Zeitschrift für Allgemeine Mikrobiologie: Band 21, Heft 6 1981 [Reprint 2021 ed.] 9783112538906, 9783112538890

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ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS HEFT 6 • 1981 • BAND 21

WEM

AKADEMIE-VERLAG

BERLIN ISSN 0044-2208

EVP 20, — M

34X12

INHALTSVERZEICHNIS

HEFT

6

Aggregation und Trennbarkeit der Enzyme des Shikimat-Pathway bei Hefen Wirkung von Wachstumsfaktoren auf die Empfindlichkeit gegen Killertoxine bei Saccharomyces cerevisiae Interaktion des Bakteriophagen 02 mit Stämmen der Gattung Oerskovia Eine temperatursensitive Mutante von Escherichia coli mit defekter Reparaturkapazität Einfluß von Dimethylsulfoxid auf die Transkription von Bakteriophagen T3-induzierter RNA-Polymerase Enzyme der Glucose-Isomerisation bei verschiedenen Mikroorganismen Zusammensetzung der Mikroorganismenpopulation im ungeschützten Fermentationsprozeß

R . BODE u n d D . BIRNBAUM

417

V . J I R K Ö u n d A . CEJKOVÄ

423

I . KLEIN, L . KITTLEB, S. KRETSCHMER,

F. Süss und U.

I R E N E MOBFIADAKIS

TAUBENECK und

439

LEB H . MUSIELSKI, W . MANN, R .

LAUE

u n d S. MICHEL

447

M . SUEKANE u n d H . IIZUKA

457

L . WÜNSCHE, K . SATTLER, N. B. GRADOVA, I . M E I N H O L D , R . H E D LICH, W . BBENDLER, H . U H L I G , G . S . RODIONOVA u n d A . I . S A I K I N A 469

475

Buchbesprechungen-

CONTENTS

OF

NUMBER

6

Aggregation and separability of the shikimate pathway enzymes of yeasts Effect of growth factor deficiency on killer toxin sensitivity of Saccharomyces cerevisiae Interaction of bacteriophage 02 with strains of the genus Oerskovia Further studies on a temperature-sensitive mutant of Escherichia coli with defective repair capacity Influence of dimethylsulfoxide on transcription by bacteriophage T3-induced RNA polymerase Enzymes of glucose isomerization in various microorganisms Composition of microorganisms in an unprotected continuous fermentation process Book Reviews

427

E . GEISS-

R . BODE a n d D . BIRNBAUM

417

J . JntKu and A.

423

ÖEJKOVA

I . KLEIN, L . KITTLER, S. KRETSCHMER,

F. Süss and

U . TAUBENECK

427

I R E N E MOBFIADAKIS a n d E . GEISS- , LER

439

H . MUSIELSKI, W . MANN, R . LAUE a n d S . MICHEL

447

M . SUEKANE a n d H . IIZUKA

457

L . WÜNSCHE, K . SATTLEB, N. B. GBADOVA, I . M E I N H O L D , R . H E D LICH, W . BRENDLER, H . U H L I G , G . S . RODIONOVA a n d A . I . S A I K I N A 469

475

ISSN 0044-2208

ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE MORPHOLOGIE, PHYSIOLOGIE, GENETIK UND OKOLOGIE DER MIKROORGANISMEN

H E R A U S G E G E B E N VON

F. Egami, Tokio G. F. Gause, Moskau 0 . Hoffmann-Ostenhof, Wien A. A. Imseneckii, Moskau G. Ivanovics, Szeged R. W. Kaplan, Frankfurt/M. F. Mach, Greifswald 1. Malek, Prag C. Weibull, Lund

unter der Chefredaktion von W. Schwartz, Braunschweig und U. Taubeneck, Jena

U N T E R MITARBEIT VON

J . H. Becking, Wageningen H. Böhme, Gatersleben M. Girbardt, Jena S. I. Kusnecov, Moskau 0 . Necas, Brno C. H. Oppenheimer, Port Aransas N. Pfennig, Göttingen I. L. Rabotnova, Moskau A. Schwartz, .Wolfenbüttel

HEFT 6 • 1981 • BAND 21

AKADEMIE-VERLAG BERLIN

REDAKTION

U. May, Jena

Die Zeitschrift für Allgemeine Mikrobiologie soll dazu beitragen, Forschung und internationale Zusammenarbeit auf dem Gebiet der Mikrobiologie zu fördern. E s werden Manuskripte aus allen Gebieten der allgemeinen Mikrobiologie veröffentlicht. Arbeiten über Themen aus der medizinischen, landwirtschaftlichen, technischen Mikrobiologie und aus der Taxonomie der Mikroorganismen werden ebenfalls aufgenommen, wenn sie Fragen von allgemeinem Interesse behandeln. Zur Veröffentlichung werden angenommen: Originalmanuskripte, die in anderen Zeitschriften noch nicht veröffentlicht worden sind und in gleicher Form auch nicht in anderen Zeitschriften erscheinen werden. Der Umfang soll höchstens Vj2 Druckbogen (24 Druckseiten) betragen. Bei umfangreicheren Manuskripten müssen besondere Vereinbarungen mit der Schriftleitung und dem Verlag getroffen werden. Kurze Originalmitteilungen über wesentliche, neue Forschungsergebnisse. Umfang im allgemeinen höchstens 3 Druckseiten. Kurze Originalmitteilungen werden beschleunigt veröffentlicht. Kritische Sammelberichte und Buchbesprechungen nach Vereinbarung mit der Schriftleitung Bezugsmöglichkeiten der Zeitschrift für Allgemeine Mikrobiologie: Bestellungen sind zu richten — in der DDR an den Postzeitungsvertrieb, an eine Buchhandlung oder an den Akademie-Verlag, D D R - 1 0 8 0 Berlin, Leipziger Straße 3 - 4 — im sozialistischen Ausland an eine Buchhandlung für fremdsprachige Literatur oder an den zuständigen Postzeitungsvertrieb — in der BRD und Berlin ( W e s t ) an eine Buchhandlung oder an die AuslieferungsstelleKUNST U N D W I S S E N , Erich Bieber, D-7000 Stuttgart 1, Wilhelmstraße 4 - 6 — in den übrigen westeuropäischen Ländern an eine Buchhandlung oder an die- Auslieferungsstelle K U N S T U N D W I S S E N , Erich Bieber GmbH, CH-8008 Zürich/Schweiz, Dufourstraße 51 — im übrigen Ausland an den Internationalen Buch- und Zeitschriftenhandel; den Buchexport, Volkseigener Außenhandelsbetrieb der Deutschen Demokratischen Bepublik, D D R - 7 0 1 0 Leipzig, Postfach 160, oder an den Akademie-Verlag, D D R - 1 0 8 0 Berlin, Leipziger Straße 3 — 4 . Zeitschrift für Allgemeine Mikrobiologie Herausgeber: I m Auftrag des Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag : Akademie-Verlag, D D R - 1 0 8 0 Berlin, Leipziger Straße 3 - 4 ; Fernruf 2 23 62 29 oder 2 23 62 21 Telex-Nr. 1 1 4 4 2 0 ; B a n k : Staatsbank der D D R , Berlin, Kto-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. UDO TAUBENECK., P r o f . D r . W I L H E L M SCHWABTZ.

Anschrift der Redaktion: Zentralinstitut für Mikrobiologie und experimentelle Therapie der Akademie der Wissenschaften, D D R - 6 9 0 0 J e n a , Beutenbergstr. 11; Fernruf: J e n a 8 8 5 6 1 4 ; TelexNr. 058621. Veröffentlicht unter der Lizenznummer 1306 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckerei „Thomas Müntzer", D D R - 5 8 2 0 B a d Langensalza. Erscheinungsweise: Die Zeitschrift für Allgemeine Mikrobiologie erscheint jährlich in einem Band mit 10 Heften. Bezugspreis je B a n d 250, —M zuzüglich Versandspesen (Preis für die D D R 2 0 0 , - M). Preis j e Heft 2 5 , - M (Preis für die D D R 2 0 , - M). Urheberrecht: Alle Rechte vorbehalten, insbesondere die der Übersetzung. Kein Teil dieser Zeitschrift darf in irgendeiner Form — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translations into foreign languages). No part of this issue may be reproduced in any form, by photoprint, microfilms or any other means, without written permission from the publishers. Erscheinungstermin: J u l i 1981 Bestellnummer dieses Heftes 1070/21/6 © 1981 by Akademie-Verlag Berlin. Printed in the German Democratic Republic AN ( E D V ) 75218

21

Zeitschrift für Allgemeine Mikrobiologie

6

1981

417-422

(Sektion Biologie der Ernst-Moritz-Arndt-Universität Greifswald, W B Molekularbiologie)

Aggregation und Trennbarkeit der Enzyme des Shikimat-Pathway bei Hefen E . BODE u n d D . BIRNBAUM

(Eingegangen am

27.10.1980)

Gel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5-dehydroquinate synthase (DHQ synthase), 5-dehydroquinase (DHQase, EC 4.2.1.10), shikimate: N A D P oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3-enolpyruvylshikimate 5-phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guiUiermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate :NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.

Die Biosynthese der aromatischen Aminosäuren ist durch das Auftreten von verschiedenen Enzymaggregaten gekennzeichnet. Dies trifft sowohl für die vom Chorismat abzweigende Tryptophan- (CRAWFORD 1 9 7 5 ) und Phenylalanin/Tyrosin-Synthese ( G I B S O N U. P I T T A E D 1 9 6 8 ) zu als auch für die enzymatischen Schritte der Bildung von Chorisminsäure aus Erythrose-4-Phosphat und Phosphoenolpyruvat. Für die fünf Enzyme (DHQ-Synthase, DHQase, Shikimat-DHG, Shikimat-Kinase, EPSP-Synthase) des Shikimat-Pathway von Neurospora crassa konnte gezeigt werden, daß sie als arom-Aggregat assoziiert sind und durch ein ,,Cluster-Gen" codiert werden ( B U R G O Y N E et al.

1 9 6 9 , G I L E S et al.

1967).

Ähnliche Befunde konnten auch für andere Pilze von A H M E D U. G I L E S ( 1 9 6 9 ) erhalten werden. Dagegen sind diese fünf Enzymaktivitäten bei Bakterien separierbar und die codierenden Gene offenbar nicht geklustert ( B E R L Y N U. G I L E S 1 9 6 9 ) . Nur Bacillus subtilis scheint bisher davon eine Ausnahme zu machen, da sowohl die Shikimat-Kinase als auch die DHQ-Synthase mit anderen Enzymen assoziert sein kann ( N A K A T S U K A S A U. N E S T E R 1 9 7 2 , H A S A N U. N E S T E R 1 9 7 8 ) . Die Enzyme des ShikimatPathway in höheren Pflanzen sind ebenfalls trennbar ( B E R L Y N et al. 1 9 7 0 ) , jedoch scheinen hier die DHQase und die Shikimat-DHG ein Enzymaggregat zu bilden ( B O U D E T U. L E C U S S A N 1 9 7 4 , K O S H I B A 1 9 7 8 ) .

Über die Organisation dieser Enzyme bei Hefen liegen bis auf Saccharomyces cerevisiae ( D E L E E U W 1 9 6 7 ) und Schizosaccharomyces pompe ( S T R A U S S 1 9 7 9 ) keine Untersuchungen vor. Wir wählten deshalb zehn weitere Hefearten aus, um deren Enzyme auf Assoziation bzw. Trennbarkeit zu überprüfen. Material

und

Methoden

Stämme und Anzucht des Zellmaterials: Für die Untersuchungen wurden folgende Hefen benutzt: Hansenula henricii CCY 38-10-2, Hansenula fabianii N R R L Y 1971, Hansenula anomala W l , Rhodosporidium toruloides IPO 0880, Rhodosporidium spaerocarpum CCY 62-1-2, Rhodotorula 27»

418

R . BODE u n d D . BIRNBAUM

rubra CCY 20-2-3, Pichia guilliermondii N R R L Y 2075, Candida utilis CCY 29-38-18, Lodderomyces elongisporus CBS 2605, Saccharomyces lipolytica CCY 29-26-13, Saccharomyces cerevisiae 15B M4. Als Vergleich wurde Neurospora crassa Gwd 188 mitverwendet. Die Anzucht erfolgte als Schüttelk u l t u r in 500-ml-Kolben zwei Tage bei 30 °C. Als Nährlösung wurde ein Hefe-Vollmedium (10 g Pepton, 10 g Glucose und 40 g Biomalz pro 1) verwendet. Enzymextrakt-Gewinnung: Die Zellen wurden aus dem N ä h r m e d i u m abzentrifugiert, gewaschen und in 0,1 m Tris-HCl (pH 7,5) m i t 1 mM Phenylmethylsulfonylfluorid aufgenommen. Der Aufschluß wurde m i t Hilfe einer X-Presse durchgeführt. Das Homogenat wurde bei 20000 g 20 min zentrifugiert u n d der Überstand als E n z y m e x t r a k t verwendet. Ionenaustauschchromatographie: Der E n z y m e x t r a k t von Hansenula henricii und Rhodosporidiurn toruloides wurde mittels DEAE-Sephadex A-50 (2 x 15 cm) aufgetrennt. Die Säule wurde m i t 0,1 M NaCl in 0,05 M Tris-HCl (pH 7,5) äquilibriert und der auf die gleiche Salzkonzentration eingestellte E n z y m e x t r a k t aufgebracht. Die Elution erfolgte m i t 200 ml eines linearen NaCl-Gradienten von 0,1—0,3 M; dabei wurden Fraktionen von 2,0 ml aufgefangen. J e 0,1 ml davon fanden f ü r die Enzymansätze Verwendung. Der Proteinspiegel wurde bei 280 n m verfolgt. Gelchromatographie: Der E n z y m e x t r a k t aller untersuchten Organismen wurde einer Gelfiltration über Sepharose 6B (2 x 45 cm) unterzogen. Die Säule wurde m i t 0,05 M Tris-HCl (pH 7,5) äquilibriert u n d F r a k t i o n e n von 2,0 ml gesammelt. Die Eichung zur Molekulargewichtsbestimmung erfolgte m i t Hilfe von Cytochrom C (MG 12500), Chymotrypsinogen (MG 25000), Ovalbumin {MG 45000), Rinderserumalbumin (MG 67000), Hefe-Alkohol-DHG (MG 144000) u n d K a t a l a s e (MG 232000). F ü r die Inkubationsansätze der einzelnen E n z y m e wurden jeweils 0,1 ml verwendet. Die Proteinelution wurde bei 280 n m verfolgt. Substratgewinnung: 3-Desoxy-D-Arabino-heptulonat-7-phosphat wurde enzymatisch mittels D A H P - S y n t h a s e aus H. henricii ( B O D E U. B I R N B A U M 1978) synthetisiert und durch Dowex 1 x 8 nach N A S S E R u. N E S T E R (1967) gereinigt. Die Synthese von 5-Dehydrochinat erfolgte chemisch nach G R E W E U. J E S C H K E (1956). Shikimat-5-Phosphat wurde enzymatisch m i t der ShikimatKinase von H. henricii gewonnen und nach K N O W L E S U. S P R I N S O N (1970) gereinigt. Die H e r k u n f t d e r anderen Substrate ist kommerziell. E n z y m a n s ä t z e : Die 1-ml-Inkubationsansätze wurden generell bei 37 °C 30 min inkubiert. 5-Dehydrochinat-Synthase: Die A k t i v i t ä t der DHQ-Synthase wurde durch den Verbrauch an D A H P , der nach B O D E U. B I R N B A U M (1978) bestimmt worden war, ermittelt. Der E n z y m a n s a t z enthielt 50 MM K - P h o s p h a t - P u f f e r (pH 7,5) ,0,1 MM NAD, 0,1 MM CoCl2 und 0,1 MM D A H P . 5-Dehydrochinase (EC 4.2.1.10): Die DHQase-Aktivität wurde durch die Veränderung der Absorption bei 234 n m , die die Bildung von 5-Dehydroshikimisäure anzeigt, gemessen (GOLLUB et al. 1967). Der S t a n d a r d a n s a t z setzte sich aus 0,1 M Tris-HCl (pH 7,5) u n d 0,5 MM Dehydrochinat zusammen. Shikimat-Dehydrogenase (EC 1.1.1.25): Es wurde die Veränderung der Absorption bei 340 n m als Maß der N A D P H - K o n z e n t r a t i o n gemessen. Zur Bestimmung der Shikimat-DHG wurde folgender Reaktionsansatz b e n u t z t : 0,1 M Tris-HCl (pH 8,9), 2 MM Cystein, 1 MM N A D P u n d 5 MM Shikimisäure. Shikimat-Kinase (EC 2.7.1.71): Die Kinase-Aktivität wurde b e s t i m m t durch die Messung des Shikimisäureverbrauchs nach G A I T O N D E u. GORDON (1958). Der E n z y m a n s a t z enthielt 0,1 M TrisHCl (pH 7,4), 5 MM MgCl 2 , 1 MM Shikimisäure u n d 5 MM A T P . 3-Enolpyruvyl-Shikimat-5-Phosphat-Synthase: Der m i t Hilfe von L D H / P K gemessene Verbrauch von Phosphoenolpyruvat diente als Maß der E P S P - S y n t h a s e - A k t i v i t ä t ( G O L L U B et al. 1 9 6 7 ) . D e r Inkubationsansatz enthielt 50 MM Citrat-Puffer (pH 5,5), 0,25 MM Shikimat-5-Phosphat und 1 MM Phosphoenolpyruvat.

Ergebnisse Die einzelnen Aktivitäten der Enzyme des Shikimat-Pathway lassen sich relativ leicht messen, nur die DHQ-Synthase-Aktivität einzelner Hefearten ist mehr oder weniger labil. Die Ergebnisse, die mit Hansenula henricii mittels Gel- und Ionenaustauschchromatographie erreicht wurden, sind in Abbildung 1 und Abbildung 2 wiedergegeben. Sie zeigen, daß sowohl die DHQ-Synthase und die Shikimat-Kinase als auch die EPSP-Synthase bei dieser Hefe separierbar sind, wogegen die Aktivität der Shikim a t - D H G immer zusammen mit der DHQase eluiert wird. Diese Enzyme bilden wahrscheinlich ähnlich wie in höheren Pflanzen einen Enzymkomplex. Die Gegenwart von Phenylmethylsulfonylfluorid sowohl während des Zellaufschlusses als auch bei der Elution, die das Wirken von Proteasen unterdrücken sollte, h a t t e auf die Trennbarkeit der Enzyme keine Wirkung. Das gleiche gilt f ü r Benzamidin, daß wir ebenfalls in einer Konzentration von 1 MM prüften.

Enzyme des Shikimat-Pathway bei Hefen

419

40 Fraktion

Abb. 1. Gelfiltration (Sepharose 6B) der Enzyme des Shikimat-Pathway von Hansenula henricii. DHQ-Synthase (A), DHQase (•), Shikimat-DHG (o), Shikimat-Kinase (•), EPSP-Synthase (•), Protein (—)

Jew/; 1/ IL ,i 0

Froktion

Abb. 2. DEAE-Sephadex-A-50-Chromatographie der Enzyme des Shikimat-Pathway von Hansenula henricii. DHQ-Synthase(B), DHQase (•), Shikimat-DHG (o), Shikimat-Kinase (•), EPSPSynthase (A), Protein ( —)

Die Molekulargewichte, die mit Hilfe der Gelfiltration für H. henricii gefunden wurden, konnten für die DHQ-Synthase mit 80000, Shikimat-Kinase mit 13000, EPSP-Synthase mit 44000 und für die DHQase/Shikimat-DHG mit 150000 bestimmt werden. Für jedes dieser Enzyme konnte nur ein Elutionsmaximum festgestellt werden; multiple Formen waren nicht nachzuweisen. Durch diese Ergebnisse bei H. henricii angeregt, wurden weitere Hefearten auf deren Organisation der Enzyme des Shikimat-Pathway überprüft. Tabelle 1 beinhaltet die Resultate, die mit Hilfe der Gelchromatographie erzielt wurden. Danach lassen sich die untersuchten Hefen in zwei Gruppen unterteilen. Die erste Gruppe umfaßt Arten, deren Enzyme separierbar sind. Hierzu zählen neben Hansenula henricii auch H. fabianii und H. anomala sowie Candida utilis, Pichia guilliermondii und Lodderomyces elongisporus. Die ermittelten Molekulargewichte sind untereinander sehr ähnlich. Die DHQase eluiert auch bei diesen Arten zusammen mit der ShikimatDHG. Ein völlig anderes Bild bieten dagegen die Ergebnisse bei Saccharomycopsis

420

R. BODE und D. BIRNBAUM Tabelle 1 Molekulargewichte der Enzyme des Shikimat-Pathway bei Hefen und N. crassa

Hansenula henricii Hansenula fabianii Hansenula anomala Candida utilis Pichia guilliermondii Lodderomyces elongisporus Saccharomycopsis lipolytica Rhodotorula rubra Rhodosporidium spaerocarpum Rhodosporidium toruloides Saccharomyces cerevisiae Neurospora crassa

DHQSynthase

DHQase

Shikimat- • DHG

ShikimatKinase

EPSPSynthase

80000 70000 70000 80000 80000 75000 270000 280000 280000 290000 280000 290000

150000 160000 160000 150000 160000 160000 270000 280000 280000 290000 280000 290000

150000 160000 160000 150000 160000 160000 270000 280000 280000 290000 280000 290000

13000 14000 14000 14000 13000 13000 270000 280000 280000 290000 280000 290000

44000 42000 40000 42000 40000 39000 270000 280000 280000 290000 280000 290000

lipolytica, Rhodotorula rubra, Rhodosporidium spaerocarpum, Rh. toruloides und Saccharomyces cerevisiae. Bei diesen Hefen konnte in keinem Falle eine Trennung der Enzymaktivitäten durch die angewandte Sepharose-6B-Elution erreicht werden. Die Molekülgrößen, die dabei ermittelt wurden, lagen zwischen 270000 und 290000. F ü r den aromMultienzymkomplex aus Neurospora crassa konnten wir mit unserer Methode eine molekulare Masse von 290000 nachweisen. Wir nehmen deshalb an, daß bei den letzteren Hefearten die fünf Enzyme des Shikimat-Pathway ebenfalls als arow-Komplex organisiert sind. Eine Ionenaustauschchromatographie, die mit dem Enzymextrakt von Rhodosporidium toruloides durchgeführt worden war, konnte unsere Annahme über die Aggregation dieser Enzyme untermauern. Auch hier fanden wir alle Enzymaktivitäten in einem Peak, der sofort bei der Erhöhung der Salzkonzentration eluierte. Diskussion Die von uns untersuchten Hefen lassen sich in Bezug auf die Enzyme des ShikimatP a t h w a y in zwei Organisationsstufen einordnen. Während die Hefen der einen Gruppe den arom-Multienzymkomplex besitzen, sind bei den anderen die Enzyme bis auf die DHQase und Shikimat-DHG voneinander trennbar. Durch den Einsatz von Proteaseinhibitoren konnten proteolytische Aktivitäten ausgeschlossen werden. A u f G r u n d d e r U n t e r s u c h u n g e n v o n AHMED U. GILES (1969), D E L E E U W ( 1 9 6 7 ) u n d

STRAUSS (1979) wurde bisher allgemein angenommen, daß die Pilze den arom-Komplex besitzen. Unsere Ergebnisse bei den drei Hansenula-Arten sowie bei P. guilliermondii, G. utilis und L. elongisporus machen deutlich, daß diese Verallgemeinerung nicht zutrifft. Die ermittelten Molekulargewichte der DHQ-Synthase dieser sechs Hefearten bewegen sich zwischen 70 und 80000 und liegen damit etwas höher als das des Enzyms d e r B a k t e r i e n (BERLYN U. GILES 1 9 6 9 ) u n d d e r h ö h e r e n P f l a n z e n (BERLYN et al. 1 9 7 0 ,

KOSHIBA 1979). Die molekulare Masse der Shikimat-Kinase mit etwa 13000 ist fast identisch mit der der Kinase in Bakterien. Multiple Formen der Shikimat-Kinase, wie sie von BERLYN U. GILES (1969) sowohl in Escherichia coli als auch in Salmonella typhimurium nachgewiesen wurden, konnten wir weder mit der Gel- noch mit der Ionenaustauschchromatographie finden. Die EPSP-Synthase dieser Hefen entspricht in ihrem Molekulargewicht (39—44000) dem Enzym der bisher untersuchten Organismen; nur bei Anabaena variabilis hat sie etwa die doppelte Größe (BERLYN et al. 1970). Die DHQase und die Shikimat-DHG dieser Gruppe von Hefen liegen offenbar als b i f u n k t i o n e l l e s E n z y m a g g r e g a t v o r , w i e e s v o n BOUDET u . LECUSSAN ( 1 9 7 4 ) b e i e i n e r

Reihe von höheren Pflanzen nachgewiesen und von KOSHIBA (1979) an

Phaseolus

Enzyme des Shikimat-Pathway bei Hefen

421

mungo bestätigt werden konnte. Auch bei Ghlamydomonas reinhardi und Physcomitrella patens wurde entsprechendes gefunden ( B E R L Y N et al. 1970). Bei den Hefen ist jedoch das Molekulargewicht des Enzymaggregats mit 150—160000 mehr als dreimal so hoch, wie bei den anderen untersuchten Organismen. Neben dem DHQase/Shikimat-DHG-Komplex konnten weitere zwei Enzyme des Shikimat-Pathway bei Organismen, die keinen arom-Komplex bilden, in Aggregaten nachgewiesen werden. So berichten N A K A T S U K A S A u. N E S T E R (1972), daß die ShikimatKinase bei Bacillus subtilis mit der DAHP-Synthase und der Chorismat-Mutase assoziert sein kann, während von H A S A N U. N E S T E R (1978) bei der gleichen Art ein trifunktioneller Komplex gefunden wurde, der die Aktivitäten der DHQ-Synthase, der Chorismat-Synthase und der Flavin-Reduktase in sich vereinigt. Ob es in den von uns untersuchten Hefen ähnliche Aggregationen gibt, kann noch nicht beantwortet werden. Es steht jedoch fest, daß die DAHP-Synthase von H. henricii mit keinem der nachfolgenden Biosyntheseenzyme einen Komplex bildet, da sie diesen gegenüber in ihren Eigenschaften große Unterschiede aufweist ( B O D E U. B I R N B A U M 1978). Die arom-Enzyme von Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica und Saccharomyces cerevisiae sind durch die von uns angewandten Verfahren nicht trennbar und besitzen ein Molekulargewicht, das mit 270000 bis 290000 bestimmt werden konnte. Der arom-Komplex von N. crassa wurde von uns mit 2 9 0 0 0 0 ermittelt; G A E R T N E R U. COLE (1977) geben 3 0 0 0 0 0 und LTIMSDEN U. COGGINS (1977) 3 3 0 0 0 0 an. Neben N. crassa konnte auch für weitere Pilze ( A H M E D U. G I L E S 1969) und Euglena gracilis ( P A T E L U. G I L E S 1979) die Existenz des orom-Multienzymaggregates durch Separationsverfahren nachgewiesen werden, während D E L E E U W (1967) bei Sacch. cerevisiae und S T R A U S S (1979) bei Schizosacch. pombe für diesen Nachweis genetische Methoden benutzten. Durch die Untersuchungen von G A E R T N E R U. COLE (1977) bzw. von L U M S D E N U. COGGINS (1977) an N. crassa konnte außerdem gezeigt werden, daß es sich beim aromKomplex um ein Homodimer eines pentafunktionellen Polypeptids mit einem ungefähren Molekulargewicht von 150000—165000 handelt. Darüber können wir bei den von uns untersuchten Hefen noch nichts aussagen. Auffallend ist aber, daß durch die Addition der molekularen Masse der ShikimatPathway-Enzyme bei den Hefen, bei denen diese separierbar sind, Werte erreicht werden, die sich um ungefähr 290000 bewegen und somit dem arom-Komplex der anderen Hefen entsprechen. Die Vorteile des arom-Komplexes können für die Zelle darin bestehen, daß ein Channeling der Intermediate der Biosynthese aromatischer Aminosäuren erfolgt. Zum einen können die gebundenen Substrate dadurch rascher und ökonomischer umgesetzt und zum anderen vor dem Angriff induzierbarer katabolischer Enzyme des Chinat-Pathway abgeschirmt werden. Letzteres trifft aber nur für N. crassa ( G I L E S et al. 1967) nicht jedoch für Sacch. cerevisiae und Euglena gracilis ( D E L E E U W 1967, P A T E L U. G I L E S 1 9 7 9 ) z u .

Daß bei den Hefen sowohl Arten vorkommen, die den arom-Komplex besitzen, als auch solche mit separablen Enzymen, ist neben den Ergebnissen einer Vielzahl anderer Untersuchungen ein weiterer Hinweis darauf, daß die äußerlich sehr ähnlichen Hefen keineswegs eine einheitliche Gruppe von Organismen bilden und kann für eine Betrachtung der Evolution der Hefen von Interesse sein. Literatur AHMED, S. I. and GILES, N. H., 1969. Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi. J. Baeteriol., 99, 231—237. BEELYN, M . B . , AHMED, S. I . a n d GILES, N . H . , 1970. O r g a n i z a t i o n of p o l y a r o m a t i c b i o s y n t h e t i c

enzymes in a variety of photosynthetic organisms. J. Baeteriol., 104, 768 —774.

422

R . BODE u n d D . BIRNBAUM

BERLYN, M. B. and GILES, N. H., 1969. Organization of enzymes in the polyaromatic synthetic pathway: separability in bacteria. J . Bacteriol., 99, 222—230. BODE, R. und BIRNBAUM, D., 1978. Die Enzyme der Biosynthese aromatischer Aminosäuren bei Hansenula henricii : 3-Desoxy-D-arabino-heptulonsäure-7-phosphat-Synthase. Biochem. Physiol. Pflanzen, 172, 2 3 3 - 2 4 3 . BOIJDET, A. M. et LECUSSAN, R., 1974. Généralité de l'association (5-déshydroquinate hydrolyase, shikimate: NADP + oxydoreductase) chez les végétaux supérieus. Planta, 119, 71 — 79. BURGOYNE, L., CASE, M. E . a n d GILES, N . H., 1969. P u r i f i c a t i o n a n d p r o p e r t i e s of t h e a r o m a t i c

(arom,) synthetic enzyme aggregate of Neurospora

crassa. Biochim. biophysica Acta, 191,

452-462.

CRAWFORD, I. P., 1975. Gene rearrangements in the evolution of the tryptophan synthetic pathway. Bacteriol. Rev., 39, 87 — 120. DE LEEUW, A., 1967. Gene-enzyme relationships in polyaromatic auxotrophic mutants in Sacchoromyces cerevisiae. Genetics, 56, 554. GAERTNER, F. H. and COLE, K. W., 1977. A cluster-gene: evidence for one gene, one polypeptide, five enzymes. Biochem. Biophys. Res. Commun., 75, 259—264. GAITONDE, M. K. and GORDON, M. W., 1958. A microchemical method for t h e detection and determination of shikimic acid. J . biol. Chemistry, 230, 1043 — 1050. GIBSON, P. and PITTABD, J., 1968. Pathways of biosynthesis of aromatic amino acids and vitamins and their control in microorganisms. Bacteriol. Rev., 32, 465—492. GILES, N . H . , CASE, M . E . , PARTRIDGE, C. W . H . a n d AHMED, S . I . , 1 9 6 7 . A g e n e c l u s t e r i n

Neu-

rospora crassa coding for an aggregate of five aromatic synthetic enzymes. Proc. Natl. Acad. Sei., 58, 1 4 5 3 - 1 4 6 0 .

GOLLUB, E., ZALKIN, H . and SPRINSON, D. B., 1967. Correlation of gene and enzymes, and studies on regulation of the aromatic pathway in Salmonella. J . biol. Chemistry, 242, 5323—5328. GBEWE, R. und JESCHKE, J., 1956. Die Synthese der 5-Dehydrochina-säure. Chem. Ber., 89, 2080—2088. HASAN, N. and NESTER, E. W., 1978. Dehydroquinate synthase in Bacillus subtilis. An enzyme associated with chorismate synthase and flavin reductase. J . biol. Chemistry, 263, 4999—5004.

KNOWLES, P . P . a n d SPRINSON, D. B., 1970. P r e p a r a t i o n of s h i k i m a t e 5-phospate. I n : COLOWICK,

S. P. and KAPLAN, N. 0 . (ed.), Meth. Enzymol. 17A, 351—352. Academic Press Inc. New York and London. KOSHIBA, T., 1978. Purification of two forms of t h e associated 3-dehydroquinate hydro-lyase and shikimate: NADP + oxidoreductase in Phaseolus mungo seedlings. Biochim. biophysica Acta, 522, 1 0 - 1 8 .

KOSHIBA, T., 1979. Organization of enzymes in the shikimate pathway of Phaseolus mungo seedlings. Plant & Cell Physiol., 20, 667—670. LUMSDEN, J . and COGGINS, J . R., 1977. The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain. Biochem. J., 161, 599—607. NAKATSUKASA, W. M. and NESTER, E. W., 1972. Regulation of aromatic amino acid biosynthesis in Bacillus subtilis 168. I. Evidence for and characterization of a trifunctional enzyme complex. ,T. biol. Chemistry, 247, 5 9 7 2 - 5 9 7 9 . NASSER, D. and NESTER, E. W., 1967. Aromatic amino acid biosynthesis: gene-enzyme relationship in Bacillus subtilis. J . Bacteriol., 94, 1706—1714. PATEL, V. B. and GILES, N. H., 1979. Purification of the arom multienzyme aggregate from Euglena gracilis. Biochim. biophysica Acta, 567, 24—34. STRAUSS, A., 1979. The genetic fine structure of the complex locus arom involved in early aromatic amino acid biosynthesis in Schizosaccharomyces pombe. Molec. Gen. Genet., 172, 233—241. A n s c h r i f t : D r . R . BODE

Ernst-Moritz-Arndt-Universität Greifswald Sektion Biologie, W B Molekularbiologie DDR-2200 Greifswald, Fr.-Ludwig-Jahn-Str. 15a

21

Zeitschrift für Allgemeine Mikrobiologie

1981

423-426

(Institute of Chemical Technology, Faculty of Food and Biochemical Technology, Prague, Czechoslovakia)

Effect of growth factor deficiency on killler toxin sensitivity of Saccharomyces cerevisiae V. JIRKU and A.

(Eingegangen

am 22.

CEJKOVA

10.1980)

The killer toxin sensitivity, as well as the levels of total sterols and individual phospholipids of Saccharomyces cerevisiae are influenced by variations in the supply of growth factors. C e r t a i n species of different y e a s t g e n e r a kill o t h e r sensitive strains b y secreting a p r o t e i n a c e o u s s u b s t a n c e , t h e "killer t o x i n " ( P H I L L I S K I R K a n d Y O U N G 1 9 7 0 , S T U M M et al. 1 9 7 7 , P A L F R E E a n d B U S S E Y 1 9 7 9 ) . T h e p r o d u c t i o n of a biologically a c t i v e killer t o x i n is usually limited t o c e r t a i n c u l t u r e conditions as t o m e d i u m composition, p H , t e m p e r a t u r e , a n d p r e s e n c e of stabilizing a g e n t s ( W O O D S a n d B E VAN 1 9 6 8 , W O O D S et al. 1 9 7 4 , I M A M U R A et al. 1 9 7 4 , Y O U N G a n d P H I L L I S K I R K 1 9 7 7 ) . T h e ability t o p r o d u c e killer t o x i n gives c e r t a i n s t r a i n s a selective a d v a n t a g e when growing in c o m p e t i t i o n w i t h sensitive strains. W e o b s e r v e d t h a t g r o w t h f a c t o r deficiency s t i m u l a t e s changes in killer t o x i n sensitivity of deficient cells. I n relation t o t h e f a c t t h a t t h e supposed t a r g e t s t r u c t u r e of killer t o x i n a c t i o n is t h e p l a s m a m e m b r a n e ( B U S S E Y a n d S H E R M A N 1 9 7 3 , S K I P P E R a n d B U S S E Y 1 9 7 7 ) we t r i e d t o c o r r e l a t e t h e t o x i n sensitivity w i t h c h a n g e s in t h e c o n t e n t of sterols a n d of individual phospholipids.

Materials

and

methods

Yeast strains and media: The killer strain Saccharomyces cerevisiae T158C (his 4C-864) and Killer toxin-supersensitive Saccharomyces cerevisiae S6 (a, ade 2 . 5 ) were kindly provided by Dr. H . B U S S E Y (McGill University, Montreal). The toxin production medium (HALVORSON, 1958) was supplemented with L-histidine, yeasts extract and peptone according to PALFREE and B U S S E Y (1979). Sensitive cells were grown in OLSON and JOHNSON (1949) synthetic medium (pH 4.7) with 10 (ig biotin, 500 ag thiamine, 50 (xg inositol, 5 mg calcium pantothenate, 1 mg pyridoxine per 1 (optimum concentrations). Deficient media (liquid and solidified) were obtained by total omission of biotin or thiamine or by 4 0 % reduction of the optimal concentrations of inositol, pantothenate or pyridoxine (60% deficiency). The media were solidified with 2 % OXOID agar No. 3 . Cultivations inliquid media were carried out at 20 °C (killer strain) or at 28 °C (sensitive strain) on a rotary water-bath shaker. Deficient cultures were inoculated with a 10 ml culture pregrown for about 20 h in the particular deficient medium. Preparation of partially purified killer toxin concentrate: Low-molecular diffusible components were removed from cell-free filtrates of the killer strain culture by rotary dialysis (FEINBERG 1976) and the filtrate was concentrated approximately 10 times by ultrafiltration under nitrogen on an AMICON PM-10 membrane using the AMICON ultrafiltration system (AMICON CO., Lexington, U.S.A.). Proteins were precipitated with (NH 4 ) 2 S0 4 (80% saturation). After 7 h the precipitate was dissolved in 2 ml of 50 mM citrate-phosphate buffer (pH 4.7) and desalted on a Sephadex G-25 column (9 X 150 mm). The protein fraction was concentrated in Aquacid I I I (SERVA) to a volume of about 1.5 ml, which was applied to a Sephadex G-75 column (9 x 1000 mm). The column was stabilized and eluted with 50 mM citrate-phosphate buffer (pH 4.7) at a flow rate of 30 ml/h. All operations were at 5 °C. Dialysis tubes SERVA C/150 were used both for dialysis and dehydration in Aquacid.

424

V . JIRKTJ a n d A . C E J K O V A

Killing activity was eluted in a single peak behind five well separated peaks of UV-absorbing material (toxin activity was associated with the first protein peak). The fractions with toxin activity were pooled, dialyzed against water, and lyophilized. The storage of toxin concentrate at 4 °C for one month did not reduce its activity. Killer toxin assay: Toxin activity in the population of Saccharomyces cerevisiae S6 was detected by use of the well method (WOODS and BEVAN 1968) under standardized conditions, using a limiting concentration of killer toxin inducing a 15 mm inhibition zone in the population of supplemented cells. Freeze-dried toxin preparation was suspended (1.2 mg • ml -1 ) in citrate-phosphate buffer (50 MM; pH 4.7). Amounts of 0.1 ml were placed in wells (4 mm) cut into agar plates containing 2 x 106 sensitive cells per ml. After 48 h incubation at 20 °C inhibition zones were measured. Analytical: The total sterol content was determined according to LONGLEY et al. (1968) using mild acid pretreatment (GONZAIES and PABKS 1977) of cells and anhydrous ergosterol for calibration. Individual phospholipids were separated and determined by two-dimensional TLC (CEJKOVA and JIRKIT 1978) combined with phosphorus estimation (MCCLABE 1970). All determinations were executed in duplicates at the time of inoculation of agar plates. Supplemented or deficient cells were harvested for analysis and inoculation of agar plates in the middle of the exponential and stationary phase of individual growth curves.

Results and

discussion

Fig. 1 illustrates that growth factor deficiency may result in changed susceptibility of yeast cells to killer toxin. The changes are dependent on the growth phase of sensitive cells and different deficiencies stimulate fairly uniform sterol content — toxin sensitivity patterns (Fig. 1 and Table 1). An increase in the content of sterols is accompanied with an enhanced susceptibility of cells to killer toxin (exponential cells). On the other hand, an increased resistance is accompanied with a decrease in the sterol content (stationary cells).

Fig. 1. Killer toxin sensitivity of exponential (•) and stationary (•) cells of Saccharomyces cerevisiae S6 determined in the case of cell populations deficient in biotin (B), thiamine (T), inositol (In), pantothenate (Pa), pyridoxine (Py), and supplemented cells (C) In view of these results one may assume that some variations in growth factor supply can stimulate the development of certain membrane alterations. In this connection the content of individual phospholipids in deficient cells has been determined and compared to that of control cells. As can be seen from Table 1, in exponential and stationary cells the respective deficiencies stimulate a uniform decrease in the content of phosphatidyl ethanolamine and a uniform increase in the content of phospatidyl choline. On the other hand, in these cells the content of phosphatidyl inositol and phosphatidyl serine is unchanged or dependent on the nature of the deficiency. Considering the role of sterols or phospholipids in the microarchitecture of biological membranes we can assume that both components play a role in membrane susceptibility to killer toxin, especially if toxin sensitivity is dependent on the degree of membrane fluidity. In this connection, our results suffer from the shortcoming that the

Killer t o x i n sensitivity of S.

cerevisiae

425

Table 1 Changes in levels of individual lipid classes s t i m u l a t e d b y g r o w t h f a c t o r deficiency in t h e supersensitive s t r a i n , Saccharomyces cerevisiae S6 Lipid class Sterols» PC»> PE PI PS

Growth phase exp stat exp stat exp stat exp stat exp stat

Deficiency (%)

Control 2.3 2.5 20.1 21.9 48.2 45.3 23.1 26.2 15.3 13.3

Biotin

Thiamine

Pantothenate

Inositol

Pyridoxine

3.2 1.9 29.6 35.1 32.3 29.5 24.2 23.1 17.4 14.5

2.8 2.0 35.4 37.2 36.1 30.5 23.5 26.2 22.3 19.1

3.6 1.5 30.2 32.2 30.0 31.4 27.1 22.3 17.2 19.5

3.0 1.6 33.1 30.3 33.5 26.8 14.2 17.1 21.2 13.3

3.4 1.5 37.2 38.1 31.6 28.7 19.3 20.0 15.8 14.2

a % of d r y cell w e i g h t ; b t h e c o n t e n t s of individual phospholipids are expressed as lipid phosphorous ((¿mol • g - 1 d r y cell weight). All values are t h e means of t w o determinations. Abreviations: P C = p h o s p a t i d y l choline, P E = p h o s p a t i d y l e t h a n o l a m i n e , P I = p h o s p a h t i d y l inositol, P S = p h o s p h a t i d y l serine

lipids of both classes are extracted from the whole, unfractioned cells and the changes in the compositions of the cytoplasmic membrane only may be masked. Similarly, it should be pointed out that killer toxin sensitivity may be determined in part by the presence of specific receptors (BUSSEY et al. 1979) in the cell wall. In view of this fact we cannot eliminate the possibility that some variation in the supply of growth factors cause an abnormality in cell wall organization. In addition, the described changes in membrane lipid composition can indirectly influence toxin action via changes in metabolic status and energy management of sensitive cells (SKIPPER and B U S S E Y 1977).

References BUSSEY, H . a n d SHERMAN, D., 1973. Y e a s t killer f a c t o r : A T P leakage a n d coordinate inhibition of macromolecular synthesis in sensitive cells. B U S S E Y , H . , SAVILLE, D . , H U T C H I N S , K . a n d P A L F R E E , R . G . E . ,

1 9 7 9 . B i n d i n g of y e a s t

killer

t o x i n t o a cell wall receptor on sensitive Saccharomyces cerevisiae. J . Bacteriol., 140, 888 — 892. CEJKOVÁ, A. a n d JIRKU, V., 1978. Changes in t h e lipid c o n t e n t during cell division of Saccharomyces cerevisiae. Folia microbiol., 23, 3 7 2 — 3 7 5 . F E I N B E R G , G., 1 9 7 6 . Modified m e t h o d s of dialysis. Biochem. J . , 153, 7 2 1 — 7 2 8 . GONZALES, R . A. a n d PARKS, L. W., 1977. Acid-labilization of sterols for e x t r a c t i o n f r o m y e a s t . Biochim. biophysica A c t a , 489, 507—509. HALVORSON, H . 0 . , 1958. Studies on protein a n d nucleic acid t u r n o v e r in growing cultures of y e a s t . Biochim. biophysica A c t a , 27, 267—276. I M A M U R A , T . , K A W A M O T O , M. a n d T A K A O K A , Y., 1974. Characteristics of m a i n m a s h infected killer y e a s t in saké brewing a n d t h e n a t u r e of its killer f a c t o r . J . F e r m e n t . Technol., 52, 293—299. L O N G L E Y , R . F . , R O S E , A. H . a n d K N I G H T S , B. A., 1968. Composition of t h e p r o t o p l a s t m e m b r a n e f r o m Saccharomyces cerevisiae. Biochem. J . , 108, 401—412. M C C L A B E , C . W . F . , 1970. A n a c c u r a t e a n d convenient organic phosphorous assay. Anal. Biochem., 39, 5 2 7 - 5 3 0 . OLSON, B. H . a n d JOHNSON, M. J . , 1949. F a c t o r s producing h i g h y e a s t yields in s y n t h e t i c media. J . Bacteriol., 67, 235—246. PALFREE. R . G. E . a n d BUSSEY, H . , 1979. Y e a s t killer t o x i n : P u r i f i c a t i o n a n d characterisation of protein t o x i n f r o m Saccharomyces cerevisiae. E u r . J . Biochem., 93, 487—493. P H I L L I S K I R K . G . a n d Y O U N G , T . W . , 1970. T h e occurrence of killer c h a r a c t e r in y e a s t s of various genera. Antonie v a n Leeuwenhoek, 41, 147 —151. S K I P P E R , N . a n d B U S S E Y , H., 1 9 7 7 . Mode of a c t i o n of y e a s t t o x i n s : E n e r g y r e q u i r e m e n t for Saccharomyces cerevisiae killer toxin. J . Bacteriol., 129, 668—677.

426

V . J I R K U a n d A . CEJKOVA

STUMM, C., HERMANS, J . M . H . , MIDDELBECK, E . J . , CROES, A . P . a n d D E V R I E S , C. J . M . L . , 1 9 7 7 .

Killer-sensitive relationships in yeasts from natural habitats. Antonie van Leeuwenhoek, 43, 1 2 5 — 1 2 8 . WOODS, D. R. and BEVAN, E. A., 1968. Studies on the nature of the killer factor produced by Saccharomyces cerevisiae. J . gen. Microbiol., 51, 115 — 126. WOODS, D. R., Ross, I. W. and HENDRY, D. A., 1974. A new killer factor produced by a killersensitive yeast strain. J . gen. Microbiol., 81, 285—289. YOUNG, T. W. and PHILLISKIRK, G., 1977. The production of a yeast killer factor in the chemostat and the effect of killer yeasts in mixed continuous culture with a sensitive strain. J . appl. Bacterid., 43, 425-436. Mailing address: Dr. V. JIRKU Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology, 166 28 Prague 6, Czechoslovakia

Zeitschrift für Allgemeine Mikrobiologie

21

6

1981

427-437

(Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena, Direktor: Prof. Dr. U . TAUBENECK)

Interaction of bacteriophage 0 2 with strains of the genus Oerskovia I . K L E I N , L . KITTLBE, S . KRETSCHMEK, F . SÜSS a n d U . TAUBENECK

(Eingegangen am

12.11.1980)

Bacteriophage 02 multiplies normally on Oerskovia turbata IMET 47153. I t has a burst size of about 100 p.f.u. per infected cell and a latent period of 100 min at 30 °C. On Oerskovia xanthineolytica IMET 47383 clear spots were formed after addition of high phage concentrations onto agar top layers. By phase contrast observation, and measurement of the optical density of infected cultures, it was found that the clearing effect on strain IMET 47383 was due to lysis-from-without. Phage 02 adsorbs and injects its DNA into cells of strain IMET 47383 but phage multiplication does not occur, and the phage DNA becomes degraded. Inhibition of phage DNA injection by the combined action of xanthotoxin — u.v. irradiation abolishes the clearing activity of phage lysates. Therefore, both adsorption and DNA injection seem to be prerequisites for the release of a lytic activity out of the phage particle, which is responsible for the clearing effect on strain IMET 47 383. In studies on taxonomic relationship among actinomycete genera, phage host range experiments have been included (BRADLEY and ANDERSON 1958, BRADLEY et al. 1961, PRAUSER and FALTA 1968). I n the course of these studies, lysis was observed that did not depend on phage multiplication and the phenomenon was called "Clearing effect" (PRAUSER 1 9 7 6 , 1 9 8 1 ) . A c c o r d i n g t o JACOBSON a n d LANDMAN ( 1 9 7 6 ) " c l e a r i n g " o f

bacterial lawns is understood as the killing of cells without phage production after addition of high-titered phage suspensions. These clearing effects were shown to be taxon-specific (PRAUSER 1981). One actinophage host system which displayed such a clearing effect was studied in more detail. Phage 0 2 isolated from soil propagates on Oerskovia turbata IMET 47153 was found to cause partially cleared spots in lawns of Oerskovia xanthinoeolytica IMET 47383. Single plaques never could be shown. From the experiments described in this paper it was concluded that the clearing effect of phage 0 2 on Oerskovia xanthineolytica IMET 47 383 was caused by lysis-from-without and that adsorption of the phage and D N A injection were prerequisites for this phenomenon. Materials

and

methods

Bacterial strains: Oerskovia turbata IMET 47153 was obtained as strain IMRU 761 from H.

LECHEVALIER (New Brunswick) and has been described by PRAUSER et al. (1970). Oerskovia xanthineolytica IMET 47383 was obtained as strain G 62 from M. LECHEVALIER (1972). In this

paper the strain numbers IMET 47153 and IMET 47383 were preferentially used. Phage: The bacteriophage 02 was isolated from soil (PRAUSER and FALTA 1968). Phage 02 was propagated on strain IMET 47153 with shaking at 30 °C using complex media M79 or CD. Phage titration was done according to the agar-layer method (ADAMS 1959) using strain IMET 47153 (about 108 cells/per overlay). Media: Complex medium M79 has been previously described (PRAUSER and FALTA 1968). CDmedium containing 0.1% K 2 HP0 4 , 0.05% NaCl, 0.03% MgS04 • 7 H 2 0, 0.001% CaC03, 0.01% FeS0 4 • 7 H 2 0, 0.5% glucose, 0.6% DIFCO proteose peptone No. 3, (agar 1.5%), aqua dest. pH 7.0 was also used.

428

I . K L E I N , L . K I T T L E R , S . KRETSCHMER, F . SÜSS a n d U . T A U B E N E C K

Electron microscopic studies: Techniques for purification of phage a n d p r e p a r a t i o n for electron microscopic observation h a v e already been described ( K L A U S et al. 1 9 7 8 ) . Adsorption studies: Cell suspensions f r o m over n i g h t cultures of b o t h strains were diluted into fresh CD-medium a n d i n c u b a t e d w i t h shaking a t 30 °C. Growth was followed b y measuring t h e optical density. A t different times samples were t a k e n f r o m t h e culture infected with phages a n d diluted. A b o u t 8 X 10 8 cells/ml were mixed w i t h phage 0 2 a t an average phage i n p u t (a.p.i.) 1 ) of O.Olp.f.u./cell. A f t e r 50 min of incubation a t 30 °C cells were sedimented b y low speed centrifugation and t h e phage t i t e r in t h e s u p e r n a t a n t was determined using t h e agar-layer m e t h o d . One-step g r o w t h e x p e r i m e n t : A b o u t 10 s cells/ml f r o m t h e late exponential phase (after 7 h of cultivation in CD-medium a t 30 °C) a n d f r o m t h e s t a t i o n a r y phase (after 14 h of cultivation in CD-medium a t 30 °C) of strain I M E T 47153 were mixed w i t h p h a g e 0 2 (a. p. i. 0.2 p. f. u./cell). Samples were t a k e n f r o m t h e suspension a t t h e t i m e s indicated in Fig. 5 a n d p l a t e d on strain I M E T 47153. A f t e r 24 h incubation a t 30 °C p.f.u. were counted. Strain I M E T 47383 (stationary cells a f t e r 18 h incubation in CD-medium a t 30 °C) was infected u n d e r t h e same conditions. P h a g e D N A break-down: 3 H-labelled phage 0 2 was mixed w i t h 8 x 10S s t a t i o n a r y phase cells of strain I M E T 47 383 (a.p.i. a b o u t 0.1 p.f.u./cell). A f t e r 60 min of adsorption cells were sedimented b y low speed centrifugation a n d washed twice w i t h fresh CD-medium t o remove free phages. A f t e r blending w i t h a mixer (type 309, MELRONEX) go remove reversibly adsorbed phages, samples of t h e cell suspension were t a k e n f r o m t h e culture a t t h e times indicated in Fig. 6, dropped onto f i l t e r p a p e r discs a n d allowed t o air-dry. 3 H-label of t h e cells was measured w i t h a Tricarb-counter Model 3 3 7 5 ( P A C K A R D I n s t r u m e n t s Company, Chicago) a f t e r 3 cycles of washing w i t h trichloracetic acid ( 5 % ) according t o M A N S a n d N O V E L L I ( 1 9 6 1 ) a n d L A U R E N T a n d V A N N I E R ( 1 9 7 3 ) . Test for lysozyme a c t i v i t y in cleared spots of phage 0 2 : Lysozyme t e s t was p e r f o r m e d as described b y T S U G I T A a n d I N O U Y E (1968). P h a g e 0 2 was dropped a t high concentration (a.p.i. 1000 p.f.u./cell) onto agar plates seeded w i t h strains I M E T 47153 a n d I M E T 47383, respectively, t o cause confluent clearing of t h e indicator lawns. T h e n samples f r o m t h e t o p layers were mixed with washed cells of Micrococcus luteus, incubated a t 37 °C, a n d t h e optical density was measured. P r e p a r a t i o n of p h a g e ghosts: P h a g e 0 2 was spun in a VAC 601 u l t r a c e n t r i f u g e (20000 r.p.m., 120 min). Then t h e phage pellets were resuspended in glycerol (3.0 M), glucose (4.0 M), a n d NaCl (4.0 M), respectively, a n d diluted 100-fold w i t h distilled water. I n separate experiments, lysates of phage 0 2 were exposed to N a 4 P 2 0 7 (final concentration 0.01 M), E D T A (0.1 M), NaC10 4 (1.5 M) or t o pH-variation (pH 3 — p H 7). H e a t t r e a t m e n t was performed a f t e r resuspension of phages in NaCl (0.15 M) a t different times a t 54 °C. Sonication of phages was performed in complex medium M79 w i t h cooling b y using t h e sonicator "SCHÖLLER SCHALL" w i t h a f r e q u e n c y of 20 k H z . T h e freezet h a w - e x p e r i m e n t was done in a m i x t u r e of ice a n d alcohol a t a t e m p e r a t u r e of — 78 °C. T h e speed of freezing was 18 °C per min a n d t h a t of t h a w i n g 29 °C per min. This freeze-thaw-cycle was rep e a t e d t h r e e times. I n a c t i v a t i o n of t h e plaque-forming ability of phage 0 2 b y combined action of x a n t h o t o x i n a n d u.v. irradiation: For irradiation experiments, a high pressure mercury l a m p (type H B O 500 V E B Berliner Glühlampenwerk) which produces primarily 365 n m wave length r a d i a t i o n was used. A WG-5 glas filter (VEB S C H O T T a n d Gen. J e n a , D D R ) was also used t o remove emissions below 300 n m . The phage suspension was irradiated in a silica cuvet a t 30 °C. X a n t h o t o x i n , a gift f r o m Prof. G. RODIGHIERO, P a d u a , was dissolved in 10% alcohol a n d added t o t h e p h a g e suspension t o give a final concentration of 50 (ig/ml. A f t e r an incubation of 30 min t h e samples were irradiated.

Results I s o l a t i o n a n d m o r p h o l o g y of p h a g e 0 2 Phage 0 2 was isolated from soil by PRAUSER and FALTA (1968) according to the specific enrichment procedure of WELSCH et al. (1963). As shown in the electron micrograph (Fig. 1) full and empty particles from a purified preparation were obtained. The hexagonal heads measured 66 nm in diameter, the tails were 134 nm long, flexible and non-contractile. The diameter of the tail was determined to be about 9 nm. The tail also possessed a base plate with spikes. Thus, it was concluded that phage 0 2 belonged t o g r o u p B of BRADLEY'S m o r p h o l o g i c a l c l a s s i f i c a t i o n (BRADLEY 1967). According t o K O U R I L S K Y (1974) we prefere t h e t e r m "average phage i n p u t " instead of multiplicity of infection.

Bacteriophage 0 3 interaction with Oerskovia

429

Fig. 1. Electron micrograph of a purified preparation of phage 0 2 particles (120,000 X)

P l a q u e morphology and clearing effect As shown in Fig. 2a, phage 0 2 formed clear plaques on Oerskovia turbata I M E T 47153. Lysogenic derivatives should not be isolated suggesting that phage 0 2 was virulent on this host. On Oerskovia xanthineolytica I M E T 47383 at high phage concentrations, clear spots were produced. At lower phage concentration the spots became more and more turbid, but single plaques did not appear (Fig. 2b). Thus, the effect

Pig. 2. a) Plaques of phage 0 2 on 0. turbata IMET 47153 b) Clearing effect of phage 0 2 on 0. xanthineolytica IMET 47 383. Spots correspond to 6 x I0 8 (1), 6 x 10' (2), 6 x 106 (3), and 6 x 105 (4) p.f.u.

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was similar to that observed after plating of different dilutions of a bacteriocin ( H A E D Y 1975). Clear spots of lysis were obtained at average inputs (a.p.i.) of about 1000 p.f.u./ cell. B y several types of control experiments it was found that the clearing effect was caused by the phage particles themselves and not by lytic or inhibitory substances of the phage lysate. Culture filtrates of strain I M E T 47153 obtained after cultivation without phage infection did not cause any clearing after transfer to plates seeded with cells of strain I M E T 47383. When phage lysates were sedimented by ultracentrifugation the supernatant was found inactive. However, the phage pellet, even when purified in CsCl gradients, was able to cause clearing after spotting onto lawns of strains I M E T 47 383.

J

A

^ 1 a> % V. ^ x / Fig. 3. Swollen cells (a) and protoplast-like particles (b) isolated from cleared areas of a lawn of 0. xanthineolytica IMET 47383 (phase contrast observation) c) cells of 0. xanthineolytica IMET 47383 (phase contrast observation) from the exponential growth phase; d) cells from the stationary growth phase

Time after inoculation (h)

Fig. 4. Growth of both Oerskovia strains and adsorption of phage 0 2 at different growth phases in liquid culture at 30 °C (CD-medium), x: growth of 0. turbata IMET 47153; growth of 0. xanthineolytica IMET 47383; A: adsorption of phage 0 2 to strain IMET 47153, a.p.i. = 0.01 p.f.u. per cell; A: adsorption of phage 0 2 to strain IMET 47383, a.p.i. == 0.01 p.f.u./cell

Bacteriophage 0 2 interaction with Oerskovia

431

In Fig. 3a material from cleared areas of a lawn seeded with strain IMET 47 383 is shown. B y phase contrast observation protoplast-like structures were visible. The hyphal tips swelled and burst after several hours of incubation, and released protoplast-like particles. In Fig. 3b normal cells without phage treatment are demonstrated. In the exponential growth phase the organism formed branched filaments of about 16 fim length. During transition to the stationary growth phase, division septa were formed and the filaments broke down into short rods of about 2 fxm length as reported b y KRETSCHMER ( 1 9 8 1 ) .

After transfer of material from cleared areas to a new lawn of strain IMET 47383, no clearing could be observed indicating that normal phage multiplication did not occur in this strain. This was further tested in the following experiments. Adsorption studies Growth curves of both Oerskovia strains cultivated at 30 °C in complex CD-medium are demonstrated in Fig. 4. There were no differences in growth parameters and morphological properties of both strains. The doubling time was about 100 min. At different times during cultivation samples were taken from both strains and tested for their ability to adsorb phage 0 2 . As shown in the lower part of Fig. 4 phages did not adsorb to both strains in the early exponential phase of growth. Strain IMET 47153 became sensitive to phage infection after 3 h of cultivation (about two generations before transition into the stationary phase). During the whole time of cultivation under stationary growth conditions we found optimal phage adsorption. The adsorption constant (ADAMS 1 9 5 9 ) was in the range of 7 . 4 X 1 0 ' 1 0 ml/ min. In contrast, strain IMET 47 383 became sensitive to phage 0 2 only about 7 hours after transition into the stationary phase of growth and the adsorption constant was lower (about K = 1.7 X 10~ U ml/min). Under the conditions of this experiment, after 50 min of adsorption 90% of input phages were fixed to strain IMET 47153 and 50% of phages to strain IMET 47383. One-step growth curve For the one-step growth experiments both strains were infected by phage 0 2 under optimal adsorption conditions. After phage infection had taken place, cells were

Time after infection (hi

Fig. 5. One-step growth curve of phage 0 2 a t 30 °C (CD-medium, a.p.i. = 0.2 p.f.u./cell). • : Cell suspensions of 0. turbata I M E T 4 7 1 5 3 , from the late exponential growth phase were diluted into fresh medium after infection; B : Stationary cell suspensions of 0. turbata I M E T 4 7 1 5 3 were diluted into fresh medium after infection; x : stationary cell suspensions of 0. xanthineolytica I M E T 4 7 3 8 3 , were diluted into fresh medium after infection 28

Z. Allg. Mikrobiol., B d . 21, H . 6

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I . K L E I N , L . KITTLER, S . KRETSCHMER, F . SUSS a n d U . TATJBENECK

transferred to fresh CD-medium to allow phage multiplication. As it is shown in Fig. 5 multiplication of phage 0 2 occurred only in strain IMET 47153. The latent period was about two hours which corresponds to the doubling time of the host, and the burst size was estimated to be 100 p.f.u./cell. Because no phage propagation could be observed with strain IMET 47 383, we tested whether phage DNA was injected after adsorption. Stationary cells were infected with 3 H-labelled phages. After 60 min of adsorption the suspension was stirred with a mixer to remove reversibly adsorbed phages. The cells were then washed and pelleted by low speed centrifugation. The 3 H-label, transferred to the cells via phage infection, was measured at different times after adsorption. From the data presented in Fig. 6 it was concluded t h a t phage 0 2 was able to infect strain IMET 47383 with low efficiency (about 25% of phage input), but the injected DNA was slowly degraded into acid soluble material.

Time after blending (min)

Fig. 6

nme ofincubatm

w

Fig. 1

Fig. 6. Degradation of 3 H-labelled phage 0 2 D N A in stationary cells of 0. xanthineolytica 47383

IMET

Fig. 7. Change of extinction of cultures of 0. turbata IMET 47383 infected by phage 0 2 in CDmedium at 30 °C. X : average phage input: 1000 p.f.u./cell (In the sample taken 2hours after incubation intact cells could not be observed any longer by phase contrast microscopy); o : average phage input: 0.5 p.f.u./cell

Lysis-from-without The observation of protoplast-like particles in cultures of strain IMET 47 383 after addition of high titer suspensions of phage 0 2 suggested that lysis-from-without was the cause of the clearing effects observed on solid medium. This assumption was further supported by the data presented in Fig. 7. Strain IMET 47153 was infected by phage 0 2 at low and high a.p.i. and the optical density of the growing cultures was followed. I n contrast to the curve obtained with a low a.p.i. addition of purified phages at a high a.p.i. caused an immediate decrease in the optical density, which was in accordance with the phenomenon of lysis-from-without. I n this type of experiment only strain IMET 47153 was used because it adsorbed phage 0 2 also in the exponential phase and

Bacteriophage 0 2 interaction with Oerskovia

433

the optical density could be followed immediately after phage addition to the growing culture. B y phase contrast observation it was found that two hours after addition of a high phage concentration intact cells were no longer visible. P r e p a r a t i o n of p h a g e g h o s t s The degradation of cell envelopes (formation of protoplast-like particles observed after infection of strain IMET 47 383 by phage 02, Fig. 3b) resembles in some ways the lytic effects observed in Proteus, Pseudomonas, Escherichia coli, and Streptomyces exposed to incomplete phages or phage ghosts (HERRIOT and BARLOW 1957, TAUBENECK 1963, 1967, ISHII et al. 1965, BAUTENSTEIN et al. 1966). Because lysozyme activity could not be found in material obtained from plates seeded with strain IMET 47383 and cleared with a high concentration of phage 0 2 (using envelopes of Micrococcus luteus as test substrate), it was assumed that internal protein(s) with endolytic activity located in the phage tail or phage ghost were responsible for the clearing effect on strain IMET 47 383. Therefore, we looked for clearing activity of phage ghosts. In order to obtain phage ghosts, phage 0 2 was exposed to different treatments known to release DNA from complete particles. Surprisingly, phage 0 2 was found to be resistant to osmotic shock (4 M glucose, 3 M glycerol, 4 M NaCl followed by 100fold dilution with aqua dest.) and treatment with chelating agents (0.1 M Na 4 P 2 0 7 , 0.1 M EDTA), 1.5 M NaC104, variation of pH (pH 3—pH 7). Repeated freezing and thawing and sonication also had a minimal effect on the plaque forming ability of phage 02. The only treatment which delivered a high yield of empty phages was heating in 0.15 M NaCl. As shown in Fig. 8, after heating at 54 °C for 17 min the phage titer was diminished about 1,000-fold. The preparation of phage ghosts, obtained after DNase-treatment of heat inactivated phages was found to be inactive in clearing tests

Fig. 8. Heat inactivation of phage 0 2 particles at 54 °C (phages were suspended in 0.15 M NaCl solution and p.f.u. were estimated on strain IMET 47153) 28«

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on strain I M E T 47383. However, b y electron microscopic analysis of the ghost preparation star-like structures were found. (Fig. 9), which were formed b y e m p t y particles sticking together b y their adsorption structures a t t h e end of t h e tails. Thus, from this experiment we could not determine whether adsorption of ghosts to phage specific receptors was the only prerequisite for clearing or whether, in addition, D N A injection was also needed in order to cause clearing b y the action of endolytic protein(s) on strain I M E T 47383.

Fig. 9. Electron micrograph of heat inactivated phage 0 2 (120,000 X) I n h i b i t i o n of D N A i n j e c t i o n A f t e r irradiation by u.v. light (365 nm) furocoumarins have been shown to induce D N A - D N A and DNA-protein cross-links (KITTLER et at. 1977). KITTLER and HRAD E C N A ( 1 9 8 0 ) described complete inhibition of injection of bacteriophage L a m b d a - D N A a f t e r exposure to the furocoumarins x a n t h o t o x i n and angelicin. Table 1 Effect of UV-activated xanthotoxin on the plaque forming ability of phage 0 2 on strain IMET 47153 and on the clearing effect on strain IMET 47383 UV dose (J/m 2 ) 0 2.6 5.2 7.8 10.4 15.6

x x x x x

103 103 103 103 103

p.f.u./ml (IMET 47153) 2.0 3.3 4.9 2.2 1.1 7.8

X X X x X X

Symbols: + + : clear spot + : turbid spot — : no clearing

10» 107 106 105 10" 102

Intensity of clearing (IMET 473.83)

++ + + — — —

Bacteriophage 0 2 interaction with

Oerskovia

435

We used xanthotoxin to find out whether the clearing activity of phage 0 2 could be abolished by the action of this furocoumarin derivative. The data presented in Table ! showam a rked decrease of the plaque forming ability of phage 02, which was correlated with the loss of clearing activity of the lysate. Control experiments showed that adsorption of phages was not diminished by the action of xanthotoxin. Thus, both the loss of plaque forming ability and the clearing activity were possibly caused by the inhibition of phage DNA injection. Discussion Phage 0 2 was found to belong to the most common morphological type of actinophage. I n size and dimensions it resembled numerous Streptomyces phages, such as 0 C 3 1 (LOMOVSKAYA et al. 1972), VP11 ( D O W D I N G 1973), VP5 ( D O W D I N G and HOPWOOD 1973), R4 (CHATER and CARTER 1980), SH10, SH11, and SH12 ( K L A U S et al. 1980, and unpublished results). The phage multiplied normally on Oerskovia turbata IMET 47153. The adsorption constant (K = 7.4- 10"10 ml/min), burst size (about 100 p.f.u./infected cell), and latent period after cultivation at 30 °C in complex medium (100 min) were in the range reported for other actinophages. On Oerskovia xanthineolytica IMET 47383, phage 0 2 did not multiply but formed clear spots at high phage concentration on agar plates. I n a first set of experiments it was found that the clearing effect was not caused by lytic enzymes or inhibitory substances present in the phage lysate. After ultracentrifugation the clearing activity was detected only in the phage pellet and not in the supernatant. In addition, the clearing effect was also observed after purification of phage 0 2 in CsCl. The finding that the clearing activity could not be transferred from one plate to another suggested that phage 0 2 did not multiply in strain IMET 47 383. Therefore, adsorption and one-step growth of phage 0 2 were studied in both alternative strains. In the course of the adsorption studies (Fig. 4)it was observed that strain IMET 47153 became sensitive to phage adsorption only in the late exponential phase of growth. Cells of strian IMET 47383 were able to adsorb phage 0 2 only after having been under stationary conditions for 7 hours, and the adsorption constant was lower than that for strain IMET 47153. Using 3 H-labelled phages it was also shown that after one hour of adsorption a considerably higher proportion of the phages were found to be reversibly attached to the cells, and could be removed by blending. About 25% of added phages were able to inject their DNA into strain IMET 47383. By the one-step-growth experiments shown in Fig. 5, the inability of phage 0 2 to multiply in strain IMET 47383 was established. I t is not yet clear if transcription or replication or both processes are blocked in this strain. Fig. 6 demonstrated a slow DNA breakdown of the injected 3 H phage DNA in strain IMET 47 383. The slow rate of DNA degradation observed did not favour the hypothesis that it was attributable to a restriction system. I t seems more likely that the unreplicated foreign DNA was degraded by a nonspecific nuclease (s). As is shown in Fig. 7, addition of a high concentration of phages to cells of strain IMET 4 7 1 5 3 harvested in the late exponential growth phase, caused an immediate decrease of the optical density of the culture. This was in accordance with the phenomenon of "lysis-from-without" (for review see S T E N T 1 9 6 3 ) , which predicts an immediate dissolution of bacteria often encountered when the multiplicity of infection is much greather than on phage per cell (DOERMAN 1 9 4 8 ) . The observation of swollen cells and protoplast-like structures by phase contrast microscopy (Fig. 3) favoured the assumption that lytic enzymes present in the phage particles (internal proteins) caused

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the clearing by cell wall digestion of strain IMET 47383. The failure to dedect lysozyme activity in material from the clearing spots was in accordance with the observation of EMEICH and STEEISINGEE ( 1 9 6 8 ) on phage T4. Thus, a different lytic enzyme was probably involved. We were not able to determine whether phage ghosts expressed clearing activity. Phage 0 2 was found to be resistant to most treatments known to desintegrate phage particles. Only heating delivered a high yield of phage ghosts, but as was shown by electron microscopy (Fig. 9), the empty particles stuck together by tail to tail interaction. Thus, the inability of these ghost preparations to cause clearing could be due to the loss of adsorption ability. Tails and incomplete particles of other phages have been previously shown to possess killing or clearing activity (TAUBENECK 1963, 1967, RAUTENSTEIN 1 9 6 6 , I S H I I et al.

1965).

In another set of experiments we asked whether phage adsorption was the only prerequisite for the clearing activity of phage 02, or if the DNA injection process was also necessary to release the lytic enzyme(s) from the phage. The combined action of 365 nm u.v. light and furocoumarin derivatives led to covalent linkages of both strands of the DNA duplex. The photoreaction with proteins was less efficient. I t has been shown for phage lambda, that after treament with xanthotoxin and u.v., there was a marked influence on DNA injection and replication, but adsorption was unaffected ( K I T T L E E and HRADECNA 1 9 8 0 ) . The inhibition of infection and replication was attributed to various types of DNA crosslinks produced in the phage heads (KITTLEE et al.

1977).

Using phage 02 similar results were obtained. As shown in Table 1, the combined action of xanthotoxin and u.v. resulted in a marked decrease of the plaque-forming bility. In addition, the clearing activity of the lysate was lost with increasing u.v. dose. I t was therefore concluded, that in addition to adsorption, phage DNA injection is a necessary condition for the clearing activity on strain IMET 47 383. Acknowledgements We are grateful to H . PRATJSER for the gift of bacterial and phage strains and for critical reading of the manuscript. We thank R. GEUTHER for help in the lysozyme test and I. ZIMMERMANN for electron microscopy. Thanks are also due to S. K L A U S for valuable suggestions in t h e course of the work and M. S. GILMOARE for reading of t h e manuscript.

References ADAMS, M. H., 1959. Bacteriophages. Interscience Publishers Inc. New York. BRADLEY, S. G. and ANDERSON, D. L., 1958. Taxonomic implication of actinophage host-range. Science, 128, 4 1 3 - 4 1 4 . B R A D L E Y , S. G., ANDERSON, D . L . and J O N E S , L . A . , 1 9 6 1 . Phylogeny of actinomycetes as revealed b y susceptibility to actinophage. Dev. Ind. Microbiol., 2, 223—237. BRADLEY, D. E., 1967. Ultrastructure of bacteriophages and bacteriocins. Bact. Rev., 31,230—314. CHATER, K . F . and CARTER, A . T . , 1 9 7 9 . A new, wide host-range, temperate bacteriophage (R4) of Streptomyces and its interaction with some restriction-modification systems. J. gen. Microbiol., 115, 4 3 1 - 4 4 2 . DOERMAN, A . H . , 1 9 4 8 . 55, 257.

Lysis and lysis inhibition with Escherichia coli bacteriophage. J . Bacteriol.,

DOWDING, J. E., 1973. Characterization of a bacteriophage virulent for Streptomyces A3(2). J. gen. Microbiol., 76, 1 6 3 - 1 7 6 .

coelicolor

DOWDING, J. E . a n d HOPWOOD, D . A., 1973. T e m p e r a t e b a c t e r i o p h a g e s for Streptomyces

coelicolor

A3(2) isolated from soil. J. gen. Microbiol., 78, 349—359. EMRICH, J. and STREISINGER, G . , 1968. The role of phage lysozym in the life cycle of phage T4. Virology, 36, 3 8 7 - 3 9 1 . HARDY, K. G., 1975. Colicinogeny and related phenomena. Bact. Rev., 39, 4, 464—515.

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a n d B A R L O W , J . L . , 1 9 5 7 . T h e protein coats or " g h o s t s " of coli phage T2. I I . T h e biological functions. J . gen. Physiol., 41, 3 0 7 — 3 3 1 . I S H I I , S . - J . , N I S H I , Y . a n d E G A M I , F . , 1 9 6 5 . T h e fine s t r u c t u r e of a pyocin. J . molecular Biol., 13,

HERRIOT, R . M .

428-431. JACOBSON, E . D .

a n d L A N D M A N , O . E . , 1 9 7 6 . I n t e r a c t i o n of bacteriophage w i t h walled a n d wallless forms of Bacillus subtilis. I n : Microbiology, p. 2 3 8 — 2 5 3 (ed. S C H L E S S I N G E R ) . K I T T L E R , L . , H R A D E C N A , Z . a n d L Ö B E R , G . , 1 9 7 7 . Photochemical induced binding of f u r o c o u m a r i n s w i t h L a m b d a p h a g e D N A in situ, s t u d i a biophys., 6 6 , 2 3 7 — 2 4 1 . K I T T L E R , L. a n d H R A D E C N A , Z . , 1 9 8 0 . Crosslink f o r m a t i o n of phage L a m b d a D N A in situ photochemically induced b y t h e f u r o c o u m a r i n derivative Angelicin. Biochim. biophysica A c t a , 607, 215-220. K L A U S , S . , SÜSS, P . , J U C H ,

C. a n d H A U C K , A., 1978. Characterization of t h e virulent a c t i n o p h a g e S2. Z. Allg. Mikrobiol., 18, 5 7 5 - 5 8 6 .

K L A U S , S . , K R Ü G E L , H . , SÜSS, F . , N E I G E N F I N D , M . , ZIMMERMANN, I . a n d TAUBENECK, U . ,

1980.

P r o p e r t i e s of t h e t e m p e r a t e actinophage SH10. J . gen. Mikrobiol., in press. K O U R I L S K Y , P H . , 1 9 7 4 . Lysogenisation b y bacteriophage A. I I . Identification of genes involved in t h e multiplicity d e p e n d e n t process. Biochimie, 56, 1 5 1 1 — 1 5 1 6 . KRETSCHMER, S., 1981. Analysis of morphogenesis of t h e nocardioform organism Oerskovia xanthineolytica. Z. Allg. Mikrobiol., 21, 235—245. L A U R E N T , S . J . a n d V A N N I E R , F . S . , 1 9 7 3 . Temperative-sensitive initiation of chromosome replication in a m u t a n t of Bacillus subtilis. J . Bacteriol., 114, 4 7 4 — 4 8 4 . LECHEVALIER, M. P., 1972. Description of a new species Oerskovia xanthineolytica and emendation of Oerskovia P R A U S E R et al.. I n t . J . Syst. B a c t e r i d . , 22, 260—264. LOMOVSKAYA,

N. D.,

MRKTUMIAN,

N. M.,

GOSTIMSKAYA,

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DANLIENKO,

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coelicolor. J . Virol.,

258-262.

a n d N O V E L L I , G . D . , 1 9 6 1 . Measurement of t h e incorporation of radioactive amino acids into p r o t e i n b y filter-paper disk m e t h o d . Arch. Biochim. Biophys., 94, 48—53. P R A U S E R , H . u n d F A L T A , R . , 1 9 6 8 . Phagensensibilität, Zellwandzusammensetzung u n d Taxonomie von Actinomyceten. Z. Allg. Mikrobiol., 8 , 3 9 — 4 6 . P R A U S E R , H . , L E C H E V A L I E R , M. P . a n d L E C H E V A L I E R , H . , 1 9 7 0 . Description of Oerskovia gen. n. t o h a r b o r ORSKOV'S motile Nocardia. Appl. Microbiol., 19, 534. PRAUSER, H . , 1976. H o s t - p h a g e relationships in nocardioform organisms. I n : T h e Biology of Nocardiae, pp. 266—284 (ed. GOODFELLOW, M., B R O W N E L L , G . H . a n d S E R R A N O , J . A.). Academic Press London-New York-San Francisco. PRAUSER, H . , 1981. Taxonspecificity of lytic actinophages t h a t do n o t m u l t i p l y in t h e cells infected. I n : Biology of Actinomycetes (ed. P U L V E R E R , G. a n d S C H A A L , K . P . ) . G u s t a v Fischer Verlag S t u t t g a r t - N e w York. MANS, R . J .

R A U T E N S T E I N , Y A . J . , S T E F A N O V , S . B . , SMIRNOVA, E . J . a n d C H A D J I M U R A T , L . N . , 1 9 6 6 . O n

fective lysogeny in Actinomyces

streptomycinii,

de-

t h e streptomycin producer. Mikrobiologia, 35,

860—863.

STENT, G. S., 1963. Molecular Biology of Bacterial Viruses. W. H . F r e e m a n a n d Company San Francisco-London. T A U B E N E C K , I L , 1 9 6 3 . Über die P r o d u k t i o n biologisch aktiver Phagenschwänze durch einen d e f e k t lysogenen Proteus mirabilis-Stamm. Z. N a t u r f o r s c h . , 18b, 9 8 9 — 9 9 1 . T A U B E N E C K , I L , 1 9 6 7 . Ü b e r i n k o m p l e t t e B a k t e r i o p h a g e n aus d e f e k t lysogenen Proteus mirabilisS t ä m m e n . Biol. Zbl., 86 (Supplement), 45—54. T S U G I T A , A . a n d I N O Y E , M . , 1 9 6 8 . Purification of bacteriophage T4-lysozyme. J . biol. Chemistry, 243, 3 9 1 - 3 9 7 . WELSCH, M., RUTTEN-PINCKAERS,

A. et S E L M A N , M . , 1 9 6 3 . Recherches sur des Streptomyces d' Afrique Centrale. IV. Isoantibiose, lysogenie et actinophages. Bull. Soc. Roy. Sei. Lg. 32, 529—573.

Mailing address: D r . I . KLEIN Z e n t r a l i n s t i t u t f ü r Mikrobiologie u n d experimentelle Therapie der A d W D D R 69 J e n a , Beutenbergstr. 11

21

Zeitschrift für Allgemeine Mikrobiologie

1981

6

439-445

(Sektion Biologie, Institut für Med. Mikrobiologie u. Epidemiologie, Bereich Medizin der WilhelmPieck-Universität Rostock, DDR, Zentralinstitut für Molekularbiologie, der Akademie der Wissenschaften der DDR, Berlin-Buch, DDR)

Further studies on a temperature-sensitive mutant of Escherichia coli with defective repair capacity I R E N E MORFIADAKIS a n d E . G E I S S L E R

(Eingegangen am

11.11.1980)

A temperature-sensitive mutant of E. coli, WG24, was studied with respect to its sensitivity to photodynamic action, its capacity to perform host controlled reactivation, and its sensitivity to transduction at elevated temperatures. Mutant cells are much more sensitive than wild type cells to photodynamic action by thiopyronine and visible light at elevated temperatures. As well defined rec mutants, WG24 cells are less able to reactivate UV irradiated Xc phages at elevated temperatures, while their ability to repair T1 phages is less impaired. Mutant cells cannot be transduced to T6 resistance at a detectable rate at elevated temperature. I t is concluded, therefore, that some rec gene carries a ts mutation in this mutant. I n an earlier paper (MORFIADAKIS et al. 1 9 7 3 ) we reported on the isolation of a temperature-sensitive repair m u t a n t W G 2 4 of Escherichia coli W 1 0 3 2 . T h e conditional defectiveness of this m u t a n t was found to affect survival after TJ V irradiation, mitomycin C t r e a t m e n t , and p h o t o d y n a m i c inactivation. These results, however, did n o t answer the question of whether the defectiveness of W G 2 4 affects the excision-dependent (uvr polA) or recombination-dependent) rec gene) repair mechanisms known to be mainly involved in the elimination of D N A lesions caused by such t r e a t m e n t (HOWARDFLANDERS 1 9 7 3 ) .

I n this paper we describe the results of testing W G 2 4 with regard to the defectiveness of functions known to be affected in E. coli by the rec genes, for example reactivation after photodynamic damage, host—cell reactivation of U V irradiated A and T 1 phages, a n d the efficiency of transduction of W G 2 4 by phage P I . Materials

and

methods

The bacterial strains used in this study are listed in Table 1. Bacteriophages: The bacteriophages T l , Ac72, Plbc(RH), and T6 were used for characterizing the isolated mutant WG24 and clarifying its function. Some of the strains listed in Table 1 and a few phage strains were kindly placed at our disposal by the following institutions: E. coli K12 Table 1 Bacterial strains used Strain E. E. E. E. E.

coli coli coli coli coli

G60 K12 K12 K12

E. coli G62

Designation W3102 WG24 C600 86 72

Relevant markers gal str r hcr + rec + gal str r hcr + rects thr leu thi As thi T6 r thr leu lac str r Tis T6s gal strr rec her

Source ECHOLS et al.

(1963)

This paper

E. coli 152 KNESER

440

IRENE MORFIADAKIS a n d E . GEISSLER

C600 by Dr. Z. HRADECNA (Institute of Biophysics, Brno, CSSR); E. coli 86 and 72, and the P I and T6 phages by Prof. H. SCHUSTER and Prof. W. MESSER (Max-Planck-Institute of Molecular Genetics, Berlin-Dahlem), Ac72 phages by Dr. S. KLAUS (Central Institute of Microbiology and Experimental Therapy, Jena, GDR). Methods: 1) Photodynamic effect: The starved, undiluted cells (titre: 4 x 108 cells/ml) in saline were treated with thiopyronine (7 (ig/ml) for 20 min in the dark at room temperature and then immediately exposed to light in an open beaker at 32 °C and 42 °C in a water bath while being agitated. The light source was a 100 W filament lamp at a distance of 35 cm and the exposure time was 32 min. Subsequently, two samples were taken from each temperature batch and immediately transferred to preheated saline (32 °C and 42 °C, respectively), further diluted, inoculated onto NB agar plates which had been preheated to 32 °C and 42 °C, respectively, and incubated overnight at these temperatures. 2) Host cell reactivation of UV irradiated T1 phages: After dilution in saline to 1—2 x 106 PFU/ ml, the lysate was irradiated in an open Petri dish (fluid level: 1 mm) for 2, 5, and 10 min with UV radiation. The samples were situated 2.10 m from the radiation source. The radiation intensity at the source was 0.1 J • m~2 sec -1 . The irradiated T1 phages were immediately added to the bacteria (titre: 1—2 x 108) which had been cultured at 25 °C, starved, and transferred to nutrient broth which was preheated to 32 °C and 42 °C, respectively, at a ratio of 1:100 and incubated at 32 °C and 42 °C, respectively, for 15 min. The bacteria-phage mixture was diluted 1:100 in nutrient broth (NB) and incubated for 60 min at 32 °C and 42 °C, respectively, following adsorption but prior to plating. Finally, the mixture was further diluted, samples were placed in test tubes with agar, transferred to NB agar plates, and incubated overnight at 32 °C and 42 °C, respectively. The strain E. coli G62 ( h r c rec~) was used as an indicator strain. 3) Host cell reactivation of UV-irradiated Ac phages: The procedure used for the T1 phages was repeated for the Ac phages. The duration of UV irradiation was 18 min. After an adsorption phase lasting 18 min the bacteria-phage mixture was immediately diluted, transferred from suitable dilutions was immediately diluted, transferred from suitable dilutions to the test tubes containing agar, pured onto preheated (32 °C and 42 °C, respectively) NB agar plates and incubated overnight at these temperatures. E. coli G62 was again used as indicator strain. 4) Transduction: The bacteria were cultured at 25 °G in trypton-yeast broth (TBY) enriched with Ca + + . This buffer solution was used for diluting the cells. The cell population which was in the stationary phase was centrifuged, washed twice in 2 X 10"3 M Ca ++ -enriched TBY, the liquid volume was reduced by 50%, and the bacteria were then preheated at 42 °C for 5 min. The cell suspension was then mixed with the appropriate P I phages (m.o.i. = 0.1) reproduced on E. coli P6 and incubated for 20 min at 42 °C while being constantly agitated. Some of the phages carried the genetic marker T6res. After adsorption the bacteria-Pi phage mixture was centrifuged and washed with TBY containing no Ca ++ , resuspended in the same medium but with half of the original cell suspension volume, split into two batches, diluted to the threefold TBY volume without Ca ++ , and incubated for 4 hours at 32 °C and 42 °C, respectively. During the first 20 min the bacteria were centrifuged three times within 25 min. Samples were subsequently taken, spread on TBY agar plates enriched with T6 phages and incubated overnight at 32 °C and 42 °C. The plate agar was enriched with T6 phages by the following procedure. A high-titre phage lysate (co. 3 X 1010 PFV/ml) was diluted with very fresh TVY. This was then placed in such quantities on the dried plates t h a t the ratio between the plated bacteria and T6 phages was about 1:20.

Results Host cell r e a c t i v a t i o n It has been shown that host cell reactivation (HCR) of UV irradiated T1 and T3 phages is controlled by uvr genes only, whereas the HCR of UV irradiated A phages depends partly on a functioning rec gene ( H A R M 1 9 6 8 ) , GEISSLER 1 9 6 8 , K N E S E R 1 9 6 8 ) . The survival of UV irradiated T1 and Ac phages plated in W G 2 4 cells was therefore studied. Fig. 1 shows the effects of different temperatures (32 °C and 42 °C) on the survival of UV irradiated T1 phages in mutant and wild type cells. The repair capacity of the

A temperature-sensitive E. coli mutant

441

Fig. 1. Host cell reactivation of UV irradiated T1 phages at 32°C and 42°C. Cells in the exponential growth phase (titre about 2 x 108/ml) were centrifuged, washed, placed in saline, starved for 90 min at 25 °C, centrifuged, resuspended in nutrient broth, and preheated at 32 °C and 42 °C, respectively, for 10 min. T1 phage lysate was diluted in saline at a titre of ca. 2 x 106 PFV/ml and irradiated with UV light (UV low pressure discharge lamp LK1000/25, Messrs. H O P F E L , Leipzig) at a distance of 2.10 m (c/. Materials and Methods for other particulars) • = reactivation in wild type W3102 at 32 °C O = reactivation in wild type W3102 at 42 °C • = reactivation in mutant cells WG24 at 32 °C A = reactivation in mutant cells WG24 at 42 °C WG 24 mutant is slightly restricted by raising the temperature to 42 °C only in the case of phages irradiated with high UV doses. The previously mentioned idea was therefore also investigated by using UV irradiated Ac phages.

Time Imml Fig. 2. Host cell reactivation of UV irradiated Ac phages in WG24 and wild type cells, respectively, at 32 °C and 42 °C. The experimental method described under Fig. 1 was used (c/. Materials and Methods for further particulars) + = reactivation in wild type W3102 at 32 °C o = reactivation in wild type W3102 at 42 °C • = reactivation in mutant cells WG24 at 32 °C A = reactivation in mutant cells WG24 at 42 °C

442

I R E N E MORFIADAKIS a n d E . G E I S S L E R

Raising the incubation temperature from 32 °C to 42 °C very slightly reduces the number of surviving phages at low UV doses if UV irradiated Ac phages are plated onto wild type cells. The inactivation curves are identical for longer irradiation times (Fig. 2). Compared with this, the mutant at 42 °C exhibited a higher sensitivity, whereas the inactivation is UV irradiated Ac phages at 32 °C corresponds to that of wild type strains. R e p a i r of p h o t o d y n a m i c l e s i o n s As results concerning the response of E. coli and Proteus mirabilis repair mutant h a v e s h o w n (BÖHME 1 9 6 8 , BÖHME a n d GEISSLEE 1 9 6 8 , HABM 1 9 6 8 , JACOB 1 9 7 3 ) , rec

gene functions are involved in this process whereas uvr gene functions are not. We therefore checked whether our mutantes temperature sensitive with respect to repair of photodynamic damage. The results show that the sensitivity of the mutant WG24 is clearly temperaturedependent (Fig. 3). Whereas 2 . 3 % of the mutant cells survived exposure even for 24 min due to treatment and incubation at 32 °C, the survival rate decreased to 2.1 X 1 0 - 4 % when the process took place at a restrictive temperature. Under identical conditions, the wild type cells exhibited almost identical inactivation at 32 °C and

Time ¡min) Fig. 3. Photodynamic inactivation of mutant and wild type cells at 32 °C and 42 °C. Bacteria in the exponential growth phase were placed in saline with thiopyronin without dilution (final concentration: 7 fig/ml), treated for 20 min in the dark at room temperature, and irradiated in a thin layer at 32 °C and 42 °C (cf. Materials and Methods). 2 X 0.1 ml bacteria suspension were then taken from each sample, each ml was placed in preheated saline (32 °C and 42 °C, respectively) further diluted, plated onto preheated (32 °C and 42 °C, respectively) plates, and incubated overnight at these temperatures. The first number is the temperature during exposure whereas the second is the temperature during dilution in saline and final incubation. Wild type (W3102): o 32 °C 32 °C, • 42 °C 42 °C Mutant (WG24): A 32 °C 32 °C, • 32 °C 42 °C, • 42 °C 42 °C, • 42 °C 32 °C

443

A temperature-sensitive E. cpli mutant

42 °C except for 1 - 2 % . Temperature changes from 32° to 42 °C and from 42 °C to 32 °C, respectively, following irradiation have no effect on cell survival, only the temperature during irradiation being decisive. Transduction efficiency Since the results obtained hitherto made it appear very likely that a rec function is involved in the case of the mutant WG24, general transduction was considered unsuitable for a further study of the WG24 strain. Transduction as well as other recombinational processes are strongly affected by defectiveness of genes also involved in D N A repair following U V irradiation (CLARK 1965, E I S E N S T A R K 1969, H O R I I and C L A R K 1973), Resistance to T6 phages was used as a feature to be transduced. I t was first necessary to investigate the spontaneous mutation rate at 32 °C and 42 °C. As shown in Table 2, the spontaneous mutation frequency (T6 Sp. Mut.) in the WG24 cells at 32 °C is about 1 X 10" 6 , whereas a rather higher mutation rate of about 3 X 10~ 6 is found in the wild type at the same temperature. The spontaneous mutation rates of both strains are reduced by 30—40% at 42 °C (Table 3). Table 2 Spontaneous T6 resistant mutants (T6 r —Sp. Mut., b) and T6 resistant transduetants (T6 r -transd., a-b) in the mutant and wild type strains under the effects of different temperatures (32 °C and 42 °C). The spontaneous mutation was tested from the initial titre at permissive and restrictive temperature, respectively. The values shown (a, b) are absolute: number of transductant+ spontaneously resistant colonies (T6 r , a) and spontaneously resistant colonies (T6 r -Sp.mut., b) related to the titre of the plated cells (c/. results) (means from 7 experiments) Strain

Temp.

T6r

(a)

T6r-Sp.Mut. (b)

T6 r -Transd. (a-b)

WG24

32 °C 4 2 °C

3.55 x 10"6 0 . 6 7 X IO" 6

0.99 x 10"6 0 . 6 5 X IO" 6

2 . 5 6 X IO" 6 0 : 0 2 x 10-«

W3102

3 2 °C 4 2 °C

6.23 x 10"6 4 . 1 3 X IO" 6

3 . 0 9 X IO" 6 1.99 X 1 0 " 6

3.13 X 10"6 2 . 1 4 X IO" 6

Table 3 Percentage reduction in spontaneous mutation rate of the mutant and wild type strains, respectively, when the temperature is changed from 32 °C to 42 °C Strain

Frequency of spontaneous T6 r mutants at 32 °C at 42 °C

Difference Absolute

WG24 (Mutant)

0.99 X 10-«

0.65 x 10~

W3102 (Wild type)

3.09 X IO- 6

1.99 X IO"6

6

0.34 x 10"

/o 6

34.3

1.10 X 10-«

35.6

In order to compare the transduction values (T6-Transd.) in the wild type and mutant strains, the spontaneous mutation rates (T6 r Sp.Mut.) must be subtracted in each case from the number of T6 resistant colonies (T6 r ) obtained experimentally (Table 2, cols. 1, b). Table 1 (cols, a, b) show that the number of transduetants is slightly reduced in the wild type (W3102) when the temperature is raised from 32 °C to 42 °C, whereas the same temperature change reduces the transduction frequency in the mutant WG24 to such an extent that the number of T6 resistant cells is of the same order as the number of spontaneous mutants.

444

IRENE MORFIADAKIS and E . GEISSLER

The transduction rates of the wild type and mutant strains at permissive temperature, and of the wild type strain at restrictive temperature agree with those stated in the literature, namely 1 X 10" s to 1 X 10~6 per phage particle. Discussion The very much increased sensitivity of the mutant cells at a restrictive temperature following photodynamic treatment is clearly due to the elevated temperature. A study involving isogenic E. coli strains which differed only in the rec allel showed that the repair of damage induced by photodynamic treatment or monofunctional alkylating agents is performed by functions controlled by the rec gene in both hcr+ and her~ cells (BÖHME and G E I S S L E R 1 9 6 8 ) . This is corroborated by the fact that rec strains are very sensitive to factors causing single strand breaks (eg. ionizing radiation), and such breaks might obviously also occur following photodynamic treatment. The results presented can, in agreement with the findings published by BÖHME and G E I S S L E E ( 1 9 6 8 ) , be interpreted by the presence of a temperature-dependent rec function. This would also agree with the different sensitivities to UV irradiation and treatment with mitomycin C as recombination enzymes participate in the post replication repair of damage which can otherwise be eliminated by excision repair(SMITH a n d M E U N 1 9 7 0 , R U P P et al.

1971).

This hypothesis was further tested with UV irradiated T1 phages and we found that the T1 phages of mutant cells can be reactivated to almost the same extent at both permissive and restrictive temperatures (Fig. 2). Since the uvr but not the rec functions are responsible for the reactivation of UV irradiated T1 phages in E. coli (GEISSLER 1968, HARM 1968, K N E S E R 1968), the uvr genes appear to be unaffected in the temperature sensitive WG24 mutant. Since it is generally known that UV irradiated A phages can be reactivated not only by excision repair but also by recombination repair, the next step was to check our conclusions by means of UV irradiated Ac phages. The survival of Ac phages in WG24 cells at 32 °C and 42 °C corresponds to the survival of Ac phages in hcr+ rec+ and hcr+ rec" host cells ( G E I S S L E R 1968). Proceeding from the fact that the inactivation of UV irradiated Ac phages in hcr+ rec cells ( G E I S S L E R 1968) is very similar to that following infection of the mutant cells at 42 °C and in view of the opinion that rec enzymes of the host also participate in the reactivation of UV irradiated Ac phages ( G E I S S L E R 1968), it can be assumed that in the case of the mutant WG24 the reduced survival of UV irradiated Ac phages is caused by a rec mutation. Since stable general transduction depends on the presence of an intact recombination system, this method can be used for indirectly ascertaining whether or not in the mutant W G 2 4 a rec function is involved (CLARK 1 9 6 7 , CLARK and MARGULIES 1 9 6 5 , EISENSTARK et al. 1 9 6 9 , HOWARD-FLANDERS and THERIOT 1 9 6 6 ) . At restrictive temperature we found almost no transduction of the mutant (ca. 0.02 X 10~6), whereas at a permissive temperature the transduction frequency of the mutant cells was almost identical to that of the wild type cells (mutant: 2.56 X 10 - 6 ; wild type: 3.13 X 10~ 6 ; cf. Table 2, col. b). Since it has already been proved that no stable general transduction takes place in recombination-defective mutants of E. coli (CLARK and MARGULIES 1 9 6 5 , HOWARDFLANDERS et al. 1 9 6 6 ) , these results support our belief that the temperature-sensitive mutation is due to some modification of the rec function of the cell. As a result of our characterization of this mutant it appears certain that the mutant possesses a temperature-sensitive repair defect and it is possible that this defect is

A temperature-sensitive E. coli m u t a n t

445

b a s e d o n a rec f u n c t i o n . W h i c h of t h e d i f f e r e n t rec l o c i (HOKII a n d CLAKK 1 9 7 3 ) is

involved in this defect is just as unknown as its relationship to the temperaturesensitive rec mutants isolated and characterized by H A L L and H O W A R D - F L A N D E R S in

1975 a n d FEEIFELDEB in

1976.

A cknowledgem We thank J .

HOFEMEISTER, S. SCHERKECK,

and

ents

M. THEILE

for critical discussions.

References BÖHME, H . ,

1968.

Absence of repair of p h o t o d y n a m i c a l l y induced d a m a g e in two m u t a n t s of w i t h increased sensitivity t o m o n o f u n c t i o n a l alkylating agents. M u t a t i o n

Proteus

mirabilis

Res., 6,

166—168.

BÖHME, H . a n d GEISSLER, E., 1968. R e p a i r of lesions induced b y p h o t o d y n a m i c action a n d b y e t h y l m e t h a n e s u l f o n a t e in E. coli. Molec. Gen. Genetics, 103, 228—232. CLARC, A. J . , 1967. The beginning of a genetic analysis of recombination proficiency. J . Cell P h y siol., 70 (Suppl. 1), 1 6 5 - 1 8 6 . CLAEC, A. J . a n d MABGULIES, A. D., 1965. Isolation a n d characterization of recombination-deficient m u t a n t s of Escherichia coli K 1 2 . Proc. N a t l . Acad. Sei. US, 5 3 , 4 5 1 — 4 5 9 . E C H O L S , H . , R E Z N I C H E K , J . a n d A D H Y A S , S., 1 9 6 3 . Complementation, recombination, a n d suppression in galactose negative m u t a n t s of E. coli. Proc. N a t l . Acad. Sei. US, 5 0 , 2 8 6 — 2 9 3 . E I S E N S T A B K , A . , E I S E N S T A R K , R . , VAN D I L L E W I J N , J . a n d R Ö R S C H , A . , 1 9 6 9 .

Radiation-sensitive

a n d recombination less m u t a n t s of Salmonella typhimurium. M u t a t i o n Res., 8 , 4 9 7 — 5 0 4 . F R E I F E L D E R , D . , 1 9 7 6 . New t y p e s of Escherichia coli recombination-deficient m u t a n t s . J . Bacteriol., 128,

681-682.

GEISSLER, E., 1968. R e a c t i v a t i o n of photodynamically i n a c t i v a t e d l a m b d a phages. Molec. Gen. Genetics, 103, 233—237.

a n d H O W A R D - F L A N D E R S , P . , 1 9 7 5 . Temperature-sensitive recA m u t a n t of Escherichia coli K-12 deoxyribonucleic acid metabolism a f t e r ultraviolet irradiation. J . Bacteriol., 121,

HALL, J . D .

892-900.

HARM, W., 1968. D a r k repair of acridine dye-sensitized photoeffects in E. coli cells a n d bacteriophage. Biochem. Biophys. Res. Commun., 32, 350—358. H O R I I , Z . I . a n d C L A R K , A. J . , 1973. Genetic analysis of t h e recA p a t h w a y t o genetic recombination in Escherichia coli K 1 2 : Isolation a n d characterization of m u t a n t s . J . molecular Biol., 80, 327-344. HOWARD-FLANDERS, P . , 1973. D N A repair a n d recombination. B r i t . Med. Bull., 29, 226—235. HOWARD-FLANDERS, P . , BOYCE, R . P . a n d THERIOT, L . , 1966. T h r e e loci in Escherichia

coli K 1 2

t h a t control t h e excision of pyrimidine dimers a n d certain other m u t a g e n p r o d u c t f r o m DNA. Genetics, 53, 1119—1136. HOWARD-FLANDERS, P . a n d THERIOT, L., 1966. M u t a n t s of Escherichia coli K-12 defective in D N A repair a n d in genetic recombination. Genetics, 63, 1137 —1150. JACOB, H . E . , 1973. Comparison of repair processes a f t e r UV- a n d p h o t o d y n a m i c action in Proteus mirabilis. studia biophysica, 36/37, 253—257. KNESER, H . , 1968. Relationship between K - r e a c t i v a t i o n a n d U V reactivation of bacteriophage. Virol., 36, 3 0 3 - 3 0 5 . M O R F I A D A K I S , I., G E I S S L E R , E . a n d T H E I L E , M . , 1973. On a temperature-sensitive m u t a n t of E. coli w i t h defective repair capacity, studia biophysica, 3 6 / 3 7 , 3 6 1 — 3 6 9 . R U P P , D . , W I L D E , C. E . , RENO, D . L . a n d HOWARD-FLANDERS, P . , 1971. E x c h a n g e s b e t w e e n D N A s t r a n d s in ultraviolet-irradiated Escherichia coli. J . molecular Biol., 6 1 , 2 5 — 4 4 . S M I T H , K . C. a n d M E U N , D . H . C., 1 9 7 0 . R e p a i r of radiation-induced d a m a g e in Escherichia coli.

I. E f f e c t of rec m u t a t i o n s on postreplication repair of d a m a g e due t o ultraviolet radiation. J . molecular Biol., 5 1 , 4 5 9 — 4 7 2 . Mailing address: D r . I . MORFIADAKIS I n s t i t u t f ü r Medizinische Mikrobiologie u n d Epidemiologie der Wilhelm-Pieck-Universität Rostock DDR-2500 Rostock, Leninallee 70

Zeitschrift für Allgemeine Mikrobiologie

21

6

1981

447-456

(Institut für medizinische und allgemeine Mikrobiologie, Virologie und Epidemiologie, Abteilung Biochemie der Viren und Mikroorganismen, Humboldt-Universität zu Berlin, D D R )

Influence of dimethylsulfoxide on transcription by bacteriophage T3-induced RNA polymerase H . M U S I E L S K I , W . MANN, R . L A U E a n d S . MICHEL

(Eingegangen

am 15. 9.

1980)

Dimethylsulfoxide (DMSO) 1 ) up to 2 5 % (v/v) does not cause irreversible alterations of T 3 D N A a t 4 2 . 5 °C as assayed by transcription with T3-specific R N A polymerase. The optimal temperature for the formation of polyanion-fesistant ternary complexes of the enzyme, T 3 DNA, and nascent R N A chains is lowered by 12.5 °C in the presence of 2 0 % (v/v) DMSO. The same solvent concentration, however, decreases the temperature optimal for T 3 R N A chain elongation by only 2.5 °C, indicating t h a t DMSO preferably affects the initiation of T 3 R N A synthesis. DMSO accelerates the loss of T3-specific R N A polymerase activity a t 4 2 . 5 °C. Nevertheless, the speed with which the binary complexes between the phage R N A polymerase and DNA are inactivated by heat (42.5 °C) is not altered in presence of 2 0 % (v/v) DMSO. The binding of T3-induced R N A polymerase to T 3 D N A in polyanion-resistant ternary complexes is influenced by DMSO which makes the enzyme accessible to the inhibitory action of polyvinyl sulfate. Elongation of T 3 R N A chains is slowed down b y 2 0 % (v/v) DMSO.

Dimethylsulfoxide (DMSO) has been shown to stimulate transcription of various DNAs by Escherichia coli RNA polymerase. This effect of the solvent is due to its action on promoter regions enhancing the formation of rifampicin-resistant binary complexes between DNA and the enzyme ( T B A V E E S 1 9 7 4 , NAKANISHI et al. 1 9 7 4 ) . In this process individual promoters are affected to different degrees (NAKANISHI et al. 1 9 7 4 ) . In a previous communication from this laboratory we reported that DMSO inhibits transcription by T3-specific RNA polymerase and that within certain solvent concentrations (below 16% (v/v)) this inhibitory action could be overcome by increasing the ionic strength (MIXSIELSKI et al. 1 9 7 9 ) . In these experiments the overall T 3 RNA synthesis was determined without differentiating between steps of T3 RNA chain initiation and T3 RNA chain elongation. Initiation of T3 RNA synthesis by T3-specific RNA polymerase requires highly specific conditions. The enzyme only recognizes efficiently initiation sites on T3 DNA (DUNN et al. 1 9 7 1 , MAITEA 1 9 7 1 , BAUTZ 1 9 7 3 ) . These regions contain some common nucleotide sequences since all RNA species synthesized by this enzyme in vitro start with pppGp(Gp)m(Ap)n (m, n 1) (MCALLISTER et al. 1 9 7 3 , MAITEA et al. 1 9 7 4 , M U S I E L S K I et al. 1 9 7 7 ) . Furthermore 7 out of 8 transcripts formed in vitro are produced in almost equal amounts independent of the ratio of DNA to T3-specific RNA polymerase, indicating that these promoter regions possess practically the same affinities for the enzyme (CHAKRABOETY et al. 1 9 7 7 ) . These findings suggest that the initiation sites have identical or closely similar nucleotide sequences. This has recently been shown for several promoter regions recognized by the closely related bacteriophage T7-induced RNA polymerase (ROSA 1 9 7 9 , O A K L E Y et al. 1 9 7 9 ) . The peculiarities of in vitro transcription by the phage-coded RNA polymerase made it desirable to investigate the influence of DMSO on the formation of ternary *) Abbreviations used: Dimethylsulfoxide DMSO, polyvinyl sulfate PVS, nucleoside triphosphate N T P 29

Z. Allg. Mikrobiol., B d . 21, H . 6

448

H . MUSIELSKI, W . MANN, R . L A U E a n d S . MICHEL

complexes between T 3 D N A , T3-specific R N A polymerase, and nascent R N A chains (initiation steps) and the process of T 3 R N A chain elongation. As will be shown below, initiation of T 3 R N A synthesis is considerably more susceptible to the solvent t h a n the elongation of already initiated R N A chains. Moreover, the strength with which the T3-induced R N A polymerase is bound to the template in t e r n a r y complexes is altered by DMSO. Materials

and

methods

T3-induced RNA polymerase was prepared according to CHARKABORTY et al. (1973) and the activity expressed in units defined by the same authors. Buffer A consisted of: 50 mmol/1 TrisHC1 (pH 7.8 at 23 °C), 20 mmol/1 MgCl2, 0.1 mmol/1 dithioerythrol, 0. 1 mmol/1 EDTA, and 0.5 mg/ml bovine serum albumin. RNA synthesis was assayed as reported previously (MUSIELSKI et al. 1977). All other reagents used are described in the preceding paper (MANN et al. 1979). DMSO concentrations are given in percent (v/v). Two kinds of experimental designs were mainly employed: Condition A was used to study the effect of DMSO on the formation of ternary complexes between T3 DNA, T3 RNA polymerase, and nascent RNA chains. Since, as it was shown earlier (MANN et al. 1979), this process mainly reflects steps involved in the initiation of T3 RNA synthesis these complexes will be called "initiation complexes". A typical protocol of an experiment of this type is presented below: 150 /A of buffer A in which 40 /tg of T3 DNA had been dissolved were incubated at the chosen temperatures for 2 min. 100 fi 1 of buffer A containing DMSO at the desired concentration were then introduced and incubations continued for 2 min. To form initiation complexes 50 /A of buffer A supplemented with 6.5 unit, of T3 RNA polymerase, ATP, GTP, and UTP, each to yield a final concentration of 0.4 mmol/1 were added. Thirty s later 50 [A of buffer A containing PVS resulting in a final concentration of 0.5 /ig/ml was pipetted into the reaction mixture to inactivate free and weakly bound RNA polymerase and to prevent reinitiation. After another 30 s aliquots of 100 /A were removed and diluted tenfold with ice-cold buffer A containing ATP, GTP, and UTP (each 0.4 mmol/1), PVS (0.5 ^g/ml), and DMSO to yield final concentrations of 2 or 3 % . These dilutions were warmed to 37 °C, and T3 RNA chain elongation started by the addition of 50 /A of buffer A supplemented with CTP and ( 3 H)UTP to give final concentrations of 0.4 mmol/1, 300 kBq/ml (740 kBq/nmol). Two min later an equal volume of 10% TCA was added and the acid-insoluble radioactivity determined (MUSIELSKI et al.

1977).

Condition B was used to investigate the effect of DMSO on initiation complexes and the elongation of T3 RNA chains. This kind of experiment was performed as follows: 100 ¡A of ice-cold buffer A containing 40 fig T3 DNA, 9 units of T3 RNA polymerase, ATP, GTP, and U T P — each NTP 0.4 mmol/1 — were warmed to 30 °C or 37 °C and kept at this temperature for 2 min to form initiation complexes. 100 fA of DMSO in buffer A yielding the desired concentration was added together with PVS (1 //g/ml) and allowed to act for 2 min. RNA chain elongation was then started by pipetting into the reaction mixture 50 [A of buffer A in which CTP, ( 3 H)UTP and PVS were dissolved to give the following final concentrations: CTP (0.4 mmol/1), ( 3 H)UTP (300 kBq/ml, 740 kBq/nmol), and PVS (0.5 ,ug/ml). Two min later the acid-insoluble radioactivity was assayed. Other experimental details are given in the legends to the figures. Results I n order to investigate the effect of DMSO on the formation of polyanion-resistant t e r n a r y complexes (initiation complexes), T 3 D N A was treated with the solvent and the complexes formed by the addition of T 3 R N A polymerase, A T P , G T P , U T P and P V S . Before starting R N A chain elongation the DMSO concentration was reduced to 2 or 3 % by dilution (Condition A). Since DMSO is known to denature D N A ( H E K S K O V I T S 1 9 6 2 ) and to inhibit transcription by T3-specific R N A polymerase ( M U S I E L S K I et al. 1 9 7 9 ) it was important for the design and interpretation of our experiments to know the concentrations of the solvent which do not cause irreversible alterations of the template properties of T 3 D N A . F o r this purpose T 3 D N A was incubated a t 4 2 . 5 °C, the highest temperature employed, for 5 min in the presence of the DMSO levels indicated. The solvent concentrations

449

Bacteriophage T3-induced B N A polymerase

were then lowered to 3% by dilution and T3 RNA synthesis assayed. Results are presented in Fig. 1. Up to 25% DMSO no irreversible changes in the template function of T3 DNA could be observed. The increased incorporation rate is due to the stimulatory action of 3% DMSO. Full template activity is regained very rapidly, i. e., within 30 s after dilution. Next the effect of DMSO on the formation of initiation complexes (Condition A) and on elongation of initiated T3 RNA chains (Condition B) was investigated (Fig. 2).

20 OMSO'%)

Fig. 1

DMS0(%)

Fig. 2

Fig. 1. Reversibility of alterations of T3 D N A caused by DMSO at 42.5 °C. 250 of buffer A in which 45 fig T3 DNA had been dissolved were incubated at 42.5 °C for 3 min. Hereafter 150 fil of buffer A containing sufficient DMSO to yield the concentrations shown was added and incubation continued for 5 min. Aliquots of 100 fd were then removed and diluted tenfold with buffer A supplemented with DMSO to give a final solvent concentration of 3%. R N A synthesis was started by introducing 50 /.