The process of sporulation in Bacillus subtilis will be used as a model system for investigating the mechanisms that control the expression of developmentally regulated genes in bacteria. Sporulation occurs in response to nutritional stress, and the initiation of sporulation is somehow controlled by the products of seven genetic loci called the spo0 loci. Of critical importance is the product of spo0A, which is believed in some way to be the agent through which the sensing of nutritional stress is transduced into a signal to activate the transcription of genes involved in the earliest stages of sporulation. The functional nature of the spo0A gene product is unknown, however, and the identity of proteins that interact directly with it to influence the initiation of sporulation remains to be established. Novel genetic approaches will be used to investigate the mechanistic nature of the spo0A protein, including the systematic isolation of mutants that sporulate efficiently under nutritional circumstances that normally inhibit sporulation very strongly. Among sporulation- enhancing mutations already characterized are missense changes in the spo0A gene itself. An attempt will be made to identify genes whose products interact with the spo0A protein by isolating extragenic suppressors of these mutations, taking advantages of a sensitive visual screen for phenotypic reversion involving the use of sporulation-specific lacZ fusions. Morphogenetic changes that take place during the middle and later stages of sporulation require the products of many additional genes. These genes are apparently activated in a temporally programmed sequence, and little is understood about the molecular mechanisms that control their activation. The nature of these mechanisms will be investigated through the detailed study of the spoIIE promoter, a very strongly expressed promoter that becomes transcriptionally active during the second hour of sporulation. A series of genetic and biochemical experiments will be carried out to identify and characterize possible trans-acting factors that interact with spoIIE to determine its regulation. In connection with the genetic investigations of sporulation initiation and spoIIE regulation, a new and general method will be developed for generating missense mutations in B. subtilis that may significantly facilitate both the genetic analysis and the cloning of mutated genes.
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