This proposal focuses on the role of CheAS plays in regulating signal transduction in bacterial chemotaxis. Evidence suggests that CheAS interacts with the protein kinase, CheAL, and a protein that regulates CheAL activity, CheW. To determine whether CheAS regulates the activity of CheAL, tethered cell assays will be used to compare the flagellar rotational behavior of cells that express varying ratios of CheAL to CheAS. To identify chemotaxis proteins with which CheAS physically interacts, dominant mutant alleles of CheAS will be isolated and conformational suppressors of these alleles identified. To explore the role of CheAS in regulating phosphate transfer, protein complexes from extracts of null cells expressing subsets of CheAL, CheAS, CheW and transducers will be immunoprecipitated using polyclonal anti-CheA antibodies and assayed for phosphate transfer by CheAL from [gamma-32P] ATP to CheAL. In addition to contributing to the long-term goal of understanding how components of signal transduction pathways interrelate such that cells elicit proper responses to environmental stimuli, it is expected this study will help to understand the regulatory role played by alternate forms of protein kinases involved in signal transduction and to the translational and structural relationships between protein products of in-phase overlapping genes.
