The phosphoenolpyruvate: glycose phosphotransferase system (PTS) catalyzes the uptake and concomitant phosphorylation of D-glucose (GIc) and many other sugars by Escherichia coli, Salmonella, Vibrios, and numerous other pathogens, especially obligate anaerobes. Since GIc is catabolized as rapidly as it is translocated, the PTS determines the rate of cell growth in GIc-Limiting media Despite an extensive literature, little is known about the molecular details of each step, and virtually nothing on how the PTS is so stringently regulated. We have recently developed a rapid quench procedure that will permit detailed biochemical studies of each step, and may ultimately lead to an understanding of how the PTS is regulated. The method has already shown that the first step, autophosphorylation of EI by PEP, is very complex because of the slow El monomer/dimer (NM) transition. EI may be a major regulator of GIc uptake via the MAD transition, and/or a recently discovered, ATP-dependent El kinase that may link PTS to the electron transport chain. Biochemical studies on both systems will be continued. One goal will be to determine the ratio of M/D during GIc uptake by membrane vesicles using fluorescence energy transfer and EI mutants. A second function of the PTS is that it mediates the """"""""glucose effect"""""""". In this signal transduction system, external GIc repressed expression of certain catabolic operons, and the protein IIP' is the effector, 111' interacts with at least 10 other proteins, including non-PTS perineases. The structures of HI' and important mutants have been well established, as has the complex between HI' and one of its targets, glycerol kinase (GK). We have now constructed 15 crr (HIC*) mutants, and these will be used to further probe interactions between this key regulatory molecule and P-BPr, and GK. For example, application of the rapid quench method to a HI' mutant has suggested a possible mechanism for the P-transfer reaction between BPr -- RIC. These studies should ultimately provide a molecular explanation for sugar uptake by bacterial pathogens.
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