MECHANISM OF CARBOHYDRATES TRANSPORT IN BACTERIA

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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. Studies in our laboratory have provided information regarding the mechanisms by which the PTS functions in catalysis and regulation of carbon metabolism. We have cloned and sequenced many of the gene encoding PTS proteins and proteins which regulate their expression, and have conducted extensive molecular genetic and biochemical analyses to define their mechanisms of action. These studies have allowed us to define the allosteric mechanism by which PTS-mediated protein phosphorylation regulates the activities of non-PTS carbohydrate permeases, catabolic enzymes and adenylate cyclase. Recently, our studies have shown that the fructose (fru) regulon is exceptionally complex and that the fru repressor (FruR) together with other fructose-specific proteins regulates transcription of hundreds of genes encoding enzymes involved in Salmonella pathogenesis and central pathways of carbon metabolism (glycolysis, the Krebs cycle, the glyoxylate shunt, gluconeogenesis, and electron transfer). The purpose of the proposed research is to extend our understanding of the fru regulon, the fructose-specific PTS proteins and their involvement in sugar transport and transcriptional regulation. Employing Salmonella typhimurium, a combined biochemical and molecular genetic approach will be taken as follows: 1. We will sequence the fruR gene and overproduce and purify FruR for physicochemical, DNA binding and in vitro transcription studies. Other proteins believed to be involved in regulation will similarly be prepared. Target genes to be studied include those of the fru regulon which are negatively regulated by FruR as well as those encoding enzymes of central metabolic pathways which are positively regulated by FruR. The possible involvement of the enzymes of the fructose PTS and protein phosphorylation will be tested. 2. We will characterize the various components of the fru regulon including cryptic genes encoding duplicate copies of PTS energy coupling proteins and permeases. We will also characterize the Enzyme IIIfru-MR-FPr and the Enzyme IIfru proteins in order to define the structure-function relationships of their various domains. X-ray diffraction studies (to be conducted collaboratively) will complement the molecular genetic and biochemical studies proposed.