Isolation and Characterization of Dominant Mutations in theBacillus subtilisStressosome Components RsbR and RsbS

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Abstract
The general stress response of Bacillus subtilis is controlled by the activity state of the sigma(B) transcription factor. Physical stress is communicated to sigma(B) via a large-molecular-mass (> 10(6)-Da) structure (the stressosome) formed by one or more members of a family of homologous proteins (RsbR, YkoB, YojH, YqhA). The positive regulator (RsbT) of the sigma(B) stress induction pathway is incorporated into the complex bound to an inhibitor protein (RsbS). Exposure to stress empowers an RsbT-dependent phosphorylation of RsbR and RsbS, with the subsequent release of RsbT to activate downstream processes. The mechanism by which stress initiates these reactions is unknown. In an attempt to identify changes in stressosome components that could lead to sigma(B) activation, a DNA segment encoding these proteins was mutagenized and placed into B. subtilis to create a merodiploid strain for these genes. Eight mutations that allowed heightened sigma(B) activity in the presence of their wild-type counterparts were isolated. Two of the mutations are missense changes in rsbR, and six are amino acid changes in rsbS. Additional experiments suggested that both of the rsbR mutations and three of the rsbS mutations likely enhance sigma(B) activity by elevating the level of RsbS phosphorylation. All of the mutations were found to be dominant over wild-type alleles only when they are cotranscribed within an rsbR rsbS rsbT operon. The data suggest that changes in RsbR can initiate the downstream events that lead to sigma(B) activation and that RsbR, RsbS, and RsbT likely interact with each other concomitantly with their synthesis.