Spo0A-dependent activation of an extended-10 region promoter in Bacillus subtilis

At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a Delta abrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the -10 region of the promoter was found to be similar to those of extended -10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended -10 region, as well as other base pairs in the -10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position -9 of Pskf produced a promoter that was active constitutively in both Delta abrB and Delta spo0A Delta abrB strains. Therefore, the base pair at position -9 of Pskf makes its activity dependent on Spo0A binding, and the extended -10 region motif of the promoter contributes to its high level of activity.