Zeitschrift für Allgemeine Mikrobiologie: Band 21, Heft 5 1981 [Reprint 2021 ed.] 9783112538883, 9783112538876

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ZEITSCHRIFT FÜR ALLGEMEINE MIKROBIOLOGIE AN INTERNATIONAL JOURNAL ON MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS HEFT 5 • 1981 BAND 21

AKADEMIE-VERLAG EVP 20,—M

BERLIN ISSN 0044-2208 34112

INHALTSVERZEICHNIS HEFT 5

Reaktion des Wildtyps und einer nicht-Stickstoffbindenden Mutante von Anabaena doliolum auf verschiedene Aminosäuren Regulation der NH 3 -Assimilation in XH 3 -limitierten

Chemostatenkulturen von Escherichia Auftreten von Bistabilität

coli ML 30:

Sammelbericht Möglichkeiten zur gezielten Manipulation der Genexpression des mikrobiellen Sekundärstoffwechsels

ASHOK KUMAR und H. D. KUMAB P . J . MÜLLER, BEATE

VON FROM-

MANSHAUSEN und H. SCHÜTZ

361

U. GRÄFE

373

Buchbesprechungen

CONTENTS

353

411

OF NUMBER 5

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids Regulation of ammonia assimilation in ammonialimited chemostat cultures of Escherichia coli ML 30: Evidence of bistability Review Possibilities for directed manipulations of gene expression of microbial secondary metabolism Book Reviews

ASHOK KUMAR and H. D. KUMAR

353

P. J. MÜLLER, BEATE VON FROMMANSHAUSEN and H. SCHÜTZ 361

U. GRÄFE

373 411

ISSN 0 0 4 4 - 2 2 0 8

ZEITSCHRIFT FÜR ALLGEMEINE MIKRO - BIOLOGIE AN

INTERNATIONAL

JOURNAL

ON

MORPHOLOGY, PHYSIOLOGY, GENETICS, AND ECOLOGY OF MICROORGANISMS

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

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

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

U N T E R M I T A R B E I T VON

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

HEFT 5 • 1981 • BAND 21

REDAKTION

U. May, Jena

Q •V \

AKADEMIE-VERLAG • BERLIN

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Zeitschrift für Allgemeine Mikrobiologie

21

1981

353-359

(Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, U. P., India)

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids ASHOK K U M A R a n d H . D . K U M A R

(Eingegangen am

23.10.1980)

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het* nif~) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 DIM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 MM. B u t at 1 n a , these amino acids were inhibitory. I n addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine a t 2 MM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N 2 -fixing blue-green lagae may reveal the regulation of the heterocyst-nitrogenase sub-system.

Normally blue-green algae synthesize all the amino acids required for their metabolic process and as such do not need supplementation of any amino acid for normal growth (CARR and W H I T T O N 1 9 7 3 ) . Earlier studies conducted on amino acid utilization gave highly variable and conflicting results and also none of the amino acids tested acted as a sole source of nitrogen for growth ( P I N T E R and PROVOSOLI 1 9 5 8 , VAST B A A L E N 1 9 6 2 ) . W Y A T T et al. ( 1 9 7 1 ) reported the utilization of arginine and glycine as nitrogen sources by a few non-nitrogen-fixing algae but these amino acids did not inhibit nitrogenase activity. N E I L S O N and DOUDOROFF ( 1 9 7 3 ) attempted to assess the ability of blue-green algae to utilize amino acids as sole sources of nitrogen. In their experiment, tests of the growth of a limited number of strains at the expense of various amino acids gave highly variable results and except for asparagine no other amino acid acted as nitrogen source. In such study, even if amino acids are simultaneously utilized, it is hard to separate the repressing effects of amino acids on heterocyst development or nitrogenase synthesis, leaving aside the possible effects of amino acid on general metabolism. However, if such study is carried out on a mutant, such as a non-nitrogen-fixing strain forming heterocysts (het+ nif~), at least the assessment of amino acids serving as nitrogen source becomes easy. In this communication we describe the effects of different amino acids on growth and heterocyst development in an Anabaena species andwe also compare the response of a non-nitrogen-fixing mutant to a few selected amino acids. Materials and methods The test organism (parent) Anabaena doliolum was isolated from a local rice field. Growth conditions and maintenance of the alga were the same as described by KUMAR and KUMAB (1980). 23»

354

ASHOK KUMAR a n d H . D. KTTMAB

The mutant het* nif~ was isolated following the method adopted by SINGH et al. ( 1 9 7 7 ) . Growth was measured by recording optical density in a BAUSCH and LOMB Spectronic-20. Heterocyst frequency was estimated by counting at least 10—12 filaments (ca. 600—1000 cells). All the amino acids were purchased from SIGMA Chemical Company, St. Louis, Missouri, U.S.A. Amino acid solutions were filter-sterilized and were added to autoclaved medium.

Results E f f e c t of d i f f e r e n t a m i n o a c i d s on g r o w t h of wild t y p e A. doliolum Table 1 shows the list of amino acids which stimulated growth. The growth stimulation by these amino acids was concentration-dependent. Out of all the amino acids, glutamine and argenine at 1 mM concentration caused the maximum growth. Asparagine, proline, serine, and aspartic acid also stimulated growth at all concentrations tested but the final growth yield was always lower than in glutamine and arginine. Table 1 Amino acids stimulating growth of Anabaena doliolum,1) Amino acids Control-AA -

Concentration (in MM)

% Growth stimulation

% Heterocyst frequency

—

5.3

L-glutamine

0.1 0.5 1.0

+ 15 +32 +56

4.6 2.8 1.2

L-arginine

0.1 0.5 1.0

+ 12 +35 +56

4.8 3.3 3.0

L-asparagine

0.1 0.5 1.0

+ 8 +20 +32

5.0 3.3 3.1

L-proline

0.1 0.5 1.0

+ 6 + 16 +26

5.3 4.8 4.8

L-serine

0.1 0.5 1.0

+ 8 +20 +32

5.5 5.2 5.0

JL-aspartic acid

0.1 0.5 1.0

+ 4 + 13 +21

5.5 5.2 4.8

—

') Growth and heterocyst frequency were estimated after 7 days and 72 hr, respectively, + % Increase over control (control 100%)

Table 2 lists amino acids which inhibited growth at a concentration as low as 0.1 mM. At 1 mM concentration of histidine and valine, there was total lysis of cells within 5 —6 days after incubation. Treatment with methionine (0.1 mM to 1 mM) caused bleaching and cultures also became yellow-green after 5 days (data not given). Table 3 includes those amino acids whose response differed at different concentrations. Alanine and lysine at 0.1 mM showed ca. 3 and 2 % increase in growth over the -control, but at higher concentrations, i.e., beyond 0.1 mM growth was inhibited. Phenylalanine, glycine, isoleucine, leucine, and tryptophan either showed no effect or caused a slight inhibition or stimulation at 0.1 mM, but the increase in concentration (i. e., -at 0.5 mM or 1 mM) caused growth inhibition. ,

Response of Anabaena

towards amino acids

355

Table 2 Amino acids wich inhibit growth1) Amino acids Control — AA"

Concentration (mM) •

—

0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0

L-histidine L-threonine L-cylteine L-valine L-glutamic acid L-methionine

% Growth ' inhibition

% Heterocyst frequency 5.3 5.3 5.0 PH NH 6.5 7.0 7.2 5.5 5.5 4.3 5.5 5.6 NH 5.4 5.8 P H 5.8 PH 5.2 6.0 6.3

—

-20 -68 Lysis -20 -68 -82 -13 -44 -72 — 18 -46 Lysis - 4 -30 -80 - 8 -18 -38

!) Growth and heterocyst frequency were estimated after 7 days and 72 hr, respectively — % Growth inhibition over control (growth in control 100%) P H — Proheterocyst NH — No heterocyst Table 3 Amino acids which inhibit growth only at high concentrations 1 ) Amino acids

Concentration (mM)

Control AA~ L-phenylalanine L-glycine L-isoleucine L-alanine L-lysine L-tryptophan L-leucine

0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0

% Growth yield

% Heterocyst frequency

100

5.3 5.5 6.0 7.5 5.0 5.5 6.0 '5.3 5.8 6.5 5.2 5.8 5.4 4.5 5.2 5.0 5.8 6.0 7.5 5.3 5.6 5.2

100 97 81 100 94 78 100 89 67 103 94 74 102 92 79 102 87 62 100 98 70

Growth and heterocyst frequency were estimated after 7 days and 72 hr, respectively

356

A s h o k Kitmab and H. D. K t j m a b

E f f e c t on h e t e r o c y s t d i f f e r e n t i a t i o n a n d f r e q u e n c y Except glutamine and arginine, other amino acids did not inhibit heterocyst formation (Tables 1—3). Even those amino acids which stimulated growth failed to repress heterocyst formation to a greater extent (viz., proline, serine and aspartic acid). Similarly, those amino acids which inhibited growth did not repress heterocyst formation and the frequency remained similar to that in unsupplemented control. Only phenylalanine and tryptophan at higher concentrations (0.5 and 1 mM) showed increase in heterocyst frequency. Histidine and glutamine acid at higher concentrations (0.5 to 1 mM) permitted the formation of only proheterocysts ; however, the spacing pattern was normal. Histidine and valine at 1 mM concentration caused lysis of filaments and so there was no formation of heterocysts. Treatment with methionine resulted in the appearance of granular inclusions in most heterocysts and also" the polar nodules were abnormally developed. G r o w t h r e s p o n s e of a n o n - n i t r o g e n - f i x i n g m u t a n t (het+ nif~) to some amino acids Figs. 1—2 show the growth response of het+ nif~ mutant towards different amino acids. The mutant grew in the presence of glutamine and arginine but its growth yield was always lower than in NH 4 + -supplemented medium. Asparagine showed a very poor growth even at elevated concentrations. Proline, serine, and aspartic acid failed to support growth.

Fig. 1. Growth response of Anàbaena doliolum het+ nif~ in medium supplemented with glutamine (3, 1 mM), arginine(x, 1 mM), andNH 4 + (o) (growth estimated after 12 days). • = parent AA, A = het+ nif-AA~ Fig. 2. Effect of 1 mM asparagine (•), 1 mM proline (O), 1 mM serine ( x ) , and 1 mM aspartic acid (A) on the growth of Anàbaena doliolum het+nif~ (growth estimated after 12 days)

E f f e c t of a m i n o a c i d s on h e t e r o c y s t p r o d u c t i o n of het+ nif

mutant

Heterocyst formation was completely repressed at a concentration of glutamine and arginine higher than 1 mM, but asparagine did not block the differentiation even at a higher concentration. Proline, serine, and aspartic acid did not repress heterocyst formation and the frequency in het+ nif~ mutant was normal (Table 4).

Response of Anabaena towards amino acids

357

Table 4 Heterocyst frequency1) of het+ nif~ mutant of A. doliolum during growth with amino acids Supplementation

Concentration (ma)

Control AA" L-glutamine L-arginine L-asparagine L-proline L-serine L-aspartic acid

0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 1.0 1.0 1.0

% Heterocyst frequency 6.22) 3.3 2.1 0.0 3.8 2.6 0.0 5.3 4.8 4.2 5.8b) 5.6b) 5.5b)

1

) Heterocyst frequency was counted after 6 days ) Heterocyst frequency was counted after 72 hr because cultures lysed after 96 hr 2

Discussion The results indicate t h a t the different amino acids affect growth and heterocyst differentiation differentially. Among all the amino acids only glutamine, arginine and asparagine were stimulatory. Though proline, serine, and aspartic acid also showed stimulation the overall contribution by these amino acids was significantly lower t h a n t h a t of the former group. Lack of definitive knowledge of the pathway of amino acid catabolism by blue-green algae makes it difficult to speculate the exact role played by those amino acids either in growth or in heterocyst differentiation. However, the data of growth and heterocyst differentiation allow us to conclude t h a t those amino acids which stimulate growth may act as nitrogen and/or carbon sources. I t has been established t h a t all the utilizable sources of combined nitrogen viz., N0 3 ~, N0 2 ~ or NH 4 + , repress heterocyst differentiation and perhaps the repressor of heterocyst production is the final metabolic product of these substrates (FOGG 1949, SINGH and SHRIVASTAVA 1968, SINGH a n d KUMAR 1971, TYAGI 1975, SINGH et al. 1977, HASELKORN 1978, MEEKS et al. 1978). T h u s , g r o w t h s t i m u l a t i o n b y g l u t a m i n e a n d asparagine

followed by repression of heterocyst frequency strongly suggests t h a t these amino acids are utilized as nitrogen source by both wild type and the m u t a n t strain. The uptake and utilization of glutamine in metabolic processes by a few blue-green algae have been reported especially those involved in ammonia assimilation process (OWNBY 1977, ROWELL et al. 1977, THOMAS et al. 1977). I t h a s b e e n e x p e r i m e n t a l l y s h o w n t h a t

the exogenous addition of glutamine reverses L-methionine-DL-sulfoximine (MSO)induced effects, otherwise MSO alone lowers the in vivo pool of glutamine. Thus available reports and our findings clearly suggest t h a t glutamine is preferentially utilized by both the strains as a nitrogen source. However, further investigation is needed to prove whether this amino acid is directly utilized or whether it is first hydrolyzed and is then utilized. Our results on the effect of arginine are consistent with the findings of earlier workers to the effect t h a t arginine supports growth of non-nitrogen-fixing forms and also inhibits

358

ASHOK KUMAR and H . D. KTJMAB

nitrogenase activity in Plectonema sp. (WYATT et al. 1971, WHEATHERS et al. 1978, NAGATAJTI and HASELKOEN 1978). WHEATHEES et al. (1978), working on an Aphanocapsa sp., have reported the actual pathway of arginine catabolism in which arginine utilization both as nitrogen and carbon sources has been shown. They have reported the existence of arginase and arginine dihydrolase pathway; the arginase pathway provides only nitrogen for the cells whereas the arginine dihydrolase pathway provides not only nitrogen but also C0 2 and adenosine-5'-triphosphate. Following the above reports, it seems that the growth stimulation and repression of heterocyst frequency in our study may probably be due to existence of arginine catabolic enzymes in both strains. Furthermore, NAGATANI and HASELKORN (1978) have reported repression of acetylene reduction following arginine treatment in Plectonema and their conclusion was that arginine-induced repression of acetylene reduction is due its metabolic utilization; this supports and augments our conclusion. I t is worth mentioning here that the arginine residue of caynophycin granules is utilized in vivo at least at a stage where the filament reaches an intracellular nitrogen depletion level and hence the observed effect by exogenous addition of this particular amino acid seems more appealing and physiologically economical. Asparagine-induced stimulation of growth and decline of heterocyst frequency may also be due to its utilization through deamination, though its catabolism needs enzymological investigation. The growth of the non-nitrogen-fixing mutant (het+ nif~) with glutamine or arginine strongly supports the idea that these amino acids were utilized as nitrogen sources. Absence of growth on other amino acids is consistent with the observed effect on parent alga, where the remaining amino acids also failed to exert significant stimulatory effects on growth. The data of heterocyst differentiation are also consistent with the proposal of utilization of glutamine or arginine as nitrogen source, because the differentiation of heterocysts remains repressed in mutant strain, as in NH 4 + supplemented medium, whereas other amino acids fail to bring about such effect on heterocyst production in the mutant alga as in the parent. The failure of growth stimulation and lack of repressive effect on heterocyst differentiation by the amino acids listed in Tables 2 — 3, may be due to the fact that these amino acids are either not deaminated or the deaminated products are insufficient to stimulate growth. In the absence of enzymological studies, it is hard to speculate the cause of growth inhibition. However, in Anabaena variabilis, valine-induced inhibition of acetolactate synthetase, the first enzyme in the biosynthetic pathway to valine and leucine, has been reported by HOOD and CAKR (1978). They have shown inhibition of threonine deaminase by isoleucine. Also, there are reports of feedback inhibition of aromatic amino acid synthesis by WEBER and BOCK (1968, 1969). I n this context, a similar type of inhibitory effect by the amino acids listed in Tables 2 —3 on biosynthetic enzymes may not be ruled out. I t is also clear from the results that the alga fails to maintain their in vivo pool level of a few amino acids at least in a condition where the demand is itself met through the biosynthetic process. The differential effect of certain amino acids as listed in Table 3 at different concentrations may be due to permeability and uptake processes in wild type alga. Our results suggest that amino acids like glutamine, arginine and asparagine may constitute important factors in understanding the regulation of heterocyst-nitrogenase subsystem. A

cknowledgements

This investigation was supported in part by the Department of Atomic Energy and Department of Science and Technology under projects BRNS/B & M/95/75 and No. ll(44)/76-SERC sanctioned to HDK. AK was a Junior Research Fellow under the project. We thank the Head of Botany Department for laboratory facilities.

Response of Anabaena towards amino acids

359

References CARR, N. G. and WHITTOK, B. A., 1973. The Biology of Blue-Green Algae. Blaekwell Scientific Publications Oxford. FOGG, G. E., 1949. Growth and heterocyst production in Anabaena cylindrica LEMM. II. In relation to carbon and nitrogen metabolism. Ann. Bot., 13, 241—259. HASELKORN, R., 1978. Heterocysts. Ann. Rev. Plant Physiol., 29, 319—344. HOOD, W. and CARR, N. G., 1978. Threonine deaminase and acetolactate synthetase in Anabaena variabilis. Biochem. J., 109, 4. KUMAR, A. and KUMAR, H. D., 1980. Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae. Curr. Microbiol., 3, 213—218. MEEKS, J . C., WOLK, C. P . , LOCKAU, W . , SCHILLING, N . , SHAFFER, P . W . a n d CHIEN, W . S . , 1 9 7 8 .

Pathways of assimilation of (13N) N2 and ( 13 NH 4 + ) by cyanobacteria with and without heterocysts. J . Bacteriol., 134, 125—130. NEILSON, A. N. and DOUDOROFF, M., 1973. Ammonia assimilation in blue-green algae. Arch. Mikrobiol., 89, 1 5 - 2 2 . NAGATANI, H. H. and HASELKORN, R., 1978. Molybdenum independence of nitrogenase component synthesis in the nonheterocystous cyanobacterium Plectonema. J . Bacterid., 134, 597—605. OWNBY, J . D., 1977. Effects of amino acids on methioninesulfoximine induced heterocyst formation in Anabaena. Planta (Berl.), 136, 277—279. PINTER, I. J . and PROVASOLI, L., 1958. Artificial cultivation of a red pigmented marine blue-green alga Phormidium persicinum. J . gen. Microbiol., 18, 190—197. ROWELL, P . , ENTICOTT, S. a n d STEWART, W . D. P . , 1977. G l u t a m i n e s y n t h e t a s e a n d nitrogenase

activity in the blue-green alga Anabaena cylindrica. New Phytol., 79, 41—54. SINGH, H. N. and SHRIVASTAVA, B. S., 1968. Studies on morphogenesis in a blue-green alga. I. Effect of inorganic nitrogen sources on deveopmental morphology of Anabaena doliolum. Canad. J . Microbiol., 14, 1341 — 1346. SINGH, H. N. and KUMAR, H. D., 1971. Physiology of heterocyst production in the blue-green alga Anabaena doliolum. I. Nitrate and light controls. Z. Allg. Mikrobiol. 11, 615—622. SINGH, H. N., LADHA, J . K. and KUMAR, H. D., 1977. Genetic control of heterocyst formation in the blue-green algae Nostoc muscorum and Nostoc linckia. Arch. Microbiol., 114, 155—159. TYAGI, V. V. S., 1975. The heterocysts of blue-green algae (Myxophyceae). Biol. Rev., 50, 247—284. THOMAS, J . , MEEKS, J . C., WOLK, C. P . , SHAFFER, P . W . , AUSTIN, S . M . a n d CHIEN, W . S . , 1 9 7 7 .

Formation of glutamine from (13N) ammonia, (13N) dinitrogen, and (14C) glutamate by heterocysts isolated from Anabaena cylindrica. J . Bacteriol., 129, 1545—1555. . VAN BAALEN, C., 1962. Studies on marine blue-green algae. Botanica Mar., 4, 129 — 139. WEBER, H. L. and BOCK, A., 1968. Comparative studies on the regulation of DAHP synthetase activity in blue-green algae. Arch. Mikrobiol., 61, 159 — 168. WEBER, H. L. and BOCK, A., 1969. Regulation of the chorismic acid branch-point in aromatic amino acid synthesis in blue-green and green algae. Arch. Mikrobiol., 66, 250—258. WHEATHERS, P . J . , CHEE, H . L . a n d ALLEN, M. M., 1978. Arginine catabolism in

Aphanocapsa

6308. Arch. Microbiol., 118, 1—6. WYATT, J . T., LAWLEY, G. G. and BARNES, R. D., 1971. Blue-green algal responses to some organicnitrogen substrates. Naturwissensch., 11, 1—3. Mailing address: Prof. H. D. KUMAR Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, U. P., India

Zeitschrift für Allgemeine Mikrobiologie

21

1981

361-372

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

Regulation of ammonia assimilation in ammonia-limited chemostat cultures of Escherichia coli ML 30: Evidence of Instability P . J . MÜLLER, B E A T E VON FROMMANNSHAUSEN a n d H . SCHÜTZ

(Eingegangen am 15. 9.1980) In a preceding paper evidence of two stationary stable states (bistability) in the specific activity of glutamine synthetase (GS) in ammonia-limited steady-state cultures of Escherichia coli ML 30 at dilution rates (D) about 0.15 h _ 1 was described (MÜLLER et al. 1977). For better understanding of the regulation mechanisms leading to GS bistability chemostat experiments were performed over a wide range of dilution rates up to D = 0.8 h - 1 . For each steady state the specific activities of GS and glutamate dehydrogenase (GDH) — the other key enzyme of the two NH 3 assimilation routes in E. coli — and in addition the remaining NH 3 concentration in the culture liquid were determined. Parallel to GS bistability two states of GDH activity and NH, concentration are found. The higher state of GS is connected with a lower GDH activity and NH 3 concentration. With rising D the GS activities decrease whereas GDH activities and NH 3 concentrations increase. Since no adenylation of the GS is detectable GS bistability seems to be regulated on the level of enzyme synthesis like GDH bistability. From the experimental findings a mathematical model is derived based on the bottle neck enzyme theory of growth. I t describes the dependence between the specific growth rates on the one hand and the specific enzyme activities and NH 3 concentration on the other. It is shown that the specific uptake rate of the limiting NH 3 and the specific growth rates, respectively, depend on the simultaneous action of two bottle neck enzymes which are connected by a regulative link. Chemostat cultures allow to investigate the enzyme regulation in microorganisms under steady-state conditions in dependence on the specific growth rate (¡x) or dilution rate (D), respectively. The situation is complicated by the possibility of bi- or multistability in the intracellular specific enzyme activities leading to different intraand extracellular metabolite concentrations (BEEGTEE 1968, 1969a und b, BEEGTEE and ROTH 1977, MÜLLER et al. 1977, 1979). The intracellular enzyme activity can be regulated on the epigenetic level, which may be indicated by experimental estimated enzyme activities, and/or on the metabolic level. More than one stable stationary state in open homogeneous enzyme systems m a y occur already in the case of substrate repression or inhibition of one enzyme (BERGTEE 1 9 6 8 , 1 9 6 9 a a n d b , D E G N 1 9 6 8 , SELKOV 1 9 6 9 a n d KNORRE et al.

1975) a n d in pure

chemical systems (FÖLLNER and GEISELER 1977). The stoichiometry of biochemical reactions as the only reason of multistability was discussed by GTJTHKE (1978). I n a preceding paper two stable stationary states in the specific activity of glutamine synthetase [GS] (measured specific activities are denoted by capital letters) in an ammonia-limited glucose-grown chemostat at D = 0.15 h _ 1 were measured (MÜLLER et al. 1977). I n addition it was shown that the initial conditions determine whether the higher or the lower level of [GS] is finally reached. The possibility of GS bistability was found during investigations on the dependence between ¡i. and the extracellular N H 3 concentration [NH 3 ] (BERGTER et al. 1977) (Previous results and predictions for

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P . J . MtriXEE, BEATE VON FROMMANNSHAUSEN a n d H . SCHUTZ

chemostat behaviour are schematically summarized in Fig. 1). A special function |i([NH3] was found which differs from a saturation function of the MooD-type (MOOD

F i g . 1. Scheme of t h e proposed correlation between t h e special function ,u([NH 3 ]) and t h e steadys t a t e behaviour (fi = D) of a NH 3 -limited chemostat culture. I t is assumed t h a t fx ([NH 3 ]) is t h e sum of a GS-dependent and a GDH-dependent function. A t t h e left side of t h e figure t h e dependence of t h e intracellular specific activities [gs] and [gdh] and t h e enzyme concentrations [GS] and [ G D H ] on t h e N H 3 concentrations is illustrated. A t t h e right hand t h e function [NH 3 ] (D), obtained b y inversion of fi ([NH 3 ]), describes t h e proposed steady-state behaviour of a chemostat. Also, t h e proposed dependencies of t h e specific activities [gs] and [gdh] and t h e enzyme concentrations [GS] and [ G D H ] are plotted. fia, m and Da, Db inclose a range of bistability. T h e points of intersection 1, 2, 3, a n d 4 mark the two states of enzyme activities a x , a 2 , a 3 , and a 4 and Ax, Av A3, and Ait and t h e two N H 3 concentrations c i ; 3 and C2;4 a t t h e specific growth r a t e juc a n d Dc, respectively

Regulation of ammonia assimilation

363

Data on regulation of ammonia assimilation in E. coli (PRIVAL et al. 1 9 7 3 ) led to the conclusion that the special function ¡JL([NH3]) is produced by regulation of the key enzymes of the two ammonia assimilation systems in E. coli. Glutamine synthetase is the key enzyme of the GS/glutamate synthase system (TEMPEST et al. 1970, 1973, BERBEBICH 1 9 7 2 , BROWN et al. 1 9 7 4 ) . The other system depends on the glutamate dehydrogenase (GDH). I t is generally accepted that the GDH-catalyzed pathway operates mainly at relatively high N H 3 concentrations because of the high -K^QDH) =

1 . 5 — 3 . 0 MM N H 3 (MILLER a n d STADTMAN 1 9 7 2 , SAKOMOTO et al. 1 9 7 5 ) . I n c o m -

parison the GS/glutamate synthase-catalyzed pathway is active at low NH 3 concentrations because of the low Km ( G S ) = 0 . 2 MM N H 3 (DENTON and GINSBUBG 1 9 7 0 ) . However, GS synthesis is repressed by higher N H 3 concentrations (MECKE and et al. 1 9 7 3 ) . A further supplement to G S regulation is a control exerted by the reversible enzyme-catalyzed adenylation and deadenylation of the G S molecule (WOOLFOLK et al. 1 9 6 7 , KINGDON and STADTMAN 1 9 6 7 ) . I t is now assumed that the first increase of the function FI([NH3]) is mainly connected with the actual intracellular specific activity (denoted by small symboles) of the GS ([gs]), which increases with increasing saturation of the enzyme by NH 3 . Theoretically the decreasing phase of ¡J. with further increasing [NH 3 ] results from a substrate inhibition and/or an inhibition by adenylation and/or by repression of GS by NH S . The following increase of \L is probably connected with an increase of the intracellular specific activity of the GDH ([gdh]), exerted by higher saturation of the enzyme by N H 3 and/or by an induction of enzyme synthesis. The special function |I([NH 3 ]) suggests a bistability in the extracellular N H 3 conecntration in the steady-state of NH 3 -limited chemostat culture. I n consequence a bistability in [GS] was predicted and already proved as mentioned above. But it was not possible to distinguish between a regulation by adenylation/deadenylation or by repression/induction mechanisms (MULLER et al. 1977) from the measured two states of [GS], The present investigation was launched to obtain information to what extent the predictions from the special function ¡J.([NH3]) for chemostat behaviour are valid. For this it was necessary to determine the specific activities [GS] and [GDH] and the N H 3 concentrations at different dilution rates in the whole range of D and especially in the range where bistability in [GS] was already proved. Moreover, the question whether adenylation of the GS molecule in both stable stationary states occurs demanded investigations. From the results a simple model was developed which demonstrated the function of two enzyme systems as simultaneously operating bottle-necks of growth (JERTJSALIMSKI 1 9 6 7 ) and which predicts the behaviour of the investigated system. HOLZER 1 9 6 6 , WOHLHUETER

Materials

and

methods

Bacterial strain and growth conditions: Escherichia coli ML 30 was used in this study. The media contained (g/1) K H 2 P 0 4 2.72, N a 2 H P 0 4 - 2 H 2 0 3.55, NaCl 5.0, N a 2 S 0 4 1.065, NH 4 C1 0.090, MgCl2 • 6 H 2 0 0.041, FeCl 3 • 6 H 2 0 0.0054, MnCI2 0.0039, glucose 3.0. Individual solutions of salts and of glucose were steam-pressure sterilized (120 °C, 20 min). The p H was adjusted to 6.8 with NaOH. The cylindrical glass chemostat vessels were of 100 or 200 ml culture volume. The culture medium was pumped through an inlet at the top of the vessel with a peristaltic pump. Exhausted medium and organisms were removed through an overflow tube, protruding from the side of the vessel. The culture medium was stirred with a glass-covered magnetic stirring bar. I n some cases the only mixing was by the bubbles of the moistened air inflow into the vessel (inflow rate = 1.65 1/min per 1 culture volume). The temperature of the cultures was maintened at 34 °C.

364

P . J. MÜLLER, BEATE YON FROMMANNSHAUSEN a n d H . SCHÜTZ

Chemostat cultures were initiated by inoculating 50 ml of an E. coli ML 30 culture with a cell concentration of about 150 mg/1 dry weight from the log phase of growth. Then medium pumping began at the chosen dilution rate. A minimum of 6—8 culture volumes after reaching the working volume was permitted to flow through the vessel before sampling began. For reaching the upper state of [GS] at D = 0.15 h"1 the dilution rate was changed by a jump from a steady state with a higher D. The lower state of GS was reached by starting the chemostates as described above or by a jump from a steady state with a smaller D to D = 0.15 h -1 . Determinations: i. Cell concentration (X): The effluent culture liquid was accumulated in a tube surrounded with ice. For determination of X the extinction at 470 nm (E170) was estimated. An E470 of 1.0 was equivalent to 0.173 g/1 dry weight. ii. Ammonia concentration in the culture liquid ([NH 3 ]): The inflow of medium was interrupted and 2—3 ml culture liquid was immediately removed from the chemostat. Bacteria were withdrawn by rapid filtration through a cooled (4 °C) filter device (membrane filter Sartorius GmbH, 0.45 fi). [NH 3 ] was measured enzymatically with glutamate dehydrogenase (DA FONSECA-WOLLHEIM et al. 1974) dissolved in glycerol (BOEHRINGER GmbH, Mannheim). The meausrements were performed with the spectrophotometer SPEKOL (CARL ZEISS Jena) equipped with a device for automatical changes of four cuvettes. The extinctions were recorded over a range of 20 min. iii. Protein concentration: Protein was determined colorimetrically with bovine serum albumin as standard according to LOWRY et al. (1951).

The cell extract was prepared in the following manner: A sample of the culture (50 — 100 ml) was removed from the vessel and rapidly cooled. The sample was centrifuged (1200 X g for 10 min at 4 °C) and the cell pellet suspended in 5 ml cooled tris-(hydroxymethyl) aminomethane buffer (0.02 mol/1, pH 7.6). The cells were disintegrated by sonic treatment at 4 °C for 100 s in 20 s periods using a 100 WATT disintegrator (Institut fur angewandte Physik, Halle, Martin-LutherUniversitat). Debris were removed by centrifugation (1200 x g for 10 min at 4 °C). The crude extract was kept at 4 °C and assayed immediately for enzyme activities. iv. Specific GS activity ([GS]): GS activity was estimated with the y-glutamyl synthetase reaction (KOHLHAW et al. 1965). The degree of adenylation was measured by the y-glutamyl transfer reaction (STADTMAN et al. 1970). Reaction blanks without adenosine 5-diphosphate and arsenate were used to correct both, Mn2+ alone (estimation of the sum of the activities of adenylated and non-adenylated GS) and the Mg2+ assay (estimation of non-adenylated GS) for glutaminase activity, which also leads to y-glutamyl hydroxamate. v. Specific GDH activity ([GDH]): GDH activity was measured spectrophotometrically with N A D P H (HIERHOLZER and HOLZER 1963).

Data analysis: For calculation the general curve fitting program ALAU (H. SCHUTZ, unpublished) was used. This program demands a problem specific subroutine, which has to deliver the actual weight W{, the weighted derivatives Vwi • d/Xi/6 In par, and the weighted differences wi (fi( — fic(i)) (par stands for any of the parameters (9)). To decide what is a good, a better and the best representation of ihe set of experimental data, a criterion over the parameter space was defined which can be minimized at least over a subspace of this space. At first sight one can think about a least square criterion of the following form: % E

-

i

Mi)2

+ A2 E ( P I B U ) I -

[ N H 3 ] E ( I ) ) 2 + a3 £

([GS]« -

[GS] C ( I ) ) 2

i i + a 4 E ([GDH] { - [GDH], (i)) 2 (1) i should be minimum (subscribt c and argument (i) mean, calculated at point i). But there are some difficulties. First the a4 should be in the same relation as the reciprocal squares of the errors of the methods for determination of ¡xi, of [NH 3 ] i( of [GS]i) and of [GDH){, respectively. But the accuracies of activity, concentration and dilution rate are hardly comparable. Secondly, and this difficulty is more serious, there exists only one model equation (7). It is possible to calculate only one of the quantities (8), regarding the other three as independent (and experimentally given). For these reasons and for the sake of simplicity the quantity (fii-MW

(2)

was chosen to be minimum as criterion. "

Results

19 Glucose-grown chemostat cultures were examined after attainment of the N H 3 limited steady-state for the specific enzyme activities [GS], [ G D H ] and the N H 3 concentration of the medium [ N H 3 ] at dilution rates up to. I ) - - 0.8 h" 1 (Tab. 1). Fig. 2

Regulation of ammonia assimilation

365

Tabelle 1 List of experimental data [GS] ((x mol • min" 1 • mg l ) estimated with : D(h^) Synthetase test 0.096 0.13 0.14 0.145 0.145 0.15 0.16 0.16 0.18 0.20 • 0.28 . 0.29 . 0.296 0.430 0.645 , 0.66 0.695 0.695 • 0.786

1.22 .1.28 1.10 1.01 3.18 2.94 0.94 3.22 1.52 3.47 0.64 0.66 0.49 0.35 .0.32 0:27 0.36 0:19 0.05

[GDH] (|j.mol • min - 1 • mg -1 )

Transferase test +Mg

++

+ Mn

—

•

2.36

++

+Mn '

.—

2.36

—

—

5.80 2.16 7.30

5.80 2.18 7.30

—

—

8.10

8.05

_

1

_

— •

—

—

.

— —

.

—

—

—

(M)'

++

—

•

• [NH 3 ]

0.07 0.82 0.93 0.94 0.49 0.50 1.10 0.59 1.25 0.65 • 3.20 3.26 ' 2.91 4.50 4.50 3.77 • 3.40 5.15 5.65

0.032 0.034 0.022 0.042 0.019 0.013 0.043 0.019 0.056 0.010 0.20 0.17 0.200.10 0.24 0.34 0.24 0.29 0.29

Fig. 2. Estimated specific activities of GS ([GS]:T), of GDH ([GDH]:«) and the NH 3 concentration of the medium ([NH3] : • ) in dependence on D. The experimental points are connected by lines according to the theoretical assumptions, reviewed in the introduction of this paper. The open points characterize the upper state of [GS] (lower state of [GDH] and [NH3])

366

P . J . M Ü L L E R , B E A T E VON F R O M M A N N S H A U S E N a n d H . SCHÜTZ

shows the effect of D on the estimated values. In the range of D = 0.14 — 0.2 h - 1 GS bistability occurs, proved with the synthetase test and also the transferase test, corrected for glutaminase activity. For identifying the range of D where bistability occurs, a chemostat was started at D = 0.6 h - 1 and after reaching of the steady state X) was jumped to Z > < 0 . 6 h - 1 . If bistability were possible here a higher GS concentration was to be expected. Because lower values of [GS] were found at D > 0.3 h - 1 , the range of bistability is restricted to values of D < 0.3 h - 1 . The higher state of [GS] ( » 3 . 2 ¡junol • m g - 1 • min - 1 in the synthetase test and s»7.0 [xmol • m g - 1 • min - 1 in the transferase test) was obtained after a jump from a steady state with a D > 0.3 h - 1 . The lower state of [GS] ( « 1 . 1 7 ¡xmol • m g - 1 • min - 1 in the synthetase test and i=a 2.3 [j.mol • mg - 1 • min - 1 in the transferase test) was reached after a jump from a lower D to D = 0.14 — 0.2 h _ 1 or after initiation of the cultures as described in "Materials and methods". N o significant differences in the GS activity of cells from both stationary states are found (estimated by the transferase test in the presence ofMn 2 + , Mg 2 + or Mg 2+ alone), showing the absence of adenylation of the GS molecule in the range of bistability (Tab. 1). Fig. 2 shows that GS-bistability is connected with a bistability in G D H activity and in N H 3 concentration. The higher state of [GS] corresponds to a value of [ G D H ] « 0.56 ¡xmol • mg" 1 • min" 1 and N H 3 » 0.015 mM, the lower state of [GS] to [ G D H ] « 1.0 [imol • m g - 1 • min - 1 and [ N H 3 ] ss 0.04 mM. With rising D the GS activity decreases while the G D H activity increases. The reciprocal relation between [GS] and [ G D H ] at all investigated dilution rates is illustrated in Fig. 3.

[6OH] jump./ mining

0 L 0

j

1

i

2

,

3. [6J] MUL mmmg

Fig. 3. Relation between the experimentally found specific activities [GS] and [GDH]. The curve is calculated from the mathematical functions, introduced in the model for the description of the dependence of [GS] and [GDH] on [NH 3 ]. One curve (full line) is calculated from the functions [GDH] ( [ N H J ) and [GS] ([NH 3 ]) (Fig. 4A, full line), the other (dashed line) curve from the functions of Fig. 4 A , indicated by dashed lines

Regulation of ammonia assimilation

367

Fig. 4 A. Dependence between [GS] ( • ) [GDH] ( • ) and [NH 3 ]. The functions [GS] ([NH S ]) and [GDH] ([NH 3 ]) are calculated by the least squares method. For calculation of the functions represented by dashed lines two points a t [NH 3 ] = 0.10 and 0.34 mM were omitted as probable experimental mistakes. The open symbols in Fig. 4 A, B, C characterize the upper state of [GS] (lower state of [GDH] and [NH 3 ]) B. The relative contribution of the GS-dependent bottle neck (fiGs/fi) in dependence on [NH 3 ] is shown. One curve (full line) was calculated with the parameter set (11), the other curve (dashed line) from (14). The symbols (A) represent values calculated from the individual experimental points with the parameter set (11) C. Dependence between ¡x and the NH 3 concentration calculated with the model. One curve (full line) is calculated from the parameter set (11), the other (dashed line) with (14). The symbols (O, • ) are experimental values. The weak relative minimum a t [NH 3 ] = 0.04 mM indicates the relative minimum proposed by the special function /tt([NH3]) for the chemostat. A more intensive minimum is not expected because the whole range of D where bistability is possible was not examined. For illustration the first maximum is designated by a free hand line ( - • - • - ) 24

Z. Allg. Mikrobiol., Bd. 21, H . 5

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P . J . M Ü L L E R , B E A T E VON F R O M M A N N S H A U S E N a n d H . S C H Ü T Z

The relationship between [GS] and [ G D H ] on one hand and the N H S concentration on the other is shown in F i g . 4 A a n d B . [ G D H ] increases nearly linearly with increasing [NH 3 ], [GS] decreases rapidly a t N H 3 concentrations about 0.05 mM and then decreases continuously with rising [NH 3 ]. The model Because the G D H and the GS-catalyzed routes are the only ammonia assimilation systems in E. coli ( B E R B B R I C H 1972), the total amount of N H 3 for growth has to flow through these systems. The results of B E R G T E K et al. (1977) and the present results supported the view that the enzymes G S a n d G D H are bottle necks of metabolism with a growth rate controlling function. F o r description of the dependence between the estimated values [GS], [ G D H ] and [NH 3 ] a t different growth rates a simple model was constructed. I t was assumed that the measured specific activities [GS] and [ G D H ] are proportional to the specific concentrations of the enzymes in the cells. The dependence of the specific growth rate ji on the specific uptake rate q of the limiting N H 3 (g NHg/h per g biomass) in the steady state of the chemostate culture is expressed in equation (3). fi=Y-q

(h-i)

(3)

Y: yield factor (g formed biomass/g N H 3 taken up) q : denotes the flow of the limiting N H 3 through the bottle neck enzymes a n d is therefore equal to the sum of the intracellular specific activities of the bottle-neck enzymes [gs] and [gdh] (4)