Structure/Function Analysis of a Bacterial Protein Kinase Phosphoprotein Phosphatase

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Over 20 different bacterial adaptive responses to environmental stimuli are regulated by a family of proteins, response regulators, sharing a conserved amino terminal domain. The activity of these response regulators is controlled by a second family of proteins sharing a conserved domain, usually located at the carboxyl terminus. Recent experiments with the purified signal transduction proteins that control nitrogen assimilation (NTR regulon), chemotaxis (Che) and osmoregulation (OMP regulon) indicate that the proteins of this second family are protein kinases and, in some cases, phosphoprotein phosphatases that regulate the adaptive responses by controlling the level of phosphorylation of the response regulators. Of these protein kinases, the best understood is NRII which regulates nitrogen assimilation by catalyzing the phosphorylation and dephosphorylation of the enhancer-binding transcription factor NRI. In this proposal, the PI presents experiments designed to further elucidate the mechanisms of the kinase and phosphatase reactions catalyzed by NRII The results of these experiments will probably be applicable to all of the homologous protein kinases. NRII has three activities: in the presence of ATP, NRII is autophosphorylated on a histidine residue. This phosphoryl group is then transferred to an aspartyl residue within the N-terminal domain of the response regulator NRI. Finally, in the presence of the signal transduction protein PII and ATP, NRII catalyzes the dephosphorylation of P-NRI. Genetic experiments are proposed that should define the requirements for nucleotide binding and the autokinase, phosphotransferase. and phosphatase activities by isolating or constructing mutants specifically deficient in these activities. In addition, experiments are proposed that should directly elucidate the identity of the autophosphorylated histidine residue, the stoichiometry of autophosphorylation, and the sites of nucleotide binding.