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. 1997 Sep;179(17):5398–5406. doi: 10.1128/jb.179.17.5398-5406.1997

Mutational characterization of promoter regions recognized by the Salmonella dublin virulence plasmid regulatory protein SpvR.

P Grob 1, D Kahn 1, D G Guiney 1
PMCID: PMC179409  PMID: 9286993

Abstract

The virulence plasmid-encoded spv regulon is essential for virulence of Salmonella dublin in mice. The spvR gene product belongs to the LysR family of transcriptional regulator proteins. SpvR induces the expression of the spvABCD operon and positively regulates its own expression. DNase I protection analysis with purified SpvR fusion proteins identified SpvR binding sites within the spvA and spvR promoters (P. Grob and D. G. Guiney, J. Bacteriol. 178:1813-1820, 1996). We have used PCR mutagenesis, combined with functional selection for reduced SpvR affinity, to define the DNA elements essential for SpvR binding. For the spvR promoter fragment, a screen for reduced expression was also applied. Sequence analysis of the resulting mutant fragments reveals that the base pair changes are clustered in distinct regions. Determination of the apparent dissociation constants of SpvR for the mutant promoters showed that the spvA LysR-type motif and the upstream palindromic sequences of both promoters play an important role in SpvR recognition.

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Selected References

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  1. Abe A., Kawahara K. Transcriptional regulation and promoter sequence of the spvR gene of virulence plasmid pKDSC50 in Salmonella choleraesuis serovar Choleraesuis. FEMS Microbiol Lett. 1995 Jun 15;129(2-3):225–230. doi: 10.1111/j.1574-6968.1995.tb07584.x. [DOI] [PubMed] [Google Scholar]
  2. Abe A., Matsui H., Danbara H., Tanaka K., Takahashi H., Kawahara K. Regulation of spvR gene expression of Salmonella virulence plasmid pKDSC50 in Salmonella choleraesuis serovar Choleraesuis. Mol Microbiol. 1994 Jun;12(5):779–787. doi: 10.1111/j.1365-2958.1994.tb01064.x. [DOI] [PubMed] [Google Scholar]
  3. Bossi L., Smith D. M. Conformational change in the DNA associated with an unusual promoter mutation in a tRNA operon of Salmonella. Cell. 1984 Dec;39(3 Pt 2):643–652. doi: 10.1016/0092-8674(84)90471-9. [DOI] [PubMed] [Google Scholar]
  4. Cadwell R. C., Joyce G. F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992 Aug;2(1):28–33. doi: 10.1101/gr.2.1.28. [DOI] [PubMed] [Google Scholar]
  5. Caldwell A. L., Gulig P. A. The Salmonella typhimurium virulence plasmid encodes a positive regulator of a plasmid-encoded virulence gene. J Bacteriol. 1991 Nov;173(22):7176–7185. doi: 10.1128/jb.173.22.7176-7185.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Chen C. Y., Buchmeier N. A., Libby S., Fang F. C., Krause M., Guiney D. G. Central regulatory role for the RpoS sigma factor in expression of Salmonella dublin plasmid virulence genes. J Bacteriol. 1995 Sep;177(18):5303–5309. doi: 10.1128/jb.177.18.5303-5309.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chikami G. K., Fierer J., Guiney D. G. Plasmid-mediated virulence in Salmonella dublin demonstrated by use of a Tn5-oriT construct. Infect Immun. 1985 Nov;50(2):420–424. doi: 10.1128/iai.50.2.420-424.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coynault C., Robbe-Saule V., Popoff M. Y., Norel F. Growth phase and SpvR regulation of transcription of Salmonella typhimurium spvABC virulence genes. Microb Pathog. 1992 Aug;13(2):133–143. doi: 10.1016/0882-4010(92)90073-w. [DOI] [PubMed] [Google Scholar]
  10. Fang F. C., Fierer J. Human infection with Salmonella dublin. Medicine (Baltimore) 1991 May;70(3):198–207. doi: 10.1097/00005792-199105000-00004. [DOI] [PubMed] [Google Scholar]
  11. Fang F. C., Krause M., Roudier C., Fierer J., Guiney D. G. Growth regulation of a Salmonella plasmid gene essential for virulence. J Bacteriol. 1991 Nov;173(21):6783–6789. doi: 10.1128/jb.173.21.6783-6789.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fang F. C., Libby S. J., Buchmeier N. A., Loewen P. C., Switala J., Harwood J., Guiney D. G. The alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11978–11982. doi: 10.1073/pnas.89.24.11978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fierer J., Eckmann L., Fang F., Pfeifer C., Finlay B. B., Guiney D. Expression of the Salmonella virulence plasmid gene spvB in cultured macrophages and nonphagocytic cells. Infect Immun. 1993 Dec;61(12):5231–5236. doi: 10.1128/iai.61.12.5231-5236.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goethals K., Van Montagu M., Holsters M. Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1646–1650. doi: 10.1073/pnas.89.5.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grob P., Guiney D. G. In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR. J Bacteriol. 1996 Apr;178(7):1813–1820. doi: 10.1128/jb.178.7.1813-1820.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guiney D. G., Fang F. C., Krause M., Libby S., Buchmeier N. A., Fierer J. Biology and clinical significance of virulence plasmids in Salmonella serovars. Clin Infect Dis. 1995 Oct;21 (Suppl 2):S146–S151. doi: 10.1093/clinids/21.supplement_2.s146. [DOI] [PubMed] [Google Scholar]
  17. Guiney D. G., Fang F. C., Krause M., Libby S. Plasmid-mediated virulence genes in non-typhoid Salmonella serovars. FEMS Microbiol Lett. 1994 Nov 15;124(1):1–9. doi: 10.1111/j.1574-6968.1994.tb07253.x. [DOI] [PubMed] [Google Scholar]
  18. Guiney D. G., Libby S., Fang F. C., Krause M., Fierer J. Growth-phase regulation of plasmid virulence genes in Salmonella. Trends Microbiol. 1995 Jul;3(7):275–279. doi: 10.1016/s0966-842x(00)88944-1. [DOI] [PubMed] [Google Scholar]
  19. Gulig P. A., Danbara H., Guiney D. G., Lax A. J., Norel F., Rhen M. Molecular analysis of spv virulence genes of the Salmonella virulence plasmids. Mol Microbiol. 1993 Mar;7(6):825–830. doi: 10.1111/j.1365-2958.1993.tb01172.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Heffernan E. J., Fierer J., Chikami G., Guiney D. Natural history of oral Salmonella dublin infection in BALB/c mice: effect of an 80-kilobase-pair plasmid on virulence. J Infect Dis. 1987 Jun;155(6):1254–1259. doi: 10.1093/infdis/155.6.1254. [DOI] [PubMed] [Google Scholar]
  22. Hengge-Aronis R. Survival of hunger and stress: the role of rpoS in early stationary phase gene regulation in E. coli. Cell. 1993 Jan 29;72(2):165–168. doi: 10.1016/0092-8674(93)90655-a. [DOI] [PubMed] [Google Scholar]
  23. Hryniewicz M. M., Kredich N. M. Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium. J Bacteriol. 1995 May;177(9):2343–2353. doi: 10.1128/jb.177.9.2343-2353.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jeong W., Kang C. Start site selection at lacUV5 promoter affected by the sequence context around the initiation sites. Nucleic Acids Res. 1994 Nov 11;22(22):4667–4672. doi: 10.1093/nar/22.22.4667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kowarz L., Coynault C., Robbe-Saule V., Norel F. The Salmonella typhimurium katF (rpoS) gene: cloning, nucleotide sequence, and regulation of spvR and spvABCD virulence plasmid genes. J Bacteriol. 1994 Nov;176(22):6852–6860. doi: 10.1128/jb.176.22.6852-6860.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kowarz L., Robbe-Saule V., Norel F. Identification of cis-acting DNA sequences involved in the transcription of the virulence regulatory gene spvR in Salmonella typhimurium. Mol Gen Genet. 1996 May 23;251(2):225–235. doi: 10.1007/BF02172922. [DOI] [PubMed] [Google Scholar]
  27. Krause M., Fang F. C., Guiney D. G. Regulation of plasmid virulence gene expression in Salmonella dublin involves an unusual operon structure. J Bacteriol. 1992 Jul;174(13):4482–4489. doi: 10.1128/jb.174.13.4482-4489.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Krause M., Fang F. C., el-Gedaily A., Libby S., Guiney D. G. Mutational analysis of SpvR binding to DNA in the regulation of the Salmonella plasmid virulence operon. Plasmid. 1995 Jul;34(1):37–47. doi: 10.1006/plas.1995.1031. [DOI] [PubMed] [Google Scholar]
  29. Krause M., Roudier C., Fierer J., Harwood J., Guiney D. Molecular analysis of the virulence locus of the Salmonella dublin plasmid pSDL2. Mol Microbiol. 1991 Feb;5(2):307–316. doi: 10.1111/j.1365-2958.1991.tb02111.x. [DOI] [PubMed] [Google Scholar]
  30. Loewen P. C., Hengge-Aronis R. The role of the sigma factor sigma S (KatF) in bacterial global regulation. Annu Rev Microbiol. 1994;48:53–80. doi: 10.1146/annurev.mi.48.100194.000413. [DOI] [PubMed] [Google Scholar]
  31. Matsui H., Abe A., Suzuki S., Kijima M., Tamura Y., Nakamura M., Kawahara K., Danbara H. Molecular mechanism of the regulation of expression of plasmid-encoded mouse bacteremia (mba) genes in Salmonella serovar Choleraesuis. Mol Gen Genet. 1993 Jan;236(2-3):219–226. doi: 10.1007/BF00277116. [DOI] [PubMed] [Google Scholar]
  32. Norel F., Robbe-Saule V., Popoff M. Y., Coynault C. The putative sigma factor KatF (RpoS) is required for the transcription of the Salmonella typhimurium virulence gene spvB in Escherichia coli. FEMS Microbiol Lett. 1992 Dec 1;78(2-3):271–276. doi: 10.1016/0378-1097(92)90039-q. [DOI] [PubMed] [Google Scholar]
  33. Rhen M., Riikonen P., Taira S. Transcriptional regulation of Salmonella enterica virulence plasmid genes in cultured macrophages. Mol Microbiol. 1993 Oct;10(1):45–56. doi: 10.1111/j.1365-2958.1993.tb00902.x. [DOI] [PubMed] [Google Scholar]
  34. Schell M. A. Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol. 1993;47:597–626. doi: 10.1146/annurev.mi.47.100193.003121. [DOI] [PubMed] [Google Scholar]
  35. Spink J. M., Pullinger G. D., Wood M. W., Lax A. J. Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes. FEMS Microbiol Lett. 1994 Feb 1;116(1):113–121. doi: 10.1111/j.1574-6968.1994.tb06684.x. [DOI] [PubMed] [Google Scholar]
  36. Tanaka K., Takayanagi Y., Fujita N., Ishihama A., Takahashi H. Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8303–8303. doi: 10.1073/pnas.90.17.8303a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Toledano M. B., Kullik I., Trinh F., Baird P. T., Schneider T. D., Storz G. Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell. 1994 Sep 9;78(5):897–909. doi: 10.1016/s0092-8674(94)90702-1. [DOI] [PubMed] [Google Scholar]

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