Regulation of Streptomyces coelicolor Development-AREA

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DESCRIPTION (provided by applicant): The broad objective of this application is to understand the molecular events that occur during development of the model bacterium Streptomyces coelicolor. The Streptomyces are common soil bacteria well-known for the prolific synthesis of bioactive molecules including the majority of antibiotics in current use. Synthesis of these secondary metabolites occurs as part of a larger developmental program in these multicellular bacteria, which culminates in the sporulation of filamentous aerial hyphae. The products of two gene clusters that have profound but different impacts on S. coelicolor development will be characterized in this study. The whiJ cluster contains genes required for the sporulation of aerial hyphae including one that encodes a putative transcription factor. WhiJ will be purified, its ability to bind DNA in vitro characterized, and the specific DNA sequence to which it binds identified using a PCR-based approach. Search of the completed S. coelicolor genome for WhiJ binding sites will thereby reveal the downstream sporulation genes regulated by WhiJ and thus elucidate the next set of molecular players in this pathway. While some gene products function to advance S. coelicolor development, others must restrict it when conditions are unsuitable. Excessive activity of the sigma factor U caused by mutation of its negative regulator abolishes differentiation presumably because the sigmaU regulon contains genes whose products halt development. The presumed complex between sigmaU and its negative regulator will be biochemically characterized using purified proteins. A transcriptional reporter will be used to identify stress conditions that activate sigmaU. Finally, members of the sigmaU regulon will be identified by genome analysis and characterization of a putative sigmaU promoter consensus sequence. The characterization of WhiJ, sigmaU and their targets will elucidate at least a partial sequence of the molecular events that mediate development in S. coelicolor and will enhance the currently incomplete understanding of this complex process. Relevance: The goal of the proposed research is to understand how Streptomyces bacteria grow and change. The more that is understood, the more likely it is that these useful bacteria can be rationally manipulated to increase the yield and variety of life-saving drugs that they make during their life cycle (such as antibiotics and anticancer drugs).