Bacteria are highly adaptable organisms capable of growth on countless carbon and nitrogen sources and of occupying an inexhaustible variety of ecological niches including, unfortunately, regions of the human body that are better left bacteria-free. The key to adaptability in bacteria is their capacity to express only those genes for enzymes and pathways that they need for maximal growth in the environment in which they find themselves. One of the major mechanisms of signal recognition leading to specific gene expression is the two-component system and its more complex variant, the phosphorelay. This proposal has the goal of understanding the mechanisms by which both an essential two-component system and the sporulation phosphorelay function, from signal ligand identity to molecular recognition between components and gene activation. Genetic methods will be used to identify signals and proteins activating sporulation sensor kinases. Proteins associating with sensor kinases will be identified. The YycG sensor kinase and YycF response regulator are essential for growth. The genes regulated by this two-component system will be identified by microarray analysis and bioinformatic techniques. Suppression, transposon and multi-copy gene expression techniques will be used to identify the role of the YycF response regulator in gene expression. The role of amino acid side chains in the surface of interaction of response regulators and phosphotransfer domains in recognition specificity will be studied by modifying the Spo0A transcription factor to a sensor kinase substrate. The structure and function of the KinA domains will be studied by domain liberation using mutants bearing specific proteolytic sites in interdomain region. Experiments are proposed to test the hypothesis that the dynamics of the loops making up the active site of Spo0F is an important determinant of sensor kinase specificity and that the dynamics may be influenced by regulating ligands that bind to the response regulator. It is believed that this combination of structural and functional studies will lead to effective anti-bacterial agents either directly or in combination as inhibitors of resistance mechanisms.
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