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Factor of
Staphylococcus aureus
(Received for publication, May 21, 1996, and in revised form, June 27, 1996)
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
factor and is
poorly active in transcription assays. Based on amino acid sequence
homology of the Bacillus subtilis vegetative
factor
A and the predicted product of the chromosomally located
plaC gene of S. aureus, it was hypothesized
that plaC could encode the vegetative
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
SA, cross-reacted with the B. subtilis
anti-
A antibody. E. coli core RNAP,
reconstituted with
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
70.
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
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
factor in S. aureus.
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