Zeitschrift für Allgemeine Mikrobiologie: Band 22, Heft 3 1982 [Reprint 2021 ed.] 9783112580783, 9783112580776

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ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS HEFT 3 • 1982 • BAND 22

AKADEMIE-VERLAG • BERLIN EVP 20,—M

ISSN 0044-2206

84112

CONTENTS

OF

NUMBER

3

Anthracyclic products Irom Streptomyces erythrochromogenes nov. sp. Biotransformation of daunomycin ( D n ) by an acellular preparation and synergism between Dn and some known antibiotics Effects of methionine sulphoximine on protein transients during heterocyst differentiation in Anabaena and Cylindrospermutn spp. Fatty acid composition of lipids of Escherichia coli W 1655 F + and its stable protoplast type L-form

NADIA M . ABDALLAH, AND M . BARBIER

Application of dielectrophoresis for preparative separation of thermotolerant yeast cells Bistability in the glucose and energy metabolism of ammonia-limited chemostat cultures of Escherichia coli ML 30 Spore differentiation in relation to certain antibiotics in the blue-green alga Nodularia spumigena

M. DEVYS 155

I. S. GROVERANDS. PURI

161

J . GUMPERT, W . SCHADE, D . K R E B S , S . B A Y K O U S H E V A , E . IVANOVA AND A S . TOSHKOV

169

G . K R A U S E , W . SCHADE, R . GLASER AND B . GRÄGER

175

P . J . MÜLLER AND B E A T E FROMMANNSHAUSEN

185

R . K . PANDEY PASAYI

AND

VON

E . R . S. TAL191

MERTENS

Regulation by repression of urease biosynthesis in Proteus rettgeri Short Notes AS-1L, a new strain of the cyanophage A S - 1 found in GDR Regulation of bistability in glucose metabolism of Escherichia coli ML 30 chemostat cultures by cyclic AMP

C . ZORN, R . DIETRICH AND H . K A L T WASSER

V . A . GORJUSIN, E . STENZ AND A . A . AVERKIEV

205

P . J . MÜLLER AND W . R Ö M E R

211

Book Reviews

215

INHALTSVERZEICHNIS

HEFT 3

Anthracycline aus Streptomyces erythrochromogenes nov. sp. Biotransformation von Daunomycin (Dn) durch einen Enzymextrakt und Synergismus zwischen Dn und einigen bekannten Antibiotica Wirkung von Metliioninsulphoximin auf das Proteinmuster während derHeterocystendifferenziel'ung bei Anabaena und Cylindrospertnum spp. Fettsäurezusammensetzung der Lipide von Escherichia coli W 1(555 F + und dessen stabiler Protoplastentyp-L-Form Anwendung der Dielektrophorese für die präparative Trennung thermotoleranter Hefezellen Bistabilität im Glucose- und Energiestoffwechsel von Ammonium-limitierten Chemostatenkulturen von Escherichia coli ML 30 Wirkung verschiedener Antibiotica auf die Sporendifferenzierung bei der Blaualge Nodularia spumigena

197

NADIA M . ABDALLAH, UND M . BARBIER

M. DEVYS 155

161

I . S . GROVER UND S . P U R I

J . GUMPERT, W . S C H A D E , D . K R E B S , S . B A Y K O U S H E V A , E . IVANOVA UND A S . TOSHKOV 169 G . K R A U S E , . W . S C H A D E , R . GLASER UND B . GRÄGER

175

P . J . MÜLLER UND B E A T E FROMMANNSHAUSEN

185

R . K . PANDEY PASAYI

UND

VON

E . R . S. TAL191

MERTENS

Derepressive Ureasebildung bei Proteus

rettgeri

Kurze Originalmitteilungen AS-1L — ein neuer, in der D D R aufgefundener Stamm des Cyanophagen AS-1 Regulation der Bistabilität im Glucosestoffwechsel von Escherichia coli ML 30-Chemostatenkulturen durch cyklisches AMP Buchbesprechungen

C . ZORN, R . D I E T R I C H UND H . K A L T WASSER 197

V . A . GORJUSIN, E . STENZ UND A . A . AVERKIEV

205

P . J . MÜLLER UND W . RÖMER

211

215

ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN

INTERNATIONAL

JOURNAL

ON

MORPHOLOGY, PHYSIOLOGY, GENETICS, A N D ECOLOGY OF

MICROORGANISMS

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

F . E g a m i , Tokio G . F . Gause, Moskau 0 . Hoffmann-Ostenhof, W i e n A . A . Imseneckii, Moskau R . W . K a p l a n , Frankfurt/M. F . Mach, Greifswald 1. Mälek, P r a g C. Weibull, L u n d

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

UNTER MITARBEIT VON

J . H . Becking, Wageningen H . Böhme, Gatersleben M. Girbardt, Jena S. I. K u s n e c o v , Moskau O. Necas, Brno C. H . Oppenheimer, P o r t Aransas N . Pfennig, Göttingen I. L . R a b o t n o v a , Moskau A . Schwartz, W o l f e n b ü t t e l

HEFT 3

1982

BAND 2 2

REDAKTION

U . M a y , Jena

A K A D E M I E - V E R L A G BERLIN

Die Zeitschrift f ü r 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 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 u n d in gleicher F o r m auch nicht in anderen Zeitschriften erscheinen werden. Der U m f a n g soll höchstens 1 y 2 Druckbogen (24 Druckseiten) betragen. Bei umfangreicheren Manuskripten müssen besondere Vereinbarungen mit der Schriftleitung und dem Verlag getroffen werden. K u r z e Originalmitteilungen über wesentliche, neue Forschungsergebnisse. U m f a n g im allgemeinen höchstens 3 Druckseiten. Kurze Originalmitteilungen werden beschleunigt veröffentlicht. Kritische Sammelberichte u n d Buchbesprechungen nach Vereinbarung mit der Schriftleitung. Bezugsmöglichkeiten der Zeitschrift f ü r Allgemeine Mikrobiologie: Bestellungen sind zu richten — in der DDR an den Postzeitungsvertrieb, an eine Buchhandlung oder an den AkademieVerlag, DDR-1086 Berlin, Leipziger Str. 3—4 — im sozialistischen Ausland an eine Buchhandlung f ü r fremdsprachige Literatur oder an den zuständigen Postzeitungsvertrieb — in der BRD und Berlin (West) an eine Buchhandlung oder an die Auslieferungsstelle K U N S T U N D W I S S E N , Erich Bieber OHG, D-7000 S t u t t g a r t 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 G m b H , CH-8008 Zürich, Dufourstraße 51 — im übrigen Ausland an den Internationalen Buch- u n d 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 f ü r Allgemeine Mikrobiologie Herausgeber: I m A u f t r a g des Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag, DDR-1086 Berlin, Leipziger Straße 3 - 4 ; Fernruf 2 2 3 6 2 2 9 oder 2236221 • Telex-Nr.'114420; B a n k : Staatsbank der D D R , Berlin, Kto.-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. U D O T A U B E N B C K , Prof. Dr. W I L H E L M S C H W A E T Z . Anschrift der R e d a k t i o n : Zentralinstitut f ü r Mikrobiologie und experimentelle Therapie der Akademie der Wissenschaften, DDR-6900 J e n a , Beutenbergstr. 11; F e r n r u f : J e n a 8 8 5 6 1 4 ; TelexNr. 058621 Veröffentlicht u n t e r 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 B a r d m i t 10 Heften. Bezugspreis je B a n d 250,— M zuzüglich Versandspesen (Preis f ü r die D D R 200, — M) Preis je H e f t 2 5 , - M (Preis f ü r 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 reprcduzieit werden. — All rights reserved (including those of translations into foreign languages). No part of this issue m a y be reproduced in any form, b y photoprint, microfilms or any other means, without written permission from t h e publishers. Erscheinungstermin: Mai 1982 Bestellnummer dieses Heftes 1070/22/3 © 1982 by Akademie-Verlag Berlin, P r i n t e d in the German Democratic Republic. AN (EDV) 75218

Zeitschrift für Allg. Mikrobiologie

22

1982

155-160

(Institut de Chimie des Substances Naturelles, CNRS, 91190 Gif sur Yvette, France)

Anthracyclic products from Streptomyces erythromogenes nov. sp. B i o t r a n s f o r m a t i o n o f d a u n o m y c i n (Dn) b y a n acellular p r e p a r a t i o n and synergism between D n a n d s o m e k n o w n antibiotics NADIA M . ABDALLAH1), M . D E V Y S a n d M . B A R B I E R

(Eingegangen

am 28.

5.1981)

The filtrate broth as well as mycelium of the new strain Streptomyces erythrochromogenes nov. sp. isolated from Saudi Arabian soil, produce the antitumor antibiotic daunomycin 1 and two anthracyclic derivatives: 7-deoxy 13-dihydrodaunomycinone 2 and 7-deoxy daunomycinone 4. The biotransformation of 1 to 2 and 4 by an acellular enzyme preparation from the strain was found to be NADPH and/or NADH dependent. Mixtures of daunomycin 1 with chloramphenicol or penicillin showed superior antimicrobial activities against Bacillus subtilis ICC strain, than the individual antibiotics. I n t h e course of screening of a n t i m e t a b o l i t e s f r o m cultures of microorganisms, a new s t r a i n , Streptomyces erythrochromogenes nov. sp., was isolated b y A B D A L L A H in 1975 f r o m t h e soil of S a u d i A r a b i a . P r e l i m i n a r y studies on cultures indicated t h e production of a m i x t u r e of a n t i b i o tics a c t i v e against G r a m - p o s i t i v e a n d some G r a m - n e g a t i v e b a c t e r i a . T h e presence of a m i n o acids a n d iron in t h e biologically a c t i v e p r o d u c t s (ABDALLAH 1975) has n o t been confirmed. F u r t h e r studies indicated t h a t t h e microorganism produced a n t h r a cyclic compounds. W e wish t o describe here t h e structure of t h e m a i n a n t h r a c y c l i c m e t a b o l i t e s of t h e new strain 8. erythrochromogenes nov. sp., biotransformation of t h e a n t i t u m o r a n t i biotic d a u n o m y c i n (Dn) b y an acellular p r e p a r a t i o n of the strain a n d t h e e f f e c t of m i x i n g D n with other k n o w n antibiotics on some selected microbes.

Materials

and

methods

The strain was cultured at 28 °C for 6 days in a jar fermentor containing 14 1 of a medium composed of 2 % soluble starch, 2 % glucose, 0.25% soya bean, 0.3% NaN0 3 , 0 . 1 % K 2 H P 0 4 , 0.05% MgS0 4 • 7 H 2 0 , 0.05% KC1 and 0.0015% F e S 0 4 • 5 H 2 0 (pH 7.0). Fermentation broth was extracted with n-butanol, evaporated under reduced pressure and re-extracted with CHCl3-MeOH (9:1) at pH 8.6. Mycelium was sonicated in methanol-water (1:1), centrifuged and the supernatant was extracted twice with CHCl3-MeOH (9:1). The extracts from filtrate and mycelium were then evaporated under vacuum to dryness, dissolved in a little volume of CHC13 and precipitated by n-hexane. 25 mg of the precipitate was dissolved in 3 ml CHCl 3 -ethyl acetate (1:1), loaded on a column of sephadex LH-20 (2.1 x 80 cm) and eluted with the same solvent. Elution pattern of the preci') Permanent address: Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, E G Y P T . 11«

156

NADIA M . A B D A L L A H , M . D E V Y S a n d M . B A R B I E R

pitate is represented in Pig. 2. Only fraction E (sub. 2) or F (sub. 4) contains one product. Fractions E and P were further purified on other columns of sephadex LH-20 and eluted with CHC13MeOH (3:2). Substances 2 and 4 were crystallized from hot »-butanol (Fig. 1). The active fractions (A, B, C and D ; Fig. 2) were combined and concentrated in vacuo to dryness. The residue was purified with the following successive chromatographies: 1) Plate of Silica gel G 1510/LS 254 thickness 1 mm (SCHLEICHER and SCHULL), Solv. A : CHC13ethanol-glacial acetic acid-water ( 1 1 9 : 4 : 5 : 1 2 ) , where anthracyclic products of weak biological activities were migrated. 0

HO

0

Fig. 1. Daunomycin 1; 7-deoxy-13-dihydrodaunomycinone 2; 7-deoxy-daunomycinone 4; isolated from S. erythrochromogenes nov. sp,

Fraction

numder

Pig. 2. Elution pattern on sephadex LH 20 of the anthracyclic products isolated from S. erythrochromogenes nov. sp. Strong activity against B. subtilis, — weak activity 2) TLC (thick) of active products (Rf = 0.0 from plate 1, Sollv.B: CHCl3-MeOH-glacial acetic acid (80:20:4), where the main biologically active product 1 was separated (Rf = 0.4). 3) Column sephadex LH-20 to purify sub. 1, Solv. C: CHCl3-MeOH 4 : 1 . With acid hydrolysis (0.1 n HC1, 85 °C, 30 min), pure active 1 gave inactive aglycone (m/e 398) and a sugar (CI 148; M + 1). Physicochemical properties of the main anthracyclic metabolites produced by the new strain 8. erythrochromogenes nov. sp.; 1, 2, 4 as well as the triacetate 8 of sub. 2, are shown in Table 1. The triacetate 3 of sub. 2 has been prepared in acetic anhydride/pyridine, 20 h at 20 °C and crystallized from benzene.

Anthracyclic products from S. erythrochromogenes nov. sp.

00

CO W c «

J a s Ü

-o T) © PÍ

¿U O CO 02 o ©

< ft ft_ S ft 5 - S t. C ca O «00 10 os ON® - IO CO t© o o ^ OC ^rt 00 .£ -

0.9 1.0

I I 1

r /

B

Key to intensity • Major

Fig. 6. Electrophoretic protein banding pattern at various intervals in ammonia-enriched medium supplemented with MSO in Cylindrospermum (A) and Anabaena (B)

Effects of methionine sulphoximine on algae

167

The protein profiles observable at 0, 2, 4, 8, 12 and 24 h in the medium lacking ammonium chloride (BM), medium containing ammonium chloride (AM) and medium containing ammonium chloride + MSO (MSOAM) are represented graphically in the Figs. 4—6. I t is evident from these figures that a profound change has occurred in the electrophoretic protein profile following transfer of culture from AM to BM. Several protein transients not decipherable at the time of shift were noticed and several others delineated at the time of shift, were not deciphered. The zymograms given in Fig. 5 suggest that protein profiles did not differ appreciably on resuspending the cultures in freshly prepared AM. The changes in Anabaena and Cylindrospermum were identical except for variations in the number of total protein transients. The upward trend in total cellular protein content was also noticed during heterocyst development. However, no such trend was evident where the cultures were suspended in AM. The electrophoretic protein bands of cultures suspended in MSOAM revealed profound changes comparable to those noticed in BM. MSOAM pattern revealed at 2 h was characterized by the presence of four protein transients not noticed in AM. The disappearance of two bands was noticed in the 4 h pattern. The pattern of MSOAM cultures harvested at 8 h revealed the presence of a new protein transient and the disappearance of another. The pattern noticed at 12 and 24 h did not differ appreciably from that at 8 h.

Discussion The failure of the heterocystous blue-green algae to develop heterocysts in ammonia-enriched medium is attributed to repression of genes controlling heterocyst development. I f such ammonia-grown cultures are shifted to medium lacking any combined nitrogen, heterocyst differentiation probably occurs due to derepression of heterocyst-forming genes (FOGG et al. 1973). Our observation of induction of heterocysts by M S O in the presence of ammonia is in agreement with those of O W N B Y ( 1 9 7 7 ) and STEWART and R O W E L L ( 1 9 7 5 ) . STEWAKT and R O W E L L ( 1 9 7 5 ) proposed that glutamine synthetase is a repressor of genes in heterocyst development and inactivation of enzyme by MSO releases the genes from repression. The observation that heterocyst formation will occur in AM if prior to resuspending it was incubated in MSOAM for a minimum period of 2 h, suggests that as soon as the genes of heterocyst development are derepressed, the potential cell (cell capable of differentiation into heterocyst) enters an irreversible phase and continues its development even in non-inductive medium. I t is pertinent to mention the observation of B R A D L E Y and CARR ( 1 9 7 6 ) who noticed that if ammonia-grown cultures of Anabaena cylindrica were transferred to nitrogen-free medium, the addition of ammonium chloride did not inhibit heterocyst differentiation if added after an interval of 5 h or later. The electrophoretic protein profiles of cultures undergoing heterocyst differentiation in MSO-supplemented medium revealed that the process is characterized by the appearance and disappearance of several protein transients. Anabaena and Cylindrospermum differed with respect to the total number of such protein bands, but the characteristic bands which appeared by 8 h incubation in B M were decipherable in both the algae in MSOAM. I t is pertinent to mention that transfer of cultures from AM to B M and consequently the formation of heterocysts are believed to be caused by derepression of genes of heterocyst development, leading to the delineation of several protein transients (FLEMING and HASELKORN 1 9 7 4 , WOOD and HASELKORN 1 9 7 7 , GROVER et al. 1 9 8 0 ) . The presence of such protein transients in M S O A M may

168

I . S. GROVER a n d S. PUBI

be a t t r i b u t e d t o t h e derepression of h e t e r o c y s t o u s g e n e s t h r o u g h i n a c t i v a t i o n of g l u t a m i n e s y n t h e t a s e as suggested b y STEWART and ROWELL (1975). T h e presence of a characteristic protein transient (Rf 0 0 . 9 5 — 0 . 9 7 ) observed o n l y in MSOAM, t e m p t s one t o conclude i t s i n v o l v e m e n t in t h e a c t i v a t i o n of g l u t a m i n e s y n t h e t a s e .

References ALLEN, M. B. and ARNON, D. I., 1955. Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena eylindrica LEMM. Plant Physiol., 30, 366—372. BRADLEY, S. and CAKE, N. G., 1976. Heterocysts and nitrogen development in Anabaena cylindrica. J . gen. Microbiol., 96, 175 — 184. DAVIS, B. J., 1964. Disc electrophoresis. I I . Methods and applications to human serum proteins. A n n . N . Y . A c a d . Sci., 121, 4 0 4 - 4 2 7 .

FLEMING, H. and HASELKORN, R., 1974. The program of protein synthesis during heterocyst differentiation in nitrogen fixing blue-green algae. Cell, 3, 159 — 170. FOGG, G . E . , STEWART, W . D . P . , F A Y , P . a n d WALSBY, A . E . , 1973. T h e B l u e - G r e e n A l g a e . A c a -

demic Press London-New York. GROVER, I. S., KAUR, R. and PURI, S., 1980. Protein transients during heterocyst differentiation in Anabaena and Cylindrospermwm sp. Z. Allg. Mikrobiol., 20, 13—21. LOWRY, 0 . H . , ROSEBROTJGH, R . J . , FARR, A . L . a n d RANDALL, R . J . , 1 9 5 1 . P r o t e i n m e a s u r e m e n t

with Folin phenol reagent. J . biol. Chem., 193, 265—275. ORNSTEIN, L., 1964. Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci., 121, 321-349.

OWNBY, J . D., 1977. Effects of amino acids on methionine sulfoximine induced heterocyst formation in Anabaena. Planta (Berl.), 136, 277—279. STEWART, W. D. P. and ROWELL, P., 1975. Effects of L-methionine-DL-sulfoximine on t h e assimilation of newly fixed NH 3 , acetylene and heterocyst production in Anabaena cylindrica. Biochem. Biophys. Res. Commun., 65, 846—856. WOOD, N.B. and HASELKORN, R., 1977. Protein degradation during heterocyst differentiation in the blue-green alga Anabaena 7120. Federat. Proc., 36, 886. Mailing address: Prof. Dr. I. S. GROVER Department of Biology, Guru Nanak Dev. University Amritsar-143005, India

Zeitschrift für Allg. Mikrobiologie

|

3

22

1982

169—174

(Akademie der Wissenschaften der D D R , Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und experimentelle Therapie, Jena 1 , D i r e k t o r : P r o f . D r . U . TAUBENECK:

und Institut für Mikrobiologie der Bulgarischen Akademie der Wissenschaften, Sofia 2 , D i r e k t o r : P r o f . D r . M . BESHKOV)

Fatty acid composition of lipids of Escherichia coli W 1655 F + and its stable protoplast type L-form J . GUMPERT1, W . SCHADE1, D . K R E B S 1 , S. BAYKOUSHEVA2, E . IVANOVA2 a n d A s . T O S H KOV2

(Eingegangen

am 3. 8. 1981)

The comparative f a t t y acid analysis of extractable and non-extractable lipids of Escherichia coli W 1655 F+ and its stable protoplast type L-form shows quantitative as well as qualitative differences. From 10 different f a t t y acids obtained 16:0, 17:0, and 1 8 : 0 are present at about t h e same quantities in the lipid fractions of the bacterial and L-form. The absence of larger amounts of 12:0, 14:0, and 14:/JOH f a t t y acids in non-extractable L-form lipids reflects the loss of the cell wall in L-form cells. 1 6 : 1 f a t t y acid was found in L-form lipids only. This qualitative difference and the 2—3 times higher content of 18:1 in L-form lipids and t h e 7 times lower content of eye 1 9 : 0 in extractable lipids of t h e L-form may be interpreted as alterations characteristic for t h e changed composition of the cytoplasmic membrane in L-form cells.

The transformation of bacteria into L-forms is accompanied by various morphologic, ultrastructural, metabolic and antigenic changes. I n the protoplast type L-forms the cytoplasmic membrane is the only barrier of the cell towards its environment. I t has to carry out m a n y functions which are associated with the complete cell envelope in normal bacterial cells, for example maintaining cell integrity and osmotic stability, protection against environmental influences, regulation of transport processes. Nearly the entire lipid content of E. coli is found in t h e outer and inner membrane of the cell envelope ( C R O N A N and V A G E L O S 1 9 7 2 ) . Changes in these structures should become visible in the f a t t y acid spectra of their lipid fractions. Comparative studies of the lipid composition in bacterial and L-forms of different gram-negative species were carried out by several authors ( C A V A B D and S C H M I T T SLOMSKA 1976, G M E I N E R a n d MARTIN 1976, K R E M B E L

1964, NESBITT a n d

LENNARZ

and M A N D E L 1 9 6 9 , W E I B U L L et al. 1 9 6 7 ) . Quantitative differences in both the lipid content as well as in its f a t t y acid composition have been found. First investigations of the bacterial and L-form of E. coli W 1 6 5 5 F + ( B A Y K O U S H E V A et al. 1980) showed t h a t the L-form contains twice as much extractable lipid and several times less non-extractable lipid than the bacterial form. The main phospholipids phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin are present in different amounts in both forms. I n this paper data concerning f a t t y acid composition of extractable and non-extractable lipids of bacterial and L-form cells will be summarized and discussed. 1965,

REBEL

Materials

and

methods

Organism and growth conditions: The stable L-form of E. coli W 1655 F+ was obtained from t h e b a c t e r i a l f o r m b y i n d u c t i o n w i t h p e n i c i l l i n (SCHUHMANN a n d TAUBENECK 1 9 6 9 , GUMPERT 12

Z. Allg. Mikrobiol., B d . 22, H . 3

170

J.

GUMPERT, W . S C H A D E , D . K R E B S , S . B A Y K O U S H E V A , E . IVANOVA a n d A s . TOSHKOV

et al. 1971). Both strains were cultivated in nutrient broth composed of meat infusion (made in ZIMET Jena from bovine meat), 1% Proteose peptone I I (DIFCO), and 0.5% NaCl. No serum was added to the cultures. The bacterial form was harvested after 18 h incubation at 37 °C on a rotatory shaker. L-form cells were incubated 42 h under the same conditions. In both cases harvested cells were in the stationary growth phase. Cells were lyophilized and washed in 0.15 M NaCI before extraction of the lipids. Extraction of lipids and analysis of fatty acids: Lipids were extracted by the method of B L I G H and D Y E R (1959) with a mixture of chloroform and methanol (1:2). The lipid fractions of two extractions were washed with aqua dest. and evaporated to dryness. For isolation of non-extractable lipids (NEL) cell debris were hydrolyzed with 2 N alcoholic KOH in a boiling water bath (2 h) and after that treated with ethylether. Methylation of the fatty acids was carried out by treatment with diazomethane. Fatty acid composition was determined by combined gas liquid chromatography — mass spectrometry of methyl esters using a J E O L J M S - D 1 0 0 mass spectrometer equipped with a JGC-20 K P gas chromatograph. The fatty acid methyl esters were separated on a 2 m X 2 mm i. d. glass column packed with 3% of OV-lOl on Gas Chrom Q (80 —100 mesh) under the following operating conditions: injector temperature 250 °C, column temperature 150 °C programmed to 220 °C at 5°/min temperature of separator 230 °C, ion source temperature 200 °C. Helium was used as carrier gas. The mass spectra were recorded at 75 eV and an acceleration voltage of 3000 V. The electron emission energy was 300 iiA. Identification of fatty acid methyl esters was confirmed by comparison of their mass spectra and their glc retention times with those of methyl ester standards. Unsaturated f a t t y acid methyl esters and cyclopropane fatty acid methyl esters were identified after selective hydrogenation according to B R I A N and GARDNER (1968). Quantitative estimations of the fatty acid methyl esters were performed on a gas-liquid chromatograph GCHF 18.3 (VEB Chromatron, Berlin) under the same operating conditions described for the GLC-MS. The percentage of each acid was calculated from the ratio of the area of its peak to the total area of all peaks. Results The results of the f a t t y acid analysis of extractable (EL) and non-extractable lipids (NEL) are summarized in Fig. 1 and Table 1. There are similarities but also Table 1 Fatty acid composition in extractable and non-extractable lipids of the bacterial (B) and stable L-form (L) of Escherichia coli W 1655 F+. Values in % calculated on the basis of peak areas. extractable lipids

Fatty acid

L

B C 12:0

»-dodecanoic (lauric) C 14:0 »i-tetradecanoic (myristic) C 14:ßOH 3-hydroxytetradecanoic (ß-hydroxymyristic) C 16:1 hexadecenoic (palmitoleic) C 16:0 ji-hexadecanoic (palmitic) eye C 17:0 methylenehexadecanoic C 17:0 w-heptadecanoic (margaric) C 18:1 octadecenoic C 18:0 M-octadecanoic (stearic) eye C 19:0 methyleneoctadecanoic ratio of unsaturated to saturated fatty acids1)

non-extractable lipids B L

0

1.9

14.0

0

3.1

4.9

15.6

6.7

0

0

27.8

6.6

0

9.5

0

8.6

53.2 25.5

54.2 20.1

17.5 3.5

46.8 9.4

0 2.9

0 6.1

1.8 2.3

0 7.0

2.9 12.5

1.4 1.7

17.0 0

14.8 0

0.69 cyclopropane fatty acids are regarded as unsaturated

0.60

0.06

0.33

Fatty acid composition of E. coli lipids

171

Fig 1. Gas-liquid chromatograms of fatty acid methyl esters in extractable lipids (left) andnonextractable lipids (right) from parent (A) and L-form (B) cells of E. coli W 1655 F+

12.

172

J . G U M P E R T , W . SCHADE, D . K R E B S , S . B A Y K O U S H E V A , E . IVANOVA a n d A s . TOSHKOV

clear differences between both E L and N E L in bacterial and L-form cells. The major component in bacterial and L-form lipids is C 16:0, amounting to about 5 4 % in E L and 46.8 or 17.5%, respectively in N E L . Further similarities are the concentrations of eye C 1 7 : 0 and C 1 8 : 0 in E L and N E L of bacterial and L-forms. An important qualitative difference is the presence of about 9 % C 1 6 : 1 in L-form lipids only. Clear quantitative differences concern the 2 — 3 times higher content of C 1 8 : 1 in both L-form lipids and the about 7 times higher content of eye C 1 9 : 0 in E L of the bacterial form in comparison to the E L of the L-form. Very striking are also the high amounts of C 12:0, C 14:0, and C 14:/JOH in the N E L of the bacterial form. These fatty acids represent more than 5 0 % of the total fatty acid content in this fraction, whereas they occur only as traces in E L and in an amount of 1 3 % in N E L of the L-form. Traces of C 1 7 : 0 could be found in both N E L fractions.

Discussion The transformation of bacterial cells into stable protoplast type L-form cells is accompanied by a more or less complete loss of the cell wall and the periplasmic space (GUMPERT et al. 1971) as well as by changes in the cytoplasmic membrane. The adaptation of L-form cells to grow in liquid media is always a relatively long process. In the course of the adaptation those L-form cell types become selected which are more resistant against unsuitable environmental influences and which are able to carry out all essential metabolic processes under these conditions. Obviously biochemical and structural changes in the cytoplasmic membrane play an important role during isolation and adaptation of stable L-forms (GMEINER and MARTIN 1976, PANOS 1 9 6 8 ) .

The fatty acid spectra of E. coli W 1655 F + and its L-form lipids are qualitatively similar to those of other E. coli strains (CRONAN and VAGELOS 1972, HOEKSTRA et al. 1 9 7 6 , ISHINAGA et al.

1 9 7 9 , R A E T Z 1 9 7 8 , S I J H E R et al.

1980). Especially C 1 6 : 0 seems

to be a major component in many E. coli strains and other gram-negative bacteria, e. g. Proteus

mirabilis

(GMEINER a n d MARTIN 1 9 7 6 , N E S B I T T a n d LENNARZ 1 9 6 5 ) .

Because its content is about 5 4 % in E L of bacterial and L-form it obviously plays an essential role as constituent of the cytoplasmic membrane in E. coli. I t does not appear to change during L-form transformation. The same conclusion can be drawn for eye C 1 7 : 0 and C 1 8 : 0 which are present in nearly the same amount in the corresponding lipid fractions of the bacterial and L-form. A remarkable qualitative difference is the presence of C 1 6 : 1 in L-form lipids exclusively. Two explanations are possible. I t is known that there can be a conversion of C 1 6 : 1 into eye C 1 7 : 0 during the transition from the exponential growth phase into the stationary growth phase (SIJHER et al. 1980). Because the L-form grows more slowly and inhomogeneously some lysis and cell propagation can occur also in later growth phases. In this case a certain portion of growing L-form cells in the culture after 42 h incubation may explain this C 1 6 : 1 content. Another explanation would be that the C 1 6 : 1 content reflects one important alteration in the composition of the L-form membrane. I t might be that this fatty acid plays an important role in strengthening the membrane structure. This is supported by the findings of NESBITT and LENNARZ (1965) in Proteus mirabilis. They obtained a 4—5 times higher content of C 1 6 : 1 in E L and N E L from L-forms in comparison to the bacterial form.

Fatty acid composition of E. coli lipids

173

Very clear quantitative differences exist in the amount of C 12:0, C 14:0, and C 14: /?OH between E L and N E L of bacterial forms as well as between N E L from bacterial and L-form. These 3 f a t t y acids represent more than 50% in the N E L of the bacterial form in comparison to 13% in that of the L-form. The conclusion that these f a t t y acids are mainly localized in the cell wall, is supported by the fact, that C 12:0, C 1 4 : 0 and C 14:/JOH are known to be components of lipid A, the characteristic cell wall lipopolysaccharide in Enterobacteriaceae ( B U R T O N and CARTER 1 9 6 4 , GOLDFINE 1 9 7 2 , RAETZ 1 9 7 8 ) .

While there are no differences in the ratio of unsaturated to saturated f a t t y acids in the E L of both cell types, the N E L of the bacterial form has very low content of unsaturated f a t t y acids (Table 1). This result confirms findings of other authors that the outer membrane in E. coli strains contains more saturated f a t t y acids than the cytoplasmic membrane (ISHINAGA et al. 1 9 7 9 , LUGTENBERG and P E T E R S 1 9 7 6 ) .

References BAYKOUSHEVA, S., IVANOVA, E . , GUMPERT, J . a n d TOSHKOV, AS., 1 9 8 0 . O n t h e l i p i d c o m p o s i t i o n

of a bacterial and stable L-form of Escherichia coli W 1655 F+. Acta microbiol. bulg., 6, 11—16. BLIGH, E. G. and DYER, W. J., 1959. A rapid method of total lipid extraction and purification. Canad. J . Biochem. Physiol., 37, 911—917. BRIAN, B. L. and GARDNER, E. W., 1968. A simple procedure of detecting the presence of cyclopropane fatty acids in bacterial lipids. Appl. Microbiol., 16, 549—552. BURTON, A. J . and CARTER, H. E., 1964. Purification and characterization of the lipid A component of the lipopolysaccharides from Escherichia coli. Biochemistry, 3, 411—418. CAVARD, D. and SCHMITT-SLOMSKA, J., 1976. Phospholipid composition of L-forms and bacteria of Proteus mirabilis and modifications observed under the action of polymyxin B. In: "Spheroplasts, Protoplasts and L-forms of Bacteria", Symp. INSERM, Vol. 64, 211—219. CRONAN, jr., J . E. and VAGELOS, P. R., 1972. Metabolism and function of the membrane phospholipids of Escherichia coli. Biochim. biophysica Acta, 265, 25—60. GMEINER, J . and MARTIN, H. H., 1976. Phospholipid and lipopolysaccharide in Proteus mirabilis and its stable protoplast L-form. Difference in content and fatty acid composition. Eur. J . Biochem., 67, 487-494. GOLDFINE, H., 1972. Comparative aspects of bacterial lipids. Advances in Microb. Physiology, 8, 1 - 5 8 . GUMPERT, J . , SCHUHMANN, E . u n d TAUBENECK, U., 1971. U l t r a s t r u k t u r d e r stabilen L - F o r m e n

von Escherichia coli B und W 1655 F+. Z. Allg. Mikrobiologie, 11, 19—33.

HOEKSTRA, D . , VAN DER LAAN, J . W . , D E L E I J , L . a n d WITHOLT, B . , 1 9 7 6 . R e l e a s e of o u t e r m e m -

brane fragments from normally growing Escherichia coli. Biochim. biophysica Acta, 455, 889 to 8 9 9 . ISHINAGA, M., KANAMOTO, R. and KITO, M., 1979. Distribution of phospholipid molecular species in outer and cytoplasmic membranes of Escherichia coli. J . Biochem., 86, 161 — 165. KREMBEL, J., 1964. Etude des lipides de la forme L stable de Proteus P 18: identification des acides gras de la fraction acetonosoluble. Pathol. Microbiol., 26, 592—600. LUGTENBERG, E. J . J . and PETERS, R., 1976. Distribution of lipids in cytoplasmic and outer membranes of Escherichia coli K12. Biochim. biophysica Acta, 441, 38—47. NESBITT, J . A. and LENNARZ, W. J., 1965. Comparison of lipids and lipopolysaccharide from the bacillary and L-forms of Proteus P 18. J . Bacteriol., 89, 1020 — 1025. PANOS, C., 1968. Comparative biochemistry of membranes from Streptococcus pyogenes and derived stable L-forms. In: "Microbial Protoplasts, Spheroplasts and L-Forms" (L. B. GUZE, Editor). The Williams & Wilkins Co, Baltimore, 154—162. RAETZ, C. R. H., 1978. Enzymology, genetics, and regulation of membrane phospholipid synthesis in Escherichia coli. Microbiol. Rev., 42, 614—659. REBEL, G. et MANDEL, P., 1969. Recherches sur les lipids des formes L dérivée du Proteus P 18 IV. Etude quantitative des lipids totaux. Ann. Inst. Pasteur, 117, 501—506.

174

J . GUMBERT, W . SCHADE, D . KREBS, S. BAYKOTJSHEVA, E . IVANOVA a n d A s . TOSHKOV

SCHTJHMANN, E. und TATTBENECK, U., 1969. Stabile L-Formen verschiedener Escherichia coliStämme. Z. Allg. Mikrobiol., 9, 297—313. SIJHER, J . S., KUSHNER, D. J . and JYER, R., 1980. Lipid composition of Escherichia coli B/r

bearing N plasmids. FEMS Microbiology Letters, 8, 51—54.

WEIBULL, C., BICKEL, W . D . , HASKINS, W . T . , MILNER, K . C. a n d RIBI, E . , 1967. C h e m i c a l , bio-

logical, and structural properties of stable Proteus L-forms and their parent bacteria. J . Bacteriol., 93, 1 1 4 3 - 1 1 5 9 .

Mailing address : Dr. J. GUMPERT Zentralinstitut für Mikrobiologie und experimentelle Therapie der AdW DDR 6900 Jena, Beutenbergstr. 11

Zeitschrift für Allg. Mikrobiologie

1982

22

175-183

(Bereich Biophysik der Sektion Biologie der Humboldt-Universität zu Berlin, Bereich Mikrobiologie der Sektion Nahrungsgüterwirtschaft und Lebensmitteltechnologie der Humboldt-Universität zu Berlin)

Anwendung der Dielektrophorese für die präparative Trennung thermotoleranter Hefezellen G . K R A U S E , W . SCHADE, R . G L A S E R u n d B .

(Eingegangen

GRÄGER

am 7. 7. 1981)

Dielectrophoresis as a new method for preparative separation of cells is being presented. This method responds to differences in the polarization of cells, which correlate with a number of complex physiological properties of cells. It is shown t h a t yeast cells of different thermotolerant properties can be differentiated by dielectrophoresis. Moreover, the application of this method permitted to separate cells with a high selectivity, which produce more biomass a t a temperature of 40 °C than the initial population. Investigations on the ability of cells to survive in an inhomogeneous a. c. field have shown that the number of dead cells a t frequencies of 10 5 cps and 10 6 cps and at field strengths (on the surface of the central electrode) below 2.4 x 10 5 V/m is small. This fact should be considered when picking out suitable separation conditions.

Die Forderung nach praxisrelevanten Zelltrennverfahren wird auf vielen Gebieten der Biologie erhoben. Ein relativ neues Verfahren, das für die präparative Zelltrennung genutzt werden kann, ist die Dielektrophorese. Die Trennung erfolgt auf Grund unterschiedlicher Polarisationseigenschaften der Zellen, die wiederum stark von physiologischen Parametern beeinflußt werden ( P L I Q U E T T 1 9 6 9 , A S A M I et al. 1 9 7 7 ) . Zellen unterschiedlichen physiologischen Zustands, wie z. B. im Extremfall lebende und tote Hefezellen (POHL U. H A W K 1 9 6 6 , M A S O N U. T O W N S L E Y 1 9 7 1 ) oder Hefezellen, die mit verschiedenen Herbiciden behandelt wurden (POHL U. C R A N E 1 9 7 1 ) zeigen deutlich nachweisbare Differenzen in ihrem dielektrophoretischen Verhalten. Das trifft auch für Zellorganellen und tierische Zellen zu (TING et al. 1 9 7 1 , C H E N 1 9 7 2 , R H O A D S 1 9 7 3 , S C H M I D T et al.

1979).

Die bisherigen Befunde haben vorwiegend analytischen Charakter. Dagegen ist es mit der im Bereich Biophysik der Sektion Biologie der Humboldt-Universität zu Berlin entwickelten Methode möglich, präparative Zelltrennungen auf Basis der Dielektrophorese vorzunehmen. Die Trennleistungen der Apparatur wurden bereits am Beispiel der Trennung eines Gemisches aus lebenden und toten Hefezellen und aus zwei naheverwandten Algenarten demonstriert (GLASER et al. 1979). In der vorliegenden Arbeit soll die Breite der Anwendungsmöglichkeiten anhand eines weiteren Beispiels, der Trennung von Hefezellen unterschiedlicher thermotoleranter Eigenschaften, verdeutlicht werden. Darüber hinaus wird der Einfluß spezieller Parameter des inhomogenen elektrischen Wechselfeldes auf die Überlebensfähigkeit des Untersuchungsmaterials gestestet. Material

und

Methoden

Zellmaterial: Die Untersuchungen erfolgten an Suspensionen aus Hefezellen der Art Saccha.romyces cerevisiae. Die untersuchten Hefezellstämme sind folgendermaßen bezeichnet:

176

G . K R A U S E , W . SCHADE, R . GLASER u n d B . GRÄGER

Ausgangsstamm Stammnummer 193 199 169 182 198 197 161 168 170

adaptierter Stamm

Herkunft S t a m m s a m m l u n g , Bereich Mikrobiologie der V E B B a c k h e f e Leipzig I n s t i t u t f ü r Gärungs- u n d G e t r ä n k e i n d u s t r i e S t a m m s a m m l u n g , Bereich Mikrobiologie d e r V E B B r a m s c h Dresden V E B B a c k h e f e Görlitz I n s t i t u t f ü r Gärungs- u n d G e t r ä n k e i n d u s t r i e I n s t i t u t f ü r Gärungs- u n d G e t r ä n k e i n d u s t r i e I n s t i t u t f ü r Gärungs- u n d G e t r ä n k e i n d u s t r i e

HU der D D R HU der D D R der D D R der D D R

193A 199A 169A 182A 198A 197A 161A 168A 170A

37,8 37,8 37,8 37,8 37,8 37,8 37,8 37,8 37,8

193A 39

Die M a r k i e r u n g A b e d e u t e t A d a p t a t i o n a n die d a n e b e n s t e h e n d e T e m p e r a t u r 37,8 °C (bzw. zusätzlich 39 °C bei S t a m m 193). Die A d a p t a t i o n d e r H e f e s t ä m m e erfolgte v o n 35 °C bis 37,8 °C stufenweise ü b e r einen Z e i t r a u m v o n 11 M o n a t e n . Z u r A d a p t a t i o n a n 39 °C w u r d e ein weiterer Z e i t r a u m v o n 6 M o n a t e n b e n ö t i g t . D a b e i w u r d e in E m e r s k u l t u r e n u n d u n t e r V e r w e n d u n g eines Melassemediums m i t Würze- u n d N ä h r s a l z z u s a t z sowie bei T e m p e r a t u r e n ü b e r 37,8 °C m i t AlkoIysatzusatz g e a r b e i t e t . Die H e r f ü h r u n g d e r H e f e s t ä m m e erfolgte v o n D a u e r k u l t u r e n a u s g e h e n d in zwei P a s s a g e n . Die erste Passage w u r d e ü b e r 72 S t d . in 5 m l F l ü s s i g k u l t u r ( u n g e h o p f t e W ü r z e , 1 0 % E x t r a k t gehalt) bei 20 °C g e f ü h r t . I n der zweiten P a s s a g e w u r d e der jeweilige S t a m m u n t e r gleichen Bedingungen auf W ü r z e s c h r ä g a g a r ( u n g e h o p f t e W ü r z e , 1 0 % E x t r a k t g e h a l t ) w e i t e r g e z ü c h t e t . N a c h d e r B e b r ü t u n g w u r d e die gewachsene H e f e k u l t u r vorsichtig m i t einer I m p f ö s e a b g e s t r e i f t u n d in destilliertes Wasser ü b e r t r a g e n . D a b e i w a r darauf zu a c h t e n , d a ß keine N ä h r b o d e n b e s t a n d t e i l e in die Lösung ü b e r f ü h r t werden. Die L e i t f ä h i g k e i t w u r d e m i t einer NaCl-Lösung (100 min) auf 20 X 1 0 - * ß - i m - i eingestellt. Nachweis ü b e r die A b t ö t u n g der Zellen: Der N a c h w e i s ü b e r die A b t ö t u n g d e r Zellen erfolgte m i t Hilfe d e r M e t h y l e n b l a u f ä r b u n g . Die M e t h y l e n b l a u l ö s u n g w u r d e n a c h der bei H L A V A C E K (1961) angegebenen Vorschrift hergestellt. Methodisch sei darauf hingewiesen, d a ß d a m i t n i c h t nachgewiesen w e r d e n k a n n , ob die so als lebend identifizierten u n g e f ä r b t e n Zellen t a t s ä c h l i c h noch v e r m e h r u n g s f ä h i g sind. Dieser Nachweis e r f o l g t e d u r c h w e i t e r e Z ü c h t u n g d e r a b g e t r e n n t e n Zellen. D a z u sind P l a t t e n m i t jeweils 1 m l d e r Zellsuspension gegossen worden, die bei 25 °C bebrütet wurden. B e s t i m m u n g der D-Werte: Die B e s t i m m u n g d e r D-Werte e r f o l g t e d u r c h g e s t e u e r t e s Heißh a l t e n v o n K a p i l l a r r ö h r c h e n bei 58 °C. J e K a p i l l a r r ö h r c h e n w u r d e n 0.02 ml Hefesuspension m i t e t w a 10 Millionen Hefezellen eingefüllt. Der zeitliche T e m p e r a t u r v e r l a u f in den K a p i l l a r r ö h r c h e n w u r d e m i t Hilfe eines K u p f e r - K o n s t a n t a n - T h e r m o e l e m e n t e s verfolgt u n d ü b e r einen X Y - K o m pensationsschreiber v o m T y p E N D I M 620/02 registriert. Die g e n a u e B e s t i m m u n g der K e i m z a h l e n v o r u n d n a c h d e r E r h i t z u n g erfolgte m i t t e l s P l a t t e n v e r f a h r e n . Die D - W e r t e w u r d e n n a c h folgender Formel berechnet: D =

^Wi

lgN0-\gN

D e z i m a l r e d u k t i o n s z e i t (die n o t w e n d i g e Zeit, die zur A b t ö t u n g von 9 0 % d e r Ausgangskeime bei k o n s t a n t e r T e m p e r a t u r erforderlich ist) iwi — w a h r e H e i ß h a l t e z e i t N 0 — Ausgangskeimzahl N — K e i m z a h l n a c h iwi D

Als w a h r e H e i ß h a l t e z e i t w u r d e näherungsweise d i e Zeit e r m i t t e l t , bei der die T e m p e r a t u r gleich oder größer als 0,25 (47'max— Z s.cer.) war. D e r e x p e r i m e n t e l l e r m i t t e l t e d u r c h s c h n i t t l i c h e Z-Wert v o n Saccharomyces cerevisiae b e t r u g 5,4 °C. (Der 2 - W e r t ist die T e m p e r a t u r e r h ö h u n g , die erforderlich ist, u m die A b t ö t u n g s z e i t auf ein Zehntel zu v e r m i n d e r n . U n t e r Tm&x wird d i e M a x i m a l t e m p e r a t u r v e r s t a n d e n , die bei der E r h i t z u n g d e r Keimsuspension erreicht wird.) B e s t i m m u n g d e r T r ü b u n g s w e r t e : Die E m p f i n d l i c h k e i t der verschiedenen U n t e r s u c h u n g s s t ä m m e gegenüber h ö h e r e n T e m p e r a t u r e n w u r d e d u r c h Vergleich des W a c h s t u m s in u n g e h o p f t e r W ü r z e ( 8 % E x t r a k t g e h a l t ) g e t e s t e t . D a z u w u r d e n u n t e r s t a n d a r d i s i e r t e n Bedingungen j e 5 Millionen Einzelzellen in 10 m l W ü r z e g e i m p f t . Die Zellvermehrung w u r d e d u r c h n e p h e l o m e t r i s c h e T r ü bungsmessung a n einem S p e k t r o p h o t o m e t e r v o m T y p S P E K O L m i t T r ü b u n g s m e ß a n s a t z v e r f o l g t . D i e Messung w u r d e u n t e r Berücksichtigung d e r notwendigen Nullwerte m i t Hilfe eines T r ü -

Dielektrophorese f ü r präparative Zelltrennung

177

bungsstandards (0,7 mg BaS0 4 /ml dest. Wasser) durchgeführt, der zum Eineichen einer 100%igen Trübung verwendet wurde. Nach einer Bebrütungszeit von 40 S t d . war bei 25 °C in jedem Fall die stationäre P h a s e erreicht. Messung des Zellvolumens der Hefezellen: Das Volumen der Hefezellen wurde m i t einem Teilchenzähl- u n d Analysengerät L A B O R S C A L E (PSL-1)-Analysator (PSA-1) (MBDICOK-Werke Budapest) bestimmt. Dabei fanden in der Regel eine Düse m i t einer Mikrobohrung von 70 [i.m und ein Meßstrom von 200 [J.A Anwendung.

Ergebnisse E i n f l u ß des i n h o m o g e n e n e l e k t r i s c h e n W e c h s e l f e l d e s auf d i e Ü b e r l e b e n s f ä h i g k e i t d e r Z e l l e n Die Überlebensfähigkeit der Zellen unter dem Einfluß des elektrischen Feldes ist von grundlegender Bedeutung für die Anwendung der Methode, insbesondere wenn vorgesehen ist, die selektierten Zellen für eine weitere Züchtung zu verwenden.

effektive Feldstärke an der Oberffache der Zentra/e/ektrode (x10sV/m) Abb. 1. Anteil abgetöteter Hefezellen (Labor-gezüchtete Hefe der A r t Saccharomyces cerevisiae, S t a m m 193) an der Gesamtzahl abgetrennter Zellen in Abhängigkeit von der Feldstärke an der Oberfläche der Zentralelektrode (mit Standardfehler des Mittelwertes, Drahtgeschwindigkeit 1,5 mm/s, Versuchszeit 8 min, Temperatur 25 °C) K u r v e a — Anteil abgetöteter Zellen bei 106 Hz (n = 10) K u r v e b — Anteil abgetöteter Zellen bei 103 Hz (n = 18)

Abbildung 1 zeigt die Abhängigkeit des Anteils toter Zellen an der Gesamtzahl abgetrennter Zellen von der Feldstärke an der Oberfläche der Zentralelektrode. Wie zu erwarten, steigt der Anteil abgetöteter Zellen mit der Feldstärke. Dabei ist zu beachten, daß die Kurven bei 103 Hz und 106 Hz stark differieren. In weiteren Experimenten wurde der Anteil toter Zellen in Abhängigkeit von der Frequenz des Wechselfeldes überprüft. Entsprechende Ergebnisse sind der Abbildung 2 zu entnehmen.

178

G . K R A U S E , W . SCHADE, R . G L A S E R u n d B . G R Ä G E R

tf 100 -1

1 101 102 103

10* 105 10

Frequenz

6

(Hz)

Abb. 2. Anteil abgetöteter Hefezellen (Labor-gezüchtete Hefe der Art Saccharomyces cerevisiae, Stamm 193) an der Gesamtzahl abgetrennter Zellen in Abhängigkeit von der Frequenz (Drahtgeschwindigkeit 1,5 mm/s, Versuchszeit 8 min, effektive Feldstärke an der Oberfläche der Zentralelektrode 1,18 x 105 V/m, n = 6, die Fehler des Mittelwertes liegen zwischen 3 und 30%)

E s wird deutlich, daß der Totzellanteil der bei niedrigen Frequenzen (10 2 Hz, 10 3 Hz) angereicherten Zellen signifikant höher liegt als bei den Zellen, die bei hohen Frequenzen abgetrennt wurden. Der Nachweis toter Zellen wurde in diesen Experimenten mit der Methode der Methylenblaufärbung erbracht. Kultivierungsversuche ergaben, daß nach dem Trennvorgang (Trennbedingungen: Feldstärke an der Oberfläche der Zentralelektrode 1,18 x 10® V/m, Frequenz 10 6 Hz) noch 7 2 % der insgesamt angereicherten Zellen vermehrungsfähig sind. Dabei sind wahrscheinlich mindestens 20% der nicht mehr vermehrungsfähigen Zellen auf die lange Verweilzeit der Hefen in destilliertem Wasser zurückzuführen. So zeigte sich, daß der Anteil kultivierbarer Hefezellen nach östündiger Inkubation in destilliertem Wasser bei Zimmertemperatur um 2 0 % vermindert ist. I m Trennexperiment betrug die Zeitspanne zwischen Suspendierung der Zellen in destilliertem Wasser bis zur anschließenden Kultivierung ca. 7 Stunden. Dielektrophoretisches Verhalten thermotoleranter Hefezellen Abbildung 3 zeigt die Temperaturabhängigkeit der dielektrischen Sammelfaktoren, et al. 1979) die von Hefezellen des Stammes 193 und des dazugehörigen adaptierten Stammes 193A 37,8 bei einer Frequenz von 10 3 Hz aufgenommen wurden. Diese Frequenz ergab sich auf Grund von Voruntersuchungen, die zeigten, daß die bei 10 3 Hz ermittelten Sammelfaktoren am empfindlichsten auf Temperaturveränderungen reagieren. Dazu wurden die Hefezellen vor Beginn eines jeden Experimentes 80 min lang bei den jeweiligen Versuchstemperaturen 25 °C, 37 °C, 40 ° C u n d 4 5 °C inkubiert. Aus den in Abbildung 3 dargestellten Ergebnissen wird deutlich, daß der temperaturempfindlichere Stamm 193 im Vergleich zu dem temperaturadaptierten Stamm 193A 37,8 auch in seinem Sammelfaktor empfindlicher auf Temperaturerhöhungen reagiert. (GLASEK

Dielektrophorese für präparative Zelltrennung

179

5 -

1 1 30

20

—i 50

1 W

Temperaiur

(°C)

Abb. 3. Abhängigkeit des Sammelfaktors von Hefezellen der Art Saccharomyces cerevisiae, Stamm 193 und 193A 37,8 von der Temperatur bei einer Frequenz von 103 Hz (mit Standardfehler des Mittelwertes, effektive Feldstärke an der Oberfläche der Zentralelektrode 1,18 X 105 V/m, Drahtgeschwindigkeit 1,5 mm/s, Versuchszeit 8 min, n für 25 °C und 37 °C = 36, n für 40 °C und 45 °C = 42) a — Kurve des adaptierten Stammes 193A 37,8 b — Kurve des Ausgangsstammes 193 Tabelle 1 Vergleich von Sammelfaktoren und Volumen von Hefezellen verschiedener Stämme Sammelf aktoren

Hefestamm 193 169 182 198 168 170 199 161 197

Ausgangsstamm

|

5,1 ± 3,6 ± 2,3 ± 1,3 ± 2,4 ± 2,5 ± 1,6 ± 0,1 ± 0,2 ±

1,2 1,3 0,2 0,3 0,3 0,3 0,6 0,01 0,02

adaptierter Stamm

1 |

1,7 ± 2,6 ± 1,3 ± 0,5 ± 1,0 ± 3,0 ± 1,2 ± 1,0 ± 0,4 ±

0,7 1,0 0,1 0,05 0,2 0,5 0,5 0,2 0,07

Verhältnis des Volumens des Ausgangsstammes zu dem des adaptierten Stammes 1:2,3 1:1,1 1:1,6 1:1,9 1:2,2 1:1,3 1:1 1:1 1:1

Versuchsbedingungen für die Bestimmung der Sammelfaktoren: Frequenz 103 Hz, Temperatur 45 °C, Feldstärke an der Oberfläche der Zentralelektrode 1,18 x 103 V/m, Versuchszeit 8 min, n = 3, die Unterschiede zwischen den Sammelfaktoren der Zellen des Ausgangsstammes und des adaptierten Stammes sind mit Ausnahme der Sammelfaktoren der Stämme 199 und 199A 37,8 signifikant (Nachweis mit dem U-Test nach M A N N und W H I T N E Y ) U m zu prüfen, ob diese Tendenz einer höheren Temperaturempfindlichkeit des Sammelfaktors nichtadaptierter Hefezellen auch bei anderen S t ä m m e n nachweisbar ist, wurden 8 weitere Hefezellstämme und die dazugehörigen adaptierten Zellen bei einer F r e q u e n z von 10 3 Hz und einer T e m p e r a t u r von 4 5 °C dielektrophoretisch untersucht. Diese in Tabelle 1 dargestellten Ergebnisse zeigen ein differenzierteres B i l d : Bei 5 der untersuchten S t ä m m e (193, 169, 182, 198, 168) liegen die Sammelfaktoren der

180

G . K R A U S E , W . SCHADE, R . GLASER u n d B . GRÄGER

adaptierten Stämme signifikant niedriger. Beim Stamm 199A wurde zwar auch ein niedrigerer Sammelfaktor ermittelt, dieser Unterschied ist aber im Vergleich zum Ausgangsstamm 199 mit einer Irrtumswahrscheinlichkeit von 0,05 nicht signifikant. Die Stämme 170, 161 und 197 zeigen eine gegenläufige Tendenz — die Sammelfaktoren der adaptierten Stämme liegen signifikant höher. Auf Grund mikroskopischer Untersuchungen ergab sich, daß die Hefezellen adaptierter Stämme teilweise größer sind. Deshalb wurden genauere Analysen des Zellvolumens der Stämme mit Hilfe eines Teilchenzählgerätes vorgenommen (Tab. 1). Vergleicht man die Volumenangaben mit den Sammelfaktoren, so fällt auf, daß gerade die Zellen, die keine Unterschiede im Volumen zwischen Ausgangsstamm und adaptiertem Stamm (199, 161, 197) aufweisen, ein abweichendes dielektrophoretisches Verhalten zeigen: Die Differenzen in den Sammelfaktoren der Stämme 199 und 199A sind nicht signifikant und die Sammelfaktoren von Zellen der nichtadaptierten Stämme 161 und 197 liegen im Gegensatz zu den anderen untersuchten Stämmen höher. Eine Ausnahme dabei bildet der Stamm 170.

Präparative Trennung thermotoleranter Hefezellen Man kann voraussetzen, daß eine Suspension temperaturadaptierter Hefezellen in Abhängigkeit vom Adaptationsstadium ein relativ breites Spektrum von Zellen enthält, die sich hinsichtlich ihres Grades an Thermotoleranz unterscheiden. Es wurde geprüft, ob es mit Hilfe der dielektrophoretischen Methode möglich ist, besonders thermotolerante Hefezellen aus einem Gemisch abzutrennen.

"5—

5k %

1

%

0

20

1

30

1

40

50

Temperatur (°C) Abb. 4. Abhängigkeit des Sammelfaktors von Hefezellen der Art Saccharomyces cerevisiae, Stamm 193 und 193A 37,8 von der Temperatur bei einer Frequenz von 106 Hz (effektive Feldstärke an der Oberfläche der Zentralelelektrode 1,18 X 106 V/m, Drahtgeschwindigkeit 1,5 mm/s, Versuchszeit 8 min, n = 30) a — Kurve des adaptierten Stammes 193A 37,8 b — Kurve des Ausgangsstammes 193

181

Dielektrophorese für präparative Zelltrennung

Die Untersuchungen erfolgten an dem an 39 °C adaptierten Zellstamm 193A bei einer Trennfrequenz von 106 Hz. Diese Trennfrequenz wurde gewählt, da hier die abzutrennenden thermotoleranten Hefezellen stärker als die Kontrollzellen angereichert werden (Abb. 4). Nach dem Trennvorgang (Trennzeit 90 min, effektive Feldstärke an der Oberfläche der Zentralektrode 1,18 X 105 V/m, Durchlaufgeschwindigkeit des Elektrodendrahtes 3 mm/s) wurden die Hefezellen aus der Sammelkammer entnommen und in 15 ml destilliertem Wasser suspendiert. Diese Zellsuspension durchlief ein zweites Mal die Trennkammer, wobei der Trennvorgang nach 8 min unterbrochen und die selektierten Zellen in 10 ml steriles destilliertes Wasser überführt wurden. Tabelle 2 D-Werte und Trübungswerte von Hefekulturen aus 10 der dielektrophoretisch abgetrennten Zellen Zellkultur

.D-Werte (s)

Trübungswert bei 40 °C(%)

Ausgangskultur 193A 39,0

16,1

11,0 ± 3,9

193A 193A 193A 193A 193A 193A 193A 193A 193A 193A

39,1 39,2 39,3 39,4 39,5 39,6 39,7 39,8 39,9 39,10

Mittelwerte aus Kulturen 1—10

15,6 12,6 15,5 13,8 14,3 17,2 15,8 26,0 17,2 22,7 17,1 ± 4,2

17,0 ± 15,0 ± 18,0 ± 21,0 ± 21,0 ± 21,0 ± 22,0 ± 21,0 20,0 ± 19,0 ±

0,5 1,9 0,5 3,5 1,4 3,2 4,5 2,1 1,9

19,5 ± 2,2

Der Hefezellstamm, aus dem die 10 Einzelzellen abgetrennt wurden, ist mit 193A 39,0 bezeichnet. Die aus den 10 Einzelzellen hergeführten Kulturen sind von 1—10 numeriert.

Die Zellsuspension ist so aufbereitet worden, daß eine Untersuchung des Wachstums bei submaximalen Temperaturen und eine Bestimmung des D-Wertes möglich waren. Da dabei mit Reinkulturen gearbeitet werden mußte, wählten wir von den 840000 züchtbaren Zellen willkürlich 10 Zellen aus, von denen jeweils eine neue Kultur gezüchtet wurde. Tabelle 2 gibt die Trübungswerte bei 40 °C und die ermittelten Z>-Werte an. Es fällt auf, daß die Biomasseproduktion bei 40 °C bei allen aus den 10 abgetrennten Zellen gezüchteten Kulturen im Vergleich zur Ausgangskultur signifikant höher ist. Das ist besonders deshalb hervorzuheben, da nur 10 der insgesamt 840000 züchtbaren Zellen mikrobiologisch untersucht wurden. Die geringen Schwankungen der Werte lassen eine gute Trenngenauigkeit hinsichtlich der Selektionen von Zellen mit höherer Biomasseproduktion bei 40 °C erkennen. Nennenswerte Veränderungen in den Trübungswerten, die bei 30 °C und 42,5 °C gemessen wurden (in Tab. nicht eingetragen), liegen nicht vor. Ob zusätzlich eine Selektion von Zellen erfolgt, die sich hinsichtlich ihres Absterbeverhaltens besonders thermotolerant verhalten, kann auf Grund des vorliegenden Faktenmaterials nicht entschieden werden. Zwar liegen die D-Werte der Kulturen 8 und 10 um ca. 41 bzw. 62% höher als der D-Wert des Stammes 193A 39,0 jedoch kann es sich hier auch um zufällige Abweichungen handeln.

182

G . K R A U S E , W . S C H A D E , R . G L A S E R u n d B . GRÄGER

Diskussion Die demonstrierten Befunde an thermotoleranten Hefezellen bestätigen, daß eine dielektrophoretische Trennung von Zellen möglich ist. Bei der hier erfolgten Abtrennung von Hefezellen des Stammes 193A 39 wird offensichtlich ein Selektionsprinzip wirksam, das auf Zellen mit solchen zellulären Parametern anspricht, die mit einer hohen Wachstumsrate bei 40 °C korrelieren. Weiterhin konnten charakteristische Unterschiede zwischen den dielektrophoretischen Eigenschaften temperaturadaptierter Hefezellen und Zellen des jeweiligen Ausgangsstammes nachgewiesen werden. Da z. Z. nur unklare Vorstellungen über den Mechanismus der Dielektrophorese bestehen, ergeben sich Schwierigkeiten bei der Erklärung des unterschiedlichen dielektrophoretischen Verhaltens von Hefezellen verschiedener Stämme. Hinzu kommt, daß sich thermotolerante Hefezellen in einer Vielzahl zellulärer Parameter unterscheiden, die die Polarisierbarkeit der Zellen beeinflussen können. Diese zellulären Eigenschaften sind aber von den hier untersuchten Stämmen nicht bekannt. Bei 5 der untersuchten Stämme wurden bei einer Frequenz von 103 Hz und einer Temperatur von 45 °C höhere Sammelfaktoren von nichtadaptierten Hefezellen gemessen als von temperaturadaptierten Hefezellen. Denkbar wäre, daß die verstärkte dielektrophoretische Sammlung temperaturempfindlicher Hefezellen eine Folge temperaturbedingter Veränderungen von Membran- und Zellwandeigenschaften ist, die jedoch beim gegenwärtigen Wissensstand nicht näher charakterisiert werden können. So wird in Diskussionen über Ursachen des dielektrophoretischen Verhaltens von Zellen im Niederfrequenzbereich sowohl die Polarisation der elektrischen Doppelschicht als auch der Zellwand im Falle von Hefezellen, Bakterien oder Algen in Betracht gezogen. Während beispielsweise Thrombocyten (RHOADS 1973) und Erythrocyten

( W I L E Y 1 9 7 0 , PESCHECK

1 9 8 0 , FOMÖENKOV U. GAVBILJUK 1 9 7 7 ,

1978)

unterhalb von 100 kHz keine Dielektrophorese aufweisen, zeigen Hefezellen und Bakterien (POHL 1973, CHEN 1972) eine relativ starke Sammlung im Niederfrequenzbereich. Die Ursache dafür wird in der Zellwand gesehen, die einen großen Beitrag zur Gesamtpolarisation der Zellen leisten soll. So zeigten z. B. EINOLF U. CAESTENSEN (1969) für verschiedene Bakterien, daß die effektive Dielektrizitätskonstante der Protoplasten um etwa 2 Größenordnungen niedriger liegt als bei den Zellen mit Zellwand. CHEN (1972) überprüfte den Einfluß von Kalium-, Calcium- und Lanthanionen auf das dielektrophoretische Verhalten von Escherichia coli. Dabei wurde eine Verminderung der Zellsammlung beobachtet, was auf eine Reduktion der Konzentration mobiler Ionen in der Zellwand zurückgeführt wurde. Im Gegensatz zum Niederfrequenzbereich werden für den MHz-Bereich Polarisationsmechanismen diskutiert, die von der Leitfähigkeit des Cytoplasmas und des die Zelle umgebenden Mediums beeinflußt werden. So führt eine Verminderung der Leitfähigkeitsdifferenz zwischen dem Zellinnern und dem Suspensionsmedium zu einer A b s c h w ä c h u n g d i e l e k t r o p h o r e t i s c h e r E f f e k t e (POHL 1978, PESCHECK 1980, WILEY 1970).

Zusammenfassend wird eingeschätzt, daß die Methode prinzipiell dazu geeignet ist, Zellen nach physiologischen Unterschieden voneinander zu trennen. Das wurde bei GLASER et al. (1979) am Beispiel der Trennung lebender und toter Hefezellen und Algen naheverwandter Arten, sowie in der vorliegenden Publikation am Beispiel der Abtrennung von Hefezellen demonstriert, die sich hinsichtlich der Eigenschaft „Vermehrungsfähigkeit bei 40 °C" besonders thermostabil verhalten. Daraus ergeben sich weitere Perspektiven für die Anwendung der Methode in der Mikrobiologie, beispielsweise für die Selektion und Züchtung industriell geeigneter Stämme.

Dielektrophorese f ü r präparative Zelltrennung

183

Literatur ASAMI, K., HANAI, T. and KOIZUMI, N., 1977. Dielectric properties of yeast cells: E f f e c t of some ionic detergents on t h e plasma membrane. J . Membrane Biol., 34, 145 —156. CHEN, C. S., 1972. On t h e n a t u r e and origins of biological dielectrophoresis. M. S. Thesis, Oklahoma S t a t e University, zitiert bei POHL 1973. EINOLF, C. W . and CAKSTENSEN, E . L., 1969. Passive electrical properties of microorganisms. IV. Studies of t h e protoplasts of Micrococcus lysodeikticus. Biophys. J . , 9, 634—641. FOMÖEKKOV, V. M. and GAVRILJUK, B. K., 1977. Dielectrophoresis of cell suspensions, studia biophysica, 65, 35—46. FOMÖENKOV, V. M. and GAVRILJUK, B. K., 1978. The s t u d y of dielectrophoresis of cells using t h e optical technique of measuring. J . Biol. Phys., 6, 29—68. GLASER, R . , PESCHECK, C . , K R A U S E , G . , SCHMIDT, K . P . u n d ROHLOFF, B . , 1 9 7 8 . V e r f a h r e n

zur

Trennung von biologischen Teilchengemischen durch Dielektrophorese. P a t e n t s c h r i f t , Aktenzeichen W P G OL N/205739, Humboldt-Univ. Berlin, Sektion Biologie.

GLASER, R . , PESCHECK, C . , K R A U S E , G . , SCHMIDT, K . P . u n d TÄUSCHER, C . , 1 9 7 9 .

Dielektrophorese

als Grundlage f ü r ein neues Verfahren zur präparativen Zelltrennung. Z. Allg. Mikrobiol., 19, 601—607. HLAVAÖEK, F., 1961. Brauereihefen: Biologie und Biochemie der Hefezellen — Die Gärung in der Brauerei — Verwertung des Nebenprodukts Hefe. V E B Fachbuchverlag Leipzig. MASON, B. D. and TOWNSLEY, P. M., 1971. Dielectrophoretic separation of cells. Canad. J . Microbiol., 17, 8 7 9 - 8 8 8 . PESCHECK, C., 1980. Das Verhalten von Zellsuspensionen in elektrischen Wechselfeldern — ein Beitrag zum Verständnis der Dielektrophorese. Dissertationsmanuskript, Humboldt-Universität Berlin. PLIQUETT, F., 1969. Biophysikalische Untersuchungen von Zellen und Geweben durch passive elektrische Verfahren. Fortschr. expt. theor. Biophysik, 11. POHL, H . A., 1973. Biophysical aspects of dielectrophoresis. J . Biol. Phys., 1, 1 — 16. POHL, H . A., 1978. Dielectrophoresis: The Behaviour of N e u t r a l M a t t e r in Nonuniform Electric Fields. Cambridge University Press. POHL, H . A. and CRANE, J . S., 1971. Dielectrophoresis of cells. Biophys. J . , 11, 7 1 1 - 7 2 7 . POHL, H . A. and HAWK, J . , 1966. Separation of living and dead cells b y dielectrophoresis. Science, 152,

647-649.

RHOADS, J . E., 1973. Dielectrophoresis of canine blood platlets. M. S. Thesis, Oklahoma S t a t e University, zitiert bei POHL 1978. SCHMIDT, K . P . , GLASER, R . PESCHECK, C . u n d K R A U S E , G . , 1 9 7 9 . D i e K e t t e n b i l d u n g s k i n e t i k

Meßparameter f ü r die Dielektrophorese von Zellen, studia biophysica, 75, 81—91.

als

T I N G , J . P . J O L L E Y , K . , B E A S L E Y , C . A . a n d POHL, H . A . , 1 9 7 1 . D i e l e c t r o p h o r e s i s o f c h l o r o p l a s t s .

Biochim. biophysica Acta, 234, 324—329. WILEY, K . L., 1970. A comparison of normal and abnormal cells using dielectrophoresis. M. S. Thesis, Oklahoma S t a t e University, zitiert bei POHL 1978. A n s c h r i f t : D r . G . KRAUSE

Zentralinstitut f ü r Arbeitsmedizin der D D R D D R 1134 Berlin, Nöldnerstraße 40—42

Zeitschrift für Allg. Mikrobiologie

22

1982

185-190

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

Bistability in the glucose and energy metabolism of ammonia-limited chemostat cultures of Escherichia coli ML 30 P . J . MÜLLER a n d BEATE VON FROMMAJSTNSHAUSEN (Eingegangen

am

2.6.1981)

Recently bistability in the pyruvate production of ammonia-limited glucose-grown Escherichia coli ML 30 chemostat cultures was evidenced. The present investigation shows that the state of higher pyruvate production is connected with a lower glycogen content than in cells in the state of lower pyruvate production. The results support the view of a regulation of bistability on the step of pyruvate consumption by the citric acid cycle. Pronounced differences in energy formation in the two states of glucose bistability are discussed.

Evidence of bistability (existence of two stable stationary states) in the pyruvate formation of ammonia-limited Escherichia coli ML 30 chemostat cultures with glucose in excess at dilution rates D = 0.1 — 0.2 h _ 1 has been presented. It was found that the state of higher pyruvate formation (state B) is correlated with a lower yield factor for glucose FX/G = X/(GR — G) ( X : steady state cell concentration, : glucose concentration in the reservoir, G: steady state glucose concentration) than the state with lower pyruvate formation (state A) (BERGTER and ROTH 1977). Because pyruvate bistability is connected with a bistability in the ammonia assim i l a t i o n s y s t e m s (BERGTER et al.

1977, MULLER a n d BERGTER 1 9 7 7 , 1 9 7 8 , MULLER

et al. 1977, 1981) it was at first assumed that a higher steady state ammonia concentration in state B activates the glucose uptake (GR — G) and pyruvate formation, leading to lower YXIG- At the other side X and in this connection also Fx/e could be affected in the same direction by the stated differences in limiting NH3 and/or by accumulation of glycogen in the cells, proved for E. coli chemostat cultures at ammonia limitation (HOLME 1958). To distinguish between these possibilities of regulation, the intracellular glycogen content, the glucose and oxygen uptake, the NH3 uptake, and the uptake of extracellular added pyruvate in each state of pyruvate formation were investigated at a dilution rate D 0.15 h - 1 . For calculation of comparable specific rates of substrate uptake and product formation a corrected cell concentration _Xcorr without glycogen is introduced. The results reflect the general property of the investigated system to exist in one of the two states characterized by profound differences in the glucose- and energy metabolism. Materials

and

methods

The culture conditions of Escherichia coli ML 30 chemostats with ammonia limitation (0.090 g/1 NH4C1 = 1.68 mmole/1 NH 3 ) and glucose (3 g/1) at a dilution rate D » 0.15 h _ 1 were described previously (BERGTER and ROTH 1977). X was estimated by absorbance at 470 nm (1A 470 = 0.173 g per 1 dry weight). 13

Z. Allg. Mikrobiol., Bd. 22, H. 3

186

P . J. MÜLLER and BEATE VON FROMMANNSHAUSEN

Pyruvate was estimated enzymatically with lactate dehydrogenase (VEB Arzneimittelwerk Dresden) by the method of CZOK and LAMPRECHT (1970), glucose enzymatically with peroxydase/ glucose oxydase and o-dianisidin (FERMOGNOST-Test, V E B Arzneimittelwerk Dresden), and the protein concentration after LOWBY et al. (1951). For estimation of glycogen the "polyglucose" of cells was hydrolyzed in acid solution to glucose (SIGAL et al. 1964). Polyglucose of E. coli is mainly composed of glycogen (PREISS 1969). The bacterial suspension was centrifuged, washed with physiological NaCl-solution and after centrifugation diluted with physiological NaCl-solution to a cell concentration of about 1 g/1. 1 ml of the cell suspension was heated with 1 ml 2 N sulphuric acid in a boiling water bath. The hydrolysate was mixed with 4 ml 2 M K 2 HP0 4 -solution and then centrifuged at 5000 x g for 10 min. The supernatants were adjusted to pH 7.0. The same procedure was done with a glucose standard (2 g/1) for estimation of the loss of glucose during hydrolysis. The loss in percent of the hydrolyzed standard was used for correction of analyses. For estimation of the 02-uptake rate of resting cells about 60 ml culture was withdrawn from the chemostat and immediately transferred to a thermostated glass vessel (50 ml volume, 34 °C), fitted with a 02-membrane electrode and a magnetic stirrer (p02-Meter M 65 F, V E B METRA, Radebeul, GDR). From the recorder traces the 02-uptake rates were calculated assuming a saturating 02-concentration of the culture of 225 JAM. Calculation of corrected cell concentrations _X corr : The measured steady state cell concentrations in each state of glucose metabolism (state A and B) 1 A and XB are different because of the different glycogen content of the cells and because of the different amount of uptaken limiting NH 3 N-R — N (2VR:NH3 concentration in the reservoir, N: steady state NH 3 concentration). For a direct comparison of the calculated specific rates it is necessary to use corrected cell concentrations without glycogen ( 2 A " , -XB°")- ^ A " and X™" are calculated from the mean values of the measured steady state concentrations X&, N x , JVB and Glys (Gly: glycogen content of cells, g glycogen/g protein) with the equations XCA0RR = XA/(1 + Prot • Gty A )

(g/1)

1 B " = XBI(1 + Prot • GIy B )

(g/1)

(1) (2)

assuming that the same yield coefficient of NH 3 is valid in the two states regarding X c o r r YXIN = X T ' I(NR N A ) = Z°B0rr I(NN NB) (g dry weight/mmole NH 3 ) (3) The protein content of the cells, corrected for glycogen, is calculated to be P f o t = 0.8418 (g protein/g dry weight). This is in the order of a value P f o t = 0.80, found in a E. coli 15 chemostat culture with acetate limitation at D = 0.381 h-1 (FORCHHAMMER and LINDAHL 1971). Calculation of differences in the energy production between state A and B: According to stoichometric coupling in glycolysis (FDP-route) and in the citric acid cycle each mole glucose, when it is completly oxidized instead of forming glycogen, gives 4 + 2 moles ATP-equivalents, 10 N A D H equivalents and 2 F A D H (2 moles A T P result from the absence of glycogen synthesis from glucose). The complete oxidation of 1 mole pyruvate is linked with the formation of 1 mole A T P , 4 moles N A D H and 1 mole F A D H . For calculating of A T P formation from N A D H and F A D H , the cases of three, two and one "site" of phosphorylation associated with pyridine nucleotide oxidation and two "sites" for flavoprotein-linked oxidation were assumed.

The differences in the specific rates of A T P formation between state A and B(KP°ATP) arecalcuattd according to ¿P,ATP = ^ r ? p y (1 + 4 • P/O + 1 • 2) - A k f % l y (6 + 10 P/O + 2 • 2) (mmole/g • h)

(4)

(P/O = 1, 2, and 3). Calculation of specific rates (mmole/g • h): The specific rates go (specific glucose uptake rate), hp, Py and &p,Gly (specific rate of pyruvate- and glycogen formation) were calculated according to ?G = =

( g B _ g). D ~ -A

Pv • D X

and ¿P> G I y = (X' = 2 or

0.8418 • S c o r r • Gly D ^ X. Xcorl)

(5) (6) (7)

187

Bistability of E. coli chemostat cultures

Results and

discussion

Regarding experimental data (Tab. 1) is it obvious that X is lower in the higher pyruvate-forming state (B) than in the lower state (A). These differences are mainly due to the different glycogen content of the cells in state A (Gly = 0.20 g/g) and state B (Gly = 0.08 g/g). Accordingly, the differences between the corrected values I A , B (without glycogen) are very small because the concentration of the limiting substrate NH 4 C1 in the reservoir was 1.68 mmole/1 in every case and the differences of uptaken N H 3 are small (Tab. 1). Also the yield coefficients for glucose Yg, calculated with 5A°,B a r e very close, indicating that the previously stated differences in I J / C result mainly from the different glycogen content of the cells. Table 1 List of experimental data (mean values of f i v e independent runs) and calculated parameters, estimated in the steady state of NH 3 -limited, glucose-grown chemostat cultures of E. coli M L 30 at D = 0.150 (0.007) h" 1 . In paranthesis the standard deviations are given (For abbreviations see Materials and Methods). Measured values and calculated parameters cell concentration, X (g/1) pyruvate concentration, P y (mmole/1) N H 3 concentration, N (mmole/1) glucose uptake, G r - G (mmole/1) glycogen content of cells, Gly (g glycogen/g protein) corrected cell concentration, J f c o r r (g/1) specific 0 2 uptake rate of resting cells (mmole/g • h), qo 2 corr qo s &P,Py (mmole/1 • h) ¿pfpy (mmole/I • h) &p,Gly (mmole/1 • h) ^P?Gly (mmole/1 • h) jG (mmole/1 • h) qa" (mmole/1 • h)

YxlG (g/mmole) 7f/g (g/mmole) Fx/N (g/mmole) YXIN (g/mmole)

state A 0.156

state B

(0.003) 0.230 (0.020) 0.0183 (0.0038) 4.99 (0.38)

0.140 2.024 0.0524 4.69

(0.002) (0.165) (0.0166) (0.34

0.201 0.133

0.085 0.130

(0.002)

10.1 11.9 0.221 0.259 0.135 0.158 4.80 5.62 0.0313 0.0267 0.0939 0.080

(0.018)

7.3 7.9 2.17 2.33 0.0622 0.0670 5.03 5.41 0.0299 0.0277 0.0860 0.080

Accumulation of glycogen in the cells is a hint to a high energy production of the cultures (CHAPMAN et al. 1 9 7 1 , D I E T Z L E R et al. 1 9 7 9 ) . Consequently, it can be assumed that in the case of low pyruvate formation the pyruvate is metabolized by the citric acid cycle, leading to an extra production of energy and glycogen, respectively. This conclusion is supported by the finding of a higher respiration rate in resting cells taken from state A. Corresponding to these results it was shown that a chemostat culture in the state A takes up added pyruvate rapidly whereas a culture in state B does not metabolize added pyruvate (Fig. 1). For a rough valutation of the difference in the ATP-production rates between the two states it was assumed that the differences of pyruvate and glycogen, respectively, flow into the citric acid cycle leading to energy ( A T P ) formation. 13*

188

P. J. MÜLLER and BEATE VON FROMMANNSHAUSEN

Fig. 1. Kinetic of pyruvate after adding of Na-pyruvate to glucose-grown, NH 3 -limited chemostat cultures of D = 0.15 h" 1 . The cultures are in a steady state (state B ) with high pyruvate production before adding pyruvate ( o ) or in a state (state A ) with low pyruvate production ( • ) . The full lines show the theoretical washing out kinetic of pyruvate

In state A 0.091 mmole/g • h glycogen ( = Aip°Gi y ) are g - h pyruvate ( = zd£p°py) are produced less than in are calculated with equation (4) using a P/O = 3 to (with P/O = 3), = 20.05 mmole/g • h (with P/O = P/O = 1).

produced more and 2.071 mmole/ state B. Energetic differences be ¿ p " T r = 27.43 mmole/g • h 2), and 12.68 mmole/g • h (with

A P/O = 0.89 in glucose-limited chemostat cultures of E. coli (HEMPFLING and MAINZER 1975) was discussed in conection with a specific influence of glucose lowering the energetic efficiency. These energetic differences are in the range of the specific ATP-production rate &P,ATP, calculated with the usual value FX/ATP = 0.01 (g dry weight/g mmole A T P ) (BAUCHOP a n d ELSDEN 1960, STOUTHAMEK a n d BETTENHAUSEN 1973, HEMPFLING a n d MAINZER 1975) a t D = 0.15 - 1 a c c o r d i n g t o

•kp.ATp =

-'ATP

¡^

=

n

r • 0.15 = 15 mmole • g _ 1 • h _ 1

0.1

(8)

The energy overproduction in state A seems not to be necessary for growth and maintenance of the culture. Thats why it is concluded that in state A the energy overproduction is consumed, besides by glycogen formation by additional energy dissipating processes, for example by so-called futile cycles (KATZ and ROGNSTAD 1976, TEMPEST 1978) g e n e r a t i n g h e a t .

Recently DIETZLER et al. (1979) described the occurrence of distinct periods of glycogen accumulation and glucose uptake during prolonged nitrogen starvation in E. coli batch cultures. During each period the rates of both glycogen synthesis and glucose utilization are constant. I t can be proposed that the bistability in pyruvate and glycogen formation in slowly growing NH 3 -limited chemostat cultures and the occurrence of the distinct periods in glucose uptake and glycogen formation are due to a common regulation mechanisms.

Bistability of E. coli chemostat cultures

189

Regarding the bistability in glucose- and energy metabolism cyclic AMP has been shown to posses a regulatory role (MÜLLER and RÖMER, 1 9 8 2 ) .

References T. a n d E L S D E N , R . , 1 9 6 0 . The g r o w t h of micro-organisms in relation t o t h e i r energy supply. J . gen. Microbiol., 23, 457—469. B E R G T E R , F . u n d R O T H , M., 1 9 7 7 . Bistabilität der P y r u v a t p r o d u k t i o n von Escherichia coli ML30 in kontinuierlicher K u l t u r . Z. Allg. Mikrobiol., 17, 3—6. B E R G T E R , F . , S C H U M A N N , H . u n d K O B U R G E R , M., 1 9 7 7 . Abhängigkeit der spezifischen W a c h s t u m s r a t e von der A m m o n i u m k o n z e n t r a t i o n bei Escherichia coli ML 30. Z. Allg. Mikrobiol., 17, BAUCHOP,

183 — 189.

A. G . , F A L L , L . a n d A T K I N S O N , D . E . , 1971. Adenylate energy charge in Escherichia coli during g r o w t h a n d s t a r v a t i o n . J . Bacteriol., 108, 1072 — 1075. CZOK, R . a n d LAMPRECHT, W., 1974. I n : " M e t h o d e n der enzymatischen A n a l y s e " ( E d i t o r : H . U . BERGMEYER), 3. Auflage, 1974. Verlag Weinheim/Bergstraße, p. 1497. D I E T Z L E R , D . N . , C . J . L A I S a n d M . P . L E C K I E , 1974. Simultaneous increases of t h e a d e n y l a t e energy charge a n d t h e r a t e of glycogen synthesis in nitrogen-starved Escherichia coli W 4597 (K). Arch. Biochem. Biophysics, 160, 14—25. CHAPMAN,

DIETZLER, D . N . , L E C K I E , M . P . , STERNHEIM, W . L . , UNGAR, J . M . , CRIMMINS, D . L . a n d

LEWIS,

J . W., 1979. R e g u l a t i o n of glycogen synthesis a n d glucose utilization in Escherichia coli during m a i n t e n a n c e of t h e energy charge. Q u a n t i t a t i v e correlation of changes in t h e r a t e s of glycogen synthesis a n d glucose utilization w i t h simultaneous changes in t h e cellular levels of b o t h glucose 6-phosphate a n d f r u c t o s e - l , 6 - d i p h o s p h a t e . J . biol. Chemistry, 2 5 4 , 8 2 7 6 — 8 2 8 7 . DIETZLER, D . N . , LECKIE, M . P . , LEWIS, J . W . , PORTER, S. E . , TAXMAN, T . L . a n d LAIS, C. J . ,

1979. Evidence of new f a c t o r s in t h e coordinate regulation of energy metabolism in Escherichia coli. E f f e c t of hypoxia, chloramphenicol succinate, a n d 2,4-dinitrophenol o n glucose utilization, glycogen synthesis, a d e n y l a t e energy charge, a n d hexose p h o s p h a t e s d u r i n g t h e first two periods of nitrogen s t a r v a t i o n . J . biol. Chemistry, 254, 8295—8307. F O R C H H A M M E R , J . a n d L I N D A H L , L . , 1 9 7 1 . G r o w t h r a t e of polypeptide chains as a f u n c t i o n of t h e cell g r o w t h r a t e in a m u t a n t of Escherichia coli 1 5 . J . Mol. Biol., 55, 5 6 3 — 5 6 8 . HEMPFLING, W . P . a n d MAINZER, S. E., 1975. E f f e c t s of varying t h e carbon source limiting g r o w t h on yield a n d m a i n t e n a n c e characteristics of Escherichia coli in continuous culture J . Bacteriol., 123, 1 0 7 6 - 1 0 8 7 . H O L M E , T . , 1958. Glycogen f o r m a t i o n in continuous culture of E. coli B . I n : "Continuous Cultivation of Microorganisms" ( E d i t o r : I. MALEK). Publishing House of t h e Czechoslovak Academy of Science, P r a g u e 1958, p. 67. K A T Z , J . a n d R O G N S T A D , R., 1976. F u t i l e cycles in t h e metabolism of glucose. I n : " C u r r e n t Topics in Cellular R e g u l a t i o n " ( E d i t o r s : B . L. H O R A C K E R a n d E. R . S T A D T M A N ) . Academic Press New York a n d London, Vol. 10, p. 238. 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 . , 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 F O L I N phenol r e a g e n t . J . biol. Chemistry, 1 9 3 , 2 6 5 — 2 7 5 . MÜLLER, P . J . u n d BERGTER, F . , 1977. U n t e r s u c h u n g e n zum transient-Verhalten ammoniumlimitierter C h e m o s t a t e n k u l t u r e n von Escherichia coli ML 30 n a c h s p r u n g h a f t e r E r h ö h u n g der V e r d ü n n u n g s r a t e . Z. Allg. Mikrobiol., 17, 131 — 137. M Ü L L E R , P . J . , IVANOVA, I . I . u n d B E R G T E R , F . , 1 9 7 7 . Bistabilität in der A k t i v i t ä t der Glutaminsynthese bei a m m o n i u m l i m i t i e r t e n C h e m o s t a t e n k u l t u r e n von Escherichia coli ML 30. Z. Allg. Mikrobiol., 1 7 , 2 2 1 - 2 2 5 . M Ü L L E R , P . J . a n d B E R G T E R , F., 1 9 7 8 . Bistability in t h e NH 3 -assimilating p a t h w a y s in Escherichia coli ML 3 0 c h e m o s t a t culture. Abstracts of t h e 12TH F E B S Meeting, Dresden, 1 9 7 8 , No. 3 9 3 0 . M Ü L L E R , P . J . , von F R O M M A N N S H A U S E N , B . a n d S C H Ü T Z , H . , 1981. Regulation of a m m o n i a assimilation in a m m o n i a - l i m i t e d c h e m o s t a t cultures of Escherichia coli ML 30: Evidence of bistability. Z. Allg. Mikrobiol., 21, 361—372. M Ü L L E R , P . J . a n d R Ö M E R , W . , 1 9 8 2 . Regulation of bistability in glucose metabolism of Escherichia coli M L 3 0 c h e m o s t a t cultures b y cyclic A M P . Z . Allg. Microbiol., 2 2 , 2 1 1 — 2 1 4 . PREISS, J . , 1969. T h e regulation of t h e biosynthesis of 1,4-glucan in bacteria a n d plants. I n : " C u r r e n t Topics in Cellular R e g u l a t i o n " ( E d i t o r : B . L. H O R E C K E R a n d E . R . S T A D T M A N ) . Academic Press New York a n d London, Vol. 1, p. 125.

190

P . J . M U L L E R a n d B E A T E VON FROMMANNSHATJSEN

SIGAL, N., CATTANEO, J. and SEGEL, J. H., 1964. Glycogen accumulation by wild type and uridine-diphosphat glucose pyrophosphorylase negative strains of Escherichia coli. Arch. Biochem. Biophysics, 108, 442. STOTJTHAMER, A. H. and BETTENHATJSEN, C., 1973. Utilization of energy for growth and maintenance in continuous and batch culture of microorganisms. A réévaluation of the method for the determination of A T P production by measuring molar growth yields. Biochim. biophysica A c t a , 301, 5 3 — 7 0 .

TEMPEST, D. W., 1978. The microbial significance of microbial growth yields: a reassessment. Trend Biochem. Sci, 180 — 184. Mailing adress : Dr. P. J. MÙLLER Zentralinstitut fiir Mikrobiologie und experimentelle Therapie der A d W D D R 6900 Jena, Beutenbergstrafie 11

Zeitschrift für Allg. Mikrobiologie

22

1982

191-196

(Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, India)

Spore differentiation in relation to certain antibiotics in the blue-green alga Nodularia spumigena M E R T E N S R . K . PANDEY a n d E . R . S. TALPASAYI

(Eingegangen

am 1.

7.1981)

Induction of spore differentiation is achieved within three days in Nodularia spumigena by incubating the cultures at 35 °C in the light. Morphologically detectable sporulation and spore germination could not occur in the presence of chloramphenicol, streptomycin and penicillin. B u t chloramphenicol-supplemented cultures developed prominent cyanophycin granules. Synthesis of these granules seems to be a non-ribosomal phenomenon.

Extremely little information exists regarding the factors which control differentiation of spores in blue-green algae (cyanobacteria). JENSEN and CLARK (1969) considered the infrastructure of the blue-green algal spore to be similar to Azotobacter cysts. But blue-green algae spores invariably contain a large number of cyanophycin and arginine (SIMON 1971). These granules range in size from 25000 to 100000 daltons and are found only in blue-green algae. Antibiotics are commonly used for the isolation of drug-resistant strains for studies on genetic recombination and transformation in blue-green algae (see LADHA and KUMAR 1978). But nobody has so far critically examined the effect of antibiotics on spore differentiation, partly because of the absence of an efficient induction method for sporulation. The present study was undertaken after formulating an induction medium for spore differentiation in Nodularia spumigena. The effect of chloramphenicol, streptomycin and penicillin on the formation and germination of spores is reported here. Materials

and

methods

Nodularia spumigena is a filamentous, heterocystous and unbranched blue-green alga which inhabits the stagnant water of rice fields. This alga is regarded as a synonym of Nostoc spumigena by DKOXJET (1978). In this alga, series of spores appear simultaneously between two heterocysts, and three stages can be distinguished during spore differentiation. First, the appearance of prominent granules. Second, the enlargement of vegetative cell, and lastly the development of a thick brown envelope around the cell. The culture methods were the same as described by PAND E Y and T A L P A S A Y I (1980). Sporulation studies were conducted in liquid medium. For the determination of sporulation frequency a minimum of 1,000 cells were counted microscopically. A cell was considered to be a spore if it was found enlarged and packed with granules. Germination studies were conducted on nutrient agar plates. Diluted spore suspension was spread and incubated in the light in the culture room. At 24 hr intervals the germinating spores were scored. Germination of the spore was identified by the protrusion of young germlings from the spore coat. The number of germinated and ungerminated spores present in 25 arbitrarily chosen microscopic fields was noted and per cent germination was calculated. Stock solutions of chloramphenicol, streptomycin sulfate and penicillin were prepared separately under aseptic conditions. The required amounts were obtained after appropriate dilutions of the respective stock in sterile distilled water and different volumes were added to the culture media to obtain the final concentration ranging from 0.1 to 1 [xg/ml.

192

R . K . P A N D E Y a n d E . R . S . TALPASAYI

SAKAGUCHI'S test for arginine (FOGG 1 9 5 1 ) was employed to stain cyanophycin granules which could be isolated from chloramphenicol-treated filaments by sonication. The amino acid composition of the isolated granules was determined after hydrolysis according to SIMON ( 1 9 7 3 ) .

Results Spore differentiation Fig. 1 shows sporulation of N. spumigena cultures grown at 25, 30 and 35 °C. Ordinarily, at the usual culture room temperature (25 °C), N. spumigena sporulates after 12 days of incubation in continuous light. This time necessary for spore formation could be considerably reduced between 30 and 35 °C where sporulation occurred within 3 days. The effects of various factors on the induction of sporulation in N. spumigena have already been described ( P A N D E Y and T A L P A S A Y I 1 9 8 0 ) .

Incubation period

(days)

Fig. 1. Effect of temperature on spore formation • 25 °C, A 30 °C, A 35 °C

E f f e c t of a n t i b i o t i c s on s p o r u l a t i o n Chloramphenicol, streptomycin and penicillin were added to freshly inoculated liquid cultures which were incubated at 35 °C. None of these antibiotics permitted sporulation to proceed even at the lowest concentration (1 fig/ml). Streptomycin and penicillin inhibited sporulation in as low as 0.1 ¡xg/ml concentration'. Chloramphenicol inhibited when more than 0.2 [i.g/ml was added. In the presence of 0.1 ¡I.g/ml of chloramphenicol, sporulation was the same as in the control. But none of the spores formed in the presence of chloramphenicol (0.1 pig/ml) had brown envelopes like in the control where most of the spores developed brown envelopes after 3 days of incubation at 35 °C in the light. Though the formation of spores was inhibited in the presence of chloramphenicol (0.2—1 ¡jig/ml), the vegetative cells were packed with granules prominent under the light microscope. I t appeared that chloramphenicol hastened the synthesis of these granules. The amino acid composition of the granules formed in presence of chloramphenicol was determined after their isolation and hydrolysis. The qualitative picture of the gross amino acid composition appearing on the chromatogram was that of aspartic acid and arginine.

193

Spore differentiation in Nodularia

Reversal of inhibition of sporulation by chloramphenicol was studied by washing off the antibiotic at different time intervals after adding it at 0 hr. Table 1 shows that as soon as chloramphenicol was withdrawn during 0 to 36 hrs, spore development proceeded uninterruptedly. Chloramphenicol inhibition was seen only if the antibiotic was added before 36 hrs. But filaments accumulated lots of cyanophycin granules in the presence of chloramphenicol at whatever stage it was added. Table 1 Effect of chloramphenicol (1 ¡xg/ml) on spore differentiation Chloramphenicol added at hrs

Results after 72 hrs Chloramphenicol added at 0 hr but washed off at different time periods and result after 72 hrs

0 hrs

4 hrs

Gr (-)

Gr (-)

N.A.

(+)

12 hrs

24 hrs

Gr (-)

Gr (-)

Gr (-)

(+)

(+)

(+)

8 hrs

36 hrs Gr

(+)

(+)

Cultures were incubated at 35 °C in light. Gr.: Cells packed up with prominent granules (cyanophycin) —): Spores not formed + ): Spores formed

E f f e c t of a n t i b i o t i c s on s p o r e g e r m i n a t i o n Table 2 shows the effect of chloramphenicol, streptomycin and penicillin on germination of spores. There was no sign of germination in any of the agar plate containing even the lowest concentration (0.2 ¡jig/ml) of antibiotics after 3 days of incubation when the control showed more than 80% germination. This observation clearly demonstrated that the antibiotic inhibited germination. The same agar plates were observed at regular intervals up to 10 days, and after 5 days, germination was found in all the plates containing up to 0.6 [xg/ml of chloramphenicol or streptomycin. Penicillin was most effective since the germination process did not proceed further after the greening of spores occurred in plates with more than 0.2 ¡i.g/ml of this drug. This step, where the spores turn green before the emergence of protuberance, was not inhibited by penicillin, while effective concentrations of streptomycin and chloramphenicol even inhibited the greening of spores. In another set of experiments the antibiotics were used in a final concentration of 1 [Ag/ml. These antibiotics were added after 4, 8, 12, 16, 20, and 24 hrs of plating and incubation of spores in the light so as to find the initial period required to attain antibiotic resistance, if any, in spore germination before the emergence of the germling. I t was found that the three antibiotics employed inhibit germination, even if added after 24 hrs of incubation of spores in the light. However, chloramphenicol was not inhibitory if added after 30 hrs of incubation of spores in the light. Reversal of inhibition was studied by the addition of antibiotics at 0 hr, and after different time intervals a portion of the liquid spore suspension was centrifuged and the spores were washed using sterile distilled water thrice and plated on agar surfaces containing basal medium. Spores germinated if chloramphenicol or penicillin were withdrawn. But spores treated with streptomycin failed to germinate even when the streptomycin was washed off (Table 3). Thus inhibitory effects of streptomycin were not reversible.

194

E . K . P A N D E Y a n d E . R . S. T A L P A S A Y I

Table 2 Effect of antibiotics on germination of spores Germination % after 3 days

5 days

7 days

9 days

82

87

87

87

00 00 00 00 00 00 00 00 00 00 00 00

52 37 32 00 46 32 29 00 00 00 00 00

55 51 50 00 50 41 38 00 12 00 00 00

55 51 50 00 50 44 41 00 44 00 00 000

Control (without antibiotics) Chloramphenicol (¡xg/ml) 0.2 0.4 0.6 0.8 Streptomycin 0.2 0.4 0.6 0.8 Penicillin 0.2 0.4 0.6 0.8

Antibiotics were added in complete growth medium and kept at 25 °C in light. Values represent rounded means of three replicate counts Table 3 Effect of antibiotics on germination and reversal of inhibition Antibiotics withdrawn after

Control Chloramphenicol Streptomycin Penicillin

4 hrs

8 hrs

12 hrs

87 87 00 87

87 87 00 87

87 87 00 87

Antibiotics were added (1 ¡¿g/ml) at 0 hr and washed off after 4, 8, 12 hrs. The cells were resuspendend in growth medium. Germination was scored after 72 hrs in the light at 25 °C. Values represent rounded means of three replicate counts

Discussion The prokaryotic nature of blue-green algae and their similarity to other Gramnegative bacteria is now one of the clear aspects. In addition, obligate autotrophy is found in these organisms. I N G R A M et al. ( 1 9 7 2 ) observed the appearance of numerous granules in the cells of Agmenellum quadruplicatum on the addition of chloramphenicol. Later, S I M O N ( 1 9 7 3 ) reported the accumulation of cyanophycin granules even in the presence of that a concentration of chloramphenicol which totally inhibited protein synthesis. Since chloramphenicol binds to 50 S subunits of 70 S ribosomes (ZAHN E E and M A S S 1 9 7 2 ) , it can be safely assumed that the synthesis of cyanophycin granules is a non-ribosomal phenomenon. Though morphologically detectable spores did not appear in chloramphenicol-supplemented cultures of N. spumigena, an increase in the size and number of cyanophycin granules was very clear under the light microscope. This increase in cyanophycin granules was not quantitatively estimated in this study but it can be stated without hesitation that chloramphenicol served as a very good trigger for the development of cyanophycin granules.

Spore differentiation in Nodularia

195

The action of chloramphenicol was also compared in this study with streptomycin which binds specifically to 30 S subunits of rihosomes ( T R A U B 1969). Cyanophycin granules were not detectable in streptomycin-treated cultures. Also, in the presence of penicillin, which is an inhibitor of cell wall synthesis in bacteria, being a substrate analogue for the enzyme involved in the termination reaction of bacterial peptidoglycan synthesis ( S T R O M J N G E R 1969), there were neither sporulation nor cyanophycin granules. In respect to the sensitivity of the germination process to chloramphenicol, streptomycin and penicillin, K U M A B and K A U S H I K (1971) noted differential sensitivity in Anahaena doliolum and Fischerella muscicola, where penicillin appeared 400 times more effective than the other two antibiotics. Spore germination in N. spumigena as found in this study, could not occur in the presence of chloramphenicol, streptomycin and penicillin. The type of differential sensitivity displayed by A. doliolum and F. muscicola was not seen in N. spumigena. When greening of spores is recognized as the first sign of germination, it is found that chloramphenicol and streptomycin inhibited even greening of spores and in this respect, these were more effective than penicillin, which did not inhibit this step. The extreme sensitivity of spore germination to these antibiotics is evidenced by the fact that even if these antibiotics were added after 4, 8, 12, 16, 20 or 24 hrs of incubation of spores in the light, the germination process was inhibited. Reversal experiments show that spores germinate when chloramphenicol or penicillin are withdrawn, but the inhibitory effects of streptomycin are not reversible. A total suppression of sporulation and reduced germination of spores at different concentrations of the antibiotics invariably indicate that fresh protein synthesis is necessary for the complete expression of spore differentiation or germination. However, the synthesis of cyanophycin granules which may be considered the first step towards spore differentiation, is found to be independent of protein synthesis. A

cknowledgements

This work was done during the tenure of a Research Fellowship under the Special Assistance Programme and forms a part of the Ph. D. thesis of R. K. P. We thank the Head of the Botany Department, Banaras Hindu University, for laboratory facilities and U. G. C. for financial assistance.

References 1 9 7 8 . Revision of the Nostocaceae with constricted trichomes. J . Cramer, Vaduz Liechtenstein. FOGG, G. E., 1951. Growth and heterocyst production in Anabaena cylindrica. The cytology of heterocysts. Ann. Bot., 15, 23—35. INGRAM, L. O., THUBSTON, E . L. and VAN BAALEN, C., 1972. Effects of selected inhibitors on growth and cell division in Agmenellum. Arch. Mikrobiol., 81, 1—12. JENSEN, T. E. and CLARK, R. L., 1969. Cell wall and coat of the developing akinete of a Cylindrospermum species. J . Bacterid., 97, 1494—1495. KUMAR, H . D . and KAUSHIK, M . , 1 9 7 1 . Studies on growth and development of two nitrogen fixing blue-green algae. II. Effect of antibiotics. Z. Pflanzen., 66, 443—452. LADHA, J . K . and KUMAR, H . D . , 1 9 7 8 . Genetics of blue-green algae. Biol. Rev., 5 3 , 3 5 5 — 3 8 6 . PANDEY, R. K. and TALPASAYI, E . R. S., 1980. Control of sporulation in a blue-green alga Nodularia spumigena MERTENS. Indian J . Bot., 3, 128 —133. SIMON, R. D., 1971. Cyanophycin granules from the blue-green alga Anahaena cylindrica: a reserve material consisting of co-polymers of aspartic acid and arginine. Proc. Natl. Acad. Sci. USA, 68, 265—267. SIMON, R. D., 1973. The effect of chloramphenicol on the production of cyanophycin granules polypeptide in the blue-green alga Anabaena cylindrica. Arch. Mikrobiol., 92, 115 —122. DROUET, F . ,

196

R. K . Pakdey and E. R. S. Talpasayi

Strominger, J. L., 1969. In: "Inhibitors—Tools in Cell Physiology Research" (Eds. Bttcher & Sies). Springer Verlag Berlin, pp. 187—207. Traub, P., 1969. In: "Inhibitors—Tools in Cell Physiology Research" (Eds. Bucher & Sies). Springer Verlag Berlin, pp. 79—99. Zahner, H. and Mass, W. K., 1972. Biology of Antibiotics, Springer Verlag New York. Mailing address: Dr. R. K. Pandey Regional Tasar Research Station Batote, 182 143, Jammu and Kashmir, India

Zeitschrift für Allg. Mikrobiologie

22

1982

197-203

(Fachrichtung Mikrobiologie der Universität des Saarlandes, Saarbrücken)

Regulation by repression of urease biosynthesis in Proteus rettgeri C. ZOEN, R . DIETRICH a n d H . KALTWASSER

(Eingegangen

am 20. 8.

1981)

Measuring the specific enzyme activity in cells of Proteus rettgeri it was shown t h a t urease formation is controlled by repression through ammonia. Derepressed synthesis of the enzyme, as initiated by the absence of ammonia, required an external nitrogen source, which m a y not only be urea, but also nitrate, glutamate or nutrient broth. In contradiction to earlier reports the observations indicated t h a t urea is not required for the synthesis of this enzyme, and t h a t , therefore, urease is not an inducible enzyme in this microorganism.

A considerable number of bacteria exhibit the capacity to form the hydrolytic enzyme urease (urea amidohydrolase [ E C 3 . 5 . 1 . 5 . ] ) and are therefore able to grow at the expense of urea as the sole nitrogen source. The presence of this enzyme, which is of taxonomic importance within the Enterobacteriaceae and other groups, is commonly demonstrated by means of urea-containing indicator media (STUART et al. 1 9 4 5 , CHRISTENSEN 1 9 4 6 ) . In most species studied so far, urease formation is subject to metabolic control ( D E T U R K 1 9 5 5 , K R Ä M E R et al. 1 9 6 7 , K A L T W A S S E R et al. 1 9 7 2 , MALOFEEVA 1 9 7 9 ) and in some bacteria the mechanism of this regulation was postulated to be an induction by urea. In the well known study of the mechanism of regulation of urease biosynthesis by MAGAÑA-PLAZA and R U I Z - H E R R E R A ( 1 9 6 7 ) , these authors came to the conclusion that "urease of Proteus rettgeri is an inducible enzyme synthesized specifically in the presence of urea". In recent investigations, however, urease formation was shown to occur in the absence of external nitrogen sources in strains of Hydrogenomonas, Bacillus and Pseudomonas, in Alcaligenes eutrophus, Paracoccus (Micrococcus) denitrificans (KRÄMER et al. 1 9 6 7 , KALTWASSER et al. 1 9 7 2 , J A N S S E N et al. 1 9 8 1 ) , in Klebsiella aerogenes (FRIEDRICH and MAGASANIK 1 9 7 7 ) and in Selencmonas ruminantium (SMITH et al. 1 9 8 1 ) , and thus to be initiated by derepression. Urease was present in these cells, when grown in the absence of its substrate, and thus the enzyme appeared not to be subject to induction by urea; its synthesis was rather controlled by repression, excerted by the reaction product, ammonia. During the present study urease formation was investigated under nitrogen limitation and in presence of urea and other nitrogen compounds, in order to gain further insight into the mechanism of urease formation in Proteus rettgeri. Materials

and

methods

Strain and media: The bacterial strain used throughout this study was obtained as a lyophylized culture from t h e Institut für Mikrobiologie der Universität Göttingen. The cells were grown a t 28 °C in a basal glycerol mineral medium (pH 7.2), containing per liter: 9 g N a 2 H P 0 4 • 12 H 2 0 , 1.5 g K H 2 P 0 4 , 0.2 g MgS0 4 • 7 H 2 0 , 1.2 mg ferric ammonium citrate, 2 0 mg CaCl 2 • 2 H 2 0 and 4.6 g glycerol. F o r stock cultures 2 0 g agar (DIFCO) and 1 g urea were added; urea was sterilized by filtration. In liquid media, urea was used a t various concentrations or replaced by other ni-

198

C. ZORN, R . DIETRICH a n d H .

KALTWASSEB

trogen sources, as otherwise indicated. For mass culturing, 3 1 of inoculated medium were aerated with air slowly passed through the vessel and distributed by a magnetic stirring bar revolving a t 6 0 0 r e v / m i n , a s d e s c r i b e d b y SCHLEGEL et al. ( 1 9 6 1 ) .

Induction experiments: In order to obtain precultures of cells containing low urease levels, cells were grown in glycerol mineral medium containing 18 mmol/1 ammonium chloride. After 1 0 to 1 4 h of incubation, the cells were harvested in a SORVALL R C 2 B centrifuge a t 4 ° C and 6 0 0 0 rpm, washed twice in sterile 0 . 0 6 6 M phosphate buffer (pH 7 . 4 ) and used to inoculate the medium. At regular intervals samples were taken for t h e determination of turbidity, concentration of protein, urea, ammonia and specific urease activity. Analytical procedures: Turbidity was measured at 436 nm in 1 cm cuvettes within the O.D. range between 0 and 0 . 3 , using a photometer S P E C T R O N I C 8 8 . Protein was determined according to S C H M I D T et al. ( 1 9 6 3 ) and urea according to M O O R E and K A U F M A N ( 1 9 7 0 ) . Ammonium was measured colorimetrically a t 546 nm, following conversion to indophenol, as described in the " B O E H R I N G E R Testfibel" ( 1 9 7 6 ) . Preparation of cell extracts: Washed cells were resuspended in phosphate buffer (2 ml/g wet weight) a t 0 °C and sonicated with a B R A N S O N Sonifier B 12 for one min per each ml a t t h e fullest output possible, when using t h e microtip. Unbroken cells and crude particles were removed by centrifugation a t 10000 g a t 4 °C. Specific urease activity: Urease was measured in most of t h e experiments by means of the coupled enzyme assay described by K A L T W A S S E R and S C H L E G E L (1966) at the optimum p H value of 7.66. In some other experiments the liberation of radioactive carbon dioxide from 14 C-urea was determined, using conventional W A R B U R G vessels, which contained the reaction mixture in the main compartment, and 0.2 ml K O H (220%) in the center well. The reaction was started by tipping 61 ¡/.moles of 14 C-urea from one of t h e side arms. After 2 to 5 min of incubation 0.2 ml of 20% phosphoric acid was added from the other side arm; subsequently the reaction mixture was shaken for 30 min in order to remove the evolved carbon dioxide. From the center well 0.05 ml K O H were transferred into 10 ml of the scintillation fluid described by SISSON (1976). Radioactivity was determined using an ISOCAP 300 scintillation counter.

Results

The e f f e c t of n i t r o g e n d e f i c i e n c y a n d u r e a s e f o r m a t i o n The effects of nitrogen deficiency and of urea on urease formation was studied in cells, which had been precultured with an excess of ammonia and therefore exhibited only low urease levels. When incubated in the absence of any nitrogen source, a small but measurable increase in urease content was detected in these cells (Fig. la). In other microorganisms under these conditions, a more pronounced derepressive urease formation was observed, which obviously occurred at the expense of stored nitrogen. In order to demonstrate such derepressed enzyme synthesis in Proteus rettgeri, it was investigated whether external nitrogen sources are able to stimulate urease formation. When urea was offered at low concentrations (Fig. 1) it was used up during the experiment and its consumption coincided with a temporary increase in urease level. Although, this might appear to be an induction by urea, it has to be pointed out, that highest levels of urease were observed in those cells to which lowest amounts of urea were available. No free ammonia was measurable under these conditions. As urea concentration was increased, more ammonia was liberated which, in turn, repressed further urease synthesis. This observation indicated that urea was serving as a nitrogen source for the synthesis of the enzyme, but not acting as an inducer in these Proteus rettgeri cells. Like in other microorganisms, urease formation was controlled by ammonia-dependent repression. This view was further supported by the experiments shown in Fig. 2. At high urease concentrations (7 mmol/1), urease formation was repressed by liberated ammonia after about 4 h, at which time large amounts of urea were still present in the medium. The available urea had obviously no inducing effect in presence of ammonia. This observation was also demonstrated in Fig. 3, when cells

Depressive urease formation in Proteus

rettgeri

199

were incubated with both ammonia and urea. Under these conditions, urease formation remained repressed as long as ammonia was available, despite the presence of urea.

4 6 lime (h) Pig, 2 Fig. 1. Increase in urease content of Proteus rettgeri cells during the incubation in nitrogen-free medium and in presence of urea at low concentrations Cells low in urease content were obtained from ammonia-containing glycerol mineral medium, washed twice and transferred into flasks containing two liters of glycerol mineral medium a = specific urease activity; b = ammonia concentration and c = urea concentration of the supernatant medium; d = protein content of the suspension Fig. 2. Formation of urease in Proteus rettgeri in presence of urea at increasing concentrations. Experimental conditions as in Fig. 1

U r e a s e f o r m a t i o n in p r e s e n c e of n i t r a t e , g l u t a m a t e a n d n u t r i e n t b r o t h I n previous experiments it was shown that urea was able to serve as a nitrogen source for urease synthesis. The following experiments demonstrated t h a t derepressed urease formation might also be supported by nitrate as a nitrogen source. In the experiment shown in Fig. 4, urease-containing cells were incubated in presence of both ammonia and nitrate. As long as ammonia was present, it was utilized for cellular growth, and it repressed urease synthesis, as can be seen from the decrease

200

C . ZORN, R . D I E T R I C H a n d H . K A L T W A S S E R

x — - x Protein

1

1

o—o

Turbidity

V3

/

-0.5

6

8

Time (h)

ft

Fig. 3. Repression of urease synthesis in Proteus rettgeri by ammonia in presence of urea. Cells low in urease content were incubated in glycerol mineral medium containing 8 mol ammonium chloride and 6 mmol of urea per liter. Other experimental conditions as in Fig. 1

Fig. 4. Derepressive formation of urease at the expense of nitrate in Proteus rettgeri. Urease was determined by means of 14 C-urea. Other experimental conditions as in Fig. 1

in specific enzyme activity. After the consumption of ammonia, however, the cells proceeded to grow and then started to produce urease at the expense of nitrate. No urea was present at all during this experiment on urease biosynthesis. Similar results were obtained when both ammonia and glutamate (Fig. 5), ammonia and nu-

201

Time (h) Fig. 5. Derepressive formation of urease at the expense of glutamate as nitrogen source in Pro tews rettgeri. Experimental conditions as described in Pig. 4

o

Jme (h) Fig. 6. Derepressive formation of urease with nutrient broth as nitrogen source in Proteus Experimental conditions as described in Fig. 4

rettgeri.

trient broth (Fig. 6) or peptone (ZORN 1979) were offered. According to these observations, the mechanism of regulation of urease biosynthesis is that of a repression by ammonia. The derepressed synthesis of the enzyme, initiated in the absence of ammonia, can be supported by various nitrogen sources, such as urea, nitrate, glu14

Z. Allg. Mikrobiol., Bd. 22, H.3

202

C . ZORN, R . D I E T B I C H a n d H . K A L T W A S S E K

tamate and nutrient broth. Urea, however, is not specifically involved as an inducer during the formation of urease in Proteus rettgeri. Discussion During experimental studies on urease formation it has to be considered that the synthesis of urease, like that of other proteins, requires a nitrogen source. Presumably for this reason, the biosynthesis of this enzyme was not studied in absence of external nitrogen sources and therefore its derepressed biosynthesis was not detected in earlier studies in Proteus rettgeri (MAGAÑA-PLAZA and RTTIZ-HERRERA 1967) and in Paracoccus (Micrococcus) denitrificans (KLECZKOWSKI et al. 1966). In these strains urease formation was subject to repression by ammonia, just as in three species of Mycobacterium, studied by IWAINSKY and SEHKT (1971). These authors also came to the conclusion that urease is inducible. They had noticed, however, that urease was not only formed in presence of urea, but also with certain amino acids, acetamide and other nitrogen sources. Therefore, like in Proteus rettgeri, repression rather than induction may be the mechanism controlling urease formation in these mycobacteria. In Pseudomonas fluorescens and Alcaligenes eutrophus (KALTWASSER et al. 1972) high levels of urease were measured after the cells had been incubated in the absence of any nitrogen source. In these strains, endogenous nitrogen compounds were obviously mobilized under nitrogen deficiency, allowing urease biosynthesis. This, however, does not seem to be the case in all microorganisms. In Proteus rettgeri (ZORN 1979) and in Bacillus strains (DIETRICH, unpublished) the derepressed synthesis of urease appeared to require external nitrogen compounds, such as nitrate, urea or peptone. Following the hydrolytic cleavage, urea does, of course, also serve as nitrogen source. The resulting ammonia subsequently leads to a temporary repression of enzyme synthesis and thus to oscillating changes in specific urease activity, as shown in Alcaligenes eutrophus (KONIG and SCHLEGEL 1967). In Proteus rettgeri only small amounts of urease were formed in absence of an external nitrogen source. The slight increase, which was observed, ceased after about 2 h of incubation at the level of 40 units per gram of protein (ZORN 1979). Urease synthesis, however, proceeded when nitrogenous compounds were offered in absence of ammonia. Not only urea, but also nitrate, glutamate, nutrient broth and peptone were able to support this enzyme biosynthesis, indicating that urea was not specifically involved as an inducer, but rather served as a nitrogen source during derepressed enzyme synthesis. Although the strain studied by MAGAÑA-PLAZA and RITIZ-HERRERA (1967) is not available for comparative studies, it might be mentioned that neither the effect of nitrogen starvation nor that of nitrate was studied by these authors. I t may therefore be assumed that, similar to other bacteria, derepressed rather than induced formation of urease occurs in Proteus rettgeri. The enzyme urease is a virulence factor in urinary-tract infections with Proteus (ROSENSTEIN et al. 1980, SENIOR et al. 1980), and the ability to form this enzyme is of taxonomic significance. In simplified test methods, ammonia evolution from urea is utilized to indicate the presence of urease in bacteria. Repeatedly these tests turned out to be unsuitable (STEWART 1965, KRAMER et al.

1967, VUYE a n d PIJCK 1973).

Since free amonia represses urease formation, it is obvious that, during growth, the ability to form the enzyme may remain undetected with such techniques. Knowledge of the mechanism of regulation of this enzyme may therefore allow to develop more reliable tests. I t may be further mentioned that only two enzyme systems, namely urease and urea carboxylase (urea:C0 2 ligase [adenosine 5'-diphosphate forming] EC 6.3.4.6) in connection with allophanate hydrolase (allophanate amidohydrolase, EC 3.5.13) (ROON and LEVENBERG 1968) are known to allow the utilization of urea

Derepressive urease formation in Proteus rettgeri

203

as a nitrogen source in microorganisms. Of these, only urease has so far been demonstrated in procaryotes. Since the ability to utilize urea as the sole source of nitrogen is not affected by ammonia repression, growth at the expense of urea may be a more reliable indication in case of uncertainty for the presence of urease in bacterial strains than conventional urease tests. References B O E H R I N G E R Test-Fibel. Mannheim: B O E H R I N G E R C H R I S T E N S E N , W . B., 1946. Urea decomposition

G m B H , Diagnostica, 1976. as a means of differentiating Proteus and paracolon cultures f r o m each other and f r o m Salmonella and Shigella types. J . Bacteriol., 52,461 —466. DETURK, W., 1955. The adaptive formation of urease by washed suspensions of Pseudomonas aeruginosa. J . Bacteriol., 70, 187 — 191. F R I E D R I C H , B . and M A G A S A N I K , B . , 1977. Urease of Klebsiella aerogenes: Control of its synthesis by glutamine synthetase. J . Bacteriol., 1 3 1 , 4 7 6 — 4 5 2 . I W A I N S K Y , H . und S E H R T , I . , 1 9 7 1 . Enzyminduktion und Stoffwechselregulation bei Mykobakterien. Zur Regulation der Urease bei Mykobakterien. Zbl. B a k t . H y g . I . A b t . Orig. A, 218, 212—223J A N S S E N , D. B., H E R S T , P. M., H O O S T E N , H . M. L. J . and VAN D E R D R I F T , C., 1981. Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in t h e regulation of t h e synthesis of NADPdependent g l u t a m a t e dehydrogenase, urease and histidase. Arch. Microbiol., 128, 398—402. KALTWASSER, H . and SCHLEGEL, H . G., 1966. NADH-dependent coupled enzyme assay for urease and other ammonia-producing systems. Anal. Biochem., 1 6 , 1 3 2 — 1 3 8 . K A L T W A S S E R , H . , K R Ä M E R , J . and CONGER, W . R . , 1 9 7 2 . Control of urease formation in certain aerobic bacteria. Arch. Mikrobiol., 81, 178 —196. K L E C Z K O W S K I , K . , H I O R T , U. und K A T I N G , H., 1 9 6 6 . Untersuchungen zum Stoffwechsel des Harnstoffes bei Mikroorganismen. I V . Adaptive Ureasebildung bei Micrococcus denitrificans BEIJ. Arch. Mikrobiol., 54, 177 — 183. K Ö N I G , C . u n d S C H L E G E L , H . G . , 1 9 6 7 . Oscillationen der Ureaseaktivität von Hydrogenomonas H 16 in statischer K u l t u r . Biochim. biophysica Acta, 139, 182 — 183. K R Ä M E R , J . , K A L T W A S S E R , H . und S C H L E G E L , H . G . , 1 9 6 7 . Die Bedeutung der Ureaserepression f ü r die taxonomische Klassifizierung von Bakterien. Zbl. B a k t . Hyg. I I . Abt., 121, 4 1 4 — 4 2 3 . M A G A N A - P L A Z A , I . and R U I Z - H E R R E R A , J . , 1 9 6 7 . Mechanisms of regulation of urease biosynthesis in Proteus rettgeri. J . Bacteriol., 9 3 , 1 2 9 4 — 1 3 0 1 . M A L O F E E V A , I . V . , 1 9 7 9 . Use of urea by purple bacteria. Mikrobiologia, 4 8 , 4 1 1 — 4 1 7 . MOORE, R . B. and K A U F E M A N , N . J . , 1 9 7 0 . Simultaneous determination of citrulline and urea using diacetylmonoxime. Anal. Biochem., 33, 163 —172. R O O N , R . J . a n d L E V E N B E R G , B., 1 9 6 8 . An adenosine triphosphate-dependent, avidin-sensitive enzymatic cleavage of urea in yeast and green algae. J . biol. Chemistry, 243, 5213—5215. ROSENSTEIN, I . , HAMILTON-MILLER, J . M . T . a n d BRUMFITT, W . , 1 9 8 0 . T h e e f f e c t of a c e t o h y d r o x a -

mic acid on t h e induction of bacterial ureases. Invest. Urol., 18, 112 —114. H . G . , K A L T W A S S E R , H . und GOTTSCHALK, G . , 1961. Ein Submersverfahren zur K u l t u r wasserstoffoxydierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch. Mikrobiol., 38, 209—222. Thiorhodaceae. S C H M I D T , K . , J E N S E N , S . L . und S C H L E G E L , H . G . , 1 9 6 3 . Die Carotinoide der I. Okenon als Hauptcarotinoid von Chromatium okenii PERTY. Arch. Mikrobiol., 46, 117 —126. S E N I O R , B. W . , B R A D F O R D , N . C . and SIMPSON, P . S . , 1980. T h e ureases of Proteus strains in relation to virulence for t h e urinary t r a c t . J . Med. Microbiol., 13, 507—512. SISSON, C . H., 1976. I m p r o v e d technique for accurate and convenient assay for biological reactions liberating UC02. Anal. Biochem., 70, 454—462. S M I T H , C . J . , H E S P E L L , R . B. a n d B R Y A N T , M. P . , 1981. Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium. Appl. Environm. Microbiol., 42, 8 9 — 9 6 . STEWART, D. J . , 1965. The urease activity of fluorescent pseudomonads. J . gen. Microbiol., 41, SCHLEGEL,

169-174. STUART, C. A . , STRATUM, A . V .

and R U S T I G A N , R . , 1 9 4 5 . F u r t h e r studies on t h e urease production by Proteus and related organisms. J . Bacteriol., 4 9 , 4 3 7 — 4 4 4 . V U Y E , A . and P U C K , J . , 1 9 7 3 . Urease activity of Enterobacteriaceae: Which medium to choose. Appl. Microbiol., 26, 850—854. Z O R N , C . , 1 9 7 9 . Einfluß der Stickstoffquelle auf die Ureasesynthese bei Proteus rettgeri. Diplomarbeit, Universität Saarbrücken. Mailing address: Prof. Dr. H . K A L T W A S S E R Fachrichtung 16.3 Mikrobiologie, Universität des Saarlandes, D-66 Saarbrücken 11 14

Zeitschrift f ü r Allg. Mikrobiologie

3

22

Kurze

1982

205-209

Originalmitteilung

(Akademie der Wissenschaften der Ukrain. SSR, Zabolotnij-Institut f ü r Mikrobiologie und Virologie, Kiev, und Karl-Marx-Universität Leipzig, Sektion Biowissenschaften, Fachbereich Pflanzenphysiologie und Mikrobiologie)

A S - I L — ein neuer, in der D D R aufgefundener S t a m m des Cyanophagen AS-1 V . A . GORJUSIN, E . STENZ u n d A . A .

(Eingegangen

am 4. 5.1981,

AVERKIEV

revidiert eingegangen am 5. 7. 1981)

I n samples, t a k e n from waters in t h e surroundings of Leipzig (GDR) in 1978, we found cyanophages in Central Europe for t h e first time. Among other cyanophages we isolated t h e new strain AS-1L. Out of 20 tested cultures of unicellular cyanobacteria seven strains belonging to t h e genus Synechococcus proved to be susceptible for this cyanophage. I n morphology AS-1L corresponds to t h e cyanophage AS-1 found in t h e U.S.A., to which it is related serologically, too. AS-1L differs from t h e other strains of AS-1 b y a shorter growth cycle, especially a shorter l a t e n t period, by t h e kinetics of inactivation by antiserum, and by a somewhat narrower p H scope of stability. Consequently t h e isolated cyanophage is to be looked a t as a new strain of t h e cyanophage AS-1. S e i t ihrer E n t d e c k u n g i m J a h r e 1963 w u r d e eine g r ö ß e r e A n z a h l v o n C y a n o p h a g e n a r t e n ( B l a u a l g e n v i r e n ) b e s c h r i e b e n (neuere Ü b e r s i c h t e n v o n S H E R M A N U . B R O W N 1978, G O R J U S I N 1980). C y a n o p h a g e n w u r d e n i n v e r s c h i e d e n e n T e i l e n der W e l t a u f gefunden, nach dem uns vorliegenden Schrifttum jedoch noch nicht in Mitteleuropa. W i r i s o l i e r t e n a u s G e w ä s s e r n der D D R n e b e n C y a n o p h a g e n der L P P - G r u p p e a u c h e i n e n n e u e n S t a m m d e s C y a n o p h a g e n A S - 1 , der m e h r e r e K u l t u r e n einzelliger C y a n o b a k t e r i e n der G a t t u n g Synechococcus b e f ä l l t . A n d e r e A S - l - S t ä m m e sind a u s d e n U S A b e k a n n t ( S A F F E R M A N et al. 1972, S H E R M A N U . C O N N E L L Y 1976). Der Cyanophage AS-1L wurde durch Zugabe von Wasserproben zu Flüssigkulturen von Cyanobakterien der Gattung Synechococcus angereichert (Methode n. SAFFERMAN 1968, B g - l l - M e d i u m n. STANIER et al. 1971, 30—35 °C, 1400 lx). Nach Filtration und wiederholten Einzelplaquepassagen konnte der reine Cyanophagenstamm isoliert werden. Die Inaktivierungstemperatur u n d pH-abhängige Stabilität der Viria bestimmten wir nach der f ü r Cyanophagen angegebenen Methode (SAFFERMAN U. MORRIS 1964) an Lysaten m i t einem Titer von 107 P F U / m l . Der Entwicklungszyklus des Cyanophagen wurde mittels der Einstufenvermehrung (SAFFERMAN et al. 1972) charakterisiert. Dazu diente eine K u l t u r von S. cedrorum 6908 mit 10s Zellen/ml unter Zusatz des Cyanophagen in einer Konzentration von 2 • 103 P F U / m l . I m Verlauf von 12 Std. wurde stündlich der Virustiter bestimmt. Die Geschwindigkeitskonstante der serologischen Virusinaktivierung wurde m i t einem Kanin2,3 • lg chenantiserum gegen AS-1L bestimmt nach K =

-

P

•D , wobei P lind P0 die Menge an

Cyanophagen nach der Zeit t des K o n t a k t e s m i t dem Antiserum beziehungsweise vor dem Kont a k t ist u n d D der Verdünnungsgrad des Antiserums (ADAMS 1 9 6 1 ) . Die morphologischen Untersuchungen erfolgten m i t einem Elektronenmikroskop UEMV-100A bei einer Beschleunigungsspannung von 75 kV. D e r C y a n o p h a g e A S - 1 L s t a m m t a u s einer M i s c h p r o b e v o n W ä s s e r n , die wir i m J u l i 1 9 7 8 a u s S e e n u n d T e i c h e n in L e i p z i g u n d seiner U m g e b u n g e n t n o m m e n h a t t e n .

206

V . A . G O R J Ü S I N , E . STENZ u n d A . A . A V E R K I E V

Außer Synechococcus cedrorum 6908 erwiesen sich bei der Prüfung des Wirtskreises für diesen Cyanophagen auch Kulturen von Anacystis nidulans 257 und 602 (beide Stämme werden jetzt zu S. leopoliensis gestellt), Anacystis spec. 6311, 8. lividus 6716 und 6717 sowie von 8. parvula 600 als sensitiv (vgl. Tab. 1). Das entspricht dem Wirtskreis des Cyanophagen AS-1. Tabelle 1 Kulturen einzelliger Cyanobakterien, die für die Prüfung der Sensitivität gegenüber dem Cyanophagen AS-IL verwendet wurden Sensitivität Sensitiv

Art Anacystis nidulans (Synechococcus leopoliensis) Anacystis spec. Synechococcus cedrorum Synechococcus lividus Synechococcus

Insensitiv

parvula

Anacystis spec. Synechococcus elongatus Synechococcus eximius Synechococcus schmidlea Synechococcus spec.

Stamm

Herkunft 1 )

257 602 6311 2 ) 6908 2 ) 6716 2 ) 6717 2 ) 600

L L M M M M L

6312 2 ) 58 6907 2 ) 726 670 535 698 747 748 753 R-2 Floyd R-6 Floyd R-13 Floyd

M L M L L L L L L L M M M

*) L = Biologisches Institut der 2danov-Universität Leningrad, M = Lehrstuhl für Genetik und Züchtung der Lomonosov-Universität Moskau ) Vertreter der typologischen Gruppe I einzelliger Cyanobakterien (STANIER et al. 1971)

2

Die Cyanophageninfektion führt in Flüssigkulturen zur Lyse, in Agarkulturen zur Bildung von Plaques. Diese erreichen in einem Rasen von 8. cedrorum, 6908 im Verlauf von 72 Std. bei 25 °C und 1000 lx einen maximalen Durchmesser von 3 bis 4 mm. Der Cyanophage AS-1L ist stabil in einem Bereich zwischen p H = 5,0 und p H = 10. Sein Temperaturinaktivierungspunkt liegt bei 57 °C. AS-IL verliert seine Infektiosität nicht, wenn die Viria für die Dauer einer Stunde einer Temperatur von 45 °C ausgesetzt werden. Bei 50 °C bleibt die Infektiosität zu 84% erhalten, bei 55 °C nur zu 5%. Aus der Einschrittvermehrungskurve (Abb. 1) ist für den Cyanophagen AS-1L eine Latenzperiode in Zellen von 8. cedrorum 6908 von nur 6,5 Stunden abzulesen. Daran schließt sich eine vierstündige Periode der Cyanophagenfreisetzung, also des Virustiteranstieges, an. Die maximale Virusmenge ist etwa 10 Stunden nach der Infektion erreicht. Die Wurfgröße beträgt annähernd 40 Viruspartikeln pro infizierte Zelle. Der Cyanophage AS-IL ist dem Cyanophagen AS-1 serologisch ähnlich, da eine Kreuzreaktion des Phagen AS-1 mit Antiserum gegen den Phagen AS-1L erfolgt. Die Untersuchung der Neutralisationskinetik der Cyanophagen AS-1L und AS-1 mit

207

AS-IL, ein neuer Cyanophage 5-

1

V

~B

T

10

11 h

Abb. 1. Einschrittvermehrungskurve für den Cyanophagen AS-1L in Synechococcus cedrorum 6908 k5-

5

10

- 1 5 min

Abb. 2. Inaktivierungskinetik der Cyanophagen AS-1 (O) und AS-1L ( # ) durch AS-lL-Antiserum. Serumverdünnung 1:1024

dem genannten Antiserum zeigte jedoch, daß beide Cyanophagen serologisch nicht identisch sind (vgl. Abb. 2). Für AS-1L beträgt die Geschwindigkeitskonstante der Inaktivierung durch AS-IL-Antiserum 108 + 18 min" 1 , für den Cyanophagen AS-1 47 ± 9 min" 1 . Die Morphologie des AS-1L-Virions entspricht der von AS-1. Die AS-lL-Phagenpartikeln besitzen einen ikosaedrischen Kopfteil mit einem Durchmesser von 86 + 5 nm. Der verhältnismäßig starre, mit einer Basisplatte versehene Schwanzteil ist 257 21 nm lang und 2?> + 3 nm breit (Abb. 3). Die Schwanzscheide ist kontraktil (Abb. 4). Von dem Cyanophagen AS-1 wurden bisher zwei Stämme bekannt. Beide wurden in den USA gefunden: der Cyanophage AS-1 in Wasser aus Klärteichen im Staate Florida (SAFFERKLAN et dl. 1972) und der Cyanophage AS-1M in Klärteichen des Staates Missouri (SHERMAN u. CONNELLY 1976). Diese Stämme unterscheiden sich voneinander im wesentlichen durch die Replikationsgeschwindigkeit. Der von uns isolierte Cyanophage stimmt morphologisch wie auch bezüglich der Inaktivierung durch Temperaturen oberhalb von 45 °C weitgehend mit den schon bekannten AS-l-Cyanophagen überein. Serologisch ist er mit AS-1 verwandt, aber

208

V . A . GOKJTJSIN, E . STENZ u n d A . A . A V E R K I E V

Abb. 3. Cyanophage AS-1L. Negativkontrastierung mit 2%igem Uranylacetat, Vergrößerung 275000fach

nicht völlig identisch. Größere Unterschiede zwischen AS-1L und den anderen Stämmen bestehen in der Dauer der Latenzperiode (ca. 6,5 Stunden im Vergleich zu 8 oder mehr Stunden) und der Lysezeiten (10 Stunden für AS-IL, 12 Stunden für AS-1M und 16 Stunden für AS-1). Außerdem sind die Viria der beiden amerikanischen Stämme in einem etwas weiteren pH-Bereich stabil als der von uns gefundene Cyanophage. Alle genannten Eigenschaften führten zu dem Schluß, daß es sich bei diesem Phagen um einen weiteren Stamm des Cyanophagen AS-1 handelt. In Übereinstimmung mit der üblichen Verfahrensweise, die Viren der Cyanobakterien mit den Anfangsbuchstaben der für sie sensitiven Wirte und mit Ziffern für die serologische Gruppe zu bezeichnen sowie unterschiedliche Stämme entsprechend ihrer Herkunft mit einem nachgestellten Buchstaben zu versehen, ergibt sich für den neuen Stamm die Benennung AS-IL. Da AS-IL-Antiserum auch gegen den Cyanophagen AS-1 wirksam ist, halten wir die Zuordnung zu dieser serologischen Gruppe für gerechtfertigt. Wir danken Herrn Prof. S. V. ÄKSTAKOV von der Lomonosov-Universität Moskau für die Überlassung des Cyanophagen AS-1 und verschiedener Kulturen von Cyanobakterien und Herrn Prof. B. V. GEOMOV von der ¿danov-Universität Leningrad für die Möglichkeit, die Kulturensammlung des Biologischen Institutes zu nutzen.

209

A S - I L , ein neuer C y a n o p h a g e

A b b . 4. Virion des C y a n o p h a g e n AS-1L mit v e r k ü r z t e r Schwanzscheide. N e g a t i v k o n t r a s t i e r u n g m i t 2 % i g e m U r a n y l a c e t a t , V e r g r ö ß e r u n g 275000fach

Literatur ADAMS, M., 1961. Bakteriofagi. Moskva GORJUSIN, V. A., 1980. Virusy nizsich r a s t e n i j . I n : I t o g i N a u k i i Techniki, Ser. Zascita R a s t e n i j , TOM 2, Moskva, 5—72. SAFFERMAN, R . S., 1968. Virus diseases in blue-green algae. I n : Algae, Man, a n d t h e E n v i r o n m e n t . P r o c . I n t . S y m p . Syracuse 1967, Syracuse, N e w Y o r k , 429—439. S A F F E R M A N , R . S . a n d M O R R I S , M . E . , 1 9 6 4 . G r o w t h characteristics of t h e blue-green algal virus L P P - 1 . J . Bacteriol., 8 8 , 7 7 1 - 7 7 5 . SAFFERMAN, R . S., D I E N E R , T . O . , DESJABDINS, P . R . a n d MORRIS, M . E . ,

1972.

Isolation

and

characterization of AS-1, a p h y c o v i r u s infecting t h e blue-green algae, Anacystie nidulans a n d Synechococcus cedrorum. Virology, 47, 105 — 113. SHERMAN, L. A. a n d BROWN, R . M., 1978. Cyanophages a n d viruses of e u k a r y o t i c algae. I n : F R A E N K E L - C O N R A T , H . a n d W A G N E R , R . R . (Editors), Comprehensive Virology, Vol. 1 2 . N e w York, 1 4 5 - 2 3 4 . SHERMAN, L. A. a n d CONNELLY, M., 1976. Isolation a n d c h a r a c t e r i z a t i o n of a c y a n o p h a g e infecting t h e unicellular blue-green algae A. nidulans a n d S. cedrorum. Virology, 72, 540—544. S T A N I E R , R . Y . , K U N I S A W A , R . , M A N D E L , M . a n d C O H E N - B A Z I R E , G., 1 9 7 1 . P u r i f i c a t i o n a n d properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev., 35, 171—205. A n s c h r i f t e n : D r . sc. E . STENZ

Sektion Biowissenschaften der K a r l - M a r x - U n i v e r s i t ä t , Fachbereich Pflanzenphysiologie u n d Mikrobiologie D D R 7010 Leipzig, T a l s t r . 33 V. A. GORJUSIN, K a n d . biol. Wiss. Z a b o l o t n i j - I n s t i t u t f ü r Mikrobiologie u n d Virologie der A k a d e m i e der W i s s e n s c h a f t e n d e r U k r a i n . S S F UdSSR 252627 Kiev-143, Ul. Zabolotnogo, 26

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Originalmitteilung

(Akademie der Wissenschaften der D D R , Forschungszentrum f ü r Molekularbiologie und Medizin, Zentralinstitut f ü r Mikrobiologie und experimentelle Therapie, Jena. Direktor: Prof. Dr. U. Taubekeck)

Regulation of Instability in glucose metabolism of Escherichia coli ML 30 chemostat cultures by cyclic AMP P. J. M ü l l e r and W. R ö m e r

(Eingegangen am 18. 6. 1981) Evidence is presented t h a t cyclic AMP is engaged in the regulation of a bistability in t h e glucose and energy metabolism of NH 3 -limited chemostat cultures of Escherichia coli ML 30. Cyclic AMP probably reverses t h e repression of the citric acid cycle by glucose favouring the state of glycogen and energy overproduction.

Evidence has been presented that NH 3 -limited chemostat cultures of E. coli ML 30 possess two possibilities (bistability) to regulate their glucose- and energy metabolism ( B e r g t e r a n d R o t h 1977, M u l l e k a n d v . FROMMAirersHAusEX 1982). T h e e x c e s s i v e l y

uptaken glucose is metabolized differently in the two steady states. State A is characterized by higher glycogen accumulation in cells and state B by pyruvate excretion. It was shown that in state A more pyruvate is metabolized by the citric acid cycle, resulting in a higher energy production and glycogen accumulation, respectively. Because it has been proposed that the synthesis of citric acid cycle and respiratory enzymes and the efficiency of energy transduction in E. coli is regulated by cyclic A M P ( M o s e s a n d S h a r p , 1970, H e m p e l i n g a n d B e e m a n 1971, H e m p e l l n g a n d M a h t z e r 1975, T a k a h a s h i 1975, D a o u d a n d H a d d o c k 1976, a n d D i m s a n d D o b r o g o s z

1977). In the present paper the role of cyclic AMP in the regulation of bistability in the glucose- and energy metabolism is investigated. The conditions of growth of t h e NH 3 -limited chemostat cultures with glucose as carbon source (3 g/1 in t h e reservoir) a t t h e investigated dilution rates D = 0.15 h _ 1 and 0.18 h" 1 , t h e methods of estimation of t h e concentrations of cells, glucose, and pyruvate were described previously. The limiting NH 3 -concentration in the reservoir ( M u l l e r et al. 1977, M u l l e r and von FromMAiTSHAiTSBjr 1982) was always 1.68 m u , allowing a cell concentration in t h e steady state X ~ 0.150 g/1 dry weight. X also depends on the glycogen content of the cells. Cyclic AMP (Fekak, Berlin) was added to t h e chemostat culture (working volume = 100 ml) in a 1 ml portion of p H = 8.0 (final concentration 10 mM). Cyclic AMP was determined on t h e basis of the protein binding method (Gelmax 1970). 10 ml samples were taken from the culture, immediately cooled by ice and filtered through a bacterial membrane filter. Additionally, t h e samples containing cyclic AMP were purified by t h e use of a prepared column of Dowex 1.

Fig. 1 shows the kinetics of cell concentrations, glucose and pyruvate after a pulse of cyclic AMP added to a NH 3 -limited chemostat culture with high pyruvate production (state B). Immediately after the pulse of cyclic AMP the cell concentration decreases, indicating growth inhibition in accordance with an earlier report (Jttdew i c e et al. 1973). The growth inhibition probably leads to an increase of the NH 3 concentration in the culture and therefore to a transient increase of glucose uptake

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Fig. 1. Kinetics of cells concentrations ( X : • ) . glucose and pyruvate (A) after a pulse of added cyclic AMP to a NH 3 limited E. coli ML 30 chemostat culture. Before the addition the culture was in a stable stationary state with high pyruvate production (state B). The dilution rate was D = 0.18 h _ 1

and pyruvate formation, respectively. Such behaviour was found to be typical after

t e r m i n a t i o n of N H 3 - l i m i t a t i o n in E. coli

(DIETZLER et al. 1 9 7 4 , MÜLLER a n d BERGTEB

1977). In the following phase the accumulated NH 3 is consumed again, leading to a more rapid growth. Here the whole pyruvate is uptaken and 28 h after the pulse of cyclic AMP the pyruvate concentration is still very low. However, theoretically a new steady state is not reached because the concentration of cyclic AMP assuming that cyclic AMP was only diluted by the inflow of fresh medium (7X working volume) was much higher ( « ¿ 0 . 1 m i ) than the measured extracellular cyclic AMP steady state concentrations (Tab. 1). A direct control of the bistability by cyclic AMP was indicated by the fact that in the state with low pyruvate production (A) the extracellular cyclic AMP concentration was twice higher (0.141 + 0.008 ;J.M) in comparison with the state B of high pyruvate formation (0.067 + 0.02 ¡AM) (Tab. 1). Both estimated values are higher than a recently published value of 0.02 ¡XM, found in a chemostat culture of E. coli K 12 at c o m p a r a b l e c o n d i t i o n s (WRIGHT et al.

1979).

According to the present results the state of lower pyruvate formation and higher glycogen accumulation is favoured by higher extracellular cyclic AMP concentrations. Possibly cyclic AMP relieves the glucose mediated repression of the citric acid cycle enzymes and energy (ATP) production, respectively. Also the finding of a higher respiration rate of resting cells taken from state A cultures (MÜLLER and VON

FROMMANSHAUSEN 1 9 8 2 ) is in a g r e e m e n t w i t h t h e s t a t e m e n t s o f HEMPFLING a n d

MAINZER (1975) of an increase of the maintenance respiration by added cyclic AMP. The same authors discussed an increase of the efficiency of respiration in glucoselimited chemostat cultures after adding cyclic AMP from P/O = 0.89 up to P/O = 2.7. Therefore, the previously calculated energy overproduction in state A (MÜLLER and VON FROMMANSHAUSEN 1982) should be composed from a fraction dependent on the extra oxidation of pyruvate and a fraction due to the higher energetic efficiency of respiration. The energy excess in state A is only partially consumed by the glycogen

Regulation of bistability in E. coli c h e m o s t a t cultures

213

Table 1 S t e a d y s t a t e c o n c e n t r a t i o n s of p y r u v a t e a n d extracellular cyclic A M P in N H 3 limited c h e m o s t a t c u l t u r e s of E. coli M L 30 (D = 0.15 ± 0.01 h- 1 ) pyruvate (g/1)

extracellular cyclic A M P ([xM)

s t a t e A (high glycogen a n d low p y r u v a t e f o r m a t i o n )

0.0066 0 0

0.148 0.147 0.144

s t a t e B (low glycogen a n d high pyruvate formation)

0.100 0.149 0.164 0.194

0.0612 0.0825 0.0920 0.0414

a c c u m u l a t i o n , k n o w n a s e n e r g y s t o r a g e p r o d u c t (CHAPMAN et al. 1 9 7 1 , DIETZLER et al. 1 9 7 4 ) . A d d i t i o n a l p r o c e s s e s of e n e r g y c o n s u m p t i o n h a v e t o b e a s s u m e d . B e c a u s e t h e s t a t e of l o w p y r u v a t e f o r m a t i o n a n d h i g h g l y c o g e n a c c u m u l a t i o n i s c o r r e l a t e d w i t h a t h r e e f o l d h i g h e r g l u t a m i n e s y n t h e t a s e a c t i v i t y (MÜLLER et al. 1977, 1981), a n d t h e N H 3 - a s s i m i l a t i o n v i a g l u t a m i n e s y n t h e t a s e is c o n n e c t e d w i t h A T P c o n s u m p t i o n (TEMPEST et al. 1 9 7 3 ) , i n t h i s s t a t e m o r e e n e r g y i s n e c e s s a r y f o r g r o w t h . A l s o t h e g l u t a m i n e s y n t h e t a s e is a p a r t of a n e n e r g y d i s s i p a t i n g f u t i l e c y c l e (PRUSINER 1 9 7 3 , TEMPEST 1 9 7 8 ) .

T h e s y n t h e s i s of g l u t a m i n e s y n t h e t a s e i s p o s i t i v e l y a f f e c t e d b y c y c l i c A M P (PRUSINER et al. 1 9 7 2 ) . T h e r e f o r e c y c l i c A M P s e e m s t o p l a y a r o l e n o t o n l y i n r e g u l a t i n g of t h e b i s t a b i l i t y of g l u c o s e m e t a b o l i s m b u t a l s o i n r e g u l a t i n g of t h e b i s t a b i l i t y of N H 3 assimilation. F r o m o u r r e s u l t s i t c a n b e a s s u m e d t h a t t h e s y n t h e s i s of c y c l i c A M P i t s e l f is a f f e c t e d b y t h e d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s of e a c h s t a t e i n a m a n n e r l e a d i n g t o a s t a b i l i z a t i o n of a c e r t a i n s t a t e .

References BERGTER, F . u n d ROTH, M., 1977. B i s t a b i l i t ä t in d e r P y r u v a t p r o d u k t i o n v o n Escherichia coli M L 30 in k o n t i n u i e r l i c h e r K u l t u r . Z. Allg. Mikrobiol., 17, 3—6. CHAPMAN, A . G., F A L L , L . a n d ATKINSON, D., 1971. A d e n y l a t e energy charge in Escherichia coli d u r i n g g r o w t h a n d s t a r v a t i o n . J . Bacteriol., 108, 1072—1086. D A O U D , M . S. a n d H A D D O C K , B. A., 1976. Electron t r a n s p o r t in m u t a n t s of Escherichia coli deficient in t h e i r a b i l i t y t o synthesize adenosine 3':5'-cyclic m o n o p h o s p h a t e a n d t h e catabolite-gene a c t i v a t o r p r o t e i n . Biochem. Soc. Trans., 4, 711—714. DIETZLER, D . N . , L A I S , C. J . a n d LECKIE, M . P . , 1974. S i m u l t a n e o u s increases of t h e a d e n y l a t e energy charge a n d t h e r a t e of glycogen synthesis in n i t r o g e n - s t a r v e d Escherichia coli W 4597 (K). Arch. Biochem. Biophysics, 160, 14—25. DILLS, S. S. a n d DOBROGOSZ, W . J . , 1977. Cyclic adenosine 3 ' , 5 ' - m o n o p h o s p h a t e r e g u l a t i o n of m e m b r a n e energetics in Escherichia coli. J . Bacteriol., 131, 854—865. GILMAN, A. G., 1970. A p r o t e i n binding assay for adenosine 3 ' , 5 ' - m o n o p h o s p h a t e . P r o c . N a t l . Acad. Sei. (USA), 67, 3 0 5 - 3 1 2 . HEMPELING, W . P . a n d BEEMAN, D. K . , 1971. Release of glucose repression of o x i d a t i v e phosphor y l a t i o n in Escherichia coli B b y cyclic adenosine 3 ' - 5 ' - m o n o p h o s p h a t e . Biochem. B i o p h y s . Res. C o m m u n . , 45, 9 2 4 - 9 2 9 . HEMPFLING, W . P . a n d MAINZER, S. E . , 1975. E f f e c t s of v a r y i n g t h e carbon source limiting g r o w t h on yield a n d m a i n t e n a n c e characteristics of Escherichia coli in continuous c u l t u r e . J . B a c t e r i d . , 123, 1076 — 1087. J U D E W I C Z , N . D . , D E R O B E R T I E S , E . M. a n d TORRES, H . N., 1 9 7 3 . I n h i b i t i o n of Escherichia coli, g r o w t h b y cyclic a d e n o s i n e 3 ' - 5 ' - m o n o p h o s p h a t e . B i o c h e m . Biophys. Res. C o m m u n . , , 52, 1257 — 1262.

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MÜLLER, P. J . und BERGTER, F., 1977. Untersuchungen zum transient-Verhalten ammoniumlimitierter Chemostatenkulturen von Escherichia coli ML 30. Z. Allg. Mikrobiol., 17, 131 — 137. MÜLLER, P . J . , IVANOVA, I . I . u n d BERGTER, F . , 1977. B i s t a b i l i t ä t in d e r A k t i v i t ä t d e r G l u t a m i n -

synthetase bei ammoniumlimitierten Chemostatenkulturen von Escherichia coli ML 30. Z. Allg. Mikrobiol., 17, 2 2 1 - 2 2 5 .

MÜLLER, P . J . , VON FROMMANSHAUSEN, B . a n d SCHÜTZ, H . , 1981. R e g u l a t i o n of a m m o n i a assimi-

lation in ammonia-limited chemostat cultures of Escherichia coli ML 30: Evidence of bistability. Z. Allg. Mikrobiol., 21, 3 6 1 - 3 7 2 . MÜLLER, P. J . and VON FROMMANSHAUSEN, B., 1982. Bistability in the glucose- and energy metabolism of ammonia-limited chemostat cultures of Escherichia coli ML 30. Z. Allg. Mikrobiol., 2 2 , 185 — 1 9 0 .

PRUSINER, S., MILLER, R . E . a n d VALENTINE, R . C., 1972. Adenosine 3',5'-cycIic m o n o p h o s p h a t e

control of the enzymes of glutamine metabolism in Escherichia coli. Proc. Natl. Acad. Sei., U S A , 69, 2922 - 2 9 2 6 .

PRUSINER, S., 1973. Glutaminases of Escherichia coli: Properties, regulation and evolution. I n : "The Enzymes of Glutamine Metabolism (Editors: PRUSINER, S. and STADTMAN, E. R.). Materials 164th Nat. Meeting Amer. Chem. Soc., 1972, p. 293—316. TAKAHASHI, Y., 1975. Effect of glucose and cyclic adenosine 3',5'-monophosphate on the synthesis of succinate dehydrogenase and isocitrate lyase in Escherichia coli. J . Biochem., 78, 1097-1100. TEMPEST, D. W., MEERS, J . L. a n d BROWN, C. M., 1973. G l u t a m a t e s y n t h e t a s e (GOGAT); A k e y

enzyme in the assimilation of ammonia by procaryotic organisms. I n : "The Enzymes of Glutamine Metabolism (Editors: PRUSINER, S. and STADTMAN, E. R.). Materials 164TH Nat. Meeting Amer. Chem. Soc., 1972, p. 167-182. TEMPEST, D. W., 1978. The biochemical significance of microbial growth yields: A reassessment. Trends Biochem. Sei., 180—184. WRIGHT, L. F., MILNE, D. P. and KNOWLES, C. J . , 1979. The regulatory effects of growth r a t e and cyclic AMP levels on carbon catabolism and respiration in Escherichia coli K-12. Biochim. biophysica Acta, 583, 73—80. Mailing address: Dr. P. J . MÜLLER Zentralinstitut für Mikrobiologie u n d experimentelle Therapie der AdW DDR 6900 Jena, Beutenbergstraße 11

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Buchbesprechungen W. BAUMEISTER and W. VOGELL (Editors), Proceedings in Life Sciences, Electron Microscopy a t Molecular Dimensions. X + 353 S., 181 Abb., 12 Tab. Berlin-Heidelberg-New York 1980. Springer-Verlag. $ 57.90. Das von BAUMEISTEE und VOGELL herausgegebene Buch enthält die Proceedings des internationalen Workshop „Regular 2-D Arrays of Biomacromolecules, Structure Determination and Assembly", der im Juni 1979 auf der Burg Gemen (BRD) stattfand. Die 39 Beiträge wurden zumeist von Autoren gestaltet, die den Fortschritt der letzten Jahre auf den Gebieten der Strukturaufklärung ausgewählter kristalliner Strukturen von Biomolekülen, der Bildaufzeichnung mit geringen, objektschonenden Elektronendosen, der Elektronenmikroskopie bei tiefen Objekttemperaturen, der computerisierten Bildverarbeitung und der experimentell-theoretischen Untersuchung 2-dimensionaler Assemblierung von Proteinen ganz wesentlich mitbestimmten. Im ersten Abschnitt („state of the art"/19 Beiträge) sind eine Reihe von strukturellen Befunden an ausgewählten, geordneten Biostrukturen (Membran- und Zellwandkomponenten, Cytochromoxidase, Ubichinon, Murein, Crotoxin, Acetylcholinrezepturen, Ribosomenkristalle, Tubulinassemblate, Bakteriophagenköpfe, Glutaminsynthetase) in zumeist eindrucksvoller Weise dargestellt. Vorgeschlagene Strukturmodelle basieren dabei oftmals nicht nur auf elektronenmikroskopischen Untersuchungen, sondern auch auf biochemischen und biophysikalischen Ergebnissen. Die 5 Beiträge über „Bildaufzeichnung" geben eine Einschätzung des Raster-TransmissionsElektronenmikroskops als attraktives Hilfsmittel für den Biologen, vermitteln Erfahrungen mit fotografischen Emulsionen und charakterisieren den Einsatz von Televisionstechniken in der Low-dose-Mikroskopie. In den 3 Beiträgen zur Thematik „Tieftemperatur-Mikroskopie" wird eindrucksvoll demonstriert, daß niedrige Probentemperaturen Zerstörungen durch den Elektronenstrahl erheblich vermindern. Mit wachsender Anwendungsbreite entsprechender Techniken ist in den nächsten Jahren zu rechnen. 7 Beiträge sind Methoden und Techniken der „Bildverarbeitung" gewidmet. Diese spielt heute eine Schlüsselrolle in der Auswertung von Elektronenmikrogrammen, die zur Vermeidung von Objektschäden mit geringen Elektronendosen aufgenommen werden müssen und oftmals ein für die unmittelbare Auswertung viel zu geringes Signal-Rausch-Verhältnis aufweisen. Mittels eleganter Methoden, die der Korrelationsmeßtechnik entlehnt wurden, gelingt nicht nur die Auswertung fragmentierter Proteinkristalle (SAXTON), sondern auch die Darstellung von einzelnen Molekülen ( K E S S E L U. a.). Zu letzterem ist allerdings eine einheitliche Orientierung der Moleküle auf dem Trägerfilm Voraussetzung. Im Abschnitt „artifizielle Assemblierung zu 2-dimensionalen Ordnungen" (5 Beiträge) werden theoretische Grundlagen und praktische Hinweise vermittelt, die für die Objektpräparation von großem Nutzen sein sollten. Hervorzuheben sind die vielen, sehr anschaulichen, hochqualitativen Abbildungen und Tabellen, die auch einem wenig sachkundigen Leser ein gutes Grundverständnis des Dargebotenen ermöglichen. Ca. 600 Literaturangaben verhelfen zu detaillierterer Information. Das Werk wird ergänzt durch ein Register mit 250 Stichwörtern. Das Buch ist sehr empfehlenswert für alle, die sich einen Überblick über Entwicklungsstand, Tendenzen und Strategien der Elektronenmikroskopie von Biomakromolekülen verschaffen möchten, und es ist von besonderem Interesse für Forschende, die Informationsgehalt und -ausbeute ihrer elektronenmikroskopischen Untersuchungen steigern möchten. W . VATER (Jena)

J . W. CORCORAN (Editor), Antibiotics, Vol. IV: Biosynthesis. X I I + 3 8 0 S., 1 6 4 Abb., 4 9 Tab. Berlin-Heidelberg-New York 1981. Springer-Verlag. DM 198,00. Seit dem Erscheinen des Bandes I I 'Biosynthesis' der Reihe 'Antibiotics' vor mehr als zehn Jahren wurden vielfältige Fortschritte bei der Aufklärung von Struktur und Biosynthese dieser mikrobiellen Sekundärmetabolite erzielt. Es ist daher zu begrüßen, daß die bis Ende 1978 publizierten Literaturangaben unter Verzicht auf einige altbekannte Antibiotica wie z.B. Chloramphenicol in einer neuen Monographie zusammenfassend referiert werden. Die experimentellen Angaben stützen sich vor allem auf Ergebnisse von Markierungsstudien mit "C-, 13C-, 3 H- und 15 N-Präkursoren, wobei aber auch Untersuchungen an zellfreien Systemen und

216

Buchbesprechungen

die E n z y m a t i k des Sekundärstoffwechsels Berücksichtigung finden. Die speziellen Kapitel betreffen sowohl die Biogenese therapeutisch wertvoller Antibiotica wie Tetracycline u n d A n t h r a cycline (C. R . H U T C H I N S O N ) , Ansamycine ( G . L A N C I N I U. M . G R A N D I ) , Aminocyclitol-Antibiotica ( C . J . PEARCE U. K . L. R H I N E H A R T , jr.), /?-Lactame ( J . O ' S U L L I V A N U. E . P . A B R A H A M ) , E r y t h r o mycin (J. W. CORCORAN), 16-gliedrige Makrolide (S. OMURA U. A. NAKAGAWA) und Peptidantibiotica (K. KURAHASHI) als auch antibiotisch wirksame mikrobielle Produkte, die erst in neuerer Zeit aufgefunden bzw. f ü r spezielle Anwendungszwecke erschlossen wurden. So f i n d e t der interessierte Leser K a p i t e l über die Polyether Lasalocid, Monensin, Salinomycin, Narasin und Lysocellin (J. W. WESTLEY), die Biosynthese von Methylenomycin A als Beispiel einer Plasmid-determinierten Antibioticumproduktion (U. H O R N E M A N N U. D. HOPWOOD), die ungewöhnlichen Makrolidantibiotica Chlorothricin, Aplasmomycin und Boromycin ( H . G . FLOSS U. C. CHANG), die Biosynthesen von Isochromanchinonen u n d aromatischen Antibiotica (H. G. FLOSS), Pyrrolo(l,4)benzodiazepine (L. H . H U K L E Y U. M . K . S P E E D I E ) , Mitomycin ( U . H O R N E M A N N ) , Streptozocin ( U . HORNEMANN) u n d Nucleosid-Antibiotica ( R . J . SUHADOLNIK). D a m i t bietet der B a n d IV von 'Antibiotics' eine breitgefächerte Information über neuere E n t wicklungen auf dem Gebiet der Biosynthese von Antibiotica u n d ist d a m i t ein wertvolles H a n d buch f ü r alle an Problemen der mikrobiellen Sekundärmetabolitproduktion interessierten Leser U . GRÄFE ( J e n a )

D. C. ELLWOOD, J . N. H E D G E R , M. J . LATHAM, J . M. L Y N C H and J . H . SLATER (Editors), Contemporary Microbial Ecology. 438 S., 81 Abb., 20 Tab. London-New York-Toronto-Syndey-San F r a n cisco. Academic Press 1 9 8 0 . $ 4 8 . 0 0 . Das Buch enthält 19 Vorträge über aktuelle Probleme der Mikrobenökologie, die beim zweiten Internationalen Symposium über Mikrobielle Ökologie 1980 in Warwick, England, gehalten wurden. Die von kompetenten Autoren v e r f a ß t e n Beiträge f ü h r e n in trendbestimmende Gebiete ein, markieren Positionen und setzen sich kritisch m i t überkommenen Anschauungen auseinander. Das breite Spektrum der behandelten Themen sei stichwortartig umrissen: Beziehungen zwischen mikrobiellen Aktivitäten und Licht, Wasser, Grenzschichten sowie extremen Umweltfaktoren ; mikrobielle Anheftungs- und Verbreitungsmechanismen ; Anpassungs- u n d Selektionsprozesse von Zellen, Populationen und Mischkulturen; Biodegradation; Energie- und Erhaltungsstoffwechsel; Zellform und F u n k t i o n ; Stoff- und Energiefluß in Böden und marinen Sedimenten; I n t e r a k t i o n e n von Mikroben m i t dem menschlichen K ö r p e r ; Pansen-Mikrobiologie, Rhizosphäre, RäuberBeute-Beziehungen, Algen-Invertebraten-Symbiosen, mikrobieller Antagonismus u n d biologische Kontrolle. Aus den anspruchsvollen u n d originellen Beiträgen lassen sich zwei Trends ableiten, erstens der Brückenschlag von der Mikrobenökologie zur Physiologie, Biochemie u n d Genetik und zweitens die in siiw-Erforschung natürlicher Ökosysteme m i t neuen Methoden. Das Buch v e r m i t t e l t eine Fülle von Anregungen und Informationen, zugleich zeigt es, wie viele Fragen der Mikrobenökologie bei tiefgründiger B e t r a c h t u n g noch ungelöst sind. E s ist nicht n u r f ü r den ökologisch arbeitenden Mikrobiologen von großem Wert. J e d e r Mikrobiologe wird es m i t Gewinn zur H a n d nehmen, da es durch die ökologische Betrachtungsweise zu einem tieferen Verständnis mikrobieller Aktivitäten beiträgt. Diese haben sich in Wechselwirkung m i t der Umwelt entwickelt u n d werden erst u n t e r diesem Aspekt verständlich. Den Herausgebern g e b ü h r t Dank, daß sie m i t der Auswahl der Beiträge und der A r t der Drucklegung eine Publikationsform f ü r Symposien gewählt haben, die wirklich neue, der disziplinaren Entwicklung des Wissenschaftsgebietes dienende Informationen bietet. W. FRITSCHE (Jena)

BRIGITTE G E D E K , 1 9 5 Abb., 3 4 Tab.

Kompendium der medizinischen Mykologie (Pareys Studientexte Berlin-Hamburg 1 9 8 0 . Verlag Paul Parey. DM 4 8 , 0 0 .

25). 3 9 5

S.,

Die Autorin des Kompendiums ist v o n der Phytopathologie und von den Mykotoxinen zur medizinischen Mykologie gelangt. Auf eine allgemeine E i n f ü h r u n g in die Mykologie und die in Frage kommenden Untersuchungsmethoden einschließlich von Methoden zur Erfassung von Mykotoxinen (131 Seiten) folgt ein spezieller Teil, der einen Überblick über die durch Pilze verursachten Erkrankungen beim Menschen und bei Haustieren gibt, unterteilt in die eigentlichen Mykosenin Pilz-Allergosen, Mykotoxikosen und einige Erkrankungen, die durch Toxine pflanzenpathogener Pilze hervorgerufen werden (Beispiel: Ergotismus, Claviceps purpureä). Alles in allem ge sehen, ein anspruchsvolles Buch, k a u m geeignet zur Einarbeitung f ü r den Anfänger, eher dagegen, bereits vorhandene Erkenntnisse und praktische E r f a h r u n g e n in einen breiteren R a h m e n zu stellen und zu ergänzen. W. SCHWARTZ (Braunschweig)

H E L M U T F R I E M E L / J O S E F BROCK

Grundlagen der Immunologie (Wissenschaftliche

Taschenbücher, Reihe

Biologie)

4., bearbeitete Auflage 1979. 224 Seiten — 80 Abbildungen — 37 Tabellen — kl. 8° — 12,50 M Bestell-Nr. 7626174 (7109)

Die Verfasser geben einen Überblick über den neuesten Erkenntnisstand auf dem Gebiet der Immunologie. Sie vermitteln Grundlagen — Antikörper, zelluläre Immunität, pathogene Immunreaktionen, Immuntoleranz, Immunsuppression und Transplantationsimmunologie — u n d stellen das immunologische Grundwissen in straffer und übersichtlicher Form dar, um immunologisch interessierte Leser in die Lage zu versetzen, die stürmische Entwicklung dieses Fachgebietes zu verfolgen und für Forschung, Lehre und Praxis nutzbar zu machen.

Bestellungen durch eine Buchhandlung

AKADEMIE-VERLAG DDR-1086 Berlin, Leipziger Str. 3—4

erbeten

Vorträge des V. Internationalen Symposiums Systemfungizide Veranstaltet durch die Sektionen Mikrobiologie und Phytopathologie der Biologischen Gesellschaft der DDR vom 9. bis 13. Mai 1977 im Schloß Reinhardsbrunn Herausgegeben von HORST LYR / CLAUS POLTER (Abhandlungen der Akademie der Wissenschaften der D D R , Abt. Mathematik — Naturwissenschaften — Technik) 1979. 371 Seiten — 191 Abbildungen — 92 Tabellen — 2 Karten — gr. 8° 58,- M Bestell-Nr. 7628014 (2001/79/2/N)

Der Band enthält 39 Vorträge von namhaften und international bekannten Spezialisten ausl2 Ländern, darunter aus der UdSSR, DDR, Polen, Ungarn, CSSR, Holland, USA, Großbritannien, Griechenland und der B R D und die wichtigsten Diskussionsbeiträge. Er vermittelt damit für jeden pflanzenphysiologisch-mikrobiologisch Interessierten und speziell für den in der landwirtschaftlichen Forschung Tätigen eine Fülle von neuen Informationen und ist eine Fundgrube für jeden, der sich mit theoretischen oder praktischen Problemen des Einsatzes von Fungiziden befaßt. Bestellungen durch eine Buchhandlung erbeten