Characterization of the primary sigma factor of Staphylococcus aureus

• Published in: The Journal of biological chemistry 1996-09, Vol.271 (36), p.21828-21834
• Main Authors: Deora, R, Misra, T K
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Base Sequence; Blotting, Western; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; Dose-Response Relationship, Drug; Escherichia coli; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Molecular Weight; N-Acetylmuramoyl-L-alanine Amidase - metabolism; Promoter Regions, Genetic; Sigma Factor - metabolism; Staphylococcus aureus - enzymology; Staphylococcus aureus - genetics; Transcription, Genetic
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.36.21828

RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays. Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S. aureus, it was hypothesized that plaC could encode the vegetative sigma factor. We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE. The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography. The purified protein, designated sigmaSA, cross-reacted with the B. subtilis anti-sigmaA antibody. E. coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S. aureus promoters hla, sea, and sec and from the E. coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E. coli sigma70. sigmaSA, when added to the purified RNAP from S. aureus, stimulated transcriptional activity of the RNAP up to 72-fold. As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs. Disruption of the plaC gene on the S. aureus chromosome was lethal. We conclude that plaC encodes the primary sigma factor in S. aureus.