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J. Biol. Chem., Vol. 280, Issue 47, 38948-38956, November 25, 2005

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Binding of the Global Response Regulator Protein CovR to the sag Promoter of Streptococcus pyogenes Reveals a New Mode of CovR-DNA Interaction^*

From the Department of Microbiology and Immunology, Emory University, Atlanta, Georgia 30322

CovR (CsrR) is a response regulator of gene expression in Streptococcus pyogenes. It regulates 15% of the genome, including the genes encoding several streptococcal virulence factors, and acts primarily as a repressor rather than an activator of transcription. We showed that in vitro, CovR is sufficient to repress transcription from the sag promoter, which directs the expression of streptolysin S, a hemolysin that can damage the membranes of eukaryotic cells and subcellular organelles. Repression was stimulated 10-fold by phosphorylation of CovR with acetyl phosphate. In contrast to binding at the has and cov promoters, which direct the expression of genes involved in capsule biosynthesis and of CovR itself, binding of CovR to Psag was highly cooperative. CovR bound to two extended regions of Psag, an upstream region overlapping the -35 and -10 promoter elements and a downstream region overlapping the translation initiation signals of the sagA gene. Each of these regions contains only a single consensus CovR binding sequence, ATTARA, which at the has promoter defines individual sites to which CovR binds non-cooperatively. At Phas and Pcov the T residues in the sequence ATTARA are important for CovR binding. However, using uracil interference experiments we find that although the ATTARA sequence in the Psag upstream region contains thymine residues important for CovR binding, important thymine residues in the Psag downstream region are located outside this sequence. Furthermore, again in contrast to its behavior at the has and cov promoters where phosphorylation of CovR leads to a 2–3-fold increase in DNA binding affinity, binding of CovR to the sag promoter was stimulated 8–32-fold by phosphorylation. We suggest that these differences in CovR binding mean that individual promoters will be repressed at different intracellular levels of phosphorylated CovR, permitting differences in the response of members of the CovR regulon to environmental and internal metabolic signals.

Received for publication, June 6, 2005 , and in revised form, September 14, 2005.

^* This work was supported in part by Public Health Service Grant AI20723 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Institutes of Health Training Grant T32-AI07470 and a United Negro College Foundation-Merck Graduate Science Research Dissertation Fellowship.

² To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-2538; Fax: 404-727-3659; E-mail: ggchurc{at}microbio.emory.edu.

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