosphate transporter is due to their structural similarities. Arsenite can enter by aqua-glyceroporins (glycerol transport proteins, like GlpF) and it may also enter by other systems [127]. In M. sedula, PitA is also low affinity-high velocity phosphate transporter which is implicated in Cu resistance and sensitivity to arsenate [179]. So, how is arsenate pumped out of the cell? Arsenate has to be reduced to arsenite, as mentioned in section 3.3, this is carried by ArsC. Then, arsenite can be transported by ArsB, this is a transmembrane protein that uses electrochemical energy to pump it out of the cell [180,181]. Some arsenic resistant systems also encode for ArsA, an oxyanion-stimulated ATPase that requires ArsB to be anchored to the inner membrane and pump arsenite out of the cell [180 182]. For reviews check [127,183]. T. thermophilus genome encodes for two arsB in the genome and one extra in one of its natural plasmids [131], as mentioned previously they are not found in an operon, these gene are scatter over the genome in ars-independent operons. Later on, one of the arsB genes was renamed arsX, and it was shown to be regulated by the ArsR/SmtB,

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these genes are adjacent to each other, but expressed from different promoters [184]. Mutants lacking arsX are more sensitive to As(V) and As(III) than the parental strain [184], interestingly the ΔarsX was also highly sensitive to cadmium, slightly more sensitive to cadmium than to arsenic (around 15-fold and 12-fold increase on sensitivity, respectively) [133]. The arsX promoter is regulated in response to arsenic and cadmium [133,184]. Several acidophilic bacteria encode for an arsB like At. caldus, At. thiooxidans, At. ferrooxidans, Leptospirillum, Acidiphilium acidophilum and Thiomonas cuprina [136 138,140,141]. In A. thiooxidans A01 arsB transcription was upregulated in response to As(III) [140]. In A. ferrooxidans ATCC 33020, heterologous expression of the arsB was able to recover arsenic resistance to a Δars E. coli strain [136]. In At. caldus strain KU the chromosomal copy of arsB is able to transport arsenite [137,138] and in strain #6 there is an additional ars operon in a transposable element, this operon has functional arsB and arsA that confer resistance only to As(III) when expressed in E. coli [138,185]. Leptospirillum groups II and III encode for arsB and only in Leptospirillum group III and group IV UBA BS species encode for an arsA [106,123]. In Leptospirillum groups II and III, the Ars proteins were identified in proteomic of an acid mine drainage [123], ArsA and ArsB were detected in the proteome of strain ML-04 exposed to As(III) [141]. In the Archaea F. acidarmanus, no arsC homologue is found in the genome, but arsB and arsR are cotranscribed and expressed in response to arsenite, there is also copy of arsA suggesting that there is an ArsAB functional pump [143,144]. Same is the case for Thermoplasma acidophilum [186] and T. volcanium, no homologues for arsB were found for Sulfolobus [2]. In M. sedula genome encodes for an arsB but no other ars gene appears to be present in this organism [95].

3.6

Conclusions and future perspectives

Resistance mechanism to metals in extremophiles is still an unexplored topic, most of the studies have focused in copper resistance for acidophilic iron-oxidizing microorganism and relating these organisms to the neutrophilic wellstablished systems. Low molecular weight thiols, BSH and GSH, and thiol-related systems have been shown to be of great importance to resist metal toxicity, as the neutrophilic counterpart. Further studies are required to understand their physiological role in metal toxicity, as well as studies for “less” common LMW thiols, as CoA, and identification of possible unknown LMW thiols, like in Leptospirillum. PolyP and nanoparticles appear to be an efficient way by which these microorganisms tolerate metals; many of these organisms encode for genes that codify for PPXs or/and PKKs, but the presence of polyP remains to be experimentally proven. For nanoparticles the problem is the opposite, many organisms have been shown to produce them, but the mechanism remains unknown and identification of the genes involved in nanoparticle biosynthesis would facilitate screening of microorganisms that can produce them. Furthermore, the role of accessory thiol proteins in metal resistance, not Trx or Grx, is still in the dark for these microorganisms. The link between LMW thiols and polyP is not clear, it appears to be related in some microorganism, but not in others. Same is the case for the link between polyP and classical resistance genes, questions remain to be answered: Are these systems complementary or independent? Does it vary in different organisms? Is it strain related?. More studies are needed to clarify the links between systems. For classical resistance systems (pumps and enzymes) more studies have been performed. Most of the studies scanned for known resistance genes and showed how similar these systems are to their neutrophilic counterparts. Extremely resistant microbes usually have transposable extra copies or plasmid carrying extra resistance genes; it has to be noted that these systems and resistance vary greatly between strains, indicating that these microbes adapt to their surrounding environments. Copper-resistance mechanisms have been focused on bioleaching microbes, but not much research has been performed in thermophiles or other acidophiles. Development of molecular tools to assess physiological roles of these genes would accelerate the research on this field. The use of proteomic and microarrays has allowed us to identify genes that respond to metal stress, many hypothetical proteins have been described in these studies, but not new resistance mechanisms have been described yet. Further studies characterizing these proteins would help to discover new mechanisms involved in metal resistance. These mechanisms might be unique and specific adaptations of these microbes to their surroundings.

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[100] Outten FW, Outten CE, Hale J, O’Halloran TV. Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. J Biol Chem 2000;275(40):31024 9. [101] Djoko KY, Xiao Z, Wedd AG. Copper resistance in E. coli: the multicopper oxidase PcoA catalyzes oxidation of copper(I) in CuICuII-PcoC. ChemBioChem 2008;9(10):1579 82. [102] Huffman DL, Huyett J, Outten FW, Doan PE, Finney LA, Hoffman BM, et al. Spectroscopy of Cu(II)-PcoC and the multicopper oxidase function of PcoA, two essential components of Escherichia coli pco copper resistance operon. Biochemistry 2002;41(31):10046 55. [103] Wiethaus J, Wildner GF, Masepohl B. The multicopper oxidase CutO confers copper tolerance to Rhodobacter capsulatus. FEMS Microbiol Lett 2006;256(1):67 74. [104] Martinez-Bussenius C, Navarro CA, Orellana L, Paradela A, Jerez CA. Global response of Acidithiobacillus ferrooxidans ATCC 53993 to high concentrations of copper: a quantitative proteomics approach. J Proteom 2017;145:37 45. [105] Christel S, Herold M, Bellenberg S, El Hajjami M, Buetti-Dinh A, Pivkin IV, et al. Multi-omics reveals the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilum. Appl Environ Microbiol 2018;84(3) e02091-17. [106] Goltsman DS, Dasari M, Thomas BC, Shah MB, VerBerkmoes NC, Hettich RL, et al. New group in the Leptospirillum clade: cultivationindependent community genomics, proteomics, and transcriptomics of the new species “Leptospirillum group IV UBA BS”. Appl Environ Microbiol 2013;79(17):5384 93. [107] Serrano-Posada H, Valderrama B, Stojanoff V, Rudin˜o-Pin˜era E. Thermostable multicopper oxidase from Thermus thermophilus HB27: crystallization and preliminary X-ray diffraction analysis of apo and holo forms. Acta Crystallogr Sect F, Struct Biol Cryst Commun 2011;67(Pt 12):1595 8. [108] Bello M, Valderrama B, Serrano-Posada H, Rudino-Pinera E. Molecular dynamics of a thermostable multicopper oxidase from Thermus thermophilus HB27: structural differences between the apo and holo forms. PLoS One 2012;7(7):e40700. [109] Barkay T, Miller SM, Summers AO. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev 2003;27(2 3):355. [110] Geesey GG, Barkay T, King S. Microbes in mercury-enriched geothermal springs in western North America. Sci Total Environ 2016;569 570:321 31. [111] Freedman Z, Zhu C, Barkay T. Mercury resistance and mercuric reductase activities and expression among chemotrophic thermophilic Aquificae. Appl Environ Microbiol 2012;78(18):6568 75. [112] Wang Y, Freedman Z, Lu-Irving P, Kaletsky R, Barkay T. An initial characterization of the mercury resistance (mer) system of the thermophilic bacterium Thermus thermophilus HB27. FEMS Microbiol Ecol 2009;67(1):118. [113] Olson GJ, Porter FD, Rubinstein J, Silver S. Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans. J Bacteriol 1982;151(3):1230 6. [114] Booth JE, Williams JW. The isolation of a mercuric ionreducing flavoprotein from Thiobacillus ferrooxidans. J Gen Microbiol 1984;130:725 30. [115] Takeuchi F, Iwahori K, Kamimura K, Negishi A, Maeda T, Sugio T. Volatilization of mercury under acidic conditions from mercury-polluted soil by a mercury-resistant Acidithiobacillus ferrooxidans SUG 2-2. Biosci Biotechnol Biochem 2001;65(9):1981 6. [116] Inoue C, Sugawara K, Kusano T. The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes. Mol Microbiol 1991;5(11):2707 18. [117] Velasco A, Acebo P, Flores N, Perera J. The mer operon of the acidophilic bacterium Thiobacillus T3.2 diverges from its Thiobacillus ferrooxidans counterpart. Extremophiles 1999;3(1):35 43. [118] Ca´rdenas JP, Valde´s J, Quatrini R, Duarte F, Holmes DS. Lessons from the genomes of extremely acidophilic bacteria and archaea with special emphasis on bioleaching microorganisms. Appl Microbiol Biotechnol 2010;88(3):605 20. [119] Orellana LH, Jerez CA. A genomic island provides Acidithiobacillus ferrooxidans ATCC 53993 additional copper resistance: a possible competitive advantage. Appl Microbiol Biotechnol 2011;92(4):761 7. [120] Sugio T, Iwahori K, Takeuchi F, Negishi A, Maeda T, Kamimura K. Cytochrome c oxidase purified from a mercury-resistant strain of Acidithiobacillus ferrooxidans volatilizes mercury. J Biosci Bioeng 2001;92(1):44 9. [121] Iwahori K, Takeuchi F, Kamimura K, Sugio T. Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans. Appl Environ Microbiol 2000;66(9):3823 7. [122] Sugio T, Fujii M, Takeuchi F, Negishi A, Maeda T, Kamimura K. Volatilization of mercury by an iron oxidation enzyme system in a highly mercury-resistant Acidithiobacillus ferrooxidans strain MON-1. Biosci Biotechnol Biochem 2003;67(7):1537 44. [123] Goltsman DSA, Denef VJ, Singer SW, VerBerkmoes NC, Lefsrud M, Mueller RS, et al. Community genomic and proteomic analyses of chemoautotrophic iron-oxidizing “Leptospirillum rubarum” (Group II) and “Leptospirillum ferrodiazotrophum” (Group III) bacteria in acid mine drainage biofilms. Appl Environ Microbiol 2009;75(13):4599 615. [124] Guo X, Yin H, Liang Y, Hu Q, Zhou X, Xiao Y, et al. Comparative genome analysis reveals metabolic versatility and environmental adaptations of Sulfobacillus thermosulfidooxidans strain ST. PLoS One 2014;9(6) e99417-e99417. [125] Schelert J, Dixit V, Hoang V, Simbahan J, Drozda M, Blum P. Occurrence and characterization of mercury resistance in the hyperthermophilic archaeon Sulfolobus solfataricus by use of gene disruption. J Bacteriol 2004;186(2):427 37. [126] Artz JH, White SN, Zadvornyy OA, Fugate CJ, Hicks D, Gauss GH, et al. 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[128] Oden KL, Gladysheva TB, Rosen BP. Arsenate reduction mediated by the plasmid-encoded ArsC protein is coupled to glutathione. Mol Microbiol 1994;12(2):301 6. [129] Ji G, Silver S. Reduction of arsenate to arsenite by the ArsC protein of the arsenic resistance operon of Staphylococcus aureus plasmid pI258. Proc Natl Acad Sci 1992;89(20):9474 8. [130] Gladysheva TB, Oden KL, Rosen BP. Properties of the arsenate reductase of plasmid R773. Biochemistry. 1994;33(23):7288 93. [131] Del Giudice I, Limauro D, Pedone E, Bartolucci S, Fiorentino G. A novel arsenate reductase from the bacterium Thermus thermophilus HB27: its role in arsenic detoxification. Biochim Biophys Acta (BBA) Proteins Proteom 2013;1834(10):2071 9. [132] Politi J, Spadavecchia J, Fiorentino G, Antonucci I, Casale S, De Stefano L. Interaction of Thermus thermophilus ArsC enzyme and gold nanoparticles naked-eye assays speciation between As(III) and As(V). Nanotechnology 2015;26(43):435703. [133] Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, et al. Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system. Microb Cell Fact 2018;17(1):78. [134] Suzuki K, Wakao N, Kimura T, Sakka K, Ohmiya K. Expression and regulation of the arsenic resistance operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli. Appl Environ Microbiol 1998;64(2):411 18. [135] Suzuki K, Wakao N, Sakurai Y, Kimura T, Sakka K, Ohmiya K. Transformation of Escherichia coli with a large plasmid of Acidiphilium multivorum AIU 301 encoding arsenic resistance. Appl Environ Microbiol 1997;63(5):2089 91. [136] Butcher BG, Deane SM, Rawlings DE. The chromosomal arsenic resistance genes of Thiobacillus ferrooxidans have an unusual arrangement and confer increased arsenic and antimony resistance to Escherichia coli. Appl Environ Microbiol 2000;66(5):1826 33. [137] Dopson M, Lindstro¨m BE, Hallberg KB. Chromosomally encoded arsenical resistance of the moderately thermophilic acidophile Acidithiobacillus caldus. Extremophiles 2001;5(4):247 55. [138] Kotze AA, Tuffin IM, Deane SM, Rawlings DE. Cloning and characterization of the chromosomal arsenic resistance genes from Acidithiobacillus caldus and enhanced arsenic resistance on conjugal transfer of ars genes located on transposon TnAtcArs. Microbiology. 2006;152(Pt 12):3551 60. [139] Tuffin IM, de Groot P, Deane SM, Rawlings DE. An unusual Tn21-like transposon containing an ars operon is present in highly arsenicresistant strains of the biomining bacterium Acidithiobacillus caldus. Microbiology 2005;151(Pt 9):3027 39. [140] Jiang H, Liang Y, Yin H, Xiao Y, Guo X, Xu Y, et al. Effects of Arsenite Resistance on the Growth and functional gene expression of Leptospirillum ferriphilum and Acidithiobacillus thiooxidans in pure culture and coculture. BioMed Res Int 2015;2015:203197. [141] Li B, Lin J, Mi S, Lin J. Arsenic resistance operon structure in Leptospirillum ferriphilum and proteomic response to arsenic stress. Bioresour Technol 2010;101(24):9811 14. [142] Tuffin IM, Hector SB, Deane SM, Rawlings DE. Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank. Appl Environ Microbiol 2006;72(3):2247 53. [143] Gihring TM, Bond PL, Peters SC, Banfield JF. Arsenic resistance in the archaeon “Ferroplasma acidarmanus”: new insights into the structure and evolution of the ars genes. Extremophiles 2003;7(2):123 30. [144] Baker-Austin C, Dopson M, Wexler M, Sawers RG, Stemmler A, Rosen BP, et al. Extreme arsenic resistance by the acidophilic archaeon ‘Ferroplasma acidarmanus’ Fer1. Extremophiles 2007;11(3):425 34. [145] Mikael Sehlin H, Bo¨rje Lindstro¨m E. Oxidation and reduction of arsenic by Sulfolobus acidocaldarius strain BC. FEMS Microbiol Lett 1992;93(1):87 92. [146] Silver S, Phung LT. Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Appl Environ Microbiol 2005;71 (2):599 608. [147] Angelov A, Liebl W. Insights into extreme thermoacidophily based on genome analysis of Picrophilus torridus and other thermoacidophilic archaea. J Biotechnol 2006;126(1):3 10. [148] Voskoboinik I, Camakaris J, Mercer JFB. Understanding the mechanism and function of copper P-type ATPases Academic Press Adv Protein Chem, 60. 2002. p. 123 50. [149] Lutsenko S, Kaplan JH. Organization of P-type ATPases: significance of structural diversity. Biochemistry 1995;34(48):15607 13. [150] Rensing C, Fan B, Sharma R, Mitra B, Rosen BP. CopA: an Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci USA 2000;97(2):652 6. [151] Zhou L, Kay KL, Hecht O, Moore GR, Le Brun NE. The N-terminal domains of Bacillus subtilis CopA do not form a stable complex in the absence of their inter-domain linker. Biochim Biophys Acta (BBA) Proteins Proteom 2018;1866(2):275 82. [152] Radford DS, Kihlken MA, Borrelly GP, Harwood CR, Le Brun NE, Cavet JS. CopZ from Bacillus subtilis interacts in vivo with a copper exporting CPx-type ATPase CopA. FEMS Microbiol Lett 2003;220(1):105 12. [153] Odermatt A, Krapf R, Solioz M. Induction of the Putative Copper ATPases, CopA and CopB, of Enterococcus hirae by Ag1 and Cu21, and Ag1 Extrusion by CopB. Biochem Biophys Res Commun 1994;202(1):44 8. [154] Solioz M, Odermatt A. Copper and silver transport by CopB-ATPase in membrane vesicles of Enterococcus hirae. J Biol Chem 1995;270 (16):9217 21. [155] Magnani D, Solioz M. Copper chaperone cycling and degradation in the regulation of the cop operon of Enterococcus hirae. Biometals 2005;18(4):407 12. [156] Franke S, Grass G, Rensing C, Nies DH. Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. J Bacteriol 2003;185(13):3804 12.

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[157] Mealman TD, Blackburn NJ, McEvoy MM. Metal export by CusCFBA, the periplasmic Cu(I)/Ag(I) transport system of Escherichia coli. Curr Top Membr 2012;69:163 96. [158] Chacon KN, Mealman TD, McEvoy MM, Blackburn NJ. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins. Proc Natl Acad Sci USA 2014;111(43):15373 8. [159] Schurig-Briccio LA, Gennis RB. Characterization of the PIB-Type ATPases present in Thermus thermophilus. J Bacteriol 2012;194 (15):4107 13. [160] Wu X, Hu Q, Hou D, Miao B, Liu X. Differential gene expression in response to copper in Acidithiobacillus ferrooxidans strains possessing dissimilar copper resistance. J Gen Appl Microbiol 2010;56(6):491 8. [161] Hu Q, Wu X, Jiang Y, Liu Y, Liang Y, Liu X, et al. Differential gene expression and bioinformatics analysis of copper resistance gene afe_1073 in Acidithiobacillus ferrooxidans. Biol Trace Elem Res 2013;152(1):91 7. [162] Wu X, Zhang Z, Liu L, Deng F, Liu X, Qiu G. Metal resistance-related genes are differently expressed in response to copper and zinc ion in six Acidithiobacillus ferrooxidans strains. Curr Microbiol 2014;69(6):775 84. [163] Navarro CA, Orellana LH, Mauriaca C, Jerez CA. Transcriptional and functional studies of Acidithiobacillus ferrooxidans genes related to survival in the presence of copper. Appl Environ Microbiol 2009;75(19):6102 9. [164] Almarcegui RJ, Navarro CA, Paradela A, Albar JP, von Bernath D, Jerez CA. Response to copper of Acidithiobacillus ferrooxidans ATCC 23270 grown in elemental sulfur. Res Microbiol 2014;165(9):761 72. [165] Ettema TJ, Brinkman AB, Lamers PP, Kornet NG, de Vos WM, van der Oost J. Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2. Microbiology 2006;152(Pt 7):1969 79. [166] Villafane AA, Voskoboynik Y, Cuebas M, Ruhl I, Bini E. Response to excess copper in the hyperthermophile Sulfolobus solfataricus strain 98/2. Biochem Biophys Res Commun 2009;385(1):67 71. [167] Vollmecke C, Drees SL, Reimann J, Albers SV, Lubben M. The ATPases CopA and CopB both contribute to copper resistance of the thermoacidophilic archaeon Sulfolobus solfataricus. Microbiology 2012;158(Pt 6):1622 33. [168] Orell A, Remonsellez F, Arancibia R, Jerez CA. Molecular characterization of copper and cadmium resistance determinants in the biomining thermoacidophilic archaeon Sulfolobus metallicus. Archaea (Vancouver, BC) 2013;2013:289236. [169] Mandal AK, Yang Y, Kertesz TM, Arguello JM. Identification of the transmembrane metal binding site in Cu1-transporting PIB-type ATPases. J Biol Chem 2004;279(52):54802 7. [170] Cattoni DI, Gonzalez Flecha FL, Arguello JM. Thermal stability of CopA, a polytopic membrane protein from the hyperthermophile Archaeoglobus fulgidus. Arch Biochem Biophys 2008;471(2):198 206. 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Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli. J Bacteriol 1980;144(1):356 65. [177] Rosenberg H, Gerdes RG, Chegwidden K. Two systems for the uptake of phosphate in Escherichia coli. J Bacteriol 1977;131(2):505 11. [178] Willsky GR, Malamy MH. Effect of arsenate on inorganic phosphate transport in Escherichia coli. J Bacteriol 1980;144(1):366 74. [179] McCarthy S, Ai C, Wheaton G, Tevatia R, Eckrich V, Kelly R, et al. Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile. J Bacteriol 2014;196(20):3562 70. [180] Dey S, Rosen BP. Dual mode of energy coupling by the oxyanion-translocating ArsB protein. J Bacteriol 1995;177(2):385 9. [181] Tisa LS, Rosen BP. Molecular characterization of an anion pump. The ArsB protein is the membrane anchor for the ArsA protein. J Biol Chem 1990;265(1):190 4. [182] Wu J, Tisa LS, Rosen BP. Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase. J Biol Chem 1992;267(18):12570 6. [183] Rosen BP. Families of arsenic transporters. Trends Microbiol 1999;7(5):207 12. [184] Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, et al. An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27. Microb Biotechnol 2017;10(6):1690 701. [185] de Groot P, Deane S, Rawlings D. A transposon-located arsenic resistance mechanism from a strain of Acidithiobacillus caldus isolated from commercial, arsenopyrite biooxidation tanks. Hydrometallurgy 2003;71(1):115 23. [186] Ruepp A, Graml W, Santos-Martinez ML, Koretke KK, Volker C, Mewes HW, et al. The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum. Nature 2000;407(6803):508 13.

Chapter 4

Halotolerant microbes and their applications in sustainable agriculture Jayant Kulkarni1,2, Sandeep Sharma3,4, Ashish K. Srivastava1,5 and Suprasanna Penna1,5 1

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai, India, 2Department of Botany, Savitribai Phule Pune

University, Pune, India, 3CSIR-Central Salt & Marine Chemicals Research Institute, Bhavnagar, India, 4Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India, 5Homi Bhabha National Institute, Mumbai, India

4.1

Introduction

It has been predicted that within the next 50 years world population will be above nine billion [1] and thus food security for the increasing population has become a major concern. The supply of food to such a huge population without putting at risk natural resources is a formidable challenge for agriculturalists and, most likely, this problem will increase over next few years [2,3]. Moreover, abiotic stresses like salinity, drought, high and low temperature, heavy metal toxicity adversely reduce the crop productivity [4]. Out of these all abiotic stresses, salinity stress is one of the major abiotic stresses which has severe effect on agricultural production across the globe. Worldwide B50% irrigated land is influenced by salinity [5]. Approximately 6 7% (800 million hectors) of land across the globe is affected by salinity and this figure is continuously growing [6]. Major factors responsible for salinization of agricultural land include irrigation with salty water, rain shortage, long term accumulation of salts in the soil and other anthropogenic activities. A change in the climatic conditions may also become a major factor that contributes to even more saline landscape [7]. If the electrical conductivity of soil is above 4 dS m21 (B40 mM NaCl) then the soil is referred to as saline. Under this condition, extra Na1 creates ionic as well as osmotic stresses in plant cells, which leads to reduction in plant overall growth and crop yield [8]. Most of the cultivated and agriculturally important crop species (referred as glycophytes) are susceptible for this NaCl concentration and results significant reduction in production [9]. Several approaches have been employed for reducing yield losses triggered by abiotic stresses (Fig. 4.1). These approaches include genetic engineering, traditional breeding, and the use of growth stimulators. Transgenic approach is a feasible option but several challenges need to be overcome including consumer acceptance and environmental concerns [10]. Although technology adoption is gaining momentum, only some countries across globe have officially approved transgenic crops [11]. Conventional plant breeding is still a popular approach to develop varieties with enhanced stress tolerance; however, this technique is time consuming (typically 8 10 years), labor intensive and many of times, leads to the loss of desirable traits. In addition, breeding techniques are not successful in many plant species [3,12]. In other hand, chemical and biological priming approach is employed to enhance abiotic stress tolerance in plants [13]. Chemical priming has been a promising approach to augment plant abiotic stress tolerance. Different chemical agents like sodium hydrosulfide, sodium nitroprusside, hydrogen peroxide, melatonin, polyamines and thiourea have been reported to confer stress tolerance in plants as they are exposed to different abiotic stresses [14]. Biological priming involves use of organic material and/or microorganism (bacteria and fungi) to develop abiotic stress tolerance in plants. These biological agents are referred to as biostimulants [15]. Plant growth promoting rhizobacteria (PGPR) and mycorrhizal fungi are major biostimulants reported to augment overall plant growth under extreme conditions [16]. Rhizosphere of a plant is a complex system and is described as hub of maximum microbial activity. Microbes including bacteria, fungi, algae, protozoa and actinomycetes, present in the rhizosphere, colonize plant roots [17]. Among all these microbes, bacteria and fungi are most abundant in the rhizosphere [18]. Rhizosphere fungi include epiphytic and endophytic species. Fungi (mycorrhizae) generate symbiotic association with plant roots and increase root Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00004-6 © 2020 Elsevier Inc. All rights reserved.

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PART | I Physiological aspects

FIGURE 4.1 Various approaches for improving abiotic stress tolerance in crop plants.

surface area, which help plants to absorb water and essential nutrients. Two types of mycorrhizae have been reported to associate with number of plant species, i.e. ecto-mycorrhizae or endo-mycorrhizae. Mycorrhizal association has been known to improve abiotic stress tolerance in plants [19]. Diverse bacterial genera have been known to present in the rhizosphere of plants like Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Enterobacter, Klebsiella, Ochrobactrum, Pseudomonas, Serratia, Variovorax, etc. and have key role in an enhancement of plant growth under stress and non-stress conditions and are term as plant growth promoting rhizobacteria (PGPR) [6,16,20]. Application of PGPR and mycorrhizal fungi is constantly increasing under stress and control condition. It also offers an eco-friendly alternative to replace the use of harmful chemical fertilizers [6,21]. PGPR are present in rhizosphere as either free-living or endophytic and colonize the roots to improve the plant growth. So far, only 2 5% of rhizospheric bacteria involved in improving plant growth and is referred as PGPR [22]. All PGPR have some distinctive characters like ability to colonize plant root surface, survival, multiplication and competence with other pathogenic microbes and must have ability to improve plant growth [23]. Various studies prove that PGPR efficiently enhances plant growth in variety of crops in different stress environments and therefore in last few years, efforts have been made to use these beneficial soil microbes to improve crop production in abiotic stress conditions [16,17,24,25]. The significance of these microorganisms for the improvement of plant stress tolerance to various abiotic stresses like salt, drought and heavy metals has been demonstrated in various crops [26]. Therefore, environmentally habitatadapted PGPR are an effective and eco-friendly way for the improvement of stress tolerance in plants [2,27]. In the present chapter, we have described the halotolerant rhizopsheric microbes as an important resource for improving salinity tolerance and sustainable agriculture.

4.2

Halotolerant biota

It has been shown that the ability of microbes to improve salinity tolerance is affected by various environmental conditions, like climate, weather, and soil structure etc. and it is also influenced by interactions with surrounding microbes [28]. PGPR isolated from saline habitat can tolerate high salt concentration and also shows plant growth promoting traits under salt stress. [29]. Therefore, halotolerant PGPR are carefully chosen based on ability to grow under high salt concentration and efficiency in showing plant growth promoting traits can significantly improve efficiency of growing crops in salinity affected environments [30]. PGPR present in the salty environment are more efficient in alleviating salinity stress in plants than PGPR isolated from non-saline areas [28]. There is absolute evidence that PGPR from rhizosphere of plants growing under harsh environments like high salinity and drought, help those plants to mitigate various abiotic stresses [31]. Therefore, rhizosphere of halophytes is the ultimate source for some halotolerant PGPR. Based on the ability to survive under salt stress, plants are categorized as glycophytes and halophytes. Halophytes are basically salt tolerant plants which generally grow near a coastal area where no other plants can survive [16]. About, 1% of the entire floras of the planet are halophytic plants [32]. Throughout evolution, these species have adapted various morphological, anatomical, and physiological strategies to grow under extreme saline conditions. Halophytes are categorized as obligate halophytes which consistently require salt to complete their life cycle and facultative

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halophytes which can grow on soil with and without NaCl [33,34]. Halophytes consume Na1 and Cl2 ions for a large portion of their osmotic change [35]. They have evolutionarily conserved strategies to grow under extreme conditions by expressing enzymes, transcription regulators and other genetic factors that function in salt responsive pathways [36]. Rhizosphere of halophytes is a rich reservoir of epiphytic and endophytic microbes that inhabit the saline ecosystem. These microbes including both fungi and bacteria are adapted to extreme environment and have ability to counter different abiotic stresses [37,38]. Based on its ability to survive under salt stress, microbes are categorized as halophilic or halotolerant. Halotolerant microbes are nonhalophilic micro-organisms that can tolerate up to 25% salt (NaCl) stress. On other hand halophilic microbes require salt for their overall growth, while non nonhalophilic microbes require less 1% NaCl [39]. (Fig. 4.2). Similar to halophytes, microbes associated with such habitats have also evolved different strategies to survive in high saline environment [40].

4.3

Rhizospheric bacteria and plant growth promotion

PGPR are a diverse group of bacteria present in rhizosphere as either free-living or endophytic and colonize the roots to improve the plant growth. Any rhizospheric bacteria to be considered PGPR must have distinctive characters like, (a) it must colonize root surface, (b) it must survive, reproduce and compete with other pathogenic microbes, (c) it must improve plant growth [23]. Therefore, only 2 5% of rhizosperic bacteria improve plant growth [22]. PGPR are potentially used as inoculants for increasing overall plant growth and crop production and provides a striking way to replace established harmful chemical fertilizers [41]. As plant releases root exudates, different types of rhizospheric bacteria are strongly attracted. Specific bacterias can effectively colonize the root surface though other selectively enters the root tissue. On the basis of this, PGPR are classified as either free living (root associative or extracellular) and endophytic (or intracellular). Free living or root associative PGPR are generally found in the rhizospheric region, while endophytic PGPR colonize inside root cell by forming specialized nodules [42]. PGPR improve plant growth by using different mechanisms which are generally categorized as direct and indirect plant growth promoting mechanisms [16]. Direct mechanism of PGPR includes synthesizing compounds necessary for plant growth or helping the uptake of essential nutrients from the soil. This uptake may aid in nitrogen fixation, phosphate solubilization, auxin synthesis, ACC deaminase activity, siderophore production etc. Indirect promotion by PGPR includes reduction or prevention of the damaging effects of pathogens on plants by generating inhibitory substances like HCN or by enhancing the natural resistance of the host plant, the phenomenon is termed as Induced Systemic Resistance (ISR) [6,43]. Another mechanism by which PGPR promote plant growth is by producing volatile organic compounds (VOC) [44]. FIGURE 4.2 Classification of microbes based on its ability to survive under NaCl.

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4.4

PART | I Physiological aspects

Stress alleviation through halotolerant rhizospheric bacteria

Different PGPR including Agrobacterium, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Klebsiella, Ochrobactrum and Pseudomonas are isolated from rhizosphere of halophytes or from saline soil [6,16]. Adaptation of bacteria for a particular stress is a complicated and it generally involves different genes [45]. Under extreme environmental conditions, these habitats adapted PGPR perform optimum metabolic processes such as enzymatic activities, membrane stability and other adaptation mechanisms to avoid stress [46]. Some of these mechanisms are found to be plasmid encoded [47]. Under salt stress, halotolerant PGPR adopt to a particular change in external osmolarity by accumulating low molecular weight hydrophilic molecules and through up take of suitable compounds from their surroundings. These compatible solutes, referred as osmolytes, also include amino acids and sugars [48,49]. For resistance of salt stress, common PGPR like Pseudomonas fluorescens induces de-novo synthesis of osmolyte, Ala, Asp, Glu, Gly, Ser and Thr in their cytosol [50]. In response to osmotic stress, there is up regulation of salt responsive genes in bacteria [51]. Halotolerant PGPR are capable of growing under high range of salinities i.e. up to 33% NaCl, they also survive in the absence of NaCl [52]. Therefore, several researchers explored halotolerant PGPR to alleviate plant salt stress [53]. Different crop plants have been successfully tested for stress tolerance using PGPR, like tomato, pepper, okra, bean, peanut and lettuce [16,25,54 58]. Halotolerant PGPR employs different mechanisms for enhancing plant growth in salt affected soil (Fig. 4.3). One of the well-known mechanisms is through lowering of induced ethylene by the action of 1-aminocyclopropane-1carboxylate (ACC) deaminase. ACC deaminase is an important enzyme, synthesized by some rhizobacteria and alters immediate ethylene precursor ACC to ammonia and α-ketobutyrate. Ultimately it results in decline of plant ethylene level which in turn continues plant growth under salt stress [16,25]. There has been increased attention on ACC deaminase synthesizing PGPR to study its effect on improving stress tolerance in crop plants [59 64]. It has been known since long that PGPR secrete different phytohormone like auxin, cytokinin and gibberellic acid [65]. Auxins (majorly IAA) produced by PGPR have significant effect on overall plant growth promotion. Major role played by IAA is root system architecture change and improvement in mineral and nutrient uptake [66]. Root system architecture includes spatial arrangement of primary and lateral roots, root system topology and the number and length of numerous diameters of roots [67]. It ultimately leads overall increase in total root surface area, and probably it improves mineral uptake [68]. Production of indole acetic acid is mediated by nitric oxide (NO) which is responsible

IAA (Auxin) production

Halophyte growing under salt stress

Changes Root architecture

ACC deaminase

Regulate stress induced ethylene level

Cytokinin production

Decreases Abscisic acid

VOC (2R, 3Rbutanediol)

Via HKT1 maintain Na+ level

Osmolyte production

Maintain osmotic potential

Sodium toxicity

+

EPS production

Binding of Na and restrict Na+ influx

Antioxidant defence

Regulate ROS and MDA

Nutrient deficiency ROS and malondialdehyde Ethylene generation

Habitat adapted PGPR

Nitrogen fixation

Increase plant available nitrogen level

P and K solubilization

Increase nutrient uptake

Siderophore production

Solubilizing various metals

Acidification of microenvironment

Increases availability of nutrients and metals

FIGURE 4.3 Mechanism of salinity tolerance induction by PGPR.

Abscisic acid Decrease in root elongation Decrease in water uptake

Salt stress

Halotolerant microbes and their applications in sustainable agriculture Chapter | 4

43

for lateral (LR) and adventitious (AR) root formation. Azospirillum brasilense is a well known candidate which has been known to mediate changes in root architecture [69] and root hairs [70]. Dobbelaere et al. [71] revealed that inoculation of wheat with IAA overproducing ipdC mutant of A. brasilense induces plant growth promotion along with changes in root architecture. It has also been shown that upon nodulation by IAA producing PGPR significantly improves abiotic stress tolerance [26,72]. For example, Egamberdieva et al. [73] reported that inoculation of IAA synthesizing Bradyrhizobium japonicum and Pseudomonas putida improves salinity tolerance in soybean by changing root architecture. Cytokinin producing PGPR induces drought tolerance in plant by decreasing abscisic acid concentration and inducing stomatal opening [24]. Ryu et al. [74] first reported secretion of some volatile organic compounds (VOC) by PGPR that increase abiotic stress tolerance. VOCs could act either by increasing essential mineral availability in the soil or by mimicking synthesis of some hormones. Under salt and drought stress VOC regulate HKT1 and maintain Na1 level and via SA pathway induces stomatal opening [28]. PGPR synthesize osmolytes and compatible solutes, that maintain osmotic potential and increases water uptake [75], and scavenges stress mediated reactive oxygen species (ROS) by activating antioxidant defense machinery of plant by inducing activity of some important enzymes like catalase (Cat), superoxide dismutase (SOD) and ascorbate peroxidase (APX) [16]. PGPR up regulated specific stress responsive genes. For example under salt stress halotolerant PGPR increases the activity of aquaporin by up regulating IP2, ZmPIP1-1, and HvPIP2-1 genes that help plant to absorb water under salt stress [76]. Halotolerant PGPR also influences activity of other genes such as HKT1 [77], ABA responsive elements (ABRE) and dehydration responsive elements (DRE) [28]. EPS producing bacteria enhance soil aggregation that ultimately increases water holding capacity as well as nutrient availability of plants [78]. EPS binds cations like Na1 and avoid Na1 accumulation in roots and its translocation into shoot [79]. PGPR improve nutrient availability of plant even under stress conditions by providing nutrients by mechanisms like nitrogen fixation, phosphate and potassium solubilization and siderophore production [6,28]. PGPR influence soil microenvironment to improve heavy metal tolerance in plants. PGPR acidify soil microenvironment, which ultimately results in change in soil pH and increases availability of nutrients and metals [60,80]. PGPR performs biomethylation and volatilization of heavy metals which increases its mobilization and availability [60,80]. Yang et al. [24] introduced the concept of Induced Systemic Tolerance (IST) for explaining how rhizobacteria induces physical and chemical modifications in host plants which ultimately improve stress tolerance.

4.5

Beneficial attributes of halotolerant PGPR

Contribution of microorganisms and its effectiveness on soil stability is found to be highly valuable for cultivating crops plants under salt affected agricultural fields [81,82]. Significant association of beneficial microorganisms has been shown under various stress environments, such as saline and alkaline areas, desert and acidic soils [83] and it is observed that the microbiota play an important role in reclamation process of agricultural field. Salt stress reduces overall plant growth by providing osmotic stress, Na1 and Cl2 toxicity, ethylene production, plasmolysis, nutrient imbalance, producing ROS and interfering with photosynthesis and it generally affect seed germination, seedling growth and vigor, flowering and fruit [1]. Halophytes are generally distributed under saline environment [84]. Rhizosphere of halophytes is a potential source for the isolation of several halotolerant microbiota or plant growth promoting halo rhizobacteria (PGPHR) that could enhance plant growth in saline soil [56,85]. Successful attempts have been made to isolate halotolerant rhizobacteria from roots of halophytic plants, such as Salicornia bigelovii, Salicornia spp., Salicornia europea, Prosopis strombulifera, Salicornia brachiate, Salicornia strobilacea and Arthrocnemum indicum [6,16,65,86 89]. In the last decade, there has been a major focus on exploring halophyte rhizosphere for its application to mitigate salt stress in crop plants (summarized in Table 4.1) [85,107 109]. Various studies have documented that PGPHR can improve salt tolerance by minimizing adverse effect of stress induced ethylene through bacterial ACC deaminase enzyme activity [110,111]. Several mechanisms have been proposed to illustrate the action of PGPHR in inducing salt tolerance response in crop plants (Fig. 4.3). PGPR reduces reactive oxygen species (ROS) by inducing plant antioxidant defense machinery, which upregulate the activity some key antioxidant enzymes [112]. Sharma et al. [16] isolated five PGPR from the rhizosphere of Arthrocnemum indicum and induce salinity tolerance in peanut, by differentially regulating expression of key enzymes in antioxidant defense machinery. Along with that PGPR also produce IAA and shows ACC deaminase activity. PGPR isolated from rhizosphere of extreme halophyte Salicornia brachiata, enhance salinity tolerance in peanut. Under salt stress inoculated plant has higher K1/Na1 ratio and Ca21 concentration as well as lesser MDA level than noninoculated plants [6,56]. Different halotolerant IAA producing rhizobacteria are isolated from rhizosphere of halophytes

44

PART | I Physiological aspects

TABLE 4.1 List of halotolerant PGPR reported to enhance salinity tolerance. PGPR

Plant

Mode of action

Reference

Bacillus spp. and Arthrobacter pascens

Wheat

Elevation of antioxidant enzymes

[90]

Bacillus megaterium

Maize

Induces expression of plasma membrane type two (PIP2) aquaporin and Increases root hydraulic conductance values

[76]

Bacillus amyloliquefaciens GB03

Arabidopsis

VOCs reduces Na1 accumulation in shoot

[77]

1

Bacillus spp. Enterobacter spp. and Paenibacillus spp.

Wheat

Decreased Na uptake

[118]

Achromobacter piechaudii

Tomato and pepper

ACC deaminase activity

[25]

Dietzia natronolimnaea

Wheat

Increases expression of proline and various antioxidants and also activate ABA signaling, SOS pathways and iron transport

[91]

Klebsiella, Pseudomonas, Agrobacterium, and Ochrobactrum

Peanut

Maintain ion homeostasis and maintain ROS level by differential expression of antioxidant genes

[16]

Enterobacter cloacae and Bacillus drentensis

Mung beans

Along with silicon synergistically maintain stomatal conductance, transpiration rate, relative water content and photosynthetic pigments

[57]

Brachybacterium saurashtrense strain, Brevibacterium casei, and Haererohalobacter spp.

Peanut

Maintain higher K1/Na1 ratio and higher Ca21, phosphorus, and nitrogen Content

[56]

Pseudomonas putida, Enterobacter cloacae, Serratia ficaria and P. fluorescens

Wheat

Maintain higher K1/Na1 ratio and higher Ca21, phosphorus, and nitrogen Content.

[92]

Alcaligenes faecalis, Bacillus pumilus and Ochrobactrum sp.

Rice

ACC deaminase activity

[93]

Azospirillum sp.

Wheat

Influence the water uptake, photosynthetic pigment contents and proline accumulation in wheat seedlings

[94]

Streptomyces sp.

Wheat

Increases nutrient uptake rate

[95]

Pseudomonas sp., Bacillus sp. and Variovorax sp.

Avocado

ACC deaminase activity

[61]

Azotobacter chroococcum

Maize

Maintain higher K1/Na1 ratio and higher Ca21 and high chlorophyll content

[96]

B. subtilis and Arthrobacter sp.

Wheat

Influence activity of antioxidant enzymes, APX, CAT and GR

[97]

P. fluorescens, P. aeruginosa and P. stutzeri

Tomato

ACC deaminase activity

[59]

Pseudomonas sp.

Eggplant

Activate antioxidant enzymes, superoxide dismutase, peroxidase and catalase

[98]

Pseudomonas. putida

Cotton

Selective absorption of Mg21, K1 and Ca21 and reduce uptake of the Na1

[99]

Pseudomonas mendocina

Lettuce

Activate antioxidant enzymes, catalase and total peroxidase

[100]

Pseudomonas syringae, Pseudomonas fluorescens and Enterobacter aerogenes

Maize

ACC deaminase activity

[62]

Pseudomonas fluorescens

Peanut

ACC deaminase activity

[64] (Continued )

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45

TABLE 4.1 (Continued) PGPR

Plant

Mode of action

Reference

Aeromonas hydrophila, Bacillus insolitus and Bacillus sp.

Wheat

Influence uptake of K1, Na1, and Ca21

[101]

Azospirillum

Maize

Increases uptake of K1 and Ca21 and maintain higher K1/Na1 ratio

[102]

P. yonginensis DCY84T

Rice

Differential regulation of AtRSA1, AtVQ9 and AtWRKY8

[103]

Burkholderacepacia SE4, Promicromonospora sp. SE188 and Acinetobacter calcoaceticus SE370

Cucumis sativus

Regulate activities of catalase, peroxidase, polyphenol oxidase, and total polyphenol

[104]

Bacillus pumilus, Pseudomonas mendocina, Arthrobacter sp., Halomonas sp., and Nitrinicola lacisaponensis

Wheat

Synthesize IAA and mediate changes in the biochemical content like, total content of carotenoids, phenolics, flavonoids, proline, reducing sugar, and soluble sugar

[105]

Klebsiella sp.

Oat (Avena sativa)

Modulated the expression profile of rbcL and WRKY1 genes

[106]

as well as from highly salty habitats [113]. Kim et al. [114] isolated IAA producing PGPR Enterobacter sp. EJ01 from Dianthus japonicus thumb, and it enhances salt tolerance in Arabidopsis and tomato. Bacillus licheniformis A2 isolated from halophyte S. fruticosa, increases salinity tolerance in peanut. It produces IAA and solubilizes insoluble phosphate and show significant increase in plant growth under salt stress. Some PGPR secrete VOCs such as 2,3- butanediol and dimethyl disulfide, that confer stress tolerance in crop plant by activating HKT1 pathway, which reduces Na1 accumulation [115]. VOC secreted by Bacillus subtilis GB03 down regulate HKT1 expression in roots, but upregulated its expression in shoot tissues; which ultimately regulate ionic homeostasis in plant [24]. Paenibacillus yonginensis DCY84T induces differential expression of AtRSA, AtWRKY8 and AtVQ9 genes under salt stress and improve salinity tolerance in Arabidopsis [103]. Nuclear-localized calcium-binding protein, AtRSA1 control the transcription of specific genes that are involved in detoxification of ROS induced by salt stress. Both AtWRKY8 and AtVQ9 are responsible of salt adaptation in plants by maintain ion homeostasis (lower cytosolic Na1/K1 ratio) [116]. AtRSA1 senses salinity induced changes and interacts with AtRITF1 (transcription factor) and it may be phosphorylated by nuclear-localized mitogen-activated protein kinases (MAPKs). AtRSA1-AtRITF1 complex is responsible for Na1 homeostasis and detoxification of salt-induced ROS [103].

4.6

Conclusions and future perspective

Increasing salinity across the globe is an alarming environmental concern for agriculture and hence it is essential to adopt eco-friendly management approaches in crop plants. In this regard, halotolerant PGPR offer a viable option to enhance plant growth in both stress and non-stress condition. Various halotolerant rhizobacteria are successfully isolated from roots of several halophytes as well as from saline soil. It has now been demonstrated that these PGPR augment growth and improve overall productivity. PGPR improve plant growth by different mechanism like producing plant hormones, maintaining ethylene level, supplying essential nutrients, controlling activity of plant pathogens, enhancing effectiveness of Induced Systemic Resistance (ISR) and improving soil structure. PGPR are found to be the best candidate to replace hazardous chemical fertilizers having various side-effects to sustainable agriculture. PGPHR reduce salinity stress in plant by increasing osmolyte accumulation in plants, regulate root architecture by producing auxin, regulate expression of salt responsive genes by producing VOC, maintain high K1/Na1 ratio and Ca21, regulate ethylene level and scavenging ROS by regulating expression of antioxidant enzymes etc. Further research at molecular level should enable elucidation of the precise plant pathways that are modulated under PGPR action. It is also important to study the bioactivity and stability of the rhizosphere microbiome. A recent study suggested a systems biology approach to look at the problem of developing microbial guild or synthetic microbial consortia having varied functional attributes

46

PART | I Physiological aspects

to alleviate stress and augment crop productivity [117]. Sustained, intensive research efforts are required to explore the multi-potentials of halotolerant rhizospheric bacteria for sustainable agriculture.

Acknowledgments JK sincerely thank DBT (Department of Biotechnology) for providing financial assistance by awarding JRF.

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Planta 2010;232(2):533 43. [77] Zhang H, Kim MS, Sun Y, Dowd SE, Shi H, Pare´ PW. Soil bacteria confer plant salt tolerance by tissue-specific regulation of the sodium transporter HKT1. Mol Plant-Microbe Interact 2008;21(6):737 44. [78] Nunkaew T, Kantachote D, Nitoda T, Kanzaki H, Ritchie RJ. Characterization of exopolymeric substances from selected Rhodopseudomonaspalustris strains and their ability to adsorb sodium ions. Carbohydr Polym 2015;115:334 41. [79] Qin Y, Druzhinina IS, Pan X, Yuan Z. Microbially mediated plant salt tolerance and microbiome-based solutions for saline agriculture. Biotechnol Adv 2016;34(7):1245 59. [80] Verma JP, Yadav J, Tiwari KN, Kumar A. Effect of indigenous Mesorhizobium spp. and plant growth promoting rhizobacteria on yields and nutrients uptake of chickpea (Cicer arietinum L.) under sustainable agriculture. Ecol Eng 2013;51:282 6. [81] Jastrow JD, Miller RM. Methods for assessing the effects of biota on soil structure. Agric Ecosyst Environ 1991;34(1 4):279 303. [82] Bargaz A, Lyamlouli K, Chtouki M, Zeroual Y, Dhiba D. Soil microbial resources for improving fertilizers efficiency in an integrated plant nutrient management system. Front Microbiol 2018;9:1606. Available from: https://doi.org/10.3389/fmicb.2018.01606. [83] Selvakumar G, Joshi P, Nazim S, Mishra P, Bisht J, Gupta H. Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high-altitude Himalayan rhizosphere. Biologia 2009;64(2):239 45. [84] Qin S, Zhang YJ, Yuan B, Xu PY, Xing K, Wang J, et al. Isolation of ACC deaminase-producing habitat-adapted symbiotic bacteria associated with halophyte Limonium sinense (Girard) Kuntze and evaluating their plant growth-promoting activity under salt stress. Plant Soil 2014;374 (1 2):753 66. [85] Ramadoss D, Lakkineni VK, Bose P, Ali S, Annapurna K. Mitigation of salt stress in wheat seedlings by halotolerant bacteria isolated from saline habitats. SpringerPlus 2013;2(1):6 (1-7). [86] Rueda-Puente E, Castellanos T, Troyo-Die´guez E, Dı´az de Leo´n-Alvarez JL, Murillo-Amador B. Effects of a nitrogen-fixing indigenous bacterium (Klebsiella pneumoniae) on the growth and development of the halophyte Salicornia bigelovii as a new crop for saline environments. J Agron Crop Sci 2003;189(5):323 32. [87] Ozawa T, Wu J, Fujii S. Effect of inoculation with a strain of Pseudomonas pseudoalcaligenes isolated from the endorhizosphere of Salicornia europea on salt tolerance of the glasswort. Soil Sci Plant Nutr 2007;53(1):12 16. [88] Sgroy V, Cassa´n F, Masciarelli O, Del Papa MF, Lagares A, Luna V. Isolation and characterization of endophytic plant growth-promoting (PGPB) or stress homeostasis-regulating (PSHB) bacteria associated to the halophyte Prosopis strombulifera. Appl Microbiol Biotechnol 2009;85(2):371 81. [89] Mapelli F, Marasco R, Rolli E, Barbato M, Cherif H, Guesmi A, et al. Potential for plant growth promotion of rhizobacteria associated with Salicornia growing in Tunisian hypersaline soils. BioMed Res Int 2013;2013 (1-14).

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[90] Ullah S, Bano A. Isolation of plant-growth-promoting rhizobacteria from rhizospheric soil of halophytes and their impact on maize (Zea mays L.) under induced soil salinity. Can J Microbiol 2015;61(4):307 13. [91] Bharti N, Pandey SS, Barnawal D, Patel VK, Kalra A. Plant growth promoting rhizobacteria Dietzianatronolimnaea modulates the expression of stress responsive genes providing protection of wheat from salinity stress. Sci Rep 2016;6:34768 (1-16). [92] Nadeem SM, Zahir ZA, Naveed M, Nawaz S. Mitigation of salinity-induced negative impact on the growth and yield of wheat by plant growth-promoting rhizobacteria in naturally saline conditions. Ann Microbiol 2013;63(1):225 32. [93] Bal HB, Nayak L, Das S, Adhya TK. Isolation of ACC deaminase producing PGPR from rice rhizosphere and evaluating their plant growth promoting activity under salt stress. Plant Soil 2013;366(1 2):93 105. [94] Zarea MJ, Hajinia S, Karimi N, Goltapeh EM, Rejali F, Varma A. Effect of Piriformosporaindica and Azospirillum strains from saline or nonsaline soil on mitigation of the effects of NaCl. Soil Biol Biochem 2012;45:139 46. [95] Sadeghi A, Karimi E, Dahaji PA, Javid MG, Dalvand Y, Askari H. Plant growth promoting activity of an auxin and siderophore producing isolate of Streptomyces under saline soil conditions. World J Microbiol Biotechnol 2012;28(4):1503 9. [96] Rojas-Tapias D, Moreno-Galva´n A, Pardo-Dı´az S, Obando M, Rivera D, Bonilla R. Effect of inoculation with plant growth-promoting bacteria (PGPB) on amelioration of saline stress in maize (Zea mays). Appl Soil Ecol 2012;61:264 72. [97] Upadhyay SK, Singh JS, Saxena AK, Singh DP. Impact of PGPR inoculation on growth and antioxidant status of wheat under saline conditions. Plant Biol 2012;14(4):605 11. [98] Fu Q, Liu C, Ding N, Lin Y, Guo B. Ameliorative effects of inoculation with the plant growth-promoting rhizobacterium Pseudomonas sp. DW1 on growth of eggplant (Solanum melongena L.) seedlings under salt stress. Agric Water Manag 2010;97(12):1994 2000. [99] Yao L, Wu Z, Zheng Y, Kaleem I, Li C. Growth promotion and protection against salt stress by Pseudomonas putida Rs-198 on cotton. Eur J Soil Biol 2010;46(1):49 54. [100] Kohler J, Herna´ndez JA, Caravaca F, Rolda´n A. Induction of antioxidant enzymes is involved in the greater effectiveness of a PGPR versus AM fungi with respect to increasing the tolerance of lettuce to severe salt stress. Environ Exp Bot 2009;65(2 3):245 52. [101] Ashraf M, Hasnain S, Berge O, Mahmood T. Inoculating wheat seedlings with exopolysaccharide-producing bacteria restricts sodium uptake and stimulates plant growth under salt stress. Biol Fertil Soils 2004;40(3):157 62. [102] Hamdia MAES, Shaddad MAK, Doaa MM. Mechanisms of salt tolerance and interactive effects of Azospirillumbrasilense inoculation on maize cultivars grown under salt stress conditions. Plant Growth Regul 2004;44(2):165 74. [103] Sukweenadhi J, Kim YJ, Choi ES, Koh SC, Lee SW, Kim YJ, et al. Paenibacillusyonginensis DCY84T induces changes in Arabidopsis thaliana gene expression against aluminum, drought, and salt stress. Microbiol Res 2015;172:7 15. [104] Kang SM, Khan AL, Waqas M, You YH, Kim JH, Kim JG, et al. Plant growth-promoting rhizobacteria reduce adverse effects of salinity and osmotic stress by regulating phytohormones and antioxidants in Cucumis sativus. J Plant Interact 2014;9(1):673 82. [105] Tiwari S, Singh P, Tiwari R, Meena KK, Yandigeri M, Singh DP, et al. Salt-tolerant rhizobacteria-mediated induced tolerance in wheat (Triticum aestivum) and chemical diversity in rhizosphere enhance plant growth. Biol Fertil Soils 2011;47(8):907 17. [106] Sapre S, Gontia-Mishra I, Tiwari S. Klebsiella sp. confers enhanced tolerance to salinity and plant growth promotion in oat seedlings (Avena sativa). Microbiol Res 2018;206:25 32. [107] Chookietwattana K, Maneewan K. Screening of efficient halotolerant phosphate solubilizing bacterium and its effect on promoting plant growth under saline conditions. World Appl Sci J 2012;16(8):1110 17. [108] Nadeem SM, Zahir ZA, Naveed M, Ashraf M. Microbial ACC-deaminase: prospects and applications for inducing salt tolerance in plants. Crit Rev Plant Sci 2010;29(6):360 93. [109] Barnawal D, Bharti N, Maji D, Chanotiya CS, Kalra A. ACC deaminase-containing Arthrobacter protophormiae induces NaCl stress tolerance through reduced ACC oxidase activity and ethylene production resulting in improved nodulation and mycorrhization in Pisum sativum. J Plant Physiol 2014;171(11):884 94. [110] Ali S, Charles TC, Glick BR. Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase. Plant Physiol Biochem 2014;80:160 7. [111] Karthikeyan B, Joe MM, Islam MR, Sa T. ACC deaminase containing diazotrophic endophytic bacteria ameliorate salt stress in Catharanthus roseus through reduced ethylene levels and induction of antioxidative defence systems. Symbiosis 2012;56(2):77 86. [112] Jha Y, Subramanian RB. PGPR regulate caspase-like activity, programmed cell death, and antioxidant enzyme activity in paddy under salinity. Physiol Mol Biol Plants 2014;20(2):201 7. [113] Siddikee MA, Chauhan PS, Anandham R, Han GH, Sa T. Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil. J Microbiol Biotechnol 2010;20(11):1577 84. [114] Kim K, Jang YJ, Lee SM, Oh BT, Chae JC, Lee KJ. Alleviation of salt stress by Enterobacter sp. EJ01 in tomato and Arabidopsis is accompanied by up-regulation of conserved salinity responsive factors in plants. Mol Cell 2014;37(2):109 17. [115] Liu XM, Zhang H. The effects of bacterial volatile emissions on plant abiotic stress tolerance. Front Plant Sci 2015;6:774 (1-6). [116] Hu Y, Chen L, Wang H, Zhang L, Wang F, Yu D. Arabidopsis transcription factor WRKY 8 functions antagonistically with its interacting partner VQ 9 to modulate salinity stress tolerance. Plant J 2013;74(5):730 45. [117] Kong Z, Hart M, Liu H. Paving the way from the lab to the field: using synthetic microbial consortia to produce high-quality crops. Front Plant Sci 2018;9:1467. Available from: https://doi.org/10.3389/fpls.2018.01467. [118] Upadhyay SK, Singh JS, Singh DP. Exopolysaccharide-producing plant growth-promoting rhizobacteria under salinity condition. Pedosphere 2011;21(2):214 22.

Chapter 5

Halophilic microorganisms: Interesting group of extremophiles with important applications in biotechnology and environment Lobna Daoud1,2 and Mamdouh Ben Ali1,2 1

Laboratory of Microbial Biotechnology and Enzyme Engineering (LBMIE), Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.

2

Astrum Biotech, Business incubator, Center of Biotechnology of Sfax (CBS), University of Sfax, Sfax, Tunisia.

5.1

Introduction

Extreme environments characterized by extreme levels of temperature, pressure, pH, salinity . . . etc., are often colonized by organisms called “extremophiles”, which are well adapted to these specific physico-chemical conditions. On the other hand, many industrial processes need more and more “unusual” biomolecules that are functional and stable at harsh conditions which result in the precipitation or denaturation of the other ones. Since extremophiles can provide new biomolecules meeting many industrial needs [1], many researchers and industries are more and more focusing on these microorganisms [2,3]. Extremophiles include many groups of organisms such as thermophiles, psychrophiles, basophiles and halophiles. Halophiles are organisms that require salt for growth, inhabiting hypersaline environments in which salinity usually exceeds the sea one up to saturation [4]. Requirement for salt and tolerance for salt should be distinguished. Unlike halophiles, halotolerant microorganisms don’t require salt for growth but can tolerate its presence in the medium even at high concentrations, exceeding 2.5 M NaCl [5]. Halophiles are distinguished from the other extremophiles by the ability to keep an osmotic equilibrium in ecosystems having highly variable ionic composition and to adapt quickly to sudden ionic changes [6]. The mechanisms that allow cells to control the internal osmotic pressure are known as osmoregulation [7]. Studies on halophiles are developing and showing many important and even unique characteristics that make them very useful and efficient bio-catalyzers for many biotechnological and industrial applications [8
12]. In this chapter, we describe many characteristics and features of halophilic microorganisms and we give their different current and potential applications.

5.2

Habitats of halophilic microorganisms

Extreme environments are defined as the media in which physicochemical conditions are far from those commonly found on Earth (temperatures between 10 and 40, near neutral pH and low salinity). These environments include hypersaline, hot and hyper-hot, alkaline, acidic and cold environments. Halophilic microorganisms harbor so-called saline and hypersaline environments that are characterized by high amounts of salts like the NaCl, KCl, MgSO4, CaSO4 and MgCl2. Seas and oceans contain about 3.5% of salts and are called saline environments. When the content of dissolved salts exceeds this amount (3.5%), the medium is then called hypersaline [4]. Hypersaline ecosystems can be of two origins: G

Oceanic origin: some environments, during geological time, are separated from the oceans. They then represent closed systems in which the evaporation of water leads to a slow concentration of the salts until saturation. Most of

Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00005-8 © 2020 Elsevier Inc. All rights reserved.

51

52

G

PART | I Physiological aspects

these systems are flat reliefs in arid or semi-arid zones, close to the oceans, like, for example, the salt marshes, sebkhas and lagoons. There, water evaporation is speedy and interchange with the sea is slow. Continental origin: it comes from the dissolution of the salts associated with rocks and geological layers caused by rainwater or run-off water. These salts are then concentrated by evaporation as in the previous case, when these waters are accumulated in impervious ponds. In this case, each salt lake chemical composition is unique [13]. As examples, the Great Salt Lake in Utah (USA) and the Dead Sea on the Border of Palestine and Jordan, with total dissolved salt concentrations greater than 300 g/L [7].

Besides the origin, the ionic composition is also an essential factor that determines the properties of the hypersaline environment. When it reflected the ionic composition of the sea, mostly during the first evaporation phases, the brines are called “thalassohaline” [7]. In other words, thalassohaline brines are the result of the evaporation of the seawater. In fact, during the seawater evaporation, the ion composition begins to change significantly up to a stage where the solubility end of CaSO4 is reached and the gypsum is formed (when the total salt concentration exceeds 100
120 g/L). In the following NaCl precipitation as halite, the ionic composition varies again and the relative K1 and Mg21 concentrations rise. Thalassohaline brines are, generally, neutral or lightly alkaline [7
8]. In the second case, the ion composition can change greatly from the seawater ionic composition, and then, the hypersaline environments are called “athalassohaline”. For example, the Dead Sea is an athalassohaline lake in which divalent cations dominate, with Mg21 and Ca21 concentrations (1.89 M and 0.45 M, respectively) higher than Na1 and K1 concentrations (1.56 M and 0.2 M, respectively). Besides, due to the high Ca21 concentration, the solubility of the sulfate is low and, consequently, the monovalent anions (Cl2 and Br2) represent more than 99.9% of the total anions. The Dead Sea is slightly acidic with a pH value around 5.8
6 [7,14]. The ionic composition of hypersaline media consists of a mixture of salts and not just NaCl. But since the majorities of these habitats are thalassohalines and contain NaCl as the major component, it is very common to exclude other ions from the calculation and consider the NaCl concentration as the salts concentration [15]. The halophilic microorganisms can also host other saline and hypersaline environments such as salted products (fish, vegetables, animals skins treated for their preservation, etc.), saline soils defined by salt amounts higher than 0.2%, and underground brines often associated with oil fields.

5.3

Classification

Halophilic organisms are found in the three life domains: Eucarya, Bacteria and Archaea. However, they are more spread in the phylogenetic trees of prokaryotes (Archaea and Bacteria). There are few microorganisms that grow over the full range of salt concentrations: from fresh water to near saturation. Among them, Halomonas elongata is a famous example. In most cases, each organism has a restricted range of salt concentrations permitting its growth. Then, some bacteria live only in saturated and near-saturated brines and can’t grow at NaCl concentrations below 15
20% (w/v). Some others are able to grow at high NaCl concentrations and other salts but with an optimal growth in the absence of salt. Such group is called halotolerant microorganisms and Staphylococcus species are a wellknown example of them. Halotolerant microorganisms can grow either in the absence or the presence of NaCl, with concentrations exceeding 10
15% or even more, and this feature is an important property often exploited in selective culture media design and diagnosis. It should be noticed that the tolerance and the requirement of salt may be temperature dependent. In fact, there are many researches reporting the improvement of salt tolerance and requirement at high temperatures [7]. The classification of halophiles was provided early and was then updated. The first classification was investigated in 1962 by Larsen [16] who defined three categories of halophilic microorganisms according to the salt concentrations range demanded for optimal growth: from 2 to 5% NaCl for weakly halophilic bacteria, 5
20% NaCl for moderate halophilic bacteria and up to 20
30% NaCl for extreme halophilic bacteria. These ranges of salt concentrations (NaCl) have been refined since by Kushner (1978, 1985) [17,18] and the obtained classification was then the most accepted one. Table 5.1 presented this classification with little modification proposed by Oren (2013) [7]. Since 2018, all documented halophilic species, along with their basic information, were gathered in a new online database named HaloDom [19]. According to HaloDom, there are actually more than 1000 halophilic species dispersed into 21.9% of Archaea, 50.1% of Bacteria and 27.9% of Eukaryotes.

Halophilic microorganisms Chapter | 5

53

TABLE 5.1 Classification of halophilic microorganisms. Category

NaCl (M)

NaCl (g/L)

Range

Optimum

Range

Optimum

Non halophilic

0
1

, 0.2

0
60

, 10

weakly halophilic

0.2
2

0.2
0.5

10
115

10
30

Moderately halophilic

0.4
3.5

0.5
2

25
200

30
115

Borderline extremely halophilic

1.4
4

2
3

80
230

115
175

Extremely halophilic

2
5.2

.3

115
300

. 175

Halotolerant

0
. 1

, 0.2

0
. 60

, 10

Haloversatile

0
. 3

0.2
0.5

0
. 175

10
30

5.4

Mechanisms of salt adaptation

When a cell is being in a high saline medium, the inside water moves to the external environment causing a decrease in the cell volume which, when too large, can cause the dormancy or the death of the cell. On the other hand, if the cell is in a medium with low osmolarity, the water flow will be in the opposite direction causing a turgor of the cell and even its break-up if its envelope elasticity limit is exceeded. For that, and to avoid these accidents, the cell must always adjust its intra-cytoplasmic osmotic pressure regarding to that of its environment. In the case of hypersaline media, the ionic composition changes over time due to water evaporation or supply (by precipitation). These sudden ionic variations push the microorganisms to adapt quickly to these new conditions. The set of mechanisms that allow cells to control this internal osmotic pressure is called osmoregulation. However, in some cases, the ability of a bacterium to grow in a hypertonic medium is a function of the strength of its envelopes, defined essentially by the thickness of the peptidoglycan layer that provides the rigidity of the cell. Generally, there are two strategies that avoid osmotic shocks in the medium: (i) the ion transport across the membrane by ion pumps or (ii) the formation or accumulation of organic molecules in the cell [4,7]. (i) Ion transport through the membrane by ion pumps This strategy is called “salt-in” strategy, used by the Halobacteriales order of aerobic Archaea and by the Haloanaerobiales order of anaerobic bacteria. It consists in the accumulation of inorganic ions at high concentrations in the cytoplasm, and this is by antiporters, pumps, ATP formation, inward transport and other systems located in the cell membrane [7]. Generally, K1 is the main intracellular cation rather than Na1, and Cl2 is the main anion. It should be noted that membranes of all halophilic microorganisms have highly active Na1/H1 antiporters which, in addition to maintaining low intracellular Na1 concentrations, have a significant role in intracellular pH regulation. The presence of molar concentrations of inorganic necessitates special adaptations of the entire intracellular enzymatic metabolism. In the “salt-in” strategy, the salt-adapted enzymes are less flexible and adaptable to changing conditions, and many of them and of structure proteins require the constant presence of salt to keep their activity and stability [20]. (ii) Synthesis or accumulation of organic molecules This strategy is adopted by most halophilic and halotolerant bacteria, except for the Haloanaerobiales, and the methanogenic halophilicarchaea. It consists in preventing high salt concentrations from reaching the cells cytoplasm, and maintaining the activity and stability of proteins and enzymes which are not created to operate at such high salinities. The low concentrations of intracellular ions are kept by their continuous pumping out of the cells. The osmotic solutes, which provide the osmotic equilibrium, are either formed by the cell or stored from the extracellular medium. [7]. These “compatible” solutes such as glycine betaine, ectoine and trehalose are with low molecular weight, highly water-soluble, neutral at physiological pH and do not disturb cell metabolism. Besides, they are unable to pass across the cytoplasmic membrane without specific transporters. The term “compatible solutes” has been introduced to describe these particular molecules. Their intracellular concentrations are changed in response to the extracellular salinity. In fact, when the salinity of the external environment is high, the osmotic solutes are accumulated in the cell by either their synthesis or their input from the external medium. In the other

54

PART | I Physiological aspects

case, when the cell faces a dilution stress, it gets rid of its osmotic solutes by their deterioration, transformation into inactive forms or excretion. The osmotic solutes permit for the “classical” enzymes which are not created adapted to salt, a high level of flexibility and adaptability leading them to keep well their activity and stability. Generally, the “Compatible” solutes are known as stabilizer agents which prevent proteins from heating, freezing and drying denaturation [7,20,21].

5.5

Structural characteristics of halophilic proteins

Halophilic proteins are characterized by their ability to keep and improve their solvation in poor water-containing environments where the other conventional proteins lose rapidly their activity and stability by denaturation, aggregation or precipitation. In fact, in such extreme environments, the hydrogen bonds between negatively charged amino-acids of proteins surfaces and water molecules become critical for maintaining a stable hydration envelope resulting in proteins stability. Since halophilic proteins contain more acidic amino-acids on their surfaces than basic amino-acids, their surface charge is negative and therefore, they keep their stability in these environments. At a structural level, the richness of halophilic proteins in acidic amino-acids was shown in few researches. For example, Dym et al. (1995) [22] and Das Sarma and Arora [4] compared the structure of Haloarcula marismortui malate dehydrogenase with its non halophilic homologue lactate dehydrogenase, and Dassarma and Dassarma (2015) [23] compared the structure model of the β-galactosidase from Halorubruml acusprofundi with the structure of β-galactosidase from Thermus thermophiles (Fig. 5.1). The halophilic proteins with resolved structures are very few [23] and are all shown in Table 5.2. FIGURE 5.1 Comparison between the structure model of the halophilic β-galactosidase from the halophilic and psychrophilic archaea Halorubrumlacusprofundi (A) and the structure of the non halophilic β-galactosidase from Thermus thermophiles (B). Red spheres represent the acidic residues; blue spheres represent the basic residues [23].

TABLE 5.2 Halophilic proteins with resolved structures. Enzymes

Origins

Malate dehydrogenase

Haloarcula/Salinibacter

Nucleotide diphosphate kinase

Halomonas/Halobacterium

α-amylase

Halothermothrix

Carbonic anhydrase

Dunaliella

Glucose dehydrogenase

Haloferax

Dihydrofolate reductase

Haloferax

Phosphatase alkaline

Halobacterium

β-glucosidase

Halothermothrix

Catalase/peroxidase

Haloferax

Rnase H1

Halobacterium

D-Mannonate

dehydratase

Chromohalobacter

Halophilic microorganisms Chapter | 5

5.6

55

Current and potential applications of halophiles

Halophilic microorganisms can survive in extreme environments where most the other microorganisms cannot grow. This implies that they are an interesting alternative for many industrial applications using environments with high salinity and/or low water activity. Indeed, the biotechnological industry has been developed considerably in the recent years in order to replace, as much as possible, the chemical industry. However, biologically manufactured products such as biofuels, biochemical and bioplastics are still very expensive because their production cost is much higher than the one of their chemical counterparts. This is due to several factors such as the rapid rise in raw material costs such as glucose extracted from starch, excessive demand for fresh water, which makes water scarcity even worse, the discontinuity of fermentations caused by contamination which weakens the production efficiency and the high cost of energy required for the sterilization of fermenters and media. To overcome these problems, research has been investigated for several years to search for microbial and/or technical alternatives of industrial biotechnology competitive to the chemical industry. The halophilic microorganisms have recently been considered to bring solutions to these problems. In fact, they can be used in hypersaline environments that greatly reduce the risk of contamination, the rate of fresh water used and the cost of sterilization at a time. For example, strains of the genus Halomonas are shown to be able to grow at high pH and salinity and at a wide range of temperatures, conditions that avoid contamination during the fermentation process and thus allow for continuous fermentation under non-sterile conditions [10]. Actually, the halophilic and halotolerant microorganisms are involved in several industrial applications such as the production of ectoine from several moderate halophilic bacteria that is applied as a stabilizer agent for enzymes and in cosmetic formulations. In addition, they produce many distinguished products that may have potential biotechnological applications, but are not yet valued on an industrial scale [9,10]. The different biotechnological applications of halophilic and halotolerant microorganisms, current and potential, are summarized in Table 5.3 and more detailed in the following.

TABLE 5.3 Current and potential industrial applications of halophilic microorganisms. Product

Application

Polyhydroxialkanoates (PHA)

Bio-materials

Polyhydroxialkanoates (PHB)

Plastics

Halomonas boliviensis Halomonas sp. TD01

Poly(hydroxybutyrate-cohydroxyvalerate) (PHBV)

Plastics, medical materials

Haloferax mediterranei

Ectoines

Protectants for proteins and cells

Ectoine

Cell membrane protection, antiageing skin protection

Halomonas elongata Halomonas salina

Hydroxyectoine

Protection of proteins against misfolding, degradation and freezing

Marinococcus M52

Amylases

Food industry

Halomonas sp. Halobacillus sp. Streptomyces sp.

Proteases

Additives in pharmaceuticals, laundry detergents, and so on

Bacillus sp. Halobacillus sp. chromohalobacter sp.

Xylanases and cellulases

Biobleaching, hydrolysis of cellulose

Streptomonospora sp. Halomonas sp.

Biosurfactants and bioemulsifiers

Solubilization of hydrophobic substrates

Halomonas spp. Natrialba sp. E21

Bioemulsifier protein PhaR β-Carotene

Represent at if producer

Halomonas sp. (recombinant) Food additive

Dunaliella spp.

glycerol

Cosmetic industries

Dunaliella spp.

Bacteriorhodopsine

Light-driven proton pump protein

Halobacterium halobium

56

PART | I Physiological aspects

5.6.1 Food fermentation Many fermentation processes in the presence of salt, are catalyzed by halotolerant microorganisms, producing characteristic taste, savor and aroma compounds. Lactobacillus plantarum plays an important role in the fermentation of pickles and sauerkraut. H. salinarum, Halococcus sp., Bacillus sp., Pseudomonas and coryneform bacteria are responsible in the production of an Asian fish sauce (in Thailand, called Nam pla) [24].

5.6.2 Production of stable enzyme Halophilic microorganisms provide various enzymes (including hydrolytic ones such as DNAases, lipases, proteases, amylases and gelatinases) able to function under conditions that precipitate or denature most of conventional proteins, such as media with high salts concentrations or high proportion of organic solvents [10,23]. Fukushima et al. [25] reported an α-amylase from a Haloarcula sp. strain (Halophilic archaea) with optimal activity at 4.3 M NaCl and 50  C, and good stability in the presence of many organic solvents such as chloroform, benzene and toluene. At less salt concentration, an α-amylase from Nesterenkonia sp. showed an optimal activity at 0.75
1 M NaCl, pH 6.5 and 45  C, which make it a good candidate for neutral and mesophilic applications at relatively high salinities [26]. The halophilic proteases were isolated from several strains such as Bacillus sp., Pseudoaltermonas sp., Salinivobrio sp., Halobacillus sp., Filobacillus sp., Chromohalobacter sp., Nesterenkonia sp. and Virgibacillus sp. [10]. A halotolerant and alkaline protease from Bacillus halodurans strain US193 (halotelerant) showed a high stability from neutral to alkaline pH values and at many temperatures and NaCl concentrations reaching 60  C and 2 M, respectively [27]. Besides, it was very stable in detergents and organic solvents which make it a successful industrial enzyme. A metalloprotease from a Salinivibrio sp. strain (moderately halophilic) was reported active at broad pH and salinity ranges varying from 6 to 10 and from 0 to 10% NaCl, respectively. It has an optimal activity at 1% NaCl, pH 8.5
9, and 60  C, so it is an alkaline and thermophilic metalloprotease with a good activity either in the existence or the deficiency of salt. This makes it with more interest in several biotechnological applications than the strict halophilic proteases [28]. Karbalaei-Heidari et al. [29] reported a protease from a Halobacillus karajensis strain (moderately halophilic), alkaline, halo-stable, thermostable. It was optimal at 0.5 M NaCl, pH 9 and 50  C and well active at salt concentrations up to 3 M NaCl, which justifies its great interest for alkaline and saline applications. The lipases from halophiles are poorly described despite their interesting biochemical characteristics making them good candidates for many industrial applications. A lipase from the halophilic archaea Natronococcus sp. strain TC6, exhibited optimal activity at 4 M NaCl, 50  C and pH 7. with high thermostability [30] thermostability. A lipase produced by a Salinivibrio sp. strain (moderately halophilic), showed a very high stability at 80  C and at different concentrations of salts such as NaCl, NaNO3, Na2 SO4 and KCl [31]. Samaei-Nouroozi et al. [32] optimized the production of a halo-alkaline lipase from Alkalibacilluss lilacus, highly active and stable at wide ranges of NaCl concentrations (0
30% (w/v)), temperature (1565  C) and pH values (4
11), and with a good tolerance to different chemical agents and organic solvents. A new lipase from a Marinobacter lipolyticus strain (moderately halophilic), cloned and expressed, showed a good activity on different kinds of esters and high effectiveness in the production of eicosapentaenoic acid (EPA), allowing it to be considered as a good candidate for the food industry [33]. The halophilic enzymes used in industrial processes are few, among them the H nuclease of Micrococcus varians subsp. halophilus, which is applied in the commercial production of a flavoring agent: the 50 -guanylic acid (50 -GMP). This enzyme breaks down RNA at 12% salt and 60  C [9].

5.6.3 Production of organic osmotic solutes “Compatible” solutes have an important role in stabilizing biomolecules such as enzymes, DNA and membranes and also the whole cells, besides the protection against stress and salts. A moderate halophilic bacterium, Halomonaselongata, is considered as an ectoin production factory with a simple recovery process. Indeed, the bacterium releases the synthesized ectoin by a hypo-osmotic shock and restarts its synthesis when the hyper-osmotic conditions are applied again [34]. The ectoin is produced industrially also by the strains Halomonassalina and Chromohalobacter salexigens. It is the most widely used compatible solute and is marketed as a protective agent for DNA, proteins and mammalian cells [35,36]. There is another compatible solute shown better than ectoin in the protection abilities which is the hydroxyectoine, and for that, it has attracted more commercial interest. The Marinococcus M52 strain is shown to convert ectoin into hydroxyectoine [10].

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Other solutes are also used such as betaine in improving the osmotic tolerance of important commercialized crops (potato, tomato, rice, and tobacco), phosphate diglycerol as a protein stabilizer and trehalose as a cryoprotectant during biomolecules lyophilization. [9].

5.6.4 Production of biosurfactants and exopolysaccharides Halophilic biosurfactants and exopolysaccharides improve the remediation of soil and water contaminated by oils. In fact, halophilic/halotolerant microorganisms that produce biosurfactants are considered as key agents in the acceleration of remediation of saline locations polluted by hydrocarbons. For example, since many oil deposits are characterized by high salinity and temperature in addition to the lack of oxygen, the oil recovery is improved by microbial biosurfactants from organisms which produce them under these severe environmental conditions [20].

5.6.5 Liposomes production Liposomes are applied in cosmetics and drugs domains as transporters of effective compounds to specific target sites. Lipids from halophilic archaea, which are with ether linkages, have more structural stability and esterase resistance than liposomes based on fatty acids [20], and, thus, they have a higher survival rate. The new ether-lipids were got from the extreme halophilic bacterium Halobacterium cutirubrum.

5.6.6 Processing of halogenated products Halogenated organic compounds have an important impact on environment since they are persistent and toxic. There are many halophilic microorganisms that tolerate these toxic compounds or even transform them. The slightly halophilic and alkalophilic bacterium Nocardioides sp. can use the 2,4-dichlorophenol, 2,4,5-trichlorophenol (from agricultural biocides) and the 2,4,6-trichlorophenol (preservative) as the unique energy source. The halophilicarchaea from the genera Halobacterium, Haloarcula and Haloferax, are adapted to high amounts of halogenated hydrocarbons (up to 1 mM), like trichlorophenols, lindane insecticides and DDT. Some moderately halophilic bacteria are also able to transform these toxic compounds like an eubacterium which can transform the formaldehyde (organic solvent) in the presence of 1
20% NaCl (w/v), aerobically [20,37].

5.6.7 Production of alternative energy Hydrogen is combustible and easily converted into electricity, so it is considered as a new and interesting source of energy. In fact, photosynthetic bacteria can produce the H2 in light using organic substances available in various biological resources. Halophilic microorganisms are reported to produce this alternative energy. Indeed, a halophilic bacterial community, including photosynthetic bacteria, showed its ability to produce H2 in a one-step culture system in the presence of 3% (w/v) NaCl and light, using raw starch directly [38].

5.6.8 Production of polyhydroxyalkanoates (PHA) Polyhydroxyalkanoates (PHAs) are a family of biocompatible and biodegradable polyesters stored by many microorganisms. PHAs are used in many industrial applications such as bioplastics, biofuels, fine chemicals and medicine. PHAs contain several types of polymers including poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), which are among those studied and are already produced on a large scale. PHB is rigid and fragile whereas PHBV is more flexible allowing more potential applications such as medical equipment, film products, disposables and packaging materials. The accumulation of PHAs was first observed in the halophiles in 1972, and since then, several halophilic microorganisms have been reported as good PHA producers with high production rates which rich 80% of the total halophile weight of the moderate halophilic Halomonas sp. TD01 for example [39]. The halophilic archaea Haloferax mediterranei produces a PHBV co-polymer with 46% of the total bacterial weight. Several other halophilic strains were stated for the production of PHBV from non-fat carbohydrates as substrates. The recombinant strain Halomonas campaniensis overexpresses PHB at 70% of its total weight, in the presence of 40 g/L NaCl, at 37  C and pH 10, and for 65 days of non-sterile and continuous fermentation. This has resulted in a gain in the sterilization system spending and an improvement in the production since it is maintained continuously for a significant period of time [10,23].

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PART | I Physiological aspects

FIGURE 5.2 Applications of halophiles/halotolerans as low-cost production cells for chemicals, materials and biofuels. Seawater can be used as a salt-containing culture medium, reducing the consumption of fresh water. Low cost substrates, including substrates from kitchen waste and/or cellulose. The non-sterile and continuous process can increase the efficiency of the process and reduce the complexity and costs of energy and process in general. Since highpressure sterilization is not necessary, low-cost materials such as plastics, ceramics and carbon steel can be used to make fermenters and piping systems to prevent corrosion of systems stainless steel due to high salt concentrations. Pollution is reduced when water is recycled during the fermentation process [10].

Fig. 5.2 [10] summarizes the many advantages of PHA production by halophilic and halotolerant microorganisms, in simplified schema, which may remain valid for other products of industrial interest synthesized by these microorganisms. These gains are proven by Koller et al. [40] who showed that the cost of PHBV production by Haloferax mediterranei is 30% lower than that obtained by the recombinant strain Escherichia coli.

5.6.9 Transfer of the halo-tolerance To recover saline lands for agricultural activities, it is interesting and useful to transfer the halotolerance feature from halophilic microorganisms to crops of agronomic value. For example; transgenic tobacco plants have gained salt stress resistance after insertion of the dnaK1 gene from Aphanothecea hlophytica (halotolerant cyanobacteria) which can grow at high salinities up to 3 M NaCl [41].

5.6.10 Production of bacteriorhodopsin with original roles Bacteriorhodopsin is a “big” protein that transforms light energy into chemical one. It is the most simple protein recognized (in 2006) capable of fulfilling this function. The simplicity of bacteriorhodopsin has made it a model for the study of bioenergetics and for membrane transport. It is also of interest for the data storage industry, since it could serve as an extremely miniaturized storage unit that can be controlled by light pulses (at a rate of one bit per molecule, a disc

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59

12 centimeters in diameter could contain 20
50 tetraoctet). The use of bacteriorhodopsin is one of the first applications of organic molecular electronics, an emerging discipline of nano-informatics [42]. Actually, there is only one company (Halotek, 2019, https://halotek.de) that sells Bacteriorhodopsin, as lyophlized powder, produced from a Halobacterium salinarum strain. This Bacteriorhodopsin is purified and can be used in structural biology, biophysics of membrane proteins, bioelectronics, study of photocycle of bacteriorhodopsin and other important applications.

5.6.11 Important role in the bioremediation Hypersaline environments, like all other ecosystems, can be affected by pollution. Besides, about 5% of industrial effluents are estimated to be saline and hypersaline. The bioremediation of these contaminated media by conventional nonextremophilic microorganisms is not valid because of their inability to efficiently transform organic pollutants at high salinities. Since halophilic microorganisms are metabolically more adapted to elevated salt concentrations, they are considered as suitable and efficient candidates for the bioremediation of contaminated hypersaline environments and saline effluents. In fact, halotolerant and halophilic bacteria are reported to degrade a wide range of contaminant compounds [43]. Among these compounds, the aromatic compounds are the most troublesome. For that, there is a big interest given to the development and optimization of new processes that treat saline environments contaminated with these compounds. As example of such environments, we found the chemical, oil refining and wood preservation industries. Aromatic compounds can also pass in the environment as an intermediate during catabolism of other chemical and/or toxic compounds. To estimate the diversity of halophilic bacteria that could transform these compounds, Garcia et al. [44] proceeded with growth-based enrichments in the presence of a number of aromatic compounds in which phenol is the major pollutant. They used a group of bacteria, adapted to a wide range of salt concentrations, and containing the genus Halomonas as the dominant bacterial genus. This study showed that the moderately halophilic bacteria, especially strains from Halomonas genus, are able to transform aromatic compounds under saline and hypersaline conditions. Another successful treatment of aromatic pollutants by species of Halomonas is that effectuated by Halomonas campisalis. It is a moderately alkalophilic halophilic bacterium, capable of degrading salicylate and benzoate at concentrations as high as 380 mg/L, at pH 9 and at high salt concentrations up to 100 g/L NaCl. The requirement of H. campisalis for high pH and salinity during its growth shows its relevance for treating saline wastewater without the need for dilution. These results confirm the potentially effective and economical application of haloalkalophilic microorganisms for treating highly alkaline and saline industrial wastewater. In addition, halophiles have proved their ability to oxidize hydrocarbons in saline ecosystems contaminated by petroleum products. In fact, huge quantities of petroleum wastewater are produced during export and petroleum extraction activities. These produced waters are with different salt concentrations, so they vary from weakly saline to highly saline. Because of their high salt and hydrocarbon content, they are a dangerous contaminant for plants, soil and groundwater, generating real environmental problems such as erosion and contamination of the aquifer. Since conventional biological treatments are not valid at high salinities, the bioremediation of these polluted waters can be effectuated by either indigenous microorganisms that transform petroleum compounds or by bio-augmentation using halophilic/halotolerant bacteria. Other aromatic compounds were also demonstrated to be degraded by halophilic bacteria. Using brine from an oil production site (Oklahoma), an enriched culture was established with benzene as the unique source of carbon [45]. Results are very interesting since the culture was able to completely degrade benzene, ethylbenzene, toluene and xylene in one to two weeks. In addition, C14 benzene was transformed into 14CO2 which suggest that the enrichment was able to mineralize benzene. Analysis of the microbial community showed that the dominant member of this halophilic culture is Marinobacter spp. It is also shown that a halophilicarchaea, strain EH4, is able to transform many molecules of n-alkanes and aromatic hydrocarbons under high salt concentrations [37,46,47]. All the same, Gauthier et al. [48] found that the bacterium Marinobacter hydrocarbonoclasticus can degrade various aromatic and aliphatic hydrocarbons, and Kuznetsov et al. [49] proved that the halotolerant bacterium Streptomyces sp., isolated from an oil field, was able to degrade crude oil. There are other bacteria that were demonstrated to degrade many polycyclic aromatic hydrocarbons (PAHs) [50]. Several other successful attempts of hydrocarbons bioremediation using halophiles have been described in the marine environment, in the Arctic environment [51,52], in the fluids of salt-rich strata [53], in Louisiana salt marshes [54,55], in a biofilm reactor [56] and in phenolic saline wastewater [57]. Other studies have focused on the decolorization of textiles azodyed with halophilic and halotolerant bacteria. Isolated from textile effluents, three bacterial strains showed an important decolorization activity on the widely used

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PART | I Physiological aspects

azodyes. Phenotypic characterization and phylogenetic analysis indicate that these strains are belonging to the genus Halomonas. They are able to decolorize azodyes at a wide range of NaCl (up to 20%), temperature (25
40  C) and pH (5
11) concentrations after 4 days of static culture incubation. They can decolorize a mixture of dyes as well as pure ones. In addition, these strains can easily grow and decolorize high dye concentrations (5000 ppm (parts per million)) and tolerate up to 10,000 ppm [58]. Nevertheless, more researches are still needed to evaluate and valorize the real potential of the halophilic microorganisms to be reproducibly applied in the bioremediation of contaminated hypersaline ecosystems and in environmental activities in general. In this context, a biotechnological strategy that can profit from the many features of halophilic and halotolerant microorganisms and be used successfully to eliminate the hardest pollutants from saline contaminated environments, is called the bioaugmentation. In fact, the bioremediation is a biological process relying on the significant capabilities of the microbial communities to transform the human-generated organic pollutants into essentially in offensive or less harmful compounds. In some cases, the indigenous microbial community of a polluted environment have not the appropriate metabolic abilities to degrade and mineralize the pollutant compounds to small stable molecules such as CH4, CO2 and H2O. Then, the inoculation of this polluted environment with specific microorganisms could be a successful bioremediation of its contamination, and this is defined as the bioaugmentation. Then, bioaugmentation represents the technique of refining the metabolic abilities of the microbial community already present in the contaminated environment (soil or other biotopes) to eliminate pollution, by introducing certain competent and specific strains or consortia of microorganisms. These introduced microorganisms with genetic diversity constituted a broader repertoire of producible biodegradation reactions.

5.7

New molecular and genomic approaches

The halophilic and halotolerant microorganisms are of great biotechnological and industrial interest. As a result, studies focused on these microorganisms are in continuous growth for the search for other enzymes and molecules of interest, and this by conventional screening methods or by new techniques of genomic and metagenomic sequencing with high throughput. In addition, since the manipulation of these microorganisms in fermenters has led to a significant reduction in the cost of production, multiple efforts are being done to invent new genetic tools (material and protocols) such as cloning and expression vectors, promoters, signal peptides and mutagenesis systems serving for expression in halophiles [10,59].

5.7.1 Development of new genetic tools for halophiles It is obvious that the genetic manipulation of microorganisms is important to develop their properties. For the halophilic microorganisms, it becomes possible essentially for the family of Halomonadaceae which contains the strains of Halomonas and other genera [59]. Indeed, heterologous expression by these hosts provides many advantages over other conventional cells such as E. coli and Bacillus: (1) members of this family are highly salt tolerant which minimizes the need for aseptic conditions for their culture, and thus causes a decrease in costs; (2) they grow and are maintained easily in the laboratory, and their nutritional requirements are not complexes; (3) the majority of them have a large variety of compounds that could be the single sources of carbon and energy. During the 15 years ago, the Argandona team (Department of Microbiology and Parasitology, University of Seville, Seville, Spain), in addition to other research teams, have been able to develop new relevant and applicable tools to facilitate handling genetics of these cells such as, for example, DNA isolation procedures, cloning and expression vectors, genetic exchange mechanisms, mutagenesis approaches, reporter genes and assays for gene expression [59]. However, this progress being interesting, did not satisfy all the conditions necessary to succeed the heterologous expression in halophiles. In fact, there are still other challenges to overcome such as the large size of the plasmids used which causes low transformation efficiency, the identification of inducible promoters and other limitations [10].

5.7.2 Genomic and metagenomic sequencing Due to the rapid advancement of synthetic biology and genomic sequencing technologies, halophilic microorganisms are increasingly sequenced. For example, the genomes of 16 strains of Halomonas have been sequenced during the years 2010
13, 7 of which are published in 2013 [10]. The genomes of other halophilic strains are also sequenced such as those belonging to the genera Halobacterium, Chromohalobacter, Haloferax, Haloarcula and Halobacillus and the

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61

TABLE 5.4 Halophilic strains having their genomes completely sequenced. Souche

Length (pb)

Proteins

Submission date

Last modification

Genome ID in NCBI

Halomonas elongata DSM 2581

4.061.300

3628

17/09/2010

30/07/2015

3125

Halobacterium hubeiense

3.130.350

3102

17/12/2015

07/01/2016

42779

Halobacterium salinarum ATCC 700922

2.571.010

2540

14/07/2000

03/08/2015

1051 (1)a

Halobacterium salinarum DSM 671

2.668.780

2592

12/02/2008

03/08/2015

1051 (2)b

Haloarcula marismortui ATCC 43049

4.274.640

4100

02/11/2004

02/08/2015

1084

Natrinema pellirubrum DSM 15624

4.309.270

3981

27/12/2012

02/12/2015

11383

Haloferax volcanii DS2

3.939.820

3734

23/03/2010

15/09/2016

1149

Halobacillus halophilus DSM 2266

4.170.010

3942

11/04/2012

01/08/2016

11352

Chromohalobacter salexigens

3.696.650

3233

11/04/2006

03/08/2015

828

1051 (1): 1st assembly of Halobacterium salinarum strain with the ID n 1051 1051 (2): 2nd assembly of Halobacterium salinarum strain with the ID n 1051

a

b

number is expected to increase considerably in the next few years. The genomes submitted in the NCBI or EMBL databases come in two forms: either fully sequenced genomes accompanied generally and often with annotation, or partial genomic sequences that can also be annotated. Table 5.4 presents some halophilic strains having their genomes completely sequenced and accessible on NCBI according to the designated “ID” addresses. In addition, metagenomic approaches can be assayed for the identification and development of new biocatalysts from uncultivated bacteria which are a mine of new and interesting proteins [60]. Indeed, these approaches are recent and make it possible to study advantages of the microbial communities of several hypersalted biotopes (lakes, ponds, . . .) which are not or little explored before. For example, recently constructed metagenomic banks for Tyrrell, Victoria and Australia lakes have uncovered two unusual genomes that reveal a new type of very small, non-cultuvable archaeal halophiles (B 0.6 μm in diameter) [61]. Metagenomic studies are also conducted on other hypersalted biotopes such as the Mallorca Pond in Spain, the Baja California Saline and the Saltron Sea (California) in the United States, the Vestfold Hills Organic Lake and the deep cold lake in the Antarctic and Utah’s Great Salt Lake in the United States [14,61]. The metagenomic studies reveal a great microbiological diversity grouping the cultivable microbes and those that cannot be cultivated. In addition, they can isolate new enzymes that may be relevant to several biotechnology applications. For example, halophilic cellulases produced by a metagenomic library from some microbial soil consortia are shown to be highly thermostable, halo-stable and alkali-stable, which favors their use in the textile, detergent and food industries [10]. The unveiling of genomic sequences information of halophiles and in silico systematic analyzes help to a better knowing of these interesting and promising microorganisms and lead to several new ways of constructing and optimizing post-genomic approaches for more industrial applications and biotechnology.

5.8

Conclusion

Halophilic microorganisms are an important group of extremophiles in terms of distribution and characteristics. In fact, they are wide spread in many large environments like the salt marshes, the lagoons, the sebkhas and the salt lakes, where the salinity exceeds 3.5% of total salt and can reach up to salt saturation (35% of salt), conditions that are unbearable by the other conventional microorganisms. In addition, they are found in the three domains of life: the Eucarya, the Archaea and the Bacteria, with more phylogenetic representation within the Bacteria domain. The bacterial groups containing at least one halophilic member are the Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes and Bacteroidetes. The Archaea contains only one exclusively halophilic family, the Halobacteriaceae, representing the most famous extremely halophilic genus: Halobacterium. The halophilic microorganisms don’t require, all, the same range of salt concentrations for optimal growth. Some ones grow best in only 0.2
0.5 M of salts such as most of marine bacteria; some others require at least 2.5 M of salts

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PART | I Physiological aspects

for optimal growth and maintenance of structural stability, like the species of Halobacterium. The most adapted classification put halophilic microorganisms in 5 groups, depending in salt concentrations ranges for best growth, which are: the slight halophiles [0.2
0.5 Msalts], moderate halophiles [0.5
2.5 M salts], borderline extreme halophiles [1.5
4 M NaCl], extreme halophiles [2.5
5.2 M salts] and the halotolerants which are non halophilic but can tolerate salts up to 2.5 M and even more. In the last case, they are called extremely halotolerants, like Staphylococcus aureus. Since the halophilic microorganisms survive in saline and hypersaline media, they ought to have distinguished mechanisms that prevent them from the harmful effects of high salinity. In fact, the high salt concentrations cause a high osmotic pressure on cell walls and then, lead to a big damage of the conventional microorganisms. For that, halophiles possess two strategies to bear this difficult condition: either strategy “salt out” which consists on the accumulation of high concentrations of inorganic ions (K 1 and Cl-) in the cytoplasm, or strategy “salt out” in which the intracellular ionic concentration is maintained low and this is by pumping the ions out of the cell and accumulating organic “compatible” solutes (betaine, ectoine, etc. . .) to provide an osmotic equilibrium. Besides the important and distinguished techniques of salt adaptation, halophilic microorganisms provide new proteins and enzymes with interesting structural characteristics, protecting them from the salt denaturation or precipitation. Indeed, halophilic proteins contain more acidic amino-acids on their surfaces than basic ones, and this makes these surfaces negatively charged, permitting then more ligation with water molecules of the direct medium. This results in better hydration and solvation of halophilic proteins even in high salt concentrations. These interesting features make halophilic microorganisms with great interest in many biotechnological and industrial applications. In fact, since the chemical industry had many bad effects on the environment and the consumer, many industries have started to avoid them and replace them or a part of them by biological products which have the same role and even better with no damage or danger neither for the consumer nor for the environment. Early, halophilic microorganisms were involved in the production of marine salt and the fermentation of many salted food especially in the Asia east. Next, they were found to produce various stable enzymes efficient in many industrial applications at harsh conditions, and organic osmotic molecules used as stabilizers for biomolecules and stress protection agents. In addition, they produce new biosurfactants and exopolysaccharides which improve the remediation of contaminated saline soil and water, and new liposomes especially those from halophilic archaea in which the ether linkages are more stable and esterase resistant than their other available ones. Moreover, they have a very important role in the production of alternative energy. Indeed, a group of halophilic bacteria was found able to use raw starch directly to produce H2 in light which is easily combustible and converted into electricity. Halophilic microorganisms are also able to provide easy and important production of polyhydroxyalkanoates that are used in bioplastics, biofuels, fine chemicals and medicine. Besides, they are distinguished by the production of an interesting protein able to convert light energy into chemical one and having an original role in the data storage industry: the Bacteriorhodopsin. One of the most attractive applications of halophiles is the bioremediation of saline and hypersaline environments (water, soil, . . .) contaminated by oily, stubborn and/or toxic compounds which are thrown by many industries. In fact, many halophilic microorganisms have demonstrated their capability to degrade aromatic compounds such as phenol, petroleum products like benzene and toluene and textile azodyes. Because of the important current and potential applications of halophiles, researches to clone the genes of interest are increasing continuously. Besides, since the manipulation of halophilic microorganisms in fermenters was proved very gainful, many efforts are being made to develop new genetic tools for expression in halophiles.

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32. [28] Amoozegar MA, Fatemi ZA, Karbalaei-Heidari HR, Razavi MR. Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004. Microbiol Res 2007;162:369
77. [29] Karbalaei-Heidari HR, Amoozegar MA, Hajighasemi M, Ziaee AA, Ventosa A. Production, optimization and purification of a novel extracellular protease from the moderately halophilicbacterium Halobacilluskarajensis. J Ind Microbiol Biotechnol 2009;36:21
7. [30] Boutaiba S, Bhatnagar T, Hacene H, Mitchell DA, Baratti JC. Preliminary characterisation of a lipolytic activity from an extremely halophilicarchaeon, Natronococcus sp. J Mol Catal B: Enzym 2006;41:21
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7. [32] Samaei-Nouroozi A, Rezaei S, Khoshnevis N, Doosti M, Hajihoseini R, Khoshayand MR, et al. Medium-based optimization of an organic solvent-tolerant extracellular lipase from the isolated halophilic Alkalibacillus salilacus. Extremophiles 2015;19:933
47. [33] Perez D, Martı´n S, Ferna´ndez-Lorente G, Filice M, Guisa´n JM, Ventosa A, et al. A novel halophilic lipase, LipBL, with applications in synthesis of Eicosapentaenoic acid (EPA). PLoS One 2011;6(8):e23325. Available from: https://doi.org/10.1371/journal.pone.0023325. [34] Sauer T, Galinski EA. Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol Bio Eng 1998;57:306
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[43] Le Borgne S, Paniagua D, Vazquez-Duhalt R. Biodegradation of organic pollutants by halophilic bacteria and archaea. J Mol Microbiol Biotechnol 2008;15:74
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24.

Chapter 6

Overview of extremophiles and their food and medical applications Jane A. Irwin Veterinary Sciences Centre, School of Veterinary Medicine, University College Dublin, Dublin, Ireland.

6.1

Introduction: what are extremophiles?

The term ‘extremophile’ describes organisms that occupy environmental niches that could be regarded as ‘extreme’ by the standards of most living organisms, particularly by mammals [1]. These environments are generally characterized by extremes of temperature, pH, salinity, dessication, hydrostatic pressure, ionizing radiation and redox potential. Some can also withstand chemical extremes (e.g. low oxygen tension) or metals in the environment. However, the concept of ‘extreme’ conditions is relative, as conditions that are optimal for most animals and plants in terrestrial habitats might be unsuitable, or even lethal, for many organisms described as extremophiles. Most extremophiles are unicellular and belong to the bacteria and archaea domains of life [2]. Some eukaryotes are adapted to extremes of temperature. These include the tardigrade, a small multicellular animal which shows remarkable resistance to a number of extreme environmental parameters, and some fish found in polar waters. The hardy tardigrade can enter a hibernation state (the ‘tun’ state) in which it can survive temperature ranging from 2272  C to 151  C, vacuum conditions, extreme dehydration, high pressure (up to 100 MPa) and exposure to ionizing radiation. Its resilence makes it an interesting model organism for space research, as it can survive the kind of environments that might be encountered in space [3]. Some commensal organisms that are important to nutrition in animals, e.g. some anaerobes that reside in the mammalian gut and the rumen of ruminants, might be considered to have some extremophilic characteristics. There are plants that are resistant to high salinity and dessication, endowing them with extremophilic characteristics. In physiological terms, extremophiles are highly diverse, and include aerobes, anaerobes, chemotrophs, chemoorganotrophs, phototrophs, chemolithotrophs and photoheterotrophs [4]. This goes in concert with highly flexible metabolic traits, including the induction of stress proteins, which enables them to survive in many different conditions [5]. Extremophiles are classified according to the nature of their environment in which they can survive and proliferate. The main categories of extremophiles and some examples are listed in Table 6.1. These include the microbes that tolerate temperature extremes, i.e. thermophiles, hyperthermophiles and psychrophiles; acidophiles and alkaliphiles, which tolerate pH extremes; halophiles, which live under conditions of high salinity; barophiles (also called piezophiles) which survive optimally under high pressures; xerophiles, which tolerate low water activity, and radioresistant organisms, which tolerate high levels of radioactivity that would kill most eukaryotic and prokaryotic organisms. Adding complexity to the definitions, some extremophiles are adapted to multiple types of extreme conditions (polyextremophiles), e.g. thermoalkaliphiles [17] or halophilic alkalithermophiles [18,19]. The archaeon Sulfolobusacidocaldaricus is polyextremophilic, growing at pH 3 and 80  C [20]. Some microorganisms can tolerate a wide pH range, along with high temperatures, for example Bacillus and Paenibacillus spp. that live in hot springs in India that grow from 20 to 80  C, and at pH values of 5 15 [21]. Many of these pH-tolerant microbes are in fact neutrophiles [22]. Many deep-sea microbes are piezophiles (barophiles) as well as being psychrophiles, as they are adapted to low temperatures (,4  C) and high pressures ( . 10 MPa, corresponding to depths of over 1000 m) that are prevalent in Earth’s oceans. This makes them psychro-piezophilic [23]. Undersea hydrothermal vents harbor an abundance of piezothermophilic life, e.g. prokaryotes like Pyrococcushorikoshii and Thermococcus profundis that withstand the high temperatures and pressures found in these undersea environments [24]. Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00006-X © 2020 Elsevier Inc. All rights reserved.

65

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PART | I Physiological aspects

TABLE 6.1 Major classes of extremophiles. Condition

Category

Growth conditions

Example

Reference

Temperature

Hyperthermophile

.80  C

Pyrolobus fumarii

[6]

Thermophile

.50 80  C

Thermus aquaticus

[7]



Psychrophile

,15 C

Psychromonas ingrahamii

[8]

Salinity

Halophile

[NaCl] .0.2 M

Halobacterium sp. NRC-1

[9]

pH

Acidophile

pH ,3

Picrophilus oshimae

[10]

Alkaliphile

pH .9

Natronococcus occultus

[11]

Hydrostatic pressure

Piezophile (barophile)

. 10 MPa

Thermococcus barophilus

[12]

Radiation

Radioresistant

High ionizing radiation

Deinococcus radiodurans

[13]

Water availability

Xerophile

Low water activity

Xeromyces bisporus

[14]

Chemicals

Metal tolerant

High metal concentration

Ferroplasma acidiphilum

[15]

Oxygen tension

Anaerobe

Low/no O2

Clostridium botulinum

[16]

The astonishing diversity of microbial life in extreme environments became apparent when metagenomic approaches and 16S DNA sequencing were developed. Much of what is known about microbiology is based on information derived from cultured microbes, but 60 99% of microbes are not easily culturable or are unculturable by standard methods [25]. This means that a great deal of microbial life amounts to ‘microbial dark matter’ which may contribute to our understanding of ecosystems and processes that are previously unknown [26].

6.2

Adaptations of extremophiles at a molecular level

There are many mechanisms that allow extremophiles to survive and flourish in extreme conditions. These include the synthesis of enzymes that can facilitate protein stability, enabling proteins to remain folded and undamaged; stable lipids; and stable DNA, which is achieved by cellular thermostabilizers, reverse gyrase and various DNA-binding proteins [4]. Acidophiles have proton pumps that allow them to keep their cytoplasm at pH values near neutral [27] and many halophiles accumulate compatible solutes that help them to maintain osmotic equilibrium. Proteins from extremophiles have a remarkable array of adaptations [28,29] that vary with the temperature and pH of the environment. Some adaptations are complementary, as illustrated by archaeal extremophiles. For example, halophiles and alkaliphiles share genome similarities, and haloalkaliphiles such as Natronomonaspharaonis, found in an extremely saline environment around pH 11, have a suite of protein adaptations that are largely halophilic rather than specific to high pH. This is accomplished by maintenance of a mildly basic cytoplasm that accumulates high salt concentrations [30]. Adaptation to high temperature and low pH often seem to go together. In general, there are three main strategies used. Thermostable proteins tend to be oligomers with large hydrophobic cores, with increases in disulfide bonds, salt bridges, and increased surface charges, along with decreases in the entropy of unfolding [28,31]. In contrast, proteins from psychrophiles tend towards fewer intra-protein stabilizing interactions, greater flexibility due to the presence of more glycine residues and fewer arginines, and fewer hydrophobic interactions [32]. Halophilic proteins have far more acidic residues on the protein surface [33], which interact with water molecules to maintain them in solution [34], fewer hydrophobic residues, and a reliance on salt to allow the proteins to fold, illustrated by the saltdependence of folding in an esterase from Haloarcula marismortui [35]. Quorum sensing mechanisms are also part of the survival strategies of various extremophiles. The systems found in mesophiles, including AI-2 based systems, are found in thermophiles, and others are found in psychrophiles and acidophiles. Radiation/oxidative stress-resistant organisms, e.g. Deinococcus radiodurans, also use AI-1 and AI-2 systems to communicate [36].

Overview of extremophiles and their food and medical applications Chapter | 6

6.3

67

Thermophiles: life at high temperature

6.3.1 Habitats and diversity Thermophiles are divided into two main categories: thermophiles, which need temperatures of 50  C or higher for optimum growth, and hyperthermophiles, which have an optimum temperature for growth over 80  C [6]. In contrast, mesophiles have optimum temperatures for growth between approximately 20 45  C and psychrophiles grow optimally at temperatures below 20  C. However, thermophilic extremophiles exhibit a remarkable range of operational temperatures, between 0  C and 120  C, and at pH values of 0 12 [37]. This means that many thermophiles are in fact polyextremophiles, i.e. thermoalkaliphiles or thermoacidophiles, and they exhibit molecular adaptations that allow them to exist in these harsh environments. Thermophiles span all three domains of life, but the majority belong to either bacteria or archaea. Which group dominates depends on the temperature: bacteria dominate between 50 90  C, whereas at temperatures over 90  C, archaea dominate, so the majority of hyperthermophiles are archaea [38]. Thermophilic microorganisms were first identified in the 1960s, when Thomas Brock isolated Thermus aquaticus, now famous as the origin of Taq DNA polymerase essential for the polymerase chain reaction, from Yellowstone National Park in the US [7]. Over the subsequent decades, thermophilic microorganisms have been isolated from numerous environments, from domestic laundry and hot water heaters [39], compost piles, silage, slag heaps and industrial processes [40], to natural environments like heated soils, geothermally heated terrestrial hot springs, geysers and submarine hydrothermal systems. Habitats heated by metabolic activity often harbor thermophilic fungi, although these cannot tolerate the extremes of heat that are optimal for some bacteria and archaea. These normally grow optimally about 45 55  C, with an upper limit of 60 62  C [41]. Thermophiles are uniquely adapted at a physiological and biochemical level to survive these extreme conditions. They include methanogens, hydrogen-generating microbes, and thermoacidophilic microorganisms [42]. The initial observations of thermoacidophiles date back to the 1970s with the discovery of Sulfolobus acidocaldarius [43] but in subsequent years, many more were discovered worldwide, particularly in solphataric environments [44] and in oil reservoirs thousands of meters underground [45]. Even Antarctica harbors thermophilic microbes, as it has warm soils, fumaroles, hot springs and hydrothermal vents [46]. The most thermophilic microorganisms are found in environments close to active volcanoes, such as solphataric fields, hot springs and deep-sea hydrothermal vents. The latter can achieve temperatures up to 400  C, and microenvironments are formed where there is a thermal and chemical gradient between the hydrothermal vent fluids released from below the Earth’s crust and the surrounding cold seawater. Some of these vents are known as ‘black smokers’ as they resemble chimneys that emit black smoke and release sulfide-rich material; others are known as ‘white smokers’, as they release minerals that are lighter in color, e.g. barium, calcium and silicon. These extremely hot, mineral-rich habitats are the realm of hyperthermophiles. The first of these hyperthermophiles to be discovered near a hydrothermal vent was Methanocaldococcus jannaschii [47], but over the intervening decades, many more archaeal and bacterial species werederived from these environments (for reviews of deep-sea vent extremophiles, see [48,49]). These habitats tend to have weakly acidic pH, as the fluids mix with seawater. Some of these microorganisms are also piezophilic, e.g. Marinitogapiezophila, which was isolated from a deep-sea vent and grew optimally at 40 MPa [50]. Among the most heat-resistant of the deep-sea hyperthermophiles known is an organism originally called strain 121 [51], now called Geogemma barossii. This Fe (III)-reducing archaeon used formate as an electron donor. It was isolated from a hydrothermal vent along the Juan de Fuca Ridge in the northeastern Pacific Ocean, grew at temperatures from 85  C to 121  C, the temperature of an autoclave, and survived 2 hours when incubated at 130  C. In comparison, Pyrolobusfumarii, reported to grow at 113  C, was inactivated after 1 hour at 121  C. The upper temperature for life was extended slightly beyond this by the successful culture of the hyperthermophilic hydrogenotrophic methanogen Methanopyrus kandleri strain 116, which could grow at 122  C under conditions of high pressure [52]. Hot terrestrial environments harbor enormous thermophile diversity. This has been shown by culture studies, partial 16 S rRNA sequencing and metagenomic approaches [53]. At present, only two bacterial families, the Aquificaceae and Thermotogaceae (including the well-characterized Thermotoga maritima), are categorised as hyperthermophiles, the others being archaea. The Sulfolobales, which are thermoacidophiles often found in terrestrial solphataric fields and mud pools, are archaea with diverse metabolic activities. Some use sulfur for autotrophic growth, some can use proteinrich substrates, and some can fix carbon dioxide [54]. Pyrococcus furiosus, a member of this order, is one of the most intensively studied hyperthermophiles. Its genome has been sequenced and it is now a commonly used organism for metabolic engineering, hosting metabolic pathways from less extreme thermophiles [54,55]. Among the Bacteria,

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PART | I Physiological aspects

Caldicellulodisruptor spp., which break down lignocellulosic biomass, and some Thermus spp. are hyperthermophilic. The diversity of anaerobic thermophiles has been reviewed by Canganella and Wiegel [56], providing a detailed list of microbial species with details of metabolism, origin and optimum growth conditions.

6.3.2 Physiology and adaptation to high temperature Most hyperthermophiles are chemolithoautotrophs, that use hydrogen or sulfur as electron donor, and most are microaerophilic [44]. A wealth of information is available regarding the genetics and genome manipulations of hyperthermophiles and thermophiles [57,58], which enables researchers to study the unique metabolic pathways they contain in more detail [59]. Maintaining membrane fluidity and stability at high temperature is a challenge. The cell membrane of thermophilic (and acidophilic) archaea is composed of tetraether lipids which span the whole membrane in a single layer, with saturated isoprenoid chains linked to glycerol or an alternative alcohol, in contrast to bilayers of acyl chains linked via esters to glycerol in bacterial cell membranes [60,61]. Consequently, cell membranes of archaea are thermostable, rigid, impermeable and resistant to oxidation. The stability and enzymatic activity of proteins from thermophiles have been studied in detail over the last 30 years, and general structural themes emerge. These include large hydrophobic cores, more salt bridges and increased surface charges, as well as more disulfide bonds [31]. An analysis of survival mechanisms of thermophiles from an omics perspective shows how multiple processes interlink to facilitate thermophile survival at high temperature [62]. At a genomic level, horizontal gene transfer has allowed organisms to exchange DNA sequences [63] conferring thermophilic characteristics (e.g. the gene encoding reverse gyrase). Thermophile genomes tend to be relatively compact in comparison to those of mesophiles [64] and have a higher GC content, while the DNA repair mechanisms are more robust in thermophiles to repair DNA, which is unstable at higher temperatures. Codon usage differs to that in mesophiles, with high frequency of A and G bases in close proximity, which leads to variation in amino acid content of proteins [65]. In general, charged residues are more frequent and polar uncharged and thermolabile amino acids are less abundant in thermophile proteins. There is a bias towards NTN codon usage as growth temperature increases in bacteria and archaea. These encode non-polar hydrophobic amino acids, suggesting that increased hydrophobicity is a contributor towards protein stabilization at high temperature [66]. The alterations in genome and amino acid sequences also contribute to added stability in the translational machinery [67] and heat shock protein gene expression is upregulated (e.g. Pyrococcus furiosus) upregulated its expression of Hsp60 and Hsp20 when the growth temperature was elevated from 90 to 105  C [68,69]. In addition, there are thermophile-specific modified nucleosides in tRNA, and Thermus thermophilus can alter its protein synthesis mechanism using these modified tRNAs and highly thermostable tRNA modification enzymes [70]. Thermophiles catalyse an enormous range of metabolic processes [56], and some, particularly the thermophilic and hyperthermophilic archaea, have modifications to their metabolic processes in comparison to bacteria. Archaea have modified variants of the Entner-Doudoroff and Embden-Meyerhof-Parmas (glycolytic) pathways, with little or no pentose phosphate pathway, which have been reviewed by [71]. In contrast, the hyperthermophilic bacterium Thermotogamaritima uses the conventional forms of these pathways [72].

6.3.3 Thermophiles in medicine and food In some cases, thermophilic bacteria can sometimes be pathogenic to humans. Among the eukaryotes, fungi tend to be the most thermophilic organisms. For example, ‘farmer’s lung’, a type of pneumonitis, is caused by thermophilic actinomycetes, e.g. Thermoactinomyces vulgaris, T. viridis, T. sacchari and Saccharopolyspora rectivirgula, which grow in moldy hay or grain in which temperatures are elevated [73]. There is a case report of Mycobacterium xenopi, a thermophilic bacterium, in a hospital’s hot water system, causing pulmonary mycobacteriosis in three out of 87 patients exposed [74]. One study found 31 clinical isolates that could grow at over 50  C [75]. The best-known application of a thermophilic enzyme in medicine, forensics and in biological research in general is DNA polymerase, isolated from thermophilic microorganisms for use in the polymerase chain reaction (PCR), which is employed to clone and amplify genes for diagnostic purposes in human and veterinary medicine. Since the initial development of this thermostable enzyme from Thermus aquaticus, now known as Taq polymerase, as a reagent for PCR [76], other thermophilic polymerases have joined it as reagents for DNA amplification [77,78]. Some thermophiles can contaminate food. Anoxybacillus flavithermus and Geobacillus spp. can contaminate and spoil powdered milk products. A. flavithermus forms spores at 55 and 60  C and forms a biofilm [79]. Geobacillusthermoglucosidans lacks lactase activity and grows poorly in skim milk, so consequently relies on A. flavithermus contamination to supply it with glucose and galactose for growth [80].

Overview of extremophiles and their food and medical applications Chapter | 6

69

TABLE 6.2 Enzymes from thermophiles with food industry and medical applications. Enzyme

Source

Application

References

DNA polymerase

Thermus aquaticus (Taq)

PCR, diagnostics, genetic analysis

[76]

Pyrococcus furiosus (Pfu)

[83]

Thermococcus litoralis (Vent)

[84]

α-Amylases

Pyrococcus spp., Thermococcus spp

Starch hydrolysis

[85]

Glucoamylases

Thermoplasma acidophilum

Glucose release from starch

[86] [87]

Picrophilus torridus Xylose isomerase

Thermotoga spp.

High fructose corn syrup production

[88]

Xylanase

Thermotoga maritima

Bread making

[89]

β-Mannanase

Aspergillus niger

Juice clarification, coffee viscosity reduction

[90]

Thermolysin

Bacillus thermoproteolyticus

Aspartame synthesis

[91]

6.3.4 Thermophilic enzymes and their applications Thermozymes have numerous applications, as their high temperature optima ensure that they resist degradation at elevated temperatures, with higher productivity and shorter process times [81,82]. A list is provided in Table 6.2. Glycosyl hydrolases are carbohydrate-degrading enzymes, which have numerous applications in the food and animal feed industries and in lignocellulose degradation [92,93]. They include α- and β-amylases, glucoamylases, α-glucosidases and pullulanases, all of which are required to fully digest starch. Cellulases and xylanases are employed in food processing [94]. Many of these enzymes have also been modified by site-directed mutagenesis to optimize their catalytic properties [82]. Proteases are also used in various industries, e.g. alcalase, a protease used to process soy meal, and thermolysin, which is used to produce the artificial sweetener aspartame [95].

6.4

Psychrophiles: life at low temperature

6.4.1 Habitats and diversity Psychrophiles are defined as organisms that can grow in cold environments. These comprise the deep sea, which covers nearly 75% of the planet, high-elevation regions, and the polar regions [96]. Sea ice containing brine and sub-glacial environments can also harbor psychrophiles. Metabolically active psychrophile bacteria have been found in the ice eutectic phase (briny liquid between ice crystals) at temperatures as low as 220  C [97]. The term ‘psychrophile’ has been split into various sub-categories, e.g. ‘psychrotroph’ and ‘psychrotolerant’ depending on the temperaturedependent growth rate [98,99]. According to Cavicchioli [100] this is misleading, as when temperature increases the rate of enzyme-catalysed reactions does likewise, due to the kinetic effect of heat, until a cellular process is compromised by an increase in temperature. Psychrophiles have traditionally been defined as organisms that cannot grow at temperatures in excess of 20  C and grow optimally at below 15  C. When applied to environmental organisms it is more difficult to define the optima, as in marine environments oligotrophic microorganisms grow slowly, compared to copiotrophic microorganisms which grow faster in nutrient-rich environments [101]. Consequently, an organism that may dominate in a particular cold, nutrient-sparse environment may fail to grow in the laboratory, or may grow at considerably higher temperatures than those at which it is normally isolated, leading to erroneous assumptions as to the true optimum conditions for its growth [100].

6.4.2 Physiological adaptation to low temperature Psychrophiles have numerous physiological and biochemical adaptations to their environment, which have been reviewed [102,103]. Various studies employing ‘omic’ technologies have revealed that some psychrophilic bacteria can downregulate oxidative metabolism in cold conditions [104,105]. In general, psychrophiles tend to repress glycolysis

70

PART | I Physiological aspects

and the TCA cycle, while shifting metabolism to alternative pathways to generate energy [103]. Psychrophilic enzymes tend to have higher activity at low temperatures, optimizing their values for kcat at the expense of Km, and be more flexible with a less stable active site [32]. Protein synthesis and folding are affected by temperature, and an understanding of these processes in terms of their thermodynamics, active site structure and requirement for chaperone proteins have expanded knowledge of how these proteins work [106]. In some cases, there is an active site which is larger and more accessible to ligands. This allows substrates to bind at lower energy costand the activation energy is lower. Other mechanisms include the production of compatible solutes, which are valuable not only for osmoprotection and protection against freezing, but also for generating energy [107]. Some bacterial species found in cold environments make polyhydroxyalkanoates, PHAs [108]. For example, in Sphingopyxis alaskensis, proteome analysis revealed that low temperatures lead to an upregulation of enzymes involved in PHA synthesis [101]. Cold conditions also induce increased production of reactive oxygen species, and some studies have shown an upregulation of antioxidant enzymes, e.g. catalase, glutathione peroxidase and superoxide dismutase under these conditions [109]. Psychrophiles can alter their cell wall components, thickening the cell wall, as seen in Exiguobacterium sibiricum, which protects it against ice formation and alterations in osmotic pressure [110].

6.4.3 Applications of enzymes and metabolites from psychrophiles According to Research and Markets [111], the world enzyme market was valued at US$7082 million in 2017 and was estimated to increase to US$ 10,519 million by 2024. A significant sector of this market is devoted to enzymes from cold-adapted organisms, which work efficiently at low temperatures. The applications of enzymes active in cold temperatures have been reviewed recently by [112]. One widespread application is in food processing, particularly milk and dairy product processing, in which cold-adapted beta-galactosidases are used to hydrolyze lactose. Lactose intolerance, which occurs when adults cannot synthesize intestinal lactase, is widespread in many human populations and causes intestinal discomfort as the intestinal microflora ferments the lactose. Betagalactosidases have widespread application [113,114] removing lactose from milk and whey. They are used to produce the low-calorie sweetener D-tagatose, and numerous cold-adapted ones have been identified and characterized. Cold-active xylanase is used for improving bread quality, and this enzyme from Pseudoalteromonas haloplanktis TAH3a has been patented (US/2008/0020088, EP1723229, WO/2005/097916) and is available commercially from the Belgian company PuratosNaamlozeVennootschap for use in increasing bread volume. In meat and fish processing, cold-adapted proteases are applied for tenderisation, and their application has been discussed in relation to enzymes from Atlantic cold-water fish [115]. Some of these enzymes are used for processing caviar, removing skin from fish fillets or squid mantle, or extracting membranes from fish [116]. Seafood processing in general makes use of various cold-adapted enzymes [117]. Cold-active lipases are made by bacteria and fungi that reside in permanently cold habitats, and by microorganisms present in refrigerated food and milk [118]. Some of these enzymes are commercially available, e.g. Lipase B from Candida antarctica [119], which is commercially available from Novozymes under the trade name Novozym 435, along with another lipase, CALB L, from the same organism. Antifreeze proteins are produced by some cold-adapted organisms. These small glycoproteins were first discovered in fish from polar waters [120], which prevent ice crystal formation in cells and body fluids. The antifreeze protein from the ocean pout fish, called ice-structuring protein, has been overexpressed in Saccharomyces cerevisiae. It is used in some of Unilever’s ice-cream brands to control ice recrystallization after freeze-thaw cycles, which could affect the ice cream’s taste and texture [121]. Some cold-adapted organisms also impact food production through their pathogenic effect on fish and animal products. For example, some cold-adapted bacteria cause disease in farmed fish, particularly those found in North Atlantic waters [122]. Cold-tolerant organisms can also spoil chilled food, potentially causing human disease. These include Pseudomonas fluorescens, which is found in raw milk [123]. Medical applications of psychrophile-derived products include diagnostics and drugs. Many marine microorganisms are found in cold water habitats, including polar seas and temperate and tropical deep waters, which have a constant temperature close to 4  C below the thermocline [124]. There have been numerous reviews of drugs and drug candidates derived from marine microorganisms, many of which are cold-adapted [125 128], and there is also particular interest in psychrophiles as a source of new antimicrobials [129]. Marine microorganisms may also be halophilic, piezophilic, and those found close to volcanic vents on or near ocean floors may be thermophilic or hyperthermophilic [130]. Deep-sea fungi produce hundreds of bioactive metabolites with activity against cancer cells, bacteria, fungi, viruses and protozoa [131]. Terrestrial cold habitats also serve as sources of cold-adapted bacteria, fungi and algae that have potential medicinal utility. In particular, the Antarctic is being explored as a source of such compounds. For example, Li

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et al. [132]. showed that a soil microbe, Oidiodendrontruncatum GW3-13, produced cytotoxic compounds, and antimycobacterials were found in Janthinobacterium sp. Ant 5-2 and Flavobacterium Ant342 [133]. Antarctic macroalgae have recently been shown to produce compounds with anti-cancer and antimicrobial properties [134].

6.5

Halophiles

6.5.1 Habitats and diversity Halophiles include bacteria, archaea and eukaryotes. One definition of halophile is that of Oren [135], who defines them as microorganisms with optimal growth at NaCl concentrations over 0.2 M. Halotolerance ranges widely, from marine organisms that grow at about 3.5% (w/v) NaCl, to moderate halophiles growing at 3 15% NaCl, to moderately halophilic organisms found at over 10% w/v NaCl (1.7 M), and extreme halophiles that grow in saturated 30% w/v NaCl solutions [136]. There are many examples of hypersaline environments worldwide: among the best characterized are the Dead Sea and Great Salt Lake, but such environments have been also found in Australia, Romania and elsewhere. Other examples are anoxic brine pools found on the bottom of the Red Sea and the Eastern Mediterranean, which display steep gradients in temperature, depth and salinity [137,138]. Our understanding of these environments has increased greatly thanks to the availability of high-throughput sequencing and metagenomic approaches [136]. They also include cold environments, e.g. brine channels in polar ice [139]. Hypersaline lakes and saltern evaporator and crystallizer ponds that produce salt from seawater provide a habitat for red halophilic archaea belonging to the family Halobacteriaceae and bacteria such as the extreme halophile Salinobacterruber [140,141], which has some archaebacterial properties. Halophilic eukaryotes, such as the unicellular β-carotene-rich alga Dunaliellasalina, are also found widely in hypersaline bodies of water. This organism is exploited for β-carotene production and employed as a model organism for studying photosynthesis [142]. The brine shrimp Artemia is known to tolerate high salt, but in general animals are not halophilic [139].

6.5.2 Physiological adaptations to high salt concentration Halophilicity can go together with other extremophilic characteristics. Halo-acidophiles and halo-alkaliphiles both exist in nature. The latter are more common, and this characteristic is sometimes combined with thermal tolerance [19] to produce halophilic alkalithermophiles. It can also be combined with cold adaptation. The Antarctic haloarchaea Halorubrum lacusprofundi and Halohastalitchfieldiae are dominant in the microbial community in permanently cold and hypersaline Deep Lake, where temperatures can drop to 220  C, and a proteomic study showed that they do so by modifying their cell envelope, which maintains osmotic balance and translation initiation, and altering their mechanisms for RNA turnover and tRNA modification [143]. The alga Dunaliella also survives in this environment and is the lake’s primary producer [144], providing nutrients for haloarchaea. There are many adaptations employed by halophilic organisms to adapt to a life in salt (see [145] for a detailed review). These include robust cell walls that can adapt their own hydrophobicity depending on the environmental NaCl, variable amounts of negatively charged polar lipids, as well as the ether linkages found in archaea, which make them less permeable to ions. Halophiles employ two main strategies to resist high salinity and water stress. One is a ‘high salt-in’ strategy. Some halophiles increase the internal osmolarity by accumulating K1 ions in the cytoplasm, requiring the expenditure of two molecules of ATP for each K1. The alternative strategy, commonly seen in Bacteria and Eukarya, is a ‘low-salt-in’ one, in which the cells accumulate compatible solutes. These small molecules, e.g. sugars, alcohols, amino acids, N-acetylated diamino acids, glycine betaine, ectoine and hydroxyectoine can be either taken from the environment or synthesized intracellularly and can act as general stress protectants. They prevent the ‘saltingout’ of proteins in the cell. Halotolerant and moderately halophilic organisms tend to use this mechanism, whereas extreme halophiles also use the ‘high salt-in’ strategy [145]. These adaptations were accompanied by certain features commonly observed in proteins from halophiles (see [146] for review). In general, such proteins had a large number of negative charges, and were more hydrophobic than their mesophilic counterparts. Sequencing of the genome of Halobacterium sp. NRC-1 revealed that the proteome is remarkably high in negative charges [147], with a median pI of 4.9 and few basic proteins, which contrasts with nonhalophilic proteomes which have an average pI close to neutral [146]. They tend to have fewer bulky hydrophobic side chains on the surface, compared to small and borderline hydrophobic residues [148]. This property aligns with the higher flexibility and surface hydration observed for halophilic proteins [149].

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Most of the information about life in high salt has been gathered from studies of organisms that reside in high NaCl. However, some organisms live in high concentrations of kosmotropic (stabilizing) salts, but others survive in high concentrations of chaotropic (destabilizing) salts, e.g. NaBr, CaCl2 and MgCl2. These include some fungi from various extreme environments, which tolerate up to 2.1 M MgCl2 or 2 M CaCl2 and could therefore be described as chaophilic [150].

6.5.3 Medical applications of molecules from halophiles Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) was first isolated from Ectothiorhodospirahalochloris [151], a phototroph which grows in up to 5 M NaCl, and has been isolated from other bacteria, which include alpha- and gamma-proteobacteria and Actinobacteria [152]. This compound, along with hydroxyectoine, its hydroxyl derivative, is widely employed as a protein stabilizer [153] and is also used for membrane protectionfrom desiccation. A German company, bitop AG (www.bitop.de), has developed a wide range of uses for osmolytes, in particular forectoine, and a technique called bacterial milking has been patented by this company and used to obtain this product from Halomonaselongata on a large scale [154]. Applications listed on bitop’s website cosmetics, food supplements, treatments for epithelial dysfunction in companion animals, formulations to treat dry eye and allergies, and skin creams. Ectoine works by a mechanism described as ‘preferential exclusion’, in which membranes expel ectoine when it is present with proteins or lipids. This increases the hydration of the membrane, and makes the lipid layers more fluid [155]. This property is exploited commercially in Hylos eye care products, used to treat ‘dry eye’ (www.hylo.de). The range of potential applications for ectoine and other osmoprotectants is considerable and is not just limited to eye and skin care [156,157]. For example, it has anti-inflammatory effects, as ectoine and 5-α-hydroxyectoine helped to relieve colitis in a rat model [158]. It was proposed that this is due to stabilization of the intestinal barrier, which could be helpful in IBD once it was shown to be safe and effective. These molecules also enhanced lung surfactants [159]. Ectoine decreases skin inflammation, as shown by a study in which an ectoine-containing cream was as effective as a non-steroidal anti-inflammatory cream and was well tolerated by patients with atopic dermatitis [160]. It can also protect skin cells from damage by ionizing radiation by absorbing ultraviolet radiation and preventing DNA strand breaks [161]. Another potential application is in the treatment of protein-folding diseases, reviewed by [162]. In several neurogenerative disorders, e.g. Alzheimer’s, Parkinson’s and Huntington’s disease, there is misfolding, aggregation and deposition of amyloid proteins in the brain [163] and ectoine and its hydroxy derivative were found to reduce formation of amyloid fibrils [164]. Ectoine-containing lozenges have also been found to be effective in treating acute viral pharyngitis [165]. Another medical application of halophiles is as a source of materials to generate nanoparticles, materials which are in clinical trials for drug and gene delivery, in vivo imaging, and in vitro diagnostics [166]. The halophile Halomonasmaura was used as a source of mauran, a polysaccharide used to encapsulate the chemotherapy drug 5fluorouracil, which was used to produce particles that were toxic towards several cancer cell lines [167,168]. Halococcussalifodinae BK3, a halophilic archaeon, was used to make spherical intracellular selenium nanoparticles [169] and tellurium nanoparticles [170], which were active against a range of Gram-negative and Gram-positive bacterial species. Halophilic archaea have also been used as sources of archaeosomes, which due to the unusual properties of their membrane lipids may potentially be used as carriers for topical delivery of drugs across the skin [171,172].

6.5.4 Halophiles and food products Their main application has been to provide compatible solutes and other compounds, e.g. beta-carotene, which can be added to food as a coloring agent or as a source of vitamin A. The green algae Dunaliellasalina and Dunaliellabardawil, found in inland aquatic saline habitats (see [142] for review) and also in coastal evaporation salt pans, such as those found in parts of India [173] produce this substance in large quantities. This helps to prevent lightinduced damage. D. salina also produces lipids containing polyunsaturated fatty acids that can be added to food products or pharmaceuticals [174]. Halophiles can also grow in foodstuffs as contaminants. Some fermented foods, particularly some from Asia, are produced with the aid of large amounts of salt. Halophilic microorganisms can grow in these while they ferment, and in some cases, the food-grade salts added can even harbor extremely halophilic archaea of the Halobacteriaceae, particularly the genera Haloarcula, Halobacterium and Halorubrum [175]. One example is the Thai anchovy fish sauce nam-pla, which is made by adding two parts of fish to one part of marine salts, adding concentrated brine, and fermenting for a year. Various halophiles have been isolated from this mixture, including

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Lentibacillushalophilus [176], Halococcusthailandensis [177] and Natrinemagari [178]. The fermented Korean fish sauce jeotgal also contained halophilic archaea [179] and the Korean fermented vegetable kimchi was found to contain an extremely halophilic bacterium, Lentibacilluskimchii, which grew in 10 30% (w/v) salt [180]. Some foods fermented with ectoine-producing microbes in the presence of salt may also contain ectoine, including the Japanese soy sauce Natto. Ectoine may help to preserve these foods, by acting as a stabilizer [181]. Halophiles (as well as acidophiles and alkaliphiles) produce exopolysaccharides [182]. These molecules have emulsifying activity and gelling properties, so they have applications in food production.

6.6

Acidophiles

6.6.1 Habitats and diversity Acidic environments with pH values ,5 are relatively common on earth, but those with pH values below 3 are far less so and are often associated with the production of sulfuric acid. These environments can often be found in volcanic and geothermal regions, including parts of New Zealand, Iceland, the Azores, and some parts of the Caribbean. Yellowstone National Park in the United States is one of the best known [183]. Some of the most acidic have been constructed by human activity, such as mining (e.g. Berkeley Pit Lake in Butte, Montana), a lake containing metal-sulfate rich and highly acidic mine waste [184], while others have arisen due to natural factors. One well-studied example is the Tinto River (Rı´o Tinto) in Spain, a river arising at the center of the Iberian Pyritic Belt. This has a pH of approximately 2.3 and a high concentration of heavy metals, iron and sulfate in the water, which makes it similar to acidic mine drainage systems like that at Berkeley Pit Lake, originally developed from an abandoned copper-pit mine [185]. Acidophiles can also be encountered in the human built environment where acidic conditions emerge: recent studies [186] have found that corroding concrete sewers with pH values of 2 4 harbor a diverse community of acidophilic microbes, the growth of which is affected by environmental H2S. Notwithstanding the acidity of these habitats, they often teem with many types of prokaryotic and eukaryotic life, in particular fungi and algae. The first one of these acid-tolerant bacteria to be identified was Thiobacillus ferrooxidans (now called Acidithiobacillus ferrooxidans) back in the 1940s [187], which was found to be involved in catalysing metal extraction. This organism is a chemolithotroph that obtains energy from the oxidation of ferrous ions by oxygen. At low pH, the re-oxidation of ferrous iron needs oxygen and the presence of acidophilic microbes. These include chemoautotrophic species, e.g. Acidithiobacillus ferrooxidans and Leptospirillum spp. A. ferrooxidans derives energy from oxidizing ferrous iron and reduced inorganic sulfur compounds at low pH [188]. It can thus generate polluting acidic metal-rich drainage waters, but in so doing it can also improve metal recovery from the leachates. Its properties allow it to colonize mineral-rich environments, and some researchers have proposed its use as a model organism for astrobiology [189]. Some acidophiles can attach to sulfide mineral surfaces and form biofilms [190]. This property has led to considerable interest in the use of acidophiles to perform biomining, and in engineering these microbes to make them more tolerant of fluctuating process conditions [191]. Tolerance of extreme pH values is often accompanied by temperature tolerance, and solphataric hot springs harbor thermoacidophiles. These can tolerate high temperatures (60 100  C), pH values ,4, and little organic material. All known thermoacidophilicarchaea tend to grow best at an extremely low pH with growth temperatures of approximately 60  C (examples include Picrophilus and Thermoplasma sp.) or, alternatively, an optimal growth temperature .80  C, but a pH above 3 [192]. Thermophilic archaea belong to both the kingdoms of Euryarchaeota and Crenarchaeota. The best characterized of the crenarchaeal thermoacidophiles include the Sulfobales, in particular the genus Sulfolobus. S. solfataricusand S. acidocaldarius are used as models for studying the metabolism of archaea, as they can be grown in the laboratory and are well characterized at a genetic level (for review of their metabolism and genetic manipulations, see [193]). The order Thermoplasmatales comprises the thermoacidophilic Euryarchaeota, and includes Picrophilus, Thermoplasma and Thermogymnomonas spp., P. torridus and P. oshimae grow optimally at 60  C and at pH values of 0.7 or below, making them the most acid-tolerant microbes known [10].

6.6.2 Physiological adaptation to low pH The existence of mechanisms for cytoplasmic pH homeostasis and pH-sensing allows bacteria to exist outside the pH ranges at which their proteins are functional. The H 1 concentration is also important for cellular bioenergetics, as ATP synthesis requires that a pH gradient must be generated across the bacterial cell membrane to produce a protonmotive force [27]. For acidophiles, which have an alkaline cytosol in comparison to the acidic external environment,

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the pH gradient is maintained by active mechanisms, with the inside being positive relative to outside [194]. Microbes have different sensors that enable them to sense pH and regulate it. They can regulate the translation of proteins that remove H1 from the cytosol, regulate surface charges, adjust membrane lipids and porins, and alter membrane H1 permeability [27].

6.6.3 Food and medicinal relevance of acidophiles P. torridus and P. oshimae can produce heat- and acid-stable glucoamylases [87]. Thermoplasmaacidophilum has a glucoamylase with maximum activity at 75  C [86]. However, these have not been commercialized. The microbial community at Berkeley Pit Lake has undergone bioprospecting [195], and this environment was found to harbor microbes that produce a range of bioactive compounds based on berkelic acid, a compound is that produced by Penicillium rubrum and other Penicillium species [184]. The secondary metabolites produced generally inhibit pathways associated with low pH and high voltage potential (Eh). Berkelic acid, as well as berkeleydiones and triones, was found to inhibit matrix-metalloprotease 3 and caspase-1 [196,197]. Other metabolites had cytotoxic properties, e.g. berkeleyones A-C [198], berkazaphilones [199], and berkeleyacetals [200]. Berkeleyacetal C exerts anti-inflammatory effects by inhibiting IL-1 receptor-associated kinase 4 [201] and NF-κB, ERK1 /2 and IRF3 signaling in macrophages and neutrophils [202]. Some acidophiles can also be pathogenic to mammals. The intracellular acidophilic bacterium Coxiella burnetii, which causes the human disease Q fever, is an intracellular pathogen. It survives within an intracellular vesicle with a pH of 4 5 that contains lysosomal proteases, at it requires low pH to activate metabolism [203]. While the human stomach has a pH of 2 3, the human gastric pathogen Helicobacter pylori survives by secreting urease, which increases the pH of its immediate environment in the mucous layer [204].

6.7

Alkaliphiles

6.7.1 Habitats and diversity Microorganisms that could grow in alkaline media were recognized as long ago as the 1920s, but the initial publication of a paper describing enzymes from one of these microorganisms was by Horikoshi [205]. He described alkaliphiles as belonging to two main groups: the alkaliphiles and the haloalkaliphiles. The former need pH values of at least 9 for growth, and grow optimally around pH 10, whereas the latterneed both alkaline pH and high salinity (up to 33%w/v NaCl) [206]. Alkali-tolerant bacteria, including many Bacillus spp. isolated from soil can grow at pH 9, but have a growth optimum pH around pH 7. Their distribution and diversity are reviewed in [207]. A third category of alkaliphile exists: the halophilic alkalithermophile. These microorganisms were defined as growing optimally at or above Na1 concentrations of 1.7 M, pH $ 8.5, and temperatures $ 50  C [17]. Haloalkaliphiles are generally isolated from extremely alkaline saline environments, which include the Rift Valley lakes in eastern Africa, e.g. Lake Magadi in Kenya, and soda lakes in the United States and China [208]. The halophilic alkalithermophiles belonging to the order Natranaerobiales have been found in environments such as the hypersaline, alkaline lakes of the Wadi AnNatrun in Egypt (reviewed by [18]). The only archaeal halophilic alkalithermophiles known are part of the order Halobacteriales, which comprises both halophilic alkaliphiles and extreme alkaliphiles [19]. Halobacteriaceae require at least 1.5 M NaCl to grow [209]. Some alkaliphiles are methanogens, e.g. Methanohalophiluszhilinaeae [210].

6.7.2 Physiological adaptation to high pH In general, alkaliphiles are dependent on alkaline environments and the presence of Na1 ions for growth, sporulation and germination [211]. There are examples of some Bacillus strains that can use K1 ions instead of Na1, and their nutritional requirements can vary. However, one common feature is the need to create a cytoplasmic pH that is close to neutral (in common with acidophiles). This is accomplished by various mechanisms. One is cell wall composition, which in alkaliphilic Bacillus spp. comprises peptidoglycans resembling those of neutrophilic Bacillus spp., but is also rich in acidic polymers, e.g. teichuronic acid [212] and poly-γ-D-glutamic acid. Bacillus halodurans C-125 also uses a Na1/H1 antiporter system and H1 expulsion driven by an ATPase to export H1 from the cytoplasm. This reduces the need for utilization of H1 by transport systems. The low H1 concentration in the external medium poses difficulties for the creation of a transmembrane pH gradient for driving ATP production. The Δψ component of the electrochemical gradient is greater than that in neutrophiles, helping to maintain H 1 near the membrane’s outer surface and contributing to ATP production [213]. One explanation for efficient ATP production in conditions where both H 1 ions and

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oxygen were limiting was the presence of a cytochrome c-associated “H 1 capacitor mechanism” to adapt to high pH conditions [214]. The halophilic alkalithermophile Natranaerobius thermophilus acidifies its cytoplasm and makes use of multiple electrogenic Na1/K1/H1 antiporters to do so. As the extracellular pH for this microorganism increases in excess of the optimum, the electrogenic antiporters cease to operate and the cytoplasm is buffered, potentially by an acidic proteome [215]. In general, alkalithermophiles with pH optima in excess of 9.5 and temperature optima that exceed 65  C are scarce. The inverse correlation between pH tolerance and heat tolerance is likely due to the fact that survival in these extreme conditions requires particular cell wall and membrane adaptations to reduce permeability to H1 and cations in general [18].

6.7.3 Applications of alkaliphile enzymes The most common application of alkaliphile enzymes is as a component for biological laundry detergents. Alkaline proteases, cellulases and lipases are used in detergents [ 206,216,217]. Cyclodextrin glycosyltransferases, which belong to the α-amylase superfamily, convert starch and oligodextrins into α (1,4)-linked oligosaccharides, known as cyclodextrins. These can be used in foodstuffs, pharmaceuticals and chemicals. These are produced by various alkaliphilic Bacillus species: for reviews, see [218,219]. Their application in foods has been reviewed by [220].

6.8

Piezophiles

6.8.1 Habitats and diversity The deep oceans include habitats that are among the most extreme on Earth. The deep-sea organisms both eukaryotic and prokaryotic need to be resistant to multiple environmental extremes. The first challenge offered by life in deep water is pressure. The definition of a piezophile (also called a barophile) is that it can grow at high pressures, with optimal growth at pressures in excess of 40 MPa. Since the pressure of water increases by 0.1 MPa for every 10 m depth, this corresponds to a depth of about 4000 m. This approximates to the average depth of Earth’s oceans. However, some parts of the ocean have depths in excess of 10,000 m, such as Challenger Deep (10,984 m) in the Mariana Trench, where microorganisms are exposed to pressure of .100 MPa [221]. A further challenge for organisms surviving at these depths is temperature. The temperature at ocean depths below the thermocline is usually below 4  C, so that most piezophilic microorganisms are also psychrophilic (piezopsychrophiles). Microorganisms in the vicinity of ‘black smoker’ underwater vents can also be exposed to high temperatures, so that these microorganisms could be defined as piezothermophiles or piezohyperthermophiles. The majority of the piezophiles isolated to date have been identified as heterotrophic, gamma-Proteobacteria on the basis of phylogenetic characterization depending on the sequences of the 5S and 16S ribosomal RNA genes [222]. Predominant genera among these are Shewanella, Photobacterium, Colwellia, Psychromonas and Moritella [223]. There is also enormous diversity in microbial communities in different parts of the ocean, as shown by [224]. They revealed, using a parallel tag DNA sequencing strategy, that the bacterial communities of deep water in the North Atlantic Ocean and diffuse flow hydrothermal vents were 10 100 times more complex in their phylogenetic diversity than any other microbial communities studied previously. Ocean trenches (deeper than 6000 m) are distinct and form what is known as the hadopelagic zone, comprising 41% of the ocean depths [225]. A study of microbial diversity in the Mariana and Kermadec trenches, both of which exceed 10,000 m in depth, revealed that many species showed similarities to members of the same genera located in other locations, but most differences were due to differences in the gamma-Proteobacteria distributed throughout the water column [226]. Most piezophiles studied to date have been cold-adapted, but there are also thermophilic piezophiles from deepsea hydrothermal vents. According to [227], of the 52 known piezophilic and piezotolerant prokaryotes isolated at that time, only 15 species (4 Bacteria and 11 Archaea) were isolated from deep-sea hydrothermal vents. Most of these belonged to the order Thermococcales. One of the best characterized piezothermophiles is Thermococcus barophilus. Strain MPT, isolated from a hydrothermal vent location at a depth of 3550 m along the North Atlantic Ridge, was piezophilic at temperatures in excess of 75  C and was an obligate piezophile at temperatures between 95 100  C [12]. Pyrococcusyayanosii is the only obligate piezophilichyperthermophilic member of the Archaea found to date. This microorganism was first isolated from a deep-sea vent and has an optimal pressure for growth of 52 MPa [228].

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6.8.2 Physiological adaptation to high pressure Studies of some piezophilic microbes have revealed the nature of adaptations to high pressure at a molecular level, reviewed by [229 233]. The effects of high pressure on other microorganisms that live at or close to atmospheric pressure, e.g. E. coli, have also provided information about the effects of high hydrostatic pressure. Subjecting E. coli to high pressure ( . 100 MPa) leads to release of cytoplasmic contents, and the liquid-crystal membrane structure is converted to a gel as the acyl chains straighten [234]. This has led to the application of pressure to kill food-borne pathogens and food spoilage microorganisms [235]. Piezophiles adjust to high pressure by packing their lipid membranes tightly and increasing their content of unsaturated fatty acids. The synthesis of the long-chain polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) is common to many piezophilic microorganisms: for example, it is produced by Shewanella strains e.g. S. benthica and S. violacea [236], Photobacterium profundum and P. frigidiphilum [237], whereas Colwellia strains produce a different PUFA, docosahexaenoic acid [238]. The low lipid melting point assists in maintaining membrane fluidity. In general, all piezophilic and psychrophilic strains analyzed have 16:1 fatty acids [229]. There are numerous other adaptations in piezophiles. Their respiratory cytochromes in the cell membrane can be altered at high pressure, e.g. in the case of S. violacea DSS12, which contains less cytochrome c in the soluble fraction and more D-type cytochrome in the membrane fraction compared to non-piezophilic species [239]. This microorganism also contained an operon that was transcribed under conditions of high pressure [240]. Lauro and Bartlett [241] reported that there were long loops present in the 16 S rRNA gene in piezophilic Shewanella species, which became more frequent as the pressure increased. Analysis of the sequences of piezophile proteins was used to show the sequence adaptation parameters for pressure adaptation in proteins. Hydrophilic and polar amino acid groups discriminated better between psychrophilic piezophilic and mesophilic piezophilic groups, whereas hydrophobic and nonpolar amino acids discriminated better for thermophilic-piezophilic groups [242]. Thermophilic piezophiles adopt other strategies in response to pressure stress. Thermococcus barophilus accumulates mannosyl glycerate, a salinity stress response osmolyte in Thermococcales, in response to thermal stress. However, this microorganism displayed an increase in mannosyl glycerate accumulation in response to sub-optimal hydrostatic pressure, and this decreased when the pressure was decreased. This suggested that this organism’s proteome was pressure-sensitive [243]. A comparison of the transcriptome of this species with that of the piezosensitive T. kodakarensis showed differences in gene expression related to carbohydrate metabolism, energy conversion, and inorganic ion metabolism, as well as replication, recombination and repair [244]. Multi-omics analyzes comparing the Pyrococcusyayanosii genome to non-obligate piezophilic Pyrococcus species showed aromatic amino acid biosynthesis pathways to be absent, and there was an increase in constitutive expression of energy metabolism with hydrogenases, along with formate metabolism. Translation machinery showed differences, with an increase in transfer RNAs and in aminoacyl tRNA synthetases [228]. To date, the specifically piezophilic properties of these microorganisms have not been widely exploited in food or medicinal applications.

6.9

Radioresistant microorganisms

6.9.1 Diversity and survival strategy Radioresistant microorganisms exist in a wide range of environments. Among these are mountain ranges and open fields exposed to high ultraviolet radiation flux, and environments contaminated with radioactive material. They have molecular defenses against ionizing radiation that enables them to withstand exposures to ionizing radiation that would be lethal to most organisms. Most vertebrate animals are killed by a whole-body exposure to 10 Gy of radiation, and 200 Gy kills most bacteria [245] but some archaea, e.g. Pyrococcusfuriosus (which is also a hyperthermophile) and Halobacterium sp. NRC-1 survive exposures to about 3000 and 5000 Gy, respectively [246,247]. Within halophilic archaea, there was wide divergence in radioresistance: a study of exposure to simulated solar radiation showed that Halobacterium salinarum NRC-1 and Halococcusmorrhuae were far more resistant to radiation than Halococcushamelinensis [248]. However, the most robustly radioresistant organisms known are bacteria from the Deinococcus-Thermus group, the best studied of which is Deinococcusradiodurans. D. radiodurans strain R1 survives chronic radiation exposure of 60 Gy/h, greater than levels found in most spent decontamination waste from nuclear reactors, and acute exposure of 10,000 20,000 Gy [245,249]. The damage is caused by reactive oxygen species, which can react with both DNA and proteins. DNA can be damaged by double-strand breaks (DSBs), as well as single-strand breaks and base modifications e.g. thymine dimers. This organism, which is also resistant to dessication, oxidizing agents, and ultraviolet radiation, has efficient mechanisms for genome assembly and repair. D. radiodurans strain R1

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was the first extremely radioresistant organism that underwent genome sequencing [250], but these studies revealed that its DNA-repair proteins appeared to be unremarkable when compared to those of other microorganisms [251]. Studies of the transcriptome in response to ionizing radiation showed that many genes were induced, not all of which appeared to be relevant to DNA repair [252], and it was unclear which genes were most important [253]. Cells can be exposed to ionizing radiation from different sources, to one or multiple forms of radiation, acute exposure or chronic. Resistance mechanisms can varyand appear to go together with the resistance to desiccation found in microorganisms from arid habitats [254]. A new paradigm for radioresistance was introduced: accumulation of Mn21 complexes in these microbes lowers the intracellular ROS concentrations that increase as a result of radiation exposure. Proteins in irradiated microbes can undergo damage to disulfide bonds and aggregate. The Mn21 complexes can prevent protein oxidation, thereby allowing the microbes to retain enough capacity to repair the genomic DSBs and reconstitute the genome, ensuring survival [255]. A comparative study of D. radiodurans with E.coli revealed that the presence of low molecular weight ‘antioxidant’ Mn21 complexes with nitrogenous compounds and orthophosphate, rather than superoxide dismutase (SodA), in D. radiodurans protected it from radiation-induced reactive oxygen species (ROS). In contrast, the SodA-rich E.coli was susceptible to damage from ROS [256]. The role of Mn21 in protection against radiation damage appears to be supported by its role in protecting human tumors from destruction by ionizing radiation. A study of levels and spatial distribution of Mn21 in 7 different human tumor types revealed that the lowest levels of Mn were found in the most radiosensitive tumors, while the least radiosensitive had the highest levels, correlating with lower patient survival [257]. Tardigrades have another mechanism that protects against radiation damage. The genome of the tardigrade Ramazzottiusvarieornatus was sequenced. Along with increased expression of stress-related genes and selective loss of some stress-related pathways, one protein, Damage Suppressor (Dsup) was found only in this organism. This DNAbinding protein also protected cultured human cells from the effects of exposure to X-rays [258].

6.9.2 Defense against ultraviolet radiation: sunscreen molecules and their applications Radioresistant microorganisms, including cyanobacteria, can produce numerous primary and secondary metabolites that protect them against high UV radiation, which damages living cells. These molecules are under investigation, and in some cases in commercial use, as drugs and protectants against radiation-induced damage. UV-B rays in sunlight are the most cytotoxic and mutagenic for human skin, causing damage leading to cancer [259], whereas UV-A can cause skin ageing and generate ROS, which can also damage DNA. These have been reviewed by [260,261] and their function as cyanobacterial compounds has been reviewed by [262]. The best known of these photoprotective compounds are scytonemin and mycosporine-like amino acids (MAAs). Scytonemin is a small molecule (Mr 544), composed of a dimer of two polycyclic chromophores synthesized when two cyclopentanone and indole rings undergo condensation. It is made only by some extremophilic cyanobacteria, including the genera Anabaena, Calothrix, Chroococcus, Lyngbya, Scytonema and Nostoc [263]. This yellow-brown to red pigment comprises part of the cyanobacterial extracellular sheath, and absorbs near-UV and blue radiation, but not the higher wavelengths of light used for photosynthesis and can reduce inhibition of photosynthesis by UV-A radiation [264]. Its mode of action involves reducing the in vivo production of ROS, as well as that of thymine dimers in DNA [265]. MAAs have a similar function. These molecules are found in cyanobacteria but are also present in some rhodophyta and in some microalgae [263]. Several of these molecules are known, including palythine, palythene, palythinol, porphyra-334, shinorine and asterina. Like scytonemin, they can absorb ultraviolet light, but their mode of action is slightly different. They can prevent formation of pyrimidine dimers in DNA [260] but they enable the cells to dissipate energy as heat, without the production of ROS [266]. These molecules provide protection against UV-B radiation, depending on their structure and intracellular localization. If localized in the cytoplasm, this protection is limited, as only 10 26% of photons are absorbed [267] whereas this protection is greater if the pigment is in the cell membranes [268]. The anti-inflammatory properties of these compounds in response to UV radiation suggest that they may have potential skin anti-aging activity, as shown by their capacity to inhibit production of COX-1 mRNA [269]. The potential of these compounds for their inclusion in a range of sunscreen and other skincare products has been reviewed by [270]. The MAA porphyra-334 from the red alga Porphyraumbilicalis is an ingredient of a product called Helioguards 365, which is available from Mibelle AG Biochemistry and has been commercialized as an anti-ageing skin product that blocks skin exposure to UV-A radiation (https://mibellebiochemistry.com/products/helioguard-365/). The French company Gelyma has also produced a product from the same organism called Helionoris that protects skin from UV-A induced damage in a similar fashion (http://www.gelyma.com/helionori.html).

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6.10

Xerophiles: life with little or no water

Xerophiles are defined as microorganisms that grow at low water activity (aw) levels. These were defined by Pitt [271] as being below 0.85. The most halophilic prokaryotes can survive in saturated NaCl, which has an aw value of less than 0.755. However, the organisms that show the highest tolerance of low aw are xerophilic fungi, which can tolerate values of 0.65 to 0.605. Many of these xerophilic fungi can colonize dried foods and survive in high-sugar environments. They can balance their outside environment by accumulating internal solutes, which allow the cells to maintain a high enough osmotic pressure for survival and growth (a similar strategy to halophiles). The most xerophilic of these food-spoilage fungi known is Xeromycesbisporus. This is capable of growth at aw 0.61 0.62 [14] and has been found in various foods, including dried fruit, fruit cake, bakery goods and animal feed [272]. Stevenson et al. [273]. investigated a range of archaea, bacteria and fungi to see if there was a common water-activity limit for archaea, bacteria and eukarya, using high-salt media for prokaryotes and high-sugar media for the fungi. They determined that the theoretical minimum values for extremely halophilic archaea and bacteria were down to 0.611 aw, while for the fungi Aspergillus penicilloides and Xeromycesbisporus theoretical water-activity growth limits of mycelia were estimated to be 0.632 and 0.636, respectively. Hyperarid desert environments are also habitats for dessication-resistant microorganisms. These environments can also have extremes of temperature (both heat and cold) and radiation from sunlight. These environments can thus serve as ideal analogs for environments found outside Earth, in particular Mars. The coldest hyperarid desert environments on Earth are the McMurdo Dry Valleys in Antarctica [274] and as such, are a good model for Martian soils. Cryptoendolithic microorganisms are the main life forms found here, e.g. Cryomycesantarcticus CCFEE. This fungus can withstand long-term dessication, temperature extremes, and high levels of radiation [275]. It withstood Martian-like conditions, including 95% CO2 and UV radiation of wavelength ,200 nm [276,277]. Studies of the Atacama Desert in Chile, which is one of the driest places on Earth, showed that microbial communities were present and after sporadic rain, biomolecules indicative of active cells and replicating genomes could be detected [278].

6.11

Metallophiles

Some microorganisms can also withstand high environmental concentrations of metals. They are notable due to the fact that they can change the physiochemical behavior of the metals in their environment, and they can regulate their biochemistry and physiology in a way that enables them to overcome the toxic effects of the metals in their environment. This makes them ideal for performing bioremediation of metal-contaminated environments [279]. This also makes them excellent candidates for bioremediating contaminants to make usable mineral materials, including nanoparticles. There are multiple mechanisms that facilitate this. Ralstonia sp. CH34 is a Gram-negative bacterium that detoxifies metal ions using inducible ion efflux systems that enable the cell to export toxic ions [280]. Ralstoniametallodurans contains a megaplasmid that contains metal resistance genes [281]. Some metallophiles are also polyextremophiles, as they are also acidophilic. This includes Ferroplasma acidophilum [15], which participates in the iron cycle of acid metal-rich environments like the Rı´o Tinto [282]. This archaeon gains energy by oxidizing ferrous iron and fixes CO2, and many of its proteins are iron-metalloproteins [283].

6.12

Conclusions

Extremophiles represent a vast resource of molecules and organisms that can be exploited for innumerable applications. Some produce enzymes that contribute to food processing, detergent formulation, biosensors, clothes manufacture, diagnostics, while others produce an array of compatible solutes and small molecules that show potential as drugs. However, their importance transcends their industrial and medical utility. They extend human knowledge about the limits of macromolecular stability and the physiochemical parameters of life. We now understand more about how living organisms can survive multiple stressors, and how adaptations that allow survival under one stress condition can also aid with survival in others. The genomic revolution has revealed more detail of the molecular foundations of these properties. Similarly, the study of extremophiles, particularly that of thermophilic archaea and chemotrophs, has expanded the understanding of the variability of energy-yielding processes that can exist. Finally, the study of extremophiles makes a valuable contribution to the developing science of astrobiology, whereby understanding how life can survive on Earth informs the ability to imagine and locate potential life in other parts of our own Solar System and beyond. Almost every imaginable habitat on Earth harbors these remarkable and resilient microorganisms. We still have a great deal to learn.

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Oxford, U.K: Eolss Publishers Co Ltd; 2004. p. 231 55. [231] Simonato F, Campanaro S, Lauro FM, Vezzi A, D’Angelo M, Vitulo N, et al. Piezophilic adaptation: a genomic point of view. J Biotechnol 2006;126:11 25. [232] Abe F. Exploration of the effects of high hydrostatic pressure on microbial growth, physiology and survival: perspectives from piezophysiology. Biosci Biotechnol Biochem 2007;71:2347 57. [233] Oger PM, Jebbar M. The many ways of coping with pressure. Res Microbiol 2010;161:799 809. [234] Braganza LF, Worcester DL. Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure. Biochemistry 1986;25:7484 8. [235] Considine KM, Kelly AL, Fitzgerald GF, Hill C, Sleator RD. High-pressure processing--effects on microbial food safety and food quality. FEMS Microbiol Lett 2008;281:1 9. [236] Fang J, Chan O, Kato C, Sato T, Peeples T, Niggemeyer K. Phospholipid FA of piezophilic bacteria from the deep sea. Lipids 2003;38:885 7. [237] Nogi Y, Masui N, Kato C. Photobacterium profundum sp. nov, a new, moderately barophilic bacterial species isolated from a deep-sea sediment. Extremophiles 1998;2:1 7. [238] Bowman JP, Gosink JJ, McCammon SA, Lewis TE, Nichols DS, Nichols PD, et al. Colwelliademingiae sp. nov., Colwelliahornerae sp. nov., Colwelliarossensis sp. nov. and Colwelliapsychrotropica sp. nov.: psychrophilic Antarctic species with the ability to synthesize docosahexaenoic acid (22: ω63). Int J Syst Evol Microbiol 1998;48:1171 80. [239] Tamegai H, Kato C. Pressure-regulated respiratory system in barotolerant bacterium, Shewanellasp. strain DSS12. J Biochem Mol Biol Biophys 1998;1:213 20. [240] Nakasone K, Ikegami A, Kato C, Usami R, Horikoshi K. Mechanisms of gene expression controlled by pressure in deep-sea microorganisms. Extremophiles 1998;2:149 54. [241] Lauro FM, Bartlett DH. Prokaryotic lifestyles in deep sea habitats. Extremophiles 2008;12:15 25. [242] Nath A, Subbiah K. Insights into the molecular basis of piezophilic adaptation: extraction of piezophilic signatures. J Theor Biol 2016;390:117 26. [243] Cario A, Jebbar M, Thiel A, Kervarec N, Oger PM. Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus. Sci Rep 2016;6:29483. Available from: https://doi.org/10.1038/ srep29483. [244] Vannier P, Michoud G, Oger P, Marteinsson VP, Jebbar M. Genome expression of Thermococcus barophilus and Thermococcus kodakarensisin response to different hydrostatic pressure conditions. Res Microbiol 2015;166:717 25. [245] Gogada R, Singh SS, Lunavat SK, Pamarthi MM, Rodrigue A, Vadivelu B, et al. Engineered Deinococcusradiodurans R1 with NiCoT genes for bioremoval of trace cobalt from spent decontamination solutions of nuclear power reactors. Appl Microbiol Biotechnol 2015;99:9203 13. [246] Di Ruggiero J, Santangelo N, Nakerdien Z, Robb F. Repair of extensive ionizing-radiation DNA damage at 95 C in the hyperthermophilic archaeon Pyrococcusfuriosus. J Bacteriol 1997;179:4643 5. [247] Kottemann M, Kish A, Iloanusi C, Bjork S, Di Ruggiero J. Physiological responses of the halophilic archaeon Halobacterium sp. strain NRC1 to desiccation and gamma irradiation. Extremophiles 2005;9:219 27. [248] Leuko S, Domingos C, Parpart A, Reitz G, Rettberg P. The survival and resistance of Halobacterium salinarum NRC-1, Halococcushamelinensis, and Halococcusmorrhuae to simulated outer space solar radiation. Astrobiology 2015;15:987 97. [249] Cox MM, Battista JR. Deinococcusradiodurans the consummate survivor. Nat Rev Microbiol 2005;3:882 92. [250] White O, Eisen JA, Heidelberg JF, Hickey EK, Peterson JD, Dodson RJ, et al. Genome sequence of the radioresistant bacterium Deinococcusradiodurans R1. Science 1999;286:1571 7. [251] Makarova KS, Aravind L, Wolf YI, Tatusov RL, Minton KW, Koonin EV, et al. Genome of the extremely radiation-resistant bacterium Deinococcusradiodurans viewed from the perspective of comparative genomics. Microbiol Mol Biol Rev 2001;65:44 79. [252] Liu Y, Zhou J, Omelchenko MV, Beliaev AS, Venkateswaran A, Stair J, et al. Transcriptome dynamics of Deinococcusradiodurans recovering from ionizing radiation. Proc Natl Acad Sci USA 2003;100:4191 6. [253] Makarova KS, Omelchenko MV, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, et al. Deinococcusgeothermalis: the pool of extreme radiation resistance genes shrinks. PLoS One 2007;2:e955. [254] Shuryak I. Review of microbial resistance to chronic ionizing radiation exposure under environmental conditions. J Env Radioact 2019;196:50 63. [255] Daly MJ. A new perspective on radiation resistance based on Deinococcusradiodurans. Nat Rev Microbiol 2009;7:237 45.

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Radiation-resistant extremophiles and their potential in biotechnology and therapeutics. Appl Microbiol Biotechnol 2013;97:993 1004. [262] Singh R, Parihar P, Singh M, Bajguz A, Kumar J, Singh S, et al. Uncovering potential applications of cyanobacteria and algal metabolites in biology, agriculture and medicine: current status and future prospects. Front Microbiol 2017. Available from: https://doi.org/10.3389/ fmicb.2017.00515. [263] Rastogi RP, Sonani RR, Madamwar D. Cyanobacterial sunscreen scytonemin: role in photoprotection and biomedical research. Appl Biochem Biotechnol 2015;176:1551 63. [264] Gao Q, Garcia-Pichel F. Microbial ultraviolet sunscreens. Nat Rev Microbiol 2011;9:791 802. [265] Rastogi RP, Sonani RR, Madamwar D. The high-energy radiation protectant extracellular sheath pigment scytonemin and its reduced counterpart in the cyanobacterium Scytonema sp. R77DM. Bioresour Technol 2014;171:396 400. [266] Conde FR, Churio MS, Previtali CM. The deactivation pathways of the excited-states of the mycosporine-like amino acids shinorine and porphyra-334 in aqueous solution. Photochem Photobiol Sci 2004;3:960 7. [267] Garcia-Pichel F, Castenholz RW. Occurrence of UV-absorbing, mycosporine-like compounds among cyanobacterial isolates and an estimate of their screening capacity. Appl Env Microbiol 1993;59:163 9. [268] Bo¨hm GA, Pfleiderer W, Bo¨ger P, Scherer S. Structure of a novel oligosaccharide-mycosporine-amino acid ultraviolet A/B sunscreen pigment from the terrestrial cyanobacterium Nostoc commune. J Biol Chem 1995;270:8536 9. [269] Suh SS, Hwang J, Park M, Seo HH, Kim HS, Lee JH, et al. Anti-inflammation activities of mycosporine-like amino acids (MAAs) in response to UV radiation suggest potential anti-skin aging activity. Mar Drugs 2014;12:5174 87. [270] Derikvand P, Llewellyn CA, Purton S. Cyanobacterial metabolites as a source of sunscreens and moisturizers: a comparison with current synthetic compounds. Eur J Phycol 2016;52. Available from: https://doi.org/10.1080/09670262.2016.1214882. [271] Pitt JI. Xerophilic fungi and the spoilage of foods of plant origin. In: Duckworth RB, editor. Water relations of foods. 1st ed. New York, USA: Academic Press; 1975. p. 273 307. [272] Leong SL, VinnerePettersson O, Rice T, Hocking AD, Schnu¨rer J. The extreme xerophilic mould Xeromycesbisporus growth and competition at various water activities. Int J Food Microbiol 2011;145:57 63. [273] Stevenson A, Cray JA, Williams JP, Santos R, Sahay R, Neuenkirchen N. Is there a common water-activity limit for the three domains of life? ISME J 2015;9:1333 51. [274] Cowan DA, Makhalanyane TP, Dennis PG, Hopkins DW. Microbial ecology and biogeochemistry of continental Antarctic soils. Front Microbiol 2014;5:154. Available from: https://doi.org/10.3389/fmicb.2014.00154. [275] Onofri S, de la Torre R, de Vera JP, Ott S, Zucconi L, Selbmann L, et al. 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Berlin, Heidelberg, Germany: Springer; 2013. p. 101 18. [280] Nies DH. Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp. CH34. Extremophiles 2000;4:77 82. [281] Mergeay M, Monchy S, Vallaeys T, Auquier V, Benotmane A, Bertin P, et al. Ralstoniametallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. FEMS Microbiol Rev 2003;27:385 410. [282] Gonza´lez-Toril E, Llobet-Brossa E, Casamayor EO, Amann R, Amils R. Microbial ecology of an extreme acidic environment, the Tinto River. Appl Env Microbiol 2003;69:4853 65. [283] Ferrer M, Golyshina OV, Beloqui A, Golyshin PN, Timmis KN. The cellular machinery of Ferroplasmaacidiphilum is iron-protein-dominated. Nature 2007;445:91 4.

Chapter 7

Applications of extremophiles in astrobiology Rebecca S. Thombre1,2, Parag A. Vaishampayan3 and Felipe Gomez4 1

Department of Biotechnology, Modern College of Arts, Science and Commerce, Pune, India, 2School of Physical Sciences, University of Kent,

Canterbury, United Kingdom, 3Biotechnology and Planetary Protection Group, NASA-Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, United States, 4Instituto Nacional de Te´cnica Aeroespacial (INTA)
Centro de Astrobiologı´a (CAB), Madrid, Spain

7.1

Introduction and historical background

The interdisciplinary field of Astrobiology studies the origin, evolution, distribution and future of life in the Universe [1]. It revolves around three fundamental questions: 1. Where do we come from? (How did life begin?) 2. Are we alone in the Universe? (Is there life beyond Earth?) 3. Where are we going? (What is the future of life on Earth?) These questions form the very backbone of Astrobiology. The field of Astrobiology research is interdisciplinary, involving Planetary scientists, microbiologist, geologist, molecular biologist, bioinformaticians, geochemists, astrophysicists, astronomers, chemists and even artists and philosophers [1]. The possibility of life in outer space had been a well debated topic for many centuries. The most noteworthy example, which may be the earliest records of astrobiology, is found in the beliefs of the Italian Dominican Friar, Giordano Bruno (1548
1600). Bruno believed that planets in outer space might foster life of their own (Cosmic pluralism) [2]. The term “Astrobiology” was first used by in 1953 by Gavriil Adrianovich Tikhov, an astronomer of Russian origin [2]. Astrobiology gained momentum after the Space age was ushered due to the launch of the first artificial satellite Sputnik in 1957. In 1960, Nobel laureate Joshua Lederberg discussed about ‘Exo-biology’ and advocated the research for the quest of life in the universe [3]. Lederberg collaborated with well-known astronomer Carl Sagan to establish ‘Exo-biology’ as a scientific discipline at National Aeronautics and Space Administration (NASA). The NASA Exobiology efforts continued after Richard (Dick) Young spearheaded the Exobiology (“Planetary Biology”) Program at NASA Headquarters in 1967 where the then focus was on the Vikings Mission to Mars [4,5]. The consecutive years saw the establishment of the International Society for the Study of the Origin of Life (ISSOL) and the NASA Exobiology program [5]. The discovery of microfossil like organic compounds in AH 84001 (Allan Hills 84001) a meteorite from Mars in 1996 further propelled the interest in Astrobiology [6]. Until the late 1990s the term ‘Exo-biology’ was more prevalent and used more often with Planetary Biology. Later, in 1998, NASA developed a dedicated institute called the NASA Astrobiology Institute (NAI) that promoted Astrobiology and ensured that Astrobiology objectives and goals formed an integral part of NASAs Space missions [1]. The first curator of NAI, Janet Morrison mentions that Lawrence J. Lafleur of Brooklyn College had written an article on “Astrobiology” in 1941 which may have been the earliest record of Astrobiology [6]. However, the term ‘Astrobiology’ became more prevalent and popular after the official establishment of NAI. During the same period in 1996, the European Space Agency (ESA) established an ESA Exobiology team to study the scope, nature and future of Exobiology and “Search for Life” in Solar system [7]. The European astrobiology community formed the European Astrobiology Network Association (EANA) in 2001 for interaction for the promotion of Astrobiology in Europe [8].

Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00007-1 © 2020 Elsevier Inc. All rights reserved.

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Currently, Astrobiology has developed into an amalgamation of interdisciplinary fields that encompass thrust areas like the quest for life and habitable zones in the Solar system, search for prebiotic life or evidences of prebiotic chemistry, studies related to evolutionary theories of origin of life like panspermia, RNA world hypothesis, abiogenesis, etc. Many philosophers and social scientists also study astrobiology in order to understand the impact it has on society and its ethical implications.

7.2

Study of extremophiles in astrobiology

One of the primary objectives of Astrobiology is to search for life on planets beyond Earth. Often the search for signs of life involves studying the habitability of planets and moons in the “Goldilock zones” or habitable zones. Such celestial bodies are the red planet Mars, Venus, Jovian moon Europa and Saturnian icy moons like Enceladus and Titan. To detect the presence of life in outer space, we need to first define the dynamic limits of survival of organismic life. Outer space has extremely harsh and inhabitable environments like extreme radiation, extreme temperatures, altered gravity and extreme salinity and nutrients. These conditions seem deleterious for the growth of life in outer space [9,10]. However, microbial life can be found inhabiting extreme inhospitable habitats on Earth like arid deserts, hot springs, deep sea hydrothermal vents, Ice covered lakes in Antarctica, Glaciers, halite crystals, sub-surface caves, rocks, acid-mine drainages, alkaline lakes and saline systems [11]. The discovery of Thermus aquaticus, the first ever extremophile, from Yellowstone National Park in the United States [12] paved way for exploring the habitability of similar extreme environments on Earth. Since then, numerous expeditions have been conducted to study the ecology, diversity and adaptations of extremophiles to stress. Extremophiles are found in all three domains of life viz. Archaea, Prokarya and Eukarya. However, major group of extremophiles belong to Archaea. By virtue of their adaptability, extremophiles thrive in such harsh environments e.g. soda lakes, deserts, acid mine waters, glaciers, etc. They are classified based on their characteristics as thermophiles (grow optimally above 55  C); radiation resistant microbes (organisms resistant to ionizing radiations); desiccation resistant bacteria; oligophiles (organisms that survive in low concentration of nutrients); alkaliphiles (optimally grow above pH 9.0); halophiles (optimally growing above 15% NaCl), acidophiles (optimally grow at pH 1.8
2.0) and many others like psychrophiles (survive in low temperatures), osmophiles (tolerant to high osmotic pressure), barophiles or peizophiles (survive at pressure more than 0.1 MPa) and metallophiles (surviving in presence of high concentration of metal). The physiological characteristics of extremophiles contribute to their biotechnological and industrial applications in production of enzymes, metabolites and biomolecules. To combat the extreme or harsh environment, they synthesize certain compounds e.g. glycine betaine in halophiles, heat shock proteins in thermophiles, polyamines in acidophiles, cold adapted enzymes in psychrophiles and biomaterials like exopolysaccharide in alkaliphiles. The biotechnological potential of extremophiles is enormous. They also contribute to environmental management e.g. bioremediation of saline, alkaline, acidic wastewaters. The exploration of extremophiles on Earth is critical in understanding their adaptation mechanisms and helps in identifying novel bio-signatures that can be used in habitable zones beyond Earth. Most of the ecological habitats of extremophiles on Earth have resemblance to planetary bodies in outer space in terms of bio-geochemistry, nutrient composition or topological similarities. These sites don’t completely resemble planetary bodies like Mars or Europa but mimic a particular character(s), condition or environmental parameter [13]. These sites are referred to as Planetary Field analogue sites (PFA) or terrestrial analogues. Studying the survival and microbial diversity of Planetary Field analogue sites provides valuable insights in understanding the evolution of life and the habitability beyond Earth [14]. The major application of these extremophiles from PFA sites and other extreme environments is its use as candidate organisms for exposure to outer space, LEO (Low Earth Orbit) or International Space Station (ISS). Numerous Astrobiology related initiatives, projects and programs were launched to study life in extreme environments. A report on “International Earth-based research program as a stepping stone for global space exploration— Earth-X” was prepared by the Committee on Space Research (COSPAR) and Panel on Exploration (PEX) in association with the European Science Foundation (ESF) [15]. The objective of this report was to support Analogue research as an international endeavor encouraging collaborations as the PFA sites are distributed around the world. A new initiative called ‘Investigating Life in Extreme Environments’ (ILEE) was launched by ESF in 2003. The main aim of this activity was to develop a coordinated, interdisciplinary approach to enhance the opportunities for studying and funding research on “Life in extreme environments” (LEXEN). Similarly, “CAREX”
the coordination action for research on study of life in extreme environments (CAREX) was one of the Seventh Framework Programme (FP7) funded by European Commission (EC) and coordinated by British Antarctic Survey and the Natural Environment Research Council (NERC). The goals of this program was studying responses, adaptation, biodiversity, bioenergetics and evolution life in extreme environments (LEXEN). The resulting roadmap for this European research on LEXEN became the basis and foundation for organizations such as COSPAR, EC, NAI and Japan Agency for Marine-Earth Science and Technology (JAMSTEC) [16]. Similar activities are warranted in India to develop a coordinated

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FIGURE 7.1 Planetary field analog sites in India. (A) Lonar lake, India, (B) salt pans from Mumbai, (C) mud volcano from Baratang, Andaman, (D) Nun Kun Massif, Zanskar, Ladakh.

program for the identification, characterization and conservation of extreme sites important for Astrobiological studies. These sites can serve as PFA sites for geological studies or testing rovers. These sites also can be used to study the presence of extreme microbial life and isolation of such candidate ‘exo-philes’ for Astrobiology studies.

7.3

Planetary field analogue sites in India and its extremophilic microbial diversity

7.3.1 Lonar lake The Lonar Crater (Latitude 19 580 , Longitude 76 360 ) is considered to be a geological and ecological enigma. It is a bowl-shaped crater with 1.83 km diameter and 130 m depth from crater rim to the water level in the lake at the center of the depression. The crater is located in Buldhana district of Maharashtra, India in the Deccan Volcanic Province [Fig. 7.1A]. The crater is believed to be formed due to meteorite impact nearly 50 thousand year ago [17
19]. Lonar lake is the only alkaline lake formed in a meteorite impact crater in basalt rock [19]. This unique basaltic rock meteorite impact crater has been considered comparable to Martian craters and hence it makes it an ideal PFA site for Martian craters [19
21]. An exhaustive study on identification of aerobic microbial diversity of Lonar lake has been performed using culturedependent and independent approach [18,19]. The extremophilic organisms isolated were mostly alkaliphiles, haloalkaliphiles and alkalitolerant like bacteria of the genera Bacillus, Arthrobacter, Micrococcus, Planococcus, Dietzia, Vagococcus, Exigobacterium, Alcaligenes, Stenotrophomonas and Paracoccus. Haloalkaliphiles related to Halomonas sp. have also been reported. Amongst other bacteria isolated were Cellulosimicrobium sp., Alkalibacillus sp. (an obligately haloalkaliphilic genus) and Thermotogales sp. (an anaerobic thermophile). Other novel organisms identified were Alkalimonas delamerensis, Halomonas campisalis, Marinobacter excellens, Marinobacter alkaliphilus, Rhodobaca bogoriensis, Roseinatronobacter monicus, and Providencia rustigianii, Rhodobacteriaceae bacterium and b-subdivision with Stenotrophomonas. Most of these organisms are obligate alkaliphiles. Phylogenetic results indicate the presence of Gram-negative bacteria related to the γ3 subdivision of Proteobacteria, Gram-positive isolates of High % G 1 C and Low % G 1 C divisions, with the majority of bacteria belonging to the Bacillus group. Several isolates representing different genera belonging to the phyla ‘Firmicutes’ (seven genera), ‘Proteobacteria’ (five genera) and ‘Actinobacteria’ (four genera) have been isolated from Lonar Lake [18]. The extremophiles from Lonar lake have been explored for numerous biotechnological applications.

7.3.2 Extremophiles from rocks, seawater and intertidal sea zones in arabian sea In recent studies, inter-tidal zones have been considered as unique terrestrial analogue sites compared to the ancient lake systems at Gale crater on Mars using various comparative parameters [22]. The parameters used to compare the inter-tidal zones as analogues for fluvio-lacustrine systems at Gale crater are pH, salinity, mineralogy, stratification, etc. [22]. Many areas along the Indian coastline, have intertidal zones and pristine habitats. These zones along the Arabian sea have salinity between 2 to 3.5%. This is comparable to salinity proposed for the ancient Martian lake system (1
2%) [22,23]. The pH is circumneutral [22] that is comparable to proposed pH of aqueous environment at Gale crater [24]. The pristine habitats lining the Arabian sea have potential sites that can be used as a model analogues sites to

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study plausibility of putative lifeforms in the fluvio-lacustrine systems on early Mars. Though there are few limitations in such analogue studies; these PFA sites help in gaining useful insights in the microbial communities thriving in environments exposed to cyclic dryness [22]. The eastern side of the Arabian sea is demarcated by the Laccadive sea lining the Western coast (Konkan coast) of India. The salinity variability in Arabian sea depends on the temporal variability, reversing monsoons, fresh water input, surface processes and ocean- atmosphere interactions at specific locations [25]. The diversity of organisms in the deep sea samples from Western side of Arabian sea have been studied previously [26]. The sites used as analogues were the intertidal zones at the coast of Chivla (16.07N, 73.45 E) and Malvan (16.07N, 73.47 E) lining the Arabian sea. The metagenomic studies of the16S rRNA gene sequences from the Arabian sea sites (Chivla and Malvan) have been studied and are depicted using abundance heatmaps from phylum, genus and species level in Fig. 7.2A
C. The heatmap depicts (Fig. 7.2A) that the most abundant taxon in Arabian seawater samples belong to phylum Firmicutes, Actinobacteria, Proteobacteria, and Cyanobacteria. Extremophilic microbial members belonging to the genus Halomonas, Marinobacter, Alcanivorax and Pseudoalteromonas were abundant in the seawater of Malvan (Fig. 7.2B). The Krona chart (Fig. 7.3A,B) depicts the relative abundance of microbial diversity from phylum to species level.

FIGURE 7.2 Heatmap showing the microbial diversity and relative abundance of bacterial taxonomies distributed among Malvan and Chivla based on PCR amplifications and high-throughput Illumina sequencing of 16 S rRNA gene sequences. (A) Comparison of reads between two samples at phylum level, (B) genus level heatmap illustrates top 20 abundance hits, (C) the plot shows the species differences between two samples. Only well annotated known taxonomy hits from Malvan and Chivla were considered for abundance comparison. The colors depict the extent of abundance in the sample as indicated in the legend. In gradient scale, the less abundant reads for particular taxon in a given metagenome are represented in green, the most abundant taxa are shown in red and the null read counts are represented in black color.

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FIGURE 7.3 (A) Krona plot depicting the overall taxonomy distribution among Arabian sea metagenome. Taxonomy abundance of Malvan sea water sample from phylum to species level. (B) Krona plot depicting the overall taxonomy distribution among Arabian sea metagenome. Taxonomy abundance of sample Chivla Sea water sample from phylum to species level. (C) Relative abundance of microorganisms from Arabian sea at species level. The 16S rRNA sequences of Malvan and Chivla seawater samples were assigned to the taxonomy using QIIME, Greengenes and SILVA 16S database. Taxa representing high abundance (top 10) among samples are shown in the bar plot, while remaining taxa are grouped in the ‘other’ category. (D) Relative abundance of microorganisms from Arabian sea at phylum level. The 16S rRNA sequences of Malvan and Chivla seawater samples were assigned to the taxonomy using QIIME, Greengenes and SILVA 16S database. Taxa representing high abundance (top 10) among samples are shown in the bar plot, while remaining taxa are grouped in the ‘other’ category.

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The extremophile, Alcanivorax venustensis was the most abundant bacteria at species level in Chivla seawater while the metagenomic datasets at species level from Malvan seawater were recruited to hitherto ‘unknown’ groups (Fig. 7.3C,D). Besides intertidal zones and deep sea samples, many rocks from pristine habitats in the Arabian sea have been explored for isolation of epilithic extremophiles. These rocks are important niche habitats and can be used in studying ‘Lithopanspermia’. Lithopanspermia is a category of panspermia theory where rock fragments act as vehicles of dispersal between planets [27]. These rocks are ejected from planets during meteorite impact and this rock can travel through space to other planets or moons [28]. It is perused that extremely resistant organisms or spores can be protectively encased in the rocks and these rocks could seed them in other moons or planets [29]. Thus studying the isolation and survival of extremophiles from rocks is imperative for conducting Lithopanspermia experiments. Scientist have previously studied the isolation of extremophiles from ancient rocks and its significance in astrobiology [30]. Extremophilic haloarchaea have been previously isolated from rock salt that is 195
250 million years old [30]. Similarly, Norton et al., 1993 have isolated Haloarcula, Halorubum and Halobacterium from deposits of Permian and Triassic era from British salt mines [31]. Denner et al. (1994) have reported the isolation of Halococcus salfodinae from ancient rock salt [32]. Halococcus dombrowskii sp. nov., an extreme halophilic archaeon has been isolated from Permo-Triassic alpine salt deposits [33]. In India, rocks from Deccan traps are well known for the presence of fossils trapped within them and are of archeological significance. Laterite rocks are commonly found in the Deccan trap area that erupted B65 million years ago [34]. The rocks submerged in Arabian sea as well as rocks from Deccan trap can be used for isolation of extremophiles of Astrobiological significance. An endolithic extremophile Natronococcus jeotgali RR17 has been reported from a laterite rock from Arabian sea (Vengurla Port) in India [9]. N. jeotgali RR17 is a haloalkaliphile that survives in medium containing upto 20% NaCl and 10% sodium carbonate. Another extremophile Halovivax asiaticus RT-5 has been reported to be isolated from laterite rock submerged in Arabian sea. This rock isolate is an extreme halophilic archaeon that grows in 25% salinity. These extremophiles were used to study the effect of space related stress on their survival [9,10]. N. jeotgali RR17 could survive in a` hypergravity stress of 223 3 g [9]. Similarly, Haloarcula argentinensis isolated from salterns could survived in microgravity environment of upto 1023 3 g [10]. Survival in altered gravity and hyperacceleration is an indispensable requirement to endure interplanetary transport in asteroids or rocks, which is imperative in litho-panspermia [35
37]. Besides lithopanspermia, survival in hypergravity is important as brown dwarfs and some sub-stellar objects have hypergravity in the range of 10
102 3 g while Jupiter has hypergravity of 2.35
2.5 3 g [9,38]. The studies related to survival of microorganisms in altered gravity, hypergravity and microgravity are imperative in understanding the fundamental questions related to dynamic limits of life [37
39]. These extreme halophiles isolated from such PFA environments can thus be used as candidate organisms or ‘exophiles’ in Astrobiology studies [40].

7.3.3 Salt deposits and saline systems in Rajasthan, Gujarat and Maharashtra Saline systems comprise of solar salterns, salt mines, salt lakes and halite deposits in caves and deserts [41]. Extremophilic halophilic archaea like Haloarcula argentinensis, Haloarcula marismortui, Haloferax alexandrines and Haloferax prahovense have been previously reported from salterns in Mumbai, India [41] (Fig. 7.1B). Prior studies report that haloarchaea can survive in fluid inclusions in halite crystals [31,33]. Infact, archaea and bacteria have been reported to be isolated from halite crystals that are 121 Ma, 112 Ma and 250 Myr [42,43]. The studies are important in Martian Astrobiology. As microbial life could be revived from ancient halite crystals on Earth, similar studies could be used to detect the evidence of life in halite deposits on Mars. It will be halophilic, or halotolerant strains which are likely to be able to be persevered in halite fluid inclusions. The Martian meteorite Nakhla has the presence of Halite in association with siderite and anhydrite [44]. Similarly, halite deposits have been identified at Meridiani Planum region in Mars [45]. The Mars Odyssey orbiter has identified chloride salt deposits of mid Noachian and Hesperian age in the southern highlands of Mars [46]. Hypersaline lakes and environments on Earth can serve as unique terrestrial analogues for halite deposits and sulfate-rich paleolakes on Mars [47]. A compilation of hypersaline environments in India and their representative extremophilic diversity is depicted in Table 7.1. Hypersaline environments rich in NaCl and magnesium salts are useful model systems for the study of life on Mars. The study of analogue environments is important for the study of extinct/extant indigenous life on Mars. This study is also imperative in planetary protection as it helps in studying the potential for contamination [63].

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TABLE 7.1 Representative extremophiles isolated from Hypersaline environments in India. No

Hypersaline

Location

Representative Genera

References

Haloarcula argentinensis, Haloarcula marismortui, Haloferax alexandrines and Haloferax prahovense Halococcus, Halorubrum, Haloarcula and Haloferax Haloferax, Halorubrum, Haloarcula, Halobacterium and Halogeometricum Geomicrobium halophilum Halobacillus kuroshimensis Virgibacillus halodenitrificans Chromohalobacter israelensis Natronobacterium sp. Geomicrobium halophilum Bacillus, Virgibacillus, Rummelibacillus, Alkalibacillus and Halobacillus Shewanella chilikensis sp. nov Streptomyces chilikensis sp. nov. Halobacillus trueperi, Shewanella algae, Halomonas venusta, Marinomonas sp. SS8

[41,48
50]

Environment 1

Solar Saltern

Mumbai

2

Solar salterns

Goa

3

Solar salters

Tuticorin, Tamilnadu

4

Sea water, Solar salterns and Saline soil

Veraval, Triveni Sangam, Bhavnagar, Khambat in Gujarat

5

Sambar salt lake

Rajasthan

6

Pulicat lake

Andhra Pradesh

7

Chilika lake

Orissa

8

Lunsu lake

Himachal Pradesh

[51
53] [54,55]

[56
58]

[59] [60] [61] [62]

7.3.4 Mud volcanoes of Andaman Mud volcanoes are typical geologic structures that are mixed with fluid, fine grain sediments, rocks or consolidated mud that are expelled from earth’s surface due to various reasons like emission of methane. In 2003, a significant eruption event caused the formation of more than 1000 mud volcanoes in the Island of Baratang in Andaman (Fig. 7.1C) [64]. Methanotrophic bacteria and archaea are dominant in soil from mud volcanoes [64]. The organisms, Cesiribacter andamanensis gen. nov., sp. nov., and Lutibaculum baratangense have been isolated from soil samples of mud volcanoes in Andamans [65,66]. The mud volcanoes from Andaman can be used as potential analogues for mud volcanoes on Mars. Mud volcanoes in Trinidad have been studied as astrobiological analogs for Martian environments [67]. Mud volcanism is pervasive in the Martian regions of Acidalia Planitia on Mars. There are more than 18,000 circular mounds in those areas which is indicative of mud volcanism [68]. Martian regions like Utopia, Isidis, Northern Borealis, Scandia and Firsoff crater of Arabia Terra have also been reported for the presence of mud volcanoes [69,70]. Mud volcanism in the Andamans can be compared as a terrestrial analogue to Martian mud volcanisms and extremophiles from these sites and serve as potential candidates for studying effect of simulated Martian environments on micro-organisms.

7.3.5 Geothermal hotsprings, cold deserts and glaciers in Leh Ladakh, Himalayas The Himalayan regions of Leh Ladakh are located in Jammu Kashmir in India. The Ladakh terrain encompasses astrobiologically important environments like cold deserts, hot springs, circumneutral springs, gullies, craters, glacial sediments, and permafrost regions (Fig. 7.1d). A comparative and morphological analysis of gullies and craters on Mars reveals that sites in Ladakh are potential terrestrial analogues of Martian craters Domoni and Maricourt [71]. Leh Ladakh is a cold desert and numerous studies have been conducted on isolation and identification of extremophilic bacteria from regions like Pangong and Chumathang lake, Indus and Zanskar River confluence and Khardungla Pass [72]. The microbial diversity studies of these areas revealed the presence of genera like Pseudomonas, Aurantimonas, Citricoccus, Cellulosimicrobium, Brevundimonas, Desemzia, Psychrobacter, Sporosarcina, Sinobaca, Stenotrophomonas and Sanguibacter [72]. The Puga hot water springs (33 13’N, 78 18’W) are located in the North Western Himalayan region of Ladakh. The water temperature is upto 84  C at the outlet and it contains high amounts of boron and sulfur [73]. The extremophile Thermococcus kodakaraensis has been reported from Chumatang hot springs (33.3 N and 78.4E) [74]. Many novel psychrophilic bacteria have been reported from Himalayan glaciers. Exiguobacterium indicum sp. nov., has been reported from the Hamta glacier of the Himalayan mountain ranges [75]. Similarly, extremophiles Paenibacillus

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glacialis sp. nov., Bacillus cecembensis sp. nov., and Cryobacterium pindariense sp. nov., have been isolated from the Kafni and Pindari glacier of the Indian Himalayas respectively [76
79]. Metagenomic studies have also been conducted to study the diversity of the brackish lake Pangong (33 430 04.59vN:78 530 48.48vE) situated at a height of 4250 m above sea level [80]. The major phyla in the sediments from Pangong were Proteobacteria and Bacteroidetes [80]. Recently, an extremophilic psychrophile, Kocuria rosea PRL-1 (Genbank accession no: MH246939.1) was isolated from Himalayan rocks and was used in the Astrobiological studies related to lithopanspermia and impact shock [81].

7.4 Extremophiles from planetary field analogue sites in Europe: astrobiological implications Europe has run several activities for the total integration of Astrobiology and research in extremophilic microbiology in planetary sciences. Europlanet (European Planetary Network) is a Pan European activity that focuses on international collaboration and development of the different fields of research in space missions, planetary sciences and astrobiology. Europlanet has been funded by the European commission and one the most relevant activity has been the study of extreme environments and Earth analogues from an astrobiological point of view. This activity has been organized in the framework of TNAs (Transnational access), which gives access to several field analog sites that provide the most realistic mimics of planetary surfaces on the Earth. Scientists are provided access to these sites to perform scientific research and testing instrumentation for space missions under planetary conditions and undertake comparative research. TNA1 Planetary Field Analogues are well-characterized terrestrial regions that have provide realistic analogues of Mars, Europa and Titan. They include deserts, permafrost, acidic regions and hydrothermal areas. The important planetary field analogue sites for Astrobiology studies are as follows:

7.4.1 Rio Tinto, Spain Rio Tinto is located in Spain and is a site in which a rock-water-biology produces acidic river water with a pH 2. The acidic pH of the water and production precipitated minerals (jarosite, goethite) depicts a distinctive geological environment that is useful for the assessment of biological and inorganic processes comparable to Mars found by the Mars Environmental Rover Opportunity (e.g. Jarosite). The river transects a pyrite-rich belt that has been used for mining since prehistoric times. Pyrite provides energy for growth of chemolithotrophic bacteria. Most organisms isolated from Rio Tinto belong to the genera Acidithiobacillus, Leptospirillum and Acidiphilium. This group of bacteria is metabolizes pyrite and oxidizes sulfur and ferrous iron parts of the mineral accompanied by the production of sulfuric acid and ferric iron like metabolic products. Other extremophilic chemolithotrophs fround in this site are Ferrimicrobium spp., Ferroplasma spp., Metallibacterium spp., Thermoplasma acidophilum, Acidisphaera spp., and Acidobacterium spp [82]. This analogue site provides an excellent example of how life may have evolved in extreme acidic conditions and may provide clues as to the type of bacterial life that may once have been present on Mars. INTA-CAB manages the region as a field analogue for the study of biogeochemical cycles and the testing of instrumentation. The Rio Tinto field analogue has a history of use for instrument testing within international cooperation’s for space missions. The field analogue is currently being used by several international partners (e.g., Wood Hole Oceanographic Institute, NASA Ames Research Centre, University of Washington at St. Louis, Honey Bee Robotics). Importantly, the Rio Tinto field analogue is not only of interest to the planetary community. Geologists, microbiologists and paleontologists work within the field analogue. The nature of the preservation of organic matter in such acidic conditions is of great interest to paleontologists and microbiologists are examining the nature and diversity of life in extreme acidic conditions. Numerous publications have discussed the wide diversity of life forms in the extreme acidic environments, long-term organic matter preservation under high oxidative stress and acidic conditions. In addition, new drilling instrumentation being developed for Mars has been tested and validated in the Rio Tinto area [82].

˚ lesund in Svalbard archipelago of Norway 7.4.2 Ny-A ˚ lesund is located on the island of Spitsbergen in the Svalbard archipelago 1200 km from the North Pole. Svalbard Ny-A includes areas in front of two glaciers that are very well suited for testing instruments for ground ice and permafrost ˚ lesund is 26.3  C (42 m.a.s.l 1961
90), and average annual precipitation mapping. The annual air temperature in Ny A is 355 mm/y. The permafrost depth is estimated to be B100 m in coastal areas and .500 m in mountainous areas.

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˚ lesund (79 N) in the Arctic region revealed the presence of The microbial diversity studies of permafrost soils of Ny-A organisms belonging to the genera Nesterenkonia, Psychrobacter, Rhizobium, Sphingopyxis, Stenotrophomonas, Rhodococcus, Sphingobacterium and Virgibacillus sp. [83]. Currently scientists from at least fifteen nations visit ˚ lesund to work on a variety of research projects. Norway, Germany, Great Britain, Italy, Japan, France, Ny-A ˚ lesund. South Korea and China have all established their own research stations in Ny-A

7.4.3 The Ibn battuta center near Marrakech, Morocco This center is an extreme desert facility used to test rover, landing systems and instruments dedicated to the Mars exploration. The desert site includes terrestrial analogues like regoliths, evaporates, sand dunes, rocky desert, and flash flood drainage. Ongoing geological analysis of the region aims to quantitatively understand the origin of environments such as mud volcanoes, evaporitic deposits, the recent stratigraphy of aeolian sediments and deflation surfaces that appear remarkable comparable to those reported from Mars. Astrobiology research is also examining potential habitats and energy sources within ancient mud volcanoes and evaporates with the goal of establishing how specific endolithic communities and arid bacterial niches function. This PFA is currently dedicated to fundamental research and instrument testing and development for future Mars exploration. The research projects are conducted by ESA missions Mars Express, ExoMars, Mars-Next and Mars Sample Return. Besides its application as a Martian PFA, this site can also be used as comparative analogue to aeolian deposits observed on Titan [84].

7.4.4 The Kamchatka Peninsula, Russia This PFA is known for its hot spring and geyser activity in Russia. This site helps in studying complex relation between volcanism and landform development, that is pertinent to planetary bodies. The hots springs also harbor extensive microbial life. The specific site for study at Kamchatka is the Uzon-Geysernaia twin caldera that forms a 9 3 18 km depression. This site originated due to large explosive eruptions in the late Pleistocene. Currently, geothermal activity is concentrated in a 0.3 3 5 km zone filled with 30 geysers, boiling springs, gas-steam jets, mudpots, small mud volcanoes, hot lakes and springs. These habitats contain a wide variety of colonies of blue-green algae and thiobacteria. Autotrophic sulfur-oxidizers of the genus Sulfurihydrogenibium (phylum Aquificae), anaerobic bacteria of genus Caldimicrobium (phylum Thermodesulfobacteria), Archaea of the genus Vulcanisaeta, organisms belonging to Nanoarchaeota and uncultured phylum of Thermoplasmataceae A10 dominate the microbial communities of Kamchatka Peninsula terrestrial hot springs [85]. The landscape of Tolbachik volcano was used in the Soviet/Russian space program for testing of Mars rovers for Mars-96 mission. The hot spring waters are rich in boron, silicon, and ammonium chloride and have high concentrations of alkali metals and ore elements. The rest of the hydrotherms are sulfate-chloride-sodium, hydrocarbonatesulfate-calcium-sodium and hydrocarbonate rich. Gas discharge includes CO2, N2, H2, H2S, CH4, and radon. Complex methane-naphthene-aromatic type hydrocarbons, possibly of biogenic nature occur in the thermal fields of the Uzon Caldera. This site is a possible analogue of prebiotic conditions and surfaces of Jovian and Saturnian moons (e.g. Europa and Titan). Kamchatka region also offers several PFA sites to Mars analogue terrains.

7.4.5 Tirez Lake, Spain Tirez Lake (39 32 N
3 210 E) is set near Toledo in the central region of Spain. It is an endorreic origin lake composed of a sun baked, dry surface of deposited salt (NaCl) covering sources of underground water. The red color is attributed to high contents of ferric iron. The hydro geochemical analyzes showed that Tı´rez waters were rich in Mg-Na-SO4-Cl brines with epsomite, hexahydrite and halite as end mineral members. The spectra of frozen Tı´rez brines analyzed by FTIR, were similar to the Galileo spectral data. The calorimetric measurements of brines indicated pathways and phase metastability for magnesium sulfate and sodium chloride crystallization which may assist in understanding the formation of Europa’s icy crust. The microbial community structure of Tirez has been studied using fluorescence “In situ” hybridization (FISH), specific DNA probes and 16 S rRNA sequencing studies. The microbial community of Tirez (sediment) was dominated by Marinobacter and Halomonas, Woloszynskia cincta, Dunaliella sp., Haloarchaea, Archaeoglobus and the halophilic sulfate reducing bacterium Desulfohalobium [86]. This environment provides a suitable analogue for chloride deposits on Mars, comparable to the layered deposits discovered at the North Pole. Studies on hypersaline environments may provide insights in our search for potential habitats in Mars and icy moons of Jupiter. Based on the comparison of its hydrogeochemistry with the geochemical features of the alteration mineralogy of meteoritic precursors and with Galileo`s NIMS data, Tı´rez Lake (Spain) is proposed as a terrestrial analogue of the Europa’s ocean.

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7.5 Micro-organisms in earth’s upper atmosphere and outer space: applications in astrobiology missions The atmosphere of Earth is a protective covering of gases that is held by gravity [87]. Our atmosphere and outer space is separated by a border called as the Ka´rma´n line, at 100 km. The atmosphere comprises of Trophosphere, Stratosphere (altitude of upto 50 km), Mesosphere (highest altitude where organisms were found), Thermosphere and Exosphere [87]. The organisms Mycobacterium luteum, Papulaspora anomala, Micrococcus albus, Circinella muscae, Aspergillus niger and Penicillium notatum have been detected in high altitude atmosphere of the mesosphere [88]. In India, a balloon experiment was conducted to study organisms from Earth’s Upper atmosphere was on 20 April 2005. A balloon from the National Scientific Balloon Facility of the Tata Institute of Fundamental Research (Hyderabad, India) was used with a cryosampler payload for collecting air samples from high-altitude (altitude 20
41.4 km). The astrobiology experiments resulted in the isolation of three novel bacteria. Janibacter hoylei sp. nov., Bacillus isronensis sp. nov. and Bacillus aryabhattai sp. nov. were reported to be isolated for the first time from air samples collected at the altitudes between 27 and 41 km [89]. Studying the organisms from Earth’s upper atmosphere provides important clues in Astrobiology. Scientists have argued that micro-organisms can travel across interplanetary space during meteoritic exchanges between the Earth and Mars [89]. Hoyle and Wickramsinghe were proponents of the theory of ‘Panspermia’ [90]. Studying the occurrence and survival of micro-organisms in Earth’s upper atmosphere (EUA), low Earth orbit (LEO) and Outer space is crucial and has implications in ‘panspermia’ and origin of life. Many organisms have been studied for their survival in LEO that reaches up to an altitude of 450 km and outer space [87]. The characteristic feature of these harsh environments is pressure, temperature, space vacuum, and radiation that comprises of ultra violet (UV), solar cosmic radiation (SCR), galactic cosmic radiation (GCR) and radiation belts trapped by the Earth’s magnetosphere [87]. The first experiments to investigate the survival of bacteria in outer space were conducted in 1967 using rocket and balloon experiments [91]. In 1972, NASA developed a sophisticated exposure device called MEED (microbial ecology equipment device) for the Apollo 16 mission [92]. MEED was used to study exposure of bacteria to space vacuum for 1.3 h and solar UV radiation with a peak wavelength of either 254 nm, 280 nm, or 300 nm. Extremely resistant spores of Bacillus subtilis were used in BIOSTACK experiments for the determination of effect of individual HZE particles along trajectories [87]. The survival of Bacillus subtilus and radiation-resistant bacterium Deinococcus radiodurans, was studied after exposure to cosmicray HZE particles and heavy and high-LET ions of GCR in the Apollo Soyuz Test Project and Spacelab 1 mission [93]. An experiment called EXOBIOLOGIE was conducted as a part of the French PERSEUS mission on the MIR station to study effects of energy-rich UV radiation and outer space on microbial spores [94]. The Russian Foton satellites [95] and the European Retrievable Carrier (EURECA) have been used to study the effect of outer space and radiations on bacterial [96]. The BIOPAN facility was developed by the European Space Agency (ESA) in 1994 [37]. It is a small retrievable capsule for short-term exposure of bacteria to outer space. Halorubrum chaoviator sp. strain Halo-G* was the first haloarchaeon exposed to space vacuum, UV and cosmic radiation embedded in clay, meteorite powder, simulated Martian soil and salt crystals. This capsule was sent in outer space using the Foton class Russian spacecraft [97]. For long-term exposure experiments, ESA developed the EXPOSE facility on the outside of the ISS (International Space Station) [98]. The EXPOSE facility could interface with EUTEF—CEPA (Columbus External Platform Adapter) for EXPOSE-E or with platform of the Russian segment of the ISS for EXPOSE-R [98]. On 7 February 2008, the EXPOSE-E mission was launched onboard NASA Space Shuttle Atlantis, STS-122. The EXPOSE-E trays had experiments that carried many different organisms on board one of which was Halococcus dombrowskii. H. dombrowskii was exposed in outer space for 559 days as part of the ADAPT (Molecular adaptation strategies of microorganisms to different space and planetary UV climate conditions) experiment in EXPOSE-E mission [33,97,98]. Extremely resistant spores of microbial spacecraft isolates B. subtilis 168 and B. pumilus SAFR-32 were also exposed in the PROTECT experiment in EXPOSE-E [99,100]. Haloarcula-G and Synechococcus were exposed to space environments in the ROSE2/OSMO experiment as a part of the EXPOSE-R mission with the Russian Progress 31-P [97,98]. An interesting experiment called SPORES (Spores in artificial meteorites) for testing lithopanspermia and interplanetary transfer was conducted as a part of European Space Agency’s EXPOSE-R mission. The facility was attached on the outside of the Russian module Zvezda of the International Space Station. In this mission spores of Bacillus subtilis 168 were exposed to outer space conditions for approximately 2 years (March 10, 2009 to February 21, 2011) [101].

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The latest experiments conducted within EXPOSE-R2 outside the Zvezda module on ISS was BIOMEX (BIOlogy and Mars EXperiment) which is an ESA/Roscosmos space exposure experiment [102]. The methanogenic archaeon Methanosarcina sp. strain SMA- 21 isolated from terrestrial permafrost was chosen due to its potential for being metabolically active on Mars [102]. In recent years, increasing efforts have been taken to study the microbiome of the ISS [103]. Studying organisms in ISS and Space craft is important due to astronaut health, integrity of space missions and planetary protection and life support systems like MELISSA (Micro-Ecological Life Support System Alternative) [103]. ISS is now used as a ‘Microbial Observatory’ to study microbial response to spaceflight conditions. Many novel organisms like Solibacillus kalamii ISSFR-015 have been reported to be isolated from a high-energy particulate arrestance filter aboard the International Space Station [104]. These studies indicate that the ISS could now be a potential site for isolation of novel species that could in turn be used in Astrobiology.

7.6 Extremophiles from space craft assembly room: applications in planetary protection Spacecraft Assembly rooms are similar to cleanrooms used in vaccine and pharmaceutical industry. Despite their cleanliness and harsh environments, many bacteria and other prokaryotes thrive in the harsh nutrient deficient cleanrooms. Studying cleanroom microbial diversity is important for the effective implementation and improvement of planetary protection practices. Planetary protection (PP) encompasses the policies, methods and practices utilized to safeguard science objectives of the missions to the planetary bodies from inadvertent contamination by terrestrial organisms from Earth as well as protection of Earth from plausible life from outer space [103,105]. The Committee on Space Research (COSPAR) is responsible for the development of planetary protection policies for the protection of Earth and other planets from biological contamination [103]. PP is imperative for the maintenance of proper conditions for future space exploration studies in order to avoid terrestrial contamination that may obscure our quest for life on other planets [105]. The study of extremophiles in clean rooms is important as these hardy micro-organisms can hitchhike via spacecraft, build in these cleanroom environments. Forward contamination is a significant concern for future missions as extremophilic bacteria may contaminate and jeopardize the scientific quest for life in habitable planetary bodies like Mars, Europa and Enceladus. Many standard microbiological methods like the NASA Spore assay (NSA), ATP assay, Live/Dead staining methods, DNA microarrays, and ‘omics’ based high throughput techniques are used for detection of microbial contamination and bioburden on spacecraft hardware. The first viability-linked metagenome assessment of the spacecraft assembly facility revealed unexplored, potentially viable diversity across a wide spectrum of the tree of life [106]. Many extremophilic and extremotolerant bacteria have been isolated from space craft assembly facilities [103]. Tersicoccus phoenicis has been isolated from the cleanroom facility where the Mars Phoenix spacecraft was assembled [107]. Extremotolerant bacteria like Acinetobacter johnsonii and Brevundimonas diminuta have been isolated from Phoenix Spacecraft assembly rooms [108]. Bacillus safensis sp. nov., and Bacillus horneckiae sp. nov., has been reported to be isolated from spacecraft and assembly-facility surfaces [109,110]. The Bacillus odysseyi sp. nov., a round-spore-forming hardy bacillus has been isolated from the Mars Odyssey spacecraft [111]. One of the extreme heat resistant spore forming bacterial strain, Paenibacillus xerothermodurans ATCC 27380, was isolated from the soil samples outside of the KSC cleanroom facility in 1973 and was recently characterized [112]. Furthermore, as the search for traces of extinct and extant life on Mars continues, the study of Clean room microbiology and implementation of PP is inevitable for preserving the integrity of future scientific exploration.

7.7

Conclusion and future outlook

Microbial life thrives in extreme conditions. Extremophiles survive in the Earths’s Upper environment, Planetary field analogue sites, Space craft assembly rooms and also in International Space Station and Outer Space. The current chapter summarizes the past and present advancements in extremophiles research in Astrobiology. The NASA Astrobiology roadmap has clearly outlines the importance of studying extremophiles and extreme terrestrial sites in Astrobiology [1]. The European AstRoMap project is the first European roadmap for astrobiology research [8]. Studying life and habitatability in extreme environment is an important goal of the European Astrobiology roadmap [8]. It is important to investigate the physical and chemical limits of survival of extreme life as it could provide useful metrics in studying habitability of extraterrestrial bodies like Mars, Europa and Enceladus. The search for life beyond Earth has been the primary objective of many space missions like the Vikings mission to Mars launched in 1976 [4]. It is important to define and understand the concept of “life” for such extraterrestrial life detection missions. A prevalent working definition of life accepted by NASA is “a self sustaining chemical system

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capable of Darwinian evolution” [113]. Biosignatures like archaeon lipid biomarkers (kerogens, hopanes, steranes), gases, organic molecules can be considered as biomarkers to detect the presence of past or present life using GC
MS or in situ microscale measurements using nanoscale secondary ion mass spectrometry (NanoSIMS) [114]. A valuable tool called as “Ladder of life detection” has been developed to guide the efforts towards the detection of microbial life using biosignatures in future space missions [114]. Infact, future sample return missions from Mars, asteroids and Icy moons will warrant different life detection methods and special mitigation strategies to avoid Earth’s ubiquitous biosphere contamination [114,115]. Besides these biosignatures and organic molecules, life detection methods also need to be developed for the detection of nucleic acids like DNA and RNA. The greatest challenge in extreme microbiology will be unraveling the mysteries of Shadow biosphere and microbial dark matter (MDM) [28]. Metagenomics, single cell genomics and parallel advancement in DNA sequencing, amplication and computing will be necessary in addition to conventional culture based axenic methods [116]. Metagenomic and single cell studying MDM help us gain valuable insights in catabolic, anabolic potentials, genomic features, molecular adaptations, stop codon reassignments and several unique features of extremophiles [28,116]. This also helps understand phylogenetic relationship and diversification of microbial lineages. Other innovative techniques like Fluorescent in situ hybridization with micro autoradiography (MAR-FISH), FISH-NanoSIMS, Raman FISH Spectroscopy, Phylogenetic microarrays with CHIP-SP can serve as potential methods to detect MDM in samples from space missions and extreme environments [116]. Finally, as the scientific fraternity gears for the future Mars2020 and ExoMars missions, we need to develop radical approaches and out of the box strategies to explore extremophiles and life and to understand life’s origin in its astrobiological context.

Acknowledgments RT thanks Europlanet for funding the Europlanet Transnational Access (TA) (17-EPN3-030) project to Tirez lake. Europlanet 2020 RI has received funding from the European Union Horizon 2020 research and innovation program under grant agreement No. 654208. Part of the research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration.

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Further reading McKay D, Gibson E, Thomas-Keprta K, Vali H, Romanek C, Clemett S, et al. Search for past life on Mars: possible relic biogenic activity in Martian Meteorite ALH84001. Science 1996;273(5277):924
30. Available from: https://doi.org/10.1126/science.273.5277.924.

Chapter 8

High-pressure adaptation of extremophiles and biotechnological applications M. Salvador-Castell1, P. Oger1 and J. Peters2,3 1

Universite´ de Lyon, CNRS, UMR, Villeurbanne, France, 2Universite´ Grenoble Alpes, LiPhy, Grenoble, France, 3Institut Laue Langevin,

Grenoble, France

8.1

Introduction

High pressure (HP) characterizes many habitats on Earth, such as deep-sea, subseafloor and continental subsurface. Deep-sea is part of the oceans that encompasses the entire biosphere below 1000 m from the water surface and present pressures higher than 10 MPa. This hydrostatic pressure originates from the weight of the water column and corresponds to 10 MPa/km [1]. The highest hydrostatic pressure detected in the ocean is approximately 110 MPa at 11,000 m depth at the Challenger Deep of Mariana Trench in the Pacific Ocean. Moreover, below the subseafloor, the pressure further increases due to the weight of the sedimentary material by roughly 15 MPa/km and ca. 28 MPa/km in oceanic rock. Such biosphere contains a substantial part of the Earth biomass, which can potentially influence global biochemistry [2
4]. All high-pressure habitats are occupied by microorganisms and other complex organisms that highly contribute to the Earth’s biomass [5,6]. Pressure impact on organisms’ growth allows to divide them in different categories. Organisms that cannot tolerate ambient pressure are designated as strict or obligate piezophiles. Inversely, facultative piezophiles are organisms that tolerate ambient pressure, but their optimal growth pressures are higher than 10 MPa (Fig. 8.1). Other organisms withstand a range of optimal pressures from ambient pressure to low hydrostatic pressures, these organisms are called piezotolerants. Finally, organisms which growth is inhibited by pressure are designed as piezosensitive [7]. Bacteria and archaea domains contain facultative and obligate piezophiles. Examples of obligatepiezophiles are the bacteria Shewanella benthica and Colwellia marinimaniae, which optimal growth pressures are 70 MPa and 120 MPa, respectively [8,9], and the archaeon Pyrococcus yayanossi, withstanding an optimal pressure of 50 MPa [10]. Obviously, piezophiles excel in sustaining pressure conditions beyond the usual limits for humans; however, the reasons for that adaptation are still debated. The technical constraints to isolate obligate piezophiles are certainly responsible for the limited attention they get. The curiosity in piezophiles has begun more than a century ago [11,12], but the technological difficulties and the need of specialized equipment have caused that high-pressure studies are not, currently, developed in most laboratories. Nevertheless, the interest on pressure and its biotechnological applications have been growing during last decades.

8.2

Effects of pressure on macromolecules and cells

Pressure alters biomolecules by changing their volume. Thermodynamically, the variation in Gibbs free energy (G) is defined by Eq. 8.1 dðΔGÞ 5 2 ΔSdT 1 ΔVdP; Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00008-3 © 2020 Elsevier Inc. All rights reserved.

(8.1)

105

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PART | I Physiological aspects

Growth rate

Piezosensitive Piezotolerant Piezophile Strict piezophile

0

20

40

60

80

100

120

140

Hydrostatic pressure (MPa) FIGURE 8.1 Schematic growth curves of microorganisms according to pressure (MPa).

where ΔS is the change in entropy, ΔV the difference in volume and T and P represent temperature and pressure, respectively. At constant temperature, dT 5 0 and thus   @G 5 ΔV: (8.2) @P T This equation, conforming to Le Chaˆtelier’s principle [13], states that an increase in pressure will cause a shift to the state that occupies the smallest volume, meaning, for example, to the unfolded state for most globular proteins, where ΔG , 0. As a result, pressure modifies the volume of the system, but not its internal energy (as temperature does). Water with its low compressibility is a crucial partner for pressure action. Notably hydration water (water bound at the surface of macromolecules) is very sensitive to pressure and it can reorganize its network under pressure implying an effect on the macromolecule [14]. Moreover, macromolecules present an extraordinary stability against pressure under low hydration conditions [15,16]. Molecular interactions result from an equilibrium of electrostatic forces, such as Van der Waals and hydrogen bonding, along with hydrophobic interactions. Relatively low pressures affect these molecular bindings, which outcome to changes on the geometry and structure of biomolecules and therefore, on their physical properties (solubility, melting point, density) and reaction rates. In contrast, high pressures above 2 GPa are needed to impact non-covalent interactions [17
19].

8.2.1 Nucleic acids Although the unfolding volume of DNA duplexes is small, pressures up to 1 GPa have, in general, a stabilizing effect on canonical DNA (e.g. with common pair bases). This stabilizing effect may be related to the decrease of hydrogen bonds’ distance. Consequently, pressure increases the duplex-single-strand transition temperature [14]. Only in specific cases of synthetic polymers (e.g. adenine
thiamine copolymers) and salt concentrations, pressure can lead to doublestranded melting [20]. Regardless, the mechanism is not the same as the heat-induced DNA melting, as under pressure, water molecules penetrate DNA base pairs destabilizing their interactions [16,21]. There is a lack of information about the effect of pressure on RNA but, generally, it has been observed that RNA is more pressure sensitive than DNA. For example, pressure induces a structure reorganization of tRNA [22,23] and it destabilizes small RNA oligomers [24]. Non-canonical pair structures (different from the usual Watson-Crick pair bases), such as G-quadruplexes or stem-loops, are less stable under pressure than canonical structures by a factor of 10 [21,25]. Although canonical DNA duplexes are stabilized by pressure, DNA—protein interactions may be perturbed due to changes in the electrostatic and hydrophobic interactions. Accordingly, pressure affects negatively all molecular reactions where DNA is involved, such as replication, transcription and recombination [26,27].

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8.2.2 Proteins Most of the knowledge about pressure effects on proteins is based on studies on globular proteins [28
30]. Some of these studies reveal that structural transitions of globular proteins due to pressure are based on a hydration mechanism that accompanies protein conformational changes. At higher pressure, the hydration degree is increased by the entrance of water into the protein cavities generating an increase of the surface in contact with the solvents, thus contributing to the volume change [31]. Pressure mainly alters tertiary and quaternary structures of proteins, but secondary structures (α-helices, β-sheets, and turns) are much less sensitive to water penetration and to destabilization by pressure. For this reason, the state unfolded by pressure may be a hydrated globular structure with large amounts of folded structure [18,32]. It should be recalled that water plays a key role on pressure denaturation, since this volume change can only be attended to proteins in solution. Dry proteins are highly stable against pressure [17,33,34]. The unfolding of many monomeric proteins begins above 200 MPa [4], however, enzymatic activities are usually modified at lower pressures. In fact, the application of pressures ,200 MPa confers higher thermostability to most proteins [33,35]. Consequently, superposing pressure and temperature application usually accelerates most of enzymatic reactions, such as hydrolases and transferase reactions [34]. As an illustration, the efficiency of coconut husk hydrolysis by cellulases from Penicillium variable is increased at 300 MPa and 50  C [36]. Moreover, reactions can already be enhanced by pressure at low temperatures [34]. For example, the activity of the enzyme polyphenoloxidase from onion is increased up to 140% at 450 MPa and 25  C [37]. Pressure generally changes the equilibrium between subunits from oligomers or between two different proteins, even at relatively low pressures (ca. 50 MPa) [38]. At this pressure, for example, ribosomes’ subunits are dissociated [27] and larger protein assemblies such as cytoskeletal proteins are disturbed resulting in reversible morphological changes [39]. However, other oligomers are more resistant to pressure such as the tetrameric urate oxidase, which dissociates at ca. 150
175 MPa [40]. In fact, a protein in its native state possesses distinct, nearly isoenergetic conformational substates, which may have similar or dissimilar functions or the same function with different rates (statistical substates). As pressure can decrease the folding rate and increase the unfolding one, it can shift the population of protein substates on the basis of their volumetric differences [41
43]. This capability allows the characterization of various intermediate substates by pressure, which may occur in the folding process [44]. Moreover, pressure can change the reaction rates, providing new information about the dynamics and reactions of proteins [32]. This was confirmed, for example, by a dynamic study of myoglobin, where it has been shown that pressure reduces protein motions and increases the structural similarities between the different conformational substates [43]. Few studies have been performed on pressure effects on non-globular proteins, such as fibrous, disordered and membrane proteins. Examples are the studies on collagen structure [45], the intrinsically disordered protein alpha synuclein [46], the Lmr transmembrane protein [47] or the ion channel MscS [48]. An important point is that the behavior of transmembrane proteins against environmental stresses is affected by the protein structure but also by its lipid surrounding [49]. Membrane proteins and membrane lipids form an ensemble; they influence mutually as a product of biochemical or environmental changes which can compromise membrane processes such as energy production or ion fluxes. The influence of the lipid matrix on the protein response to pressure has been studied for few proteins [50
52]. For example, the transporter efficiency of the tryptophan permease Tat2 from yeast cells is affected due to a modification of membrane fluidity under HP [53]. Therefore, membrane integrative studies are necessary to better describe membrane protein behavior.

8.2.3 Phospholipids Lipids, and specially their hydrocarbon chains possess a high compressibility [54]. When pressure is applied on a phospholipid bilayer, the acyl chains are straightened resulting in a thicker and more ordered bilayer. Namely, pressure induces the lipid phase transition from a liquid-crystalline (phase essential for the biological function of the membrane) to a more rigid phase called the gel phase. Additionally, pressure can also promote the apparition of new phases, such as interdigitated phases or non-lamellar phases (i.e., cubic or hexagonal) [54
58]. Nevertheless, all lipids do not have the same sensitivity to pressure. For example, lipids with longer hydrophobic chains are higher responsive to pressure. This may result in a phase separation in domains on membranes formed by a mixture of lipids [59]. Pressure may also have an impact on more complex macromolecules, such as lipoproteins. Recent studies on human plasma lipoproteins under HHP revealed a reduced flexibility and higher compressibility of its triglyceride rich form, the form associated to pathological health conditions [60,61] compared to the native one.

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8.2.4 Cells Surprisingly, pressure is the unique physical parameter capable of inducing heat- and cold-shock proteins’ as a cell response to the same stress. Escherichia coli exposed to 53 MPa induces 55 proteins, 11 heat-shock and 4 cold-shock proteins among them. E. coli may try to neutralize the damage produced by pressure at different cell levels, such as stability of macromolecules and membrane functionality [62]. As mentioned above, pressures up to 100 MPa affect most of the cellular functions such as enzymatic reactions, gene expression, cell motility and morphology, and the cell membrane itself (Fig. 8.2). Since pressure is transmitted through a fluid, it is uniformly transmitted (Pascal’s law) over the whole cell and therefore, it makes it difficult to identify the main cause of cell death. Moreover, pressure-induced cell inactivation relies upon the nature of microorganism and its physiological conditions, such as water content and salt presence. Overall, eukaryotes are more pressure-sensitive than prokaryotes and piezosensitive bacilli and spiral-shaped bacteria are inactivated at lower pressures than cocci [38,63]. For instance, pressures above 150 MPa usually reduce the viability of mammalian cells and may induce cell death by apoptosis from 200 MPa on or through a necrotic-like pathway at 300 MPa [64]. On the other hand, bacteria cocci may resist to much higher pressure variations, for example, Staphylococcus aureus cell inactivation begins at 350 MPa [63]. In addition, most Gram-negative bacteria seem to be less resistant to pressure than Gram-positive [65]. Gramnegative bacteria possess a much more complex membrane which makes it a target for pressure damage [66]. Finally, microorganisms in the exponential growth phase present lower pressure-tolerance than in their stationary growth phase [67,68]. For example, exponential-phase cells may present a filamentous shape under pressure which can disrupt the

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FIGURE 8.2 Above, illustration of effects on different cell macromolecules: (A) lipids, (B) multimer proteins, (C) proteins, (D) flagella, (E) DNA (red) and tRNA, (F) RNA translation. Adapted from P.M. Oger, M. Jebbar, The many ways of coping with pressure, Res Microbiol 161 (2010) 799
809. https://doi.org/10.1016/j.resmic.2010.09.017. Below, common pressure effects on cellular macromolecules (black) and cells (red).

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membrane functions [27,69]. Moreover, stationary-cells have the capability to synthesize stress-response proteins to adapt and, therefore, better resist to different harsh conditions [70]. Spores present formidably high resistance to harsh environments, likely due to their structure with numerous protective layers and their low water content [71]. Interestingly, relatively moderate pressures (50
300 MPa) cause the germination of a dormant spore. Though, higher pressures are often less effective to induce germination [72]. Pressure alone is not very effective to inactivate bacterial spores and a treatment together with temperature is necessary [73].

8.3

Pressure adaptation in piezophiles

The biodiversity of piezophiles is huge [5,74
76]. Organisms adapted to pressure include unicellular bacteria, archaea, eukaryotesas invertebrates, fishes and even deep diving marine mammals [77
79]. For example, large invertebrates like mussels, crabs and shrimps inhabit hydrothermal vents and some marine mammals can be exposed to almost 20 MPa without presenting any negative symptom [80,81]. There are differences in the microorganisms adapted to HP, as some are also adapted to low temperatures (psychrophiles) and others to high temperatures (hyperthermophiles), which increases the piezophilic diversity. Hyperthermophilic and piezophilic organisms are found near vent sites, where temperatures range from 350  C to 2  C in only few cm distance. Altogether, this represents a reserve of microorganisms with great potential for technological and pharmaceutical applications, such as new enzymes, antibiotics or cancer cell line active derivatives. Most piezophiles identified in the deep sea are bacteria, they are psychrophiles and piezophiles. However, some archaea reside in hydrothermal vents, being hyperthermophiles and piezophiles [82] (Fig. 8.3). Psychrophiles and hyperthermophiles piezophiles had follow different pathways to adapt to temperature and therefore, their pressure adaptation process may also be distinct. Beside temperature adaptation, piezophiles are acclimated to salt presence, i.e. they are slight halophiles, and finally, some piezophiles are also adapted to nutrient limitation, i.e. designated as oligotrophs [4]. Consequently, piezophiles may adopt a common strategy to cope with various stresses simultaneously and therefore it’s tricky to identify and separate specific pressure-adaptation pathways from other stress adaptation [83]. Additionally, not all pressure-adaptation mechanisms are deleted at ambient pressure, and therefore the homologue piezosensitive organism may use the same mechanism [84], which hinders a possible comparison between mechanisms from piezophile and a similar pressure-sensitive organism. Extremophiles have developed great capabilities to adapt to harsh and even fluctuating conditions (e.g. temperature, pressure, composition of the host rocks) thanks to synthesis of unique macromolecules, such as extracellular polysaccharides [85], lipids [86], proteins [87] and even specialized organs [88]. These macromolecules adapted to extreme environments, such as high pressure, present a high potential to develop new biotechnological applications.

8.3.1 Genomes To date, it has not been possible to detect any piezospecific gene. Consequently, it is not possible to determine if an organism is piezophile by molecular techniques, it is necessary to do cultivation approaches and to determine the growth rates at different pressures. However, it has been found that the pressure regulated operons ORFs 1
3 are distributed among different piezophilic Shewanella species [89]. The high genetic manageability and hyper-responsiveness to pressure of the piezophile Photobacterium profundum strain SS9 has made it a reference for researchs on pressure adaptation. Studies on its RecD gene, responsible for a DNA-binding protein, indicates that this gene may have an important role for piezoadaptation together with its role of DNA metabolism and cell division [90,91]. This conclusion is sustained by the discovery of a pressure-sensitive mutant of SS9 that lacks the RecD gene and, besides, by the capability of E. coli to divide normally under HP after the transfer of this gene. Recently, it has been described that the gene Ypr153w is possibly responsible for the tryptophan permease’s Tat2 stability in Saccharomyces cerevisiae under pressure. It is a gene which has also been identified in other related species as Debaryomyces and Candida strains which have been isolated from sediment samples of deep sea floors [92]. Another possible HP adaptation could be the 16s rRNA longer stems found in strains from Photobacterium, Colwellia and Shewanella [93].

8.3.2 Proteins Relatively few enzymes from piezophiles have been studied under pressure. Although there are no apparent differences between the crystal structures of an enzyme from a piezophile and its piezosensitive homologue, there is a

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120 MPa C. marinimaniae

110 MPa

100 MPa S. benthica KT99

90 MPa

Optimal growth pressure

C. hadalensis

80 MPa M. yayanosii Rhodobacterales bacterium PRT1

70 MPa

60 MPa

M. jannaschii

S. benthica DF172F/DB21MT-2 Colwellia sp. strain MT41 C. piezophila S. benthica DB172R P. hadalis

S. benthica DB5501/DB6705/DB6906 Psychromonas sp. strain CNPT3

50 MPa P. piezophila

40 MPa

M. piezophila

T. barophilus P. ferrophilus

P. thermophilus

D. profundus S. benthica F1A P. profundum SS9 P. profunda S. piezotolerans WP3 D. hydrothermalis

P. pacificus T. guayamasensis

20 MPa

A. fermentas

S. psychrophila Carnobacterium sp. strain AT7 Carnobacterium sp. S. piezotolerans WP2 strain AT2

T. japonicus T. aggregans

T. lithotrophica

10 MPa

10ºC

P. kukulkanii

T. peptonophilus D.abyssi

S.violacea M. abyssi

P. profundum DSJ4

P. yayanosii

M. thermolithotrophicus T. piezophilus

Moritella sp. strain PE36 M. profunda

30 MPa

M. japonica

P. kaikoae S.benthica DB6101

P.abyssi

M. kandleri

T. eurythermalis

S. profunda D. piezophilus

20ºC

30ºC

40ºC

50ºC

60ºC

70ºC

80ºC

90ºC

100ºC

110ºC

Optimal growth temperature

FIGURE 8.3 Optimal growth temperature and pressure for all idenfied piezotolerant and piezophile bacteria (blue) and archaea (red). A. represents Anoxybater; C. represents Colwellia; D. represents Desulfovibrio except for Dermacoccus abyssi; M. represents Moritella except for Marinitoga piezophila, Methanocaldococcus jannaschii, Methanococcus thermolithotrophicus and Methanopyrus kandleri; P. represents Psychromonas except for Piezobacter thermophilus, Profundionmas piezophila, Photobacterium profundum, Paleococcus ferrophilus, Paleococcus pacificus, Pyrococcus abyssi, Pyrococcus yayanosii and Pyrococcus kukulkanii; S. represents Shewanella; T. represents Thermococcus except for Thioprofundum lithotrophica and Thermosipho japonicus. Adapted from M. Jebbar, B. Franzetti, E. Girard, P. Oger, Microbial diversity and adaptation to high hydrostatic pressure in deep-sea hydrothermal vents prokaryotes, Extremophiles 19 2015 721
40. https://doi.org/10.1007/s00792-015-0760-3. References for C. marinimaniae (Kusube M, Kyaw TS, Tanikawa K, Chastain RA, Hardy KM, Cameron J, et al. Colwellia marinimaniae sp. nov., a hyperpiezophilic species isolated from an amphipod within the challenger deep, Mariana Trench. Int J Syst Evol Microbiol 2017;67:824
31), T. piezophilus (Dalmasso, C., Oger, P., Selva, G., Courtine, D., L’Haridon, S., Garlaschelli, A., et al. (2016). Thermococcus piezophilus sp. nov., a novel hyperthermophilic and piezophilic archaeon with a broad pressure range for growth, isolated from a deepest hydrothermal vent at the Mid-Cayman Rise. Syst. Appl. Microbiol. 39, 440
44. https://doi.org/10.1016/j.syapm.2016.08.003) and P. kukulkanii (Dalmasso, C., Oger, P., Selva, G., Courtine, D., L’Haridon, S., Garlaschelli, A., et al. (2016). Thermococcus piezophilus sp. nov., a novel hyperthermophilic and piezophilic archaeon with a broad pressure range for growth, isolated from a deepest hydrothermal vent at the Mid-Cayman Rise. Syst. Appl. Microbiol. 39, 440
44. https://doi. org/10.1016/j.syapm.2016.08.003).

variation in the stability between both enzymes caused by a difference in flexibility and hydration of the proteins [94]. Most molecular motion studies about pressure adaptation have been done in vitro, or investigating, for example, molecular dynamics [95]. Nevertheless, nowadays in vivo studies have gained in importance thanks to, for example, neutron scattering and NMR experiments that can examine timescales from few nanoseconds to hundreds of milliseconds [96
98]. It has been shown that some proteins are involved in HP adaptation as well as in adaptation to other stresses (Hsp60, Hsp70, OmpH, RecA, F1F0 ATPases, Cct and Tat2) [99]. A system highly studied under pressure is the Omp/ Tox system. The proteins ToxS and ToxR from P. profundum SS9 are responsible for regulating the genes that encode the membrane proteins OmpH, OmpL and OmpI. Pressure reduces the abundance and the activity of ToxR, which therefore upregulates the protein OmpH among others, the system acts as a piezometer. Regardless, the systems ToxS and ToxR do not confer HP adaptation and their role under pressure is not clear [11,100].

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The protein adaptation to extreme conditions is a balance between the imperative stability (higher number of bounds) to be functional and the flexibility (lower number of bounds) to be capable to adapt to different conditions [101]. One of the most studied enzymes is dihydrofolate reductase (DHFR). Studies comparing DHFR from the piezosensitive E. coli and from the facultative psychro-piezophile bacteria Moritella profunda reveals that applying pressure decreases EcDHFR’s activity and increases MpDHFR’s activity up to 50 MPa before diminishing its activity at higher HP. MpDHFR seems to have higher sensitivity to pressure due to its higher flexibility [102]. Such flexibility may explain the higher absolute activity of piezophile proteins [103]. However, most of the studies are done in protein-isolated solutions, which differs from their native state. An innovative quasi-elastic neutron scattering study examined the dynamics from whole cells of the piezophile Thermococcus barophilus and the piezosensitive Thermococcus kodakarensis microorganism under atmospheric pressure and 40 MPa. This study revealed that the HP adaptation on whole cells is based on an overall higher proteins’ flexibility within the cells and, in addition, on the modification of their hydration water layers [97]. Proteins from piezophiles may have a larger total volume of small internal cavities, which makes the protein more compressible and less sensitive to distortion caused by pressure [103]. Moreover, the presence of more cavities of small size allows water penetration at HP and consequently increases the hydration but, as seen in MpDHFR, cavities are not big enough to cause the protein denaturation but allow the protein to be more flexible. The presence of more small cavities could decrease the amount of water molecules contained in each cavity (a volume of 15A3 is necessary for a single water molecule and an increase of approximately 45A3 is required for each extra molecule [104]). It is important to consider that cavities are not mere “packing defects” but that they play a role in conformational changes and in controlling binding and catalysis of the proteins [104,105]. Generally, monomeric proteins are more resistant to pressure than oligomeric proteins. However, it has been shown that multimeric proteins may be adapted to resist pressure. For example, studies on the hyperthermophile and piezophile TET3 peptidase from Pyrococcus horikoshii indicate that the protein multimerizes into a dodecamer structure instead of conserving its classical barrel-shape multimer conformation. Dodecamer multimerization protects the hydrogen bonding between the different subunits and increases its stability against temperature and pressure up to 300 MPa [106]. A general extrinsic cell response to pressure-stress is the presence of piezolytes and other low weight organic compounds called osmolytes (e.g., sugars and amino acids) to protect cell macromolecules, such as proteins, from pressure modification [107] and therefore adapt their dynamics. Some piezophiles accumulate these low-weight molecules in response to an increase in pressure and others to a decrease, indicating in the latter case that the growth at lower pressure than optimal is perceived as a stress for these piezophiles [86]. For example, trimethylamine oxide (TMAO) is a pressure co-solute that helps proteins to remain active under HP in certain fishes and crustaceans, while the hyperthermophilic and piezophilic Thermococcus barophilus accumulates mannosyl-glycerate when grown in sub-optimal conditions (ambient pressure) [108]. Few studies have been done on pressure adaptation of higher complex pluricellular organisms. For example, it is thought that the regulation of N-methyl-D-aspartate receptor (NMDR), a cell membrane protein found in nerve cells, is responsible for the absence of the high-pressure nervous syndrome (HPNS) on deep dive mammalians [81,109]. The regulation of this protein may be done by modulating its interaction with lipids, for example by the presence of cholesterol, and thanks to the protein’s particular tertiary structure in piezo-tolerant organisms.

8.3.3 Membrane lipids Cells have the capability to customize their cell membrane lipid composition metabolically to maintain it in a functional liquid crystalline phase with specific functional physicochemical properties, such as fluidity, permeability and membrane curvature, in spite of environmental stresses. This process is known as homeoviscous adaptation [110]. Eukarya and Bacteria possess different lipids from those in Archaea but their homeoviscous adaptations have similarities (Fig. 8.4). Eukaryal and bacterial lipids are composed by straight hydrocarbon chains linked by ester bonds on 1,2-sn-glycerol and a phosphodiester-linked polar group or sugar. On the other hand, archaeal lipids have isoprenoid hydrocarbon chains bound by ether bonds on 2,3-sn-glycerol. Partly, the adaptation of archaea to extreme conditions may thus rise from their particular lipid structure [111]. The common routes of lipid adaptation of deep-sea microorganisms’ membranes are the change of the acyl chain length, the addition or removal of mono-unsaturated lipids and the change in the polar head groups [86,112]. Longer acyl chains translate into more rigid membranes, in contrary adding just one unsaturation to lipid chains makes the membrane more permeable and larger head groups increase the membrane fluidity by disrupting the membrane packing.

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Eukaryote/Bacteria

Archaea O

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+

N

FIGURE 8.4 Different kind of homeoviscous adaptation to change membrane permeability according to environmental conditions. Blue: mechanisms present in eukaryote and bacteria; red: mechanisms present in archaea; blue and red: adaptation mechanism found in all domains. At left, bacteria-type phospholipids, for example, one lipid present in cell membranes is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE (1), if organism change its lipid head group (phosphoethanolamine) to a bigger one (for example phosphatidylcholine, DPPC (2)) it will increase the membrane permeability. Another homeoviscous adaptation is to change the chain length, bacteria and eukarya can do it by adding or deleting two carbons, for example deleting two carbons from (2) would give to 1,2-dimyristoyl-sn-glycero-3-phosphocholine DMPC (3). Archaea may also change acyl chain length but with groups of 5 carbons (its subunit is the isoprene). Finally, membrane permeability also increases by adding unsaturations in the hydrophobic regions of lipids, it will lead to, for example, 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPC (4) and to all-cis-docosa-4,7,10,13,16,19hexa-enoic acid (5). Monounsaturated lipids may also be present in archaea, but it is still controversial. Hydrophobic region of lipids from archaea are based on isoprenoid structures (5 carbons) and they possess a variety of head groups. Archaea has the capability to create glycerol dialkyl glycerol tetraethers GDGT (dipolar lipids) with cyclopentane rings, which will decrease membrane permeability, as in (6). Dipolar lipids in absence of cyclopentane rings are represented in (7) and glycerol tribiphytanyl glycerol tetraethers GTGT (dipolar lipids crosslinked) in (8). Archaea also presents monopolar lipids (10) designed as dialkyl glycerol diether DGD and it exists monopolar lipids crosslinked (9) which will be less permeable. For further details, see [86].

In addition, psychrophilic bacteria present polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid and eicosapentaenoic acid, which, just as lipids with one unsaturation, increase the permeability of the membrane under low temperatures [113]. The function of PUFAs is not clear, one of the hypotheses is that they may play a role in cell division under HP, as demonstrated for Shewanella violacea [114]. Another hypothesis is that PUFAs, which also are highly specialized lipids from animals’ cell membranes, are produced by bacteria as an interaction with deep-sea animals [72]. Finally, another possibility is that, in addition to increase membrane permeability as mono-unsaturated lipids do, they may have an effect on energy production and conservation [115]. Besides that, archaea possess tetraether lipids, which may form a less permeable monolayer instead of the common bilayer. In addition, some archaeal species comprise lipids with cyclopentane rings and isoprenoid chains that are crosslinked, which decreases membrane permeability. The change in the different ratios from di- and tetra-ether lipids and the presence of cyclopentanes and crosslinked chains modifies as well the properties of the cell membrane. Only two studies have been done to examine the lipid composition under pressure: one on Methanocaldococcus jannaschii and another on T. barophilus [116,117]. Both present an increment in the diether: tetraether lipid ratio to counteract the increase in rigidity provoked by pressure on the cell membrane.

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Pressure biotechnological applications

HP application is mostly used in food processing since it does not affect non-covalent interactions (at least up to 2 GPa) and it can inactivate bacteria and viruses without changing markedly nutrients and flavors of food. Furthermore, pressure can change the reaction rates, which may favor the extraction of the required product [19]. Besides, pressure may be used for diverse biotechnological and biopharmaceutical applications, for example to explore new therapies [118,119], conserve vaccines [121], improve cryopreservation [121] or for orthopedics’ surgery [122].

8.4.1 Food industry 8.4.1.1 Food preservation HP (400—600 MPa) inactivates microorganisms, like yeast, molds and viruses. It affects the cell at different levels, such as nutrient transport and cell reproduction, lead to cell death [73,123]. Moreover, HP hardly affects low-molecular weight compounds (e.g. amino acids, vitamins and flavor molecules), so organoleptic and nutritional properties are only slightly modified [124]. Nevertheless, HP alone cannot inactivate bacterial spores and thus an association with other parameters, such as pH, chemicals or thermal processes may be needed. Nevertheless, pressure reduces considerably the working temperature, as 70  C instead of 180  C is enough to inactivate spores if it is combined with 600 MPa [73]. Such decrease in temperature can help preserve quality and minimize off-flavor generations. Therefore, HP techniques are useful as a complement on thermal processes but also to inactivate microorganisms on products where temperature cannot be applied. As an illustration, high pressure pasteurization of cold-pressed juices eliminates pathogens without impairment of its fresh-like qualities increasing the shelf life of the product [125,126]. HP extends the shelf life of a high variety of food products. For example, fresh shrimps treated at 435 MPa have a shelf life of 15 days, three times longer than the shelf life of the untreated shrimps [127]. Similarly, fresh cheese treated at 300
400 MPa has a shelf life at 4  C of 14
21 days, which is greatly higher than the 7 days for the untreated cheese [128]. Food is a complex matrix and inactivation efficiency depends on different factors as treatment conditions, microorganisms to inactivate and its food matrix characteristics. For example, meat treated at 300 MPa has a cooked like appearance but if it is processed at 100
200 MPa and 60  C, it is more tender than the untreated meat [129]. Consequently, inactivation conditions must be defined for every food product. HP is not efficient for low water content food (such as flour) or food with high content of air bubbles and if the food needs to be wrapped before treatment only plastic packaging is acceptable, as packaging material needs a compressibility of at least 15% [123].

8.4.1.2 Pre-treatment HP may also be applied as a pre-treatment. On the one hand, it has been demonstrated that the application of sublethal HP on cells gives them cross-resistance to other stresses. This opens the possibility to improve the survival of microorganisms of health interest during food processing and preservation. For example, the most studied probiotic, Lactobacillus rhamnosus, is more resistant to heat after an application of 100 MPa for 10 min [130], this cross resistance may be important to maintain it after milk processing. On the other hand, pre-treatment can be useful to facilitate the extraction of internal nutritional components. HP increases cell permeability, thus facilitating the mass transfer rate, and finally, increasing the release of extracts. It has been shown that the time extraction of caffeine from green tea leaves is reduced from 20 h to 1 min if a pressure of 500 MPa is applied; extraction of anthocyanin from red grape skin is increased by 23% by applying 600 MPa of pressure and the extraction yield of gingenosides from Panax quinquefolium root increases linearly between 100 MPa and 500 MPa [131].

8.4.2 Allergenicity and digestibility Several proteins can provoke allergic reactions caused by an immune disorder on the IgE binding. Because HP tends to denature proteins, it has been shown to induce a modification of their allergenicity [132], both on protein solutions and on food systems. For example, pressures of 300
700 MPa reduce the allergenicity of a ginkgo seed protein and of soybean allergens [133]. Another interesting example is the use of HP together with proteases to obtain hypoallergenic rice [134,135]. However, the effect of HP on allergenicity is not universal. There is no allergenicity change caused by pressure on almonds, or on the protein Mald1 from apples [136,137]. Mald1 native state possess a high internal cavity

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occupied by water [138] and therefore, pressure may not be able to conform significant hydration changes since the protein is already highly hydrated. HP may not only influence the allergenicity of the food products but can also help to increase its digestibility by exposing inaccessible sites of proteins and, thereby, enhancing the efficiency of protein hydrolysis. For example, the time required for proteolysis of β-lactoglobulin, the major allergen in cow’s milk, is reduced from 48 h to 20 min at 200 MPa [139].

8.4.3 Medical applications 8.4.3.1 Antiviral vaccines Several viruses are inactivated or dissociated by pressure. Under pressure, the atomic contacts among subunits are superseded by interactions with the solvent and therefore once pressure is released, viruses cannot come back to their native form. For example, pressure inactivates picornaviruses by causing the lack of VP4 from the intern capsid [140]. Both viruses with polyhedral and helicoidal symmetry are sensitive to pressure. Even so, not all viruses are equally reactive to pressure. For example, the foot-and-mouth-disease picanovirus is highly sensitive, but poliovirus is much more resistant to HP [140]. Interestingly, viruses re-associate under their fusogenic state under pressure, a less infectious and highly immunogenic form [140
142]. This is why high pressure has been suggested for antiviral vaccine development. It has been demonstrated that immunization against HP-inactivated virus is equally effective as against intact virus and has a higher immunity response than isolated viral subunits [41,143].

8.4.3.2 Bacterial ghosts Bacterial ghosts are empty cell envelopes, they are usually obtained by the expression of a lysis gene that lead to the cell material leakage. As their cell surface is not affected, they retain immunogenic properties and they are usually used as delivery systems for subunit or DNA based vaccines [144,145]. Antigen-presenting cells, like dendritic cells and macrophages, interacts very effectively with these bacterial ghosts. HP allows a new method to produce them, but in contrary to bacterial ghosts produced by lysis gene, HP-produced bacterial ghosts retains their cell material. This may be an advantage as it will not be necessary to insert the macromolecules to deliver after cell lysis. For example, HP bacterial ghosts have been obtained by applying a pressure of 100 MPa for 15 min to E. coli [146].

8.4.3.3 Vaccine preservation Most vaccines are heat labile, which confers problems of access to them as they need to be kept under refrigeration during their transport and preservation. HP may be a solution to this problem since it could confer heat resistance to attenuated virus. For example, it has been demonstrated that pressures of 310 MPa stabilizes attenuated poliovirus, one of the most heat labile vaccine, at temperatures of 37  C [120].

8.4.3.4 Cryopreservation Oocyte cryopreservation by vitrification is one technique used to maintain women’s fertility but blastocyst formation rate after this process is still low due to the production of ROS components, therefore techniques to improve it are being studied. As remarked above, sublethal HP stress makes the cells more resistant to thermal treatments and oocytes are not an exception. For example, pig oocytes, bovine and mouse blastocysts show a higher resilience against cryopreservation after receiving a non-lethal HP treatment [147
149]. Moreover, it has been demonstrated that HP treatment (20
40 MPa for 90
120 min) of bull and boar spermatozoa before cryopreservation preserve their viability, motility and fertility [121].

8.4.4 Biotechnological applications 8.4.4.1 Bio-purification An antigen may be purified from its medium by affinity chromatography due to a steric recognition with an antibody linked to a matrix. The recognition causes an increase in molecular volume and, as pressure causes a volume decrease, it could be useful to apply pressure to dissociate the product of interest without using drastic elution processes which reduce the lifetime of matrices [150]. This has been demonstrated for the recovery of β-galactosidase: four 15 min cycles of 150 MPa at 4  C recovers 32% of E. coli β-galactosidase compared with the 46% recovered by adding a

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solution of pH 5 11 [151]. Although the product yield is lower when using HP, the method is simpler and has a lower impact on matrices than the current elution process. HP ability to disrupt immune complexes has been proven on anti-prostate specific antibody from its antigen [151,152], its dissociation was increased by 22
37% when pressures from 140 to 550 MPa were applied. Pressure may also optimize the dissociation of amphiphilic biomolecules from a fixed adsorbent: 80% of Triton-X can be recovered form a bed absorption if a pressure of 250 MPa is applied on the system [153]. Finally, as pressure can dissociate aggregates, it may be used for the retrieval of proteins from inclusion bodies, i.e. aggregates of incompletely folded proteins. Traditionally, to separate proteins from inclusion bodies it is necessary to use high concentrations of agents that destroy the spatial structure of proteins with a necessary subsequent difficult refolding. However, a pressure of 240 MPa is effective to dissociate the inclusion bodies of endostatins and a subsequent application of 40 MPa induces the refolding of 78% of the protein [154].

8.4.4.2 Modulation of cell activity Already relative low applied pressures can enhance the cell activity to our profit, as for example observed at 10 MPa for ethanol production by Saccharomyces cerevisiae [155] which occurs three times faster than at atmospheric pressure. Another example is on the fermentation by Clostridium thermocellum [156]. This Clostridium transforms cellobiose to biofuels but also synthesizes additional non-desired products (e.g. acetate, H2, CO2). When the fermentation happens under pressure of 7 or 17 MPa, the microorganism modifies the metabolic pathways and shifts the production to desired metabolites, reaching an increase of 60-fold. However, as HP is considered a stress for most cells, it will translate into the expense of additional energy for cell maintenance and growth, reducing the product yield. For example, HP reduces the fermentation rate of lactic acid fermentation due to the inhibitory effect on the growth of Streptococcus thermophilus, Lactobacillus bulgaricus and Bifidobacterium lactis [157]. To avoid the loss of efficiency rates under HP, efforts are made to enhance the resistance of mesophilic microorganisms to HP, leading to organisms with higher performance under HP [158].

8.5

Biotechnological applications of piezophiles

There are more than 3000 enzymes identified to date and most of them are used for biotechnological applications. Nevertheless, these enzymes are not enough to respond to the new technological challenges that appear each day [159]. One of the problems is the stability of the enzymes under industrial conditions, so it is necessary to find enzymes which are highly resistant to harsh conditions and here deep-sea enzymes may play a major role. Pressure-stable enzymes are able to conduct biocatalysis under HP, modifying therefore specific enzymatic reactions, and have even higher thermostability. For example, Biolabss has already commercialized a DNA polymerase from a hyperthermophile and piezophile Pyrococcus, which presents a half-life of 23 hours at 95  C (Deep Vent DNA Polymerase, Catalog #M0258L, New EnglandBioLabs, Inc) [160]. Moreover, piezophilic enzymes may possess different properties than their surface homologues, which may open new possibilities for industry [161]. The market for industrial enzymes is growing every year and the exploitation of extremozymes is a huge and mostly unexplored resource [162]. As we have seen, lipids from extremophiles are unique. Archaea in particular contain lipids which confer to the cell a highly stable and impermeable membrane. The unique stability may be used in biotechnological or pharmaceutical applications, for example to protect therapeutic peptides from the harsh environment of the gastrointestinal tract [163,164]. Additionally, lipids from many piezophilic bacteria comprise omega 3-PUFAs within their cell membrane, which are precursors of hormones in many animals. Consequently, it could be used for treatment of hypertriglyceridemia diseases and clinical studies for this purpose have already been approved [161]. The high marine biodiversity has woken up the interest to search new compounds with biopharmaceutical potential [165]. Marine derived molecules may present beneficial functions as, for example, antitumor potential, pain reliever, or antimicrobial activities [161,164,165]. For instance, studies have identified some bioactive compounds from marine echinoderms (such as the piezotolerant Cucumaria frondosa) with antiproliferative, antimetastatic and immunomodulatory activities [166].

8.6

Conclusion and future perspectives

All pressure-specific impacts allow to modify macromolecules and cells in unique ways. For example, HP is capable to alter reaction rates since it induces a shift towards the state with the smallest volume. Moreover, such volume

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modification may be also useful for purification of chromatography systems and, since HP is able to change protein structures, to decrease food allergenicity and produce antiviral vaccines. Pressure affects most of cell functions and among them, increases cell permeability, which can be advantageous to facilitate food extraction. Furthermore, application of non-lethal pressures provides the cell with resistance to other stress, such as temperature. This characteristic has been largely studied as a pre-treatment for probiotics, vaccines’ preservation and oocytes’ cryopreservation. Finally, employment of lethal pressures inactivates undesired microorganisms without affecting covalent bonds of nutrients, such technique is already used by food industry to increase products’ shelf life. Food industry was pioneer in using pressure to inactivate microorganisms or as a pre-treatment and, although HP processing still constitutes a minority, the development of HP equipment and the increase in manufacturers have boost the number of HP industrial machines to more than 300 and this global market to $9.8 billion in 2015 [123]. Nevertheless, pressure capability does not stop here. Promising applications, such as antiviral vaccines, the use of pressure for bio-purification or to modify cell activities has led to a greater interest on this physical parameter. In addition, piezophilic organisms open a range of possibilities to use pressure-adapted molecules, and for example, give access to highly thermostable enzymes, which may even possess favorable enzymatic rates. Moreover, they provide a major opportunity to find new bioactive molecules for medicine and biotechnological applications, such as antitumor or pain reliever molecules found in marine biosphere.

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Chapter 9

Fructanogenic halophiles: a new perspective on extremophiles ¨ ner Gu¨lbahar Abaramak*, Onur Kırtel* and Ebru Toksoy O Bioengineering Department, IBSB—Industrial Biotechnology and Systems Biology Research Group, Marmara University, Istanbul, Turkey

9.1

Introduction

Halophiles are microorganisms that require certain concentrations of salt to survive, and they are found in both Eubacterial and Archaeal domains of life. In Eubacteria, halophiles are a very heterogeneous group, having members in at least eight different phyla. In Archaea however, halophilism is strictly limited to the members of the Haloarchaea class and the ‘Nanohaloarchaeota’ subphylum [1]. According to their degrees of salt requirements, halophiles are classified into three groups: slight (0.34 0.85 M salt), moderate (0.85 3.4 M salt), and extreme halophiles (3.4 5.1 M salt) [2]. There are also many halotolerant Eubacteria in nature, which do not rely on salt to thrive but can withstand significant concentrations of it. Halohilic Archaea are of special interest among halophiles since they dominate the most hypersaline environments on Earth, and many of them can even survive salt concentrations close to saturation levels [3]. Halophilic Archaea are found under the class of Haloarchaea (also formerly known as Halobacteria) within the Archaeal domain of life, which descend from methanogenic Archaea [4]. Throughout their evolution they developed strategies to adapt to salt concentrations that are lethal to most mesophilic organisms. The first and most commonly observed osmoadaptation strategy in Haloarchaea is the cytoplasmic accumulation of K1 ions, which is also called the salt-in strategy. Most Haloarchaea can accumulate high concentrations of cytoplasmic K1 while exporting Na1 ions to the extracellular space, thus balancing the osmotic pressure of the cell [5]. Another strategy, usually seen in most halophilic Eubacteria and some Haloarchaea is the synthesis of compatible solutes like betaine and ectoine in the cytoplasm to achieve osmoregulation [6]. Another intriguing aspect of Haloarchaea is that they adopted an aerobic lifestyle unlike any other class of Archaea. This is most probably due to extensive gene transfer events happened between them and their halophilic Eubacterial neighbors. As Haloarchaea adopted hundreds of gene families from Eubacteria, this drastically changed their metabolism, especially the genes in their carbohydrate utilization subsystems [7]. One of those gene families express Glycoside Hydrolase J (GH-J) clan enzymes, which take part in the synthesis and breakdown of fructose polymers called fructans. Intriguingly, GH-J clan genes are present only in the Haloarchaea class, and not in other Archaeal lineages [8]. Thus, this book chapter aims to give an insight into these Haloarchaeal GH-J clan enzymes and potential roles fructans synthesized by them in hypersaline environments.

9.2

Fructans

Fructans are polysaccharides made of fructose that are already present in a plethora of both Gram-negative and Grampositive Eubacteria, some flowering plants and a limited number of fungi, while they are completely absent in animals [9]. Organisms that are able to accumulate fructans are called fructanogenic organisms. In nature, mainly two types of fructans are found: levans and inulins. Levans are fructose polymers that carry fructofuranosyl moieties bound with β-2,6 linkages that make up the backbone of the polysaccharide with varying number of branches at β-2,1 linkage positions. They are produced by various Gram-positive and Gram-negative Eubacterial genera such as Bacillus, Brenneria, Clostridium, Erwinia, Gluconacetobacter, Lactobacillus, Pseudomonas, Streptococcus, Zymomonas, etc., and are *These authors have contributed equally to this work. Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00009-5 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 9.1 Structures of fructans found in nature. Agavins and graminans are specific to plants. Hexagon: glucose, pentagon: fructose.

usually functional components of the biofilms produced by these microorganisms; providing water to the biofilm structure, acting as storage carbohydrates, being virulent factors against plants or even releasing small diffusible fructans that may take part in signaling processes [10]. The other main type of fructan, inulin, carries fructofuranosyl residues bound with β-2,1 linkages on the polysaccharide backbone, and may show β-2,6 branching [11]. Although found in substantial amounts in plants such as chicory and Jerusalem artichoke, to date, inulin synthesis by microorganisms has only been demonstrated in Gram-positive genera such as Bacillus, Lactobacillus, Leuconostoc, Streptococcus, and Weissella [12,13]. It is still not fully understood why compared to levan, inulin is synhtesized by a much more limited number of Eubacterial species. However, the first thing that draws the attention is that while most levan-producer Eubacteria are plant-associated species, most inulin-producer microorganisms are lactic acid bacteria, living inside or in close contact with animals. Apart from levan and inulin, there are also some plant-specific fructans such as agavins (found in Agave spp.) [14] and graminans (mostly found in cereals [15]), which show more complex branching patterns. Since this chapter focuses on fructans from halophilic microorganisms, plant-specific fructans will no longer be discussed hereafter. Fig. 9.1 shows the chemical structures of common fructans found in nature. It is noteworthy to indicate that although levans and inulins are found in both Eubacteria and plants, the difference in their molecular weights is huge: microbial fructans usually have very high degrees of polymerization (DP, up to 10,000 fructose units), while plant fructans are much shorter (DP is usually below 100), which greatly affects their physicochemical properties and functionalities [10].

9.3

Microbial fructan synthesis mechanism

Synthesis of fructans in microorganisms relies on the action of fructosyltransferases (FTs) called Glycoside Hydrolase 68 (GH68) family enzymes. This family includes two different enzymes, namely inulosucrase (EC 2.4.1.9) and levansucrase (EC 2.4.1.10), which are responsible from the synthesisinulin and levan, respectively. Fructan biosynthesis happens in three stages and starts with sucrose, the most common fructosyl donor. A GH68 family enzyme first cleaves the fructose moiety of sucrose (hydrolysis), then transfers it to another sucrose molecule (transfructosylation), creating either 6-kestotriose (a levan-type trisaccharide) or 1-kestotriose (an inulin-type trisaccharide). Successive transfers of fructose residues to these molecules create elongating chains of levans or inulins (polymerization) while releasing glucose to the medium [16]. Almost all GH68 family enzymes are extracellular and crystal structures of six of them have been solved: Bacillus megaterium levansucrase (PDB ID: 3OM2), Bacillus subtilis levansucrase (PDB ID: 1OYG), Erwinia amylovora levansucrase (PDB ID: 4D47), Erwinia tasmaniensis levansucrase (PDB ID: 6FRW), Gluconacetobacter diazotrophicus levansucrase (PDB ID: 1W18), and Lactobacillus johnsonii inulosucrase (PDB ID: 2YFR). In addition to GH68 family enzymes, the other fructan-active enzyme family is the Glycoside Hydrolase 32 (GH32) family. The best known enzyme belonging to this family is invertase/β-fructofuranosidase, which mainly

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hydrolyzes sucrose but may also show transfructosylation activity at high sucrose concentrations, producing fructooligosaccharides (FOS) [17]. Other well-known members of this family are levanases and inulinases, which hydrolyze levans and inulins, respectively, either in an endo- or exo- fashion [18,19]. In addition to Eubacteria, a wide range of fungal species also harbor GH32 family enzymes, which can easily utilize fructans as energy sources [20]. GH68 and GH32 family enzymes together make up the GH-J clan. GH-J clan enzymes allharbor a 5-bladed β-propeller domain surrounding the catalytic core. In addition to this, a β-sandwich domain is also encountered in GH32 family enzymes, which may be taking part in substrate recognition processes. All enzymes from the GH-J clan show Koshland retaining mechanism: the configuration of the substrate molecula is not altered during catalysis [16].

9.4

Fructanogenic halophiles

Fructanogenic organisms are found under several domains of life [1]. However, presence of fructans in halophiles have long been neglected. Presence of such multifunctional polymers in hypersaline habitats may lead to discovery of yetunknown dynamics in those environments. First reports on possibly fructan-producing halophilic Eubacteria date back to 1957 and 1985 [21,22]. Researchers have isolated Gram-negative, non-motile, aerobic rods from “ropy” herring brine and observed that these microorganisms produce polysaccharides mainly composed of fructose. However, the techniques used at that time could not exclude the possible presence of other monomeric sugars in the polymer structure. In 2009, Halomonas smyrnensis AAD6 was isolated from the soil of C¸amaltı Saltern in Western Turkey, and identified as the first halophilic levan producer [23]. Levan produced by this moderately halophilic bacterium was characterized as a highly linear polymer with a molecular weight of over 2 3 106 Da [24], which is equal to more than 12,000 DP. In 2015, another fructanogenic halophilic bacterium, Chromohalobacter japonicus BK AB18, isolated from a mud crater in Indonesia, was shown to be able to produce levan efficiently [25]. Another fructanogenic bacterium, Bacillus licheniformis BK AG21, was among the species isolated from the same mud crater [26], however strains of this species are usually halotolerant but not halophilic. In another study, three different Bacillus strains, namely Bacillus endophyticus SH, Bacillus subtilis WA and Bacillus subtilis MO, were isolated from honey [27]. All strains showed maximal growth in the presence of 4 8% NaCl and produced fructans. The same isolates also showed osmotolerant characteristics, being able to grow at sucrose concentrations up to 20%. Although fructans from various Eubacterial and plant sources have been studied upon for over a century now, studies focusing on fructanogenic halophiles started to appear in the literature only in the last decade, and fructanogenic Archaea have never been investigated. As discussed in our previous studies, putative GH-J clan genes that are responsible for fructan biosynthesis are present in many Archaeal species [8,28]. What’s interesting is that they only appear within the halophilic Haloarchaea class, but not in other Archaeal classes. Haloarchaea are known to have adopted hundreds of chimeric gene families from Eubacteria, and putative GH68 family genes have also been predicted to be among those genes [7]. Thus, the best possible explanation to the presence of these genes in Archaea is that they appeared in the genomes of these microorganisms together with other gene families during their adaptation to an aerobic lifestyle, as a result rendering them to be efficient utilizers of various carbohydrates. Although it is not possible to foresee if these putative GH68 family genes are functional without in vitro experiments, their preservation in more than 50 different species of Haloarchaea from all three orders of the Haloarchaea class (Halobacteriales, Haloferacales and Natrialbales) is a promising indicator that fructanogenic traits are not exceptional but rather widespread in extremely halophilic Archaea. To further support this hypothesis, some properties of the putative Haloarchaeal GH68 family enzymes are discussed in the following section.

9.5

Putative GH68 family enzymes of haloarchaea

It is imperative to remember that the functionalities of GH-J clan enzymes can be altered drastically even by single amino acid mutations. For instance, the residue R360 in Bacillus subtilis levansucrase (H304 in H. smyrnensis AAD6 levansucrase; GenBank accession number AGG11046.1) is known to be crucial for the polymerization process. Replacing this residue with a smaller amino acid can result in a complete change in the product spectrum [29,30], for instance synthesizing FOS instead of long-chain fructans. Other critical amino acids in levansucrases that take part in the catalysis are as follows (residue numbers are given according to their homologues in the levansucrase of H. smyrnensis AAD6): D47, the nucleophile; W130, substrate-binding residue; R201, forms a salt bridge with the transition state stabilizer; D202, the transition state stabilizer; E286, the general acid/base catalyst. When the structures of putative Haloarchaeal GH68 family enzymes are investigated via multiple sequence alignment, it is observed that the critical residues mentioned above are strictly conserved in almost all of them (Fig. 9.2).

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FIGURE 9.2 Constraint-based multiple sequence alignment of a selection of GH68 family proteins from Eubacteria (Erwinia amylovora, Bacillus subtilis, Halomonas smyrnensis AAD6) andArchaea (Haloarcula amylolytica JCM 13557, Halogeometricum limi, Natronococcus jeotgali DSM 18795). Amino acid residues that are known to play a role in catalysis are shown in red. Alignment was carried out via NCBI COBALT web tool.

Most enzymes from halophilic microorganisms show activity at salt concentrations that would easily denature most mesophilic enzymes. It is known that halophilic proteins that are exposed to high salt concentrations in their natural environments (for instance, an extracellular enzyme in a hypersaline lake, or an intracellular protein that is subjected to molar concentrations of accumulated cytoplasmic K1 ions) are known to have developed strategies to stay soluble and active under those conditions. The best known strategy is evolving increased numbers of negatively charged(acidic) residues (D and E) on the surface of the proteins, thus attracting water molecules and preserving the hydration shell [5,31]. For instance, H. smyrnensis AAD6 levansucrase was reported to harbor a total number of 64 negatively chargedand 30 positively charged amino acids, and a theoretical pI value of 4.35 [32]. Majority of these acidic residues were far from the catalytic core and exposed outside on the protein surface, thus enabling this enzyme to show activity at almost saturated NaCl concentrations while the maximum activity was observed at 3.5 M NaCl. A similar trend is also observed in putative Haloarchaeal GH68 family enzymes. For instance, ratio of negatively charged to positively charged residues of putative GH68 family enzymes from Haloarchaeal members Halostella salina, Halogeometricum rufum and Natrialba taiwanensis are 71:34, 77:34, and 69:30, respectively (according to ExPASy ProtParam, http://web.expasy. org/protparam/). For levansucrases from mesophilic Eubacterial species such as Erwinia amylovora and Pseudomonas syringae, these ratios are 54:35 and 53:34, respectively. This clearly shows that as the environments in which these microorganisms thrives in increase in salt content (in other words, going from mesophiles to halophiles or vice versa), these enzymes evolve higher numbers of acidic residues to stay active and soluble under such circumstances. Presence

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of significantly increased numbers of acidic residues on putative Haloarchaeal GH68 family enzymes may indicate that the salt concentration in the environment still exerts an effective selection pressure on these proteins, increasing the possibility that those relative genes are actively transcribed, synthesizing fructans. Fructan biosynthesis requires the presence of a fructosyl donor, which is sucrose in most cases. If Haloarchaea are effectively producing fructans in their natural environments, that means sucrose should be present. Although being the most frequently encountered group, Haloarchaea are not the only residents of hypersaline habitats. They are usually accompanied by a plethora of Eubacteria, viruses, unicellular photosynthetic Eukarya, some fungi, and even some insects [4]. In most cases, extremely halophilic algae are the only autotrophic organisms in such habitats, with extremely halophilic Dunaliella spp. being the best known ones, some species being able to thrive at salt concentrations close to saturation levels [33,34]. It is possible that sucrose produced by these algae may effectively be used by some Haloarchaea to synthesize fructans as part of a yet-unknown symbiosis. Haloarchaea may use those polysaccharides as part of their biofilms, making use of them in a similar manner to Eubacteria. Such phenomenons have never been experimentally investigated before, but remain as an intriguing aspect that may reveal some unknown dynamics in hypersaline environments. Another thing to keep in mind that most Haloarchaea do not harbor any putative GH-J clan genes, meaning that possible functions of fructans in those microorganisms may be compensated by other polymeric substances. Also, Haloarchaea that harbor putative GH68 family genes do not necessarily require sucrose to be present as a carbon source, which means that fructans are not essential for their survival, but may be mediators in some symbiotic interactions.

9.6

Levan and levansucrase from Halomonas smyrnensis AAD6

Halomonas smyrnensis AAD6 is a moderately halophilic bacterium from C ¸ amaltı Saltern Area on the western coast of Turkey. This bacterium is the first halophilic fructan producer reported in the literature, and effectively converts sucrose into levan at high titers [35]. Levansucrase from H. smyrnensis (Hs_Lsc) is a 416 amino acid long protein. Its theoretical pI value was calculated to be 4.35. The enzyme is synthesized by the bacterium to the extracellular space, and do not carry a predictable N-terminal signal peptide, like most other levansucrases from Gram-negative Eubacteria. It shows 68% sequence identity with E. amylovora levansucrase (PDB ID: 4D47). To investigate deeper on the properties of this halophilic levansucrase, the enzyme was recombinantly expressed and purified in a previous study [32]. The purified enzyme was used for some characterization studies and revealed some interesting aspects. Firstly, at least 1.5 M NaCl is required for the enzyme to be active, and it shows maximal activity at 3.5 M NaCl, which is a unique property among levansucrases characterized to date. In substrate specificty studies it was revealed that Hs_Lsc polymerizes sucrose into levan at rather low substrate concentrations (0.15 M sucrose), while catalysis was shifted from polymerization to transfructosylation (FOS production instead of long-chain levan) at elevated subsbtrate concentrations (1.5 M sucrose), which is a common trait among levansucrases [36]. Hs_Lsc could produce a wide range of FOS; namely 1-kestotriose, 6-kestotriose, 1-kestotetraose, 6-kestotetraose, blastose, inulobiose, levanbiose, and neokestose. Increasing NaCl concentration in the medium did not have an effect on the profile of saccharides synthesized, but increased their concentrations in the final solution. Levansucrases can also transfer fructose to other saccharides than sucrose, producing valuable oligosaccharides [37]. Hs_Lsc was able to use galactose, lactose, cellobiose and L-(1)-arabinose as fructosyl acceptors, producing fructosylated versions of these saccharides, which makes this enzyme a valuable source for the production of prebiotic food additives. Additionally, it was observed that Hs_Lsc could use raffinoseboth as a fructosyl acceptor and donor, catalysing the synthesis of melibiose or three different raffinose-type oligosaccharides. Activity of Hs_Lsc was maximal at 37  C and pH 5.9, while at 15  C polymerization (levan formation) was more prominent. Intriguingly, the enzyme was still active at sub-zero temperatures and produced levan, thanks to reaction medium still being in liquid form due to its high salt content. Unlike other levansucrases reported in the literature, Hs_Lsc was very vulnerable to the presence of most divalent cations: presence of even 1 mM Co21, Cu21, Fe21, and Ni21 completely abolished its activity, while 1 mM EDTA enhanced it 1.3-fold. An in silico genome-scale model of H. smyrnensis AAD6 was constructed to improve its levan productivity [38]. The interrelations between its metabolic pathways and levan biosynthesis mechanisms were elucidated by this model. Results showed that metabolic pathways of glycolysis, fructose-mannose metabolism and pentose-phosphate pathway had significant relationships with levan biosynthesis. The major finding of this study was the significant effect of mannitol on synthesis of levan. Supplementing 30 g/L of mannitol to the sugar-based medium increased levan production by two-fold. Also, supplementation of mannitol increased sucrose hydrolysis rate, while fructose uptake rate was decreased.

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Halomonas levan and its chemically modified forms were used to investigate their anti-tumor activity to develop polysaccharide-based anticancer therapeutics [39]. MCF-7 (human breast adenocarcinoma), A549 (human lung adenocarcinoma), AGS (human gastric adenocarcinoma), and HepG2/C3A (human liver hepatocellular carcinoma)cell lines were used to assess anti-cancer activities of Halomonas levan and its chemically modified forms. Results showed that introducing aldehydes to linear levan structure exhibited greatly improved anti-cancer activity. Levan derivatives had high anti-cancer activity and low cytotoxicity to normal cells, and exhibited enhanced caspase 3/7 activity. These features makes them promising anti-cancer agents. In another study with Halomonas levan, its antiproliferative effect on MCF-7 cell lines were investigated. The study showed that antiproliferative effect of levan depends on time and concentration. Cell apoptosis and oxidative stress were related with its antiproliferative effect. An increase in the percentage of cells in the sub-G1 phase and an increase in caspase 3/7 positive cell percentage indicate that cell apoptosis was increased by Halomonas levan. mRNA expressions of p27 and p53 were also increased. Sulfated Halomonas levan exhibits anticoagulation and heparin mimetic activity [40]. Anticoagulation inhibitory activity of sulfated Halomonas levan was shown to be superior to heparin. The study also showed that increasing sulfation degrees of levan increases anticoagulant activity. Sulfated levan is a promising biomaterial with heparin mimetic activity and high biocompatibility for cardiac tissue engineering applications such as tissue scaffolds and functional, bioactive thin films. Halomonas levan is biodegradable and biocompatible. Also, it shows prebiotic, hypocholestrolamic, cellproliferating, irritation-reducing effects and also is a promising component that can be used as a coating material for drugs. Halomonas levan can be used as a carrier system for peptide-/protein-based [41] or antibiotic drugs such as encapsulated vancomycin [42]. These studies point out that halophilic microorganisms show unique traits that are not observed in their mesophilic counterparts, and halophiles can effectively be utilized for novel applications related to human health and food in the future where biotechnological processes will depend on the usage of saline water in a world with ever-decreasing freshwater supplies.

9.7

Conclusions and future directions

Apart from their various functionalities in natural phenomena, fructans are also attractive polysaccharides for humanrelated applications thanks to their interesting physicochemical and physiological characteristics. Being indigestible fibers, both levan and inulin show promising prebiotic effects in the gut, enhancing the numbers of beneficial microorganisms [43,44]. In the functional food additives market, inulin and inulin-type FOS still have the largest market share, and their prebiotic effects are well-established. However, inulin production in industrial scale still relies on plant-based inulin. Levan production on the other hand is still an expensive bioprocess and requires the development of costeffective strategies [35]. Halophiles have proven to be promising organisms for obtaining fructans at high titers with lower production costs thanks to usage of non-sterile, saline production media, and their fructosyltransferase enzymes exhibit many unique characteristics. To further discover the full potential of fructans both in human-related applications and their possible functionalities in nature, fructanogenic Haloarchaea are promising candidates.

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Microbial invertases: a review on kinetics, thermodynamics, physiochemical properties. Process Biochem 2015;50(8):1202 10. [21] Lindeberg G. Lævan-forming halophilic bacteria. Nature 1957;180(4595) 1141-1141. [22] Magnu´sson H, Mo¨ller A. Ropiness in the brine of sugar-salted herring. Int J Food Microbiol 1985;1(5):253 61. ¨ ner E, et al. High level synthesis of levan by a novel Halomonas species [23] Poli A, Kazak H, Gu¨rleyenda˘g B, Tommonaro G, Pieretti G, Toksoy O growing on defined media. Carbohydr Polym 2009;78(4):651 7. ¨ ner E. Effective stimulating factors for microbial levan production by Halomonas [24] Kazak Sarilmiser H, Ates O, Ozdemir G, Arga K, Toksoy O smyrnensis AAD6T. J Biosci Bioeng 2015;119(4):455 63. [25] Nasir D, Wahyuningrum D, Hertadi R. Screening and characterization of levan secreted by halophilic bacterium of halomonas and chromohalobacter genuses originated from Bledug Kuwu Mud Crater. Proc Chem 2015;16:272 8. [26] Wahyuningrum D, Hertadi R. Isolation and characterization of levan from moderate halophilic bacteria Bacillus licheniformis BK AG21. Proc Chem 2015;16:292 8. [27] Abdel Wahab W, Saleh S, Karam E, Mansour N, Esawy M. Possible correlation among osmophilic bacteria, levan yield, and the probiotic activity of three bacterial honey isolates. Biocatal Agric Biotechnol 2018;14:386 94. ¨ ner E, Van, den Ende W. The fructan syndrome: evolutionary aspects and common themes among plants and [28] Versluys M, Kirtel O, Toksoy O microbes. Plant Cell Environ 2017;41(1):16 38. [29] Chambert R, Petit-Glatron M. Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis. Biochem J 1991;279(1):35 41. [30] Ortiz-Soto M, Rivera M, Rudin˜o-Pin˜era E, Olvera C, Lo´pez-Munguı´a A. Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties. Protein Eng Des Select 2008;21(10):589 95. [31] Lenton S, Walsh D, Rhys N, Soper A, Dougan L. Structural evidence for solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations. Phys Chem Chem Phys 2016;18(27):18054 62. ¨ ner E. Levansucrase from Halomonas smyrnensis AAD6T: first [32] Kirtel O, Mene´ndez C, Versluys M, Van den Ende W, Herna´ndez L, Toksoy O halophilic GH-J clan enzyme recombinantly expressed, purified, and characterized. Appl Microbiol Biotechnol 2018;102(21):9207 20. [33] Oren A. The ecology of Dunaliella in high-salt environments. J Biol Res—Thessaloniki 2014;21(1):23. [34] Henley W, Cobbs M, Novoveska´ L, Buchheim M. Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake. J Phycol 2018;54(4):483 93. ¨ , Toksoy O ¨ ner E. Development of a cost-effective production process for Halomonas levan. [35] Erkorkmaz B, Kırtel O, Ate¸s Duru O Bioprocess Biosyst Eng 2018;41(9):1247 59. [36] Santos-Moriano P, Fernandez-Arrojo L, Poveda A, Jimenez-Barbero J, Ballesteros A, Plou F. Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions. J Mol Catal B: Enzym 2015;119:18 25. [37] Li W, Yu S, Zhang T, Jiang B, Mu W. Recent novel applications of levansucrases. Appl Microbiol Biotechnol 2015;99(17):6959 69. ¨ , Arga K, Toksoy O ¨ ner E. The stimulatory effect of mannitol on levan biosynthesis: lessons from metabolic systems analysis of [38] Ates O Halomonas smyrnensis AAD6T. Biotechnol Progress 2013;29(6):1386 97. ¨ ner E. Investigation of anti-cancer activity of linear and aldehyde-activated levan from Halomonas smyrnensis [39] Kazak Sarilmiser H, Toksoy O AAD6T. Biochem Eng J 2014;92:28 34. [40] Erginer M, Akcay A, Coskunkan B, Morova T, Rende D, Bucak S, et al. Sulfated levan from Halomonas smyrnensis as a bioactive, heparinmimetic glycan for cardiac tissue engineering applications. Carbohydr Polym 2016;149:289 96. ¨ ner E, Akbu˘ga J. Levan-based nanocarrier system for peptide and protein drug delivery: optimization and influence [41] Sezer A, Kazak H, Toksoy O of experimental parameters on the nanoparticle characteristics. Carbohydr Polym 2011;84(1):358 63.

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¨ ner E, Akbu˘ga J. Development and characterization of vancomycin-loaded [42] Sezer A, Kazak Sarılmı¸ser H, Rayaman E, C ¸ evikba¸s A, Toksoy O levan-based microparticular system for drug delivery. Pharmaceut Dev Technol 2015;22(5):627 34. [43] Vandeputte D, Falony G, Vieira-Silva S, Wang J, Sailer M, Theis S, et al. Prebiotic inulin-type fructans induce specific changes in the human gut microbiota. Gut 2017;66(11):1968 74. [44] Hamdy A, Elattal N, Amin M, Ali A, Mansour N, Awad G, et al. In vivo assessment of possible probiotic properties of Bacillus subtilis and prebiotic properties of levan. Biocatal Agric Biotechnol 2018;13:190 7.

Chapter 10

Applications of sulfur oxidizing bacteria Kavita Rana1, Neerja Rana2 and Birbal Singh3 1

University of Horticulture and Forestry, Nauni, India, 2University of Horticulture and Forestry, Nauni, India, 3Indian veterinary Research Institute-

Regional Station, Palampur, India

10.1

Introduction

Being one of the essential nutrients, sulfur subsidizes much more than that of biogeochemical cycling. It occurs in both organic and inorganic amalgamation with three states of oxidation viz., 22 (reduced organic Sulfur and sulfide), 0 (Sulfur in elemental form) and 16 (sulfate) are most substantial in environment. Sulfates that are used in other biological processes are formed as a result of sulfur oxidation, which is most important step in sulfur cycle. The sulfur cycle consists of oxidative and reductive sides. Sulfate on the reductive side functions as an electron acceptor in metabolic pathways used by a wide range of microorganisms and is converted to sulfide. On the oxidative side, reduced sulfur compounds such as sulfide serve as electron donors for phototrophic or chemolithothrophic bacteria which convert these compounds to elemental sulfur or sulfate [1]. A situation in which the reductive and oxidative sides of this cycle are not in balance could result in accumulation of intermediates such as sulfur, iron sulfide and hydrogen sulfide. In the overall cycling of sulfur, microorganisms perform an imperative role. Microflora has the greatest potential for both mineralization and successive conversions of states of sulfur according to requirement. Thus this transforamation of sulfur from inorganic to organic pool is wholly caused by this microbial biomass. These makeovers in a biogeochemical cycle are due to many oxidation and reduction reaction. For example, sulfur oxidizing bacteria (SOB) oxidize H2S, a reduced form of sulfur to sulfate. The mentioned transformations are carried out by sulfate reducing bacteria as well as some other specific ones. These processes are energy generating where thiosulfate or elemental sulfur act both as electron donor and electron acceptor which leads to the formation of sulfide and sulfate pool [2]. The bacteria of the sulfur cycle, specifically sulfate reducing and sulfide oxidizing bacteria play an instrumental role in many environmental and industrial settings. There are somewhat hindrances in environmental or processing problems due the activities of certain bacteria. Though, exploitation of their activities under meticulous circumstance could resolve problems come across in the petroleum and mining industries. For instance, corrosion is caused by Sulfur reducing bacteria attributable to production of H2S in oil reservoirs during production, handling and conveyance of crude and by products of petroleum. Moreover releases of H2S from manure pits, biogas generation from animal dung are featured to the activity of sulfate reducing bacteria [2]. On the other side sulfur oxidizing bacteria in combination sulfur reducing bacteria can unravel environmental glitches encountered by mining activities like acid mine drainage. Apart from this impact of biological treatment of acid mine drainage, problems like bioleaching of recalcitrant minerals, exclusion of H2S, treatment of sour gases and sulfide polluted water from various industrial waste are cured by sulfur oxidizing bacteria [3] through unwanted oxidation of minerals and waste rocks [4].

10.2

Oxidation behavior of sulfur oxidizing bacteria

Sulfur bacteria are capable of using inorganic sulfur compounds, including sulfide, elemental sulfur (S0), thiosulfate (HS2O32 -), and sulfite (HSO32 -), as their energy source. The sulfur oxidizing bacteria are found in almost every nook and corner of environments where there is availability of reduced Sulfur compounds [5]. The Sulfur oxidizing bacteria are gram negative bacteria. They are classified into two types based on their metabolic function viz., chemolithotrphs and photoautotrophic. Chemolithotrophs are the ones those grow on supplement of oxidizable sulfur compounds such Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00010-1 © 2020 Elsevier Inc. All rights reserved.

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PART | I Physiological aspects

as Thiobacillus neapolitanus, Thiobacillus thioxidans (extreme acidiophiles), Thiobacillus thiospora, Thiobacillus denitrificans (facultative denitrifiers), Thiobacillus halophilus (halophiles) and Thiobacillus ferrooxidans (acidophilic ferrous iron-oxidizers). Photoautotrophic are the ones those require light as energy source includes Thiobacillus aquaesulis (moderate thermophiles), Thiomicrospira thvasirae, Thiobacillus novellus, Paracoccus denitrificans Thiobacillus acidophilus (acidophiles) [6]. Other categories of sulfur oxidizer is gliding Sulfur oxidizing which include species that carry out sulfide oxidation in rice fields such as Chlorobium, Chromatium and Beggiotta and the non-filamentous include sulfolobus and Thiospira [7]. The sulfides serve as electron donor for both photoautotrophic or chemolithotrophic bacteria to convert elemental sulfur or sulfide as given in Eqs. 10.1 and 10.2 [6]

(10.1 and 10.2)

There are direct and indirect methods that carry out oxidation of both organic and elemental sulfur. In the direct method chemolithotrophic or photoautotrophic sulfur oxidizing bacteria convert sulfide (electron donor) to sulfate or sulfur [8]. Co2 and oxygen (aerobic) or nitrate and nitrite (anaerobic) are used as terminal electronacceptor by photoautotrophic and chemolithotrophs, respectively [8]. In the indirect method ferric iron act as oxidizing agent which carry out chemical oxidation of reduced sulfur whereas iron oxidizing bacteria regenerates ferric ion to keep ongoing the process [9].

10.3

Photoautotrophic oxidation

Green sulfur bacteria and the purple sulfur bacteria such as Cholrobium and Allochromatium, respectively carry out anaerobic photoautotropic sulfide oxidation process [10]. For CO2 reduction in photosynthetic reaction they make use of H2S as an electron donor. Multiple respiratory activities of sulfur oxidizing bacteria oxidize bulk of hydrogen sulfide formed by dissimilatory sulfur reduction via direct reaction with oxygen resulting into formation of transitional products like sulfur and thiosulfate [8]. Cholorobiacea, a phototropic green sulfur bacteria have cholorosome which are light harvesting system, help them to grow adjacent to sulfide production zone that have low light intensities and water level. Sulfide is oxidized to elemental sulfur outside the cell by such sulfur oxidizing bacteria which is further oxidized to sulfate [10]. Certain genera of purple sulfur oxidizing bacteria are Chromatium, Thioalkalicoccus, Thiorhodococcus, Thiocapsa, Thiocyctis, Thiococuus, Thiospirillum, Thiodictyon and Thiopedia. Apart from these Ectothiorhodospira, Halorhodospira and Thiorhodospira are special as they produce suphur outside the cell [10].

10.4

Chemolithotrophic sulfide oxidation

Colorless chemolitotrophic sulfur oxidizing bacteria does not contain bacteriochlorophyll so as the name. For example Thermothrix, Beggiotoa, Thioplaca, Thiobacillus, Achromatium, Thiomicrospira, Acidithiobacillus, Thiosphaera and Thiothrix [6]. These oxidizers survive by detoxifying hydrogen peroxide, produced metabolically [11]. They are able to grow in a habitat having reduced inorganic sulfur compounds, sulfur and thiosulfate and compounds like dimethylsulfide and methanethiol due their diverse ecological, morphological and physiological properties. Aerobic chemolithotrophs having oxygen as terminal electron acceptor whereas anaerobic ones grow using nitrite or nitrate as terminal electron acceptor. Thiobacillus denitrificans oxidize thiosulfate and sulfide at the expense of nitrate [12]. Sulfur released from degradation of proteins of decayed plants and animals gets accumulated in soil. It is then oxidized aerobically or anaerobically to sulfate and then to sulfur. This sulfur is used to produce H2S (Eq. 10.3) thereby gets incorporated in the process of anaerobic sulfate reduction. H2S further oxidized to sulfate under aerobic condition by sulfur oxidizing bacteria. (10.3) As said earlier the behavior of sob’s in agriculture is much more predominant but has immense application in other fields as well. Some of them are discussed ahead in the chapter.

Applications of sulfur oxidizing bacteria Chapter | 10

10.5

133

Enzyme responsible for sulfur oxidation

Sulfide oxidase is the major enzyme responsible for sulfide ion oxidation [13]. Sulfide oxidase has two identical subunits in it i.e. N-terminal domain and C-terminal domain that differ in their cofactors. The N-terminal domain has heme as a cofactor surrounded by five alpha helices with three adjacent antiparallel beta sheets. The C-terminal has molydopterin cofactor encircled by thirteen beta sheets and three alpha helices. Molybdopterin has Mo (VI) at its middle welded by sulfur and two terminal oxygen [6]. Sulfatase is another important enzyme found in Serratia that hydrolyzes organic suphate [13]. Arthrobacter species and Bacillus species are the major genera that oxidize sulfide with the help of former described sulfide oxidase enzyme. The one which is found in Arthrobacter had been potentially used in waste water treatment due to their cell bound nature and elite activity at broader range of pH [13].

10.6

Applications

10.6.1 Sulfur oxidizing bacteria in biogeochemical cycling To regulate the energy flow in the system, the availability and biogeochemical cycling of C and N is must. Though sulfur is minor element but vital for life. Productivity of ecosystem can be affected by iogeochemistry of sulfur as it forms the largest organic sulfur pool. This organic sulfur is present in bound state that must undergo mineralization to inorganic sulfur so as to become readily available (mainly as sulfate) from soil and microbes for plant uptake. Sulfur oxidizing bacteria plays a significant role in this transformation from one form (inorganic sulfur) to another form (organic sulfur) in the biosphere. Various sulfur oxidizing bacteria carry out the process of oxidizing sulfur compounds to sulfate (SO32). It has been comprehended that on increase in the sulfur content of the organic pool by addition of elemental sulfur fertilizer also need sulfur oxidizing bacteria to oxidize it to make it available to the plant for nutrition. Such amendments are necessary in agricultural soils because of the presence of sulfur oxidizers that gets moved up on addition of sulfur. Thiobacilli performs the maximum oxidation and transformation in soil thus regarded as most efficient genus of sulfur oxidizing bacteria. Tourna et al. [14].

10.6.2 Bioleaching With the increase in global demand for base metals, mining companies are turning to the extraction of low quality and extra composite ores to accomplish the call of the hour. For low grade metal extraction, heap leaching and heap bioleaching are ideal technologies for oxidized ores and sulfidic ores, respectively [15]. Bioleaching is a novel biotechnological process of dissolving (leaching) sulfide from its ore and extraction is easily done by acidification as well as solubilization. It is low cost operative and modest technology [16]. Sulfur compounds are converted to sulfuric acid by these sulfur oxidizing bacteria thereby acidifying soils and mobilizing metal ions [17]. Acidothiobacillus ferrioxidans and Acidithiobacillus thioxidans are acidophilic sulfur oxidizing bacteria that transform metal ores of sulfide to low quality sulfates. Sulfur oxidizing bacteria are used to excerpt metals from poor quality ores, trace elements and mineral compounds. Aspergillus niger has been used in the extraction of not only slphur from its ores by direct method as discussed aboube, but also of iron (Fe), tin (Sn) and silver (Ag) by indirect method, due to its good leaching efficacy. At lower pH i.e. 2; sulfur oxidizing bacteria such as Thiobacillus ferrooxidans dissolve pyrite (FeS2) and chalcopyrite (CuFeS2) by order of 5 6 magnitude (Eq. 10.4) as compared to chemical oxidation (Eq. 10.5). Also by indirect bioleaching several harmful compounds are rendered less toxic sulfide minerals by oxidation [18].

(10.4 and 10.5)

10.6.3 Bioremediation With the advent of industrialization contamination of soil and water has increased due to industrial effluents, agricultural and domestic activities. Now, it has become necessary to eliminate heavy metals that are toxic to human and environmental health [19]. Bioleaching is an established technology for bioremediation of soil where SOB play role in

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extraction of toxic metal to harmless metal ions [17]. Sulfides in oilfield waters are undesirable due to their toxicity, corrosiveness and propensity to form insoluble metal sulfides. Therefore, the remediation of sulfide in such habitat is dependent on sulfur oxidizing species such as Thiobacillus thioparus; which oxidize sulfides produces by sulfur reducing bacteria [20]. Sulfide is generated during anaerobic treatment of high sulfide waste water usually from rubber factories and landfill covers soil [21]. This process also generates a huge amount of biogas that is heavily contaminated with H2S and does not meet the standard of biogas composition. So, as to reduce this amount of sulfide and hydrogen sulfide (H2S); sulfur oxidizing bacteria are of immense help that are being used as terminal electron acceptor. For example:- Thiobacillus thioxidans can assimilate with CO2 by utilizing the energy produced when it oxidizes sulfur compound, is known to oxidize inorganic sulfur compound in the presence of water as expressed by the following equation.

(10.6 -10.8)

10.6.4 Biofilteration Industries such as petrochemical and cellulose industries produce total reduces Sulfur compound that are odorous and neurotoxic in nature are oxidized using sulfur oxidizing microorganism. These industrial emissions are very high in temperature and need to be oxidized before release. Some hyper thermophilic microorganisms for example Sulpholobus metalica at higher temperature can oxidize these compounds and is generally used in biofilteration technology [22]. The reduced sulfur composites present in industrial gas emission are tarnished with the help of sulfur oxidizing bacteria. Biotreatment of boiler incineration, petroleum filtering, smelting and composting facilities at elevated temperatures are finished by means of biofiters impregnated with sulfur oxidizers [23]. The immobilization of the desulfurizing bacteria by forming biofilm on certain packing polymer is an quick and economical process under extreme temperature conditions [24]. SOB biofilm on titanium alloy has been used in biotechnology process such as sulfide contaiminated waste water reatment. For example genus belonging to the Acidothiobacillus viz., A.thiooxidans and A. ferooxidans [25].

10.6.5 Biofertilizers Sulfuric acid (H2SO4) is produced by biochemical sulfur oxidation. This sulfuric acid leads to decrease in pH of the soil and dissolution of calcium carbonate (CaCO3) in alkaline soil having calcareous nature. Sulfuric acid also mineralizes other plant nutrients, especially phosphorous for healthy plant growth. Soil fertility has been increased by exploiting Thiobacillus thioxidans strain because of its sulfur solubilizing character [26]. With its oxidation behavior its makes obtainability of easy to get to sulfur by transforming non- available sulfur and its combinations. As it also lowers the soil pH, can be well utilized to treat saline and alkaline soil of better cultivation. Thiobacillus thioparous and Thiobacillus thioxidans strains oxidizes hydrogen sulfide and sulfur in soil, which is a dominant form that is taken up by plants and other useful microbes thathelp in nourishment of plants [7]. They also improve the saline alkaline soil [27]. The biological oxidation brings about changes in soil structure by decreasing pH, salt content and increasing positive microbial activities. The overall change in soil fertility help plants to withdraw better nutrition and organic matter [28].

10.6.6 Bio controlling agent Oxidation of sulfur compounds has been known to control certain plant pathogens by balancing the sodium content in the soil [29]. Potato scab and rot of sweet potato caused by Streptomyces scabies and Streptomyces ipomea, respectively can be controlled by inoculating sulfur amended soil with Thiobacillus under acidic conditions [26].

10.6.7 Deodorization Sulfur reduction produces fouling smell due to release of H2S which is very unpleasant. Deodorization of this smelling gas can be done biologically by using sulfur oxidizing microorganism. Cholorobium limicola, F. thiosulatophilum, Xanthomonas species, Pseudomonas putida and chemoautotrophic Thiobacillus are such bacteria that are exploited for

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135

biological deodorization process by oxidizing H2S. At very low microbial count they have high affinity and high removal rate of H2S at very modest nutritional prerequisite offer an advantage. Thiobacillus, Acidothiobacillus, Acidothiobacillus ferooxidans, Thiobacillus thioparus and Thiobacillus denitrificans have been reported in H2S removal. The physiology of such microbes has greatly improved the economics of the process of removal of H2S from any effluent. Therefore, they are important as can oxidize sulfide to sulfur at ambient temperature and neutral pH in waste water.

10.6.8 Rubber recycling A worldwide environmental threat has been posed by waste rubber material such as vehicle tires and unutilized rubber raw material. The sulfur cross linking in rubber polymer due to vulcanization has creates a serious problem in recycling of rubber material. Unlike plastic and glass it is difficult to melt and reshape rubber due to these cross links which give strength and exceptional inflexible properties to it. The SOB Acidothiobacillus helps in recycling vulcanized rubber by breaking these cross links and also expands the physical properties of reprocessed rubber [30].

10.6.9 Biosensor Due to the rapid pollution of water bodies caused by heavy metals, etc., make it necessary to monitor their toxicity level in water environment so as to protect natural ecosystem and human health. As there are various toxic compound in water bodies, posing threat too aquatic life, usage of one single analytical instrument will not be helpful in the era of running development [31]. Genus Thiobacillus as sulfur oxidizing bacteria can be exploited to monitor toxicity of metal ions by measuring electrical conductivity (EC) and pH. It produces H2SO4 (sulfuric acid) under aerobic condition which decrease pH and increase EC of the medium that can be easily detected. On another side activity of sulfur oxidizing bacteria decresea in presence of toxic chemical thus there is no formation of H2SO4 meaning thereby no change in pH and EC in the water medium [32].

10.7

Conclusions

Considering sulfur pollution, predominantly caused by sulfur in reduced form as suphides due to their toxic and odorous physiognomies. There has been recent increase in research of applications of biological method for sulfide removal to develop new technologies biofilters, biosensors, biofertilizers, etc. In this chapter we have tried to accumulate as much as information on sulfur oxidizing bacteria and their probable exploitation for better sustainability.

References [1] Robertson LA and Kuenen JG. The colourless sulphur bacteria. In Dworksin M, Falkow S, Rosenberg E, Schleifer KH, Stackerbrandt E (Eds.). The prokaryotes b 2; 2006. 985 1011. [2] Tang K, Baskaran V, Nemati M. Bacteria of the sulphur cycle: an overview of microbiology, biokinetics and their role in petroleum and mining industries. Biochemical Eng J 2009;44:73 94. [3] Predicala B, Nemati M, Stade S, Lague¨ C. Control of H2S emission from swine a. manure using Na-nitrite and Na-molybdate. J Hazard Mater 2008;154(1 3):300 9. [4] Baskaran V, Nemati M. Anaerobic reduction of sulfate in immobilized cell bioreactors, using a microbial culture originated from an oil reservoir. J Biochemical Eng 2006;31(2):48 159. [5] Rawat R, Rawat S. Colorless sulfur oxidizing bacteria from diverse habitats. Adv Appl Science Res 2015;6(4):230 5. [6] Bahera BC, Mishra RR, Dutta SK, Thatoi HN. Sulphur oxidizing bacteria in mangrove ecosystem: a review. Afr J Biotechnol 2014;13:2897 907. [7] Vidyalakshmi R, Paranthaman R, Bhakyaraj R. Sulphur oxidizing bacteria and pulse nutrition a review. World J Agric Sci 2009;5(3):270 8. [8] Kuenen JG. Colourless sulphur bacteria and their role in the sulphur cycle. Plant Soil 1975;43:49 76. [9] Pagella C, Faveri DM. H2S gas treatment by iron bioprocess. Chem Eng Sci 2000;55:2185 94. [10] Madigan MT, Martinko JM. Brock biology og microorganisms. 11th ed. Upper Saddle River, NJ: Prentice Hall; 2006. [11] Larkin JM, Strohl WR. Beggiatoa, Thiothrix and Thioploca. Annu Rev Microbiol 1983;37:341 67. [12] Cardoso RB, Sierra-Alvarez R, Rowlette P, Flores ER, Gomez J, Field JA. Sulfide oxidation under chemolithoautotrophic denitrifying conditions. Biotechnol Bioeng 2006;95:1148 57. [13] Mohapatra BR, Gould WD, Dinardo O, Papavinasam S, Revie RW. Optimization of cultural condition and properties of immobilized sulphide oxidase from Arthrobacter Species. J Biotechnol 2006;124:523 31.

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[14] Tourna M, Maclean P, Condron L, Callaghan MO, Wakelin SA. Links between sulphur oxidation and sulphur-oxidising bacteria abundance and diversity in soil microcosms based on soxB functional gene analysis. FEMS Microbiol Ecol 2014;88(3):538 49. [15] Watling HR, Shiers DW, Collinson DM. Extremophiles in mineral sulphide heaps: some bacterial responses to variable temperature. Acidity Solut Composition Microorg 2015;. [16] Roy S, Roy M. Bioleaching of heavy metals by sulfur oxidizing bacteria: a review. Int Res J Environ Sci 2015;4(9):75 9. [17] Maini G, Sharman AK, Sunderland G, Knowles CJ, Jackman SA. An integrated method incorporating sulfur-oxidizing bacteria and electrokinetics to enhance removal of copper from contaminated soil. Env Sci Technol 2000;34:1081 7. [18] Cvetkovska Vesna T, Milena C. Bioleaching of Zn-Pb-Ag sulphidic concentrate, 15th International Research/Expert Conference. Trends Dev Machinery Associated Technol 2011;681 4. [19] Ramola Singh B, Ajay. Heavy metal concentrations in pharmaceutical effluents of industrial area of Dehradun (Uttarakhand), India. J Env Anal Toxicol 2013;3. [20] Ivanov MV. Microbiological processes in the formation of sulfur deposits. In: Kuznetsov SI, editor. Israel Program for Scientific Translations, vol. 298; 1968. [21] He R, Xia FF, Bai Y, Wang J, Shen DS. Mechanism of H2S removal during landfill stabilization in waste biocover soil, an alternative landfill cover. J Hazard Mater 2012;67 75. [22] Emky VR, Nathaly RT, Leslie A, Germa´n A, Homero U. Characterization of a hyperthermophilic sulphur-oxidizing biofilm produced by archaea isolated from a hot spring. Electron J Biotechnol 2017;25:58 63. [23] Zhang LL, Lin J, Liu J. Biological technologies for the removal of sulfur containing. Microbiol Biotechnol 2015;31:1501 15. [24] Sakai H, Kurosawa S. Exploration and isolation of novel thermophiles in frozen enrichment cultures derived from a terrestrial acidic hot spring. Extremophile 2016;20:207 14. [25] Cwalina B, Dec W, Michalska JK, Kik MJ. Initial stage of the biofilm formation on the NiTi and Ti6Al4V surface by the sulphur-oxidizing bacteria and sulphate-reducing bacteria. J Mater Sci: Mater Med 2017;28:173. [26] Priyanka S, Sivaji M, Sridar R. Isolation and characterization of novel multifunctional sulphur oxidizing bacterium (SOB) and its use as biofertilizer. Int Sci J 2014;28 34. [27] Shuochao B, Qing W, Xinhua B, Zijian W. Characters of saline-alkali soil in Western Jilin and biological treatment. J Pure Appl Microbiol 2013;809 12. [28] Baoa S, Wang Q, Baoc X, Lia M, Wangd Z. Biological treatment of saline-alkali soil by Sulfur-oxidizing bacteria. Bioengineered 2016;7 (5):372 5. [29] Kertesz MA, Mirleau P. The role of soil microbes in plant sulphur nutrition. J Exp Botany 2004;44:1939 45. [30] Katrina B. Sulphur utilizing microorganism in biotechnological application- Rubber recycling and vanadium reduction. [Dissertation]. Department of Biotechnology, Lund University; 2003. [31] Oha SE, Hassana Sedky HA, Ginkelb SWV. A novel biosensor for detecting toxicity in water using sulfur-oxidizing bacteria. Sens Actuators B: Chem 2011;154:17 21. [32] Gurung A, Kang WC, Shin BS, Cho JS, Oh SE. Development of an online sulfur-oxidizing bacteria biosensor for the monitoring of water. Toxic Appl Biochem Biotechnol 2014;174:2585 93.

Further reading Watling HR. The bioleaching of sulphide minerals with emphasis on copper sulphides—a review. Hydrometallurgy 2006;84:81 108.

Chapter 11

Physiological and genomic perspective of halophiles among different salt concentrations Ashish Verma1, Sachin Kumar1 and Preeti Mehta2 1

Microbial Type Culture Collection & Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh, India, 2DBT-IOC

Centre for Advanced Bioenergy Research, Research & Development Centre, Indian Oil Corporation Limited, Sector-13, Faridabad, India

11.1

Introduction

Extremophiles belong to members of all domains of life such as prokaryotes and eukaryotes which can survive in extreme and harsh conditions due to their adaptation strategies at both the intra- and extracellular levels. Considering the distribution pattern of extremophiles among the domains of life, the prokaryotes (Bacteria and Archaea) represents the significant player followed by some members of eukaryotes (protists and multicellular forms) [1]. Extremophiles are classified based on the environment in which they survive such as different range of temperature (thermophiles and hyperthermophiles), pH (acidophiles and alkaliphiles), hydrostatic pressure (barophiles and pieziophiles), gravity (hyper-gravity and hypo-gravity), desiccation (xerophiles) and salinity (halophiles). The members of extremophiles can also tolerate a range of hostile conditions and can be termed as multi- or polyextremophiles [2
4]. Approximately .70% of the Earth’s surface is covered with seawater which consists of 3.5% of dissolved salts, while the elevated salt concentrations are usually reported from near shore environments and the natural inland salt lakes consisting of 27
34% of total dissolved salts. The different gradients of salinity are also reported from man-made multi-pond solar saltern systems where the exchange of open sea water results in evaporation during the successive summer seasons resulting in crystallization of salts. The adaptability of prokaryotic life has been well established from the seawater salinity to NaCl saturated brines [5
8]. The hypersaline environments have been distinguished as thalassohaline and athalassohaline based on salt composition, dominating ions and pH conditions. Some of the hypersaline environments have salinity conditions equivalent to seawater (so-called thalassohaline environments) and are mainly dominated by ions belonging to sodium and chloride with their pH ranging from neutral to slightly alkaline during the first stages of evaporation [7,8]. With the successive evaporation events, the ionic concentration changes due to precipitation of gypsum and associated minerals. These NaCl saturated thalassohaline brines mostly appears as bright red owing to several pigmented microorganisms. The niche or environment which reflects the ionic concentration like seawater can be attributed to Great Salt Lake, Utah and its water can be classified as thalassohaline [9]. In one of the other hypersaline environments (so-called athalassohaline environments), Mg21 and Ca21 ion concentration exceeds than that of Na1 and K1 ions with relatively low pH (around 6.0) as represented by Dead Sea brine and thus represents the ionic composition to be different from seawater [9]. Additionally, alkaline athalassohaline represented by soda lake brines are distributed over a diverse range of geographic locations such as India, China, California, East Africa, Egypt, Nevada and elsewhere [9]. These soda lake brines are dominated by monovalent ions and high pH ($10
11) with the solubility of divalent cations below the detection limit [9].

11.2

Classification and evolutionary relationships among halophiles

Several classification systems are available with respect to salinity range and optimum required for the prokaryotes, but the classification given by Donn Kushner, 1978, 1985 [10,11] is very well accepted. According to the classification Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00011-3 © 2020 Elsevier Inc. All rights reserved.

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TABLE 11.1 Classification of microorganisms based on response to salt. Class of prokaryotes with respect to salinity

Salinity conditions

Examples

Non-halophilic

,0.2 M

Bacteria thriving in freshwater

Halotolerant

Able to tolerate salt; if tolerates . 2.5 M, then considered as extremely halotolerant

Staphylococcus sp.

Slight halophile

0.2
0.5 M

Bacteria constituting marine habitats

Moderate halophile/ borderline extreme halophile

0.5
2.5 M/1.5
4.0 M

Salinivibrio costicola, Halomonas elongate, Tamilnaduibactersalinus/Halorhodospira halophila

Extreme halophile

2.5
5.2 M

Type species of genus Halobacterium, Halobacterium salinarum, Halomarinaoriensis, Salinibacter ruber (member of Bacteroidetes)

FIGURE 11.1 Evolutionary relationship of three domains of life based on 16 S rRNA gene sequence comparison. The members of Bacteria and Archaea shown in bold with bold lines represents the members of borderline extreme halophiles and extreme halophiles.

scheme, an organism can be considered as non-halophile, halotolerant and halophilic (Table 11.1). Based on properties such as minimum salt requirement, optimum salt requirement and the upper salinity tolerance found in the prokaryote world, it is sometime impossible to define a sharp boundary for the classification of what a halophile is. This continuum of salinity range in case of halophile group has led to its classification ranging from slight, moderate, borderline extreme and extreme halophile (Table 11.1). As per the classification scheme, there are a total of six different classes each with specific properties towards salinity conditions (Table 11.1). The different category of prokaryotes has been divided based on the optimum salinity requirement in the microbiological media (Table 11.1). The present classification is based on the requirement of NaCl concentration and thus requires revision as some of the organisms do survive especially in athalassohaline environment where the importance of other ions such as Mg21 and Ca21 are more relevant [12]. Some of the environments belonging to Dead Sea, Mediterranean Sea and deep-sea hypersaline brines are rich in magnesium/calcium ions, thereby supporting the claim of classification revision which is based solely on NaCl response [9]. The members of halophiles have been well distributed among all the three domains of life (Fig. 11.1; [13]). The evolutionary relationship among the three domains of life based on small subunit phylogenetic gene marker (16S rRNA gene in Bacteria/Archaea and 18S rRNA gene in Eukarya) showed well differentiated clades, and the members of halophiles represented in bold with bold line in the phylogenetic tree can grow at $ 15% NaCl concentration (Fig. 11.1).

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In the present Fig. 11.1, both the halophilic and non-halophilic members of prokaryotes have been shown with different salt requirements and tolerance. Among the Bacteria, the members of halophiles have been well distributed among the phyla Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Proteobacteria and Spirochaetes [14]. Within the Archaea, the microorganisms which can tolerate more salinity conditions have been found. The members of order Halobacteriales (family Halobacteriaceae) are known to have the taxa which cannot survive if supplemented with NaCl concentration ,2.5
3.0 M and are thus are known as extreme halophiles [10]. The concentration of NaCl is very much important for the optimum growth and structural stability of members belonging to family Halobacteriaceae and in many case irreversible damage and cell lysis may occur if suspended in solutions containing ,1.0
2.0 M salt. There are some reports in literature where the members of family Halobacteriaceae were isolated with relatively low-salt requirement. The specific members of taxa belonging to Halofrex, Haladaptatus and Halosarcina have been isolated from low salt spring (7
10 g/L) in Okhlahoma [15
17]. All these three taxa isolated from the low-salt environment have the lowest salinity boundary in the range of 47
76 g/L, but the optimal salinity conditions and the maximum salinity tolerance remain at par with the other members of family Halobacteriaceae. Similarly, the members of halophilic or highly halotolerant representatives were reported from methane producing microbes belonging to class Methanotherma of order Methanosarcinales. The members of order Methanosarcinales are well distributed and cosmopolitan in anaerobic environments [18]. Till now, the halophilic methanogens have been reported only from order Methanosarcinales, but recently only a member of deep phylogenetic lineage of extremophilic methanogens were reported from hypersaline lake which formed a class level lineage “Methanonatronarchaeia” closely related to class Halobacteria [19]. The members of halophilic taxa have been reported from the order Halobacteriales, Methanosarcinales and class Methanonatronarchaeia of Euryarchaeota, while no halophilic member have been reported from Crenoarchaeota [19]. The study of eukaryotic microorganism with respect to their distribution and physiological adaptation has not been well studied from the high salt environments when compared to members from bacterial and archaeal domains. The unicellular green algae belonging to genus Dunaliella, brine shrimps (Artemia salina, Artemia franciscana), meristematic fungus (Trimmatostroma salinum) and black yeast (Hortea werneckii) have been reported from saltern brine and the hypersaline environments [20
22]. Similarly, other members belonging to Eukarya living in high-salt environment have been even more neglected, irrespective of their existence for a long time [23,24]. Several different halophilic flagellates were characterized from the Korean saltern ponds [25], out of which Pleurostomum flabellatum (optimum 300 g/L; [26]) and Halocafetaria seosinensis (optimum 150 g/L; [27]) have been characterized in-depth. Within the evolutionary relationships of bacterial and archaeal domains based on 16S rRNA gene sequences (Fig. 11.1), three groups of prokaryotes belonging to order Halobacteriales, family Halobacteriaceae and Halomonadaceae were found to be coherent at both the phylogenetic and physiological scale consisting entirely the members of halophiles. The family Halobacteriaceae [28] and Halomonadaceae [29] consists majority of members belonging to aerobic heterotrophs with some facultative aerobe members. The third group belonging to order Halanaerobiales consists of anaerobic fermentative bacteria and mostly live by fermentation of sugars and amino acids [13]. Many salt requiring species have been validly named and characterized from the family Halobacteriaceae ( . 90 species), Halomonadaceae ( . 60 species), while least number of species are reported from order Halanaerobiales (,25 species) (http://www.bacterio.net/classifphyla.html). Both the classification and the phylogenetic view of halophiles can be traced back only to culturable isolates, and thus signifies a greater void of halophilic diversity which have not been isolated till date. Most of the members of halophiles which have been characterized till date are being difficult to isolate and maintain because of their slow growth patterns on solid media. The optimum growth patterns of some of the isolates normally take 3
4 weeks for proper growth which somehow hinders the isolation of extremely halophilic strains. This view was further supported by genomic and metagenomics studies of different hypersaline environments such as Great Salt Lake, Dead Sea, saltern evaporation and crystallizer ponds which clearly showed that many halophilic prokaryotes still await to be isolated and characterized [30,31]. But the isolation of these uncultivated halophiles are still possible using appropriate skill and patience using a combination of culture dependent and high throughput cultivation approaches [32
34].

11.3

Mechanism of salt adaptation in halophiles

The survival of microorganisms in the hypersaline environment requires the cells to maintain its intracellular osmotic pressure to be at least isosmotic or hyperosmotic [5,35]. Moreover, the regulation of lost water in the salt rich environment through the permeable biological membranes is not energetically feasible through active energy-dependent inward transport. To adapt to such harsh salinity conditions, the halotolerant/halophilic microorganisms utilize two different adaptation strategies. The first strategy known as “high-salt-in” involves the accumulation of molar concentration of

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K1 and Cl2, while the second strategy of halo adaptation known as “low-salt-in” involves the role of organic osmolytes which are either produced internally by cells itself or gets accumulated from the medium [36].

11.3.1 ‘‘High-salt-in’’ strategy This strategy has been well utilized by a number of halophilic representatives of domain Bacteria, aerobic Salinibacter ruber and anaerobic members of order Halanaerobiales [13,37]. The members of aerobic Archaea of order Halobacteriales also utilizes this strategy to survive in the high osmotic salt concentration [28]. The members of order Halanaerobiales are more related to halophilic aerobic Archaea based on physiological and biochemical properties, rather than members of aerobic halophilic Bacteria which mainly utilize the ‘low-salt-in’’ or organic solute strategy. Till now, no organic solute strategy has been reported among the members of order Halanaerobiales [38
40]. The estimation of molar concentration of different ions among the members of order Halanaerobiales, Halanaerobium sp. and Halobacteroides sp. further revealed that K1 formed the key cation in the rapidly growing cells [38,41]. Among the members utilizing ‘high-salt-in’’ strategy, a number of different ion transporters and transport proteins have been reported to act in a concerted manner for the maintenance of peculiar ionic concentration gradient in the cell’s cytoplasm and membrane [42]. The main purpose of these transport proteins lies in the maintenance of high concentration of K1 and Cl2 ions along with acidic conditions inside the cell membrane, and the same has been shown among the members of order Halobacteriales (Fig. 11.2). In the order Halobacteriales, the extrusion of protons (A) are mainly accompanied by electron transport chain utilizing oxygen or other electron acceptors, thereby generating an electrochemical gradient with positive outside (acidic) and negative inside (alkaline). The members of taxa containing retinal protein (bacteriorhodopsin) in their membrane may also generate proton electrochemical gradient using light energy [13,41,42] (B). The generated proton electrochemical gradient is the main driving force for meeting the energy demands of the cell. The energy required for the ATP production in the cell is controlled by the inward flux of H1 ions through ATP synthase complex coupled with phosphorylation of ADP with inorganic phosphate (C). The ATP synthase pump can also work in the reverse direction when the ATP production occurs through substrate level phosphorylation. The anaerobic growth of taxa belonging to Halobacterium salinarum and members of order Halanaerobiales can occur by fermentation of arginine and sugars/amino acids respectively [13,43]. Most of the membranes of halophiles which use “high-salt-in” strategy try to maintain higher molar concentration of K1 as compared to Na1 ions inside the cell. The analysis of cell membranes among halophiles indicated high activities of Na1/H1 antiporter inside the cell; [44] (D). Moreover, the Na1/H1antiporter also support the regulation of intracellular pH [9]. The gradient of sodium ions established through Na1/H1 antiporter can also be utilized to drive endergonic processes such as transport of amino acids and other compounds in halophilic Bacteria and Archaea, thereby serves as an energy source; [14] (E). Due to more negative-inside membrane potential, there occurs K1 accumulation inside the cell via uniport system; [45] (F). As the molar concentration of Na1 was decreased due to Na1/H1 antiporter(d), K1 enters the cell to maintain the electroneutrality. The molar concentration of Cl2 ion plays an important role in general growth and cell-division inside the intracellular compartment of cell. During the cell growth, the net flux of K1 concentration provides net gain in intracellular volume [46]; (F). Two different active chloride pumps have also been identified; the first one is lightindependent symport with Na1; [47] (G), while the second is light-dependent retinal protein, halorhodopsin; [41,46] (H). The ‘high-salt-in’’ strategy adaptations make the cell rigorously dependent on the constant amount of high salt FIGURE 11.2 Ion movements across the cell membrane of order Halobacteriales. The details about the different processes shown in the figure have been described in text (A
H).

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concentration in order to maintain the structural integrity and viability [48
50]. In some members of order Halobacteriales, the salt concentration is so important that many proteins and enzyme denature if suspended in solutions below 1
2 M salt. Similarly, the intracellular enzymes of members of order Halanaerobiales also function optimally in presence of salt rather than salt-free medium [51
53]. The higher concentration of salts inside the cell can result in the distortation of protein structure because of enhancement of hydrophobic interactions, charge shielding within or between macromolecules and salt hydration [54]. The analysis of genomic sequences of order Halobacteriales showed that most proteins contain a large repertoire of acidic amino acids (Glu and Asp) than the basic amino acids (Lys and Arg). Similarly, the genomic sequences of Salinibacterruber encodes more percentage of acidic amino acids similar in line with the members of order Halobacteriales in spite of being phylogenetically unrelated to each other [55,56]. The analysis of cellular protein in members of order Halanaerobiales was also found to be acidic in nature adapted to ‘high-salt-in’’ strategy [38,57]. Both the strategies i.e., ‘high-salt-in’’ and the acidic residues complement each other to survive and maintain the structural integrity and viability of cell in hypersaline environments. The more of the negative charges on the side chain of protein can make the structure unstable unless the salts in the form of cations are added to stabilize the acidic groups of proteins. This strategy plays an important role in the survival of cells in hypersaline environments. The role of molar concentration of salts does not solely limited to charge-shielding mechanism, considering that the concentration required are higher [58,59]. The maximal charge-shielding have been achieved already in as low as 0.1
0.5 M or even lower molar concentration of divalent cations. Among the acidic amino acids, glutamate has the highest water binding capacity and has greater implication in maintenance of proper hydration shell for functional protein [9]. The members of order Halobacteriales are further characterized by low content of hydrophobic amino acid residues such as Ser and Thr [58]. The combination of both the strategies i.e., weak hydrophobic interactions among protein molecules and the limited content of hydrophobic residues participates in the structural integrity of proteins and enzymes in high salt concentration. The ‘high-salt-in’’ strategy adaptation has been reported mainly from the prokaryotes surviving at salt concentration around 15%, but this adaptation can be widespread over a range of saline conditions. Further, the distribution of acidic proteome has not been restricted to microbial communities isolated from high salt concentration, but has also been reported from lower salt concentration at around 9% [60].

11.3.2 ‘‘low-salt-in’’ strategy This strategy has been used by most halotolerant/halophilic representatives of Bacteria and methanogenic Archaea to cope up with the external osmotic pressure by maintaining much lower concentration of salt than the outside medium [14,61]. The osmotic balance in this adaptation mechanism is mainly maintained by the organic solutes or osmolytes which are mainly produced by cell or either transported from the media. Different types of organic solutes corresponds to polyols, sugars, amino acids, betaines, ectonies and in some cases peptides [62]. The distribution of organic solutes among the Bacteria, Archaea and Eukarya concluded that Archaea prefers negatively charged solutes, while the Bacteria and Eukarya prefer to accumulate neutral compatible solutes [63,64]. These organic solutes do not affect cellular processes except modulating the individual enzymatic activities, due to which they are labeled as ‘compatible solutes’’ [65]. These compatible solutes form a significant portion of osmolytes in the cell’s cytoplasm. Their percentage distribution in the cell’s cytoplasm helps to maintain the overall architecture and viability of the cell, which are all important for survival and cell proliferation. A number of analytical methods such as 13C/1H NMR studies of cell extracts, HPLC, refractive index, anion exchange chromatography and pulse amperometric detection have been used till date to detect the presence of osmotic solutes accumulated in the halophilic/halotolerant microorganisms [66
75]. Organic osmolytes are mostly soluble, polar and generally divided into three different chemical categories: (i) zwitterionic, (ii) uncharged, and (iii) anionic solutes. The structure, distribution along with their references of some of the selected zwitterionic, uncharged and anionic organic solutes (carboxylates, phosphate and sulfate) is mentioned in Tables 11.2
11.4. The zwitterionic organic solutes are accumulated and synthesized in a number of bacterial and archaeal groups and plays a role in osmotic balance. These organic solutes are not stored at high concentration as they represent the intermediates in the protein biosynthesis pathways. The list of the important organic solutes belonging to this category has been mentioned in Table 11.2. The molecules which are polar but lacking any charge are well represented in members of eukaryotes and to some extent in halophilic bacteria. Glycerol represents as one of the prevalent osmolyte in marine and halophilic Dunaliella, halotolerant yeast Debaryomyces sp. and black yeast Hortea sp. [98,99]. Similarly, myo-inositol represents one of the uncharged polar osmolytes along with glycerol and neither of these is present in Bacteria and Archaea except Eukarya,

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TABLE 11.2 Structure and distribution of some zwitterionic organic osmolytes within the prokaryotes. Solute

Distribution

References

Glycine betaine

Halotolerant and halophilic bacteria, phototrophic Halorhodosporahalochloris, aerobic chemoheterotrophic bacteria, methylotrophic bacteria, Methylarculaterricola, Methylophaga sp., Sporosarcina pasteurii, Brevibacterium sp., Chromohalobacter sp.

[67,76
78]

Ectoine

Halotolerant and halophilic bacteria, Heterotrophic Gammaproteobacteria (Vibrio cholera, Halorhodosporahalochloris, Halomonas elongate, H. variabalis, Chromohalobacter salexigens, C. israelensis, Methylophaga alcalica, Halobacillus halophilus, Micrococcus sp., Bacillus spp., Rhodovulum sulfidophilum, Brevibacterium sp.

[79
81]

Hydroxy Ectoine

Halomonas elongata, Nocardiopsis halophile, Sporosarcina sp.

[82]

β-glutamine

Methanogenic anaerobic Archaea (Methanohalophilusportucalensis, Methanosarcina thermophile, Methanothermusthermolithotropicus)

[69]

TABLE 11.3 Structure and distribution of selected uncharged organic osmolytes within the prokaryotes. Solute

Distribution

References

α-Glucosylglycerol

Marine and fresh water cyanobacteria (Pseudomonas mendocina, P. pseudoalkaligenes, Stenotrophomonas, Microcyctis firma)

[83]

Trehalose

Cyanobacteria, Halorhodospira spp., Sulfolobus solfataricus, Thermoproteus tenax, Rhodothermusobamensis, Desulfovibrio halophilus, Thermoplasma acidophilum

[78,84,85]

Sucrose

Cyanobacteria, Proteobacteria

[86,87]

TABLE 11.4 Structure and distribution of selected anionic organic osmolytes (carboxylates, phosphate, sulfate) within the prokaryotes. Solute

Distribution

References

α-glutamate

Many halophilic bacteria and methanogens (Halomonas elongata, Halobacterium salinarum, Methanophilus portucalensis

[88]

β-glutamate

Methanogenic Archaea (Metahnothermococcus thermolithotropicus)

[88
91]

β-hydroxybutyrate

Methylarcula marina, Methyl arculaterricola, Photobacterium profundum

[79,92]

α-mannosylglycerate

Methanothermus sp., Rhodothermus sp.

[93
95]

α-diglycerol phosphate

Archaeoglobus sp.

[96]

Sulfotrehalose

Natronococcus occultus, Natronobacterium sp.

[97]

Archaeoglobus sp., Pyrococcus sp., Thermotoga maritima

[70,71,81]

0

di-myo-inositol-1,1 -phosphate

while the negatively charged derivatives are accumulated by Archaea. The relevant information about some of the selected uncharged organic solutes in halophilic/halotolerant bacteria has been mentioned in Table 11.3. The cells of halophilic microorganisms have negative potential inside along with high intracellular K1 ions. The anionic organic solutes help in balancing the effects of high intracellular K1 and regulate the osmotic pressure. The anionic osmolytes used for the osmotic balance can have carboxylate, phosphate and sulfate as the supply of negative charge. The list of some of the selected anionic organic solutes has been given in Table 11.4. Most of the prokaryotic cells contain cocktails of these organic solutes for maintaining the osmotic balance in the cell rather than restricted to a

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single osmolyte [62]. The cations such as Na1 and K1 are also intertwined together with these organic solutes and can further contribute to osmotic balance. It has been shown that the synthesis of organic compatible osmolytes as compared to ‘high-salt-in’’ strategy is energetically more costly and thus offers much more flexibility to survive in a range of different salt concentration without the requirement of high degree adaptation of the intracellular enzymes [100,101].

11.4

Extracellular hydrolytic enzymes from haloarchaea

The enzymes (biocatalysts) produced from the microorganisms have been regarded as the better alternative to chemical synthesis due to decreased secondary reactions, better chemical precision and efficient production of single stereoisomer with lower environmental footprint [102]. The microbial enzymes are currently being used in number of industrial processes due to its less energy demand for enzymatic reactions and the ability to function in the non-aqueous media. Due to the role of microbial enzymes in number of industrial and biotechnological processes, the search for a number of novel enzymes capable of surviving and operating at harsh industrial conditions are highly sought after by number of researchers around the globe. Extremophilic microorganisms are known to survive in harsh environmental conditions ranging from extremes of temperature, salinity, pH, pressure and combinations thereof and can thus represents a natural source of enzymes which can operate at such harsh conditions at industrial/pilot scale [103,104]. The high salt conditions further being regarded as a boon for the halophilic enzymes, as the steps of purification and sterilization can be taken care of for the applied use, since the unwanted contaminating proteins and biomolecules remain inactive under such conditions. Moreover, many halophilic enzymes can operate at low water potential in the presence of organic solvents [105]. The modification of halophilic enzymes such as immobilization are sometimes required to protect the enzymes from the harsh conditions such as temperature and organic solvents, which can further increase the standard quality parameters during operational processes [106,107]. The enzymes being purified and characterized from halophilic Archaea have been in trend for the past many years and continues to be discovered considering their significance and use in number of industrial processes. Table 11.5 summarizes the comprehensive list of extracellular enzymes being produced by haloarchaea along with their NaCl concentration for optimal activity and molecular mass of purified biocatalysts.

11.5

Genomic insights into halophilic prokaryotes

Before the advent of whole genome sequencing of halophilic prokaryotes, the classical studies have contributed significantly in the understanding of adaptive mechanisms. The notable discoveries include the S-layer glycoprotein, ether lipids (branched), bacteriorhodopsin (light-driven proton pump) and other biosynthetic and metabolic activities operating at high salinity conditions [126
129]. These key discoveries further intrigued several researchers around the world to analyze the genomic cockpit of haloarchaeal strains for survival strategies at the gene/proteome level and different metabolic pathways. Most of the members of haloarchaea belonging to class Halobacteria have been genome sequenced as draft, while some of them as complete. Among the class Halobacteria, a number of strains belonging to order Halobacteriales (total 168), Haloferacales (total 165) and Natrialbales (total 88) genomes have been sequenced as of Feb, 2019 (https://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/Halobacteria). The genomic insights from these haloarchaeal strains are not possible as most of them have been submitted as draft genome, while only a handful of them have been completely sequenced. The complete genome sequencing of a haloarchaeal strain, Halobacterium sp. NRC-1 was first completed in year 2000 [130
132], and is widely distributed in number of hypersaline environments [133]. The complete genome sequences of other halophiles have also been sequenced: Haloarcula marismortui isolated from Dead Sea [134], Halofrex volcanii from Dead Sea mud [135], Halorubrumlacus profundi from Antarctic lake [136], Natronomonas pharaonis from soda lake [137] and square archaeon Haloquadratum walsbyi isolated independently from Spanish and Australian solar saltern [138]. The case studies of one of the borderline extreme halophile i.e., square archaeon Haloquadratum walsbyi (optimum growth at 3.1 M NaCl concentration) and extremely halophile (optimum growth at 4.3 M NaCl concentration) have been briefly discussed with respect to their survival mechanisms, genomic content, cellular processes and metabolic pathways.

11.5.1 Case study of square archaeon Haloquadratumwalsbyi The cells of members of this haloarchaea are square, non-motile and pigmented which dominate the most thalassic environment (seawater derived) with an abundance of 107 cells per mL. The genomic insights into the mechanism of its

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TABLE 11.5 List of some hydrolytic enzymes produced from members of haloarchaea. Enzyme

Haloarchaea

Amylase

Halobacterium salinarum

Molecular mass (kDa)

References

0.8

NDa

[108]

Halorubrum xinjiangense

4

60

[109]

Haloferax mediterranei

3

58

[110]

4
5

50

[111]

Haloarcula hispanica

Optimum NaCl (M)

Haloarcula sp. strain S-1

4.3

70

[112]

Natronococcus amylolyticus

2.5

74

[113]

Agarases

Halococcus sp. 197 A

3.5

55

[114]

Cellulase

Haloarcula sp. G41

3

36

[115]

Haloarcula sp. LLSG7

3.4

ND*

[116]

Chitinase

Halobacterium salinarum NRC-1

1

ND*

[117]

Lipase/ esterase

Haoarcula sp. G41

2.6

45

[115]

Haloarcual marismortui

0.5
5

50

[118]

Natronococcus sp. TC6

4

ND

[119]

Halobacterium sp. NRC-1

5

ND

[120]

Haloferax mediterranei

-

41

[121]

Halogeometricumborinquense strain TSS101

3.4

86

[122]

Haloferax lucentensis strain VKMM 007

4.3

57.8

[123]

Halobacterium sp.

2.6

43

[124]

β-xylanase

Halorhabdusuathensis

0.150.43

45

[125]

β-xylosidases

Halorhabdus utahensis

0.15

67

[125]

Protease

a

ND-Not determined.

survival in sub-lethal concentration of MgCl2 and the high solar irradiance points to different modifications and the survival mechanism being adopted in its genome. For H. walsbyi, 2 circular replicons were mapped, a 3,132,494 bp chromosome and a plasmid PL47 consisting of 46,867 bp [138]. The mechanisms which are being used by the strain to maintain the optimal water potential within the cell and at the cell surface due to concomitant high salinity have been mentioned below: 1. The main factor which helped in the survival of square archaeon H. walsbyi in low water activity is the presence of largest archaeal protein termed as halomucin (Hmu1) with its 27,000 nucleotide long gene (9159 amino acid long protein). This giant protein share the amino acid composition and domain organization when compared to animal mucin [139]. Some of the domains of halomucin gene are responsible for the post-translational modifications which may contribute to overall negative charge and thereby create an aqueous shield covering the cells. The synthesis of sialic acid by sialic acid biosynthesis gene (neuA and neuB) and poly-gamma-glutamate capsule by bacterial type poly-gamma-glutamate biosynthesis protein complex CapBCA can led to protection against desiccation and can further contribute to rigidity and maintenance of unique square cells of archaeon H. walsbyi [140]. 2. The presence of arid climate characterized by low precipitation levels and intense exposure to light can led to generation of proton gradient using photo-reactive rhodopsin proteins to harness light energy. In case of H. walsbyi, two proton pumping bacteriorhodopsin (BpoI and BpoII) and one chloride pumping halorhodopsin are present [138]. Both contain all conserved amino acids essential for retinal binding and the ion translocation. 3. In the presence of high Mg21 concentration, the availability of phosphate became limiting for growth of H. walsbyi as the phosphate tend to form insoluble complex with the divalent cation. The genomic analysis of H. Walsbyi showed the presence of gene clusters which allow uptake of phosphonates and the cleavage of carbon-phosphonate

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bond. The transporters such as PitA1-3, Phn2ECD, PstABC and PpK are common in several haloarchaea, while the bacterial type proteins, an ABC-type phosphonate uptake system and the phosphotransferase dependent dihydroxyacetone kinaseare involved in phosphonate metabolism. The metabolic pathways of phosphonate metabolism are induced in the absence of phosphate [141,142]. The operons SqdB and GalEare the key enzymes in the sulfolipid biosynthesis, which plays an important role in replacing phospholipid during phosphate limitation in several photosynthetic bacteria [143,144]. 4. The prokaryotes are able to survive even in the hostile conditions because of their ability to alter or modify their genomic content for their survival. The similar fact have been seen in members of haloarchaea with generally high G 1 C content (60
70%) in order to adapt to high levels of UV irradiation inhypersaline habitats. The thymidine dimer formation due to UV induced mutation in case of AT rich genome’s has led to selection of GC rich genomes in the members of haloarchaea. In some bacterial strains, the lower G 1 C content holds an advantage due the decreased demand for nitrogen, as the structure of guanine incorporates one additional nitrogen as compared to rest of the nucleotides [145,146]. However, the low GC content (47.9%) of H. walsbyi cannot be attributed to nitrogen as the natural source of its isolation is not devoid of nitrogen. It has been hypothesized that the adaptation to extremely rich MgCl2 is responsible for the shift to AT rich genome. Such a shift has been reported in H. walsbyi, which can be attributed to the over-stabilization effect of Mg21 on the DNA duplex thereby interfering with the central dogma processes. Thus, the over-stabilization effect of Mg21 can be seen as the long term evolutionary adaptation along with lowering of genomic G 1 C content [138]. 5. The presence of long intergenic spacer regions ( . 1000 bp) in the genome of H. walsbyi had led to low coding density of 76% as compared to prokaryotes and other members of haloarchaea with 86
91% coding density [124]. This low coding density along with pseudogenes, insertion sequence (IS) transposases and the drift towards the AT rich genome points to the fact that the genome of H. Walsbyi is shrinking possibly due to its growth in a restrictive and specific environment coupled with lack of competitive growth from the rest of other species [138]. Due to the regular desiccation in the summer periods of these environments, it might lead to evolutionary bottleneck favoring genome degradation.

11.5.2 Case study of Halobacterium sp. NRC-1 The study of Halobacterium sp. NRC-1 is advantageous as its generation time is 6 hours at 42  C in rich organic broth, genetically tractable due to high efficiency transformation and availability of good selection cloning and expression vectors [147
149]. The complete genome sequence of Halobacterium sp. NRC-1 consists of 3 circular replicons, a 2,571,010 bp genome and 2 large megaplasmids i.e., pNRC200 and pNRC100 of size 350,000 bp and 200,000 bp respectively [131,150,151]. Most of the protein coding genes were found to be unique (2532 out of 2630) with acidic proteome [152]. The lateral gene transfer has been speculated to be responsible for the presence of substantial number of protein coding genes to be of bacterial origin [153]. The two megaplasmids pNRC200 and pNRC100 codes for 40 genes essential for cell viability such as DNA polymerase, thioredoxin reductase, cytochrome oxidase, multiple TATA binding proteins (TBP), transcription factors and are thus suggested to be essential “minichromosomes” [130]. Some of the insights from the genomic properties, extremophilic lifestyle and cellular processes of Halobacterium sp. NRC-1 are as follows: 1. Two types of gene systems have been widely distributed, with the first one focusing on their success in extreme environment with the modifications resulting in purple membrane, gas vesicles, anaerobic physiology and DNA repair while the other one targets the fundamental eukaryotic processes of central dogma except translation [154]. The purple membrane contains the complex of proteins known as bacterio-opsin, (a member of bop gene product) and retinal chromophore. These complex proteins uncover the cells to grow and survive in high radiation conditions, which thereby help in the survival during microaerobic and stressful conditions [128,155]. The gas vesicles on the other hand are the buoyant protein structures which allow the cells to float and thus increasing their access to illumination and oxygen. The genes responsible for the biogenesis of gas vesicles are encoded by large gene clusters on the minichromose pNRC100 [156
158]. The ability to survive in an anoxic environment in the hypersaline environment can also be attributed to bifunctional DMSO/TMAO reductase encoded by dmsREA-BCD operon [159]. The transcript levels of the dms operon is strongly regulated by anoxic conditions as shown by whole genome oligonucleotide microarray and transcript level [159]. 2. The replication process has been found to be present on both the chromosomal and the extrachromosomal replicon and provides the first opportunity to isolate the self-replicating replicons from an archaeon [160,161]. The presence

146

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of repH gene on both the pNRC200 and pNRC100 plasmids are responsible for the replications of both replicons [130
132]. The replicating sequences have been utilized for the development of both expression and shuttle vectors and further have been shown to harbor resistance and repair genes from both the bacteria and eukaryotic homologs [149,162]. The UV resistance is mediated by the presence of two phr gene homologs in the genome coding for the presence of cyclobutane pyrimidine dimer photolyase activity [140]. A number of genetic markers have been developed on the extrachromosomal segments such as mevinolin resistance (Mevr) and selectable ura3 gene for the utilization of gene knockouts and replacements [141
143]. An upstream allele of Halofrex volcanii i.e., mva gene is present in Mevr marker responsible for the biosynthesis of branched chain lipid [144]. 3. The direct gene replacements and knockout methods are the first described case for this specific archaeon, which exploits ura3 as selectable marker. The ura3 codes for orotidine 5’-phosphate decarboxylase required for pyrimidine biosynthesis [145]. The isoelectric point analysis of the proteome showed low average pI (B4.9), which was further confirmed through analysis of other halo-archaeal genome sequences [154]. The acidic proteome provided deep insight into the survival of halo-archaeal strains in hyper saline environments [130
132,153]. Some of the proteins belonging to a transcription factor (TbpE) and a topoisomerase subunit (GyrA) were shown to have higher surface negative charge when compared to homologs in non-halophilic organisms using Column charge calculation. Some of the genes have been found to be of bacterial origin and somehow confirms the concept of lateral gene transfer. The components of electron transport chain were found to have the gene organization identical to homologous E. coli operons coding for subunits of complex proteins such as nuo for NADH dehydrogenase, cox for cytochrome oxidase and men gens for menaquinone bio-synthesis [153]. This has suggested that haloarchaea have adapted to oxidizing environment by acquiring the components of electron transport chain from bacteria [132]. One more example of LGT has been attributed to arginylsynthetase (argS) gene in the plasmid pNRC100 responsible for protein synthesis [130].

11.6

Concluding remarks

Halophiles are being considered as the type of extremophiles which can survive extreme salinity conditions in a wide range of environments. Archaea are known to be the dominating group in these salinities rich environment as compared to bacterial counterparts due to their various adaptation and survival strategies. The discovery of number of halophilic enzymes from the haloarchaea further signifies the importance of harsh environmental conditions in the selection of novel genes and metabolic pathways. Efforts are further needed to utilize the true potential of extremozymes, which is generally possible with the discovery of novel halophilc members and the subsequent screening, optimization and purification strategies. The cultivation of such novel halophilic members from the hypersaline environment is not a regular exercise, but represents a daunting task as is evident from the cultivation of Walbsy’s square haloarchaeon which took almost 20 years to cultivate. The cultivation of halophilic members thus require extra efforts right from designing of media supplemented with vitamins, trace metals, salts, signaling molecules, co-culturing, novel isolation techniques and the much needed skill and patience. Further, the genomic analysis of haloarchaeal members provided an insight into the adaptation strategies, metabolic processes, transport proteins and various cellular processes in the past two decades. So, the complete genome sequencing of halotolerant/halophilic strains need to be taken up on a large scale to further understand the role of genes and proteins in its genomic cockpit which is generally lacking from draft genome sequencing projects. So, the efforts for the isolation of novel halophilc prokaryotes from different geographic habitats, study of their adaptation strategies, halophilic enzymes and the complete genome analysis represents an exciting time for the biology of halophiles in the nearby future.

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2):21
6. [120] Camacho RM, Mateos-Dı´az JC, Diaz-Montan˜o DM, Gonza´lez-Reynoso O, Co´rdova J. Carboxyl ester hydrolases production and growth of a halophilic archaeon, Halobacterium sp. NRC-1. Extremophiles 2010;14(1):99. [121] Stepanov V, Rudenskaya G, Revina L, Gryaznova YB, Lysogorskaya E, IYu F, et al. A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins. Biochemical J 1992;285(1):281
6. [122] Vidyasagar M, Prakash S, Litchfield C, Sreeramulu K. Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101. Archaea 2006;2(1):51
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93. [126] Mescher MF, Strominger JL. Structural (shape-maintaining) role of the cell surface glycoprotein of Halobacterium salinarium. Proc Natl Acad Sci 1976;73(8):2687
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8. [130] Ng WV, Ciufo SA, Smith TM, Bumgarner RE, Baskin D, Faust J, et al. Snapshot of a large dynamic replicon in a halophilic archaeon: megaplasmid or minichromosome? Genome Res 1998;8(11):1131
41. [131] Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD, et al. Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci 2000;97(22):12176
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99. [133] Baxter BK, Litchfield CD, Sowers K, Griffith JD, Dassarma PA, Dassarma S. Microbial diversity of great salt lake. Adaptation to life at high salt concentrations in Archaea. Bacteria, and eukarya: Springer; 2005. p. 9
25. [134] Baliga NS, Bonneau R, Facciotti MT, Pan M, Glusman G, Deutsch EW, et al. Genome sequence of Haloarcula marismortui: a halophilic archaeon from the Dead Sea. Genome Res 2004;14(11):2221
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24. [136] Goo YA, Roach J, Glusman G, Baliga NS, Deutsch K, Pan M, et al. Low-pass sequencing for microbial comparative genomics. BMC Genomics 2004;5(1):3. [137] Falb M, Pfeiffer F, Palm P, Rodewald K, Hickmann V, Tittor J, et al. Living with two extremes: conclusions from the genome sequence of Natronomonas pharaonis. Genome Res 2005;15(10):1336
43. [138] Bolhuis H, Poele EMT, Rodriguez-Valera F. Isolation and cultivation of Walsby’s square archaeon. Environ Microbiol 2004;6(12):1287
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[141] White AK, Metcalf WW, Two C-P. Lyase operons in Pseudomonas stutzeri and their roles in the oxidation of phosphonates, phosphite, and hypophosphite. J Bacteriol 2004;186(14):4730
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52. [149] Berquist BR, Mu¨ller JA, DasSarma P, DasSarma S. Geneticsystems for halophilicarchaea. In: Oren A, Rainey F, editors. Methods in microbiology. Elsevier/Academic Press; 2005. p. 148
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50. [154] DasSarma S, Berquist BR, Coker JA, DasSarma P, Muller JA. Post-genomics of the model haloarcaeon Halobacterium sp. Saline Syst 2006;2:3. [155] Baliga NS, Kennedy SP, Ng WV, Hood L, DasSarma S. Genomic and genetic dissection of an archaeal regulon. Proc Natl Acad Sci 2001;98 (5):2521
5. [156] Jones JG, Hackett NR, Halladay JT, Scothorn DJ, Yang C-f, Ng W-l, et al. Analysis of insertion mutants reveals two new genes in the pNRC100 gas vesicle gene cluster of Halobacterium halobium. Nucleic Acids Res 1989;17(19):7785
93. [157] Jones JG, Young DC, DasSarma S. Structure and organization of the gas vesicle gene cluster on the Halobacterium halobium plasmid pNRC100. Gene 1991;102(1):117
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92. [159] Mu¨ller JA, DasSarma S. Genomic analysis of anaerobic respiration in the archaeon Halobacterium sp. strain NRC-1: dimethyl sulfoxide and trimethylamine N-oxide as terminal electron acceptors. J Bacteriol 2005;187(5):1659
67. [160] Ng WL, Dassarma S. Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100. J Bacteriol 1993;175(15):4584
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66. [162] DasSarma S, Kennedy SP, Berquist B, Victor Ng W, Baliga NS, Spudich JL, et al. Genomicperspective on the photobiology of Halobacterium species NRC-1, a phototrophic, phototactic, and UV-tolerant haloarchaeon. Photosynth Res 2001;70:3
17.

Further reading Roberts MF. Organic compatible solutes of halotolearnt and halophilic microorganisms. Saline Syst 2005;1:5. Mira A, Ochman H, Moran NA. Deletional bias and the evolution of bacterial genomes. Trends Genet 2001;17(10):589
96.

Chapter 12

CRISPR/Cas system of prokaryotic extremophiles and its applications Richa Salwan1, Anu Sharma2 and Vivek Sharma2 1

Department of Basic Sciences, College of Horticulture and Forestry, (Dr. YSP- University of Horticulture and Forestry), Neri, Hamirpur (HP), India,

2

University Centre for Research and Development, Chandigarh University, Chandigarh, India

12.1

Introduction

Since the unraveling of central dogma in biological sciences, scientific efforts to endeavor new technologies for understanding the biological role of a gene through efficient and precise editing have gained special attention. At present, the technological inventions have made gene or genome level editing across simple prokaryotes to complex eukaryotic organisms significantly easy. These technological interventions are revolutionary due to their immense role both in basic and applied aspects of biomedical [1], agricultural, generation of renewable energy from plant biomass to several other sectors [2
14]. The genome editing tools include the DNA-binding domains of transcription such as zinc-finger nucleases (ZFNs) [15
17] and transcription activator-like effector nucleases (TALENs) [18
20] is fused to nucleases such as FokI (Fig. 12.1B). The FokI through its nuclease site-specific activity generates a double strand break (DSB)

FIGURE 12.1 Overview of genetic tools used in recombination. (A) Conventional recombination involves acquiring gene from other parent through recombination. (B) Zinc finger nuclease (ZFNs) and transcription activator like nuclease (TALENs) are a group of a DNA-binding transcription factors, fused to FokI nucleases. The FokI through its nuclease site-specific activity, creates a double strand break (DSB) near to binding sites of ZFNs and TALENs. Then strands are repaired either using nonhomologous end joining (NHEJ) or homology-directed repair (HDR). (C-D); CRISPR-Cas system uses RNA-guided nuclease. Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00012-5 © 2020 Elsevier Inc. All rights reserved.

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near to binding sites. Then cleaved strands are repaired either using non-homologous end joining (NHEJ) or homologydirected repair (HDR) [21]. The transcription activator-like effectors (TALEs) are novel DNA-binding proteins, exclusive to certain plant pathogens. The TALENs are easy to design but compared to their counterpart ZFNs, the large size of TALENs is drawback. However, the major concern with both ZFNs and TALENs is their off-target action [21]. Since these tools involves cloning and fusion of transcription factor and nuclease for protein
DNA interaction, therefore targeting another site, necessitates cloning of new gene impede the use of ZFNs and TALENs for highthroughput application. Initially, discovered as a part of hereditary and adaptive process of prokaryotes immunity, it contain clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas) which acts together to protect against attacking viruses and foreign plasmids [22]. The genome/gene uses a combination Cas systems and CRISPR. In comparison to ZFNs and TALENs, CRISPR-Cas system uses RNA-guided nuclease [1]. The CRISPR array represents, genomic sensors or imprints which contains information of foreign DNA in the form of spacers, acquired serially. These serially acquired DNA fragments help in developing immunological memory in prokaryotes genome which are hereditary [23]. The guide RNA are acquired by bacteria from bacteriophages through “protospacers” and incorporated into their genomes as imprints which are then used as short guide RNAs, to target foreign DNA sequence [21] (Fig. 12.1C). The spacer elements are incorporated into repeats were initially reported in Escherichia coli genome as unusual repetitive DNA sequence during exploration of genes involved in phosphate metabolism [24]. In archaea, these DNA fragments were reported first time from Haloferax mediterranei [25]. In subsequent studies these DNA fragments were found to be distributed in several bacterial and archaeal genomes [24]. Here, the specificity is determined by base pairing of gRNA on complimentary target site which is facilitated by adjacent motif (PAM), found on the genome [5,7,8,26,27]. Cas9 system is more adaptive because by just changing gRNA sequence, they can be reprogrammed to target new site on the genome, hence are ideal and better suited platform for high-throughput applications. The acronym CRISPR was used in 2002, after initial discovery in bacterial and archaea genome [28]. The sequence comparison between spacer region of CRISPR and plasmids, bacteriophages, finally led to elucidation of CRISPR, as an immune response against external DNA [24]. Still the initial effort went unnoticed until three researchers [26,29,30] explored Cas proteins, previously found associated with DNA repair of hyperthermophilic archaea [31] were exclusively associated to CRISPR system [3]. It is now quite evident that how CRISPR and Cas proteins act together and provide acquired immunity to bacteria against foreign DNA of invading bacteriophage and plasmids. This system works analogue to eukaryotic RNA interference (RNAi) [32]. In general, the organization of CRISPR array contains a number of noncontiguous direct repeats which are separated by sequences known as spacers (Fig. 12.2). These spacers are present near to cas genes, are variable in their sequence

FIGURE 12.2 Overview of the CRISPR/Cas mechanism. Here, guide RNA, acquired by bacteria during immunization from “protospacers” is incorporated into their genomes (spacer) as imprints in the CRIPSR array. During, immunity CRISPR repeat-spacer array is transcribed into a pre-crRNA that is processed into mature crRNAs or guide RNA, to target foreign DNA sequence complementary to the protospacer.

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and are acquired from foreign DNA processing during adaptation (Fig. 12.2). The cas encoding genes having active domains of nucleases, helicases, polymerases, and nucleotide-binding proteins [29]. The comparative genomics, computational tools coupled to advance biochemistry have played vital role in culminating the origin, structural composition, insights into mechanism and evolution of CRISPR systems [24]. The repeats within CRISPR array are highly conserved and ranges between 23 to 47 bp in size. The spacers are derived from plasmids and bacteriophages and the size ranges between 21 to 72 bp in size [28]. The protospacer-adjacent motif (PAM) in the foreign DNA, rather than CRISPR loci of the host genome is vital to distinguish ‘self’ and ‘non-self’ [33]. In general, the CRISPR loci contains, a total of less than 50 repeat/spacer units but repeat/spacer up to 375 has been reported in Chloroflexus sp. CRISPR locus of up to 18 loci have been identified in the genome of Methanocaldococcus jannaschii, contributing to over more than 1% of total genome size [34]. CRISPRs array in general are located on the genome, rarely some have been identified on plasmids [30,35,36]. The production of phase variants in Streptococcus thermophiles, alter the repeat-spacer units in CRISPR loci in polarized way, starting from the leader end [4,37] and hence led to hyper variability CRISPR loci in different strains [26,29]. The role of the spacer units in developing bacterial resistance was established by altering its content where it was shown that how acquisitions or deletion of spacer can lead to the development of novel phage resistance [4,28]. Since its discovery as a part of adaptive immunity of bacteria and archaea, presently CRISPR/Cas offers immense potential for gene(s)/genome level editing which have immense potential in both basic and industrial fields related to the biological, agricultural and other industrial applications. Moreover, the variants of CRISPR/Cas such as dCas and CRISPR/Cas, to recruit fluorescent labeled protein to target DNA location, can be used for gene regulation and cell imaging. Here in this book chapter, attempt has been made to understand the basic organization of CRISPR-cas system in bacteria and how its variants from extremophiles can be used for editing genes of industrial importance under extreme environmental conditions.

12.2

Organization of CRISPR/Cas in bacteria

The sensor-based microbial immunity in genomic loci is comprised of two adjacent regions CRISPR and its-associated Cas system, through RNA guided immune structure, provides immunity against foreign biological entities such as phages and plasmids [4,38,39]. The initial genomic region a CRISPR array, contains interspersed repetitive sequences known as CRISPR. The second domain of CRISPR array represents CRISPR-associated genes known as Cas genes. The cas encoded protein are major machine which enables the CRISPR loci to serially incorporate between CRISPR repeat sequences 30- to 84-bp DNA elements known as “spacers” from invading bacteriophages and plasmids [5,27]. The DNA corresponding foreign plasmid is termed as “protospacers” [23]. The structural characteristics of CRISPR array, downstream of cas genes include a unique leader sequence of few hundred base pairs followed by multiple short direct repeats and non-repetitive spacer elements between these repeats. CRISPRs play an important role in the reorganization of chromosomes, deployment of DNA, overhauling and regulation and replicon separation [32,40
43]. Although CRISPR system is sequence specific and heritable still, it can adopt new invaders [22]. In general, all of the so far discovered CRISPR-Cas systems of diverse bacteria, possess similar features which consists repeats of identical units between spacers of variable type [44,45]. The cas proteins play important role in acquiring novel spacer elements from foreign DNA, developing phase resistance by targeting newly invasive genetic elements using spacer unit as memory unit. Therefore, although the information for invaders are spacer encoded and are a component of CRISPR system. These informations are printed and made available to Cas machinery through transcription. During transcription, a noncoding sequence and low-complexity region made up A/T-rich, located immediate upstream of the first repeat which is also known as CRISPR leader act as a promoter for transcribing CRISPR array into the pre-crRNA [36,46]. The spacer unit is capable of pairing with sense as well as antisense DNA and hence allow it to even target dsDNA. In Pyrococcus, CRISPR complex is composed of crRNA and Cas proteins and catalyze the complementary-dependent cleavage of invader RNA under in vitro conditions [47] (Fig. 12.2). So far, a total of 25 cas gene families have been identified which represents eight gene members (cas1, cas2, cas4, cas6, cas7) with nuclease action, nine members (cas5) with repeat-associated mysterious proteins and two members (cas3) with DNA/RNA polymerase/helicase action [32,48,49]. The biological functions of other cas gene families is still unexplored [50]. Subsequently, the pre-crRNA is cleaved into specific small RNA entity known as spacer which is located adjacent to two partial repeats in CRISPR array [28,46,51,52]. The vast diversity of CRISPR/Cas systems are capable of targeting both DNA and RNA. Initially, after recognition and adaptive immunization, the spacer is integrated at the leader end between two adjacent repeat units of CRISPR locus and this process is known as protospacer [37]. In majority, a small unit of conserved

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FIGURE 12.3 The type I system contain cas3 gene which possess both nuclease and helicase domains whereas type II 2 CRISPR-Cas systems is one the most explored and widely studied system. It consists a duplex of crRNA involved in recognizes the invading DNA and the tracrRNA which base pair with the crRNA. Type III-A system uses a multi subunit system Csm and a CRISPR RNA (crRNA) for interference to cleave the active cognate DNA.

nucleotides protospacer adjacent motif (PAM), in vicinity of protospacer is considered vital and facilitate Cas proteins in foreign DNA acquisitions [53,54]. In the second step, RNA polymerase mediated transcription of CRISPR loci leads to precursor RNA (pre-crRNA) which is subsequently cleaved into much smaller CRISPR RNAs (crRNAs). These crRNAs either alone in combination with tracr RNA are known as guide RNAs (Fig. 12.3) [51,52] or prokaryotic silencing RNA (psiRNAs) guide the cas proteins [32,47] to specifically recognize and complementarity pair with invader’s nucleotides [51]. The lack of PAM at the CRISPR loci in the host genome play important role in the recognition of self from nonself-foreign DNA [55]. After binding to the target site, endonucleases activity in cas proteins, leads to a double-stranded break in DNA (DSB) followed by DNA overhauling at desire site [56
60] (Fig. 12.2).

12.3

Classification of CRISPR cas system

Depending upon the CRIPSR array and cas proteins organization, CRISPR cas system is categorized into two classes and six subtypes [44,45,61,62]. The class 1 represents type I, III and IV, uses a complex of different proteins whereas class 2 representing type II, V and VI, uses single cas protein to target and degrade the foreign DNA. The type I, II and III are widely distributed both in bacteria and archaea and contains signature Cas3, Cas9, and Cas10 protein, respectively [45,61,62] (Fig. 12.3). The type I are additionally divided into six subtypes (IA-F). The presence of PAM, near to protospacer is known to improve the efficiency of both type I and II systems and help in distinguishing ‘self’ and ‘nonself’ [26,33,37,54]. The PAM is located at located at either 5’ or 3’ end of the protospacer and mostly 2
5 nt long [54,63]. Cas9 protein, contains a phosphate lock loop which interact with the target-strand DNA, directly near to the PAM. Moreover, this interaction is known to kink the DNA and facilitate the DNA strand separation at local level, an essential feature required for the formation of the gRNA
DNA duplex [45]. Since it is well evident now that CRISPR system operates through the cooperative action of different cas proteins. The mature crRNA, contains spacer sequence which helps in targeting foreign genome and hence acts as a guide whereas a component of crRNA is involved in the interaction with Cas proteins and other components. For type II CRISPR system, a noncoding trans-activating CRISPR

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RNA (tracrRNA) hybridizes itself with the crRNA sequence and then acts guide RNA for Cas9 binding to target site followed by Cas9-mediated target degradation [6,7] (Fig. 12.3). The distribution of total CRISPR loci in the genome differs from 1 to 18 and number of repeat units in CRISPR array can vary from 2 to 374. According to the CRISPR database (http://crispr.i2bc.paris-saclay.fr), from a total of 232 archaea and 6782 bacterial analyzed genome, confirmed CRISPRs have been identified in 202 archaea and 3059 bacterial respectively. A detail list of information, related to literature and vectors area available at http://www.addgene. org/crispr/. CRISPR-Cas9 through 20 b long gRNA specifically bind the complementary protospacer DNA in the genome. Cas9 system generates double stranded DNA break, exactly 3 base pairs upstream of the PAM sequence. During this process, CRISPR-Cas9 undergo several conformational changes [64] and binding of a gRNA to target site, transforms the CRISPR-Cas9 system to its active form. After binding to the protospacer element, gRNA with the help of the PI domain of cas9 which interact to PAM, interrogates the DNA strands and provides the specificity to cas9 [64
66]. The CRISPR-cas 9 system of Streptococcus pyogenes, revolutionized the genome editing modification since its inception. Cas9 contains a bilobe structure with a nuclease (HNH) domain and α-helical recognition (RuvC) domain [65
67]. The HNH nuclease domain target and degrade the complementary DNA to the guide RNA. To degrade the target DNA, the HNH nuclease and RuvC nuclease domain uses a single and two metal mechanisms [65
67]. The RuvC domain contains regions, involved in the recognition of heteroduplexes gRNA
target template and precise identification of cognate sgRNA [1,65
67]. Mutation in anyone one of domain i.e. HNH or RuvC domain, leads to a nickase Cas9 (nCas9) which can target only one DNA strand [10,11,68,69]. The pair of nCas9s can be used to target both DNA strands, for creating DSB which further increases the specificity of Cas9-based genes (s) editing [10,11,68,69]. On the other side mutation in nuclease domains, creates a deactivated cas9 (dCas9) which lack ability, to break DNA strands but are capable of binding DNA-through gRNA. The fusion of deactivated Cas9 with other effectors, can be explored for epigenetic and site-specific gene modulations without degrading target DNA [70
73]. Alternatively, by simply using sgRNA, capable of pairing new DNA site, the Cas9 can be retargeted for another gene editing. The PAM sequences are widely distributed in the genome and are present after every 8 bp within the genome and even Cas9s from other species can recognize different PAMs which allow different Cas systems such as SaCas9 [74], St1Cas9, and NmCas9 [75] to target any gene of interest [1]. The engineering of already prevailing Cas9 systems, led to the establishment of new variants of Cas9 with different PAM sequences [76,77]. Moreover, the base-dependent interactions are known to determine the Cas9 interaction with PAM [1]. The SpCas9 system interact specifically with 5’-NGG-’PAM. The presence of two arginine residues at position 1333 and 1335 on cas protein, interact with GG on PAM site, present on non-target strand. The other Cas9 system specifically interact with 5’-NNGRRT-’PAM sequence PI domain contains multiple residues i.e. asparagine at positons 985 and 986 and aspartate at positions 986 and 1015 which interact with GRRT [65]. On the side, Cpf1 does not need tracrRNA. Here, crRNA alone guide the system to cleave the target strand. Further, Cpf1 creates a staggered DSB and recognize different PAM sequences, compared to Cas9 system [78]. The domain analysis revealed that Cpf1 owns a single RuvC- like domain [78]. Other classification of class 2 CRISPR systems have been given by Shmakov et al. [79]. Cas3 protein is reported for type I whereas Cas9 are reported for type II. Cas10 protein is reported for type III. The multi-meric effector complexes of type I and type III systems, are respectively termed as CRISPR-associated complex and the Csm/Cmr complex are structurally comparable and evolutionarily related. Another uncharacterized type IV system lacks adaptation module of nucleases Cas1 and Cas2. The effector modules of subtype III-B system is known to utilize spacers, produced by type I systems. similar to subtype III-B systems, type IV system uses crRNAs from different CRISPR arrays. In another classification, each type is further classified into subtypes such as I-A to F and U, type III into A-D subclasses based on the additional signature [80].

12.3.1 Type I CRISPR system The type I system is most wildly distributed system [61,62], represents 6 subtypes (IA
F) and contain cas3 gene (Fig. 12.3). The ca3 possess both nuclease and helicase domains. In some subtypes like A, B, and D of type CRIPSR, distinct genes encode nuclease and helicase domain. For each subtypes, the specific variant of cas proteins interacts with crRNA and then interrogate the target complementary template. Initially discovered in E. coli K12 (type I-E), tcrRNA-guided surveillance complex system is composed of five functional cas proteins [81]. The Cas6 unit of surveillance guide, was previously known as CasE or Cse3, is an endoribonuclease, cleave the precursor CRISPR RNA into matured 61-nt crRNAs [51,82]. The crRNA and Cas6e components are also necessary for the assembly and stability of Cse1, Cse2, Cas5 and Cas7 [83].

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12.3.2 Type II CRISPR or gRNA-Cas9 complex system The type II 2 CRISPR-Cas system is one the most explored and widely studied system for genome editing. It is categories into categorized into two subtype (A-B). In former, the cas component is encoded by cas1, cas2, cas9, and csn2 genes whereas cas4 is responsible for the type II. Here, a duplex of crRNA involved in recognizes the invading DNA and the tracrRNA, base pair with the crRNA [4
8,47]. The signature Cas9 gene belonging to type IIA encodes a multifunctional protein, capable of converting precursor crRNA into mature RNA and also target the foreign DNA [6,7]. The tracrRNA is essential prerequisite for precursor RNA processing and target recognition. Compared to other CRISPR types, the processing of crRNA/gRNA require trans-activating crRNA (tracrRNA). In Streptococcus pyogenes, the tracrRNA is located upstream on the complementary strand of the CRISPR
cas loci [6]. The Cas9 protein contains nucleases are classified in three subclasses: type II-A, type II-B, and type II-C [13,84
86]. The small size of Sa Cas9, permit it to avoid the delivery issues of large CRISPR systems such as SpCas9. In addition to SpCas9 and SaCas9, other orthologs such as from Neisseria meningitidis are also explored [75,87
90]. St1Cas9 system engineered as dCas9 variants is being used for gene regulation study [1]. The fusion of crRNA and tracrRNA results, formation of dsRNA that is responsible for cleaving the RNase III [83]. Beside this, several other cas proteins Cas12a (Cpf1), and Cas13a (C2c2), Cas12b (C2c1) in type V, and Cas13b (C2c3) in type VI have been identified [24].

12.3.3 Type III CRISPR system Although type III CRISPR-Cas systems are one of the most common RNA-guided adaptive immunity system in bacteria and archaea but their biological action on target site and cleavage mechanism are not fully understood. The Type III-A system such as Csm complex of S. epidermidis can targets DNA and RNA [47,83,91,92]. The type III-A CRISPR system of T. thermophiles a thermophilic bacterium are attractive tool for gene editing due to their better stability at high temperature. Type III system are most common in archaea and are subdivide into type IIIA and III-B (32). Both of these systems have cas6 and cas10. Cas6 is CRISPR-specific and cas10 is involved in target interference. There are report that type III-A of S. epidermidis target DNA (21) and type III-B systems of Pyrococcus furiosus and S. solfataricus target RNA [47,83,91,92]. Type III-A systems is intriguing, known to use a multi subunit system Csm and a CRISPR RNA (crRNA) for interference to cleave the transcriptionally active cognate DNA. A number of studies on archaeal CRISPR type III systems mediated cleavage of mRNA degradation (Zebec et al., 2014) and targeting of RNA and DNA are available [50] (Peng et al., 2015).

12.4

CRISPR-Cas system in extremophiles

Although CRISPR/Cas9 is one of most explored genome editing tool but its applications in thermophiles are restricted. Despite the advantages for these microbes for industrial and scientific applications. the genetic engineering tools for thermophilic microorganisms are limited. A combination of genomics and mathematical model based analysis of several hundred prokaryotic genomes, it was found that CRISPR-Cas systems are highly widespread in thermophiles, compared to their mesophiles counterpart [93]. The genomic island of thermophiles and their counterpart mesophiles share almost similar genes with some variations [94]. CRISPRs which represents the genomic hall mark of the prokaryotes and constitute over 40% CRISPR loci in response to immunity against viruses and phages [95]. The genetic diversity of CRISPR loci have shown a distinct pattern in Archaean, Methanocaldococcus jannaschii and thermophilic bacterial strains Streptococcus thermophilus [88,89]. Some of CRISPR loci in the genome of Thermoanaerobacter tengcongensis and Thermus thermophilus HB8 are known to share identity with megaplasmids [3,35]. The variation in spacer elements have been reported for five strains of Thermotoga neapolitana [41,50]. A Type III Csm complex (TthCsm) system active at 65  C has been identified from Thermus thermophiles [80]. The endonuclease activity of TthCsm, mediated by crRNA can target RNA and transcriptionally active DNA [23]. The genetic tools for a large number of thermophiles are available but their limited efficiency impede with the proper exploitation of extremophile such as thermophiles. A temperature controlled recombination/counter selection tool has been developed for moderate thermophile genome editing [96,97]. The SpCas9 is active only below 42  C, therefore using a combination of homologous recombination, SpCas9 based counter-selection and elevated moderate temperature i.e. 28  C, cas9 system has been developed for facultative thermophiles. However, for obligate thermophiles, CRISPR- Cas type-IIC system (thermoCas9) which uses RNA-guided DNA-endonuclease of Geobacillus thermodenitrificans T1230, a thermophilic

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bacterium has been characterized. This system is active between 20 and 70  C [96,97]. The thermoCas9 has been deployed for the genome editing and silencing of a thermophilic Bacillus smithii ET 13831inudtrially important strain at 55  C [96,97].

12.5

CRISPR/Cas system of halophilic archaea

Initially, the halobacteria are known to lack CRISPR-Cas mediated immunity. In recent studies, sequences matching to the PAM have been recognized in Haloferax volcanii archaea. Several of these motifs are found active in triggering the defense responses. The selection of protospacer DNA from the invader appears only for a few PAM sequences [22]. It is observed that over half of the haloarchaea species have the same CRISPR/Cas system [22]. In Haloferax, the CRISPR/cas system is composed of three CRISPR RNAs and eight Cas proteins. The phylogenetic analysis revealed that it belongs to the type I-B CRISPR/Cas systems group. However, due to limited availability of CRISPR loci for halophiles in databases, the comparative study for Haloferax revealed only two matches. One of the match differed only at four positions to the environmental sample collected from a salt-lake in Australia. Moreover, low number of spacers were found reported which could be due to the limited haloarchaeal viruses. Another reason of low spacers in the array is that the DS2 strain was isolated from the Dead Sea a log time back in 1974 [94]. In another study, on Haloquadratum walsbyi revealed eight matches for space sequences and the number of PAM sequences (seven) is identical to PAM sequences reported for Haloferax. Likewise, a CRISPR/Cas type I-B system for Haloferax, Hqr. Walsbyi, contains CRISPR repeat sequences which are very similar to Haloferax [22]. The comparison the CRISPR database (crispr.u-psud.fr/crispr/, July 2012) showed that Cas proteins of haloarchaea all belong to the type I-B CRISPR/Cas group [22].

12.6

Delivery methods

Based on the type of application, several methods for Cas9 systems are available which uses different types of vector systems. The choice of components and their entry depends on the assay and cell type. A number of vectors encoding CRISPR components are accessible and can be obtained from Addgene (https://www.addgene.org/) or from other sources. Based on Cas9-encoding vector, an appropriate complementary sgRNA-vector can be selected. For targeted action of dCas9, cloning of B100 bp short sgRNA followed by their introduction into desired cell type is required [98
100]. For pooled screening, it is good to develop and use stable Cas9 or dCas9 system in cell lines either using lentiviral or retroviral vectors, prior to entry of a lentiviral sgRNA pool [101]. However, vector systems which can encode Cas9 and sgRNA is preferred method [98,102]. The non-viral based methods, explore Cas9-encoding plasmids or Cas9 mRNA or hydrodynamic injections for genome editing or efficient gene corrections or mutations [103
106]. Another viral or nonviral delivery method known as purified Cas9
sgRNA ribonucleoproteins (RNPs) can enhance the efficiency, avoids undesired genomic aberrations and decrease the off-target effects whereas for normalization in CRISPRi/a, it is good to use one or multiple non target sgRNAs as negative control.

12.7

Applications

The gRNA-directed CRISPR-Cas system has been successful explored for microorganisms, plants, and animal genome modifications. The Cas9-mediated gene-editing systems are widely used to understand the role of specific genes, and explore their applications both in academic and industrial sectors [13,14,107]. The variants of cas9 and other CRISPR systems such as nuclease-deactivated Cas9 (dCas9) has been developed and fused to either transcription modulators and epigenetic modifications [70
73]. In addition to their primary role in genome editing, the fusion of cas proteins fluorescent proteins expand the usage of this technology for imaging of gene loci in living cells directly [108,109,110] (Fig. 12.4). Applications of dCas9 has been explored for targeting RNA [1,30,111] and proteins interaction with loci [112]. In plant biology, the CRISPR loci and Cas systems have gained widespread attention to target genes [113
115]. The Cas9 platform are currently deployed for large-scale genome-wide knockout studies which were previously unfeasible [102,116
119]. The modification of dCas9, the sgRNA can be used for recruiting transcriptional regulators [120
122]. The fusion of sgRNAs to orthogonal protein-interacting RNA RBP aptamers, can be used to achieve multimodal regulation i.e. both the activation and repression simultaneously [121]. Since its discovery, CRISPR-Cas9-based genome editing tools have transformed basic research and applied research in biology. However, the established Cas9 systems is not suitable for applications which demands enhanced stability,

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FIGURE 12.4 Overview of CRISPR based genome editing technology in basic research.

at high temperature. Therefore, ThermoCas9 derived from the thermophilic Geobacillus thermodenitrificans T12 strain is functional in a temperature range of 20
70  C. Moreover, the ThermoCas9 permits less spacer-protospacer mismatches, compared to SpCas9 and hence more precise. ThermoCas9 system is found effective for transcriptional silencing and deletion at 55  C in Bacillus smithii and deletion at 37  C in Pseudomonas putida [96,97]. ThermoCas9 variants area powerful genome engineering tool and open new possibilities of Cas9 technologies in novel applications under harsh conditions. Microbial fermentation of renewable resources such as biomass into biofuels and other natural products is expanding at great pace. However, the production costs of these processes under extreme conditions demands better and effective strains. The ability of thermophilic organisms to grow and ferment at high temperatures, can reduce the production cost and lower the contamination risk even without using nonsterilized conditions. Further, the regulation of sporulation gene such as sigF during in industrial fermentations is offer potential for commercialization.

12.8

Conclusion and future directions

Initially discovered as palindromic segments, CRISPR-Cas are genomic sensors of bacteria, which confers immunological memory and adaptive immunity by acquiring foreign DNA fragments. The gRNA-directed CRISPR-Cas is presently method of preference for genome editing of microorganisms, plants, and animal genome modifications. The foreign acquired DNA fragments guide the cas proteins and targets, foreign DNA upon infection. The CRISPR/Cas system undoubtedly holds immense potential for genome/gene(s) editing [123
130] but there are several limitations. For example, the mesophilic nature of Cas-endonucleases, limits application of CRISPR-Cas technologies below 42  C [96,97,131] and excluded in obligate thermophiles which demands high elevated temperatures. For example, ThermoCas9 system of thermophilic bacterium G. thermodenitrificans strain T12 isolated from compost is active in a wide temperature range of 20
70  C, compared to mesophilic orthologue SpCas9 which is active in narrow range i.e. 25
44  C. This extended temperature range, permits ThermoCas9 use where DNA manipulation needs to be performed at 20
70  C or under harsh environmental conditions [96,97]. Another drawback of CRISPR is off-target activity, which can result unwanted and potential pathological consequences. The off-target effects of Cas9 [132], due to the 20-bp sgRNA with only 3 bp PAM on the genome, may leads to elsewhere pairing [133
135]. These problems can be addressed by exploring genome of extremophile for genome/gene editing tool box. The off target activity could cleave a number of individual genes. To mitigate the off target effects, a number of approaches have been deployed which includes isolation of alternate specific and more stringent CRISPR-Cas from other strains of archaea or bacteria and developing rational gRNAs [135], use of dCas9 enzymes for the introduction of single strand break, instead of DSBs [10,11]. The target site degradation by the CRISPR- Cas system, needs PAM downstream to the protospacer element where gRNA binds the target. These PAM sequence differs in number and composition of nucleotides across the bacterial strains [136]. For example, the PAM sequences in two systems i.e. SpCas from S. pyogenes

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and StCas from S. thermophiles need 5-NGG-30 and 5-NNAGAAW-30 nulcoetide sequences as PAM recognition site respectively. Here, the possibility of these 8 bp, present in StCas from S. thermophiles may be difficult throughout the genome and hence can reduce the offset activity remarkably [136]. Moreover, the complete gene deletion using CRISPR/Cas9, demands mutation of all alleles of the gene hence screening in such cases is often challenging. The deletion of several genes may leads to lethal effect on the host. The abundant dark matter islands in prokaryotic genomes particularly extremophiles, often a rich and potential source of novel genes and new types of CRISPR-Cas systems array, for genome engineering [94]. Additional strategies such as optimized sgRNA design [118,135,137], use of paired nCas9s, paired dCas9-FokI nucleases [138,139] or decreasing the amount of the Cas9
sgRNA complex etc. Hsu et al., [135,140], can improve SpCas9 specificity. Another concern associated with improved specificity is that it may cost efficiency. The CRISPR efficiency, can be enhanced significantly by deploying multiple transcriptional activators which can upregulate the gene transcription. Use of multiple sgRNAs, for recruiting multiple dCas9 activators, along the promoter have been developed for transcriptional activation of genes [122,141,142]. For example, a dCas9-VP64 in combination with modified sgRNA, fused with MS2 RNA aptamers, can be used for recruiting a pair of cognate RNA-binding proteins such as p65 and HSF1 (MCPp65-HSF1) [65,122], results enhancement in the efficiency for large scale genomic screening [122]. In another method, based on combined use of dCas9 system with multipeptide array, has been explored for loading multiple VP64 activator modules to the binding site of a single dCas9 unit [142,143]. The genome engineering tool such as CRISPR/Cas9, permits us to make optimum use nature’s technological tools which has been improved for billions of years through natural evolution. The careful address of the concerns among bioethicists, policymakers, and the public, about how to ethically and responsibly use gene editing technology, for human welfare, will benefit the scientific research [144
149] and our society. In addition to the scope for improvements, its ability to edit genome, without any question offers vast potential, for editing the genomes of different organisms. The identification of Cas9 in thermophilic bacteria such as ThermoCas9 variants, as potent genome engineering tool, for thermophilic and mesophilic organisms, offer vast potential and open new possibilities of Cas9 technologies in novel applications under harsh conditions.

Acknowledgments The authors are thankful to Chandigarh University for providing necessary infrastructure and SEED Division, Department of Science and Technology, GOI for providing financial benefits (SP/YO/125/2017 and SEED-TIASN-0232018) during the completion of this work.

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[141] Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, et al. Highly efficient Cas9-mediated tran- scriptional programming. Nat Methods 2015;12:326
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61.

Further reading Koonin EV, Makarova KS. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol 2013;10:679
86. O’Connell MR, Oakes BL, Sternberg SH, East-Seletsky A, Kaplan M, Doudna JA. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 2014;516:263
6.

Chapter 13

Lipases/esterases from extremophiles: main features and potential biotechnological applications Valentina De Luca1 and Luigi Mandrich2 1

Institute of Protein Biochemistry, National Research Council, Naples, Italy, 2Research Institute on Terrestrial Ecosystem, National Research Council,

Naples, Italy

13.1

Introduction

The organisms that live in extreme environments are called extremophiles, they have achieved a series of adaptations to live in extreme conditions of temperatures, pH, high salinity, high pressure, and in consequence are indicated for the chemical-physical characteristic to which are adapted, for instance thermophiles for high temperature, halophiles for high salinity and so on [1]; in Table 13.1 a classification of the different types of extremophiles is reported. The organisms of each class of extremophiles have developed specific adaptation to overcome the particular environmental conditions, for example thermophilic and hyperthermophilic organisms have proteins showing a more compact structure by the reduction of loops length and the increase of charged amino acids [1,2], moreover they present a different composition of the plasmatic membranes [1,3]. Psychrophilic organisms show opposite features, in fact they have proteins rich

TABLE 13.1 Extremophiles classification. Chemical-physical parameters Temperature

Name

Range of adaptation 

Class of organisms

Hyperthermophiles

Growth . 80 C

Archaea, bacteria

Thermophiles

Growth 55 80  C

Archaea, bacteria

Mesophiles

Growth 15 55  C

Eucaryotes, bacteria, plants



Psychrophiles

Growth ,15 C

Algae, bacteria, plants

Alkaliphiles

pH . 9

Archaea, bacteria, eucaryotes

Acidophiles

pH , 3

Archaea, bacteria, eucaryotes

Salinity

Halophiles

High salt concentration

Algae, archaea, bacteria

Pressure

Piezophiles

High pressure

Archaea, bacteria

Radiation

Radiotolerant

UV resistant

Bacteria

Oxygen

Anaerobe

Not tolerant O2

Archaea, bacteria

H2O

Xerophiles

Low availability of H2O

Plants, yeasts, bacteria

Rocks

Endoliths

Live inside rock and coral

Algae, archaea, bacteria, yeasts

Metals

Metalotolerants

High metal concentration

Algae, plants, bacteria

pH

The classification has been based on the chemical-physical parameters at which the organisms are tolerant, are also reported the range of conditions of adaptation and the class organisms identified to be tolerant to these extreme conditions.

Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00013-7 © 2020 Elsevier Inc. All rights reserved.

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in α-helices and polar groups which allow an increase of protein flexibility; membranes are more fluid and contain the antifreeze proteins that maintain liquid the intracellular solutions [4]. The radiotolerant organisms have many and efficient DNA repair enzymes [5]. Metalotolerant organisms contain specific enzymatic systems to reduce ions and generate metal precipitate [6]. Here, we report the main characteristics and the classification of enzyme from extremophiles, with particular interest for esterases and lipases that are target of many studies for basic knowledge about these enzymes, but also for their potential in industrial applications.

13.2

Structural features and classification of esterases/lipases

The hydrolase superfamily is the most numerous group of enzymes that have as distinctive features the use of water to break chemical bonds of the substrates and the same fold, called α/β-hydrolase fold [7]. All the enzymes have a canonical triad of catalytic residues (nucleophile-His-acidic residues), evolved to hydrolyze different substrates more efficiently (Fig. 13.1). In the enzyme classification (EC) by number, hydrolases are indicated as EC3, and are further classified on the bases of the bonds on which they act, in: EC 3.1: ester bonds (esterases, lipases, phosphoesterases) (Fig. 13.2A); EC 3.2: glycosidic bonds (glycosidases) (Fig. 13.2B); EC 3.3: ether bonds (ether hydrolases, epoxide hydrolases) (Fig. 13.2C); EC 3.4: peptide bonds (proteases, peptidases) (Fig. 13.2D); EC 3.5: carbon-nitrogen bonds (amino hydrolases, amidases) (Fig. 13.2E); EC 3.6: acid anhydrides (anhydride hydrolases, NucleotideTPase) (Fig. 13.2F); EC 3.7: carbon-carbon bonds (oxaloacetases, acylpyruvate hydrolases) (Fig. 13.2G); EC 3.8: halide bonds (alkylhalidases, haloacetate dehalogenases) (Fig. 13.2H); EC 3.9: phosphorus-nitrogen bonds (phosphoamidases) (Fig. 13.2I); EC 3.10: sulfur-nitrogen bonds (sulfohydrolases) (Fig. 13.2J); EC 3.11: carbon-phosphorus bonds (phosphonatase) (Fig. 13.2K); EC 3.12: sulfur-sulfur bonds (trithionatehydrolase) (Fig. 13.2L); EC 3.13: carbon-sulfur bonds (carbon disulfide hydrolase) (Fig. 13.2M). As said before hydrolases utilize water to break chemical bonds of the substrates (Fig. 13.2) following the scheme reported in Fig. 13.3A. In general, the hydrolysis of substrate leads to the release of the acid and basic components of substrate, AOH and B-H of Fig. 13.3A. The reaction mechanism is divided into two steps, in the first step the hydroxyl group of the catalytic Serprovides to the nucleophilic attack to broken the bound between the two substrate components, with release of the basic component A-OH (Fig. 13.3B D). In the second step, the water molecule is polarized by the catalytic His (step 1 of Fig. 13.3E), promoting the attack on the acid component of substrate bond to the serine (step 2 3 of Fig. 13.3E), with consequent release of the acid component B-H and restoring of catalytic His (Fig. 13.3F). Among the superfamilies belonging to the hydrolases, our interest is focused on esterases and lipases. These enzymes are very important for their versatility in the cells but also for the numerous biotechnological applications in which they are currently used. Lipases and esterases are differentiated on their substrate specificity; in particular, esterases hydrolyze short acyl chain esters water soluble, whereas lipases hydrolyze long acyl chain glycerol-esters that are water insoluble [8]. All these enzymes are characterized to have the α/β fold and the triad of catalytic residues: histidine, serine and acid residues (aspartic or glutamic acid). By the comparison of the amino acid sequences, esterases and lipases are divided in four families: the Choline esterase family (C-family), the Hormone-sensitive lipase family (H-family) and the lipoprotein lipase family (L-family); all the other enzymes not included in these three classes are grouped in the X family. In the amino acid sequence the catalytic residues follow the order is nucleophilic-acidic-histidine, the nucleophilic residues is Ser that together to His is always presents in all three group, whereas as acid residue in H and L families is present Asp, and only in C-family Glu. The α/β fold has a core composed of a central 8 parallel β-strands, which are twisted FIGURE 13.1 α/β-hydrolase fold. Topological diagram of a canonical α/β-hydrolase fold, in red arrows are indicated the strands and in green cylinders the helices. The positions of the catalytic residues are indicated.

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FIGURE 13.2 Substrates of the hydrolases. General structure of substrates of the hydrolases are reported, in red are indicated the bonds cleaved/ cutted by the specific hydrolases: (A) esters and phosphoglycerides for EC 3.1; (B) glycosides for EC 3.2; (C) ethers and epoxides for EC 3.3; (D) peptides/proteins for EC 3.4; amines and carboxyamines for EC 3.5; carboxyacid anhydrides and nucleotides triphosphates for EC 3.6; oxaloacetate and 3-acyl-pyruvate for EC 3.7; haloacetates and bromochloromethane for EC 3.8; N-phosphocreatine for EC 3.9; N-sulfo-D-glucosamine for EC 3.10; phosphonoacetaldheyde for EC 3.11; trithionate for EC 3.12; carbon disulfide for EC 3.13.

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FIGURE 13.3 General reaction mechanism of hydrolases. ( A) Scheme of reaction catalysed by hydrolases; (B) schematic representation of active site of hydrolases, with indication of residues involved in catalysis (Ser-His-Glu); (C) the serine hydroxyl group promotes the nucleophilic attack on the A-B substrate; (D) the first step leads to the release of the basic component of substrate, keeping the acid water component of substrate bound to the catalytic serine; (E and F) in the second step the water molecule leads to the release of the acid component of substrate, restoring the initial serine in the active site.

and enclosed by helices on each side(six or more helices). In Fig. 13.4 the scheme of the structures of the three families H, L and C is reported; for the X family, being heterogeneous, it is not possible to indicate a general structural scheme (ESTHER database at bioweb.ensam.inra.fr/ESTHER). The C-family includes cholinesterases from vertebrate and invertebrate, lipase from fungi, many esterases and some non-enzymatic proteins. The H-family includes lipases and carboxylesterases from vertebrate and bacteria; the name H-family or HSL is derived from the Hormone Sensitive Lipase from human, which was the first sequence included in this group. Human HSL show amino acid sequence homology with several bacterial proteins but not with proteins belonging to the other groups. The L-family includes lipases from vertebrate and bacteria, lipoprotein lipases, lecithin-cholesterol acyl-transferases and related non-enzymatic proteins. Many lipases are characterized also for the presence of a mobile sub-domain, called lid, which can assume two positions: a closed conformation, if the lid closes and prevents to the substrate to enter in the active site, and the open conformation, if the lid allows to the substrate to enter in the active site. The opening of the lid is modulated by the presence of micellar substrate at the interface with the catalytic site [9,10]. In Fig. 13.5 is reported the scheme of the open/closed conformation of the lid. Among esterases/lipases superfamily there are enzymes coming from organisms living in extremophilic environments, these enzymes are also called extremozymes, and they have the peculiarity to be active only in extreme conditions. As previously reported, they differ from mesophilic enzymes in term of amino acids composition and 3D structure, whereas the catalytic triad and the α/β fold are conserved. At the moment, in literature have been reported about 850 papers on extremophilic esterases/lipases, the first was published in 1966, but in the last 20 years have been published 70% of total papers, indicating the increasing interest for these type of extremozymes. A further differentiation can be made on the different types of tolerance that these extremozymes shown, in Fig. 13.6 is reported the subdivision of the papers by classes of extremophilic esterases/ lipases. The most studied are the thermophiles with about 75% of the papers, followed by halophiles (15%) and psychrophiles (8%), few papers are on acidophiles (1.5%) and alkalophiles (1%) and only 1 paper on barophiles (these data are referred up to January 2019).

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FIGURE 13.4 Schemes of structures of esterase/lipases superfamily. Following the classification reported in ESTHER database (bioweb.ensam. inra.fr/ESTHER) it has been reported the scheme of structures of the three subfamilies in which are divided: (A) choline esterase family (C-family); (B) hormone-sensitive family (H-family); (C) lipoprotein lipase family (L-family). In red arrows are indicated the strands and in green cylinders the helices. The positions of the catalytic residues are indicated.

13.3

Thermophilic esterases/lipases

The organisms adapted to survive at high temperature are characterized to live from 45  C up to 122  C, respect to mesophilic organisms they show many differences at molecular level, and principally they are archaea and eubacteria.

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FIGURE 13.5 Schematic representation of lipase lid activation by substrate. In many lipases the lid domain closes the access to the active site, in presence of micellar substrates the lid changes its conformation opening the way for the catalytic site to the substrate.

FIGURE 13.6 Graphic representation of distribution of the papers published on extremophilic esterases/lipases. The total number of papers published is about 850 (updated to January 2019), and they are divided by color: red for thermophilic enzymes (642 papers), green for halophiles (127 papers), blue for psychrophiles (63 papers), yellow for acidophiles (12 papers), violet for alkalophiles (8 papers) and light blue for barophiles (1 paper).

The main differences respect to mesophlic organisms are at DNA, membranes and proteins level, in fact the GCcontent of the coding regions correlates with the temperature, in fact at increasing temperature increase the GC-content [11]. The membranes have different composition with respect to the mesophilic counterpart. In fact, many studies have revealed that the membranes of thermophilic organisms contain higher percentages of lipids stable to the temperatures, in particular ether lipids [12] and esters with long acyl chains [12,13]; hereas the percentage of branched chains decrease [12,13]. The main evolutionary difference of the adaptation at high temperatures concerns proteins, because high temperatures are one of the main denaturing agents, and the thermophilic organisms to survive have proteins that intrinsically are resistant to high temperatures, in fact they are stable over time, can be stored for long time and exploiting their heat resistance are easily to purify; all these features allowed and facilitated the use of thermophilic enzymes in industry. How is obtained this resistance? Many studies has been made to elucidate what are the determinants of protein thermostability, and several aspects have been reported to be involved; analysis were made at amino acids composition level and by three-dimensional structure comparison with the mesophilic and psychrophilic counterparts. The thermophilic proteins have different mechanisms to increase their stability, in fact at amino acidic level has been observed the decrease of polar residues, decrease of cysteine and deamidation sites (glutamine and asparagine), increase of charged residues and increase of proline in loop regions [14 16]; at structural level has been observed the decrease of loops length, increase of ion pairs, hydrophobic and electrostatic interactions, decrease of number and volume of internal protein cavities [17 19]. It’s important to note that comparative analysis conducted on huge number of proteins give only

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general trends but not rigorousness differences among thermophiles and mesophiles proteins, for this reason to highlight better the differences in amino acid composition or structures among thermophilic and mesophilic proteins it is necessary consider protein belonging to a restrict group or families of proteins. This is the case of esterases belonging to the HSL family, which were identify as the determinants of thermostability [20]; more interestingly has been demonstrated that to increase the protein stability besides the increase of number of ion pairs it has been observed an increase of the number of electrostatic interactions and the pathways of interaction that stabilize the charged residues on protein surface [2,21]. In the case of HSL family three proteins were analyzed: the mesophilic brefeldin A esterase (BREFA) [22], the thermophilic EST2 [23] and the hyperthermophilic AFEST [17]; the number of ion pairs found at cut-off distance of ˚ were 16 for BREFA, 14 for EST2 and 18 for AFEST, and at 6 A ˚ cut-off distance was 22 for BREFA, 22 for EST2 4A and 21 for AFEST [2]. It’s clear from this analysis that the number of ion pairs do not seem a determinant for thermostability, but by analyzing the pathways of interaction among charged residues on protein surface, the data of number of ion pairs and electrostatic interactions are in correlation with the increasing of thermostability. Using mutagenesis analysis it has been demonstrated that some critical charged residues involved in large pathways of interactions are changed in consequence change the thermostability [21]. The increasing knowledge about thermophilic enzymes in terms of activity, specificity and stability, made these enzymes of great interest in biotechnological applications. Actually many lipases and esterases has been isolated and from thermophilic sources, their potential applications are from food to pharmaceutical industries, their strengths are: high thermal stability, high half-life and high stability to organic solvents [24].

13.4

Psychrophilic esterases/lipases

Psychrophilic organisms are found in regions characterized by low temperatures, such as ocean deep, polar regions, high mountains and perennial glaciers; principally they are bacteria, archaea, yeasts and algae. Equally to high temperatures, low temperatures are hard denaturing conditions and in this view psychophilic organisms have adopted a series of modifications to survive and live optimally at low temperatures. A special case is represented by Antarctic fish, which are evolutionarily separated from the other species by the Antarctic Circumpolar Current that flows from west to east around Antarctica. These fishes are different from the other organisms at level of immune system, circulatory system, for the presence of antifreeze glycoproteins or ice-binding proteins (IBPs), as well as have proteins adapted to low temperatures [25]. The function of the IBPs is different from the proteins psychrotolerant, because IBPs mediate freeze tolerance, ice adhesion and inhibition of ice re-crystallization, which is the process of formation of big ice crystals that causes dehydration and cellular damage. IBPs bind to specific axes of ice and inducing a microcurvature, allowing the growth of ice in a restricted area between the adsorbed IBP and the curved surface. From a thermodynamic point of view the association of water molecules is more difficult (Kelvin effect), in this way the water freezing temperature FIGURE 13.7 Schematic representation of ice-binding proteins IBPs. Generally IBPs are small proteins, showing different structures. (A) Type I IBPs, only α-helices; (B) β-solenoid type IBPs, only β-sheet; (C) mechanism of action of IBPs to avoid ice formation, in red are indicated the IBPs; (D) schematic representation of the protection mechanism of alga, in red are indicated the IBPs; (E) schematic representation of the protection mechanism of bacteria, in red are indicated the IBPs; (F) schematic representation of the protection mechanism of fish, IBPs (red circles) cover the ice particles, limiting their growth.

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decreases in a non-colligative manner, leading an hysteresis between the freezing and the melting temperature [26] (Fig. 13.7). The molecular and functional diversity of IBPs is considerable, in fact they have evolved independently by organisms that belong to different biological kingdoms and are hosted in different niches. By the classification of their crystal structures IBPs are divided into 11 different folds, all sharing a common structural strategy coherent with the need to fold and to operate in the cold. The IBPs stabilize their structure not by their hydrophobic core but through disulfide bonds and networks of hydrogen bonds [27] (Fig. 13.7A C). The ice-binding sites present two main features: are flat and hydrophobic, without charged residues and containing repeated amino acid sequences, related to their ability to organize ice-like water on specific IBP surfaces [28]. In Fig. 13.7D F is reported a representative scheme of IBPs and the mechanisms adopted by algae, bacteria and fish to maintain liquid water at low temperature by IBPs. Low temperatures solidify the membranes which lead to loss of functionality. The solution to overcome this problem is in the membrane composition, in fact psychrophilic organisms have high content of lipid with short acyl chain length, polyunsaturated and simply unsaturated fatty acids, methylated branched fatty acids, and high ratio of lipopolysaccharides, compared to the mesophilic counterpart [29]. Regarding the adaptation of psychrophilic proteins, the main differences observed are: decrease of hydrophobic surface residues [29], decrease of aromatic interaction [29], decrease of disulfides and salt bridges [29], decrease of arginine and proline content [29], increase of glycine at active site [29], increase of size and number of enzyme cavities [30], and increase of polar residues (Asn, Cys, Gln, Ser, Thr, Tyr) [30]. All these differences are aimed to achieve high flexibility at level of protein structure, but for this reason the thermal stability of psychrophilic proteins at temperatures around 37  C is strongly compromised [31]. Till today, many psychrophilic esterases and lipases has been isolated and studied principally to understand the molecular determinants for the cold adaptation, their applications are in all the operation conditions in which substrates are sensible to the temperatures, such as food, organic synthesis, animal feed, textile, detergent and beverage industries [32].

13.5

Other extremophilic esterases/lipases

13.5.1 Halophiles These organisms are adapted to live at high concentrations of salt (maximum 5 M NaCl) [33], principally are archaea and bacteria [34]. Their ability is to maintain the osmotic balance accumulating salt at isotonic concentrations with the environment [35]. The main adaptation of halophilic proteins is at protein surface level by increasing the number of negative residues to prevent their precipitation, but this adaptation confer stability also in condition of low water content [35]. Several lipases and esterases have been isolated from halophilic organisms, their putative applications are under characterization [36,37].

13.5.2 Alkalophiles/acidophiles These organisms are adapted to live in conditions of high or low pH [38]. Their ability is to maintain internal pH near the neutrality by proton pumps, in this light the proteins do not need of particular adaptation, excluding those in the periplasmatic space. Few lipases are isolated to be adapted at high pH value, and they will be used in the detergent preparation for the hydrolysis of fats, where usually are used condition of high pH values [39].

13.6

Running and potential applications for extremophilic esterases/lipases

Since the industrial enzymes market is estimated to reach in several billion of US $ and being constantly growing, there is an increasing interest for new enzymes; among these many lipases and esterases are used in industrial applications. In particular, lipases are used in food and detergents industries, other possible applications are in organic synthesis, in pharmaceuticals and in biofuel production. In Table 13.2 are listed the most significant application for esterases and lipases.

13.6.1 Detergent The first application of extremophilic esterases/lipases in industry has been possible by the heterologous expression of these enzymes in E. coli and yeast and was from Novo Nordisk (Denmark) that introduces the first thermophilic lipase used in detergent industry, the Lipolase from Thermomiceslanuginosus, a thermophilic fungus [40]. The idea to use a thermophilic lipase derived from the practical use in combination with detergents, because this lipase is resistant to

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TABLE 13.2 Biotechnological application of esterases and lipases. Industry

Type of enzyme

Action

Range of activity

Detergency

Thermophilic

Dissolving fats by hydrolysis

pH 7 11 Temperature 20 70  C

Psychrophilic Food

Thermophilic

Modification of fats and oil for baked food and emulsifiers

Temperature 40 70  C

Solvent-tolerant Biodiesel

Solvent-tolerant

pH 7 9

Transesterification reaction with methanol and ethanol

pH 8 10 Temperature 20 60  C

Alkalophilic Thermophilic Cocoa butter

Thermophilic

Interesterification of cocoa butter-type triacylglycerols

pH 4 7 Temperature 40 50  C

Drug

Oleochemical

Solvent-tolerant

Resolution of racemic solution of acids and alcohols by

pH 4 7

Stereospecific

Esterification and transesterification

Temperature 40 50  C

Thermophilic

Glycerolysis for soap production

pH 4 8 Temperature 40 -70  C

Solvent-tolerant Dairy

Thermophilic

Increase of flavor development of cheese

pH 4 7 Temperature 40 70  C

Extremophilc esterases and lipases are very interesting for their potential in biotechnological application, actually they are used in detergent, food, cocoa butter and drug preparation, but very promising are biodiesel and dairy applications.

temperatures and detergents, another advantage derive from washing at high temperature because increasing the temperature increase the solubility of fats (making substrate more accessible to the enzyme) and the catalytic activity of the enzyme [41]. Regarding the use of lipases as detergent, many studies, also derived from the market analysis, have indicated that the use of psychrophilic lipase would give a number of advantages, such as lower cost by reducing the energy utilized, and at lower temperatures the colored fabrics are more protected. Esterases and lipases required for the detergent industry have specific features, prevalently are from alkalophilic or thermophilic organisms, and they must be active at high pH (10 11), high temperature (30 60  C), stable to detergents and have low substrate specificity to hydrolase a range of different fats [39].

13.6.2 Food Many flavor compounds are used in food and beverages industries, they are esters obtained naturally by esterification or transesterification reactions, and being natural compounds are extracted by natural sources. The growing demand has lead to an increase in the costs of these compounds and therefore alternative systems have been searched for their production, such as the use of biotechnologies by esterases and lipases synthesis, also from extremophiles [42 44]. More specifically, a screening of halophilic lipases has been made to find enzymes able to improve the flavor of a fish sauce very used in Southeast Asia and obtained by salted fish [36]. The halophilic lipase LipBL from Marinobacterlipolyticus SM19 has been used to hydrolyze olive and fish oil, resulting in the enrichment of eicosapentanoic acid (EPA), an omega-3 fatty acid that have a high nutritional value [45]. In the contest of food production, we can consider also the production of cocoa butter, which is used in chocolate industry. Chocolate contain about 30% of cocoa butter, but for the increasing demand of product tends to be very expansive, for this reasons it has been developed an alternative system to obtain cocoa butter-like triacylglyceroles. The process has been developed using Lipozime, from Novo Nordisk (Denmark), with a mixture of palm-oil and stearoylglycerol esters [46]; the Uniliver Group in Holland have used this system to produce and use cocoa butter-like.

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13.6.3 Biodiesel With the term biodiesel or biofuel are indicated methyl and ethyl esters of monoalkyl fatty acids, which are analogous to that derived from petroleum and used as fuel. Biodiesel is obtained by transesterification reaction of vegetable oils with ethyl or methyl alcohol, in physical condition of high pH and temperatures. To reduce the waste of chemical reaction of transesterification and the energy of the process, several lipases (psychrophiles, mesophiles and thermophiles) have been tested on vegetable oils to this aim, in particular the psychrophilic lipase LipB68 from Pseudomonas fluorescens 868 at 20  C [47], and the thermophilic lipase from Thermomices lanuginose TL at 40  C [48].

13.6.4 Drug Esterase and lipases are mainly used for enantioselective reactions for the preparation of chiral intermediate for pharmaceuticals [49,50], or the resolution of racemic solutions of drugs, such as the (S)-ibuprofen that is the only active form and is purified from the R-form by stereoselective hydrolysis of lipase [51]. Anti-inflammatory drugs, such as indomethacin, naproxen, etodolac, ketoprofen, and ibuprofen are used in human diseases, their efficacy is limited by the low solubility in water, it is studied that the formation of sugar esters increases drug solubility [52]. These results have been obtained by using the lipase from the psychrophilic organism Candida antarctica, which catalyse the transesterification of glucose with vinyl esters of indomethacin, ketoprofen, and etodolac [53]. Moreover the psychrophilicesterase EstO from Pseudoalteromonasarctica is able to hydrolyze some of the molecules above reported (naproxen, ketoprofen and ibuprofen) and increase their solubility [54].

13.6.5 Oleochemical Oleochemistry is the study of vegetable and animal oils. Oleochemical processes are principally applied in making of soaps and cosmetics; the main oleochemical substances are fatty acids, fatty alcohols, fatty amines, sugars and glycerol esters. The reaction associate to the oleochemical industry are alcoholysis, acidolysis, hydrolysis and glycerolysis, and they require high temperature and pressure. In this light, the development of enzymatic processes can reduce the production costs of oleochemical compounds, to this aim several studies of lipases applied in oleochemistry have been made; the main features of these enzymes are solvent and thermo-tolerant [55].

13.6.6 Dairy In dairy industry, exogenous lipases are widely used during cheese making, but the origin of the enzymes is prevalently mesophilic and from safe sources, being the final products used for human nutrition. Lipases act on milk fats by hydrolysis or synthesis of new esters, which will give the characteristic flavors to the cheeses. Lipases are present in raw milk, where they are attached to the fat globes but during the handling and pasteurization of milk, they are destroyed. For this reason, it is necessary to add lipases into milk. Normally, lipases are present in rennet [56,57], but to increase the final flavor of cheese, other lipases can be added. The main sources of lipase are lambs and calves; exogenous lipases are also used in dairy industry for over 40 years [58], but only recently it has been reported the use of lipases from plants [59] and a thermophilic esterase as potential additive in cheese making [60]. In this case it has been tested the ability of the esterase EST2 from Alicyclobacillus acidocaldarius [61] to hydrolyze and synthesize esters and thioesters in milk and cheese model, in comparison to one of the most used Lactic Acid Bacteria (LAB) Lactococcuslactis, from which EstAthe only esterase is present [62]. In both cases EST2 resulted more active than the EstA [60]. When EST2 has been used in cheese making, have revealed its potential for biotechnological use because the results have indicated that EST2 intensify the release of flavor compounds by increasing the lipolysis up to 30% and the relative amount of short- and medium- chain fatty acids, respect to the control cheese [63].

13.7

Conclusion and future

Esterases and lipases are enzymes present in every living organism, together to proteases and polymerases are most important groups of enzymes used in biotechnologies, in particular enzymes from extremophiles for their intrinsic features are the most promising and used in industrial processes. Since in nature, it is not always possible to find enzymes that make specific reactions as request from industry, therefore in most cases to fill this gap it has been used as strategy site direct or random mutagenesis to modify the specificity and/or to increase the catalytic activity of an extremophilic esterases/lipases towards a specific substrate, but maintaining the characteristics of stability, solvent-tolerance and pH

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stability of the starting enzyme. In literature, there are many examples of extremozymes mutagenized for the optimization in a particular process or reaction [33,64], including esterases and lipases [65,66], and this can be considered a promising strategy for the future of enzymes application in industry.

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BMC Bioinforma 2010;11(Suppl 11):S7. [12] Koga Y. Thermal adaptation of the archaeal and bacterial lipid membranes. Archaea 2012; article ID789652: 7 pages. [13] Pond JL, Langworthy TA. Effect of growth temperature on the long-chain diols and fatty acids of Thermomhicrobiumroseum. J Bacteriol 1987;169(3):1328 30. [14] Haney PJ, Badger JH, Buldak GL, Reich CI, Woese CR, Olsen GJ. Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species. Proc Natl AcadSci USA 1999;96:3578 83. [15] Vieille C, Zeikus GJ. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. MicrobiolMolBiol Rev 2001;65(1):1 43. [16] Pace CN. Single surface stabilizer. Nat StructBiol 2000;7(5):345 6. [17] De Simone G, Menchise V, Manco G, et al. The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobusfulgidus. J MolBiol 2001;314(3):507 18. 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A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormonesensitive lipase. J Mol Biol 2000;303(5):761 71. [24] Mandrich L, De Santi C, de Pascale D, Manco G. Effect of low organic solvents concentration on the stability and catalytic activity of HSLlike carboxylesterases: analysis from psychrophiles to (hyper)thermophiles. J Mol Catal B: Enzymatic 2012;82:46 52. [25] Beers JM, Iayasundara N. Antarcticnotothenioidfish: what are the future consequences of ‘losses’ and ‘gains’ acquired during long-term evolution at cold and stable temperatures? JExpBiol 2015;218(12):1834 45. [26] Nutt DR, Smith JC. Dual function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations. J AmChemSoc 2008;130:13066 73. [27] Davies PL. Ice-binding proteins: a remarkable diversity of structures for stopping and starting ice growth. Trends Biochem Sci 2014;39 (11):548 55. 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[30] Mandrich L, de Pascale D. Microbial marine community as source of hydrolytic enzymes. Study on thermal adaptation of esterases and lipases from marine micro-organisms (Chapter 4). In: Nemeth AD, editor. The marine environment. Nova Science Publishers, Inc; 2011. p. 1 15. [31] Siddiqui KS. Defying the activity stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability. Crit Rev Biotechnol 2017;37(3):309 22. [32] Ramnath L, Sithole B, Govinden R. Classification of lipolyticenzymes and their biotechnological applications in the pulpingindustry. Can J Microbiol 2016;63(3):179 92. [33] Demirjian DC, Morı´s-Varas F, Cassidy CS. Enzymes from extremophile. CurrOpinChemBiol 2001;5(2):144 51. [34] Kamekura M. Diversity of extremely halophilic bacteria. Extremophiles 1998;2:289 95. [35] Hough DW, Danson MJ. Extremozymes. CurrOpinChemBiol 1999;3:39 46. [36] Kanlayakrit W, Boonpan A. 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Enzymatic synthesis of geraniol and citroneilolesters by direct esterification in n-hexane. Biotechnol Lett 1993;15:1211 15. [43] Karrachaabouni M, Puivin S, Touraud D, Thomas D. Enzymatic synthesis of geraniol esters in a solvent free system by lipases. Biotechnol Lett 1996;79:1083 8. [44] Gonzalez-Navarro H, Braco L. Lipase-enhanced activity in flavour ester reactions by trapping enzyme conformers in the presence of interfaces. BiotechnolBioeng 1998;59:122 7. [45] Perez D, Martin S, Fernandez-lorente G, Filice M, Guisan JM, Ventosa A, et al. A novel halophilic lipase, LipBL, showing high efficiency in the production of eicosapentaenoic acid (EPA). PLoS One 2011;6(8):e23325. [46] de Castro HF, Anderson WA. Fine chemicals by biotransformation using lipases. Quimica Nova 1995;18(6):544 54. [47] Luo Y, Zheng Y, Jiang Z, Ma Y, Wei D. A novel psychrophilic lipase from Pseudomonas fluorescens with unique property in chiral resolution and biodiesel production via transesterification. 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Chapter 14

Thermostable Thermoanaerobacter alcohol dehydrogenases and their use in organic synthesis ¨ rlygsson Sean M. Scully and Jo´hann O Department of Natural Resource Sciences, University of Akureyri, Akureyri, Iceland

14.1

Introduction

The oxidation of hydroxyl groups and the corresponding reduction of carbonyls are among the most essential reactions in synthetic organic chemistry. Traditional approaches to these redox reactions often require stoichiometric quantities of hydride transfer reagents or oxidants such as heavy metals, respectively. Beyond the environmental impact of such methods, these reagents seldom offer a high-degree of regio- and stereocontrol demanded by modern applications. The use of enzymes often offer a high degree of selectivity although they frequently have poor stability due to thermal inactivation, contact with solvents, or the loss of a cofactor thus making their use in aqueous or organic solvent systems problematic [1]. As a result, the use of thermostable enzymes from extremophiles offer unique tools to accomplish reactions under conditions that mesophilic enzymes cannot handle as thermozymes have been shown to have greater stability in organic solvents making them useful catalysts for synthesis [2
4]. The use of NAD(P)-dependent oxidoreductases, such as the highly chemo- and enantioselective alcohol dehydrogenases isolated from numerous microorganisms, to facilitate enantioselective hydrogen transfer reactions has been extensively reported in the literature [5
10]. The stereospecific reduction of ketones to their corresponding enantiopure alcohols is among the most critical aspects of producing chiral chemical building blocks [11]. The secondary alcohol dehydrogenases (SADHs) of Thermoanaerobacter species are noteworthy for their high thermostability and high degree of enantiomeric discrimination during hydride transfer reactions. The chiral alcohols formed by these SADHs are often necessary precursors for the stereospecific synthesis of pharmaceutical agents as well as other applications demanding a high degree of stereospecificity [12]. The specificity of Thermoanaerobacter’s ADHs, particularly their secondary ADHs (TSADHs) which are specific for their action on secondary hydroxyl groups and ketones, makes them potentially useful synthetic tools for asymmetric reductions although their use has not widely proliferated into routine use to the same degree as other commercially available enzymes. Nonetheless, TSADHs, present some excellent selectivities for chiral alcohol synthesis and other potentially useful chemistries have recently been demonstrated. This chapter hopes to bridge the gap between the role of TSADHs in microbial physiology and the applications in synthesis while more generalized reviews on the use of ADHs for chiral alcohol production can be found at the following references [13
16]. Herein, the use of thermostable NADP-dependent alcohol dehydrogenases from Thermoanaerobacter species is reviewed in the context of bioreduction with an emphasis on the structural characteristics responsible for their thermostability and their use in synthetic applications as well as improvements to TSADHs using genetic modification techniques.

14.2

Thermoanaerobacter ADHs and their role in physiology

The genus of Thermoanaerobacter falls within Cluster V of class Clostridia with the type species being T. ethanolicus, a highly ethanologenic strict anaerobe isolated from a geothermal pool at Yellowstone National Park [17]. The genus currently consists of 19species with standing in nomenclature [18]. As with T. ethanolicus, many of the other species Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00014-9 © 2020 Elsevier Inc. All rights reserved.

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within the genus of Thermoanaerobacter are highly ethanologenic and can degrade hexoses, pentoses, and disaccharides as well as polysaccharides such as amylose, pectin, and xylan. Most of the research efforts on the genus have centered on ethanol production, due to Thermoanaerobacter species’ ability to degrade a wide-range of carbohydrates as well as for their inherent thermotolerance which makes them potentially useful bioprocessing organisms [19,20]. There has also been interest in the thermostable enzymes of Thermoanaerobacter species, such as xylanases [21], amylases, pectinases [22], pullulanases [23,24], and glycotransferases [25
27] in addition to their ADHs. Like many other fermentative organisms, Thermoanaerobacter species use the Embden-Meyerhoff-Parnas (EMP) pathway which generates pyruvate as a key intermediate. Ultimately, fermentation products often consist of a mixture of acetate, lactate, hydrogen, ethanol, and CO2. The presence of multiple alcohol dehydrogenases responsible for ethanol formation has been extensively scrutinized in ethanol producing organisms and in the case of Thermoanaerobacter strains, they often possess up to three separate ADHs: AdhA, AdhB, and AdhE, each with differing activities as summarized in Fig. 14.1. The AdhA of T. ethanolicus has the highest activity towards ethanol and other primary alcohols while AdhB has activity towards ethanol and secondary alcohols. The presence of multiple ADHs with differences in substrate selectivity and cofactor preference may serve a role in NADPH generation. Recent studies suggest that the presence of AdhE is critical for achieving high ethanol titers while T. pseudoethanolicus adh B gene encodes a secondary ADH although there is evidence that its natural substrate is acetyl-CoA [28,29]. Studies on the metabolic role of specific ADHs have investigated mutant strains lacking one or more ADH-coding genes. The separate deletion ofT. mathranii’s adhA and adhB revealed that they had similar ethanol yields to the wild type [30]. A study of T. ethanolicus (JW200), which like T. pseudoethanolicus, contains three ADHs (AdhA, AdhB, and AdhE), found that the deletion of either adhA or adhB increased ethanol yields by up to 70% [31]. Thus, the primary role of AdhB seems to be acetaldehyde reduction activity while AdhE has high acetyl-CoA reduction activity. Beyond the different physiological roles of TADHs, primary ADH (PADHs) and SADHs are active at different times during the growth cycle. Interestingly, the expression of specific ADHs depends on the growth phase of the culture with the SADH being produced early in the culture’s growth while the PADH is produced at later stages of the cultivation. A study by Phillips and coworkers revealed that the SADH of T. ethanolicus (JW200) is highly expressed early in the growth phase with maximum activity for 2-PrOH after 8 hours and slowly decreases until 24 hours into the fermentation [32]. Alternately, the PADH slowly increased over the course of the fermentation reaching a maximum Glucose

Proposed carbon and flow for T. pseudoethaonlicus

Gly colysis

LDH

D-Lactate

H2

Pyruvate

NAD+ NADH H2-ase

Fdox

CoASH

2H +

PF OR

Fdred

CO2

NADH FNOR

NAD + O

Pi CoASH

2° AD H

CoA S Acetyl-CoA

Ethanol

2X NADP+

NADH PTA

Acetyl Phosphate

NAD+

NADPH 2° ADH

ALDH 1° ADH

ADP ATP

NADH NAD+ AK

Acetylaldehyde

Acetate

1° ADH

FIGURE 14.1 Role of multiple alcohol dehydrogenases in Thermoanaerobacter in fermentative pathways. Modified from Burdette D, Zeikus JG. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 Adh) as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioes. Biochem J 1994;302:163
70.

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activity at the start of the stationary phase. Controlling the growth temperature has been used to manipulate whether the SADH or PADH is dominant; T. ethanolicus cells grown at 50  C harvested after 22 hours predominately gave SADH while those grown at 60  C gave PADH [33]. This gives researchers a convenient and facile methodology to produce either PADH or SADH with some degree of selectivity.

14.3

Structure and thermostability

It is crucial to understand the structural features of Thermoanaerobacter ADHs that are critical to their operation as enantioselective and thermostable catalysts that distinguish them from similar mesophilic ADHs that cannot operate at elevated temperatures. Bryant et al. described two ADHs from T. ethanolicus with two different substrate specificities [32]. As noted previously, one of the ADHs, PADH, was selective for primary alcohols with the highest activities towards ethanol and isopropanol. Lamed and Zeikus described the presence of a NADP-dependent secondary alcohol dehydrogenase in T. brockii, T. pseudoethanolicus, and to a lesser extent T. thermohydrosulfuricus, with no similar activity being present in Cl. thermocellum or T. acetoethylicus [34]. The ADHs of Thermoanaerobacter are classified as tetrameric medium chain zinc-containing alcohol dehydrogenases with molecular weights around 40 kDa and use either NAD or NADP as a cofactor. The majority of studies have focused on the SADH from T. ethanolicus (TeSADH), T. pseudoethanolicus (TpSADH), and T. brockii (TbSADH). The preliminary characterization of TeSADH revealed that it is highly thermostable with a preference for secondary alcohols and uses NADP as a cofactor [35] as evidenced by the rates of the oxidation of secondary alcohols being higher than that of primary alcohols. TbSADH showed a clear substrate preference for 2-butanol and a high degree of thermal stability [36]. The high-resolution crystal structures of TADHs have given insight into their configuration and function, providing a basis for further engineering the substrate and stereospecificity of TADHs as well as revealing the basis for their thermostability while serving as a useful model for understanding other TSADHs.

14.3.1 Structure and binding pocket specificity TbSADH was first sequenced in the late 1980s [37] and subsequent crystallographic studies of its apo- and holoenzyme forms have given unprecedented insight into its structure and function. Korkin et al. described the overall 3D structure of TbSADH although with the SADH of the mesophile Cl. beijerinckii [38]. Subsequent papers discussed the details of TbSADH’s structure; comparison of the crystal structures of Cl. beijerinkcii’s SADH (CbSADH) and TbSADH reveals that the two enzymes are highly similar in terms of their overall shape and have 75% similarity to one another at the sequence level [39]. The similarity between TpSADH and TbSADH is 99.4% as they differ by only 3 residues [40] while similar unpublished results by Laivenieks and coworkers have shown that TbADH and TeSADH are identical [14]. The configuration of the substrate binding site as well as the cofactor binding pocket have an impact on the enantioselectivity of ketone reductions. Like many other ADHs, the NADP(H) binds in a Rossmanfold [41] while four conserved residues (Gly198, Ser199, Arg200, and Tyr218) make contact with the 20 -phosphate group of NADP(H) with alterations of Gly199 by site directed mutagenesis (SDM)switching cofactor preference to NADH [39]. The residues Ser199, Arg200, and Tyr21 hydrogen bond with the oxygen atoms of the phosphate group. The binding site contains a number of nonpolar and polar residues which interact with residues from another monomer. The C1 of 2-butanol is very close (within the van der Waal’s distance) to a number of residues within the binding site, namely His59, Ala85, Trp110, Asp150, and Leu294. The active site also has a crevice which has an opening lined with non-polar residues (Ile49, Leu107, Trp110, and Tyr267) as well as Cys283 and Met285 from another subunit. The zinc ion is directly coordinated by Cys37, Thr38, Met337, and Met151 in addition to Asp150 although it is not in direct contact with the zinc ion [42]. Similar to the active site of other ADHs such as horse liver ADH (HLADH), the zinc ion of TbSADH is tetracoordinated by three highly conserved residues, Cys37, His59, and Cys174, with Asp40, Glu60, Ser39, and Ser113 being involved in a second coordination sphere although not directly associated with the zinc ion [39]. These highly conserved residues (Cys37, His59, or Asp150) are involved in coordinating the Zn ion as evidenced by their mutation to other residues resulting in a loss of Zn21 from the active site of TbSADH [43]. Furthermore, mutants in which Cys37, His59, or Asp150 was replaced with a non-metal coordinating residue (Ala) resulted in a loss of activity which suggests that these residues within the active site of TbSADH are critical for Zn21 coordination [43]. The Asp150 residue is turned towards the active carbon atom of NADP(H) such that it can form hydrogen bonds with the hydrogen atom involved in hydride transfer reactions [39]. Later studies showed that although Glu60 is highly conserved, it is not

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essential for catalysis, however, the catalytic efficiency of enzymes with E60A and E60D substitutions were lower than that of the native TbSADH [44]. Further analysis of the 3D structure of TbSADH revealed that the active site contains two non-polar pockets with one forming a larger cavity than the other [39,42,45]. The TbSADH binding pocket can accommodate small aliphatic ketones up to approximately seven carbon atoms in size [32,46]. Large ketones, such as acetophenone, are not readily reduced. The smaller pocket can accommodate methyl or ethyl groups while the most sterically bulky group attached to the ketone associates with the larger pocket. During reductions, the cofactor directs the hydride to either the re- or siface of the carbonyl to yield the S or R product, respectively, depending on the CIP order in accordance with Prelog’s rules with most ketones yielding the Salcohol with the exception of small ketones (R1 is C3 or smaller) that give the R alcohol. The stereochemistry of the ketones reduced by TSADHs is dependent upon how the substrate is placed within the catalytic site; to address the Prelog selectivity observed by TSADHs, Keinan suggested a model which describes the catalytic site as having two pockets, one large and one small. The SADH of Thermoanaerobacterbrockii obeys Prelog’s rule except when small ketones are being reduced as exemplified by 2-butanone which gives (2R)-butanol [47]. This is discussed further in Section 14.4. The ability of ADHs to distinguish between the diastereotopic protons of reduced cofactors can be broadly classed as those that displace the pro-R hydrogen atom from the C4-position or the pro-S hydrogen [48]. 1H NMR studies revealed that TbSADH transfers the pro-R hydrogen atom at the C4 position of the cofactor (NADPH) including when the stereochemistry of the product is the S alcohol [49]. This observation further supports the notion that substrate size affects how the substrate is situated within the active site pocket. Interestingly, this high degree of stereospecificity with respect to the hydride transfer was preserved at high temperatures (70  C). NMR observations using deuterated analogs show that D atom is shifted with respect to the Re-face of NADP by TeSADH which further supports these conclusions [50].

14.3.2 Thermal stability of TADHs The features giving rise to thermostability in proteins have been the subject of intense investigation in part due to the improved utility of enzymes capable of tolerating elevated temperatures. An enzyme is considered thermostable if it meets one of two criteria: a high defined transition temperature (Tm), typically above the thermophilic boundary (55  C), or a high half-life (t1/2) at a designated temperature [51]. For example, TbSADH is thermostable at 80  C but shows a decrease in catalytic function at 86  C and is inactive at 98  C [36]. This is a much higher thermostability than exhibited by most other ADHs which are generally thermolabile with yeast ADH being unstable at 25  C [52] whereas TeSADH has a t1/2 of 1.7 h at 90  C [40]. One general feature for increased thermostability involves stabilization of quaternary structure [38,43]. Interestingly, TbADH’s tetrameric form is not essential for its activity with some catalytic activity being present when the protein is incomplete which could account for its activity at higher temperatures where some of its native structure is lost [53]. Thermozymes are more rigid making them more tolerant to the presence of organic solvents than their mesophilic counterparts. The high thermal stability of TSADHs can likely be attributed to the general features exhibited by other thermozymes. Common problems with operating enzymes at higher temperatures include denaturation, deamination, and the reduction of cysteine bridges or other thermolabile residues. For example, at extreme temperatures and pH, asparagine and glutamine deaminate to aspartate and glutamate residues [54]. Thiols, such as cysteine, are easily oxidized in air particularly in the presence of divalent metal cations [54]. Thus, adaptations to high temperatures must address these challenges. Some common features of thermostable enzymes include helix stabilization, more favorable stabilizing interactions such as strategically placed salt bridges while removing destabilizing unpaired ionic amino acid residues, increasing the number of interactions between domains, a more densely packed hydrophobic cores, and ensuring surface-presenting residues are not prone to thermodegration [51]. Thermozymes are often very similar to their mesophilic counterparts in that the primary sequence of amino acids is 40
85% similar, their three dimensional structures are superposable, and the catalytic mechanism by which they operate is conserved [55]. Analysis of proteins from thermophiles and their mesophilic homologs has revealed that there is no general strategy for increasing thermal stability [56]. However, several features are generally attributed to increases in thermostability; these features can be explained in terms of changes to primary, secondary, and tertiary structure, thermodynamic properties, and (chemical) interactions within a protein, as briefly discussed above. A number of studies have examined the structural features of TADHs finding similar overall trends which explain their enhanced thermal stability. The ADHs isolated from Thermoanaerobacter have a highly similar primary structure, compared to their mesophilic counterparts such as other mesophilic solventogenic Clostridia, such as Cl. beijerinckii,

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yet TADHs have enhanced thermostability [57]. Studies comparing Cl. beijerinckii’s SADH (1KEV) with TbSADH (1YKF) reveal a number of features that explain why these two ADHs differ in their thermotolerance. Both enzymes are homotetramers with similarly broad substrate preferences but differ in their thermal stability by 26  C. Comparative studies examining TbSADH and the highly similar ADH from Cl. beijerinckii have shed some light on the features responsible for its increased thermostability despite being 75% similar on the basis of primary AA sequence [58]. The authors suggested that the eight additional proline residues of TbSADH as well as a number of larger hydrophobic residues being substituted for smaller residues might provide the structural basis for the enhanced thermophily of TbSADH [58]. Subsequent work demonstrated that the features responsible for the enhanced thermophily are likely structural elements distributed throughout the enzyme [59]. Ultimately, the thermostable nature of TbSADH is attributable to more efficient packing of the molecule, improved alpha helix stability, added ion pairs, as well as additional hydrogen bonds [57]. Work that examined the features of TbSADH found that substituting its cofactor binding domain with that of CbSADH substantially reduced the thermostability of TbSADH although examination of the crystal structure found that there were no dramatic changes indicating that the decline in thermal stability is likely the result of subtle changes to the primary sequence [60]. Another study was conducted independently to understand the key structural nature of TbSADH in comparison with CbADH. Comparisons of the structure of TbSADH (1BXZ) and of the similar mesophilic CbADH found that TbSADH is smaller and more compact than CbADH and has botha more nonpolar surface and more proline and alanine residues which stabilize alpha helices [42]. Furthermore, some serine residues within the protein’s core are replaced by more nonpolar residues (such as Thr) which still allows for the formation of hydrogen bonds. TbSADH, while having fewer hydrogen bonds due to having fewer buried polar residues within monomeric subunits, has more salt bridges and van der Waals forces among monomers thus increasing the stability of the tetramer. Additionally, a comparative study of multiple ADHs found that the TbSADH has a number of highly conserved extra proline residues present on surface loops which likely contribute to its increased thermal stability [61].

14.4

Biocatalysis using thermostable TADHs

The thermostability, solvent tolerance, and high degree of enantiomeric discrimination has led to interest in the use of TADHs for a number of synthetic applications. Two potentially useful reactions that can be carried out with Thermoanaerobacter strains are the reduction of carboxylic acids to their corresponding primary alcohol [62
65] and the asymmetric reduction of ketones using their SADHs [6,8,13,14]. The chemistry of the latter has received considerably more attention with most of the work reported in the literature focused on the use of purified TSADHs for enacting biotransformations although some work on whole-cell catalysis has been described. Generally, ketones are reduced using 2-PrOH as source of reducing potential for cofactor regeneration and as a co-solvent as summarized in Fig. 14.2. These reactions are typically performed at ambient temperature with no special precautions to avoid atmospheric oxygen. A wide variety of substrates have been reduced to their corresponding alcohol with the S alcohol being the dominant product with the exception of small ketones which show the opposite selectivity. Beyond linear aliphatic ketones, TSADHs have proved to be useful tools for the production of asymmetric ketones including cycloalkanes [50], nonconjugated unsaturated and bifunctional saturated ketones [66], chloro ketones [67], 2-oxo-carboxylic acid esters [68], and ethynyl ketones and ethynylketoesters [69]. Wild-type TSADHs are largely limited to accepting linear aliphatic and alicyclic ketones [70] although there are conflicting reports of acetophenone reduction. The reduction of small aliphatic ketones by TSADHs generally follows Prelog’s rule and thus yields the corresponding S alcohol. One exception to this is when small ketones, such as 2-butanone, 3-methyl-2-butanone, or methyl TADH

O R1

OH

R2

R1 NADPH

NADP+ OH

O NADPH Recycling

R2

FIGURE 14.2 A general scheme for enantioselective ketone reduction using a TSADH using 2-PrOH for in situ cofactor regeneration.

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[H- ] OH

O R1

TSADH

R2

NADPH Small pocket

NADP+

R1

R2

FIGURE 14.3 A model for the substrate binding site of TbSADH explaining the nature of its S selectivity. Modified from Keinan E, Hafeli EK, Seth KK, Lamed R. Thermostable enzymes in organic synthesis. 2. Asymmetric reduction of ketones with alcohol dehydrogenase from Thermoanaerobium brockii. J Am Chem Soc 1986;108:162
69.

Large pocket

cyclopropyl ketone, are used as substrates as they yield the corresponding R alcohol by TbSADH [47]. A noteworthy size-dependent reversal of enantioselectivity was observed in which a ketone possessing two small alkyl groups yielded the corresponding R alcohol while methylketones in which the other substituent was C4 or larger followed Prelog’s rule giving the Salcohol. Later work using the SADH of T. ethanolicus yielded similar results with ethynyl ketones with alkyl groups up to C3 giving anti-Prelog selectivity [69]. To explain the change in stereopreference, Keinan proposed a model in which the ADH has an active site with two pockets in which the fitting of the two groups into these pockets determines selectivity [47] as shown in Fig. 14.3. The small pocket has a higher affinity for the smaller alkyl groups (up to 3 C atoms) of the ketone. Generally, linear substrates were reduced more quickly than their branched chain counterparts with ketones with side chains of 5 carbons or greater giving high enantiomeric purity for the S alcohol. The reduction of hex-1-en-5-one gave a low enantiomeric excess (ee) of 53% with the S alcohol being the dominant product. Interestingly, Hex-1-yn-5one gave the corresponding R alcohol with poor ee. Large ketones such as acetophenone were not accepted as substrates by TbSADH under the reaction conditions although placing two or more methylene residues between the reaction center and the phenyl group yielded the corresponding alcohol with high ee. A number of the synthons were used for the total synthesis of macrolide pheromones. One of the synthons, (S)-methyl-8-hydroxynonanoate was subsequently used in the total synthesis of (S)-ferrulactone [71]. Zheng and coworkers reported the reduction of aliphatic and cyclic ketones using immobilized TeSADH [50]. While the enantiomeric purity of the reduction of aliphatic ketones up through 2-pentanone was low, products of higher optical purity were obtained on 2- and 3-hexanone, 2-heptanone, 6-methyl-5-heptene-2-one, and 2-octanone. Acetophenone, 2,4-pentanedione, and 1-phenyl-1,3-butanedione were converted despite previous reports indicating that beta-ketoesters could not be reduced [72]. The authors were able to produce high purity ( . 99% ee) (1S,3S)-(1) 2 3methyl-cyclohexanone from the rac-cyclic ketone by taking advantage of the faster rate of reduction of the S enantiomer and stopping the reaction at 30% conversion. While most studies have worked with purified TADHs, several instances of the use of whole cells of T. brockii have been reported including the enantioselective reduction of 3-oxo-valerate to the corresponding S alcohol with an ee of 93% [73]. The use of T. brockii for the reduction of C4-C8 oxoesters gave the corresponding alcohol in good yield (50
80%) and higher enantiomeric purity as compared to yeast-mediated reductions [74]. These examples highlight that whole cell systems can sometimes result in a larger range of substrates being utilized albeit at the expense of ease of workup. Beyond the use of straightforward batch reactions which can suffer from phenomena such as substrate inhibition, the use of continuous systems has also been explored. The enantioselective transformation of several ketones and oxoacid esters using T. brockii grown in continuous culture using pulse or shift techniques to increase substrate concentration without impacting cell growth has been reported [68]. The authors achieved substrate concentrations greater than 10 g/L during acetone and 2-pentanone reduction although the later gave a racemic mixture due to temperature effects [75]. During the course of the 2-pentanone reduction, the amount of ethanol in the system decreased suggesting that the ketone was serving as an alternative electron acceptor. Attempts with 2-octanone resulted in cell lysis at concentrations as low as 1.3 g/L with 2-formyl-propionic acid also being inhibitory at 2.5 g/L but still resulted in an enantioselective reduction.

14.5

Enzyme improvement

Given the limited size of the substrate that can be accommodated by the active site of TSADHs, there is recognition of the importance of expanding the size of substrates that can be accepted to accommodate larger ketones such as those

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bearing a phenyl ring or other bulky groups. As the propensity of TSADHs for following Prelog’s rules limits their use as only one enantiomer is available, having a route to both enantiomers has obvious advantages. There have been considerable efforts devoted to engineering TSADHs in order to both expand their substrate specificity to accept larger molecules, such as those with aromatic moieties, as well as to reverse their enantiopreference. The availability of crystal structures and an understanding of the role of specific residues within the active site has greatly aided efforts to design mutants with the desired features. There are three general targets for improving the utility of TSADHs: improved substrate spectra, altered stereopreference, and changes in cofactor specificity. Based on knowledge of the active site, a number of amino acid residues have been targeted in an effort to achieve the outlined aims. There are two general strategies for the modification of enzymes: random mutagenesis and rational design. A better understanding of the influence of changes to the structure of TSADHs makes rational design easier through site directed mutagenesis (SDM). The use of these rational protein engineering strategies to introduce changes to allow expanded substrate utilization or alter the stereopreference of the enzyme have yielded excellent results. Given the large number of potential permutations, clever strategies to minimize the size of libraries while ensuring adequate coverage are needed. While early work focusing on generating TSADHs serine mutations using traditional mutagenesis [29], more modern approaches have employed site-directed mutagenesis and rational design strategies [14] as well as Triple-Code Saturation Mutagenesis (TCSM) [76,77].

14.5.1 Altering cofactor preference There has been little work on altering the cofactor preference of TADHs from NADP to NAD; this is particularly important due to the cost associated with using NADP(H) as a cofactor. It was reported by Bogin et al. that the alteration of Gly198 to Asp via site directed mutagenesis altered the cofactor preference from NADPH to NADH but resulted in a reduction in enzymatic activity. Based on the crystallographic work of TbSADH, Korkhin et al. proposed that further substitutions, Y218F, S199G, and R200G, may improve utilization of NADH as a cofactor [39]. Given the use of efficient NADP recycling, the utility of using NAD or other analogs may ultimately prove to be of limited utility.

14.5.2 Altering stereoselectivity One of the major drawbacks to the use of TADHs is that the reactions generally favor the formation of alcohols according to Prelog’s rule and are thus limited to producing the corresponding Salcohol with the exception of ketones with small sidechains. As there is a need to be able to produce both enantiomers of a given alcohol, catalytic tools capable of anti-Prelog selectivity are essential. Thus, engineering TADHs which have reversed selectivity is of great synthetic value. To this end, the use of mutagenesis to enhance substrate specificity and alter stereoselectivity patterns has been applied in the case of TeSADH. The alteration of TeSADH’s Ser39 residue to Thr had a dramatic impact on the rates of oxidation of chiral alcohols [78]. Wild type TeSADH has a preference for the oxidation of (S) 2 2-pentanol while the S39T mutant preferentially oxidizes (R)-butanol and 2-pentanol. Other work with a C295A mutation resulted in a larger small pocket leading to a reversal of the stereochemical preference towards isobutyl and butyl ketones [28]. A mutant TeSADH with anti-Prelog selectivity was created by converting Ile86 within the small pocket of the active site to an alanine residue [79]. This alteration converted what was once the small pocket of wild type TeSADH into the larger pocket resulting in the ability to accommodate larger substrates as well as flipped enantioselectivity (Fig. 14.4). 1H NMR spectroscopy on NADP1 incubated with deuterium-labeled 2-PrOH confirmed that I86A indeed delivered the hydride to the pro-R position. Further work on TeSADH focusing on Cys295, which is situated in the small binding pocket of the active site, conversion to an Ala residue resulted in an enzyme with broader substrate specificity and altered enantioselectivity [28]. A double mutant of TeSADH (I86A/C295A) demonstrates not only anti-Prelog selectivity but has a wider substrate specificity including aromatic ketones such as meta-substituted acetophenones [70]. The double mutant accepts both m- and TeSADH(I86A)

O

OH

O R1

R2

R1

R2 NADPH

Small pocket

Large pocket

O

NADP+

R2 R1 R-Alcohol OH

FIGURE 14.4 Use of a Thermoanaerobacter ethanolicus mutant with anti-prelog selectivity.

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p- substituted aromatic ketones and leads to alcohols with high enantiomeric purity although the substrate conversion is incomplete. Further work by Nealon et al. to expand the small pocket of TeSADH by converting M151 to Ala resulted in change in the stereospecificity towards some ketones, namely R1 5 (CH3)2CHCH2 and R1 5 t Bu, which gave the R alcohol as opposed to the S alcohol formed by the wild-type [80].

14.5.3 Altering substrate specificity Targets for the enantioselective reduction of ketones have largely focused upon designing ADHs that accept larger substrates such as those bearing one or more aromatic rings. Generally speaking, the basis for enantiomeric discrimination often relies upon steric or electronic factors, thus making the synthesis of small, nearly symmetrical ketones difficult. Consider the challenges associated with a ketone in which its two substituents flanking the carbonyl carbon are too similar, as is the case with ketones such as tetrahydrofuran-3-one. There has been considerable work done to expand the catalytic promiscuity of TeSADH to include ketones with aromatic moieties for the synthesis of chiral aromatic alcohols using modern protein engineering techniques based on insights into the structure of TADHs and the nature of their binding pocket. Small alterations, such as single substitutions, can have dramatic effects on the substrate spectra and the enantioselectivity of an enzyme. Given the position of W110 in the large pocket and the placement of Met151 and Thr153 in the small pocket, these are logical targets for expanding the size of the ketones that can be reduced. To address the enantioselective reduction of challenging ketones with similar groups flanking the carbonyl carbon, Sun and co-workers turned to a TCSM strategy to generate a library of TbADH mutants with the reduction of tetrahydrofuran3-one and related derivatives [77]. The strategy involved creating two mutant libraries with each focusing on one site for saturation mutagenesis: site A focused on A85/I86/L294/C295 while the other focused on A85/I86W110/L294 with the expectation that library A would yield R-selective mutants. Ultimately, screening tetrahydrofuran-3-one derivatives revealed that mutations at positions 85, 86, and 294 gave a number of R-selective mutants. Two promising mutants, SZ2074 (I86N/C295N) and SZ2172 (I86V/W110L/L294Q), were used for the reduction of 50 mL of 100 mM tetrahydrofuran-3-one which fully converted the ketone to R and S alcohols with 99% and 94% ee, respectively. Agudo et al. generated a mutant library of TbSADHs using directed evolution via combinational active-site saturation test (CAST) focusing on six residues (S39, A85, I86, W110, Y267, and C295) as targets for saturation mutagenesis [81]. These mutants were then evaluated for the selective reduction of 4-alkylidene cyclohexane derivatives. The I86 mutants yielded the S alcohol while the W110 mutants gave the R alcohol for the reduction of 4-(bromomethylene) cyclohexanone and 4-ethylidenecyclohexanone. Scale-up of the reduction of 4-(bromomethylene)cyclohexanone with the W110T mutant afforded 81% yield of the R alcohol and I86A gave 84% of the Salcohol. A large number of the mutants in this study were able to reduce acetophenone as well as cyclic ketones with 4 and 8 membered rings. The use of site-saturation mutagenesis was used to generate a mutant library of TeSADH W110 which lies within the large pocket of the active site [82]. Several of those mutants successfully reduced phenylacetone, 1-phenyl-2butanone, and 4-phenyl-2-butanone to their corresponding Salcohols with high conversion and enantiomeric purity. Work with a double mutant of TeSADH (I86A/C295A) showed anti-Prelog specificity for aryl ketones. Further efforts to expand the small binding pocket of TeSADH have involved targeting Met151 and Thr153 for replacement with a smaller Ala residue [70,80]. As previously mentioned, some alterations in the stereopreference of the resultant mutants were observed with the M151A and T153A mutants showing lower selectivity for small ketones. Integrating these changes to form the triple mutants I86A/M151A/C295A and I86A/T153A/C295A resulted in enzymes with altered specificity for substituted acetophenones with the I86A/T153A/C295A being better able to convert 30 OCH3 acetophenone derivatives as well as a slight improvement for 40 Cl derivative. The I86A/M151A/C295A mutant gave poorer conversions. Larger ketones, such as substituted 2-tetralols, are another target for reduction with 4 mutants of TeSADH [83]. The mutants with an expanded active site showed a higher conversion of the ketone substrate and generally resulted in an alcohol with high enantiopurity. Reductions yielded the corresponding Salcohol, with the exception of 5-methoxy-2tetralone and 8-methoxy-2-tetralone, likely due to the improveddocking of the substrate inside the expanded active site. Further work by [84] on the double mutant W110A/I86A TeSADH demonstrated an expanded capacity to accommodate larger aromatic ketones which can often serve as building blocks for synthesis.

14.6

Conclusions and future directions

While work on Thermoanaerobacter ADHs has given promising results, the majority of the work reported has focused on T. brockii or T. pseudoethanolicus and on the enantioselective reduction of small ketones. Other members of the

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genus may have ADHs with altered selectivities, such as for larger or aromatic substrates, that would make worthy targets for future studies. It is likely that further metabolic engineering efforts targeting mutations allowing larger substrates to be reduced will be explored as well as targeting anti-Prelog selectivity to ensure that routes to multiple enantiomers are available. Other limitations such as limited substrate loadings can be overcome using enzyme immobilization techniques as well as customizable solvent blends, potentially including ionic liquids. Ultimately, the alcohol dehydrogenases of Thermoanaerobacter are powerful tools for enantioselective reactions creating chiral centers. Their thermostable features give them enhanced stability over their mesophilic counterparts allowing for their use in organic solvents. While the substrate range of wild type TSADHs is limited, genetic engineering has allowed for the reduction of larger ketones as well as, in some cases, allowing for anti-Prelog selectivites.

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[29] Burdette D, Zeikus JG. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 Adh) as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioes. Biochem J 1994;302:163
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11. ˚ , Wehtje E, Adlercreutz P, Mattiasson B. The enantiomeric purity of alcohols formed by enzymatic reduction of ketones can [46] Yang H, Jo¨nsson A be improved by optimisation of the temperature and by using a high co-substrate concentration. Biochim Biophys Acta
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an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol Biofuels 2008;1(1):7. [54] Creighton TE. Proteins. 2nd ed. New York: W.H. Freeman and Company; 1993. [55] Vieille C, Zeikus JG. Thermozymes: identifying molecular determinants of protein structural and functional stability. Trends Biotechnol 1996;14:183
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the structural key to thermal stability in bacterial alcohol dehydrogenases. Protein Sci 1999;8:1241
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805.

Chapter 15

Biotechnological platforms of the moderate thermophiles, Geobacillus species: notable properties and genetic tools Keisuke Wada1 and Hirokazu Suzuki2,3 1

Research Institute for Sustainable Chemistry, Department of Materials and Chemistry, National Institute of Advanced Industrial Science and

Technology (AIST), Hiroshima, Japan, 2Faculty of Engineering, Tottori University, Tottori, Japan, 3Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan

15.1

Introduction

The genus Geobacillus comprises aerobic or facultative anaerobic bacteria capable of endospore formation. Cells are gram-positive, rod-shaped, and chained occasionally (Fig. 15.1). Growth occurs preferentially at temperatures ranging between 55
C and 70
C, but not below 35
C or above 76
C; therefore, Geobacillus spp. are moderate and obligate thermophiles. Since hyperthermophiles that propagate even at .100
C have been identified, researchers may exhibit low interest in Geobacillus spp.; however, the species are of biotechnological importance in terms of the following two factors: (i) Geobacillus spp. have been a key source of thermostable proteins that serve as robust enzymatic catalysts or biomimetic structures [1]. Among 758 references published between 2001 and 2018 with titles containing the term Geobacillus, .400 focus on proteins from the species (Web of Science; http://apps.webofknowledge.com). Considering the genus Geobacillus was reclassified from the genus Bacillus in 2001 [2], there are much more relevant references. (ii) Geobacillus spp. are often practical as hosts for various bioprocesses, particularly for bioproduction and FIGURE 15.1 Microscope photograph of Geobacillus kaustophilus HTA426. Cells were stained using Gram’s solution. The scale bar indicates 10 μm.

Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00015-0 © 2020 Elsevier Inc. All rights reserved.

195

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bioremediation. Such applications (whole-cell applications) are facilitated by their excellent metabolism and cellular propagation; moreover, growth properties at elevated temperatures provide several advantages over organisms that are only propagated under moderate temperatures [3]. In this chapter, we first overview the history, classification, habitats, and general aspects of Geobacillus spp.; subsequently, notable properties and genetic tools of Geobacillus spp. are highlighted to explore their potential in whole-cell applications and recent advances of biotechnological platforms for the species.

15.2

Overview of the genus Geobacillus

Aerobic or facultative anaerobic endospore formers were roughly categorized into the genus Bacillus during early classification; however, currently, numerous strains have been reclassified into other or new genera including the genus Geobacillus [4]. Since the reclassification activities have led to confusion regarding the nomenclature of Geobacillus spp., we first review the history and transitional classification of the genus in the following subsections. Habitats and general features of Geobacillus spp. are also examined.

15.2.1 History In 1917, an endospore former with the capacity to propagate between 45
C and 76
C was isolated from spoiled samples of canned corn [5]. The strain was designated as Bacillus stearothermophilus. Although a similar bacterium, termed Bacillus coagulans, has been isolated from spoiled samples of evaporated milk [6], the strain grew more rapidly at 37
C than 55
C; therefore, B. stearothermophilus was distinguished from B. coagulans based on its optimum growth temperature. Gordon and Smith analyzed 216 isolates of endospore formers and categorized 87 and 73 isolates as B. stearothermophilus and B. coagulans, respectively [7], which confirmed that B. stearothermophilus was widely distributed in nature. B. stearothermophilus was isolated even from cool environments [8,9]. The observation is peculiar considering B. stearothermophilus is an obligate thermophile that hardly grows below 45
C [5]. Consequently, obligate thermophiles in cool environments were described as alien bacteria [9]. Similar strains were increasingly identified and transiently classified into several species, including Bacillus acidocaldarius [10], Bacillus kaustophilus [11], Bacillus pallidus [12], Bacillus thermocloaceae [13], Bacillus thermoglucosidasius [14], and Bacillus thermoleovorans [15]. Phylogenetic analyses based on 16S rRNA gene sequences were subsequently performed to reveal that B. stearothermophilus is distinct from the genus Bacillus [16] and forms a separate phylogenetic cluster with B. kaustophilus and B. thermoglucosidasius [17]. In 2001, several species were validly reclassified from the genus Bacillus to the new genus Geobacillus [2] along with Geobacillus caldoxylosilyticus from the genus Saccharococcus [18].

15.2.2 Species placed under the genus Geobacillus Sixteen species are presently described in the genus Geobacillus with a bacteriological code (Table 15.1). Certain strains initially defined as B. stearothermophilus (later G. stearothermophilus) may be other species because the nomenclature was roughly assigned during early classification (see above) [17]. This view is supported by a phylogenetic analysis based on 16S rRNA genes, which suggests that G. stearothermophilus strains K1041 and NUB3621 are Geobacillus thermodenitrificans and Geobacillus thermoglucosidasius, respectively [30]. Based on recN similarities, G. stearothermophilus NUB3621 belongs to G. caldoxylosilyticus [31]. G. stearothermophilus ATCC 8005 has already been recategorized as G. kaustophilus ATCC 8005 [32]. Although organisms are commonly classified based on partial genotypic and phenotypic properties, the approach is suggested to be inadequate for classifying novel strains into G. stearothermophilus because of their phenotypic diversity [33]. Suspicious classification is reported even for other species. Geobacillus debilis [34], Geobacillus pallidis [12], and Geobacillus tepidamans [35] were reclassified into the genera Caldibacillus [24], Aeribacillus [36], and Anoxybacillus [24], respectively. Geobacillus gargensis was incorporated into Geobacillus thermocatenulatus [37,38]. In addition, recN sequences suggest that Geobacillus thermantarcticus is placed under G. thermoglucosidasius [31]. A comparative analysis of genome sequences suggests that G. kaustophilus strains HTA426 and GBlys are not monophyletic [39]. Aliyu et al. employed genome-wide metrics (i.e., average amino acid identity, average nucleotide identity, and digital DNA-DNA hybridization) to classify Geobacillus spp., suggesting that G. kaustophilus and Geobacillus thermoleovorans are monophyletic and that G. caldoxylosilyticus, G. thermoglucosidasius, G. thermantarcticus, and Geobacillus toebii are reclassified into a novel genus, Parageobacillus, along with G. stearothermophilus NUB3621 [19]. Geobacillus yumthangensis has been recently reported as a novel species [40]. The following species have been effectively published but are not validly

Biotechnological platforms of the moderate thermophiles, Geobacillus species Chapter | 15

197

TABLE 15.1 Geobacillus spp. validly described with bacterial codes. Species

Type strain

Optimum growth conditions


Temperature ( C) G. caldoxylosilyticus

DSM 12041

65 (4375)

a

Synonym

Reference

Parageobacillus caldoxylosilyticus

[1921]

pH 6.87.2

Saccharococcus caldoxylosilyticus G. galactosidasius

DSM 18751

70 (5075)

6.07.0

[22]

G. icigianus

DSM 28325

6065 (5075)

6.57.0

[23]

G. jurassicus

DSM 15726

5860 (4565)

7.07.2

[24,25]

G. kaustophilus

DSM 7263

6065 (3765)

6.07.2

G. lituanicus

DSM 15325

5560 (5570)

6.5

G. stearothermophilus

DSM 22

50 (4576)

6.09.0

G. subterraneus

DSM 13552

5560 (4570)

6.07.8

G. thermantarcticus

DSM 9572

60 (3780)

6.0

Bacillus kaustophilus

[2,11,26] [27]

B. stearothermophilus

[2,5,24] [2,24]

B. thermantarcticus

[19,24]

P. thermantarcticus G. thermocatenulatus

DSM 730

60 (3780)

B. thermocatenulatus

[2,24]

G. gargensis G. thermodenitrificans

DSM 465

50 (5070)

7.0

B. thermodenitrificans

[2,24]

G. thermoglucosidasius

DSM 2542

50 (4060)

8.0

B. thermoglucosidasius

[2,19,24]

G. thermoglucosidans P. thermoglucosidasius G. thermoleovorans

DSM 5366

5565 (4275)

6.27.5

B. thermoleovorans

[2,15]

G. toebii

DSM 14590

60 (3770)

7.5

P. toebii

[19,24,28]

G. uzenensis

DSM 13551

5560 (4565)

6.27.8

G. vulcani

DSM 13174

60 (3772)

6.0

[2,24] B. vulcani

[2,29]

a

The numbers in parentheses indicate temperature ranges at which growth was observed.

described under bacteriological code regulations: Geobacillus anatolicus [41], Geobacillus bogazici [42], Geobacillus kaue [42], Geobacillus mahadia [43], Geobacillus thermopakistaniensis [44], Geobacillus thermoparaffinivorans [45], Geobacillus uralicus [46], and Geobacillus zalihae [47]. In the following sections, the 16 species with bacteriological codes are described as Geobacillus to avoid confusion, even if original articles had adopted other nomenclatures.

15.2.3 Diverse habitats and their implications Table 15.2 summarizes isolates described as Geobacillus spp. Their habitats are remarkably diverse, including not only hot environments but also cool or ambient environments such as temperate soils, deep-sea sediments, ocean core, foods, wood slime, plants, seaweeds, and air samples. Notably, Geobacillus spp. are abundant in soils in which temperatures never exceed 27
C [111]. Since Geobacillus spp. are detected even in the air and rainwater, the organisms apparently diffuse through atmospheric bridges and settle on diverse environments in the geosphere [112,113]. Considering Geobacillus spp. are obligate thermophiles that can form endospores, it is likely that they exist as endospores in ambient environments and reproduce in narrow hot environments [114]. Cells reproduced in hot environments eventually form endospores and diffuse again via atmospheric bridges. Cells reproduce when the endospores reach appropriate environments; however, they remain viability as endospores for ages, which explain why Geobacillus spp. are frequently identified from numerous locations including cool environments. In addition, it is possible that Geobacillus spp. reproduce slightly in ambient environments because isolates from such environments

198

PART | II Biotechnological aspects

TABLE 15.2 Isolates described as Geobacillus spp. Strain

Origin

G. icigianus G1w1

Hydrothermal outlet

Notable ability

Reference

G. jurassicus DS1

Oilfield

Oil degradation

[25]

G. kaustophilus A1

Geothermal field

Arsenic resistance

[49]

G. kaustophilus DSM 7263

Pasteurized milk

[50]

G. kaustophilus HTA426

Deep-sea sediment

[51]

G. kaustophilus TERINSM

Oily soil

Paraffinic wax utilization

[52]

G. pallidus C5

Oilfield

Tyrosol degradation

[53]

G. pallidus DSM 16016

Temperate soil

G. pallidus XS2

Oilfield

G. stearothermophilus 10

Hot spring

[50]

G. stearothermophilus 22

Hot spring

[55]

G. stearothermophilus A1

Milk powder

Biofilm formation

[33,56]

G. stearothermophilus BR219

River sediment

Phenol degradation

[57]

G. stearothermophilus DSM 458

Sugar beet juice

Antimicrobial substance producer

[58]

G. stearothermophilus JPL_T2a

Clean room

[59]

G. stearothermophilus NUB3621

Soil

[50]

G. stearothermophilus UCP 986

Effluent

Azo dye degradation

[60]

G. stearothermophilus XL-65-6

Rotting wood

Cellulose degradation

[50]

G. subterraneus DSM 13552

Oilfield

Hydrocarbon utilization

[50]

G. subterraneus Ge1

Hot spring

G. subterraneus K

Oilfield

Hydrocarbon utilization

[62]

G. tepidamans HB1

Active sludge

Poly(vinyl alcohol) degradation

[63]

G. thermocatenulatus DSM 730

Thermal pipe

G. thermodenitrificans DSM 465

Sugar beet juice

Denitrification

[50]

G. thermodenitrificans DSM 101594

Compost

Pectin degradation

[64]

G. thermodenitrificans ET 144-2

Compost

Transformable with electroporation

[65]

G. thermodenitrificans NG80-2

Oilfield

Hydrocarbon degradation

[66]

G. thermodenitrificans OS27

Seaweed

Seaweed degradation

[67]

[48]

[34] Hydrocarbon degradation

[54]

[61]

[50]

G. thermodenitrificans T12

Compost

Lactate fermentation

[68]

G. thermoglucosidasius C56-YS93

Hot spring

Biomass degradation

[69]

G. thermoglucosidasius DSM 2542

Soil

G. thermoglucosidasius M10EXG

Compost

Ethanol tolerance

[70]

G. thermoglucosidasius NY05

Compost

Crystal formation

[7173]

G. thermoglucosidasius PB94A

Hemp bast fiber

Pectin degradation

[74]

G. thermoglucosidasius TNO-09.020

Dairy plants

Biofilm formation

[75]

[50]

G. thermoglucosidasius W-2

Oil reservoir

Organosulfur degradation

[76]

G. thermoleovoransstrains B23

Oilfield

Hydrocarbon degradation

[77]

G. thermoleovorans CCB_US3_UF5

Hot spring

[78] (Continued )

Biotechnological platforms of the moderate thermophiles, Geobacillus species Chapter | 15

199

TABLE 15.2 (Continued) Strain

Origin

Notable ability

Reference

G. thermoleovorans DSM 5366

Soil

Hydrocarbon utilization

[50]

G. thermoleovorans DSM 15393

Volcanic area

G. thermoleovorans N7

Hot spring

G. thermoleovorans SGAir0734

Air sample

G. thermoleovorans T70

Soil

G. thermopakistaniensis MAS1

Hot spring

[50] Protease producer

[79] [80]

Hydrocarbon degradation

[81] [44]

G. toebii L1

Compost

Hydrocarbon degradation

[82]

G. uzenensis DSM 13551

Oilfield

Hydrocarbon utilization

[50]

G. zalihae T1

Palm oil mill effluent

Lipase producer

[47]

Geobacillus sp. 8

Soil on oil pool

Geobacillus sp. 1017

Oil reservoir

Hydrocarbon utilization

[84]

Geobacillus sp. 12AMOR1

Hydrothermal vent

Starch degradation

[85]

Geobacillus sp. ARS4

Temperate soil

Arsenic resistance

[86]

Geobacillus sp. CAMR5420

Culture collection

Hemicellulose degradation

[87]

Geobacillus sp. DC3

Temperate wood slime

Lignocellulose degradation

[88]

Geobacillus sp. EPT3

Hydrothermal field

Superoxide dismutase producer

[89]

Geobacillus sp. FW23

Oil well

Geobacillus sp. GHH01

Temperate soil

Geobacillus sp. GWE1

Sterilization oven

Geobacillus sp. H6a

Hot spring

Vitamin B6 producer

[94]

Geobacillus sp. ID17

Hot soil

Gold nanoparticles accumulation

[95]

[83]

[90] Lipase producer

[91] [92,93]

Geobacillus sp. JF8

Compost

Polychlorinated biphenyls degradation

[96]

Geobacillus sp. LC300

Contaminant

Rapid growth

[97]

Geobacillus sp. LEMMY01

Hot soil

Geobacillus sp. Sah69

Soil

Carbohydrate degradation

[99]

Geobacillus sp. SBS-4S

Hot spring

Lipase producer

[100]

Geobacillus sp. SG-01

Compost

Denitrification

[101]

Geobacillus sp. SH-1

Deep oil well

Oil degradation

[102]

Geobacillus sp. T1

Temperate soil

Cellulose utilization

[103]

Geobacillus sp. T6

Hot spring

[104]

Geobacillus sp. WCH70

Wood compost

[105]

Geobacillus sp. WSUCF1

Compost

Lignocellulose degradation

[106]

Geobacillus sp. XT15

Oilfield

Hydrocarbon utilization

[107]

Geobacillus sp. Y412MC52

Hot spring

Xylan degradation

[108]

Geobacillus sp. ZGt-1

Hot spring

Antibacterial peptide producer

[109]

Geobacillus sp. ZY-10

Oilfield

Crude oil degradation

[110]

[98]

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PART | II Biotechnological aspects

often exhibit a unique capacity to reproduce in the respective ambient habitats (Table 15.2). A good example is G. thermodenitrificans OS27 isolated from seaweed, which utilizes polysaccharides abundant on the seaweed surface. Similarly, numerous isolates from oily environments and composts can degrade oil compounds and hemicelluloses, respectively. When a microorganism inhabits a niche, it has to adapt to numerous environmental factors; [4] therefore, diverse habitats of Geobacillus spp. imply that they possess excellent capacity for environmental adaptation. Intriguingly, evidence suggest that Geobacillus spp. evolved from Bacillus spp. via remarkable genomic diversification through horizontal gene transfer [114,115], and that Geobacillus spp. can rapidly diversify their genomes using inductive mutations and transposable elements when subjected to environmental stress [4,116]. The observation indicates that Geobacillus spp. have diversified their niches by environmental adaptation via an active genetic alteration to acquire different properties that confer selective advantages.

15.2.4 Cellular characterization Geobacillus spp. are aerobic or facultatively anaerobic bacteria that grow preferentially at neutral or moderate alkaline conditions and at temperatures generally between 55
C and 65
C (Table 15.1). Growth occurs on wide range of nutrients. Some strains grow in media with minimal nutrients, which contain only a carbon source and inorganic elements [117,118]. Some display motility and swarm on agar surfaces [114]. Some lyse under nutrient starvation; however, most other isolates form endospores on solid media under laboratory conditions [114]. Similarly to other thermophilic species, Geobacillus spp. potentially produce polyamines [119,120], which are aliphatic compounds with positive charges and that facilitate the stabilization of nucleic-acid structures at elevated temperatures via ionic interactions [121]. Cellular fatty acids mainly comprise 13-methyltetradecanoic acid (iso-C15:0), 14-methylpentadecanoic acid (isoC16:0), and 15-methylhexadecanoic acid (iso-C17:0) [2,24].

15.2.5 Genomic features Complete and draft genome sequences have been published for 27 and 67 strains, respectively. The genome sizes range between 3.4 and 4.0 Mb, which are substantially smaller than those of mesophiles that are phylogenetically related to the genus Geobacillus (e.g., B. subtilis 168; 4.2 Mb). This is consistent with a negative correlation observed between genome size and growth temperature of microorganisms [122], and can be explained by the energy burden on DNA replication at elevated temperatures [123]. In contrast, there is no clear difference in GC contents between Geobacillus spp. (42%55%) and B. subtilis 168 (44%). Although higher GC contents facilitate stable base-pair formation at elevated temperatures, it is known that GC contents are not correlated with optimum growth temperatures in prokaryotes [124]. Geobacillus genomes contain genes that potentially respond to cold or heat shocks. In fact, numerous proteins are induced in response to heat shock in G. stearothermophilus NUB3621 [125] and to cold shock in G. stearothermophilus TLS33 [126]. Such observations imply that Geobacillus spp. could maintain cellular functions at both elevated and ambient temperatures, which is consistent with the view that Geobacillus spp. reproduce even in ambient environments (see above). Hemicellulose utilization genes often assemble in Geobacillus genomes and form the echD-npd island [127]. This feature is unique to Geobacillus genomes.

15.3

Genetic tools for Geobacillus spp.

Genetic tools facilitate rational and effective modification of microorganisms for use in whole-cell applications. When researchers seek to genetically modify a microorganism, plasmid transformation would be explored first as it is the most straightforward and essential modification. Plasmids capable of autonomous replication facilitate the efficient expression of homologous and heterologous genes in microorganisms, whereas integration plasmids allow gene insertion and gene disruption in chromosomes via homologous recombination. Once plasmid transformation is achieved, researchers may explore additional genetic tools, such as selection markers, promoters, and reporter proteins, while constructing more convenient plasmids and host strains. They can also determine whole genome sequences, which provide important information for the modification or improvement of microorganisms. The following subsections describe genetic tools developed for Geobacillus spp., including tools that have not been described in previous reviews [1,128,129].

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201

TABLE 15.3 Natural plasmids for which replicons are used for plasmid transformation of Geobacillus spp. Antibiotic resistancea

Plasmid

Origin

pBC1

Bacillus coagulans

[130]

pBST1

G. stearothermophilus NRRL 1102

[131]

pGTD7

Geobacillus sp. 610

[132]

pSTK1

G. stearothermophilus TK015

pTB19 pTB20

Thermophile (putative Geobacillus sp.) Thermophile (putative Geobacillus sp.)

Reference

[133,134] R

R

Km , Tet

[135137]

R

[135]

R

Tet

pTHT15

Thermophile (putative Geobacillus sp.)

Tet

[137139]

pUB110

Staphylococcus aureus

KmR

[135137,140142]

a

KmR and TetR denote that the plasmid confers kanamycin and tetracycline resistance, respectively, on the host.

15.3.1 Plasmid replicons A natural plasmid contains a region essential for autonomous replication. The region termed replicon generally functions in a narrow group of organisms; therefore, identifying a plasmid functional in a microorganism is important for the achievement of its genetic modification. Table 15.3 shows natural plasmids that have been demonstrated to replicate in Geobacillus spp. The pTB19 and pTB20 plasmids were isolated from thermophilic bacilli that were potentially Geobacillus spp [135]. pTB19 can be used to transform G. stearothermophilus CU21, and has demonstrated to stably replicate in the transformant even at .60
C. pTB19 was further analyzed to reveal two replicons (repA and repB) [143], which have been used to construct several plasmids [135,136,143146]. Other plasmids whose replicons function in Geobacillus spp. include pBST1 [131], pTHT15 [137,138], pSTK1 [133], and pGTD7 [132]. These also originate from thermophilic bacilli. In addition, two replicons from mesophilic plasmids (pUB110 from Staphylococcus aureus [140] and pBC1 from Bacillus coagulans [147149]) have been demonstrated to function in Geobacillus spp. Since pUB110 hardly replicates in Geobacillus spp. at .68
C, its derivatives are used as temperature-sensitive suicide plasmids that facilitate gene knockout via the allele-coupled exchange [150]. Geobacillus spp. often harbor large circular plasmids whose sizes range between 32.7 and 91.2 kb. The plasmids contain potential replicons of two major classes. One type is identified in pBt40 (origin; length in kb: Geobacillus sp. JF8; 39.7), pGARCT (Geobacillus sp. 12AMOR1; 32.7), pGS18 (G. stearothermophilus 18; 62.8), pGt35 (Geobacillus sp. LC300; 38.4), pHTA426 (G. kaustophilus HTA426; 47.9), pLDW-1 (G. thermoleovorans KCTC 3570; 48.7), and pLDW-3 (G. lituanicus N-3; 51.6). The other type of replicon is shared by pGeo12a (G. thermodenitrificans T12; 58.8), pLDW-2 (G. thermoleovorans ID-1; 91.2), pLW1071 (G. thermodenitrificans NG80-2; 57.5), and pNCI002 (G. thermoglucosidasius NCIMB 11955; 47.9). Such replicons could be exploited to stably maintain a number of heterologous genes in Geobacillus spp. although there are no relevant studies.

15.3.2 Antibiotic resistance markers A plasmid designed for microbial genetic modification generally contains an antibiotic resistance gene as the selection marker, which facilitates the distinction of transformants carrying the plasmid from other cells in the presence of the antibiotic. pTB19 was originally identified as a plasmid that conferred resistance to tetracycline and kanamycin on B. subtilis [135]; however, it was subsequently reported to confer both resistance even on G. stearothermophilus CU21 [136]. Similarly, pC194 and pUB110 from a mesophile (S. aureus) conferred chloramphenicol and kanamycin resistance, respectively, on the same strain [136]. Although antibiotic resistance genes on such plasmids actually served as selection markers for the identification of plasmid transformants, the selection required prolonged incubation over five days at 48
C. This partially arose from the inadequate functioning of antibiotic resistance genes at higher temperatures that were suitable for the cellular reproduction of Geobacillus spp. Consequently, researchers generated mutant genes that conferred antibiotic resistance at elevated temperatures. Representative studies have been carried out on the kanamycin resistance gene in pUB110. The gene encodes for kanamycin nucleotidyltransferase (KNT) that

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PART | II Biotechnological aspects

functions at ,55
C [136]. Matsumura and Aiba screened KNT variants that conferred kanamycin resistance at elevated temperatures on G. stearothermophilus CU21 and identified the D80Y and T130K variants that functioned at .63
C [151]. Liao et al. incubated KNT gene at elevated temperatures in G. stearothermophilus NRRL 1174 and generated a mutant gene for the D80Y/T130K variant via intracellular mutagenesis [131]. This variant functions even at 69
C, allowing us to select plasmid transformants via incubation at 60
C within one day. The gene, termed TK101, has been applied extensively as a selection marker in Geobacillus spp. In addition, a mutant termed catE1 has been generated from the chloramphenicol resistance gene in pC194, which functions at 60
C more effectively than the parent gene in Geobacillus spp [152154]. Wada et al. generated a mutant of the thiostrepton resistance gene from Streptomyces azureus [155]. The mutant, termed tsrH258Y, conferred thiostrepton resistance on G. kaustophilus HTA426 at 55
C although the parent gene could not.

15.3.3 Counterselection markers Orotate phosphoribosyltransferase (PyrE) and orotidine 50 -phosphate decarboxylase (PyrF) are involved in the biosynthesis of uracil-related metabolites while converting 5-fluoroorotic acid into toxic metabolites; therefore, ΔpyrE and ΔpyrF mutants are auxotrophic for uracil and resistant to 5-fluoroorotic acid, whereas cells carrying pyrE and pyrF are prototrophic for uracil and sensitive to 5-fluoroorotic acid (Fig. 15.2). Since this enables the positive selection of both ΔpyrF mutants carrying pyrF marker (selection) and their derivatives that eliminate the pyrF marker (counterselection), the pyrF marker can be repeatedly used in ΔpyrF mutants for gene deletion and integration in chromosomes (Fig. 15.3). Similarly, the pyrE marker can be used in ΔpyrE mutants as a counterselectable marker. The systems have been established in G. thermoglucosidasius NCIMB 11955 [150] and G. kaustophilus HTA426 [156]. In addition, bgl gene that encodes for thermostable β-glucosidase serves as a counterselection marker in G. thermoglucosidasius NCIMB 11955 [157]. Since the bgl product degrades 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside into a pigment that is toxic to the thermophile, cells that eliminate the bgl marker efficiently grow on media supplemented with 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside, whereas bgl-carrying cells cannot.

15.3.4 Recombinant plasmids Escherichia coli has historically served as a common host for DNA manipulation; therefore, plasmids that can shuttle between E. coli and Geobacillus spp. have been developed extensively (Table 15.4). The plasmids frequently contain replicon and selection marker regions from pBR322 or pUC plasmids for DNA manipulation in E. coli. Plasmids developed earlier employ the kanamycin resistance gene from pUB110 as the selection marker in Geobacillus spp.; however, recent plasmids widely use TK101, which confers kanamycin resistance at higher temperatures. pGAM46 contains pyrF as a counterselectable marker but not a replicon functional in Geobacillus spp., as it was constructed for marker-free integration of heterologous genes in ΔpyrF mutants derived from G. kaustophilus HTA426 [156]. pGKE70 is wholly integrated via a single crossover (Campbell-type) recombination at the trpE locus of G. kaustophilus HTA426 [160]. FIGURE 15.2 PyrE and PyrF reactions in the biosynthesis of pyrimidine-related nucleotides (A) and in the metabolism of 5fluoroorotic acid (B). (A) PyrE and PyrF collaboratively convert orotic acid into uridine 50 -monophosphate, which is used to synthesize pyrimidine-related nucleotides. Although both enzymes are essential for de novo synthesis of nucleotides, cells deficient in pryE and/or pyrF can grow in the presence of uracil because uracil phosphoribosyltransferase directly synthesizes uridine 50 -monophosphate from uracil. (B) PyrE and PyrF jointly convert 5-fluoroorotic acid to 5-fluorouridine 50 monophosphate, which further converts into toxic metabolites; therefore, 5-fluoroorotic acid is toxic to cells carrying pryE and pyrF but not to cells deficient in pryE and/or pyrF.

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203

FIGURE 15.3 pyrF-based counterselection in ΔpyrF mutants. ΔpyrF mutants (host and revertant cells) are auxotrophic for uracil and resistant to 5-fluoroorotic acid; therefore, they can grow on minimal media supplemented with uracil and 5-fluoroorotic acid but not in the absence of uracil. In contrast, transformants carrying pyrF marker are prototrophic for uracil and sensitive to 5-fluoroorotate; therefore, transformants can grow on minimal media without uracil but not in the presence of 5-fluoroorotic acid. This facilitates positive selection for both the introduction and elimination of the pyrF marker in ΔpyrF mutants.

TABLE 15.4 Recombinant plasmids constructed for the genetic modification of Geobacillus spp. Geobacillus spp.

Plasmid Replicon pBST16 pBST22

pBST1 pBST1

Marker

Escherichia coli

a

R

Km (pUB110) R

Km (TK101)

Replicon

Marker

pBR322

AmpR (pBR322)

pUC

R

Reference

a

[131]

R

R

[158]

R

R

Amp (pUC19), Cm (pC194)

pG1AK

pBST1

Km (TK101)

pUC

Amp (pUC18), Km (TK101)

[154]

pG1C

pBST1

CmR (catE1)

pUC

CmR (catE1)

[154]

pG1K pG2K

pBST1 pUB110

pGAM46

Km (TK101)

pUC

R

Km (TK101)

pUC

pyrF (G. kaustophilus) R

pGKE70 pIH41

R

Km (TK101) pTHT15

R

pUC

Cm (pC194)

pUB110

CmR (pC194), KmR (pUB110)

pPL401 pRP9 pSTE12

pBC1 pUB110 pBC1 pTHT15

R

Km (pUB110) Cm (pC194) R R

Cm (pC194) R

Tet (pTHT15) R

KmR (pTB19), TetR (pTB19)

pUCG3.8

pBST1 pBST1

R

Km (pUB110)

[150]

R

Amp (pBR322)

[50,65,68]

Cm (pC194), Km (pUB110)

Km (TK101)

pUCG18

[137]

pBR322

pSTK1

pUB110

[160]

Amp (pUC19)

R

pTB19 (repA)

pTMO31

[156,159]

R

Amp (pUC19)

[137,142]

pUC

R

pTB53

pTB19 (repB)

[154]

R

[147]

pSTE33

pTB913

Km (TK101)

R

pBC1

pNW33N

[154]

R

Cm (pC194), Tet (pTHT15)

pLW05

pUB110

Km (TK101)

R

pLM6

pMTL62110

pUC

R

[161] pUC pUC pUC

R

Amp (pUC19), Cm (pC194)

[162]

R

[133,163]

Amp (pUC19) Amp (pUC19)

[135]

Km (pTB19) Km (pUB110) R

Km (TK101) R

Km (TK101) R

[148]

R

R R

R

[143] pUC pUC pUC

R

[164]

R

[154,163,165]

Amp (pUC19) Amp (pUC18) R

[154,166]

R

Km (TK101)

pUCG3.8bgl

pBST1

Km (TK101), bgl (Thermus thermophilus)

pUC

Km (TK101)

[157]

pUCK7

pGTD7

KmR (TK101)

pUC

AmpR (pUC19)

[132]

a pyrF and bgl encode orotidine 50 -phosphate decarboxylaseand β-glucosidase, respectively. AmpR, CmR, KmR, and TetR denote ampicillin, chloramphenicol, kanamycin, and tetracycline resistance genes, respectively. The plasmids or organisms in parentheses indicate the origin of the marker gene. TK101 and catE1 are mutant genes derived from KmR (pUB110) and CmR (pC194), respectively, to function at higher temperatures.

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PART | II Biotechnological aspects

The event disrupts trpE, which is essential for tryptophan biosynthesis; therefore, the appropriate integration can be simply confirmed based on tryptophan auxotrophy. pMTL62110 was constructed for gene knockout via allele-coupled exchange in G. thermoglucosidasius NCIMB 11955 [150]. The plasmid employs the pUB110 replicon and is unstable at .65
C. pMTL62110 is used as follows: (i) homology arms flanking a target gene are cloned in pMTL62110; (ii) the plasmid is maintained stably at 52
C in G. thermoglucosidasius NCIMB 11955; (iii) a double crossover occurs between the arms and the chromosome to produce mutants that delete the target gene; (iv) the plasmid is eliminated from cells by incubation at 60
C; (v) the resultant cells frequently contain a mutant that deletes the target gene but does not carry the plasmid. The plasmid provides a platform for relatively easy gene deletion and integration in G. thermoglucosidasius NCIMB 11955 and potentially in other strains. pUCG3.8bgl was also designed for allele-coupled exchange in G. thermoglucosidasius NCIMB 11955 [157]. This plasmid contains the bgl gene, as a counterselection marker, and pBST1 replicon for autonomous replication in Geobacillus spp. The replicon is relatively stable at elevated temperatures than pUB110 replicon; however, cells that eliminate pUCG3.8bgl can be selected on solid media supplemented with 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside due to the bgl marker.

15.3.5 Protoplast transformation Several Geobacillus spp. have been shown to be transformable using protoplast procedures (Table 15.5). Imanaka et al. introduced pTB19 into G. stearothermophilus CU21, which was constructed from G. stearothermophilus ATCC 12980 via the elimination of an intrinsic plasmid [136]. The transformation was essentially achieved using a protoplast procedure established for B. subtilis and generated transformants with .105 efficiencies (per μg DNA). In addition, the procedure was effective for the introduction of pUB110 and pTB19 derivatives into G. stearothermophilus CU21 [136,143145,151]. G. stearothermophilus CU21 cannot grow in minimal medium; therefore, Zhang et al. explored strains that could grow in minimal medium and accept exogenous plasmids through a protoplast procedure [141]. Consequently, they identified G. stearothermophilus SI1 and eliminated its intrinsic plasmid to generate strain SIC1, which accepted pUB110 and pTB19 derivatives with 104106 frequencies. G. stearothermophilus NRRL 1174 and G. stearothermophilus NUB3621 are also transformable [131,137,148,158]. Notably, NUB3621 protoplasts were regenerated by incubation at 50
C for 12 h followed by 60
C for 48 h, with .72% regeneration rates, and accepted several plasmids with 102108 frequencies. The regeneration is much more rapid and efficient when compared with regeneration of protoplasts from G. stearothermophilus strains CU21 and NRRL 1174.

15.3.6 Electroporation Table 15.5 shows Geobacillus spp. that are transformable with electroporation. Narumi et al. collected 67 strains of G. stearothermophilus and examined whether they were transformable with exogenous plasmids via electroporation [142]. The approach identified a transformable strain termed K1041, which accepted several plasmids with 104106 efficiencies (per μg DNA) [133,142,162]. Bosma et al. employed a similar approach to identify G. thermodenitrificans strains ET 144-2 and ET 251 [65]. Electroporation procedures have also been established for G. thermodenitrificans DSM 465 [149] and G. thermoglucosidasius strains C56-YS93 [171], DL44 [154,165], DSM 2542 [149], and NCIMB 11955 [166]. G. thermodenitrificans T12 accepted pNW33N withan efficiency of 1.7 3 104 [68], and G. stearothermophilus NUB3621 accepted pUCK7 with an efficiency of 1.4 3 102 [132]. G. kaustophilus CER5420, G. stearothermophilus K1041, G. thermoglucosidasius DL44, and G. thermoleovorans DSM 14791 are transformable with pG1K although the efficiencies vary remarkably and range between 101 and 105 [154].

15.3.7 Conjugative plasmid transfer Natarajan and Oriel have reported the conjugative transfer of pAM120A from E. coli to G. stearothermophilus BR219 [169]. pAM120A cannot replicate in Geobacillus spp. but contains Tn916 transposon with a thermostable α-amylase gene. The transfer resulted in chromosomal integration of α-amylase gene potentially via Tn916 transposition from the plasmid. Several strains are shown to be transformable with autonomously replicating plasmids via conjugation with E. coli (Table 15.5). Miyano et al. have reported that B. subtilis performs conjugative plasmid transfer to G. kaustophilus HTA426 [168]. In conjugation among mesophiles, it is often difficult to distinguish between transconjugants and donor cells when they share a selection marker; however, Geobacillus spp. can be readily separated from mesophilic donors simply by incubation at an elevated temperature.

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205

TABLE 15.5 Geobacillus spp. transformable with exogenous plasmids. Straina

Procedure

Proven replicon

Reference

G. kaustophilus ATCC 8005

Conjugation

pBST1

[167]

G. kaustophilus CER5420

Electroporation

pBST1

[154]

G. kaustophilus HTA426

Conjugation

pBST1, pSTK1

[163,168]

G. stearothermophilus 10

Conjugation

pBST1

[167]

G. stearothermophilus BR219

Conjugation

G. stearothermophilus CU21 (ATCC 12980)

Protoplast

G. stearothermophilus K1041

[169] pTB19 (repA), pTB19 (repB), pUB110

[136,143145,151,167]

Electroporation

pBST1, pSTK1, pTHT15, pUB110

[133,142,154,162]

G. stearothermophilus NRRL 1174

Protoplast

pBST1, pUB110

[131,158]

G. stearothermophilus NRRL B-4419

Conjugation

pBST1

[167]

G. stearothermophilus NUB3621 (NUB36)

Electroporation

pBC1, pGTD7, pTB19 (repB), pTHT15, pUB110

[132,137,148,170]

pTB19 (repA), pTB19 (repB), pUB110

[141,146]

Conjugation

Protoplast G. stearothermophilus SIC1 (SI1)

Protoplast

G. stearothermophilus XL-65-6

Conjugation

pBST1

[167]

G. subterraneus DSM 13552

Conjugation

pBST1

[167]

G. thermodenitrificans DSM 465

Electroporation

pBC1

[149]

G. thermodenitrificans ET 144-2

Electroporation

pBC1

[65]

G. thermodenitrificans ET 251

Electroporation

pBC1

[65]

G. thermodenitrificans OS27

Conjugation

pBST1

[67]

G. thermodenitrificans T12

Electroporation

pBC1

[68,149]

G. thermoglucosidasius C56-YS93

Electroporation

pBST1

[171]

G. thermoglucosidasius DL44 (DL33)

Electroporation

pBST1, pUB110

[154,165,172]

G. thermoglucosidasius DSM 2542

Conjugation

pBC1, pBST1

[149,167,173]

Electroporation G. thermoglucosidasius NCIMB 11955

Electroporation

pUB110, pBST1

[150,157,166]

G. thermoglucosidasius TN

Electroporation

pBST1

[174]

G. thermoleovorans B23

Conjugation

pBST1

[167]

G. thermoleovorans DSM 5366

Conjugation

pBST1

[167]

G. thermoleovorans DSM 14791

Electroporation

pBST1

[154]

G. uzenensis DSM 13551

Conjugation

pBST1

[167]

a

The wide-type strain is indicated in parentheses when the strain is a laboratory derivative.

15.3.8 Strategic circumvention of restriction-modification (RM) systems Microorganisms commonly harbor enzymatic systems that selectively digest exogenous DNA. The systems, which aretermed RM systems, essentially play an important role in the protection of microorganisms from infection by bacteriophages that have DNA genomes. RM systems are classified into four major categories [175]. Type II consists of restriction endonuclease and DNA methylase. Endonuclease digests exogenous DNA at specific sites but not endogenous DNA that has been methylated by methylase. Types I and III digest exogenous DNA in a similar manner; however, they form protein complexes including endonuclease and methylase subunits. Lastly, type IV solely consists of endonuclease that digests exogenous DNA with heterologous methylation. RM systems digest not only bacteriophage

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PART | II Biotechnological aspects

DNA but also exogenous DNA that researchers artificially introduce into cells; therefore, plasmid transformation is generally inefficient for microorganisms that harbor RM systems. This can be observed in the plasmid transformation of G. stearothermophilus K1041. The strain accepted exogenous plasmids with 104108 efficiencies (per μg DNA) when the plasmids had been preliminarily propagated in the strain; however, the efficiency drastically decreased when the plasmid had been propagated in E. coli, probably because G. stearothermophilus K1041 harbors an RM system that digests E. coli DNA [133,142,162]. A similar observation was made in G. kaustophilus HTA426, which accepted exogenous plasmids from E. coli that carried a methylase gene (dam) but negligibly from dam2 strains [163]. dam is responsible for 50 -G6mATC-30 methylation at 50 -GATC-30 sites where 6 A indicates N6-methyladenine. This suggests that G. kaustophilus HTA426 harbors an RM system that digests plasmids without 50 -G6m ATC-30 methylation; in fact, an HTA426-derivative deficient in possible RM genes efficiently accepted exogenous plasmids from dam2 strains [123]. Since RM systems are frequently identified in Geobacillus genomes, researchers should explore ways of circumventing RM systems to facilitate the efficient transformation of Geobacillus spp. We note that RM systems can be strategically circumvented using plasmids that artificially imitate DNA methylation of the target organism [175].

15.3.9 Genetic elements to control gene expression Table 15.6 summarizes promoters reported to function in Geobacillus spp. The amy promoter upstream of α-amylase gene in G. stearothermophilus MK232 was used to produce a heterologous protein in G. stearothermophilus SIC1 [146]. Although the productivity was relatively low, the report was notable as a pioneering study on the useful promoters that function in Geobacillus spp. The ldh promoter from G. stearothermophilus NCA1503 is expressed under oxygen limitation [157,165]; therefore, it is extensively used to express heterologous genes in G. thermoglucosidasius in fermentative biofuel production [150,157,165,173]. Reeve et al. have reported that the rplS promoter directs stronger expression compared with ldh promoter in G. thermoglucosidasius DL44 [154]. To fine-tune the gene expression, they further generated 20 variants of rplS promoter that exhibited varying expression levels spanning a 100-fold range along withfour sequences that functioned as ribosome binding sites with different efficiencies. A similar approach was employed to generate 16 variants of groES promoter from Geobacillus sp. GHH01 [171]. The expression level of the variants spanned a 76-fold range in G. thermoglucosidasius C56-YS93. Pogrebnyakov et al. have reported pfl and xylA promoters and six sequences that function as ribosome binding sites with different efficiencies [171]. The pfl promoter is probably constitutive, whereas the xylA promoter is slightly inducible by xylose under the control of the XylR regulator. In G. thermoglucosidasius NUCIB 11955, heterologous glucoside hydrolases were produced under the control of βglu and 2n38 promoters [166]. The βglu promoter functions as an inducible promoter in response to cellobiose. The 2n38 promoteris constitutive and was generated from the upp promoter of G. thermoglucosidasius NUCIB 11955. Zhou et al. used recA promoter to modify G. thermoglucosidasius 95A1 [176]. glpD and P43 promoters function in G. thermoglucosidasius DSM 2542 [173], and uppT12 and pta promoters serve as constitutive promoters in G. thermodenitrificans T12 and G. thermoglucosidasius DSM 2542, respectively [68,149]. Blanchard et al. reported heterologous protein production in G. stearothermophilus NUB3621 using surP promoter [170]. The promoter is induced by sucrose when coexpressed with surT, potentially via antitermination by the surT product. In G. stearothermophilus NUB3621, RHIII promoter served as a constitutive promoter [170]. The gk704, gk1859, gk1894, gk1899, gk1907, gk2150, and sigA promoters are characterized in G. kaustophilus HTA426 [156,159]. Among these promoters, the gk704 promoter is inducible by maltose and facilitates protein production in G. kaustophilus HTA426 and G. thermodenitrificans OS27 [67,159].

15.3.10 Reporter proteins Fluorescence proteins have been used extensively as reporter proteins in numerous organisms. Although their expression is generally inefficient at elevated temperatures because of inefficient protein folding, superfolder green fluorescence protein (sfGFP) is applicable in Geobacillus spp [154,170,171]. Frenzel et al. have recently reported sfGFP variants that function more efficiently at .55
C in Geobacillus spp [149]. A red fluorescence protein also functions in Geobacillus spp., albeit less efficiently compared with sfGFP at .50
C [154]. Catechol 2,3-dioxygenase (PheB) from G. stearothermophilus DSM 6285 is a different type of reporter protein [68,154,172]. The enzyme converts catechol into 2-hydroxymuconic semialdehyde, which can be detected using quantitative colorimetric assay [172]. PheB functions even under oxygen-deficient conditions in contrast to fluorescence proteins, which require molecular oxygen for protein maturation; therefore, PheB reporter is applicable in cells during anaerobic culture. Other reporter proteins include β-galactosidase from Geobacillus spp. and α-galactosidase from G. stearothermophilus NUB3621 [159,170,173]. Both proteins can be detected using commercially available chromogenic substrates.

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TABLE 15.6 Promoters demonstrated to function in Geobacillus spp. Promoter

Origin

Products from downstream gene(s)

P2n38

G. thermoglucosidasius NUCIB 11955

Uracil phosphoribosyltransferase

Inducer

Reference [166]

Pamy

G. stearothermophilus MK232

α-Amylase

[146]

Pgk704

G. kaustophilus HTA426

Maltose utilization system

Maltose

[67,159]

Pgk1859

G. kaustophilus HTA426

Cellobiose utilization system

Lactose

[159]

Pgk1894

G. kaustophilus HTA426

myo-Inositol utilization system

myo-Inositol

[159]

Pgk1899

G. kaustophilus HTA426

myo-Inositol utilization system

myo-Inositol

[159]

Pgk1907

G. kaustophilus HTA426

L-Arabinose

utilization system

Pgk2150

G. kaustophilus HTA426

D-Galactose

utilization system

PglpD

G. thermoglucosidasius NG80-2

Glycerol-3-phosphate dehydrogenase

Pβglu

G. thermoglucosidasius NUCIB 11955

Phosphotransferase system

PgroES

Geobacillus sp. GHH01

Chaperonin

[171]

Pldh

G. stearothermophilus NCA1503

L-Lactate

[150,157,165]

PP43

Bacillus subtilis

Cytidine/deoxycytidine deaminase

[173]

Ppfl

G. thermoglucosidasius C56-YS93

Pyruvate/formate lyase

[171]

Ppta

G. thermoglucosidasius DSM 2542

Phosphate acetyltransferase gene

[149]

PrecA

G. thermoglucosidasius 95A1

RecA

[176]

PRHIII

G. stearothermophilus NUB3621

Ribonuclease

[170]

PrplS

G. thermoglucosidasius DL44

Ribosomal protein

[154]

PsigA

G. kaustophilus HTA426

Sigma factor

[159]

PsurP

G. stearothermophilus NUB3621

Sucrose phosphotransferase

[170]

PuppT12

G. thermodenitrificans T12

Uracil phosphoribosyltransferase

[68]

PxylA

G. thermoglucosidasius C56-YS93

Xylose isomerase

[159] D-Galactose

[159] [173]

Cellobiose

dehydrogenase

D-Xylose

[166]

[171]

15.3.11 Protein secretion Extracellular protein production potentially increases productivity since protein concentrations are not limited in the extracellular environment in contrast to intracellular spaces. This also expands opportunities for microorganisms to degrade macromolecular and/or insoluble materials that cells cannot absorb. Extracellular production of heterologous proteins was observed when xylanase T-6 from G. thermoglucosidasius NG80-2 was produced in G. thermoglucosidasius C56-YS93 [171], and when α-amylase from G. stearothermophilus CU21 was produced in G. kaustophilus HTA426 [159]. An endoglucanase from a hyperthermophilic archaeon (Pyrococcus horikoshii) was secreted from G. kaustophilus HTA426, suggesting that archaeal secretion signals function in Geobacillus spp [159]. BartosiakJentys et al. achieved the extracellular production of heterologous glycoside hydrolases in G. thermoglucosidasius NCIMB 11955, where the enzyme was fused with a secretion signal peptide of heterologous endo-1,4-β-xylanase [166]. Excluding the above example, there is no other report of extracellular protein production in combination with a heterologous signal.

15.4

Geobacillus spp. that have potential in whole-cell applications

Geobacillus spp. can be used for whole-cell applications that are performed at elevated temperatures, which can kill or at least prevent animal pathogens including all viruses from active proliferation [3]. Since virulent pathogens commonly exist in nature and may increase when cultured under moderate conditions, Geobacillus spp. serve as hosts for the safe bioconversion of crude biomass. In addition, bioprocesses that proceed at elevated temperatures have several

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advantages as follows [3]: (i) numerous chemicals increase diffusion, ionization, and solubility; (ii) solution decreases density, surface tension, and viscosity; (iii) higher metabolic activities can be expected; (iv) process temperatures can be maintained without a cooling system; (v) volatile products are spontaneously removed; (vi) the solubility of molecular oxygen decreases; (vii) bioprocesses can be stopped at room temperature. In the following subsections, we describe progressive studies on whole-cell applications of Geobacillus spp. and promising strains that have notable properties.

15.4.1 G. caldoxylosilyticus T20 Organophosphorus compounds contain stable bonds that covalently link phosphorus and carbon atoms, and they are widely distributed in nature. Some of the compounds have been industrially synthesized for use as herbicides, as exemplified by glyphosate, which has been historically produced under the trade name, Roundup, and is splayed extensively on farmlands. Since glyphosate has some clinical manifestations upon exposure to humans (e.g., hypotension and irritation) [177], it is important to degrade glyphosate that persists in farmlands. Obojska et al. screened thermophiles that exploited organophosphorus substrates and identified G. caldoxylosilyticus T20 from a water sample from a domestic heating system [178]. The strain grows preferentially at 60
C and exploits diverse organophosphonates including glyphosate as a source of phosphorus. Although the strain degrades organophosphonate only under phosphate starvation conditions, it has great potential as an organophosphonate degrader and a source of thermostable enzymes for the degradation of organophosphorus compounds.

15.4.2 G. kaustophilus HTA426 G. kaustophilus HTA426 was isolated from a mud sample from the bottom of the Challenger Deep in the Mariana Trench (10,897 m in depth) [51]. The strain grows in the presence of 3% NaCl but not under a hydrostatic pressure of .30 MPa; therefore, it seems that the strain existed as an endospore in the deep-sea environment. In consistent with the previous observation [179], G. kaustophilus HTA426 from the RIKEN BioResource Center (Tsukuba, Japan; JCM 12893) does not produce endospores [117]. This may be attributable to successive cultures that resulted in the repression of endospore formation. G. kaustophilus HTA426 is the first strain among Geobacillus spp. for which the complete genome sequence was determined [180]. Associated genetic tools have also been developed including a transformation procedure [163], promoters [159], a counterselectable system [156], and genetically tractable strains [123,181]. G. kaustophilus HTA426 induces mutagenesis when exposed to antibiotics to produce antibiotic-resistant cells more rapidly and frequently compared with stress-induced mutagenesis reported in other microorganisms [116]. The phenotype offers a novel approach for the generation of thermostable enzyme variants, termed thermoadaptation-directed enzyme evolution, which was first used to generate mutant genes coding for thermostable variants of B. subtilis PyrF [160], and sebsequently, to generate genes for thermostable rRNA methyltransferase and chloramphenicol acetyltransferase that are responsible for resistance to thiostrepton and chloramphenicol, respectively [152,153,155].

15.4.3 G. stearothermophilus ATCC 12978 Cement formation in the presence of ureolytic bacteria improves cement performance (e.g., strength and durability) via microbial CaCO3 mineralization [182]. A similar observation was reported for G. stearothermophilus ATCC 12978, which improved the strength and water absorption capacity of cement-sand mortar when cells were mixed during mortar formation [183]. The greatest improvement was observed at a cell concentration of 109 cfu/mL. The authors attributed the phenomenon to pore structure modification by wollastonite (CaSiO3), which is biologically formed by G. stearothermophilus ATCC 12978.

15.4.4 G. stearothermophilus NUB3621 G. stearothermophilus NUB36 has been used to generate numerous derivatives via spontaneous mutations or chemical mutagenesis with N-methyl-N0 -nitro-N-nitrosoguanidine [184]. One derivative, termed NUB3621, lacks RM genes and efficiently accepts exogenous plasmids using a protoplast procedure. The efficiency is notably high; for example, transformation efficiencies with pLW05 and pTHT15 are 2 3 108 and 3 3 108 (per μg DNA), respectively [137]. Because of the genetic tractability, G. stearothermophilus NUB3621 has previously been subjected to reverse genetic analysis [185188]. In particular, genes induced by heat shock were intensively analyzed including genes for molecular

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chaperons (groESL and dnaK) [189,190], a transcription regulator (hrsA) [191], and transporters (glnH and glnQ) [185]. More recently, the strain has attracted interest as a metabolic engineering host [170].

15.4.5 G. thermocatenulatus 11 Nylon 12 is a synthetic polymer that has been industrially produced for global fiber and plastic applications. The polymer is highly resistant to biodegradation and causes serious environmental pollution; therefore, the efficient degradation of nylon polymers remains a challenge. G. thermocatenulatus 11 was isolated from a soil sample as a thermophilic nylon degrader [192]. The strain grows preferentially at 55
C and degrades nylons 12 and 66, although not nylon 6, where nylons 12 and 66 decrease over 20 days in molecular weight from 41,000 to 11,000 and from 43,000 to 17,000, respectively. Considering the capacity to degrade nylons is uncommon, G. thermocatenulatus 11 could increase the prospects of nylon biodegradation.

15.4.6 G. thermodenitrificans OS27 Seaweeds can grow rapidly in the sea without freshwater, fertilizer, or arable land. Although cellulose and hemicellulose in terrestrial plants are highly resistant to degradation because being surrounded by robust lignin, seaweeds contain no or less lignin. In this light, seaweeds are a promising feedstock for biofuel production; however, seaweeds are rich in unique polysaccharides that are hardly degraded by common glycoside hydrolases. G. thermodenitrificans OS27 was isolated from seaweed as a thermophilic seaweed degrader and was analyzed with the draft genome sequence [67]. In contrast to a related strain [66,193], G. thermodenitrificans OS27 cannot degrade long-chain alkanes but can exploit diverse seaweed polysaccharides such as carrageenan and fucoidan. Since ethanol production was not observed during anaerobic incubation of the strain, G. thermodenitrificans OS27 may be impractical for the application of biofuel production; however, the strain has great potential as a genetic resource of thermostable enzymes for the degradation of seaweed polysaccharides.

15.4.7 G. thermodenitrificans T12 Lactic acid is a precursor for the production of bioplastics and can be produced via microbial fermentation. Daas et al. isolated 94 strains of Geobacillus spp. from compost samples, and characterized their properties to identify thermophiles that participate in fermentation activities and are transformable using an electroporation procedure [68]. The approach identified G. thermodenitrificans T12, which efficiently produces lactic acid from both C6 and C5 sugars, and can degrade xylan. The genome sequence of G. thermodenitrificans T12 has been determined [194], and an endoxylanase encoded in the genome was characterized to reveal its enzymatic properties [195]. G. thermodenitrificans T12 was further modified with heterologous endoglucanase to generate a derivative that is more promising for lactate production from terrestrial plant biomass [196].

15.4.8 G. thermoglucosidasius DSM 2542 G. thermoglucosidasius DSM 2542 serves not only as the type strain of the species but also as a platform for biofuel production since the strain can be genetically modified and the genome sequence has been published [173,197]. Lin et al. modified G. thermoglucosidasius DSM 2542 to express homologous or heterologous genes for 2-ketoisovalerate decarboxylase, ketol-acid reductoisomerase, and acetolactate synthase under the control of the ldh promoter [173]. The resultant strain produced isobutanol from D-glucose and cellobiose with a yield of 0.09 and 0.02 g/g, respectively, within 48 h at 50
C. Zhou et al. disrupted genes for lactate and formate biosynthesis, which potentially decreases ethanol production, and expressed a heterologous gene for pyruvate decarboxylase (pdc) [176]. The resultant strain produced ethanol from D-glucose with a higher yield (0.38 g/g) but exhibited poor growth, potentially because of the insufficient production of acetyl-CoA under microaerobic conditions; therefore, they generated mutants that demonstrated improved growth rates. A resultant mutant efficiently produced ethanol from D-glucose with a yield of 0.43 g/g, which is remarkable, considering 0.51 g/g is the maximum in theory. Gene mutations in the mutant were found in aprt and spoIIIAA that encoded for adenine phosphoribosyltransferase and stage III sporulation protein AA, respectively. The aprt mutation is considered important for the recovery of growth rate because aprt-targeted deletion had a similar effect. In addition to alcohols, G. thermoglucosidasius DSM 2542 can produce hydrogen gas using carbon monoxide as an electron donor [198,199]. The strain produces 2.47 mmol of hydrogen while consuming 2.28 mmol of carbon

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monoxide when incubated with atmospheric air at 1 bar for 84 h. The mechanism of hydrogen production remains unknown but potentially involves carbon monoxide dehydrogenase and hydrogenase.

15.4.9 G. thermoglucosidasius M10EXG G. thermoglucosidasius M10EXG was isolated from a compost sample [70]. A phylogenetic analysis based on 16S rRNA genes suggests that the strain is closely related to G. thermoglucosidasius DSM 2542; however, the two strains vary based on several properties (e.g., motility, endospore formation, growth temperature, and sugars utilized as nutrients). Most notably, G. thermoglucosidasius M10EXG exhibits tolerance to 10% ethanol although thermophilic anaerobes are generally sensitive to .5% ethanol and even an ethanol-tolerant variant generated from Clostridium thermocellum is sensitive to .8% ethanol [70]. G. thermoglucosidasius M10EXG produces ethanol when anaerobically cultured with D-glucose as the sole carbon source. Tang et al. analyzed cells under ethanol-producing conditions by 13 C-metabolic flux analysis [200], which can quantitatively trace how cells convert a carbon-13 labeled substrate into other metabolites using a comprehensive mass spectrometric analysis for numerous metabolites [201204]. The analysis revealed that the strain produced high amounts of organic acids (e.g., formate, lactate, and acetate) in addition to ethanol, suggesting that ethanol production could be improved via a genetic modification to block pathways leading to the production of organic acids. Tang et al. further employed in silico metabolic analysis to predict key factors that enhance ethanol production. The result suggests that lactate and acetate production are the key factors that affect ethanol production and that ethanol production may be increased three-fold when their production is inhibited.

15.4.10 G. thermoglucosidasius NCIMB 11955 G. thermoglucosidasius NCIMB 11955 can ferment C5 and C6 sugars to produce ethanol; therefore, it has been studied extensively for ethanol production applications based on biomass-derived feedstocks [164,205,206]. Associated genetic tools have been developed in addition to its genome sequence being published [207]. Cripps et al. generated derivative strains and showed that some produced ethanol more efficiently compared with the wide-type strain [164]. A derivative termed TM242 is deficient in genes for acetate and lactate production while enhancing the expression of the pyruvate dehydrogenase gene. This strain produced ethanol from D-glucose within 7.5 h at ayield of 0.47 g/g, which corresponds to 92% of the maximum yield in theory [164]. TM242 can utilize hydrolysates of Palm kernel cake, which contain high amounts of fermentable mixed sugars rich in mannose oligomers. TM242 utilized hydrolysates to produce ethanol with a yield of 0.47 g/g at 60
C for 48 h, suggesting that TM242 is a promising ethanol producer that can directly utilize oligosaccharides in addition to C5 and C6 sugars [206]. Geobacillus spp. have no gene for pyruvate decarboxylase, although the gene potentially enables a simpler production of ethanol from pyruvate; therefore, Van Zyl et al. examined to express pyruvate decarboxylase gene from Gluconobacter oxydans in G. thermoglucosidasius NCIMB 11955 [208]. The resultant strain produced ethanol from D-glucose with a yield of 0.35 g/g at 45
C for 48 h. Although productivity is less than in TM242, higher productivity may be achieved when pyruvate decarboxylase functions more efficiently at elevated temperatures.

15.4.11 G. thermoglucosidasius NY05 G. thermoglucosidasius NY05 was isolated from compost and found to extracellularly produce magnesian calcite in the presence of certain minerals [71]. Evidence suggests that endospores function as nuclei for crystal formation [72,73]. Calcite crystals formed by G. thermoglucosidasius NY05 could be used as novel phosphors because of their distinct fluorescence property, which is excited by lights at 260400 nm while emitting fluorescence at 350600 nm [71]. It is noteworthy that the crystal is produced without rare metals in contrast to conventional relevant phosphors.

15.4.12 G. thermoglucosidasius PB94A G. thermoglucosidasius PB94A was isolated from a plant sample based on its capacity to utilize pectin as the sole carbon source [74]. The strain is moderate alkaliphilic and produces no cellulase while degrading pectin; therefore, it is useful for the purification of cellulose fibers that are industrially produced from plant feedstocks through an alkaline process. Jua´rez et al. reported that green flax fibers were produced with less pectin contamination when the production process involved incubation with G. thermoglucosidasius PB94A under mild alkaline conditions [209].

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15.4.13 Geobacillus sp. LC300 Cordova et al. isolated a thermophilic contaminant from a culture of the hyperthermophile Thermus thermophilus [97]. The strain, termed Geobacillus sp. LC300, utilizes both C5 and C6 sugars and grows with high cell densities. The growth is notably rapid; for example, it grew on D-glucose with a doubling time of 19 min, which is a third lower than that of E. coli. Since Geobacillus spp. exist even in clean rooms [59], it is not surprising that the strain had contaminated a laboratory reagent or glass in the form of endospores; however, it is equally possible that Geobacillus sp. LC300 inhabited a niche for T. thermophilus and had evolved to utilize nutrients more rapidly than T. thermophilus for survival in the environment. To investigate why Geobacillus sp. LC300 can rapidly propagate, Cordova et al. cultured the strain in the presence of C5 or C6 sugars as the sole carbon sources and analyzed intracellular metabolism using 13 C-metabolic flux analysis [210,211]. The analysis revealed that Geobacillus sp. LC300 catabolized sugars via the pentosephosphate pathway more efficiently than the thermophiles T. thermophilus and Rhodothermus marinus. The pathway plays an important role in the maintenance of intracellular oxidoreduction potential via the production of NADPH from NADP1 but, with regard to bioproduction, active catabolism via the pentosephosphate pathway is disadvantageous because it discharges carbon dioxide in the course of converting 6-phosphogluconate into ribulose 5phosphate. When researchers can construct a derivative strain deficient in the pentosephosphate pathway while being able to produce NADPH without relying on the pathway, it may serve as an excellent host that rapidly produces certain metabolites using C5 and C6 sugars as feedstock.

15.4.14 Geobacillus sp. XT15 Geobacillus sp. XT15 was isolated from by-product water generated during crude oil production [107]. The strain grows on oil components while dispersing large oil drops potentially due to the action of biosurfactants secreted from cells. The strain produced 7.7 g/L acetoin and 14.5 g/L 2,3-butanediol when cultured with D-glucose and spray-dried corn steep liquor powder as a nitrogen source. Since both C5 and C6 sugars are utilized by Geobacillus sp. XT15, the strain may be useful in the production of acetoin and 2,3-butanediol from terrestrial plant biomass.

15.5

Conclusion and perspective

Geobacillus spp. exhibit notable properties that are useful in whole-cell applications. Numerous genetic tools have facilitated rational genetic modification of Geobacillus spp.; moreover, recent advances in omics analyzes that can wholly determine gene sequences (genomics), gene expressions (transcriptomics), protein amounts (proteomics), intracellular metabolites (metabolomics), and metabolic fluxes (fluxomics) facilitate the estimation of efficient modifications that enhance the identified useful properties of Geobacillus spp. Indeed, there is already a biotechnological platform to generate practical strains that are adopted for industrial whole-cell applications. In addition, it may be possible to modify Geobacillus spp. using evolutionary approaches because Geobacillus spp. have an excellent capacity to adapt to environments due to high genetic plasticity. In contrast to rational genetic modification based on omics analyses evolutionary approaches can potentially alter Geobacillus spp. only by exposing cells to stressors without fatal impacts on metabolism. In the future, the combination of rational modification based on omics analysis and evolutionary modification based on adaptive mutagenesis may serve as a much more efficient approach for the modification of Geobacillus spp.

Acknowledgments This work was funded by the following organizations: Japan Society for the Promotion of Science (Grant number: 17K06925); Nagase Science and Technology Foundation; and the Institute for Fermentation, Osaka, Japan.

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Chapter 16

Thermophiles and thermophilic hydrolases Shilpi Ghosh1, Khusboo Lepcha1,2, Arijita Basak1 and Ayan Kumar Mahanty1 1

Department of Biotechnology, University of North Bengal, Raja Rammohunpur, Siliguri, India, 2Department of Microbiology, University of North

Bengal, Raja Rammohunpur, Siliguri, India

16.1

Introduction

Extremophiles have the remarkable capability to survive in extreme ecological niche characterized by extremes of temperature, pH and salinity. Among these, thermophiles are organisms with relatively higher temperature habitat of at least 60
C [13]. Under natural conditions, they are found in various geothermal zones such as, deep sea, deserts, hot springs, and volcanic area rich in sulfur and toxic metals and other hot environments, like composting sites and biogas plants [14]. Depending on their growth temperature, thermophiles are divided into three classes, namely, moderate thermophiles, which optimally grow at 4565
C; extreme thermophiles, which grow between 6590
C; and hyperthermophiles that optimally grow at and above 90
C [2]. Thermophiles are reported from all the three domains of life i.e. eukarya, bacteria, and archaebacteria, however, most of them belong to bacteria and archaebacteria. In general, moderate thermophiles are primarily bacteria, whereas majority of hyperthermophiles are archaea [2,4,5]. It has been reported that thermophilic bacteria originated in mesophilic habitat and at a later stage they colonized the high temperature environments; on the other hand, thermophilic archaea emerged in hot environments [5,6]. The ability of thermophiles to survive in extreme habitat impart them high genomic, proteomic and metabolic flexibility that in turn make them interesting and challenging platforms for discovery of industrially valuable products like thermostable proteins and enzymes, antibiotics, hormones etc. The enzymes produced by the thermophilic and hyperthermophilic organisms develop unique structural and functional properties of high thermal stability and temperature optima at or above 70
C [711]. With the exuberant growth of biotechnology industries, there has been considerable increase in the requirement of thermotolerant enzymes due to their feasibility to the processes involved. Thermostable enzymes with the ability to degrade biopolymers such as xylanases, amylases, cellulases, lipases, and proteases are particularly important in paper, pulp, pharmaceutical, food, chemical and waste-treatment industries [8,9].

16.2

Discovery and diversity of thermophiles

During late 19th century, with the development of pasteurization technique by Louis Pasteur, it was almost established that microorganisms can be safely killed at temperatures between 80 and 100
C [12]. However, several thermophiles were isolated from natural hot environments later on and with their discovery it became almost established that certain prokaryotes not only have the potential to tolerate but also require temperatures exceeding 80100
C for optimal growth. The initial characterization of thermophiles was limited to spore-forming aerobes such as Bacillus stearothermophilus and anaerobes such as Clostridium thermosaccharolyticum [7]. In true sense, first thermophilic bacterium and first hyperthermophilic archaeon were Thermus aquaticus (Topt 70
C) and Sulfolobus acidocaldarius (Topt 80
C), respectively. Both of them were isolated from extremely hot and acidic spring of Yellowstone National Park (YNP) and their aerobic nature of growth with high energy yield appeared to be essential to resist thermal deconstruction [13,14]. The prejudice was further supported by significantly lower growth temperature of anaerobic thermophilic methanogens and consequently, the presence of anaerobic thermophiles within boiling terrestrial and marine environments was thought to be highly unlikely [15,16]. However, several anaerobic, lithotrophic thermophiles such as, Methanothermus Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00016-2 © 2020 Elsevier Inc. All rights reserved.

219

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PART | II Biotechnological aspects

fervidus [17], Thermoproteales [18] were later isolated from anaerobic environments that grew at temperature up to 97
C. This was followed by isolation of several anaerobic hyperthermophiles belonging to bacterial genera like Aquifex, Thermotoga, Thermocrinis, Thermosipho, and archaeal genera like Desulfurococcus, Acidianus, Staphylothermus, Metallosphaera, Stygiolobus, Thermofilum, Thermoproteus, Pyrobaculum, Thermococcus, Ferroglobus, Thermosphaera, Ignicoccus, Thermodiscus, Pyrodictium, Pyrolobus, Archaeoglobus, Methanothermus, Pyrococcus, Methanopyrus, Nanoarchaeum and Candidatus korarchaeum [15,19]. Among archaea the record of significantly high growth temperature (90100
C) is held by Pyrococcus furiosus, P. fumarii and Methanopyrus kandleri, whereas in bacteria Anaerocellum thermophilum, Caldibacillus cellulovorans, and Fervidobacterium pennavorans exhibit the highest growth temperature of 75 2 80
C (Table 16.1, 2079). Until now, no hyperthermophilic microorganisms in the domain eukarya have been reported. Initial studies on thermophiles required their isolation and cultivation at higher temperatures. Although the techniques for isolation have been improved, they have limited implication in studying the microbial diversity of thermophilic ecosystem [2]. Recent development of high throughput DNA sequencing techniques has enabled the metagenomic analysis of gene encoding 16S rRNA and thus providing a greater insight into the microbial diversity of several high temperature ecosystems [2]. The first metagenomics based research on thermophilic environment happens to be the microbial diversity analysis of Jim’s Black Pool Hot Spring situated in YNP [80]. Further the discovery next generation sequencing (NGS) methods has resulted in comprehensive understanding of the community structure of several thermophilic environments, including hot springs [80,81], deserts [82], compost [83,84], biogas plant [8587], hydrocarbon reservoirs [88,89], and hydrothermal vents [90,91].

16.3

Thermophilic adaptations

Thermophiles have been the attractive and challenging system for scientific community since their discovery, with interests in unraveling the physicochemical and molecular basis of thermophily. They are known to adapt to higher temperature by altering their membrane composition and hence, fluidity. They keep their cellular machinery stable and active in a cooperative mode integrating genomics and proteomics. Moreover, they must possess an efficient DNA repair system in order to prevent the temperature induced genome damage [4,92].

16.3.1 Membrane level adaptations The biological membranes play important roles in cellular communication and energy transduction. High environmental temperature is known to increase the membrane permeability and fluidity. Under such conditions membranes are required to maintain its liquid crystalline state; and semi permeability to small molecules and ions in order to generate proton motive force and pH homeostasis. Thermophilic bacteria regulate their membrane fluidity primarily by altering phospholipid composition like increasing the amount of branched chain iso-fatty acids, long chain fatty acids, and saturated fatty acids [4,92,93]. The archaeal membrane phospholipids contain saturated isoprenoid chains that remain linked to the glycerol backbone by the chemically resistant ether linkage instead of the ester linkage present between fatty acids and glycerol in case of most bacterial membranes [4,92]. Moreover, the hyperthermophilic archaea are reported to possess membrane spanning dimeric dibiphytanylglycerol nonitol tetraethers (nonitolcaldarchaeols) and dibiphytanyldiglycerol tetraethers (caldarchaeols) that provide a high degree of rigidity to the cell membrane [94].

16.3.2 Genome level adaptations The structural and functional integrity of the genetic material are essential to control the fidelity of replication, transcription and translation processes [3,7]. Bacterial and archaeal hyperthermophiles exclusively express reverse gyrase, which enhance the melting temperature (Tm) of DNA by introducing positive supercoils in an ATP dependent reaction [95,96]. The hyperthermophilic archaea possess DNA with relaxed to slightly positively supercoiled topology and this unique topological state is due to the combined action of reverse gyrase and DNA topoisomerase II that removes negative supercoils [97]. In a hyperthermophilic archaea Thermococcus kodakarensis, deletion of the reverse gyrase encoding gene from the chromosome resulted in slower growth of the mutant strain, demonstrating the critical role of reverse gyrase to thermophily [3,97]. In addition, some small basic proteins are reported to increase the Tm of DNA by associating with chromatin. For example, the ‘7 kDa DNA binding’ (Sul7d) proteins from the archaeal order Sulfolabales, such as Sso7d and Sac7d impart thermostability to the DNA and also promote the annealing of complementary strands of DNA above the Tm value [98,99].

Thermophiles and thermophilic hydrolases Chapter | 16

221

TABLE 16.1 Growth characteristics of thermophilic organisms. Sl. no.

Organism

Optimum temperature

Growth physiology

Reference no.

1

Anaerocellum thermophilum

75
C

Strictly anaerobic

[20]

2

Acidobacterium capsulatum

65
C

3 4 5 6

Alicyclobacillus acidocaldarius Anaerocellum thermophilum Anoxybacillus flavithermus Anoxybacillus kamchatkensis NASTPD13

Facultative anaerobic

[21]




Strictly anaerobic

[22]




Strictly anaerobic

[23]




Facultative anaerobic

[24]




Facultative anaerobic

[25]




60 C 74 C 60 C 65 C

7

Bacillus licheniformis

70 C

Facultative aerobic

[26]

8

Bacillus licheniformis

70
C

Facultative aerobic

[27]

9

Bacillus pumilus

70
C

10 11 12 13

Bacillus stearothermophilus Bacillus thermoleovorans ID-1 Bacillus vallismortis RG 07 Burkholderia ubonensis SL-4

Endospore forming aerobic

[28]




Facultative aerobic

[29]




Facultative aerobic

[30]




Aerobic

[31]




Obligate aerobic

[32]




65 C 74 C 65 C 65 C

14

Caldicellulosiruptor saccharolyticus

70 C

Anaerobic

[33]

15

Caldanaerobacter subterraneus

75
C

Anaerobic

[34]

16

Caldibacillus cellulovorans

80
C

17

Caldicellulo siruptorbescii

Aerobic or Facultative anaerobic

[35]




Anaerobic

[36]




70 C

18

Caldicellulosiruptor saccharolyticus

69 C

Extremely thermophilic anaerobic

[37]

19

Cellulosimicrobium cellulans CKMX1

60
C

Xylanolytic anaerobic

[38]

20

Clostridium thermocellum

(5568)
C

21 22 23

Clostridium thermosulfurogenes Dehalococcus restrictus Desulfitobacterium frappieri TCE1

Anaerobic

[39]




Strictly anaerobic

[40]




Strictly anaerobic

[41]




Strictly anaerobic

[42]




60 C 63 C 65 C

24

Fervidobacterium islandicum AW-1

70 C

Extremely thermophilic anaerobic

[43]

25

Fervidobacterium pennavorans Ven5

80
C

Extremely thermophilic anaerobic

[44]

26

Geobacilluss tearothermophilus

70
C

Aerobic or Facultative anaerobic

[45]

27

Geobacilluss tearothermophilus

(6080)
C

Aerobic or Facultative anaerobic

[46]




28

Geobacillus thermocatenulatus MS5

(6065) C

Aerobic or Faculatative anaerobic

[47]

29

Meiothermus taiwanensis WR-220

65
C

Aerobic

[48]

30

Methanopyrus kandleri

110
C

Obligate anaerobic

[49]




31

Methanothermobacter thermoautotrophicus

(6070) C

Obligate anaerobic

[50]

32

Nocardiopsissp B2

70
C

Strictly aerobic

[51]

33

Pyrococcus furiosus

100
C

Hyperthermophilic anaerobic

[52]

34

Pyrococcus furiosus

.72
C

Thermophic anaerobic

[53] (Continued )

222

PART | II Biotechnological aspects

TABLE 16.1 (Continued) Sl. no.

Organism

Optimum temperature

Growth physiology

Reference no.

35

Pyrococcus abyssi

96
C

Hyperthermophilic anaerobic

[54]

36

Pyrococcus horikoshii

98
C

37 38 39 40

Rhodothermus marinus Rhodothermus marinus Staphylococcus aureus ALA1 Streptomyces thermocerradoensis I3

Hyperthermophilic anaerobic

[55]




Obligate aerobic

[56]




Obligate aerobic

[57]




Facultative anaerobic

[58]




Anaerobic

[59]




70 C 80 C 60 C 75 C

41

Sulfolobus solfataricus

85 C

Obligate aerobic

[60]

42

Thermoanaerobacter ethanolicus

60
C

Anaerobic

[61]

43

Thermoanaerobacter subterraneus

65
C

Anaerobic

[62]

Anaerobic

[63]

44

Thermoanaerobacter tengcongensis




75 C


45

Thermoanaerobacter thermohydrosulfuricus

(6070) C

Obligate anaerobic

[64]

46

Thermoanaerobacterium saccharolyticum NTOU1

63
C

Anaerobic

[65]

47

Thermococcus kodakaraensis

85
C

Obligate anaerobic

[66]




48

Thermococcus litoralis

.72 C

Anaerobic

[67]

49

Thermomonosporafusca

75
C

Aerobic or Facultative anaerobic

[68]

50

Thermoplasma acidophilum

60
C

51 52 53 54

Thermosyntrophalipolytica Thermotaga maritima Thermotaga neapolitana Thermotoga.naphthophila

Facultative anaerobic

[69]




Anaerobic

[70]




Anaerobic

[71]




Anaerobic

[72]

Anaerobic

[73]

75 C 80 C 80 C


80 C


55

Thermotoga maritima

7682 C

Anaerobic

[74]

56

Thermotoga lettingae

65
C

Anaerobic

[75]

57

Thermotoga petrophila

80
C

Anaerobic

[73]

Aerobic

[76]

Obligate aerobic

[77]

75 C

Aerobic

[78]




Aerobic

[79]

58 59 60 61

Thermus aquaticus Thermus caldophilus Thermus caldophilus Thermus thermophila




.75 C


(7077) C


.75 C

The comparison of thermophile and mesophile genomes has shown that GC content of thermophiles, like Thermus sp. strain CCB_US3_UF1 (68.60%) and Thermus thermophilus ATCC 33923 (69.40%), is higher compared with mesophiles like Geobacillus kaustophilus (52.10%), suggesting a correlation between GC content and overall growth temperature [100,101]. However, some thermophiles are reported to contain genomes with similar or even lower GC content, like Caldicellulosiruptor hydrothermalis having genome with only 35% GC has optimum growth temperature of 70
C [101]. Hence, the GC content of the genome doesn’t seem to be universally correlated with the thermophily. On the other hand, GC content of tRNAs and rRNA strongly correlates with the optimum growth temperature (OGT) that might support efficient translation of sufficient number proteins by the thermophiles for maintaining functional performance at higher temperature [102,103].

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In thermophiles purine loading of mRNA increases as a survival response resulting in their greater values of Chargaff differences, a measure of purine loading, as compared with non-thermophiles [96,104]. Such base bias directly influences the usage of amino acids in the proteins with increased frequency of lysine (Lys), arginine (Arg) and glutamate (Glu), and decreased number of uncharged polar amino acids, like glutamine (Gln), asparagine (Asn), threonine (Thr) and serine (Ser), in the proteome of thermophiles [105]. A combinatorial study by Farias and Bonato (2003) reported the ratio of (Glu 1 Lys)/(Gln 1 His) as an indicator of protein thermal stability with the values, above 4.5, below 2.5 and between 3.2 and 4.6 for hyperthermophiles, mesophiles and thermophiles, respectively [106]. Thermophilic archaea and bacteria are reported to produce unusual long-chain (homocaldopentamine, caldopentamine and caldohexamine) and branched-chain (tris-(-3-aminopropyl) ammonium, N4-aminopropylspermidine) polyamines that are shown to stabilize the DNA structure and contribute to the regulation of gene expression. The archaea like, Pyrobaculum aerophilum and Hyperthermus butylicus have long-chain polyamines, while T. kodakarensis has only branched-chain polyamines [107]. In addition, some methanogenic bacteria and Pyrococcus woesei contain salts like tripotassium cyclic-2,3-diphosphoglycerate and di-myo-inositol-1,10 -phosphate, respectively, which are shown to stabilize DNA under in vitro conditions [108].

16.3.3 Proteome level adaptations Comparative studies on protein homologs from thermophiles and mesophiles indicated their similarities in amino acid sequence (4085%), three dimensional structure and catalytic mechanisms [10,109]. However, a closer examination revealed several differences in amino acid sequence and structural characteristic contributing to the thermotolerance property of thermophilic proteins. Among these the most common are increased prevalence of hydrophobic residues with branched side chains, an increased proportion of charged residues, surface loop deletion and tight packing. In addition, the impact of electrostatic interactions such as ion pair networks and hydrogen bonds, disulfide bonds and hydrophobic interactions are mostly stabilizing for thermophilc proteins [109111].

16.3.3.1 Amino acid composition The genome sequencing of several thermophilic and hyperthermophilic organisms followed by their availability in the genome database has resulted in considerable increase in information on amino acid sequences and structural characteristics of thermophilic proteins. A comparative proteome analysis revealed that a higher ratio of polar charged versus polar uncharged amino acids being the signature of the thermophiles [112]. The amino acids Ser, glycine (Gly), Lys, and Asp residues in thermophilic proteins are often replaced with Thr, alanine (Ala), Arg, and Glu, respectively [112]. These substitutions increase the inner core hydrophobicity and reduce the external surface hydrophobicity that imparts stability to the thermophilic proteins [110,113]. The substitution of Glu stabilizes a protein due to its relatively higher conformational entropy. Also, the increased side chain length of Glu allows relatively more hydration and thus lowers the disolvation penalty on protein folding [114]. Also Gln and Asn being thermolabile in nature, their lower abundance reduces the susceptibility of thermophilic proteins to backbone cleavage, deamination and oxidation at higher growth temperature of thermophiles [110,115]. The occurrence of hydrophobic residues, like proline (Pro), isoleucine (Ile), and valine (Val) are higher in thermophiles compared with mesophiles, which in turn increases the hydrophobicity and rigidity of the proteins [111,116].

16.3.3.2 Surface and core distribution of amino acids The proteins are known to modify their molecular surfaces according to their subcellular location in terms of amino acid composition and similarly, a greater difference in amino acid composition of surfaces rather than interior of thermophilic and mesophilic proteins can be reasonably expected. For thermophilic proteins, the average surface area distribution was found to be lower for Ala and Pro and higher for phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and Met. Ala and Pro having fixed configuration are thought to bury the more non-polar area into the core for thermal stabilization. On the other hand, placement of aromatic side chains on the surface may reduce fluctuations in folding of the proteins at higher growth temperatures of thermophiles. In contrast to the surface, a relatively lower difference in amino acid sequence occurs between interior of the thermophilic and mesophilic proteins [111,115]. A structural comparison of 127 homologous thermophilic and mesophilic protein groups has shown the predominance of stabilizing ion-pairs on the surface of the former group. The ion-pairs are relatively unaffected by higher temperature and therefore, their presence on the protein surfaces have been recognized as an efficient mechanism to increase thermostability from evolutionary point of view [118].

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16.3.3.3 Ion pair networks, hydrogen bonds and aromatic interactions Ion-pair networks and hydrogen bonds are important forces to achieve thermostability of proteins. Although the charged amino acids are important for electrostatic interactions, the increase in charge contribution to the protein folding stabilization generally do not depend on the number of ionizable amino acid residues rather than their location within the protein structure [114]. Due to higher electrostatic free energy the buried ion-pairs have destabilizing effect on protein structure unless they undergo favorable interactions with other ion-pairs to form the ion-pair network. Thermophilic proteins are known to enhance the electrostatic interactions by the formation of such network within their structures through optimum placement of charged amino acid residues without disrupting the core residues characteristics of the protein families [115,117]. Furthermore, the extensive inter-subunit ion-pair networks and H-bonds can be a general strategy for manipulation of thermostability of multimeric enzymes. The ion-pair network content, and H-bonds between amino acid side chains has been reported to increase in the monomers of most of the multimeric thermophilic proteins and at their interface [111]. Homology modeling and structural comparison of glutamate dehydrogenase (GDH) from Pyrococcus furiosus (Topt 100
C) and Thermococcus litoralis (Topt 88
C) were performed, in order to assess the primary stabilizing factors of thermophilicity [118]. Although, the hexameric GDH from two sources showed 87% amino acid sequence homology, they differed in thermal stability at 104
C by 16-folds. The inter-subunit ion-pair network was markedly reduced in the enzyme with lower stability from T. litoralis and substitution of two amino acids by site directed mutagenesis restored the network with four-fold improvement of stability at 104
C [118]. Though aromatic interactions are weak non-covalent forces between aromatic amino acid side chains, can act together to contribute in the folding and stability of proteins. Aromatic interactions like π-π, cation-π, and Phe-Phe interactions have significant role in thermal stability [110,119]. In a study, the role of aromatic interactions in thermostability of 24 families of thermophilic proteins was investigated. Most of the protein families possessed higher number of aromatic residues as compared to their mesophilic homologs. Ten protein families had greater number of surface exposed aromatic clusters [120]. The stability effect of Phe-Phe interaction relies on its location within the protein structure and it shows more stabilizing effect on the C-terminus rather than on the central region of the helix. The aromatic clusters were also found to be important in the stability of hair pin loops [110,119].

16.3.3.4 Hydrophobic interactions and disulfide bonds Hydrophobic interaction has dominant role in protein folding and in general, thermophilic proteins have relatively higher hydrophobicity than that of their mesophilic homologs [110,116]. Though the study of Kumar et al. [115] showed an almost similar hydrophobicity of thermophilic and mesophilic proteins, the research works of Takano et al. [5] reported the major contribution of hydrophobic interactions in thermostability of archaeal proteins. Furthermore, hydrophobic cores have been recognized as target for protein engineering and studies have proved that the protein stability could be altered significantly by mutations in the hydrophobic core [121]. Disulfide bond, a much stronger covalent force, can stabilize the proteins by decreasing the entropy of folding [122,123]. Although cysteine (Cys) and disulfide bonds show susceptibility to destruction at higher temperature; several disulfide bond containing thermophilic proteins with thermal stability at $ 100
C have been characterized. In these proteins disulfide bridges can be a strategy for stabilization; and their solvent accessibility and conformational environment can protect the disulfide bridges against thermal destruction [10]. Thermophilic archaea belonging to the Crenarchaeal branch are reported to be universally rich in disulfide bonds [123]. The role of disulfide bond and hydrophobic interaction in protein thermostability was further confirmed by a study on glucoamylase from Aspergillus awamori. The thermal stability of the enzyme was significantly improved by the engineering of disulfide bonds and by the substitution of G396 and G407 with Ala [124].

16.3.3.5 Secondary structures The presence and abundance of α-helices are shown to play a role in protein stability. Thermophile proteins contain a significantly greater fraction of amino acids in the form of α-helix and these α-helices lack proline residues [115]. Furthermore, the substitution of Ala and Leu with amino acids having a higher tendency to form α-helix, changes the composition of α-helices of thermostable proteins [113]. A study on the comparison of thermophilic and mesophilic RecA proteins suggested that the increased fraction of amino acids with higher propensity to form helical structure and the interaction between side chains are the main factors contributing to thermostability. In addition, the combined stability of

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helices, instead of individual helix plays important role in thermostability [125]. Engineering of α-helix stability has been implicated to generate industrially important enzymes with the ability to function at higher temperatures [113].

16.3.3.6 Protein packing and folding The conformational stability of proteins is attributed to a compromise between the catalytic flexibility and conformational rigidity. The conformational rigidity is determined by compactness, which is a measure of fractions of polar and buried surface area, the loop lengths, and decrease in the number and volume of cavities within the protein structures [114,116]. Thermophiles are reported to have higher levels of Ile, Ala, and Pro that provide tighter packing in hydrophobic cores and extra stability to loops [116]. Though Kumar et al. [115] found very similar compactness values for thermophilic and mesophilic proteins, the research work by Glyakina et al. [126] showed closer packing of amino acids in the water accessible surface of thermophilic proteins. Also, protein stability has been engineered by the introduction of proline residues in order to decrease the entropy of the side chain in the folded state [18].

16.4

Thermophilic enzymes

The stability temperature of enzymes belonging to thermophilic and hyperthermophilic organisms is not essentially the same as organism’s growth temperature (OGT). Although most of the enzymes from these organisms have temperature optima around OGT of the organisms, their cell bound or extracellular enzymes have optimum activity at temperature higher than OGT [10,110]. The thermostable enzymes are attractive for various biotechnological industries due to their higher thermal stability and feasibility to the processes involved. The enzyme catalyzed reactions carried out at higher temperature has additional advantage of reduced viscosity and therefore, increased diffusion of the substrates resulting in higher product yield. Thermostable enzymes can also serve as model for determining the thermostability mechanism at molecular level [711]. Research works have shown that thermophilic enzymes are novel catalysts with applications in the industrial sectors. Thermophilic hydrolases catalyzing the degradation of biopolymers like amylases, lipase, pullulanases, xylanases, proteases, and cellulases play important role in food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries. The following section describes some of the industrially important thermostable enzymes and their applications.

16.4.1 Amylases Amylases catalyze the hydrolysis of the α-1,4-glucosidic bonds of starch, and other related polysaccharides to release glucose, maltose and maltotriose units. Thermostable amylases are desirable in the textile, food, detergent and paper industries [127129]. Bacillus licheniformis produced a highly thermotolerant α-amylase (BLA) which served as a model for understanding the structural basis of thermostability. Like other α-amylases, the enzyme (469 amino acids) contained three domains A, B and C, among which domain B (104206 amino acids) appeared to have important role in thermostability [130]. A total 175 variants of Bacillus licheniformis α-amylase were created and analyzed in vitro for thermostability. The replacement of three Asn residues at positions 172, 188 and 190, with other amino acids led to considerable increase in thermostability in comparison to the native enzyme. The highest thermal stabilization was achieved by substituting Phe by Asn 190 [131]. Nathan and Nair [132] engineered a repression free catabolite-enhanced overexpression of BLA from a strong self-inducible promoter of pstSCAB operon, for cost effective production of the enzyme. The scheme resulted in glucose enhanced expression of the BLA with optimum activity at 90
C and a wide pH range of 510 [132]. The amino acid sequence comparison of BLA with Bacillus amyloliquefaciens α-amylases (BAA) showed the prevalence of two amino acid (EG and AA) insertions in BAA group α-amylases and were identified as key factors for decreased thermal stability. The insertion mutations were thought to cause conformational disturbances that weakened the calcium binding affinity and consequently decreased the thermostability [133]. A thermophilic β-amylase encoding gene of Clostridium thermosulfurogenes was cloned into Bacillus subtilis. The amino acid sequence of the translated protein showed lesser number of polar amino acids and more Cys residues, when compared with mesophilic enzyme [134]. In another research work, an extremely thermophilic and obligately anaerobic bacterium, Dictyoglomus thermophilum, produced multiple amylases extracellularly. The recombinant amylases expressed in E. coli exhibited temperature optima of 7080
C and showed different pattern of starch hydrolysis [135]. In a recent study, a Flavobacteriaceae Sinomicrobium α-amylase (FSA) closely related to archaeal α-amylase but evolutionarily distinct from bacterial amylase has been characterized. The highly expressed recombinant enzyme showed

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weak thermostability, however, the site directed mutagenesis based introduction of disulfide bonds resulted in significant improvement in stability and activity of the enzyme at higher temperature [136]. Although most of thermophilic enzymes are derived from bacteria, several thermophilic amylases are reported from fungi such as Rhizomucor pusillus, Paecilomyces variotti [137,138]. Two genes encoding novel glucoamylase (RpGla) and amylase (RpAmy) was cloned from thermophilic fungus Rhizomucor pusillus. The co-expression of RpGla and RpAmy in Pichia pastoris resulted in dramatic improvement in amylase activity [139].

16.4.2 Proteases The catalytic activity of proteases hydrolyzes the proteinaceous materials into constituent amino acids. They represent the largest group of enzymes with applications in biotechnology industries such as textile, food, leather, detergent, and pharmaceutical industries. Depending upon the catalytic mechanisms they are classified as aspartic proteases, serine proteases, cysteine proteases or metallo proteases. Thermotolerant proteases are promising in various industries due to their robustness and broad substrate specificity [140,141]. Subtilisins are serine proteases that comprise the largest group of commercial proteolytic enzymes [141]. They have drawn increasing attention because of their implication in determining the molecular basis of protein thermal stability. Several thermostable subtilisin proteases are reported from archaea such as, stetterlysin from Thermococcus stetteri [142], pyrolysin from Pyrococcus furious [143], Ak.1 protease from Bacillus sp. Ak.1 [144], Tk-SP, Tk-1689, and Tk subtilisins from Thermococcus kodakaraensis KOD1 [145] and aqualysin I from Thermus aquaticus TY-1 [146], and proteolysin from Coprothermobacter proteolyticus [141]. Among these, Tk-subtilisin, Tk-SP and pyrolysin showed temperature optima of 90, 100 and 115
C and half-lives of 50, 100 and 240 min at 100
C, respectively [145,147,148]. Homology modeling studies of three-dimensional structures of pyrolysin and stettrlysin with subtilisins from the mesophilic bacteria showed a correlation of higher thermal stability with increased number of ion-pair networks and aromatic interactions [142]. In another research work, the importance of ion-pairs in the high temperature stability of aqualysin was determined by site directed mutagenesis of the participating amino acids. Out of several mutants produced D183N showed markedly lower thermostability and altered secondary structure [146]. Aqualysin and proteolysin are expressed as precursor proteases. Aqualysin contains amino terminal pre-prosequence (127 amino acids), the protease (281 amino acids), and a carboxy-terminal pro-sequence (105 residues). Further studies using E. coli as expression host indicated that both the N- and C-terminal signal sequences are removed by proteolytic activity of aqualysin itself, converting the precursor enzyme to active form [149]. Pyrolysin showed four peptide inserts (IS8, IS27, IS29 and IS147) in the catalytic domain, of which three inserts (IS147, IS27, IS8) were found to be either important or essential for efficient maturation of the enzyme at high temperature [143]. Three novel alkaline and thermophilic serine proteases namely, EI, EII and EIII, from Pseudomonas aeruginosa demonstrated optimum activity at 60
C. Among these proteases, EIII presented the highest proteolytic activity toward wheat distiller dry grain with soluble (DDGS) protein enabling release of 63% of the total glycine, which was 2.2-fold higher than that with commercial Pronases [150]. Keratins are water insoluble waste of poultry, slaughter house and leather-processing industries that are extremely resistant to proteolysis by many proteases. Keratinases are reported to be produced by several thermophilic and extremely thermophilic bacteria, including Fervidobacterium pennivorans, F. islandicum AW-1, F. thailandense FC2004T, Thermoanaerobacter keratinoplilus and Thermoanaerobacter sp. strain 1004-09. T. keratinoplilus, (Topt 70
C) possessed extracellular and intracellular proteases optimally active at 85
C, pH 8.0 and 60
C, pH 7.0, respectively [151]. F. pennivorans, F. islandicum and Sp. FA004 have been identified as fast feather degraders and they produced keratinases with optimal activities between 80100
C and were stable at 7080
C for days [43,152]. Thermophilic proteases encoding genes have been cloned and expressed in mesophilic bacteria. Thermicine, a novel subtilisin-like protease from Thermoanaerobacter yonseiensis KB-1 (DSM 13777) was expressed in E. coli. The recombinant enzyme showed optimum activity at 92.5
C and pH 9.0, and had half-life of 30 h at preincubation temperature of 80
C [153]. In another research work, the proteolysin gene from Coprothermobacter proteolyticus was expressed in E. coli and the purified enzyme showed activity up to 80
C [150]. In addition, the metagenomic analysis of thermophilic environments has identified novel protease producing microoganisms. For example, the genome of Geobacillus thermoleovorans strain N7 (MCC 3175), isolated from Paniphala Hot Spring, West Bengal, India, harbored several genes encoding multiple proteases with industrial importance [154].

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16.4.3 Cellulases Cellulosic biomass is the most abundant biopolymer on earth that can be hydrolyzed to fermentable sugars by the activity of cellulases. Cellulases are complementary enzymes complex consisting of (i) exoglucanases that act on cellulose from either reducing or non-reducing ends to release cellobiose units, (ii) endoglucanases that act randomly on cellulose chains internally to produce cellulose oligosaccharides and cellobioses, and (iii) β-glucosidases hydrolyze the cellulose oligosaccharides and cellobioses to glucose molecules [155157]. Cellulases are required for several biotechnology industries including biofuels, food, brewing, textiles, detergent, paper and pulp, and animal feeds. The enzyme is also being used for waste management, extraction of pigments, olive oil compounds and other bioactive molecules from plant materials [158]. Thermotolerant cellulolytic enzymes are particularly attractive candidates for application in plant biomass depolymerization. They allow effective depolymerization of chemical pretreated cellulosic biomass for extended time period [159]. Cellulases are either integrated into multi-enzymatic complexes called cellulosomes or secreted as free enzymes. The cellulosome can be directly targeted to cellulosic substrate for efficient release of sugars [155,157]. During past several cellulase producing thermophiles have been reported. Caldicellulosiruptor bescii, a hyperthermophilic anaerobe isolated from a hot springs in Kamchatka, Siberia, expressed highly active cellulase [160]. The bacterium showed the ability to depolymerize cellulosic substrate without chemical or enzymatic pretreatment and the cellulolytic ability was due to the extracellular production of multi-modular and multi-functional enzymes called CelA, which outperformed commercially available cellulases [161]. Recently, Kahn et al. (2019) reported the designing of a “designer cellulosome” system using cellulolytic enzymes of C. bescii, which showed activity and stability at 75
C [162]. In another study, a β-glucosidase encoding gene (Dtur_0462) from hyperthermophilic bacterium Dictyoglomus turgidum was expressed in E. coli. The monomeric enzyme exhibited optimum activity at pH5.4 and 80
C and it retained 70% activity after 2 h of preincubation at 70
C. The enzyme with high glucose and ethanol tolerance was suitable for industrial production of ethanol [163]. Similarly, some novel archaeal thermostable cellulases were also identified. Cellulase F1 obtained from oil reservoir metagenome contained two separate cellulase modules, probably formed by combining two different archaeal cellulases. The fusion enzyme showed remarkably higher thermostability and activity as compared to commercially available enzymes [164]. Similarly, a thermophilic archaea Sulfolobus shibatae produced an endo-1,4-β-D-glucanase and the encoding gene was cloned in E. coli. The expressed enzyme exhibited maximum activity at 95100
C. The enzyme exhibited remarkable tolerance to higher temperature and 100% activity was noted after 1 h incubation at 75, 80 and 85
C while 98%, 90% and 84% of original activity was noted after 2 h incubation at 75, 80 and 85
C, respectively [165]. Apart from thermophilic bacteria and archaea, several thermophilic fungi are reported to express endoglucanases. The enzyme from Thielavia terrestris [166], Chaetomium thermophilum [167], and Sclerotinia sclerotiorum [168], had optimum temperature between 50 and 70
C and they retained substantial activity after preincubation at 8090
C. The endoglucanase from Thielavia arenaria XZ7 expressed in Pichia pastoris exhibited extra ordinary thermostability at 90 and 100
C. Out of seven disulfide bonds present in the protein, C12C14 bond was found to be critical for thermal adaptation [169].

16.4.4 Xylanases Xylanases catalyze the hydrolysis of heterogeneous hemicellulosic polysaccharide made of xylan, xyloglucans and mannans, constituting the plant cell wall [170]. Xylanases have industrial applications in the clarification of fruit juice, removal of xylans from kraft-pulp for making paper, improvement of rumen digestion, production of chemicals and fuel from cellulosic biomass, and in the brewing process [171]. Several thermophilic and hyperthermophilic microorganisms isolated from marine and terrestrial solfataric fields, hot springs and decaying organic debris undergoing selfheating have been reported to produce xylanases. These include thermophilic bacteria, like Bacillus stearothermophilus [172], Rhodothermus marinus [173], Thermomyces lanuginosus [174], and Thermoascus aurantiacus [175], and hyperthermophilic archaea such as Thermococcus zilligii [176], Sulfolobus solfataricus [60] and Pyrodictium abyssi [9]. Most of these thermophilic xylanases are members of glycoside hydrolase (GH) families 10 and 11 of carbohydrate active enzymes [177]. Amino acid sequence alignment, X-ray crystallography and mutagenesis studies have revealed that enhanced thermal stability of xylanases are attributed to several minor modifications, including the presence of tandem repeats of thermostability domains [178], a greater number of charged amino acids in the surface [179], a greater number of ionpairs and hydrogen bonds [180], the presence of disulfide linkages particularly in the α-helix, and N- and C-terminals

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[181] and aromatic residues forming ‘sticky patches’ on the surface [182]. In general, thermophilic xylanases having lower molecular weight have stretches of about ten (B10) amino acid residues incorporated into their chains, the most noticeable being between the 3rd β- sheet and the 5th α-helix [183]. Thermotolerant xylanases have drawn attention due to longer half-life at higher temperatures. Geobacillus sp. strain WSUCF1 produced xylanase with exceptional high thermostability with 18 and 12 days of half-lives at 60 and 70
C, respectively [184]. Xylanase from Clostridium thermocellum contained thermostabilizing noncatalytic domains which exhibited sequence homology with a thermostable domain of xylanase A of Thermoanaerobacterium saccharolyticum [185]. In another study Hachem et al. (2003) reported the role of calcium in maintaining the thermostability of GH10 xylanases [173]. The xylanase from Thermotoga thermarum was expressed in E. coli BL21 (DE3). The recombinant protein showed high thermal stability over temperature range of 5590
C and exhibited enhanced thermostability upon the addition of 5 mM Ca21. It was concluded that metal ion maintained the structural stability, probably by binding to the two CBMs present in the enzyme [186]. The molecular characterization of extracellular multidomain endoβ-1,4-xylanase (Xyn10B) from Caldicellulosiruptor lactoaceticus revealed the presence of one GH10 catalytic domain (CD), three N-terminal family-22 CBMs, two S-layer homology (SLH) modules in the C-terminal and two family-9 CBMs. Although the functions of these domains and modules being different, they had synergistic effect on thermostability, substrate binding, and hydrolysis of xylan component of lignocellulose [187]. The researches based on construction of thermophilic xylanases by mutagenesis have also gained wide attention. A thermostable xylanase was designed by site directed mutagenesis of N-terminal region of an endoxylanase (GH11) produced by a mesophile Talaromyces cellulolyticuss and the genetically engineered enzyme exhibited .20
C higher melting temperature without loss of specific activity when compared with wild type [188]. Similarly Han et al. (2017) have reported favorable mutations at the C-terminal residues of a GH11 family xylanases from ruminal fungus Neocallimastix patriciarum. The mutant XynMUT showed 14% more retention of residual activity as compared with the wild type, after 1 h incubation at 80
C and consequently, it retained about 50% of maximum velocity after 1 h incubation at 95
C and also had improved kinetic parameters. Molecular dynamic studies revealed that the enhanced activity and thermostability of Xyn-MUT could be attributed to the opening of the active site cleft for efficient substrate binding and enhanced hydrogen bond pairing, respectively [189]. A comparative study on thermophilic (TfxA) and mesophilic (SoxB) xylanases showed the importance of two N-terminus regions in the thermostability of TfxA. Introduction of seven amino acids from the N-terminal of TfxA into SoxB resulted in outperformance of SoxB over thermophilic TfxA [190]. Recent studies have also attributed thermophilic xylanse along with other associated enzymes in consolidated bioprocessing (CBP), which can be employed for achieving hydrolytic enzymes production, cellulose degradation and microbial fermentation in single step. In a study, CBP was implicated for production of butanol from xylan via the butanol-ethanol pathway using a newly isolated Thermoanaerobacterium sp. M5 [191].

16.4.5 Lipases Lipase (triacylglycerol acyl hydrolases; EC 3.1.1.3) catalyzes the hydrolysis of triacylglycerides to glycerol, esters and fatty acids at the oil-water interface, as well as the reverse reaction of synthesis of ester from fatty acid and glycerol. They are ubiquitously distributed in animals, plants and prokaryotes [192,193]. Lipases from thermophiles have received considerable attention for various industrial applications, including organic synthesis, hydrolysis of fats and oils, modification of fats, wood pulp processing, biofuel production and management of sewage. Thermostable lipases requiring higher operation temperature allow higher reactivity, higher product yield due to enhanced substrate solubility, lower viscosity and fewer contamination problems [193]. Anoxybacillus kamchatkensis and A. flavithermus isolated from the high temperature factory effluent produced lipases with application in treatment of waste at oil mills [194]. Similarly, Geobacillus thermoleovorans produced lipase with optimum activity at 62
C and the enzyme showed the ability to degrade animal carcass-associated fats in tannery wastes [195]. Thermolipase from Rhizomucor sp. was used for degradation of fatty membrane of tea leaves for better release of catechins for improvement of tea quality [196]. T. thermophilus and B. licheniformis showed the capability of producing alkaline lipases for commercial detergent production [197]. Lipase encoding genes from several thermophiles, like Geobacillus sp. JM6 [198], Geobacillus sp. EPT9, Bacillus sp. HT19 [199], Actinomadura sp. S14 [200], Bacillus thermocatenulatus [201] have been cloned and expressed in mesophilic strains. Geobacillus sp. TW1 isolated from hot spring of China, produced a highly thermostable lipase with significant activity up to 90
C. The enzyme expressed in E. coli had properties similar to the native ones [202]. In another study, two novel lipase encoding genes from extremely thermophilic and strictly anaerobic bacteria, Thermoanaerobacter thermohydrosulfuricus and Caldanaerobacter subterraneus subsp. Tengcongensis were expressed

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in E. coli. The enzymes showed activity in the temperature range of 4090
C and their half-life at 75
C was about 48 h [34]. A novel lipase gene lip256 was cloned from Bacillus sp. HT19 isolated from hot spring. The amino acid sequence of the lip256 translation product showed ,32% homology with other esterases and contained a unique motif (GTSAG) that differed from other clusters in the lipase superfamily. The purified lipase (Topt 70
C) showed high tolerance to organic solvents, like glycerol, butyl-alcohol, pyridine, urea, and acetonitrile [199]. Disulfide bridges are one of the most common post translational modifications in protein structures and have been identified to improve lipase stability [203]. The thermal stability of lipase B from Candida antartica (CalB-WT) was improved by introduction of a new disulfide bond. Among the five pairs of amino acids selected for mutation to Cys, one pair CalB A162C-K308C achieved significantly greater thermostability while maintaining the catalytic efficiency. Moreover, the half-life of the mutant CalB A162C-K308C was 4.5 times higher than that of CalB-WT [204].

16.5

Conclusion

Thermophilic bacteria and archaea have the capability to colonize environments that were previously considered inhabitable for survival. Recent development of metagenomic analysis techniques has provided a greater understanding of the ecological diversity of several high temperature environments. However, the true diversity of thermophiles has yet to be explored thoroughly. Thermophiles have been the attractive and challenging system with interests in unraveling the molecular bases of thermophily, which involves thermostabilization of biological structures and different classes of macromolecules including genome and proteome. In recent years the availability of genome sequence data for many thermophilic and hyperthermophilic organisms have provided greater insight into sequence and structural information for thermophilic proteins. During past several thermophilic hydrolases have been purified and characterized. The cloning of hydrolase encoding genes from thermophiles and their overexpression in mesophilic system has allowed better utilization of biological resources, besides providing insight to our understanding of molecular basis of thermophilic adaptation. Moreover, the structural information of thermophilic proteins have been utilized in protein engineering for designing superior biocatalysts with novel properties. With the flourishing industrial growth, the requirement of enzymes with thermophilic property has increased due to their higher stability and activity at higher temperature as well as compatibility to industrial processes. Among the thermophilic hydrolases, biopolymer hydrolyzing enzymes such as cellulases, xylanases, amylases, proteases, and lipases have potential applications in chemical, food, paper, pulp, waste-treatment and pharmaceutical industries. The future research in the field of thermophile and thermophilic hydrolases is fascinating and unlimiting.

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Chapter 17

Effects of single nucleotide mutations in the genome of multi-drug resistant biofilm producing Pseudomonas aeruginosa Sanjay Gunabalan, Chew Jactty and Babu Ramanathan Department of Biological Sciences, School of Science and Technology, Sunway University, Kuala Lumpur, Malaysia

17.1

Introduction

Multidrug resistant (MDR) bacteria are constantly evolving in clinical settings and thus reducing the efficacy of even major antibiotics used for treatment. Pseudomonas is one such multi drug resistant saprophytic bacteria and the genus includes approximately 140 species in which 25 are related to human infections. The major Pseudomonads associated with opportunistic infections are P. aeruginosa, P. fluorescens, P. putida, P. cepacia, P. stutzeri, P. maltophilia, and P. putrefaciens. Among all, P. aeruginosa plays a major role in nosocomial infections acquired in hospitals and ranking it as the third recurrent pathogen causing the disease [1]. Patients with cystic fibrosis (CF) have a critical load of P. aeruginosa colonization within their lungs but however for P. cepacian, can spread to other regions and trigger complications such as endocarditis, necrotizing vasculitis, pneumonia, wound infections, and urinary tract infections. P. aeruginosa is a gram negative, rod shaped and aerobic bacteria which has flagellum and usually inhabiting in the soil. Aquatic conditions may also aid as reservoirs for the pathogen and these conditions may further help in imparting drug resistance to antibiotics contained within the antibiotic resistant genes [2]. Various antibiotics that have different mode of actions to penetrate P. aeruginosa cells have been reported. Antibiotics such as aminoglycosides (e.g., Gentamicin) attaches to the ribosome’s 30 S subunit and prevent synthesis of protein. The quinolones (e.g., Ciprofloxacin) targets the DNA gyrase and attaches to the A subunit that organizes the chromosomal structure within cells. Antibiotics such as β-lactams (e.g., Imipenem and Meropenem) targets the outer cytoplasmic membrane and interferes with the peptidoglycan-assembling transpeptidases. Antibiotics such as Coloymisin and Colistin interrupts the function of barrier by attaching to the phospholipids in the cytoplasmic membrane [3]. A number of mechanisms of resistance have been extensively studied in P. aeruginosa (Table 17.1) such as, 1) Outer membrane as barrier, 2) efflux systems, 3) antibiotic inactivation and modification, 4) target change and biofilm formation. In patients with cystic fibrosis, P. aeruginosa may form an alginate which surrounds and holds the cells in aggregates, leading to biofilm formation. Besides that, they also attach to antibiotics like aminoglycosides due to their cationic properties which will eventually prevent diffusion across the membrane [8]. The outer membrane also serves as a barrier preventing large molecules as well as small hydrophilic molecules from entering into the cell. Hence, antibiotics like β -lactams and quinolones possessing small hydrophilic molecules has to enter via porin channel. However, with the absence of porin OprD protein which is known for uptake of positive charged amino acids, is shown to increase rate of resistance and minimum inhibitory concentration [9] thus making it difficult for the antibiotics to effectively control the bacteria. On the other hand, for polymixins and aminoglycosides, bind to the lipopolysaccharide (LPS), and then resist their mode of action. OprH a protein on the outer membrane is overexpressed which in turn prevents binding of antibiotics on LPS [10].

Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00017-4 © 2020 Elsevier Inc. All rights reserved.

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TABLE 17.1 Antibiotic resistant mechanisms and known genes involved in the resistance of P. aeruginosa. Resistant mechanism

Resistant gene ID

Gene name

Protein

Reference

Involved in Kreb’s cycle and overexpressed during stationary growth phase

PNSNP32

gltA gene

Type II citrate synthase

[4]

CDS for tadZ within WCI encoding adherence pili

PNSNP27

tadZ

TadZ

[4]

Loss of function mutation

T247P

mutS

MutS

[5]

Mutator phenotype

H469R

mutL

MutL

[5]

Resistance nodulation cell division (RND)

PA0424

mexR

MDR operon repressor

[6]

Maybe involved in drug resistance

PA2020 stop codon

mexZ

ArmZ

[7]

TABLE 17.2 Key mutations reported in antibiotic resistant genes of P. aeruginosa multidrug resistant isolates. Gene ID

Gene/protein affected

Nucleotide change/SNP position

Amino acid change

Reference

PA1179

PhoP

A41G

H14R

[7]

PA1580

Type II citrate synthase

T1719347G

D to A

[4]

PA2018

RND efflux transporter

T2208200G

N1036T

[18]

PA3596

Putative methylated DNA protein

C4032021A

C to F

[4]

PA2019

RND efflux membrane fusion protein

A2211441G

W358R

[18]

PA4303

TadZ

G4827417T

P to Q

[4]

PA2020

MexZ

G2213177A

W167a

[18]

PA4218

ArmP

G4722060A

L267F

[18]

PA5471

ArmZ

T6159991C

I237V

[18]

Unknown

mexB

468009

a

[19]

a

Mutation resulted in a stop codon.

P. aeruginosa has four major efflux pump systems that are essential for resistance [11] such as, mexAB-oprM, mexXY-oprM, mexCD-oprJ and mexEF-oprN. They all work differently. e.g., β-lactams and quinolones are excluded due to the role of mexAB-oprM while mexXY-oprM excludes aminoglycosides and mexEF-oprN targets quinolones and carbapenems. The expression of mexAB and mexXY, leads to intrinsic resistance to many antibiotics [11]. Antibiotics may also not be very effective due to overexpression of ampC, those that affects cephalosporins and extended spectrum plasmid mediated enzymes(ESBLs) against penicillin [12]. Resistance due to nucleotide mutations is common and extensively reported, for example, the mutation in gene gyrA that codes for the target enzyme A subunit indirectly impacts quinolones [13]. As such, antibiotics and physical biochemical approaches has been inefficient for the treatment of P. aeruginosa infections. The pathogen may also be able to persist the infection due to chromosomal derepression of β lactamases and prolonged antibiotic accumulation over time in biofilm and hence unable to be effective [14]. There are different strategies for identifying these antibiotic resistant genes and factors such as mutations that may have a crucial role in antibiotic resistance. With genomics and the next generation genome sequencing tools, we have access to the core and accessory genomes of various pathogens. In P. aeuruginosa, the accessory gene pool has been proven vital for its persistence in harsh environments. Besides, it is known to promote virulence [15] as well as code genes that contribute to antibiotic resistance [16]. Comparing experimental evolution studies and whole genome sequencing (WGS) can provide information on antibiotic resistance [17]. WGS provides a confirmed catalog of genetic polymorphisms such as SNP variants (Table 17.2) as well as helps to connect epidemiology data to genome evolution, structural biology of pathogens and content of genes in the genome of organisms. Eventually, this strategy supports to

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gain information of biological markers that are responsible for antibiotic resistance and different virulence factors that are governed within the organism [20].

17.2

β-Lactam resistance

β-Lactam resistance is due to inactivation of β-lactamases. Penicillinases are one of the most common antibiotics to class molecular A-serine β-lactamases such as PSE, TEM and CARB families, of which PSE is most predominant [21]. Extended spectrum β-lactamases does not grant resistance to carbapenem, but the presence of different classes of carbapenem hydrolyzing enzymes do. P. aeruginosa contains blaAmpC that produces different arrays of class C β-lactamase [22]. β-lactamase gives resistance to various molecules like aminopenicillins, first and second generation cephalosporins [23]. However, if mutation leads to overproduction, AmpC can play a huge role in resistance. On the other hand, dacB that plays a role in ampC expression by encoding the production of penicillin binding protein 4(PBP4) [24]. Therefore, the deactivation of PBP4 results in high levels of AmpC and β-lactam resistance. Also, the gene essential in regulation of AmpC is ampD and the deactivation of ampD will potentially repress the ampC expression [25]. In experiments conducted to analyze the phenotypic and genotypic changes of P. aureginosa in Cystic Fibrosis (CF) and bronchiectasis patients, the strain PAHM4 showed the hypermutation due to mutS mutation [26]. PAHM4 developed resistance to penicillin, cephalosporin and meropenem due to mutations in genes like dacB, ampD and penicillin binding protein. PAHM4 is known for access production of alginate due to a mutation in mucA gene generating a stop codon hence producing mutant allele known as mucA22 which is mucoid by nature contributing to large amount of alginate production [27]. Non-synonymous SNPs were identified in genes that are significant in β-lactam resistance such as transporter (PA4218), transcriptional regulators (PA1184, PA0032, PA5293 and PA2383) and protein (PA3693) and essential in reducing antibiotic intake and permeability of the cell wall [28].

17.3

Fluoroquinolone resistance

A major point mutation in GyrA that results in an amino acid substitution (D87Y) in the quinolone resistant gene was known to cause fluoroquinolone resistance in many CF patients [29]. Resistance to fluoroquinolones in P. aeruginosa is found only in chromosomal gene mutation which target DNA gyrase (gyrA and gyrB) for fluoroquinolones as well as topoisomerase IV(parC and parE) and over expression of efflux pumps [11]. They are usually found in quinolone resistant deciding region (QRDR) in gyrACF [30 32] reporting strains which have mutation in that region to have peak resistant levels in gyrA and topoisomerase IV gene parC [33,34].

17.4

Aminoglycoside resistance

Aminoglycoside resistance usually includes enzymatic inactivation via modification of chemicals and they contain enzymes that phosphorylate, acetylate and adenylate the antibiotic. Besides resistance may be induced due to 16S rRNA being methylated via methylase encoding gene rmtA. Five ribosomal methyltransferase enzymes were detected (RmtA, RmtB, RmtC, RmtD, and ArmA) and their respective genes were found to associate with mobile genetic elements [35 39]. Resistance to aminoglycosides were reported by inducing mutation in the AmgRS two component system(TCS) in Pseudomonas isolates resistant to aminoglycosides [40]. The researchers identified missense mutation in sensor areas, AmgS of the AmgRS TCS. Previous studies have also revealed eight-day exposure of wild type P. aeruginosa to peroxide increased recovery of amikacin resistant mutants which are known to be pan-aminoglycoside resistant. The AmGRS operon produces membrane stress reactive TCS, related to intrinsic resistance to aminoglycosides. Lab extract from genome sequencing showed pan-aminoglycoside resistance in strain K2979 with various mutations which includes amgS being substituted resulting in 182C change in AmgS sensor kinase. Inducing this mutation and then reverting it to previous state showed that amgS mutation is responsible for resistance in K2979 strain. Of 37 mutations, a transition at nucleotide 544 from C to T causes Arg to Cyst substitution in amino acid R182C, hence produces sensor component for AmGRS TCS playing a role in intrinsic resistance [40].

17.5 Target efflux pumps (before jumping to each system give 2-3 lines details about this) Gram-negative bacteria remain clinically important and gains much focus both in clinical and community settings. Efflux pumps play a crucial role in the multidrug antibiotic resistance in many gram-negative bacteria including P. aeruginosa. Four multidrug efflux pump systems have been well characterized in P. aeruginosa such as the MexA-MexB-

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OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM. These efflux pumps possess different substrate specificities, and their activity can be controlled by factors such as., low pH, inoculum concentration and bacterial growth in stationary-phase Studies have showed that the prevalence of efflux pump overproduction in clinical strains of P. aeruginosa may range from 14% to 75% [41]. Having that in mind, the current treatment regime for bacteria that overproduce efflux pumps is to use antibiotic combinations that targets different pump systems may be a reasonable strategy.

17.5.1 MexAB-OprM Efflux pump contributes to resistance to quinolones and B-lactams. Regulatory loci play a crucial role in MexAB-OprM being expressed, such as mexR, nalD and nalC which all regulate the operon in a negative manner [42]. Regulatory gene mutation triggers derepression of mexO hence upregulating MexAB-OprM expression [43,44]. Besides, high expression of MexAB-OprM is also resulted by protein PA3719 interacting with MexR and reducing the repression of MexR and indirectly leading to high expression of MexAB-OprM.

17.5.2 MexXY-OprM Aminoglycoside, fluoroquinolone and cephalosporin antibiotic resistance is majorly seen because of high expression of MexXY-OprM. Previous studies have shown MexXY-OprM provides resistance to P. aeruginosa to specific substance capable of mexXY operon induction leading to expression, due to defect in protein formation [45]. Activation of mexXY is dependent on expression of unknown functioning gene PA5471 which paired with another nonessential gene PA5470 producing a peptide releasing factor [46]. Presence of antibiotics prevents the translation hence disabling the formation of secondary mRNA structures and thus the PA5471 gene is expressed and mexXY is further activated [47]. Both CF and non-CF strains with overexpression of MexXY-OprM showed the containment of mutation which inactivates the mexZ gene [48].

17.5.3 MexCD-OprJ Mutation in nfxB gene triggers MexCD-OprJ to be overproduced, thus enhances its resistance to fluoroquinolones, however, they do not contribute to intrinsic resistance [49]. Overexpression of MexCD-OprJ from mutants showed four to sixteen-fold increased resistance to fluoroquinolones, macrolides and cephalosporin in clinical isolates compared to wild type strain. Similarly, two- to eight-folds of increase to tetracyclin and chloramphenicol resistance was observed. However, for β-lactams and aminoglycosides, resistance levels reduces and increasing susceptibility from two to thirty two folds suggesting their rare presence in clinical surroundings [50 53].

17.5.4 MexEF-OprN Overexpression of MexEF-OprN and mutated mexS gene encoding oxidoreductase was reported in clinical isolates, however there is no evidence of clinical isolates carrying mexS gene mutation in MexEF-OprN overexpressed strains. Overproduction of MexEF-OprN and MexXY(OprA) in PA7 strain inhibits the growth of P. aeruginosa as was observed with non-uniform phenotype and growth on agar plates. Similarly, increased susceptibility to aminoglycosides was due to MexEF-OprN-overproducing nfxC mutants disrupt the efflux system. Besides, valine at position 155 of MexSPA7 may be responsible for enhanced action of MexEF-OprN. Additionally, MexEF-OprN was shown to overexpress in nfxC multidrug resistant strains that have resistance to fluoroquinolones, chloramphenicol, trimethoprim and imipenem [54,55]. MexEF-OprN is positively regulated by mexT gene product which is known to reduce the expression of OprD expression in strains of nfxC. Mutation in gene PA2491 in the mexT gene stream, displayed increased expression of mexEF-oprN hence reducing synthesis of OprD similar to nfxC mutants. PA2491 was reported to be a oxidoreductase/dehydrogenase homologue that is regulated by MexT positively [55] hence the absence in activity for the PA2491 gene triggers the expression of mexEF-oprN and thus playing an important role in antibiotic detoxification.

17.6

Antibiotic resistance and bacterial phenotype in biofilm formation

Comparison of Pseudomonas antibiotic resistant bacterial colonies to wild type colonies showed rough colony phenotype known as rough small colony variant (RSCV) [56]. The clinical isolate showed higher resistance to kanamycin

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than wild type and also resistant to amikacin, carbenicillin, gentamicin, tobramycin and tetracyclin. Eventhough their colonies were smaller compared to wild type it was not an indication of slow growth as both generation time were similar in liquid medium. RSCV also showed a higher level of attachment to glass and the surface of poly-vinyl chloride PVC plates due to interaction between hydrophobic molecules in bacteria. RSCV was found to be agglutinated at lower salt concentration meaning higher levels of surface hydrophobicity. PCR analysis on fourteen strains using pvrR gene specific probe showed prominence in pvrR. pvrR overexpression and showed a drop in resistance on kanamycin as well as drop in level of attachment to PVC. A deletion in pvrR showed increased resistance to kanamycin which shows that pvrR is a regulator that can either increase resistance or susceptibility besides controlling formation of biofilm. Strains with P2 phenotype has high collagenase activity, ability to swarm, high synthesis of pyocyanin in liquid content and greater ability to destroy tissues in comparison to strains which has P1 phenotype. P2 phenotypic strains contains mexT and mexZ mutations having a part in functioning of MexEF-OprN [56] where MexT is known to be a mutational “hot spot” for P. aeruginosa [57]. Mucoidy is an essential phenotype observed during chronic colonization [58] in many CF patients. In comparison with RSCV, the small colony variants (SCV) also show similar characteristics when compared to wild type P. aeruginosa strain. Collectively, phenotypic characters were highly expressed such as increased resistance to antibiotics, increased formation of biofilm, returning to wild-type morphotypes, reduced propagation, as well as slow automatic growth in aggregation [59,60]. Cyclic-di-GMP (c-di-GMP) known as the second intracellular messenger [61] is discovered to be the switch of SCV phenotypes such as formation of biofilm, exopolysaccharide synthesis and decreased motility [62 67]. The phenotypic switch in which antibiotic resistant and auto-aggregative of RSCV from strain PA14 to wild type susceptible strains have been discovered to be phenotypic variant regulator (PvrR) harboring EAL domain phosphodiesterase (PDE) is involved in hydrolysis of c-di -GMP. Besides, elevated levels of c-di-GMP is shown to cause two EPS encoding loci of P. aeruginosa strains to auto aggregate and hyperadhere [68 70]. However, the down regulation of a cytochrome c ccoN (PA4133) showed correlation in forming SCV hence the enhanced resistance to aminoglycosides [71]. Cells with persistent connective properties were known to be involved in biofilm connected infections and further contributes to failure in treatment [72]. Additionally, single point mutations have been attributed towards resistance such as SNP mutations in the mexT triggers the P2 phenotype. Single nucleotide mutation from C to A at position 2807731 in the NC002516 genome found in the mexT gene created in frame stop codon thus resulting in a nonfunctional truncated protein [73]. The inactivation of pilY1 gene had led to the development of wild type strains of PA14 and PAO1 to lose their twitching and motility as well as promoting stationary growth phase [74]. Besides, insertion of transposon between PA0716 and PA0717 was showed to increase resistance towards carbenicillin and down regulation of rhlA gene triggers smaller amount of rhamnolipid production and hence decreasing swarming motility and missing channels in PAO-SCV biofilms [75,76]. On the other hand, down regulation of gene cytochrome c ccoN gene (PA4133) played a role in SCV formation hence causing aminoglycoside resistance [71]. Phage genes that are down regulated in PAO-SCV when compared to wild type contributed to lower production of biofilms and hence antibiotic resistance [77].

17.7

Conclusion

P. aeruginosa is a multidrug resistant bacteria and many of the clinical isolates develop resistance to several leading antibiotics in clinical practices. Single nucleotide mutations are known to contribute for antibiotic resistance and in this chapter, we have explored different antibiotic resistant mechanisms and the role of single nucleotide polymorphisms in antibiotic resistance within the multi-drug resistant biofilm forming Pseudomonas clinical isolates. We focused in genes that play a crucial role in antibiotic resistance mechanisms such as genes affecting resistance to β-lactams, fluoroquinolones, aminoglycosides and efflux pumps. The information reported in this chapter may provide insights in the role of SNPs in antibiotic resistance and further the knowledge for effective strategies to control the multi-drug resistant P. aeruginosa.

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[62] Malone JG, Jaeger T, Spangler C, Ritz D, Spang A, Arrieumerlou C, et al. YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa. PLoS Pathog 2010;6(3):e1000804. [63] D’Argenio DA, Calfee MW, Rainey PB, Pesci EC. Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants. J Bacteriol 2002;184(23):6481 9. [64] Hickman JW, Tifrea DF, Harwood CS. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci U S A 2005;102(40):14422 7. [65] Kuchma SL, Brothers KM, Merritt JH, Liberati NT, Ausubel FM, O’Toole GA. BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14. J Bacteriol 2007;189(22):8165 78. [66] Kulasekara BR, Kulasekara HD, Wolfgang MC, Stevens L, Frank DW, Lory S. Acquisition and Evolution of the exoU Locus in Pseudomonas aeruginosa. J Bacteriol 2006;188(11):4037 50. [67] Meissner A, Wild V, Simm R, Rohde M, Erck C, Bredenbruch F, et al. Pseudomonas aeruginosa cupA-encoded fimbriae expression is regulated by a GGDEF and EAL domain-dependent modulation of the intracellular level of cyclic diguanylate. Env Microbiol 2007;9(10):2475 85. [68] Friedman L, Kolter R. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J Bacteriol 2004;186(14):4457 65. [69] Jackson KD, Starkey M, Kremer S, Parsek MR, Wozniak DJ. Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation. J Bacteriol 2004;186(14):4466 75. [70] Matsukawa M, Greenberg EP. Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development. J Bacteriol 2004;186(14):4449 56. [71] Schurek KN, Marr AK, Taylor PK, Wiegand I, Semenec L, Khaira BK, et al. Novel genetic determinants of low-level aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2008;52(12):4213 19. [72] Brooun A, Liu S, Lewis K. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2000;44(3):640 6. [73] Olivas AD, Shogan BD, Valuckaite V, Zaborin A, Belogortseva N, Musch M, et al. Intestinal tissues induce an SNP mutation in Pseudomonas aeruginosa that enhances its virulence: possible role in anastomotic leak. PLoS One 2012;7(8):e44326. [74] Bohn Y-S, Brandes G, Rakhimova E, Horatzek S, Salunkhe P, Munder A, et al. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection. Mol Microbiol 2008;71(3):730 47. [75] Davey ME, Caiazza NC, O’Toole GA. Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J Bacteriol 2003;185(3):1027 36. [76] Caiazza NC, Shanks RM, O’Toole GA. Rhamnolipids modulate swarming motility patterns of Pseudomonas aeruginosa. J Bacteriol 2005;187 (21):7351 61. [77] Whiteley M, Bangera MG, Bumgarner RE, Parsek MR, Teitzel GM, Lory S, et al. Gene expression in Pseudomonas aeruginosa biofilms. Nature 2001;413(6858):860 4.

Chapter 18

Understanding the structural basis of adaptation in enzymes from psychrophiles Mahejibin Khan CSIR-Central Food Technological Research Institute-Resource Centre, Lucknow, India

18.1

Introduction

Temperature is one of the major factors for survival of any organism on the earth. Approximately 70% area of Earth is covered by ocean which maintain an average temperature of 2 10  C. The vast landmasses are sporadically cold and some regions are permanently cold, or even frozen [1,2]. Other colder region on earth include, Antarctic continent, the Arctic ice floes and the deep-sea waters of the oceans surrounding them that cover nearly one third of the earth’s surface, the mountains, glacier regions and their caves. However, cold environments are no barriers to microbial growth, as they survive and flourish very well in low temperature and even in ice. A number of microorganisms belonging to different groups have been isolated from low temperature (4 8  C) regions. Such microorganisms are generally classified as psychrotolerant, because they can grow and survive in the cold environment, but grow better at 25 35  C. Microorganisms that grow only at 15  C or below, and are usually present in permanently cold environments are defined by Morita as True psychrophiles [3]. These microorganism may exist in super-cooled cloud droplets in high altitude, in permafrost conditions or even in the most amazing locations such as in the Antarctic subglacial below 3500 m of ice having temperatures much lower than 0  C [4,5]. The size of majority of the microbes taken from the glacier is less than 1 μm, which is smaller than the size (1 10 μ) of commonly occurring bacteria. Some of these bacteria are even so small that they passed through filters with 0.2-μm pores [6]. Below freezing, some of the organisms can survive but metabolically they are not much active. Perhaps more interesting are the organisms that can live, grow and metabolize at low temperatures, between 1 and 15  C. Even though low temperature causes the denaturation of biomolecules and ice crystals formation within the cell leads to the structural damage. Psychrophiles manages these challenges and thrive in the cold habitat because of the unique lipid constituents of their cell membrane, ability to transport the substrates across the membrane, rapid synthesis of cryoprotectant such as antifreeze, cold shock and cold tolerant proteins [7 10] and by producing cold adapted enzymes. It has been found that the number of unsaturated fatty acids is more in the cell membranes, allowing them to remain fluid and transport solutes. When there is sudden downshift in temperature, a number of modifications in cellular physiology have been observed such as, (i) change in membrane fluidity, (ii) secondary structures of nucleic acids gets stabilized that results in reduced efficiency of transcription and mRNA translation, (iii) change in folding efficiency of some proteins, and (iv) hindered ribosome function. Berry and Foegeding and many other authors reviewed microbial response to cold shock and their adaptation in detail [5,11 13].

18.2

Cold adapted enzymes

Enzymes are the elementary component of cell metabolism and carried out all biochemical reactions within the cell. At low temperature, reaction rate of biochemical processes catalyzed by enzymes reduced exponentially [12]. It has been studied that a decrease of 10  C in reaction temperature, slow down the reaction rate up to three times. Therefore, a shift in mesophillic enzyme from 37 to 10  C results in 20 to 60 times reduction in their catalytic efficiency [13]. Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00018-6 © 2020 Elsevier Inc. All rights reserved.

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But surprising, psychrophillic enzymes active at a temperatures below 210/ 2 15  C have also been recognized [14]. Another interesting feature of psychrophilic bacteria growing at around 0 5  C is showing same generation time as those of microorganisms growing at 37  C. Metabolically active microorganism have been reported even at 220  C in the sea-ice [15,16]. Low temperature loosens the hydrophobic interactions that play a vital role in protein folding and stability [17] and cause unfolding of protein. At a temperature below 215  C, complete denaturation of proteins occurs. Thermodynamically, whole process is favored at low temperature [18,19]. Hence, it is obvious that psychrophilic enzymes have evolved some mechanisms to deal with the reduced biochemical reaction rates intrinsic to low temperatures. There are group of enzymes that are not only stable and active at low temperature, but also function effectively under more than one extreme condition such as, acidic or basic pH, high or low temperature, nonaqueous medium or in low water concentration. These enzymes are referred as polyextremohilic enzymes. General characteristics of psychrophilic enzymes as defined by different researchers [20,21] are (a) they are optimally active at the temperature below 20  C which is lower than their mesophilic and thermophilic counterpart; (b) they are weakly stable and generally get denatured at 37  C; (c) they maintain their high catalytic activity at low temperature by modifying their Kcat and Km values.

18.3

Structure-function relationship of cold adapted enzymes

Enzymes are proteinaceous in nature and three-dimensional structure of enzymes is highly prone to fluctuation of physical factors such temperature, pressure, salinity and therefore, marginally stable. However, all the atoms in protein structure are folded to form a single static rigid structure and functions as a whole but every atom is constantly in motion. Other than these small movements, a large conformational change in protein structure takes place which is usually essential for enzymes to be active. To offset the enhanced structural rigidity at lower temperatures, psychrophillic enzyme have low thermal stability in respect to their mesophilic orthologues [22,23]. Protein stability, flexibility, surface residues, solvent exposed residues etc. are some of the crucial factors that play a crucial role in maintaining the enzyme integrity and activity at low temperature. Flexibility/rigidity in term of proteins is the property which is used to define internal degree of freedom of atoms with in a molecule. It is the measure of protein’s ability to modify its conformation in response to the fluctuating environmental conditions [24,25]. Various non-covalent interaction such as hydrogen bonds, hydrophobic interactions, salt bridges and weak van der Waals forces contribute to provide the stability to the native structure of an enzyme [26,27]. Protein flexibility governs the functional interactions between enzyme and substrate and plays an important role in enzyme catalysis. Thermal fluctuations induce many changes in the structural organization of proteins which include conformational changes and rotations, movement of side chains within domain and also changes in quaternary structural. To maintain catalytic activity at low temperature equivalent to that of a protein which functions at warmer temperature, the psychrophilic proteins are generally more flexible and easily embrace structural modification required to function efficiently. This process involves reduction in enthalpy-driven interactions which results in decrease in protein rigidity and enhanced flexibility [28]. Therefore, a balance between enzyme stability-flexibility is the main property on which a protein relies for its activity under stressed conditions. A number of in vitro and in vivo studies carried out using various in silico and biophysical techniques have provided insight into the structural-functional relationship of cold-adapted enzymes and proved that these enzymes are generally more plastic or flexible [29]. These features of the psychrophilic enzymes help them to interact in a better way with substrates and reasoned for their lower activation energy (Ea) requirements, lower thermostability, and higher catalytic rate (kcat), compared with mesophilic and thermophilic homolougs [30]. Various studies carried out on extremophilic enzymes revealed that psychrophilic enzymes originated from different sources or in different conditions adopt different mechanisms, therefore, no general theory for cold adaptation of enzyme has been given [31]. But certain modification in cold adapted enzymes have been proposed which suggest that cold adapted enzymes are often more flexible or folded up less tightly and also bind to their substrates more efficiently when compared with their mesophilic homologs. Structural adaptations that allow them to work more efficiently at low temperature are a. b. c. d.

Smaller and less hydrophobic amino acids as compared to mesophiles and thermophiles homolougs Reduced number of salt bridges More clustering in glycine residues to provide local mobility Fewer proline residues in loops improve chain flexibility between secondary structures

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e. Low number of arginine residues results in lower number of ion pairs, salt bridges, hydrogen bonds and weak aromatic interactions as compared to their mesophilic enzymes. Therefore, poor binding of stabilizing cofactors, reduce the packing within the enzyme and favor unfolding of proteins f. Several solvent-exposed residues and ion pairs are replaced by non-polar groups on protein surface, and surrounding medium which increases the overall negative charges and favor interactions with the solvent. g. Ion binding. Co-factors or metal ion binding stabilize the proteins structure more than any other weak interaction such as H bond or even disulfide bond. Zn ions or Ca ions have strong binding affinity and can bridge many secondary structures. Very low binding affinity has been found in all the psychrophilic enzymes with any of bivalent ions. It could be due to the substitution of more polar amino acid with the less or non-polar amino acid in the conserved region of protein. (Asn - threonine in psychrophiles) and clustering of Gly around the ligand [32]. All these factors contribute in the attainment of flexibility of protein at near zero degrees and help them to optimize the kcat/Km ratio, which can be achieved either by increasing kcat and reducing Km or by modifying both Kcat and Km [33]. Therefore, most of the psychrophilic enzymes having higher catalytic efficiency as compare to their mesophilic or thermophilic homolougs display better local flexibility [13,34]. It is also a well established fact that reactions catalyzed by psychrophilic enzymes at low temperature have lower enthalpy and more negative entropy of activation than their mesophilic homologs [35,36]. Hence, make the reaction rate less dependent on temperature. However, overall activation free energies are mostly similar as at warmer temperature [37]. Molecular mechanism of cold adaptation in malate dehydrogenase enzyme from Aquaspillium arcticum, which is a psychrophilic enzyme, was elucidated by Kim and their co-workers [38] in 1999. Crystal structure analysis of Aa-MDH identified a Glycine residue (Gly227) in Aa-MDH which was not conserved in MDH family. Comparative study of the crystal structures of Aa-MDH and MDH from Thermus flavus (Tf MDH) revealed that Gly227 was replaced by alanine in Tf MDH. As glycine residues do not form side chains and therefore provide more flexibility and high catalytic efficiency to Aa MDH at low temperatures. Furthermore, Aa-MDH was also characterized by higher positive potential in the surface around the substrate binding site. To understand the cold adaptive response of a DNA repairing enzyme Uracil DNA glycosylase, Olufsen et al. [39] conducted the molecular simulation studies and compared UDG from Atlantic cod (cUDG) and its homologs human UDG which is a warm temperature enzyme. The finding resulted in the identification of an important loop Leu272 loop that is involved in DNA recognition. Comparative studies revealed that in cUDG structure, Leu272 loop is the most flexible part than that of human counterpart. Experimental data of kcat/Km values further confirms the correlation of structural flexibility well with the cold adaptation. Comparative X-ray structure analysis of three cold adapted enzymes, trypsins, citrate synthase, and AHA, with their hyperthermophile homolog revealed the absence of Ille clusters at the subunit of cold adapted enzymes. The branched structure of Ille supports the tight packing inside the core and therefore, stabilizes a protein under thermophilic conditions [34]. Furthermore, Siddiqui et al. [40] demonstrated the induction of mesophilic-like character in α-amylase isolated from psychrophilic Pseudoalteromonas haloplanktis (AHA), by replacing lysine with homo-arginine. These results supported the significance of lysine in maintaining the cold-adapted properties. In order to understand the structure- function and flexibility relationship of enzymes functional at different temperature regime, Georlette et al. [41] studied the comparative three dimensional structure of thermophillic, mesophillic and pscychrophillic DNA ligase in terms of structural permeability, stability, surface hydrophobicity, and thermal folding/ unfolding. The finding demonstrated a well established link between enzyme stability and flexibility with temperature. Psychrophillic DNA ligase showed higher flexibility and low stability specifically in the active site at low to moderate temperature with high catalytic efficiency. Papaleo et al. [42] studied the molecular basis of cold adaptation in elastase family of enzyme through molecular dynamics simulations. Researchers compared the intramolecular and protein-solvent interaction, secondary structure and molecular flexibility of cold active and mesophilic elastase and found that transition in protein-solvent interactions is not the evolutionary trait. Furthermore, amino acid present around the pocket or active site showed higher flexibility in the psychrophilic enzyme, however, regions distant from functional sites were characterized by high rigidity. In another study, Chiuri et al. [43] compared two carbonic anhydrases Ice-CA and BCAII adapted for different thermal conditions. Detailed structural- functional analysis of Ice-CA confirmed the higher flexibility in the part of enzyme that regulated the folding and unfolding of the catalytic region. A remarkable increase in rigidity was observed in the lower region on enzyme which is supposed to maintain the conformation, when compared with BCAII. In a comparative study of trypsin from Atlantic salmon and its mesophilic counterpart using molecular dynamics (MD) simulations [44], more flexible amino acid residues were reported in close vicinity of the active site parts in Atlantic salmon. It is important to note that, the active site is the region which differs significantly in terms of amino acid composition between psychrophilic and mesophilic proteins. Diana et al. [45], examined high resolution crystal structures of a non-redundant set of 20 psychrophilic

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enzymes with their homologous mesophilic enzyme. Sequence identity between psychrophilic and mesophilic enzyme was 35 76%. Detailed analysis of cavities and void volumes was done to evaluate the amino acid residue present around the cavities. Larger cavity size surrounded with more hydrophilic amino acid groups was evident in psychrophilic enzymes than their mesophilic counterparts. These results indicate that more water molecules are present within the cavities of psychrophilic enzymes which support the conformational dynamics of enzymes and results in higher enzymatic activity under the cold environment. Moreover, a significantly more flexible secondary structure was also reported in psychrophilic proteins than their mesophilic counterparts. In another study Siglioccolo et al. [46] also studied the B’-values of both psychrophilic and mesophilic proteins possessing common secondary structures (α helices, β-sheets, and turns) and observed that β sheets and turns of psychrophilic proteins displayed better flexibility than helices. To elucidate the molecular adaptation and strategy of cold adaptation in β glucosidase EaBglA enzyme isolated from Exiguobacterium antarcticum B7 Zanphorlin et al. [47] carried out in solution X Ray crystallographic and molecular dynamics simulations studies of cold active β glucosidase and compared finding with the thermoactive homologs HoBglA, of the same enzyme. EaBglA was characterized by more number of ala and threonine residues, which forms less side chains and H bonds, and lower number of charged residues such as arginin, less salt bridges and more hydrobhobic residues on solvent exposed regions and lower thermal stability as compared to HoBglA. A more remarkable strategy for cold adaptation observed is the formation of tetrameric configuration. EaBglA tetramers increased the surface flexibility near the catalytic region and are reported not only to stabilize the native structure but also enhance the enzymatic activity to many folds. In the earlier studies, oligomerization of GH1 family enzymes was reported only in hyperthermohilic. Therefore, based on these studies, it can be concluded that GH1 enzymes forms multimers under both extreme high and low temperature depending on selection pressure. But mode of adaptation may be different. Park et al. [48], identified a cold active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas ˚ . When arctica PAMC 21717, and resolved the crystal structure of its catalytic domain (CD) at a resolution of 1.4 A the resolved structure was compared with subtilisin Carlsberg (PDB code: 1YU6), a remarkable difference was observed in substrate pocket size, pocket volume and active site length of the two enzymes. Pro21717-CD S2 and S4 pocket volumes was larger which suggests that Pro21717-CD can accommodate a wide range of substrates. Bigger substrate pocket size and active site length of Pro21717-CD facilitates batter substrate-binding site. Furthermore, a prominent increase in the loop content of Pro21717-CD was also found in secondary structures analysis, compared with subtilisin Carlsberg. In a recent report [49], conformational changes towards the enhanced flexibility in cold adapted β-galactosidase were confirmed through site directed mutagenesis. Wherein six amino acid residues; four buried in active site and two located on surface that were supposed to function for low temperature adaptation of β-galactosidase were replaced with amino acid conserved in mesophilic homolouges. Five out of six substitutions resulted in alteration in internal charge, packing, hydrogen bond formation which ultimately affected the structural flexibility and enzyme activity at low temperature. N251D, I299L, F387L, I476V and V482L mutations resulted in reduced substrate binding and reaction rate at lower temperature. These results suggests that (,1%) residues in protein structure are responsible for shifting the enzyme activity toward cold temperatures. Smalas et al. proposed the role of intra-molecular hydrogen bonds in stabilizing the proteins, and suggested the low occurrences of H bond in cold-adapted proteins [31]. In the earlier studies [50] carried out on crystal structures of salmon and bovine trypsins, it was observed that two β-barrel domains present in between the N-terminal and C-terminal domain helix and closely situated to the catalytic residues, were loosely packed because of the absence of a number of H-bonds in the cold-adapted enzyme. Lower numbers of polar (Ser, Thr, Asn and Gln) residues contributing in hydrogen-forming formation were also reported in digestive enzymes from cold-adapted fish [51]. In another study [52] more than 100 simulations were carried out to compare the transition and reactant states in bovine trypsin BT and psychrophilic anionic salmon trypsin AST enzymes as models. The results did not show any differences in the overall RMSFs values in the reactant and transition state of protein backbone which was calculated ˚ and 0.66 A ˚ for BT and 0.61 A ˚ and 0.65 A ˚ for AST, respectively. However, local differences were observed in 0.65 A conserved amino acid Tyr97 and Asp150 on the protein surface. Tyr97 is present in the Nβ5-Nβ6 loop, and Asp150 is situated in autolysis loop both are conserved in different cold-adapted trypsins. Tyr97 and Asp150 from AST were highly flexible than their corresponding BT residues. Hence, it is obvious that even though both psychrophilic and mesophilic enzymes possess a relatively rigid surfaces but there are modification in the surface regions of the cold-adapted enzyme which makes them softer than the mesophilic protein. Further studies of crystal structures of both BT [53] and ˚ ) revealed the presence of large amount of surface bound water molecules. AST [54] at ultra-high resolution (0.75 1.0 A These water molecules interact with polar amino acid such Asn, Gln, Ser and Thr, available at protein surface.

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Therefore, it is evident from the finding that the point mutation (Asn97 in BT to Tyr97 in AST) of polar residues with less polar or non polar residue in cold adapted enzyme softens the surface by disrupting the H bond. In addition to structural flexibility, electrostatic potential is also an important measure of enzyme activity and adaptation to different environmental conditions. It defines the enzyme interaction with the substrate. A change in electrostatic potential in and around the active site of cold adapted enzymes such as trypsin [55,56], malate dehydrogenase [38], uracyl-DNA glycosylase [54] and citrate synthase [57] etc and their mesophilic and thermophilic counterparts has been reported.

18.4

Conclusion and future directions

Therefore, it can be concluded that psychrophies are an important resource of a variety of cold active enzymes that are being used in various biotechnological and industrial application. Under low temperature conditions, most of the enzyme tries to increase their flexibility through the structural modification, i.e change in amino acid composition in and around the active site. Modification in surface exposed residues and reduction in weak intermolecular interaction as H-bonds has also been used as strategy to cold adaption. To achieve the high reaction rate, psychrophilic enzymes have been evolved to maintain Kcat/Km balance such that their rate of reaction become temperature-independent.

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Crystal structure of a cold- active protease (Pro21717) from the psychrophilic bacte˚ resolution: Structural adaptations to cold and functional analysis of a laundry deterrium, Pseudoalteromonas arctica PAMC 21717, at 1.4 A gent enzyme. PLoS One 2018;13(2):e0191740. Available from: https://doi.org/10.1371/journal.pone.0191740. [49] Laye VJ, Karan R, Kim J-M, Pecher WT, DasSarma P, DasSarma S. Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase. Proc Natl Acad Sci U.S.A. 2017/11/06. National Academy of Sciences. 2017;114 (47):12530 5. Available from: https://www.ncbi.nlm.nih.gov/pubmed/29109294. [50] Smala˚s A, Heimstad E, Hordvik A, Willassen N, Male R. Cold adaption of enzymes: Structural comparison between salmon and bovine trypsins. Proteins: Struct, Funct, Genet 1994;20(2):149 66. [51] Gudmundsdo´ttir E, Spilliaert R, Yang Q, Craik C, Bjarnason J, Gudmundsdo´ttir A. Isolation and characterization of two cDNAs from atlantic cod encoding two distinct psychrophilic elastases. Comp Biochem Physiol Part B: Biochem Mol Biol 1996;113(4):795 801.

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˚ qvist J, Brandsdal BO. Enzyme surface rigidity tunes the temperature dependence of catalytic rates. Proc Natl Acad Sci U S A [52] Isaksen GV, A [Internet] 2016;113(28):7822 7 2016/06/27. Available from: https://www.ncbi.nlm.nih.gov/pubmed/27354533. [53] Liebschner D, Dauter M, Brzuszkiewicz A, Dauter Z. On the reproducibility of protein crystal structures: five atomic resolution structures of trypsin. Acta Crystallogr 2013;69:1447 62 D. [54] Leiros HKS, McSweeney SM, Smala˚s AO. Atomic resolution structures of trypsin provide insight into structural radiation damage. Acta Crystallogr 2001;57:488 97 D. [55] Brandsdal BO, Smalas AO, Aqvist J. Electrostatic effects play a central role in cold adaptation of trypsin. FEBS Lett 2001;499:171 5. [56] Gorfe AA, Brandsdal BO, Leiros HK, Helland R, Smalas AO. Electrostatics of mesophilic and psychrophilic trypsin isoenzymes: Qualitative evaluation of electrostatic differences at the substrate binding site. Proteins 2000;40:207 17. [57] Russell RJ, Gerike U, Danson MJ, Hough DW, Taylor GL. Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium. Structure 1998;6:351 61.

Chapter 19

Molecular and functional characterization of major compatible solute in Deep Sea halophilic actinobacteria of active volcanic Barren Island, Andaman and Nicobar Islands, India Balakrishnan Meena1, Lawrance Anburajan1, Nambali Valsalan Vinithkumar1, Ramalingam Kirubagaran2 and Gopal Dharani2 1

Atal Centre for Ocean Science and Technology for Islands, National Institute of Ocean Technology, Port Blair, India, 2Marine Biotechnology

Division, Ocean Science and Technology for Islands Group, Ministry of Earth Sciences, Government of India, Chennai, India

19.1

Introduction

Industrial biotechnology has been developed substantially in the past years with an aim to partially replace petroleum based chemical industry [1
3]. However, bio-based products such as biochemical, bioplastics and biofuels are still too expensive due to their high production cost compared with chemical counterparts [4]. High production cost of bioprocessing is mainly associated with the following issues; the price of raw materials (substrates), e.g. glucose from hydrolysis of starch, has increased very fast [5]. Bioprocessing requires large amount of fresh water, making water shortage even worse [6]. Most of the fermentation processes (bioprocessing) are run discontinuously to avoid microbial contamination, which results in low production efficiency. Intensive sterilization of the fermenters and piping systems as well as the medium is a very expensive process [7]. The investment to purchase stainless steel bioprocess facilities is heavy. Finally, procedures to maintain contamination free and batch processes make the bioprocessing very complicated, leading to increasing cost for biochemical production. To make industrial biotechnology as competitive as the chemical industry, it is vital to develop low cost bioprocessing technology. In such technology, low energy and fresh water consumptions as well as contamination free continuous fermentation processes are required in addition to low cost substrates. Halophilic microorganisms have recently been re-discovered to possess advantages for the above-mentioned desirable properties [7]. Halophiles (salt-loving) are referred to those microorganisms that require salt (NaCl) for growth, and they can be found in all three domains of life
Archaea, Bacteria and Eukarya [8]. Halophiles can be found in hypersaline environments which are widely distributed in various geographical areas on earth, such as saline lakes, salt pans or salt marshes [9]. According to the salt concentration for optimal growth, halophiles can be roughly divided into two groups, moderate and extreme halophiles [10,11]. A moderate halophile grows at salt concentration of 3
15% (w/v) and can tolerate 0
25% (w/v) [10]. A large number of phylogenetic subgroups contain many types of halophilic bacteria, most of which belongs to the family Halomonadaceae (class Gammaproteobacteria) [12]. Halophilic microorganisms have developed two basically different mechanisms to cope with ionic strength and the considerable water stress, namely the “salt-incytoplasm” mechanism and the organic-osmolyte mechanism. Organisms following the salt-in-cytoplasm mechanism adapt the interior protein chemistry of the cell to high salt concentration [13]. The osmotic adjustment of the cell can be achieved by raising the salt concentration (KCl) in the cytoplasm according to the environmental osmolarity [14].

Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00019-8 © 2020 Elsevier Inc. All rights reserved.

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In contrast, microorganisms applying the organic-osmolyte strategy keep their cytoplasm, to a large extent, free of KCl and the design of the cell’s interior remains basically unchanged. Instead, organisms of this group accumulate uncharged, highly water-soluble, organic compounds in order to maintain an osmotic equilibrium with the surrounding medium [8,15]. The organic osmolyte mechanism is widespread among bacteria and eukaryotes and also present in some methanogenic archaea [12,14]. Organic osmolytes are of diverse chemical structure comprising different types of sugars (e.g. trehalose, polyols, amino acids (proline) and their derivatives (ectoine, glycine betaine)) and are accumulated inside the cell either by de novo synthesis or by uptake from the surrounding environment. These non-ionic, highly water-soluble molecules do not disturb the metabolism, even at high cytoplasmic concentrations, and are thus suitably named compatible solutes [16]. Compatible solutes can act as stabilizers for biological structures and allow the cells to adapt not only to salts but also to heat, desiccation, cold and freezing conditions [17], allowing the halophile to grow at around pH 10 and over 50  C [18]. These types of solutes have physico-chemical properties that distinguish them from other types of organic compounds, and similar types of low-molecular weight compounds have been selected during the course of evolution in all three domains of life to fulfill cellular functions as cytoprotectants [19
24]. One of the most widely distributed compatible solutes on Earth is ectoine. This compound, and many other compatible solutes, is not only employed as an effective osmostress protectant, but also provides cytoprotection against challenges posed by external environment in growth temperature and hydrostatic pressure, attributes that lead to the description of particular compatible solutes as thermolytes and piezolytes, respectively [25
32]. A hallmark of compatible solutes is their preferential exclusion from the immediate hydration shell of proteins [33], an effect largely caused by unfavorable interactions between these solutes and the protein backbone [34
36]. This preferential exclusion [33] leads to an uneven distribution of compatible solutes in the cell water and therefore generates a thermodynamic driving force that acts against the denatured and aggregated state of proteins. Hence, proteins are forced to adopt a compact and well-folded state under intracellular unfavorable osmotic and ionic conditions to minimize the number of excluded compatible solute molecules from surfaces [34
36]. Consequently, the accumulation of compatible solutes not only has beneficial effects on cellular hydration and maintenance of turgor, but also promotes the functionality of macromolecules (proteins and membranes, and protein: DNA interactions) under otherwise activity-inhibiting conditions [37
43]. The function-preserving property of compatible solutes has attracted considerable biotechnological interest, and the term “chemical chaperones” was coined in the literature [44,45] to reflect the beneficial effects of these compounds as protein stabilizers and protectants for entire cells [46
49]. The function of compatible solutes as chemical chaperones will certainly contribute to their role as protectants against extremes in either high or low temperatures for microorganisms [26
29,50
55], an underappreciated physiologically important attribute of these types of solutes. For instance, the hyperthermophile Archaeoglobus fulgidus cannot grow at 90 oC in a chemically % defined minimal medium [56], despite the fact that this archaeon synthesizes the extremolyte diglycerol phosphate in response to heat stress, an excellent stabilizer of protein function at high temperature [31,57]. However, the addition of 1 mM glycine betaine to the growth medium and its import via the heat stress inducible ProU ABC transporter efficiently rescued growth of A. fulgidus at the extreme temperature of 90  C [56]. In other words, glycine betaine can act as an effective thermoprotectant for a hyperthermophile. Similarly, a defect in the molecular chaperone DnaK that causes thermo-sensitivity of E. coli at 42 oC, can be functionally rescued by an external supply of the compatible solutes L-proline, glycine betaine and by% the glycine betaine biosynthetic precursor choline [28,50]. Furthermore, a broad spectrum of compatible solutes serves as thermoprotectants at the cutting upper (about 52 oC) and lower (about 13 oC) temperature boundaries for growth of Bacillus subtilis in a chemically % % defined minimal medium [26,27,58,59]. Although originally coined for unusual compatible solutes produced by microorganisms that live in habitats with extreme temperature, salt and pH profiles [60], the term extremolyte can be applied to these types of solutes in general [20,30,32,60,61]. This is exemplified by the above-cited example of the impressive thermoprotection of A. fulgidus by the “ordinary” compatible solute glycine betaine. Within the domain of the bacteria, important representatives of compatible solutes are the amino acid L-proline, the trimethylammonium compound glycine betaine and its analogue arsenobetaine, proline-betaine, trehalose, glucosyl glycerol, sulfur-containing dimethylsulfoniopropionate (DMSP), and the tetrahydropyrimidines ectoine and 5-hydroxyectoine. Many halophilic bacteria accumulate ectoine or hydroxyectoine as the predominant compatible solutes. Other intracellular compatible solutes include amino acids, glycine betaine and other osmotic solutes accumulated in small amounts [10,54,62].

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255

List of osmolytes and its applications. Application

Omolytes

Protection of biological macromolecules and protection of oxidative protein damage (LDH) Stabilization of enzymes against thermal stress and freeze drying Stabilization of recombinant nuclease Enzyme stabilization against heating, freezing, and drying Protection of LDH against heat and freeze-thawing Protection against proteolytic cleavage of antibodies Thermostabilization of proteins Thermostabilization of rubredoxin Cutinase unfolding and stabilization Inhibition of insulin amyloid formation Protection against freeze-thaw stress of immunotoxins Reduction of vascular leak syndrome in immunotoxin therapy Expression of functional recombinant proteins Stabilization of retroviral vaccines Reduction of apoptotic cell death induced by Machado-Joseph disease gene product Inhibition of aggregation and neurotoxicity of Alzheimer’s betaamyloid Protection of cell stabilization of E. coli during drying and storage Induction of thermotolerance in E. coli Protection of P. putida against anhydrobiotic stress Osmoprotection of lactic acid bacteria Stabilization of tobacco cells against hyperosmotic stress Block of UVA-induced ceramide release in human keratinocytes Protection of human RBC membranes Protection of mitochondrial DNA in human dermal fibroblasts Protection of the skin barrier against water loss and drying Protection of skin immune cells against UV radiation Reduction of UV-induced sunburn cells Prevention of UVA-induced photoaging Cytoprotection of keratinocytes

Hydroxyectoine

19.2

Mannosylglycerate Mannosylglycerate Ectoine Ectoine Ectoine Diglycerol phosphate Diglycerol phosphate Mannosylglycerate Ectoine Hydroxyectoine Hydroxyectoine Hydroxyectoine Mannosylglycerate, Hydroxyectoine Ectoine Ectoine, Hydroxyectoine Ectoine; Hydroxyectoine Hydroxyectoine Hydroxyectoine Ectoine Ectoine Ectoine Ectoine Ectoine Ectoine Ectoine Ectoine Ectoine Ectoine

Ectoine
a major compatible solute in halophilic eubacteria

Ectoine, a small organic molecule, occur widely in aerobic, chemoheterotrophic, and halophilic organisms that enable them to survive under extreme conditions. These organisms protect their biopolymers (bio membranes, proteins, enzymes, and nucleic acids) against dehydration caused by high temperature, salt concentration, and low water activity by substantial ectoine synthesis and enrichment within the cell. The organic osmolyte, ectoine and hydroxyectoine are amphoteric, water-binding, organic molecules. They are generally compatible with the cellular metabolism without adversely affecting the biopolymers or physiologic processes and are so-called compatible solutes. Ectoine [(4S)-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was originally discovered in the extremely halophilic phototrophic purple sulfur bacterium Ectothiorhodospira halochloris (now taxonomically re-classified as H. halochloris) by Louis and Galinski [62]. This seminal discovery was followed by the detection of a hydroxylated derivative of ectoine, 5-hydroxyectoine [(4S,5S)-2-methyl-5-hydroxy-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] by Inbar and Lapidot in [63] in the Gram-positive soil bacterium Streptomyces parvulus. Ectoine and 5-hydroxyectoine can chemically be classified as either heterocyclic amino acids or as partially hydrogenated pyrimidine derivatives [63,64]. Both ectoine and 5-hydroxyectoine were initially viewed as rare naturally occurring compatible solutes (e.g., in comparison with the almost universally distributed glycine betaine molecule). However, improved screening procedures using HPLC analysis and, in particular, 13C-natural abundance NMR spectroscopy revealed their widespread synthesis in bacteria in response to high salinity and pressure [30,65]. Ectoine producers can be found within a physiologically and taxonomically diverse set of microbial species [66
69]. Today, ectoine is known to be one of the most ubiquitously distributed compatible solutes in halophilic eubacteria. The identification of ectoine biosynthetic genes (ectABC) [62] and of the gene coding for the ectoine hydroxylase (ectD) [70
72] proved to be a major step forward for an in silico assessment in the distribution of ectoine/5-hydroxyectoine biosynthesis in bacteria and archaea [67,68]. Producers of

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ectoines are primarily found among members of the bacterial domain [66,67,73] and in restricted number of the Archaea [68].

19.3

Physicochemical properties of ectoine

Like other compatible solutes [35,36], ectoine and 5-hydroxyectoine are low-molecular mass compounds that are highly soluble in water (about 4 M at 20  C) [74], thereby allowing the amassing of these compounds to near molar concentrations in severely osmotically stressed microbial cells [66,69,75]. A variety of biophysical techniques have been used to study the effects of ectoine on the hydration of proteins and cell membranes and on interactions mediated via hydrogen bonding. Collectively, these data revealed that ectoine is excluded from the monolayer of dense hydration water around soluble proteins and from the immediate hydration layer at the membrane/liquid interface [39,74]. Ectoine enhances the properties of hydrogen bonds in aqueous solutions and thereby contributes to the dynamics and stabilization of macromolecular structures. Ectoine possesses a negatively charged carboxylate group attached to a ring structure that contains a delocalized positive charge. The resulting interplay between hydrophilic and hydrophobic forces influences waterwater and water-solute interactions [76] and thereby exerts strong effects on the hydration of ectoine itself, the binding of ions and the influence on the local water structure [77
81]. Molecular dynamics simulations have indicated that ectoine and 5-hydroxyectoine are strong water-binders and are able to accumulate seven and nine water molecules, respectively, around them at a distance smaller than 0.6 nm [80]. Collectively, the physico-chemical attributes of ectoine allow a physiologically adequate hydration of the cytoplasm upon their osmostress-responsive accumulation, afford effects on the local water structure, and also exert a major protective influence on the stability of proteins and the functionality of macromolecules [46,47,74,82,83].

19.4

Osmolytic properties of ectoine

Ectoine and 5-hydroxyectoine are produced by microorganisms in response to true osmotic stress, and not just in response to increases in the external salinity [84]. In cases where the build-up of ectoine/5-hydroxyectoine pools has been studied in more detail, there is often a linear relationship between the cellular content of these solutes and the external salinity/osmolarity [84
86]. This finding implies that bacterial cells can perceive incremental increases in the degree of the environmentally imposed osmotic stress, can process this information genetically/physiologically, and can then set its ectoine/5-hydroxyectoine biosynthetic capacity in a finely tuned fashion to relieve the constraints imposed by high-osmolarity on cellular hydration, physiology, and growth [19,87
89]. High-osmolarity-dictated increases in the cellular ectoine pools are largely accomplished through osmotically-responsive increases in the transcription of the ectoine/5-hydroxyectoine biosynthetic genes, although there might be post-transcriptional effects as well. Attesting to the role of ectoine as a potent osmostress protectant is the finding that the disruption of the ectABC biosynthetic genes causes osmotic sensitivity [90,91] and the genetic disruption of the gene (ectD) for the ectoine hydroxylase in Chromohalobacter salexigens impairs the ability to cope effectively with high growth temperature extremes [71]. In microorganisms that are capable of synthesizing both ectoine and 5-hydroxyectoine, a mixture of these two solutes is frequently found. Interestingly, such a 1:1 mixture (0.5 mM each) provided the best salt and heat stress protection to Streptomyces coelicolor when it was added to the growth medium [72]. However, there are also microorganisms that seem to produce almost exclusively 5-hydroxyectoine during osmotic stress and different growth phases of the culture [92,93].

19.5

Biosynthesis of ectoine

Ectoines are common in aerobic heterotrophic eubacteria. The entry molecule in ectoine biosynthesis is aspartate semialdehyde, which is an intermediate in amino acid metabolism. The aldehyde is converted to L-2,4-diaminobutyric acid, which is then acetylated to from Nγ-acetyldiaminobutyric acid (NADA). The final step is the cyclization of this solute to form ectoine. Ectoine synthesis is carried out by the process of three genes: ectABC. The ectA gene codes for diaminobutyric acid acetyltransferase; ectB codes for the diaminobutyric acid aminotransferase and ectC codes for ectoine synthase [15]. Ectoine is synthesized from aspartate-semialdehyde, the central intermediate in the synthesis of amino acids belonging to the aspartate family (Fig. 19.1). Ectoine formation comprises three enzymatic steps [62,90,94,95]. First, aspartate-semialdehyde is transaminated to 2,4-diaminobutyric acid (DABA) with glutamate as amino-group donor. The transamination is catalyzed by DABA transaminase ectB. EctB is a 421-residue protein with a molecular mass of

Molecular and functional characterization of major compatible solute Chapter | 19

O

H+3N

O O– O–

O L-aspartate

aspartokinase (Cs) 2.7.2.4 H+3N ATP

ADP

O O P O– O– O–

O L-aspartyl-4-phosphate

–

O

aspartate-semialdehyde dehydrogenase (Cs) 1.2.1.11 H+3N

H+ NADPH

NADP+ phosphate

257

H O–

O L-aspartate-semialdehyde O–

H+3N

O

O–

O L-glutamate

diaminobutyrate aminotransferase (Cs): Cs-ectB 2.6.1.76

O

O O–

O– O 2-oxoglutarate

NH

H+3N

O

H+3N

O–

O N-acetyl-L-2,4-diaminobutanoate

H2O

NH3+

diaminobutyrate acetyltransferase (Cs): Cs-ectA 2.3.1.178

Coenzyme A H+

O

O–

O L-2,4-diaminobutanoate

CoA acetyl-CoA

ectoine synthase (Cs): Cs-ectC 4.2.1.108 O O–

HN

NH+

ectoine

FIGURE 19.1 Ectoine biosynthesis pathway. http://biocyc.org/META/NEW-IMAGE?type 5 PATHWAY&object 5 P101-PWY&detail-level 5 4, 18.06.2016.

46.1 kDa, which requires K 1 for its transaminase activity and for protein stability. Gel filtration experiments with purified protein from H. elongata indicate that the DABA aminotransferase ectB might form a homohexamer in the native state. Then, an acetyl group is transferred to DABA from acetyl-CoA by DABA-Nγ-acetyltransferase ectA in order to synthesize Nγ-acetyl-L-2,4-diaminobutyric acid. EctA is a 192-residue protein with a calculated molecular mass of 21.2 kDa. Finally, ectoine synthase ectC catalyzes the cyclic condensation of Nγ-acetyl-2,4-diaminobutyric acid, which leads to the formation of ectoine. EctC is a 137-residue protein with a calculated molecular weight of 15.5 kDa with a pI value of 4.9. The ectC protein belongs to the enzyme family of carbon-oxygen lyases. In vitro experiments with purified ectC revealed that ectoine-synthase activity and affinity to its substrate are strongly affected by NaCl. Under certain stress conditions (e.g. elevated temperatures) H. elongata converts some of the ectoine to 5- hydroxyectoine [96] by ectoine hydroxylase (EctD). The ectoine hydroxylase EctD consists of 332 amino acids and has a molecular weight of 37.4 kDa. The ectD protein is a member of an oxygenase subfamily within the nonhemecontaining, iron (II)- and α- ketoglutarate-dependent dioxygenase superfamily. Ectoine hydroxylase was shown to catalyze the direct hydroxylation of ectoine to 5-hydroxyectoine [72]. The genes for ectoine biosynthesis ectABC are clustered together and can be found only 5 kb from the termination point of chromosome replication [97]. The ectD gene encoding the hydroxylase for hydroxyectoine synthesis is located apart from the ectABC cluster. Recently, Schwibbert and coworkers mapped the transcriptional initiation sites of ectABC and found two promoters in front of ectA and one upstream of ectC [97]. Upstream of ectA a putative σ70 promoter and an osmotically inducible σ38 promoter [98] were found, while in front of ectC a σ54-controlled promoter is located. σ54-controlled promoters are often involved in transcription of nitrogen-regulated genes [99,100]. The transcriptional regulation of ectABC by an osmoregulated σ38 promoter and a σ54 promoter is in agreement with physiological observations made with other bacteria, such as Corynebacterium glutamicum and Halorhodospira (formerly Ectothiorhodospira) halochloris. In these organisms, it was shown that synthesis of the compatible solutes proline and glycine betaine, respectively, is not only

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PART | II Biotechnological aspects

determined by salinity but also by nitrogen supply [101,102]. Ectoine biosynthesis is regulated at the level of transcription and enzyme activity [103]. Transcriptome analysis of H. elongata revealed an increase of mRNA from ectA, ectB, and ectC with increasing sodium chloride concentration from 0.6% (0.1 M) to 12% (2 M) NaCl. Similar results were obtained from proteome analysis that showed an increase in ectABC protein at elevated salinities. Contribution of expression and enzyme activity of ectABC to osmoregulated ectoine synthesis was investigated with cells of H. elongata that have been treated with chloramphenicol (Cm) to abolish protein biosynthesis [103]. Cultures of H. elongata with and without Cm growing at 1% NaCl (0.17 M) and 4% NaCl (0.68 M) were exposed to an osmotic upshift. The salt concentration of the cultures growing at 1% NaCl was increased to 4% NaCl and in the cultures growing at 4% NaCl to 8% NaCl. The Cm treated cells grown at a low salt concentration of 1% NaCl failed to synthesize sufficient ectoine in response to the salt shock that raised the salinity to 4% NaCl.

19.6

Transport of ectoine

Osmolyte transporters also play an important role in the osmotic response. Some of these transporters are very specific and serve to retrieve any solute released by cells. Others have evolved to scavenge solute or osmolyte precursors so that the more wasteful biosynthetic resources of the cell are not used. Recent years have witnessed progress in identifying and characterizing the proteins responsible for uptake of the osmolytes glycine betaine and ectoine from the medium. In other cases putative transporter genes have been identified but no accumulation of the solute is observed. Ectoine that is provided in the medium can be internalized by some microorganisms. Growth of halotolerant Brevibacterium sp. JCM 6894 is stimulated by exogenous ectoine or hydroxyectoine [104]. In H. elongata the transporter for ectoine and hydroxyectoine (TeaA, TeaB, TeaC) is similar to members of the tripartite ATP-independent periplasmic transporter family (TRAP-T) [105]. The Ks (ect) is 21.7 μM, indicating a high affinity for external ectoine. The role of this transporter appears to be recovery of ectoine leaked from the cell. Marinococcus halophilus also can transport external ectoine. In this cell, the EctM gene product is a BCCT family member [106]. In the same manner, a proteomic analysis of Sinorhizobium meliloti in medium that was supplemented with ectoine detected increased synthesis of ten proteins, eight of which were identified by MALDI-TOF analysis of peptides from the two-dimensional gels [107]. Five of these belong to the same gene cluster (localized on the pSymB megaplasmid), whose components code for the ATP-binding cassette transporter ehu (ectoine/hydroxyectoine uptake). Another cluster of genes (eutABCDE) would produce proteins capable of ectoine catabolism. The net result of exposing S. meliloti to ectoine is to enhance the production of proteins to internalize and use any of these molecules that escape the cell.

19.7

Industrial production of ectoine

The industrial-scale production scheme for ectoine relies on the highly salt-tolerant Gammaproteobacteria, H. elongata as a natural cell factory [60,97]. It exploits the massive production of ectoine under high-salinity growth conditions [97] and their non-specific release from the producer cells via the transient opening of mechanosensitive channels upon a severe osmotic down-shock [66,69,76]. Ectoine can easily be synthesized chemically [108,109]. However, large-scale chemical synthesis of ectoine is not competitive with biotechnological production because of the need for high prized precursors such as diaminobutyric acid. The first process that had been developed for the biotechnical production of ectoine was the so-called bacterial milking procedure [110]. Ectoine has a maximum solubility of 6.5 mol/kg water at 25  C [76] and is accumulated up to molar concentration inside the cytoplasm. Halophilic bacteria such as H. elongata thriving in a high saline environment have to be attuned to a sudden decline in salinity caused by rainfall or flooding. In this situation the cell has to cope with a cytoplasm that has a significantly higher osmolarity (or lower chemical potential) than the surrounding environment causing a sudden influx of water (since water is freely permeable across the cell membrane). In order to avoid bursting, the cell has to release ectoine and other compatible solutes from the cytoplasm, probably through mechanosensitive channels, of which H. elongata possesses three MscS and one MscK. H. elongata combines several features making it a reliable industrial producer strain: it grows robustly on a wide variety of substrates it is safe as also indicated by its use in food processing [111] and can achieve high cell densities ( . 40 g dry weight/l corresponding to .10 g/L ectoine). The bacterial milking process exploits the ability of H. elongata to release ectoine in response to dilution stress to the medium. Cells of H. elongata are grown in a fed-batch fermentation process at a salinity of 10% NaCl (1.7 M) until a high cell density has been reached. Then, an osmotic down shock from 10% to 2% NaCl is applied. As a result, approximately 80% of the cytoplasmic ectoine is released to the culture medium. The excreted ectoine is then recovered from the medium by cross-flow filtration and further purified by cation exchange chromatography and crystallization. After harvesting, the filtered cell mass is reused and brought back into high saline

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259

(10% NaCl) culture medium for another round of ectoine synthesis. Within 10 hours the bacterial milking can be repeated. A significant increase in productivity could be achieved by replacing the fed-batch process with a continuous fermentation [112].

19.8

Biotechnological applications of ectoine

The excellent function-preserving attributes of ectoine have attracted considerable attention to their exploitation in the fields of biotechnology, skin care, and medicine [60,66,69,113
115]. High demand for ectoine for practical purposes has led to an industrial-scale production process that exploits H. elongata as a natural and engineered cell factory, delivering ectoine on the scale of tons [60,66,69]. Data reported in the literature [116,117] estimate a worldwide production level of ectoine of about 15,000 tons per annum, which putatively have an estimated sales value of approximately 1000 US Dollars kg21. However, another study reports a price for ectoine at between about 14,000 and 18,000 Euro kg21 [118]. Of the extremolytes currently considered for practical applications [114], ectoine and 5-hydroxyectoine certainly have the greatest potential for sustained commercial exploitation [60,66,69,113,115]. It is outside the scope of this overview to address in depth the biotechnological production of ectoine in natural and synthetic microbial cell factories, or to describe in detail the varied practical applications for these compounds. The properties of osmolytes make them suitable for a variety of uses in biotechnology as long as one can generate reasonable quantities either in vivo or in vitro. Induction of osmolytes in cells can increase protein folding, so that engineering osmolyte biosynthesis genes in an organism should improve its salt tolerance. The trick is to couple osmolyte production to salt stress. For in vitro uses, large amounts of pure solutes are needed. In many cases, the solutes can be supplied by ‘bacterial milking’. Both ectoine and hydroxyectoine have been produced in large quantities using H. elongata [110]. This process is the basis of the German biotechnology company Bitop http://www.Bitop.de/sources/html/e/index.htm that has developed preparative methods for many of the unique osmolytes produced by microorganisms.

19.8.1 Chemical chaperones for protein folding Insoluble or misfolded overexpressed proteins can often be partially denatured and refolded in the presence of osmolytes. A specific example is the use of osmolytes to enhance the yield of folded, functional cytotoxic proteins directed to the periplasm of E. coli [83]. Cells grown in 4% NaCl with 0.5 M sorbitol and supplemented with 10 mM betaine can accumulate large amounts of the target protein in the periplasm. Protein is released by freeze-thaw cycles. Both high osmotic strength and added compatible solutes (betaine and sorbitol) are necessary for high yields of protein. In the same vein, ectoine, betaine, trehalose, and citrulline have been shown to inhibit insulin amyloid formation in vitro [119]. This observation may provide directions for designing small molecules to inhibit myelin formation associated with neurodegenerative disorders.

19.8.2 Enhancing PCR Several osmolytes (betaine, ectoine) have been shown to be useful in PCR amplification of G-C-rich (72.6%) DNA templates with high Tm. In particular, ectoine was shown to outperform regular PCR enhancers; it works by reducing the DNA Tm [120]. Interestingly, hydroxyectoine increases the Tm of duplex DNA. However, the optimal solute for these experiments is homoectoine (4,5,6,7-tetrahydro-2-methyl-1H-[1,3]-diazepine-4-carnoic acid), a synthetic derivative of ectoine with the ring expanded by one carbon. For betaine the effective range of solute is 0.5
2.0 M; for ectoine much less (0.25
0.5 M) is needed for the same effect. It would be intriguing to see what effect DIP type solutes have on PCR since they are synthesized by hyperthermophiles above 80  C.

19.8.3 Cryo-protection of microorganisms Organic osmolytes have also been used as cryo-protectants. In a recent study, the ability of betaine to act as a cryoprotectant during freezing of diverse bacteria was examined. Betaine is often much better than two common cryoprotectant mixtures, serum albumin and trehalose/dextran and ectoine, particularly under conditions simulating long term storage [121]. It is better than the other treatments at preserving long term viability for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. Betaine is as effective as glycerol for liquid nitrogen freezing of halophilic archaea, and neutrophilic Fe-oxidizing bacteria.

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19.8.4 Use in cosmeceuticals and pharmaceuticals The ability of osmolytes to aid in protecting cells from diverse stresses has led to the use of ectoine, in the cosmeceutical industry. Ectoine has been shown to protect skin from UVA-induced cell damage [122]. Based on this, RonaCaret Ectoin, produced by Merck KgaA, Darmstadt, is presently in use as a moisturizer in cosmetics and skin care products. Osmolytes have not been developed as reagents in the pharmaceutical industry, in part because as ‘compatible solutes’ they interact minimally with cellular machinery. However, their ability to stabilize biomolecules may have some very specific uses. As an example, the German company Bitop in collaboration with researchers at the Cologne University Clinic is exploring the use of these solutes in certain cancer therapies where they may protect tissues against vascular leak syndrome, a severe side effect of anti-cancer agents.

19.8.5 Generation of stress-resistant transgenic organisms Insertion of genes for osmolytes into non-halotolerant organisms should increase their ability to withstand salt stress. Plants are a good target for these types of experiments since they are often exposed to drought conditions that would concentrate salt. A few reports of transgenic plants suggest that eventually this strategy might be useful. Arabidopsis thaliana transformed with a choline oxidase gene (needed to synthesize betaine) from Arthrobacter globiformis has a significantly improved tolerance of salt stress along with improved cold and heat tolerance [123]. Transgenic tobacco with E. coli betA and betB genes has also been constructed. This modified plant exhibits better salt and cold tolerance [124]. Inserting the H. elongata ectABC genes also confers hyperosmotic tolerance on cultured tobacco cells [125]. This was shown to increase the hyperosmotic tolerance of cultured cells, although only a small amount of ectoine accumulated. Other recent work to introduce genes for synthesizing osmolytes in plants [126] as a way to improve stress tolerance has, so far, not led to high accumulation of the osmolytes. Further developments await a determination of what limits osmolyte levels in plant cells.

19.8.6 Ectoine based products in market The first commercial use of ectoine was as a skin care ingredient [127,128] and this application still plays a major role, especially in sun protection and anti-aging products where ectoine is widely used [113,129,130]. Recently, the use of ectoine in health care products has become of increasing importance. Buenger and Driller [122] and [131], revealed the ability of ectoine to inhibit the early UV radiation-induced ceramide signaling response in human keratinocytes. Mitigating effect of ectoine on inflammatory conditions of human skin was also examined. Pre-treatment of keratinocytes with ectoine, reduced the number of sunburn cells, prevented the decline in Langerhans cells [132] and decreased UV-induced DNA single strand breaks [133]. In vitro studies on ectoine´s beneficial effects for human skin are confirmed by clinical trials on skin aging [134]. Here it could be demonstrated that a formulation with already good skin care properties could be significantly improved by the addition of 2% ectoine, which led to superior skin hydration, skin elasticity and skin surface structure. The mitigating effect of ectoine was not only observed in skin but also in inflammatory conditions of other epithelia. For lung epithelia, it was shown that ectoine protects against nanoparticleinduced airway inflammation [135]. For nasal and eye epithelia in allergic conjunctivitis, ectoine containing nasal spray and eye drop products relieved effectively the hallmark symptoms of rhino conjunctivitis with treatment effects similar to those of antihistamines, steroids and leukotriene modifiers, with no side effects [136]. Based on this, wide ranges of ectoine-based medical device products for the treatment of allergies (allergy nasal sprays and eye drops), skin inflammatory conditions like atopic dermatitis, treatment for dry eye, dry nose and rhinosinusitis have been developed, successfully and tested in clinical trial [137] and commercialized. Due to the excellent safety profile in combination with clinically proven efficacy in the treatment of inflammatory conditions of epithelia, wider use of ectoine can be envisaged in the future. Potential future applications include the treatment of epithelial derived inflammatory diseases, especially nanoparticle induced, lung inflammation [138], colitis [139] and tissue protection in ischemia [140,141]. Cosmetic formulations based on natural marine resource-derived ingredients are a good marketing argument, although this resource is still poorly exploited. The potential applications of osmolytes derived from the halophilic eubacteria promises a bright future for the cosmetics industry that is constantly looking for innovation. Therefore, a wide variety of marine natural products have received increased attention, especially those derived from micro- and macro-algae and deep sea halophilic eubacteria. However, their potential is far from being fully exploited, especially for deep sea-inhabiting marine organisms that remain to be described. Once the valuable species are clearly identified,

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it will remain to optimize the mode of production/extraction of the molecules of interest and to perform tests to ensure their effectiveness and their safety for cosmetic applications.

19.9 Molecular and functional characterization of ectoine in deep sea halophilic actinobacteria, nocardiopsis alba All the above research studies clearly explained about the importance and market value of compatible solutes including ectoine. To the best of our knowledge, no report on functional characterization of ectoine biosynthesis genes from deep sea actinobacteria, Nocardiopsis alba. In this study ectoine biosynthesis gene cluster (ectABC) from Nocardiopsis alba NIOT-DSB14 was characterized and determined the diversity and phylogenetic relationship of ectA, B and C genes with other eubacteria. Nocardiopsis alba strain NIOT-DSB14 isolated from deep sea sediment was grown in starch casein agar (SCA) medium added with nalidixic acid (25 μg/mL; HiMedia, India) to inhibit the fast growing Gram-negative bacteria. Sample inoculated plates were incubated at room temperature (28 6 2  C) for 21 days and the colonies were recognized by their characteristic chalky to leathery appearance on SCA plates. Individual colonies were selected and sub cultured in SCA slants for further characterization studies. The genomic DNA of Nocardiopsis alba NIOT-DSB14 was extracted according to Kutchma et al [142]. and 1 μL of genomic DNA was used for PCR amplification. EctABC gene cluster of Nocardiopsis alba was amplified by PCR using gene specific primers. PCR was performed in 50 μL of reaction mixture which contained 50 ng of genomic DNA, 0.5 μM of each primer, 200 μM each of dNTP (MBI Fermentas, USA), 1.25 U of Pfu DNA polymerase (MBI Fermentas), 1 3 Pfu buffer; 2.5 mM of MgSO4 and remaining autoclaved Millipore water. Amplification was performed in a Master cycler (Eppendorf, Germany) with the following conditions; initial denaturation at 94  C for 3 min, followed by 30 repeated cycles of 94  C for 30 s, 50  C for 1 min and 72  C for 2 min and final extension at 72  C for 5 min. The PCR amplified product was analyzed on 1.5% agarose gel along with DNA molecular weight marker (MBI Fermentas) and documented in gel documentation system (UVP BioSpectrum Imaging system, USA). The ectABC PCR amplicons were purified by MinElute Gel purification Kit (Qiagen, Germany) and cloned into pDrive (Qiagen), according to the manufacturer’s instructions. The pDrive-ectABC construct was transformed into E. coli JM109 (recA1, endA1, gyrA96, thi-1, hsdR17 (rK-mk 1 ), e14-(mcrA
), supE44, relA1, Δ(lac-proAB)/F0 [traD36, proAB 1 , lac Iq, lacZΔM15]). White colonies were selected for PCR amplification with vector primers M13f-M13r (MBI Fermentas) and the clones with the correct insert as judged by size were sequenced on an ABI PRISM 377 genetic analyzer (Applied Biosystems Inc., USA). The recombinant plasmid pDrive-ectABC construct were double digested with SacI and BamHI (MBI Fermentas) and purified by MinElute Gel purification Kit. The purified ectABC gene was recloned into pQE30 expression vector (Qiagen), which had previously been digested and purified. The resulting recombinant expression vector pQE30ectABC cassette was transformed into E. coli M15 (pREP4). A single colony of the recombinant culture was inoculated into 5 mL of LB broth containing 100 μg/mL of ampicillin and 25 μg/mL of kanamycin, and incubated overnight at 37  C. About 2  5 mL of the culture was transferred into 50 mL of LB containing 100 μg/mL of ampicillin and 25 μg/ mL of kanamycin and incubated at 37  C, until OD600 value reached 0  6. Isopropyl-β-D-thiogalactoside [IPTG] (MBI Fermentas) was then added into the culture at the final concentration of 1 mM and was continuously incubated at 37  C for 4 h. The induced bacterial cells were harvested by centrifugation and resuspended in 1 3 SDS-PAGE sample buffer and lysed in boiling water bath for 3 min. The cells were centrifuged at 4000g for 20 min and the supernatant was checked for expression of soluble proteins. The expression of the target proteins were analyzed by SDS-PAGE as described by Laemmli [143], The molecular mass was estimated by SDS-PAGE with protein ladder (Sigma-Aldrich, USA). Enzyme extraction was performed as described previously [95]. Briefly, the cells obtained from a 100 mL culture were resuspended with 1 mL of 50 mM Tris HCl buffer (pH 8.0) and treated with 20 μg of lysozyme at 37  C for 5 min. The mixture was then incubated with 0.5 mg of DNase I and 2.0 mg of RNase A at 37  C for 10 min. The cell lysate was then treated with 1 mM phenylmethylsulfonyl fluoride [PMSF] (Sigma-Aldrich, USA), 0.5 mM EDTA, 0.1 M NaCl and centrifuged at 10,000 g for 20 min. The protein concentration was determined according to the method of Bradford [144], with bovine serum albumin as the standard. The enzyme activity of 2,4-diaminobutyric acid (DABA) aminotransferase (ectB) was assayed as described previously [95]. DABA aminotransferase activity was determined by the amount of glutamate produced in the reverse reaction. Briefly, 100 μL reaction mixture consisting of 5 mM 2-oxoglutarate, 10 mM DABA, 10 μM pyridoxal phosphate (PLP), 50 mM Tris HCl buffer (pH 8.5), and 25 mM KCl was incubated at 25  C for 30 min, and the reaction was stopped by boiling the mixture for 5 min.

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The concentration of the released glutamate was determined by L-glutamic acid kit of Roche (R-Biopharm GmbH, Germany). Activity of DABA acetyltransferase (ectA) was assayed by acylation assay as described previously [145]. Briefly, the reaction mixture contained 0.3 mM Ellman’s reagent [5,50 -Dithiobis(2-nitrobenzoic acid)] in 60 mM Tris HCl buffer (pH 8.5), 0.4 mM NaCl, 2 mM acetyl-coenzyme A and 30 mM diaminobutyrate. A coupled spectrometric test was employed. The acylation activity of ectA was analyzed using a coupled spectrometric test with the detection at 410 nm. Ectoine synthase (ectC) activity was performed as described previously [95]. Briefly, the reaction was carried out in a 100 μL mixture consisting of 10 mM ADABA, 0.6 M NaCl, 1 mM DABA, 50 mM Tris
HCl buffer (pH 9.5). The reaction mixture was incubated at 15  C for 10 min and stopped by 0.3% of trifluoroacetic acid (TFA). The amount of ectoine produced by the ectABC gene cluster was determined by HPLC [104]. Ectoine was detected in a HPLC system (Waters 4707) using an NH2 column (LiChroCART 250-4 NH2; Merck, NJ, USA) at 30  C with an acetonitrile-water (85%, v/v) as the mobile phase at a flow rate of 1.0 mL/min. Purified ectoine synthase was detected by UV at 210 nm. The identification and quantification of ectoine were carried out using ectoine (Sigma-Aldrich) as the standard. The nucleotide sequences obtained were compared against database sequences using BLAST provided by NCBI (http://www.ncbi.nlm.nih.gov) and were aligned and clustered using CLUSTAL-X version 1.81 program [146]. The output alignments were imported into the GeneDoc program (http://www.psc.edu/biomed/genedoc/) and BioEdit version 7.05 program (www.mbio.ncsu.edu/BioEdit/) to calculate the percent identities among the nucleotide and amino acid sequences. The molecular masses and the theoretical pI values of the polypeptides were predicted using the ProtParam tool (http://www.expasy.org/tools/protparam.html). The sequences generated in this study have been deposited in the GenBank database under the accession numbers MK613836, MK613837 and MK613838.

19.10 PCR amplification, cloning and sequencing of ectoine biosynthesis genes The ectA, B and C genes encode the diaminobutyric acid acetyltransferase, diaminobutyric acid aminotransferase and ectoine synthase respectively. Together these proteins constitute the ectoine biosynthetic pathway. The ectoine biosynthesis genes ectA, B and C from deep sea bacteria, Nocardiopsis alba were PCR amplified and encoded by polynucleotides of 471 bp, 1266 bp and 378 bp (Fig. 19.2A and B). The ectA, B and C genes encodes polypeptides of 156, 421 and 125 amino acids with calculated molecular masses of 17,291 45,809, 13,789 Daltons. The amplicons were purified from the agarose gel and cloned into pDrive cloning vector. The positive clones were selected and screened for the presence of ectoine biosynthesis genes by PCR using specific primers. The recombinant transformants of ectA, B and C genes were also confirmed by double digestion with SacI and SmaI restriction enzyme, which released full gene along with flanking region of the vector.

19.11 Molecular characterization of ectoine biosynthesis genes The recombinant expression vector pQE30-ectABC cassette was transformed into E. coli M15(pREP4). The expression of the ectoine biosynthetic genes was confirmed by determining the activity of the individual enzymes. The functional activity of ectA protein was determined by acylation assay. The acylation activity in the expressed cells was 2.3 mU/mg,

FIGURE 19.2 (A) Agarose gel electrophoresis of ectoine biosynthesis genes. Lane a: ectA amplicon (471 bp), Lane b: 100 bp DNA ladder. (B) Agarose gel electrophoresis of ectoine biosynthesis genes. Lane a: 100 bp DNA ladder, Lane b: ectB amplicon (1266 bp), Lane c: ectC amplicon (378 bp).

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which is three times more than that of control cells. Aminotransferase assay was employed to determine the activity of ectB protein. The aminotransferase activity of the expressed cells was 4.1 mU/mg, which is considerably higher than that of uninduced cells. The ectoine synthase activity of the expressed cells was also in the higher side 5.3 mU/mg, which is higher than that of uninduced cells. The ectoine biosynthesis genes ectA, B and C have been functionally characterized in Marinococcus halophilus, Bacillus pasteurii, Methylomicrobium alcaliphilum and Chromohalobacter salexigens [62,85,145,147]. Expression of the ectoine biosynthesis genes was analyzed by SDS-PAGE electrophoresis. The lysates of induced cells showed two clear expressed bands with molecular masses of 45 kDa and 13 kDa that correspond to ectB and ectC, which was not present in non-induced cells. The expressed protein band of ectA gene was not detectable in SDS-PAGE due to its instability. The instability of the ectA has already been reported [145].

19.12 Sequence analysis of ectA, B and C genes The ectA, B and C sequences from Nocardiopsis alba NIOT-DSB14 were analyzed with reported amino acid sequences of other eubacteria viz. Marinococcus halophilus (GenBank Accession No. U66614), Bacillus alcalophilus (DQ471210), Bacillus pasteurii (AF316874), Virgibacillus salexigens (AY935521), Virgibacillus pantothenticus (AY585263), Halobacillus dabanensis (DQ108975), H. elongata (AF031489) using Clustal X program. Homology search of the nucleotide and deduced amino acid sequence was performed using BLAST program. The amino acid analysis revealed that the ectA gene encoded protein belongs to the GNAT family. The ectA gene encodes proteins of 156 amino acids with the pI value of 4.65. The amino acid sequences of ectA showed a high similarity with other bacteria: B. alcalophilus, 53% identity with 155 aa overlap. H. dabanensis, 36% identity with 140 aa overlap; V. salexigens, 33% identity with 141 aa overlap; V. pantothenticus, 23% identity with 140 aa overlap; B. pasteurii, 31% identity with 154 aa overlap; H. elongata, 26% identity with 156 aa overlap and M. halophilus, 63% identity with 146 aa overlap. The amino acid analysis of ectA revealed that, the amino acid sequence of B. alcalophilus and M. halophilus has the maximum identity of 50% and 63% respectively with that of Nocardiopsis alba NIOT-DSB14. All the other sequences had less than 40% similarity with the amino acid sequence of Nocardiopsis alba NIOT-DSB14. The amino acid analysis of ectB gene suggests that the encoded protein belongs to the GabT family. The ectB gene encodes proteins of 421 amino acids with the pI value of 5.01. Among the ectoine biosynthesis genes, the amino acid sequences of ectB gene showed highest similarity with other bacteria compared to ectA and C genes. B. alcalophilus sequence has 79% identity with 413 aa overlap; H. dabanensis, 56% identity with 403 aa overlap; V. salexigens, 57% identity with 404 aa overlap; V. pantothenticus, 55% identity with 394 aa overlap; B. pasteurii, 45% identity with 313 aa overlap; H. elongata, 45% identity with 418 aa overlap and M. halophilus has 56% identity with 407 aa overlap. The amino acid sequence analysis revealed that, only B. alcalophilus has the maximum identity of 71% with the amino acid sequence of Nocardiopsis alba NIOT-DSB14. The amino acid analysis of ectC gene suggests that the encoded protein belongs to the ectoine synthase. The ectC gene encodes proteins of 125 amino acids with the pI value of 5.09. The amino acid sequences of ectC also revealed a considerable homology with ectoine synthase from other eubacteria. B. alcalophilus sequence has 70% identity with 118 aa overlap; H. dabanensis, 52% identity with 118 aa overlap; V. salexigens, 45% identity with 108 aa overlap; V. pantothenticus, 49% identity with 118 aa overlap; H. elongata, 45% identity with 120 aa overlap; B. pasteurii, 56% identity with 119 aa overlap and M. halophilus has 58% identity with 114 aa overlap. The amino acid sequence of B. alcalophilus and M. halophilus has the maximum similarity of 70% and 58% with the amino acid sequence of Nocardiopsis alba NIOT-DSB14 than other eubacteria.

19.13 Phylogenetic tree construction and analysis of ectoine biosynthesis genes Phylogenetic tree analysis of nucleotide and amino acid sequences of ectA revealed a single cluster pattern for Nocardiopsis alba NIOT-DSB14, Streptomyces sp. (GenBank accession No. KU902741), Streptomyces sp. (KU902749), Streptomyces sp. (KU902752) and Streptomyces sp. (KU902751). The phylogenetic tree of nucleotide and amino acid sequences of ectB gene also revealed the grouping of Nocardiopsis alba and Streptomyces sp., in a single cluster as that of ectA. Nucleotide sequences of ectC gene in Nocardiopsis alba was clustered separately from other eubacteria, however in the amino acid sequence analysis, Nocardiopsis alba and Streptomyces sp., were grouped in the single cluster. On phylogenetic analysis, ectC gene was found to have highest similarity between the actinobacterial species while compared to the ectA and ectB genes. Based on phylogenetic analysis, Nocardiopsis alba and Streptomyces sp. were found to be clustered together for all the genes (Fig. 19.3A
C). The bacterial species switched to different clusters at nucleotide and amino acid level indicates the divergence among the organisms and the degree of

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(A)

Streptomyces sp. KU902741 (ectA)

(B)

Streptomyces albulus JF427576 (ectB)

Streptomyces sp. KU902751 (ectA) Streptomyces sp. KU902799 (ectB)

Streptomyces sp. KU902752 (ectA) Streptomyces rimosus KC876560 (ectB)

Streptomyces sp. KU902749 (ectA)

FIGURE 19.3 (A) Phylogenetic tree analysis of ectoine biosynthesis genes. Amino acid sequences of ectA gene. (B) Phylogenetic tree analysis of ectoine biosynthesis genes. Amino acid sequences of ectB gene. (C) Phylogenetic tree analysis of ectoine biosynthesis genes. Amino acid sequences of ectC gene.

N. alba NIOT-DSB14 (ectB)

N. alba NIOT-DSB14 (ectA)

0.5

0.2

(C)

N. alba NIOT-DSB14 (ectC)

Micromonospora sp. JN038178 (ectC)

Enhygromyxa salina KU237243 (ectC)

Streptomyces ansochromogenes KF170351 (ectC) 0.2

divergence in the sequences. Even though the ectoine biosynthesis pathway is evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic eubacteria [147].

19.14 Concluding remarks In this chapter, we report the importance of compatible solutes and heterologous expression of ectoine biosynthetic genes from the deep sea halophilic actinobacteria, Nocardiopsis alba NIOT-DSB14 in E. coli M15. The engineered E. coli strain has potential industrial application since it produces ectoine at high rates and can avoid the complex down streaming process associated with the conventional bioprocess. On phylogenetic analysis, the ectA, B and C genes of Nocardiopsis alba were found to be highly conserved among the eubacterial and actinobacterial species. The ectB gene was found to have highest similarity between actinobacterial species compared to the ectA and ectC genes. The genes involved in the biosynthesis of ectoine in Nocardiopsis alba NIOT-DSB14 was comparatively well conserved compared to other eubacterial and actinobacterial species both at nucleotide and amino acid level.

Acknowledgments The authors gratefully acknowledge the financial support given by the Earth System Sciences Organization (ESSO), Ministry of Earth Sciences (MoES), Government of India, New Delhi to conduct the research. The authors are thankful to Dr M. A. Atmanand, Director, ESSO-National Institute of Ocean Technology (ESSO-NIOT), MoES, Chennai, for constant support and encouragement to perform this research.

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97.

Chapter 20

Antarctic microorganisms as sources of biotechnological products Tarcı´sio Correa and Fernanda Abreu Paulo de Go´es Microbiology Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

20.1

Introduction

For many years, Antarctica remained an unexplored continent. In 1961, the Antarctic Treaty System (ATS) was implemented and established this continent as a scientific preserve. Currently, ATS regulates international relations within this complex and fragile environment. Despite the challenging access to this extreme environment, the number of publications about the diversity/bioprospection of microorganisms in this continent has increased significantly over the years (Fig. 20.1). Antarctic continent is home to a substantial diversity of marine and continental environments. Extreme abiotic conditions of Antarctic environments like cold, high salinity, and ultraviolet light incidence play selective pressures over bacterial and fungal aquatic and terrestrial communities [2,3]. Antarctic microorganisms had evolved survival strategies to resist such harsh conditions, and these include but are not limited to cold-tolerant enzymes, antimicrobials, and antifreezing components [4]. As depicted in Fig. 20.2, bioactive compounds are known to be produced in a diversity of microbial habitats, such as sediments of lakes and bays, seawater, soils, and permafrost [4,5].

FIGURE 20.1 Global publication records (January 1, 1980
May 29, 2019) for Antarctic microbial diversity. The search using keywords “Antarctic” and “Antarctica” combined with “microorganism(s),” “bacterium(a),” “fungus(i),” and “yeast(s)” was performed on Scopus database. Selection of data was done as described in Ref. [1]. Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00020-4 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 20.2 Diversity of Antarctic environments and bioactive compounds obtained from Antarctic microbiota.

20.2

Bioprospection of microbial derived bioactive compounds in Antarctica

In this chapter, we focus on Antarctic-derived-enzymes, which are by far the most explored microbial products from Antarctica, but we also give some insights in drug discovery, antifreezing biomolecules, and nanotechnology.

20.2.1 Enzymes Extremophilic enzymes are preferred for use in diverse industrial processes because of their stability under harsh conditions [6]. While thermophilic enzymes are advantageous because they preserve activity under high temperatures, psychrophilic enzymes can dispense the use of heat in productive processes, since their activity is relatively high at low temperatures [6]. This property would be economically beneficial because energy consumption costs for heat generation are saved. Moreover, when heating is avoided, degradation of thermolabile products in bioreactors is prevented [6]. Here, we focus on describing purified, partially purified, or extracellular enzymes with potential to industrial application from microorganisms isolated from different Antarctic environments. Most enzymes display maximum activities in the range 30 C
37 C, but they are able to significantly retain their activities in temperatures as low as 5 C
15 C. Table 20.1 summarizes isolated Antarctic enzymes and their potential applications.

20.2.1.1 Discovery and purification The first step of discovery of cold-adapted enzymes is the isolation and identification of producing microorganisms from environmental samples. One classic approach for the enzyme discovery is the detection of substrate degradation in diagnostic growth media by microorganisms. In a search for proteolytic enzymes [25], water samples were collected

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TABLE 20.1 Enzyme classes isolated from Antarctic microorganisms, their activity and potential uses. Enzyme class

Source microorganisms

Higher classification

Activity

Application

Reference

Photoliase

Hymenobacter sp. UV-11

Flavobacteria

UV-induced DNA damage repair

Formulation of dermatological cosmetics

Marizcurrena et al. [7]

Leucine dehydrogenase

Pseudoalteromonas sp. ANT178

Gammaproteobacteria

Conversion of branched chain amino acids into alpha-ketoacids

Pharmaceutical synthesis

Wang et al. [8]

Homoserinelactonase

Planococcus versutus L10.15

Firmicutes

Degradation of homoserine-latones (quorum-sensing mediators)

Inhibition of phytopathogens

See-Too et al. [9]

β-Galactosidase

Halorubrum lacusprofundi

Archaea

Hydrolysis of β-galactosides into monosaccharides

Production of lactose-free dairy products

Laye et al. [10]

Pseudoalteromonas sp. 22b

Gammaproteobacteria

Turkiewicz et al. [11]

Alicyclobacillus acidocaldarius

Firmicutes

Gul-Guven et al. [12]

Flavobacterium sp.

Flavobacteria

Arthrobacter sp.

Actinobacteria

Alcohol dehydrogenase

Enantioselective oxidation of alcohols

Pharmaceutical and agrochemical synthesis

Araujo et al. [13]

Dephosphorylation of 50 and 30 ends of DNA and RNA phosphomonoesters

Molecular biology (cloning, probes preparation)

Guthrie et al. [14]

Alkaline phosphatase

TAB5

Keratinase

Lysobacter sp. A03

Gammaproteobacteria

Proteolysis of keratin

Recycling of poultry wastes into livestock feed

Pereira et al. [15]

Chitinase

Lecanicillium muscarium

Ascomycota

Hydrolysis of chitin into N-acetylglucosamine dimers

Control of fungal infections and treatment of chitin-rich waste

Barghini et al. [16]

Sanguibacter antarcticus

Actinobacteria

Xylanases

Cladosporium sp.

Ascomycota

Hydrolysis of β1-4 linkages of xylan

Biobleaching of paper and pulps

Gil-Dura´n et al. [18]

Lipases

Janibacter sp. R02

Actinobacteria

Hydrolysis of acylglicerols, release of fatty acids

Biodiesel production; bioremediation; and detergents

Castilla et al. [19]

Pseudomonas sp. AMS8

Gammaproteobacteria

Ganasen et al. [20]

Geomyces sp. P7

Ascomycota

Florczak et al. [21]

Park et al. [17]

(Continued )

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TABLE 20.1 (Continued) Enzyme class

Source microorganisms

Higher classification

Activity

Application

Reference

Amylases

Nocardiopsis sp. 7326

Actinobacteria

Breakdown of starch polysaccharides

Baking and brewing and food industries

Zhang and Zeng [22]

Tetracladium sp.

Ascomycota

Carrasco et al. [23]

Geomyces pannorum

Ascomycota

He et al. [24]

from an Antarctic lake and aliquots were plated onto Luria
Bertani solid agar. Petri dishes were incubated at 4 C to obtain isolated colonies. Then, isolated colonies were streaked onto minimal milk medium for the detection of proteaseproducing colonies through formation of milk-degradation halo during incubation. It is worth highlighting the importance of incubation at low temperatures for mimicking natural psychrophile conditions at searching cold-active enzymes. A qualitative screening of multiple hydrolytic activities of different yeast strains cultivated from soil, bay, and lake samples collected on King George Island revealed 12 strains produced extracellular proteases when incubated at 8 C but not at 20 C [26]. Among isolated bacteria, Guehomyces pullulans showed the most diverse enzyme production profile at psychrophilic conditions, with the presence of proteases, esterases, amylases, pectinases, and inulinases being detected. This finding corroborates the idea that the bioprocesses for enzyme production themselves benefit from psychrophile characters of Antarctic microorganisms. Enrichment techniques are also used for the screening of certain types of microbial biocatalysts. In these techniques, media compositions are changed so that the survival and growth of microorganisms that are capable of producing a particular type of enzymes are stimulated over other microorganisms. As an example, microorganisms from sediments and soils from King George Island were isolated in media containing (RS)-1-(phenyl)ethanol as the carbon source [23]. This approach led to the isolation of organisms producing enantioselective alcohol-degrading oxidases or dehydrogenases either by selecting constitutively producing microorganisms or by inducting formation of these oxidative enzymes. Classical screening techniques have led to the discovery of most enzymes isolated from Antarctic microbiota, including the notable enzymatic product Antarctic phosphatase derived from psychrophilic strain TAB5 and marketed by New England Biolabs Inc [14,27]. On the other hand, a significant portion of enzymes may not be detected by classic screening due to lack of cultivability of some producing microorganisms [27]. In this sense, genome-based discovery can be useful for enzyme discovery as it dispenses cultivation steps during screening [27]. A typical workflow for identification and laboratory-scale production of cold-tolerant enzymes is described in Fig. 20.3 and comprises the following: (i) screening of degrading enzyme
producing bacteria or fungi, usually by the detection of substrate-degradation halo formed during growth of inoculated organism from an environmental sample on agar plate; (ii) isolation of substrate-degrading colony and cultivation in enrichment liquid media under low temperature (4 C
15 C); (iii) cell concentration and lysis in the case of intracellular enzymes or supernatant concentration in the case of extracellular enzymes; and (iv) multistep purification of lysate or concentrated supernatant (e.g., precipitation and affinity and ionic chromatography). More recently, molecular tools are often used for phylogenetic analyses and enzyme-coding gene identification, cloning, and construction of expression vectors for recombinant production.

20.2.1.2 Activity retention Enzymes obtained from Antarctic microorganisms have been reported as thermostable [12,21,19,25], halotolerant [19,28], acidophilic [12], alkaliphilic [20], organic-solvent-tolerant [20], in addition to cold-adapted activity. Despite that, little is known about biochemical adaptation of biocatalysts in Antarctic environment. One study analyzed β-galactosidase from Halorubrum lacusprofundi (archaeon) from deep-lake sediment and found a small number of differences in enzyme amino acid sequence underlies stability and activity retention [10]. H. lacusprofundi β-galactosidase have been described as polyextremophile, maintaining activity under low temperatures and high salinity and even elevated concentrations of highly oxidative perchlorate salts [29], making this enzyme a good

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273

FIGURE 20.3 A typical workflow describing steps of Antarctic enzymes discovery process.

model for understanding superstability mechanisms. The alignment of β-galactosidases sequences of closely related mesophilic archaea allowed the identification of conserved amino acid residues that differed from those of H. lacusprofundi. Only a small number (,1%) of residues differed from mesophilic enzyme and these were attributed to the retention of enzymatic activity in cold and highly saline environment. Nonconserved residues were substituted by conserved ones in β-galactosidase sequences by site-directed mutagenesis to generate mutated enzymes. Kinetics studies of mutated and wild-type β-galactosidases indicated that those minor differences in amino acid residues are responsible for the reduced surface charge, which loosens binding of water molecules, permitting greater flexibility in low temperatures; the increased negative internal charge, which prevents aggregation in cold and high salinity and the decreased internal amino acid packing, possibly enhancing substrate binding to active site [10].

20.2.1.3 Enzymes for biorefinery and biodiesel production The ability of some microorganisms to grow feeding on complex biopolymeric substrates relies on the secretion of digestive enzymes that degrades these polymers into smaller fragments that can be taken up by the cells [30]. For this reason, many of enzymes used for industrial degradation of such matrixes have been obtained from microorganisms isolated from sources rich in that substrate. In this context, a keratinolytic bacterium Lysobacter sp. A03 has been isolated from decomposing feathers from penguins inhabiting Elephant Island, Antarctica [15]. This bacterium was then cultivated in feather-meal broth, from where a cold-active keratinase was partially isolated [15]. As in the case of keratin, polymeric substrates are usually available from renewable and low-cost sources and may be employed as substrate for biotechnological production of hydrolytic enzymes. Crustacean shells are rich in chitin, an N-acetyl-glucosamine polymer, which can be used as substrate for chitinolytic microorganisms [31]. An optimized large-scale (3 L) cultivation of shrimp-shell degrading Antarctic fungus Lecanicillium muscarium was developed for cold-tolerant chitinase production [16]. The developed bioprocess led to a chitinase production of 243 U/L, with an enzymatic activity described as an endochitinase (i.e., cleaving nonreducing ends of chitin chain). Complimentarily, an endochitinase, which cleaves internal sites of chitin generating shorter fragments, was isolated from Sanguibacter antarcticus [17]. This bacterium was isolated from Antarctic seawater and the purified enzyme retained 40% and 60% of its activity at 0 C and 10 C, respectively. Another example of hydrolytic enzymes are xylanases, which degrades

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β1-4 linkages of xylan, a major component of plant cell walls [32]. Degradation products of xylan varies depending on the hydrolysing enzyme [32]. A thermolabile endoxylanase (a type of xylanase that releases xylooligosaccharides) from sponge-associated fungus Cladosporium sp. has been heterologously expressed and purified [18]. The purified enzyme showed higher activity on arabinose-rich xylans (rye arabinoylan and wheat arabinoxylan). The advantage of applying cold-active degrading enzymes in industrial processes is the reduction of production costs deriving from heat generation. Processes of conversion of poultry waste into reusable materials (for keratinase), treatment of chitin-rich wastes (for chitinases), and pulp and paper bleaching (for xylanases) would benefit from the hydrolytic activities at low or moderate temperatures. Lipases catalyse the hydrolysis of long-chain triacylglycerols yielding free fatty acids, glycerol, and mono- and diglycerols [30]. These enzymes have been applied in food, detergents, and pharmaceutical industries, probably making lipase the most important enzyme class for industrial catalyzes. In biodiesel production, lipases catalyse two-step transesterifications of vegetable, animal, and algal oils [33]. Lipases from Antarctic microorganisms present unusual stability profiles. A lipase from soil bacterium Janibacter sp. R02 presents a thermophilic, halophilic, and alkaliphilic profile [19]. Its optimum activity occurs in pH 8
9, at 80 C, at a 10 mM concentration of NaCl/KCl mixture with a higher affinity for short-chain butyrate (C4) than for heptanoate (C7) and oleate (C18). A cold-tolerant lipase purified from Pseudomonas sp. AMS8 showed good stability in the presence of organic solvents. It displayed 92%, 109%, and 88% of its control (i.e., absence of organic solvents) activity in the presence of 25% (v/v) xylene, octane, and methanol, respectively [20]. Due to the tolerance for usually toxic organic solvents, a simpler method for purifying Pseudomonas sp. AMS8 lipase has been developed [34]. The method consists of a two-step (from aqueous phase to an organic phase and back to an aqueous phase) liquid
liquid extraction using a mixture of toluene and Triton X-100, a non-ionic surfactant, as organic phase. The extraction recovered 43% of the enzymatic activity when performed at 10 C—for comparison purposes, gel-filtration chromatography recovers 23% of activity. This extraction method dispenses the need of onerous chromatographic steps, making purification more suitable for industrial settings.

20.2.1.4 Enzymes for pharmaceuticals and cosmetics production As discussed earlier, Antarctic environment acts as pressure to produce biomolecules that enable organisms’ survival in that extreme environment. In the case of UV light, its incidence in Antarctic region is more intense due to ozone layer depletion [2]. Photolyase is a class of enzymes with the ability of repairing UV-damaged DNA [7]. UV lights induced the formation of pyrimidine dimers, impairing the transcription process by RNA polymerase and ultimately leading to skin cancer [7]. A photolyase gene has been identified in genome of lake bacterium Hymenobacter sp. UV-11 [7]. Heterologous expression of this gene in Escherichia coli allowed the purification of the recombinant enzyme preserving its UV-photoprotective activity. Recombinant photolyase could remove pyrimidine dimers of UV-irradiated DNA and repair UV-induced DNA damage in CHO and HaCat cells. The application of the Antarctic photolyase in the dermatological formulations could generate valuable product with skin cancer preventive action. A leucine dehydrogenase from the Antarctic sea-ice bacterial strain Pseudoalteromonas sp. ANT178 was purified after heterologous expression in an E. coli DE3 vector [8]. This enzyme catalyzes the conversion of branched chain amino acids (with the highest specificity for leucine), to their corresponding α-ketoacids by oxidative deamination. This type of catalysis is important for pharmaceutical industry because the conversion products (e.g., L-tert-leucine) are used as active ingredients in pharmaceutical industry [8]. The cold-active leucine dehydrogenase obtained from Pseudoalteromonas sp. ANT178 showed a maximum activity at 30 C and pH 9. However, the enzyme retained 40% of its activity at 0 C and 65% at 15 C. The most prominent example of L-tert-leucine derivative is atazanavir, one of most prescribed antiretroviral drugs for the treatment of HIV infection [35].

20.2.1.5 Enzymes for agriculture and brewing N-Acylhomoserine-lactones are a group of signaling molecules used in quorum-sensing of bacterial communities [9]. The Antarctic bacterium Planococcus versutus L10.15 is known to produce the enzyme N-acylhomoserine-lactonase, which degrades homoserine-lactones at high rates [9]. The optimum degrading activity is at 28 C, but the enzyme retains 60% of this activity at 18 C. Thus P. versutus N-acylhomoserine-lactone might be used in quorum-quenching (i.e., interrupting quorum-sensing communication between bacteria) for inhibiting some plant diseases. Soft-rot disease in cabbage is caused by phytopathogen Pectobacterium carotovorum, the phytopathogeny mechanism of which relies on homoserine-lactones-based quorum-sensing. In one experiment, P. versutus N-acylhomoserine-lactone was able to inhibit rotting of Chinese cabbage caused by P. carotovorum. This inhibition was due to quorum-quenching, which attenuated plant-tissue degradation by the phytopathogen [9]. In another approach, enzymes from Antarctica

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275

microorganisms have been used in the brewing process optimization. Amylases are responsible for the degradation of starch into mono- and oligosaccharides and are of value for different industries, including brewing and baking [30]. Two of most important enzymes of this class are α-amylases, which break down α-(1,4) links within polysaccharide chain, and glucoamylases, which cleave nonreducing ends of starch chains, releasing glucose [30,36]. Beer production demands enzymes as adjuncts for brewing processes and amylases may be employed at various steps of brewing process and they improve fermentability properties of wort [36]. As brewing fermentation processes take place at relatively low temperatures: lagers at 10 C
15 C and ales at 15 C
20 C, the use of cold-adapted enzymes becomes beneficial. A cold-active α-amylase was obtained from the actinomycete Nocardiopsis sp. 7326, which was isolated from Antarctic deep-sea sediment [22]. While α-amylase activity maximum occurs in 35 C, more than 70% of that is maintained at 20 C and 35% at 0 C. In addition, a cold-adapted glucoamylase was isolated from Antarctic soil yeast Tetracladium sp. through heterologous expression [23]. Activity of this enzyme was optimum at 30 C, retaining more than 50% of this activity at 22 C. As most glucoamylases present enzymatic optimum at 45 C
50 C, Antarctic glucoamylase would provide high catalytic activity in industrial process with low-temperature requirements.

20.2.1.6 Immobilization of Antarctic-derived enzymes As discussed throughout this section, Antarctic enzymes are excellent catalysts at low and moderate temperatures, with promising industrial applications. However, industrial processes require high-yielding biocatalysts to be reused and this is achieved by immobilization techniques [37]. Immobilization improves physical
chemical stability, allowing enzymes to remain active after multiple cycles [37]. Immobilization of cold-active enzymes could improve feasibility of low-temperature processes by dispensing energy consumption and recovering usually expensive biocatalysts. An α-amylase from Geomyces pannorum, a psychrotolerant fungus isolated from continental Antarctica, was covalently immobilized onto iron-oxide magnetic nanoparticles, resulting in an evident stability enhancement [24]. Both soluble and immobilized amylase displayed an activity optimum at 40 C. When immobilized, the enzyme retained more than 40% of its optimal activity at 20 C and 70 C, while soluble enzyme activity was below 25% in either case. In addition, 90% of its activity was retained after three cycles and 60% was retained after eight cycles of reutilization. In another case, chitosan beads were used as support for covalent immobilization of β-galactosidase from Pseudoalteromonas sp. 22b, isolated from the digestive system of Antarctic krill Thysanoessa macrura [11,26]. Retention of enzyme activity after immobilization was 53%, with lactose hydrolysis being carried at 4 C for 48 h. From that a recycling system for continuous lactose hydrolysis (1 mL/min) was developed with a column packed with enzyme-functionalized beads. The system reached a steady efficiency of 93% of lactose hydrolysis at 18 h of operation. The activity of continuous lactose hydrolysis lasted for 40 days at 15 C.

20.2.2 Drug discovery 20.2.2.1 Antimicrobial drug discovery Antarctica’s extreme environments with different abiotic characteristics is home to a large variety of microbial species [3]. This combination makes Antarctica a promising site for obtention of bioactive compounds due to the diversity of metabolic pathways resulting from evolutionary adaptation to cold and nutrient-limited environments [38]. Production of extracellular antimicrobial compounds as secondary metabolites is assumed to be an adaption strategy by conferring producing microorganisms a competitive advantage. In one study, fungal strains isolated from marine environments showed production of metabolites with antimicrobial action against important human pathogens [39]. Cultivation of isolated strains at low temperatures positively influenced the production of antimicrobial substances: Atradidymella sp. showed higher production at 4 C, while Pseudogymnoascus sp. and Penicillium flavigenum more intense production at 15 C. This finding highlights the adaptative ability in producing and excreting antimicrobials at polar environments and the role of temperature in regulating production of these bioactive compounds. Table 20.2 summarizes antimicrobial activities of compounds isolated from Antarctic microbiota and Fig. 20.4 depicts the structures of these compounds. As most identified antimicrobial molecules are excreted during microbial growth, preliminary characterization on their biological activity is performed using cell-free fermentation media containing metabolic products. Using this approach, a search of antimicrobial-producing strains from Antarctic permafrost identified six Bacillus sp. strains with relevant antimicrobial activity against pathogenic bacteria [5]. Among them, Bacillus safensis showed strong inhibition of multidrug-resistant Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus INA 00761. Fermentation broth of halophilic bacterium Nocardioides sp. A1 isolated from soil samples from Antarctica also showed strong activity against Bacillus subtilis and rice-phytopathogen Xanthomonas oryzae [46]. Purification, structural characterization,

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TABLE 20.2 Antimicrobial and antiviral components derived from Antarctic microorganisms. Source microorganism Pseudomonas sp. BTN1

Component(s) Rhamnolipids C1 and C2

Test microorganism(s)

Activity measure

Burkholderia cenocepacia

MIC

Values (μg/ mL) 3.12 (C1 and C2)

Reference Tedesco et al. [40]

1.56 (C1) and 3.12 (C2)

Staphylococcus aureus Aequorivita sp.

Aminolipids

Methycilin-resistant S. aureus

IC50

22

Chianese et al. [41]

Penicillium sp. SCSIO 05705

Questiomycin A

Mycobacterium tuberculosis

MIC

0.83

Wang et al. [42]

Streptomyces griseus

Frigocyclinone

Bacillus subtilis DSM 10

MIC

4.6

Bruntner et al. [43]

15.3

S. aureus Spiromastix sp.

Aspergillus ochraceopetaliformis

Spiromastixone J

Ochraceopone A (oa), asteltoxin (as) and isoasteltoxin (ia)

Methycilin-resistant S. aureus

IC50

1.0

Methycilin-resistant Staphylococcus epidermidis

0.5

Vancomycin-resistant Enterococcus faecalis

2.0

H1N1 H3N3

IC50

0.10 (as) and 0.23 (ia)

Niu et al. [44]

Wang et al. [45]

5.5 (oa), 0.28 (as), and 0.35 (ia)

Their activity against selected test strain is listed as minimal inhibitory concentration (MIC) or IC50 as available from the respective reference.

and further studies of bioactivities of isolated compounds may then proceed in the case of confirmed positive activity in those preliminary tests. Partial characterization of organic extract of Nocardioides sp. A1 growth medium revealed active substances consisted mainly of glycolipids and/or lipopeptides [46]. This discovery is in accordance with the described fact antimicrobial activities of lipidic compounds from natural sources [47]. Different antimicrobial lipid
based substances from Antarctic microorganisms have been isolated. Rhamnolipids, a particular class of bacterial lipids, are structurally composed by a rhamnose sugar moiety bound to a 10- to 12-carbon long lipid chain [40]. Rhamnolipids named C1 and C2 have been purified from the marine-sediment bacterium Pseudomonas sp. BTN1 obtained from Antarctica samples and displayed antibacterial activity against Burkholderia cenocepacia isolated from patients with cystic fibrosis and a standard strain of S. aureus [40]. Aminolipids are another special class of microbial lipids. Their structural skeleton comprises a fatty acid amide chain esterified with a second fatty acid chain through their C3 hydroxy groups [41]. Three aminolipids bearing an N-terminal glycine residue were isolated from shallow-sea-sediment bacterium Aequorivita sp. [41]. These three compounds were effective against methycilin-resistant S. aureus (MRSA). Apart from lipids, a variety of compounds have also been described from Antarctic microorganisms with potential to be used in treatments of bacterial infections of great public health concern, including in cases of antibiotic resistance. Alkaloids questiomycin A from soil Penicillium strain SCSIO 05705 displayed potent antituberculosis activity [42]. Antibacterial activity H37Ra strain of Mycobacterium tuberculosis was close to that of antituberculosis antibiotic isoniazid, used as positive control [42]. Frigocyclinone, an antibiotic, the structure of which consists of a four-ring moiety attached to an aminodeoxysugar through a C-glycosidic linkage to an aminodeoxysugar, was isolated from the soil actinomycete Streptomyces griseus strain NTK 97 [43]. It has been described as a good inhibitor of B. subtilis DSM 10 and S. aureus DSM 20231.

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FIGURE 20.4 Structure of selected antimicrobial metabolites isolated from Antarctic microorganisms.

Spiromastix sp. fungus from deep-sea sediment produces spiromastixones, the skeleton of which consists of two chlorinated benzene rings linked through an ester and an ether bridges [44]. Structurally related spiromastixones A
O present variable degree of antimicrobial activity against multidrug-resistant clinical strains [44]. Among them, spiromastixone J is the most promising of isolated compounds with a 16-fold more potent inhibition of MRSA and 4-fold better activity against methycilin-resistant Staphylococcus epidermidis than the control antibiotic levofloxacin. While spiromastixone J also resulted in strong inhibition of two vancomycin-resistant Enterococcus sp. isolates, levofloxacin, had minor effect. Antarctic bioactive molecules are effective to pathogens other than bacteria. Three compounds with antiviral activity were extracted from fermentation broth of Aspergillus ochraceopetaliformis SCSIO05702 isolated from an Antarctica’s soil sample [45]. These compounds, sequiterpenoids ochraceopone A, asteltoxin, and isoasteltoxin, were more potent against H1N1 and H3N3 viruses than antiviral drug oseltamivir [45]. As we described here, Antarctic microbiome is a source of structurally diverse compounds. Harsh conditions in that continent drives genetic-level evolutionary adaptations leading to diversified metabolic pathways [38]. The drugsynthetizing property of psychrophiles constitutes a biotechnological potential to transpose the ability to eliminate competitors in natural habitats to generate more efficient medicines to inhibit/kill human pathogens, especially when treatments no longer respond to available therapies.

20.2.2.2 Anticancer drug discovery Screening of antibiotics from natural sources has led to discovery of efficient drugs for treatment of cancers [48]. Commonly, antibiotics that made their ways to cancer chemotherapy present DNA-binding action interfere with transcription or replication of DNA and block cell replication [48]. For that reason, microbial secondary metabolites from environmental samples have inhibitory cross action against cancer cells. Thus, metabolite-producing Antarctic microorganisms become good candidates for anticancer drug search. Table 20.3 summarizes anticancer activities of compounds isolated from Antarctic microbiota and Fig. 20.5 depicts the structures of these compounds.

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TABLE 20.3 Components derived from Antarctic microorganisms and their IC50 against selected cell lines. Source microorganism

Component(s)

Cell line(s)

IC50 (μg/ mL)

Hs578T (breast cancer)

B7a

HeLa (breast cancer)

B5a

A375 (melanoma)

B14a

1-Chloro-3β-acetoxy-7-hydroxytrinoreremophil-1,6,9-trien-8-one

HL60 (leukemia)

3.34 6 0.05

A549 (lung carcinoma)

3.45 6 0.03

Streptomyces sp. SCO736

Antartin

A549

4.45

U87 (brain cancer)

8.06

Penicillium sp. SCSIO 05705

Meleagrin

K562 (myelogenous leukemia)

1.54 6 0.21

U937 (lymphoma)

1.18 6 0.22

Botryidiopsidaceae sp. (microalga)

Penicillium sp. PR19N-1

Ethanolic extract

Neoxaline

Oidiodendron truncatum GW3-13

Chetracin B

Melinacidin IV

Bacillus sp. N11-8

PBN11-8 (polypeptide)

K562

1.73 6 0.13

U937

2.13 6 0.14

HCT8

0.009 6 0.04

Bel-7402

0.002 6 0.03

HCT8

0.038 6 0.05

Bel-7402

0.004 6 0.02

Bel-7402

1.56

HepG2 (liver cancer)

1.57

Panc-28 (pancreatic cancer)

1.73

Reference Suh et al. [49]

Wu et al. [50]

Kim et al. [51]

Wang et al. [42]

Li et al. [52]

Zheng et al. [53]

a

Approximately calculated from data available on paper.

In vitro studies against cancer cell lines have demonstrated the anticancer potential of metabolites present in cell lysates and extracts as well as in culture supernatants of Antarctic microorganisms. In a screening study [54], fermentation broths from cultivation of seven microbial strains isolated from Antarctic seawaters were tested in cytotoxicity assays against several cell cancer lines. The most promising results of this screening were inhibitions between 35% and 68% of viability of human hepatocellular carcinoma cells from Bel7402 line [54]. Although most studies describe antitumoral metabolites from fungi and bacteria, Antarctic microalgae are also promising sources of these compounds. Dried biomass of Antarctic freshwater microalga Botryidiopsidaceae sp. was submitted to extraction of bioactive compounds with ethanol as extractive solvent [49]. The microalgal extract displayed a selective antiproliferative action (i.e., no effect observed on noncancerous cell lines) against different human cancer cell lines. The most evident results were against Hs578T and HeLa (breast cancer) and A375 (human melanoma) cell lines, which were inhibited in a concentration- and time-dependent manner. The anticancer activity of this extract is at least partially due to induction of apoptosis. Treatment of HeLa cells increased expression of p53 and caspase-3 (proapoptotic proteins) and decreased expression of Bcl-2 (antiapoptotic protein) [49]. In addition, it was observed an impaired invasion and migration capacities through basement membrane [49]. Sesquiterpenes are a class of natural products of 15-carbon skeleton synthesized by biochemical pathways in plants and marine organisms [55]. These small molecules are of great interest for drug discovery because of their varied

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FIGURE 20.5 Structures of compounds isolated from Antarctic microorganism with described anticancer activity.

biological activities, with good potential for cancer treatment, and availability from natural sources [56]. Isolation and identification of excreted secondary metabolites belonging to sesquiterpenoid class was achieved for deep-sea-sediment fungus Penicillium sp. PR19N-1 [50]. Among identified metabolites, a novel chlororinated eremophilane-type sesquiterpene 1-chloro-3β-acetoxy-7-hydroxy-trinoreremophil-1,6,9-trien-8-one (molecular formula C14H15ClO4) was the only one with cancer-inhibiting property. HL60 (human leukemia) and A549 (human lung carcinoma) cell lines suffered moderate inhibition when treated with that compound [50]. Another anticancer compound, Antartin, a zizaane-type sesquiterpene, was purified from Streptomyces sp. SCO736 fermentation broth [51]. Treatment of cells from A549 and H1299 (lung cancer) and U87 (brain cancer) lines with 10 μg/mL antartin led to almost complete proliferation inhibition. For lung cancer lines the formation of tumorigenic foci was prevented, indicating reversal of capacity of cells to aggregate into tumors. Anticancer activity of antartin against lung cancer is underpinned by the suppressed expression of Ki-67 (proliferation marker) and cyclins and cyclin-dependent kinases (cell-cycle-regulation factors) [51]. Microbial anticancer compounds of other classes have also been identified microorganisms isolated from Antarctica’s samples. Alkaloids meleagrin and neoxaline from soil Penicillium strain SCSIO 05705 displayed potent cytotoxic activity against U937 (lymphoma) and K562 (myelogenous leukemia) [42]. Chetracin B and melinacidin IV, belonging to epipolythiodioxopiperazine class, were isolated from soil fungus Oidiodendron truncatum GW3-13 [52]. Chetracin B showed cytotoxic activity against HCT-8 (human colorectal tumor) and Bel-7402 (hepatocellular carcinoma), while melinacidin IV inhibited Bel-7402 cells at nanomolar concentrations. Activities of both Penicillium and O. truncatum metabolites were more potent than that of classic chemotherapy drug paclitaxel, which was used as a positive control in proliferation assays. An extracellular polypeptide was identified from fermentation broth of marine Bacillus sp. N11-8 [53]. The peptide PBN11-8 displayed significant cytotoxicity against Bel-7402, HepG2 (liver cancer), and Panc-28 (pancreatic cancer). Bel-7402 inhibition was further studied and a reduction in the migration and invasion abilities of tested cells was observed. The expression of integrin β1, an important mediator of cell survival, proliferation, differentiation, and migration in cancer [57], was also downregulated in the presence of PBN11-8. The peptide also hindered the expression of matrix metalloproteinases, which participate in metastasis process by degrading extracellular matrix [53].

20.2.3 Ice-binding proteins One adaptive mechanism for survival in extremely cold regions is the synthesis of molecules that inhibits ice formation inside cells [58]. Ice-binding proteins (IBPs) have the unique capacity to interact with ice surface and prevent growth of

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ice crystals [58,59]. In polar marine environments, water temperature is commonly under 0 C as salinity decreases seawater freezing point. In order to survive in such cold environments, IBPs are synthetized, which is essential for keeping tissue integrity in Arctic and Antarctic fishes as ice crystal formation could cause cell disruption [58]. Due to this ability, these proteins have potential for application in process where freezing damage is undesirable such as food preservation and cell storage [58]. Despite more commonly described in fishes, antifreezing proteins may also be isolated from Antarctic psychrophile microorganisms. Here, we describe some of microbial sources of these antifreezing agents and discuss some potential applications. Cold-induced IBP expression has been observed in the Antarctic yeast Glaciozyma antarctica [60]. A set of nine different genes coding for IBPs were overexpressed as temperature decreased from 15 C to 212 C. Structural analysis of IBPs from bacteria that survived a freezing
thawing selection test revealed ice-binding regions contains multiple and regularly spaced threonine residues [59]. IBPs from Sphingomonas GU1.7.1, Plantibacter GU3.1.1, and Pseudomonas AFP5.1 were isolated and used as cryopreservatives of cucumber and zucchini. The fruits were treated with IBPs in a concentration of 0.1 mg/mL and frozen at 220 C for 16 h. While disrupted cell walls and dead cells were observed in nontreated fruits, IBP-treated tissues retained cell viability and cell wall integrity. In another approach, an IBP from Flavobacterium frigoris was supplemented in freezing medium along with DMSO for preservation of microalga Isochrysis galbana [61]. Cells viability after thawing was greater in comparison to DMSO used as the only cryopreservative. Thermal hysteresis (TH) is the parameter used for measurement of cryoprotective activity and corresponds to the difference between freezing and melting points. IBPs from marine flagellate Pyramimonas gelidicola [62] and diatom Chaetoceros neogracile [63] have close TH values, which are also similar to fish IBPs. Ice crystals formed in the presence of IBPs from all microorganisms presented here showed a hexagonal shape, which is related to antifreezing activity. These results reinforce the potential of IBP to be used as cryoprotective agents for food preservation and cell culture purposes.

20.3

Nanoparticles

Besides organic molecules, some microorganisms are excellent producers of inorganic nanoparticles through biomineralization processes [64]. Application of nanoparticles is present in diverse fields in technology. Microbial synthesis of nanoparticles is considered green chemistry because it does not use expensive and toxic chemicals, which are often necessary for synthetic production [64]. Quantities of produced nanoparticles may be increased by scaling up cultivation from lab flasks to 100-L bioreactors [64]. Despite underexplored, Antarctic biomineralizing microorganisms have been described and constitute a promising platform for the synthesis of these nanomaterials [64].

20.3.1 Cadmium nanoparticles Peroxide-resistant psychrophile strains of Pseudomonas spp. were grown from Antarctic samples through cultivation under in oxidative stress conditions [65]. These bacteria synthetize semiconductor cadmium sulfide (CdS) nanoparticles when cultured at 15 C in a medium supplemented with H2O2 and CdCl2. Two tellurium-resistant Pseudomonas sp. and one Psychrobacter sp. that also produce CdS nanoparticles were isolated through tellurite enrichment [66]. For these strains, production is better achieved at 28 C. The uptake of Cd ions from environments at relatively low temperatures characterizes these microorganisms as excellent candidates for bioremediation of heavy-metal-contaminated waters and soils in polar and subpolar regions [66]. In addition to removal of these pollutants, minerals formed from captured metals can be recovered as these microorganisms accumulate these components in form of nanoparticles. These nanostructures range in size from 10 to 40 nm as measured in observation of producing cells by transmission electron microscopy. Semiconductor nanoparticles from Antarctic bacteria have emission peaks between 500 and 600 nm when excited at 400 nm [65]. The fluorescence properties of CdS enable nanoparticles to be used as quantum dots in medical image diagnostics.

20.3.2 Iron-oxide nanoparticles A peculiar group of iron-mineralizing prokaryotes has been identified in extreme aquatic environments, including Antarctica [67]. The so-called magnetotactic bacteria synthetize iron-oxide nanoparticles, either magnetite (Fe3O4) or greigite (Fe3S4) depending on the biomineralizing species. These structures, known as magnetosomes, are disposed in a single or multiple chain along bacterial cell long axis [67]. The iron mineral is in the nanoparticle core and is surrounded by a biological membrane, possibly derived from cell’s inner membrane [68]. Magnetotactic bacteria constitute

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FIGURE 20.6 Transmission electron microscopy of a magnetotactic coccus from marine sediments from Monsiment Cove, King George Island. Arrow indicates one of the magnetosome chains.

a morphologic, phylogenetically and metabolically diverse group. Phylogenetic analyses based on the gene sequence encoding the 16S rRNA have led to the identification of magnetotatic bacteria in the phylum Proteobacteria (classes Alpha-, Delta-, Gamma- and, more recently, Beta- and Zetaproteobacteria), in the phylum Nitrospirae, in the superphylum Planctomycetes
Verrucomicrobia
Chlamydiae and possibly in the candidate phylum Latescibacteria [69]. Magnetotactic cocci (Fig. 20.6) from Antarctica marine sediments belong to the Alphaproteobacteria class and produce elongated magnetite magnetosomes disposed in two or four chains or nonorganized in chains [70]. Dimensions of aligned magnetosomes ranged between 117
102 nm in length and 67
72 nm in width. Unlike chemical synthesis processes (i.e., iron precipitation), microbial production of magnetite yields nanoparticles with finely controlled size, morphology, and chemical purity [68]. These unique characteristics are result of a genetically controlled biomineralization process and enable magnetosomes to be used in a variety of applications [1]. Purified magnetosomes have been employed as support for enzyme immobilization, nanoformulations for magnetically driven drug delivery, alternating magnetic field
induced hyperthermia agents for cancer treatments, contrast agents for magnetic resonance imaging, among others [1].

20.4

Conclusion and future directions

This brief review about Antarctica’s microbial metabolic content/products showed the inestimable value of this extreme environment as a scientific preserve and biotechnological repository. Hopefully, future studies on psychrophilic/psychrotolerant microorganisms and their enzymes will allow the development of new technologies, processes, and products. In this sense, future research should focus on the upscaling of cultivation of psychrophiles, high-yield purification of bioactive products, and genetic engineering for an optimized production of Antarctic biotechnological products.

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Chapter 21

The secretomes of extremophiles Eyad Kinkar and Mazen Saleh Department of Biology, Laurentian University, Sudbury, ON, Canada

21.1

Introduction

The term “extremophiles” refers to microorganisms that inhabit extreme environments and includes members of both the bacteria and the archaea. Bacterial systems with regards to their secretomes have been treated extensively in the literature but not so the archaea. This chapter will therefore focus the discussion on the secretomes of archaeal extremophiles. Archaea is the third domain of life and is characteristically divided into two major phyla, the Euryarchaeota and the Crenarchaeota [1]. Since 2003, new archaea have been discovered distinct from those of the two major phyla and were proposed to have their own phyla, including the Nanoarchaeota [2] and the Korarchaeota [3]. Some members of this domain are recognized for their ability to thrive in extreme conditions, including high salinity, temperatures, extreme pH, and others. Euryarchaeota exhibits high phenotypic diversity and includes methanogens, halophiles, thermoacidophiles, and some hyperthermophiles. The diversity of Crenarchaeota appears to be limited to hot and aquatic environments [1,4]. Archaea share some similarities from other domains in life but more so from the bacteria rather than the eukaryotes. Research has shown that archaea mimic most of the metabolic pathways that are found in bacteria and can be either heterotrophs or autotrophs [5]. The archaeal cell wall differs substantially from the bacterial cell wall in that they do not have a proper peptidoglycan. Archaeal cell walls can either be composed of heteropolysaccharides or pseudomurein but not the peptidoglycan [6]. Researchers have shown that most archaeal cells are surrounded by a single glycerolipid membrane that consists of lipids with linked ethers of glycerol backbone to a repeated isoprenyl group (C40), while the bacteria glycerolipids are made of esters of fatty acids and glycerol. In extreme environments, it is thought that the C40 isoprenoid acyl chains that form the monolayer are more effective at stabilizing the membrane [7,8]. Protein trafficking has been extensively studied in bacteria and eukarya but lagged in the third domain of life. Despite the presence of unique physiological adaptations in, research has shown that the transport of proteins across membranes operates by common principles in all domains of life [1,7 9]. The majority of secreted proteins are expressed as preproteins with an N-terminal cleavable signal peptide. This signal is required for targeting to the cytoplasmic membrane and subsequent translocation beyond the membrane. These signal sequences can be of two types: I and II [8 10]. The former targets proteins to the general secretory pathway (Gsp) and the latter to the twin-arginine translocation (Tat) pathway (Fig. 21.1) [11]. The Gsp pathway (SecYEG) translocates proteins in an extended unfolded conformation, whereas the Tat pathway translocates proteins, particularly those with redox cofactors, in a folded conformation. Upon translocation across the membrane specialized peptidases cleave the signal sequence and release the mature protein [12]. Research has shown that most, if not all, members of the archaea possess a surface layer (S-layer) that is different from the bacterial S-layer because it is anchored directly to the cytoplasmic membrane [8,9]. This chapter will discuss and analyze the substrates of the two well-characterized protein translocation systems described earlier (the SecYEG and the Tat pathways). It is important to define the secretome before discussing it and for our purposes, we will define the secretome as the fraction of proteins that are translocated across the cytoplasmic membrane and contain either Type I, Type II, or Tat signal sequences, according to Fig. 21.2.

21.2

The Sec pathway

The Sec complex in bacteria is known as SecYEG complex, and in eukaryotes it is known as Sec61p. In archaea the SecGhomolog is nonessential and is more homologous to the Sec61β subunit of the eukaryotes than to the bacterial Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00021-6 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 21.1 A schematic of the proposed organization of the Tat (1) and the Sec (2) pathways in archaea. The pilin secretion system is also shown for comparison. It is important to note that for archaea, there are no SecG homologs, but rather a homolog of the eukaryotic Sec61β component. Archaea also lack a SecA homolog.

Type I signal sequence M

N-Domain

H-Domain

C-Domain AXA

Mature protein

H-Domain

C-Domain AXG

C Mature protein

C-Domain AXA

Mature protein

Type II signal sequence M

N-Domain

Tat signal sequence M

(S/T)RRXFLK

H-Domain

FIGURE 21.2 General features of the Type I and Type II signal sequences. The arrow identifies the signal peptidase cleavage site. The AXA concensus shown is not universal. For the Type II signal sequences the Cys in the N-terminus of the mature protein immediately follows the cleavage site. The N-domain varies in length but is typically dominated by polar or positively charged amino acids. The H-domain also varies in length but is typically between 12 and 15 amino acids long. It is dominated by hydrophobic and neutral amino acids (I,L,V,A,G,S,W,M,F,T) and lacks any charged amino acids. The C-domain contains the cleavage site (AXA) and is much shorter than the H-domain. This domain varies greatly in composition, including the actual cleavage site.

SecG [13]. The β notation is therefore used on occasions to signify the translocation system of archaea as SecYEβ. Components of the Sec translocation system in archaea show homologies to components of both the bacteria and the eukarya [14,15]. As shown in Table 21.1, one of the signal recognition particle (SRP) components in archaea (SRP19) is more homologous to the component in eukaryotes, whereas the other component, Ffh, is typical of what is found in bacteria [14,15]. Similarly, the RNA component of the SRP in archaea is designated as 7S, as in eukaryotes, whereas in Escherichia coli it is 4.5S and in Bacillus subtilis it is scRNA [16,17]. Once the N-terminal signal sequence emerges from the ribosome during translation, the binding of this sequence by the SRP can arrest the translation process. In cotranslational translocation the entire complex of SRP nascent polypeptide ribosome is targeted to the cytoplasmic membrane and docks onto the SecYEG translocon via the SRP receptor [12,18]. Other proteins can be targeted to the translocon posttranslationally and with the aid of SecB, a cytoplasmic partner of the translocon. The bacterial SRP is a ribonucleoprotein complex composed of a homolog of eukaryotic SRP54 known as fifty-four homolog (Ffh), a GTPase, and 4.5S RNA, with its receptor being the FtsY (filamentous temperature-sensitive Y) [19,20]. In archaea, this general

The secretomes of extremophiles Chapter | 21

287

TABLE 21.1 Comparison of the components of the membrane translocation system (for class I signal sequences) in the endoplasmic reticulum and those in the cytoplasmic membrane in bacteria and archaea. Component

Endoplasmic reticulum

Escherichia coli

Bacillus subtilis

Archaea

Sec translocase

Sec61αβγ

SecYEG

SecYEG

SecYEβ

Sec-associated subunits

Sec62/63, Sec71/72, TRAM

SecDFYajC

SecDFYajC

SecDF

Motor ATPase

Bip

SecA

SecA

TatA, TatB, TatC, TatE

TatAdCd, TatAyCy

TatA, TatC

Tat translocase SRP RNA

7S

4.5S

scRNA

7S

SRP protein components

SRP54, SRP10, SRP9/14, SRP68/72

Ffh

Ffh, HBsu

Ffh, SRP19

SRP receptor

α,β

FtsY

FtsY

FtsY

SRP, Signal recognition particle.

secretory pathway is conserved in all members examined to date, but unlike bacteria, they do not express the chaperone SecA. It is still unknown how archaea translocate secreted protein through their cell wall, since research has shown that archaea lack a few translocation factors that are found in bacteria and eukaryotes, including ATPases SecA, YidC, Kar2p/BiP, TRAM, and Sec63 [21].

21.3

The Tat pathway

The Tat pathway is known as a Sec-independent system because ultimately secreted proteins that utilize this pathway possess twin-arginine signal sequences [22]. In bacteria, this system is used to transport secreted proteins across the inner membrane. On the other hand, in chloroplast, it was found that proteins are directly imported from the stroma. For both bacteria and chloroplasts, the Tat pathway requires three major membrane-bound components for a successful translocation, including TatA, TatB, and TatC (bacteria) or their chloroplast equivalents, Tha4, Hcf106, and cpTatC, respectively [23,24]. The Tat system utilizes the proton motive force to energize the process of translocation, whereas in the SecYEG system, ATP is used. Bacterial genetic analysis has shown that some microorganisms surprisingly do not encode for TatB, including archaea, and rely heavily on TatA and TatC to secret folded proteins [25,26]. Substrates of the Tat system contain an N-terminal signal sequence similar in many aspects to that of the Sec substrates (Fig. 21.2). The most distinguishing feature however is a double Arg in the N-domain of the signal sequence. The Tat system preferentially selects for prefolded proteins containing a cofactor [11,24]. Ng et al. [27] have suggested that Halobacterium sp. NRC-1 expresses two isoforms of TatC known as TatC1 and TatC2. TatC1 is predicted to have six membrane-spanning domains and N-terminal cytoplasmic domain that is composed of about 100 amino acid residues. TatC2 protein is predicted to have two domains that share 45% identity linked by two membrane-spanning domains each of which is similar to TatC proteins [27]. Surprisingly, the cytoplasmic region of the linker shows 40% identity to the TatC1 cytoplasmic domain. On the other hand, research could not identify TatB homologs in this archaeon [24,27]. In Gram-negative bacteria, as few as 6% of all secreted proteins are directed to this pathway but in some halophilic archaea in silico prediction showed that the Tat pathway is utilized to secret as much as 90% of their secreted proteins [24,27]. Recently, it has been shown that the Tat pathway is important of bacterial infection in animals and plants, which raises a few questions toward archaeal pathogenicity. With recent discoveries of archaea in the human microbiome, it will be interesting to see what research will discover in the role that these microorganisms play within the microbiome [28,29].

21.4

The signal sequence

The Sec-dependent signal sequence is known as class I N-terminal signal sequence and can be between 15 and 38 residues long, with some exceptions. It is divided into three domains: the N-domain, the H-domain, and the C-domain (Fig. 21.2). The N-domain is between two and eight residues long and mainly dominated by polar or positively charged residues. The H-domain is the middle region that is dominated by neutral or hydrophobic residues and can be as long as 15 residues. The C-domain is dominantly made up of polar residues and contains the signal peptidase recognition site [12,30 32].

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Unlike the Tat system, Sec-dependent substrates do not carry a consensus sequence within their signal sequence. Genetic analysis has indicated that Tat-secreted proteins possess a consensus sequence in a form of (S/T)-R-R-X-F-LK, where X can be any polar residue and K is not shown to be consistent in all Tat-secreted proteins [12,24,26]. Class II signal sequences share some similarities to class I N- and H-domains but the C-domain contains a conserved lipobox consensus sequence in the form of L-P-X-T-G-C, where signal peptidase II cleaves just before the cysteine residue (Fig. 21.2). Once the cleavage occurs, the cysteine becomes modified with a lipid and anchors the protein to the membrane [33,34]. Class III signal peptides are related to prepilin-like proteins and carry a positively charged N-domain and a hydrophobic H-domain as in Class I and II signal sequences. However, for prepilin-like proteins, the N-domain is truncated once the signal peptidase recognizes the (I/L)(P/A)-X-TG consensus motif, thus allowing the H-domain to remain part of the mature protein, as it plays a role in the pilin structure assembly [35]. Class IV signal sequences direct substrates to the ABC-type transporters and typically lack the H-domain. Some have been shown to possess a double glycine motif by the cleavage site [36]. Representative Type I signal sequences from Halobacterium salinarum NRC-1, Methanobacterium lacus, Natrinema sp. J7-2, and Thermococcus nautili are shown in Table 21.2. For simplicity, only a small number of predicted secreted proteins are shown, but they provide the essential features of the signal sequences and allow for comparison between the different archaea selected in this example. The identification of putative Type I signal sequences in this example was carried out using the ExProt program and the Gram-positive signal sequences as a training dataset [37]. Qualitative assessments of these sequences reveal that the sequences among the selected archaea are similar in some aspects and different in others. ExProt allows for a number of variations in the prediction of the cleavage site but it is evident from Table 21.2 that there seems to be preference for cleavage following A or G in the C-domain of the signal sequences. Other features include the relatively frequent occurrence of R in the N-domain. This observation was in fact made earlier and was proposed to be due to the fact that R occurs in higher frequency in archaea [34]. Similar analysis and presentation for lipoproteins (Type II signal sequences) for these archaea is shown in Table 21.3. For these sequences, it was observed that there is relatively less variation in the sequences than observed for the Type I signal sequences presented in Table 21.2. This was also observed to be the case when the entire secretomes were compared. In identifying secreted proteins, whether it is carried out experimentally or in silico, membrane proteins are often among those identified. Experimentally, membrane proteins will be isolated from the secreted protein fraction (culture supernatant) because they are released during loss of membrane fragments as the microbes divide and age/lyse. This will be the case even if the fraction is collected during the exponential growth phase. For the in silico analysis, membrane proteins will also be among those identified as secreted proteins because of the similarities between the Type I signal sequences and the membrane anchor sequences found in integral membrane proteins.

21.5

Secretomes of archaea

Genomic and proteomic analysis of archaea lagged behind that of the bacteria primarily due to difficulties in cultivating the microorganisms. Genome sequences for a limited number of archaea became available 20 years ago and experimental genomic and proteomic work was also limited. There have been a few studies early on making use of bioinformatics analysis to try and fill gaps in this information. The study of Saleh et al. [34] reported on the in silico analysis of 24 archaeal genomes. The putative secretomes reported in that study ranged from 6% of total open reading frames (ORF) for Methanopyrus kandleri to 19% for Halobacterium sp. NRC-1. For comparison to bacterial secretomes, it was noted by another study that the archaeal secretomes tend to be smaller in size [34]. For some bacteria the secretomes made up over 35% of the total ORF (Pseudomonas aeruginosa PAO1 and Thiobacillus denitrificans ATCC 25259). The study of Saleh et al. also pointed out that archaea appear to invest in more lipoproteins as compared to bacteria (in terms of fraction of secretomes). In one instance, as many as 81 putative lipoproteins were predicted for Methanosarcina acetivorans. There are those however that appear to show the opposite phenomenon, with as a few as 1 predicted lipoprotein (Pyrobaculum aerophilum and Nanoarchaeum equitans). Other features of archaeal signal sequences highlighted in that study include the abundance of the amino acids Thr, Val, and Gly within the H-domain, whereas in bacteria, this region of the signal sequences is dominated by Leu and Ile. Other amino acid variations include the abundance of the amino acid Arg but reduced frequency of Lys, as compared to bacterial proteins in general. Other observations to note include the fact that the typical Tat substrate consensus of (S/T)-R-R-X-F-L-K is not conserved in archaea and modifications to the amino acids S/T, F, L, & K are much more tolerated than in bacteria [34]. Surprisingly, almost all the aligned predicted lipoprotein signal sequences contain a twin arginine sequence which may suggest that they are transported through the Tat pathway. Some recent reports on experimental analysis of secreted proteins from a number of archaea have shed new light on the nature of signal sequences in these

TABLE 21.2 Representative class I signal sequences as identified by ExProt. Archaea

Gene ID

Sequence

Annotation

Halobacterium salinarum NRC-1

graD2

MQAIVVAAGRGTRMGPLTETRPKPLVPVAGATLLEHVLDAAA GVV

Glucose-1-phosphate thymidylyltransferase

rpl15e

MARSFYSHIKEAWEDPDDGKLA ELQ

50S ribosomal protein L15E

ilvE1

MRYHVNGALVDAADATVSVRDRGFQYGDAAFETMRA YGG

Branched-chain amino acid aminotransferase

flaB1

MFEFITDEDERGQVGIGTLIVFIAMVLVAA IAA

Flagellin B1 precursor

flaB2

MVLVAAIAAGVLINTAGYLQSKGSATG EEA

Flagellin B2 precursor

ADZ09689.1

MKKNKEQIKTFESYRKNYPIEIQKRLDQIQEAIKSA VPE

Domain of unknown function DUF1801

ADZ09694.1

MKPENFDVKGADVSWCPGCGNFSILSNLKNVLA DLE

Pyruvate ferredoxin/flavodoxin oxidoreductase

Methanobacterium lacus

Natrinema sp. J7-2

Thermococcus nautili

ADZ09697.1

MENENFKAMVSNIARKGFKNKQTLGFAVVAPLIVLIVLGYMVTMA GYT

ABC-2 type transporter

ADZ09708.1

MDPIYSNIAANKRWTYLFFLFYALMLG AIG

protease htpX

ADZ09712.1

MLGFNTIYAGSGYDSSFYNVRNYTG

Heat shock protein DnaJ domain protein

AFO56238.1

MREWDAMVPDSLRTALIVVTLGSLLVLSSVATGAGA VSS

Putative lipoprotein carrier protein, LolA

AFO55867.1

MMDGSSRPTVFVVLMTLLLVTSPVVAATG SLT

Subtilisin-like serine protease

AFO57928.1

MKRSRRNVLRTASTLSAIVAGVGVGTA AAT

PKD domain containing protein

AFO59077.1

MGATSGTATPAHSLAASDGTDDS

PKD domain containing protein

AFO57659.1

MSLWRSRRTGYYLALVAVTTVVSTLVYNYGMA TLE

TrkA-N domain protein

AHL22014.1

MPARVVGEEKVKLKKSFVKPWMG IKY

Formatehydrogenlyase subunit 6 (chain I)

AHL22022.1

MVSILKRGLAIITLLIIGYWLAQGLA GVP

Multisubunit Na 1 /H 1 antiporter, MnhB subunit

AHL22026.1

MEEASRASKFAYTFIVLFILWLVVTA SLD

Multisubunit Na 1 /H 1 antiporter, MnhE subunit

AHL22230.1

MKFCPKCGNLMLPDRKKKVWVCRVCGYEEPFDEEKDRDKT RIT

DNA-directed RNA polymerase

AHL22239.1

MARWNVCSYCGREFEPGTGKMFVRNDGRVLFFCSSKCEKYYFMG RNP

Ribosomal protein L24e

TLR

YVD

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TABLE 21.3 Representative class II signal sequences as identified by ExProt. Archaea

Gene ID

Sequence

Annotation

Halobacterium salinarum NRC-1

ugd

MRVSIVGSGYVGTTVAACFTD

UDP-glucose dehydrogenase

prrC

MRRRTYLSLVGSAAAAGTAGCLGV

Regulatory protein

htrA

MPSRRDVLRLGAGVLAAGTAGCTDT

Serine proteinase

fbr

MQRRREFLQATGAALAAVGLAGCSDS

Cytochrome-like protein

imp

MPTEHTRRRFLQATGATSIAALAGCAGG

Immunogenic protein

ADZ08296.1

MERSIHECDVLVIGSGGAGCRAA

L-Aspartate

ADZ08759.1

MVKIMTRNPNHELYKLITISSLLIISCLLT

Signal transduction histidine kinase

ADZ09152.1

MKITIVGTGYVGLVTGACFSE

Nucleotide sugar dehydrogenase

ADZ09182.1

MERGQIYRCNTCGNMFELINVGGGTSSCCGT

Desulfoferrodoxin

ADZ09207.1

MKIVDGGVCAVNGVLAAGACDDD

Arginine biosynthesis bifunctional protein ArgJ

AFO55363.1

MTRRSVLAATGVGATTALAGCIGG

TRAP transporter solute receptor, TAXI family

AFO55498.2

MSNDRTWTRRNVLRTGGAIAGISAMAGCLDS

Ferrichrome-binding protein

AFO55704.1

MRRRTFVGAVGGSAAAGVAGCLTR

ABC transporter periplasmicbinding protein thiB

AFO56206.1

MSDRIGAPGLGLSRREFVAATGGAAALTGLAGCTGQ

Multicopper oxidase type 2

AFO56675.1

MNRRGFLRRTATIGAVGTVGTAGCLEG

Fe21 transport protein

AHL21682.1

MKKLTTLALVAVLVLSMVVAGCIGG

TRAP-type uncharacterized transport system

AHL21774.1

MRKALALLVILLVVSVAGCIGT

ABC-type Fe31-hydroxamate transport system

AHL21887.1

MKKMSFLLVALLLLATVGAGCITS

Uncharacterized protein conserved in archaea

AHL21999.1

MRTWIPLLIIALLIGSGCISG

Phospholipid-binding protein

AHL22002.1

MKKAGVLLLLLVLLGFSAGCIGS

Thiol-disulfide isomerase and thioredoxins

Methanobacterium lacus

Natrinema sp. J7-2

Thermococcus nautili

oxidase

microorganisms. Proteomic analysis of the secretome of Natrinema sp. J7-2 has demonstrated that up to 30% of proteins with predicted (using TATFIND) Tat signal sequences can be experimentally detected in the culture medium [38]. It was surprising to see such a large number of predicted Tat substrates (142 in total), but more surprising was their finding that 87 of the 142 predicted Tat substrates also have the Type II signal sequence (lipoproteins). Experimentally, 30 of the 87 lipoproteins were detected [38]. For Sec-dependent signal sequences the results were not as agreeable. In comparing the predictions of these signal sequences using SignalP trained on either the eukaryotic signal sequences, Gram-positive, or Gram-negative bacterial signal sequences, experimental detection rates were the highest for those predicted to be Sec substrates using the SignalP trained on Gram-positive sequences. This confirms earlier conclusions from bioinformatic analyses [34]. In this manner the authors reported on 46 putative Sec substrates, of which 10 were detected experimentally. Three interesting Sec substrates in that report, which were also predicted as Sec substrates by ExProt, are a lipoprotein carrier protein Lo1A superfamily, a subtilisin-like serine protease, and a PKD domain containing protein (Table 21.2). For Tat substrates, our in-house program ExProt identifies 25 putative secreted proteins (Table 21.4). Only four of these were defined as possible lipoproteins. These included one hypothetical protein, a phosphate-binding protein, an Fe21 transport protein, and a multicopper oxidase Type II. All four lipoproteins with a Tat signal sequence were identified in the culture supernatant of Natrinema sp. J7-2 [38]. For non-Tat putative lipoproteins, ExProt identifies a total of

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291

TABLE 21.4 Putative Tat substrates as identified by ExProt from Halobacterium salinarum NRC-1. Gene ID

Sequence

Annotation

celM

MDDARRTFLDELLAVHSPTGHEAAA QRV

Endoglucanase

dmsA

MSDTDLNATRRDVLKSGAVAAVGLSGGGLLSTLQEADDSDTA GDA

Dimethylsulfoxide reductase

ibp

MSEESGFRRRTFLAATGAATLSGLAGCSGMLGSNSST DDR

Iron-binding protein

phr2

MPAAQPPGMQLFWHRRDLRTTDNRGLAAAAPGVTA VDG

Photolyase/cryptochrome

proX

MMDTPEHASTSSRRQLLGMLAAGGTTAVAGCTTITGGGDTTSTGG GDS

Putative ABC transporter

imp

MPTEHTRRRFLQATGATSIAALAG CAG

Immunogenic protein

pcy

MHRRAFLAGGTTLSVGVLAG CIG

Plastocyanin homology

chi

MPHDRRSYLRTSSAVIASLLAASTPTSAA DTP

Chitinase

rbsC2

MSQDTPRWRRAIARLVDASVKERLVISVAALVLA VAV

Ribose ABC transporter permease

hcpA

MSAMGRAPDRRTFLRSAVAGGLAAIAG CTD

Halocyanin precursor like

sub

MRQTRRTFMKTAAAAIGGLSATTQPVTAEGVRDDQFLVDTQSATS DAW

Subtilisin homolog

hpb

MDRRNFLKTAGAAGTIGISGLSG

Possible phosphate-binding protein

dppD

MFKYAIVPYSRMSDDTVSRRGFLKAAGAATVVATSTAGCTDSGGG GDG

Dipeptide ABC transporter ATPbinding

AAG18883.1

MTRGFYIGRFQPFHTGHRRVIEQIATEVDELVVGIGSAGDS HSA

Conserved hypothetical protein

AAG19088.1

MGGSSVPPMISVLERRTCGRALSVAA SRG

Conserved hypothetical protein

AAG19934.1

MSRLSKSQVIRRYYLYRATARPGFHYAIYTFFLLFNGLSYT QIG

Conserved hypothetical protein

AAG20219.1

MLARQRARRDTTGGCAMSSGIIPVVATIIA LGV

Conserved hypothetical protein

AAG20486.1

MRRRDYLRAVGGGATGVAAAG CLQ

Conserved hypothetical protein

AAG19338.1

MADFDRREFLKLAGGTVGASLVAG CSS

Conserved hypothetical protein

AAG20014.1

MHRRQFLTGLGISLGGATVGSSTRARTVITDTTAATA DDR

Conserved hypothetical protein

AAG18667.1

MPTENSRRGSTSEQSSCTVPANA PPS

Hypothetical protein

AAG19449.1

MMACTRRKALAAVGTTLSLSG CAR

Hypothetical protein

AAG19261.1

MNSDQRGVPRREFLKAAVAIGGASALSA CLG

Hypothetical protein

AAG19346.1

MNRRAFLTASAGLGSTAALAG CLG

Hypothetical protein

AAG20412.1

MRTRRQFLATTTSLTTVGLLAG CAR

Hypothetical protein

CLG

63, as opposed to 87 reported by Feng et al. [38]. It is logical to conclude that the secretome of a microorganism reflects the specific physiology of the microorganism with respect to its needs for carbon and nitrogen sources, specifically. Natrinema sp. J7-2 is capable of growth on gluconate, glycerol, or acetate as sole carbon sources [39]. This is reflected by the ability of this microorganism to secrete polysaccharide hydrolases and uptake systems. It is also capable of utilizing ammonium ions as a source of nitrogen. This is reflected by the presence of various ammonium transport proteins in the secretome of this microorganism [38,39] (Table 21.5). A similar study was carried out on another heterotrophic archaeon, Pyrococcus furiosus. This organism is a hyperthermophile and analysis of its secretome uncovered new information regarding the link between the secretome and environmental adaptations. Protein identification of the culture supernatant for this archaeon grown on starch produced a list of 15 secreted proteins with different N-terminal signal sequences, although a total of 58 proteins were identified in this fraction [40]. Of the 15 identified with predicted signal sequences, 7 show typical Type I signal sequences, 5 with Type II, and 3 with Type III signal sequences. The latter refers to prepilin signal peptides. Secreted proteins with Type I signal sequences included solute-binding proteins, alpha amylase, amylopullulanase, and hypothetical proteins. Those with a Type II signal sequence were made up mostly of solute-binding proteins. The remaining 43 proteins in the culture supernatant did not contain recognizable cleavable signal sequences. The

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TABLE 21.5 Putative Tat substrates as identified by ExProt from Natrinema sp. J7-2. Gene ID

Sequence

Annotation

AFO55499.1

MDPNPWAGMPVVRRSYALRFLAALVVIVLIVGLFGGSIYA QTG

Chemotaxis sensory transducer

AFO55661.1

MMADELKRRLVHASGSGLVALYLLA NYL

Dolichol kinase-like protein

AFO56206.1

MSDRIGAPGLGLSRREFVAATGGAAALTGLAG CTG

Multicopper oxidase type 2

AFO56445.2

MPPRRRGHGTRLLMALVGCLLLLWSLALAAISYWALSALRASA PDP

Peptidase M48 Ste24p

AFO56675.1

MNRRGFLRRTATIGAVGTVGTAG CLE

Fe21 transport protein

AFO56833.1

MRSTQLGRFRGGISRRTFIAATGGTGLTGLAG

AFO56838.1

MMADNQFGRDAVSRRKFIAAAGITGTVAA AGC

Phosphate ABC transporter

AFO57217.2

MPGDNNQVSRRRFMKSAGSATVVATAA SSV

Family 5 extracellular solute-binding protein

AFO57665.1

MTYPQLNRRHVLQAIGATGAVAVAGCLGGDAQG SST

Nitrite reductase, copper-containing

AFO57824.1

MSIRSVAKKDFLDVRRSKSVWIVSGLYALLVVCFFYLG

ABC-2 type transporter

AFO57925.1

MKRTRRRVIRNASLVTAALAGVPAVSA ADA

Chitinase

AFO57936.1

MNDRSRRKLLQSAGATTTLGLAGCLDGFTGSGGAGDA VRA

Family 1 extracellular solute-binding protein

AFO58178.1

MGRDTTGQCDRARLRRRSFLAAASASTAGAVA VTG

Family 1 extracellular solute-binding protein

AFO58305.1

MTTELSRRRFVDVLTATVIAAYVLVALGTAVSTTDSAA ACS

Protoheme IX farnesyltransferase

AFO58542.1

MGNDATEHEAPTRREYVKYGGTVVGGGLLAGCAGLSDSDS TPE

Putative iron transport protein

AFO58828.1

MAEYEGLDRRGLLGLLGASATAGIAGCLGGNRPGSG

Lipoprotein

AFO58894.1

MRRRELLAGIGSVGVLTGGVGVVLGGVPSFG DEP

CLG

Phosphate binding protein

QSG

GDD

Alkyl hydroperoxide reductase

AFO59339.1

MTMSEQDGAGVSRRSVLLSAGAAAGMLGLGGHSA LQS

Trimethylamine-N-oxide reductase

AFO55568.1

MGPGLDERSSGGSHSRWHVTRREALVALGGAALAASTGPSKTTMA ADR

Hypothetical protein

AFO56200.1

MTDDNSNRFGLGRRPVLGGLAGAVATGAVGTAAA SRQ

Hypothetical protein

AFO56873.1

MTMSGARGSRRPLPNGCRKLLALALGLAVIGSLVATGAGA SAS

Hypothetical protein

AFO57003.1

MMGGETRREPPTRRDALKYGMTLTAGVTISG CSD

Hypothetical protein

AFO59109.1

MVEFTRRKLMASSAAAAIGMGALGTASA ADT

Hypothetical protein

AFO56200.1

MTDDNSNRFGLGRRPVLGGLAGAVATGAVGTAAASRQQHDGSTA DTI

Hypothetical protein

AFO56792.1

MWIAAIDRRVPMATARRRVASRALASNRSKTVITA AVA

Hypothetical protein

vast majority of those proteins were annotated as metabolic enzymes, including oxidoreductases. Another proteomic study on an archaeon was that of Palmieri et al. [41]. The study was conducted on Aeropyrum pernix K1, another hyperthermophilic. The archaeon was cultured in a commercial broth (marine broth, Difco) with sodium thiosulfate. The exoprotein fraction, making up those proteins released to the culture medium, contained 40 proteins. In that study, bioinformatic analysis of the proteome of A. pernix K1 using SignalP for prediction of Type I signal sequences provided a total of 278 putative secreted proteins (16% of the proteome). Interrogation of the proteome for Tat-like signal sequences using the TATFIND program predicted nine proteins only [41].

21.6

Conclusion and future directions

The general secretory system evolved early in cells and is conserved in all domains of life. This is unsurprising as cells’ survival was dependent on their ability to communicate with their environments and exploit environmental resources

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for nutrients. There are a few differences in the nature and number of factors associated with this secretion system, but the essential components are common: cytoplasmic factors (chaperones/SRP), membrane-associated factors (SRP receptor/signal peptidase/ATPase), and a membrane channel. In archaea the SecG component is replaced with Secβ, a component of the Sec complex with the same function essentially as SecG. The variations in the secretion-associated factors in archaea potentially reflect the physiological adaptations of these extremophiles; as cellular proteins change in their amino acid composition, for example, changes in the secretion-associated factors must also change accordingly to maintain function. We continue to see an increase in the number of archaea genomes being sequenced. This surely adds to our knowledge of archaeal physiology, and for protein secretion specifically. Bioinformatics has also been instrumental in exposing the nature of proteins elaborated by members of the archaea in different environments and culture conditions. The secretome may play a more active role in industrial applications in the future. Industry already exploits a number of archaeal metabolites and enzymes for a variety of applications, including the use of compatible solutes in cosmetics, enzymes in processes requiring extremozymes, and others in optical switches and holography.

References [1] Forterre P. Evolution of the Archaea. Theor Popul Biol 2002;61:409 22. [2] Huber H, Hohn MJ, Rachel R, Fuchs T, Wimmer VC, Stetter KO. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 2002;417:63 7. [3] Elkins JG, Podar M, Graham DE, et al. A korarchaeal genome reveals insights into the evolution of the Archaea. Proc Natl Acad Sci USA 2008;105:8102 7. [4] Woese CR, Kandlert O, Wheelis ML. Towards a natural system of organisms: proposal for the domains. Proc Natl Acad Sci USA 1990;87:4576 9. [5] Huber R, Huber H, Stetter KO. Towards the ecology of hyperthermophiles: biotopes, new isolation strategies and novel metabolic properties. Elsevier 2000;24:615 23. [6] Kandler O, Ko H. Cell wall polymers in Archaea (archaebacteria). Cell Mol Life Sci 1998;54:305 8. [7] Pohlschroder M, Pfeiffer F, Schulze S, Farid M, Halim A. Archaeal cell surface biogenesis. FEMS Microbiol Rev. 2018;42:694 717. [8] Ellen AF, Zolghadr B, Driessen AMJ, Albers S. Shaping the archaeal cell envelope. Hindwi Publ Corp 2010;2010:1 13. [9] Sa M, Sleytr UWEB. S-Layer proteins. J Bacteriol 2000;182:859 68. [10] Szabo Z, Pohlschroder M. Diversity and subcellular distribution of archaeal secreted proteins. Front Microbiol 2012;3:1 14. [11] Yen MR, Tseng YH, Nguyen EH, Wu LF, Saier MH. Sequence and phylogenetic analyses of the twin-arginine targeting (Tat) protein export system. Arch Microbiol 2002;177:441 50. [12] Lycklama A Nijeholt JA, Driessen AJM. The bacterial Sec-translocase: structure and mechanism. Philos Trans R Soc B Biol Sci. 2012;367:1016 28. [13] Kinch LN, Saier MH, Grishin NV. Sec61β—a component of the archaeal protein secretory system. Trends Biochem Sci 2002;27:170 1. [14] Alavian CN, Ritland Politz JC, Lewandowski LB, Powers CM, Pederson T. Nuclear export of signal recognition particle RNA in mammalian cells. Biochem Biophys Res Commun 2004;313:351 5. [15] Sinning I, et al. Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation. Nat Commun 2015;6:1 11. [16] Pohlschro¨der M, Dilks K, Hand NJ, Rose RW. Translocation of proteins across archaeal cytoplasmic membranes. FEMS Microbiol Rev 2004;28:3 24. [17] Yuan J, Zweers JC, Van Dijl JM, Dalbey RE. Protein transport across and into cell membranes in bacteria and archaea. Cell Mol Life Sci 2010;67:179 99. [18] Randall LL, Hardy SJS. SecB, one small chaperone in the complex milieu of the cell. Cell Mol Life Sci 2002;59:1617 23. [19] Seluanov A, Bibi E. FtsY, the prokaryotic signal homologue, is essential for biogenesis of membrane proteins. Biochemistry 1997;272:2053 5. [20] Neumann-Haefelin C. SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein. EMBO J 2002;19:6419 26. [21] Albers SV, Szabo´ Z, Driessen AJM. Protein secretion in the Archaea: multiple paths towards a unique cell surface. Nat Rev Microbiol 2006;4:537 47. [22] Chaddock AM, et al. A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the delta pH-dependent thylakoidal protein translocase. EMBO J 2018;14:2715 22. [23] Lee PA, Tullman-Ercek D, Georgiou G. The bacterial twin-arginine translocation pathway. Annu Rev Microbiol 2006;60:373 95. [24] Robinson C, Bolhuis A. Protein targeting by the twin-arginine translocation pathway. Nat Rev Microbiol 2001;2:350 6. [25] Bolhuis A, et al. TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli. J Biol Chem 2001;276:20213 19. [26] Mori H, Cline K. A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase. J Cell Biol 2002;157:205 10.

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[27] Ng WV, et al. Genome sequence of Halobacterium species NRC-1. Proc Natl Acad Sci USA 2000;97:12176 81. [28] Dridi B, Henry M, El Khe´chine A, Raoult D, Drancourt M. High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol. PLoS One 2009;4:1 6. [29] Drancourt M, et al. Evidence of archaeal methanogens in brain abscess. Clin Infect Dis 2017;65:1 5. [30] Akita M, Sasaki S, Matsuyama S, Mizushima S. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem 1990;265:8164 9. [31] Papanikou E, Karamanou S, Economou A. Bacterial protein secretion through the translocase nanomachine. Nat Rev Microbiol 2007;5:839 51. [32] Luirink J, Sinning I. SRP-mediated protein targeting: Structure and function revisited. Biochim Biophys Acta—Mol Cell Res. 2004;1694:17 35. [33] Storf S, et al. Mutational and bioinformatic analysis of haloarchaeal lipobox-containing proteins. Hindwi Publ Corp 2010;2010:1 11. ˜ . Indicators from archaeal secretomes. Microbiol Res 2010;165:1 10. [34] Saleh M, Song C, Nasserulla S, Leduc LGA [35] Dramsi S, Trieu-cuot P, Bierne H. Sorting sortases : a nomenclature proposal for the various sortases of Gram-positive bacteria. Elsevier 2005;156:289 97. [36] Michiels J, Dirix G, Vanderleyden J, Xi C. Processing and export of peptide pheromones and bacteriocins in Gram-negative bacteria. Trends Microbiol 2001;9:164 8. [37] Saleh MT, Fillon M, Brennan PJ, Belisle JT. Identification of putative exported/secreted proteins in prokaryotic proteomes. Gene 2001;269:195 204. [38] Feng J, Wang J, Zhang Y, Du X, Xu Z, Wu Y, et al. Proteomic analysis of the secretome of haloarchaeon Natrinema sp. J7-2. J Proteome Res 2014;13:1248 58. [39] Feng J, Liu B, Zhang Z, Ren Y, Li Y, Gan F, et al. The complete genome sequence of Natrinema sp. J7-2, a haloarchaeon capable of growth on synthetic media without amino acid supplements. PLoS One 2012;7:e41621. [40] Schmid G, Mathiesen G, Arntzen MO, Eijsink VGH, Thomm M. Experimental and computational analysis of the secretome of the hyperthermophilic archaeon Pyrococcus furiosus. Extremophiles 2013;17:921 30. [41] Palmieri G, Cannio R, Fiume I, Rossi M, Pocsfalvi G. Outside the unusual cell wall of the hyperthermophilic archaeon Aeropyrum pernix K1. Mol Cell Proteom 2009;8:2570 81.

Chapter 22

Carbonic anhydrase from extremophiles and their potential use in biotechnological applications Claudiu T. Supuran1 and Clemente Capasso2 1

Sezione di Scienze Farmaceutiche, Dipartimento Neurofarba, Universita` degli Studi di Firenze, Florence, Italy, 2Istituto di Bioscienze e Biorisorse,

CNR, Napoli, Italy

22.1

Extremophiles

Extremophiles come from Bacteria, Archaea, and Eukarya domains. Those from Bacteria and Archaea are represented entirely by microorganisms, whereas within the Ekarya domain, they are mainly algae forming lichens, fungi, protozoa, including the organisms adapted to live at a very low temperature (e.g., the Antarctic fish) or creatures tolerant to low, high temperatures, and high doses of radiation, such as the microscopic invertebrates, known as Tardigrades [1]. The extremophiles populate life-challenging niches and, thus, are exposed to hot or cold temperatures, high concentrations of salt, as well as acid or alkaline conditions, toxic waste, organic solvents, heavy metals, high pressure, or other environmental situations considered unfavorable for life [2]. Generally, five branches typify the extremophiles, which are as follows: (i) thermophiles, organisms living at temperature $ 45
C, which are subdivided into moderate thermophiles (45
C70
C), extreme thermophiles growing optimally at temperature $ 70
C, and hyperthermophiles, organisms growing at very high temperatures (optimal temperature $ 80
C); (ii) psychrophiles, organisms adapted at low temperatures; (iii) acidophiles and alkaliphiles, organisms tolerating high acidic or basic pH values, respectively; (iv) barophiles, organisms that grow under high pressure; and (v) halophiles, organisms that tolerate high concentration of salt [2,3]. To cope with these extreme habitats, the extremophiles evolved biomolecules adapted to operate under such harsh environmental conditions (temperatures, salinity, pH, pressure, and solvent). It is important to stress that the mesophilic counterparts cannot survive at all the conditions previously mentioned [4,5]. The macromolecules of the extremophiles are characterized mainly by high stability and optimal enzymatic activity offering unique biotechnological advantages over the mesophilic enzymes (optimally active at 25
C50
C), which are often not well suited for the harsh reaction conditions required in industrial processes. As a result, extremophiles are a source of biocatalysts, which can be extensively used in biotechnological purposes, such as esterases/lipases, glycosidases, aldolases, nitrilases/amidases, phosphatases, racemases, thermostable DNA polymerases, and enzymes used in the biofuel production or mining processes [5].

22.2

Bacterial carbonic anhydrases

A pivotal reaction of the central bacterial metabolism is the CO2 hydration/dehydration reaction. This reaction is connected with numerous metabolic pathways, such as photosynthesis and carboxylation reactions, and biochemical processes such as pH homeostasis, secretion of electrolytes, and transport of CO2 and bicarbonate [6,7]. Moreover, the interconversion of CO2 and HCO2 3 occurs spontaneously at a slow rate and is precisely fine-tuned in all living organisms to maintain the equilibrium between dissolved inorganic carbon dioxide (CO2), carbonic acid (H2CO3), bicarbon
22 ate ðHCO2 Þ, and carbonate CO [811]. The survival of the microorganism is dependent on the availability of 3 3 these metabolites because they are essential for the biosynthesis and energy metabolism of the microbe [12]. The CO2 hydration/dehydration is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1) [1317], which are categorized into eight genetically distinct families (or classes), named α-, β-, γ-, δ-, ζ-, η-, Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00022-8 © 2020 Elsevier Inc. All rights reserved.

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FIGURE 22.1 Distribution of CAs in Gram-negative bacteria. The α-CA is a periplasmic protein able to convert the diffused CO2 inside the periplasmic space into bicarbonate, whereas the cytosolic β- and γ-CAs are capable of feeding of CO2 and bicarbonate the central bacterial metabolism.

and ɵ-CAs. [1317]. Up to date, it has been demonstrated that the bacterial genome encodes only three of these classes: α-, β-, and γ-CAs [1723]. In Fig. 22.1 the simple but physiologically crucial interconversion of carbon dioxide and water into bicarbonate and 1 protons is shown ðCO2 1 H2 O " HCO2 3 1 H Þ [13,17,2429], as well as the transport of carbon dioxide and bicarbonate assisted by bacterial CAs. The bicarbonate indispensable for the bacterial metabolic processes cannot be provided through the uncatalyzed naturally occurring CO2 hydration/dehydration reaction because, at physiological pH, the rate is too low (kcat hydration 5 0.15 s21 and kcat dehydration 5 50.0 s21), whereas the catalyzed reaction has a rate of 104106 s21 [21,30]. It has been speculated that in Gram-negative bacteria, the α-CA, characterized by an N-terminal signal peptide, is able to convert the atmospheric and/or metabolic CO2 diffused in the periplasmic space, while β- or γ-classes have a cytoplasmic localization and are responsible for CO2 supply for carboxylase enzymes, pH homeostasis, and other intracellular functions ensuring the survival and/or satisfying the metabolic needs of the microorganism [31,32]. It is interesting to note that bacteria show an intricate distribution pattern of the CA-classes. It is true that the bacterial genome encodes for the three CA-classes (α, β, and γ), but it is also common to identify bacteria, the genome of which encodes only for one or two CA-classes and, rarely, for any CAs [21,33]. Recently, it has been demonstrated that the primary structure of the β-CAs identified in the genome of the pathogenic Gram-negative bacteria, such as Helicobacter pylori, Vibrio cholerae, Neisseria gonorrhoeae, Streptococcus salivarius present at the N-terminal part a secretory signal peptide of 18 or more amino acid residues [21,30]. Intriguingly, the CAM enzyme, which is a γ-CA, contained a short putative signal peptide at its N-terminus, too. Since the signal peptide is essential for the translocation across the cytoplasmic membrane in prokaryotes, probably the β- or γ-CAs characterized by the presence of a signal peptide might coexist in the periplasmic space together with the α-CAs [21,30].

22.3

Carbonic anhydrases in extremophilic bacteria

Since many bacteria are incredibly abundant in environments that are hostile to all other forms of life, our groups focused their scientific attention on the presence of CAs in thermophilic microorganisms. The study of the biochemical and physical properties of the “extreme” bacterial CAs has led to the discovery of molecular features, which make them different from those of the mesophilic counterpart allowing their use in biotechnological fields generally typified by conditions deleterious for the enzyme activity [4,34]. As results, the extreme CAs appeared exciting candidates to be used in industrial and medical applications, such as the postcombustion carbon capture process and the realization of artificial lungs and biosensors [23,3546]. Thus CA enzymes can be considered as a biotechnological multitasking

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superfamily because the CA-classes are potentially able to fight both the increase of CO2 in the atmosphere produced by the anthropogenic activities and for ameliorating the human health because of their biomedical interests. In this context, two CAs, identified in the genome of Sulfurihydrogenibium yellowstonense and Sulfurihydrogenibium azorense and belonging to the α-CA class, have been investigated by our groups [4,4754]. The CO2 hydratase activity of SazCA (kcat 5 4.40 3 106 s21) was the highest among the extreme and other known CAs described in the literature. In fact, up to that time, the most active CA was the human isoform hCA II with a kcat 5 1.40 3 106 s21. Moreover, SazCA with a kcat/KM value of 3.5 3 108 M21 s21 is the second most efficient enzyme after the superoxide dismutase. SspCA also showed an excellent catalytic activity for the same reaction, with a kcat value of 9.35 3 105 s21 and a kcat/KM value of 1.1 3 108 M21 s21. Fascinating was the behavior of SspCA at high temperature (Fig. 22.2). As shown in Fig. 22.2, SspCA resulted to be highly thermostable retaining an excellent catalytic activity when heated for a prolonged period (more than 180 min) to 100
C [4,47,49,5155]. The SspCA and SazCA X-ray tridimensional structures resolved by our groups provided the rationale at molecular level for their thermostability: they have a dimeric structure characterized by high compactness, a higher content of secondary-structural elements, an increased number of charged residues on the protein surface, and a significant number of ionic networks with respect to the mesophilic counterparts [4,34]. Fig. 22.3 shows the 3D fold of the SspCA.

FIGURE 22.2 SspCA thermostability. The enzyme was incubated for a different time at the temperatures indicated on the X-axis and assayed using CO2 as substrate. Each point is the mean 6 SEM of three independent determinations. Incubation time: 30, 60, 120, and 180 min.

FIGURE 22.3 The dimeric structure of SspCA with a monomer in blue and the other one in red.

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22.4

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Potential use of extreme carbonic anhydrases in biotechnological applications

22.4.1 Biosensors As described in the literature, various biosensors based on CAs have been obtained. For example, those for checking the toxic effects of zinc on marine life [56] or measuring sulfanilamide pharmaceutical residues in biological and environmental samples [56]. In this context, the thermostable α-CAs, SspCA and SazCA, could be used for ameliorating the stability of these biosensors. Intriguing, natural CA-based biosensors exist, and they are represented by the microorganism living in extreme environments, which are employed in detecting leaks of CO2 from storage areas by evaluating their CA gene and protein expression [57].

22.4.2 Artificial lungs Recently, the human CA has been covalently immobilized to the surface of hollow fiber membranes, which are used in the realization of artificial lungs [58] for accelerating the removal of carbon dioxide from blood for the treatment of acute respiratory failure [58]. These CA-based artificial lungs were subjected to an inadequate transfer of CO2 across the hollow fiber membrane, which might be improved by increasing the rate of the blood mixing. Unfortunately, this strategy denatures the mesophilic CA immobilized on the fiber membrane because of the shearing force. To overcome this limitation the thermostable CAs identified in the genome of thermophilic bacteria could be used to obtain more efficient artificial lungs.

22.4.3 Post-combustion carbon dioxide capture The increase in the atmosphere of the CO2 concentration developed by the anthropogenic activities and the greenhouse effects of this gas has stimulated the scientific community to focus their research on various solutions for lowering the CO2 emission. As demonstrated by the existence of numerous patents, one elegant and, above all, an eco-compatible strategy is the post-combustion carbon dioxide capture through the use of “robust” CAs (biomimetic strategy). The biomimetic approach represents an exciting strategy for CO2 capture. It allows CO2 conversion to water-soluble ions, offering many advantages over other methods, for example, its eco-compatibility and the possibility to use the enzyme for multiple cycles. Moreover, since thermophilic proteins are thermostable and thermoactive with respect to the mesophilic counterpart, they are preferred in environments characterized by hard conditions, such as those of the carbon capture process (high temperature, high salinity, and extreme pH). In addition, there are some disadvantages using the free enzyme in solution because the repeatable usage of the biocatalyst is limited, and generally it is not possible to recover the catalyst from the reaction mixture. Fortunately, these disadvantages can be eliminated immobilizing the enzyme on specific supports. The term “immobilized enzymes” is referred to enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously [59]. The immobilization of enzymes on solid supports is historically very important for overcoming their general instability in harsh operational conditions and their low shelf-life, as well as the need for their recycling more times [59,60]. Furthermore, the physical separation of the biocatalyst from the reaction mixture avoids the protein contamination of the products. Although a reduction in reaction rates sometimes occurs, because the enzyme cannot mix freely with the substrate or a particular conformational change is needed for the biocatalyst efficiency, there are many examples of increased activity and stability of immobilized enzymes [61]. Many chemical or physical methods for the enzyme immobilization are currently available, from the physical adsorption to the covalent coupling on supports [59,60], such as adsorption (ionic interaction, hydrogen bonds, and van der Waal forces), covalent binding, cross-linking, copolymerization, and encapsulation (membrane confinement) (Fig. 22.4). As shown in Table 22.1, the matrixes or supports commonly used for immobilization of enzymes or whole cells are grouped in two major categories: (i) organic (natural polymers and synthetic polymers) and (ii) inorganic materials [59,60]. The characteristics of the matrix are crucial in determining the performance of the enzyme and the system functionality. For example, the ideal support should have properties that include physical resistance to compression, hydrophilicity, inertness toward enzymes, biocompatibility, resistance to microbial attack, and availability at low cost. The necessity to use the biocatalyst repeatedly and continuously led to the immobilization of the recombinant SspCA on polyurethane (PU) foam, a prepolymer of polyethylene glycol [62], onto supported ionic liquid membranes (SMLs), to realize a system able to selectively separate and transform CO2 [63]. Furthermore, the immobilization onto magnetic support for recovering the biocatalyst from the bioreactor effortlessly and practically, for example, through the use of a magnet, was also proposed for these thermostable CAs [64].

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FIGURE 22.4 Schematic representation of the enzyme immobilization methods.

TABLE 22.1 Classification of the supports used in the enzyme immobilization. Classification of supports Organic Natural polymers Polysaccharides: cellulose, dextrans, agar, agarose, and chitin, alginate Proteins: collagen and albumin Carbon Synthetic polymers Polystyrene Other polymers: polyacrylate polymethacrylates, polyacrylamide, polyamides, vinyl, and allyl-polymers Inorganic Natural minerals: bentonite and silica Processed materials: glass (nonporous and controlled pore), metals, and controlled pore metal oxides

22.5

SspCA immobilization

22.5.1 Polyurethane foam Our groups selected the extreme SspCA to realize a three-phase bioreactor (gas, liquid, and solid), which was filled with the recombinant SspCA immobilized on PU, a prepolymer of polyethylene glycol [62]. The calculated specific activity of the PU-SspCA was similar to that of the free enzyme and the PU-SspCA specific activity remained constant up to 1 month, while that of the free catalyst decreased slightly. Once immobilized, PU-SspCA adsorption capacity has been verified in the laboratory using a three-phase trickle-bed bioreactor [62]. The gas phase was a mixture of N2/CO2 (20% by volume) injected from the bottom of the bioreactor, the aqueous phase was the distilled water pumped from

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the top, and the solid phase was the enzyme immobilized on PU. The CO2 consumption was monitored using a CO2 analyzer [62]. The results obtained using the lab-scale bioreactor previously described showed that the immobilized PU-SspCA is capable of converting about 5% or 10% (depending on the amount of immobilized enzyme) of CO2 from a gas mixture, the initial concentration of which was 20%. In the absence of the catalyst the spontaneous CO2 conversion was about 2%. These results confirm that the CA from extremophile is an excellent candidate to be used in postcombustion carbon capture. Besides, Russo et al. using the free SspCA in solution and the protocol based on CO2 absorption experiment in a stirred cell apparatus determined the kinetics of SspCA for the CO2 hydration reaction and the long-term catalyst resistance to high temperatures. The results showed that SspCA has a first-order kinetic constant at 25
C of 9.16 3 106 L mol21 and a half-life of 53 and 8 days at 40
C and 70
C, respectively [65]. The experiments were carried out using as liquid phase an alkaline solution of 0.5 M Na2CO3/0.5 M NaHCO3 buffer, pH 9.6. Interestingly, the SspCA long-term stability at high temperatures, as demonstrated by these results, is compatible with the operating conditions of a typical absorption unit used for capturing CO2 from flue gas and coupled with a vacuumstripping unit for solvent regeneration and CO2 recovery.

22.5.2 Ionic liquid membranes (supported ionic liquid membranes) The immobilization of CA onto the membranes selectively facilitates the CO2 absorption and separation. Membranes have been extensively used for CO2 sequestration from gaseous streams. Generally, the membrane is in the alkaline water for CO2 absorption and the CO2, pumped through the feed side, contacts the membrane-immobilized CA producing bicarbonate in aqueous solution. The use of liquid membranes is affected by the problem of the water because it evaporates even at relatively low temperatures [66]. To overcome this issue, SLMs in which a solvent is immobilized inside the porous structure of the supporting membrane by capillary forces have been used in gas separation applications. Neves et al. provided an innovative concept for the removal of CO2 from flue gas streams, using biomimetic SILMs containing a bovine CA that enhances the selective transport of CO2 [67]. The operating temperature of combustion gases may easily exceed the optimal temperature for an enzyme used to the capture process. For this reason, our groups impregnated the SLM membranes with the thermostable SspCA to realize a system cable of selectively separate and transform CO2 [63]. The results showed that the SML membranes with the immobilized SspCA had high permeability toward CO2 at high temperatures (up to 100
C) and a reasonable transport selectivity toward CO2 [63]. This strategy offers, between the others, new insights into the realization of an efficient and competitive system to be used in the postcombustion carbon capture process.

22.5.3 Magnetic particles Recently, our groups decided to immobilize SspCA onto magnetic support for recovering the catalyzer from the bioreactor effortlessly and practically, for example, through the use of a magnet. For this purpose the enzyme was covalently immobilized by the carbodiimide activation onto the surface of homemade magnetic particles (Fe3O4) prepared by coprecipitating ferric sulfate and ferrous chloride with aqueous ammonia solution [64]. The biocatalyst can be recovered from the reaction mixture directly applying a magnet or an electromagnet field because of the strong ferromagnetic property of the magnetite. The behavior of the thermostable CA immobilized on the magnetic particles is impressive. The residual activity of the bound SspCA remained constant (100%) at both 50
C and 70
C for all the incubation time (80 h). The stability of the mesophilic enzyme is improved with the immobilization but diversely from that of the thermostable free SspCA. Moreover, comparing the long-term stability of the free and bound SspCA or bCA (the mammalian enzyme) at 25
C and after an incubation time of 30 days, the residual activity of the bound SspCA respect to the free catalyst was of 100%. The covalent immobilization of SspCA directly onto the surface of the magnetic particles increased the stability and the long-term storage and quickly allowed the recovery of the biocatalyst from the reaction mixture.

22.5.4 In vivo immobilization Unfortunately, the strategies aforementioned in Sections 22.5.122.5.3 may discourage the extensive utilization of enzymes in industrial applications because of the high costs connected to the biocatalyst production and purification, and the expenses for the preparation of the immobilization support. This limitation can be easily overcome by the direct in vivo immobilization of SspCA [68]. This system, known as one-step immobilization procedure, consists in the overexpression of SspCA directly onto the surface of bacterial hosts. It was realized by using the ice nucleation protein (INP) from the Gram-negative bacterium Pseudomonas syringae [68]. Escherichia coli cells were transformed with a construct composed by a chimeric gene resulted by the fusion of a signal peptide, the P. syringae INP domain (INPN, Ice Nucleation Protein N-terminal domain), and the gene encoding for the thermostable α-CA (SspCA) (Fig. 22.5). The

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construct of Fig. 22.5 (top of the figure) triggered the overexpression and the immobilization of the thermostable SspCA directly on the outer membrane of the microorganism (bottom of the figure) [68]. The anchored SspCA was active and efficiently overexpressed on the bacterial surface of E. coli. Moreover, it was stable and active for 15 h at 70
C and days at 25
C [68]. Such a system, with respect to the magnetic Fe3O4 particles, drastically reduces the costs needed for the enzyme purification, immobilization, and those for the support preparation. Besides, the biocatalyst can be recovered by a simple centrifugation step from the reaction mixture. The strategy of the INPN-SspCA obtained by engineering E. coli could be considered as a brilliant strategy to be used for the biomimetic capture of CO2 and other biotechnological applications in which a highly effective, thermostable catalyst is needed. Recently, the improvement of the thermostability of SspCA was realized by using a novel protein-tag system, the ASLtag [69]. The anchored SspCA was fused to the thermostable variant of the alkylguanine-DNA-alkyl-transferase (H5) from the hyperthermophilic archaea on Sulfolobus solfataricus [69]. The chimeric H5-SspCA was efficiently overexpressed on the bacterial surface of E. coli (Fig. 22.6). Using CO2 as the substrate, the hydratase activity of all the SspCA (free and

FIGURE 22.5 Fragment of the expression vector encoding the chimeric membrane-bound SspCA (top). OM with the immobilized SspCA (in blue; PDB ID: 4G7A) (bottom). Legend: pelB, the signal sequence for the periplasmic translocation of the protein (21 amino acid residues); INPN domain (204 amino acid residues); Spacer (five amino acid residues); SspCA: the thermostable CA (226 amino acid residues); His-Tag: histidines at the C-terminus (6 amino acid residues); OM, bacterial outer membrane. The catalytic reaction of SspCA (the hydration/ dehydration of CO2) is also shown.

FIGURE 22.6 In vivo immobilization of SspCA (in blue; PDB ID: 4G7A) and fusion with H5 (in green; PDB ID: 6GA0). The catalytic reaction of SspCA (the hydration/dehydration of CO2) and H5 (the conversion of the fluorescent O6-benzyl-guanine derivative, BG-FL, in the free guanine and the fluorescent benzyl-guanine derivative, B-FL, covalently linked to the active site of H5) are also shown.

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FIGURE 22.7 Protonography of SspCA and H5-SspCA immobilized on the bacterial cell surface. Filled green, white, and black arrows represent the ASLtag-SspCA, INPN-SspCA, and the free SspCA, respectively.

FIGURE 22.8 Fluorescence microscopy of Escherichia coli BL21(DE3) cells transformed with pET-22b/INPN-SspCA (left) or with pET-ASLtagSspCA (right). The cells were incubated with BG-FL and then analyzed by fluorescence microscopy. Images show AlexaFluor488 (green). As expected, the fluorescence is only evidenced in bacterial cell transformed with the ASLtag system.

immobilized) was investigated in solution [59]. The CO2 hydratase activity of the membrane-bound SspCA with and without H5 did not show any differences. Otherwise, the activity of SspCA was compared by using the protonography, which is a technique able to reveal the hydrogen ions produced by the hydratase activity reaction as a yellow band on the SDSPAGE. The protonography results showed that all the forms of SspCA (the two membrane-bound ones and the free enzyme) had comparable enzyme activity (Fig. 22.7). The fluorescent microscopy evidenced the overexpression of the protein on the bacterial external surface (Fig. 22.8). Fig. 22.9 shows the SspCA or H5-SspCA residual activity as a function of temperature. The activity of the SspCA and H5-SspCA remained almost constant at 25.0
C and 50.0
C (panels A and B). In contrast, it is readily apparent that at higher temperatures (70
C) SspCA and H5-SspCA behave differently (panel C): the residual activity of SspCA started to decline rapidly after 2 h, getting a value of about 60% after 14 h of incubation; whereas the stabilizing effect of H5 on the SspCA showed a residual activity of about 85% and remained almost constant for the rest of the time indicated in the figure. These results demonstrated that the anchoring ASLtag system enhanced the SspCA stability of about 20%, even if both anchored enzymes continued to work for several hours at temperatures considered prohibitive. Capasso’s group demonstrated that the free SspCA showed a residual activity of 20% when heated at 70.0
C for 15 min [68]. This aspect is crucial in the context of the postcombustion carbon capture process, which requires temperatures ranging from 40.0
C to 60.0
C [65]. Fig. 22.9 (bottom of the figure) shows the residual activity for the CO2 hydration reaction for SspCA and H5-SspCA when the whole cells were treated at different temperatures for a very long period (up to 10 days). At 25.0
C the SspCA residual activity started to decrease after 4 days and reached a value of about 70% after 10 days, while H5-SspCA remained almost constant. At 50.0
C and 70.0
C the residual activity of SspCA decreased up to 40% and 20%, respectively (panels B and C),

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FIGURE 22.9 Thermostability (top) and long-term stability (bottom) of immobilized SspCA and H5-SspCA on the bacterial surface. Measures were carried out at indicated temperatures, by using aliquots of the whole cells incubated at the time shown on the X-axis. Legend: Continuous line, membrane-bound H5-SspCA; dashed line, membrane-bound SspCA. Each point is the mean of three independent determinations.

whereas H5-SspCA showed a residual activity of about 60% and 40%, respectively (panels B and C). All these data confirmed that the presence of a thermostable protein-tag between the INPN anchoring domain and the SspCA significantly improved the long-term stability and the storage of this CA. The ASLtag system may thus be considered as a brilliant strategy to further increase the thermostability of proteins to be used in biotechnological applications, in which a highly active and thermostable catalyst is needed.

22.6

Conclusion

Thermostable CAs are enzymes that tolerate high temperature and compared to the mesophilic counterparts generally can also tolerate high salinity and extreme pH values. The discovery of thermostable CAs allowed their use in biotechnological field, characterized by conditions deleterious for the enzyme activity [4,34]. SspCA, identified in the genome of the bacterium S. yellowstonense [4,47,49,5155] resulted to be a highly active catalyst for the CO2 hydration reaction and extremely thermostable, maintaining an excellent catalytic activity even if heated for a prolonged period (up to 180 min). The carbon capture through the biomimetic approach was pursued by immobilizing the thermostable SspCA

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onto the PU, magnetic particles, and SMLs. Intriguing is the in vivo immobilization of SspCA, which was carried out using the ASLtag system, which efficiently overexpressed the chimeric H5-SspCA onto to the bacterial cell surface. Moreover, by investigating at different temperatures the activity of the membrane-bound SspCA or H5-SspCA, it has been demonstrated an enhancement in terms of the thermal stability of the chimeric protein (H5-SspCA). The H5-SspCA obtained by the ASLtag system constitutes a valid strategy for further increasing the thermostability of proteins, for processes in which a highly effective, thermostable catalyst is needed. In conclusion, the extreme CAs could have a pivotal role in fighting the increase of CO2 in the atmosphere caused by the anthropogenic activity or in improving the human health since they can be used in some medical applications (e.g., artificial lungs).

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[56] El Harrad L, Bourais I, Mohammadi H, Amine A. Recent advances in electrochemical biosensors based on enzyme inhibition for clinical and pharmaceutical applications. Sensors (Basel) 2018;18(1). [57] Hicks N, Vik U, Taylor P, Ladoukakis E, Park J, Kolisis F, et al. Using prokaryotes for carbon capture storage. Trends Biotechnol 2017;35 (1):2232. [58] Arazawa DT, Oh H-I, Ye S-H, Johnson Jr. CA, Woolley JR, Wagner WR, et al. Immobilized carbonic anhydrase on hollow fiber membranes accelerates CO(2) removal from blood. J Memb Sci 2012;404-404:2531. [59] Tosa T, Mori T, Fuse N, Chibata I. Studies on continuous enzyme reactions. I. Screening of carriers for preparation of water-insoluble aminoacylase. Enzymologia 1966;31(4):21424. [60] Zhou Z, Hartmann M. Progress in enzyme immobilization in ordered mesoporous materials and related applications. Chem Soc Rev 2013;42 (9):3894912. [61] Mohamad NR, Marzuki NH, Buang NA, Huyop F, Wahab RA. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol Biotechnol Equip 2015;29(2):20520. [62] Migliardini F, De Luca V, Carginale V, Rossi M, Corbo P, Supuran CT, et al. Biomimetic CO2 capture using a highly thermostable bacterial alpha-carbonic anhydrase immobilized on a polyurethane foam. J Enzyme Inhib Med Chem 2014;29(1):14650. [63] Abdelrahim MYM, Martins CF, Neves LA, Capasso C, Supuran CT, Coelhoso IM, et al. Supported ionic liquid membranes immobilized with carbonic anhydrases for CO2 transport at high temperatures. J Membr Sci 2017;528:22530. [64] Perfetto R, Del Prete S, Vullo D, Sansone G, Barone CMA, Rossi M, et al. Production and covalent immobilisation of the recombinant bacterial carbonic anhydrase (SspCA) onto magnetic nanoparticles. J Enzyme Inhib Med Chem 2017;32(1):75966. [65] Russo ME, Olivieri G, Capasso C, De Luca V, Marzocchella A, Salatino P, et al. Kinetic study of a novel thermo-stable alpha-carbonic anhydrase for biomimetic CO2 capture. Enzyme Microb Technol 2013;53(4):2717. [66] Trachtenberg MC, Cowan RM, Smith DA, Horazak DA, Jensen MD, Laumb JD, et al. Membrane-based, enzyme-facilitated, efficient carbon dioxide capture. Energy Procedia 2009;1:35360. [67] Neves LA, Afonso C, Coelhoso IM, Crespo JG. Integrated CO2 capture and enzymatic bioconversion in supported ionic liquid membranes. Sep Pur Tech 2012;97:3441. [68] Del Prete S, Perfetto R, Rossi M, Alasmary FAS, Osman SM, AlOthman Z, et al. A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli. J Enzyme Inhib Med Chem 2017;32(1):11208. [69] Merlo R, Del Prete S, Valenti A, Mattossovich R, Carginale V, Supuran CT, et al. An AGT-based protein-tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane. J Enzyme Inhib Med Chem 2019;34(1):4909.

Chapter 23

Understanding the protein sequence and structural adaptation in extremophilic organisms through machine learning techniques Abhigyan Nath1 and S. Karthikeyan2 1

Department of Biochemistry, Pt. Jawahar Lal Nehru Memorial Medical College, Raipur, India, 2Department of Computer Science, Institute of

Science, Banaras Hindu University, Varanasi, India

23.1

Introduction

Extremophilic organisms are those organisms that can thrive successfully in extremes of environmental conditions, such as thermophilic and psychrophilic—thriving in extremes of higher and lower temperatures, respectively; halophilic—in high salt conditions; and piezophilic—in high pressure environments (Yayanos [1] coined the term “piezophilic,” earlier they were known as barophiles [2]). Based on their habitat, the piezophilic organisms are further classified into psychrophilic
piezophilic (cold temperature loving piezophiles), mesophilic
piezophilic (normal temperature loving piezophiles) and thermophilic piezophiles (high temperature loving piezophiles) [3]. Alkaliphiles thrive in high alkaline environments, while Acidophiles can successfully survive below pH 2.0. Polyextremophiles can be defined as those organisms that can survive the vicissitudes of different extremophilic surroundings (e.g., psychrophilic
piezophilic organism). They show adaptation at various levels to accommodate changes to suit their specific niche. The molecular basis of thermophilic adaptation is most well studied as compared to the other extreme environmental adaptations. Recent years have also witnessed an increased interest in the study of psychrophilic and halophilic proteins as well [4]. Analysis of the extremophilic protein sequences can reveal the molecular basis of adaptation parameters responsible for protein structural stability and function preservation at extremes of environmental surroundings, which can subsequently be utilized for designing structurally stable proteins of industrial interest in the laboratories in a cogent way. The computational studies on the evolution of extremophilic proteins that are being discussed in this chapter can be broadly classified into two parts—machine learning
based studies (involving prediction modeling and rule generation) and sequence statistical analysis. The type of data being used in the investigation decides the type of methods that can be further applied. Broadly the analytical methods can be divided into sequence-only methods and structure-based methods. The structural coordinate data of proteins are very little as compared to sequence data. As a consequence, the methods that are utilizing a vast amount of available sequence data for their predictive modeling and statistical analysis are more widespread as compared to the methods that are dependent on tertiary structural data alone.

23.2

Databases

Prokaryotic Growth Temperature Database (PGTdb) [5] is an excellent source for obtaining the growth temperature data of extremophilic organisms. PGTdb consists of optimal growth temperature, its range and it also classifies each organism into a particular temperature category. Based on growth temperature, all the organisms in the database are classified into four categories: hyperthermophiles are those organisms having growth temperature greater than 80 C, thermophiles are those having growth temperature in the range .45 C
80 C, mesophiles in the range of 20 C
45 C, Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00023-X © 2020 Elsevier Inc. All rights reserved.

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and psychrophilic organisms having less than 20 C. The complete genomes and proteomes of extremophilic organisms can be downloaded from the NCBI FTP repository. The protein sequences of extremophilic organism can be obtained from the popular databases such as UNIPROT [6] and NCBI protein database. The corresponding structural data of the protein sequences are available from the PDB database [7]. Apart from the abovementioned databases, there are some specialized databases such as ProTherm [8] that is a database compiling numerous parameters of thermodynamic importance such as change in enthalpy for proteins and their mutants. It also provides information about few structural properties, for example, secondary structure elements and residue-specific solvent accessibility information. ProtDataTherm [9] consists of protein sequences categorized into protein families, which are further classified into psychrophilic, mesophilic, and thermophilic for each protein family. Both ProTherm and ProtDataTherm can prove to be very useful in engineering of enhanced stable proteins. HaloWeb [10], HprotDB [11], and HaloDom [12] are the dedicated repositories providing genomic information about archaeal, bacterial, and eukaryotic halophiles.

23.3

Machine learning

The primary aim is to achieve the superior generalization ability (which is defined as the performance of the machine learning procedure on the unseen data) for machine learning
based prediction methods for obtaining high prediction accuracy. The prediction of extremophilic proteins can be framed as a binary classification task. In a binary classification problem, the positive class is the class of interest, for example, in our case it will be the set of extremophilic protein sequences and the negative class will comprise all the nonextremophilic protein sequences. The general schematic representation of a supervised machine learning
based prediction protocol is given in Fig. 23.1.

23.3.1 Machine learning platforms A number of machine learning platforms with a plethora of algorithms for preprocessing, clustering, classification, etc. are now available. Prominent among them is the java-based machine learning platform WEKA [13]. It is a very easily usable package even for novice users without programming experience due to the presence of well-documented user guide as well as user-friendly graphical user interface (GUI). Other well-known platforms are KNIME [14] and RapidMiner [15]. There are also many R packages available for the implementation of machine learning algorithms, for example, H2O [16] and Rattle (GUI available) [17]. Python programming language also provides a number of modules for implementing various machine learning algorithms and the prominent among them is the scikit-learn [18].

23.3.2 Feature extraction and representation The mining of pertinent measureable attributes/features from the proteins and representing them in a fixed length feature vector is a salient task in developing a machine learning
based prediction model. The features are some measurable properties of a protein sequence, for example, the simplest feature of a protein is its composition of 20 amino acids residues. The way the protein sequences are fed into the machine learning algorithm determines the final outcome of the accuracy of the trained model. A wide range of servers and tools are available for calculating a plethora of features from protein sequences, for example, PROFEAT [19], PseAAC-builder [20], Pse-in-One [21], iFeature [22], propy (python package) [23], and protr (R package) [24].

Testing data

Training data with class Build model

Machine learning algorithm

Evaluate

FIGURE 23.1 Schematic representation of a supervised machine learning protocol.

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FIGURE 23.2 A sample ARFF file structure for binary classification problem.

One of the popular formats of feature input file that is widely recognized by almost every machine learning platform is the Attribute-Relation File Format (ARFF) [25]. The structure of an ARFF file for a binary classification problem is shown in Fig. 23.2. An ARFF file can be separated into two parts—the header region and the data region. The header region comprises the title followed by the description of the types of features used to represent each of the instances. The data part consists of the numerical features and the class information for each of the instances.

23.3.3 Feature selection Feature selection involves choosing the right feature subset from the full set of extracted feature set for the purpose of enhancing the prediction accuracies of the machine learning algorithms. In certain cases the number of extracted features increases to an extent which affects the algorithm training time as well as the prediction accuracy (the curse of dimensionality). The full set of features may contain irrelevant, uninformative, and redundant features, which increase the overall dimensionality of the feature vector resulting in increasing computational complexity without giving any classification advantage. Training a machine learning algorithm with an optimal nonredundant and informative subset of features has many benefits such as shorter training time, decrease in overfitting, higher prediction accuracy, less computational overhead, and simpler prediction models. Feature selection approaches can be broadly classified into wrapper methods, which involve the addition or removal of features to a specific classifier until further improvement in performance evaluation metrics stops, and filter methods, which utilize class label information for the calculation of a discriminatory score for each of the attributes. Top scoring attributes can then be selected for building the final prediction model. Many of the feature selection algorithms such as Chi-squared [26], OneR [27], and ReliefF [28]. provide a ranking of features in accordance with their importance in classification between the two classes. The feature selection algorithm should always be applied on the training set and the selected features should be evaluated on the testing set so as to avoid over optimistic performance evaluation metrics.

23.3.4 Model performance validation 23.3.4.1 Types of validation method for testing the performance of trained machine learning models There are basically three different validation methods in practice for testing and obtaining confidant estimates of performances of trained machine learning models: 1. Separate training/validation sets: When the dataset is large, then splitting the dataset into mutually exclusive training and testing set is preferred. The testing data should ideally have the same distribution as the training set for the proper evaluation of the trained model. If the dataset is small, splitting the dataset into separate training/testing set leaves very small amount of data, which may not be adequate in most of the cases for complete learning. 2. K-fold cross-validation: In this method of evaluation the training dataset is separated into K-folds (K-partitions, where K is an integer). K 2 1 folds are allocated in the training set and the left out single fold is allocated in the testing set. All the K-folds are roughly of the same size. The procedure continues until all the folds are allocated once in the testing set. Ten- and fivefold cross-validation are most commonly employed.

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3. Leave one out cross-validation: This method of evaluation is most suitable when the dataset is small. In this method a single instance (sample) is allocated as a test case and the rest of the instances (samples) are allocated in the training set. The procedure continues until all the instances are allocated once as a test case.

23.3.5 Model performance evaluation metrics Sensitivity: It states the percentage of correctly predicted extremophilic proteins. Sensitivity 5

TP 3 100 ðTP 1 FNÞ

(23.1)

Specificity: It states the percentage of correctly predicted nonextremophilic proteins. Specificity 5

TN 3 100 ðTN 1 FPÞ

(23.2)

Accuracy: It states the percentage of correctly predicted extremophilic and nonextremophilic proteins. Accuracy 5

TP 1 TN 3 100 TP 1 FP 1 TN 1 FN

(23.3)

Area under ROC (receiver operating characteristic) (AUC): AUC [29,30] is one of the threshold-dependent metrics, which is used to characterize ROC curves. The possible values of AUC range from 0 to 1. An AUC value of 1 is considered best for the trained prediction model. Matthews correlation coefficient (MCC): It is an evaluation parameter for binary classification problems with values ranging from 21 to 11. A value closer to 11 is considered good for the trained prediction model [31]. ðTP 3 TNÞ 2 ðFP 3 FNÞ MCC 5 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðTP 1 FNÞðTP 1 FPÞðTN 1 FPÞðTN 1 FNÞ

(23.4)

g-Means: The geometric mean of sensitivity and specificity is called as the g-means [32]. It combines both sensitivity and specificity to give a balanced opinion about accuracy. The higher the value (i.e., closer to 100) the better is the balance between the accuracy of the of the two classes. pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi g-means 5 Sensitivity 3 Specificity (23.5)

23.4

Statistical analysis for inferring the molecular basis of extremophilic adaptation

The simplest method for finding the protein’s stability factors is the consensus method. This method involves a comparison of homologous sequences (extremophilic proteins and their nonextremophilic counterparts) using a multiple sequence alignment and then replacement of nonconsensus residues is performed by most probable amino acid residues [33,34]. For a comparative analysis using various sequence and structural features an independent sample t-test is most commonly used [35
39]. The general formula for applying the independent sample t-test is given in the following equation: Sextremophilicðsequence featuresÞ 2 Snonextremophilicðsequence featuresÞ t 5 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi    ffi Varextremophilic =nextremophilic 1 Varnonextremophilic =nnonextremophilic

(23.6)

where Sextremophilic (sequence features) is the means of sequence-based features of extremophilic proteins sequences, Snonextremophilic (sequence features) is the means of sequence-based features of nonextremophilic proteins sequences, Varextremophilic is the variances of sequence-based features of extremophilic protein sequences, Varnonextremophilic is the variances of sequence-based features of nonextremophilic protein sequences, nextremophilic is the total number of extremophilic proteins, nnonextremophilic is the total number of nonextremophilic proteins. The sequence features can range from amino acid composition to amino acid solvent accessibility, etc. One of the important structural features for the relative protein structure analysis between extremophilic and nonextremophilic organisms is the solvent accessibility [38,39]. It is expressed as the area drawn out by the center of a probe

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311

FIGURE 23.3 A sample DSSP output.

sphere, which is used as a representative of a solvent molecule [40]. It is generally expressed in percentages as relative solvent accessibility [36] and is defined by the following equation: Rel S Ai 5

S Ai 3 100 Max S Ai

(23.7)

where S_Ai is the solvent accessibility of the ith amino acid residue, Rel_S_Ai is the relative solvent accessibility of the ith amino acid residue, Max_S_Ai is the maximum solvent accessibility of the ith amino acid residue. The maximum solvent accessibility values are usually obtained from a representative tripeptide of Gly-X-Gly or Ala-X-Ala where X is the amino acid residue of interest. The raw solvent accessibility values can be obtained by parsing the pdb file of a protein through the DSSP program [41]. The sample output of DSSP is shown in the following figure. The solvent accessibility values are provided under the ACC column (Fig. 23.3). In cases where the structures of extremophilic proteins and their nonextremophilic counterparts are available, the solvent accessibility
based comparative analysis can be performed giving clues about the possible bias of sequence properties in the different regions of the tertiary structure. Using some appropriate relative solvent accessibility cutoff, the amino acid residues in a protein structure can be divided into two classes (buried and exposed) or into three classes (buried, intermediate, and exposed). This gives the advantage of investigating the dominant amino acid residues present on the surface or in the other regions of a protein structure. Apart from amino acid composition, the distribution of secondary structure elements and physicochemical properties can also be investigated in the buried and exposed regions among extremophilic and nonextremophilic protein pairs.

23.5

Inferences from preceding methods

Machine learning
based prediction methods, which could be used to discriminate between a mesophilic protein sequence from its extreme environment counterpart, has been developed by many previous research works. A plethora of machine learning algorithms has been used for developing accurate machine learning
based prediction models. Most of the machine learning algorithms behave like black boxes that do not provide any information about the

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interaction between the different features. One could not infer how the learning algorithm has discriminated the mesophilic protein sequence from its extremophilic counterpart. To mitigate the shortcomings of the black box
based approach, a white box approach using rule induction algorithms has also been used. Comparison of protein sequences and structures of extremophilic organisms with its nonextremophilic counterpart is the basis of comparative elucidation of the extremophilic parameters responsible for the preservation of stability and function. In thermophiles, comparative protein analysis revealed substitution of glycine, serine, lysine, and aspartic acid by alanine, threonine, arginine, and glutamic acid [42,43] and also higher aliphatic index. Overall increase in hydrophobic amino acid residues is observed that is a contributor to hydrophobic effect and is responsible for increasing the stability of thermophilic proteins [44,45]. Thermolabile enzymes undergo deamination at elevated temperature, which forms the basis for their avoidance for asparagine [46]. An increase in amino acid residues contributing to the ionic interaction, for example, arginine and glutamic acid are also observed. Aromatic amino acids contributing toward stabilizing cation
pi interactions are also found to be overrepresented. Critical differences in dipeptide composition (DPC) and counts are also observed between extremophilic and their nonextremophilic counterparts. Instead of sequence features such as AAC and DPC, other attributes that can accommodate sequence order effects such as pseudo amino acid composition may also be extracted. In Ref. [47] machine learning
based discriminating model was developed for different categories of piezophilic organisms. The model construction consisted of three stages; the first stage used a machine learning algorithm to filter the strong instances (i.e., the correctly predicted piezophilic and nonpiezophilic sequences). The next stage consisted of using a rule induction algorithm (PART [48] rule induction algorithm) to generate human interpretable rules. The last stage involved the analysis of rules. Further an implementation of feature ranking was performed to infer the importance of each amino acid residue composition in discriminating between the two classes. The following amino acids were observed to be important in different piezophilic groups [psychrophilic piezophilic (PP), psychrophilic nonpiezophilic (PNP), mesophilic piezophilic (MP), mesophilic nonpiezophilic (MNP), thermophilic piezophilic (TP), and thermophilic nonpiezophilic (TNP)]: PP/PNP—glutamine, glutamic acid, asparagine, and lysine; MP/MNP—lysine, threonine, and asparagine; TP/TNP— arginine, lysine, and isoleucine In terms of physicochemical properties, hydrophobic and polar amino acids in PP/PNP and MP/MNP group and hydrophilic, polar, hydrophobic, charged and nonpolar amino acid groups in TP/TNP have higher discriminative abilities. The study also observed that the discriminatory power of amino acid composition increased with increasing temperature range (i.e., PP , MP , TP). Tiwari et al. [49] further improved the prediction accuracy (average accuracy of 77.10%) of piezophilic protein sequences from nonpiezophilic sequences using fuzzy-rough feature selection and an optimally balanced dataset. Machine learning was also implemented for developing prediction models for classifying psychrophilic proteins from nonpsychrophilic proteins [50,51]. The trained model achieved an overall accuracy of 70.5% using the Rotation Forest algorithm. Feature ranking using a number of filter-based algorithms (Chi-squared, ReliefF, etc.) indicated the importance of serine, lysine, glutamic acid, and alanine in classification. Metpally and Reddy [35] also observed similar results, they found overrepresentation of serine, aspartic acid, threonine, alanine in coiled regions. The voting-based model developed in Ref. [36] for halophilic protein prediction achieved an accuracy of 90% on a hold out testing set using the same dataset as used in Ref. [52]. Further three different discretization methods were employed with the PART rule induction algorithm for enhanced prediction and interpretable rules. In halophilic proteins, isoleucine, lysine, and serine are avoided and aspartic acid and glutamic acid are preferred. Lower preference for average charge, hydrophobicity, and average bulkiness and higher preference for average flexibility and average polarity are also observed among the halophilic proteins. Till now very limited computational approaches have been explored for understanding the adaptation parameters of ionizing-radiation-resistant bacteria (IRRB). IRRB have the potential for the detoxification of mercury, toluene, and other radioactive wastes [53,54]. The prediction of IRRB was formulated as a binary classification problem with multiple instance learning (MIL) approach. In MIL, class labels are not provided to individual samples (instances) but to the bags of instances [55]. The training of a learning algorithm proceeds with a training set of bags of instances with class labels. The bags can be classified into either positive (IRRB) or negative (non-IRRB).

23.6

Conclusion

The identification and unraveling of the molecular basis of extremophilic adaptation is far from complete. Accurate in silico methods are being developed on a regular basis using novel algorithms and statistical analysis. Machine

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learning
based prediction and statistical analysis of preference/avoidance of sequence and structural features are one of the most prevalent analysis methods used in the research community. As more sequence and structural data are being deposited every day and with the advent of specialized extremophilic databases, the computational methods will prove to provide a cost-effective authenticated solution (i.e., becoming more accurate). Bioinformatics and computational
based protein adaptation analysis to extremes of environment would facilitate the design of stable proteins for different industrial and academic needs. Knowledge of sequence and structural parameters will also facilitate in engineering of nonextremophilic to extremophilic proteins with the desired property.

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93. [47] Nath A, Subbiah K. Insights into the molecular basis of piezophilic adaptation: Extraction of piezophilic signatures. J Theor Biol 2016;390:117
26. [48] Frank E. Witten IH and science UoWDoC. Generating accurate rule sets without global optimization. Department of Computer Science, University of Waikato; 1998. [49] Tiwari AK, Shreevastava S, Subbiah K, Som T. Enhanced prediction for piezophilic protein by incorporating reduced set of amino acids using fuzzy-rough feature selection technique followed by SMOTE. Singapore: Springer Singapore; 2018. p. 185
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Chapter 24

Exploration of extremophiles genomes through gene study for hidden biotechnological and future potential Pijush Basak1, Arpita Biswas2 and Maitree Bhattacharyya1,2 1

Jagadis Bose National Science Talent Search, Kolkata, India, 2Department of Biochemistry, University of Calcutta, Kolkata, India

24.1

Introduction

At which extreme condition a few microorganism can grow and survive by adapting their cellular machinery is called the extremophilic condition. As such type of microbial community grows in extremophilic condition, they show their potential to remodulate their cellular machinery to survive in extreme environment such as temperature, pH, and salt concentration. Survival and propagation range of extremophilic microorganism can differ from permafrost ice region to hot spring region, from the deepest ocean to the highest point of atmosphere, from high salinity to low salinity region, and from deserts to humid region. The extremophiles belong to all three forms of life: eukarya, bacteria, and archaea [1]. There are mainly two classes of extremophiles; one can flourish only in extreme conditions and the other class of extremophiles can tolerate certain types of extreme conditions. Environmental extremophiles have evolved themselves to adjust in low-temperature range (psychrophiles), very high temperature range (thermophiles), high ion and salt concentration (halophiles), acidic condition (acidophiles), pressure (piezophiles), whereas microorganisms present in human body have evolved their biochemical pathways to nourish within host tissues [2]. Nowadays, increasing use of fertilizers, herbicides, insecticides, and intense pollution increases day-to-day environmental challenges. This encouraged to do further research on protein adaptation mechanism of extremophiles which can further help us in improvising crop, industrial waste management, and also in recycling process. Protein adaptation study is important for both understanding the stress tolerance mechanism in crop/plant and sustainable agriculture production [3]. As an example, γ-amminobutyric acid helps legumes to cope with salt stress [4]. For survival in extreme conditions, extremophiles require protein adaptation besides secondary metabolite production or cellular stability. As an example, halophiles possess a sodium potassium pump on their cell membrane to combat with extreme oxidative stress [5]. It means that the change in protein sequence leads to emerge protein characteristics to work under greater extremes. Nowadays, many extremophiles have a large use in the field of biotechnology, industry, and many other [6]. Few examples of industrial and research use of extremophiles include DNA polymerases that are used for polymerase chain reaction (PCR) [7], various enzymes involved in biofuels [8], and carotenoids that are used in food industry and cosmetic industries [9]. They are also useful to produce antibiotic, anticancer, and antifungal drug [10]. However the production of extremoenzymes from extremophiles could replace many enzymes for making the reactions more efficient and cost-effective [11]. In the era of “omics” tools and metagenomics, to know about the variability and complexity of these extremophile communities is not a huge matter. Research on the genomes of bacteria and archaea provided a key role in many fields. For an example, their evolutionary role in biogeochemical cycle, pathogenesis, and antimicrobial resistance opens a new door for researchers. Metagenome sequencing, with other “omic” technologies, such as transcriptomics (measuring of mRNA transcript levels), proteomics (study of the protein complement), and metabolomics (study of cellular metabolites), give a new leap to systems biology techniques which make the combination study of the functions and interactions of the microbial community within, and with, the environment more easier [12]. Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00024-1 © 2020 Elsevier Inc. All rights reserved.

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Types and characteristics of extremophiles

24.2.1 Temperature adaptation Very few places in the earth are considered to be hot springs, whereas most of the portions in the earth possess very low temperature throughout the year. Depending on the survival temperature they can be classified in two classes: thermophiles (in hot springs) and psychrophiles (in permafrost region). Their adaptation characteristics are also different. Naturally, enzymatic reactions such as transcription, translation, protein folding misfolding, slowdown in low temperature. Psychrophiles have to adapt with extremity by minimizing thermal barrier, maximizing viscosity or they have to develop different biochemical pathways. As an example, Moritella profunda have adapted themselves to the deepest part of the sea and have an optimal growth at 2 C [13]. In psychrophilic enzymes the number of arginine decreases, whereas the number of alanine increases. Actually in the protein structure, alanine (ala) and lysine (lys) replaced glutamic acid (glu) and arginine (arg), respectively. Another amino acid valine (val) in beta strands is replaced by alanine (ala) in alpha-helices. Such type of amino acid mutations helps them to survive in the extreme low temperature [14]. On the other hand, the optimum temperature for growing in the case of the thermophiles is from 50 C to 70 C and the same of hyperthermophiles is up to 105 C. These two types share almost similar adaptation mechanism. At such extreme temperature, irreversible unfolding of the proteins takes place which exposes the hydrophobic cores causing aggregation [15]. The proteins and enzymes of thermophiles have the properties to adapt with the extremes. Compared to mesophilic variants of thermophilic enzyme, increased disulfide bond and salt bridging are important characteristics as they destabilize the mesophilic enzymes due to hydrophobic interaction [16].

24.2.2 pH adaptation Naturally, microorganism can grow in neutral pH but few extremophiles can grow from acidic lake to high saline lake. Extremophiles that can grow below pH 3 are called acidophiles and that can grow at higher pH are called alkaliphiles. Basically, most of the acidophiles are thermophiles, so their adaptation style is like thermophiles. At very low pH the charges of many polar-charged residues are changed due to protonation. They decrease the permeability of cell membrane to maintain proton gradient properly across the cytoplasm having efficient proton pump [17]. Acidophiles have adapted their membrane pore size to be decreased which also decreases the membrane permeability. As an example, carboxylesterase in Ferroplasma acidiphilum has a pH optimum of approximately 2. Acidophiles can maintain pH homeostasis by cytoplasm buffering which helps them to keep the intracellular pH constant. To improve the intracellular damage when proton penetrates the cell membrane, acidophiles first increase their buffering capacity to release proton as a mechanism of pH homeostasis (Fig. 24.1). Proteins present in the community of extreme acidophile, including F. acidiphilum, have high portion of iron-containing proteins that at very low pH can function as a “rivet” [18]. FIGURE 24.1 Mechanism of pH homeostasis; acidophiles have to evolve their cell membrane highly impermeable to delay the proton influx into the cell. pH is retained by active proton transporter (violet). Genome sequencing also indicated that the acidophiles have secondary transporter (green) for proton than mesophiles. Organic acids acting as uncoupler also maintain cell pH by degrading themselves by heterotrophic acidophiles. Acidophiles also maintain their pH by potassium transporter with the help of ATPase pump.

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A vitamin B6-dpendent enzyme, alkaliphilic phosphoserine aminotransferase can form homodimer. This enzyme is structurally similar with its mesophilic analogue that differs in increased hydrophobic interaction and hydrogen bonds at the interface of dimer, and in alkaline atmosphere the stability is provided by the amino acid residues that are negatively charged [19].

24.2.3 Salt adaptation Extremophiles that have evolved their cellular machinery in cell cytoplasm to struggle with varying salt concentration are called halophiles. As salt plays a vital role in solubility, stability and confirmation of protein halophiles have to adapt with the extreme for surviving. The entry of inorganic salt (like NaCl) in halophilic organism which lives in extremely salt lake (like dead sea) is prevented and small organic osmolytes are synthesized to maintain osmotic pressure within the cell. Water is not fully available to internal protein as most of the salt and water are remaining like an ionic lattice at extreme salt concentration that is higher than 0.1 M [20]. These trigger the hydrophobic amino acids to aggregate by losing hydration. Hence, hydrophobic interactions are increased in protein by high salt concentration [21]. Like thermophiles, halophiles have greater salt bridge than other proteins and majority of them have heat labile adaptation to fight against temperature. It is already reported earlier that in hyposaline conditions, halophiles contain a unique protein named P45 to protect denaturation. By forming complexes with halophilic malate dehydrogenase, P45 can also resist salt-dependent deactivation of malate dehydrogenase [22]. In halophiles, transcription binding protein (TBP), TATA-box-binding protein shows expanded interaction with DNA in contrast to their mesophilic counterparts in high salt concentration. This happens due to the change in interaction kinetics of DNA-binding proteins in halophiles, which occurs by mutagenesis of the existing protein and it can be reversed to its mesophilic counterpart [23].

24.2.4 Pressure adaptation The organisms living under extremely high hydrostatic pressure are called piezophiles. It was first discovered in mixed culture from the deep sea. Since hydrothermal vents were discovered in deep sea, it revealed adaptations at both high temperature and pressure conditions. Marine organisms prefer environments that have a high range of temperature and pressure (1 C 300 C and 0.1 110 MPa, respectively). The first isolated piezophiles were reported in 1979 [24] and till now many piezophiles have been isolated with various optimum grow pressure. General adaptations of piezophiles include the presence of a hydrophobic core with the preference for smaller amino acids. Moreover, bacteria in deep sea and proximal to the hydrothermal vents have pressure-sensing operon system with their growth regulated by both temperature and pressure [25]. For example, Shewanella benthica, a deep-sea isolate, displays piezotolerant growth at 4 C (when growth rate remains constant from 0.1 to 50 MPa) but displays piezophilic growth when the temperature shifts to 10 C (resulting in a shift of the optimal growth rate to 70 MPa) [26,27]. In piezophiles, multimerization of protein helps them to survive in extreme environment by the hydrogen bonding between protein subunits. Some thermophilic adaptations, which include increasing basic amino acids, are also present in the proteins of extremophiles.

24.3

Survival strategy to combat cold stress

On this planet, more than 80% of biosphere is below 5 C and the gene study and physiology of the microorganisms to fight with this environment are yet to be known. In comparison to the high temperature adaptation mechanism of archaea and bacteria, there is lack of information about low temperature adaptation mechanism and also differences in the cold adaptation mechanisms of archaea and bacteria [28].

24.3.1 Membrane fluidity Microbes face problems at temperatures which are either higher or lower than their optimal temperature to preserve the functioning of their macromolecules such as nucleic acid, proteins, and lipids. Fluidity of bacterial membrane depends on the membrane’s phase-transition temperature (Tm) which is the temperature required to shift the membrane from liquid to gel phase. At a temperature below Tm, the phospholipid bilayer becomes frozen and the hydrocarbon chains become perpendicular to lipid bilayer, whereas above Tm, motility of phospholipid bilayer increases [29]. For maintaining the membrane fluidity beneath growth temperature, microbes adopt specific modifications for lowering their Tm [30]. Various targets modified by bacteria for cold adaptation in physiological and stress conditions have been

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identified. They are short chain fatty acids, branched chain fatty acids, unsaturated fatty acids, glycolipids, uncommon polar lipids, and lastly carotenoids [31]. Till now there are only two confirmed species of archaeal psychrophiles (optimum growth temperature at or less than 15 C); Cenarchaeum symbiosum (Topt 5 10 C) [32] and Methanogenium frigidum (Topt 5 15 C) [33]. Like bacteria, they also undergo some lipid adaptation to increase membrane fluidity such as the number of pentacyclin, unsaturated diethers, tetraether:diether ratio, and isoprenoid hydroxylation [34].

24.3.2 Protein synthesis and cold-accustomed protein Psychrophiles adapted many temperature-sensitive mechanisms at transcription and translation level to low temperature. The involved enzymatic activities in synthesis of protein have activity with low stability, a general trait for coldadapted enzyme. It is assumed that the nucleic-acid-binding proteins that are naturally unfavorable with low temperature play a key role in cold adaptation of psychrophiles [35]. Nucleic-acid-binding proteins are assumed to be coded by the five unique genes that are identified in the genome of archaea resistance to cold stress [36]. RNA helicase removes secondary structure formed in Antarctic archaeon at 4 C shows higher accumulation at low temperature in comparison to higher temperature like 23 C [37]. Depending upon the physiological significance, proteins preset in psychrophiles can be distinguished in four groups. They are cold shock proteins (CSPs) which are overexpressed only after a large temperature downshift, CSPs with optimal expression after mild shocks, cold acclimation proteins (CAPs) which are overexpressed after all cold shocks, and late CAPs proteins which are present at the high concentrations but only in a low temperature of 4 C [38]. Hence, CAPs protein plays a significant role for cold-adapted microorganism by maintaining both their cell cycle and growth at low temperature. In the case of mesophiles, some of the CAPs proteins of cold-adapted bacteria act as CSPs like RNA chaperon CSPA [39].

24.3.3 Structural adaptation of cold-active enzyme Proteins and enzymes present in thermophiles and mesophiles have rigid structure compared to psychrophiles as psychrophilic enzymes are more flexible. Comparative studies from the X-ray crystallographic structure of wild-type α-amylase and psychrophilic α-amylase determine the possible determinants of cold adaptation, which concludes that the less rigid protein core, with less interdomain interactions, is the major factor for the conformational flexibility of the enzyme which allows enzyme efficiency in cold environments [40]. Another thing about psychrophilic enzyme comes to highlight that the catalytic cavity appears to be broader and more attainable to the ligands than mesophilic enzymes and this is due to the deletion of residues from the active side border and substitute of the side chains with small residues at the active site. It enhances the release of the reaction products. Electrostatic potential of active site of those enzymes is also improved to attract the ligands and channel the substrate toward the catalytic cavity [41].

24.3.4 Mutational study Mutational studies of cold-active enzymes also help in determining the adaptive feature of them. As an example, one disulfide bond and weak interactions are present in pancreatic α-amylase but absent in psychrophilic α-amylase. These mutants already have confirmed the role of disulfide bond and weak interaction in stability of protein [42]. Various mutational studies about the cold-active enzyme can help us to know about the effect of mutation o the enzymes.

24.4

Bioactive natural products by extremophiles

Although a very small fraction of microorganism is culturable (,1%), extremophile community has a remarkable versatility and complexity among them. The recent advancement of metagenomics tools allowed current research toward a new leap [43]. Depending on the role of extremophiles in biogeochemical cycle and their evolution, their pathogenesis and antimicrobial resistant and genetic determinants, biotechnological potential of the extremophile increases. With the help of proteomics (study of protein complement), transcriptomics (measuring of mRNA transcript levels), and metabolomics (study of cellular metabolites), the limitation of single organism genetic compliment has been resolved [12]. Till now .5000 full metagenomes and 16,000 amplicon libraries have been sequenced and analyzed which have opened up many unique elements from extremophiles communities [12].

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24.4.1 Gene study Two approaches are used to study the genome of extremophiles and their phyla. One is cultivation-dependent approach and the other is cultivation independent. Extremophiles were one of the first microbial communities with their genomes in sequence as their genome size is small and phylogeny is branched. Metagenomics and single-cell genomics are the part of cultivation-independent approach [44]. Genome study of bulk DNA with its environment is called metagenomics, whereas the genome study of a single cell at a particular time is called single-cell genomics. Metagenomics is done by various bioinformatics tools and the process of sample preparation is easier than single-cell genomics. Metagenomics can easily be applied for any sample from which enough amount of DNA can be eluted. In comparison to the earlier process, single-cell genomics have to undergo various steps such as DNA isolation, multiple DNA amplification (MDA), and amplification by PCR followed by sequencing [44]. The first full metagenome of acidophile biofilm was sequenced [45] and acid mine drainage (AMD) metagenomes revealed about the low diversity of microbial community due to the dissolution of pyrite (pH ranges in between 0.5 and 1.5) catalyzed by microbial community [44]. Although deeper Sanger sequencing based on nucleotide word frequency allowed construction of a complete composite genome of the Archaeal Richmond Mine acidophilic nanoorganism (ARMAN-2) lineage, first a few small biofilm metagenomic contigs consisting of novel SSU rRNA gene sequences were recovered [46]. Two related lineage ARMAN-4 and ARMAN-5 are also explored by sequencing of DNA of small purified fraction of biofilm amplified by MDA. These three ARMAN genomes encode almost TCA cycle and are supposed to be able to do aerobic respiration [47]. The ssu rRNA phylogenetics development efficiently disclosed the diversity and complexity of prokaryotic phylotypes of thermophiles [48]. Community metagenomics play significant role in metabolic capacity in the systems which are not accessible to in situ studies. For an example, sulfur-cycling genes are dominated in the most abundant genera in sulfidic deep-sea hydrothermal communities [49]. Ammonia-oxidation genes (amoA) are most opulent in ammonia-rich region but N-assimilation (like urea utilization) mechanisms were also incriminated [50,51]. De novo sequencing of metagenomic samples obtained from hypersaline regions resulted in the discovery of an unknown class, the Nanohaloarchaea [52]. Detailed functional annotation of a hypersaline metagenome has shown capacity of simple carbon- and nitrogen biogeochemical cycling, but as an energy source various bacteriorhodopsins use light [53]. Water samples collected from ultradeep mine were analyzed and have shown the presence of various nitrogencycling gene (such as NifH, NPD, NarV, NifK, NifD, and NifE) [54].

24.4.2 Bioactive natural products There are two types of metabolites. The bioactive metabolites have peptide or macrolide-like structure or both [55] and the other metabolites are alkaloid class of compounds. These are called bioactive natural products [56]. Cyanobacteria growing in diverse ecosystem play a key role in biogeochemical cycles particularly can produce secondary metabolites. Cyanobacteria isolated from fresh water, marine, terrestrial habitats, Antarctic, and hot spring can produce polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) [55]. Microbial natural products produced by two biosynthetic pathways; one is nonribosomal synthesis (by the giant multidomain enzyme, NRPS) and another one is ribosomal synthesis (by synthesis and posttranslational modification). NRPS is consisting of modules that are responsible for the incorporation of single amino acid and catalyzes the production of peptide both in lower eukaryotes and prokaryotes. Succession of modules corresponds to the order of amino acids in the peptide chain [57]. Macrolides are formed by PKSs that resemble NRPS in their modular nature. In comparison to the other enzymes that synthesize peptide, PKS activated, gathered, and modified various carboxylic acids. The maximum set of an individual PKS module domain is almost identical to the animal fatty acid synthase (FAS) [58] and it is composed of acyltransferase, ketosynthase, ketoreductase, enoyl reductase, dehydratase, and acyl carrier protein (ACP) domains [59]. The NRPS, FAS, and PKS termination modules must consist of thioesterase domain as their most downstream domain and catalyze the discontinuation of the full length peptidyl or acyl chain [60] from the adjacent downstream (methyl)malonyl-S-ACP/aminoacyl-S-peptidyl carrier protein domain [61,62]. A major portion of cyanobacterial pathways in fresh water, marine, terrestrial, and hot springs on the basis of NRPS and PKS gene clusters is the persistent mixture of PKS and NRPS modules. The products of NRPS and PKS pathways in fresh water are microsystin, anatoxin, and anabaenopeptin; in marine water are barbamide, jamaicamide, and curacin; and in terrestrial habitats are nostopeptolide, nostocyclopeptide, cryptophysin, etc. [63]. Structure of the products of NRPS and PKS biosynthesis is shown in Fig. 24.2A and B.

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FIGURE 24.2 Products of NRPS and PKS pathway in fresh water (A) and in marine water (B). NRPS, Nonribosomal peptide synthetase; PKS, polyketide synthase.

Cyanobacterial biosynthetic enzymes already have opened a great prospective in the branch of synthetic biology and biotechnology for moderations of existing leading compounds and for the generation of new components library [64]. Natural products of cyanobacteria are mainly focused on toxin and particularly on hepatotoxic microcystin I [63]. But natural bioactive products of cyanobacteria by NRPS and PKS pathways are also used in antiprotease, antiviral and antifungal activities, multidrug-resistance reversal, and lastly cytotoxic activities [65].

24.5

Biotechnological use of extremophiles

Nowadays, in the field of biotechnology and industry, few extremophiles/extremozymes have large use. Extremozymes are such type of enzymes that are derived from extremophiles and are capable of withstanding the extreme condition of industrial process. Classical biocatalysts are stable in moderate temperature and condition which are not such successful in industrial use but the extremozymes are capable of catalyzing in high pressure, acidic or alkaline condition, and also in higher temperature (from 0 C to 140 C) [6] (Fig. 24.3). Extremophiles have huge importance in many of the fields such as research areas, food and dairy production, biofuel formation, mining industry, production of antibiotic, anticancer, antifungal drug, also in bioelectricity production [11] (Fig. 24.4).

24.5.1 Polymerase chain reaction PCR would not have been possible without the enzyme DNA polymerase from the thermophiles named Thermus aquaticus, Pyrococcus furiosus, and Thermococcus litoralis, which are also known in molecular biology field as Taq [66], Pfu [67], and Vent [68], respectively. Without PCR, many advanced research in the field of biosciences are impossible. Hence, the invention of DNA polymerase enzyme is like a blessing for bioscientists. The use of those DNA polymerase enzymes in PCR is more commercial and is also allowed the automation of PCR as this is thermostable enzyme.

24.5.2 Biomining The application of extremophilic enzymes can be observed in biomining, also known as bioleaching which is a mobilization process of metals from their oxides and sulfides using microorganism. It is more eco-friendly and secured process than the traditional one [69]. From an earlier literature, there are two mechanisms for bioleaching [70 72]: one is direct leaching and the other is indirect leaching. In direct leaching, electrons are directly transferred to the cells that are attached with mineral surface from metal sulfide which does not seem to exist. In indirect leaching, iron(III) ions that oxidize the metal sulfides are produced by iron(II) that oxidizes microorganism attached to mineral surface or planktonic [69].

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FIGURE 24.3 Comparison between extremozymes and classical biocatalyst (extremozymes have wider range than classical biocatalyst).

From another article, it is known that worldwide copper production is 15% and that of gold is 5% [73]. Many other metals such as zinc, silver, nickel, and uranium are also mined by biomining techniques. Mainly acidophiles such as Acidithiobacillus sp. and Ferroplasma sp. are used in this process [1,69]. Biominig can cause AMD that produces during sulfide oxidation and begins to leach out of mine [69]. But the possibility of producing AMD can be reduced by using thermophiles instead of acidophiles.

24.5.3 Biofuel production Instead of using diminishing supply of fossil fuel, there is another way to produce fuels by using biomass such as corn, wheat, and sugar cane. Biofuels can be classified into two generations. Biofuels of first generation are easily derived from starches, hydrolyzed sugars, and oils produced from available crops, and the biofuels of second generation are obtained from hydrolysis resistant lingo cellulosic material. Biobutanol, bioethanol, hydrogen, and methane are used as biofuels which can be produced by microorganisms. As many biofuel formation steps need extreme temperature and pH, mesophilic enzymes used in traditional methods are easily replaced by extremophiles. For an example, methane is biologically produced by methanogens [8]. To produce ethanol a thermophile named Thermoanaerobacterium saccharolyticum uses hemicellulose and pentose sugar as starting material [74].

24.5.4 Industrial use In food industries, starch is used as a source of glucose and fructose and the conversion is occurred through steps such as liquefaction of raw starch and enzymatic saccharification [75]. Key enzyme of these processes is α-amylase along with fungal glucoamylase which targets α-1,4 glycosidic backbone of starch [76]. The first acid stable archeal α-amylase has an optimum temperature of 100 C and was found from P. furiosus [77]. Using maltohexose as a substrate in high pressure, α-amylase produces maltotriose instead of maltose and maltotetrose [77]. Dunaliella salina, a halophilic alga, produces β-carotene, a red-/orange pigment present in carrot, pumpkin, and halophilic microorganism used in baking process (food coloring) and emulsion (confectionary) [11]. Proteases and lipases are basically used in laundry detergent to remove protein-based strain [78] for their stability in low temperature and alkaline environment. Proteases are also used for baking, cheese making, and brewing.

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FIGURE 24.4 Use of various extremophiles in their respective field of industry.

Pectin is a major element of middle lamella in plant cell wall. Pectin mainly comprises α-1,4-linked D-galacturonic acid residues. Pectin lyases mainly hydrolyse polygalacturonase [79]. Thermostable pectinases can be used for producing fruit juice or wine [80]. Enzymatically, enhanced pectinases are used in food preparation as dietary fibers [81]. Pectinases and xylanses are also used in paper industry [2]. Bacteriorhodopsin from halophilic archaea Halobacterium salinarum used to produce artificial retina, photochromic dyes, and bioelectronics [2,82].

24.5.5 Medicinal aspects Besides mesophilic microorganisms [83], extremophiles are also used to produce antibiotic, antifungal, and antitumor medicines [84]. Extremophiles can generate antimicrobial peptides and diketopiperazine which can act on blood coagulation functions and also having antibacterial, antifungal, and antiviral properties [85]. These were discovered in halophiles such as Haloterrigena hispanica and Natronococcus occultus that can activate and disable quorum-sensing pathways (an intriguing pathway of drug resistant pathogen like Pseudomonas aeruginosa) [86]. So it can be used in treatment of drug-resistant P. aeruginosa. Many species of halophilic archaea generate polyhydroxyalkanoates, a miscellaneous group of polyester which is used to make bioplastic that is water resistant and biodegradable. They are used as a storage of carbon in the case of microbial cells [81,87].

24.6

Conclusion

Extremophiles are unique in nature by thriving in extreme and stress conditions, including adverse range of temperature, pH, salt concentration, radioactivity, and pressure. In this chapter, we have tried to discuss about the protein adaptation of the extremophiles to thrive in extremes, their genomic study, and their commercial use in many fields. Some amino

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acid compositions are needed to be changed remaining active in extremes which can change charges, and hydrophobicity and pH of the cell. Due to specialized adaptations, sometimes they fail to survive in moderate environments. The organisms occurring in extremophilic conditions have quicker rate of evolution due to their higher frequency of horizontal gene transfer in comparison to mesophiles [88]. Sequencing of genomes in these organisms uncovered a greater potential for generation of bioactive natural products. These genomic approaches could be used for designing novel compounds and their applications in medical aspects. They have enhanced stability and specificity to use them as biocatalyst and in biotransformation. The discovery of Taq polymerase is now considered as a pillar in PCR reactions that can withstand extreme protein denaturing conditions. Like this, many extremozymes consisting of cellulases, proteases, amylases lipases, etc. have high commercial value in research and industries. Some extremozymes were seen to be produced in vitro by mesophilic organism such as Escherichia coli. Economic value of the extremozymes should be increased in large scale. Lastly, the rapid development of biotechnological “-omics” tools can emerge biocatalyst with desired properties for future green industries.

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Chapter 25

The ecophysiology, genetics, adaptive significance, and biotechnology of nickel hyperaccumulation in plants Anthony L. Ferrero1, Peter R. Walsh1 and Nishanta Rajakaruna1,2 1

Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA, United States, 2Unit for Environmental Sciences and

Management, North-West University, Potchefstroom, South Africa

25.1

Introduction

Plants are challenged by various environmental stressors that interfere with their biochemical and physiological processes. Some heavy metals have critical physiological roles [1 3] in plants, but elevated concentrations of these essential heavy metals or exposure to other heavy metals, which lack any known physiological roles, may result in stress via interference with either the function of enzymes or information-coding DNA or RNA in cells [4 6]. Some plants are more metal-tolerant than others, and thus heavy metal tolerance has been central to understanding adaptive evolution in plants [7 12] as well as the development of biotechnological fields such as phytoremediation and phytomining [13 16]. Some soils, including serpentine soils [17], have naturally high levels of heavy metals, and plants that can withstand these metal-rich soils are known as metallophytes [18,19]. Metallophytes tolerate metals via exclusion or accumulation. Most metallophytes exclude metals by chelation, binding ions to organic acids or other ligands, or by sequestration, storing metals within vacuoles of root cells where they cannot interrupt key cellular processes [20]. Accumulators, on the other hand, concentrate metals in plant parts, especially the epidermal tissues of leaves, whereas excluders generally keep shoot metal levels consistently low over a wide range of soil metal concentrations. Some metallophytes, termed “hyperaccumulators” [21 23], take up and sequester considerably high concentrations of metals in their aboveground tissues, often beyond thresholds that would be lethal to most plants [3]. Among these metal-hyperaccumulating plants, those that take up nickel (Ni) have received much attention both in terms of basic and applied research [20,24 26]. Plants require Ni as a micronutrient for nitrogen (N) and plant antioxidant metabolism [27 29]. Urease (EC 3.5.1.5, urea amidohydrolase) is perhaps the most important of the several known Ni requiring enzymes in higher plants [30]. Nickel functions as a cofactor to enable urease to catalyze the conversion of urea into ammonium (NH1 4 ), which plants can use as a source of N. Thus, urea conversion is impossible without Ni. Nickel-deficient plants develop leaf chlorosis and leaf tip necrosis [31], symptoms that can be prevented by Ni application, which increases leaf urease activity and prevents urea accumulation [32]. Thus, in generally N-poor soils such as serpentine [33], Ni may be particularly important for N acquisition and metabolism. The effects of Ni on Ni-hyperaccumulating plants (i.e., those that take up .1000 µg Ni g21 dry leaf tissue) [22] have not received much attention beyond the well-studied phenomenon of the role of Ni in plant defense against pathogens and herbivores [34,35]. Enhanced growth [36 39] and increased flowering [40] of some metal hyperaccumulator plants in the presence of higher Ni concentrations have previously been reported, but no physiological mechanisms have been suggested. The growth-stimulating effect may stem from direct beneficial effects of Ni on N metabolism or from indirect effects resulting from a potential role of Ni-containing urease in supporting plant pathogen defense [41]. Plants that hyperaccumulate Ni are equipped with physiological mechanisms for both increased uptake and tolerance. For example, Ingle et al. [38] report constitutively high expression of the histidine biosynthetic pathway in the Ni-hyperaccumulating Alyssum lesbiacum (Brassicaceae). Nickel hyperaccumulation is a worldwide phenomenon Physiological and Biotechnological Aspects of Extremophiles. DOI: https://doi.org/10.1016/B978-0-12-818322-9.00025-3 © 2020 Elsevier Inc. All rights reserved.

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spanning a vast diversity of higher plant families and taxa native to many metal-enriched ultramafic habitats [42 45] and is presumably mediated by the primary Fe21 or Zn21 transporters found within plant roots [46,47]. This chapter highlights current knowledge of the physiological mechanisms of Ni uptake, tolerance, and hyperaccumulation in plants and their underlying genetic bases and discusses the use of Ni-hyperaccumulating plants in biotechnological endeavors, especially in the use of plants to remediate Ni-contaminated soils (i.e., phytoremediation) and to mine Ni (i.e., phytomining/agromining).

25.2

Physiology: mechanisms of Ni uptake, translocation, chelation, and storage

Nickel is an essential micronutrient; however, it is toxic to most plants even at low concentrations [48 50]. The physiological mechanisms underlying metal accumulation are not fully understood, but recent studies have begun to tease apart the ways in which some species are able to tolerate and accumulate Ni [26,49]. Hyperaccumulation begins below ground in the rhizosphere, where Ni is taken up into roots from the soil. The bioavailability of soil Ni depends on the interaction of various chemical, physical, and biological processes in the soil. Low pH appears to be critical for making metals such as Ni more bioavailable [51,52] and some hyperaccumulating species secrete protons [53] to acidify the rhizosphere, thereby increasing Ni bioavailablity [54]. Soil bacteria may also contribute to this process by releasing compounds that make metals more bioavailable for plant uptake [55,56]. Once Ni is bioavailable and is taken up into a root, it is quickly chelated by various amino acids and organic acids and translocated until it can be properly stored, all while moderating the ion’s toxic effects on cellular processes [20,26,49].

25.2.1 Uptake The first step in the hyperaccumulation process is uptake of Ni from soil by roots. Nickel appears to be taken up primarily as Ni21, and a significant decrease in Ni uptake by Berkheya coddii (Asteraceae; Fig. 25.1H) has been reported upon addition of chelating agents to soil that reduce bioavailable Ni21 concentration [57]. Nickel uptake has been shown to increase under more acidic (low pH) conditions and decrease under more basic conditions [49], likely as a result of increased Ni solubility as H1 concentrations rise [52,58,59]. Uptake as Ni21 is important, as soluble Ni can enter roots by coopting uptake mechanisms of cations of similar charge and size (such as Zn21 or Fe21). For example, serpentine endemic Ni accumulators have been found to preferentially accumulate Zn21 in the presence of both Ni and Zn [60,61]. This suggests that a high-affinity transport mechanism for Zn is also used to take up Ni into root cells as opposed to Ni-specific transport mechanisms. Similarly, Fe21 transporters have been implied in Ni uptake [46,47]. In Arabidopsis thaliana (Brassicaceae), Ni is absorbed via the Fe uptake system, which is mediated by the iron-regulated transporter 1 (IRT1), the protein responsible for the primary Fe uptake mechanism in roots [47]. Excess Ni accumulation results in the induction of IRT1 expression, suggesting that the first stage of Ni accumulation may be facilitated by IRT1. Similarly, recent work on hyperaccumulators Odontarrhena bracteata and O. inflata (Brassicaceae) found that Ni uptake in O. inflata could be induced by Fe [62], suggesting a link between uptake of both metals. However, more work is necessary to fully understand the mechanisms of Ni uptake and the relationship between the accumulation of Ni and other divalent cations.

25.2.2 Chelation After Ni is brought into the root, it must be quickly chelated and complexed in order to reduce its toxic effects [39,63,64]. This appears to be done by both amino acids and organic acids [20] with the former acting primarily in roots and shoots and the latter acting primarily in leaf and shoot tissues [65,66]. Numerous studies have established free histidine as a primary chelating agent for Ni in root tissue [38,50,67,68]. Free histidine concentration in roots was found to be greater in an accumulator species, A. lesbiacum, compared to that of a nonaccumulator species, Brassica juncea (Brassicaceae; [66]). In addition, an increase in free histidine concentration was positively correlated with increased uptake of Ni in A. lesbiacum [66]. Histidine was also found to increase the mobility of Ni in roots of Noccaea caerulescens (Brassicaceae; Fig. 25.1D) to make it more available for xylem loading and radial transport [50]. Histidine biosynthetic pathways were also found to be more active in the Ni hyperaccumulator A. lesbiacum than in a nonaccumulating congener A. monatum [38]. This strongly suggests an important role for histidine in the chelation and detoxification of Ni in roots. Similarly, nicotinamine, an amino acid similar in structure to histidine, may play a role in the process of Ni chelation in hyperaccumulators [69,70]. In N. caerulescens (Fig. 25.1D), Ni exposure triggers the accumulation of nicotinamine in roots and subsequent formation of Ni nictotinamine complexes within the xylem sap, both of which did not occur when

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nonaccumulator Thlaspi arvense (Brassicaceae) was exposed to Ni, strongly suggesting that nicotinamine may act as a ligand in Ni chelation and transport in some hyperaccumulators [70]. Further, in N. caerulescens (Fig. 25.1D), a strong positive correlation between concentration of Ni and nicotinamine in leaf tissues was also observed, in addition to a negative correlation between foliar Ni and Fe concentrations, suggesting that Ni and Fe may compete for chelation by nicotinamine [71]. There is also evidence that organic acids are the primary complexing agents within leaf and stem tissue in some accumulators. Montarge`s-Pelletier et al. [63] used X-ray spectrography to demonstrate that across three hyperaccumulating species of Brassicaceae, Leptoplax emarginata, Odontarrhena chalcidica (Brassicaceae; Fig. 25.2B), and N. caerulescens (Fig. 25.1D), malic acid was the main ligand for Ni in leaf tissue, while in stem tissue citric acid was the primary ligand. In the South African hyperaccumulator Berkheya coddii (Fig. 25.1H), chelidonic acid was found to be the major chelating agent of Ni [72]. Nickel is also often unchelated while moving through nonliving xylem cells; Alves et al. [73] found that the primary species of Ni found in xylem sap of the hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum (Brassicaceae) was hydrated Ni21. This shows that there is much variability in the form of Ni within a plant and along the steps of Ni translocation. Unlike hyperaccumulators of other metals, such as cadmium and zinc, Ni hyperaccumulators do not use peptides such as phytochelatins or metallothionines (see Ref. [74] for a review on phytochelatins and for metallothionines see Ref. [75]) to complex and detoxify Ni. This lack of specific peptide chelators, paired with Ni’s coopted, low-affinity import mechanism, illustrates how the Ni hyperaccumulation process may not be a Ni-specific gene driven phenomenon [47,60,61].

25.2.3 Transport Another important aspect of the hyperaccumulation process is how the Ni is moved from roots through shoots to leaf epidermal cells. There is contrasting evidence for the relative importance of translocation in hyperaccumulation. FIGURE 25.1 A selection of Ni hyperaccumulators from around the world. (A) Bornmuellera kiyakii (Brassicaceae; Turkey); (B) Cochlearia aucheri (Brassicaceae; Turkey); (C) Noccaea jauberti (Brassicaceae; Turkey); (D) Noccaea caerulescens (Brassicaceae; Somerset, UK); (E) Justicia lanstyakii (Acanthaceae; Niquelaˆndia, Brazil); (F) Phyllanthus chryseus (Phyllanthaceae; Yamanigu¨ey, Cuba); (G) Berkheya zeyheri (Asteraceae; South Africa); and (H) Berkheya coddii (Asteraceae; South Africa); Photos courtesy (A) (F) Roger Reeves and (G) (H) Delia Oosthuizen.

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FIGURE 25.2 Ni hyperaccumulators used in agromining. (A) Phyllanthus rufuschaneyi (Phyllanthaceae) in field and pot experiments in Sabah (photo courtesy of Antony van der Ent); (B) Odontarrhena chalcidica (Brassicaceae) in field experiments in Albania (photo courtesy of Aida Bani); and (C) Streptanthus polygaloides (Brassicaceae) in a natural population in California (photo courtesy of Anthony Ferrero).

Some studies have found transport proteins to be among those consistently overexpressed in hyperaccumulators [77]. In contrast, research on Thlaspi goesingense (Brassicaceae) found that Ni translocation rates from roots to stem xylem were conserved between accumulators and nonaccumulators [77]. The process of Ni translocation between cell types appears to be another example of Ni coopting existing metal transport mechanisms, as there is evidence that Ni may be transported via the same efflux proteins used for Fe and Zn transport [78,79]. Some candidate transporters are in the Zrt/Irt-like proteins (ZIP) and NRAMP families, which are known to be low-specificity transport proteins for Zn and other transition metals [80]. There is evidence for root-to-shoot translocation of Ni being mediated by Fe-transporter in Alyssum species, as Fe deficiency appears to increase concentrations of Ni found in aboveground tissues [62]. The final step in Ni transport is moving the ion from the cytoplasm of a sink cell into the vacuole for storage. It has been shown in Alyssum lesbiacum that Ni may be pumped into the vacuole using secondary active transport through an H1/Ni21 antiporter driven by a proton gradient created by V-ATPase proton pumps [81]. This could be an example of a Ni-specific transport mechanism, but the exact protein has yet to be characterized. Further research into the action and regulation of these proteins, and the genes that encode them, could reveal the nature of Ni transport, homeostasis, and detoxification [24].

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25.2.4 Localization and storage The final steps in the physiological processing of Ni by plants are localization to specific sink tissues and storage of Ni within these cells. There is evidence that during the localization process, hyperaccumulators transport Ni through phloem tissue, as visualized by X-ray analysis of Senecio coronatus (Asteraceae) roots [82] and phloem sap analysis of N. caerulescens (Fig. 25.1D), which shows bidirectional transport of Ni chelated with malate [83]. In addition, work on hyperaccumulating trees from Borneo, Rinorea bengalensis (Violaceae; Fig. 25.3) and Phyllanthus balgooyi (Phyllanthaceae; Fig. 25.4), also has shown Ni to be present in high concentrations in phloem sap (Figs. 25.3 and 25.4), 7.9% and 16.9%, respectively, and it likely is chelated with citrate [84,85,86]. Nickel mobility in phloem is also supported indirectly by research that shows the presence of Ni in flowers and seeds [87 90]. Reproductive tissues are major phloem sinks and the elevated Ni concentrations found there implicate the phloem as an important sink. Further, the ecological implications of Ni localization in leaves have led to numerous studies on multiple hyperaccumulating species. These studies have indicated that Ni is primarily localized in vacuoles and cell walls of leaf epidermal cells [82,91]. Streptanthus polygaloides (Brassicaceae [92]; Fig. 25.2C) and O. chalcidica ([93] Fig. 25.2B) tissues were analyzed using scanning electron microscopy and energy-dispersive X-ray probing and were shown to have the greatest Ni concentration in leaf epidermal cell vacuoles, suggesting a role for Ni in herbivory or pathogen defense ([94]; see the following sections on adaptive significance of Ni hyperaccumulation). Similar studies on the hyperaccumulator T. goesingense have also provided evidence for Ni storage in leaf epidermal cell walls and to a lesser extent in vacuoles, complexed with citrate and histidine [84,85]. Similarly, energy-dispersive X-ray microanalysis of Thlaspi montanum var. siskiyouense (Brassicaceae; now Noccaea fendleri subsp. siskiyouensis) shows Ni localization in subsidiary cells that surround guard cells, but not in guard cells or in other more elongated epidermal cells [91]. Cell walls, cuticles, and epidermal trichomes can also store high concentrations of Ni (e.g., [77,78,88,95,96 98]). Furthermore, FIGURE 25.3 Phloem tissue of Rinorea bengalensis (Violaceae; Malaysia) contains over 4% Ni. Plant (top left), leaves (top right), and piece of bark showing Ni-enriched phloem (bottom). Photo courtesy of Antony van der Ent.

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FIGURE 25.4 The New Caledonian endemic tree Pycnandra acuminata can accumulate an astonishing 25 wt.% nickel in its blue latex. Photo courtesy of Antony van der Ent.

Ni enrichment has been recorded in leaf tips, likely due to secretion of excess Ni via guttation [99], as well as in latex (e.g., Pycnandra acuminata, Sapotaceae [100] Fig. 25.4). Once Ni is stored in a manner that limits its toxic and oxidative effects, plants use Ni for a variety of biological functions [101], some of which may increase overall fitness [40] as discussed in the following section on the adaptive significance of Ni hyperaccumulation.

25.3

Why hyperaccumulate nickel?

The ubiquity of Ni hyperaccumulation poses an interesting question about the evolutionary factors driving this unusual phenomenon. Since the “discovery” of Ni hyperaccumulation in 1948 [102], numerous hypotheses have been brought forth to explain the adaptive significance of Ni hyperaccumulation in plants, given that this process is energetically costly and that plants exhibiting this trait risk effects of Ni toxicity [103,104]. There is currently little consensus on the adaptive significance of Ni or other metal hyperaccumulation on a wide evolutionary scale; however, four main hypotheses have been presented with mixed experimental evidence, each stating that Ni hyperaccumulation plays a role in one or more of the following phenomena: elemental defense, nutrition, elemental allelopathy, and drought tolerance [34,105 107].

25.3.1 Elemental defense Thus far, the prominent hypothesis for the adaptive significance of Ni (and other metal or metalloid) hyperaccumulation in plants is the elemental defense hypothesis, which states that intracellular storage of high concentrations of heavy metals can prevent, deter, or provide resistance against unadapted pests, pathogens, and herbivores, which may feed on hyperaccumulators [94,105,108]. There is experimental evidence demonstrating deterrence of herbivory by multiple hyperaccumulating taxa, notably S. polygaloides ([109] Fig. 25.2C), Alyssum spp. [110], and Berkheya coddii ([35,42,111] Fig. 25.1H). The

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evolutionary and physiological mechanisms behind the utilization of Ni toxicity for defense against herbivory vary by taxon; however, two main explanations describe the relationship between Ni concentration and plant tissue toxicity. According to the defensive enhancement hypothesis, greater concentrations of Ni provide more effective defense from herbivory, which provides selective pressure to hyperaccumulate Ni in higher quantities [94,108]. Alternatively, as described by the joint effects hypothesis [112], some plants produce organic molecules that can enhance the effect of Ni toxicity in either additive or synergistic ways. For example, the presence of nicotine enhances the toxicity of Ni, resulting in higher deterrence than either of the substances alone [113]. Therefore, plants have two main strategies for increasing the toxicity of their tissues for purposes of elemental defense: accumulate more metals or synthesize chemicals with joint effects. Despite the potential toxic effects of heavy metals and other plant-produced chemicals, many specialist insects exist which feed on hyperaccumulating plants. One well-studied example is Melanotrichus boydi (Hemiptera: Miridae), a monophagous specialist herbivore on the Ni hyperaccumulator S. polygaloides, which, in host-choice studies, consistently prefers S. polygaloides over other nonhyperaccumulating plants found in its native range [114]. M. boydi sustains an elevated body Ni concentration of .700 µg Ni g21 [115]; however, there is little evidence that this trait offers the plant bug significant defense against its predators or pathogens. Boyd and Wall [116] demonstrated that Ni can be transferred up the food chain from high-Ni content insects to predators, resulting in moderate Ni accumulation in the third trophic level. However, predators varied in reaction to elevated body Ni concentration resulting from consumption of M. boydi, and only the crab spider Misumena vatia (Araneae: Thomisidae) showed adverse effects, resulting in significantly decreased survivorship when fed M. boydi, in comparison to Ni-absent, control diets. Thus, in this case, elevated body Ni concentration in a monophagous herbivore is most likely a consequence of consumption of high-Ni plant tissues, rather than a trait specifically selected for the purpose of defense [117]. Boyd et al. [118] demonstrated decreased susceptibility to two pathogens; a fungus (Alternaria brassicicola) and a bacterium (Xanthomonas campestris) in high-Ni-containing S. polygaloides, suggesting Ni can act as an antimicrobial agent in leaf tissues. In addition, Ni-treated Alyssum spp. show resistance to fungi Pythium mamillatum and P. ultimatum, which cause damping-off disease of seedlings [119]. In both instances, plants grown on Ni-amended soil consistently outperformed those grown in the absence of Ni. Although these results are promising, more research is required to elucidate the relationship between Ni hyperaccumulation and resistance to pathogens commonly found in a given plant’s native range [120].

25.3.2 Nutritional demand The nutritional demand hypothesis suggests that hyperaccumulating species have a physiological requirement for high levels of Ni, due to the extensive use of Ni in certain biochemical pathways. Nickel is required as a micronutrient in most plants [101] and Ni deficiency can result in leaf chlorosis and leaf tip necrosis [31]. Nickel-hyperaccumulating plants maintain tissue concentrations of the metal high above the sufficient amount for most plants, in quantities indicative of macronutritional demand. The presence of Ni has been demonstrated to enhance flowering of the hyperaccumulator Alyssum inflatum (Brassicaceae; [40]); however, the biochemical pathway responsible has yet to be elucidated. Nickel is used as a cofactor in multiple plant enzymes, most importantly in urease, which converts urea to ammonium, thereby making nitrogen (N) available to a plant [30,41]. Since serpentine soils that host these hyperaccumulator plants are generally N-poor [33], it is possible that the elevated Ni requirement is due to greater urease activity. Experiments on soybeans show that Ni supplementation improves the growth of urea-fed plants while having no apparent effect on nitrate-fed plants [121], suggesting that higher Ni concentration could be a result of greater levels of N fixation from urea. Nickel accumulation can also interfere with the uptake and homeostasis of other heavy metals such as copper and iron [78], because each of these ions have similar chemical properties and often share a transporter protein. Thus, due to its interaction with multiple micronutrients and macronutrients, Ni may be required in large quantities to regulate tissue concentrations of key elements found in serpentine soils.

25.3.3 Elemental allelopathy Since the high shoot Ni concentrations common in hyperaccumulators are much higher than can be tolerated by most intolerant plants without experiencing Ni toxicity, it is possible that Ni deposition in surface soil could result in the deterrence or toxicity of intolerant plants, conferring a competitive advantage for hyperaccumulators. Zhang et al. [122] assessed the effects of high-Ni leaves shed by O. chalcidica by measuring both the change in soil Ni concentration over time and the germination rate of natural competitors in Ni-enriched soils. Results showed that, while Ni was quickly released from O. chalcidica biomass causing an increase of phytoavailable Ni in the soil, Ni concentration also rapidly decreased due to chelation by Fe and manganese (Mn) oxides and silicates, which are both commonly found in serpentine soils. Addition of O. chalcidica biomass, moreover, did not demonstrate any significant effect on seed germination

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of eight different competing herbaceous species. Thus, these results do not support the hypothesis that elemental allelopathy via leaf litter provides a competitive advantage to hyperaccumulators via direct toxicity to competitors. It is possible that tissue deposition can result in allelopathy via phytoenrichment of the soil, which alters the microbial community and the resulting fitness of a competing plant species [123]. A study of S. polygaloides (Fig. 25.2C) and nonendemic serpentine congener S. tortuosus (Brassicaceae) showed that the latter experienced decreased germination rates and fruit development in high-Ni soil, while S. polygaloides had its greatest reproductive success in high-Ni soil [87]. Studies that aim to draw cause effect relationships between leaf litter of hyperaccumulators and soil Ni phytoenrichment have produced mainly speculative results, however, because it is difficult to demonstrate that leaf litter is directly causing soil Ni enrichment, as opposed to plants being predisposed to grow in naturally Ni-enriched soils [124,125]. However, a recent study by Adamidis et al. [126] shows that Ni-resistant decomposers in serpentine soil contribute to the accelerated breakdown of high-Ni plant litter, suggesting a potential selective advantage for the metal-hyperaccumulating plants through litter decomposition on serpentine soils. High-Ni concentration in floral tissues can cause Ni hyperaccumulators to host distinct floral visitor communities by selecting for specific pollinators (i.e., elemental filter hypothesis [127]). It is hypothesized that transfer of Ni to intolerant plants via pollinators carrying floral offerings, such as pollen, could lead to elemental allelopathy [128], as Ni is known to cause detrimental effects on plant reproductive systems [87]. However, thus far, there has been no support for elemental allelopathy via hetero-specific pollen transfer from Ni hyperaccumulators to Ni-intolerant plants [128].

25.3.4 Drought tolerance Serpentine soil, by virtue of its shallow depth, poor soil structure, and high porosity can maintain exceptionally low water content at field capacity. Thus, serpentine soils in Mediterranean climates are subject to prolonged drought conditions, and there is a significant selective pressure for drought tolerance among serpentine-tolerant plants [129]. Since Ni often is stored in the leaf epidermis, including in guard cells of stomata [92], some researchers have hypothesized that Ni may act as an osmoticum for metal hyperaccumulators, adjusting the water potential of the plant so that it is able to maintain a favorable water potential gradient under dry conditions [105]. Studies of the Ni hyperaccumulator Stackhousia tryonii (Stackhousiaceae) showed enhanced Ni hyperaccumulation with increasing levels of drought stress, suggesting a possible role for Ni in osmotic adjustment [130]. A similar study investigating the effects of poly ethylene glycol (PEG)-simulated drought on Cleome heratensis (Cleomaceae) reported that Ni exposure was necessary for optimum performance in drought conditions; however, it was unclear if this was an adaptation to Ni or drought [131]. Whiting et al. [132] stated that there was no strong evidence that Ni hyperaccumulation augmented drought tolerance for O. chalcidica (Fig. 25.2B) and that metal concentration had little effect on the rate of evapotranspitation or the osmolality of leaf-sap extracts obtained from the plant. Similarly, experiments on Hybanthus floribundus (Violaceae), a nickel-hyperaccumulating shrub violet, failed to elucidate a role of Ni in osmotic adjustment [133]. Investigations into the effects of other hyperaccumulated elements, such as zinc (Zn) or selenium (Se), on drought tolerance have failed to produce solid evidence that elemental hyperaccumulation results in a physiological advantage under dry conditions [134]. Zinc hyperaccumulation has no significant effect on drought tolerance in N. caerulescens at the whole-plant level, and most evidence shows that Zn accumulation is constitutively expressed, rather than activated as a stress response [132]. However, Hajiboland et al. [135] demonstrated that Se-treated wheat exhibited higher hydraulic conductivity than control groups, suggesting an improvement of water uptake capacity.

25.4 Genetics of nickel accumulation Although many of the physiological mechanisms driving the process of Ni hyperaccumulation have been coopted from metal uptake and tolerance mechanisms known for metals such as Zn and Fe [46,78 80], the heritability and selection mechanisms for the hyperaccumulation trait also imply an underlying genetic basis. A number of techniques have been utilized to better understand the genetics of hyperaccumulation [102,136,137], including comparative population genome sequencing [138,139], identification of target genes from key proteins involved in hyperaccumulation ([80,136] Table 25.1), and phylogenetic analyses [140 143]. In the future, continued experimentation, using RNA sequencing and mutated or knocked out target genes [77], will be extremely valuable in elucidating the ways in which certain genes and gene products contribute to the hyperaccumulation phenotype [24].

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TABLE 25.1 Known genes and gene products associated with Ni hyperaccumulation. Gene

Protein

Function

Taxa

Citation

Tjznt1, Tjznt2

TjZNT1, TjZNT2

Regulate Zn, Fe, and Ni transport

Thlaspi japonicum

[80]

TjNramp4

TjNRAMP4

Transport of metals across vacuolar membrane

T. japonicum

[80]

PgIREG1

PgIREG1

Vacuolar sequestration of metals in tonoplast cells

Psychotria gabriellae

[144]

NcIREG2

NcIREG

Fe and Ni sequestration in vacuoles

Noccaea caerulescens

[76]

TcYSL3

TcYSL3

Transports nicotinamine-bound Ni and Fe

N. caerulescens

[145]

HMA3, HMA4

HMA3, HMA 4

P-type ATPase ion pumps

N. caerulescens

[146]

NcIRT1

NcIRT1

Maintenance of homeostasis in Fe and Ni uptake

N. caerulescens

[146]

NcZIP10

NcZIP10

Transport of Ni, Zn, and Fe

N. caerulescens

[146]

PDF2.3

PDF2.3 Defensin peptides

Binding and transport of Ni

N. caerulescens

[146]

ALMT12

ALMT12

Malate transporter

N. caerulescens

[146]

TgMTP1

TgMTP1t2

Transport of metals into the vacuole

T. goesingense

[147]

BjYSL7

BjYSL7

Nicotinamine-bound Ni and Fe influx transporter

Brassica juncea

[196]

BjCET3, BjCET4

BjCET3, BjCET4

Cation efflux transporter proteins

B. juncea

[197]

TcNAS

TcNAS

Nicotinamine biosynthesis

N. caerulescens

[70]

HSN1

HSN1

Histidine biosynthesis

Arabidopsis thaliana

[149]

TgSAT-c

SAT

Glutathione biosynthesis

T. goesingense

[153]

AtIRT1

AtIRT1

Ni and Fe uptake transporter

A. thaliana

[47]

Most known genes that are vital to the process of Ni hyperaccumulation are either (1) ion transporters, which assist in the uptake of Ni from the environment or (2) involved in the biosynthesis and feedback pathways of Ni-chelating molecules.

25.4.1 Identification of target genes involved in Ni hyperaccumulation by transporters A primary area of research on the genetics underlying Ni hyperaccumulation involves the identification and characterization of target genes that directly or indirectly influence a plant’s ability to tolerate, transport, and accumulate Ni. One of the key distinctions between hyperaccumulators and metal-tolerant species is the tendency for hyperaccumulators to localize heavy metals in leaf epidermal cells, whereas metal-tolerant species tend to exclude metals from aboveground tissue [20]. This implies an important role for transport proteins in facilitating root-to-shoot translocation of heavy metals, as well as the characterization of the hyperaccumulation phenotype [144]. Membrane proteins such as IRT and ZIP have been shown to play important roles mediating Ni import and translocation [46,47]. Proliferation of next generation sequencing technologies and genetic manipulation tools is making it easier than ever for researchers to locate impactful genes. For example, a study of the genomes of A. serpyllifolium populations found on and off of serpentine soil shows high DNA polymorphism at loci encoding natural resistance associated macrophage proteins (NRamp) and iron-regulated (IREG) transporter proteins, which are known to influence Ni tolerance [138]. This presents a potential example of local genetic adaptation, as opposed to phenotypic plasticity, resulting in Ni hyperaccumulation. A recent cross-species analysis of hyperaccumulator RNA sequences revealed that, across five genera, high IREG/ferroportin transporter expression was a conserved quality [77]. In addition, the study quantified the necessity of the NcIREG2 gene for hyperaccumulation in N. caerulescens (Fig. 25.1D), as transgenic plants with reduced expression of NcIREG2 had decreased Ni accumulation. The continued engineering of transgenic hyperaccumulator models will be a vital study tool in the future for understanding the genetic mechanisms of Ni hyperaccumulation.

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Most genetic studies to date have utilized model organisms such as Arabidopsis and yeast to find influential DNA sequences or proteins and subsequently look for homologs and measure their expression activity in the hyperaccumulator in question. Alternatively, others have examined genes from accumulators and tested their expression patterns in model organisms. While a Ni-specific transport protein has yet to be discovered [8], most studies of Ni hyperaccumulation genetics have focused on integral membrane metal transporter proteins. For example, in Thlaspi japonicum, three genes presumed to be Ni transporters have been cloned [80]: ZIP transporters TjZnt1 and TjZnt2, and an NRamp transporter gene, all of which were homologous to genes known to influence metal transport in other species. When yeast were transformed with these genes and exposed to Ni21, the yeast expressing ZIP transporters showed increased tolerance to Ni while those transformed with an NRamp transporter showed an increase in Ni concentration and sensitivity, suggesting a role for these proteins and genes in Ni accumulation [8]. Similar methods were used on Psychotria gabriellae (Rubiaceae) to determine if PgIREG1, which encodes an IREG family metal transporter protein that is thought to be responsible for sequestration of metal into vacuoles, was a candidate gene influential in Ni accumulation [136]. The authors observed a two and a half times higher rate of expression of PgIREG1 in P. gabriellae than in a nonaccumulating congener, P. semperflorens. In addition, a Yellow Stripe-Like (YSL) transporter family gene in Thlaspi caerulescens (syn. N. caerulescens; Fig. 25.1D), TcYSL3, increased Ni/Fe-nicotinamide chelate uptake in yeast, indicating TcYSL3 expression may serve as a key phenotype in metal uptake and accumulation in T. caerulescens [145]. A comparison of expression levels of many different genes across different ecotypes of the same species found a number of candidate genes that could be impacting accumulation [146], the most significant genes being those encoding HMA3 and HMA4 (both ATPase transporters) as well as the genes coding for IRT1 and ZIP10 proteins. Metal tolerance protein (MTP) family cation efflux transporters are also important in conferring Ni tolerance. In T. goesingense, enhanced expression of the TgMTP1 gene, producing the TgMTP cation efflux protein, was observed under hyperaccumulation and also was found to confer Ni tolerance in yeast, suggesting a role for the gene in Ni accumulation [147]. Further, two genes from B. juncea encoding MTP family proteins, BjCET3 and BjCET4, were found to contribute to Ni homeostasis in yeast cells, suggesting that cation efflux transporters (coded for by BjCET genes) may aid in Ni localization and tolerance [148]. Also, in B. juncea, the gene encoding another YSL family transporter protein (BjYSL7) was upregulated manyfold upon the addition of NiCl2 to the soil solution, implying a role for BjYSL7 in the import and storage of Ni. Taken together, these studies show that much of the genetic regulation for Ni hyperaccumulation relies on regulation of transport proteins. However, transport is only one part of the accumulation process, and additional genes and proteins influencing the process of hyperaccumulation have also been identified.

25.4.2 Identification of target genes involved in Ni hyperaccumulation by chelators While much research has focused on identifying target genes for metal transport proteins, some research has been directed at elucidating the expression pathways for known chelators of Ni. For example, in N. caerulescens (Fig. 25.1D), the nicotinamide synthase gene (TcNAS) was important in Ni chelation to nicotinamide by producing nicotinamide in leaves and facilitating its transport to roots, although TcNAS expression did not change in response to Ni addition [70]. Studies on Arabidopsis to investigate the histidine biosynthetic pathway [149] identified eight different enzymes important in histidine synthesis, including HISN1, which is normally inhibited by histidine. However, transgenic A. thaliana mutants that constitutively produce HISN1 exhibit elevated levels of free histidine as well as significantly greater tolerance to Ni, suggesting an important role for HISN genes in regulation of Ni uptake and tolerance [38]. Although most studies on Ni hyperaccumulation have found little to no link with phytochelatins, in Rauvolfia serpentina (Apocynaceae) culture, expression of the short peptide and phytochelatin (y-Glu-Cys)2 4-Gly could be induced via introduction of Ni [150]. This suggests that Ni may directly influence phytochelation expression in some plants; however, more work needs to be done to firmly establish a broader importance of phytochelatins in Ni tolerance. Another interesting biochemical pathway involved in Ni hyperaccumulation is glutathione (GSH) synthesis. GSH is an antioxidant believed to be important in the process of decreasing the oxidative stress commonly induced by plant uptake of heavy metals [151,152]. In T. goesingense and transgenic Arabidopsis, GSH concentrations increased in response to addition of Ni [153]. Serine acytyl-transferase (SAT), which produces glutathione, was found to have greater activity in response to Ni, and plants overexpressing the gene encoding SAT and TgSAT-m had increased Ni tolerance, suggesting that there is an important genetic component to GSH-mediated Ni tolerance.

25.5

Phytoremediation and agromining

Due to their highly unusual physiology, metal hyperaccumulators hold the potential for exciting new methods of ecofriendly mining and bioremediation [154]. Ni-hyperaccumulating plants are being used for phytoextraction (for reviews

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see Refs. [155 157]), which involves growing hyperaccumulators on metalliferous soils and subsequently removing the plant biomass from the site, either with the goal of harvesting valuable metals (i.e., phytomining or agromining) [154,158] or to clean metal-contaminated soils (i.e., phytoremediation) [157]. Nickel hyperaccumulators are excellent candidates for phytoremediation and agromining because Ni has relatively high monetary value in today’s market, it can be efficiently extracted from ash [159,160], and a number of Ni hyperaccumulators have sufficiently high tissue Ni concentrations to potentially support the economic viability of the practice [97,161,162]. It is hypothesized that long-term cropping of Ni hyperaccumulators can gradually detoxify mildly to moderately Ni-contaminated soils [163,164] and thus make such soils available for other agricultural uses. The time frame required for ample phytoextraction of heavy metals resulting in rehabilitation of contaminated soils is referred to as the “extraction period,” and varies largely depending on the concentration of metals in the soil and efficiency of extraction [165]. The main limitations to practical use of hyperaccumulators for phytoremediation relate to long extraction periods [166] and the costs of operation for these potentially multidecade endeavors. Successful longterm phytoremediation projects rely on income from biomass yield and element recovery to cover the cost of crop maintenance [167] and for this reason, soil remediation is often considered an advantageous by-product of agromining. Since both processes aim to extract toxic metals from the soil with high efficiency and most recent literature emphasizes the viability of commercial agromining with soil cleanup as a potential consequence, we will mostly focus on the logistics of agromining in this chapter. In conventional mining practices, ores are extracted from the earth and then smelted to yield pure metals. Since this is an energetically and monetarily expensive operation, for the practice to be economically viable the ore must contain a minimum level of target metals, termed the “mineralogical barrier” (sensu [168]). Estimating the threshold for economic viability of Ni extraction via traditional strip-mining processes is complicated because (1) Ni is often produced as a by-product while mining for other metals, such as copper, and (2) differing mineralization processes of Ni indicate varying thresholds for economic viability. The proposed mineralogical barrier for laterite, one of the most common rock types mined for Ni, is approximately 3000 mg kg21 [169], which means that this is the lowest Ni concentration for which conventional mining practices may produce an adequate yield of Ni to support ongoing extraction. Many Nihyperaccumulating plants grow naturally on serpentine soils with Ni concentrations well below 1500 mg kg21, and multiple field trials for phytoextraction have been performed on these relatively low-Ni soils [170,171], which means that they could be potentially acceptable for agromining on a wide scale. Critically high-concentration ore deposits are limited to small areas and many of these deposits are being depleted due to continuous mining [169], while subeconomic ultramafic soils are more widespread and largely unused for direct human economic benefits [14]. Thus, low-Ni concentration soils inherently lend themselves better to agromining than strip-mining. The ideal soils for commercial agromining have the highest possible phytoavailable Ni while still maintaining a moderate pH range (between 4 and 7); have an acceptable level of soil structure, depth, and water-holding capacity; and are found on relatively flat land [165]. Such soils are found throughout the Mediterranean, as well as in subtropical areas such as New Caledonia and Sabah, Malaysia [161,172,173]. Nkrumah et al. [169] estimated the gross monetary yield of an extensive agromining practice to be approximately US$1980 ha21, which is higher than the estimated monetary yields for common crops on fertile soils, such as corn in the United States, which yields approximately US $1400 ha21 in today’s market at the current price of US$3.68 per bushel [175]. In addition, these crops would not displace any preexisting farms, except for those on metalliferous soils, which would naturally yield a suboptimum harvest of most food crops. The economic viability of agromining on disturbed serpentine soils, therefore, is noteworthy, especially in areas dominated by ultramafic soils that offer little agricultural potential. Increasing the efficiency and monetary yield of agromining has become a hot topic in applied ecology in recent years, prompting investigations into various techniques to improve shoot Ni concentration or biomass of hyperaccumulating plants [176]. Basic regimens have been established for irrigation, pest control, and fertilization in agromining practices [174], which vary by the species being used. All these practices aim at increasing plant survivorship and biomass; however, greater shoot biomass in the plant is usually accompanied by lower shoot Ni concentrations [177]. In contrast, soil enrichment with excess nitrate can enhance IRT1 expression in the roots of Arabidopsis spp., thereby increasing the rate of Ni import [178]. In addition, soil amendment with Ca is useful, as foliar Ca and Ni concentrations are positively correlated in many instances [86], and soil Ca levels commonly become depleted due to periodic removal from the field when harvesting bioore [174]. Utilization of select rhizobacterial inoculants could increase Ni yield by enhancing shoot biomass [179]; however, there is little information available concerning the specific physiology of advantageous soil symbionts. Nitrogen fertilization promotes the growth of a healthy bacterial community in the rhizosphere of O. chalcidica (Brassicaceae; syn. O. chalcidica; Fig. 25.2B) by alleviating the deleterious effects of Ni toxicity on the microbes, resulting in an increase in both shoot Ni concentration and biomass [180]. Cocropping of multiple hyperaccumulator species inoculated with naturally occurring rhizobacteria

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results in significant increase of Ni uptake by the plants and an increase in diversity of the microbial community, highlighting the importance of using multispecies covers in agromining operations [181]. An excellent strategy to improve levels of soil nitrogen and microbial enzymatic activity at the same time involves cocropping Ni hyperaccumulators with legumes, as demonstrated with O. chalcidica grown in conjunction with Lupinus albus (Fabaceae), which proves beneficial to both species and provides an opportunity to produce food and Ni simultaneously [182]. Further investigations of legumes tolerant of ultramafic soils are necessary to optimize this regimen for practical agromining [183]. Genetic engineering provides a cutting-edge option to optimize agromining by producing modified hyperaccumulators designed specifically to take up maximum amounts of Ni. Upregulation or overexpression of key genes important for Ni tolerance and accumulation is the common strategy for producing strains ideal for agromining [184]. Generally, target genes are selected based on their presumed role in ion transport, metal chelation, or metal detoxification [185]. Although some novel genetic modifications have been made to enhance metal tolerance and uptake in some plant species [20], no instances of genetic manipulation directly aimed at increasing Ni hyperaccumulation for the purposes of commercial agromining have been described to date. One excellent case study on implementation of agromining at an economically viable level is that of O. chalcidica grown on ultramafic vertisols in Albania (Fig. 25.2B [177]), resulting from a series of field experiments performed between 2005 and 2014, optimizing procedures for Ni phytoextraction. The steps followed for agromining with O. chalcidica are as follows: plant density of 4 plants m22; plowed ultramafic soil with nonlimiting conditions of Ni availability; and adequate fertilization with N, P, K, S, and Ca. Results demonstrated an improvement in yield from 1.7 to 105 kg Ni ha21 after optimization, but little decrease in soil Ni concentration was recorded, although it is expected that long-term agromining would cause depletion of soil Ni ([177], Table 25.2). Nickel yield and overall efficiency of the process have increased due to development and subsequent optimization of an economically feasible hydrometallurgical process for producing ammonium nickel sulfate hexahydrate salt, which can be extracted from O. chalcidica ash at a purity of 88.8% [159], or even 99.1% [160]. Another pioneering study of a serpentine quarry in northwest Spain explored the viability of four herbaceous Brassicaceae annuals—Odontarrhena serpyllifolia, O. chalcidica, Bornmuellera emarginata, and N. caerulescens—as candidates for use in agromining on mine tailings [158]. Quarries and mine tailings are particularly unfavorable environments for plants due to shallow, underdeveloped soil with low water-holding capacity, limited organic matter content, and nutrient deficiency [165], all of which are common symptoms of “serpentine syndrome,” a set of characteristics that makes ultramafic soils physiologically demanding habitats for plant growth [188]; these stressors are much more extreme in the case of disturbed ultramafic settings such as quarries and mines. In this study, plots were amended with composted sewage sludge or inorganic NPK fertilizers, and while biomass yields increased for both treatments, compost was much more effective. This was possibly due to a three- to six-fold increase in bacterial density, which is known to positively affect shoot biomass in Ni hyperaccumulators [189]. All species except O. chalcidica exhibited a decrease in aboveground Ni concentration following the addition of compost; however, the significant increase in biomass resulted in the greatest total yield of Ni. O. chalcidica exhibited the greatest overall yields of Ni, making it a favorable candidate for agromining in the Mediterranean region [158]. In subtropical regions, shrubs and trees, rather than herbaceous annuals, are utilized for prospective agromining practices. The first tropical “metal farm” in Sabah, Malaysia is using Phyllanthus rufuschaneyi (Phyllanthaceae; Fig. 25.2A), which yields high-Ni concentrations, often .6500 mg Ni kg21 [161]. High plant biomass and accompanying shoot Ni concentration provide much promise for the development of commercial agromining in subtropical regions using this

TABLE 25.2 Economically important Ni hyperaccumulators. Species

Shoot Ni conc. (g kg21)

Ni yield (kg ha21)

Native region

Citations

Odontarrhena chalcidica

11.5

105

Temperate—Mediterranean

[177]

Ophrys bertolonii

7

72

Temperate—Mediterranean

[186]

Tropical—Sabah

[161]

a

Phyllanthus rufuschaneyi

25

250

Streptanthus polygaloides

10

100

Mediterranean—California

[21,187]

Berkheya coddii

10

100

Temperate—South Africa

[58]

Shoot Ni concentration, biomass, and total Ni yield are based on greatest values from experimental data. Only taxa tested in agromining field experiments are included. a Suggested value based on field experiments conducted to date.

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taxon, which could achieve a yield of 250 kg Ni ha21([156] Table 25.2). If replicated on a wide scale, this would be the greatest yield of Ni via agromining yet to be recorded anywhere in the world. The species has other favorable qualities in addition to high-Ni concentration and biomass, such as a multiple stems and rapid regeneration after harvest. However, some uncertainties exist concerning the wide-scale propagation of P. rufuschaneyi—the species is susceptible to fungal infections under shade, and its dependence on a specialized pollinator (Epicephala moths) poses a barrier for its broader geographic use [190]. There are potentially many more Ni hyperaccumulators to be discovered from around the world and floristic surveys should be encouraged to document such species before they are lost from their native habitats (i.e., serpentine outcrops) that are undergoing drastic changes resulting from agriculture, deforestation, mining, exotic species invasions, and atmospheric deposition of previously limiting macronutrients such as nitrogen [191]. For agromining to be successful at a worldwide scale, and without posing the environmental risks associated with moving hyperaccumulators from one region to another for agromining [20], region-specific plants will have to be utilized after carefully studying their biology and ecology and testing them in small-scale trials prior to additional testing on a larger scale [155]. In this regard, the Global Hyperaccumulator Database (http://hyperaccumulators.smi.uq.edu.au/collection/) is an important resource for identifying region-specific Ni hyperaccumulators.

25.6

Conclusion

Plants can tolerate nearly every environment on Earth and have evolved adaptations to deal with extreme abiotic and biotic stressors. The unique nutritional and osmotic challenges associated with ultramafic soils are no different, and plants growing on these soils have developed traits to cope with limited essential nutrients, high heavy metal content, and low calcium to magnesium ratios [33]. Heavy metal hyperaccumulation, defined as .1000 mg metal kg21 dry weight for most metals (Ref. [24] and also see Ref. [192]), is a phenomenon born out of these harsh edaphic conditions and exhibited by more than 500 species worldwide [193]. Nickel is by far the most commonly accumulated element and, as a result, much of the literature on hyperaccumulation concerns Ni [194]. Recent studies have begun to fully elucidate the physiological mechanisms plants use to take up, detoxify, transport, and store bioavailable Ni from the soil. The unique capacity of hyperaccumulators to take up Ni into the roots seems to be dependent on a number of factors, namely, soil pH, the presence of other heavy metals, the bioavailable Ni concentration, and the efficiency of pumps and transport proteins in root epidermal cell membranes [53,144]. Nickel is chelated by various organic and amino acids upon entry to a root and translocated into aboveground tissue via xylem, where it is then primarily localized into leaf epidermal cell walls and vacuoles or to phloem tissue [66,82,89,84,88,91]. The Ni hyperaccumulation process seems to draw heavily on existing transport and homeostasis mechanisms for other metals, namely, Zn and Fe, indicative of more recent evolution of the trait [47,58,61]. There are four main hypotheses for the adaptive significance of Ni hyperaccumulation, the most supported being the elemental defense hypothesis [105], which states that plants with elevated tissue Ni concentrations are better protected from unadapted pathogens, pests, and herbivores. Other hypotheses are that Ni hyperaccumulation evolved because certain species require high levels of Ni to carry out metabolic functions [31,40], Ni is involved in elemental allelopathy to inhibit the growth of nearby plants [81], and Ni is used as an osmoticum, aiding in drought tolerance [84,88]. The genes, expression patterns, and proteins that create this unique phenotype are slowly being discovered. So far a handful of genes involved in Zn and Fe transmembrane transport have been shown to play a role in Ni tolerance and accumulation by facilitating movement of Ni into aboveground tissues [80,136,145,146]. Further, genes and gene products for the biosynthetic pathways controlling Ni chelator concentration and localization may also be involved in Ni transport and detoxification [38,149,153]. Innovations in genetic sequencing and gene manipulation tools are making it ever easier to determine the genetic factors behind Ni hyperaccumulation and subsequently alter these factors to create transgenic strains of hyperaccumulators for biotechnological application (but see Refs. [20,155] for environmental risks associated with genetic modification). Agromining is an expanding field and the efficiency of the practice is gradually increasing with greater interest in improving the economic viability of this new green technology [154]. Optimized irrigation, fertilization, land management, and Ni extraction techniques ([160,161,177]) have demonstrated a highly realistic prospect of implementing large-scale agromining operations in Mediterranean and tropical regions of the world. In addition, it is possible that long-term phytoextraction could result in rehabilitation of contaminated soils or mine wastes, due to depletion of toxic metals [164,165]. Nickel hyperaccumulation is a fascinating adaptation to ultramafic soils worldwide and its study from physiological, ecological, genetic, and biotechnological perspectives has provided unique insights into the ways in which plants cope with harsh soil environments. Continued research will bring about more discoveries about the nature of Ni hyperaccumulation and its wide-ranging applications.

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Acknowledgments We thank Dr. Bob Boyd, Auburn University, AL, USA for constructive comments of an earlier draft of the manuscript and Antony van der Ent, Roger Reeves, Aida Bani and Delia Oosthuizen for providing images for Figs. 25.1 25.4. Funding from the Frost Undergraduate Research Program of the College of Science and Mathematics and the Biological Sciences Department, California Polytechnic State University, San Luis Obispo as well as the Garden Club of America (The Joan K. Hunt and Rachel M. Hunt Summer Scholarship in Field Botany to ALF) is gratefully acknowledged.

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Further Reading Harrison S, Rajakaruna N, editors. Serpentine: The Evolution and Ecology of a Model System. Los Angeles, CA: University of California Press; 2011. Noller BN, Barnabas A, Mesjasz-Przybylowicz J, Callahan DL, Przybylowicz WJ, Harris HH, et al. Nickel biopathways in tropical nickel hyperaccumulating trees from Sabah (Malaysia). Sci Rep 2017;7:1 21. Available from: https://doi.org/10.1038/srep41861. Rajakaruna N, Boyd RS, Harris TB, eds. (2014). Plant Ecology and Evolution in Harsh Environments. Nova Science Publishers, Inc., NY, USA. 426p.

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A A-type ATPases, 24 A549 cell line, 128, 278
279 AA. See Amino acid (AA) Aa-MDH, 247
248 AAC, 312 ABA responsive elements (ABRE), 43 Abiotic stresses, 39 ABRE. See ABA responsive elements (ABRE) ACC deaminase. See 1-Aminocyclopropane-1carboxylate deaminase (ACC deaminase) Accumulators, 327 Acetophenone, 188 Achromatium, 132 Acid mine drainage (AMD), 17, 319 Acid rock drainage (ARD), 17 Acidi(thio)microbium spp., 24
25 Acidianus, 14, 219
220 Acidic amino-acids, 54, 141 Acidic hot springs, 18 Acidic sulfur springs, 17 Acidihiobacillus T3.2, 30 Acidimicrobium, 14, 26
27 Acidimicrobium ferrooxidans, 24
26, 134 ATCC 33020, 31 Acidiphilium, 18, 96 Acidiphilium acidophilum, 31 Acidiphilium multivorum AIU 301, 28 Acidisphaera spp., 96 Acidithiobacillus biosynthesis of CdS QDs, 26 Acidithiobacillus caldus (At. caldus), 31 ATCC 51756 and SM-1, 26
27 strain KU, 31 strains ATCC 51756 and SM-1, 29 Acidithiobacillus ferrivorans SS3 (At. ferrivorans SS3), 29 Acidithiobacillus ferrooxidans (At. ferrooxidans), 4
5, 19, 24, 26
27, 31, 73 ATCC 53993 and ATCC 23270, 29 E15, 27, 30 GR gene, 25 strains, 30 TFI 29 MerA, 27 Tn5037, 30 Acidithiobacillus sp., 14, 18, 24
26, 96, 132, 321 strains, 26 T3.2, 27

Acidithiobacillus thiooxidans (At. thiooxidans), 25
26, 31, 133
134 A01, 28 arsB transcription, 31 Acidobacteria, 6
7 Acidobacterium spp., 17, 96 Acidophiles, 14, 18, 65
66, 73
74, 295, 315
316, 316f food and medicinal relevance, 74 habitats and diversity, 73 physiological adaptation to low pH, 73
74 physiological adaptations of extremophiles, 17 Acidophilic bacteria, 31 Acidothiobacillus, 134
135 Acidothiobacillus ferooxidans, 133
135 Acinetobacter johnsonii, 99 Acinetobacter sp., 5 ACP domains. See Acyl carrier protein domains (ACP domains) Actinobacteria, 92, 139 Actinomadura sp. S14, 228
229 Actinomycetes, 39
40 Activation energy (Ea), 246
247 Acyl carrier protein domains (ACP domains), 319 Acylated homoserine lactones (AHL), 18
19 Adaptations of extremophiles at molecular level, 66 Adaptive immunity, 157 ADHs. See Alcohol dehydrogenases (ADHs) Adventitious root formation (AR root formation), 42
43 Aequorivita sp., 276 Aerobic halophilic Bacteria, 140 Aeropyrum pernix, 28 AfMerC, 30 Agavins, 123
124 Agriculture, enzymes for, 274
275 Agrobacterium, 42 Agromining, 336
339 Ni hyperaccumulators used in, 330f AGS (human gastric adenocarcinoma cell lines), 128 AHA. See Pseudoalteromonas haloplanktis (AHA) AHL. See Acylated homoserine lactones (AHL) AI-1. See Autoinducer-1 (AI-1) Ala-X-Ala, 311

Alanine (Ala), 223, 316 Alcalase, 69 Alcaligenes, 91 Alcanivorax, 92 Alcanivorax venustensis, 94 Alcohol dehydrogenases (ADHs), 184 Aldehyde, 256 Algae, 15, 39
40 Alicyclobacillus acidocaldarius, 28, 178 LAA1, 26
27 Alkalibacillus sp., 91 Alkalibacilluss lilacus, 56 Alkalimonas delamerensis, 91 Alkaline phosphatases, 5 proteases, 75 Alkaliphiles, 5, 14, 65, 74
75, 91, 295, 307, 316 applications, 75 habitats and diversity, 74 physiological adaptation of extremophiles, 17 to high pH, 74
75 Alkaliphilic phosphoserine aminotransferase, 317 Alkaloids meleagrin, 279 Alkalophiles/acidophiles, 176 Allergenicity, 113
114 Allivibrio fischeri, 18
19 Allochromatium, 132 Alpha synuclein, 107 α-amylase, 56, 225, 274
275, 321 α-diglycerol phosphate, 5
6 α-galactosidase, 206 α-helices, 224
225 5-α-hydroxyectoine, 72 α-proteobacteria, 14 α/β-hydrolase, 170 Alternaria brassicicola, 332
333 Alternative energy production, 57 Alyssum inflatum, 333 Alyssum lesbiacum, 328
330 Alyssum serpyllifolium subsp. lusitanicum, 328
329 Ambient environments, 197
200 AMD. See Acid mine drainage (AMD) AmgS sensor kinase, 239 Amino acid (AA), 5
6, 225, 248
249 analysis of ectA gene, 263

349

350

Index

Amino acid (AA) (Continued) of ectB gene, 263 of ectC gene, 263 L-proline, 254
255 sequence, 227
228 surface and core distribution, 223 1-Aminocyclopropane-1-carboxylate deaminase (ACC deaminase), 42 activity, 41 Aminoglycosides, 237, 240 resistance, 239 Aminolipids, 276 Aminopenicillins, 239 Aminotransferase assay, 262
263 Ammonia-oxidation genes (amoA), 319 AmpC gene, 239 amy promoter, 206 Amylases, 5, 225
226, 274
275 Anabaena, 77 Anaerobes, 219
220 Anaerobic bacteria, 97, 195
196 Anaerocellum thermophilum, 219
220 Anionic organic solutes, 141
143 Anoxybacillus flavithermus, 68, 228 Anoxybacillus kamchatkensis, 228 Antarctic biomineralizing microorganisms, 280 fish, 175
176, 295 glucoamylase, 274
275 macroalgae, 70
71 microalgae, 278 microbiome, 277 microorganisms bioprospection of microbial derived bioactive compounds, 270
280 diversity of Antarctic environments and bioactive compounds, 270f global publication records for Antarctic microbial diversity, 269f nanoparticles, 280
281 photolyase, 274 Antarctic-derived enzymes, immobilization of, 275 Antarctic Treaty System (ATS), 269 Antartin, 278
279 Antibiotics, 237 resistance in biofilm formation, 240
241 resistance markers, 201
202 Anticancer drug discovery, 277
279 components derived from Antarctic microorganisms, 278t structures of compounds isolated from Antarctic microorganism, 279f Antifreeze proteins, 70 Antifreezing proteins, 280 Antigen-presenting cells, 114 Antiinflammatory drugs, 178 Antimicrobial drug discovery, 275
277 antimicrobial and antiviral components, 276t structure of selected antimicrobial metabolites, 277f Antimicrobial lipid
based substances, 276 Antiviral vaccines, 114 Aphanothecea hlophytica, 58

APX. See Ascorbate peroxidase (APX) Aqualysin, 226 Aquaspillium arcticum, 6, 247
248 Aquaspirillum, 18 Aquifecae thermophiles, 27 Aquifex, 219
220 Aquificaceae, 67
68 Aquificae, 6
7, 13
14 AR root formation. See Adventitious root formation (AR root formation) Arabidopsis, 335
336 Arabidopsis thaliana, 260, 328 Arabinose-rich xylans, 273
274 Archaea(l), 3, 13
14, 17, 52, 90, 140
141, 285 genera, 219
220 lipids, 111 membrane phospholipids, 220 secretomes of, 288
292 Archaeal Richmond Mine acidophilic nanoorganism lineage (ARMAN-2 lineage), 319 Archaebacteria, 3 Archaeoglobus, 97, 219
220 Archaeosomes, 72 Archeoglobus fulgidus, 29
30 ARD. See Acid rock drainage (ARD) Area under curve (AUC), 310 ARFF. See Attribute-Relation File Format (ARFF) Arginine (Arg), 223, 316 Arginylsynthetase gene (argS gene), 146 argS gene. See Arginylsynthetase gene (argS gene) ARMAN-2 lineage. See Archaeal Richmond Mine acidophilic nanoorganism lineage (ARMAN-2 lineage) Aromatic compounds, 59 interactions, 224 arsA, 31 arsB, 30
31 arsC, 27
28, 31 ArsD, 27
28 Arsenate (As(V)), 27
28 Arsenic (As), 27
28, 30
31 Arsenite (As(III)), 24, 27
28, 30 ArsR, 27
28, 31 arsX, 30
31 Artemia. See Brine shrimp (Artemia) Artemia franciscana, 139 Artemia salina. See Sea Monkey (Artemia salina) Arthrobacter, 13, 39
40, 91, 133 Arthrobacter globiformis, 260 Arthrocnemum indicum, 43
45 Artificial lungs, 298 Ascorbate peroxidase (APX), 43 ASLtag system, 300
302 Asn. See Asparagine (Asn) asoA, 28 asoB, 28 Asparagine (Asn), 223 Aspergillus awamori, 224

Aspergillus niger, 98, 133 Aspergillus ochraceopetaliformis SCSIO05702, 277 Aspergillus penicilloides, 78 Aspergillus spp., 5
6 Asterina, 77 Astrobiology applications in astrobiology missions, 98
99 extremophiles from planetary field analogue sites in Europe, 96
97 from space craft assembly room, 99 study, 90
91 historical background, 89
90 micro-organisms in EUA and outer space, 98
99 PFA sites in India and extremophilic microbial diversity, 91
96 Asymmetric ketones, 187 Athalassohaline, 52 environments, 137 Athalassohaline, 52 environments, 137 Atmosphere of Earth, 98 Atradidymella sp., 275 AtRITF1 transcription factor, 45 AtRSA gene, 45 AtRSA1 gene, 45 AtRSA1-AtRITF1 complex, 45 ATS. See Antarctic Treaty System (ATS) Attribute-Relation File Format (ARFF), 309, 309f AtVQ9 gene, 45 AtWRKY8 gene, 45 AUC. See Area under curve (AUC) Aurantimonas, 95
96 Autoinducer-1 (AI-1), 18
19 Autoinducer-2 (AI-2), 18
19 Auxins, 42
43 synthesis, 41 Azospirillum, 39
40, 42 Azospirillum brasilense, 42
43 Azotobacter, 39
40, 42

B BAA. See Bacillus amyloliquefaciens α-amylases (BAA) Bacillithiol (BSH), 23
24 Bacillus alcalophilus, 263 Bacillus amyloliquefaciens α-amylases (BAA), 225 Bacillus aryabhattai sp. nov, 98 Bacillus cecembensis sp. nov., 95
96 Bacillus cellulosilyticus, 6 Bacillus cereus, 5 Bacillus clausii, 19 Bacillus endophyticus SH, 125 Bacillus halodurans, 19 Bacillus halodurans C-125, 74
75 Bacillus horneckiae sp. nov., 99 Bacillus isronensis sp. nov, 98 Bacillus licheniformis, 5
6, 225, 228 A2, 43
45

Index

BK AG21, 125 Bacillus megaterium, 18 levansucrase, 124
125 Bacillus odysseyi sp. nov., 99 Bacillus pasteurii, 262
263 Bacillus pseudofirmus, 18
19 Bacillus pumilus SAFR-32, 98
99 Bacillus safensis, 275
276 Bacillus safensis sp. nov., 99 Bacillus smithii, 161
162 ET 13831, 160
161 Bacillus sp., 14
15, 39
40, 42, 56, 60, 65, 74
75, 91, 123
124, 133 Bacillus sp. HT19, 228
229 Bacillus sporothermodurans, 18 Bacillus stearothermophilus, 25, 196
197, 219
220, 227 Bacillus subtilis, 19, 98, 225
226, 254
255, 275
276, 285
287 168, 98
99 GB03, 45 levansucrase, 125 MO, 125 WA, 125 Bacillus thermocatenulatus, 228
229 Bacillus thermophilus, 13
14 Bacteria(l), 14, 39
40, 52, 139, 141 carbonic anhydrases, 295
296 CRISPR/Cas organization in, 157
158 genera, 219
220 ghosts, 114 lipids, 111 milking, 72 nanoparticle biosynthesis, 25
26 phenotype in biofilm formation, 240
241 Bacterio-opsin, 145 Bacteriorhodopsin, 58
59, 143
144 Bacterioruberin, 5
6 Bacteroidetes, 95
96, 139 Barophiles. See Piezophiles Beggiotoa, 132 Bel-7402 inhibition, 279 Berkazaphilones, 74 Berkeleyacetals, 74 Berkeleydiones, 74 Berkeleyones A-C, 74 Berkelic acid, 74 Berkheya coddii, 328
329, 332
333 Beta-carotene, 72
73 β-galactosidase, 206, 272
273 Beta-galactosidases, 70 β-glucosidase, 5
6, 227 β-lactams, 238 resistance, 239 β-proteobacteria, 14 Betaine, 56
57, 259 Bicarbonate (HCO32), 295
296 Bicarbonate indispensable, 296 Bifidobacterium lactis, 115 Bio controlling agent, SOB in, 134 Bio-based products, 253 Bio-purification, 114
115 Bioactive compounds, 269 Bioactive natural products, 318
320

gene study, 319 Bioaugmentation, 60 Biocatalysis model for substrate binding site of TbSADH, 188f using thermostable TADHs, 187
188 Biocatalysts, 295 Biodiesel enzymes for biodiesel production, 273
274 extremophilic esterases/lipases in, 178 BioEdit version 7.05 program, 262 Biofertilizers, SOB in, 134 Biofilteration, SOB in, 134 Biofuels, 5, 55 production, 321 G. thermoglucosidasius, 206 seaweeds for, 209 Biogeochemical cycling, SOB in, 133 Biolabss, 115 Bioleaching. See Biomining Biological membranes, 220 Biological priming approach, 39 BIOlogy and Mars EXperiment (BIOMEX), 98
99 Biomining, 320
321 SOB in, 133 Biomolecules, 51 BIOPAN facility, 98 Bioplastics, 5, 55 Biopolymers, 219, 225, 255 Bioprocessing cost, 253 Bioprospection of microbial derived bioactive compounds, 270
280 drug discovery, 275
279 enzymes, 270
275 ice-binding proteins, 279
280 Biorefinery production, enzymes for, 273
274 Bioremediation, 59
60 SOB in, 133
134 Biosensors, 298 SOB in, 135 Biosphere, 105 BIOSTACK experiments, 98 Biostimulants, 39 Biosurfactants, 5
6, 57 Biosynthesis of ectoine, 256
258, 257f Biotechnological applications of ectoine, 259
261 HP in, 114
115 bio-purification, 114
115 modulation of cell activity, 115 of piezophiles, 115 Biotechnological potential of extremophiles, 5
6 Biotechnologies, 177, 227 Bitop AG, 72, 259 BjCET3, 336 BjCET4, 336 BjYSL7, 336 Black smokers, 67 Black yeast (Hortaea werneckii), 5, 139, 141
142 BLAST, 262 Bornmuellera emarginata, 338

351

Botryidiopsidaceae sp., 278 Bradyrhizobium, 42 Bradyrhizobium japonicum, 42
43 Brassicaceae annuals, 338 Brassica juncea, 328
329 BREFA. See Brefeldin A esterase (BREFA) Brefeldin A esterase (BREFA), 174
175 Brenneria, 123
124 Brevibacterium sp. JCM 6894, 258 Brevundimonas, 95
96 Brevundimonas diminuta, 99 Brewing, enzymes for, 274
275 Brine shrimp (Artemia), 71 BSH. See Bacillithiol (BSH) Burkholderia, 39
40 Burkholderia cenocepacia, 276 2,3-Butanediol, 45

C c-di-GMP. See Cyclic-di-GMP (c-di-GMP) C-domain, 287 C-family. See Choline esterase family (Cfamily) C-metabolic flux analysis, 210 13 C-natural abundance NMR spectroscopy, 255
256 C-terminal domain, 133 C14 benzene, 59 Cadmium (Cd), 24 Cadmium nanoparticles, 280 Cadmium sulfide (CdS), 280 QDs, 25
26 CALB L, 70 Calcium carbonate (CaCO3), 134 Caldanaerobacter subterraneus, 228
229 Caldarchaeols, 220 Caldibacillus cellulovorans, 219
220 Caldicellulodisruptor spp., 67
68 Caldicellulosiruptor bescii, 227 Caldicellulosiruptor hydrothermalis, 222 Caldicellulosiruptor lactoaceticus, 228 Caldimicrobium, 97 Calothrix, 77 CAM enzyme, 296 Candida, 5
6, 109 Candida antarctica, 70, 178, 229 Candidatus korarchaeum, 219
220 CAPs. See Cold acclimation proteins (CAPs) Carbon, 5 source, 127 Carbon capture process, 298
300 postcombustion, 296, 300
302 Carbon dioxide (CO2), 60, 295
296 Carbon-carbon bonds, 170 Carbon-nitrogen bonds, 170 Carbon-phosphorus bonds, 170 Carbon-sulfur bonds, 170 Carbonate (CO322), 295
296 Carbonic acid (H2CO3), 295
296 Carbonic anhydrases (CAs), 295
296 bacterial, 295
296 distribution in gram-negative bacteria, 296f extreme, 298

352

Index

Carbonic anhydrases (CAs) (Continued) in extremophilic bacteria, 296
297 Carboxylesterases, 170
172 CAREX. See Coordination action for research on study of life in extreme environments (CAREX) CAs. See Carbonic anhydrases (CAs) Cas genes. See CRISPR associated genes (Cas genes) Catalase (Cat), 43 Catalytic domain (CD), 228, 248 Catalytic residues, 170 Catechol 2, 3-dioxygenase (PheB), 206 Cation-diffusion facilitator family (CDF family), 24 Caudovirales, 15 Cb. tepidum phosphatases, 26 CBP. See Consolidated bioprocessing (CBP) CbSADH. See Clostridium beijerinkcii’s SADH (CbSADH) Cct protein, 6 CD. See Catalytic domain (CD) CDF family. See Cation-diffusion facilitator family (CDF family) CdS. See Cadmium sulfide (CdS) Cell membrane of thermophilic archaea, 68 Cell viability, 145
146 Cellobiohydrolase, 5 Cellovibrio mixtus, 18 Cells, pressure effects on, 105
109 Cellular characterization of Geobacillus sp., 200 sequestration, 23
24 by thiol systems, 24
25 Cellulases, 5, 69, 75, 225, 227 Cellulomonas spp., 6 Cellulosic biomass, 227 Cellulosimicrobium, 91, 95
96 Cellulosomes, 227 Cenarchaeum symbiosum, 318 Centibacterium arsenoxidans, 28 CEPA. See Columbus External Platform Adapter (CEPA) Cephalosporin, 240 CF. See Cystic fibrosis (CF) Chaetoceros neogracile, 280 Chaetomium thermophilum, 227 Chalcopyrite (CuFeS2), 133 Chelation of Nickel, 328
329 Chemical chaperones, 254 for protein folding, 259 Chemical priming approach, 39 Chemolithotroph, 73, 131
132 Chemolithotrophic sulfide oxidation, 132 Chemotaxis in extremophiles, 18
19 Chemotaxonomic methods, 13 Chenopodium quinoa, 5 Chi-squared algorithm, 309, 312 Chiral alcohols, 183, 189 Chiral aromatic alcohols, 189 Chlamydomonas, 3, 15 chloramphenicol (Cm), 256
258 Chlorobium, 26 Chloroflexi, 6
7, 18

Chloroflexus aurantiacus, 28 Chloroflexus sp., 156
157 Choline esterase family (C-family), 170
172 Cholorobiacea, 132 Cholorobium limicola, 134
135 Cholrobium, 132 Chromatium, 132 Chromohalobacter, 6, 56, 60
61 Chromohalobacter japonicus BK AB18, 125 Chromohalobacter salexigens, 56
57, 256, 262
263 Chroococcus, 77 Circinella muscae, 98 Citrate synthase, 247
248 Citricoccus, 95
96 Cladophialophora, 3 Cladosporium sp., 273
274 Class 2 CRISPR systems, 159 Class I N-terminal signal sequence, 287 Classical screening techniques, 272 Cleome heratensis, 334 Cloning, 156 of ectoine biosynthesis genes, 262 Clostridia, 186
187 Clostridium, 123
124 Clostridium beijerinckii, 186
187 Clostridium beijerinkcii’s SADH (CbSADH), 185 Clostridium bifermentans, 18 Clostridium lituseburense, 18 Clostridium thermocellum, 115, 228 Clostridium thermosaccharolyticum, 219
220 Clostridium thermosulfurogenes, 225
226 Clustal X program, 263 CLUSTAL-X version 1.81 program, 262 Clustered regularly interspaced short palindromic repeats (CRISPR), 156 Clustered Regularly Interspaced Short Palindromic Repeats and CRISPRassociated proteins (CRISPR-CAS), 7, 156f applications, 161
162 CRISPR based genome editing technology, 162f classification, 158
160 type I CRISPR system, 158f, 159 type II CRISPR system, 160 type III CRISPR system, 160 delivery methods, 161 in extremophiles, 160
161 genetic tools in recombination, 155f of halophilic archaea, 161 organization in bacteria, 157
158 CoA. See Coenzyme A (CoA) Cocoa butter, 177 Codon usage, 68 Coenzyme A (CoA), 24
25 Cofactor preference alteration for enzyme improvement, 189 Cold acclimation proteins (CAPs), 318 Cold adapted enzymes, 245
246, 249 structure-function relationship of, 246
249 Cold shock proteins (CSPs), 318 Cold temperature loving piezophiles, 307

Cold-accustomed protein, 318 Cold-active lipases, 70 Cold-active xylanase, 70 Cold-adapted beta-galactosidases, 70 Cold-adapted enzymes, 70, 270
272, 274
275 Cold-adapted proteases, 70 Cold-induced IBP, 280 Colistin, 237 Coloymisin, 237 Columbus External Platform Adapter (CEPA), 98
99 Colwellia marinimaniae, 5, 105 Colwellia sp., 5, 13
14, 75
76, 109 Combat cold stress, survival strategy to, 317
318 membrane fluidity, 317
318 mutational study, 318 protein synthesis and cold-accustomed protein, 318 structural adaptation of cold-active enzyme, 318 Committee on Space Research (COSPAR), 90, 99 Community metagenomics, 319 Comparative genomics, 6 Compared UDG from Atlantic cod (cUDG), 247
248 Compatible solutes, 53
54, 56
57, 69
70, 141, 254, 259 Conjugative plasmid transfer, 204 Consolidated bioprocessing (CBP), 228 Continental subsurface, 105 Conventional plant breeding, 39 Coordination action for research on study of life in extreme environments (CAREX), 90
91 copA gene, 26, 29
30 copAB proteins, 29 CopB, 29
30 copM, 29
30 Copper (Cu), 24, 26
27, 29
30 Copper nanoparticles (CuNP), 25
26 Coprothermobacter proteolyticus, 226 copT, 29
30 copY, 29
30 copZ, 29
30 Corynebacterium, 24
25 Corynebacterium glutamicum, 256
258 Coryneform bacteria, 56 Cosmeceuticals, ectoine in, 260 Cosmetic formulations, 260
261 Cosmetics production, enzymes for, 274 COSPAR. See Committee on Space Research (COSPAR) Counterselection markers, 202 PyrE and PyrF reactions, 202f pyrF-based counterselection, 203f COX-1 mRNA, 77 Coxiella burnetii, 74 Crab spider (Misumena vatia), 332
333 Crenarchaeota, 285 Crenoarchaeota, 139 CRISPR. See Clustered regularly interspaced short palindromic repeats (CRISPR)

Index

CRISPR associated genes (Cas genes), 7, 156
157 Cas10, 160 Cas6, 160 Cas6e, 159 Cas9-mediated gene-editing systems, 161 CasE, 159 CRISPR RNAs (crRNAs), 157
160 CRISPR-Cas9-based genome editing tools, 161
162 crRNAs. See CRISPR RNAs (crRNAs) Crustacean shells, 273
274 Cryo-protection of microorganisms, 259 Cryobacterium pindariense sp. nov., 95
96 Cryomyces antarcticus CCFEE, 78 Cryopreservation, 114 Cryoprotectant, 245 Cryptobiosis, 4 Crystallizer ponds, 139 Cse3, 159 CSPs. See Cold shock proteins (CSPs) Cucumaria frondosa, 115 cUDG. See Compared UDG from Atlantic cod (cUDG) CueO, 26
27 Curtobacterium luteum, 5 CusA, 29 cusABCF operon, 29 CusB, 29 CusC, 29 CusF, 29 Cyanidioschyzon merolae, 3 Cyanobacteria, 3, 15, 92, 139 Cyanobacterial biosynthetic enzymes, 320 Cyclic 2,3-diphosphoglycerate, 5
6 Cyclic-di-GMP (c-di-GMP), 241 Cyclodextrin glycosyltransferases, 75 cysJIHDNG (cysteine biosynthetic pathway), 25 Cysteine (Cys), 23
25, 224 Cys295 residue, 189
190 desulfurase, 25 residues, 225
226 synthase, 25 Cystic fibrosis (CF), 237, 239 Cytochrome c ccoN gene, 241 Cytochrome c-oxidase, 24 Cytochrome oxidase, 6 Cytokinin, 42
43 producing PGPR, 43 Cytoprotectants, 254

D DABA. See 2,4-Diaminobutyric acid (DABA) dacB gene, 239 Dairy industry, extremophilic esterases/lipases in, 178 Damage Suppressor (Dsup), 77 dCas9. See Deactivated cas9 (dCas9) DDGS protein. See Distiller dry grain with soluble protein (DDGS protein) De-novo synthesis, 42 Deactivated cas9 (dCas9), 159, 161

Dead Sea brine, 52, 137
139 Debaryomyces sp. See Halotolerant yeast (Debaryomyces sp.) Decolorization of textiles, 59
60 Deep Sea, 105 deep sea-inhabiting marine organisms, 260
261 fungi, 70
71 halophilic actinobacteria molecular and functional characterization of ectoine in, 261
262 sequence analysis of ectA, B and C genes, 263 hydrothermal vents, 67 hypersaline brines, 137
138 Defensive enhancement hypothesis, 332
333 Defluviitalea phaphyphila, 24
25 Dehydration responsive elements (DRE), 43 Deinococcus, 13
14 Deinococcus peraridilitoris, 5 Deinococcus radiodurans, 19, 66, 76
77, 98 Deinococcus-Thermus group, 6
7, 24, 76
77 Denaturing gradient gel electrophoresis, 13 Dendritic cells, 114 Deodorization, SOB in, 134
135 Desemzia, 95
96 “Designer cellulosome” system, 227 Desulforudis audaxviator, 4
5 Desulfurococcus, 219
220 Desulfuromonas, 13
14 Detergent, extremophilic esterases/lipases in, 176
177 DHFR. See Dihydrofolate reductase (DHFR) 2,4-Diaminobutyric acid (DABA), 256
258, 261
262 Dianthus japonicus thumb, 43
45 2,4-Dichlorophenol, 57 Dichotomicrobium, 14 Dictyoglomus thermophilum, 225
226 Dictyoglomus turgidum, 6, 227 Dienococcus radiodurans, 5 Dietzia, 91 Digestibility, 113
114 Dihydrofolate reductase (DHFR), 6, 111 Dimethyl disulfide, 45 Dimethylsulfoniopropionate (DMSP), 254
255 Dipeptide composition (DPC), 312 Direct gene replacements, 146 Distiller dry grain with soluble protein (DDGS protein), 226 Disulfide bonds, 224 Diversity acidophiles, 73 eukaryotic, 4 of extremophiles, 14
15 of thermophiles, 219
220 Diversity acidophiles, 73 eukaryotic, 4 of extremophiles, 14
15 of thermophiles, 219
220 DMSP. See Dimethylsulfoniopropionate (DMSP) DNA, 76
77

353

DNA-binding domains of transcription, 155
156 DNA-DNA hybridization, 13 gyrase, 239 isolation, 319 polymerase, 6, 68, 315 probes, 13 repair enzymes, 6 dnaK1 gene, 58 Docosahexaenoic acid, 111
112 Dodecamermultimerization, 111 Double-strand break (DSB), 76
77, 155
158 DPC. See Dipeptide composition (DPC) DRE. See Dehydration responsive elements (DRE) Drought tolerance, 334 Drug discovery from Antarctic microorganisms anticancer drug discovery, 277
279 antimicrobial drug discovery, 275
277 extremophilic esterases/lipases in, 178 DSB. See Double-strand break (DSB) Dsup. See Damage Suppressor (Dsup) Dunaliella, 3, 5
6, 15, 71, 97, 127, 139, 141
142 Dunaliella bardawil, 72
73 Dunaliella salina, 71
73, 321

E Ea. See Activation energy (Ea) EaBglA enzyme, 248 EANA. See European Astrobiology Network Association (EANA) Earth biomass, 105 Earth’s upper atmosphere (EUA), 98 micro-organisms in, 98
99 EC. See Electrical conductivity (EC); Enzyme classification (EC); European Commission (EC) ectD. See Ectoine hydroxylase (ectD) Ecto-mycorrhizae, 39
40 Ectoine(s), 5
6, 56
57, 72, 254
256 based products in market, 260
261 biosynthesis, 256
258, 257f molecular characterization of, 262
263 PCR amplification, cloning and sequencing of, 262 phylogenetic tree construction and analysis of, 263
264, 264f biotechnological applications, 259
261 chemical chaperones for protein folding, 259 cryo-protection of microorganisms, 259 enhancing PCR, 259 generation of stress-resistant transgenic organisms, 260 use in cosmeceuticals and pharmaceuticals, 260 ectoine-containing lozenges, 72 industrial production, 258
259 molecular and functional characterization, 261
262 osmolytic properties of, 256

354

Index

Ectoine(s) (Continued) physicochemical properties of, 256 sequence analysis of ectA, B and C genes, 263 synthase, 262 transport, 258 Ectoine biosynthesis gene cluster (ectABC), 255
256, 260
261 PCR amplicons, 261 Ectoine hydroxylase (ectD), 255
258 Ectoine/hydroxyectoine uptake transporter (ehu transporter), 258 Ectothiorhodospira. See Halorhodospira Ectothiorhodospira halochloris, 72, 255
256 Efflux pumps and transporter, 29
31 arsenic, 30
31 copper, 29
30 mercury, 30 EH4 strain, 59 ehu transporter. See Ectoine/hydroxyectoine uptake transporter (ehu transporter) Eicosapentaenoic acid (EPA), 56, 76, 111
112, 177 Electrical conductivity (EC), 135 Electrochemical gradients, 19 Electroporation, 204 Geobacillus spp. transformable with exogenous plasmids, 205t Electrostatic potential, 249 Elemental allelopathy, 333
334 Elemental defense hypothesis, 332
333 Elemental sulfur (S0), 131
132 Embden-Meyerhoff-Parnas pathway (EMP pathway), 68, 184 Enantiopure alcohols, 183 Enantioselective hydrogen transfer reactions, 183 Endo-β-1,4-xylanase, 228 Endo-mycorrhizae, 39
40 Endochitinase, 273
274 Endoglucanase, 5, 227 Endoliths, 5 Energy, 55, 57, 60 Engineering TADHs, 189 Enhancing PCR, 259 Enrichment techniques, 272 Enterobacter, 39
40 Enterobacter sp. EJ01, 43
45 Entner-Doudoroff pathways, 68 Environmentally habitat-adapted PGPR, 40 Enzymatic detoxification, 26
28 arsenic, 27
28 copper, 26
27 mercury, 27 Enzyme classification (EC), 170 Enzymes, 5
6, 183, 245
246, 270
275, 318 activity retention, 272
273 for agriculture and brewing, 274
275 from Antarctic microorganisms, 271t for biorefinery and biodiesel production, 273
274 discovery and purification, 270
272 extraction, 261
262

immobilization of Antarctic-derived enzymes, 275 improvement, 188
190 altering cofactor preference, 189 altering stereoselectivity, 189
190 altering substrate specificity, 190 for pharmaceuticals and cosmetics production, 274 responsible for sulfur oxidation, 133 EPA. See Eicosapentaenoic acid (EPA) EPS producing bacteria, 43 Ergothioneine, 25 Erwinia, 123
124 Erwinia amylovora, 126
127 Erwinia tasmaniensis levansucrase, 124
125 ESA. See European Space Agency (ESA) ESBLs. See Extended spectrum plasmid mediated enzymes (ESBLs) Escherichia coli, 18
19, 25
27, 30, 58, 60, 108, 156, 202
204, 226, 274, 285
287 cells, 300
302 fluorescence microscopy, 302f ESF. See European Science Foundation (ESF) Ester bonds, 170 Esterases, 5, 170
172 ESTHER database, 170
172 Ether bonds, 170 Ether lipids, 143 Etodolac, 178 EUA. See Earth’s upper atmosphere (EUA) Eubacterium, 57 Eucarya, 52 Euglena, 3, 15 Eukarya, 14, 90, 139, 141 Eukaryal lipids, 111 Eukaryotic diversity, 4 Eukaryotic extremophiles, 3
4 EURECA. See European Retrievable Carrier (EURECA) European Astrobiology Network Association (EANA), 89 European Commission (EC), 90
91 European Retrievable Carrier (EURECA), 98 European Science Foundation (ESF), 90 European Space Agency (ESA), 89, 98 Europlanet, 96 Euryarchaeota, 285 Exigobacterium, 91 Exiguobacterium antarcticum, 6 Exiguobacterium antarcticum B7, 248 Exiguobacterium indicum sp. nov., 95
96 Exiguobacterium sibiricum, 69
70 Exiguobacterium undae Su-1, 5 Exo-biology, 89 EXOBIOLOGIE experiments, 98 Exoglucanases, 227 ExoMars, 97 Exophiala, 3 Exopolyphosphatases, 26 Exopolysaccharides, production of, 57 Exponential-phase cells, 108
109 EXPOSE-E mission, 98
99 ExProt program, 288 class I signal sequences, 291t

class II signal sequences, 290t ExProt program, 288 class I signal sequences, 291t class II signal sequences, 290t Exremophilic microbes, 15 Extended spectrum plasmid mediated enzymes (ESBLs), 238 Extracellular hydrolytic enzymes from haloarchaea, 143 polypeptide, 279 protein, 207 Extraction period, 337 Extreme environments, 51 Extreme halophiles, 139 Extreme thermophiles, 295 ‘Extreme’ conditions, 65 “Extreme” bacterial CAs, 296 ionic liquid membranes, 300 magnetic particles, 300 potential use in biotechnological applications artificial lungs, 298 biosensors, 298 classification of supports used in enzyme immobilization, 299t enzyme immobilization methods, 299f post-combustion carbon dioxide capture, 298 in vivo immobilization, 300
302, 301f Extremolytes, 5
6 Extremophiles, 3, 13, 51, 65
66, 137, 169
170, 219, 285, 295, 315, 322f bioactive natural products, 318
320 biotechnological potential of, 5
6 biotechnological use, 320
322 biofuel production, 321 biomining, 320
321 industrial use, 321
322 medicinal aspects, 322 PCR, 320 classes of, 66t classification, 169t CRISPR-Cas system in, 160
161 eukaryotic extremophiles, 3
4 extremophilic esterases/lipases, 176 running and potential applications for, 176
178 isolated from Hypersaline environments in India, 95t metagenomics and WGS, 6
7 pH adaptation, 316
317 pressure adaptation, 317 prokaryotic extremophiles in diverse habitats, 4
5 salt adaptation, 317 study in astrobiology, 90
91 survival strategy to combat cold stress, 317
318 temperature adaptation, 316 Extremophilic condition, 315 Extremophilic enzymes, 270 Extremophilic microbes, 4
5 Extremophilic microorganism, 143, 315 Extremophilic organisms, 307 databases, 307
308

Index

inferences from preceding methods, 311
312 machine learning, 308
310 sample DSSP output, 311f statistical analysis for inferring molecular basis of extremophilic adaptation, 310
311 Extremotolerant organisms, 3 Extremozymes, 5, 172, 320, 321f

F F1F0 ATPases protein, 6 Facultative piezophiles, 105 Fatty acid synthase (FAS), 319 Fatty acids, 13 Feature extraction and representation, 308
309 Feature selection, 309 Fermentation processes, 253 Fermentative organisms, 184 Ferrimicrobium, 14, 96 Ferrimicrobium acidiphilum, 4
5, 24
25 Ferroglobus, 219
220 Ferroplasma, 14, 17
18, 26
27, 96, 321 Ferroplasma acidarmanus, 19, 25 Ferroplasma acidiphilum, 316 Ferroplasma acidophilum, 78 Ferrovum, 14 Fervidobacterium islandicum AW-1, 226 Fervidobacterium pennavorans, 219
220, 226 Fervidobacterium thailandense FC2004T, 226 Fervidobacterium thiosulatophilum, 134
135 Fibrobacter succinogenes, 6 Filamentous temperature-sensitive Y (FtsY), 285
287 Filobacillus sp., 56 Firmicutes, 6
7, 24
25, 92, 139 Fischerella, 18 FISH. See Fluorescence “in situ” hybridization (FISH) Flavobacteriaceae Sinomicrobium a-amylase (FSA), 225
226 Flavobacterium, 13
14 Flavobacterium Ant342, 70
71 Flavobacterium frigoris, 280 Flexibility/rigidity of proteins, 246
247 Fluorescence “in situ” hybridization (FISH), 97 Fluorescence proteins, 206 5-Fluoroorotic acid, 202 Fluoroquinolone, 240 resistance, 239 5-Fluorouracil, 72 FokI gene, 155
156 Food extremophilic esterases/lipases in, 177 fermentation, 56 HP in food industry, 113 food preservation, 113 pre-treatment, 113 Foot-and-mouth-disease picanovirus, 114 Forward contamination, 99 FOS. See Fructooligosaccharides (FOS) Free living PGPR, 41 Freeze-thaw cycles, 70

Frigocyclinone, 276 Fructanogenic halophiles, 125 fructans, 123
124 levan and levansucrase from Halomonas smyrnensis AAD6, 127
128 microbial fructan synthesis mechanism, 124
125 putative GH68 family enzymes of haloarchaea, 125
127 Fructanogenic organisms, 123
124 Fructans, 123
124 biosynthesis, 127 Fructooligosaccharides (FOS), 124
125 Fructosyl donor, 127 Fructosyltransferases (FTs), 124
125 FSA. See Flavobacteriaceae Sinomicrobium aamylase (FSA) FTs. See Fructosyltransferases (FTs) FtsY. See Filamentous temperature-sensitive Y (FtsY) Fungi, 3, 39
40 Fusion of transcription factor, 156

G g-Means, 310 G-quadruplexes, 106 Galactic cosmic radiation (GCR), 98 γ-proteobacteria, 14 GCR. See Galactic cosmic radiation (GCR) GDH. See Glutamate dehydrogenase (GDH) Geitlerinema, 18 Gel phase, 107 GeneDoc program, 262 General secretory pathway (Gsp), 285 Genetic elements to control gene expression, 206 promoters demonstration to function in Geobacillus spp., 207t Genetic polymorphisms, 238
239 Genetic tools for Geobacillus spp., 200
207 antibiotic resistance markers, 201
202 conjugative plasmid transfer, 204 counterselection markers, 202 electroporation, 204 genetic elements to control gene expression, 206 plasmid replicons, 201 protein secretion, 207 protoplast transformation, 204 recombinant plasmids, 202
204, 203t reporter proteins, 206 strategic circumvention of RM systems, 205
206 for halophiles, 60 Genetics of nickel accumulation, 334
336 target genes identification by chelators, 336 by transporters, 335
336 Genome, 109 -based discovery, 272 editing tools, 155
156 Genome level adaptations, 220
223

355

Genomic island (GI), 25, 27 Genomic(s) and evolution of extremophiles, 18 insights into halophilic prokaryotes, 143
146 case study of Halobacterium sp. NRC-1, 145
146 case study of square archaeon, 143
145 hydrolytic enzymes produced from members of haloarchaea, 144t sequencing of halophilic microorganisms, 60
61 Geobacillus, 14
15 Geobacillus bogazici, 196
197 Geobacillus caldoxylosilyticus, 196
197 Geobacillus caldoxylosilyticus T20, 208 Geobacillus gargensis, 196
197 Geobacillus kaue, 196
197 Geobacillus kaustophilus, 196
197, 222 Geobacillus kaustophilus HTA426, 202
204, 208 Geobacillus mahadia, 196
197 Geobacillus pallidis, 196
197 Geobacillus species, 195
200 cellular characterization, 200 diverse habitats and implications, 197
200, 198t genetic tools for, 200
207 genomic features, 200 Geobacillus sp. EPT9, 228
229 Geobacillus sp. JM6, 228
229 Geobacillus sp. LC300, 211 Geobacillus sp. XT15, 211 Geobacillus spp., 68 history, 196 microscope photograph of Geobacillus kaustophilus HTA426, 195f potential in whole-cell applications, 207
211 G. caldoxylosilyticus T20, 208 G. kaustophilus HTA426, 208 G. stearothermophilus ATCC 12978, 208 G. stearothermophilus NUB3621, 208
209 G. thermocatenulatus 11, 209 G. thermodenitrificans OS27, 209 G. thermodenitrificans T12, 209 G. thermoglucosidasius DSM 2542, 209
210 G. thermoglucosidasius M10EXG, 210 G. thermoglucosidasius NCIMB 11955, 210 G. thermoglucosidasius NY05, 210 G. thermoglucosidasius PB94A, 210 Geobacillus sp. LC300, 211 Geobacillus sp. XT15, 211 species placed under genus, 196
197 Geobacillus spp. validly described with bacterial codes, 197t Geobacillus stearothermophilus, 4
5 Geobacillus stearothermophilus ATCC 12978, 208 Geobacillus stearothermophilus CU21, 201
202, 204

356

Index

Geobacillus stearothermophilus MK232, 206 Geobacillus stearothermophilus NUB3621, 196
197, 208
209 Geobacillus stearothermophilus SIC1, 206 Geobacillus stearothermophilus strains, 196
197 Geobacillus stearothermophilus V cysteine desulfurase gene (iscS), 25 Geobacillus tepidamans, 196
197 Geobacillus thermantarcticus, 196
197 Geobacillus thermocatenulatus, 196
197 Geobacillus thermocatenulatus 11, 209 Geobacillus thermodenitrificans, 196
197 Geobacillus thermodenitrificans OS27, 197
200, 209 Geobacillus thermodenitrificans T12, 209 Geobacillus thermodenitrificans T1230, 160
161 Geobacillus thermoglucosidasius, 196
197 Geobacillus thermoglucosidasius DSM 2542, 209
210 Geobacillus thermoglucosidasius M10EXG, 210 Geobacillus thermoglucosidasius NCIMB 11955, 202
204, 210 Geobacillus thermoglucosidasius NY05, 210 Geobacillus thermoglucosidasius PB94A, 210 Geobacillus thermoleovorans, 196
197, 228 Geobacillus thermoleovorans strain N7, 226 Geobacillus thermopakistaniensis, 196
197 Geobacillus toebii, 196
197 Geobacillus uralicus, 196
197 Geobacillus yumthangensis, 196
197 Geobacillus zalihae, 196
197 Geobacillusanatolicus, 196
197 Geobacillusdebilis, 196
197 Geobacillusthermoglucosidans, 68 Geogemma barossii, 67 Geomyces pannorum, 275 Geothermal hotsprings, cold deserts and glaciers in Leh Ladakh, Himalayas, 95
96 Geothermal zones, 219 Geothermobacterium ferrireducens, 3 GG. See Glucosylglycerate (GG) GH. See Glycoside hydrolase (GH) GI. See Genomic island (GI) Gibberellic acid, 42
43 Gibbs free energy, 105
106 gk704 promoter, 206 Glaciecola, 13
14 Glaciozyma antarctica, 280 Global Hyperaccumulator Database, 339 Gloeocapsa spp., 3 Glucoamylases, 274
275 Gluconacetobacter, 123
124 Gluconacetobacter diazotrophicus levansucrase, 124
125 Gluconobacter oxydans, 210 Glucosylglucosylglycerate, 5
6 Glucosylglycerate (GG), 5
6 Glutamate, 141 Glutamate (Glu), 223 Glutamate dehydrogenase (GDH), 224

Glutamic acid (glu), 316 L-Glutamic acid kit, 261
262 Glutamine (Gln), 223 Glutaredoxin system, 25 Glutathione (GSH), 23
24 GSH S-transferases, 25 synthesis, 336 Glutathione amide, 24 Glutathione reductase (GR), 25 Glutathionylspermidine, 24 Glycerol, 141
142 Glycine (Gly), 223 Glycine betaine, 254
255 Glycophytes, 39
41 Glycoside hydrolase (GH), 227 GH-J, 123
125 GH32 family, 124
125 GH68 family enzymes, 124
125, 126f Glycosidic bonds, 170 Glycosyl hydrolases, 69 Glyphosate, 208 Goldilock zones, 90 GR. See Glutathione reductase (GR) Gram-negative bacteria, 108
109, 239
240, 287 Gram-negative Eubacteria, 123
124 Gram-positive Eubacteria, 123
124 Gram-positive signal sequences, 288 Gram-positive soil bacterium, 255
256 Graminans, 123
124 Graphical user interface (GUI), 308 Great Salt Lake, 139 Greigite (Fe3S4), 280
281 gRNA-Cas9 complex system. See Type II CRISPR systems gRNA-directed CRISPR-Cas system, 161 GSH. See Glutathione (GSH) Gsp. See General secretory pathway (Gsp) 5’-Guanylic acid (5’-GMP), 56 Guehomyces pullulans, 270
272 GUI. See Graphical user interface (GUI) Guide RNA, 156
158 GyrA gene, 239

H 1

H NMR spectroscopy, 189 H-domain, 287 H-family. See Hormone sensitive lipase (HSL) H 1 concentration, 73
74 H5-SspCA, 300
302 protonography of, 302f thermostability and long-term stability, 303f Habitable zones, 90 Haladaptatus, 139 Halanaerobiales, 139
140 Halanaerobium sp., 140 Halide bonds, 170 Halo-alkaline lipase, 56 Halo-G* (Halorubrum chaoviator sp. strain), 98 Halo-tolerance transfer, 58 Haloalkaliphiles, 14
15, 74, 91 Haloarchaea, 14
15, 97, 123

extracellular hydrolytic enzymes from, 143 putative GH68 family enzymes of, 125
127 Haloarchaeal GH-J, 123 Haloarcula, 6, 14, 56
57, 60
61, 72
73, 94 Haloarcula-G, 98
99 Haloarcula argentinensis, 94 Haloarcula marismortui, 5, 54, 66, 94, 143 Halobacillus, 56, 60
61 Halobacillus dabanensis, 263 Halobacillus halophilus, 18
19 Halobacillus karajensis strain, 56 Halobacteria, 123, 139, 143, 161 Halobacteriaceae, 74, 139 Halobacteriales, 74, 125, 139, 143 Halobacterium, 14, 57, 60
61, 72
73, 94 Halobacterium cutirubrum, 57 Halobacterium salinarum, 56, 58
59, 140
141, 322 Halobacterium salinarum NRC-1, 76
77, 288, 290t putative Tat substrates as identified by ExProt from, 291t Halobacterium sp. NRC-1, 71, 76
77, 145
146, 287
288 Halobacteroides sp., 140 Halocafetaria seosinensis, 139 Halococcus, 14, 56 Halococcus dombrowskii, 98
99 Halococcus dombrowskii sp. nov., 94 Halococcus hamelinensis, 76
77 Halococcus morrhuae, 76
77 Halococcus salfodinae, 94 Halococcus thailandensis, 72
73 Halococcussa lifodinae BK3, 72 HaloDom (online database), 52 Haloferacales, 125, 143 Haloferax, 6, 14, 57, 60
61 Haloferax alexandrines, 94 Haloferax mediterranei, 57
58, 156 Haloferax prahovense, 94 Haloferax volcanii, 161 Halofrex, 139 Halofrex volcanii, 5, 143 Halogenated product processing, 57 Halogeometricum rufum, 126
127 Halohastalitch fieldiae, 71 Halomicrobium, 6 Halomonadaceae, 60, 139, 253
254 Halomonas, 14
15, 55, 59
61, 91
92, 97 Halomonas anticariensis, 18
19 Halomonas campaniensis, 57 Halomonas campisalis, 59, 91 Halomonas elongata, 52, 72, 258
259, 263 Halomonas elongata ectABC genes, 260 Halomonas eurihalina, 18
19 Halomonas levan, 128 Halomonas maura, 72 Halomonas smyrnensis AAD6, 125 levan and levansucrase from, 127
128 levansucrase, 126
127 Halomonas sp. TD01, 57 Halomonaselongata, 56
57 Halomonassalina, 56
57 Halomucin (Hmu1), 144

Index

Halophiles, 5
6, 51, 65, 71
73, 123, 176, 253
254, 315, 317 classification and evolutionary relationships among, 137
139 microorganisms classification based on response to salt, 138t extracellular hydrolytic enzymes from haloarchaea, 143 and food products, 72
73 genomic insights into halophilic prokaryotes, 143
146 habitats and diversity, 71 medical applications of molecules from, 72 physiological adaptations of extremophiles, 17 physiological adaptations to high salt concentration, 71
72 salt adaptation mechanism in, 139
143 Halophilic archaea, 59, 72, 123, 143 CRISPR/Cas system of, 161 bacteria, 14
15, 254
255, 258
259 cellulases, 6
7 enzymes, 143 Eubacteria, 123 eukaryotes, 71 genomic insights into halophilic prokaryotes, 143
146 microorganisms, 5, 51, 253
254 applications of halophiles/halotolerans, 58f classification, 52 current and potential applications of halophiles, 55
60 habitats, 51
52 halophilic proteins with resolved structures, 54t halophilic β-galactosidase, 54f industrial applications, 55t new molecular and genomic approaches, 60
61 salt adaptation mechanisms, 53
54 structural characteristics of halophilic proteins, 54 proteases, 56 proteins, 66 Halophilic aerobic Archaea, 140 Halophilism, 123 Halophytes, 40
41, 43 Haloquadratum, 6 Haloquadratum walsbyi. See Square archaeon (Haloquadratum walsbyi) Halorhodospira, 132, 256
258 Halorubrum, 6, 72
73, 94 Halorubrum lacusprofundi, 5, 54, 71, 143, 272
273 Halosarcina, 139 Halostella salina, 126
127 Haloterrigena hispanica, 322 Halothiobacillus, 14 Halotolerance, 5 Halotolerant microbes beneficial attributes of halotolerant PGPR, 43
45

halotolerant biota, 40
41 rhizospheric bacteria and plant growth promotion, 41 stress alleviation through halotolerant rhizospheric bacteria, 42
43 microorganisms, 52 Halotolerant yeast (Debaryomyces sp.), 109, 141
142 Halovivax asiaticus RT-5, 94 Harbor enzymatic systems, 205
206 Harundinacea, 19 HB8 strain, 28 HB27 strain, 28 HDR. See Homology-directed repair (HDR) Heavy metals, 327, 339 HeLa cells, 278 Helicobacter pylori, 74 Helioguards 365, 77 Helionoris, 77 HepG2/C3A (human liver hepatocellular carcinoma cell lines), 128 High pressure (HP), 105 adaptation in piezophiles, 109
112 genomes, 109 membrane lipids, 111
112 proteins, 109
111 biotechnological applications, 113
115 of piezophiles, 115 effects of pressure on macromolecules and cells, 105
109 nucleic acids, 106 phospholipids, 107 proteins, 107 growth curves of microorganisms, 106f habitats, 105 High salt-in strategy, 17, 71, 139
141 Ion movements across cell membrane of Halobacteriales, 140f High temperature loving piezophiles, 307 High-Ni concentration in floral tissues, 334 High-pressure nervous syndrome (HPNS), 111 Highly salt-tolerant Gammaproteobacteria, 258
259 Histidine, 328
329 HKT1 gene, 43, 45 HL60 cell line, 278
279 HLADH. See Horse liver ADH (HLADH) Hmu1. See Halomucin (Hmu1) HNH nuclease domain, 159 HoBglA enzyme, 248 Homeoviscous adaptation, 111, 112f Homoectoine, 259 Homologues systems, 25 Homology modeling, 226 Homology-directed repair (HDR), 155
156 Hormone sensitive lipase (HSL), 170
172 Horse liver ADH (HLADH), 185
186 Hortaea werneckii. See Black yeast (Hortaea werneckii) Hot springs, 67 HP. See High pressure (HP) HPNS. See High-pressure nervous syndrome (HPNS) Hs578T cell, 278

357

HSL. See Hormone sensitive lipase (HSL) Hs
Lsc. See Levansucrase from H. smyrnensis (Hs
Lsc) Hsp20 gene, 68 Hsp60 protein, 6, 68 Hsp70 protein, 6 Human cancer cell lines, 278 HvPIP2
1 gene, 43 Hybanthus floribundus, 334 Hybridization cloning, 6 Hydration water, 106 Hydrocarbons bioremediation, 59 Hydrogen, 57 bonds, 224 Hydrogen sulfide (H2S), 133
134 Hydrogenivirga sp., 27 Hydrogenobacter, 14, 18 Hydrogenobaculum sp., 27 Hydrolase superfamily, 170 Hydrophobic amino acid residues, 141 interactions, 224 Hydrophobicity, 68 Hydroxyectoine, 56
57, 72, 255
256, 259 Hymenobacter sp. UV-11, 274 Hyperaccumulators of Ni, 327
328, 329f, 330f Hyperarid desert environments, 78 Hypersaline, 123 ecosystems, 51
52 environments, 51, 137, 139 Hyperthermophiles, 67
68, 219, 295 Archaeoglobus fulgidus, 254
255 Hyperthermophilic AFEST, 174
175 Hyperthermophilic archaeon (Pyrococcus horikoshii), 65, 111, 207, 219
220, 227 Hyperthermus butylicus, 223 Hypoliths, 5

I IAA, 42
43 Ibn battuta center near Marrakech, Morocco, 97 IBPs. See Ice-binding proteins (IBPs) Ibuprofen, 178 Ice nucleation protein (INP), 300
302 Ice re-crystallization, 175
176 Ice-binding proteins (IBPs), 175
176, 175f, 279
280 Ice-structuring protein, 70 Idiomarina, 13 iFeature, 308 Ignicoccus, 219
220 ILEE. See Investigating Life in Extreme Environments (ILEE) Immobilization, 143 Immobilized enzymes, 298 Immobilized TeSADH, 188 In silico genome-scale model of H. smyrnensis AAD6, 127 In silico metabolic analysis, 210 In vivo immobilization, 300
302, 301f Indomethacin, 178 Induced Systemic Resistance (ISR), 41

358

Index

Induced Systemic Tolerance (IST), 43 Induction algorithm, 312 of stress proteins, 65 Industrial biotechnology, 253 Inorganic polyphosphates (polyP), 26 INP. See Ice nucleation protein (INP) INP domain (INPN), 300
302 INTA-CAB, 96 Intensive sterilization, 253 Inter-tidal zones, 91
92 International Society for the Study of the Origin of Life (ISSOL), 89 International Space Station (ISS), 90, 98
99 Intertidal sea zones, extremophiles from, 91
94 Intra-molecular hydrogen bonds, 248 Intracellular compatible solutes, 254
255 Inulinases, 124
125 Inulins, 123
124 Inulosucrase, 124
125 Invertase/β-fructofuranosidase, 124
125 Investigating Life in Extreme Environments (ILEE), 90
91 Ion pair networks, 224 Ion transport through membrane by ion pumps, 53 Ionic composition, 52
54 Ionic liquid membranes, 300 Ionizing-radiation-resistant bacteria (IRRB), 312 IP2 gene, 43 IPTG. See Isopropyl-β-D-thiogalactoside (IPTG) IREG transporter proteins. See Iron-regulated transporter proteins (IREG transporter proteins) Iridoviridae, 15 Iron (Fe), 5, 133 uptake system, 328 Iron-oxide nanoparticles, 280
281 Iron-regulated transporter (IRT), 335 IRT1, 328 Iron-regulated transporter proteins (IREG transporter proteins), 335 IRRB. See Ionizing-radiation-resistant bacteria (IRRB) IRT. See Iron-regulated transporter (IRT) Isoenergetic conformational substates, 107 Isoleucine (Ile), 223 Isopropyl-β-D-thiogalactoside (IPTG), 261 ISR. See Induced Systemic Resistance (ISR) ISS. See International Space Station (ISS) ISSOL. See International Society for the Study of the Origin of Life (ISSOL) IST. See Induced Systemic Tolerance (IST)

J Janibacter hoylei sp. nov., 98 Janibacter sp. R02, 274 Janthinobacterium sp. Ant 5
2, 70
71 Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 90
91 Joint effects hypothesis, 332
333

K K-fold cross-validation, 309 Kamchatka Peninsula, Russia, 97 Kanamycin nucleotidyltransferase (KNT), 201
202 Ka´rma´n line, 98 Kelvin effect, 175
176 Keratinases, 5, 226 Keratins, 226, 273
274 1-Kestotriose, 124
125 6-Kestotriose, 124
125 Ketones, 186
187 Ketoprofen, 178 Klebsiella, 39
40, 42 KNIME, 308 Knockout methods, 146 KNT. See Kanamycin nucleotidyltransferase (KNT) Kocuria rosea PRL-1, 95
96

L L-family. See Lipoprotein lipase family (Lfamily) Lactic Acid Bacteria (LAB), 178 Lactobacillus, 123
124 Lactobacillus bulgaricus, 115 Lactobacillus johnsonii inulosucrase, 124
125 Lactobacillus plantarum, 56 Lactobacillus rhamnosus, 113 Lactococcuslactis, 178 Lactose intolerance, 70 Lateral root formation (LR root formation), 42
43 Laterite, 337 ldh promoter, 206 Le Chaˆtelier’s principle, 106 Leave one out cross-validation, 310 Lecanicillium muscarium, 273
274 Lentibacillus halophilus, 72
73 Lentibacilluskimchii, 72
73 LEO. See Low Earth orbit (LEO) Leptolyngbya, 18 Leptoplax emarginata, 328
329 Leptospirillum, 14, 17
18, 24, 26
27, 31, 73, 96 group IV UBA BS species, 26
27 Leptospirillum ferriphilum, 17, 25
26 L. ferriphilumT, 26
28 ML-04, 27
29 Leptospirillum ferrooxidans, 19 Leu272 loop, 247
248 Leucine dehydrogenase, 274 Leuconostoc, 123
124 Levanases, 124
125 Levans, 123
124 from Halomonas smyrnensis AAD6, 127
128 Levansucrase, 124
125 Levansucrase from H. smyrnensis (Hs
Lsc), 127 Lichens, 3 Life in extreme environments (LEXEN), 90
91

Ligases, 5 Ligation-independent cloning, 6 Lipases, 5
7, 75, 170
172, 225 catalyse, 274 from halophiles, 56 lipase B, 70 Lipases/esterases from extremophiles biotechnological application of, 177t extremophilic esterases/lipases alkalophiles/acidophiles, 176 halophiles, 176 psychrophilic esterases/lipases, 175
176 running and potential applications for, 176
178 biodiesel, 178 dairy, 178 detergent, 176
177 drug, 178 food, 177 oleochemical processes, 178 structural features and classification, 170
172 graphic representation of distribution of papers, 174f lipase lid activation by substrate, 174f thermophilic esterases/lipases, 173
175 Lipid(s), 107, 276 from halophilic archaea, 57 profiling, 13 Lipopolysaccharide (LPS), 237 Lipoprotein lipase family (L-family), 170
172 Lipoproteins, 107, 288 Liposomes production, 57 Lipozime, 177 Lithopanspermia, 94 Lithotrophic thermophiles, 219
220 Lmr transmembrane protein, 107 LMW thiols. See Low molecular weight thiols (LMW thiols) Lonar Crater, 91 Lonar lake, 91 Low Earth orbit (LEO), 90, 98 Low molecular weight thiols (LMW thiols), 24
25 Low salt, organic solute-in strategy, 17 “Low-salt-in” strategy, 139
143 anionic organic osmolytes, 142t uncharged organic osmolytes, 142t zwitterionic organic osmolytes, 142t LPS. See Lipopolysaccharide (LPS) LR root formation. See Lateral root formation (LR root formation) Lupinus albus, 337
338 Lutibaculum baratangense, 95 Lux S operon, 18
19 LuxI-LuxR, 18
19 Lyngbya, 77 Lysine (Lys), 223, 316 Lysobacter sp. A03, 273

M MAAs. See Mycosporine-like amino acids (MAAs)

Index

Machine learning, 308
310, 312 feature extraction and representation, 308
309 feature selection, 309 machine learning
based prediction methods, 311
312 machine learning
based studies, 307 model performance evaluation metrics, 310 validation, 309
310 platforms, 308 supervised machine learning protocol, 308f Macrolides, 319 Macromolecules, 254 pressure effects on, 105
109 Macrophages, 114 Magnetic particles, 300 Magnetite (Fe3O4), 280
281 Magnetosomes, 280
281 Magnetotactic bacteria synthetize iron-oxide nanoparticles, 280
281 Malate dehydrogenase, 6 Mald1 protein, 113
114 Mannose, 5
6 Mannosylglyceramide, 5
6 Mannosylglycerate, 5
6 MAPKs. See Mitogen-activated protein kinases (MAPKs) Marine derived molecules, 115 Marine microorganisms, 15, 70
71 Marine organisms, 317 Marine volcanic vents, 17 Marinitogapiezophila, 67 Marinobacter alkaliphilus, 91 Marinobacter excellens, 91 Marinobacter hydrocarbonoclasticus, 59 Marinobacter lipolyticus strain, 56 Marinobacter spp., 59, 92, 97 Marinobacterlipolyticus SM19, 177 Marinococcus halophilus, 258, 262
263 Marinococcus M52 strain, 56
57 Mars Express, 97 Mars Sample Return, 97 Mars-Next, 97 Martian meteorite Nakhla, 94 Matthews correlation coefficient (MCC), 310 Maximum solvent accessibility values, 311 MCC. See Matthews correlation coefficient (MCC) MCF-7 (human breast adenocarcinoma cell lines), 128 MD simulations. See Molecular dynamics simulations (MD simulations) MDA. See Multiple DNA amplification (MDA) MDH from Thermus flavus (Tf MDH), 247
248 MDR bacteria. See Multidrug resistant bacteria (MDR bacteria) Medical applications, HP in, 114 antiviral vaccines, 114 bacterial ghosts, 114 cryopreservation, 114 vaccine preservation, 114 Mediterranean climates, 334

Mediterranean Sea, 137
138 MEED. See Microbial ecology equipment device (MEED) Melanin, 5
6 Melanotrichus boydi, 332
333 MELISSA. See Micro-Ecological Life Support System Alternative (MELISSA) Membrane fluidity, 317
318 level adaptations, 220 lipids, 107, 111
112 proteins, 107, 335 mer operons, 25, 27 merA, 27 merACR, 27 MerB, 27 MerC, 30 merCAR, 27 Mercury (Hg), 24, 27, 30 MerE, 30 MerF, 30 Meristematic fungus (Trimmatostroma salinum), 139 merP, 27 MerR, 27 merRBC, 27 merRCA, 27 merRTPA operon, 27 merT, 27, 30 Mesophiles, 67 Mesophilic BREFA, 174
175 Mesophilic nonpiezophilic (MNP) organism, 312 Mesophilic piezophilic (MP) organism, 307, 312 Metabolically active microorganism, 245
246 Metabolomics, 15, 318 Metagenome sequencing, 315 Metagenomics, 6
7, 13, 15, 18, 220, 319 sequencing of halophilic microorganisms, 60
61 Metal tolerance, 338 Metal tolerance protein (MTP), 336 Metal toxicity, 23
25 Metal-phosphate complexes, 26 Metallibacterium spp., 96 Metalloenzymes, 295
296 Metallomics, 6 Metallophiles, 78 Metallophytes, 327 Metallosphaera, 219
220 Metallosphaera sedula, 25
26 Metallothioneins (MT), 23
24 Metallothionines, 328
329 Metalotolerant organisms, 169
170 Methane (CH4), 60 Methanobacterium lacus, 288, 290t, 291t Methanocaldococcus jannaschii, 67, 112, 156
157, 160 Methanocella, 18 Methanogenic bacteria, 223 Methanogenium frigidum, 318 Methanogens, 18 Methanohalophilus zhilinaeae, 74

359

Methanomassiliicoccus, 18 Methanonatronarchaeia, 139 Methanopyrus, 219
220 Methanopyrus kandleri, 3, 13, 219
220, 288 strain 116, 67 Methanosarcina acetivorans, 288 Methanosarcina sp. strain SMA-21, 98
99 Methanosarcinae, 26 Methanosarcinales, 139 Methanotherma, 139 Methanothermus, 219
220 Methanothermus fervidus, 219
220 Methanothrix, 19 Methycilin-resistant Staphylococcus epidermidis, 277 Methylarcula, 14 Methylomicrobium alcaliphilum, 262
263 Mevinolin resistance (Mevr), 145
146 MexAB-OprM, 240 MexCD-OprJ, 240 MexEF-OprN, 240 MexXY-OprM, 240 Micro-Ecological Life Support System Alternative (MELISSA), 98
99 Microalgae, 3, 15 Microbes, 14, 24, 39
40, 65, 317
318 Microbial anticancer compounds, 279 bioprospection of microbial derived bioactive compounds, 270
280 communities, 18 analysis, 6
7 dark matter, 66 diversity, 15 enzymes, 143 fermentation of renewable resources, 161
162 fructan synthesis mechanism, 124
125 habitats, 269 natural products, 319 synthesis of nanoparticles, 280 taxonomy, 13 Microbial ecology equipment device (MEED), 98 Microbial Observatory, 98
99 Microbiota, 43 Micrococcus, 13, 91 Micrococcus albus, 98 Micrococcus varians subsp. halophilus, 56 Microflora, 131 Microfossil, 89 Microorganisms, 4
5, 4f, 13, 74, 245, 316 cryo-protection of, 259 in earth’s upper atmosphere and outer space, 98
99 MIL. See Multiple instance learning (MIL) MinElute Gel purification Kit, 261 Mineralogical barrier, 337 Minichromosomes, 145
146 Misumena vatia. See Crab spider (Misumena vatia) Mitogen-activated protein kinases (MAPKs), 45 Mn21 complexes, 77

360

Index

Mobility of metals, 23
24 Model performance evaluation metrics, 310 validation, 309
310 Molecular chaperons, 208
209 Molecular dynamics simulations (MD simulations), 247
248, 256 Molecular interactions, 106 Molecular markers, 13 Molecular mechanism of cold adaptation, 247
248 Molecular phylogenetic analysis, 6
7 Molybdopterin, 133 MON-1 strain, 27 Monomeric proteins, 111 Morganella, 25
26 Morganella psychrotolerans, 25
26 Moritella, 13
14, 75 Moritella profunda, 6, 111, 316 MP/MNP, 312 MpDHFR, 111 MscS ion channel, 107 MSH. See Mycothiol (MSH) MT. See Metallothioneins (MT) MTP. See Metal tolerance protein (MTP) Mud volcanoes of Andaman, 95 Multi-functional enzymes, 227 Multi-omics, 76 Multidrug resistant bacteria (MDR bacteria), 237 Multiple DNA amplification (MDA), 319 Multiple instance learning (MIL), 312 Mutagenesis studies, 227
228 Mycobacterium luteum, 98 Mycobacterium tuberculosis, 276 Mycobacterium xenopi, 68 Mycorrhizae, 39
40 Mycorrhizal fungi, 39 Mycosporine-like amino acids (MAAs), 5
6, 77 Mycothiol (MSH), 24
25 myo-inositol, 141
142

N N-Acylhomoserine-lactones, 274
275 N-domain, 287 N-methyl-D-aspartate receptor (NMDR), 111 N-terminal cleavable signal peptide, 285 N-terminal domain, 133 NAD(P)-dependent oxidoreductases, 183 NADA. See Nγ-acetyldiaminobutyric acid (NADA) NAI. See NASA Astrobiology Institute (NAI) Nanoarchaeum, 219
220 Nanoarchaeum equitans, 288 Nanohaloarchaea, 319 Nanoparticles (NP), 25
26, 280
281 cadmium nanoparticles, 280 iron-oxide nanoparticles, 280
281 Naproxen, 178 NASA. See National Aeronautics and Space Administration (NASA) NASA Astrobiology Institute (NAI), 89

NASA Spore assay (NSA), 99 National Aeronautics and Space Administration (NASA), 89 Natranaerobiales, 74 Natranaerobius thermophilus, 74
75 Natrialba magadii, 19 Natrialba taiwanensis, 126
127 Natrialbales, 125, 143 Natrinema sp. J7
2, 288, 290t, 291t Natrinemagari, 72
73 Natronococcus jeotgali RR17, 94 Natronococcus occultus, 19, 322 Natronococcus sp. strain TC6, 56 Natronomonas, 6, 14 Natronomonas pharaonis, 5, 66, 143 Natural Environment Research Council (NERC), 90
91 Natural plasmid, 201 Natural resistance
associated macrophage proteins (NRamp), 335 families, 329
330 transporter, 335
336 nCas9. See Nickase Cas9 (nCas9) NCBI protein database, 307
308 NcIREG2 gene, 335 Neisseria gonorrhoeae, 259 Neisseria meningitidis, 160 Neocallimastix patriciarum, 228 Neoxaline, 279 NERC. See Natural Environment Research Council (NERC) Nesterenkonia sp., 56, 96
97 Next generation sequencing (NGS), 18, 220 genome sequencing tools, 238
239 nfxB gene, 240 NGS. See Next generation sequencing (NGS) NHEJ. See Non-homologous end joining (NHEJ) Ni-resistant decomposers, 333
334 Nickase Cas9 (nCas9), 159 Nickel (Ni), 327
328, 333 chelation, 328
329 effects on Ni-hyperaccumulating plants, 327
328 hyperaccumulators, 329f, 330f localization and storage, 331
332 transport, 329
330 uptake, 328 Nickel hyperaccumulation, 327
328, 332
334 drought tolerance, 334 economically important Ni hyperaccumulators, 338t elemental allelopathy, 333
334 elemental defense hypothesis, 332
333 genetics of nickel accumulation, 334
336 known genes and gene products, 335t nutritional demand hypothesis, 333 phytoremediation and agromining, 336
339 Nickel-deficient plants, 327 Nicotinamide synthase gene (TcNAS), 336 Nitrate, 5 Nitric oxide (NO), 42
43 Nitrogen fertilization, 337
338

fixation, 41 Nitrosomonas, 14 NMDR. See N-methyl-D-aspartate receptor (NMDR) NO. See Nitric oxide (NO) Nocardioides sp., 57 Nocardiopsis alba, 261
264 Nocardiopsis sp. 7326, 274
275 Noccaea caerulescens, 328
329, 336, 338 Non-covalent interaction, 246
247 Non-globular proteins, 107 Non-homologous end joining (NHEJ), 155
156 Non-synonymous SNPs, 239 None thiol, extracellular and intracellular complexation, 25
26 inorganic polyphosphates, 26 NP, 25
26 Nonitolcaldarchaeols, 220 Nonribosomal peptide synthetase (NRPS), 319, 320f Normal temperature loving piezophiles, 307 Nostoc, 77 NP. See Nanoparticles (NP) NRamp. See Natural resistance
associated macrophage proteins (NRamp) NRPS. See Nonribosomal peptide synthetase (NRPS) NSA. See NASA Spore assay (NSA) Nuclease-dCas9, 161 Nucleic acids, 106 Nucleotide sequences, 262 Nutritional demand hypothesis, 333 ˚ lesund in Svalbard archipelago of Ny-A Norway, 96
97 Nylon 12, 209 Nγ-acetyldiaminobutyric acid (NADA), 256

O Obligate piezophiles, 105 Oceanic origin of hypersaline ecosystems, 51
52 Oceanobacillus iheyensis, 18
19 Ochrobactrum, 39
40, 42 Ochromonas, 3, 15 Odontarrhena bracteata, 328 Odontarrhena chalcidica, 328
329, 331
334, 337
338 Odontarrhena inflata, 328 Odontarrhena serpyllifolia, 338 OGT. See Optimum growth temperature (OGT); Organism’s growth temperature (OGT) Oidiodendron truncatum GW3
13, 70
71, 279 Oleochemical processes, 178 Oleochemistry, 178 Oligotrophs, 5 Omega 3-PUFAs, 115 Omic based approaches, 6 Omic technologies, 69
70 Omp/Tox system, 110 OmpH protein, 6

Index

OneR algorithm, 309 Oocyte cryopreservation, 114 Open reading frames (ORF), 288 Opitutus terrae, 18 OprD protein, 237 OprH protein, 237 Optimum growth temperature (OGT), 222 ORF. See Open reading frames (ORF) Organic compounds, 254 Organic molecules, 280 synthesis or accumulation of, 53
54 Organic osmolytes, 139
141, 255, 259 anionic, 142t mechanism, 254 uncharged, 142t zwitterionic, 142t Organic osmotic solute production, 56
57 Organic-osmolyte strategy, 253
254 Organism’s growth temperature (OGT), 225 Organophosphorus compounds, 208 Orotate PyrE, 202 Orotidine 50 -phosphate decarboxylase (PyrF), 202 Osmolytes, 42, 111, 259 transporters, 258 Osmolytic properties of ectoine, 256 Osmoregulation, 51, 53
54 Osmotic shocks, 53
54 Osmotic solutes, 53
54 Outer space, micro-organisms in, 98
99 Oxidants, 183 Oxidation behavior of SOB, 131
132 Oxidoreductases, 183

P P-type ATPases, 24, 29 PA5471 gene, 240 PADHs. See Primary ADH (PADHs) Paecilomyces variotti, 226 Paenibacillus glacialis sp. nov., 95
96 Paenibacillus spp., 65 Paenibacillus xerothermodurans ATCC 27380, 99 Paenibacillus yonginensis DCY84T, 45 PAHs. See Polycyclic aromatic hydrocarbons (PAHs) Palythene, 77 Palythine, 77 Palythinol, 77 PAM. See Protospacer adjacent motif (PAM) Panax quinquefolium, 113 Panel on Exploration (PEX), 90 ‘Panspermia’ theory, 98 Papulaspora anomala, 98 Paracoccus, 91 Paracoccus denitrificans, 131
132 Parageobacillus, 196
197 PART rule induction algorithm, 312 Pasteurization technique, 219
220 PcoA, 26
27 PCR. See Polymerase chain reaction (PCR) pdc. See Pyruvate decarboxylase (pdc) PDE. See Phosphodiesterase (PDE)

Pectin, 322 Pectinases, 5, 322 Pectobacterium carotovorum, 274
275 Peizophiles, 14
15 physiological adaptations of extremophiles, 18 Penicillinases, 239 Penicillium flavigenum, 275 Penicillium notatum, 98 Penicillium rubrum, 74 Penicillium sp. PR19N-1, 278
279 Penicillium variable, 107 2,4-Pentanedione, 188 Peptide bonds, 170 Peroxidases, 5 Peroxide-resistant psychrophile strains of Pseudomonas, 280 PEX. See Panel on Exploration (PEX) PFA sites. See Planetary Field analogue sites (PFA sites) pfl promoter, 206 PgIREG1, 335
336 PGPHR. See Plant growth promoting halo rhizobacteria (PGPHR) PGPR. See Plant growth promoting rhizobacteria (PGPR) PGTdb. See Prokaryotic Growth Temperature Database (PGTdb) pH adaptation, 316
317 homeostasis, 316f Pharmaceuticals ectoine in, 260 enzymes for, 274 PHAs. See Polyhydroxyalkanoates (PHAs) PHB. See Poly (3-hydroxybutyrate) (PHB) PHBV. See Poly (3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV) Phe. See Phenylalanine (Phe) Phe-Phe interaction, 224 Phenol, 59 Phenotypic methods, 13 Phenotypic variant regulator (PvrR), 240
241 1-Phenyl-1,3-butanedione, 188 Phenylalanine (Phe), 223 Phenylmethylsulfonyl fluoride (PMSF), 261
262 Phloem nickel mobility in, 331
332 tissue of R. bengalensis, 331f Phosphate solubilization, 41 source, 5 Phosphate specific transport (Pst), 30 Phosphodiesterase (PDE), 241 Phosphodiesters di-myoinositol-1,1’-phosphate, 5
6 Phospholipids, 107 Phosphoribosyltransferase (PyrE), 202 Phosphorus-nitrogen bonds, 170 Photoautotrophic oxidation, 131
132 Photobacterium, 75, 109 Photobacterium profundum, 5
6, 19, 76 strain SS9, 109

361

Photolyase, 274 Photosynthesis and carboxylation reactions, 295
296 phr gene, 145
146 Phycodnaviridae, 15 Phyllanthus balgooyi, 331
332 Phyllanthus rufuschaneyi, 339 Phylogenetic gene marker, 137
138 Phylogenetic method, 13 Phylogenetic tree construction, 263
264, 264f Physiological adaptations of extremophiles, 15
18, 16f acidophiles, 17 alkaliphiles, 17 halophiles, 17 peizophiles, 18 psychrophiles, 15
17 thermophiles, 17 Phytochelatins, 328
329, 336 Phytoremediation, 336
339 Pi transport (Pit system), 30 Pichia pastoris, 226
227 Picornaviruses, 114 Picrophilus, 14, 17, 73 Picrophilus torridus, 3, 13, 29, 73
74 Piezolytes, 111 Piezophiles, 5, 65, 75
76, 295, 307, 315, 317 biotechnological applications of, 115 habitats and diversity, 75 physiological adaptation to high pressure, 76 pressure adaptation in, 109
112 Piezophilic organism, 307 Piezotolerants, 105 Pit system. See Pi transport (Pit system) PitA, 30 PKS. See Polyketide synthase (PKS) Planetary Field analogue sites (PFA sites), 90 extremophiles in Europe, 96
97 Ibn battuta center near Marrakech, Morocco, 97 Kamchatka Peninsula, Russia, 97 ˚ lesund in Svalbard archipelago of Ny-A Norway, 96
97 Rio Tinto, Spain, 96 Tirez Lake, Spain, 97 in India and extremophilic microbial diversity, 91
96 extremophiles from rocks, seawater and intertidal sea zones, 91
94 geothermal hotsprings, cold deserts and glaciers, 95
96 Lonar lake, 91 mud volcanoes of Andaman, 95 salt deposits and saline systems, 94 Planetary protection (PP), 99 Planococcus, 5
6, 91 Planococcus versutus L10.15, 274
275 Plant growth promoting halo rhizobacteria (PGPHR), 43 Plant growth promoting rhizobacteria (PGPR), 39 beneficial attributes of halotolerant PGPR, 43
45 direct promotion mechanism, 41

362

Index

Plant growth promoting rhizobacteria (PGPR) (Continued) environmentally habitat-adapted PGPR, 40 halotolerant, 40, 42, 44t salinity tolerance induction mechanism by, 42f Plant(s), 327 cells, 39 growth promotion, 41 Plasmids, 202
204 natural, 201t replicons, 201 Pleurostomum flabellatum, 139 PLP. See Pyridoxal phosphate (PLP) PMSF. See Phenylmethylsulfonyl fluoride (PMSF) Poecilia mexicana, 4 Polar residues, 176 Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), 57 Poly (3-hydroxybutyrate) (PHB), 57 Polycyclic aromatic hydrocarbons (PAHs), 59 Polyextremohilic enzymes, 245
246 Polyextremophiles, 3, 272
273, 307 Polyextremophilic organism, 4 Polyhydroxyalkanoates (PHAs), 57 production, 57
58 Polyketide synthase (PKS), 319, 320f Polymerase chain reaction (PCR), 68, 315, 320 amplification, 262 Polyols, 5
6 Polyphosphatase (PPX), 26 Polyphosphate kinase (PPK), 26 Polysaccharides, 183
184 Polyunsaturated fatty acid (PUFA), 76, 111
112 Polyurethane foam (PU), 299
300 Porphyra umbilicalis, 77 Porphyra-334, 77 Post-combustion carbon dioxide capture, 298 PP. See Planetary protection (PP) PPK. See Polyphosphate kinase (PPK) PPX. See Polyphosphatase (PPX) Precursor RNA (pre-crRNA), 157
158 Prelog’s rule, 187
189 Pressure adaptation, 317 Pressure-stable enzymes, 115 Primary ADH (PADHs), 184
185 Pro. See Proline (Pro) PROFEAT, 308 Prokarya, 90 Prokaryotes, 137, 145 resistance mechanisms in, 23f Prokaryotic cells, 142
143 Prokaryotic communities, 15 Prokaryotic extremophiles in diverse habitats, 4
5 Prokaryotic Growth Temperature Database (PGTdb), 307
308 Prokaryotic silencing RNA (psiRNAs), 157
158 Proline (Pro), 223 Propy (python package), 308 Prosopis strombulifera, 43

ProtDataTherm, 308 Proteases, 5
7, 69, 225
226 Protein(s), 13, 107, 109
111, 318 chemical chaperones for protein folding, 259 flexibility, 246
247 packing and folding, 225 protein-isolated solutions, 111 protein-tag system, 300
302 protein
DNA interaction, 156 secretion, 207 synthesis, 318 and folding, 69
70 thiols, 25 trafficking, 285 Proteobacteria, 6
7, 92, 95
96, 139, 280
281 Proteolysin, 226 Proteome level adaptations, 223
225 amino acid composition, 223 hydrophobic interactions and disulfide bonds, 224 ion pair networks, hydrogen bonds and aromatic interactions, 224 protein packing and folding, 225 secondary structures, 224
225 surface and core distribution of amino acids, 223 Proteomics, 6, 15, 318 ProTherm, 308 Protists, 3, 15 Proton efflux systems, 17 electrochemical gradient, 140 Protonography, 300
302 SspCA and H5-SspCA, 302f Protoplast transformation, 204 Protospacer adjacent motif (PAM), 157
158 Protospacers, 157
158 Protozoa, 39
40 ProtParam tool, 262 protr (R package), 308 ProU ABC transporter, 254
255 Providencia rustigianii, 91 Pse-in-One, 308 PseAAC-builder, 308 Pseudoaltermonas haloplanktis, 19 Pseudoalteromonas, 56, 92 ANT178, 274 Pseudoalteromonas arctica, 6, 178 PAMC 21717, 248 Pseudoalteromonas haloplanktis (AHA), 6, 247
248 TAH3a, 70 Pseudogymnoascus sp., 275 Pseudomonas, 5
6, 13, 24
26, 39
40, 42, 56, 95
96, 123
124, 237 AMS8 lipase, 274 Pseudomonas aeruginosa, 18
19, 226, 237, 322 aminoglycoside resistance, 239 antibiotic resistance and bacterial phenotype in biofilm formation, 240
241 antibiotic resistant mechanisms and known genes, 238t

ATCC 27853, 275
276 β-Lactam resistance, 239 fluoroquinolone resistance, 239 key mutations reported in antibiotic resistant genes of, 238t PAO1, 288 target efflux pumps, 239
240 Pseudomonas cepacia, 237 Pseudomonas fluorescens, 42, 70, 237 P. fluorescens 868, 178 Pseudomonas putida, 42
43, 134
135, 161
162, 237 Pseudomonas putrefaciens, 237 Pseudomonas stutzeri, 237 Pseudomonas syringaei, 126
127, 300
302 psiRNAs. See Prokaryotic silencing RNA (psiRNAs) Pst. See Phosphate specific transport (Pst) Psychotria gabriellae, 335
336 Psychrobacter, 95
97 Psychroflexus, 13
14 Psychromonas, 13
14, 75 Psychrophiles, 4
5, 13
14, 69
71, 245, 277, 295, 315
316, 318 applications of enzymes and metabolites from, 70
71 cold adapted enzymes, 245
246 habitats and diversity, 69 physiological adaptation of extremophiles, 15
17 to low temperature, 69
70 structure-function relationship of cold adapted enzymes, 246
249 Psychrophilic bacteria, 245
246 Psychrophilic enzymes, 245
246, 270 Psychrophilic esterases/lipases, 175
176 biotechnological application, 177t ice-binding proteins, 175f Psychrophilic microorganisms, 4 Psychrophilic nonpiezophilic (PNP) organism, 312 Psychrophilic organisms, 169
170 Psychrophilic piezophilic (PP) organism, 312 Psychrophilic proteins, 176 Psychrophilic
piezophilic organism, 307 Psychrophillic DNA ligase, 247
248 Psychrotolerant, 275 Psychrotrophs, 4 PU. See Polyurethane foam (PU) PUFA. See Polyunsaturated fatty acid (PUFA) Pullulanases, 225 Purified Cas9
sgRNA RNPs, 161 Putative GH68 family enzymes of haloarchaea, 125
127 PvrR. See Phenotypic variant regulator (PvrR) Pycnandra acuminata, 331
332, 332f Pyramimonas gelidicola, 280 PyrE. See Phosphoribosyltransferase (PyrE) Pyridoxal phosphate (PLP), 261
262 Pyrite (FeS2), 133 Pyrite oxidation, 15 Pyrobaculum, 219
220 Pyrobaculum aerophilum, 223, 288 Pyrobaculum calidifontis, 14
15

Index

Pyrococcus, 115, 219
220 Pyrococcus furiosus, 5, 19, 25, 67
68, 76
77, 160, 219
220, 224, 292, 320 Pyrococcus horikoshii. See Hyperthermophilic archaeon (Pyrococcus horikoshii) Pyrococcus woesei, 223 Pyrococcus yayanossi, 5, 75
76, 105 Pyrodictium, 219
220 Pyrodictium abyssi, 4
5, 227 Pyrolobus, 219
220 Pyrolobus fumarii, 3, 67, 219
220 Pyrolysin, 226 Pyruvate decarboxylase (pdc), 209
210 Pythium mamillatum, 332
333 Python programming language, 308

Q Quantum dots (QDs), 25
26 Quinolone resistant deciding region (QRDR), 239 Quinolones, 238 Quinones, 13 Quorum sensing (QS), 18
19 mechanisms, 66

R Radiation belts, 98 Radioresistant ionizing radiation, 5 Radioresistant microorganisms, 76
77 defense against ultraviolet radiation, 77 diversity and survival strategy, 76
77 Radiotolerant organisms, 169
170 Ralstonia metallodurans, 78 Ralstonia sp. CH34, 78 Ramazzottius varieornatus, 77 Random mutagenesis, 189 RAPD, 13 RapidMiner, 308 Rational design, 189 Rauvolfia serpentina, 336 16S rDNA, 13 Reactive oxygen species (ROS), 43
45, 77 RecA protein, 6 RecD protein, 6, 109 Receiver operating characteristic (ROC), 310 Recombinant DNA technology, 6 Recombinant plasmids, 202
204, 203t pDrive-ectABC, 261 Recombinant SspCA, 299
300 Recombinant transformants, 262 ReliefF algorithm, 309, 312 Renewable resources, 161
162 repH gene, 145
146 Replication process, 145
146 Reporter proteins, 206 Resistance mechanism cellular sequestration by thiol systems, 24
25 efflux pumps and transporter, 29
31 enzymatic detoxification, 26
28 none thiol, extracellular and intracellular complexation, 25
26 in prokaryotes, 23f

Restriction enzymes, 5 Restriction fragment length polymorphism (RFLP), 13 Restriction-modification systems (RM systems), 205
206 strategic circumvention of, 205
206 Retinal chromophore, 145 RFLP. See Restriction fragment length polymorphism (RFLP) Rhamnolipids, 276 Rheinheimera, 13 Rhizobium, 96
97 Rhizomucor pusillus, 226 Rhizomucor sp., 228 Rhizosphere bacteria, 41 of halophytes, 41, 43 of plant, 39
40 rhlA gene, 241 Rhodobaca bogoriensis, 91 Rhodobacteriaceae, 91 Rhodococcus, 96
97 Rhodococcus erythropolis, 18 Rhodospirillum, 14 Rhodothermus marinus, 211, 227 Rhodovibrio, 14 Ribonucleoproteins (RNPs), 161 Ribosomal methyltransferase enzymes, 239 Rinorea bengalensis, 331
332 phloem tissue of, 331f Rio Tinto, Spain, 96 RM systems. See Restriction-modification systems (RM systems) RNA helicase, 318 polymerase, 274 RNA interference (RNAi), 156 RND superfamily. See Root, nodulation, cell division superfamily (RND superfamily) RNPs. See Ribonucleoproteins (RNPs) “Robust” CAs, 298 ROC. See Receiver operating characteristic (ROC) Rocks, extremophiles from, 91
94 Root, nodulation, cell division superfamily (RND superfamily), 24 Root associative PGPR, 41 ROS. See Reactive oxygen species (ROS) Roseinatronobacter monicus, 91 Rough small colony variant (RSCV), 240
241 RpAmy gene, 226 RpGla gene, 226 rplS promoter, 206 16S rRNA, 109, 137
138, 138f, 220, 239 RSCV. See Rough small colony variant (RSCV) Rubber recycling, SOB in, 135 Russian Foton satellites, 98 RuvC domain, 159

S S-layer. See Surface layer (S-layer) S-layer glycoprotein, 143

363

S-layer homology (SLH), 228 (S)-ibuprofen, 178 (S)-methyl-8-hydroxynonanoate, 188 Saccharomyces cerevisiae, 6, 70, 109, 115 Saccharopolyspora rectivirgula, 68 SADH from T. brockii (TbSADH), 185
186 SADH from T. ethanolicus (TeSADH), 185 SADH from T. pseudoethanolicus (TpSADH), 185 SADHs. See Secondary alcohol dehydrogenases (SADHs) Salicola, 14
15 Salicornia bigelovii, 43 Salicornia brachiata, 43
45 Salicornia europea, 43 Salicornia spp., 43 Salicornia strobilacea, 43 Saline, 39 environments, 51 systems in Rajasthan, Gujarat and Maharashtra, 94 Salinibacter, 14
15 Salinibacter ruber, 140 Salinity, 39 Salinivobrio sp., 56 Salinobacter ruber, 71, 141 Salt adaptation, 53
54, 317 “high-salt-in” strategy, 140
141 “low-salt-in” strategy, 141
143 mechanism in halophiles, 139
143 deposits in Rajasthan, Gujarat and Maharashtra, 94 stress, 39, 43 “Salt-in-cytoplasm” mechanism, 253
254 “Salt-in” strategy, 53, 123 Saltern evaporation, 139 Sanguibacter, 95
96 Sanguibacter antarcticus, 273
274 SAT. See Serine acytyl-transferase (SAT) SCA. See Starch casein agar (SCA) Sclerotinia sclerotiorum, 227 SCR. See Solar cosmic radiation (SCR) SCV. See Small colony variants (SCV) Scytonema, 77 Scytonemin, 5
6, 77 SDM. See Site directed mutagenesis (SDM) SDS-PAGE, 261 electrophoresis, 262
263 Sea Monkey (Artemia salina), 4, 139 Seafood processing, 70 “Search for Life” in Solar system, 89 Seawater, extremophiles from, 91
94 Sec pathway, 285
287, 286f Second intracellular messenger. See Cyclic-diGMP (c-di-GMP) Secondary alcohol dehydrogenases (SADHs), 183 Secretomes, 285 of archaea, 288
292 class I signal sequences, 289t class II signal sequences, 290t putative Tat substrates, 291t Sec pathway, 285
287, 286f signal sequence, 287
288

364

Index

Secretomes (Continued) Tat pathway, 287 Secretomics, 6 SecYEG pathway, 285
287 Selenium (Se), 334 Senecio coronatus, 331
332 Sensitivity, 310 Sensor-based microbial immunity, 157 Separate training/validation sets, 309 Sequence-based features, 310 Sequencing of ectoine biosynthesis genes, 262 Serine (Ser), 223 Ser39 residue, 189 Serine acytyl-transferase (SAT), 336 Serpentine outcrops, 339 soils, 327, 333
334 syndrome, 338 Serratia, 39
40 Sesquiterpenes, 278
279 Seventh Framework Programme (FP7), 90
91 sfGFP. See Superfolder green fluorescence protein (sfGFP) sgRNA, 159, 161 Shewanella, 6, 13
14, 19, 75
76, 109 Shewanella benthica, 5, 76, 105 Shewanella violacea, 76, 111
112 DSS12, 76 Shinorine, 77 Short peptide, 336 Sialic acid biosynthesis gene, 144 Siderophore production, 41 sigF gene, 161
162 Signal recognition particle (SRP), 285
287 SRP
nascent polypeptide
ribosome, 285
287 Silver (Ag), 133 Silver nanoparticles (AgNp), 25
26 Single nucleotide mutation, 241 Single-cell genomics, 319 Sinobaca, 95
96 Sinorhizobium meliloti, 258 Site directed mutagenesis (SDM), 185, 189 Site-saturation mutagenesis, 190 SLH. See S-layer homology (SLH) SLMs. See Supported ionic liquid membranes (SLMs) Small colony variants (SCV), 241 SOB. See Sulfur oxidizing bacteria (SOB) SOD. See Superoxide dismutase (SOD) Sodium-motive force, 19 Soft-rot disease, 274
275 Soil bacteria, 328 Solar cosmic radiation (SCR), 98 Solibacillus kalamii ISSFR-015, 98
99 Solphataric fields, 67 Solvent accessibility, 310
311 values, 311 Space craft assembly room, extremophiles from, 99 Spacers, 156
157 SpCas9 system, 159
161 Specificity, 310 Sphingobacterium, 13, 96
97

Sphingopyxis, 96
97 Sphingopyxis alaskensis, 69
70 Spirochaetes, 139 Spiromastix sp., 277 Spiromastixones, 277 Spores, 109 Spores in artificial meteorites (SPORES), 98
99 Sporosarcina, 95
96 Square archaeon (Haloquadratum walsbyi), 5, 143, 161 case study of, 143
145 SRP. See Signal recognition particle (SRP) SspCA, 297, 300
302 dimeric structure, 297f protonography of, 302f thermostability, 297f and long-term stability, 303f ssu rRNA phylogenetics development, 319 St1Cas9 system, 160 Stabilizer agents, 53
54 Stable enzyme production, 56 Stackhousia tryonii, 334 Staphylococcus aureus, 108, 201 INA 00761, 275
276 Staphylococcus species, 52 Staphylothermus, 219
220 Starch casein agar (SCA), 261 Stationary-cells, 108
109 Stem-loops, 106 Stenotrophomonas sp., 5, 18, 91, 95
97 Stereoselectivity alteration for enzyme improvement, 189
190 Strain 121, 67 Strategic circumvention of RM systems, 205
206 Streptanthus polygaloides, 331
334 Streptococcus, 123
124 Streptococcus pneumoniae, 259 Streptococcus pyogenes, 159
160 Streptococcus thermophiles, 156
157 Streptococcus thermophilus, 115, 160 Streptococcus thermosulfidooxidans genome, 29 strain ST, 28 Streptomyces coelicolor, 256 Streptomyces griseus strain NTK 97, 276 Streptomyces ipomea, 134 Streptomyces parvulus, 255
256 Streptomyces scabies, 134 Streptomyces sp., 59, 263
264 Stress, 108 alleviation through halotolerant rhizospheric bacteria, 42
43 induction of stress proteins, 65 stress-resistant transgenic organisms generation, 260 Structural adaptation of cold-active enzyme, 318 Stygiolobus, 219
220 Subsea-floor, 105 Substituted 2-tetralols, 190 Substrate specificity alteration for enzyme improvement, 190

Subtilisins, 226 Sucrose, 124
125 Sulfatase, 133 Sulfated Halomonas levan, 128 Sulfates, 131 Sulfide oxidase, 133 Sulfides, 131
134 Sulfite (HSO32), 131
132 Sulfobacillus, 14 Sulfolobales, 67
68, 220 Sulfolobus, 13
14, 26
27, 131
132 Sulfolobus acidocaldarius, 14
15, 67, 73, 219
220 Sulfolobus shibatae, 227 Sulfolobus solfataricus, 25
26, 29
30, 73, 227 Sulfolobus tokodaii, 28 Sulfolobusacido caldaricus, 65 Sulfur, 131 bacteria, 131
132 compounds, 25 cycle, 131 oxidation, 131 reducing bacteria, 131 Sulfur oxidizing bacteria (SOB), 4
5, 131 applications, 133
135 chemolithotrophic sulfide oxidation, 132 enzyme responsible for sulfur oxidation, 133 oxidation behavior, 131
132 photoautotrophic oxidation, 132 Sulfur-nitrogen bonds, 170 Sulfur-sulfur bonds, 170 Sulfuric acid (H2SO4), 134
135 Sulfurihydrogenibium, 97 Sulfurihydrogenibium azorense, 297 Sulfurihydrogenibium yellowstonense, 297 Sulpholobus metalica, 134 Sunscreen molecules and applications, 77 Superfolder green fluorescence protein (sfGFP), 206 Superior generalization ability, 308 Superoxide dismutase (SOD), 43, 77 Supported ionic liquid membranes (SLMs), 300 Surface layer (S-layer), 285 Synechococcus, 98
99

T T-RFLP. See Terminal restriction fragment length polymorphism (T-RFLP) t-test, 310 TADHs. See Thermoanaerobacter ADHs (TADHs) D-Tagatose, 70 TALENs. See Transcription activator-like effector nucleases (TALENs) TALEs. See Transcription activator-like effectors (TALEs) Taq DNA polymerase, 5, 67 Taq polymerase, 68 Tardigrade, 4, 65, 77, 295 Target efflux pumps, 239
240 MexAB-OprM, 240 MexCD-OprJ, 240

Index

MexEF-OprN, 240 MexXY-OprM, 240 Targeted metagenomics, 18 Tat pathway. See Twin-arginine translocation pathway (Tat pathway) Tat2 protein, 6 TATA binding proteins (TBP), 6, 145
146 TATA-box-binding protein (TBP), 317 TATFIND program, 288
290 Taxonomy of extremophiles, 13
14 TBP. See TATA binding proteins (TBP); TATA-box-binding protein (TBP) TbSADH. See SADH from T. brockii (TbSADH) TcNAS. See Nicotinamide synthase gene (TcNAS) TCS. See Two component system (TCS) TCSM. See Triple-Code Saturation Mutagenesis (TCSM) Tellurite, 24 Temperature, 245 adaptation, 316 Tengcongensis, 228
229 Terminal restriction fragment length polymorphism (T-RFLP), 13 Terrestrial analogues, 90 Tersicoccus phoenicis, 99 TeSADH. See SADH from T. ethanolicus (TeSADH) Tetraether lipids, 112 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidine carboxylic acid. See Ectoine(s) Tetrameric urate oxidase, 107 Tf MDH. See MDH from Thermus flavus (Tf MDH) TFA. See Trifluoroacetic acid (TFA) TgMTP cation efflux protein, 336 TH. See Thermal hysteresis (TH) Thai anchovy fish sauce nam-pla, 72
73 Thalassohaline brines, 52 environments, 137 Thermaerobacter subterraneus, 28 Thermal hysteresis (TH), 280 Thermales, 13
14 Thermicine, 226 Thermoactinomyces vulgaris, 68 Thermoadaptation-directed enzyme evolution, 208 Thermoanaerobacter, 25
26, 183 Thermoanaerobacter 1004
09 strain, 226 Thermoanaerobacter ADHs (TADHs), 183
185 role of, in physiology, 183
185 thermal stability of, 185
186 Thermoanaerobacter brockii, 185 Thermoanaerobacter ethanolicus, 183
185 Thermoanaerobacter keratinoplilus, 226 Thermoanaerobacter pseudoethanolicus, 184
185 Thermoanaerobacter secondary ADHs (TSADHs), 183 Thermoanaerobacter tengcongensis, 4
5, 160

Thermoanaerobacter thermohydrosulfuricus, 228
229 Thermoanaerobacter yonseiensis KB-1, 226 Thermoanaerobacterium saccharolyticum, 228, 321 Thermoanaerobacterium sp. M5, 228 Thermoascus aurantiacus, 227 ThermoCas9, 160
162 Thermococcales, 75
76 Thermococcus, 219
220 Thermococcus barophilus, 75
76, 111
112 Thermococcus kodakaraensis, 76, 95
96, 111, 220 KOD1, 226 Thermococcus litoralis, 25, 224, 320 Thermococcus nautili, 288, 290t, 291t Thermococcus profundis, 65 Thermococcus stetteri, 226 Thermococcus viridis, 68 Thermococcus zilligii, 227 Thermocrinis, 219
220 Thermodesulfobacter, 13
14 Thermodiscus, 219
220 Thermofilum, 219
220 Thermogymnomonas spp., 73 Thermolabile endoxylanase, 273
274 Thermomices lanuginosus, 176
178 Thermomonospora sp., 25
26 Thermomyces lanuginosus, 227 Thermophiles, 5, 67
69, 160
161, 219, 295, 315 discovery and diversity of, 219
220, 221t enzymes from, 69t genomes, 68 habitats and diversity, 67
68 in medicine and food, 68 physiological adaptations of extremophiles, 17 physiology and adaptation to high temperature, 68 thermophilic enzymes and applications, 69 Thermophilic adaptations, 220
225 genome level adaptations, 220
223 membrane level adaptations, 220 proteome level adaptations, 223
225 archaea, 219 bacteria, 219, 227 bacterium, 219
220 β-amylase, 225
226 enzymes, 225
229 amylases, 225
226 cellulases, 227 lipases, 228
229 proteases, 226 xylanases, 227
228 EST2, 174
175 esterases/lipases, 173
175 fungi, 15 Geobacillus thermodenitrificans T12 strain, 161
162 microorganisms, 4
5, 67 piezophiles, 76, 307 proteases, 226

365

proteins, 174
175, 224 Thermophilic nonpiezophilic (TNP) organism, 312 Thermophilic piezophilic (TP) organism, 312 Thermoplasma, 14, 18, 73 Thermoplasma acidophilum, 13
14, 74, 96 Thermoplasmataceae A10, 97 Thermoproteales, 219
220 Thermoproteus, 219
220 Thermosipho, 219
220 Thermosphaera, 219
220 Thermostable CAs, 298, 300, 301f enzymes, 183, 186, 219, 225 NADP-dependent alcohol dehydrogenases, 183 polymerases, 5 proteins, 66 Thermostable TADHs biocatalysis using, 187
188 enzyme improvement, 188
190 structure and thermostability, 185
187 structure and binding pocket specificity, 185
186 thermal stability of TADHs, 185
186 Thermoanaerobacter ADHs and role in physiology, 183
185 Thermothrix, 132 Thermotoga, 219
220 Thermotoga maritima, 4
5, 19, 67
68 Thermotoga neapolitana, 5, 160
161 Thermotoga thermarum, 228 Thermotogaceae, 67
68 Thermotogae, 13
14 Thermotogales sp., 91 Thermotolerant, 13
14 cellulolytic enzymes, 227 enzymes, 219 xylanases, 228 Thermozymes, 69, 186 Thermus, 13
14, 19, 28, 67
68 Thermus aquaticus, 5, 13
14, 28, 67
68, 90, 219
220, 320 TY-1, 226 Thermus brockianus, 18 Thermus thermophilus, 4
5, 18, 26
29, 54, 68, 160, 211, 228 ATCC 33923, 222 BSH, 24 genome, 30
31 Thielavia arenaria XZ7, 227 Thielavia terrestris, 227 Thioalkalicoccus, 132 Thiobacilli, 133 Thiobacillus, 4
5, 132, 134
135 Thiobacillus acidophilus, 131
132 Thiobacillus aquaesulis, 131
132 Thiobacillus denitrificans, 131
132, 134
135 ATCC 25259, 288 Thiobacillus ferrooxidans, 73, 131
133 Thiobacillus halophilus, 131
132 Thiobacillus neapolitanus, 131
132 Thiobacillus novellus, 131
132 Thiobacillus thioparus, 4
5, 133
135

366

Index

Thiobacillus thiospora, 131
132 Thiobacillus thioxidans, 131
134 Thiocapsa, 132 Thiococuus, 132 Thiocyctis, 132 Thiodictyon, 132 Thiols, 186 cellular sequestration by thiol systems, 24
25 LMW thiols, 24
25 protein thiols, 25 Thiomicrospira, 132 Thiomicrospira thvasirae, 131
132 Thiomonas cuprina, 31 Thiomonas sp., 28 Thiopedia, 132 Thioplaca, 132 Thioredoxin reductase (TR), 6, 25 Thioredoxins (Trx), 25 Thiorhodococcus, 132 Thiorhodospira, 132 Thiosphaera, 132 Thiospira, 131
132 Thiospirillum, 132 Thiosulfate (HS2O32-), 131
132 Thiothrix, 132 Thlaspi arvense, 328
329 Thlaspi goesingense, 329
330 Thlaspi japonicum, 335
336 Thlaspi montanum var. siskiyouense, 331
332 Threonine (Thr), 223 Thysanoessa macrura, 275 Tin (Sn), 133 Tirez Lake, Spain, 97 TMAO. See Trimethylamine oxide (TMAO) TNA1 Planetary Field Analogues, 96 TNAs. See Transnational access (TNAs) TnAtcArs genes, 28 Toxitolerant organic solvents, 5 ToxR protein, 110 ToxS protein, 110 TpSADH. See SADH from T. pseudoethanolicus (TpSADH) TR. See Thioredoxin reductase (TR) tracrRNA. See Trans-activating CRISPR RNA (tracrRNA) Trained machine learning models, 309
310 Trans-activating CRISPR RNA (tracrRNA), 158
160 Transcription activator-like effector nucleases (TALENs), 155
156 Transcription activator-like effectors (TALEs), 155
156 Transcription factors, 6 Transcriptome analysis of H. elongata, 256
258 Transcriptomics, 6, 15, 318 Transformation procedure, 208 Transgenic approach, 39 Transnational access (TNAs), 96 TRAP-T. See Tripartite ATP-independent periplasmic transporter family (TRAP-T)

W

Trehalose, 5
6 Trianionic pyrophosphate, 5
6 2,4,5-Trichlorophenol, 57 2,4,6-Trichlorophenol, 57 Trifluoroacetic acid (TFA), 262 Trimethylamine oxide (TMAO), 111 Trimmatostroma salinum. See Meristematic fungus (Trimmatostroma salinum) Triones, 74 Tripartite ATP-independent periplasmic transporter family (TRAP-T), 258 Triple-Code Saturation Mutagenesis (TCSM), 189 Trp. See Tryptophan (Trp) Trx. See Thioredoxins (Trx) Trypanothione, 24 Trypsins, 247
248 Tryptophan (Trp), 223 TSADHs. See Thermoanaerobacter secondary ADHs (TSADHs) Twin-arginine translocation pathway (Tat pathway), 285, 286f, 287 Two component system (TCS), 239 Type I CRISPR systems, 158f, 159 Type II CRISPR systems, 159
160 Type II signal sequences, 288 Type III CRISPR systems, 159
160 Tyrosine (Tyr), 223

Weissella, 123
124 WEKA (java-based machine learning platform), 308 WGS. See Whole genome sequencing (WGS) White smokers, 67 Whole genome sequencing (WGS), 6
7, 238
239 Whole-cell applications G. caldoxylosilyticus T20, 208 G. kaustophilus HTA426, 208 G. stearothermophilus ATCC 12978, 208 G. stearothermophilus NUB3621, 208
209 G. thermocatenulatus 11, 209 G. thermodenitrificans OS27, 209 G. thermodenitrificans T12, 209 G. thermoglucosidasius DSM 2542, 209
210 G. thermoglucosidasius M10EXG, 210 G. thermoglucosidasius NCIMB 11955, 210 G. thermoglucosidasius NY05, 210 G. thermoglucosidasius PB94A, 210 Geobacillus sp. LC300, 211 Geobacillus sp. XT15, 211 Geobacillus spp. potential in, 207
211 Wild-type TSADHs, 187 Wollastonite (CaSiO3), 208 Woloszynskia cincta, 97

U

X

Ultra violet (UV), 98 defense against ultraviolet radiation, 77 lights, 274 Umbilicaria cylindrica, 3 Uncharged polar amino acids, 223 Uncharged solutes, 141 Unicellular green algae, 139 UNIPROT protein database, 307
308 Unsaturated fatty acids, 245 ura3 codes, 146 Uracil DNA glycosylase, 247
248 Urease, 327 Usnea antarctica, 3 UV. See Ultra violet (UV)

V Vaccine preservation, 114 Vagococcus, 91 Valine (Val), 223 Variovorax, 39
40 Verrucomicrobia, 18 Vibrio, 13 Vibrio harveyi, 18
19 Viral communities, 15 Virgibacillus pantothenticus, 263 Virgibacillus salexigens, 263 Virgibacillus sp., 56, 96
97 Vitamin B6-dpendent enzyme, 317 Volatile organic compounds (VOC), 41, 43, 45 Vulcanisaeta, 97

X-ray crystallography, 227
228 Xanthomonas campestris, 332
333 Xanthomonas oryzae, 275
276 Xanthomonas species, 134
135 Xeromyces bisporus, 78 Xerophiles, 5, 65, 78 xylA promoter, 206 Xylanases, 5
7, 69, 225, 227
228, 322

Y Yellow Stripe-Like transporter (YSL transporter), 335
336 Yellowstone National Park (YNP), 219
220 Ypr153w protein, 6, 109 YSK, 28

Z Zinc (Zn), 334 hyperaccumulation, 334 metal resistance, 24 Zinc-finger nucleases (ZFNs), 155
156 ZmPIP1
1 gene, 43 Zooplankton, 3 Zrt/Irt-like proteins (ZIP), 329
330, 335
336 Zwitterionic organic solutes, 141 Zymomonas, 123
124