Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Dec;173(23):7481–7490. doi: 10.1128/jb.173.23.7481-7490.1991

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.

C S Dattananda 1, K Rajkumari 1, J Gowrishankar 1
PMCID: PMC212513  PMID: 1938945

Abstract

Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region of proV also exhibited partial derepression of proU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression.

Full text

PDF
7484

Images in this article

7486FIG. 2
on p.7486
7486FIG. 3
on p.7486

Selected References

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

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Auble D. T., Allen T. L., deHaseth P. L. Promoter recognition by Escherichia coli RNA polymerase. Effects of substitutions in the spacer DNA separating the -10 and -35 regions. J Biol Chem. 1986 Aug 25;261(24):11202–11206. [PubMed] [Google Scholar]
  3. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Csonka L. N. Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev. 1989 Mar;53(1):121–147. doi: 10.1128/mr.53.1.121-147.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dattananda C. S., Gowrishankar J. Osmoregulation in Escherichia coli: complementation analysis and gene-protein relationships in the proU locus. J Bacteriol. 1989 Apr;171(4):1915–1922. doi: 10.1128/jb.171.4.1915-1922.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dayn A., Malkhosyan S., Duzhy D., Lyamichev V., Panchenko Y., Mirkin S. Formation of (dA-dT)n cruciforms in Escherichia coli cells under different environmental conditions. J Bacteriol. 1991 Apr;173(8):2658–2664. doi: 10.1128/jb.173.8.2658-2664.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Drlica K. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol Rev. 1984 Dec;48(4):273–289. doi: 10.1128/mr.48.4.273-289.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. Haniford D. B., Pulleyblank D. E. Transition of a cloned d(AT)n-d(AT)n tract to a cruciform in vivo. Nucleic Acids Res. 1985 Jun 25;13(12):4343–4363. doi: 10.1093/nar/13.12.4343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Hsieh L. S., Rouviere-Yaniv J., Drlica K. Bacterial DNA supercoiling and [ATP]/[ADP] ratio: changes associated with salt shock. J Bacteriol. 1991 Jun;173(12):3914–3917. doi: 10.1128/jb.173.12.3914-3917.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hulton C. S., Seirafi A., Hinton J. C., Sidebotham J. M., Waddell L., Pavitt G. D., Owen-Hughes T., Spassky A., Buc H., Higgins C. F. Histone-like protein H1 (H-NS), DNA supercoiling, and gene expression in bacteria. Cell. 1990 Nov 2;63(3):631–642. doi: 10.1016/0092-8674(90)90458-q. [DOI] [PubMed] [Google Scholar]
  19. Jacobson A. B., Good L., Simonetti J., Zuker M. Some simple computational methods to improve the folding of large RNAs. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):45–52. doi: 10.1093/nar/12.1part1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jaworski A., Higgins N. P., Wells R. D., Zacharias W. Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo. J Biol Chem. 1991 Feb 5;266(4):2576–2581. [PubMed] [Google Scholar]
  21. Jovanovich S. B., Record M. T., Jr, Burgess R. R. In an Escherichia coli coupled transcription-translation system, expression of the osmoregulated gene proU is stimulated at elevated potassium concentrations and by an extract from cells grown at high osmolality. J Biol Chem. 1989 May 15;264(14):7821–7825. [PubMed] [Google Scholar]
  22. Kawula T. H., Orndorff P. E. Rapid site-specific DNA inversion in Escherichia coli mutants lacking the histonelike protein H-NS. J Bacteriol. 1991 Jul;173(13):4116–4123. doi: 10.1128/jb.173.13.4116-4123.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Marsh P. Ptac-85, an E. coli vector for expression of non-fusion proteins. Nucleic Acids Res. 1986 Apr 25;14(8):3603–3603. doi: 10.1093/nar/14.8.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. May G., Dersch P., Haardt M., Middendorf A., Bremer E. The osmZ (bglY) gene encodes the DNA-binding protein H-NS (H1a), a component of the Escherichia coli K12 nucleoid. Mol Gen Genet. 1990 Oct;224(1):81–90. doi: 10.1007/BF00259454. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. McClellan J. A., Boublíková P., Palecek E., Lilley D. M. Superhelical torsion in cellular DNA responds directly to environmental and genetic factors. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8373–8377. doi: 10.1073/pnas.87.21.8373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McFall E. cis-acting proteins. J Bacteriol. 1986 Aug;167(2):429–432. doi: 10.1128/jb.167.2.429-432.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Overdier D. G., Olson E. R., Erickson B. D., Ederer M. M., Csonka L. N. Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA. J Bacteriol. 1989 Sep;171(9):4694–4706. doi: 10.1128/jb.171.9.4694-4706.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Park S. F., Stirling D. A., Hulton C. S., Booth I. R., Higgins C. F., Stewart G. S. A novel, non-invasive promoter probe vector: cloning of the osmoregulated proU promoter of Escherichia coli K12. Mol Microbiol. 1989 Aug;3(8):1011–1023. doi: 10.1111/j.1365-2958.1989.tb00252.x. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Ramirez R. M., Villarejo M. Osmotic signal transduction to proU is independent of DNA supercoiling in Escherichia coli. J Bacteriol. 1991 Jan;173(2):879–885. doi: 10.1128/jb.173.2.879-885.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Shure M., Pulleyblank D. E., Vinograd J. The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity. Nucleic Acids Res. 1977;4(5):1183–1205. doi: 10.1093/nar/4.5.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Suzuki M. SPXX, a frequent sequence motif in gene regulatory proteins. J Mol Biol. 1989 May 5;207(1):61–84. doi: 10.1016/0022-2836(89)90441-5. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Wells R. D. Unusual DNA structures. J Biol Chem. 1988 Jan 25;263(3):1095–1098. [PubMed] [Google Scholar]
  41. Wu H. Y., Shyy S. H., Wang J. C., Liu L. F. Transcription generates positively and negatively supercoiled domains in the template. Cell. 1988 May 6;53(3):433–440. doi: 10.1016/0092-8674(88)90163-8. [DOI] [PubMed] [Google Scholar]
  42. Yang J., Pittard J. Molecular analysis of the regulatory region of the Escherichia coli K-12 tyrB gene. J Bacteriol. 1987 Oct;169(10):4710–4715. doi: 10.1128/jb.169.10.4710-4715.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES