Nitrogen Catabolite Repression of the L-Asparaginase of. Bacillus licheniformis. KENDRA J. GOLDEN AND ROBERT W. BERNLOHR*. Biochemistry Program ...
JOURNAL OF BACTERIOLOGY, Nov. 1985, p. 938-940
Vol. 164, No. 2
0021-9193/85/110938-03$02.00/0 Copyright C 1985, American Society for Microbiology
Nitrogen Catabolite Repression of the L-Asparaginase of Bacillus licheniformis KENDRA J. GOLDEN AND ROBERT W. BERNLOHR* Biochemistry Program, Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802 Received 17 June 1985/Accepted 22 August 1985 We report the presence of a single L-asparagine aminohydrolyase activity (EC 3.5.1.1) in extracts of BaciUus licheniformis AS. The synthesis of the enzyme was apparently under nitrogen catabolite repression control.
activity resulted in the formation of 1 nmol of aspartic acid per min from asparagine at 37°C. Protein concentrations were determined by the method of Kalb and Bernlohr (10). The enzyme exhibited one rather broad pH optimum centered at pH 7.3, and subsequent experiments were conducted at this pH. Mutants of B. licheniformis A5 that were readily isolated (9) were unable to grow on L-asparagine as a nitrogen source and were found to lack asparaginase. In assays for asparaginase activity in these mutants, it was found that activity was less than 1% of the wild-type level regardless of the nitrogen source on which the cells were grown. Thus, both induced and basal levels of activity were similarily affected. This result suggested that there is only one asparaginase enzyme present in B. licheniformis, in contrast to Escherichia coli, which produces two asparaginase enzymes (2-4, 6). Preliminary kinetic studies with crude extracts of the organism grown on L-proline and glucose yielded an apparent Km value of about 55 mM for L-asparagine. The low affinity of the enzyme for Lasparagine was expected, since it would serve to ensure that asparagine is hydrolyzed only when asparaginase is derepressed under nitrogen starvation conditions. To demonstrate nitrogen catabolite repression of the asparaginase, B. licheniformis A5 was grown in media containing various combinations of nitrogen and carbon sources. Table 1 shows the steady-state level of activity of asparaginase in extracts of cells grown in these media. It was found that when the medium contained a readily utilizable nitrogen source and nitrogen was not limiting, the level of asparaginase activity was low (see experiments 1 to 3, Table 1). L-Asparagine addition to the medium did not induce asparaginase activity under any circumstance, indicating that basal levels of asparaginase were capable of providing sufficient nitrogen for cellular growth (experiments 4 to 8, Table 1). Furthermore, nitrogen was not limiting when L-asparagine served as the sole nitrogen source, or even as the sole nitrogen and carbon source (experiments 5 and 6). Under nutritional conditions in which the nitrogen source was apparently the limiting factor for growth, the level of asparaginase activity increased by as much as 100-fold (experiments 9 to 13, Table 1). When the cells were grown on sodium L-glutamate as the sole nitrogen and carbon source, the conditions were apparently limiting for carbon, and enzyme levels were low (experiment 15). This was clearly indicated by the demonstration that ammonia addition did not increase growth rate or asparaginase levels (experiment 16). When glucose served as the carbon source and sodium L-glutamate served as the nitrogen source (experiment 17), growth was more rapid, and the enzyme was derepressed.
In gram-negative bacteria, yeasts, and fungi, a regulatory mechanism termed nitrogen catabolite repression exists (1, 8, 11-13, 18) in which organisms growing on a preferred nitrogen source repress the synthesis of unneeded catabolic enzymes. When a poor nitrogen source is present, however, catabolic enzymes are derepressed, providing the cell with ammonia. In gram-positive bacteria, examples of nitrogen catabolite-repressed enzymes are lacking (15, 17). Previously, this laboratory reported the presence of several nitrogen-metabolizing enzymes in Bacillus spp., including histidase, arginase, and alanine dehydrogenase. The cellular level of these enzymes was dependent on the presence of inducer, although each remained at induced levels in the presence or absence of ammonia, a preferred nitrogen source (16). The activities of the enzymes glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were found to depend on the nature of the nitrogen source present in the medium, but no apparent correlation between the level of activity and nitrogen source was observed (16). This laboratory has also reported the presence in Bacillus licheniformis A5 of L-glutaminase (5), which was induced in the presence of glutamine whether or not ammonia was present in the growth medium. It was concluded that the above enzymes were not under the control of nitrogen catabolite repression. The L-asparaginase of B. licheniformis, however, which catalyzes the formation of aspartate and ammonium ion from asparagine, is apparently controlled by a different mechanism than that of the previously reported enzymes. It is this enzyme and its regulation on which we report here. B. licheniformis A5 was grown in minimal A salts medium (7) with various carbon and nitrogen sources as indicated. Cultures were grown for at least 15 generations, with shaking at 37°C. Cells were harvested at midlogarithmic phase (determined turbidimetrically with a Klett-Summerson photoelectric colorimeter) by centrifugation at 10,000 x g for 15 min. After two washings with 100 mM Tris hydrochloride (pH 8.4) containing 1 M KCl, the cells were suspended in 10 mM Tris hydrochloride (pH 8.4) containing 10 mM mercaptoethanol, broken by sonic oscillation (Biosonik IV; Bronwill) for a total of 5 min, and centrifuged (20 min at 40,000 x g). The supernatant solution was decanted to yield an extract containing approximately 8 to 10 mg of protein per ml. Asparaginase activity was assayed by measuring [14C]aspartic acid production from [14C]asparagine by the method of Prusiner and Milner (14), except that Tris phosphate (pH 7.3) replaced Tris hydrochloride. A total of 1 U of *
Corresponding author. 938
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NOTES
TABLE 1. Steady-state level of asparaginase in cells grown on various nitrogen sourcesa Glucose Expt
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16 17 18
19 20 21 22
Nitrogen source (mM)
Urea(10) Casamino Acids (0.6%) Ammonium sulfate (10) Ammonium sulfate (10) plus L-asparagine (20) L-Asparagine (20) L-Asparagine (34) L-Asparagine (34) plus ammonium sulfate (10) L-Asparagine (34) plus sodium sulfate (17) L-Proline (20) L-Proline (20) plus ammonium sulfate (10) Potassium nitrate (20) L-Alanine (20) L-Ormithine (20) L-Arginine (20) Sodium L-glutamate (34) Sodium L-glutamate (34) plus ammonium sulfate
(17)
Sodium L-glutamate (20) Sodium L-glutamate (20) plus ammonium sulfate (10) Sodium L-aspartate (34) Sodium L-aspartate (34) plus ammonium sulfate (10) Sodium L-aspartate (20) Sodium L-aspartate (20) plus ammonium sulfate (10)
presen
Doubling Asparaginase time (U/mg of
mM)
(min)
+
54 51 53 54
protein) 6 ± 0.2 12 ± 2 14 ± 6 18 ± 5
-
54 57 65
22 ± 4 13 ± 0.4 12 ± 0.3
-
69
7± 2
+
66 49
354 ± 41 15 ± 1
+ +
+
+
301 268 185 126 3 4
-
84 116 80 57 216 231
+ +
87 48
227 ± 16 2 ± 0.7
-
315 225
9 ± 0.4 13 ± 1
+ +
66 50
243 ± 11 5 ± 0.9
+ + + +
± ± ± ± ± ±
71 50 39 35 1 1
a Cells were harvested one doubling time before the beginning of the stationary phase. Doubling times may vary (±8%). Data are averages from duplicate assays on at least two separate cultures.
Under this condition, nitrogen was limiting for growth. When ammonia was added to a growth medium containing glucose and sodium L-glutamate (experiment 18), however, nitrogen was not limiting, and the asparaginase level was low. The same phenomenon occurred with sodium Laspartate (experiments 19 to 22). Asparaginase was derepressed only when glucose was present as the carbon source and sodium L-aspartate became nitrogen limiting for growth. Finally, when B. licheniformis AS was grown on L-proline and glucose, asparaginase activity was derepressed (experiments 9 and 10, Table 1). Addition of ammonium sulfate to the same medium strongly repressed synthesis. This confirmed that nitrogen depletion allowed derepression of the enzyme, and corroborated the evidence for nitrogen catabolite repression control of the synthesis of this enzyme. The regulation of the asparaginase enzyme of B. licheniformis is of particular interest in terms of nitrogen metabolism in gram-positive bacteria. Nitrogen catabolite repression refers to the ability of microorganisms to bypass the synthesis of unneeded nitrogen-metabolizing enzymes when the organism is growing efficiently on an easily utilized nitrogen source. Nitrogen catabolic enzymes are derepressed only when a readily metabolized nitrogen source is
939
unavailable. Although this phenomenon has been documented in gram-negative organisms, yeasts, and fungi (1, 8, 11-13, 18), it has not been clearly demonstrated in grampositive bacteria. The asparaginase from B. licheniformis reported here is regulated in a manner resembling nitrogencatabolic enzymes in gram-negative bacteria and in fact appears to be an example of nitrogen catabolite repression in a gram-positive organism. This form of nitrogen-dependent control is inducer independent. As such, it would serve as an example of gratuitous derepression control. It may be expected that other nitrogencatabolic enzymes would be regulated in a similar manner. This form of control is energetically inefficient, however, and the inducer-dependent mechanism (seen for histidase, arginase, etc.; 16) would be preferred. Consequently, the role of this type of nitrogen catabolite repression in metabolism or sporulation in Bacillus spp. is not evident at this time. This research was supported in part by grant PCM 8121902 from the National Science Foundation. We thank Alison Depto for the isolation of mutants. LITERATURE CITED 1. Brenchley, J. E., D. M. Bedweil, S. M. Dendinger, and J. M. Kuchta. 1980. Analysis of mutations affecting the regulation of nitrogen utilization in Salmonella typhimurium, p. 79-93. In J. Mora and R. Palacios (ed.), Glutamine: metabolism, enzymology, and regulation. Academic Press, Inc., New York. 2. Campbell, H. A., L. T. Mashburn, E. A. Boyse, and L. J. Old. 1967. Two L-asparaginases from Escherichia coli B. Their separation, purification, and antitumor activity. Biochemistry 6:721-730. 3. Cedar, H., and J. H. Schwartz. 1967. Localization of two L-asparaginases in anaerobically grown Escherichia coli. J. Biol. Chem. 242:3753-3755. 4. Cedar, H., and J. H. Schwartz. 1968. Production of Lasparaginase II by Escherichia coli. J. Bacteriol. 96:2043-2048. 5. Cook, W. R., J. H. Hoffman, and R. W. Bernlohr. 1981. Occurrence of an inducible glutaminase in Bacillus licheniformis. J. Bacteriol. 148:365-367. 6. Del Casale, T., P. Sollitti, and R. H. Chesney. 1983. Cytoplasmic L-asparaginase: isolation of a defective strain and mapping of ansA. J. Bacteriol. 154:513-515. 7. Donohue, T. J., and R. W. Bernlohr. 1978. Effect of cultural conditions on the concentrations of metabolic intermediates during growth and sporulation of Bacillus licheniformis. J. Bacteriol. 135:363-372. 8. Dunlop, P. C., G. M. Meyer, and R. J. Roon. 1980. Nitrogen catabolite represssion of asparaginase II in Saccharomyces cerevisiae. J. Bacteriol. 143:422-426. 9. Fields, P. I., H. J. Schreier, A. L. Saha, and R. W. Bernlohr. 1984. Use of the 13-lactamase inhibitor clavulanic acid in the isolation of auxotrophic mutants of Bacillus licheniformis. J. Bacteriol. 159:803-804. 10. KaIb, V. F., and R. W. Bernlohr. 1977. A new spectrophotometric assay for protein in cell extracts. Anal. Biochem. 82:362-371. 11. Marzluf, G. A. 1981. Regulation of nitrogen metabolism and gene expression in fungi. Microbiol. Rev. 45:437-461. 12. Meile, L., L. Soldati, and T. Leisinger. 1982. Regulation of proline catabolism in Pseudomonas aeruginosa PAO. Arch. Microbiol. 132:189-193. 13. O'Reilly, T., and D. F. Day. 1983. Effects of cultural conditions on protease production by Aeromonas hydrophila. Appl. Environ. Microbiol. 45:1132-1135. 14. Prusiner, S., and L. Milner. 1970. A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase, and asparaginase. Anal. Biochem. 37:429-438. 15. Schreier, H. J., S. H. Fisher, and A. L. Sonenshein. 1985.
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NOTES
Regulation of expression from the ginA promoter of Bacillus subtilis requires the glnA gene product. Proc. Natl. Acad. Sci. USA 82:3375-3379. 16. Schreier, H. J., T. M. Smith, and R. W. Bernlohr. 1982. Regulation of nitrogen catabolic enzymes in Bacillus spp. J. Bacteriol. 151:971-975.
J. BACTERIOL.
17. Sonenshein, A. L. 1985. Recent progress in metabolic regulation of sporulation, p. 185-193. In J. A. Hoch and P. Setlow (ed.), Molecular biology of microbial differentiation. American Society for Microbiology, Washington, D.C. 18. Tyler, B. 1978. Regulation of the assimilation of nitrogen compounds. Annu. Rev. Biochem. 47:1127-1162.
