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. 1993 Jan;175(2):519–527. doi: 10.1128/jb.175.2.519-527.1993

Repressor binding to a regulatory site in the DNA coding sequence is sufficient to confer transcriptional regulation of the vir-repressed genes (vrg genes) in Bordetella pertussis.

D T Beattie 1, M J Mahan 1, J J Mekalanos 1
PMCID: PMC196167  PMID: 8419298

Abstract

Five TnphoA fusions to vir-repressed genes (vrg genes) have been identified in the respiratory pathogen Bordetella pertussis. A comparison of vrg DNA sequences suggests a consensus DNA element within the coding regions of four of five vrg genes. To determine the role of this DNA sequence in vrg regulation, a nucleotide substitution mutation in the conserved region of vrg-6 was isolated. This mutant showed constitutively high levels of expression in the absence of antigenic modulators, MgSO4 and nicotinic acid, suggesting that this DNA element may be a control site for vrg repression. Moreover, Northern (RNA) analysis and transcriptional fusion analysis suggest that vrg genes are regulated at the transcriptional level. To determine whether sequences in the coding region were sufficient to respond to antigenic modulation, a vrg-6::TnphoA promoter deletion plasmid that contained a heterologous promoter driving the expression of vrg-6 coding sequences from the vrg-6 translation start site to the TnphoA fusion junction was constructed. This heterologous construct responded to modulators in a vir-dependent fashion, indicating that sequences upstream of the coding sequence are not required for antigenic modulation. Southwestern (DNA-protein) analysis and mutational studies suggest that the vrg consensus DNA sequence is specifically recognized by a 34-kDa vir-activated gene (vag) product, whose binding results in down-regulation of vrg transcript levels. We conclude, at least for the vrg::TnphoA fusion strains, that a site on the DNA that corresponds to a consensus sequence located in the vrg coding region is sufficient to confer the transcriptional regulation (repression) of vrg genes when B. pertussis strains are grown under nonmodulating conditions.

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Selected References

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

  1. Aricó B., Miller J. F., Roy C., Stibitz S., Monack D., Falkow S., Gross R., Rappuoli R. Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6671–6675. doi: 10.1073/pnas.86.17.6671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beattie D. T., Knapp S., Mekalanos J. J. Evidence that modulation requires sequences downstream of the promoters of two vir-repressed genes of Bordetella pertussis. J Bacteriol. 1990 Dec;172(12):6997–7004. doi: 10.1128/jb.172.12.6997-7004.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beattie D. T., Shahin R., Mekalanos J. J. A vir-repressed gene of Bordetella pertussis is required for virulence. Infect Immun. 1992 Feb;60(2):571–577. doi: 10.1128/iai.60.2.571-577.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DiRita V. J., Mekalanos J. J. Periplasmic interaction between two membrane regulatory proteins, ToxR and ToxS, results in signal transduction and transcriptional activation. Cell. 1991 Jan 11;64(1):29–37. doi: 10.1016/0092-8674(91)90206-e. [DOI] [PubMed] [Google Scholar]
  6. DiRita V. J., Parsot C., Jander G., Mekalanos J. J. Regulatory cascade controls virulence in Vibrio cholerae. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5403–5407. doi: 10.1073/pnas.88.12.5403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Finn T. M., Shahin R., Mekalanos J. J. Characterization of vir-activated TnphoA gene fusions in Bordetella pertussis. Infect Immun. 1991 Sep;59(9):3273–3279. doi: 10.1128/iai.59.9.3273-3279.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. Huh Y. J., Weiss A. A. A 23-kilodalton protein, distinct from BvgA, expressed by virulent Bordetella pertussis binds to the promoter region of vir-regulated toxin genes. Infect Immun. 1991 Jul;59(7):2389–2395. doi: 10.1128/iai.59.7.2389-2395.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Idigbe E. O., Parton R., Wardlaw A. C. Rapidity of antigenic modulation of Bordetella pertussis in modified Hornibrook medium. J Med Microbiol. 1981 Nov;14(4):409–418. doi: 10.1099/00222615-14-4-409. [DOI] [PubMed] [Google Scholar]
  13. Irani M. H., Orosz L., Adhya S. A control element within a structural gene: the gal operon of Escherichia coli. Cell. 1983 Mar;32(3):783–788. doi: 10.1016/0092-8674(83)90064-8. [DOI] [PubMed] [Google Scholar]
  14. Knapp S., Mekalanos J. J. Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis. J Bacteriol. 1988 Nov;170(11):5059–5066. doi: 10.1128/jb.170.11.5059-5066.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LACEY B. W. Antigenic modulation of Bordetella pertussis. J Hyg (Lond) 1960 Mar;58:57–93. doi: 10.1017/s0022172400038134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller J. F., Mekalanos J. J., Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989 Feb 17;243(4893):916–922. doi: 10.1126/science.2537530. [DOI] [PubMed] [Google Scholar]
  19. Miller J. F., Roy C. R., Falkow S. Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli. J Bacteriol. 1989 Nov;171(11):6345–6348. doi: 10.1128/jb.171.11.6345-6348.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miller S. I., Landfear S. M., Wirth D. F. Cloning and characterization of a Leishmania gene encoding a RNA spliced leader sequence. Nucleic Acids Res. 1986 Sep 25;14(18):7341–7360. doi: 10.1093/nar/14.18.7341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mooi F. R. Virulence factors of Bordetella pertussis. Antonie Van Leeuwenhoek. 1988;54(5):465–474. doi: 10.1007/BF00461865. [DOI] [PubMed] [Google Scholar]
  22. Oehler S., Eismann E. R., Krämer H., Müller-Hill B. The three operators of the lac operon cooperate in repression. EMBO J. 1990 Apr;9(4):973–979. doi: 10.1002/j.1460-2075.1990.tb08199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rappuoli R., Aricó B., Scarlato V. Thermoregulation and reversible differentiation in Bordetella: a model for pathogenic bacteria. Mol Microbiol. 1992 Aug;6(15):2209–2211. doi: 10.1111/j.1365-2958.1992.tb01395.x. [DOI] [PubMed] [Google Scholar]
  24. Rolfes R. J., Zalkin H. Autoregulation of Escherichia coli purR requires two control sites downstream of the promoter. J Bacteriol. 1990 Oct;172(10):5758–5766. doi: 10.1128/jb.172.10.5758-5766.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ronson C. W., Nixon B. T., Ausubel F. M. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell. 1987 Jun 5;49(5):579–581. doi: 10.1016/0092-8674(87)90530-7. [DOI] [PubMed] [Google Scholar]
  26. Roy C. R., Falkow S. Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon. J Bacteriol. 1991 Apr;173(7):2385–2392. doi: 10.1128/jb.173.7.2385-2392.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roy C. R., Miller J. F., Falkow S. The bvgA gene of Bordetella pertussis encodes a transcriptional activator required for coordinate regulation of several virulence genes. J Bacteriol. 1989 Nov;171(11):6338–6344. doi: 10.1128/jb.171.11.6338-6344.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scarlato V., Aricò B., Prugnola A., Rappuoli R. Sequential activation and environmental regulation of virulence genes in Bordetella pertussis. EMBO J. 1991 Dec;10(12):3971–3975. doi: 10.1002/j.1460-2075.1991.tb04967.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scarlato V., Prugnola A., Aricó B., Rappuoli R. Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6753–6757. doi: 10.1073/pnas.87.17.6753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stibitz S., Yang M. S. Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis. J Bacteriol. 1991 Jul;173(14):4288–4296. doi: 10.1128/jb.173.14.4288-4296.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weiss A. A., Falkow S. Genetic analysis of phase change in Bordetella pertussis. Infect Immun. 1984 Jan;43(1):263–269. doi: 10.1128/iai.43.1.263-269.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun. 1983 Oct;42(1):33–41. doi: 10.1128/iai.42.1.33-41.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Weiss A. A., Hewlett E. L. Virulence factors of Bordetella pertussis. Annu Rev Microbiol. 1986;40:661–686. doi: 10.1146/annurev.mi.40.100186.003305. [DOI] [PubMed] [Google Scholar]
  35. Weiss A. A., Melton A. R., Walker K. E., Andraos-Selim C., Meidl J. J. Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes. Infect Immun. 1989 Sep;57(9):2674–2682. doi: 10.1128/iai.57.9.2674-2682.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  37. von Gabain A., Belasco J. G., Schottel J. L., Chang A. C., Cohen S. N. Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. Proc Natl Acad Sci U S A. 1983 Feb;80(3):653–657. doi: 10.1073/pnas.80.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]

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