A number of genes including those for the major degradative enzymes of Bacillus subtilis appear to be controlled by several regulatory genes which may comprise a global regulatory system. The regulatory genes, sacU, sacQ, prtR, and hpr pleiotropically affect the synthesis of several enzymes including levansucrase, sacB, and alkaline protease, aprE. The target of these regulatory genes on either sacB or aprE is at least 100 basepairs upstream of the promoter for the genes and they stimulate transcription from the normal start site. Studies are proposed to isolate the only remaining uncloned regulatory gene, sacU and characterize mutations in the gene. The molecular basis for the phenotypes of mutations in each gene will be probed. Other genes in which mutation leads to an alteration of this regulatory network will be isolated and characterized. The site of action of the regulatory genes will be determined for the aprE (subtilisin) promoter. Extensive deletion analyses from both directions will be used to define the target sites. Site-directed and cassette mutagenesis experiments will further implicate important bases. Messenger RNA will be isolated from mutant strains and transcription start sites found by primer extension to determine if all the regulatory mutations stimulate transcription from the normal start site. Northern analysis will ascertain if messenger RNA levels in all mutants are elevated. Large quantities of purified proteins will be produced in expression vectors. These proteins will be used in binding studies and footprints analyses to determine which proteins bind to the targets and where. Studies are proposed using lac fusions to each regulatory gene to uncover the regulatory interactions among them. The relationship between these genes and catabolite repression will be probed.
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