UBC Theses and Dissertations
The effects of the crsA mutation in the major vegetative sigma factor [sigma]-A on the regulation of sporulation initiation in Bacillus subtilis Dixon, Laurie G.
The crsA mutation is located within the gene for the major vegetative sigma factor of Bacillus subtilis, σ[sup A]. The presence of this mutation results in alterations in the regulatory events controlling sporulation initiation, such that spore formation proceeds despite the presence of inhibitory concentrations of glucose. In an effort to more fully understand the mechanisms of glucose repression of sporulation, the effects of the crsA mutation on sporulation gene expression were examined. The in vivo promoter activity of genes involved in the initial stages of sporulation was examined in the crsA mutant using promoter-lacZ fusion constructs. The observed patterns of gene expression indicated that key regulatory checkpoints in the sporulation initiation pathway were bypassed in the presence of the mutant σ[sup A]. The activity of genes encoding phosphorelay proteins was altered, suggesting the inappropriate activity of the sporulation sigma factor, σ[sup H]; as well, both the expression of the operon encoding the transition state regulator SinR, and the expression of spo genes negatively regulated by SinR, were altered. Analysis of spoOA promoter expression suggested that transcription from the vegetative promoter of spoOA was increased in crsA mutant strains. Analysis of both the expression from and the sporulation frequency of a spoOA promoter mutant supported this observation, and implicated altered spoOA expression in the glucose resistant sporulation phenotype of the crsA mutant. Comparative in vitro transcription assays were performed using wild type and crsA mutant RNA polymerases, providing evidence that transcription from the σ[sup A]dependent spoOA promoter by the crsA mutant RNA polymerase was increased over that seen with the wild type enzyme. The data presented herein suggested that the alteration of spoOA gene expression was a direct effect of the crsA mutation in σ[sup A] . This increase in spoOA expression, combined with inappropriate σ[sup H] activity and altered sin expression, resulted in changes in the expression patterns of key genes involved in the initiation of sporulation, overcoming regulatory checkpoints at which sporulation would normally be repressed by glucose. These data indicate that prevention of SpoOA accumulation and negative regulation of σ[sup H] activity are important in the mechanism of glucose inhibition of sporulation.
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