Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin

Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790, USA 1 Laboratory of Biochemistry, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20982, USA 2 Author for correspondence: Radheshyam K. Jayaswal. Tel: +1 309 438 5128. Fax: +1 309 438 3722. e-mail: drjay{at}ilstu.edu Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying ß-galactosidase activity in an msrA promoter:: lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H 2 O 2 . Northern blot analysis revealed that the size of the msrA transcript was 2·3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA . 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 msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus 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 H 2 O 2 compared to the parent strain, but not to oxacillin. Keywords: PilB, oxidative stress, MsrA, antibiotic stress Abbreviations: MBC, minimal bactericidal concentration; Met(O), methionine sulfoxide