Approaches are described to determine how the individual proteins of the primary iron transport system of Escherichia coli, that mediated by the siderophore enterobactin (Ent), function in the transmembrane passage of iron. A knowledge at the molecular level of how Ent is exported and how FeEnt is imported is basic to our overall goal of understanding bacterial iron assimilation, including its regulation and how iron transport processes are integrated into overall cell metabolism. This information, in turn, is crucial to an understanding of the role of iron in microbial infections. The outcome of many infections is determined by the ability of pathogens to acquire iron from an enviroment that is being rendered increasingly deficient in available iron by several host mechanisms. (Ent is produced by a variety of enteric pathogens in addition to E. coli, including Klebsiella pneumoniae, Salmonella typhimurium, and Shigella sonnei.) The genes specifically required for the Ent system of iron transport are clustered; work to determine the number of these genes, their order, and their polypeptide products will be completed. The processes and relevant gene products then to be studied are as follows: (i) export of Ent from cell (EntDEFG, as Ent synthetase), (ii) transport of FeEnt through the outer membrane (FepA), (iii) passage of iron through the periplasm and cytoplasmic membrane (FepB, FepC), (iv) release of iron from Ent in the cytosol (Fes) and (v) possible roles of FepB and FepC in other iron transport systems. The proteins will be localized and characterized with respect to such properties as enzymatic activities, pore-forming abilities, and binding affinities for other proteins, Ent and FeEnt. Recombinant DNA, cross-linking, in vitro DNA-directed protein synthesis and immunological procedures in conjunction with standard genetic techniques will be used to accomplish these goals.
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