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Journal of Bacteriology, August 2003, p. 4291-4297, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4291-4297.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Expression of the Secondary Sigma Factor {sigma}X in Streptococcus pyogenes Is Restricted at Two Levels

Jason A. Opdyke, June R. Scott, and Charles P. Moran, Jr.*

Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322

Received 25 March 2003/ Accepted 9 May 2003

Secondary RNA polymerase sigma factors in many bacteria are responsible for regulating a vast range of processes including virulence. A protein ({sigma}X) in the gram-positive human pathogen Streptococcus pyogenes (the group A Streptococcus or GAS) was recently shown to function in vitro as a secondary sigma factor. We report here the isolation of a mutant in which both sigX genes are inactivated, show that {sigma}X functions in GAS cells, and show that the amount of {sigma}X is controlled at two levels. Primer extension analysis indicates that sigX transcription is low in GAS cells grown in Todd-Hewitt yeast broth, and immunoblot assays with a {sigma}X-specific polyclonal antibody demonstrate that the protein does not accumulate in these cells. To increase the level of sigX transcription in GAS, we constructed a strain that constitutively expresses the sigX gene from a heterologous promoter. Expression of sigX from this promoter led to transcription of the {sigma}X-dependent cinA promoter in GAS cells. We found that expression of the sigX gene in a clpP mutant strain resulted in greater accumulation of {sigma}X protein, which resulted in higher levels of transcription from the {sigma}X-dependent promoters cinA, smf, and cglA. In addition, a clpP mutant containing sigX only at its wild-type loci on the chromosome generated more transcription from the {sigma}X-dependent cinA promoter than did the wild-type parental strain. Therefore, {sigma}X activity in GAS is limited by low-level transcription of the sigX structural genes and by clpP, which appears to negatively regulate {sigma}X accumulation.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322. Phone: (404) 727-5969. Fax: (404) 727-3659. E-mail: moran{at}microbio.emory.edu.

Journal of Bacteriology, August 2003, p. 4291-4297, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4291-4297.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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