Carbon and Nitrogen Repression of Arginine Catabolic

Vol. 137, No. 1

JOURNAL OF BACTERIOLOGY, Jan. 1979, p. 189-196 0021-9193/79/01-0189/08$02.00/0

Carbon and Nitrogen Repression of Arginine Catabolic Enzymes in Bacillus subtilis SIMON BAUMBERG'* AND COLIN R. HARWOOD2 Department of Genetics, University of Leeds, Leeds LS2 9JT, England,' and Department of Microbiology, University of Newcastle upon Tyne, Newcastle upon Tyne NEI 7RU, England. Received for publication 7 November 1978

Specific activities of arginase and ornithine aminotransferase, inducible enzymes of arginine catabolism in Bacillus subtilis 168, were examined in cells grown with various carbon and nitrogen sources. Levels of these enzymes were similar in arginine-induced cultures whether glucose or citrate was the carbon source (in contrast to histidase), suggesting that carbon source catabolite repression has only limited effect. In media with combinations of nitrogen sources, glutamine strongly. repressed induction of these enzymes by proline or arginine. Ammonium, however, only repressed induction by proline and had no effect on induction by arginine. These effects correlate with generation times in media containing these substances as sole nitrogen sources: growth rates decreased in the order glutamine-arginine-ammonium-proline. Similar phenomena were observed when glutamine or ammonium were added to arginine- or proline-grown cultures, or when arginine or proline were added to glutamine- or ammoniumgrown cultures. In the latter cases, an additional feature was apparent, namely a surprisingly long transition between steady-state enzyme levels. The results are compared with those for other bacteria and for eucaryotic microorganisms. Control of arginine metabolism in the grampositive bacterium Bacillus subtilis is of interest because it shows elements of similarity with control systems found in yeast (10). It is not known, however, to what extent such similarities apply to mechanisms at the molecular level. Arginine is able to supply the nitrogen requirement of B. subtilis. After uptake, arginine not required directly for protein synthesis may be converted to glutamate by a series of three enzymically catalyzed steps (Fig. 1). At least two of the three enzymes involved, arginase and ornithine aminotransferase, are induced in the presence of L-arginine, L-ornithine, and L-citrulline (10). The last reaction in this pathway, the conversion of A'-pyrroline 5-carboxylate to glutamate, is shared with the proline catabolic pathway (Fig. 1). It is not known whether these pathways use the same or independent catabolic enzymes for this step (it now appears that an earlier report [7] showing the presence of two catabolic A'-pyrroline 5-carboxylate dehydrogenase enzymes referred to a strain of B. lichenformis rather than B. subtilis [17]). This may not be the sole route of arginine utilization in B. subtilis; Broman et al. (1) have reported a deiminase pathway in B. lichenformis, similar to that found in pseudomonads, which is apparently subject to strong glucose and oxygen repression (V. Stalon, personal communication).

B. subtilis synthesizes arginine from glutamate by a series of eight enzymically catalyzed steps identical to those found in Escherichia coli (30). At least five of these enzymes have been shown to be repressed in the presence of arginine (10, 18, 30); however, no repression has been observed in the presence of the intermediates ornithine and citrulline (10), in contrast to the closely related B. lichenformis (15) or the unrelated E. coli (20). Consequently, when B. subtilis is grown in the presence of ornithine or citrulline, both the biosynthetic and catabolic enzymes of arginine metabolism are present at an elevated level. Futile cycling between ornithine and arginine that might result is prevented by a specific interaction between the biosynthetic ornithine carbamoyltransferase and arginase, which results in the inhibition of ornithine

carbamoyltransferase activity (11). Arginase activity is unaffected. A similar control mechanism has been found in yeast (22). Preliminary studies on the control of arginine metabolism in B. subtilis indicate that its regulation is complex. We have isolated a large number of mutants which are resistant to the arginine analog arginine hydroxamate (10). Most were tound to have reduced levels of arginase and ornithine aminotransferase activity under a variety of conditions in which these enzymes are normally induced. Some mutants are simulta-

189

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BAUMBERG AND HARWOOD proline

p~yrroline 5carboxylate

J. BACTERIOL. carbamyl

phosphate

aspartate

oxoglutarate

\111'.6 urea

FIG. 1. Pathways of arginine biosynthesis and catabolism in Bacillus subtilis. Enzymes are: AGA, arginase; GS, glutamine synthetase; OAT, ornithine aminotransferase; OCT, ornithine carbamoyltransferase; and PCD,A'-pyrroline 5-carboxylate dehydrogenase.

neously affected in their biosynthetic and catabolic enzymes, implying that in B. subtilis, as in yeast, these pathways have controlling elements in common (10, 32). Reports that the levels of arginine catabolic enzymes in various microorganisms are altered by the choice of carbon or nitrogen sources have led us to study the effects of various carbon and nitrogen sources on the control of these enzymes in B. subtilis. For example, the arginine catabolic pathway of Pseudomonas fluorescens, which proceeds via citrulline and which is normally induced by arginine, is not inducible in the presence of glucose (25). This is also the case for the arginine catabolic pathway of B. licheniformis, which is identical to the pathway found in B. subtilis (14). In contrast, no glucose repression of these enzymes has been reported for yeast; instead they have been found not to be induced in the presence of ammonium (32). Here we describe experiments to determine conditions under which levels of arginine catabolic enzymes of B. subtilis are affected by the carbon or nitrogen sources employed. MATERIALS AND METHODS Strains. The bacterial strain used throughout this work was EMG50 (4), a prototrophic transformant of B. subtilis 168. Stocks were kept on nutrient agar at 4°C or in nutrient broth containing 10% (vol/vol) glycerol at -20°C. Media. Nutrient media were either Lemco (L) broth, Oxoid or tryptone soya (TS) broth which contained, per liter: tryptone (Oxoid), 17 g; soya peptone (Oxoid), 3 g; sodium chloride, 5 g; dipotassium hydrogen orthophosphate, 2.5 g; and glucose, 2.5 g; pH 7.0. Modified minimal medium (MMM) contained, per liter: dipotassium hydrogen orthophosphate, 14 g; potassium dihydrogen orthophosphate, 6 g; and triso-

dium citrate, 2H20, 1 g; pH 7.0. It was sterilized by autoclaving at 121°C for 15 min, and then magnesium sulfate solution was added aseptically to a final concentration of 0.02%. In certain experiments where the nitrogen source was other than ammonium sulfate, potassium sulfate (1.74 g/liter) was added; however, no differences were found when the latter was omitted. Glucose was added to a final concentration of 0.5%. Nitrogen sources, which were sterilized by membrane filtration (invariably, in the case of glutamine) or by autoclaving, were added to a final concentration of 10 or 15 mM. MMM-Cit (MMM without trisodium citrate) or MMM-Glu (MMM without glucose) were used where glucose (0.5%) or citrate (0.1%), respectively, was the sole carbon source. Culture methods. All liquid cultures were grown at 37°C by shaking in an orbital incubator (Gallenkamp) at 200 to 250 rpm. Culture volumes usually did not exceed 20% of the volume of the flask. Nutrient (L or TS) broth cultures were inoculated directly from stock plates; minimal cultures were inoculated from either L or TS broth cultures. (i) Carbon source experiments. Portions (20 ml) of MMM-Cit or MMM-Glu with appropriate nitrogen source and contained in 100-ml flasks were inoculated to an absorbance (at 500 nm) of approximately 0.03 with a washed suspension of EMG50 grown in L broth. Cultures were harvested on reaching an absorbance of approximately 0.6 by adding chloramphenicol (to 100 ,ug/ml) and centrifuging at 17,000 x g for 15 min at 4°C. Enzyme assays were performed on frozen-thawed cells, resuspended in either 0.05 M glycine-NaOH buffer, pH 9.5, or 0.1 M potassium phosphate buffer, pH 7.0 (Harwood, Ph.D. thesis, University of Leeds, Leeds, England, 1974). (ii) Nitrogen source experiments. Portions (25 ml) of MMM with the required nitrogen source and contained in 250-ml flasks were inoculated to an absorbance at 500 nm of approximately 0.04 from a washed suspension of EMG50 grown in L broth. The cultures were grown to an absorbance of 0.3 to 0.5 and were then diluted fivefold into fresh, prewarmed me-

VOL. 137, 1979

CONTROL OF ARGININE CATABOLISM IN B. SUBTILIS

dium of the same composition to extend the exponential-growth phase. When these cultures reached an absorbance of 0.4 to 0.6, chloramphenicol was added, and the cultures were harvested as above. (iii) Kinetics of repression experiments. The kinetics of arginase and ornithine aminotransferase repression were determined by measuring the activities of these enzymes in cultures containing arginine or proline as the sole nitrogen source before and after the addition of either glutamine or ammonium. A starting culture, inoculated from a TS broth culture, was grown for 16 h in MMM containing either arginine or proline as the sole nitrogen source. This was used to inoculate a prewarmed 250-ml flask containing 25 ml of medium of the same composition. When the culture was well in exponential growth (absorbance at 600 nm, approximately 0.4), it was diluted 20-fold into 380 ml of the same medium, prewarmed, and contained in a 2,000-ml flask. By this stage the culture had been maintained in exponential growth for at least 10 generations. Samples (6 ml) were taken at various times until an absorbance of 0.15 was reached. The culture was then divided equally between three prewarmed, 2,000-ml flasks. Glutamine was added to one flask, ammonium sulfate was added to another, and extra arginine or proline was added to the third to compensate for the extra nitrogen added to the first two cultures, each nitrogen source being present finally at 10 mM. Further 6-ml samples were removed from all three cultures until an absorbance of 0.6 to 0.7 was reached. All samples were pelleted by centrifugation for 90 s in a Microfuge B centrifuge (Beckman-RIIC) at 1,100 x g and at ambient temperature, with chloramphenicol added as above. The pellets were stored at -20°C. Arginase, ornithine aminotransferase, and protein assays were carried out on the pellets after they had been thawed and resuspended in 0.1 M potassium phosphate buffer, pH 7.0. (iv) Kinetics of induction experiments. The kinetics of arginase induction were determined by measuring enzyme activity in cultures containing ammonium or glutamine as sole nitrogen source before and after the addition of arginine or proline. A starting culture, inoculated from a nutrient agar plate, was grown for 16 h in MMM containing ammonium sulfate as the sole nitrogen source. The cells were centrifuged and resuspended in MMM (without a nitrogen source) and inoculated into 100-ml lots of MMM containing the appropriate nitrogen source at 15 mM, in 250-ml Erlenmeyer side-arm flasks. Absorbance was monitored in a Klett-Summerson colorimeter with a no. 66 filter, the initial reading being 5 to 15. At a Klett reading of approximately 25, arginine or proline was added to 15 mM. At various times before and after the latter additions, two 1.8-ml portions were removed and centrifuged in a Jobling Quickfit bench microcentrifuge at 14,000 x g. One pellet was resuspended in glycine-NaOH buffer (for arginase assay), and the other was suspended in potassium phosphate buffer (for protein assay), both suspensions being stored at -20°C until required. Enzyme assays. Arginase (L-arginine ureohydrolase, EC 3.5.3.1), ornithine aminotransferase (L-ornithine: 2 oxoglutarate aminotransferase, EC 2.6.1.13) and ornithine carbamoyltransferase (carbamoylphos-

191

phate: L-ornithine carbamoyltransferase, EC 2.1.3.3) assays were performed on frozen-thawed cell suspensions as described previously (10), except that in the kinetics of repression experiments, arginase assays were carried out on potassium phosphate buffer suspensions diluted at least fivefold in glycine-NaOH buffer. The presence of up to 4 mM phosphate buffer in the reaction mixture has no significant effect on its pH or on arginase activity. Histidase (histidine ammonia-lyase, EC 4.3.1.3), which catalyzes the conversion of histidine to urocanate, was assayed in phosphate-resuspended frozen-thawed cells by measuring the rate of urocanate formation through its increased absorption at 277 nm (molar extinction coefficient, 18,000), by the method of Chasin and Magasanik (2). Enzyme specific activities for arginase, histidase, and ornithine carbamoyltransferase are given in micromoles of product formed per hour per milligram of protein, and those for ornithine aminotransferase are given in optical density units corresponding to product formed per hour per milligram of protein. Protein assay. Protein determinations were made on frozen-thawed extracts in either potassium phosphate or glycine-NaOH buffers by the original method of Lowry et al. (19) or the modification of this method by Hartree (9) in the case of some kinetic experiments. Bovine serum albumin fraction V (BDH) standards were run with each assay. Chemicals. Inorganic chemicals were analytical grade. Unless otherwise stated, organic compounds were obtained from BDH (AnalaR grade) or from Sigma.

RESULTS Effect of carbon source. EMG50 was grown on MMM-Cit or MMM-Glu to determine the effect of carbon source on the ability of arginine to induce arginase and ornithine aminotransferase. Parallel cultures with the same carbon sources, but with histidine as the nitrogen source, were included because of the demonstration by Chasin and Magasanik (2) that glucose represses the induction by histidine of the histidine catabolic enzymes in B. subtilis. Table 1 shows the generation times and enzyme activities of EMG50 on the various combinations of carbon and nitrogen sources. Of the nitrogen sources, arginine permitted the fastest growth on both carbon sources, followed by ammonium and then histidine. In the case of arginine and ammonium, growth was faster on glucose than citrate; with histidine as the nitrogen source, however, growth was faster on citrate. The table also provides the explanation for this and confirms the earlier work of Chasin and Magasanik by showing that although glucose has only a 2.7-fold effect on induction of arginase and no effect on that of ornithine aminotransferase in the presence of arginine, it reduces the induced level of histidase 38-fold. The resulting low level of histidase is presumably responsible for the low growth rate on histidine as the sole

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BAUMBERG AND HARWOOD

TABLE 1. Generation times and specific activities of arginase, ornithine aminotransferase, and histidase for EMG50 grown on various carbon and nitrogen sources'

TABLE 2. Generation times and specific activities of arginase, ornithine aminotransferase, and ornithine carbamoyltransferase for EMG50 grown on various nitrogen sourcesa

Enzyme sp act Carbon source

Nitrogen source

Generation time

Arg-

(mm) inase Citrate

Glucose

Enzyme sp act

Orni

Ammonium Arginine Histidine

250 140 270

9 360 ND

Ammonium

170

8

thine amino-

transferase 0.1 1.0 ND 0.1

Gener-

Histidase

Nitrogen source

om-

ation time

(min)

Arginase

1

NDb 270 1

ND 0.9 135 69 Arginine 7 ND 600 ND Histidine cit(carbon in source, MMM-Glu were grown 'Cultures rate) or MMM-Cit (carbon source, glucose) with the appropriate nitrogen source. Culture conditions, enzyme assay methods, and definitions of unit of enzyme specific activity are given in the text. 'ND, Not determined.

nitrogen source in the presence of glucose as compared with citrate; the much smaller reduction in levels of the arginine catabolic enzymes in glucose medium accompanies an actual improvement in growth rate over that in citrate medium. Effect of nitrogen source. Ammonium represses the induction by arginine of the arginine catabolic enzymes of the yeast S. cerevisiae (32). Wiame found that S. cerevisiae grows equally well on ammonium, glutamate, or glutamine as the sole nitrogen source (generation time, 120 min), but slightly more slowly on arginine (generation time 150 min). It therefore appears possible that ammonium repression functions to ensure preferential, utilization of the most efficient nitrogen source from a mixture of nitrogen sources. We therefore devised experiments to determine the effects of ammonium and some other nitrogen sources on these enzymes in B. subtilis 168. EMG50 was grown in MMM with either ammonium, glutamate, glutamine, arginine, or proline as the sole nitrogen source. The generation times given in Table 2 show that glutamine is the preferred nitrogen source, followed by arginine, then ammonium and, finally, glutamate or proline (although some variation in absolute magnitude was observed on repeat occasions, the relative values for the different nitrogen sources from a given inoculum follow the order indicated). Growth rates for ammonium- and arginine-grown cultures are higher here for MMM containing both citrate and glucose than in Table 1 for MMM containing only one or other of these compounds; this suggests that the

Ammonium Arginine Glutamate

50 48 74

Glutamine

42 72 46

Proline Arginine + am-

16 100 6 3 96 110

On-

trans-

thine carbamoyl-

ferase

ferase

0.3 1.6 0.1 0.08 2.1 1.85

1.25 0.3 1.5

thine

a.no

1.1 1.0

0.06

monium Arginine + glu-

0.1 ND' 115 1.2 tamate 0.05 0.3 10 40 Arginine + glutamine 1.2 4 0.2 Proline + am- ND monium ND 6 0.2 Proline + gluta- ND mine a Cultures grown in MMM (containing citrate + glucose) with the appropriate nitrogen source. Culture conditions, enzyme assay methods, and definitions of unit of enzyme specific activity are given in the text. 'b ND, Not determined.

necessity either for generation of intermediary metabolites from glucose or for sugar synthesis from citrate may depress growth rate. Growth rates in arginine + ammonium or arginine + glutamine were reproducibly higher than in arginine or glutamine alone; this may be because in these cases the poorer nitrogen source provides some metabolite less readily obtained from the better nitrogen source so that the combination permits faster growth than either singly. The enzyme activities show that proline, as well as arginine, acts as an inducer of arginase and ornithine aminotransferase (as shown previously for B. lichenifornis [15]), although it does not repress omithine carbamoyltransferase. The terms "induce" and "repress" are only used here operationally, because we do not know which metabolite is active directly in the regulatory mechanism. A striking feature is that glutamine antagonized induction by arginine or proline, whereas ammonium antagonized induction by proline but not by arginine. Ornithine carbamoyltransferase remained repressed in arginine + glutamine cultures. Kinetics of repression experiments. Cultures were grown in MMM with arginine or proline as sole nitrogen source, and glutamine or

VOL. 137, 1979


ammonium was added in mid-exponential growth; samples were withdrawn both before and after addition of the second nitrogen source and assayed for arginase and ornithine aminotransferase (details above). Generation times decreased after addition of the second nitrogen source in accord with the generation times given in Table 2-only slightly for the arginine-grown cultures, and from 73 to 48 min (+glutamine) or 55 min (+ammonium) for the proline-grown cultures. Plots of enzyme activity per milliliter of culture against absorbance of culture (which was shown to be proportional to protein per milliliter of culture under the conditions used) are presented in Fig. 2. Control cultures and those where ammonium was added to an argininegrown culture showed a linear relationship of enzyme activity per milliliter of culture to absorbance. Addition of glutamine to an argininegrown culture or of either glutamine or ammo-

193

nium to a proline-grown culture resulted in greatly reduced or no further enzyme synthesis. These results therefore are in accord with those of Table 2. It is interesting that in all three cases of repression, whereas arginase activity continued to increase slowly for at least 1.5 generations after addition of glutamine or ammonium, ornithine aminotransferase activity showed no effect for about half a generation, increasing in parallel with the control, but then abruptly leveled off. Kinetics of induction experiments. Cultures are grown in MMM with glutamine or ammonium as the sole nitrogen source, and arginine or proline was added in mid-exponential growth; samples were withdrawn both before and after addition of the second nitrogen source and assayed for arginase (details above). Results are shown in Fig. 3. Addition of arginine to an ammonium-grown culture resulted in induction of arginase (although there is again a delay of 0.5 to 1.0 generations before any effect is noted); addition of arginine to a glutamine-grown culture or of proline to ammonium- or glutaminegrown cultures yielded no increase in rate of enzyme synthesis. Proline is still however capable of inducing arginase if present as sole nitrogen source. These results therefore are in accord with those of Table 2 and Fig. 2.

DISCUSSION Whereas the induced level of histidase is much lower when glucose rather than citrate is the sole carbon source, the same does not seem to

I

II.

be true of arginase or ornithine aminotransferase, which show at most a 2.7-fold reduction. a4 The simplest way of describing this result is that histidase, but not arginase or ornithine aminoa transferase, is subject to strong carbon source catabolite repression during rapid growth on glucose by comparison to slower growth on citrate. It must be borne in mind, of course, that there is as yet no reason to suppose that this a(02 phenomenon is mechanistically related to cyclic AMP-mediated catabolite repression as deCL? Oa. scribed in the enterobacteria (6) or Pseudomonas aeruginosa (28). This difference in conABSORBANCE (600nm) FIG. 2. Kinetics of repression of arginase and or- trol between histidase and the arginine enzymes, nithine aminotransferase by glutamine or ammo- first reported by Chasin and Magasanik (2), is nium. EMG50 was grown in MMM (containing cit- not easy to interpret. rate + glucose) with arginine (a,b) or proline (c,d) as The difference between B. subtilis and B. the sole nitrogen source. Glutamine or ammonium licheniformis with respect to the arginine catawas added as shown by the arrows. Samples were bolic enzymes (15), which in the latter organism withdrawn before and after addition of the second show glucose repression, can perhaps be related and assayed nitrogen source (as shown by arrows) for arginase (a,c) and ornithine aminotransferase to physiological differences between the two; in (b,d). Symbols: 0, arginine orproline as sole nitrogen particular B. licheniformis is a facultative anaerobe rather than a strict aerobe (8). It may be source; A, arginine or proline + ammonium; 0, arginine or proline + glutamine. Further details in the noted that the alternative catabolic pathway described by Broman et al. (1) for B. lichenifortext. d

04

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J. BACTERIOL.


I9

5

I-

3

-

a

l/

b

4-

2

/

;/'a /

-

3z/

-

/

LAJ

uAJ

,

X

1_

60

2 0

A 80

100

ABSORBANCE IKKn unks) FIG. 3. Kinetics of induction of arginase by glutamine or ammonium. EMG50 was grown in MMM (containing citrate + glucose) with glutamine or ammonium as the sole nitrogen source (except for b, [U] where proline was sole nitrogen source). Arginine (a) or proline (b) was added to some cultures as shown by the arrows. Samples were withdrawn before and after addition of the second nitrogen source and assayed for glutamine; 0, glutamine, then arginine or proline added at arrow; A, arginase. Nitrogen sources: ammonium; A, ammonium, then arginine or proline added at arrow; U, proline alone. Further details in the 0,

text.

mis is also strongly repressed during rapid growth on glucose (1, 16). This may reflect a role in generation of ATP under adverse conditions of energy provision, because ATP may be formed by the successive action of arginine deiminase, the catabolic ornithine carbamoyltransferase, and the reported carbamate kinase (12). The requirement for diminished oxygen availability for derepression (1) is also consistent with such a hypothesis. This pathway probably also occurs in B. subtilis (V. Stalon, personal communication). In B. subtilis 168, arginase and ornithine aminotransferase are markedly repressed by various nitrogen sources, in contrast to the rather minor effects of carbon sources. Glutamine antagonizes induction of these enzymes by either arginine or proline; ammonium only antagonizes induction by proline. These results correlate with growth rate on these substances as the sole nitrogen source: glutamine is a better nitrogen source by this criterion than arginine or proline, and ammonium is a better nitrogen source than proline but a worse one than arginine. The correlation could reflect growth rate-dependent levels of some nitrogen-containing metabolite with a central regulatory function, like cyclic 3',5'-AMP for carbon metabolism in E. coli; however, this is only speculation at present. The data presented here do not exclude transport phenomena from a role in repression; for instance, addition of glutamine to an arginine-grown culture could

result in diminished arginine uptake. That the level of ornithine carbamoyltransferase remains repressed in this case (Table 2), however, suggests that the intracellular arginine concentration remains appreciable. Further work is needed on this point. The significance of proline induction of arginase and ornithine aminotransferase is not clear. It may be that this has no adaptive value but reflects the molecular mechanism of induction of enzymes in the convergent arginine and proline catabolic pathways, many similar instances being found, for example, in the pseudomonads (3). It is possible, as suggested before (7), that A'-pyrroline 5-carboxylate or glutamic y-semialdehyde, at which the pathways converge, is the true inducer. From the kinetic experiments, the repressing effect of glutamine or ammonium on induced cultures (Fig. 2) and the inducing effect of arginine on noninduced culture' (Fig. 3) seem surprisingly delayed. It is again possible that uptake mechanisms are involved. The phenomenon resembles the delay in derepression, and in subsequent re-establishment of repression by arginine, of the biosynthetic enzyme N-acetylornithine 8-transaminase after arginine deprivation of a B. subtilis arginine auxotroph, as reported by Vogel and Vogel (31). The preference of B. subtilis for glutamine or arginine over ammonium as sole nitrogen source, as judged by growth rate, is in interesting con-

VOL. 137, 1979


trast to yeast (32) and Aspergillus (13), where ammonium seems to be preferred. The ecology of B. subtilis in soil (26, 27) suggests that it is adapted to the utilization therein of organic nitrogenous compounds. In yeast (32) and Aspergillus nidulans (24), it is again ammonium that represses enzymes involved in catabolism of nitrogenous compounds. However, in Neurospora crassa, it has recently been reported that glutamine represses the enzymes of arginine catabolism (23, 29). The apparent importance of glutamine in regulation of arginase and ornithine aminotransferase tempts speculation on the possible role of glutamine synthetase in the mechanisms of regulation of nitrogen source utilization enzymes. This has been clearly documented by Magasanik's group for Klebsiella aerogenes (21). In B. subtilis, it has recently been reported (5) that certain mutants altered in glutamine synthetase also appear to be altered in the regulation of synthesis of this enzyme itself. It has not so far been possible to show any alteration in control of arginine enzymes in these mutants (D. R. Dean, personal communication; C. R. Harwood,

unpublished data). ACKNOWLEDGMENTS We are grateful to J.-M. Wiame and V. Stalon for helpful discussion and permission to describe unpublished results. We also thank D. I. Vernon and M. Ward for excellent technical assistance. Part of this work was supported by an MRC project grant G/970/367/B to S. B. LITERATURE CITED 1. Broman, K., V. Stalon, and J.-M. Wiame. 1975. The duplication of arginine catabolism and the meaning of the two ornithine carbamoyltransferases in Bacillus licheniformis. Biochem. Biophys. Res. Commun. 66: 821-827. 2. Chasin, L. A., and B. Magasanik. 1968. Induction and repression of the histidine-degrading enzymes of Bacillus subtilis. J. Biol. Chem. 243:5165-5178. 3. Clarke, P. H., and L. N. Ornston. 1975. Metabolic pathways and regulation, p. 131-340. In P. H. Clarke and M. H. Richmond (ed.), Genetics and biochemistry of Pseudomonas. John Wiley & Sons, New York. 4. Clowes, R. C., and W. Hayes. 1968. Experiments in microbial genetics, p. 196. Blackwell Scientific Publications, Oxford. 5. Dean, D. R., J. A. Hoch, and A. I. Aronson. 1977. Alteration of the Bacillus subtilis glutamine synthetase results in overproduction of the enzyme. J. Bacteriol. 131:981-987. 6. de Crombrugghe, B., B. Chen, W. B. Anderson, M. E. Gottesman, R. L. Perlman, and I. Pastan. 1971. Role of cyclic adenosine 3',5'-monophosphate receptor protein in the initiation of lac transcription. J. Biol. Chem. 246:7343-7348. 7. De Hauwer, G., R. Lavall6, and J.-M. Wiame. 1964. etude de la pyrroline deshydrogenase et de la regulation du catabolisme de l'arginine et de la proline chez Bacillus subtilis. Biochim. Biophys. Acta 81:257-269. 8. Gibson, T., aud R. E. Gordon. 1974. Genus I. Bacillus,

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