Expression of the Secondary Sigma Factor σ X in Streptococcus pyogenes Is Restricted at Two Levels

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Abstract
Secondary RNA polymerase sigma factors in many bacteria are responsible for regulating a vast range of processes including virulence. A protein (σ 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 σ X functions in GAS cells, and show that the amount of σ 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 σ 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 σ 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 σ X protein, which resulted in higher levels of transcription from the σ 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 σ X -dependent cinA promoter than did the wild-type parental strain. Therefore, σ X activity in GAS is limited by low-level transcription of the sigX structural genes and by clpP , which appears to negatively regulate σ X accumulation.