Spo0A-Dependent Activation of an Extended −10 Region Promoter in Bacillus subtilis

Abstract

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, P skf , controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of P skf 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 P skf activity in a Δ abrB strain. Here we investigate the mechanism of P skf 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 P skf produced a promoter that was active constitutively in both Δ abrB and Δ spo0A Δ abrB strains. Therefore, the base pair at position −9 of P skf makes its activity dependent on Spo0A binding, and the extended −10 region motif of the promoter contributes to its high level of activity.