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 fo r the ferric enterobactin siderophore complex and colicins B and D. A foreign antigenic determinant inserted into selected FepA sites allowe d mutational analysis of receptor function and in situ immunological t racking of specific protein domain's with respect to the bacterial cel l compartment, Immunoblot analysis of bacterial proteins using an epit ope-specific antibody detected the peptide determinant in the receptor fusions. The impact of the insertions on FepA function was examined b y ferric enterobactin-mediated iron uptake experiments and colicin sen sitivity tests. In all cases, FepA retained biological activity despit e introduction of the foreign sequence. To further develop the topolog ical model of FepA, the peptide-specific antibody was used to localize epitope-carrying FepA domains in intact bacterial cells and their iso lated membranes. One epitope resided in a region on the exterior of th e cell, at the surface of the FepA protein, while other epitopes appea red to be localized to the periplasm or within the outer membrane.