Mutational Analysis of RsbT, an Activator of theBacillus subtilisStress Response Transcription Factor, σB

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Abstract
σB, the stress-activated σ factor ofBacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway. Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for σBinduction. Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established. We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities. Eleven of eighteenrsbTmutations blocked σBinduction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation inBacillus subtilis. Four of the five most carboxy-terminal mutations yieldedrsbTalleles whose products were undetectable inB. subtilisextracts. Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity. Only when the substitutions extended into a region of kinase homology was σBinduction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate σBand displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay. These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.

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