Sporulation is a mechanism that microorganisms have developed for survival under adverse conditions. The process follows an energy-intensive pathway with the activation or repression of numerous specific genes in a temporal and spatial sequence resulting in the production of a highly differentiated structure. The phosphorelay is a complex signal transduction system that governs the onset of sporulation initiation in Bacillus subtilis by responding to a myriad of positive and negative signals and integrating them into the most appropriate response. Negative inputs on the response regulators SpoOF and Spo0A of the phosphorelay are interpreted by the Rap and Spo0E families of phosphatases, respectively. The phosphatase genes are induced by physiological conditions antithetical to sporulation. Additionally, each Rap activity is inhibited by a specific pentapeptide which is generated from the processing of corresponding phr gene products. The aim of this proposal is the detailed molecular, biochemical andI structural characterization of phosphatase activities and Phr peptide processing and inhibition. Additionally, we propose experiments that will provide a global view of the complex regulatory network that governs the cell's physiology through the action of the phosphatases. The widespread presence of Rap and Spo0E phosphatases among Gram-positive bacilli will allow our findings to be extrapolated to poorly understood pathogenic species, for which a better knowledge of basic physiological mechanisms seems to be an urgent need. Genetic and biochemical approaches are proposed with the aim of identifying the enzyme(s) involved in Phr peptide processing. The kinetic parameters of RapA interaction with its inhibitor PhrA and its substrate Spo0F-P will be investigated by biochemical means that will allow us to define the mechanism of pentapeptide inhibition. The proteins of the Spo0E family of phosphatases will be biochemically characterized and the genetic and physiological factors controlling their genes' transcription investigated. The role of the Rap phosphatases not involved in sporulation will be investigated by microbiological and genetic approaches