Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jul;178(13):3978–3981. doi: 10.1128/jb.178.13.3978-3981.1996

A glutamate-dependent acid resistance gene in Escherichia coli.

B M Hersh 1, F T Farooq 1, D N Barstad 1, D L Blankenhorn 1, J L Slonczewski 1
PMCID: PMC232665  PMID: 8682809

Abstract

Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 isolates screened, 3 Tet(r) strains were identified as extremely acid sensitive (<0.1% survival at pH 2.5 for 2 h). Flanking sequences of the Tn10 inserts were amplified by inverse PCR. The sequences encoded a hydrophobic partial peptide of 88 residues. A random-primer-generated probe hybridized to Kohara clones 279 and 280 at 32 min (33.7 min on the revised genomic map EcoMap7) near gadB (encoding glutamate decarboxylase). The gene was designated xasA for extreme acid sensitive. xasA::Tn10 strains grown at pH 7 to 8 showed 100-fold-less survival in acid than the parent strain. Growth in mild acid (pH 5 to 6) restored acid resistance; anaerobiosis was not required, as it is for acid resistance in rpoS strains. xasA::Tn10 eliminated enhancement of acid resistance by glutamic acid. xasA was found to be a homolog of gadC recently sequenced in Shigella flexneri, in which it appears to encode a permease for the decarboxylated product of GadB. These results suggest that GadC (XasA) participates in a glutamate decarboxylase alkalinization cycle to protect E. coli from cytoplasmic acidification. The role of the glutamate cycle is particularly important for cultures grown at neutral pH before exposure to extreme acid.

Full Text

The Full Text of this article is available as a PDF (246.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Auger E. A., Redding K. E., Plumb T., Childs L. C., Meng S. Y., Bennett G. N. Construction of lac fusions to the inducible arginine- and lysine decarboxylase genes of Escherichia coli K12. Mol Microbiol. 1989 May;3(5):609–620. doi: 10.1111/j.1365-2958.1989.tb00208.x. [DOI] [PubMed] [Google Scholar]
  3. Barth M., Marschall C., Muffler A., Fischer D., Hengge-Aronis R. Role for the histone-like protein H-NS in growth phase-dependent and osmotic regulation of sigma S and many sigma S-dependent genes in Escherichia coli. J Bacteriol. 1995 Jun;177(12):3455–3464. doi: 10.1128/jb.177.12.3455-3464.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohannon D. E., Connell N., Keener J., Tormo A., Espinosa-Urgel M., Zambrano M. M., Kolter R. Stationary-phase-inducible "gearbox" promoters: differential effects of katF mutations and role of sigma 70. J Bacteriol. 1991 Jul;173(14):4482–4492. doi: 10.1128/jb.173.14.4482-4492.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Foster J. W., Bearson B. Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy. J Bacteriol. 1994 May;176(9):2596–2602. doi: 10.1128/jb.176.9.2596-2602.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Foster J. W., Hall H. K. Adaptive acidification tolerance response of Salmonella typhimurium. J Bacteriol. 1990 Feb;172(2):771–778. doi: 10.1128/jb.172.2.771-778.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Foster J. W., Hall H. K. Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium. J Bacteriol. 1991 Aug;173(16):5129–5135. doi: 10.1128/jb.173.16.5129-5135.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gorden J., Small P. L. Acid resistance in enteric bacteria. Infect Immun. 1993 Jan;61(1):364–367. doi: 10.1128/iai.61.1.364-367.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hall H. K., Karem K. L., Foster J. W. Molecular responses of microbes to environmental pH stress. Adv Microb Physiol. 1995;37:229–272. doi: 10.1016/s0065-2911(08)60147-2. [DOI] [PubMed] [Google Scholar]
  10. Halling S. M., Simons R. W., Way J. C., Walsh R. B., Kleckner N. DNA sequence organization of IS10-right of Tn10 and comparison with IS10-left. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2608–2612. doi: 10.1073/pnas.79.8.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kleckner N., Bender J., Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. doi: 10.1016/0076-6879(91)04009-d. [DOI] [PubMed] [Google Scholar]
  12. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  13. Kolter R., Siegele D. A., Tormo A. The stationary phase of the bacterial life cycle. Annu Rev Microbiol. 1993;47:855–874. doi: 10.1146/annurev.mi.47.100193.004231. [DOI] [PubMed] [Google Scholar]
  14. Lange R., Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol. 1991 Jan;5(1):49–59. doi: 10.1111/j.1365-2958.1991.tb01825.x. [DOI] [PubMed] [Google Scholar]
  15. Lee I. S., Slonczewski J. L., Foster J. W. A low-pH-inducible, stationary-phase acid tolerance response in Salmonella typhimurium. J Bacteriol. 1994 Mar;176(5):1422–1426. doi: 10.1128/jb.176.5.1422-1426.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lin J., Lee I. S., Frey J., Slonczewski J. L., Foster J. W. Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J Bacteriol. 1995 Jul;177(14):4097–4104. doi: 10.1128/jb.177.14.4097-4104.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McCann M. P., Kidwell J. P., Matin A. The putative sigma factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli. J Bacteriol. 1991 Jul;173(13):4188–4194. doi: 10.1128/jb.173.13.4188-4194.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mulvey M. R., Loewen P. C. Nucleotide sequence of katF of Escherichia coli suggests KatF protein is a novel sigma transcription factor. Nucleic Acids Res. 1989 Dec 11;17(23):9979–9991. doi: 10.1093/nar/17.23.9979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Médigue C., Bouché J. P., Hénaut A., Danchin A. Mapping of sequenced genes (700 kbp) in the restriction map of the Escherichia coli chromosome. Mol Microbiol. 1990 Feb;4(2):169–187. doi: 10.1111/j.1365-2958.1990.tb00585.x. [DOI] [PubMed] [Google Scholar]
  20. Rudd K. E., Miller W., Werner C., Ostell J., Tolstoshev C., Satterfield S. G. Mapping sequenced E.coli genes by computer: software, strategies and examples. Nucleic Acids Res. 1991 Feb 11;19(3):637–647. doi: 10.1093/nar/19.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Slonczewski J. L., Rosen B. P., Alger J. R., Macnab R. M. pH homeostasis in Escherichia coli: measurement by 31P nuclear magnetic resonance of methylphosphonate and phosphate. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6271–6275. doi: 10.1073/pnas.78.10.6271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Small P., Blankenhorn D., Welty D., Zinser E., Slonczewski J. L. Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. J Bacteriol. 1994 Mar;176(6):1729–1737. doi: 10.1128/jb.176.6.1729-1737.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Smith D. K., Kassam T., Singh B., Elliott J. F. Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci. J Bacteriol. 1992 Sep;174(18):5820–5826. doi: 10.1128/jb.174.18.5820-5826.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Triglia T., Peterson M. G., Kemp D. J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988 Aug 25;16(16):8186–8186. doi: 10.1093/nar/16.16.8186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Watson N., Dunyak D. S., Rosey E. L., Slonczewski J. L., Olson E. R. Identification of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH. J Bacteriol. 1992 Jan;174(2):530–540. doi: 10.1128/jb.174.2.530-540.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Way J. C., Davis M. A., Morisato D., Roberts D. E., Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984 Dec;32(3):369–379. doi: 10.1016/0378-1119(84)90012-x. [DOI] [PubMed] [Google Scholar]
  28. Yoshida T., Yamashino T., Ueguchi C., Mizuno T. Expression of the Escherichia coli dimorphic glutamic acid decarboxylases is regulated by the nucleoid protein H-NS. Biosci Biotechnol Biochem. 1993 Sep;57(9):1568–1569. doi: 10.1271/bbb.57.1568. [DOI] [PubMed] [Google Scholar]
  29. Zilberstein D., Agmon V., Schuldiner S., Padan E. Escherichia coli intracellular pH, membrane potential, and cell growth. J Bacteriol. 1984 Apr;158(1):246–252. doi: 10.1128/jb.158.1.246-252.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES