| Secondary sigma factors are global regulators of transcription in most bacteria, however no secondary sigma factor has previously been identified in the Gram positive pathogen Streptococcus pyogenes. The annotated genome sequence of GAS contains two identical open reading frames encoding a protein that is 40% identical to ComX in Streptococcus pneumoniae, which functions as a secondary sigma factor. The GAS protein, which we call σX, was cloned and purified from Escherichia coli. σX was tested in vitro to determine if it functions as a secondary sigma factor. We demonstrate in vitro that σX directs Bacillus subtilis core RNA polymerase to transcribe GAS promoters; these are similar to the cin-box sequence in S. pneumoniae, which is necessary for ComX directed expression in vivo. The σX-dependent transcripts initiate directly downstream from the cin-box sequence, and two individual point mutations in this conserved sequence abolish the ability of σX RNA polymerase to use these promoters. A GAS strain containing a deletion of both copies of sigX was isolated, and we found that sigX is dispensable for growth under standard laboratory conditions. Primer extension analysis indicates that sigX transcription is low in GAS cells when grown in Todd Hewitt Yeast broth, and immuno-blot assays with a σX-specific polyclonal antibody demonstrate that the protein does not accumulate in GAS cells. A strain constitutively expressing sigX was constructed, and transcription from cin-box promoters was studied. We demonstrate that constitutive expression of sigX in GAS cells is sufficient for transcription from cinbox promoters, cinA, smf and cglA. A clpP mutant constructed in the sigX over expression strain leads to increased accumulation of the σX protein, which in turn results in higher levels of transcription from σX-dependent promoters. A clpP mutant containing sigX only at its wild-type locus on the chromosome produces a small amount of transcript from the σX-dependent cinA promoter. Therefore, σ X activity in GAS is limited by low level transcription of the sigX structural genes and clpP appears to negatively regulate σX accumulation. |
