1887

Abstract

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.

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2001-11-01
2019-12-08
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