New family of regulators in the environmental signaling pathway which activates the general stress transcription factor sigma(B) of Bacillus subtilis

Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma (B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma (B), and this kinase activity is magnified in vitro by the RsbR p rotein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is red undant with other unidentified regulators. A search of the translated B. su btilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory rol es were investigated using three different approaches. First, genetic analy sis found that null mutations in four of the six paralogous genes have mark ed effects on the sigma (B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes wh ich appear to encode a split paralog. Second, biochemical analysis found th at YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the Rsb T environmental signaling kinase, as had been previously shown for RsbR, wh ich is phosphorylated on two threonine residues in its C-terminal region. B oth residues are conserved in the three phosphorylated paralogs but are abs ent in the ones that were not substrates of RsbT: YetI and YezB, each of wh ich bears only one of the conserved residues; and YtvA, which lacks both re sidues and instead possesses an N-terminal PAS domain. Third, analysis in t he yeast two-hybrid system suggested that all six paralogs interact with ea ch other and with the RsbR and RsbS environmental regulators. Our data indi cate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmenta l stress signaling pathway; (ii) YtvA acts as a positive regulator; and (ii i) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulator s whose loss increases sigma (B) activity more than 400-fold in unstressed cells.