TOPOLOGICAL ANALYSIS OF THE ESCHERICHIA-COLI FERRICHROME-IRON RECEPTOR BY USING MONOCLONAL-ANTIBODIES

Ferrichrome-iron transport in Escherichia coli is initiated by the out er membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (Mabs) were isolated to examine the surface accessibility of FhuA seq uences and their contribution to ligand binding. The determinants of 3 2 of the MAbs were mapped to eight distinct regions in the primary seq uence of FhuA by immunoblotting against (i) five internal deletion Fhu A proteins and (ii) four FhuA peptides generated by cyanogen bromide c leavage. Two groups of MAbs bound to FhuA in outer membrane vesicles b ut not to intact cells, indicating that their determinants, located be tween residues 1 and 20 and 21 and 59, are exposed to the periplasm. O ne of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located bet ween amino acids 321 and 381, is cell surface exposed. This Mab and fo ur others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially di stinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of Fh uA sequences which lie external to the outer membrane may adopt a tigh tly ordered organization with little accessible linear sequence.