Zeitschrift für Allgemeine Mikrobiologie: Band 21, Heft 10 1981 [Reprint 2021 ed.] 9783112580745, 9783112580738

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ZEITSCHRIFT FÜR ALLGEMEINE MIKRO-BIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS HEFT 10 • 1981 BAND 21

AKADEMIE-VERLAG • BERLIN ISSN 0044-2208

EYP 20,— M

INHALTSVERZEICHNIS HEFT

10

Allgemeine Regulation der Aminosäurebiosynthese in Ilansenula henricii

R . B O D E UND D . B I R N B A U M

705

Septenbildung und Hemmung der RNA-Synthese bei einer Mutante von Salmonella typhimurium

E . HERRERO, R . GUERRERO, H . W O L F - W A T Z UND S . N O R M A R K

715

Untersuchungen zur Frage einer Laugung von Erzen mit heterotrophen Mikroorgamismen — Entwicklung eines Screening-Verfalirens

D . KLAGES, I . M E Y E R , W . SCHWARTZ UND R . N Ä V E K E

729

Torulopsis ethanolitolerans n. sp. nolitolerans var. minor n. var.

etha-

J O H A N N A R YBÄBOVÄ, F . S T R O S UND ANNA K0CK0VÄ-KRAT0CHVIL0VÄ

739

Wirkung von Äthanol auf das Temperaturprofil von Saccharomyces cerevisiae

N . VAN U D E N UND H . D A C R U Z D U ARTE

743

Leukaemomycin-geblockte Mutanten des myces griseus und ihre Pigmente

CHRISTINA WAGNER, CORNELIA STENGEL, INGE ERITT, G . SCHUM A N N UND W . F . F L E C K

751

Kurze

und T.

Strepto-

Originalmitteilung

Myxobakterien flächen

( M y x o b a c t e r a l e s ) auf

Blattober-

761

G. RÜCKERT

Buchbesprechungen

765

CONTENTS O F N U M B E R 10 General regulation of amino acid biosynthesis in Hansenula henricii

R . BODE a n d D . BIRNBAUM

A mutant of Salmonella typhimurium with an abnormal septation pattern associated with an inhibition of RNA synthesis

E . HERRERO, R . GUERRERO, W O L F - W ATZ a n d S . N O R M A R K

Development of a screening method in relation to the leaching of metals with carbon heterotrophie microorganisms

D . KLAGES, I . M E Y E R , W . SCHWARTZ AND R . N Ä V E K E 729

Torulùpsis ethanolitolerans n. sp. nolitolerans var. minor n. var.

J O H A N N A R Y B Ä Ü O V Ä , F . S T R O S AND A N N A KOCKOVA-KRATOCHVILOVA 739

and T.

etha-

705 H. 715

Effects of ethanol on the temperature profile of Saccharomyces cerevisiae

N . VAN U D E N ARTE

Leukaemomycin-blocked mutants of griseus and their pigments

CHRISTINA WAGNER, CORNELIA STENGEL, INGE ERITT, G . SCHUMANN AND W . F . F L E C K 751

Short

Streptomyces

743

Note

Myxobacteria ( M y x o b a c t e r a l e s ) on leaf surfaces Book

AND H . DA C R U Z D U -

Reviews

G. RÜCKERT

761 765

ISSN 0044-2208

ZEITSCHRIFT FÜR ALLGEMEINE MIKRO- BIOLOGIE

HERAUSGEGEBEN VON

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

unter der Chefredaktion von AN INTERNATIONAL JOURNAL ON

W. Schwartz, Braunschweig

MORPHOLOGY, PHYSIOLOGY, GENETICS,

und

AND ECOLOGY OF MICROORGANISMS

U. Taubeneck, Jena UNTER M I T A R B E I T 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 10 1981

BAND 21

REDAKTION

U. May, Jena

AKADEMIE-VERLAG BERLIN

Die Zeitschrift für Allgemeine Mikrobiologie soll dazu beitragen, Forschung und internationale Zusammenarbeit auf dem Gebiet der Mikrobiologie zu fördern. Es werden Manuskripte aus allen Gebieten der allgemeinen Mikrobiologie veröffentlicht. Arbeiten über Themen aus der medizinischen, landwirtschaftlichen, technischen Mikrobiologie u n d 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 l 1 ^ 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, a n eine Buchhandlung oder an den Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3 - 4 — im sozialistischen Ausland an eine Buchhandlung für fremdsprachige Literatur oder an den zuständigen Postzeitungsvertrieb — in der BRD und Berlin (West) an eine Buchhandlung oder an die Auslieferungsstelle KUNST U N D WISSEN, Erich Bieber, OHG, D-7000 Stuttgart 1, Wilhelmstraße 4 - 6 — in den übrigen westeuropäischen Ländern an eine Buchhandlung oder an die Auslieferungsstelle KUNST UND WISSEN, 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 Republik, DDR-7010 Leipzig, Postfach 160, oder an den Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3—4. Zeitschrift für Allgemeine Mikrobiologie Herausgeber: I m Auftrag des Verlages v o n einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3 - 4; Fernruf 2 23 62 29 oder 2236221 Telex-Nr. 114420; Bank: Staatsbank der D D R , Berlin, Kto-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. U D O T A U B E N E C K , Prof. Dr. W I L H E L M S C H W A R T Z . Anschrift der Redaktion: Zentralinstitut für Mikrobiologie u n d experimentelle Therapie der Akademie der Wissenschaften, DDR-6900 Jena, Beutenbergstr. 11; Fernruf: Jena 885614; TelexNr. 058621. Veröffentlicht unter der Lizenznummer 1306 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckerei „Thomas Müntzer", DDR-5820 Bad Langensalza. Erscheinungsweise: Die Zeitschrift f ü r Allgemeine Mikrobiologie erscheint jährlich in einem Band mit 10 Heften. Bezugspreis je Band 250, —M zuzüglich Versandspesen (Preis f ü r die D D R 2 0 0 , - M). Preis je 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: Dezember 1981 Bestellnummer dieses Heftes 1070/21/10 © 1981 by Akademie-Verlag Berlin. Printed in the German Democratic Republic AN (EDV) 75218

Zeitschrift für Allgemeine Mikrobiologie

21

10

1981

705-713

(Sektion Biologie der Ernst-Moritz-Arndt-Universität Greifswald)

Allgemeine Regulation der Aminosäurebiosynthese in Hansenula henricii R . BODE und D . BIRNBAUM

(Eingegangen

am 13. 2. 1981)

The general control of amino acid biosynthesis was investigated in Hansenula henricii. B v limitation for single amino acids in wild type strain and mutants no derepression of enzymes was caused. In prototrophic revertants however, obtained from auxotrophic mutants (his, pdx) enzyme activities were 2 — 17 times higher than in the wild type. In these revertants enzymes of the biosynthetic pathways for tryptophan, phenylalanine, tyrosine, arginine, histidine, cysteine, methionine, asparagine, threonine, and isoleucine were derepressed. The amino acid pool pattern of the revertants is completely different from that in the wild type strain. A discussion of possible mechanisms of general control of amino acid biosynthesis in H. henricii is presented.

Eine entscheidende Rolle im Stoffwechsel der Organismen spielt die rasche und wirksame Regulation der Genexpression, die auf unterschiedlichen Ebenen erfolgen kann. Von C A E S I O T I S VI. L A C Y ( 1 9 6 5 ) und C A R S I O T I S et al. ( 1 9 7 0 ) wurde bei Neurospora crassa ein Regulationssystem entdeckt, das „cross pathway regulation" bzw. „Allgemeine Kontrolle der Aminosäure-Biosynthese" genannt wurde und sich in einer Dereprimierung von verschiedenen Aminosäure-Biosyntheseenzymen unter Aminosäure-Mangelbedingungen äußert. Ähnliche Ergebnisse konnten auch bei Saccharomyces cerevisiae (SCHÜRCH et al. 1 9 7 4 , N I E D E R B E R G E R 1 9 7 7 ) , Euglena gracilis ( L A R A u. M I L L S 1 9 7 3 ) und Aspergillus nidulans ( P I O T R O W S K A 1 9 8 0 ) gefunden werden. Die Möglichkeit von gemeinsamen Kontrollelementen der Aminosäure-Synthese wird aber auch bei Prokaryonten diskutiert ( N E S T E R et al. 1 9 7 4 , B R O W N et al. 1 9 7 7 , 1 9 7 8 ) . Von uns konnten prototrophe Revertanten von his- und jwfx-Mutanten der Hefe Hansenula henricii isoliert werden, die größere Mengen an Anthranilsäure und Tryptophan ausschieden ( B O D E et al. 1 9 8 0 ) . Bei Untersuchungen der Tryptophan-Biosyntheseenzyme hatten wir feststellen können, daß der Level zweier Enzyme gegenüber der Wildtyp-Aktivität sehr stark angehoben war. Diese Ergebnisse deuteten auf das Vorhandensein einer allgemeinen Regulation der Aminosäure-Biosynthese in dieser Hefe hin. Um dieses Phänomen gründlicher zu untersuchen und auch im Hinblick auf andere Biosynthesewege zu überprüfen, wurden die vorliegenden Untersuchungen durchgeführt. Material

und

Methoden

Stämme: Für die Untersuchungen wurden folgende Stämme der Hefe Hansenula henricii verwendet (BODE et al. 1980): Wildstamm CCY 38— 10—2: Hg 4 6 — aus dem Wildstamm gewonnene auxotrophe Äis-Mutante; Hg 48 — aus dem Wildstamm gewonnene auxotrophe prf.r-Mut.ant;;; Hg 46-1 — aus Hg 46 hervorgegangene prototrophe Revertante; Hg 48-2 — aus Hg 48 hervorgegangene protrophe Revertante. Kulturbedingungen: Die Anzucht der Hefen erfolgte als Durchlüftungskultur bis zum Erreichen des Beginns der stationären Phase bei 30 °C. Als Nährlösung wurde das Minimalmedium nach T A N A K A et al. (1967) mit 1 % Glucose und 2 mg Biotin/1 verwendet. Für die Anzucht von Hg 46 und Hg 48 wurde mit 100 mg/1 Histidin bzw. 10 mg/1 Pyridoxin supplementiert. 45*

Zeitschrift für Allgemeine Mikrobiologie

21

10

1981

705-713

(Sektion Biologie der Ernst-Moritz-Arndt-Universität Greifswald)

Allgemeine Regulation der Aminosäurebiosynthese in Hansenula henricii R . BODE und D . BIRNBAUM

(Eingegangen

am 13. 2. 1981)

The general control of amino acid biosynthesis was investigated in Hansenula henricii. B v limitation for single amino acids in wild type strain and mutants no derepression of enzymes was caused. In prototrophic revertants however, obtained from auxotrophic mutants (his, pdx) enzyme activities were 2 — 17 times higher than in the wild type. In these revertants enzymes of the biosynthetic pathways for tryptophan, phenylalanine, tyrosine, arginine, histidine, cysteine, methionine, asparagine, threonine, and isoleucine were derepressed. The amino acid pool pattern of the revertants is completely different from that in the wild type strain. A discussion of possible mechanisms of general control of amino acid biosynthesis in H. henricii is presented.

Eine entscheidende Rolle im Stoffwechsel der Organismen spielt die rasche und wirksame Regulation der Genexpression, die auf unterschiedlichen Ebenen erfolgen kann. Von C A E S I O T I S VI. L A C Y ( 1 9 6 5 ) und C A R S I O T I S et al. ( 1 9 7 0 ) wurde bei Neurospora crassa ein Regulationssystem entdeckt, das „cross pathway regulation" bzw. „Allgemeine Kontrolle der Aminosäure-Biosynthese" genannt wurde und sich in einer Dereprimierung von verschiedenen Aminosäure-Biosyntheseenzymen unter Aminosäure-Mangelbedingungen äußert. Ähnliche Ergebnisse konnten auch bei Saccharomyces cerevisiae (SCHÜRCH et al. 1 9 7 4 , N I E D E R B E R G E R 1 9 7 7 ) , Euglena gracilis ( L A R A u. M I L L S 1 9 7 3 ) und Aspergillus nidulans ( P I O T R O W S K A 1 9 8 0 ) gefunden werden. Die Möglichkeit von gemeinsamen Kontrollelementen der Aminosäure-Synthese wird aber auch bei Prokaryonten diskutiert ( N E S T E R et al. 1 9 7 4 , B R O W N et al. 1 9 7 7 , 1 9 7 8 ) . Von uns konnten prototrophe Revertanten von his- und jwfx-Mutanten der Hefe Hansenula henricii isoliert werden, die größere Mengen an Anthranilsäure und Tryptophan ausschieden ( B O D E et al. 1 9 8 0 ) . Bei Untersuchungen der Tryptophan-Biosyntheseenzyme hatten wir feststellen können, daß der Level zweier Enzyme gegenüber der Wildtyp-Aktivität sehr stark angehoben war. Diese Ergebnisse deuteten auf das Vorhandensein einer allgemeinen Regulation der Aminosäure-Biosynthese in dieser Hefe hin. Um dieses Phänomen gründlicher zu untersuchen und auch im Hinblick auf andere Biosynthesewege zu überprüfen, wurden die vorliegenden Untersuchungen durchgeführt. Material

und

Methoden

Stämme: Für die Untersuchungen wurden folgende Stämme der Hefe Hansenula henricii verwendet (BODE et al. 1980): Wildstamm CCY 38— 10—2: Hg 4 6 — aus dem Wildstamm gewonnene auxotrophe Äis-Mutante; Hg 48 — aus dem Wildstamm gewonnene auxotrophe prf.r-Mut.ant;;; Hg 46-1 — aus Hg 46 hervorgegangene prototrophe Revertante; Hg 48-2 — aus Hg 48 hervorgegangene protrophe Revertante. Kulturbedingungen: Die Anzucht der Hefen erfolgte als Durchlüftungskultur bis zum Erreichen des Beginns der stationären Phase bei 30 °C. Als Nährlösung wurde das Minimalmedium nach T A N A K A et al. (1967) mit 1 % Glucose und 2 mg Biotin/1 verwendet. Für die Anzucht von Hg 46 und Hg 48 wurde mit 100 mg/1 Histidin bzw. 10 mg/1 Pyridoxin supplementiert. 45*

706

R . BODE u n d D . BIRNBAUM

Gewinnung des E n z y m e x t r a k t e s : Der Aufschluß der Hefezellen, die nach der K u l t i v i e r u n g abzentrifugiert u n d gewaschen wurden, erfolgte m i t Hilfe einer X-Presse in 0,1 M Tris-HCl (pH 7,5). Das Homogenat wurde bei 20000 g 20 min abzentrifugiert u n d der Überstand mittels Sephadex G—25 von niedermolekularen Bestandteilen befreit. Die dabei verwendeten Gelsäulen wurden äquilibriert u n d die E n z y m e eluiert mit dem gleichen Puffern, die auch f ü r die Bestimmung der einzelnen E n z y m a k t i v i t ä t e n benutzt wurden. Tabelle 1 Bestimmungsmethoden der untersuchten E n z y m e Enzym 3-Desoxy-D-arabino-heptulonsäure-7-phosp h a t (DAHP)-Synthase Dehydrochinat-Synthase Dehydrochinase Shikimat-DHG 3-Enolpyruvyl-Shikimat-5-Phosphat (EPSP)-Synthase Chinat-DHG Anthranilat-Synthase Anthranilat-Phosphoribosyl(PR)-Transferase Phosphoribosyl-Anthranilat(PRA)-Isomerase Indolglycerin-Phosphat(IGP)-Synthase Tryptophan-Synthase Tryptophan-Aminotransferase Chorismat-Mutase Phenylalanin-Aminotransferase Tyrosin-Aminotransferase Prephenat-Aminotransferase P r e p h e n a t -Dehydratase Arogenat-DHG Histidinol-DHG Phosphoribosyl-ATP-Synthase y-Cystathionase Sulfit-Reduktase Cystein-Synthase ß - Cystathionase N-Acetylglutamat-Kinase Acetylornithin-Aminotransferase Ornithin-Acetyltransferase Ornithin-Aminotransferase Ornithin-Carbamyltransferase Argininosuccinase Arginase Homoserin-DHG Formamidase a-Aminoadipat-Reduktase Asparagin-Synthetase Glutamin-Synthetase Glutamat-DHG Threonin-Deaminase Leucin-Aminotransferase Isoleu ein-Aminotransferase Valin-Aminotransferase Alanin-DHG alkalische Phosphatase Glutamat-Oxalacetat-Aminotransferase Glucose-ß-Phosphat-Isomerase Glucose-6-Phosphat-DHG Isocitrat-DHG Fumarase

EC-Nummer

Autor

4.1.2.15

BODE et al. (1980)

4.2.1.10 1.1.1.25

B O D E U. B I R N B A U M B O D E U. B I R N B A U M B O D E U. B I R N B A U M

(1981) (1981) (1981)

B O D E U. B I R N B A U M B O D E U. B I R N B A U M

(1981) (1981)

4.1.3.27 2.4.2.18 4.1.1.48 4.2.1.20 2.6.1.27 5.4.99.5 2.6.1.5

1.1.1.23 2.4.2.e 4.2.99.9 1.8.1.2 4.2.1.22 2.7.2.8 2.6.1.11 2.3.1.35 2.6.1.13 2.1.3.3 4.3.2.1 3.5.3.1. 1.1.1.3 3.5.1.9 1.2.1.31 6.3.1.1 6.3.1.2 1.4.1.2 4.2.1.16 2.6.1.6 2.6.1.e 1.4.1.1 3.1.3.1 2.6.1.1 5.3.1.9 1.1.1.49 1.1.1.42 4.2.1.2

BODE et al. (1980) BODE et al. (1980) BODE et al. (1980) BODE et al. (1980) BODE et al. (1980) B O D E U. B I R N B A U M

(1978)

BODE et al. (1980) BODE BODE BODE BODE BODE

U. U. U. U. U.

B I R N U A U M (1978) B I R N B A U M (1978) B I R N B A U M (1978) BIRNBAUM (1979 a) BIRNBAUM ^ 9 7 9 a)

MARTIN etal. MARTIN etal.

(1971) (1971)

FLAVIN u . SLAUGTER ( 1 9 7 1 ) Y O S H I M O T O et al. ( 1 9 7 1 ) K R E D I C I I u. B E C K E R (1971)

GUGGENHEIM (1971) J A U N I A U X et al. J A U N I A U X et al.

(1978) (1978)

DENES (1970) J E N K I N S U. T S A I ( 1 9 7 0 )

SCHIMKE ( 1 9 7 0 a ) RATNER (1970) SCHIMKE ( 1 9 7 0 b ) DATTA U. GEST ( q 9 7 0 ) B O D E U. B I R N B A U M (1979b) CHATTOO U . S H E R M A N ( 1 9 7 9 )

RAVEL (1970)

R o w s et al. (1970) D O H E R T Y (1970) H A T F I E L D U. U M B A R G E R ( 1 9 7 1 ) J E N K I N S U. T A Y L O R ( 1 9 7 0 ) J E N K I N S U. T A Y L O R ( 1 9 7 0 ) J E N K I N S U. T A Y L O R ( 1 9 7 0 ) Y O S H I D A U. F R E E S E ( 1 9 7 0 ) S C I I U R R U. Y A G I L ( 1 9 7 1 ) B E R G M E Y E R (1970) B E R G M E Y E R (1970) J A U N I A U X et al. ( 1 9 7 8 ) B E R G M E Y E R (1970) B E R G M E Y E R (1970)

A m i n o s ä u r e b i o s y n t h e s e - R e g u l a t i o n in H.

707

henricii

E n z y m b e s t i m m u n g e n : Die B e s t i m m u n g der E n z y m a k t i v i t ä t e n erfolgte n a c h d e n Angaben in der Tabelle 1. P r o t e i n b e s t i m m u n g : Die P r o t e i n k o n z e n t r a t i o n der E n z y m e x t r a k t e w u r d e n a c h LOWRY et al. (1951) b e s t i m m t . R i n d e r s e r u m a l b u m i n diente als Eichprotein. B e s t i m m u n g des Aminosäurepools: Die K o n z e n t r a t i o n a n freien A m i n o s ä u r e n w u r d e n a c h DELFOBGE et al. (1975) m i t d e m MIKBOTECHNA A u t o m a t i s c h e n Aminosäure-Analysator b e s t i m m t (Dr. H . LUBS, I n s t . f. Med. Genetik der Univ. Greifswald, d a n k e n wir f ü r die A n f e r t i g u n g der Analysen). Die B e s t i m m u n g v o n T r y p t o p h a n erfolgte nach BODE et al. (1980).

Ergebnisse Von uns konnte schon in einer früheren Arbeit berichtet werden, daß die Aktivität der Anthranilat-Synthase und der PR-Transferase in den untersuchten Revertanten Hg 46-1 und Hg 48-2 gegenüber der des Wildstamm.es von H. henricii wesentlich erhöht ist (BODE et al. 1980). Die Untersuchung aller Enzyme der Aromatenbiosynthese mit Ausnahme der Chorismat-Synthase zeigt, daß neben den obengenannten Enzymen nur noch die Shikimat-Kinase eine erhöhte spezifische Aktivität bei den Revertanten aufweist (Tab. 2). Die Dereprimierungsfaktoren, die dabei erreicht werTabelle 2 A k t i v i t ä t der Biosyntheseenzyme aromatischer Aminosäuren von H. Enzym DAHP-Synthase Dehydrochinat-Synthase Dehydrochinase Shikimat-DHG Chinat-DHG Shikimat-Kinase EPSP-Synthase Chorismat-Mutase Prephenat- Dehydratase Phenylalanin-Aminotransferase Prephenat-Aminotransferase Tyrosin-Aminotransferase Arogenat-DHG Anthranilat-Synthase I A n t h r a n i l a t - S y n t h a s e I2II2 PR-Transferase PRA-Isomerase IGP-Synthase Tryptophan -Synthase 2

CCY 38-10-2 pkat/mg

H g 46 RA1)

H g 46-1 RA

110 18 45 950 300 80 15 450 250 2900 1500 2400 63 0,4 2,0 1,4 21 23 85

0,9 1,0 nd nd nd 1.1 nd 1,2 nd nd nd nd nd 1,1 0,8 0,9 0,9 0,9 1,0

1,0 1,1

1,0 1,1 0,9 3,5

1,0

1,4 1,1 1,2 1,1

1,0

1,2 6,5 5,2 7,0 0,9 0,9 0,8

henricii H g 48 RA

H g 48-2 RA

1,0

1,0 1,1

nd nd nd nd 0,8 nd 1,4 nd nd nd nd nd 0,9 1,1 1,1 1,1 1,1

1,0

1,0

0,9 0,9 4,0 1,1 1,1

1,0

1,1 1.1 1,1 1,1 7,5 5,0 5,7 0,9 0,9 1,0

) R A — relative A k t i v i t ä t , W i l d s t a m m R A = 1,0, n d — n i c h t b e s t i m m t

den, sind etwas geringer als die der Anthranilat-Synthase und PR-Transferase. Die Aktivität der anderen fünfzehn Biosyntheseenzyme der aromatischen Aminosäuren unterscheidet sich in den untersuchten Stämmen nur unwesentlich voneinander. Um weitere, in den Revertanten eingetretene Veränderungen zu erfassen, wurden Enzyme anderer Aminosäuresynthesen in die Untersuchungen einbezogen. Wie aus Tabelle 3 deutlich wird, ist die Dereprimierung einzelner Enzyme in den Revertanten nicht auf die Aromatenbiosynthese beschränkt, sondern tritt auch bei einer Reihe weiterer Enzyme auf. Das betrifft Enzyme aus den Biosynthesewegen der Aminosäuren Histidin, Cystein, Methionin, Arginin, Asparagin, Threonin und Isoleucin. Aus der Tabelle ist weiterhin zu entnehmen, daß auch hier nicht alle Enzyme eines Biosyn-

708

R . BODE u n d D . BIRNBAUM

Tabelle 3 Aktivität von Aminosäure-Biosyntheseenzymen von H. henricii Enzym Histidinol-DHG PR-ATP-Synthetase Sulfit-Reduktase y-Cystathionase Cystein-Synthase ß-Cystathionase N-Acetyl-glutamokinase Acetylornithin-Aminotransferase Ornithin-Acetyltransferase Ornithin-Carbamyltransferase Argininosuccinase Homoserin-DHG a-Aminoadipat-Reduktase Asparagin-Synthetase Glutamin-Synthetase Glutamat-DHG Glutamat-Oxalacetat-Aminotransf. Threonin-Deaminase Leucin-Aminotransferase Isoleucin-Aminotransferase Valin-Aminotransferase Alanin-DHG 1

CCY 38-10-2 pkat/mg

Hg 46 RA

Hg 46-1 RA

Hg 48 RA

Hg 48-2 RA

13 3 100 2 1 9 100 140 35 1100 190 13 210 45 160 440 200 300 3000 2800 1100 10

0,9 0,8 nd 1,1 nd 0,9 nd nd nd 1,0 nd nd nd 1,2 nd nd nd 1,1 1,1 nd nd nd

5,2 3,8 1,0 15 12 4,4 0,9 2,5 0,9 2,2 1,0 0,9 1,0 9,0 1,1 1,1 1,0 3,7 1,1 1,1 1,2 1,0

1,0 0,9 1,1 1,0 nd 1,0 nd 0,9 nd nd nd nd nd nd nd nd nd nd 1,0 nd nd nd

5,0 4,0 1,0 17 10 4,2 0,8 2,3 0,8 2,3 1,0 1,0 1,0 9,5 1,2 1,0 1,0 3,5 1,2 1,1 1,0 0,9

) R A — relative Aktivität, Wildstamm R A = 1,0, nd — nicht bestimmt

Tabelle 4 Aktivität von katabolen Enzymen des Aminosäure-Stoffwechsels, von Enzymen des Kohlenhydrat Stoffwechsels und der alk. Phosphatase von H. henricii Enzym Tryptophan-Aminotransferase Formamidase Ornithin-Aminotransferase Arginase Glucose-6-Phosphat-DHG Glucose-6-Phosphat-Isomerase Isocitrat-DHG Pumarase Alkalische Phosphatase 1

CCY 38-10-2 pkat/mg 2000 120 25 2100 10000 3000 220 1300 800

Hg 46 RA 0,8 0,9 1,0 nd 0,9 nd 1,1 nd nd

Hg 46-1 RA

Hg 48 RA

Hg 48-2 RA

1,1 0,6 1,1 1 1 1,0 0,9 0,9 1,1 1,1

1,0 0,9 0,9 nd nd nd 1,0 nd nd

1,0 0,5 1,1 1,0 1,2 1,0 0,9 1,1 1,0

) R A — relative Aktivität, Wildstamm R A = 1,0, nd = nicht bestimmt

theseweges dereprimiert in den Revertanten vorliegen. So haben von den fünf untersuchten Arginin-Enzymen lediglich zwei eine veränderte Aktivität. Die Dereprimierungsfaktoren für die einzelnen Enzyme sind aber nicht identisch, sondern unterscheiden sich stark voneinander. So wird für die Ornithin-Carbamyltransferase in Hg 46-1 ein Faktor von 2,2 beobachtet, während dieser für die y-Cystathionase des gleichen Stammes 15 beträgt. Dagegen ist die Erhöhung der spezifischen Aktivität der Enzyme der beiden Revertantenstämme, die von unterschiedlichen Mutanten abstammen, fast identisch. Die Dereprimierung scheint sich nur auf anabole Enzyme der Aminosäurebiosynthesen zu beschränken und keine katabolen Enzyme zu erfassen. In Tabelle 4 sind vier katabole Enzyme aufgeführt, die im Tryptophan- bzw. Argininstoffwechsel eine Rolle

709

Aminosäurebiosynthese-Regulation in H. henricii

spielen. Daneben sind in der Tabelle Ergebnisse der Untersuchungen an vier Enzymen des C-Stoffwechsels u n d an der alkalischen Phosphatase enthalten. Keines dieser E n z y m e weist eine wesentliche Veränderung der spezifischen A k t i v i t ä t auf. Die Dereprimierung einzelner Enzyme in II. henricii ist nur auf die verwendeten R e v e r t a n t e n begrenzt u n d t r i t t weder im Wildstamm noch in den M u t a n t e n auf. D u r c h Limitierung einzelner Aminosäuren mit Hilfe von Aminosäureanaloga konnte bei einigen untersuchten E n z y m e n des Wildstammes, die bei den R e v e r t a n t e n im dereprimierten Zustand vorliegen, keine Dereprimierung erreicht werden (Tab. 5). Eine Veränderung der E n z y m a k t i v i t ä t e n bei den S t ä m m e n H g 46 u n d H g 48 unter Histidin- bzw. Pyridoxin-Limitierung konnte ebenfalls nicht beobachtet werden (BODE et al. 1980). Aus Tabelle 5 k a n n auch e n t n o m m e n werden, daß Amitrol die Synthese der Anthranilat-Synthase und der Histidinol-DHG reprimiert. Das erstere E n z y m wird auch in Gegenwart von 5-Methyltryptophan, das zweite in Gegenwart von Thienylalanin in geringerer Menge gebildet. Die Dereprimierung verschiedener Enzyme der Biosynthese von Aminosäuren sollte sich, wenn sie generell eine E r h ö h u n g des Substratdurchflusses ermöglicht, in einem erhöhten Aminosäurepool in der Zelle niederschlagen. Tabelle 6 verdeutlicht unsere A n n a h m e . Bis auf die Aminosäuren Threonin, Ornithin u n d Lysin ist der Tabelle 5 Relative spezifische Aktivität von Aminosäure-Biosyntheseenzymen des Wildstammes von H. henricii nach unterschiedlicher Anzucht Anzuchtmedium Enzym

Anthranilat-Synthase Threonin-Deaminase Histidinol-DHG /2-Cystathionase Ornithin - Carb amyltrf.

K

0,5 m M Amtirol

1,0 1,0 1,0 1,0 1,0

0,2 1,0 0,2 1,0 1,0

!

i

0,25 m M 5-Methyl-Trp

0,25 m M Thienyl-Ala

0,5 1,2 0,8

1,0 1,2 0,4 1,0 1,0

1,0

0,8

Tabelle 6 Aminosäurepool von H. henricii (in UM) Aminosäure Asparaginsäure Glutaminsäure Asparagin/Glutamin Cystein Threonin Serin Glycin Alanin Valin Cystathionin Isoleucin Leu ein Tyrosin Phenylalanin Ornithin Lysin Histidin Arginin Tryptophan

CCY 38-10-2

Hg 46-1

15,2 33,7 7,8 1,5 3,1 3,5 1,3 19,8 3,1 0,0 1,5 2,7 2,5 1,2 4,7 18,3 2,6 12,6 0,2

36,1 98,5 24,2 1,8 0,9 11,8 6,0 44,2 18,4 1,4 4,8 3,8 4,2 2,0 2,3 16,8 11,3 39,1 1,4

Hg 48-2 35,2 116,4 28,6 2,0 0,7 12,7 5,0 48,3 17,5 1,8 4,4 3,3 4,4 2,1 2,5 15,5 10,8 36,9 1,2

710

R . BODE und D. BIRNBAUM

Zellpool in den untersuchten Revertanten Hg 46-1 und Hg 48-2 gegenüber dem. Wildstamm CCY 38-10-2 erhöht. Sowohl Threonin als auch Ornithin werden von Enzymen als Substrat benutzt, die in den Revertanten dereprimiert vorliegen, so daß die niedrigere Konzentration an diesen Aminosäuren verständlich wird. Cystathionin ist nur in den Revertanten nachzuweisen. Die Erhöhung der Menge einzelner Aminosäuren ist nicht gleich; so beträgt die Konzentration von Tyrosin im Stamm Hg 48-2 nur das l,7fache der des Wildstammes, während die Histidinkonzentration auf das 4,2fache erhöht wurde. Beim Vergleich der Revertanten untereinander fällt dagegen wie schon bei den Dereprimierungsfaktoren die annähernde Gleichheit der Zellpools der einzelnen Aminosäuren auf. Diskussion Das Vorkommen des Phänomens der allgemeinen Kontrolle der Aminosäurebiosynthese läßt sich in H. henricii mit den bisher bekannten Methoden nicht nachweisen. Aminosäure-Limitierung mit Hilfe der verwendeten Antimetabolite Amitrol, 5Methyltryptophan und Thienylalanin führt im Wildstamm dieser Hefe zu keiner Dereprimierung der untersuchten Enzyme. Von SCHÜRCH et al. ( 1 9 7 4 ) , W O L F N E R et al. ( 1 9 7 5 ) und D E L F O R G E et al. ( 1 9 7 5 ) konnten die beiden erst genannten Analoga bei Saccharomyces cerevisiae erfolgreich zur Erhöhung des Levels von Enzymen angewandt werden, die auch in unsere Untersuchungen an H. henricii einbezogen waren. In bestimmten Aminosäure-Mangelmutanten von 8. cerevisiae läßt sich unter Aminosäurelimitierung ebenfalls die allgemeine Kontrolle nachweisen; es werden Enzyme der Biosynthesewege von Tryptophan, Phenylalanin, Tyrosin, Histidin, Arginin, Lysin, Leucin, Valin, Isoleucin, Serin, Prolin und Methionin beeinflußt (NIEDERBERGER 1977). Von uns wurde schon in einer früheren Arbeit darauf hingewiesen, daß die untersuchten Mutanten Hg 46 und Hg 48 von H. henricii unter limitierenden Wachstumsbedingungen keinen erhöhten Level der Anthranilat-Synthase und PR-Transferase aufweisen (BODE et al. 1980). Wir nehmen deshalb an, daß sich das Regulationssystem der allgemeinen Kontrolle in H. henricii von dem in 8. cerevisiae oder aber in Neurospora crassa (CARSIOTIS et al. 1 9 7 4 ) und in Aspergillus nidulans (PIOTROWSKA 1 9 8 0 ) in bestimmter Weise unterscheidet. Daß dieses Regulationssystem auch in H. henricii existiert, konnte erst mit Hilfe von speziell selektierten prototrophen Revertanten des Stammes Hg 46 (his) und Hg 48 (pdx) nachgewiesen werden. Diese Revertanten exkretieren Tryptophan und Anthranilsäure in das Medium (30 bzw. 200 mg/1) (BODE et al. 1980). Da die PR-Transferase und zum Teil auch die Anthranilat-Synthase die begrenzenden Enzyme der Tryptophan synthese führte. Deshalb ist eine Ubereinstimmung derAktivitätserhöhung einiger Enzyme bei den so isolierten Stämmen unterschiedlicher Mutanten nicht außergewöhnlich. Der Mutantentyp (prototrophe, Anthranilsäure- u. Tryptophan-ausscheidende Revertanten von his- bzw. pdx-Mutanten), der mit unserer Methode bei H. henricii erzeugt worden ist, ist mit den cdr-Mutanten von 8. cerevisiae vergleichbar (NIEDERBERGER 1977). Beide besitzen für eine Reihe von Aminosäurebiosyntheseenzymen höhere spezifische Aktivitäten. Der Level einiger anderer untersuchter Enzyme blieb konstant im Vergleich mit den Wildtypstämmen. Bei einem Vergleich der Levelerhöhung von Enzymen speziell des Tryptophan-, Arginin- und Histidin-Biosyntheseweges zwischen 8. cerevisiae und H. henricii stellten wir fest, daß die untersuchten Histidinenzyme bei beiden Arten dereprimiert vorliegen. Dagegen ist die Argininosuccinase-Aktivität von H. henricii im Unterschied zu 8. cerevisiae nicht erhöht. Ähnlich sind die Verhältnisse im Tryptophan-Biosyntheseweg. Bei Ii. henricii unter-

A m i n o s ä u r e b i o s y n t h e s e - R e g u l a t i o n in H.

henricii

711

liegen nur die Anthranilat-Synthase und die PR-Transferase der allgemeinen Kontrolle, während in S. cerevisiae noch die IGP-Synthase und Tryptophan-Synthase hinzukommen. Auch die Enzymmengen der a-Aminoadipat-Reductase und der Ilv-Aminotransferase sind im Unterschied zu S. cerevisiae bei II. henricii nicht erhöht. Die Untersuchungen an einer Reihe anderer Biosyntheseenzyme von II. henricii zeigt, daß der allgemeine Regulationsmechanismus noch weitere Aminosäurebiosynthesen beeinflußt. Insgesamt konnten durch die Enzymuntersuchungen für die Biosynthesen der Aminosäuren Tryptophan, Phenylalanin, Tyrosin, Arginin, Iiistidin, Cvstein, Methionin, Asparagin, Threonin und Isoleucin das Vorhandensein diese Art der Regulation in H. henricii bestätigt werden. Offenbar wird aber auch der Zell-Pool an Valin, Alanin, Glycin, Serin, Asparaginsäure und Glutaminsäure in den Revertantenzellen erhöht, so daß angenommen werden kann, daß auch die Biosynthesewege dieser Aminosäuren durch die allgemeine Kontrolle beeinflußt werden. Es bleibt die Frage offen, inwieweit von diesem Regulationsmechanismus auch katabole Enzyme der Aminosäure-Biosynthese oder anderer Biosynthesewege betroffen werden können. Die Anzahl der untersuchten Enzyme reicht nicht aus, um hier eine schlüssige Aussage zu treffen. Es kann nur festgestellt werden, daß in II. henricii von neun untersuchten Enzymen keines der allgemeinen Kontrolle unterliegt. Dagegen berichteten DELEORGE et al. (1975) über eine Dereprimierung der Fumarase und der katabolischen Glutamat-DHG bei S. cerevisiae. Der Ort und der Mechanismus der allgemeinen Kontrolle sind bisher weder bei cerevisiae noch bei N. crassa bekannt. Es wird sowohl die Transkriptionsebene als auch die Translationsebene in Erwägung gezogen (DELFORGE et al. 1 9 7 5 , MESSENGUY 1979).

Unsere Schlußfolgerungen, die wir auf Grund unserer Ergebnisse an H. henricii ziehen, können ebenfalls nur hypothetischen Charakter tragen. Wichtig erscheint uns jedoch, daß 1. aus zwei unterschiedlichen Mutanten {Ms bzw. pdx) ähnliche prototrophe Revertanten hervorgegangen sind, die sich durch einen erhöhten Level einer Reihe von Aminosäure-Biosyntheseenzyme auszeichnen, 2. die Selektion dieser Revertanten unter gleichen Bedingungen, nämlich erhöhter Tryptophanproduktion, erfolgte, 3. nur aus etwa zwei Prozent der von uns untersuchten auxotrophen Mutanten dieser Revertantentyp entstand, 4. nur ein geringer Prozentsatz der Revertanten sowohl der his- als auch der prfx-Mutanteri (5 bzw. 15%) erhöhte Enzymlevel besaß und 5., daß sowohl im Wildstamm als auch in den beiden Mutanten Hg 46 und Hg 48 unter limitierenden Wachstumsbedingungen keine Dereprimierung der untersuchten Enzyme auftrat. Daraus leiten wir den Schluß ab, daß die Veränderung, die zur Prototrophie der Mutante führte, identisch ist mit der Ursache der Levelerhöhung einiger Enzyme. Dies ist erklärbar, wenn wir annehmen, daß die Mutation, die zur Histidin- bzw. Pyridoxin-Auxotrophie führte, nicht eine vollständige Inaktivierung des betroffenen Enzyms bewirkte (erste Untersuchungen weisen darauf hin!), sondern daß diese Enzyme noch zum Teil aktiv sein können. Die Aktivität reicht aber nicht aus, um die Zellen vermehrungsfähig zu halten. Die Mutation, die zur Prototrophie führte, könnte die Transkriptions- bzw. Translationsrate f ü r die defekten Enzyme erhöht haben. Durch den erhöhten Level wurde die Auxotrophie überwunden. Der gleiche Effektor könnte auch für die Steigerung der Expressionsrate bei einer weiteren Anzahl von Genen verantwortlich sein. Dies kann zum Beispiel durch Erhöhung der mRNA-Transkription durch Beeinflussung der RNA-Polymerase-BindungamPromotor geschehen (STEPHENS et al, 1975). Ein gleicher Effekt wäre durch Beeinflussung der post-transkriptionalen bzw. Translations-Kontrolle möglich (REVEL U. GBONER 1978, OCHOA U. D E HARO 1979). Inwieweit Effektormoleküle die Ausprägung der allgemeinen Kontrolle initiieren, bleibt weiteren Untersuchungen vorbehalten.

712

R . BODE u n d D . BIRNBAUM

Aus evolutionistischer Sicht ist von Interesse, daß verschiedene Gene für Aminosäurebiosyntheseenzyme wahrscheinlich identische Regulationsmöglichkeiten besitzen, die bei entsprechenden Bedingungen in der Zelle in Aktion treten können. Es scheint so, daß für H. henricii im Gegensatz zu S. cerevisiae oder N. crassa diese allgemeine Kontrolle im Wildstamm nicht mehr vorhanden ist, sondern erst durch entsprechende Mutationen wirksam wird. Literatur BERGMEYER, H . U., 1970. Methoden der enzymatischen Analyse. Akademie-Verlag Berlin. BODE, R. u n d BIRMBAUM, D., 1978. Die E n z y m e der Biosynthese aromatischer Aminosäuren bei Hansenula henricii-. Die Aminotransferasen. Biochem. Physiol. P f l a n z e n , 173, 44—50. BODE, R . u n d BIRNBAUM, D., 1979a. Die E n z y m e der Biosynthese aromatischer Aminosäuren bei Hansenula henricii: Nachweis u n d Charakterisierung der E n z y m e des Pretyrosin-Weges. Z. Allg. Mikrobiol., 19, 8 3 - 8 8 . BODE, R . u n d BIRNBAUM, D., 1979b. Die Formamidase von Hansenula henricii: Isolierung, Charakterisierung u n d Regulation multipler F o r m e n . Biochem. Physiol. Pflanzen, 174, 26 — 38. BODE, R . , BÖTTCHER, F . a n d BIRNBAUM, D . , 1980. I s o l a t i o n a n d c h a r a c t e r i z a t i o n of a n t h r a n i l a t e

excreting m u t a n t s of Hansenula henricii. Cell. Molec. Biol., 26, 615 — 620. BODE, R . u n d BIRNBAUM, D., 1981. Eigenschaften und Regulation der E n z y m e des P a t h w a y von Hansenula henricii. Biochem. Physiol. Pflanzen, 176, 331—343.

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LARA, J . C. a n d MILLS, S. E., 1973. Regulation of L - t r y p t o p h a n biosynthesis in Euglena gracilis. Abstr. Meet. Amer. Soc. Microbiol., Ì41 (P5). L O W R Y , 0 . H . , R O S E B R O U G H , N . J . , F A R R , A . L . and R A N D A L L , R . J . , 1 9 5 1 . P r o t e i n m e a s u r e m e n t w i t h t h e Folin phenol reagent. J . biol. Chemistry, 193, 265- 275. MARTIN, R . G., BEBBERICH, M . A . , AMES, B . N . , DAVIS, W . W . , GOLDBERGER, R . F . a n d

YOURNO,

J . D., 1971. E n z y m e s a n d i n t e r m e d i a t e s of histidine biosynthesis in Salmonella typhimurium. In: Meth. E n z y m o l . X V I I B , 3 — 4 4 (COLOWICK, S . P . a n d K A P L A N , N. 0 . , Editors). Academic Press New Y o r k . MESSENGUY, F., 1979. Concerted repression of t h e synthesis of t h e arginine b i o s y n t h e t i c enzymes b y aminoacids: a comparison between t h e regulatory mechanisms controlling aminoacid biosynthesis in b a c t e r i a a n d in yeast. Molec. Gen. Genet., 169, 85—95. NESTER, E . W . , DALE, B . , MONTOYA, A . a n d VOLD, B . , 1974. Cross p a t h w a y r e g u l a t i o n of t y r o s i n e

a n d histidine synthesis in Bacillus subtilis. Biochemical, genetic, a n d t r a n s f e r R N A studies. Biochim. biophysica Acta, 361, 59 — 72. NIEDERBERGER, P . , 1977. Die Allgemeine Kontrolle der Aminosäure-Biosynthese bei Saccharomyces cerevisiae. Dissertation E T H Z Nr. 5882. J u r i s D r u c k & Verlag Zürich. OCHOA, S. a n d DE HARO, C., 1979. Regulation of p r o t e i n synthesis in eukaryotes. A n n . R e v . Biochem., 48, 5 4 9 - 5 8 0 . PIOTROWSKA, M., 1980. Cross-pathway regulation of o r n i t h i n e c a r b a m o y l t r a n s f e r a s e synthesis in Aspergillus nidulans. J . gen. Microbiol., 116, 3 3 5 - 339. R A T N E R , S . , 1 9 7 0 . Argininosuccinase (steer liver). I n : Meth. E n z y m o l . X V I I A , 3 0 4 — 3 0 9 (COLOWICK;, S . P. a n d K A P L A N , N. 0 . , Editors). Academic Press New Y o r k . RAVEL, J . M., 1970. Asparagine s y n t h e t a s e (Lactobacillus arabinosus). I n : Meth. E n z y m o l . X V I I A , 7 2 2 — 7 2 6 (COLOWICK, S . P . , a n d K A P L A N , N. 0 . , Editors). Academic Press New Y o r k . R E V E L , M . and G R O N E R , Y . , 1 9 7 8 . Post-transcriptional a n d translational controls of gene expression in eukaryotes. A n n . R e v . Biochem., 47, 1079 — 1126. R O W E , W. B., R O N Z I O , R . A., W E L L N E R , V. P . a n d M E I S T E R , A., 1970. G l u t a m i n e s y n t h e t a s e (sheep brain). I n : Meth. E n z y m o l . X V I I A , 9 0 0 - 9 1 0 (COLOWICK, S. P . a n d KAPLAN, N. 0 . , Editors). Academic Press New Y o r k . SCHIMKE, R . T., 1970a. Ornithine c a r b a m y l t r a n s f e r a s e ( M y c o p l a s m a ) . I n : Meth. E n z y m o l . X V I I A , 295 — 297 (COLOWICK, S. P . a n d KAPLAN, N . O., E d i t o r s ) . Academic Press New Y o r k . SCHIMKE, R . T., 1970b. Arginase (rat liver). I n : M e t h . E n z y m o l . X V I I A , 3 1 3 - 3 1 7 (COLOWICK, S. P . and KAPLAN, N. 0 . , Editors). Academic P r e s s New Y o r k . S C H U R C H , A . , MIOZZARI, J . a n d H Ü T T E R , R , 1 9 7 4 . R e g u l a t i o n of t r y p t o p h a n biosynthesis in Saccharomyces cerevisiae : mode of action of 5 - m e t h y l - t r y p t o p h a n a n d 5 - m e t h y l - t r y p t o p h a n - s e n s i t i v e m u t a n t s . J . Bacteriol., 117, 1131 — 1140. SCHURR. A. a n d YAGIL, E., 1971. Regulation a n d characterization of acid a n d alkaline p h o s p h a t a s e in yeast. J . gen. Microbiol., 65, 2 9 1 - 3 0 3 . STEPHENS, J . C., ARTZ, S. W . a n d AMES, B . N . , 1975. G u a n o s i n e 5 ' - d i p h o s p h a t e

3'-diphosphate

(ppGpp) : positive effector for histidine operon t r a n s c r i p t i o n a n d general signal for a m i n o acid deficiency. P r o c . N a t l . Acad. Sci. USA, 72, 4 3 8 9 - 4 3 9 3 . T A N A K A , A . , O H I S H I , N. a n d F U K U I , S., 1967. Studies on t h e f o r m a t i o n of v i t a m i n s a n d t h e i r f u n c t i o n in h y d r o c a r b o n f e r m e n t a t i o n P r o d u c t i o n of v i t a m i n B 6 b y Candida albicans in h y d r o c a r b o n m e d i u m . J . F e r m e n t . Techn., 45, 617 — 623. W O L F N E R , M . , Y E P , D . , M E S S E N G U Y , G . a n d F I N K , G . R . , 1 9 7 5 . I n t e g r a t i o n of a m i n o acid biosynthesis i n t o t h e cell cycle of Saccharomyces cerevisiae. J . Mol. Biol., 96, 273 — 290. YOSHIDA, A. a n d FREESE, E., 1970. L-Alanin dehydrogenase (Bacillus subtilis). I n : Meth. E n z y m o l . X V I I A , 176 — 181 (COLOWICK, S. P . a n d KAPLAN, N. O., Editors). Academic Press N e w Y o r k . YOSHIMOTO, A., N A I K I , N. a n d SATO, R . , 1 9 7 1 . Sulfite r e d u c t a s e (bakers yeast). I n : M e t h . E n z y mol. X V I I B , 5 2 0 - 5 2 8 (COLOWICK, S. P . a n d K A P L A N , N . 0 . , Editors). Academic P r e s s New York. A n s c h r i f t : D r . R . BODE

E r n s t - M o r i t z - A r n d t - U n i v e r s i t ä t Greifswald Sektion Biologie, W B Molekularbiologie D D R - 2 2 0 0 Greifswald, J a h n s t r a ß e 15a

Zeitschrift für Allgemeine Mikrobiologie

21

10

1981

715-728

(Departemento de Microbiologia, Facultad de Ciencias, Universidad Autonoma, Bellaterra, Barcelona, Spain 1 ) and Department of Microbiology, University of Umea, S-901 87 Umea, Sweden 2 ))

A mutant of Salmonella typhimurium with an abnormal septation pattern associated with an inhibition of RNA synthesis E . HERRERO 1 ), R . GUERRERO 1 ), H . WOLF-WATZ 2 ) a n d S. NORMARK2)

(Eingegangen

am

21.11.1980)

A mutant strain of S. typhimurium that is disturbed in the regulation of cell division and macromolecular synthesis is described. The life cycle of the mutant can be divided into two discrete stages. When growing in rich medium at a low cell density, cell division is inhibited and the cells filament at the same time as the relative amount of RNAshows a continuous increase. However, a t a certain stage, RNA synthesis stops and the filaments start to septate resulting in chain-formation. These chains can thereafter segregate into individal cells of unit cell length. The accumulation of RNA is rather due to a regulatory defect in the synthesis of the stable RNA species than to an unusual stability of messenger RNA (mRNA)as the half life of mRNA was estimated to 2.3 minutes during the period of R N A accumulation. Latter inhibition of RNA synthesis affects only stable species of RNA. The ppGpp pools of the strain did not fluctuate during growth, showing t h a t inhibition of RNA synthesis is not correlated to changes in the level of ppGpp. Different treatments that reduce the level of transcription such as sublethal concentrations ofrifampicin, a shift-down or high concentrations of nalidixic acid, all induced cell division of filamentous cells, suggesting that there exists an intimate relationship between macromolecular synthesis and cell division. The behaviour of this mutant fits best with the proposed hypothesis t h a t the biomass to volume ratio is of importance in the regulation of cell division in bacteria.

Cell division is linked to several other physiological events in bacteria. In a not well characterized way it is coupled to DNA replication. Thus, thermosensitive dna

mutants of E. coli are blocked in division at the restrictive temperature (HIROTA

et al. 1 9 6 8 ) . Also the studies of mutants such as recA, lexA, tif, and zab show that cellular events seemingly so different as cell division, UV-mutagenesis, repair of DNA damages and prophage induction have common pathways (see the reviews of RADMAN 1 9 7 5 and WITKIN 1 9 7 6 ) . Furthermore, mutants of E. coli and 8. typhimurium are known to have alterations in the early pathways of septation (VAN DE PUTTE et al.

1 9 6 4 , CIESLA et al. 1 9 7 2 , RICARD a n d HIROTA 1 9 7 3 , ALLEN et al. 1 9 7 4 , SANTOS a n d D E ALMEIDA 1 9 7 5 , SPRATT 1 9 7 7 ) , while other types of mutants are altered in the structuration of septum material (WALKER et al. 1 9 7 5 , NORMARK et al. 1 9 7 6 ) or in the final steps of separation of already formed septa (NORMARK and WOLF-WATZ 1 9 7 4 ) .

One mutant is known (SPRATT 1977) in which the altered protein has been localized as a component of the cytoplasmic membrane that acts as a target for low concentrations of penicillin, a drug known to inhibit cell division.

Several studies have pointed out the relation between bacterial cell division and the kinetics of cell elongation. While total cell biomass grows exponentially during the cell cycle, the rate of cell elongation is apparently linear, with a sharp doubling in elongation rates at a discrete stage of the cell cycle (SARGENT 1 9 7 5 , DONACHIE et al. 1 9 7 6 , KOPPES et al. 1 9 7 8 ) . As a consequence of this, cell density fluctuates during the cell cycle (POOLE 1 9 7 7 ) , with a maximum just prior to the time when the rate of elongation doubles (ZARITSKY and PRITCHARD 1 9 7 3 , ROSENBERG et al. 1 9 7 8 ) . It has been postulated that this fluctuation of the cell density controls the kinetics of septum

Zeitschrift für Allgemeine Mikrobiologie

21

10

1981

715-728

(Departemento de Microbiologia, Facultad de Ciencias, Universidad Autonoma, Bellaterra, Barcelona, Spain 1 ) and Department of Microbiology, University of Umea, S-901 87 Umea, Sweden 2 ))

A mutant of Salmonella typhimurium with an abnormal septation pattern associated with an inhibition of RNA synthesis E . HERRERO 1 ), R . GUERRERO 1 ), H . WOLF-WATZ 2 ) a n d S. NORMARK2)

(Eingegangen

am

21.11.1980)

A mutant strain of S. typhimurium that is disturbed in the regulation of cell division and macromolecular synthesis is described. The life cycle of the mutant can be divided into two discrete stages. When growing in rich medium at a low cell density, cell division is inhibited and the cells filament at the same time as the relative amount of RNAshows a continuous increase. However, a t a certain stage, RNA synthesis stops and the filaments start to septate resulting in chain-formation. These chains can thereafter segregate into individal cells of unit cell length. The accumulation of RNA is rather due to a regulatory defect in the synthesis of the stable RNA species than to an unusual stability of messenger RNA (mRNA)as the half life of mRNA was estimated to 2.3 minutes during the period of R N A accumulation. Latter inhibition of RNA synthesis affects only stable species of RNA. The ppGpp pools of the strain did not fluctuate during growth, showing t h a t inhibition of RNA synthesis is not correlated to changes in the level of ppGpp. Different treatments that reduce the level of transcription such as sublethal concentrations ofrifampicin, a shift-down or high concentrations of nalidixic acid, all induced cell division of filamentous cells, suggesting that there exists an intimate relationship between macromolecular synthesis and cell division. The behaviour of this mutant fits best with the proposed hypothesis t h a t the biomass to volume ratio is of importance in the regulation of cell division in bacteria.

Cell division is linked to several other physiological events in bacteria. In a not well characterized way it is coupled to DNA replication. Thus, thermosensitive dna

mutants of E. coli are blocked in division at the restrictive temperature (HIROTA

et al. 1 9 6 8 ) . Also the studies of mutants such as recA, lexA, tif, and zab show that cellular events seemingly so different as cell division, UV-mutagenesis, repair of DNA damages and prophage induction have common pathways (see the reviews of RADMAN 1 9 7 5 and WITKIN 1 9 7 6 ) . Furthermore, mutants of E. coli and 8. typhimurium are known to have alterations in the early pathways of septation (VAN DE PUTTE et al.

1 9 6 4 , CIESLA et al. 1 9 7 2 , RICARD a n d HIROTA 1 9 7 3 , ALLEN et al. 1 9 7 4 , SANTOS a n d D E ALMEIDA 1 9 7 5 , SPRATT 1 9 7 7 ) , while other types of mutants are altered in the structuration of septum material (WALKER et al. 1 9 7 5 , NORMARK et al. 1 9 7 6 ) or in the final steps of separation of already formed septa (NORMARK and WOLF-WATZ 1 9 7 4 ) .

One mutant is known (SPRATT 1977) in which the altered protein has been localized as a component of the cytoplasmic membrane that acts as a target for low concentrations of penicillin, a drug known to inhibit cell division.

Several studies have pointed out the relation between bacterial cell division and the kinetics of cell elongation. While total cell biomass grows exponentially during the cell cycle, the rate of cell elongation is apparently linear, with a sharp doubling in elongation rates at a discrete stage of the cell cycle (SARGENT 1 9 7 5 , DONACHIE et al. 1 9 7 6 , KOPPES et al. 1 9 7 8 ) . As a consequence of this, cell density fluctuates during the cell cycle (POOLE 1 9 7 7 ) , with a maximum just prior to the time when the rate of elongation doubles (ZARITSKY and PRITCHARD 1 9 7 3 , ROSENBERG et al. 1 9 7 8 ) . It has been postulated that this fluctuation of the cell density controls the kinetics of septum

716

E . HERRERO, R . GUERRERO, H . WOLF-WATZ a n d S. NORMARK

formation. W i t h this in mind, it can be expected t h a t mutants altered in R N A or protein accumulation would be unbalanced in the ratio of surface area t o biomass, resulting in alterations in cell division. This paper describes a mutant of Salmonella typhimurium t h a t , at low population density, shows an abnormal high accumulation of R N A , which is associated with a blocked cell division. Treatments that affect R N A transcription supress the inhibition of division, suggesting an intimate relationship between transcription control and cell division. Materials

and

methods

Bacterial strains: Strains used in this work are indicated in Table 1, with source and relevant genotype. The parental strain was JL1205. From it, the mutant strain JL1302 was obtained by treatment with N-methyl-N'-nitro-N-nitrosoguanidine and selected as resistant to IJV irradiation at 313 nm after incorporation of BrUdr (INGRAHAM et al., in preparation). Table 1 Bacterial strains Strain Salmonella typhimurium

Origin or derivation

~F~pyrC cdd cod tpp udp thy ~E~pyrC cdd cod tpp udp thyO F ~thr F'lac+proA+B+/pyrC cdd cod tpp udp thy F'lac+proA+B+/pyrC cdd cod tpp udp thyW Tlac+proA+B+lthr

JL1205 JL1302 UA101 UA1013 UA1014

UA1015

Escherichia CSH28

Genotype

coli

~F'lac+proA+B+/A(lac pro) trp pyrF his strA thi

supF

J . L . INGRAHAMW M u t a g e n i z a t i o n of J L 1 2 0 5

Spontaneous from wild type LT2 UA1015 x

JL1205

UA1015 x

JL1302

CSH28 x UA101

Cold Spring Harbor Collection

W University of California, Davis (b) Plus a mutation affecting cell division, as described in the text Growth media: Bacteria were usually grown in L B medium supplemented with medium E (VOGEL and BONNER 1956), 0.2% of glucose and 20 [xg/ml of thymidine. As minimal medium, AB medium was used, supplemented with 0.2% glucose, thymidine (20 ng/ml) and uracil (10 (ig/ml). Viable counts were determined in minimal AB medium solidified with 1.5% agar (DIFCO). Dilutions were made in 0.9% NaCl. Temperature of growth was 37 °C. Absorbance of cultures was recorded at 550 nM when grown in L B medium and at 450 nm when grown in minimal AB medium, using a BECKMAN spectrophotometer (model DU-2). Shift-down experiments were performed by filtering a volume of cells using a SARTORIUS membrane filter (pore size 0.45 (im); cells were washed with suspension medium at 37 °C and quickly resuspended in the same volume of new medium preincubated at 37 °C. Materials: Ampicillin, nalidixic acid, rifampicin, isopropyltiogalactoside (IPTG), and ortonitrophenylgalactopiranoside (ONPG) were obtained from SIGMA Chemical Co., St. Louis, Mo., USA. Radioactive thymidine, uracil and ortophosphate were purchased from the Radiochemical Centre, Amersham, Bucks., England. Determination of cell number, cell size and septa per cell: Samples were taken from the cultures and cells were fixed in 0.1% formaldehyde at 0 °C. Total cell number was determined with a PETROFF-HAUSER counter. Both a filament and a chain was counted as a single cell. The absorbance (at 550 nm)/10 9 cells ratio was taken as a relative measure of the cell size for cells grown in L B medium. It is assumed that the morphology and density of cells does not alter their absorbance. Septa per cell were determined by counting the number of septa observed in at least 200 cells sing a ZEISS phase contrast microscope. A septum was considered only when a clear refractive constriction was observed within the cytoplasm.

8. typhimurium-mutant

with an abnormal septation pattern

717

Measurement of DNA and RNA accumulation: Cells were prelabelled for at least two generations in the case of the parental strain JL1205 and overnight in the case of the m u t a n t strain JL1302, in LB medium with 10 [xg/ml of cold thymidine and labelled with 3 H-thymidine (10 fxCi/ml, specific radioactivity 17 Ci/mmole) and 14C-uracil (1 [xCi/ml, specific radioactivity 59 mCi/mmole) at 37 °C. Cells were then resuspended in fresh labelled medium with the same composition and incubated at 37 °C. At appropriate times samples of 0.2 ml were taken, added to 3 ml of ice-cold 10% trichloroacetic acid (TCA) and kept overnight at 0 °C. Each sample was filtered on a WHATMAN GF/C filter, washed with 25 ml of ice-cold 5% TCA and dried with 20 ml of acetone. Radioactivity was counted in a liquid scintillation counter Nuclear Chicago Mark I, using toluene-based scintillation liquid. Rate of RNA synthesis: From a culture of cells in LB medium with nonradioactive uracil (2 (xg/ml) at 37 °C, samples of 1 ml were taken at appropriate times and placed in preincubated tubes with 3 H-uracil (final radioactivity, 20 ¡xCi/ml). Incorporation was stopped after 2 min by adding 0.2 ml of the mixture to 3 ml of 10% TCA at 0 °C. Washing and counting of radioactivity was as described above. Synthesis of stable RNA: Basically the method of PATO and VONMEYENBTOG (1970) was followed. From a culture of bacteria in LB medium with uracil (2 ¡xg/ml) at 37 °C, samples were taken when indicated in each experiment (time 0) and added to preincubated flasks containing 3 H-uracil (20 (xCi/ml, specific activity 45 ¡xCi/[xmole), rifampicin (200 [xg/ml) and nalidixic acid (100 [xg/ml). The latter drug was added to prevent any residual incorporation of 3 H-uracil into DNA despite t h a t the strains used were Thy" and could not convert uracil into thymine nucleotides. Samples of 0.2 ml were taken at intervals during the next 20 min and acid-precipitable radioactivity was determined. In a parallel control experiment residual incorporation of 3 H-uracil into RNA was calculated during inhibitory conditions. Rifampicin and nalidixic acid were therefore added at time 0, 3 H-uracil 5 min later and samples were taken from this moment. Final level of radioactivity indicates the amount of RNA polymerase molecules engaged, a t time 0, in the synthesis of stable RNA. Induction of lac messenger RNA and measurement of its stability: In order to measure induction of lac mRNA cells were incubated in LB medium at 37 °C; samples of 1 ml were taken and added to preincubated tubes containing IPTG (final concentration 1 MM). Synthesis of lac mRNA was allowed during 8 min after which one drop of toluene was added. After strong agitation, tubes were incubated at 37 °C during two hours to allow evaporation of toluene. Finally /S-galactosidase activity was determined as described by PAKDEE et at. (1959). Induced activity was calculated by substracting from the total activity the enzyme activity present in 1 ml of culture without addition of inducer. To determine the half-life of induced lac mRNA, IPTG (1 MM) was added to 10 ml of cells growing in LB medium at 37 °C. Incubation was allowed during 3 min and all subsequent initiations of lac mRNA synthesis were inhibited by addition of rifampicin (200 |xg/ml) at time 0. Rifampicin was rapidly mixed by strong agitation during 10 seconds and samples of 1 ml were taken during the following 10 seconds. Cells were toluenized and, after evaporation of toluene, /9-galactosidase activity was determined. Conjugation: Transfer of an F'/oc+ proA+ B+ plasmid from E. coli to 8. typhimurium strains was done by spreading about 108 cells from a culture of the F~ recipient strain (in the late exponential phase) on minimal medium plates with 0.2% lactose as the only source of carbon. About 106 cells of donor strain E. coli CSH 28 (F+ Lac+) were spotted on the lawn of recipient cells and plates were incubated at 37 °C during 2—3 days. Exconjugants were tested for their Lac+ phenotype by streaking them on lactose minimal medium in two successive rounds. Finally, one of them was chosen as a Lac+ derivative. Determination of GTP and ppGpp pools: Cells were grown a t 37°C in MOPS medium (NEIDHARDT etal. 1974) supplemented with adenine, guanine, cytosine, and uracil (10 (xg/ml each); thymidine (20 (xg/ml); thiamine, biotin and pantothenic acid (all at 2 |xg/ml); K H 2 P 0 4 0.3 MM and 32 P-ortophosphate (40 (xCi/ml). To determine the pool size of GTP and ppGpp at different times during growth, samples of 100 |xl were taken, added to 100 (xl of formic acid (2 M) and left a t 0 °C for 30 min. After sedimenting bacterial debris, 100 ¡xl of the extracts were applied on PEI-cellulose plates and developed in NaH 2 P0 4 1.5 M at pH 3.4. Location of nucleotides was determined by autoradiography with X-ray films; spots were cut out and radioactivity was determined with toluene-based scintillation fluid. Results

G r o w t h p a t t e r n of s t r a i n J L 1 3 0 2 To obtain mutants altered in the incorporation of BrUdr into DNA, the mutant strain JL1302 was obtained from JL1205 as a derivative resistant to radiation at 313 nm

718

E . H E R R E R O , R . GTJERRERO, H . W O L F - W A T Z a n d S. N O R M A R K

after BrUdr treatment (INGRAHAM et al., in preparation). During exponential growth in glucose minimal medium at 37 °C JL1302 has a doubling time of 110 min, compared to 50 min for the parental strain. Strain JL1302 formed colonies of normal size on solid glucose minimal medium while on L B agar only small colonies grew after 48 hours of incubation at 37 °C. The growth pattern in liquid L B medium was therefore studied. To follow the morphological evolution of cells in a standardized fashion, cells from an overnight culture were diluted (approximately 1/80) into fresh medium at 37 °C after which several parameters were followed; absorbance (as an indicator of total cell volume), total number of cells, viable cells, septa per cell and cell size (AS50/109 cells).

Time

(ftfs)

Fig. 1. Growth pattern of mutant strain JL1302 in LB medium at 37 °C. (a) Absorbance at 550 nm (o), total number of cells per ml (A) and viable number of cells per ml (A), (b) Number of septa per cell ( • ) and cell size ( • ) (calculated from the ratio A550/109 cells) The growth pattern of strain JL1302 is depicted in Fig. 1. Two periods can be distinguished. Initially, absorbance of the culture increases exponentially with a doubling time of 80 min, while total cell number increases very slowly. Thus, filamentation of cells occurs, with a low frequency of septa per cell (about 0.1). A t this stage almost 80% of cells are viable. A t a certain stage the filaments initiate division resulting in a marked increase in the rate of cell formation. Two aspects must be considered, however: (1) The rate of formation of septa is higher than the rate of separation and, as a consequence, the average number of septa per cell increases to approximately 0.5 (cells with multiple septa will be named chains). (2) A fraction of segregating cells are not viable; therefore, the proportion of viable cells in the culture decreases continuously. This is one of the reasons why doubling time of the whole biomass in the culture decreases continuously.

8. typhimurium,-mutant

with an abnormal septation pattern

719

While strain JL1302 shows this complex pattern in rich medium, parental strain JL1205 has a normal exponential growth in L B medium with a constant index A550/109 cells of 0.30 and a doubling time of 34 min (data not shown). I t can be concluded that strain JL1302 is a mutant altered in the process of division when growing in rich media at low population densities.

Time (hrsl Pig. 2. Effect of dilution of chain-forming cells of mutant strain JL1302. Cells were grown in L B medium at 37 °C and, at A 550 of about 0.7, they were diluted into fresh medium preincubated at the same temperature. The arrow indicates the time of dilution, (a) Absorbance ( • , o ) and total number of cells per ml (A, A). (b) Cell size (A, A), (c) Septa per cell ( • , • ) . Close symbols and continuous lines correspond to the untreated control culture; open symbols and dashed lines correspond to the diluted culture

The decrease in growth rate at high densities of the culture is not due to the exhaustion of some components of the medium as dilution of septated cells in the second stage of growth into fresh medium did not restore the initial growth pattern. In fact, biomass increased at a slow rate whereas cells divided at a high rate (Fig- 2). Thus, the stage at which filaments start to divide is not reversible. I t can be noted from the figure, that the number of septa per cell in the rediluted culture did not increase further. This means that, at low densities of the culture, separation of septa is as rapid as the initiation of division, which is in contrast to high cell densities. S y n t h e s i s of m a c r o m o l e c u l e s in r i c h m e d i u m A dependence of growth on the richness of the medium has been observed in mutants altered in the accumulation of macromolecules (Normark and W o l f - W a t z 46

Z. Allg. Mikrobiol., Bd. 21, H . 10

720

E. HERRERO, R. GUERRERO, H. WOLF-WATZ and S. NORMARK

1 9 7 4 , NORMABK et al.

1 9 7 6 , ISAKSSON a n d TAKATA 1 9 7 8 , TAKATA a n d ISAKSSON 1 9 7 8 ) .

I t was therefore of interest to follow t h e synthesis of D N A and R N A in t h e m u t a n t when growing in L B medium. F i g . 3(a) demonstrates t h e accumulation of ^ - t h y m i dine and 1 4 C-uracil into D N A and R N A , respectively. During t h e filamentous stage of growth both D N A and R N A accumulates a t a higher rate t h a n t h e increase of absorbance. At t h e transition point, there is a complete inhibition of further accumulation of R N A , without D N A being affected. Thus, two situations exist in t h e m u t a n t with respect to R N A synthesis which parallel t h e morphological abnormalities. At low densities, r a t e of R N A synthesis accelerates and t h e cells filament, whereas at high densities R N A accumulation stops and filaments begin t o divide.

Fig. 3. RNA and DNA synthesis of mutant strain JL1302. Cells were grown at 37 °C in L B medium supplemented with cold thymidine (10 (xg/ml), 3H-thymidine (10 (j.Ci/ml, 17 Ci/mmole) and 14Curacil (1 (xCi/ml, 59 mCi/mmole). At different times samples were taken out and radioactivity incorporated into DNA and RNA was measured, (a) Untreated culture, (b) Culture treated with ampicillin (1 (j.g/ml), added at the time indicated by arrow. Symbols: absorbance (•), total number of cells per ml (O), cpm incorporated into DNA (A) and cpm incorporated into RNA (A) T h e coincidence between morphological and macromolecular abnormalities points t o a relation between division and R N A synthesis in t h e m u t a n t . T o see if inhibition of R N A synthesis is dependent on t h e initiation of division, D N A and R N A accumulation were followed when septum formation was inhibited b y low concentrations of ampicillin (Fig. 3(b)). During these conditions ampicillin completely inhibited division of t h e filaments b u t did n o t alter t h e increase in biomass of the t r e a t e d culture. D N A synthesis was n o t affected but R N A accumulation stopped almost completely a t t h e transition point of growth rate, as in t h e u n t r e a t e d culture. A small increase in R N A accumulation was observed ( 2 4 % ) , b u t it was small compared to t h e increase of t o t a l cell volume ( 3 0 0 % ) . I t must therefore be concluded t h a t inhibition of R N A synthesis a t t h e critical point is n o t caused b y t h e induction of division. Accumulation of R N A can be due t o : either i) an abnormal stability of messenger R N A ; or ii) a too high level of R N A synthesis. T o discriminate between these two

8. typhimurium-mut&nt

with an abnormal septation pattern

721

possibilities half life of lac m R N A was determined in strain UA1014 (a F ' L a c + derivative of m u t a n t JL1302 obtained by transfer of a F'lac* pro A + B + episome from E. coli CSH28 to S. typhimurium UA101 and from this to JL1302). Calculated from t h e deceleration t o synthesize /3-galactosidase in t h e presence of inhibitory concentrations of rifampicin, t h e lac m R N A half life was found to be 2.3 min (Fig. 4). This figure is similar to t h e half life of most messenger molecules in E. coli ( P E D E R S E N et al. 1978). When t h e rate of R N A synthesis was measured b y giving pulses of 3 Huracil for only 2 min (Fig. 5) it was found t h a t t h e amount of R N A synthesized per unit of absorbance increased during the filamenting stage of growth up till maximum value was reached which occurred a t the transition point. At this point, t h e rate of R N A synthesis decreased abruptly to a value of only 10% of t h e maximum.

5 10 75 0 3 6 9 Time (min) Time (min) Pig. 4

2

4 6 Time (hrs) Fig. 5

Pig. 4. F u n c t i o n a l half life of lac messenger R N A in s t r a i n UA1014, a Lac+ d e r i v a t i v e of m u t a n t J L 1 3 0 2 . Cells were grown in L B m e d i u m a t 37 °C; w h e n t h e a b s o r b a n c e of t h e c u l t u r e w a s a b o u t 0.3 (filamenting stage) a n a l i q u o t w a s t a k e n o u t a n d lac m R N A synthesis was induced w i t h I P T G (1 mM) d u r i n g 3 m i n . A t t i m e 0 r i f a m p i c i n (200 (j.g/ml) w a s a d d e d t o t h e induced c u l t u r e , samples were t a k e n o u t a n d t h e a c t i v i t y of /S-galactosidase w a s e s t i m a t e d , (a) A m o u n t of /3-galactosidase synthesized a t d i f f e r e n t times, (b) R e m a i n i n g c a p a c i t y t o synthesize /3-galactosidase Fig. 5. R a t e of i n c o r p o r a t i o n of 3 H - u r a c i l into m u t a n t s t r a i n J L 1 3 0 2 . Cells were grown a t 37 °C in L B m e d i u m s u p p l e m e n t e d w i t h cold uracil (2 (i.g/ml). A t d i f f e r e n t t i m e s s a m p l e s of 1 ml were t a k e n o u t a n d a pulse of 3 H - u r a c i l (20 [iCi/ml, 45 [j.Ci/(xmoIe) was given d u r i n g 2 min. R a d i o a c t i v i t y i n c o r p o r a t e d i n t o R N A , normalized t o t h e a b s o r b a n c e of t h e c u l t u r e , is shown. D a s h e d line corr e s p o n d s t o t h e increase in a b s o r b a n c e of t h e c u l t u r e

L e v e l of m e s s e n g e r a n d r i b o s o m a l R N A s y n t h e s i s a t d i f f e r e n t s t a g e s of growth rate When the m u t a n t is in the chain-forming stage absorbance increases b u t R N A accumulation stops. As t h e m R N A of the m u t a n t does not show an abnormal halflife, it seems clear t h a t m R N A synthesis must continue during this stage of growth to allow protein synthesis. Therefore, the low remaining level of R N A synthesis observed in Fig. 5, m a y be due to m R N A synthesis. In order to demonstrate t h a t m R N A was synthesized during this stage, we induced /3-galactosidase at different times during growth of the m u t a n t in LB medium and the rate of /3-galactosidase synthesis was scored for. Strains UA1013 and UA1014, Lac + derivatives of t h e parental and m u t a n t strains, respectively, were used (both behave as the original strains with respect to other phenotypic characters). At different times samples were taken out a n d the amount of /S-galactosidase synthesized 8 min after induction was estimated. Results are shown in Fig. 6. The capacity to synthesize t h e enzyme (normalized to t h e absor46*

722

E . H E R R E R O , R . GUERRERO, H . W O L F - W A T Z a n d S. NORMARK

bance of t h e culture) increased along time. However, t h e rate of synthesis during t h e filamenting stage was higher t h a n t h a t of the p a r e n t ; this result was in concordance with t h e observed abnormal accumulation of R N A . F u r t h e r m o r e , during t h e chainforming stage of t h e m u t a n t , /3-galactosidase was induced indicating t h a t lac m R N A was synthesized, even t h o u g h there was a very low level of R N A synthesis (Fig. 5). The result presented above suggested t h a t t h e observed inhibition of R N A synthesis affected r R N A a n d t R N A , t h a t is, the stable species of R N A . The a m o u n t of R N A polymerase engaged in t h e transcription of stable R N A chains in both stages of growth of the m u t a n t was measured (see Fig. 7). I t was shown t h a t during t h e chain-forming stage in sharp contrast t o t h e filamenting stage there was almost no synthesis of stable R N A . Fig. 7 does not show a typical "Q c u r v e " representing degradation of m R N A as has been

s h o w n b y o t h e r s (PATO a n d VON MEYENBURG 1970, PEDERSEN

1976). We did not observe a m a x i m u m followed b y a step down in TCA precipitable material of labelled uracil. Our strains were not hyperpermeable for rifampicin. Therefore, it is possible t h a t t h e drug did not m o m e n t a r y bind to its target molecule thereby creating a situation where we were not able t o detect any decay of m R N A .

A55Q Fig. 6

Time ( min} Fig. 7

Fig. 6. Induction of lac m R N A in strains UA1013 and UA1014, Lac+ derivatives of parental and mutant strains. Cells were grown in L B medium at 37 °C. At different times samples (1 ml) were taken out, lac R N A synthesis was induced with IPTG (1 mil) during 8 min and /S-galactosidase activity was calculated. Induced enzyme activity per unit of absorbance is shown with respect to the absorbance of the culture in parental ( • ) and mutant ( X ) strains Fig. 7. Level of stable rRNA synthesized during the filamenting and chain-forming stages of strain JL1302. At time 0 3 H-uracil (20 (j.Ci/ml), (45 jxCi/^mol) was added to a culture sample grown in LB medium with cold uracil (3 |ig/ml) at 37 °C. Simultaneously rifampicin (200 (J-g/ml) and nalidixic acid (100 ¡Jtg/ml) were added. Radioactivity incorporated into R N A was calculated. • : sample taken at the filamenting stage (A 550 of 0.35), sample taken at the chain-forming stage (A 550 of 0.80)

R e l a t i o n b e t w e e n R N A a c c u m u l a t i o n a n d d i v i s i o n in J L 1 3 0 2 As described above, in strain JL1302 acceleration of R N A synthesis and inhibition of cell division occur simultaneously and b o t h effects are t u r n e d off a t the same time. If t h e inhibition of division was due to an accumulation of R N A we argued t h a t t r e a t m e n t of strain JL1302 with sublethal concentration of rifampicin which partially inhibits R N A synthesis might also suppress inhibition of cell division. M u t a n t cells were therefore grown in L B medium containing 8 ¡J.g/ml of rifampicin (Fig. 8). During these conditions initial doubling time was 128 min, although it increased with time. Evolution of the number of cells parallelled the biomass of t h e culture; therefore, cell

S. typhimurium-mutant

with an abnormal septation pattern

723

size is almost constant during t h e population cycle, with an index A 5 3 0 /10 9 cells of about 0.3, a figure similar to t h e parental strain growing in L B medium. T h i s result demonstrates t h e suppressive effect of rifampicin on t h e inhibition of cell division. However, viable number of cells remained constant, indicating t h a t cells, although viable in t h e culture, died when plated. I t can be seen in F i g . 8, t h a t , a t high cell densities, there was an increase in t h e frequency of septa per cell t o a value of 0.35. As t h e increase in cell number was normal, this increase must reflect a decline in t h e rate of separation of formed septa.

Time ( hrs) Fig. 8. Growth pattern of mutant strain JL1302 in the presence of sublethal concentrations of rifampicin. Cells were grown in L B medium and supplemented with rifampicin (8 (xg/ml) at 37 °C. (a) Absorbance (•) and total number of cells per ml (A), (b) Number of septa per cell (o) and the cell size (A) T h e above results suggest t h a t R N A accumulation alters t h e process of division of t h e m u t a n t . Several other t r e a t m e n t s are known which affect t h e r a t e of R N A transcription such as shift-down to poorer media (MOLIN et al. 1977) or t r e a t m e n t of cells with high concentrations of nalidixic acid (SMITH et al. 1978). T h e effect of both t r e a t m e n t s on cells in t h e filamenting stage was studied (Fig. 9). Shift-down from L B t o glucose minimal medium induced division of t h e filaments after a lag of about 2 0 min. T h e increase in absorbance of t h e culture arrested after t h e shift-down a t least for t h e following 70 min, while t h e size of cells rapidly decreased. Nalidixic acid, besides inhibiting D N A replication, at high concentrations is known to block transcription b y inhibiting D N A gyrase a c t i v i t y , necessary to form transcriptional loops (SMITH et al. 1978). T r e a t m e n t of strain J L 1 3 0 2 in t h e filamenting stage with nalidixic acid (100 ¡I,g/ml) slightly induced division of cells; in f a c t , t h e rate of cell division during the following 4 0 min was higher t h a n in nontreated cells. After 80 min, cell division was inhibited because no chromosome replication was going on. Thus, partial inhibition of transcription induced division of filamenting cells of strain J L 1 3 0 2 .

724

E . HERRERO, R . GUERRERO, H . W O L F - W A T Z a n d S. NORMARK

S §

!

« 05 Qc

SO

-40

0 40 Time (min)

80

J„

Fig. 9. Effect of shift-down and nalidixic acid on filaments of strain JL1302 grown in L B medium at 37 °C. At time 0 a culture of JL1302 at A 55(l of 0.25 was divided and (a) 10 ml of cells were filtered, washed with 20 ml of minimal medium and resuspended in 10 ml of glucose minimal medium preincubated at 37 °C; (b) To 10 ml of the culture nalidixic acid (100 (¿g/ml) was added. Absorbance and total number of cells were followed. ( • , o ) : control culture not treated; (A, A ) : shift-down treated cells; ( • , • ) : nalidixic acid treated cells. Absorbance (closed symbols) and total number of cells per ml (open symbols) were taken as one unit at time 0

The coupling between R N A synthesis and cell division as found in the mutant could possibly be related to changes in the ppGpp pool. Therefore, intracellular levels of ppGpp were measured in the parental and mutant strains growing in MOPS medium supplemented with 32P. It seems clear that the ppGpp level did not differ significantly between both strains (Table 2). While the GTP level remained rather constant during different stages of growth, the concentration of ppGpp slightly increased in both strains at late stages but not enough to account for inhibition of R N A synthesis (during the stringent response ppGpp levels increase more than ten times over basal levels when R N A synthesis is inhibited). It seems therefore likely that inhibition of R N A synthesis and induction of cell division in strain JL1302 is not a result of a change in the ppGpp pool. Table 2 GTP and ppGpp pools in parental and mutant strains Strain JL1205 (parental) JL1302 (mutant)

Stage of growth Exponential Near stationary( a ) Filamenting Chain-forming Near stationaryW

GTP (pmole/A450) 359.4 381.3 222.0 213.4 295.1

ppGpp (pmole/A450) 6.6 15.9 5.8 11.8 19.3

ppGpp/GTP .018 .042 .026 .055 .065