Bacteria respond to environmental stress by activating transcription of genes to modify the organism for the new environment. It has recently become apparent that general mechanistic response prevails in which an external signal induces the transcriptional change through a so-called two component regulatory switch. These component systems consist of a histidine containing protein kinase that when activated by ATP, after external stimulus, will phosphorylate a particular region, usually the' amino-terminal domain of a regulatory protein which in turn influences the transcription of genes. Such a system is present in Bacillus subtilis and controls the transition from dormant to sporulating forms. This proposal is designed to determine the precise mechanism by which the kinase activates the response regulators (RRs) in B. subtilis. The kinase (KinA) and two RR proteins (SpoOF and SpoOA) have been identified and expressed in milligram quantities in Escherichia coli and an in vitro phosphorylation system has been developed. Using synthetic peptide analogs, mutant forms of the RR proteins, various chemical analytical techniques, hydrolytic cleavage and X-ray crystallographic analysis it is intended to definitively describe the structure of SpoOF/SpoOA and their phosphorylated counterparts. In contrast to the phosphorylated CheY from Salmonella typhimurium the phosphorylated SpoOF has a long half life (> 12 h). Thus the site of phosphorylation will be identified and this derivative will also be crystallized to allow conformational comparisons with the free protein. Structural studies will then be extended to the more complex SpoOA. Genetically engineered truncated minimal histidine containing regions of KinA will be generated and crystallized and molecular graphics studies will be employed to, determine interactions between conserved SpoOF/SpoOA-KinA regions. The! phosphorylation reactions will be analyzed kinetically, nucleotide binding sites, kinetic constants and participation of metal ions determined. In addition, the occurrence of reverse phosphorylation reactions will be examined and conformational changes will be revealed by fluorescence, UV/Vis and CD spectrometric measurements. From these observations a detailed picture of kinase-RR interactions will be developed and placed in the context of overall B. subtilis regulation. The results will then be, used to consolidate the more general theory of two component switches forming the basis of bacterial regulatory responses to environmental changes.
