Expression of the secondary sigma factor σX in Streptococcus pyogenes is restricted at two levels

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.