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Volume 271, Number 36, Issue of September 6, 1996 pp. 21828-21834
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the Primary sigma  Factor of Staphylococcus aureus

(Received for publication, May 21, 1996, and in revised form, June 27, 1996)

Rajendar Deora and Tapan K. Misra

From the Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612

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 sigma A 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 sigma SA, cross-reacted with the B. subtilis anti-sigma A antibody. E. coli core RNAP, reconstituted with sigma SA, 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 sigma 70. sigma SA, 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 sigma SA-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.


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