Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Jun;176(12):3638–3645. doi: 10.1128/jb.176.12.3638-3645.1994

The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation.

J Mellies 1, R Brems 1, M Villarejo 1
PMCID: PMC205553  PMID: 8206842

Abstract

The proU operon of Escherichia coli encodes a high-affinity glycine betaine transport system which is osmotically inducible and enables the organism to recover from the deleterious effects of hyperosmotic shock. Regulation occurs at the transcriptional level. KMnO4 footprinting showed that the preponderance of transcription initiated at a single primary promoter region and that proU transcription activation did not occur differentially at alternate promoters in response to various levels of salt shock. Mutational analysis confirmed the location of the primary promoter and identified an extended -10 region required for promoter activity. Specific nucleotides within the spacer, between position -10 and position -35, were important for maximal expression, but every mutant which retained transcriptional activity remained responsive to osmotic signals. A chromosomal 90-bp minimal promoter fragment fused to lacZ was not significantly osmotically inducible. However, transcription from this fragment was resistant to inhibition by salt shock. A mutation in osmZ, which encodes the DNA-binding protein H-NS, derepressed wild-type proU expression by sevenfold but did not alter expression from the minimal promoter. The current data support a model in which the role of the proU promoter is to function efficiently at high ionic strength while other cis-acting elements receive and respond to the osmotic signal.

Full text

PDF
3639

Images in this article

3641Image
on p.3641

Selected References

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

  1. Barr G. C., Ni Bhriain N., Dorman C. J. Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase. J Bacteriol. 1992 Feb;174(3):998–1006. doi: 10.1128/jb.174.3.998-1006.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Carpousis A. J., Gralla J. D. Interaction of RNA polymerase with lacUV5 promoter DNA during mRNA initiation and elongation. Footprinting, methylation, and rifampicin-sensitivity changes accompanying transcription initiation. J Mol Biol. 1985 May 25;183(2):165–177. doi: 10.1016/0022-2836(85)90210-4. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Chan B., Busby S. Recognition of nucleotide sequences at the Escherichia coli galactose operon P1 promoter by RNA polymerase. Gene. 1989 Dec 14;84(2):227–236. doi: 10.1016/0378-1119(89)90496-4. [DOI] [PubMed] [Google Scholar]
  7. Clark J. M., Beardsley G. P. Functional effects of cis-thymine glycol lesions on DNA synthesis in vitro. Biochemistry. 1987 Aug 25;26(17):5398–5403. doi: 10.1021/bi00391a027. [DOI] [PubMed] [Google Scholar]
  8. Culham D. E., Lasby B., Marangoni A. G., Milner J. L., Steer B. A., van Nues R. W., Wood J. M. Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP. J Mol Biol. 1993 Jan 5;229(1):268–276. doi: 10.1006/jmbi.1993.1030. [DOI] [PubMed] [Google Scholar]
  9. Dattananda C. S., Rajkumari K., Gowrishankar J. Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene. J Bacteriol. 1991 Dec;173(23):7481–7490. doi: 10.1128/jb.173.23.7481-7490.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Defez R., De Felice M. Cryptic operon for beta-glucoside metabolism in Escherichia coli K12: genetic evidence for a regulatory protein. Genetics. 1981 Jan;97(1):11–25. doi: 10.1093/genetics/97.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Diderichsen B. cur-1, a mutation affecting a phenotype of sup+ strains of Escherichia coli. Mol Gen Genet. 1980;180(2):425–428. doi: 10.1007/BF00425858. [DOI] [PubMed] [Google Scholar]
  12. Dinnbier U., Limpinsel E., Schmid R., Bakker E. P. Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations. Arch Microbiol. 1988;150(4):348–357. doi: 10.1007/BF00408306. [DOI] [PubMed] [Google Scholar]
  13. Druger-Liotta J., Prange V. J., Overdier D. G., Csonka L. N. Selection of mutations that alter the osmotic control of transcription of the Salmonella typhimurium proU operon. J Bacteriol. 1987 Jun;169(6):2449–2459. doi: 10.1128/jb.169.6.2449-2459.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elliott T. A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon. J Bacteriol. 1992 Jan;174(1):245–253. doi: 10.1128/jb.174.1.245-253.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Göransson M., Sondén B., Nilsson P., Dagberg B., Forsman K., Emanuelsson K., Uhlin B. E. Transcriptional silencing and thermoregulation of gene expression in Escherichia coli. Nature. 1990 Apr 12;344(6267):682–685. doi: 10.1038/344682a0. [DOI] [PubMed] [Google Scholar]
  17. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hayatsu H., Ukita T. The selective degradation of pyrimidines in nucleic acids by permanganate oxidation. Biochem Biophys Res Commun. 1967 Nov 30;29(4):556–561. doi: 10.1016/0006-291x(67)90521-9. [DOI] [PubMed] [Google Scholar]
  20. Higgins C. F., Dorman C. J., Stirling D. A., Waddell L., Booth I. R., May G., Bremer E. A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli. Cell. 1988 Feb 26;52(4):569–584. doi: 10.1016/0092-8674(88)90470-9. [DOI] [PubMed] [Google Scholar]
  21. Ide H., Kow Y. W., Wallace S. S. Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro. Nucleic Acids Res. 1985 Nov 25;13(22):8035–8052. doi: 10.1093/nar/13.22.8035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Keilty S., Rosenberg M. Constitutive function of a positively regulated promoter reveals new sequences essential for activity. J Biol Chem. 1987 May 5;262(13):6389–6395. [PubMed] [Google Scholar]
  23. Kumar A., Malloch R. A., Fujita N., Smillie D. A., Ishihama A., Hayward R. S. The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an "extended minus 10" promoter. J Mol Biol. 1993 Jul 20;232(2):406–418. doi: 10.1006/jmbi.1993.1400. [DOI] [PubMed] [Google Scholar]
  24. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  26. Laimins L. A., Rhoads D. B., Epstein W. Osmotic control of kdp operon expression in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Jan;78(1):464–468. doi: 10.1073/pnas.78.1.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Lucht J. M., Bremer E. Characterization of mutations affecting the osmoregulated proU promoter of Escherichia coli and identification of 5' sequences required for high-level expression. J Bacteriol. 1991 Jan;173(2):801–809. doi: 10.1128/jb.173.2.801-809.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. May G., Faatz E., Lucht J. M., Haardt M., Bolliger M., Bremer E. Characterization of the osmoregulated Escherichia coli proU promoter and identification of ProV as a membrane-associated protein. Mol Microbiol. 1989 Nov;3(11):1521–1531. doi: 10.1111/j.1365-2958.1989.tb00138.x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Milner J. L., Grothe S., Wood J. M. Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12. J Biol Chem. 1988 Oct 15;263(29):14900–14905. [PubMed] [Google Scholar]
  32. Overdier D. G., Csonka L. N. A transcriptional silencer downstream of the promoter in the osmotically controlled proU operon of Salmonella typhimurium. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3140–3144. doi: 10.1073/pnas.89.7.3140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Owen-Hughes T. A., Pavitt G. D., Santos D. S., Sidebotham J. M., Hulton C. S., Hinton J. C., Higgins C. F. The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression. Cell. 1992 Oct 16;71(2):255–265. doi: 10.1016/0092-8674(92)90354-f. [DOI] [PubMed] [Google Scholar]
  34. Prince W. S., Villarejo M. R. Osmotic control of proU transcription is mediated through direct action of potassium glutamate on the transcription complex. J Biol Chem. 1990 Oct 15;265(29):17673–17679. [PubMed] [Google Scholar]
  35. Ramirez R. M., Prince W. S., Bremer E., Villarejo M. In vitro reconstitution of osmoregulated expression of proU of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1153–1157. doi: 10.1073/pnas.86.4.1153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Record M. T., Jr, deHaseth P. L., Lohman T. M. Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction. Biochemistry. 1977 Nov 1;16(22):4791–4796. doi: 10.1021/bi00641a005. [DOI] [PubMed] [Google Scholar]
  37. Rhoads D. B., Waters F. B., Epstein W. Cation transport in Escherichia coli. VIII. Potassium transport mutants. J Gen Physiol. 1976 Mar;67(3):325–341. doi: 10.1085/jgp.67.3.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Richey B., Cayley D. S., Mossing M. C., Kolka C., Anderson C. F., Farrar T. C., Record M. T., Jr Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression. J Biol Chem. 1987 May 25;262(15):7157–7164. [PubMed] [Google Scholar]
  39. Roberts T. M., Kacich R., Ptashne M. A general method for maximizing the expression of a cloned gene. Proc Natl Acad Sci U S A. 1979 Feb;76(2):760–764. doi: 10.1073/pnas.76.2.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roe J. H., Record M. T., Jr Regulation of the kinetics of the interaction of Escherichia coli RNA polymerase with the lambda PR promoter by salt concentration. Biochemistry. 1985 Aug 27;24(18):4721–4726. doi: 10.1021/bi00339a002. [DOI] [PubMed] [Google Scholar]
  41. Sasse-Dwight S., Gralla J. D. Footprinting protein-DNA complexes in vivo. Methods Enzymol. 1991;208:146–168. doi: 10.1016/0076-6879(91)08012-7. [DOI] [PubMed] [Google Scholar]
  42. Schmitt R. Analysis of melibiose mutants deficient in alpha-galactosidase and thiomethylgalactoside permease II in Escherichia coli K-12. J Bacteriol. 1968 Aug;96(2):462–471. doi: 10.1128/jb.96.2.462-471.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  44. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  45. Sippel A., Hartmann G. Mode of action of rafamycin on the RNA polymerase reaction. Biochim Biophys Acta. 1968 Mar 18;157(1):218–219. doi: 10.1016/0005-2787(68)90286-4. [DOI] [PubMed] [Google Scholar]
  46. Spassky A., Rimsky S., Garreau H., Buc H. H1a, an E. coli DNA-binding protein which accumulates in stationary phase, strongly compacts DNA in vitro. Nucleic Acids Res. 1984 Jul 11;12(13):5321–5340. doi: 10.1093/nar/12.13.5321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Spears P. A., Schauer D., Orndorff P. E. Metastable regulation of type 1 piliation in Escherichia coli and isolation and characterization of a phenotypically stable mutant. J Bacteriol. 1986 Oct;168(1):179–185. doi: 10.1128/jb.168.1.179-185.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stirling D. A., Hulton C. S., Waddell L., Park S. F., Stewart G. S., Booth I. R., Higgins C. F. Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems. Mol Microbiol. 1989 Aug;3(8):1025–1038. doi: 10.1111/j.1365-2958.1989.tb00253.x. [DOI] [PubMed] [Google Scholar]
  49. Sutherland L., Cairney J., Elmore M. J., Booth I. R., Higgins C. F. Osmotic regulation of transcription: induction of the proU betaine transport gene is dependent on accumulation of intracellular potassium. J Bacteriol. 1986 Nov;168(2):805–814. doi: 10.1128/jb.168.2.805-814.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tanaka K., Muramatsu S., Yamada H., Mizuno T. Systematic characterization of curved DNA segments randomly cloned from Escherichia coli and their functional significance. Mol Gen Genet. 1991 May;226(3):367–376. doi: 10.1007/BF00260648. [DOI] [PubMed] [Google Scholar]
  51. Ueguchi C., Mizuno T. The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor. EMBO J. 1993 Mar;12(3):1039–1046. doi: 10.1002/j.1460-2075.1993.tb05745.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Varshavsky A. J., Nedospasov S. A., Bakayev V. V., Bakayeva T. G., Georgiev G. P. Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein. Nucleic Acids Res. 1977 Aug;4(8):2725–2745. doi: 10.1093/nar/4.8.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES