GENE EXPRESSION DURING SPORULATION

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It is proposed to analyze the control of expression of three Bacillus subtilis sporulation loci, spoIIA, spoVA and spoVI, chosen so as to elucidate the controls of the whole network of gene expression during sporulation. Transcriptional fusions of the spo locus control region to the E. coli lac Z gene will be used to provide an easy assay for expression. They will be used to test the effect of addition of nutrients (catabolites) on gene expression; toluenized cells may be used to overcome permeability barriers. Translational spo-lac Z fusions will be studied to see if genes within an operon can sometimes be regulated independently of each other; particular attention will be paid to spoIIAC which codes for an RNA polymerase sigma-factor-like protein. Controlled amplification of the number of copies of spo-lac Z transcriptional fusions, by use of integrational vectors, will be used to titrate gene copy number against enzyme activity. Use of orthogonal-field-alternation gel electrophoresis will enable accurate determination of copy numbers over a wide range. This approach will be used to see if, in different genetic and physiological backgrounds, expression is subject to repression, activation, or is unregulated. The effect of amplification of the intact spoIIAC gene on other sporulation events will also be tested. Strains harbouring multiple copies of spo-lac fusions will be used to isolate negative and constitutive mutants so as to identify new genes that regulate spo gene expression, and in particular genes that exercise temporal control. Mutations will be sought using integratable plasmid, transposon, and chemical mutagenesis. The regulatory genes will be cloned so as to analyze them in depth. Further studies will identify the products of regulatory genes and their site(s) on the spo loci.