Epitope insertions define functional and topological features of the Escherichia coli ferric enterobactin receptor.
The outer membrane protein FepA of Escherichia coli is the receptor for the ferric enterobactin siderophore complex and colicins B and D. A foreign antigenic determinant inserted into selected FepA sites allowed mutational analysis of receptor function and in situ immunological tracking of specific protein domains with respect to the bacterial cell compartment. Immunoblot analysis of bacterial proteins using an epitope-specific antibody detected the peptide determinant in the receptor fusions. The impact of the insertions on FepA function was examined by ferric enterobactin-mediated iron uptake experiments and colicin sensitivity tests. In all cases, FepA retained biological activity despite introduction of the foreign sequence. To further develop the topological model of FepA, the peptide-specific antibody was used to localize epitope-carrying FepA domains in intact bacterial cells and their isolated membranes. One epitope resided in a region on the exterior of the cell, at the surface of the FepA protein, while other epitopes appeared to be localized to the periplasm or within the outer membrane.
Armstrong
S K
SK
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354.
McIntosh
M A
MA
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial; D004758:Enterobactin / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D000939:Epitopes; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C029489:enterobactin receptor; D004758:Enterobactin