1887

Abstract

A  : :  reporter was constructed in strains Newman and 8325-4, whereby the level of tetracycline resistance reflected the activity of the promoter. Wild-type strains carrying a single copy of this construct exhibited a low level of tetracycline resistance, suggesting that the promoter is weak. Spontaneous mutants that grew at higher tetracycline concentrations were isolated. Some were due to point mutations in the promoter that led to increased expression of the gene. The promoter was identified by primer extension analysis and −35 and −10 elements were assigned. The promoter regions from the tetracycline-resistant mutants were sequenced and several had base changes within or adjacent to the −35 box. Three created the consensus −35 sequence TTGACA. The mutant promoters were fused to . β-Galactosidase activity was six- to ninefold higher in the mutant strains compared to the wild-type. The wild-type gene was placed under the control of the mutant promoters. ClfB expression was higher than the corresponding wild-type strains and the protein was present on bacteria from the stationary phase instead of being confined to the exponential phase. Therefore, mutations in the promoter that cause changes in the −35 region produce a stronger promoter that is capable of increased transcription and, as a result, increased expression of ClfB.

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2003-01-01
2019-10-14
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References

  1. Barne, K. A., Bown, J. A., Busby, S. J. W. & Minchin, S. D. ( 1997; ). Region 2·5 of the Escherichia coli RNA polymerase σ70 subunit is responsible for the recognition of the ‘extended −10’ motif at promoters. EMBO J 16, 4034–4040.[CrossRef]
    [Google Scholar]
  2. Brunner, M. & Bujard, H. ( 1987; ). Promoter recognition and promoter strength in the Escherichia coli system. EMBO J 6, 3139–3144.
    [Google Scholar]
  3. Busby, S. & Ebright, R. H. ( 1994; ). Promoter structure, promoter recognition, and transcription activation in prokaryotes. Cell 79, 743–746.
    [Google Scholar]
  4. Chan, P. F., Foster, S. J., Ingham, E. & Clements, M. O. ( 1998; ). The Staphylococcus aureus alternative sigma factor σB controls the environmental stress response but not starvation survival or pathogenicity in a mouse abscess model. J Bacteriol 180, 6082–6089.
    [Google Scholar]
  5. Cheung, A. L., Eberhardt, K. J. & Fischetti, V. A. ( 1994; ). A method to isolate RNA from gram-positive bacteria and mycobacteria. Anal Biochem 222, 511–514.[CrossRef]
    [Google Scholar]
  6. Cheung, A. L., Schmidt, K., Bateman, B. & Manna, A. C. ( 2001; ). SarS, a SarA homolog repressible by agr, is an activator of protein A synthesis in Staphylococcus aureus. Infect Immun 69, 2448–2455.[CrossRef]
    [Google Scholar]
  7. Deora, R. & Misra, T. K. ( 1996; ). Characterization of the primary sigma factor of Staphylococcus aureus. J Biol Chem 271, 21828–21834.[CrossRef]
    [Google Scholar]
  8. Deora, R., Tseng, T. & Misra, T. K. ( 1997; ). Alternative transcription factor σSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar. J Bacteriol 179, 6355–6359.
    [Google Scholar]
  9. Duthie, E. S. & Lorenz, L. L. ( 1952; ). Staphylococcal coagulase: mode of action and antigenicity. J Gen Microbiol 6, 95–107.[CrossRef]
    [Google Scholar]
  10. Foster, T. J. ( 1998; ). Molecular genetic analysis of staphylococcal virulence. Methods Microbiol 27, 433–454.
    [Google Scholar]
  11. Harley, C. B. & Reynolds, R. P. ( 1987; ). Analysis of E. coli promoter sequences. Nucleic Acids Res 15, 2343–2361.[CrossRef]
    [Google Scholar]
  12. Hartford, O., O'Brien, L., Schofield, K., Wells, J. & Foster, T. J. ( 2001; ). The Fbe (SdrG) protein of Staphylococcus epidermidis HB promotes bacterial adherence to fibrinogen. Microbiology 147, 2545–2552.
    [Google Scholar]
  13. Hawley, D. K. & McClure, W. R. ( 1983; ). Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 11, 2237–2255.[CrossRef]
    [Google Scholar]
  14. Höök, M. & Foster, T. J. ( 2000; ). Staphylococcal surface proteins. In Gram-Positive Pathogens. Edited by V. A. Fischetti, R. P. Novick, J. J. Feretti, D. A. Portnoy & J. I. Rood. Washington, DC: American Society for Microbiology.
  15. Josaitis, C. A., Gaal, T., Ross, W. & Gourse, R. L. ( 1990; ). Sequences upstream of the −35 hexamer of rrnB P1 affect promoter strength and upstream activation. Biochim Biophys Acta 1050, 307–311.[CrossRef]
    [Google Scholar]
  16. Kaatz, G. W., Seo, S. M. & Ruble, C. A. ( 1993; ). Efflux-mediated fluoroquinolone resistance in Staphylococcus aureus. Antimicrob Agents Chemother 37, 1086–1094.[CrossRef]
    [Google Scholar]
  17. Kobayashi, N., Taniguchi, K. & Urasawa, S. ( 1998; ). Analysis of diversity of mutations in the mecI gene and mecA promoter/operator region of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother 42, 717–720.
    [Google Scholar]
  18. Kreiswirth, B. N., Löfdahl, S., Betley, M. J., O'Reilly, M., Schlievert, P. M., Bergdoll, M. S. & Novick, R. P. ( 1983; ). The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305, 709–712.[CrossRef]
    [Google Scholar]
  19. Kullik, I., Giachino, P. & Fuchs, T. ( 1998; ). Deletion of the alternative sigma factor σB in Staphylococcus aureus reveals its function as a global regulator of virulence genes. J Bacteriol 180, 4818–4820.
    [Google Scholar]
  20. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  21. LeClerc, J. E. & Istock, N. L. ( 1982; ). Specificity of UV mutagenesis in the lac promoter of M13/lac hybrid phage DNA. Nature 292, 596–598.
    [Google Scholar]
  22. Maguin, E., Duwat, P., Hege, T., Ehrlich, D. & Gruss, A. ( 1992; ). New thermosensitive plasmid for gram-positive bacteria. J Bacteriol 174, 5633–5638.
    [Google Scholar]
  23. McAleese, F. M., Walsh, E. J., Sieprawska, M., Potempa, J. & Foster, T. J. ( 2001; ). Loss of clumping factor B fibrinogen binding activity by Staphylococcus aureus involves cessation of transcription, shedding and cleavage by metalloprotease. J Biol Chem 276, 29969–29978.[CrossRef]
    [Google Scholar]
  24. McDevitt, D., Wann, E. R. & Foster, T. J. ( 1993; ). Recombination at the coagulase locus in Staphylococcus aureus: plasmid integration and amplification. J Gen Microbiol 139, 695–706.[CrossRef]
    [Google Scholar]
  25. McDevitt, D., Francois, P., Vaudaux, P. E. & Foster, T. J. ( 1994; ). Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol Microbiol 11, 237–248.[CrossRef]
    [Google Scholar]
  26. McNamara, P. J., Milligan-Monroe, K. C., Khalili, S. & Proctor, R. A. ( 2000; ). Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus. J Bacteriol 182, 3197–3203.[CrossRef]
    [Google Scholar]
  27. Meyer, B. J., Maurer, R. & Ptashne, M. ( 1980; ). Gene regulation at the right operator (OR) of bacteriophage lambda. II. OR1, OR2, and OR3: their roles in mediating the effects of repressor and cro. J Mol Biol 139, 163–194.[CrossRef]
    [Google Scholar]
  28. Moran, C. P., Jr, Lang, N., LeGrice, S. F. J., Lee, G., Stephens, M., Sonenshein, A. L., Pero, J. & Losick, R. ( 1982; ). Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet 186, 339–346.[CrossRef]
    [Google Scholar]
  29. Ní Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Höök, M. & Foster, T. J. ( 1998; ). Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus. Mol Microbiol 30, 245–257.[CrossRef]
    [Google Scholar]
  30. Novick, R. P. ( 1967; ). Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology 33, 155–166.[CrossRef]
    [Google Scholar]
  31. Ouyang, S., Sau, S. & Lee, C. Y. ( 1999; ). Promoter analysis of the cap8 operon, involved in type 8 capsular polysaccharide production in Staphylococcus aureus. J Bacteriol 181, 2492–2500.
    [Google Scholar]
  32. Patti, J. M., Allen, B. A., McGavin, M. J. & Höök, M. ( 1994).; MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 45 , 585 –617.
    [Google Scholar]
  33. Ponnambalam, S., Webster, C., Bingham, A. & Busby, S. ( 1986; ). Transcription initiation at the Escherichia coli galactose operon promoters in the absence of the normal −35 region sequences. J Biol Chem 261, 16043–16048.
    [Google Scholar]
  34. Pribnow, D. ( 1975; ). Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. J Mol Biol 99, 419–443.[CrossRef]
    [Google Scholar]
  35. Record, M. T., Reznikoff, W. S., Craig, M. L., McQuade, K. L. & Schlax, P. J. ( 1996; ). Escherichia coli RNA polymerase (Eσ70), promoters, and the kinetics of the steps of transcription initiation. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, vol. I, pp. 792–820. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
  36. Ross, W., Gosink, K. K., Salomon, J., Igarashi, K., Zou, C., Ishihama, A., Severinov, K. & Gourse, R. L. ( 1993; ). A third recognition element in bacterial promoters: DNA binding by the α-subunit of RNA polymerase. Science 262, 1407–1413.[CrossRef]
    [Google Scholar]
  37. Salim, B. S., Dunman, P. M., McAleese, F. M. & 7 other authors. ( 2003; ). The role of Rot in the regulatory network of Staphylococcus aureus virulence genes. J Bacteriol (in press).
  38. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  39. Schaller, H., Gray, C. & Herrman, K. ( 1975; ). Nucleotide sequence of an RNA polymerase binding site from the DNA of bacteriophage fd. Proc Natl Acad Sci U S A 72, 737–741.[CrossRef]
    [Google Scholar]
  40. Takanami, M., Sugimoto, K., Sugisaki, H. & Okamoto, T. ( 1976; ). Sequence of promoter for coat protein gene of bacteriophage fd. Nature 260, 297–302.[CrossRef]
    [Google Scholar]
  41. Tegmark, K., Karlsson, A. & Arvidson, S. ( 2000; ). Identification and characterisation of SarH1, a new global regulator of virulence gene expression in Staphylococcus aureus. Mol Microbiol 37, 398–409.[CrossRef]
    [Google Scholar]
  42. Waldvogel, F. A. ( 1995; ). In Principles and Practice of Infectious Diseases, 4th edn, pp. 1754–1777. Edited by G. L. Mandel, J. E. Bennett & R. Dolin. New York: Churchill Livingstone.
  43. Wann, E. R., Gurusiddappa, S. & Höök, M. ( 2000; ). The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen. J Biol Chem 275, 13863–13871.[CrossRef]
    [Google Scholar]
  44. Zhang, S. & Stewart, G. C. ( 2000; ). Characterization of the promoter elements for the staphylococcal enterotoxin D gene. J Bacteriol 182, 2321–2325.[CrossRef]
    [Google Scholar]
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