The beta-barrel domain of FhuADelta5-160 is sufficient for TonB-dependent FhuA activities of Escherichia coli.
FhuA in the outer membrane of Escherichia coli serves as a transporter for ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and phi80. The previously determined crystal structure reveals that residues 160-714 of the mature protein form a beta-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1-159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuADelta5-160, that was incorporated in the outer membrane. Cells that synthesized FhuADelta5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of ferrichrome supported growth of a tonB mutant that synthesized FhuADelta5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuADelta5-160. However, growth of a FhuADelta5-160 tonB wild-type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of ferrichrome binding sites provided by the cork. FhuADelta5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuADelta5-160 depended on TonB, although the mutant lacks the TonB box (residues 7-11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of ferrichrome through FhuADelta5-160. FhuADelta5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the beta-barrel to release ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of ferrichrome through the open channel of FhuADelta5-160, interaction of TonB with the beta-barrel is sufficient to release ferrichrome from the residual binding sites at the beta-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and phi80.
Braun
M
M
Mikrobiologie/Membranphysiologie, Universität Tübingen, D-72076 Tübingen, Germany.
Killmann
H
H
Braun
V
V
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001430:Bacteriocins / Q000494:pharmacology; D001692:Biological Transport; D003087:Colicins / Q000494:pharmacology; D003090:Coliphages / Q000472:pathogenicity; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism* / Q000821:virology; D029968:Escherichia coli Proteins; D005291:Ferrichrome / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D011991:Receptors, Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012294:Rifamycins / Q000494:pharmacology; D017384:Sequence Deletion
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001430:Bacteriocins; D003087:Colicins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008565:Membrane Proteins; D011991:Receptors, Virus; D012294:Rifamycins; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli; C054846:CGP 4832; C020411:microcin; D005291:Ferrichrome; C015657:albomycin