In Gram-positive bacteria such as species of Bacillus, Streptococcus and Staphylococcus the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports and phosphorylates a variety of sugars such as glucose, fructose, mannitol, lactose and sucrose. Glucose and other sugar substrates of the PTS (PTS sugars) repress synthesis of carbohydrate catabolic enzymes, inhibit uptake of non-PTS sugars, and stimulate efflux of both PT'S and non-PT'S sugars. Our current evidence suggests that these processes are regulated by a metabolite-activated, ATP-dependent protein kinase that phosphorylates seryl residue 46 in the phosphocarrier protein of the PTS, HPr. In heterofermentative lactobacilli such as L. brevis which lack an intact PTS, HPr and the HPr kinase/phosphatase system are present, most likely functioning in the regulation of carbohydrate transport and metabolism. This regulatory system is altogether lacking from enteric bacteria such as E. coli. In the proposed research we will combine physiological, biochemical and molecular genetic approaches to establish the molecular mechanism(s) and physiological significance of these protein kinase-mediated regulatory processes. First, we will conduct cellular and vesicular transport experiments as well as in vitro transcription-translation coupled experiments to define the role of HPr(ser) phosphorylation in transport regulation and catabolite repression in B. subtilis and L. brevis. Second, we will overexpress the gluconate permease gene (gntP) of B. subtilis, prepare inside-out vesicles enriched for GntP, and demonstrate a direct interaction between this permease and the various HPr derivatives which are believed to control its activity. Third, we will isolate and characterize mutants defective in the HPr(ser) kinase/phosphatase regulatory system of B. subtilis and define the physiological consequences of the loss of the system. Fourth. we will clone the gene which encodes the HPr(ser) kinase (ptsk) from B. subtilis in order to allow primary structural determination of this protein and facilitate subsequent molecular genetic analyses. Finally, we will continue to collaborate with other laboratories to define the 3-dimensional structures of critical proteins in this regulatory cascade and their phosphorylated derivatives. This research is expected to reveal the molecular details and physiological consequences of novel protein kinase-mediated regulatory mechanisms in prokaryotes. The knowledge gained should be applicable to the control of pathogenic bacteria. The protein kinase-mediated regulatory processes under study are expected to be relevant to analogous processes in eukaryotic organisms.
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