Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in in response to cell-wall-active antibiotics. In the present study, the gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of was studied by assaying β-galactosidase activity in an promoter:: fusion strain and by Northern blot analysis. Transcription of was increased by oxacillin; but not by a variety of other stresses including HO. Northern blot analysis revealed that the size of the transcript was 23 kb, considerably larger than the 531 nt ORF. The transcription start site was mapped 25 nt upstream of the start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of . The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the downstream region indicated that the was transcribed as part of a polycistronic message. Interestingly, purified PilB was shown to possess ∼∼28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to HO compared to the parent strain, but not to oxacillin.


Article metrics loading...

Loading full text...

Full text loading...



  1. Abrams, W. R., Weinbaum, G., Weissbach, L., Weissbach, H. & Brot, N. (1981). Enzymatic reduction of oxidized alpha-1-proteinase inhibitor restores biological activity. Proc Natl Acad Sci USA 78, 7483-7486.[CrossRef] [Google Scholar]
  2. Augustin, J., Rosenstein, R., Wieland, B., Schneider, U., Schnell, N., Engelke, G., Entian, K. D. & Götz, F. (1992). Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur J Biochem 204, 1149-1154.[CrossRef] [Google Scholar]
  3. 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]
  4. Hassouni, M. E., Chambost, J. P., Expert, D., Gijsegem, F. V. & Barras, F. (1999). The minimal gene set member msrA, encoding peptide methionine sulfoxide reductase, is a virulence determinant of the plant pathogen Erwinia chrysanthemi. Proc Natl Acad Sci USA 96, 887-892.[CrossRef] [Google Scholar]
  5. Hayes, C. S., Illades-Aguiar, B., Casillas-Martinez, L. & Setlow, P. (1998).In vitro and in vivo oxidation of methionine residues in small, acid-soluble spore proteins from Bacillus species. J Bacteriol 180, 2694-2700. [Google Scholar]
  6. Horsburgh, M. J., Clements, M. O., Crossley, H., Ingham, E. & Foster, S. J. (2001). PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus. Infect Immun 69, 3744-3754.[CrossRef] [Google Scholar]
  7. Kili, A. O., Herzberg, M. C., Meyer, M. W., Zhao, X. & Tao, L. (1999). Streptococcal reporter gene-fusion vector for identification of in vivo expressed genes. Plasmid 42, 67-72.[CrossRef] [Google Scholar]
  8. Kreiswirth, B. N., Lofdahl, 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]
  9. Kuroda, M., Ohta, T., Uchiyama, I. & 34 other authors (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357, 1225–1240.[CrossRef] [Google Scholar]
  10. Levine, R. L., Berlett, B. S., Moskovitz, J., Mosoni, L. & Stadtman, E. R. (1999). Methionine residues may protect proteins from critical oxidative damage.Mech Ageing Dev 107, 323-332.[CrossRef] [Google Scholar]
  11. Lowther, W. T., Brot, N., Weissbach, H. & Matthews, B. W. (2000). Structure and mechanism of peptide methionine sulfoxide reductase, an ‘anti-oxidation’ enzyme. Biochemistry 39, 13307-13312.[CrossRef] [Google Scholar]
  12. Mead, D. A., Szczesna-Skorupa, E. & Kemper, B. (1986). Single-stranded DNA ‘blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng 1, 67-74.[CrossRef] [Google Scholar]
  13. Mei, J. M., Nourbakhsh, F., Ford, C. W. & Holden, D. W. (1997). Identification of Staphylococcus aureus virulence genes in a murine model of bacteraemia using signature-tagged mutagenesis. Mol Microbiol 26, 399-407.[CrossRef] [Google Scholar]
  14. Miller, J. M. (1972).Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  15. Moreno, J. J. & Pryor, W. A. (1992). Inactivation of alpha 1-proteinase inhibitor by peroxynitrite. Chem Res Toxicol 5, 425-431.[CrossRef] [Google Scholar]
  16. Moskovitz, J., Rahman, M. A., Strassman, J., Yancey, S. O., Kushner, S. R., Brot, N. & Weissbach, H. (1995).Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J Bacteriol 177, 502-507. [Google Scholar]
  17. Moskovitz, J., Weissbach, H. & Brot, N. (1996). Cloning and expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins. Proc Natl Acad Sci USA 93, 2095-2099.[CrossRef] [Google Scholar]
  18. Moskovitz, J., Berlett, B. S., Poston, J. M. & Stadtman, E. R. (1997). The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo. Proc Natl Acad Sci USA 94, 9585-9589.[CrossRef] [Google Scholar]
  19. Moskovitz, J., Flescher, E., Berlett, B. S., Azare, J., Poston, J. M. & Stadtman, E. R. (1998). Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress. Proc Natl Acad Sci USA 95, 14071-14075.[CrossRef] [Google Scholar]
  20. Moskovitz, J., Berlett, B. S., Poston, J. M. & Stadtman, E. R. (1999). Methionine sulfoxide reductase in antioxidant defense. Methods Enzymol 300, 239-244. [Google Scholar]
  21. Moskovitz, J., Poston, J. M., Berlett, B. S., Nosworthy, N. J., Szczepanowski, R. & Stadtman, E. R. (2000). Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity. J Biol Chem 275, 14167-14172.[CrossRef] [Google Scholar]
  22. Novick, R. P. (1991). Genetic systems in staphylococci.Methods Enzymol 202, 587-636. [Google Scholar]
  23. Novick, R. P., Edelman, I. & Lofdahl, S. (1986). Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes. J Mol Biol 192, 209-220.[CrossRef] [Google Scholar]
  24. Pfeltz, R. F., Singh, V. K., Schmidt, J. L., Batten, M. A., Baranyk, C. S., Nadakavukaren, M. J., Jayaswal, R. K. & Wilkinson, B. J. (2000). Characterization of passage-selected vancomycin-resistant Staphylococcus aureus strains of diverse parental backgrounds. Antimicrob Agents Chemother 44, 294-303.[CrossRef] [Google Scholar]
  25. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  26. Schenk, S. & Laddaga, R. A. (1992). Improved methods for electroporation of Staphylococcus aureus. FEMS Microbiol Lett 94, 133-138.[CrossRef] [Google Scholar]
  27. Singh, V. K., Jayaswal, R. K. & Wilkinson, B. J. (2001). Cell wall-active antibiotic induced proteins of Staphylococcus aureus identified using a proteomic approach. FEMS Microbiol Lett 199, 79-94. [Google Scholar]
  28. Sun, H., Gao, J., Ferrington, D. A., Biesiada, H., Williams, T. D. & Squier, T. C. (1999). Repair of oxidized calmodulin by methionine sulfoxide reductase restores ability to activate the plasma membrane Ca-ATPase. Biochemistry 38, 105-112.[CrossRef] [Google Scholar]
  29. Taha, M. K., Larribe, M., Dupuy, B., Giorgini, D. & Marchal, C. (1992). Role of pilA, an essential regulatory gene of Neisseria gonorrhoeae, in the stress response. J Bacteriol 174, 5978-5981. [Google Scholar]
  30. Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988). Pilin expression in Neisseria gonorrhoeae is under both positive and negative transcriptional control. EMBO J 7, 4367-4378. [Google Scholar]
  31. Vogt, W. (1995). Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radic Biol Med 18, 93-105.[CrossRef] [Google Scholar]
  32. Vriesema, A. J., Dankert, J. & Zaat, S. A. (2000). A shift from oral to blood pH is a stimulus for adaptive gene expression of Streptococcus gordonii CH1 and induces protection against oxidative stress and enhanced bacterial growth by expression of msrA. Infect Immun 68, 1061-1068.[CrossRef] [Google Scholar]
  33. Wizemann, T. M., Moskovitz, J., Pearce, B. J., Cundell, D., Arvidson, C. G., So, M., Weissbach, H., Brot, N. & Masure, H. R. (1996). Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens. Proc Natl Acad Sci USA 93, 7985-7990.[CrossRef] [Google Scholar]
  34. Xiong, A. & Jayaswal, R. K. (1998). Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococcus aureus. J Bacteriol 180, 4024-4029. [Google Scholar]
  35. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error