Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1996 Jan 2;15(1):102–109.

Phosphorylated BvgA is sufficient for transcriptional activation of virulence-regulated genes in Bordetella pertussis.

P Steffen 1, S Goyard 1, A Ullmann 1
PMCID: PMC449922  PMID: 8598192

Abstract

In Bordetella pertussis the expression of virulence factors is coordinately regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal transduction family, BvgS being the transmembrane sensor and BvgA the regulator. Activation of virulence gene expression requires phosphorylation of BvgA. On the basis of observed differences in the regulation of individual genes, the existence of accessory regulators has been postulated. They were supposed to be necessary for expression of genes encoding adenylate cyclase toxin (cya) and pertussis toxin (ptx), but not required for the expression of fha, encoding filamentous hemagglutinin. To clarify this issue we investigated the mechanism of activation of the BvgAS-controlled genes by performing in vitro run-off transcription experiments. We show, using purified RNA polymerase of B.pertussis, that phosphorylated BvgA is sufficient for transcriptional activation of the major virulence genes, thus providing good evidence that BvgA regulation operates directly with the transcription initiation machinery at the promoters of the virulence genes without a requirement for accessory activators. In addition, our results indicate that activation of the different promoters may involve distinct mechanisms. We suggest that the previously observed differences in regulation of individual virulence-associated genes reflect differences in the phosphorylation state of BvgA.

Full text

PDF
102

Images in this article

Selected References

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

  1. Amann E., Ochs B., Abel K. J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988 Sep 30;69(2):301–315. doi: 10.1016/0378-1119(88)90440-4. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Boucher P. E., Menozzi F. D., Locht C. The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis. J Mol Biol. 1994 Aug 19;241(3):363–377. doi: 10.1006/jmbi.1994.1513. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Burgess R. R., Jendrisak J. J. A procedure for the rapid, large-scall purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry. 1975 Oct 21;14(21):4634–4638. doi: 10.1021/bi00692a011. [DOI] [PubMed] [Google Scholar]
  6. Carbonetti N. H., Fuchs T. M., Patamawenu A. A., Irish T. J., Deppisch H., Gross R. Effect of mutations causing overexpression of RNA polymerase alpha subunit on regulation of virulence factors in Bordetella pertussis. J Bacteriol. 1994 Dec;176(23):7267–7273. doi: 10.1128/jb.176.23.7267-7273.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coote J. G. Antigenic switching and pathogenicity: environmental effects on virulence gene expression in Bordetella pertussis. J Gen Microbiol. 1991 Nov;137(11):2493–2503. doi: 10.1099/00221287-137-11-2493. [DOI] [PubMed] [Google Scholar]
  8. Elliott T., Geiduschek E. P. Defining a bacteriophage T4 late promoter: absence of a "-35" region. Cell. 1984 Jan;36(1):211–219. doi: 10.1016/0092-8674(84)90091-6. [DOI] [PubMed] [Google Scholar]
  9. Elliott T., Kassavetis G. A., Geiduschek E. P. The complex pattern of transcription in the segment of the bacteriophage T4 genome containing three of the head protein genes. Virology. 1984 Dec;139(2):260–282. doi: 10.1016/0042-6822(84)90373-8. [DOI] [PubMed] [Google Scholar]
  10. Feng J., Atkinson M. R., McCleary W., Stock J. B., Wanner B. L., Ninfa A. J. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J Bacteriol. 1992 Oct;174(19):6061–6070. doi: 10.1128/jb.174.19.6061-6070.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goyard S., Ullmann A. Analysis of Bordetella pertussis cya operon regulation by use of cya-lac fusions. FEMS Microbiol Lett. 1991 Jan 15;61(2-3):251–256. doi: 10.1016/0378-1097(91)90561-n. [DOI] [PubMed] [Google Scholar]
  12. Gross R., Rappuoli R. Positive regulation of pertussis toxin expression. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3913–3917. doi: 10.1073/pnas.85.11.3913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ishihama A. Protein-protein communication within the transcription apparatus. J Bacteriol. 1993 May;175(9):2483–2489. doi: 10.1128/jb.175.9.2483-2489.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ishihama A. Role of the RNA polymerase alpha subunit in transcription activation. Mol Microbiol. 1992 Nov;6(22):3283–3288. doi: 10.1111/j.1365-2958.1992.tb02196.x. [DOI] [PubMed] [Google Scholar]
  15. Kolb A., Busby S., Buc H., Garges S., Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. doi: 10.1146/annurev.bi.62.070193.003533. [DOI] [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. Laoide B. M., Ullmann A. Virulence dependent and independent regulation of the Bordetella pertussis cya operon. EMBO J. 1990 Apr;9(4):999–1005. doi: 10.1002/j.1460-2075.1990.tb08202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lonetto M., Gribskov M., Gross C. A. The sigma 70 family: sequence conservation and evolutionary relationships. J Bacteriol. 1992 Jun;174(12):3843–3849. doi: 10.1128/jb.174.12.3843-3849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lukat G. S., McCleary W. R., Stock A. M., Stock J. B. Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):718–722. doi: 10.1073/pnas.89.2.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miller J. F., Johnson S. A., Black W. J., Beattie D. T., Mekalanos J. J., Falkow S. Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene. J Bacteriol. 1992 Feb;174(3):970–979. doi: 10.1128/jb.174.3.970-979.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Minton N. P. Improved plasmid vectors for the isolation of translational lac gene fusions. Gene. 1984 Nov;31(1-3):269–273. doi: 10.1016/0378-1119(84)90220-8. [DOI] [PubMed] [Google Scholar]
  23. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  24. Reznikoff W. S. The lactose operon-controlling elements: a complex paradigm. Mol Microbiol. 1992 Sep;6(17):2419–2422. doi: 10.1111/j.1365-2958.1992.tb01416.x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Roy C. R., Miller J. F., Falkow S. Autogenous regulation of the Bordetella pertussis bvgABC operon. Proc Natl Acad Sci U S A. 1990 May;87(10):3763–3767. doi: 10.1073/pnas.87.10.3763. [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., Aricò B., Rappuoli R. DNA topology affects transcriptional regulation of the pertussis toxin gene of Bordetella pertussis in Escherichia coli and in vitro. J Bacteriol. 1993 Aug;175(15):4764–4771. doi: 10.1128/jb.175.15.4764-4771.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Scarlato V., Aricó B., Domenighini M., Rappuoli R. Environmental regulation of virulence factors in Bordetella species. Bioessays. 1993 Feb;15(2):99–104. doi: 10.1002/bies.950150205. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Schneider G. J., Tumer N. E., Richaud C., Borbely G., Haselkorn R. Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120. J Biol Chem. 1987 Oct 25;262(30):14633–14639. [PubMed] [Google Scholar]
  33. Stibitz S. Mutations in the bvgA gene of Bordetella pertussis that differentially affect regulation of virulence determinants. J Bacteriol. 1994 Sep;176(18):5615–5621. doi: 10.1128/jb.176.18.5615-5621.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Strickland M. S., Thompson N. E., Burgess R. R. Structure and function of the sigma-70 subunit of Escherichia coli RNA polymerase. Monoclonal antibodies: localization of epitopes by peptide mapping and effects on transcription. Biochemistry. 1988 Jul 26;27(15):5755–5762. doi: 10.1021/bi00415a054. [DOI] [PubMed] [Google Scholar]
  36. Uhl M. A., Miller J. F. Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1163–1167. doi: 10.1073/pnas.91.3.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES