Zeitschrift für Allgemeine Mikrobiologie: Band 21, Heft 7 1981 [Reprint 2021 ed.] 9783112477960, 9783112477953

Citation preview

ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS HEFT 7 • 1981

Ä EVP 20,—M

BAND 21

AKADEMIE-VERLAG • BERLIN ISSN 0044-2208 34112

INHALTSVERZEICHNIS HEFT 7 Vergleich der toxischen Wirkung von 2-Desoxy-Dglucose und 2-Desoxy-2-fluor-D-hexose au! Zellen und Protoplasten von Saccharomyces cereinsiae Regulation der Steroid-16 a-Hydroxylierung bei Streptomyces

P. BIELY, L. P. RJAZANOVA und A . B. TSIOMENKO 489 J. DtuGOfisKi und L. SEDLACZEK

499

olivoviridis

Charakterisierung von Strukturelementen in den Lipopolysacchariden von Pasteurella multocida Zusammenhang zwischen Koloniemorphologie und Polysaccharidgehalt in zellwandmodifizierten M u tanten von Candida spec. „ H " Eine neue extrazelluläre Proteinase aus Bacillus pumilus Amylasen aus Schwanniomyces castellii Propionatbildung bei Rhodospirillum rubrum, unter anaeroben Bedingungen Mikrobiologische Anwendung elektrischer Feldeffekte. III. Stimulation der Hefeprotoplastenfusion durch elektrische Feldimpulse

W . ERLER, H . FEIST, K . D . FLOSSMANN, B . JACOB und A . PILARSKI 507 R . KÖLBLIN und S. BIRKENBEIL 519,

R . 0 . OKOTORE, E . O. AKINRIMISI UND M. O. OJO 531 K . OTENG-GYANG, G. MOULIN UND P. GALZY 537 H . VOELSKOW UND G. SCHÖN 545 H . WEBER, W . FÖRSTER, H . - E . JACOB und H . BERG

555 563

Buchbesprechungen

CONTENTS OF N U M B E R 7 A comparison of the toxic effects of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-hexoses on Saccharomyces cerevisiae cells and protoplasts Regulation of steroid 16 a-hydroxylation in Streptomyces olivoviridis Characterization of structure elements in the lipopolysaccharides of Pasteurella multocida Connection between morphology of the colonies and polysaccharides content of cell wall-modified Candida spec. , , H " mutants A novel extracellular proteinase from Bacillus pumilus A study of the amylotic system of castelii

Schwanniomyces

Propionate formation in Rhodospirillum rubrum under anaerobic dark conditions Microbiological implications of electric field effects. I I I . Stimulation of yeast protoplast fusion by electric field pulses Book Reviews

P . BIELY, L . P . RJAZANOVA and A . B . TSIOMENKO 489 J. DLTTGOI&SKI and L . SEDLACZEK

499

E . ERLER, H . FEIST, K . D . FLOSSMANN, B . JACOB and A . PILARSKI 507 R . KÖLBLIN and S. BIRKENBEIL 519

R . O. OKOTORE, E . 0 . AKINRIMISI AND M. O. OJO 531 K . OTENG-GYANG, G. MOULIN AND P . GALZY 537 H . VOELSKOW AND G. SCHON 545 H . WEBER, W . FORSTER, H . - E . JACOB AND H . BERG 555

563

ISSN 0044-2208

ZEITSCHRIFT FÜR ALLGEMEINE MIKRO -BIOLOGIE MORPHOLOGIE, PHYSIOLOGIE, GENETIK UND OKOLOGIE DER MIKROORGANISMEN

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

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

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

U N T E R MITARBEIT VON

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

HEFT 7 • 1981 BAND 21

REDAKTION

U. May, Jena

AKADEMIE-VERLAG BERLIN

Die Zeitschrift f ü r Allgemeine Mikrobiologie soll dazu beitragen, Forschung und internationale Zusammenarbeit auf dem Gebiet der Mikrobiologie zu fördern. Es werden Manuskripte aus allen Gebieten der allgemeinen Mikrobiologie veröffentlicht. Arbeiten über Themen aus der medizinischen, landwirtschaftlichen, technischen Mikrobiologie u n d aus der Taxonomie der Mikroorganismen werden ebenfalls aufgenommen, wenn sie Fragen von allgemeinem Interesse behandeln. Zur Veröffentlichung werden angenommen: Originalmanuskripte, die in anderen Zeitschriften noch nicht veröffentlicht worden sind und in gleicher Form auch nicht in anderen Zeitschriften erscheinen werden. Der U m f a n g soll höchstens Druckbogen (24 Druckseiten) betragen. Bei umfangreicheren Manuskripten müssen besondere Vereinbarungen mit der Schriftleitung und dem Verlag getroffen werden. Kurze Originalmitteilungen über wesentliche, neue Forschungsergebnisse. U m f a n g im allgemeinen höchstens 3 Druckseiten. Kurze Originalmitteilungen werden beschleunigt veröffentlicht. Kritische Sammelberichte und 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 A K A D E M I E VERLAG, DDR-1080 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 Auslieferungstelle 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 Schweiz, Dufourstraße 51 — im übrigen Ausland an den Internationalen Buch- und Zeitschriftenhandel; den Buchexport, Volkseigener Außenhandelsbetrieb der Deutschen Demokratischen Republik, DDR-7010 Leipzig, Postfach 160, oder an den Akademie-Verlag, DDR-1080 Berlin, Leipziger Straße 3—4. Zeitschrift f ü r Allgemeine Mikrobiologie Herausgeber: I m Auftrag des Verlages von einem internationalen Wissenschaftlerkollektiv herausgegeben. Verlag: Akademie-Verlag, DDR-1080 Berlin, Leipziger Straße 3 - 4 ; F e r n r u f : 2236222 oder 2236221 • Telex-Nr!: 114420; B a n k : Staatsbank der D D R , Berlin, Kto.-Nr.: 6836-26-20712. Chefredaktion: Prof. Dr. U . TATTBENECK, P r o f . D r . W . SCHWARTZ.

Anschrift der Redaktion: Zentralinstitut f ü r Mikrobiologie u n d 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 885614; TelexNr. 058621 Veröffentlicht unter der Lizenznummer 1306 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Gesamtherstellung: V E B Druckerei „Thomas Müntzer", DDR-5820 B a d Langensalza. Erscheinungsweise: Die Zeitschrift f ü r Allgemeine Mikrobiologie erscheint jährlich in einem Band mit 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 F o r m — durch Photokopie, Mikrofilm oder irgendein anderes Verfahren — ohne schriftliche Genehmigung des Verlages reproduziert werden. — All rights reserved (including those of translations into foreign languages). No p a r t of this issue m a y be reproduced in a n y form, b y photoprint, microfilms or any other means, without written permission f r o m t h e publishers. Erscheinungstermin: Oktober 1981 Bestellnummer dieses Heftes 1070/21/7 © 1981 b y Akademie-Verlag Berlin, Printed in t h e German Democratic Republic. AN (EDV) 75218

Zeitschrift für Allgemeine Mikrobiologie

21

1981

489-497

(Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Czechoslovakia 1 and Institute of Biochemistry and Physiology of Microorganisms, Academy of Sciences of USSR, Pushchino, USSR 2 )

A comparison of the toxic effects of 2-deoxy-D-glucose and 2-deoxy-2-fluoro-D-hexoses on Saccharomyces cerevisiae cells and protoplasts P . BIELY 1 , L . P . RJAZANOVA 2 a n d A . B . TSIOMENKO 2

( Eingegangen am 18. 12.1980) The toxicity to the cells and protoplasts of Saccharomyces cerevisiae of the sugar analogues modified at carbon 2 increases in the order 2-deoxy-n-glucose (DG), 2-deoxy-2-fluoro-D-glucose (FG) and 2-deoxy-2-fluoro-D-mannose (FM). The fluorohexoses, similarly as DG, behave generally as analogues of both glucose and mannose, depending on the hexose used as a carbon source in the medium. Relative inhibitions of glucan and mannan synthesis in protoplasts were found to be dependent more on glucose and mannose used as the growth support than on the type of the sugar analogue. Certain degree of structural relationship of fluorohexoses to the corresponding natural hexoses was reflected in their effects on growth of intact cells. Growth on glucose was inhibited most effectively by FM, growth on mannose by FG. The data obtained support the view that the sugar analogues interfere mainly with the glucose-mannose interconversion catalyzed by hexosephosphateisomerases. A comparison of the effects of fluorohexoses and DG on the synthesis of extracellular invertase and intracellular a-glucosidase and alkaline phosphatase in protoplasts pointed to the fact that all three sugar analogues tested also participate in metabolic control of enzyme synthesis.

Non-fermentable analogues of glucose and mannose modified at carbon 2, 2-deoxyD-glucose (DG), 2-deoxy-2-fluoro-D-glucose (FG) and 2-deoxy-2-fluoro-D-mannose (FM) are known as potent inhibitors of growth of the yeast Saccharomyces cerevisiae (HEREDIA et al. 1964, BIELY et al. 1 9 7 1 , 1 9 7 3 ) . The analogues are taken up by the cells and phosphorylated to 6-phosphates by hexokinases (CRAMER and WOODWARD 1952, BESSEL et al. 1972). The phosphates further enter the metabolic pathways of glucose and mannose in which the six-carbon chains of the natural hexoses are preserved. All three sugar analogues mentioned were shown to be incorporated into sugar nucleotides and cell wall polysaccharides of S. cerevisiae (BIELY and BAUER 1 9 6 0 , 1 9 6 8 , KRATKY et al. 1975, LEHLE and SCHWARZ 1976, SCHMIDT et al. 1978). The extent of their incorporation into cell wall polysaccharides is rather low, however, their phosphate esters and nucleotides are accumulated inside the cells and interfere severely in the synthesis of structural cell wall polysaccharides (FARKAS et al. 1969, Kuo and LAMPEN 1972, KRATKY et al. 1975, TSIOMENKO et al. 1978). The inhibition of polysaccharide synthesis results in lysis of growing cells due to disturbance of balanced cell wall g r o w t h (JOHNSON 1968, BIELY et al. 1973).

Studies devoted to the effects of DG (BIELY et al. 1971, KRATKY et al. 1975) and FG et al. 1973) on growth and some metabolic activities of S. cerevisiae cells and protoplasts showed that FG is more toxic than DG. Particularly the synthesis of cell wall glucan was found to be extremely FG-sensitive (BIELY et al. 1973). Similar studies with FM have not been done yet. We considered it of interest to compare the effects of FG and FM on S. cerevisiae cells and protoplasts in order to find out to what extent the two 2-deoxy-2-fluoro-D-hexoses behave in this yeast as analogues of the corresponding natural hexoses. For this purpose the effects of FG and FM were inves(BIELY

321

490

P . B I E L Y , L . P . RJAZANOVA a n d A . B . T S I O M E N K O

tigated in media containing glucose or mannose as the carbon source. DG, effects of which were shown to be dependent on whether glucose or mannose was used as the growth support (KRATKY et al. 1975), was included in this study for comparison. Materials

and methods

Yeast a n d culture conditions: Saccharomyces cerevisiae CCY 21-4-13, was grown on a reciprocating shaker a t 27 °C in a s y n t h e t i c medium consisting of 2 % glucose or mannose, 0.67% yeast nitrogen base,0.2% asparagine, a n d 0 . 5 % K H 2 P 0 4 , w i t h o u t or w i t h various addition of DG, F G or FM. For p r e p a r a t i o n of protoplasts, t h e cells were grown in t h e R I D D E R m e d i u m ( T S I O M E K K O et al. 1975). G r o w t h was followed by measuring t h e absorbance a t 420 n m of t h e cell suspensions in 0.2 cm wide cuvettes. Chemicals: 2-Deoxy-D-glucose (DG) was f r o m K O C H - L I G H T (England), 2-deoxy-2-fluoro-Dglucose (FG) a n d 2-deoxy-2-fluoro-D-mannose (FM) were generous gifts f r o m Prof. A . B. F O S T E R a n d D r . J . H . W E S T W O O D (Chester B e a t t y Research I n s t i t u t e , London). Mannose f r e e of glucose was f r o m S H U C H A R D T ( U S A ) . D-[U- 14 C]glucose (specific r a d i o a c t i v i t y 100 Ci/mol) was f r o m I n s t i t u t e for Research, Development and P r o d u c t i o n of Radioisotopes (Prtfha, Czechoslovakia) a n d D - [ U - 1 4 C ] mannose (specific r a d i o a c t i v i t y 230 Ci/mol) f r o m A M E R S H A M (England). P r e p a r a t i o n a n d cultivation of p r o t o p l a s t s : P r o t o p l a s t s were prepared f r o m exponential phase cells grown in t h e R I D D E R m e d i u m b y t h e procedure given elsewhere ( B I E L Y et al. 1973). A f t e r two washings w i t h 0.8 M m a n n i t o l solution in 0.1 M p h o s p h a t e buffer (pH 6), t h e protoplasts were suspended in t h e s y n t h e t i c m e d i u m containing 2 % glucose or mannose supplied w i t h 0.7 M mannitol a n d grown a t 30 °C u n d e r occasional stirring. Concentration of protoplasts in t h e media was 2 X 107 per ml. Their stability was followed b y d e t e r m i n a t i o n of a c t i v i t y of -nitrophenyla-D-glucopyranoside a n d jj-nitrophenyl p h o s p h a t e as s u b s t r a t e s as described b y K R A T K Y et al. (1975). One u n i t of a c t i v i t y of t h e enzymes is defined as t h e a m o u n t liberating f r o m t h e corresponding s u b s t r a t e 1 ¡¿mole of j)-nitrophenol in 1 min. U p t a k e of glucose: This was followed b y d e t e r m i n a t i o n of glucose in appropriately diluted growth media b y t h e glucose-oxidase t e s t (BOEHRINGER, Mannheim, F R G ) . Measurement of r a d i o a c t i v i t y : R a d i o a c t i v i t y of glucan fibrils collected on glass-fiber discs, a n d t h a t of m a n n a n - p r o t e i n s dried on filter p a p e r strips, was measured on a Nuclear Chicago c o u n t e r using a toluene scintilation fluid containing 6 g of P P O a n d 0.75 g of P O P O P in 1 liter.

Results E f f e c t of s u g a r a n a l o g u e s on cell g r o w t h The effect of DG, FG and FM on growth of S. cerevisiae in glucose and mannose medium is shown in Fig. 1 and Fig. 2. Growth on both hexoses was inhibited more intensively by fluorohexoses than by DG. Growth on glucose was almost completely blocked by FM. On the other hand, FG was found to be the most effective inhibitor of the cell growth on mannose. Microscopic observation of the cultures in the presence of sugar analogues revealed that the lag-phase, reduction of the growth rate as well as later stop of the growth were accompanied by extensive lysis of the population. No viable cells were found, for instance, after 80 hours in the glucose medium containing FM.

Effect of sugar analogues on yeast

491

The resumption of the growth observed after long lag-phase is a result of multiplying of selected individuals which are resistant to the starting concentration of the analogue. It is of interest to note that a population of cells resistant to 0.05% FG in glucose medium was unable to grow in the same medium containing 0 . 0 5 % FM. The same concentration of FM as that of FG is more toxic to the cells grown on glucose.

Time (hrs) Fig. 1. Effect of sugar analogues on growth of S. cerevisiae in 2 % glucose medium. Control (o), 0.05% DG (•), 0.02% FG (•), 0.05% FG (A), 0.02% FM (•), 00,5% FM (A)

Time ( hrs)

Fig. 2. Effect of sugar analogues on growth of S. cerevisiae in 2 % mannose medium. Symbols as in Fig. 1

492

P . B I E L Y , L . P . RJAZANOVA a n d A . B . TSIOMENKO

E f f e c t on wall p o l y s a c c h a r i d e s y n t h e s i s in p r o t o p l a s t s The results of the growth experiments suggested that FG is more antagonistic to mannose than to glucose, and FM more antagonistic to glucose than to mannose. These observations led us to examine the effects of fluorohexoses on the synthesis of wall polysaccharides in protoplasts grown on glucose and mannose. A convenient way to follow the formation of cell wall polysaccharides in yeast, separating the lytic effects of the sugar analogues on growing cells, is to use protoplasts. Table 1 shows that the formation of glucan fibrils on the surface of protoplasts as well as the secretion of mannan-proteins to the medium (considered here for a measure of their synthesis) Table 1 Effect of DG, FG and PM on cell wall glucan and mannan synthesis in protoplasts of 8. cerevisia grown in 2 % glucose or 2 % mannose medium. Protoplasts were grown for 3 hours in the absence and in the presence of sugar analogues (0.02%), then the radioactivity of isolated glucan fibrile and mannan-proteins was determined. Radioactivity of control samples was taken for 100%s Carbon source

Sugar analogue

Polysaccharide Glucan

Mannan

Glucose

DG FG FM

% of control 80 75 46 54 37 26

Mannose

DG FG FM

59 44 40

88 72. 65

Fig. 3. Inhibition of cell wall glucan (Part A) and mannan (Part B) synthesis in protoplasts grown in 2 % glucose medium in the presence of 0 . 0 3 % DG (•), FG (A) and FM (A). Control protoplasts (O)

Effect of sugar analogues on yeast

493

was reduced most by FM, regardless the hexose used as a carbon source in the medium. The inhibition of glucan formation by all three analogues tested was greater in the medium containing mannose. Synthesis of mannan appears to be more sensitive in glucose medium. Time course of synthesis of glucan and mannan by protoplasts grown in 2 % glucose medium in the presence of 0.03% sugar analogues (Fig. 3) further demonstrated that FM is the most potent inhibitor, particularly that of mannan formation. E f f e c t on g l u c o s e u p t a k e In the same experiment as that presented in Fig. 3, we followed the uptake of glucose by protoplasts (Fig. 4). The uptake was inhibited by all three sugar analogues to a much lesser extent than the formation of wall polysaccharides. Up to 3 hours, there was no substantial difference among the effect of DG, FG and FM. Later on, however, the uptake of glucose became remarkably reduced by FM, when compared to the effects of FG and DG.

Time (hrs)

Pig. 4. Glucose uptake by protoplasts grown in the'absence (o) and in the presence of 0.03% DG (•), F G (A), and FM (A)

E f f e c t on s y n t h e s i s of e x t r a c e l l u l a r a n d i n t r a c e l l u l a r e n z y m e s Effect of sugar analogues on synthesis of invertase, as a typical extracellular glycoprotein enzyme of yeast, was followed only in protoplasts grown on mannose, because the strain of 8. cerevisiae used synthesizes about twenty times more invertase in mannose than in glucose medium (KRÂTKY et al. 1975). As can be seen in Fig. 5, FM again appears to be the most potent inhibitor. Extremely strong is also the FM impediment in the synthesis of intracellular non-glycosylated enzyme, a-glucosidase (Fig. 6), regardless the hexose used as the growth support. Similarly as reported in studies with DG (Kuo and LAMPEN 1 9 7 2 , KRATKY etal. 1 9 7 5 ) , alkaline phosphatase, as another intracellular enzyme, was not very much influenced by FG and FM (not shown).

494

P . B I E L Y , L . P . R J A Z A N O V A a n d A . B . TSIOMENKO

03

0

J

2

3 Time

4

S

! h r s )

Fig. 5. Effect of sugar analogues (0.03% concentration) on the formation of extracellular invertase in protoplasts grown in 2% mannose medium. Control (o), DG ( • ) , FG (a), F M (A)

0

7

2

3

4

5

7

2

3

if

S

T i m e ( h r s )

Fig. 6. Effect of sugar analogues (0.03% concentration) on a-glucosidase synthesis in protoplasts grown on 2 % glucose (Part A ) and 2 % mannose (Part B). Control (o), DG ( • ) , FG (A), FM (A)

Effect of sugar analogues on yeast

495

In protoplasts grown on mannose for 3 hours we have compared the activities of invertase and a-glucosidase as a function of concentration of DG and FM (Fig. 7). In spite of the fact that both enzymes differ in cellular localization and in the presence of carbohydrate moiety, the relative inhibition of synthesis of both enzymes was found to be the same in the presence of DG and FM, the latter analogue being again a more effective inhibitor. In comparison to FM, about double concentration of DG is required to bring about the same inhibition of invertase or a-glucosidase synthesis.

Tig. 7. Synthesis of invertase (Part A) and a-glucosidase (Part B) in protoplast as a function of DG ( o ) and FM (•) concentration. Protoplasts were grown in 2 % mannose medium for 3 hours, and then the enzyme activities were determined

Discussion The results of the present study suggest that the mechanism of inhibitory action of DG, FG and FM on growth and metabolism of S. cerevisiae is of a similar or of the same nature. The sugar analogues differ only in the intensity of their toxic effects. The same extent of inhibition of all followed activities in cells and protoplasts can be achieved by lower concentration of fluoroanalogues than by DG. Their effects are, in general, dependent on whether the cells and protoplasts were grown on glucose or mannose. Such a phenomenon has been first observed in a study with DG (KRATKY et al. 1975) and was interpreted in the view that DG, via its metabolites, can act as an analogue of both glucose and mannose, depending on the exogeneous hexose. Essentially the same is the behaviour of fluorohexoses. The cells do not seem to recognize sufficiently the fluorine atom at carbon 2 as a hydroxyl group, so that FG and FM do not exhibit a strict antagonism to the corresponding hexoses. This is in a consonance with metabolic transformations of FG and FM in the cells of the investigated yeast strain (SCHMIDT et al. 1978). Both fluoroanalogues enter the metabolic pathways of glucose and mannose leading to synthesis of cell wall polysaccharides.

496

P . B I E L Y , L . P . RJAZANOVA a n d A . B . TSIOMENKO

Certain degree of antagonism of fluorohexoses to the corresponding natural hexoses was observed, however, in the growth experiments with intact cells. The fluorinated analogues inhibited growth by inducing cell lysis more intensively when the cells were grown in the medium containing not the corresponding natural hexoses, but their 2-epimers. These findings do not correlate well with the effects of fluorohexoses on t h e synthesis of cell wall polysaccharides in protoplasts. Relative amounts of glucan and mannan synthesized were found to be more dependent on the hexose used as a main carbon source than on the type of the fluoroanalogue. All these observations represent therefore a strong evidence t h a t fluorohexoses, similarly as DG, interfere mainly with glucose-mannose interconversion catalyzed by hexosephosphate isomerases. Inhibition of sugar uptake, due to accumulation of phosphorylated metabolites, as suggested by K u o and LAMPEN ( 1 9 7 2 ) , certainly plays additional role in the mechanism of repression of some basic metabolic activities in the cells. The view t h a t the mechanism of toxic effects of fluorohexoses is of the same nature as t h a t of DG, finds further support in the comparison of inhibition of invertase and a-glucosidase synthesis in protoplasts in the presence of DG and FM. Strikingly close correlation between the inhibition of invertase and a-glucosidase synthesis by these analogues is in a consonance with earlier conclusions of KRATKY et al. ( 1 9 7 5 ) , t h a t the synthesis of invertase in the presence of sugar analogues is controlled more by protein synthesis t h a n by glycosylation. Metabolites of fluorohexoses, similarly as those of DG, not only inhibit t h e enzyme reactions of the corresponding metabolites of glucose and mannose, b u t also interfere with metabolic control of enzyme synthesis, simulating thus the role of the corresponding metabolites of natural hexoses (catabolic repression). The reason for different toxicity of the three sugar analogues tested remains unknown. Under the assumption t h a t the extent of the inhibitory effects is proportional to intracellular concentration of phosphorylated metabolites of the sugar analogues, one can relate the highest toxicity of FM to the fact t h a t of 6-phosphates of DG, FG and FM, only FM-6-phosphate is not a substrate for glucose-6-phosphate dehydrogenase (BESSEL and THOMAS 1 9 7 3 ) . This enzyme could play a significant role in reduction of intracellular concentration of the toxic phosphate esters. I t has been already shown t h a t 2-deoxy-D-gluconic acid can be formed in t h e cells of S. cerevisiae on account of the decrease of other DG metabolites (BAUER and BIELY 1 9 6 8 ) . I n a study with fluorohexoses (SCHMIDT et al. 1978) it has been erroneously concluded t h a t aldonic acid or its 6-phosphate was not formed from FG. I t has been overlooked t h a t t h e used fluorohexoses tritiated by the methods of EVANS et al. ( 1 9 7 4 ) were exclusively [l- 3 H]-labelled, so t h a t the conversion of FG to fluorogluconic acid would be accompanied by removal of the radioactive label. Acknowledgements One of the authors (P. B.) is indebted to the scientists of the Institute of Biochemistry and Physiology of Microorganisms, Academy of Sciences USSR, and particularly to Prof. I. S. KTJLAEV, for their hospitality and advice.

References S. and B I E L Y , P., 1968. Metabolism of 2-deoxy-D-glucose by baker's yeast. II. Formation of 2-deoxy-D-D-gluconic acid. Coll. Czechoslov. Chem. Commun., 33, 1 1 6 5 — 1 1 7 3 . B E S S E L , E . M . , FOSTER, A . B . and WESTWOOD, J . H . , 1 9 7 2 . The use of deoxyfluoro-D-glucopyranoses and related compounds in a study of yeast hexokinase specificity. Biochem. J., 128, 199 to 204. BATJEB,

E f f e c t of sugar analogues on yeast

497

a n d T H O M A S , P . , 1 9 7 3 . T h e effect of s u b s t i t u t i o n a t C - 2 of D-glucose 6-phosphate on t h e r a t e of d e h y d r o g e n a t i o n b y glucose 6 - p h o s p h a t e dehydrogenase (from y e a s t a n d r a t liver).

BESSEL, E . M .

B i o c h e m . J . , 181, 83—89.

a n d BAXTER, § . , 1 9 6 6 . Metabolism of 2-deoxy-D-glucose b y b a k e r ' s yeast. I . Isolation a n d identification of p h o s p h o r y l a t e d esters of 2-deoxy-D-glucose. Coll. Czechoslov. Chem.

BIELY, P .

C o m m u n . , 32, 1588—1594. BIELY, P . , KRATKY, Z . , KOVARIK, J . a n d BAUER,

1971. E f f e c t of 2 - d e o x y g l u c o s e o n cell w a l l

EVANS, E . A . ,

WARRELL, D . C.,

f o r m a t i o n in Saccharomyces cerevisiae a n d its relation t o cell g r o w t h inhibition. J . B a c t e r i d . , 107,121-129. BIELY, P., KOVARIK, J . a n d BAUER, §., 1973. Lysis of Saccharomyces cerevisiae w i t h 2-deoxy-2fluoro-D-glucose, an inhibitor of t h e cell wall glucan synthesis. J . B a c t e r i d . , 115, 1108 — 1120. C R A M E R , F . B . a n d W O O D W A R D , G . E . , 1 9 5 2 . 2-Deoxy-D-glucose as an a n t a g o n i s t of glucose in y e a s t f e r m e n t a t i o n . J . F r a n k l i n Inst., 258, 3 5 4 — 3 6 0 . SHEPPAJRD, H . C . ,

TURNER, J . C.

and

1974.

New

approach

to

specific labeling of organic compounds w i t h t r i t i u m . Catalyzed exchange in solution w i t h t r i t i u m gas. J . Labelled Compounds, 10, 569—587. F A B K A S , Y . , SVOBODA, A . a n d B A U E R , 1 9 6 9 . I n h i b i t o r y effect of 2-deoxy-D-glucose ot t h e f o r m a t i o n of t h e cell wall in y e a s t protoplasts. J . Bacteriol., 98, 744—748. H E R E D I A , C. F . , D E L A F U E N T E , G . a n d SOLS, A., 1 9 6 4 . Metabolic studies w i t h 2-deoxyhexoses. I. Mechanism of inhibition of g r o w t h a n d f e r m e n t a t i o n in b a k e r ' s y e a s t . Biochim. biophysica Acta, 8 6 , 2 1 6 — 2 2 3 . JOHNSON, B. F., 1968. Lysis of y e a s t cell walls induced b y 2-deoxyglucose a t t h e i r sites of glucan synthesis. J . Bacteriol., 95, 1169 — 1172. K R A T K Y , Z . , B I E L Y , P . a n d B A U E R , § . , 1 9 7 5 . Mechanism of 2-deoxy-D-glucose inhibition of cellwall polysaccharides a n d glycoprotein biosynthesis in Saccharomyces cerevisiae. E u r . J . Biochem., 54, 459 - 4 6 7 . K u o , S.-C. a n d LAMPEN, J . O., 1972. I n h i b i t i o n b y 2-deoxy-D-glucose of synthesis of glycoprotein enzymes b y p r o t o p l a s t s of Saccharomyces-. R e l a t i o n t o inhibition of sugar u p t a k e a n d m e t a bolism. J . Bacteriol., I l l , 419—429. L E H L E , L . a n d S C H W A B Z , R . T . , 1 9 7 6 . F o r m a t i o n of dolichol m o n o p h o s p h a t e 2-deoxy-D-glucose a n d its interference w i t h t h e glycosylation of m a n n o p r o t e i n s in yeast. E u r . J . Biochem., 67, 239-245 S C H M I D T , M. F . , B I E L Y , P . , K R A T K Y , Z .

a n d S C H W A R Z , R . T . , 1 9 7 8 . Metabolism of 2-deoxy-2fluoro-D[ 3 H] glucose a n d 2-deoxy-2-fluoro-D-[ 3 H] m a n n o s e in y e a s t s a n d chick-embryo cells. E u r o p . J . Biochem., 87, 55 — 68. T S I O M E N K O , A. B . , D M I T R I E V , V . V . , VAGABOV, B . M. a n d K U L A E V , I . S., 1 9 7 8 . Regeneration of t h e y e a s t cell wall. I n : E x p e r i m e n t a l S t u d y of D e v e l o p m e n t of Microorganisms (In Russian), p p . 1 2 4 — 1 2 8 . A k a d e m i a N a u k SSSR, Puscino. T S I O M E N K O , A. B . , VAGABOV, V . M . a n d K U L A E V , I . S., 1975. E f f e c t of 2-deoxyglucose on syntesis a n d secretion of acid p h o s p h a t a s e a n d m a n n a n in y e a s t p r o t o p l a s t (in Russian). Dokl. A k a d . N a u k SSSR, 220, 250 - 2 5 3 . Mailing address: D r . P . BIELY I n s t i t u t e of Chemistry Slovak A c a d e m y of Sciences D u b r a v s k a cesta 809 33 Bratislava, Czechoslovakia

Zeitschrift für Allgemeine Mikrobiologie

21

7

1981

499-506

(Institute of Microbiology, University of Lodz, 90-237 Lodz, Poland)

Regulation of steroid 16a-hydroxylation in Streptomyces olivoviridis J . DMTGONSKI a n d L . SEDLACZEK

(Eingegangen

am 11.

11.1980)

The steroid 16 ¡ÍH> CS

s S 3

02

J>» "3

® ® ® ® M s"i .2 ® 5 tcaS ccoS O m 03 0 C3 S ^ 0) "T Iah a« i í. t+4 03 A -gjs s " M ì t -öjs a s cS g g 3 e O «2 l>lj S 8 | l l I •SS 8 8 « S S fi s ? J2 g uC TS — ® FRS^ ü 8 M O 8 M fi ¿ WJM 0Q 8 MM

IN

The amylolytic system of Schwanniomyces

541

Optimum p H The optimum p H was determined with buffers varying in p H from p H 3.0 to p H 9.0. For the p H values of 3.0 to 6.0, we used a citrate buffer, from 5.5 to 8.0 a phosphate buffer, and from 8.0 to 9.0 a boric acid-borax buffer. These buffers had no influence on the enzymes. All three enzymes have an optimum p H at 6.0. Optimum temperature The optimum temperature (Fig. 4) is approxmatively 60 °C for all three enzymes.

Time (mini

0

10

20 JO Time Iminl Fig. 4. Effect of temperature on 6 g l y c o s i d e bond. T h e p r e s e n c e of t h e s e t h r e e e n z y m e s c o u l d r e s u l t i n b e t t e r h y d r o l y s i s of s t a r c h b y t h i s s t r a i n t h a n b y o t h e r s . I t w o u l d b e i n t e r e s t i n g t o f i n d o u t if t h e 2 g l u c o a m y l a s e s a r e i n d u c i b l e b y t h e s a m e s u b s t r a t e s or a s s h o w n i n Candida pelliculosa (KAWAMURA a n d SAWAI 1 9 6 8 ) b y d i f f e r e n t s u b s t r a t e s . S t u d i e s are u n d e r w a y t o f i n d p o s s i b l e w a y s of i n c r e a s i n g t h e p r o d u c t i o n of all 3 e n z y m e s a n d t h u s t h e b i o m a s s p r o d u c t i o n b y o p t i m i z a t i o n of c u l t u r e c o n d i t i o n s . References E., S C H M I D T , P . a n d S T O R K , H . , 1 9 7 4 . Methods of E n z y m a t i c Analysis, I . E d n , 1196 — 1201. Academic Press L o n d o n . BERNFELD, P . , 1955. M e t h o d s in Enyzmology, 149. Academic Press New Y o r k . E B E R T O V A , H . , 1966a. Amylolytic enzymes of Endomycopsis capsularis. I . F o r m a t i o n of t h e amylolytic system in cultures of Endomycopsis capsularis. Folia microbiol., 11, 14—20. EBERTOVA, H . , 1966b. Amylolytic enzymes of Endomycopsis capsularis. I I . A s t u d y of t h e properties of isolated a - a m y l a s e , amyloglucosidase, m a l t a s e transglucosidase. Folia microbiol., 11, 422—438. G A S D O R F , H . , A T H A S A M P O N I V A , P . , D A K , V . a n d S M I L E Y , K . , 1 9 7 0 . P a t t e r n s of action of glucoa m y l a s e isoenzyme f r o m Aspergillus sp. on glycogen. C a r b o h y d r a t e Res., 42, 147 —156. HATTORI, Y., 1961. Studies on amylolytic enzymes produced b y Endomyces sp. I . P r o d u c t i o n of extracellular a m y l a s e b y Endomyces sp. Agric. biol. Chem., 25, 737—743. H A T T O R I , Y . a n d T A K E U C H I , I . , 1 9 6 1 . Studies on amylolytic enzymes produced b y Endomyces sp. I I . P u r i f i c a t i o n a n d general properties of amyloglucosidase. Agric. biol. Chem., 25, 895—901. HATTORI, Y. a n d TAKEUCHI, I . , 1962. Studies on amylolytic enzymes produced b y Endomyces sp. I I I . Hydrolysis of s t a r c h a n d glucosyl saccharides w i t h amyloglucosidase. Agric. biol. Chem., 26, 3 1 6 - 3 2 2 . K A W A M U R A , S . a n d S A W A I , T . , 1 9 6 8 . Differential induction of t w o glucoamylases in Candida pelliculosa. Agric. biol. Chem., 32, 114 — 116. M O U L I N , G . e t G A L Z Y , P . , 1 9 7 8 a. E t u d e d e L'a-amylase de la paroi d e Pichia burtonii BOIDIN. Z. Allg. Mikrobiol., 18, 2 6 9 - 2 7 4 . M O U L I N , G . e t G A L Z Y , P . , 1978b. R e m a r q u e sur la régulation d e la biosynthese de l ' a - a m y l a s e de Pichia burtonii B O I D I N . Z . Allg. Mikrobiol., 1 8 , 329—333. M O U L I N , G . a n d G A L Z Y , P . , 1978c. Amylase a c t i v i t y of Torulopsis ingeniosa D I M M B N A . Folia microbiol., 23, 423—427. M O U L I N , G . a n d G A L Z Y , P . , 1979. S t u d y of an a m y l a s e a n d its regulation in Lipomyces starkeyi. Agric. biol. Chem., 4 3 , 1 1 6 5 - 1 1 7 1 . O T E N G - G Y A N G , K . , 1 9 7 9 . E t u d e de levures amylolytiques en v u e d e la p r o d u c t i o n d e proteines d' organismes unicellulaires. Thèse de 3ème cycle. U.S.T.L. Montpellier F r a n c e . SMITH, B. W . a n d ROE, J . H . , 1949. A p h o t o m e t r i c m e t h o d for t h e d e t e r m i n a t i o n of a - a m y l a s e in blood a n d urine w i t h use of t h e s t a r c h iodine color. J . biol. Chemistry, 7 , 5 3 — 5 9 . T O R A B A L L A , J . C. a n d E I T I N G T O N , M . , 1 9 6 7 . Action of u r e a a n d certain o t h e r a m i d e r e a g e n t s on crystalline porcine p a n c r e a t i c amylase. Arch. Biochem. Biophysics, 119, 519. WABBURG, O. a n d CHRISTIAN, W., 1939. Isolation a n d crystallization of proteins of t h e oxidatives f e r m e n t a t i o n s enzymes. Biochem. Z., 303, 40—68. Y A M A S A K I , Y . , S U Z U K I , Y . a n d O Z A W A , J . , 1977. P u r i f i c a t i o n a n d properties of t w o f o r m s of glucoa m y l a s e f r o m Pénicillium oxalicum. Agric. biol. Chem., 41, 755—762. BERMEYER,

H . U . , BERNT,

Mailing a d d r e s s : Prof. Dr. G . G A L Z Y Chaire de Genetique e t Microbiologie E.N.S.A. - I . N . R . A . 34060 Montpellier — Cedex, F r a n c e

Zeitschrift für Allgemeine Mikrobiologie

21

7

1981

545-553

(Institut Biologie I I der Universität, Mikrobiologie, D-7800 Freiburg, Federal Republic of Germany)

Propionate formation in Rhodospirillum rubrum under anaerobic dark conditions H . V O E L S K O W a n d G . SCHÖN

(Eingegangen

am

20.11.1980)

Experiments with 14C labelled propionyl-CoA, methylmalonyl-CoA and succinyl-CoA showed t h a t these compounds are intermediates of propionate synthesis in fermentative metabolism of Bhodospirillum rubrum. The rate of propionate and succinate production is dependent on the C 0 2 concentration of the medium. There is, however, no evidence for a transcarboxylation, and high concentrations of propionate in the medium did not inhibit propionate synthesis as is the case in Propionibacteria. PEP-carboxykinase (EC 4.1.1.32) and propionyl-CoA-carboxylase (EC 6.4.1.3) showed high activities, whereas the other two PEP-carboxylases (EC 4.1.1.31, EC 4.1.1.38), and the pyruvate-carboxylase (EC 4.1.1.1.) showed only very low activity. I t is probable that in pyruvate fermentation metabolism of R. rubrum no specific enzymes are activated for propionate formation and all enzymes are still present from aerobic or photo trophic preculture. I n a n a e r o b i c d a r k m e t a b o l i s m of Bhodospirillum rubrum p y r u v a t e is b r o k e n down m a i n l y t o f o r m a t e a n d a c e t y l - C o A ( J U N G E R M A N N a n d SCHÖN 1 9 7 4 , SCHÖN a n d V O E L S K O W 1 9 7 6 , G O R K E L L a n d U F F E N 1 9 7 7 , V O E L S K O W a n d SCHÖN 1 9 7 8 ) . D e p e n d e n t on t h e g r o w t h conditions, however, p r o p i o n a t e also o c c u r r e d as a n e n d p r o d u c t . R e l a t i v e l y large quantities of p r o p i o n a t e a r e f o r m e d especially in resting cells, in cultures which a r e growing slowly on p y r u v a t e m e d i u m , or when f r u c t o s e is given as s u b s t r a t e or f u m a r a t e as a n e x t r a electron a c c e p t o r ( K O H L M I L L E R a n d G E S T 1 9 5 1 , SCHÖN a n d B I E D E R MANN 1 9 7 2 , 1 9 7 3 , G Ü R G Ü N et al. 1 9 7 6 . ) T h e present e x p e r i m e n t s seek t o d e t e r m i n e t h e p a t h w a y of p r o p i o n a t e synthesis in order t o o b t a i n some i n f o r m a t i o n a b o u t t h e signif i c a n c e of p r o p i o n a t e f o r m a t i o n for a n a e r o b i c d a r k g r o w t h of R. rubrum. Materials

and

methods

Bhodospirillum rubrum strains FJ (DSM 1 ) 1068) and Ha (DSM 1 ) 107) were precultured anaerobically in the light or aerobically in the dark on malate medium (SCHÖN 1968) for about 18 h (O.D.623,I-0CM = 0.8). The conditions for anaerobic dark culture with pyruvate (and/or 14 C-pyruvate) were as described previously (SCHÖN and VOELSKOW 1976). In most cases N 2 was streamed through the culture vessels during the experiment except in the 1 4 C0 2 -experiments where the vessels were closed after the addition of K H 1 4 C 0 3 , which had the same specific activity as the 14 C-pyruvate (8,000 dpm/nmol C02).

Cell-free extracts were produced in phosphate buffer (0.1 mol/1 K 2 H P 0 4 , 0.5 g/1 MgS0 4 • 7 H 2 0 , pH 8.0) using an AMINCO French press (140 • 10 6 Pa). Buffer volume was twice that of the bacterial wet weight. The unbroken cells were removed by centrifugation (20 min, 11,500 • g). The experiments with succinyl-CoA (1.4- 14 C), methylmalonyl-CoA (methyl- 14 C), and propionyl-CoA (1- 14 C) were performed in THUNBERG tubes (0.7 ml extract, 1.3 ml phosphate buffer). Anaerobic conditions were produced by repeated evacuations and additions of Ar. In between 0.1 ml pyruvate (0.1 mol/1) was added. The CoA-compounds (0.1 ml: 0.25 fiCi/ml; 0.5 — 1.0 mg/ml) were added from the side arm to the extract in the main compartment after incubation of the tubes for Abbrevations: Carboxylases see Fig. 1, O.D. = optical density; P H B A = poly-ß-hydroxybutyric acid; P F L = pyruvate formate lyase; P D H = pyruvate dehydrogenase complex r

) DSM = Deutsche Sammlung von Mikroorganismen, Göttingen

546

H . VOELSKOW a n d G . SCHÖN

3 min in a shaking waterbath (30 °C) and after 5, 10 or 15 min the reaction was stopped by heat inactivation (100 °C, 2 min). Breakdown of L- 14 C-pyruvate and production of "C-endproducts were determined b y isoionic chromatography (THATJER et al. 1970, SCHON and VOELSKOW 1976)

and in a scintillation counter (PACKARD). For determination of the carboxylase activities the cells were washed twice (Tris-HCl-buffer 50 mmol/1, MgS0 4 • 7 H 2 0 0.5 g/1, pH 7.0), broken in a FRENCH press (140 • 10 6 P a ; buffer: TrisHC120 mmol/1, E D T A 1 mmol/1, glutathione [red] 0.5 mmol/1, pH 7.5; buffer: cell wet weight 4 : 1 ) . Whole cells and cell debris were eliminated by centrifugation (48,000- g; 1 h). The suspension stirred together with Dowex-Cl" (p.a., 1 x 8, 200 — 400 mesh; 2 g pro 10 ml) to eliminate nucleotides, then filtered and used for the enzyme determinations. The test conditions for the P E P - and pyruvate carboxylases differ in the literature (Lit. see: COOPER a n d B E N E D I C T 1 9 6 8 , E V E R S 1 9 7 5 , FRINGS a n d SCHLEGEL 1 9 7 1 , K O H L E R et al.

1976, PAYNE

and MORRIS 1969, SCRUTTON 1974). Therefore, in preliminary experiments t h e influence of CoA,

acetyl-CoA, Mg , Mn , Co and the pH-value on these enzyme activities in B. rubrum cell extract was examined. The modified test conditions are summarized in Table I. Propionyl-CoA 2+

2+

2+

carboxylase was determined as described b y OLSEN and MERRICK (1968).

Table 1 Test conditions for propionyl-CoA-carboxylase (Prop-CoA-C, EC 6.4.1.3), PEP-carboxykinase (PEP-CK, EC 4.1.1.32 = G T P : oxalacetate carboxylate [transphorylating]), PEP-carboxylase (PEP-C, EC 4.1.1.31 = orthophosphate: oxalacetate carboxylyase [phosphorylating]), P E P carboxy-transphosphorylase (PEP-CT, EC 4.1.1.38 = pyrophosphate: oxalacetate carboxylyase [transphosphorylating]), pyruvate carboxylase (Pyr-C, EC 4.1.1.1.). Test solutions 1) Tris-HCl-buffer 1 mol/1, glutathione 50 mmol/1 (red) 2) MgS0 4 50 m m o l / l + A T P 100 mmol/1 3) MgS0 4 50 mmol/1 + A T P 20 mmol/1 4) MnS0 4 15 mmol/1 5) GDP 20 mmol/1 6) P0 4 -buffer 100 mmol/1, pH 7.0 7) Na-pyruvate 50 mmol/1 8) P E P (mono-K-salt) 50 mmol/1 9) Na-propionate 100 mmol/1 10) Coenzyme-A 10 mmol/1 11) NADH 15 mmol/1 + acetyl-phosphate 50 mmol/1 12) Malatedehydrogenase 60 i. u./ml + phosphotransacetylase 50 i. u./ml 13) NaHC0 3 100 mmol/1 + Na 2 1 4 C0 3 2 (xCi/ml l

Prop.-CoA-C

P E P - C K PEP-C

PEP-CT

Pyr-C

0.1, pH 7.0

0.1, pH 8.5

(ml) 0.1, pH 8.0 0.1 —

0.1, pH 7.0

0.1, pH 7.5

—

—

—

—

—

—

0.1

0.1 0.1a)

0.1

— —

—

—

—

— —

0.1») 0.1

—

0.1 —

—

—

0.1a) —

—

0.1a) —

0.1 —

—

0.1 — — —

0.1 a ) — —

—

0.1 0.1

0.1 0.1

0.1 0.1

0.1 0.1

—

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

) controls without this solution

The test solutions were put in scintillation vials. Before 1 4 C0 2 was added to the vials to start the reaction the volume was made up to 1.0 ml with distilled water, the cell extract added (0.1 ml and 0.2 ml twice for testing and twice as controls), and the vials put into a water bath (30 °C). The reactions were stopped by addition of 0.2 ml HC10 4 (8%) after 10 min (PEP-CK), 20 min (Prop-COA) or 30 min (PEP-C, Pyr-C). 2 drops silicon-antifoam (1:1000 diluted, RoTH-Karlsruhe) were then added and air streamed for 30 min through the solution to eliminate all unfixed 1 4 C0 2 . The incorporated 1 4 C0 2 was measured in the same vials after addition of 18 ml Triton-toluene scintillation mixture (VOELSKOW and SCHÖN 1978). The quench correction was determined by the automatic external standard (A.E.S.). Protein was determined by a modified Folin-phenol method (LOWRY et al. 1951). Standard for calculation was bovine albumin, fract. V (CALBIOCHEM.). The optical density (O.D.) of the suspension was measured at 623 nm (1.0 cm) in an EPPENDORF photometer. Enzymes and coenzymes were obtained from BOEHRINGER, Mannheim, N 2 (reinst) and Ar (special) were supplied by MESSER GRIESHEIM (Düsseldorf). 14 C-pyruvate and 1 4 C0 2 were purchased from AMERSHAM BÜCHLER (Braunschweig) and the "C-CoA-derivatives from N E N Chemicals (Dreieichenhain).

Propionate formation in Rhodospirillum

547

Results Cell e x t r a c t s of B. rubrum were incubated anaerobically in the dark w i t h unlabelled p y r u v a t e as substrate, in addition 1 4 C-labelled p y r u v a t e or labelled probable C o A intermediates of p r o p i o n a t e synthesis were present in the medium ( T a b . 2 and T a b . 3). T h e main product of p y r u v a t e metabolism in the e x t r a c t was propionate w i t h o n l y a l i t t l e succinate and f o r m a t e being f o r m e d ( T a b . 2). P r o p i o n y l - C o A was 9 0 % conv e r t e d t o p r o p i o n a t e in 5 minutes ( T a b . 3). N o succinate was produced f r o m this Table 2 Pyruvate utilization and product formation in cell-free extracts of B. rubrum (FJ under anaerobic dark conditions (phosphate buffer, pH 8.0) ; preculture : phototrophic in malate medium Time of incubation (30 °C) 0 min 10 min 20 min

pyruvate

propionate

succinate

formate

|j.mol/ml 5.00 2.04 1.47

0.00 2.68 3.16

0.00 0.05 0.13

0.00 0.19 0.25

Table 3 Propionate and succinate formation from some coenzyme-A derivatives (14C-labelled) in cell-free extracts of R. rubrum (Pi).

Time of incubation (30 °C)

Radioactivity (in % of the added CoA-compound) recovered in the fractions of succinate

94.9 96.4 94.7

0.0 0.0 0.0

0.6 0.8 0.7

0.0 15.6 40.0

0.0 2.2 1.8

6.3 67.0 34.7

21.5 25.8 26.7

72.9 61.9 67.6

1.3 1.7 1.1

3.6 6.3

23.4 32.4

1.0 6.4

6.7 6.5 5.8

81.7 89.6 80.5

6.6 8.3 8.0

0.5 0.8

0.4 0.5

0.7 0.8

14 C-propionyl-CoA

5 min 10 min 15 min controls I II III

14C-succinyl-CoA

5 min 10 min 15 min controls II III

14 C-methyl-malonyl-CoA

5 min 10 min 15 min controls II III

all other fractions

propionate

The extract was incubated with unlabelled pyruvate (10 mmol/2 ml) anaerobically in the dark. Further conditions as experiments in Tab. 2. control I : distilled water instead of cell extract at 0 min control I I : distilled water instead of cell extract, incubation 5 min control I I I : incubation 10 min with heat inactivated cell extract (100 °C, 2 min) Remaining radioactivity was lost on the chromatography column.

548

H. VOELSKOW and G. SCHON

compound. I n controls using heat inactivated cell extracts propionate occurred in much lower quantities. From succinyl-CoA the main product was succinate, although again about 2 5 % was converted to propionate. This was a considerably higher amount than in the controls. With methylmalonyl-CoA as additional substrate the product was more than 8 0 % succinate with small amounts of propionate, although the concentration of the latter was 10 times that found in the controls. These results suggest that these CoA-derivatives are intermediates in propionate synthesis and that it is unlikely that propionyl-CoA is a precurser for succinate synthesis under this conditions. Propionate should then be formed in fermentative metabolism of R. rubrum via malate and succinate as in propionibacteria. I n contrast to the propionibacteria, however, the C 0 2 concentration in the medium had a considerable influence on propionate and succinate synthesis (Tab. 4). I n dense suspensions (strain Ha and F x ) where pyruvate was substrate either C 0 2 was continuously removed by a N 2 stream, or C 0 2 from fermentation was not removed, or, in a third experiment, K H C 0 3 was added. I t could be confirmed that propionate synthesis increased with increasing C 0 2 content (up to 50 K H C 0 3 mmol/1). In strain Ha (which produces small amounts of succinate) synthesis of succinate was also increased by a high C 0 2 concentration in the medium. Formate production was not influenced by C0 2 , although splitting of formate b y the hydrogen lyase was completely inhibited by high C 0 2 partial pressure (50 mmol KHC0 3 /]). Table 4 Influence of C 0 2 on the propionate and succinate production from pyruvate in R. rubrum (strain Fj and Ha) [product formation (fxmol) pro pyruvate metabolized ([xmol)] Strain :

Pi Ha Ha

KHCO3 added

C 0 2 removed by N 2 stream

C 0 2 not removed

12.5 mmol/1

propionate succinate

0.16

0.26

0.28

—

—

—

—

propionate succinate

0.12 0.03

0.19 0.03

0.28 0.04

0.32 0.08

production of

°05

1.0

50 mmol/1 0.2

tj /0 O-D-eaamtlOcm)

Fig. 1. Change in the propionyl-CoA carboxylase from phototrophic cultures during growth, and ( X = propionate, o = malate) in the medium. about

activity in cell-free extracts of R. rubrum (Fj) dependence of the activity on the substrate The log-phase ended at an O.D. 6 2 3 (i.o cm) of 1.1

Propionate formation in

549

Rhodospirillum

That C0 2 is involved in succinate and propionate formation is also shown by the strong uptake of 1 4 C0 2 into these compounds on fermentation of pyruvate (Fig. 2). The unlabelled pyruvate in the medium and the formate produced from pyruvate showed also labelling through exchange reactions. Specific labelling (dpm/mol product) of propionate, after 6 h culture with substrate exhaustion, was however 2—3 times as high as in the case of pyruvate and formate radioactivity, and succinate specific labelling again double that of propionate. C0 2 uptake, the formation of oxalacetate from pyruvate, probably occurs via a carboxylation of P E P - C K . In anaerobic dark cultured cells this carboxylase showed the highest activity of the PEP-carboxylases and the pyruvate carboxylase, although changing from phototrophic to dark fermentative metabolism the activity at first fell (Fig. 2). On substrate exhaustion a further inactivation was noticeable. In aerobic or phototrophic cultured cells P E P - C K had about the same activity and this was again about 10 times that of the other PEP-carboxylases and pyuruvate carboxylase (Tab. 5).

\JB00

§• 1000 -

0

2

6

10

%

C0i - addition ('moles/mi1

n

Fig. 2. Dependence of the 1 4 C0 2 incorporation, during the anaerobic dark fermentation (6 h) of R. rubrnrn (Ha) with pyruvate as substrate, in propionate (•), succinate ( + ), formate (o), and pyruvate ( X ) on the C0 2 -concentration in the medium Table 5 Activity of some carboxylating enzymes in cell-free extracts of R. rubrnrn ( F J from oxidative or phototrophic culture (at the beginning of the stationary growth phase) on malate medium (O.D.623nm, 1.0cm = 1.1) (standard deviation s was calculated from 6 determinations) Activity (nmol • mg protein"1 • min" 1 ) Carboxylase ) 1

PEP-CK PEP-C PEP-CT Pyr.-C Prop.-CoA-C

phototrophic 50.58 5.41 2.27 0.91 8.48

± ± ± ± ±

0.96 0.08 0.42 0.02 0.34

oxidative 50.67 3.20 3.09 0.50 5.69

± ± ± ± ±

1.57 0.57 0.22 0.12 0.61

*) Abbrev. see Tab. 1

Activity of propionate carboxylase varied considerably depending on growth phase and substrate of the preculture (Fig. 1, Fig. 3). On change from phototrophic to anaerobic dark culture the activity fell somewhat or remained constant (Fig. 3). When the substrate was exhausted the activity increased in all cases (5 Exp.) by 35—50% of the activity occurring during pyruvate breakdown in the cells. In some cases, however, a later decrease was observed. An attempt was made to inhibit propionate production in fermentation metabolism by addition high concentrations of propionate or succinate (Tab. 6). Neither 12.5 mmol/I

550

H . VOELSKOW a n d G . SCHÖN

nor 50 mmol/1 propionate or succinate in the medium had any influence on the formation rate of propionate. The rate of propionate synthesis was also unchanged on inhibition of p y r u v a t e formate lyase with hypophosphite (5 mmol/1), although the absolute amount of propionate produced (pro p y r u v a t e decomposed) was higher, on account of inhibition of formate production. Under these conditions higher C 0 2 production

/ ( M ) " — " " O o

o'

A

Prop-Co AC

^ 18

h

210

*PEP-CT ¿"~ r*" r"'

'

s

Light Dark Anaerobic culture

Fig. 3. ¡Activities of propionyl-CoA, pyruvate and PEP carboxylases at the end of an anaerobic light culture of B. rubrum ( P I ) in a malate medium with ( N H 4 ) 2 S 0 4 and after shifting the cells to anaerobic dark conditions with pyruvate as substrate (with ( N H 4 ) 2 S 0 4 , O . D . 6 2 3 , ( l . o c m ) = about 1.1) : Activity of the propionyl-CoA carboxylase from cells precultured phototrophically in a malate medium (M) or propionate medium (P) and then incubated anaerobically in the dark with pyruvate (without (NH 4 ) 2 S0 4 , O.D.623, l.ocm about 10.4). In both experiments the pyruvate was exhausted in about 5 hours. [Abbrev. of the carboxylases see table 1, p. 546] Table 6 The influence of propionate (50 mmol/1), succinate (50 mmol/1) and hypophosphite (5 mmol/1) on the propionate formation in pyruvate fermentation of R. rubrum (Ha) Additions propionate succinate hypophosphite control

pyruvate utilization (¿mol/ml • h 2.4 2.9 1.3 2.7

propionate production (zmol/ml • h 0.35 0.38 0.38 0.37

Propionate formation in Bhodospirillum

551

indicated that instead of breakdown of pyruvate to formate an increased decarboxylation of pyruvate by the pyruvate dehydrogenase complex occurred. Although in this way more NADH was produced from pyruvate the propionate formation rate showed no increase. Discussion In anaerobic dark cultures of B. rubrum propionate synthesis occurs, as suggested by K O H L M I L L E R and G E S T ( 1 9 5 1 ) and SCHÖN and B I E D E R M A N N ( 1 9 7 2 , 1 9 7 3 ) in a similar way to the case of Propionibacteria and not via the acrylate pathway as in Clostridium propionicum or in the rumen ( A L L E N et al. 1 9 6 4 , P R I N S and VAN D E R M E E R 1 9 7 6 , S W I C K a n d WOOD 1 9 6 0 , W A L L N Ö F E R a n d B A L D W I N 1 9 6 7 , WOOD et al.

1963,

1969).

Pyruvate is probably not directly carboxylated to oxalacetate but converted to P E P by a phosphoenolpyruvate synthase, which is present in E. rubrum (BUCHANAN and E V A N S 1 9 6 6 ) . The next reaction is the carboxylation of P E P by the PEP-carboxykinase (EC 4 . 1 . 1 . 3 2 ) . After oxalacetate, the further intermediates are malate, fumarate, succinate (succinyl-CoA and methylmalonyl-CoA). As the enzymes of the tricarboxylic acid cycle have been demonstrated in B. rubrum ( E I S E N B E R G 1 9 5 3 ) a conversion of oxalacetate to succinate is possible. The further enzymes for propionate synthesis are also present as propionate can be converted to succinate with methylmalonyl-CoA as an intermediate ( K N I G H T 1 9 6 2 , O L S E N and M E R R I C K 1 9 6 8 ) . In phototrophic metabolism the propionyl-CoA carboxylase is functioning in the opposite direction as is the case of the PEP-CK. In phototrophic culture with a high ATP level in the cells the P E P - C K is catalysing the formation of P E P from oxalacetate for biosynthesis ( K L E M M E 1 9 7 6 ) . In fermentation metabolism with a low ATP level this enzyme couldprobably function in the other direction forming oxalacetate. The strong dependence on C0 2 partial pressure is evidence against a transcarboxylation and favours the view that — as in Veillonella (NG and HAMILTON 1 9 7 3 ) — free C 0 2 is involved in the pathway of propionate synthesis ( K O H L M I L L E R and G E S T 1 9 5 1 , SCHÖN and B I E D E R M A N N 1 9 7 3 , GÜRGÜN et al. 1 9 7 6 ) . This view point is supported by the high degree of specific labelling of propionate and succinate by 1 4 C 0 2 . Whereas pyruvate formate lyase, the key enzyme for fermentative pyruvate breakdown is activated and strongly synthesized in anaerobic dark culture, propionate production was more or less constant under these conditions (SCHÖN and V O E L S K O W 1 9 7 6 , VOELSKOW a n d SCHÖN 1 9 7 8 ) .

Neither inhibition by high concentrations of propionate, as in Propionibacteria and SAKHAROVA 1 9 7 4 ) , nor an increase in propionate synthesis on inhibition of pyruvate formate lyase (when the activity of pyruvate dehydrogenase complex increased, SCHÖN and J A C O B S 1 9 7 9 ) could be observed. Thus propionate synthesis in pyruvate cultures seems to be an unimportant side pathway in fermentative energy metabolism and the relevant enzymes are already present from the aerobic or phototrophic metabolism (SCHÖN and V O E L S K O W 1 9 7 6 , V O E L S K O W and SCHÖN 1 9 7 8 ) . This is also confirmed by inhibition experiments with chloramphenicol. Thus in fermentation metabolism with pyruvate as substrate propionate formation is probably without much importance, as no net ATP production occurs. Furthermore, under good fermentative conditions pyruvate is normally broken down to acetyl-CoA and formate, then formate is split further to C0 2 and H 2 by most strains of B. rubrum, so that excess reducing equivalents are eliminated as H 2 (SCHÖN and V O E L S K O W 1 9 7 6 , V O E L S K O W (IBRAGIMOVA

a n d SCHÖN 1 9 7 8 , 1 9 8 0 ) .

I t is interesting that on exhaustion of pyruvate in the medium the propionyl-CoA carboxylase rises as the breakdown of endogenous reserves begins. A relatively high ; 36 Z. Allg. Mikrobiol., B d . 21, H. 7

552

H . VOELSKOW a n d G. SCHON

propionate production is also found on breakdown of endogenous polysaccharides, where fructose instead of pyruvate is the substrate (SCHON and BIEDERMANN 1 9 7 2 ) or where fumarate is given as an extra electron acceptor. With fructose and fumarate in the medium the biosynthetic activity of the cells is at least twice of that, which could be observed in suspensions without fumarate. During fermentation of fructose or especially where fumarate is available as electron acceptor, propionate formation probably plays a more important role in removing excess reducing equivalents than in pyruvate fermentation. The relevance of fumarate in the energy metabolism and growth of R. rubrum under anaerobic dark condition is being investigated. In Fig. 4 the probable main pathways of pyruvate, fructose, and reserve material (polysaccharide) metabolism in R. rubrum under anaerobic dark conditions are summarized. rmm-p— -\pomcm ¡MAO' IMP (K [FelllJMSOJMAO] J) pathway e'-Acceptox W-CK f } OXALAUTATE—MALATE"~FUMAm '

\

SUCCINATE

\

isucam-coA)

ACETnPHOSPHATE

J

anabolic metabolism IMElHYL-MALONYL-CoA1 •CO,

\ACETATE\

\PP0PI0NATE\

Fig. 4. Main p a t h w a y s of f r u c t o s e (polysaccharide) a n d p y r u v a t e m e t a b o l i s m in R. rubrum (F u n d e r anaerobic d a r k conditions (formation of b u t y r i c acid, long chain volatile acids a n d acetoine a r e n o t shown). P F L = p y r u v a t e f o r m a t e lyase (catabolic metabolism), P D H = p y r u v a t e dehydrogenase complex (mainly anabolic metabolism), F H L = f o r m a t e h y d r o g e n lyase, P S = p y r u v a t e s y n t h e t a s e , P E P - C K = P E P - c a r b o x y k i n a s e , F e I I I = K 3 [Fe(CN) 6 ], DMSO = dimethylsulfoxide, TMAO = trimethylamine-N-oxid

Acknowledgements W e wish to t h a n k CH. BEGGS for help in p r e p a r a t i o n t h e p a p e r . T h e work was s u p p o r t e d b y g r a n t s (Scho 169) f r o m t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , Bonn — B a d Godesberg.

References ALLEN, S. H . G., KELLERMEYER, R . W., STJERNHOLM, R . L. a n d WOOD, H . G., 1964. P u r i f i c a t i o n a n d properties of enzymes involved in t h e propionic acid f e r m e n t a t i o n . J . B a c t e r i d . , 8 7 , 1 7 1 —187. BUCHANAN, B. B. a n d EVANS, N.C. W., 1966. T h e synthesis of phosphoenol-pyTuvate f r o m p y r u v a t e a n d A T P b y e x t r a c t s of p h o t o s y n t h e t i c bacteria. Biochim. Biophysic. Res. Commun., 22, 484-487. COOPER, T. G. a n d BENEDICT, C. R . , 1968. P E P - c a r b o x y l a s e exchange reaction in p h o t o s y n t h e t i c bacteria. P l a n t Physiol., 48, 788—792. EISENBERG, M. A., 1953. T h e tricarboxylic acid cycle in Bhodospirillum rubrum. J . biol. Chemistry, 208, 8 1 5 - 8 3 6 . EVERS, A. K . , 1975. A n i m p r o v e d m e t h o d for assay of carboxylation enzymes. Anal. Biochem., 64, 606-608.

Propionate formation in

Rhodospirillum

553

FRINGS, W. und SCHLEGEL, H. G., 1971. Zur Synthese von C 4 -Dicarbonsäuren aus P y r u v a t durch Hydrogenomonas eutropha Stamm H 16 . Arch. Mikrobiol., 79, 204—219. GORRELL, T. E. and UFFEN, R. L., 1977. Fermentative metabolism of pyruvate b y Rhodospirillum rubrum, after anaerobic growth in darkness. J . Bacteriol., 181, 533—543. GÜRGÜN, V., KIRCHNER, G. und PFENNIG, N. 1976. Vergärung von Pyruvat durch sieben Arten phototropher Purpurbakterien. Z. Allg. Mikrobiol., 16, 573—586. IBRAGIMOVA, S. J . and SAKHAROVA, Z. V., 1974. The inhibiting action of sodium propionate on Propionibacterium shermanii. Microbiology, 43, 12 — 16. JUNGERMANN, K . and SCHÖN, G., 1974. Pyruvate formate lyase in Rhodospirillum rubrum H a adapted to anaerobic dark conditions. Arch. Mikrobiol., 99, 109 —116. KLEMME, H. J . 1976. Undirectional inhibition of phosphoenolpyruvate carboxykinase from Rhodospirillum rubrum by ATP. Arch. Microbiol., 107, 189—192 (1976). KNIGHT, M., 1962. The photometabolism of propionate by Rhodospirillum rubrum. Biochem. J . , 84, 170—185. KÖHLER, G . - H . , RINDT, K . - P . u n d OHMANN, E . , 1 9 7 6 . D i e B i l d u n g a k t i v e r P y r u v a t - C a r b o x y l a s e

aus Apoenzym lind Biotin in Rhodopseudomonas

sphaeroides. Biochem. Physiol. Pflanzen, 169,

99-104.

KOHLMILLER, E . F. and GEST, H., 1951. A comparative study of the light and dark fermentations of organic compounds by Rhodospirillum rubrum. J . Bacteriol., 61, 269—282. LOWBY, 0 . H . , ROSEBROUGH, N . J . , FABR, 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 t h e Folin phenol reagents. J . biol. Chemistry, 198, 265—275. NG, S. K . C. and HAMILTON, I. R., 1973. Carbon dioxide fixation by Veillonella parvula M4 and its relation to propionic acid formation. Canad. J . Microbiol., 19, 715—723. OLSEN, I. and MERRICK, J . M., 1968. Identification of propionate as an endogenous C0 2 acceptor in Rhodospirillum rubrum and properties of purified propionyl coenzyme A carboxylase. J . Bacteriol., 95, 1774 — 1778. PAYNE, J . and MORRIS, J.G., 1969. Pyruvate carboxylase in Rhodopseudomonas sphaeroides. J . gen. Microbiol., 59, 97—101. PRINS, R.A. and VAN DER MEER, P., 1976. On the contribution of the acrylate pathway to t h e formation of propionate from lactate in the rumen of cattle. Antonie van Leeuwenhoek, 42, 25—31. SCHÖN, G., 1968. Fructoseverwertung und Bacteriochlorophyllsynthese in anaeroben Dunkel- und Lichtkulturen von Rhodospirillum rubrum. Arch. Mikrobiol., 63, 362—375. SCHÖN, G. und BIEDERMANN, M., 1972. Bildung flüchtiger Säuren bei der Vergärung von P y r u v a t und Fructose in anaerober Dunkelkultur von Rhodospirillum rubrum. Arch. Mikrobiol., 85, 77—90.

SCHÖN, G. and BIEDERMANN, M., 1973. Growth and adaptive hydrogen production of Rhodospirillum rubrum (FJ) in anaerobic dark cultures. Biochim. biophysica Acta, 304, 65—75. SCHÖN, G. und JACOBS, E., 1976. Aktivität und Funktion des Pyruvat-Dehydrogenase Komplexes bei der Adaptation von Rhodospirillum rubrum an anaerobe Dunkelbedingungen. Abstr. Verhandl. 37. Tagung der Deutschen Gesellschaft für Hygiene und Mikrobiol., p. 32. SCHÖN, G. and VOELSKOW, H., 1979. P y r u v a t e fermentation in Rhodospirillum rubrum a f t e r transfer from aerobic to anaerobic conditions in t h e dark. Arch. Microbiol., 107, 87—92. SCRTJTTON, M. C., 1974. Properties of t h e activation of phosphoenolpyruvate carboxylase from Escherichia coli by acryl derivatives of coenzyme A. F E B S Letters, 48, 145—148. SWICK, R. W. and WOOD, H. G., 1960. The role of transcarboxylation in propionic acid fermentation. Proc. n a t . Acad. Sei. USA, 46, 28—41. THAITER, R . K . , RTJPPRECHT, E . a n d JUNGERMANN, K . , 1 9 7 0 . S e p a r a t i o n of

14

C-formate from C02-

fixation metabolites by isoionic-exchange chromatography. Analyt. Biochem., 38, 461—468. VOELSKOW, H . and SCHÖN, G., 1978. P y r u v a t e fermentation in light-grown cells of Rhodospirillum rubrum during adaptation to anaerobic dark conditions. Arch. Microbiol., 119, 129 — 133. VOELSKOW, H. and SCHÖN, G., 1980. H 2 production of Rhodospirillum rubrum during adaptation to anaerobic dark conditions. Arch. Microbiol., 125, 245—249. WALLNÖFER, P. and BALDWIN, R. L., 1967. P a t h w a y of propionate formation in Bacteroides ruminicola. J . Bacteriol., 93, 504—505. WOOD, H . G . , ALLEN, S . H . G . , STIERNHOLM, R . a n d JACOBSON, B . , 1963. T r a n s c a r b o x y l a s e I I I .

Purification and properties of methyl-malonyl-oxalacetic transcarboxylase containing tritiated biotin. J . biol. Chemistry, 238, 547—556.

WOOD, H . G . , DAVIS, J . J . a n d WILLARD, J . M . , 1969. P h o s p h o e n o l p y r u v a t e

carboxytransphos-

phorylase from Propionibacterium shermanii. I n : Methods in Enzymology, Vol. 13,p.: 297—309 (Editor: LOWENSTEIN, J . M.). Academic Press New York and London. Mailing address: Prof. Dr. G. SCHÖN Institut Biologie I I der Universität Mikrobiologie Schänzlestr. 1 D-7800 Freiburg

36*

21

Zeitschrift für Allgemeine Mikrobiologie

1981

555-562

(Akademie der Wissenschaften der D D R , Forschungszentrum fur Molekularbiologie und Medizin, Zentralinstitut fiir Mikrobiologie und experimentelle Therapie, J e n a , Direktor: Prof. Dr. U . TATJBENECK)

Microbiological implications of electric field effects III. Stimulation of yeast protoplast fusion by electric field pulses H . W E B E R , W . F O R S T E R , H . - E . JACOB a n d H . B E R G

(Eingegangen

am

18.12.1980)

Prototrophic colonies could be selected on minimal medium after mixing of protoplasts from diauxotrophic mutants of t h e yeasts Saccharomycopsis lipolytica and/or Lodderomyces elongisporus and treatment with polyethylene glycol ( P E G ) in the presence of calcium chloride. This is t h e result of protoplast fusion and complementation of auxotrophic deficiencies. Under identical conditions an electric field pulse in the (is-range applied via an electric discharge to the protoplastP E G mixture resulted in a drastic enhancement of t h e protoplast fusion r a t e . The presence of polyethylene glycol was demonstrated to be a prerequisite for fusion in this case, too. The frequency of hybrid formation detected as prototrophic colonies could be increased in t h e case of intraspecific fusion a t initial electric field strengths between 2.5 and 5 kV • c m - 1 . The application of an electric field pulse of proper strength and duration to a yeast protoplast suspension turned out to be a more effective tool in production of fusion products than conventional methods. L a r g s numbers of parasexual hybrids for different selection programmes in yeast genetics and for industrial purpose m a y be delivered by this technique.

The complete removal of the cell wall of microorganisms including yeasts by means of lytic enzymes results in formation of protoplasts. Under specific conditions protoplasts of different strains or species of yeast can be induced to fuse by polyethylene glycol as an fusogenic agent and to form somatic hybrids ( F E R E N C Y et al. 1 9 7 5 , ANNE and PEBERDY

1 9 7 5 , PROVOST et al.

1978,

STAHL 1 9 7 8 ,

SPATA a n d W E B E R

1980).

Protoplast fusion and vector-mediated transformation (HICKS et al. 1978) provide genetics and molecular biology of yeasts with a technology to explore aspects of research on lower eukaryotes previously not possible. Since the introduction of these techniques several attempts have been made to improve different steps of the fusion process. Up to now fusion frequencies were reported to be very low, usually in the range of 10" 5 to 10" 4 . Electric field effects on cell suspensions, especially the influence of short electric field pulses on the permeability of biological membrane systems, have often been described. Since the pioneer work of S A L E and HAMILTON ( 1 9 6 7 ) with bacteria and yeasts as well as with bacterial protoplasts, studies with higher eukaryotic cells have demonstrated the phenomenon of dielectric breakdown of membranes, which is accompanied by drastic permeability changes (NEUMANN and ROSENHECK 1 9 7 2 , CROWLEY 1 9 7 3 , ZIMMERMANN et al. 1 9 7 3 , 1 9 7 6 , COSTER a n d ZIMMERMANN 1 9 7 5 , TSONG and K I N G S L E Y 1 9 7 5 , KINOSITA and TSONG 1 9 7 7 , JACOB et al. 1 9 8 1 ) . While the me-

chanism of this breakdown is not yet fully understood, its dependence on pulse form, critical membrane potential difference, ionic strength and temperature is ascertained. Taking into account this knowledge we started in 1978 with experiments on protoplast fusion in the PEG-Ca 2 + system in order to try to enhance the fusion frequency of different yeast species by electric field pulse treatment. First positive results we

556

H . WEBER, W . FORSTEE, H . - E . JACOB a n d H . BERG

could present at the Vth Internat. Symp. on Bioelectrochemistry and Bioenergetics (JACOB et al. 1979), at the Vth Internat. Symp. on Yeasts (WEBER et al. 1980) and at t h e 1 s t G O R D O N R e s e a r c h C o n f e r e n c e o n B i o e l e c t r o c h e m i s t r y (BERG et al. 1980). Independently of us S E N D A et al. (1979) described the electrically stimulated fusion

of plant protoplasts, which were in close contact one with another and which were stimulated b y two microelectrodes with a current of 10 ¡iA and 5 ms duration. Meanwhile N E U M A N N et al. (1980) reported cell fusion of Dictyostelium induced b y high electric impulses (without P E G ) and ZIMMERMAN» and SCHEURICH (1981) described t h e fusion of protoplasts of Vicia faba b y combining the method of dielectrophoresis (POHL 1978) with the application of short electric field pulses. Neither S E N D A nor the other authors describing cell fusion resulting from electric stimulation proved the viability and hybrid character of their fusion products. I n this paper we present the first genetic evidence of protoplast fusion induced b y an electric field pulse in the presence of P E G .

Materials

and

methods

Strains: Haploid diauxotrophic m u t a n t strains of the yeasts Saccharomycopsis lipolytica, and Lodderomyces elongisporus with the following phenotypes were used (Table 1): Double mutants from 8m. lipolytica have been induced by ultraviolet light and subsequent crosses from wild type strains (YB 423-12, CX 39-34) which were kindly supplied bei J . BASSEL (Berkeley), m u t a n t s of Lodderomyces elongisporus were kindly provided by R. KOLBLIN (Jena). Table 1 Phenotypes of diauxotrophic m u t a n t s used in fusion experiments Mating type S accharomycopsis lipolytica, Lodderomyces elongisporus

Mutant phenotype

Strains designation

B A A

arg, ade met, ade ilv, lys

S 64 S 113 26-10

_

ade, his

H 236

Media: Yeast strains were maintained on slants with yeast extract-pepton-glucose-agar medium (YEPG) and cultivated in liquid Y E P G medium on a reciprocal shaker. The fusion mixture or hybrids were plated on solidified minimal medium with the following composition: N H 4 H 2 P 0 4 (0.5%), K H 2 P 0 4 (0.25%), MgS0 4 • 7 H 2 0 (0.1%), glucose (1%), Ca(N0 3 ) 2 • 4 H 2 0 (20 mg/1), PeCI 3 -6 H 2 0 (2 mg/1), H 3 B0 4 (0.5 mg/1), MnS0 4 • H 2 0 (0.4 mg/1), ZnS0 4 (0.4 mg/1), CaCl2 0.1 mg/1), CuSO„ • 5 H 2 0 (0.1 mg/1), K J (0.1 mg/1), thiamine (100 (¿g/1); p H was adjusted to 6.5. For protoplasts all media were stabilized osmotieally with 1.4 M sorbitol. Protoplast formation: Formation of protoplast was carried out in modification of the method described by MAY (1971): Yeasts were harvested in the logarithmic growth phase. After washing t h e cells were treated in 10 ml 2-mercaptoethanol solution (2 ,ul ME in 50 ml Tris-EDTA solution), collected after 10 min by centrifugation and resuspended in preparation medium (phosphate-citrate-buffer, p H 6.0), washed twice and again resuspended in preparation medium with snail enzyme (160 mg of lyophilized gastric juice of Helix pomatia per 1 g yeast wet weight in 50 ml preparation medium with 1.4 M sorbitol). The suspension was gently moved on a reciprocal shaker a t 28 °C for 30 min. Protoplast formation was checked under the microscope and stopped when maximum of protoplast liberation had been achieved. Protoplasts were separated from all debris, intact cells, and snail enzyme by centrifugation, followed by resuspension in 1.4 M sorbitol. The process was repeated twice as before.

557

Microbiological implications of electric field effects. I I I .

Protoplast fusion: Fresh protoplast suspensions of p a r t n e r strains in 1.4 M sorbitol containing a b o u t 107 —108 protoplasts/ml were mixed to obtain a protoplast mixture in 1:1 proportion. The mixture was centrifuged (10 min, 500 g), t h e s u p e r n a t a n t removed and 2 ml of polyethylene glycol solution (40% P E G , MW 6000) containing 0.01 M CaCl 2 and 1.4 M sorbitol were added. T h e suspension for fusion was incubated a t 28 °C for 30 min. I n t h e case of t h e electric field pulse t r e a t m e n t t h e identical protoplast suspension was placed between two stainless steel electrodes in a cylindric discharge chamber and a high voltage capacitor was discharged through t h e electrolytic suspension a t room t e m p e r a t u r e . A f t e r subsequent incubation (30 min a t 28 °C) samples of 0.1 ml of t h e protoplast mixture were plated on osmotically stabilized minimal medium for selection of protrophs. Controls were carried o u t for estimation of regeneration frequency, back m u t a t i o n , ratio of intact cells to protoplasts a f t e r snail enzyme digestion, and formation of hybrids b y fusion w i t h o u t electric field t r e a t m e n t . Electric discharge circuit: A high voltage capacitor of C = 2 piE can be charged u p to 20 kV. I t s discharge through t h e cylindric discharge chamber with a diameter of 3 cm, an electrode distance of 0.4 cm, a n d a volume of 2.8 ml is triggered b y a spark gap. The t i m e constant of t h e exponential electric discharge under t h e given ionic conditions is about 40 ¡xs. The discharge results in a heating of t h e protoplast suspension of about 1 °C a t an initial electric field strength of 12 kV • c m - 1 with a heating time constant of 20 (xs. Initial electric field strengths in t h e fusion experiments were u p t o 20 kV • cm" 1 .

Results and

discussion

The discharge of a high voltage capacitor through a suspension of mixed protoplasts of different yeast strains in the presence of PEG and Ca 2+ -ions strongly enhances the fusion of protoplasts normally occurring under these conditions as detected by selection of prototrophic colonies on solid minimal medium. In our experiments the number of prototrophic colonies formed from protoplast suspensions to which an electric field pulse of proper strength had been applied was much higher than from unaffected suspensions (see Fig. 1 and Tab. 2). These colonies are the result of complementation of auxotrophic deficiencies of the parental strains. The latter are not able to grow and to form colonies on unsupplemented minimal medium. The optimal field strength in the case of intraspecific fusion is about 3 to 5 kV • c m - 1 for Sm. lipolytica (Tab. 2b, c). An enhancement of the number of colonies on minimal medium by a factor of 20 to 35 was found. For intergeneric fusion higher field strengths were necessary for optimal action (Tab. 2a). Here with 10 kV • c m - 1 and 80-fold increase of the number of colonies was observed. Individual colonies, isolated from plates of fusion experiments were maintained over a long period. Hybrids resulting from intraspecific fusion have been shown to be very stable, whereas intergeneric fusion products after few passages segregated to one of the auxotrophic parental strains. Table 2 E f f e c t of electric field pulse t r e a t m e n t on intergeneric (a) and intraspecific (b, c) fusion of yeast protoplasts. Number of colonies formed per plate (0.1 ml protoplast suspension)on minimal medium. Numbers represent mean values of five plates each. Electric field strength kV • cm" 1 0 (control) 1.25 2.5 3.75 5.0 10.0 15.0 20.0

a strains S 64/H 236 8

622 160 66

b strains S 113/26-10 28

553 8 1

c strains S 113/26-10 6 7 47 21 62

558

H . W E B E R , W . FORSTER, H . - E . JACOB a n d H . B E R G

Control

Fig. 1. Formation of prototrophic colonies on minimal medium without (control) and after electric field pulse in presence of P E G . Parental strains: Saccharomycopsis lipolytica S 113 and 26-10

As demonstrated in Fig. 1 the hybrids differ sharply in colony size which can be observed not only for hybrids resulting from field-induced fusion, but also in hybrids induced by P E G alone (control). They have not yet been studied cytologically and genetically in detail. The different colony size may be explained by loss of single chromosomes or by multiple fusion. As studied so far, in the selected colonies only uninuclear hybrid cells were observed confirming that fusion of nuclei did occur after fusion of protoplasts. Prerequisites for cell fusion are a sufficient high protoplast titer, the formation of a very close contact between protoplasts, the diminishing of the negative surface charges, and a breakdown of the cell membranes. Addition of P E G and Ca 2 + ions to a protoplast suspension results in a rapid and strong agglutination due to dehydration and charge neutralization of the protoplast surface. B y means of scanning electron microscopy it could be demonstrated that protoplasts during P E G action are touched one into each other. When P E G was partly removed by washing, impressions resulting from adjacent protoplasts during P E G treatment could be detected (Fig. 2). I n our electric field pulse experiments it was found that the presence of P E G in high

Microbiological implications of electric field effects. I I I .

559

Fig. 2. Effect of P E G t r e a t m e n t on protoplast morphology (Saccharomycopsis lipolytica). (a) — Protoplast in sorbitol stabilized medium before t r e a t m e n t ; (b) —Protoplasts after addition of P E G ; (c)—Protoplasts immediately after partial removing of P E G by washing. Impressions from a d j a c e n t protoplasts (arrow) are preserved. Bar represents 1 [¿m Table 3 E f f e c t of PEG-concentration on protoplast fusion a t a given field strength (3.75 kV • cm - 1 ). Fusion strains S 113 and 26-10 P E G concentration (%)

number of colonies per plate

without P E G

10

25 40

enough concentrations (30 to 40%) is essential for the drastic enhancement of the protoplast fusion rate (Tab. 3). Without addition of PEG to the yeast protoplast mixture no hybrids were detectable after the electric field pulse. The same negative result was obtained when PEG was added immediately after the electric field pulse

560

H. WEBER, W. FORSTER, H.-E. JACOB and H. BERG

treatment. This means that the necessary close contact between protoplasts is brought about by PEG, while the elctric field pulse additionally supports the membrane labilization and breakdown processes as decisive steps in the fusion event. This is in line with the well-established drastic conductivity and permeability increase of cell membranes in the case of dielectric breakdown. As may be seen from Table 2 the optimal initial electric field strength for fusion enhancement is rather different for the intraspecific and intergeneric fusion experiments. At least two factors might be responsible for this result. First, we observed a different sensitivity among different yeast species and even strains with respect to an electric field pulse (Tab. 4). Table 4 Sensitivity of different yeast species and strains with respect to an electric field pulse of 20 kV • cm - 1 (Results of two experiments, intact cells) Species S. cerevisiae S. lipolytica S. cerevisiae L. elongisporus Sm. lipolytica

strain H H H H H

192 222 246 218 194

survivors (%) at 20 kV • cm7 18 28 39 68

11 14 27 44 82

Usually, some inactivation of intact yeast cells is caused by an electric discharge (SALE and H A M I L T O N 1967, BERG et al. 1978, JACOB et al. 1981). In corresponding

experiments the rate of survivors at a given field strength of 20 kV • c m - 1 varied between 75 and 10% for the test objects in Table 4. This obviously may also have some influence on the optimal field strength for fusion of protoplasts from different strains or species. Second, we found a correlation between protoplast regeneration and electric field pulse treatment (Tab. 5). Interestingly the electric field strength for optimal regeneration and fusion of protoplasts coincides (Tab. 5 and Tab. 2c). This enhancement of regeneration rate might be different for the strains and species used, which might affect the observed optimal field strengths for fusion. For estimation of fusion rates these data on regeneration have to be taken into account. In most fusion experiments the fusion rate is expressed as the relation of prototrophic colonies on minimal medium to colonies on complete medium (control of regeneration). Because of the mentioned differences in regeneration we prefer to express the fusion frequency as the number of prototrophs related to the number of protoplasts potentially able to regenerate and to form colonies, i.e., the protoplast titer in the fusion experiment. By application of an optimal electric field pulse the fusion rate could be increased in our experiments from 10~5—10"4 up to 10~3 to 10~2. It is very probable that the overall fusion rate (without selection) was much higher. Table 5 Regeneration of mixed protoplasts on complete medium influenced by electric field impulses. Number of colonies formed from 10- protoplasts per plate. Strain: 26 — 10 Electric field strength (kV • cm"1) 0 (control) 1.25 2.5 5.0

number of colonies per plate 9 24 51 21

Microbiological implications of electric field effects. III.

561

Comparing the method of electric stimulation of cell fusion described above with other such methods ( S E N D A et al. 1 9 7 9 , N E U M A N N et al. 1 9 8 0 , Z I M M E R M A N N and S C H E U R I C I I 1 9 8 1 ) one main difference seems to be the way how to establish the necessary close contact between the fusion partners prior to electric stimulation. While we used only a chemical treatment (PEG, Ca2+), S E N D A uses combined chemical and mechanical means (Ca 2+ , micromanipulator, microelectrodes). N E U M A N N achieves cell agglutination by rolling the cell suspension in plastic tubes and uses neither PEG nor Ca 2+ ions prior to the electric treatment (one should keep in mind, however, that his object is rather different from ours). Z I M M E R M A N N dielectrophoretically induces adhesion of protoplasts one to another (achieved by a high frequency, inhomogeneous electric field) after which cell fusion caused by a high electric field pulse occurs. I t must be underlined t h a t up to now neither of the authors mentioned demonstrated t h a t his fusion products, which were observed microscopically, were able to reproduce, as we did. In eukaryotes like most fungi or higher plants the formation of the cell wall (regeneration) after fusion of protoplasts and nuclei is an essential condition for cell multiplication and/or selection of hybrids. This condition is fulfilled in our fusion experiments with the combined application of PEG, Ca 2+ ions, and a high electric field pulse, which allows to obtain large quantities of parasexual (somatic) hybrids. Trying to classify the different methods of electric stimulation of cell fusion we would give the following scheme: 1. large scale production of hybrids; fast contacting of cells (this work) large scale production; slow contacting of cells ( N E U M A N N ) 3 . low scale production; fast contacting of cells ( Z I M M E R M A N N ) 4 . lowest scale production; slow contacting of cells ( S E N D A ) . 2.

I t is an open question if stimulation by low level pulsating current according to in connection to fusion might be useful, too. Perhaps the most elegant and promising way could be the purely electric fusion method (3.) if developped to a large scale output of hybrids. This may be achieved by application of some kind of flow system with dielectrophoretic contacting of the fusion partners and repeated application of high electric field pulses ( P O H L 1 9 7 8 , PILLA (1980)

G L A S E R et al.

1 9 7 9 , ZIMMERMANN a n d SCHEURICH

1981).

Summarizing we would like to propose that the stimulation of the fusion of eukaryotic cells or protoplasts by electric field pulses will be a suitable method to improve modern genetic techniques like combination of whole genomes by fusion of protoplasts or transformation of yeasts by plasmids with inserted definite DNA sequences. A

cknowledgements

We are grateful to Mrs. FRAKZL and and Mrs. HAMANN for excellent technical assistance. Thanks are also due to G . BARTH and R . KOLBLIN for providing with mutant strains and Mrs. OTTO for typing the manuscript.

References and P E B E R D Y , J . F . , 1 9 7 5 . Conditions for induced fusion of fungal protoplasts in polyethylene glycol solutions. Arch. Microbiol., 105, 2 0 1 — 2 0 5 . BERG, H . , FORSTER, W . , JACOB, H.-E., JUNGSTAND, W . and MTTHLIG, P . , 1 9 7 8 . Electric field effects on single cells, studia biophysica, 74, 31 —32. BERG, H., FORSTER, W . , JACOB, H.-E. and W E B E R , H., 1980. Electric field pulses on yeast cells and protoplasts and their genetic implications. Lecture at the 1st Gordon Research Conference of Bioelectrochemistry, Tilton,, N. H., 8. 8. 1980. ANNE, J.

562

H . W E B E R , W . FÖRSTER, H . - E . JACOB a n d H . BERG

COSTER, H . G. L. and ZIMMERMANN, U., 1975. The mechanism of electrical breakdown in t h e membranes of Valonia utricularis. J . Membrane Biol., 22, 73—90. CROWLEY, J . M., 1973. Electrical breakdown of bimolecular lipid membranes as an electromechanical instability. Biophys. J., 13, 711—724. F E R E N C Z Y , L . , K E V E I , F . and SZEGEDT, M . , 1 9 7 5 . High frequency fusion of fungal protoplasts. Experientia, 31, 1028 — 1030. G L A S E R , R . , P E S C H E K , C H . , K R A U S E , G . , S C H M I D T , K . P . und T E U S C H E R , L . , 1 9 7 9 . Dielektrophorese als Grundlage f ü r ein neues Verfahren zur präparativen Zelltrennung. Z. Allg. Mikrobiol., 19, 601—607.

HICKS, J . B., HINNEN, A. and PINK, G. R., 1978. Properties of yeast transformation. Cold Spring Harbor Symp. Quantit. Biology, X L I I I , 1305 — 1313. J A C O B , H.-E., F Ö R S T E R , W . , W E B E R , H . and B E R G , H., 1979, 1980. Electric field effects on single cells. Posters a t t h e V t h I n t e r n a t . Symp. on Bioelectrochem. Bioenerg., Weimar, and 1st Gordon Research Conference of Bioelectrochemistry, Tilton, N. H . J A C O B , H . - E . , F Ö R S T E R , W . and B E R G , H . , 1 9 8 1 . Microbiological implications of electric field effects. I I . Inactivation of yeast cells and repair of their cell envelope. Z. Allg. Mikrobiol., 21, 225-233. K I N O S I T A , K . and T S O N G , T . Y., 1977. Formation and resealing of pores of controlled sizes in h u m a n erythrocyte membrane. Nature, 268, 438—441. MAY, R., 1971. Isolationsbedingungen f ü r Zellkerne aus Hefeprotoplasten. Z. Allg. Mikrobiol., 11, 1 3 1 - 1 4 2 . NEUMANN, E. and ROSENHECK, K., 1972. Permeability changes induced b y electric impulses in vesicular membranes. J . Membrane Biol., 10, 279—290. N E U M A N N , E . , G E R I S C H , G . and O P A T Z , K . , 1 9 8 0 . Cell fusion induced by high electric impulses applied to Dictyostelium. Naturwissenschaften, 6 7 , 4 1 4 — 4 1 5 . PILLA, A. A., 1980. Electrochemical information transfer a t cell surfaces and junctions: Application to t h e s t u d y and manipulation of cell regulation. I n : Bioelectrochemistry (Editors: K E Y S E R , H . and G U T M A N N , F . ) . Plenum Press New York-London, p. 3 5 3 — 3 9 6 . PROVOST, A . , B O U R G U I G N O N , C., F O U R N I E R , P . , R I B E T , A . M. and H E S L O T , H . , 1 9 7 8 . Intergeneric hybridization in yeast through protoplast fusion. F E M S Microbiol. Letters, 3 , 3 0 9 — 3 1 2 . S A L E , A. J . H . and H A M I L T O N , W . A., 1 9 6 7 . Effects of high electric fields on microorganisms. I . Killing of bacteria and yeasts. Biochim. biophysica Acta, 1 3 8 , 7 8 1 — 7 8 8 . S E N D A , M . , T A K E D A , J., A B E , S . and N A K A M U R A , T . , 1979. Induction of cell fusion of plant protoplasts by electrical stimulation. P l a n t & Cell Physiol., 20, 1441—1443. SPATA, L. and WEBER, H., 1980. A s t u d y on protoplast fusion and parasexual hybridization of alcane utilizing yeasts. Advances in Protoplast Research. Akademia: Kiado, Budapest, 131 — 137 and Pergamon Press Oxford. STAHL, U., 1978. Zygote formation and recombination between like mating types in t h e yeast Saccharomycopsis lipolytica b y protoplast fusion. Molec. gen. Genet., 160, 111—113. T S O N G , T . Y. and K I N G S L E Y , E., 1975. Hemolysis of h u m a n erythrocyte induced b y a rapid temperature jump. J . biol. Chemistry, 250, 786—789. W E B E R , H., F Ö R S T E R , W . , J A C O B , H.-E. and B E R G , H., 1 9 8 0 . E n h a n c e m e n t of yeast protoplast fusion by electric field effects. Proceedings V t h Intern. Symposium on Yeasts, London (Ontario); Pergamon Press, in press. Z I M M E R M A N N , U . , S C H U L Z , J . and P I L W A T , G . , 1 9 7 3 . Transcellular ion flow in Escherichia coli B and electrical sizing of bacteria. Biophys, J., 13, 1 0 0 5 — 1 0 1 3 . ZIMMERMANN,.U., PILWAT, G . , BECKERS, F . a n d RIEMANN, F . , 1976. E f f e c t s of e x t e r n a l e l e c t r i c a l

fields on cell membranes. Bioelectrochem. Bioenerg., 3, 58—83. and S C H E U R I C H , P., 1 9 8 1 . High frequency fusion of p l a n t protoplasts b y electric fields. Planta, 151, 2 6 - 3 2 . Mailing address: Dr. H . WEBER Zentralinstitut f ü r Mikrobiologie und experimentelle Therapie der AdW DDR-69 J e n a , Beutenbergstr. 11

ZIMMERMANN, U .

Zeitschrift für Allgemeine Mikrobiologie

21

8

1981

563-566

Buchbesprechungen J . ADAM (Herausgeber), Mathematik und Informatik in der Medizin. 272 S., 82 Abb., 71 Tab., 7 Taf. Berlin 1980. VEB Verlag Volk und Gesundheit. M 13,80. Nach zwei einführenden Kapiteln über Funktionen sowie Aussagenlogik und Mengenlehre folgen drei auf medizinische Anwendungen orientierte Kapitel über mathematische Modelle, Informatik und Statistik. Das Kapitel über Statistik vermittelt elementare Kenntnisse und befähigt den Studierenden zu eigener Bearbeitung kleiner statistischer Analysen. Die vorhergehenden Kapitel geben lediglich einen Einblick in vorhandene Möglichkeiten ohne bereits Fähigkeiten zu eigener Problem analyse zu vermitteln. Somit spiegelt das Buch die momentanen Schwerpunkte der Anwendung von Mathematik und Rechentechnik in Kliniken wider. Neben der Darstellung einfacher mathematischer Modelle wäre eine grobe Übersicht über mathematische Modelle in der Medizin, sofern sie für die Klinik relevant sind, vorteilhaft gewesen, um stimulierend auf die Forschung und Mathematikanwendung Einfluß zu nehmen. Bei einer Neuauflage wären Mängel in der Abb. 3.12. zu beheben. Jedes Kapitel schließt mit einer Reihe von Aufgaben, wodurch das Selbststudium unterstützt wird. Dem Anliegen des Herausgebers, mit diesem Buch den Unterricht im Fach Mathematik im Medizinstudium zu unterstützen, ist sehr gut entsprochen worden. Das Buch kann für Medizinstudenten der ersten Semester empfohlen werden sowie für alle in medizinischen Berufen Tätige, die sich elementares Handwerkszeug für statistische Untersuchungen aneignen wollen. R. GTJTHKE (Jena)

J . C. AYRES, J . 0 . MÜNDT a n d W . E . SANDINE, Microbiologv of F o o d s . X I I + 708 S., 134 A b b . ,

66 Tab. 20 Tafeln. San Francisco 1980. W. H. Freeman and* Company. £ 10.80.

Die in den letzten Jahrzehnten erfolgte rasche Entwicklung der Biotechnologie hat auch deutliche Auswirkungen auf die Gewinnung, Verarbeitung und Haltbarmachung von Nahrungs- und Gemißmitteln gezeigt. Der heutige Wissensstand auf diesem Gebiet wird im vorliegenden Titel in einer zwar gedrängten, aber informativen Form dargestellt. Das Buch ist in vier voneinander unabhängige Teile gegliedert. In den sechs Kapiteln des ersten Teils werden dem Leser die notwendigen Voraussetzungen zum Verstehen der taxonomischen Einordnung und der Physiologie der für die Lebensmittelmikrobiologie wichtigen Bakterien, Hefe und Schimmelpilze gegeben sowie die hauptsächlichen nichtbiologischen Verfahren zur Haltbarmachung von Lebensmitteln dargestellt. Es folgen in den vier Kapiteln des zweiten Teils die Herstellungstechnologien für die verschiedenen alkoholischen Getränke, Sauermilchgetränke und Sauergemüse einschließlich der bei uns nur wenig bekannten, im asiatischen und pazifischen Raum aber weit verbreiteten Soßenspeisen, die durch spezielle Fermentationen aus Sojabohnen, Reis bzw. anderen heimischen pflanzlichen Rohstoffen bereitet werden. Die Mikrobiologie von Gewürzen, kohlenhydrathaltigen Nahrungsmitteln, Früchten und verschiedenen Gemüsearten sowie von Milch-, Fleisch- und Eiprodukten wird in den zehn Kapiteln des dritten Teils abgehandelt. Fragen der allgemeinen Lebensmittelhygiene und die durch aufgenommene Nahrungsmittel möglichen Intoxikationen sind Gegenstand der drei Kapitel des letzten Teils. Der vielfältige Stoff wird vorteilhaft gestrafft und gut verständlich dargeboten. Die Ausführungen werden durch übersichtliche technologische Fließschemata, instruktive Strichzeichnungen, zahlreiche Tabellen und photographische Abbildungen wirkungsvoll unterstützt. Jedem Kapitel ist ein ausführliches Verzeichnis der betreffenden relevanten Literatur angefügt. Die Verfasser berücksichtigen verständlicherweise die Lebensmittelgesetzgebung der USA, so daß in einigen Fällen, beispielsweise bei der Weinbereitung und der Haltbarmachung von Lebensmitteln, Abweichungen von den in anderen Ländern verbindlichen diesbezüglichen Rechtsvorschriften festzustellen sind. Nach Meinung der Autoren soll dieser Titel nur eine Einführung für Studierende der Mikrobiologie bzw. Lebensmitteltechnologie sein; er dürfte aber auch den in diesen Fachdisziplinen der technischen Mikrobiologie Erfahrenen wertvolle Hinweise und Anregungen geben. H . BOCKER (Jena)

564

Buchbesprechungen

U. F Ö R S T N E R and G. T. W. W I T T M A N N , Metal Pollution in t h e Aquatic Environment. 486 S., 102 Abb., 94 Tab., Berlin-Heidelberg-New York 1979. Springer-Verlag. DM 98,00. Gesundheitsschädigungen durch homöopathische Dösen von Metallen im Trinkwasser f ü h r t e n zu einer intensiven Untersuchung der Gewässer unserer Erde. Lag in der ersten H ä l f t e des 20. J a h r h u n d e r t s das Schwergewicht des Interesses auf rein geochemischem Gebiet, ist nun die Abprodukt- und Abwasserproblematik, die Belastung der Umwelt mit organischen wie anorganischen Elementen und Verbindungen zu einem Problem nicht n u r der großen Industriestaaten herangewachsen. F Ö R S T N E R U. W I T T M A N N ist zu danken, daß die nun umfangreiche, weitgestreute Literatur auf dem Gebiet der Gewässerbelastung mit Metallen zusammengetragen wurde (über 2000 Literaturangaben im Anhang). Es werden folgende Problemkreise behandelt: Toxische Metalle, die Metallkonzentration im Fluß-, See- und Meerwasser, der Metallgehalt der Sedimente und der Transfer Fest-Flüssigphase sowie die Schwermetallkonzentration und die Anreicherung in den Wasserorganismen. Von besonderer praktischer Bedeutung sind die Untersuchungen über das Verhalten der Spurenmetalle bei der Trinkwasseraufbereitung und der Abwasserbehandlung. Durch bakteriologische Aktivitäten werden Spurenelemente mobilisiert und Metalle wie Hg, As, Se, P b in die Methylform ü b e r f ü h r t . Die Toxizität dieser Verbindungen unterstreicht die Bedeutung und die Gefahren der Umweltverschmutzung mit Metallen. Das vorliegende Buch liefert eine solide, umfassende Grundlage, frei von emotionellen Tendenzen, zur Beurteilung und Bearbeitung dieser T h e m a t i k G. PROFT ( J e n a )

M. G I B B S and E. L A T Z K O (Editors), Photosynthethesis I I . Photosynthetic Carbon Metabolism and Related Processes. Vol. 6: Encyclopedia of P l a n t Physiology, New Sériés (A. P I R S O N and M. H . Z I M M E R M A N N (Eds.). X X + 578 S., 75 Abb. Berlin-Heidelberg-New York 1979. Springer-Verlag. DM 198,00. I n diesem B a n d der neuen großen Enzyklopädie der Pflanzenphysiologie werden die Prozesse der C0 2 -AssimiIation, ihre Regulation und die Interaktionen mit dem Atmungs- und Stickstoffstoffwechsel behandelt. Der bereits 1977 erschienene Band Photosynthesis I enthielt die Prozesse des photosynthetischen Elektronentransports und der Phosphorylierung. Die Bände berücksichtigen auch mikrobielle Prozesse, soweit sie zum Gesamtbild beitragen und f ü r die vergleichende biologische Betrachtung von Bedeutung sind. So enthält auch der vorliegende Band eine Reihe von Beiträgen zu mikrobiologischen Problemen, teils in gesonderten Kapiteln, teils in pflanzenphysiologischen Abschnitten. Sie lassen sich aus mikrobiologischer Sicht um drei Themenkreise gruppieren 1. C0 2 -Assimilation. O H M A N N gibt einen klaren Überblick über die autotrophe C0 2 -Assimilation durch prokaryotische Mikroorganismen, B U C H A N A N behandelt die Ferredoxin-abhängige C0 2 Fixierung bei der bakteriellen Photosynthese und stellt in diesem Zusammenhang die Rolle des reduktiven Carboxylsäure-Zyklus f ü r die Aminosäure- und Kohlenhydratsynthese dar. 2. Interaktionen zwischen Photosynthese und Atmung. E V A N S und C A R R g ehen auf diese Beziehungen bei Mikroalgen ein, W I E S S N E R f a ß t die Prozesse der Photoassimilation organischer Verbindungen durch Algen zusammen. 3. Beziehungen zwischen N 2 -Bindung und Photosynthese. S T E W A R T behandelt diese Beziehungen bei Mikroorganismen und geht auf das stickstoffbindende System in den Heterocysten von Cyanobakterien ein. QUEBEDEAUX behandelt die Beziehungen der symbiontischen Stickstoffbindung zur Photosynthese höherer Pflanzen. Alle Beiträge geben einen ausgezeichneten Einblick in den derzeitigen Erkenntnisstand und demonstrieren die Fortschritte der letzten J a h r e . Gute Schemata illustrieren diese Ausführungen. Durch die Darstellung der genannten Prozesse im R a h m e n des Gesamtsystems der Photosynthese werden sehr tiefgründige Informationen über photosynthetische Leistungen von Purpurbakterien, Cyanobakterien und Mikroalgen vermittelt. W . F R I T S C H E (Jena)

F . A. G U N T H E R and J . D. G U N T H E R (Editors), Residue Reviews. Residues of Pesticides and Other Contaminants in t h e Total Envirenment. V I I I + 154 S., 10 Abb., 21 Tab. New York-HeidelbergBerlin 1979. Springer-Verlag. DM 39,50. I n h a l t dieser Review-Serie sind zusammenfassende und kritische Beiträge problematik von Pesticiden und anderen Fremdstoffen. Diese Problematik ist tionen zwischen Mikroben und Fremdstoffen in der Umwelt verbunden. I n allen vorliegenden Bandes spielen mikrobiologische Aspekte eine Rolle. A. E. M C C A N N

zur Rückstandseng m i t Interakvier Artikeln des und D. R . C U L L I -

Buchbesprechungen

565

zeigen m i t dem Beitrag über den E i n f l u ß von Pesticiden auf die Algenflora des Bodens, d a ß vor allem die Herbicide, welche die P h o t o s y n t h e s e h e m m e n , die Algenflora beeinflussen. Die Autoren betonen, d a ß wesentlich m e h r Felduntersuchungen notwendig sind, u m zu Aussagen über das A u s m a ß der E f f e k t e in der U m w e l t zu k o m m e n . Eine ähnliche P r o b l e m a t i k wird in dem folgenden Beitrag von D. T A Y L O R über die W i r k u n g von Quecksilber auf das aquatische Leben deutlich. Trotz zahlreicher g u t f u n d i e r t e r L a b o r b e f u n d e über lethale u n d sublethale E f f e k t e von Quecksilber auf aquatische Organismen ist eine Ü b e r t r a g b a r k e i t auf natürliche Ökosysteme n u r bedingt möglich. U. a. sind die mikrobiellen Reaktionen, die die K o n z e n t r a t i o n e n der sehr toxischen organischen Quecksilberverbindungen in der U m w e l t beeinflussen, unzureichend u n t e r s u c h t . R . ENGST u n d Mitarbeiter stellen den derzeitigen Entwicklungsstand des Lindanstoffwechsels dar, wobei sie eingehend die zahlreichen O x y d a t i o n s p r o d u k t e berücksichtigen, die durch Mikroorganismen, P f l a n z e n u n d Tiere gebildet werden. P . G R A N D - J E A N u n d T. N I E L S E N geben einen umfassenden Überblick über U m w e l t - u n d Gesundheitsaspekte von Organobleiverbindungen, die vor allem als Treibstoffzusätze in die U m w e l t gelangen. Alle Beiträge e n t h a l t e n klare Aussagen über den derzeitigen E r k e n n t n i s s t a n d . Die umfassend ausgewertete L i t e r a t u r ist in z. T. umfangreichen tabellarischen Übersichten a u f b e r e i t e t . Ungelöste F r a g e n werden aufgezeigt. D a m i t wird auch der Mikrobiologe u n m i t t e l b a r angesprochen. Die Review-Artikel informieren n i c h t nur, sie t r a g e n zur E n t w i c k l u n g der interdisziplinären Zusammenarbeit zur Lösung der R ü c k s t a n d s p r o b l e m a t i k bei. W . F R I T S C H E (Jena) MORE

P . L A N G E u n d K . W Ö H R M A N N , Genetisches G r u n d p r a k t i k u m . 154 S., 70 Abb., 19 T a b . S t u t t g a r t N e w Y o r k 1979. G u s t a v Fischer Verlag. D M 24,00. Die A u t o r e n wenden sich m i t dieser P r a k t i k u m s a n l e i t u n g an Anfänger eines Biologiestudiums u n d a n Schüler in den Oberstufen der Gymnasien. I m ersten Teil des Buches behandeln sie die V e r s u c h s o b j e k t e u n d ihre K u l t u r m e t h o d e n , u n d im zweiten werden n a c h k u r z e n theoretischen B e t r a c h t u n g e n die einzelnen Versuche beschrieben. Versuchsobjekte sind Bakterien (Escherichia coli, Bacillus subtilis), Pilze (Sordaria, Ascobolus, Saccharomyces), I n s e k t e n (Drosophila) u n d Blütenp f l a n z e n (Mais, Erbse, Phlox, W u n d e r b l u m e u. a.). Die Versuche beinhalten DNA-Isolierung, Mitose, Meiose, Mutagenese, E r b g ä n g e u n d Spaltungsverhältnisse, L e t a l f a k t o r e n , Genkopplung u n d Genkartierung, extrakaryotische Vererbung u n d Geschlechtsbestimmung. O b j e k t e u n d Versuche sind so ausgewählt, d a ß elementare genetische Prinzipien demonstriert werden können, u n d der T e x t erscheint wissenschaftlich zuverlässig u n d didaktisch geschickt formuliert. Allerdings d ü r f t e die etwas eigenwillige Definition der MoRGAN-Einheit auf den Seiten 105 u n d 106 eher Verwirrung als K l a r h e i t stiften. D a s Buch k a n n n i c h t n u r S t u d e n t e n , sondern auch Kursleitern zur Vorbereitung genetischer P r a k t i k a empfohlen werden, doch scheinen d e m Rezensenten t r o t z unzulänglicher Kenntnisse über die biologischen Lehrpläne der Gymnasien letztere m i t ihm etwas überf o r d e r t zu sein. H . M A L K E (Jena)

A.RIETH, Süßwasserflora von Mitteleuropa, B a n d 4 : X a n t h o p h y c e a e , 2. Teil. 147 S., 61 Abb. J e n a 1980. V E B G u s t a v Fischer. M 68,00. Der vorliegende B a n d 4 der „Süßwasserflora" b r i n g t die im allgemeinen als monotypisch angesehene Familie der Vaucheriaceae, deren systematische Zuordnung noch i m m e r problematisch ist. Hier werden sie den Xanthophyceae als eigene Ordnung Vaucheriales nachgestellt. Die terrestrische G a t t u n g Asterosiphon, in gewisser Hinsicht zwischen den Botrydiales u n d Vaucheriales v e r m i t t e l n d , wird anhangsweise b e h a n d e l t . Vaucheria zerfällt in 12 Sektionen m i t e t w a 57 Arten, von denen 23 im Gebiet aquatische u n d feuchte, binnenländische S t a n d o r t e bewohnen. Die restlichen 8 halophilen A r t e n der Meeresküsten u n d die Masse der 26 f ü r E u r o p a noch n i c h t nachgewiesenen A r t e n sind in den Bestimmungsschlüsseln m i t berücksichtigt. Die Darstellung zeichnet sich durch sorgfältige, den Variabilitätsbereich der T a x a umgrenzende Diagnosen, durch auf klar abgrenzbaren Merkmalen f u ß e n d e Bestimmungsschlüssel u n d durch zahlreiche, n a t u r g e t r e u e u n d aussagekräftige Abbildungen (in der Regel Originale) aus. Die langjährigen Untersuchungen, die der Verfasser an lebendem Material vom natürlichen S t a n d o r t u n d aus K u l t u r e n angestellt h a t , drücken dem T e x t ihren Stempel auf. Selbstverständlich ist die einschlägige L i t e r a t u r eingehend (das Literaturverzeichnis u m f a ß t r u n d 150 Titel; dabei ist die im T e x t zitierte L i t e r a t u r n i c h t wiederholt) berücksichtigt. D e m Benutzer d ü r f t e lediglich die eigenwillige, aber stets begründete Behandlung nomenklatorischer F r a g e n Schwierigkeiten bereiten. I m allgemeinen Teil finden sich Angaben über Morphologie u n d Anatomie des vegetativen u n d sexualreifen Thallus, die Ontogenie (unter ausführlicher Berücksichtigung der sexuellen F o r t pflanzung), über Vorkommen, K u l t u r m e t h o d e n u n d Parasiten sowie Hinweise auf die systematische Abgrenzung der Vaucheriales u n d ihre U n t e r s u c h u n g u n d Bestimmung. Der spezielle Teil bringt, wie üblich, die Bestimmungsschlüssel f ü r alle Taxa, die Diagnosen u n d Abbildungen. A u s s t a t t u n g

566

Buchbesprechungen

und Druck sind sehr gut. Insgesamt eine ausgezeichnete, den gegenwärtigen Stand unserer Kenntnisse adäquat widerspiegelnde Arbeit, die die „Süßwasserflora" würdig fortsetzt. Auf ein paar geringfügige „Mängel" sei hingewiesen: In der Legende zu Figur 57 (S. 131) fehlt der Text zu „ m " (Chloroplasten). Vaucheria walzi, deren Schreibweise auf S. 96 ausdrücklich begründet wird, erscheint im Bestimmungsschlüssel (S. 86) als F. walzii. Mißverständlich ist die Unterordnung der var. nuoljae und var. major (als „Formen" von V. terrestris; vgl. S. 91) unter F. terrestris var. terrestris (S. 87). Nicht ganz klar ist die Formulierung „ F . pachyderma ... var. chittagoensis ... Möglicherweise zur Art zu ziehen" (S. 54). Zu welcher Art ? S. J . CASPER (Jena)

J . P. THOMPSON and V. B. D. SKERMAN, Azotobacteraceae: The Taxonomy and Ecology of the Aerobic Nitrogen-Fixing Bacteria. X X I + 4 1 7 S., 13 Abb., 48 Tab. London-New York-TorontoSydney-San Francisco 1979. Academic Press. § 32.00. Wegen ihrer auffallend großen Zellen, der Fähigkeit zur Bildung von Cysten und, damit verbunden, einem ausgeprägten Entwicklungszyklus zogen Vertreter dieser Familie schon immer das Interesse der Mikrobiologen auf sich. Ein weiterer Grund für ihre prominente Rolle ist ihr Modellcharakter für die Untersuchung der Biochemie der N 2 -Fixierung. So gesehen, stellt die Familie eine relativ gut untersuchte Gruppe dar, deren Taxonomie und speziell die Verwandtschaft zwischen den Genera kontrovers ist, wenngleich auch die Probleme, verglichen etwa mit den „coryneformen Bakterien", noch überschaubar sind. Die vorliegende Monographie ist der Versuch einer Gesamtdarstellung der Taxonomie und Ökologie, basierend auf der numerisch-taxonomischen Untersuchung von 151 Stämmen (davon 39 Neuisolaten). Das erste Kapitel behandelt die gesamte taxonomische Literatur beginnend mit BEIJERINCK (1901), der die erste Art dieser Familie beschrieb. Dabei werden nicht nur die nomenklatorischen, wechselnden Positionen der einzelnen Taxa verfolgt, sondern auch die Originaldiagnosen der jeweils neuen Taxa sowie Klassifikationssysteme der verschiedenen Autoren aufgeführt. Im zweiten Kapitel „Material und Methoden" ist für den methodisch interessierten Leser vor allem die Codierung der 230 Merkmale in „binary attributes", „ordered multistate attributes" und „disordered multistate attributes" sowie die Anwendung verschiedener Programme (MULTIBET speziell für die Analyse von Daten mit gemischten Merkmalen geeignet; GOWER) zur Klassifizierung von Bedeutung. Das dritte Kapitel enthält die Ergebnisse der ökologischen Untersuchungen in bezug auf das Vorkommen von Azotobacter spp. und Beijerinckia spp. in den verschiedenen Biotopen in Australien, die jedoch einen verhältnismäßig kleinen Anteil am Buch ausmachen. Die Ergebnisse der numerischtaxonomischen Analyse dagegen sind in aller Ausführlichkeit (Datenmatrix, Dendrogramm, dreidimensionale Vektoren-Analyse) dargestellt, so daß sie etwa ein Drittel des Buches ausmachen. Diese Großzügigkeit, die bei Publikationen numerisch-taxonomischer Analysen in Zeitschriften aus Platzgründen kaum möglich ist, trägt entschieden zur Durchschaubarkeit der Untersuchungen bei. Im vierten Kapitel wird dann ein Klassifikationssystem der Gattungen, Arten und Unterarten vorgeschlagen. Danach werden 7 Gattungen unterschieden: Azotobacter BEIJERINCK 1901 (5 Arten); Azomonas WINOGRADSKY 1938 (2Arten); Azotomonas STAPP 1940 (2Arten);Beijerinckia DERX 1950 (4 Arten) und Derxia JENSEN et al. 1960 (1 Art). Da weder Azotobacter paspali DÖBEREINER 1966 noch Azotobacter macrocytogenes JENSEN 1955 Ähnlichkeit mit der bisherigen Gattung Azotobacter noch mit einer der anderen 4 Gattungen aufweisen, wird die Errichtung zweier neuer, monotypischer Gattungen vorgeschlagen: Azomonotrichon gen. nov. mit A. macrocytogenes comb. nov. und Azorhizophilus gen. nov. mit A. paspali comb. nov. Diese Konzeption weicht von der Klassifikation, wie sie im BERGEY (8. Aufl.) für die Familie Azotobacteraceae durch JOHNSTONE für Azotobacter und Azomonas und durch BECICING für Beijerinckia und Derxia gegeben wird, ab. In bezug auf ein Familienkonzept der Azotobacteraceae und vermuteter Verwandtschaften zwischen den Gattungen kommen die Autoren zu dem Ergebnis, daß 1. Azotomonas STAPP 1940 kein N2 fixiert und aus der Familie ausgeschlossen werden sollte und 2. die übrigen 6 Gattungen durchaus nicht so eng miteinander verwandt sind. Die Abgrenzung einer separaten Familie Azotobacteraceae beruht nur auf der Fähigkeit der N 2 -Fixierung und nicht auf einer erwiesenen oder hinreichend gesicherten Verwandtschaft. Aus Gründen der Zweckmäßigkeit sprechen sie sich jedoch für die Aufrechterhaltung der Familie aus, allerdings unter Ausschluß der Gattung Azotomonas STAPP. Ein fünftes Kapitel mit den Bestimmungsschlüsseln für die Gattungen und Arten sowie selektiven Anreicherungsmethoden und Beschreibungen der Medien schließt die Monographie ab. Dem taxonomisch orientierten Mikrobiologen bleibt der Wunsch, daß dieses lobenswerte Beispiel einer monographischen Bearbeitung Schule macht und andere Spezialisten anregt, in anderen Bakteriengruppen oder -familien Ähnliches zu tun. J . MEYER (Jena)

Eine neue Zeitschrift

ACTA BIOTECHNOLOGICA Journal of microbial, biochemical and bioanalogous technology Herausgegeben von M. Ringpfeil und G. Vetterlein Unter Mitarbeit eines internationalen Wissenschaftlerkollektivs Je Heft 96 Seiten — 16,7 x 24 cm — jährlich erscheinen 4 Hefte — je Heft 30,— M Bestell-Nr. 1094

Die „Acta Biotechnologica" veröffentlicht in Englisch, Deutsch oder Russisch Übersichtsbeiträge, Originalarbeiten, Kurzmitteilungen und Berichte aus dem Gesamtgebiet der Biotechnologie. Die Zeitschrift soll die Herausbildung der Biotechnologie als neues, einheitliches Wissensgebiet fördern. Zur Biotechnologie gehören die mikrobiologische Technologie, die biochemische Technologie und die Technologie der Gewinnung und Anwendung bioanaloger Reaktionssysteme. Der technologische Charakter der Zeitschrift wird dadurch gewährleistet, daß mikrobiologische, biochemische!, chemische, physikalische und mathematische Arbeiten den technologischen Bezug eindeutig aufweisen.

Bestellungen durch eine Buchhandlung oder den Postzeitungsvertrieb erbeten

Akademie-Verlag DDR-1080 Berlin, Leipziger Str. 3—4

Immunologie Ein Nachschlagewerk Herausgegeben von Gerhard Bundschuh/ Burkhard Schneeweiss 2., bearbeitete Auflage 1978. X L I I I , 503 Seiten - 103 Abbildungen - gr. 8° - Leinen 4 2 , - M Bestell-Nr. 7618887 (6135)

Die Immunologie hat in den letzten Jahren eine stürmische wissenschaftliche Entwicklung erfahren. Sie reicht bereits in alle Fachgebiete der Biologie und Medizin hinein. 65 Fachvertreter der Biologie und Medizin geben mit diesem Immunologischen Wörterbuch eine rasche und fundierte Information über alle wesentlichen, immunologisch relevanten Begriffe. Sie werden nach einem einheitlichen Schema abgehandelt: Nach Aufzählung der wichtigsten Synonyma folgt die Definition, bei umfassenderen Begriffen die Darstellung ihrer theoretischen Grundlagen sowie die Beschreibung ihrer praktisch-medizinischen Bedeutung. Instruktive Skizzen, Tabellen usw. unterstützen eine übersichtliche Orientierung.

Bestellungen durch eine Buchhandlung erbeten

Akademie-Verlag DDR-1080 Berlin, Leipziger Str. 3 - 4