SarA represses agr operon expression in a purified in vitro Staphylococcus aureus transcription system - PubMed (original) (raw)

SarA represses agr operon expression in a purified in vitro Staphylococcus aureus transcription system

S K Chakrabarti et al. J Bacteriol. 2000 Oct.

Abstract

Mutation and genetic complementation studies suggested that two chromosomal loci, agr and sar, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner in Staphylococcus aureus. We purified recombinant T7-tagged SarA from Escherichia coli and determined its effect on transcription from several S. aureus promoters by using purified RNA polymerase reconstituted with either sigma(A) or sigma(B) from S. aureus. Of the seven sigma(A)-dependent promoters that we tested, SarA repressed transcription from agrP2, agrP3, cna, sarP1, and sea promoters and did not affect sec and znt promoters. Furthermore, SarA had no effect on transcription from the sigma(B)-dependent sarP3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.

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Figures

FIG. 1

FIG. 1

Binding of SarA to the cna promoter. EMSA was done as described in the text.

FIG. 2

FIG. 2

Effect of SarA protein on transcription from the cna promoter. pMP7-cna and pMP7-zntPR DNA (Table 1) were used as templates for the determination of transcriptional activities from the cna and znt promoters (indicated at the top), respectively. The specific transcripts are indicated by arrows. Lane M, RNA markers (sizes are in nucleotides).

FIG. 3

FIG. 3

SarA protein affects transcription of several specific ςA-dependent promoters in a dose-dependent manner. Plasmid DNA containing the specific promoter along with a partial or complete gene adjacent to the promoter, as indicated in Table 1, was used as the template. The different promoter DNAs used in each lane are noted at the top. The specific transcripts are indicated by arrows. Lane M, RNA markers (sizes are in nucleotides).

FIG. 4

FIG. 4

Effect of SarA on transcription from the sar promoters. pALC561 DNA containing the entire sar operon was used as the template. pMP7-zntPR DNA was used as a control in lanes 1 to 4. RNA polymerase reconstituted with either ςA (lanes 1 to 4) or ςB (lanes 5 to 7) from S. aureus, in the presence of different concentrations of SarA, was used for the transcription reactions. Lane M, RNA markers (sizes are in nucleotides).

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