Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Apr;171(4):1915–1922. doi: 10.1128/jb.171.4.1915-1922.1989

Osmoregulation in Escherichia coli: complementation analysis and gene-protein relationships in the proU locus.

C S Dattananda 1, J Gowrishankar 1
PMCID: PMC209840  PMID: 2649478

Abstract

The proU locus in Escherichia coli encodes an osmotically inducible transport system for two substrates, glycine betaine and L-proline, whose intracellular accumulation represents an important component in the physiology of osmoregulation. Several osmoresponsive proU::lac mutants were isolated and tested for complementation with plasmids carrying different functional regions of proU. Three classes of mutations were identified which were physically mapped to distinct regions of DNA from this locus. Tn1000-insertion mutagenesis of cloned proU DNA also yielded three phenotypic classes of mutations whose physical distribution approximately corresponded with those of the chromosomal mutants above. Three proteins, of Mr 44,000, 35,000, and 33,000, were shown to be products of proU, and the last of these was localized to the periplasmic space. The data indicate that proU is an operon with three genes, designated in order proV, proW, and proX, encoding respectively the gene products above. All three genes were shown to be necessary for exhibition of the proU-mediated osmoprotective effects of both glycine betaine and L-proline in E. coli.

Full text

PDF
1915

Images in this article

Selected References

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

  1. Ames G. F. Bacterial periplasmic transport systems: structure, mechanism, and evolution. Annu Rev Biochem. 1986;55:397–425. doi: 10.1146/annurev.bi.55.070186.002145. [DOI] [PubMed] [Google Scholar]
  2. Ames G. F., Prody C., Kustu S. Simple, rapid, and quantitative release of periplasmic proteins by chloroform. J Bacteriol. 1984 Dec;160(3):1181–1183. doi: 10.1128/jb.160.3.1181-1183.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev. 1983 Jun;47(2):180–230. doi: 10.1128/mr.47.2.180-230.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barron A., Jung J. U., Villarejo M. Purification and characterization of a glycine betaine binding protein from Escherichia coli. J Biol Chem. 1987 Aug 25;262(24):11841–11846. [PubMed] [Google Scholar]
  5. Barron A., May G., Bremer E., Villarejo M. Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli. J Bacteriol. 1986 Aug;167(2):433–438. doi: 10.1128/jb.167.2.433-438.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bassford P., Beckwith J. Escherichia coli mutants accumulating the precursor of a secreted protein in the cytoplasm. Nature. 1979 Feb 15;277(5697):538–541. doi: 10.1038/277538a0. [DOI] [PubMed] [Google Scholar]
  7. Bremer E., Silhavy T. J., Weinstock G. M. Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli. J Bacteriol. 1985 Jun;162(3):1092–1099. doi: 10.1128/jb.162.3.1092-1099.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cairney J., Booth I. R., Higgins C. F. Osmoregulation of gene expression in Salmonella typhimurium: proU encodes an osmotically induced betaine transport system. J Bacteriol. 1985 Dec;164(3):1224–1232. doi: 10.1128/jb.164.3.1224-1232.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cairney J., Booth I. R., Higgins C. F. Salmonella typhimurium proP gene encodes a transport system for the osmoprotectant betaine. J Bacteriol. 1985 Dec;164(3):1218–1223. doi: 10.1128/jb.164.3.1218-1223.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Casadaban M. J., Chou J. In vivo formation of gene fusions encoding hybrid beta-galactosidase proteins in one step with a transposable Mu-lac transducing phage. Proc Natl Acad Sci U S A. 1984 Jan;81(2):535–539. doi: 10.1073/pnas.81.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  12. Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chumley F. G., Menzel R., Roth J. R. Hfr formation directed by tn10. Genetics. 1979 Apr;91(4):639–655. doi: 10.1093/genetics/91.4.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Csonka L. N. A third L-proline permease in Salmonella typhimurium which functions in media of elevated osmotic strength. J Bacteriol. 1982 Sep;151(3):1433–1443. doi: 10.1128/jb.151.3.1433-1443.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dunlap V. J., Csonka L. N. Osmotic regulation of L-proline transport in Salmonella typhimurium. J Bacteriol. 1985 Jul;163(1):296–304. doi: 10.1128/jb.163.1.296-304.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Faatz E., Middendorf A., Bremer E. Cloned structural genes for the osmotically regulated binding-protein-dependent glycine betaine transport system (ProU) of Escherichia coli K-12. Mol Microbiol. 1988 Mar;2(2):265–279. doi: 10.1111/j.1365-2958.1988.tb00028.x. [DOI] [PubMed] [Google Scholar]
  17. Giaever H. M., Styrvold O. B., Kaasen I., Strøm A. R. Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli. J Bacteriol. 1988 Jun;170(6):2841–2849. doi: 10.1128/jb.170.6.2841-2849.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gowrishankar J. Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation. J Bacteriol. 1985 Oct;164(1):434–445. doi: 10.1128/jb.164.1.434-445.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gowrishankar J., Jayashree P., Rajkumari K. Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance. J Bacteriol. 1986 Dec;168(3):1197–1204. doi: 10.1128/jb.168.3.1197-1204.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gowrishankar J. Nucleotide sequence of the osmoregulatory proU operon of Escherichia coli. J Bacteriol. 1989 Apr;171(4):1923–1931. doi: 10.1128/jb.171.4.1923-1931.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gowrishankar J., Pittard J. Molecular cloning of pheR in Escherichia coli K-12. J Bacteriol. 1982 Oct;152(1):1–6. doi: 10.1128/jb.152.1.1-6.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gowrishankar J. proP-mediated proline transport also plays a role in Escherichia coli osmoregulation. J Bacteriol. 1986 Apr;166(1):331–333. doi: 10.1128/jb.166.1.331-333.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Grothe S., Krogsrud R. L., McClellan D. J., Milner J. L., Wood J. M. Proline transport and osmotic stress response in Escherichia coli K-12. J Bacteriol. 1986 Apr;166(1):253–259. doi: 10.1128/jb.166.1.253-259.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  25. Higgins C. F., Sutherland L., Cairney J., Booth I. R. The osmotically regulated proU locus of Salmonella typhimurium encodes a periplasmic betaine-binding protein. J Gen Microbiol. 1987 Feb;133(2):305–310. doi: 10.1099/00221287-133-2-305. [DOI] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Landfald B., Strøm A. R. Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol. 1986 Mar;165(3):849–855. doi: 10.1128/jb.165.3.849-855.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Le Rudulier D., Strom A. R., Dandekar A. M., Smith L. T., Valentine R. C. Molecular biology of osmoregulation. Science. 1984 Jun 8;224(4653):1064–1068. doi: 10.1126/science.224.4653.1064. [DOI] [PubMed] [Google Scholar]
  29. May G., Faatz E., Villarejo M., Bremer E. Binding protein dependent transport of glycine betaine and its osmotic regulation in Escherichia coli K12. Mol Gen Genet. 1986 Nov;205(2):225–233. doi: 10.1007/BF00430432. [DOI] [PubMed] [Google Scholar]
  30. Measures J. C. Role of amino acids in osmoregulation of non-halophilic bacteria. Nature. 1975 Oct 2;257(5525):398–400. doi: 10.1038/257398a0. [DOI] [PubMed] [Google Scholar]
  31. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  32. Milner J. L., McClellan D. J., Wood J. M. Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12. J Gen Microbiol. 1987 Jul;133(7):1851–1860. doi: 10.1099/00221287-133-7-1851. [DOI] [PubMed] [Google Scholar]
  33. Nossal N. G., Heppel L. A. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem. 1966 Jul 10;241(13):3055–3062. [PubMed] [Google Scholar]
  34. Perroud B., Le Rudulier D. Glycine betaine transport in Escherichia coli: osmotic modulation. J Bacteriol. 1985 Jan;161(1):393–401. doi: 10.1128/jb.161.1.393-401.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yancey P. H., Clark M. E., Hand S. C., Bowlus R. D., Somero G. N. Living with water stress: evolution of osmolyte systems. Science. 1982 Sep 24;217(4566):1214–1222. doi: 10.1126/science.7112124. [DOI] [PubMed] [Google Scholar]

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

RESOURCES