The long-term goal of this project is to understand at the molecular level the mechanisms that control synthesis and activity of the enzymes that convert acetyl CoA to glutamate in Bacillus subtilis. This pathway, which is critical for generation of energy, reducing power, and biosynthetic building blocks, is also the junction between carbon and nitrogen metabolism and plays a key regulatory role in bacterial differentiation. Genes that encode the enzymes of this pathway, citrate synthase, aconitase, isocitrate dehydrogenase, and glutamate synthase, have all been cloned and have been shown to be regulated at the level of transcription.
The specific aims of this project are to identify and isolate the proteins that regulate each of these genes, to deduce the intracellular metabolites that control activity of each regulatory protein and to reconstruct each regulatory system in vitro. One of these proteins has already been identified; GltC, the positive regulator of glutamate synthase synthesis, is a member of the LysR family. Since relatively little is known about how this family of proteins contacts DNA and stimulates transcription, the regions of GltC that contribute to DNA binding, effector binding, multimerization, and interaction with RNA polymerase will be targets for mutagenesis and genetic selection. The B. subtilis system is the primary paradigm for studies of Gram-positive bacteria and prokaryotic differentiation. Fundamental studies of B. subtilis gene expression, regulation of metabolism, and response to the environment are highly informative about the biology of related pathogenic bacteria and provide a means of studying issues of universal biological importance in an organism that is easily manipulated physiologically and genetically.
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