Control of Sigma B Activity in B subtilis

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DESCRIPTION: (provided by applicant): In response to environmental stress, sigma B, a transcriptional regulator of B. subtilis, is released from an inhibitory association with an anti-sigma B protein (RsbW) to activate expression of the bacterium's general stress regulon. The stress-generated signal that activates sigma B is unknown; however, recent evidence implicates the bacterium's ribosome and a small GTP binding protein (Obg) in this process. The proposal seeks to determine the roles of the ribosome and Obg in the stress induced activation of sigma B. Both Obg and the non-essential ribosome protein L11 are needed for environmental stress to activate sigma B. We will perform directed and random alterations of the coding sequences for each of these proteins to identify regions or activities that modify the inducibility of sigma B and determine how these changes influence each of these proteins' other known functions. The analyses should identify regions of Obg and L11 that are important for their biochemical activities and suggest which of these properties are required for sigma B induction. Several components of the stress activation cascade (RsbR, S and T) have been observed to cofractionate with ribosomes. This putative association will be examined, using velocity centrifugation and gel filtration analyses under conditions that are likely to cause partial or total dissociations. The specific ribosome fraction components and Rsb proteins that are involved in the associations will be identified. The types of complexes found and the identities of proteins directly involved could give clues as to the role of the association in stress signaling and sigma B induction. Finally, a detailed mutational analysis will be undertaken of rsbT, the most upstream positive regulator in the sigma B stress induction pathway and the gene whose product is the most likely to be directly influenced by stress signaling. It is anticipated that the changes in rsbT and their resulting phenotypes will provide a test of RsbT's proposed activities as well as identify sites where stress directed signals alter RsbT activity. This work will not only explore the fundamental biological question of how cells recognize and react to hostile environments, but, given the presence of sigma B and its principal regulators in the human pathogens Staphylococcus aureus, Listeria monocytogenes and Mycobacterium tuberculosis, it may have practical applications, identifying weaknesses in these pathogens' stress adaptation responses to host defenses.