The signal transduction pathway for the initiation of sporulation in Bacillus subtilis consists of a sequential series of phosphorylation reactions, termed a """"""""phosphorelay"""""""", catalyzed by two kinases, KinA and KinB, and a phosphoprotein phosphotransferase coded for by the spoOB gene. The end product of the phosphorelay is the phosphorylation of the SpoOA transcription factor which activates it to repress certain genes and activate the transcription of others. The genes controlled by SpoOA control whether the cell divides or initiates the sporulation process. The actual signal transduction event is activation of either kinase to phosphorylate itself in an autophosphorylation reaction. This is followed by a series of phosphotransfer reactions where the phosphate group is transferred from kinase to SpoOF. SpoOF-P is the substrate for a phosphoprotein phosphotransferase, SpoOB, which removes the phosphate group from SpoOF and transfers it to SpoOA. This phosphorelay is controlled by several mechanisms. The level of the SpoOA and SpoOF components of the relay are controlled by the level of SpoOA-P in a positive feedback loop. The flux of phosphate through the pathway is controlled in some unknown way by the product of the spoOE gene. Finally, the kinases which are activated to kick off the flow of phosphate to the phosphorelay are either activated or inhibited by metabolic and/or environmental effectors of unknown origin. The focus of this proposal is to study the control of the phosphorelay. Effectors for KinA and KinB will be sought in order to understand the incoming signals that activate the phosphorelay. Negative control of sporulation by the spoOE gene will be studied by producing quantities of SpoOE protein to test in vitro the activity of this protein on the component reactions of the phosphorelay. Further mutants will be isolated that identify genes for negative regulatory components on this pathway. The relationship of a GTP-binding protein, Obg, to the phosphorelay will be investigated. Mutants will be obtained in Obg, including a temperature-sensitive mutant and mutants in the GTP-binding site with predictable characteristics. The oligopeptide transport system also is required for the initiation of sporulation, although the actual peptide being transported is obscure. A mutant search has been proposed that will isolate suppressors of this pathway, allowing the identification of the site of action of this system. A search will be carried out to identify all genes positively controlled by SpoOA-P.
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