Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop.
Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy-dependent process mediated by the FhuA protein. A FhuA deletion derivative lacking residues 322 to 355 (FhuA delta322-355) forms a permanently open channel through which ferrichrome diffused. This finding led to the concept that the FhuA protein forms a closed channel that is opened by input of energy derived from the electrochemical potential across the cytoplasmic membrane, mediated by the Ton system. In this study, we constructed various FhuA derivatives containing deletions inside and outside the gating loop. FhuA delta322-336 bound ferrichrome and displayed a residual Ton-dependent ferrichrome transport activity. FhuA delta335-355 no longer bound ferrichrome but supported ferrichrome diffusion through the outer membrane in the absence of the Ton system. FhuA delta335-355 rendered cells sensitive to sodium dodecyl sulfate and supported diffusion of maltotetraose and maltopentaose in a lamB mutant lacking the maltodextrin-specific channel in the outer membrane. Cells expressing FhuA delta70-223, which has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only weak ferrichrome uptake, suggesting formation of a nonspecific pore through the outer membrane. FhuA delta457-479 supported Ton-dependent uptake of ferrichrome. None of these FhuA deletion derivatives formed pores in black lipid membranes with a stable single-channel conductance. Rather, the conductance displayed a high degree of current noise, indicating a substantial influence of the deletions on the conformation of the FhuA protein. FhuA also supports infection by the phages T1, T5, and phi80 and renders cells sensitive to albomycin and colicin M. Cells expressing FhuA delta322-336 were sensitive to albomycin and colicin M but were only weakly sensitive to T5 and phi480 and insensitive to T1. Cells expressing FhuA delta335-355 were resistant to all FhuA ligands. These results indicate different structural requirements within the gating loop for the various FhuA ligands. Cells expressing FhuA delta457-479 displayed a strongly reduced sensitivity to all FhuA ligands, while cells expressing FhuA delta70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resistant. It is concluded that residues 335 to 355 mainly determine the properties of the gate with regard to FhuA permeability and ligand binding.
Killmann
H
H
Mikrobiologie II, Universität Tübingen, Germany.
Benz
R
R
Braun
V
V
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000502:physiology; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003087:Colicins / Q000494:pharmacology; D003090:Coliphages / Q000502:physiology; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005291:Ferrichrome / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D008024:Ligands; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000502:physiology; D017433:Protein Structure, Secondary; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D007473:Ion Channels; D008024:Ligands; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011991:Receptors, Virus; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; D005291:Ferrichrome; C015657:albomycin; D012967:Sodium Dodecyl Sulfate