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Analysis of the Spo0A(257V) MUTANT OF Bacillus subtilis Turner, Barbara Marion

Abstract

In response to a deteriorating environment, Bacillus subtilis cells are capable of several alternate survival strategies including motility, competence development, secretion of proteases and surfactants, and sporulation. Members of the response regulator family of proteins play key roles regulating entry into these alternate states. In the case of sporulation, the master regulator is SpoOA. SpoOA initiates the onset of sporulation by direct or indirect activation or repression of transcription of over 500 genes within the SpoOA regulon. The A257V mutation within SpoOA was previously identified as a mutation which abolishes the ability of B. subtilis cells to sporulate. In vivo, the mutation prevents transcription activation at both the oA-dependent spoIIG operon promoter and the oH-dependent spoIIA operon promoter, yet does not affect the ability of SpoOA to repress transcription at the abrB promoter. In this thesis I investigated the biochemical properties of SpoOA(A257V) to determine how the A257V mutation uncouples transcription activation from repression to lead to a sporulation negative phenotype. I demonstrated that the protein is phosphorylated efficiently by a reconstituted phosphorelay in vitro. I showed that SpoOA(A257V) can both repress transcription and activate oA-dependent transcription in vitm, although at a reduced level compared to wild type SpoOA. I showed that the A257V mutation did not affect the ability of SpoOA to recognize and bind specific sequences within promoter DNA, but rather that the reduction in transcription activation and repression in vitro could be attributed to a modest decrease in the apparent binding affinity of the mutant protein. While the reduction in apparent binding affinity could explain the in vitro results, it did not account for the complete lack of sporulation in spoOA(A257V) B. subtilis cells. Analysis of SpoOA expression in wild type and mutant strains indicated that SpoOA(A257V) expression was decreased as compared to SpoOA. To discriminate between mechanisms controlling the amount of SpoOA in vivo, strains were constructed which overexpressed wild type or mutant SpoOA proteins and used to test activation of oA and oH-dependent promoters in vivo. Results from these experiments were inconclusive; the levels of induced protein may have been insufficient to activate stage II sporulation genes.

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