Mechanistic studies on the activation of a cell-specific transcription factor in Bacillus subtilis

Regulation of gene transcription often involves complex mechanisms that control the activity of individual transcription factors. This thesis addresses the question of how a cell-specific transcription factor is activated during sporulation in B. subtilis. The process of sporulation commences with the conversion of a growing cell into two dissimilar-sized compartments called the mother cell and the forespore. A series of four cell-specific transcription factors are activated in a spatially and temporally restricted fashion in one or the other compartment to orchestrate gene expression during the course of development. This thesis primarily focuses on the mechanism by which a cell-specific transcription factor called σF is activated in the forespore, but also examines the timing of activation of the other cell-specific transcription factors.; The activation of σF is regulated by an inhibitory protein, the antisigma factor SpoIIAB, which binds to σF to hold it in an inactive complex of stoichiometry 2 to l. The σ F factor is released from SpoIIAB in the forespore by the action of the anti-antisigma factor SpoIIAA. Moreover, SpoIIAB is also a protein kinase that inactivates SpoIIAA by phosphorylating it on a serine residue. I present evidence based on the construction of heterodimeric mutant forms of SpoIIAB that support a docking model for the release of σF. In the model, SpoIIAA docks on one of the two subunits of SpoIIAB in the SpoIIAB ₂:σF₁ complex, displacing σ F from the complex. I also present evidence that phosphorylation is not required for the displacement of σF and possibly occurs after σF has been released.; This thesis also examines the timing of transcription factor activation by utilizing fusions to genes for fluorescent proteins GFP and RFP in concert with fluorescent microscopy. This assay has made it possible to monitor the relative timing of sigma factor activation in individual cells. The results show that σG is activated in the forespore approximately one hour later than σF.