Threonine phosphorylation of modulator protein RsbR governs its ability toregulate a serine kinase in the environmental stress signaling pathway of Bacillus subtilis

The sigma(B) transcription factor of the bacterium Bacillus subtilis contro ls the synthesis of over 100 general stress proteins that are induced by gr owth-limiting conditions. Genetic evidence suggests that RsbR modulates the phosphorylation state of the RsbS antagonist in the signaling pathway that regulates sigma(B) activity in response to environmental stresses that lim it growth. According to the current model, the phosphorylated RsbS antagoni st is unable to complex RsbT, which is then released to initiate a signalin g cascade that ultimately activates sigma(B). Here, we show that the RsbR p rotein itself has no kinase activity but instead stimulates RsbS phosphoryl ation by the RsbT serine kinase in vitro. We further show that in addition to its previously known serine kinase activity directed toward the RsbS ant agonist, purified RsbT also possesses a threonine kinase activity directed toward residues 171 and 205 of the RsbR modulator. Threonine residues 171 a nd 205 were each found to be important for RsbR function in vivo, and phosp horylation of these residues abolished the ability of RsbR to stimulate Rsb T kinase activity in vitro. These results are consistent with a model in wh ich RsbR modulates the kinase activity of RsbT directed toward its RsbS ant agonist in vivo, either specifically in response to environmental signals o r as part of a feedback mechanism to prevent continued signaling. (C) 1999 Academic Press.