ISOLATION OF EARLY SPORULATION GENES

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Spore formation in Bacillus subtilis is an elaborate adaptation to life in a poor nutritional environment. This process can be divided into two regulatory phases. The initiation phase can be viewed as the immediate response to a deteriorating environment, leading to expression of a small set of genes, some of which are likely to be involved in turning on other genes; this phase is followed by a prolonged period of differential gene expression that is regulated in time and space. This latter phase is regulated at least in part by changes in the sigma factor component of RNA polymerase; to some extent this regulation can be seen to respond to morphological cues. The initiation phase is much less well understood. The major clues relevant to this aspect of the problem are a strict correlation between initiation of sporulation and a drop in the intracellular concentration of GTP and the existence of certain mutations that appear to define genes whose products are involved in this regulation. The central questions can be framed as follows: a) What is the mechanism by which a drop in GTP induces sporulation? b) What is the order of the earliest regulatory events? and, c) What are the roles of putative regulatory gene pro- ducts? This proposal seeks to address these questions by identifying a class of genes expressed at very early times of sporulation, analyzing the regulation of those genes, and identifying the proteins responsible for their regulation. A complementary project deals with regulation of the gene for aconitase and other carbon-regulated genes. Since carbon limitation is an important factor in inducing sporulation, a clear understanding of the mechanism by which certain genes respond to the available carbon source is likely to provide important information about early sporulation events. The specific aims of the proposal are: (1) Create cDNA probes that are enriched for early sporulation sequences and use them to isolate and study early sporulation genes; (2) analyze the regulation of the aconitase gene by identifying regulatory sites in the promoter region and proteins that interact with those sites; (3) isolate and study a class of genes that is turned on when the carbon source becomes limiting for growth; and, (4) determine the mechanism by which a change in the pool of GTP leads to early sporulation gene expression.