SRF OPERON--REGULATION AND ROLE IN GENETIC COMPETENCE

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Bacteria possess complex signal transduction networks that govern the cells' response to environmental changes. In Bacillus subtilis, such a network controls processes that are activated when a growth-limiting environment is encountered. Systems which sense conditions of high cell density and starvation converge to activate the expression of the srf operon, encoding the enzyme complex that catalyzes the biosynthesis of the lipopeptide antibiotic, surfactin, and encoding a regulator of genetic competence, ComS, which is required for the expression of genes that function in DNA import. Studies of srf regulation and function will enhance our understanding of how bacteria regulate the production of toxins and antibiotics, and the process of genetic transformation, which is one way bacteria acquire genes that confer drug resistance. Mutations that alter the nutrition-dependent regulation of srf expression and sporulation, called ggr (glucose-glutamine-dependent regulation), result in an elevated concentration of sigma-H, encoded by spoOH and required for the production of the pheromone Csf (competence stimulating factor) which activates srf transcription. The ggr mutations also confer a Crs (catabolite resistant sporulation) phenotype. The ggr loci will be isolated and characterized to determine their roles in cell density- and nutritional control of srf expression. The comS gene, located within and out-of-frame with the srfB gene of the srf operon, is subject to regulation that is separate from that which controls srf transcription initiation. This regulation requires sinR, which also functions in the cell's decision to undergo sporulation or competence development. The region(s) of srf that functions in sinR- dependent control will be identified. Other mutations that effect specifically comS expression will be isolated and characterized by nucleotide sequence determination, and by introducing the mutations into sinR mutants or cells that overexpress sinR. The intracellular location and the cell type distribution (competent or non-competent cell) of ComS will be determined using anti-ComS antibody. The structure of ComS and its possible role in controlling the activity of DegU (a response regulator-like protein that functions in the contor of degradative enzyme production and competence), ComK (the transcriptional activator of competence genes) or MBA and B (homologues of ATP-dependent proteases and heat shock proteins, and negative regulators of ComK) will be investigated by mutational analysis and protein-protein crosslinking experiments. The identification of genes that encode ComS- binding proteins will be isolated using the yeast two-hybrid system.