Alternative transcription factor sigma(SB) of Staphylococcus aureus: Characterization and role in transcription of the global regulatory locus sar

A homolog of the multiple-stress-responsive transcription factor sigma(B) of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter, A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated sigma(SB), indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis sigma(B)-dependent P-B promoter of the sigB operon was stimulated by sigma(SB) in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted sigma(B)-dependent P-B promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins, Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary sigma factor sigma(SA), while that of the P3 promoter is dependent on sigma(SB). As determined by primer extension studies, the 5' end of the sigma(SB)-initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5' end of the cellular RNA.