Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fraley, C. D. Articles by Matin, A. Search for Related Content PubMed PubMed Citation Articles by Fraley, C. D. Articles by Matin, A.

 Previous Article  |  Next Article 

Journal of Bacteriology, August 1998, p. 4287-4290, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The Escherichia coli Starvation Gene cstC Is Involved in Amino Acid Catabolism

C. D. Fraley, J. H. Kim, M. P. McCann,^§ and A. Matin^*

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305

Received 14 January 1998/Accepted 3 June 1998

	ABSTRACT

Top Abstract Text References

Escherichia coli strains mutant in the starvation gene cstC grow normally in a mineral salts medium but are impaired in utilizing amino acids as nitrogen sources. They are also compromised in starvation survival, where amino acid catabolism is important. The cstC gene encodes a 406-amino-acid protein that closely resembles the E. coli ArgD protein, which is involved in arginine biosynthesis. We postulate that CstC is a counterpart of ArgD in an amino acid catabolic pathway. The cstC upstream region contains several regulatory consensus sequences. Both ^S and ⁵⁴ promoters are probably involved in cstC transcription and appear to compete with each other, presumably to match cstC expression to the cellular amino acid catabolic needs.

	TEXT

Top Abstract Text References

Escherichia coli differentiates into a resistant cellular state in response to starvation due to the expression of 30 to 80 starvation genes (12-14). We report here on the role of an E. coli starvation gene, cstC (map position, 38.2 min) that we described previously (2, 9).

Bacterial strains and plasmids used are listed in Table 1. Cultures were grown in Luria-Bertani broth or in M9 supplemented with D-glucose as described previously (10). All experiments were done at least twice.

View this table: [in this window] [in a new window]   TABLE 1.   E. coli strains and plasmids used in this study

cstC is involved in amino acid catabolism. The cstC-lacZ transcriptional fusion strain, AMS96, demonstrated wild-type growth in LB or glucose-M9 medium, but it was impaired in using amino acids as sole nitrogen sources. While the wild type had doubling times of 4.5 h with L-ornithine and 7.5 h with N--acetyl-L-ornithine or L-arginine as nitrogen source, the doubling time of the mutant in glucose-L-ornithine medium was 23 h, and it did not grow with L-arginine as nitrogen source. The mutant was also impaired in starvation survival, where amino acid catabolism is important (3): at 125 h after the exhaustion of ammonium from glucose-M9 medium, the wild-type culture showed 60% viability, but AMS96 showed only 4% viability.

To further explore if the cstC gene in fact had a role in amino acid catabolism, the gene and contiguous region (Fig. 1) were cloned from the Kohara E. coli miniset collection, using 1.6-kb PstI-BglII ³²P-labeled fragment originally from pAMC3 (2) as probe. The desired DNA was obtained from phage 328 and cloned into pBluescript II KS(+), generating pAMC162. Sequencing of the PstI and XmnI region (Fig. 1) showed that the 5' end of the cloned fragment corresponded to nucleotide (nt) 99 of the xthA gene (19), which is transcribed divergently to cstC. A putative open reading frame (ORF) spanning nt 418 to 1635 (Fig. 1) exhibited a strong DNA homology to E. coli N--acetylornithine--aminotransferase, the product of the argD gene (map position, 75.1 min). The derived amino acid sequence of the cstC ORF revealed a protein of 406 amino acids which, when shifted three residues relative to ArgD, exhibited ca. 60% identity and 91% similarity to the latter (Fig. 2). ArgD belongs to class III of the pyridoxal phosphate (PLP)-dependent aminotransferases; its putative cofactor binding site occurs at Lys²⁵⁵ (8). CstC also contains a lysine at this position in the homology alignment (Fig. 2).

View larger version (6K): [in this window] [in a new window]   FIG. 1.   The PstI-EcoRI fragment containing the cstC gene cloned from Kohara phage 328 in pAMC162. The diagram includes information derived from work discussed in the text (the cstC start codon, the site of kan cassette insertion in AMS349 [Table 1], the Mu dX fusion joint, and the cstC translational stop codon). Numbering is in relation to the cstC sequence deposited in GenBank (accession no. U90416); the PstI site is 127 nt upstream of the first nucleotide of this sequence.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   FASTA (16) amino acid sequence alignment of the putative cstC ORF with ArgD. Boxes indicate identical residues; shaded residues indicate conservative substitutions. Note that relative to ArgD, the cstC ORF is displaced three residues to the right. The asterisk over the lysines at positions 252 and 255 of the cstC ORF and ArgD, respectively, denotes the putative PLP binding site.

Phage Mu dX had inserted in strain AMS35 (and AMS96) near the 3' end of the cstC gene (Fig. 1). As this may not have generated a complete loss-of-function mutation, an additional mutation was constructed by inserting a kanamycin (kan) cassette just after Ala⁹⁶ in the CstC polypeptide (Fig. 2), as described previously (6); the Ala⁹⁶ region is vital for the function of amino acid aminotransferases, being involved in subunit dimerization, and thus active-site formation, as well as PLP binding (5). The resulting strain, AMS349, exhibited a phenotype similar to that of AMS96. We thus assume that both AMS35 and AMS349 are loss-of-function mutants.

As opposed to the role for CstC in amino acid catabolism as suggested by the above experiments, ArgD is involved in arginine biosynthesis. However, many closely related enzymes carry out similar biochemical reactions but with equilibria favoring opposite directions, and given the phenotype of the cstC mutants, we hypothesize that CstC may be a counterpart of ArgD in a catabolic pathway for amino acids. Indeed, the E. coli genome sequence in the cstC region (1), as well as biochemical studies presented in an accompanying report (20) indicate that cstC (astC [20]) is the first gene in a five-gene operon (astCADBE). This operon encodes the ammonium-producing arginine succinyltransferase (AST) pathway, which probably catabolizes arginine and other amino acids.

The cstC upstream region contains several regulatory sequences. Computer analyses revealed several readily recognizable consensus sequences upstream of the cstC ORF (Fig. 3): two each for cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) and NR_I binding sites, one for an integration host factor binding site, and consensus sequences for three promoters, ⁷⁰, ^S, and ⁵⁴. The putative carbon and ammonium starvation regulatory sites overlap, with the ^S promoter residing within the ⁵⁴ promoter and the proximal cAMP-CRP site (nt 107 to 123) located within the two NR_I sites.

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Sequence of the upstream cstC regulatory region from nt 1 of the deposited sequence to the putative start codon. Letters above or below indicate deviations from the consensus sequences, under- or overscores can be any nucleotide, and X denotes where a base should be deleted to obtain the consensus sequence. Abbreviations for the putative sites: CAP/CRP, cAMP-CRP consensus sequence (4); IHF, integration host factor (7); NRI, nitrogen regulatory protein binding site (11); RpoN, 54 promoter region (17); RpoS, 10 region of the S promoter region (24); 10 and 35, E70 promoter sequences (18); S.D., Shine-Dalgarno sequence (22).

We used induction of the cst-lacZ fusion in appropriate mutant backgrounds to assess the roles of ^S and ⁵⁴ in cstC expression. Under ammonium-sufficient growth conditions (i.e., with NH₄⁺ as nitrogen source), cstC was positively regulated by ^S, as its expression decreased about twofold in an rpoS strain (AMS352) (Table 2), but its expression was not negatively affected in an rpoN strain (AMS351). If anything, the presence of ⁵⁴ in the cells attenuated expression: -galactosidase production was moderately but reproducibly lower in AMS351. Replacing NH₄⁺ with one of several amino acids as nitrogen source (Table 2), thereby generating ammonium-limited conditions, induced cstC expression, but the roles of the two sigma factors were reversed, with ⁵⁴ acting as the positive regulator and ^S attenuating expression. Thus, strain AMS351 showed a 3-fold decrease whereas AMS352 showed a 2.5-fold increase in -galactosidase production under these conditions (Table 2). Qualitatively similar results were obtained during total ammonium starvation (data not shown).

View this table: [in this window] [in a new window]   TABLE 2.   cstC-lacZ expression in different backgrounds during ammonium-sufficient and ammonium-limited conditions

As amino acids are a valuable cellular resource, especially under starvation conditions, a pathway like the AST pathway must be carefully regulated, and the complex regulatory region upstream of the cstC gene shows that this is indeed the case. Primer extension start site analysis showed that both ⁵⁴ and ^S promoters are used in cstC transcription (6a), and the fusion studies discussed above show that when one of these two promoters becomes dominant under a given condition, the other assumes an attenuating role. This competition may be designed to accurately match the expression of the AST pathway to the condition-specific needs of the cell for amino acid catabolism.

	ACKNOWLEDGMENTS

We thank R. Eakins and L. Kozar for help with sequencing and computer analyses, respectively, B. Cormack and K. Wilson for advice, L. Reitzer for providing bacterial strains, and Sara White for expert secretarial help.

This work was supported by grants from the National Science Foundation (ECE86-13227 and DCB-9207101), National Institutes of Health (GM 42159), U.S. EPA (R823390), and Western Region Hazardous Substance Research Center (to A.M.), and ALCOA (to C.D.F.).

	FOOTNOTES

^* Corresponding author. Mailing address: D317 Sherman Fairchild Science Building, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305-5402. Phone: (650) 725-4745. Fax: (650) 725-6757. E-mail: a.matin{at}forsythe.stanford.edu.

Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307.

Present address: Department of Food Science & Technology, College of Agriculture, Gyeongsang National University, Chinju, Korea 660-701.

^§ Department of Biology, St. Joseph's University, Philadelphia, PA 19131.

	REFERENCES

Top Abstract Text References

1.	Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The completed genome sequence of Escherichia coli K-12. Science 277:1453-1474[Abstract/Free Full Text].
2.	Blum, P. H., S. B. Jovanovich, M. P. McCann, J. E. Schultz, S. A. Lesley, R. R. Burgess, and A. Matin. 1990. Cloning and in vivo and in vitro regulation of cyclic AMP-dependent carbon starvation genes from Escherichia coli. J. Bacteriol. 172:3813-3820[Abstract/Free Full Text].
3.	Bockman, A. T., C. A. Reeve, and A. Matin. 1986. Stabilization of glucose-starved Escherichia coli K-12 and Salmonella typhimurium LT2 by peptidase-deficient mutants. J. Gen. Microbiol. 132:231-235[Abstract/Free Full Text].
4.	Busby, S. J. W. 1986. Positive regulation in gene expression. Symp. Soc. Gen. Microbiol. 39:51-77.
5.	Danishefsky, A. T., J. J. Onnufer, G. A. Petsko, and D. Ringe. 1991. Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase. Biochemistry 30:1980-1985[Medline].
6.	Fraley, C. D. 1998. Ph.D. dissertation. Stanford University, Stanford, Calif.
6a.	Fraley, C. D., and A. Matin. Unpublished data.
7.	Friedman, D. I. 1988. Integration host factor: a protein for all reasons. Cell 55:545-554[Medline].
8.	Heimberg, H., A. Boyen, M. Crabeel, and N. Glansdorff. 1990. Escherichia coli and Saccharomyces cerevisiae acetylornithine aminotransferases: evolutionary relationship with ornithine aminotransferases. Gene 90:69-78[Medline].
9.	Kim, J. H., E. Auger, R. Soly, and A. Matin. 1993. E. coli starvation genes: cloning and analysis of pexA, abstr. I-52. In Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993. American Society for Microbiology, Washington, D.C.
10.	Lomovskaya, O. L., J. P. Kidwell, and A. Matin. 1994. Characterization of the 38-dependent expression of a core Escherichia coli gene, pexB. J. Bacteriol. 176:3928-3935[Abstract/Free Full Text].
11.	Magasanik, B. 1996. Regulation of nitrogen utilization, p. 1344-1356. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 1. ASM Press, Washington, D.C.
12.	Matin, A., E. A. Auger, P. H. Blum, and J. E. Schultz. 1989. Genetic basis of starvation survival in nondifferentiating bacteria. Annu. Rev. Microbiol. 43:293-316[Medline].
13.	Matin, A. 1996. Role of alternate sigma factors in starvation protein synthesisnovel mechanisms of catabolite repression, p. 494-505. In 14th Forum in Microbiology.
14.	Matin, A., M. Baetens, S. Pandza, C.-H. Park, and S. Waggoner. Survival strategies in the stationary phase. In E. Rosenberg (ed.), Microbial ecology and infectious disease, in press. American Society for Microbiology, Washington, D.C.
15.	McCann, M. P., J. P. Kidwell, and A. Matin. 1991. The putative  factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli. J. Bacteriol. 173:4188-4194[Abstract/Free Full Text].
16.	Pearson, W. R., and D. J. Lipman. 1988. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85:2444-2448[Abstract/Free Full Text].
17.	Reitzer, L. J., and B. Magasanik. 1985. Expression of glnA in Escherichia coli is regulated at tandem promoters. Proc. Natl. Acad. Sci. USA 82:1979-1983[Abstract/Free Full Text].
18.	Reznikoff, W. S., and W. R. McClure. 1986. E. coli promoters, p. 1-33. In W. Reznikoff, and L. Gold (ed.), Maximizing gene expression. Butterworth Publishers, Stoneham, Mass.
19.	Saporito, S. M., B. J. Smith-White, and R. P. Cunningham. 1988. Nucleotide sequence of the xth gene of Escherichia coli K-12. J. Bacteriol. 170:4542-4547[Abstract/Free Full Text].
20.	Schneider, B. L., A. K. Kiupakis, and L. J. Reitzer. 1998. Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coli. J. Bacteriol. 180:4278-4286[Abstract/Free Full Text].
21.	Schultz, J. E., G. I. Latter, and A. Matin. 1988. Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli. J. Bacteriol. 170:3903-3909[Abstract/Free Full Text].
22.	Stormo, G. D. 1986. Translation initiation, p. 195-224. In W. Reznikoff, and L. Gold (ed.), Maximizing gene expression. Butterworth Publishers, Stoneham, Mass.
23.	Ueno-Nishio, S., K. C. Backman, and B. Magasanik. 1983. Regulation at the glnL-operator-promoter of the complex glnAG operon of Escherichia coli. J. Bacteriol. 153:1247-1251[Abstract/Free Full Text].
24.	von Ossowski, I., M. R. Mulvey, P. A. Leco, A. Borys, and P. C. Loewen. 1991. Nucleotide sequence of Escherichia coli katE, which encodes catalase HPII. J. Bacteriol. 173:514-520[Abstract/Free Full Text].

---

Journal of Bacteriology, August 1998, p. 4287-4290, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Tocilj, A., Schrag, J. D., Li, Y., Schneider, B. L., Reitzer, L., Matte, A., Cygler, M. (2005). Crystal Structure of N-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway of Escherichia coli. J. Biol. Chem. 280: 15800-15808 [Abstract] [Full Text]  
• Vijayakumar, S. R. V., Kirchhof, M. G., Patten, C. L., Schellhorn, H. E. (2004). RpoS-Regulated Genes of Escherichia coli Identified by Random lacZ Fusion Mutagenesis. J. Bacteriol. 186: 8499-8507 [Abstract] [Full Text]  
• Kiupakis, A. K., Reitzer, L. (2002). ArgR-Independent Induction and ArgR-Dependent Superinduction of the astCADBE Operon in Escherichia coli. J. Bacteriol. 184: 2940-2950 [Abstract] [Full Text]  
• Oh, M.-K., Rohlin, L., Kao, K. C., Liao, J. C. (2002). Global Expression Profiling of Acetate-grown Escherichia coli. J. Biol. Chem. 277: 13175-13183 [Abstract] [Full Text]  
• Reitzer, L., Schneider, B. L. (2001). Metabolic Context and Possible Physiological Themes of {sigma}54-Dependent Genes in Escherichia coli. Microbiol. Mol. Biol. Rev. 65: 422-444 [Abstract] [Full Text]  
• Zimmer, D. P., Soupene, E., Lee, H. L., Wendisch, V. F., Khodursky, A. B., Peter, B. J., Bender, R. A., Kustu, S. (2000). Nitrogen regulatory protein C-controlled genes of Escherichia coli: Scavenging as a defense against nitrogen limitation. Proc. Natl. Acad. Sci. USA 97: 14674-14679 [Abstract] [Full Text]  
• Lu, C.-D., Abdelal, A. T. (1999). Role of ArgR in Activation of the ast Operon, Encoding Enzymes of the Arginine Succinyltransferase Pathway in Salmonella typhimurium. J. Bacteriol. 181: 1934-1938 [Abstract] [Full Text]  
• Schneider, B. L., Kiupakis, A. K., Reitzer, L. J. (1998). Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli. J. Bacteriol. 180: 4278-4286 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fraley, C. D. Articles by Matin, A. Search for Related Content PubMed PubMed Citation Articles by Fraley, C. D. Articles by Matin, A.