title: Transport activity of FhuA, FhuC, FhuD, and FhuB derivatives in a system free of polar effects, and stoichiometry of components involved in ferrichrome uptake.
abstract: The Escherichia coli fhu operon, composed of the fhuA, C, D, and B genes, is essential for the utilization of ferric siderophores of the hydroxamate type and for the uptake of the antibiotic albomycin. We have had difficulty studying the effects of missense mutations in individual plasmid-encoded transport genes because appropriate test strains were not found: all isolated chromosomal mutations in either one of the fhu genes (with a complete loss of function) negatively influenced the expression of other fhu genes in the operon. In order to analyze Fhu mutant proteins in a system free of polar effects, we constructed a plasmid-encoded gene cassette system by introducing unique restriction sites that allowed precise cloning of individual fhu genes. The fhu cassette operon expressed in a chromosomal fhu deletion mutant enabled us to evaluate the transport activity of mutated FhuA, FhuC, FhuD or FhuB derivatives. In addition, we found that transport across the outer membrane (via FhuA, TonB, ExbB, D) rather than transport across the cytoplasmic membrane (via FhuC, D, B) was rate limiting. The stoichiometry of the components involved in the uptake of iron(III) hydroxamates seems to be important for proper functioning.
authors.affiliation: Mikrobiologie/Membranphysiologie, Universität Tübingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Mademidis
authors.forename: W
authors.initials: W
authors.lastname: Köster
mesh_terms: D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005291:Ferrichrome / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D009876:Operon; D033902:Periplasmic Binding Proteins; D011991:Receptors, Virus / Q000235:genetics*; D013997:Time Factors
chemical_list: D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; C066629:FhuC protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; D011991:Receptors, Virus; C069968:fhuB protein, E coli; C069969:fhuD protein, E coli; D005291:Ferrichrome; C015657:albomycin