The phosphoenolpyruvate: sugar phosphotransferase system (PTS) is a complex enzyme system which in Salmonella typhimurium and Escherichia coli catalyzes the concomitant transmembrane transport and phosphorylation of its sugar substrates. It also regulates the utilization of a variety of carbon sources not taken up via the PTS-catalyzed group translocation mechanism. Recent studies in our laboratory have provided new information regarding the mechanisms by which the PTS functions in catalysis and regulation of sugar uptake. Specifically, we have purified the mannitol permease (EnzymeII-mtl) to homogeneity, characterized and reconstituted its transport function in an artificial membrane, cloned and sequenced its structural gene, and established the mechanism by which the PTS controls the activities of other permeases. The purpose of the proposed research is to expand upon our understanding of hexitol transport, to further our current hypotheses and to establish novel regulatory mechanisms. Both biochemical and genetic approaches will be taken. 1. Structure of Enzyme II-mtl and mechanism of mannitol transport. We will take the approaches of the immunochemist, the protein chemist and the molecular geneticist to try to correlate structure of Enzyme II-mtl with its function and regulation. We will also use molecular genetic approaches to study its biogenesis. 2. Comparative Studies on the Structure of the Enzyme II-gut-IIIgut pair and the mechanism of glucitol transport. The glucitol-specific PTS enzymes will be studied by biochemical and molecular genetic approaches. Comparisons with the mannitol system will be emphasized. 3. Mechanism of PTS-mediated control of glycerol kinase in E. coli, S. typhimurium and B. subtilis. Studies concerned with the regulation of glycerol kinase by Enzyme IIIg+-c of the PTS will be emphasized in E. coli, S. typhimurium and B. subtilis. These studies will be extended to adenylate cyclase and various permeases. 4. Mechanism of PTS-mediated control of the utilization of amino acids and Krebs cycle intermediates. Involvement of the crrB and fruR genes. Genetic techniques and biochemical approaches will be applied in attempts to define the molecular details of a novel regulatory mechanism. Newly discovered proteins of the PTS which may be involved in regulation will be characterized.
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