In Gram-positive bacteria such as Streptococcus pyogenes, S. lactis and S. mutans, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports and phosphorylates a variety of sugars including glucose, fructose, mannitol, lactose and sucrose. Extensive evidence suggests that the rates of sugar uptake and efflux are controlled by ATP-dependent protein phosphorylation. An HPr-kinase which phosphorylates a seryl residue in HPr, and an HPr(ser)-P phosphatase have been identified in vitro and implicated in regulation. In order to characterize this novel bacterial regulatory mechanism we propose to take a straightforward biochemical approach. Specifically we plan to: 1. Isolate milligram quantities of P-(ser)HPr and free HPr so that we can compare their interactions with the other proteins of the PTS (Enzyme I, Enzyme III(glc), Enzyme III(lac), and the membrane-associated Enzyme II complexes). 2. Purify both the HPr kinase and the HPr(ser)P phosphatase. 3. Attempt to discover the physiologically relevant sugar-P phosphatase which is activated either by P(ser)HPr or by a protein kinase-catalyzed phosphorylation event. 4. Determine the mechanism by which the Enzymes II of the PTS catalyze efflux of free sugar. 5. Examine other bacteria for protein kinases which may regulate carbohydrate transport and metabolism. These studies should lead to a concrete biochemical concept of protein-kinase mediated transport regulation in bacteria and allow application of genetic techniques to the problem.
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