title: Diffusion through channel derivatives of the Escherichia coli FhuA transport protein.
abstract: FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports [Fe3+]ferrichrome, the antibiotics albomycin and rifamycin CGP 4832, and mediates sensitivity of cells to the unrelated phages T5, T1, phi80 and UC-1, and to colicin M and microcin J25. The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infection by phage T5. The FhuA crystal structure reveals 22 antiparallel transmembrane beta-strands that form a beta-barrel which is closed by a globular N-terminal domain. FhuA still displays active transport and sensitivity to all ligands except microcin J25 when the globular domain (residues 5-160) is excised and supports weakly unspecific diffusion of substrates across the outer membrane. Here it is shown that isolated FhuADelta5-160 supported diffusion of ions through artificial planar lipid bilayer membranes but did not form stable channels. The double mutant FhuADelta5-160 Delta322-336 lacking in addition to the globular domain most of the large surface loop 4 which partially constricts the channel entrance, displayed an increased single-channel conductance but formed no stable channels. It transported in vivo[Fe3+]ferrichrome with 45% of the rate of wild-type FhuA and did not increase sensitivity of cells to antibiotics. In contrast, a second FhuA double mutant derivative which in addition to the globular domain contained a deletion of residues 335-355 comprising one-third of surface loop 4 and half of the transmembrane beta-strand 8 formed stable channels in lipid bilayers with a large single-channel conductance of 2.5 nS in 1 m KCl. Cells that synthesized FhuADelta5-160 Delta335-355 showed an increased sensitivity to antibiotics and supported diffusion of maltodextrins, SDS and ferrichrome across the outer membrane. FhuADelta5-160 Delta335-355 showed no FhuA specific functions such as active transport of [Fe3+]ferrichrome or sensitivity to the other FhuA ligands. It is concluded that FhuADelta5-160 Delta335-355 assumes a conformation that is incompatible with any of the FhuA functions.
authors.affiliation: Mikrobiologie/Membranphysiologie, Universität Tübingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Braun
authors.forename: Helmut
authors.initials: H
authors.lastname: Killmann
authors.forename: Elke
authors.initials: E
authors.lastname: Maier
authors.forename: Roland
authors.initials: R
authors.lastname: Benz
authors.forename: Volkmar
authors.initials: V
authors.lastname: Braun
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002455:Cell Division / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005291:Ferrichrome / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000378:metabolism; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011134:Polysaccharides; D011991:Receptors, Virus; D011994:Recombinant Proteins; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D005291:Ferrichrome; C008315:maltodextrin