The initiation of sporulation in Bacillus subtilis is regulated by the phosphorelay signal transduction system. The goal of the phosphorelay is the production of SpoOA~P. The level of SpoOA~P reflects the recognition and integration of a series of metabolic, environmental and cell cycle signals which regulates the initiation of sporulation. A family of aspartate-phosphatases (Rap) provides a mechanism for regulating the phosphate flow in the phosphorelay by specifically dephosphorylating the SpoOF~P intermediate response regulator. Rap phosphatases are induced by physiological states which are alternative to sporulation. RapA activity is controlled by the product of the phrAgene. The PhrA C-terminal pentapeptide directly inhibts RapA phosphatase activity. Production of the pentapeptide inhibitor is regulated by an export-import control circuit which yields the pentapeptide inhibitor form internalized by the OppA system. The focus of this proposal is the characterization of the RapA-PhrA system modulating the phosphorelay. The role of the export-import circuit that controls the formation of active peptide inhibitor will be investigated by characterization of the enzymes involved in peptide processing. We will investigate whether a regulatory hierarchy is the basis for timing modulation of phosphatase activity by the PhrA peptide. The enzymes involved in peptide processing will be identified and characterized. Peptide localization studies will be carried out in order to define the peptide role in cell communication. The interaction of PhrA peptide with RapA will be investigated biochemically via competitive binding assays and surface plasmon resonance, and genetically by generating RapA-RapB hybrid proteins. The mechanisms of interaction of RapA with SpoOF will be studied by the surface plasmon resonance method and with a genetic approach. Genetic and biochemical approaches will be used for the identification of the target of RapC.
