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Mutational Analysis of RsbT, an Activator of the Bacillus subtilis Stress Response Transcription Factor, ^B

Robyn L. Woodbury, Tingqiu Luo, Lindsay Grant, and W. G. Haldenwang^*

Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, Texas 78229-3900

Received 28 November 2003/ Accepted 26 January 2004

^B, the stress-activated factor of Bacillus 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 ^B induction. 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 eighteen rsbT mutations blocked ^B induction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis. Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B. subtilis extracts. 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 ^B induction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate ^B and 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.

Present address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322.

Present address: IBIS Therapeutics, Carlsbad, CA 92008.

Present address: NIH/NCI, Advanced Technology Center, Gaithersburg, MD 20892.

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