In Gram-positive bacteria such as species of Streptococcus, Staphylococcus, Bacillus and Lactobacillus, the phosphoenolpyruvate: sugar phosphotransferase system (PTS) transports and phosphorylates a variety of sugars including glucose, fructose, mannitol, lactose and sucrose. Extensive evidence suggests that in all of these bacteria 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 characterized in vitro and implicated in regulation. In order to further characterize this novel bacterial regulatory mechanism we propose to take a combined biochemical genetic approach. Specifically we plan to: 1. Utilize a newly developed exchange assay in order to define the effect of HPr(seryl) phosphorylation on the interactions of HPr with all of the relevant proteins of the PTS. 2. Isolate and characterize mutants defective in the PTS regulatory system in order to define the physiological functions of the system. 3. Clone the genes which encode the HPr(ser) kinase and phosphatase in order to allow primary structural determination of these proteins and to facilitate subsequent molecular genetic analysis. 4. Further examine the mechanisms of sugar efflux via the Enzymes II of the PTS. 5. Determine the physiological function of HPr(ser) phosphorylation in heterofermentative lactic acid bacteria which have been reported to lack an intact PTS. 6. Define the mechanism by which Enzyme III 1ac of Staphylococcus aureus regulates lac operon expression.
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