title: Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12.
abstract: The ferrichrome-iron receptor of Escherichia coli K-12 is FhuA (M(r), 78,992), the first component of an energy-dependent, high-affinity iron 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 albomycin. To probe the topological organization of FhuA which enables recognition of these different ligands, we generated a library of 16 insertion mutations within the fhuA gene. Each insertion spliced a 13-amino-acid antigenic determinant (the C3 epitope of poliovirus) at a different 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 proteins were subjected to flow cytometric analysis using anti-FhuA monoclonal antibodies. Such analysis showed that (i) the chimeric proteins were properly localized and (ii) the wild-type FhuA protein structure had not been grossly altered by insertion of the C3 epitope. Twelve of sixteen strains expressing FhuA.C3 proteins were proficient in ferrichrome transport and remained sensitive to FhuA-specific phages. Three FhuA.C3 proteins, with insertions after amino acid 321, 405, or 417 of FhuA, were detected at the cell surface by flow cytometry using anti-C3 antibodies. These three chimeric proteins were all biologically active. We conclude that amino acids 321, 405, and 417 are surface accessible in wild-type FhuA.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, Canada.
authors.forename: G S
authors.initials: GS
authors.lastname: Moeck
authors.forename: B S
authors.initials: BS
authors.lastname: Bazzaz
authors.forename: M F
authors.initials: MF
authors.lastname: Gras
authors.forename: T S
authors.initials: TS
authors.lastname: Ravi
authors.forename: M J
authors.initials: MJ
authors.lastname: Ratcliffe
authors.forename: J W
authors.initials: JW
authors.lastname: Coulton
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002213:Capsid / Q000032:analysis / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins; D002462:Cell Membrane / Q000737:chemistry; D000939:Epitopes / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D017955:Poliovirus / Q000276:immunology; D017510:Protein Folding; D011991:Receptors, Virus / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D036022:Capsid Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C442540:VP1 protein, Poliovirus