1887

Abstract

Heat-labile enterotoxin, a major virulence determinant of enterotoxigenic , is encoded by the operon. To elucidate the molecular mechanism by which the heat-stable nucleoid-structural (H-NS) protein controls transcription of , the authors constructed an transcriptional fusion and performed -galactosidase analysis. The results showed that H-NS protein exerts fivefold repression on transcription from the promoter at 37 °C and 10-fold repression at 22 °C. Two silencer regions that were required for H-NS-mediated repression of expression were identified, both of which were located downstream of the start site of transcription. One silencer was located between +31 and +110, the other between +460 and +556, relative to the start site of transcription, and they worked cooperatively in repression. DNA sequences containing the silencers were predicted to be curved by analysis and bound H-NS protein directly . Repression of transcription by H-NS was independent of promoter strength, and the presence of H-NS protein did not affect promoter opening , indicating that repression was achieved by inhibiting promoter clearance or blocking transcription elongation, probably via DNA looping between the two silencers.

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2005-04-01
2020-07-14
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References

  1. Atlung T., Ingmer H. 1997; H-NS: a modulator of environmentally regulated gene expression. Mol Microbiol24:7–17[CrossRef]
    [Google Scholar]
  2. Badaut C., Williams R., Arluison V., Bouffartigues E., Robert B., Buc H., Rimsky S. 2002; The degree of oligomerization of the H-NS nucleoid structuring protein is related to specific binding to DNA. J Biol Chem277:41657–41666[CrossRef]
    [Google Scholar]
  3. Beloin C., Dorman C. J. 2003; An extended role for the nucleoid structuring protein H-NS in the virulence gene regulatory cascade of Shigella flexneri. Mol Microbiol47:825–838[CrossRef]
    [Google Scholar]
  4. Black R. E. 1993; Persistent diarrhoea in children of developing countries. Pediatr Infect Dis J12:751–761[CrossRef]
    [Google Scholar]
  5. Bustamante V. H., Santana F. J., Calva E., Puente J. L. 2001; Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression. Mol Microbiol39:664–678[CrossRef]
    [Google Scholar]
  6. Casadaban M. J. 1976; Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol104:541–555[CrossRef]
    [Google Scholar]
  7. Dattananda C. S., Rajkumari K., Gowrishankar J. 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. J Bacteriol173:7481–7490
    [Google Scholar]
  8. Dersch P., Kneip S., Bremer E. 1994; The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment. Mol Gen Genet245:255–259
    [Google Scholar]
  9. DiRita V. J., Parsot C., Jander G., Mekalanos J. J. 1991; Regulatory cascade controls virulence in Vibrio cholerae. Proc Natl Acad Sci U S A88:5403–5407[CrossRef]
    [Google Scholar]
  10. Dole S., Nagarajavel V., Schnetz K. 2004; The histone-like nucleoid structuring protein H-NS represses the Escherichia coli bgl operon downstream of the promoter. Mol Microbiol52:589–600[CrossRef]
    [Google Scholar]
  11. Dorman C. J. 2004; H-NS: a universal regulator for a dynamic genome. Nat Rev Microbiol2:391–400[CrossRef]
    [Google Scholar]
  12. Evans D. G., Pierce N. F, Evans D. J. Jr. 1973; Differences in the response of rabbit small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli. Infect Immun7:873–880
    [Google Scholar]
  13. Falconi M., Prosseda G., Giangrossi M., Beghetto E., Colonna B. 2001; Involvement of FIS in the H-NS-mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli. Mol Microbiol42:439–452[CrossRef]
    [Google Scholar]
  14. Gourse R. L., Ross W., Gaal T. 2000; UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition. Mol Microbiol37:687–695[CrossRef]
    [Google Scholar]
  15. Haack K. R., Robinson C. L., Miller K. J., Fowlkes J. W., Mellies J. L. 2003; Interaction of Ler at the LEE5 (tir) operon of enteropathogenicEscherichia coli. Infect Immun71:384–392[CrossRef]
    [Google Scholar]
  16. Hase C. C., Mekalanos J. J. 1998; TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci U S A95:730–734[CrossRef]
    [Google Scholar]
  17. Higgins D. E., Nazareno E., DiRita V. J. 1992; The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators. J Bacteriol174:6974–6980
    [Google Scholar]
  18. Jordi B. J., Higgins C. F. 2000; The downstream regulatory element of the proU operon of Salmonella typhimurium inhibits open complex formation by RNA polymerase at a distance. J Biol Chem275:12123–12128[CrossRef]
    [Google Scholar]
  19. Kieny M. P., Lathe R., Lecocq J. P. 1983; New versatile cloning and sequencing vectors based on the bacteriophage M13. Gene26:91–99[CrossRef]
    [Google Scholar]
  20. Krukonis E. S., Yu R. R., Dirita V. J. 2000; The Vibrio cholerae ToxR/TcpP/ToxT virulence cascade: distinct roles for two membrane-localized transcriptional activators on a single promoter. Mol Microbiol38:67–84[CrossRef]
    [Google Scholar]
  21. Lucht J. M., Dersch P., Kempf B., Bremer E. 1994; Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli. J Biol Chem269:6578–6586
    [Google Scholar]
  22. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. 1983; Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature306:551–557[CrossRef]
    [Google Scholar]
  23. Messing J. 1983; New M13 vectors for cloning. Methods Enzymol101:20–78
    [Google Scholar]
  24. Miller J. H. 1974; Experiments in Molecular Genetics pp352–355 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Murphree D., Froehlich B., Scott J. R. 1997; Transcriptional control of genes encoding CS1 pili: negative regulation by a silencer and positive regulation by Rns. J Bacteriol179:5736–5743
    [Google Scholar]
  26. Nataro J. P., Kaper J. B. 1998; Diarrheagenic Escherichia coli. Clin Microbiol Rev11:142–201
    [Google Scholar]
  27. Nye M. B., Pfau J. D., Skorupski K., Taylor R. K. 2000; Vibrio cholerae H-NS silences virulence gene expression at multiple steps in the ToxR regulatory cascade. J Bacteriol182:4295–4303[CrossRef]
    [Google Scholar]
  28. Overdier D. G., Csonka L. N. 1992; A transcriptional silencer downstream of the promoter in the osmotically controlled proU operon of Salmonella typhimurium. Proc Natl Acad Sci U S A89:3140–3144[CrossRef]
    [Google Scholar]
  29. Porter M. E., Dorman C. J. 1994; A role for H-NS in the thermo-osmotic regulation of virulence gene expression in Shigella flexneri. J Bacteriol176:4187–4191
    [Google Scholar]
  30. Praszkier J., Wilson I. W., Pittard A. J. 1992; Mutations affecting translational coupling between the rep genes of an IncB miniplasmid. J Bacteriol174:2376–2383
    [Google Scholar]
  31. Rimsky S. 2004; Structure of the histone-like protein H-NS and its role in regulation and genome superstructure. Curr Opin Microbiol7:109–114[CrossRef]
    [Google Scholar]
  32. Rimsky S., Zuber F., Buckle M., Buc H. 2001; A molecular mechanism for the repression of transcription by the H-NS protein. Mol Microbiol42:1311–1323
    [Google Scholar]
  33. Rowe B., Taylor J., Bettelheim K. A. 1970; An investigation of travellers' diarrhoea. Lancet1:1–5
    [Google Scholar]
  34. Sambrook J., Fritsch E. F., Maniatis T. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  35. Schlör S., Riedl S., Blass J., Reidl J. 2000; Genetic rearrangement of the regions adjacent to genes encoding heat-labile enterotoxin (eltAB) of enterotoxigenic Escherichia coli strains. Appl Environ Microbiol66:352–358[CrossRef]
    [Google Scholar]
  36. Schneider D. A., Ross W., Gourse R. L. 2003; Control of rRNA expression in Escherichia coli. Curr Opin Microbiol6:151–156[CrossRef]
    [Google Scholar]
  37. Schnetz K. 1995; Silencing of Escherichia coli bgl promoter by flanking sequence elements. EMBO J14:2545–2550
    [Google Scholar]
  38. Skorupski K., Taylor R. K. 1997; Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli. Mol Microbiol25:1003–1009[CrossRef]
    [Google Scholar]
  39. Spangler B. D. 1992; Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev56:622–647
    [Google Scholar]
  40. Trachman J. D., Maas W. K. 1998; Temperature regulation of heat-labile enterotoxin (LT) synthesis in Escherichia coli is mediated by an interaction of H-NS protein with the LT A-subunit DNA. J Bacteriol180:3715–3718
    [Google Scholar]
  41. Trachman J. D., Yasmin M. 2004; Thermo-osmoregulation of heat-labile enterotoxin expression by Escherichia coli. Curr Microbiol49:353–360[CrossRef]
    [Google Scholar]
  42. Ueguchi C., Suzuki T., Yoshida T., Tanaka K., Mizuno T. 1996; Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS. J Mol Biol263:149–162[CrossRef]
    [Google Scholar]
  43. Ussery D. W., Hinton J. C., Jordi B. J.. 7 other authors 1994; The chromatin-associated protein H-NS. Biochimie76:968–980[CrossRef]
    [Google Scholar]
  44. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene19:259–268[CrossRef]
    [Google Scholar]
  45. Yamamoto T., Tamura T., Yokota T. 1984; Primary structure of heat-labile enterotoxin produced by Escherichia coli pathogenic for humans. J Biol Chem259:5037–5044
    [Google Scholar]
  46. Yang J., Hwang J. S., Camakaris H., Irawaty W., Ishihama A., Pittard A. J. 2004; Mode of action of the TyrR protein: repression and activation of the tyrP promoter of Escherichia coli. Mol Microbiol52:243–256[CrossRef]
    [Google Scholar]
  47. Yu R. R., DiRita V. J. 2002; Regulation of gene expression in Vibrio cholerae by ToxT involves both antirepression and RNA polymerase stimulation. Mol Microbiol43:119–134[CrossRef]
    [Google Scholar]
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