GENETIC INSERTION AND EXPOSURE OF A REPORTER EPITOPE IN THE FERRICHROME-IRON RECEPTOR OF ESCHERICHIA-COLI K-12

The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iro n uptake pathway. FhuA is also the cognate receptor for bacteriophages T5, T1, phi 80, and UC-1, for colicin M and microcin 25, and for albo mycin. To probe the topological organization of FhuA which enables rec ognition of these different ligands, we generated a library of 16 inse rtion mutations within the fhuA gene. Each insertion spliced a 13-amin o-acid antigenic determinant (the C3 epitope of poliovirus) at a diffe rent position within FhuA. Immunoblotting of outer membranes with anti -FhuA and anti-C3 antibodies indicated that 15 of 16 FhuA.C3 proteins were present in the outer membrane in amounts similar to that observed for plasmid-encoded wild-type FhuA. One chimeric protein with the C3 epitope inserted after amino acid 440 of FhuA was present in the outer membrane in greatly reduced amounts. Strains overexpressing FhuA.C3 p roteins were subjected to flow cytometric analysis using anti-FhuA mon oclonal antibodies. Such analysis showed that (i) the chimeric protein s were properly localized and (ii) the wild-type FhuA protein structur e had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferr ichrome transport and remained sensitive to FhuA-specific phages. Thre e FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using ant i-C3 antibodies. These three chimeric proteins were all biologically a ctive. We conclude that amino acids 321, 405, and 417 are surface acce ssible in wild-type FhuA.