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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022203-03
Application #
3133051
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-09-01
Project End
1989-03-31
Budget Start
1987-09-01
Budget End
1989-03-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78713
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Staab, J F; Earhart, C F (1990) EntG activity of Escherichia coli enterobactin synthetase. J Bacteriol 172:6403-10
Coderre, P E; Earhart, C F (1989) The entD gene of the Escherichia coli K12 enterobactin gene cluster. J Gen Microbiol 135:3043-55
Staab, J F; Elkins, M F; Earhart, C F (1989) Nucleotide sequence of the Escherichia coli entE gene. FEMS Microbiol Lett 50:15-9
Elkins, M F; Earhart, C F (1989) Nucleotide sequence and regulation of the Escherichia coli gene for ferrienterobactin transport protein FepB. J Bacteriol 171:5443-51
Elish, M E; Pierce, J R; Earhart, C F (1988) Biochemical analysis of spontaneous fepA mutants of Escherichia coli. J Gen Microbiol 134:1355-64
Pierce, J R; Earhart, C F (1986) Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake. J Bacteriol 166:930-6