The β‐barrel domain of FhuAΔ5‐160 is sufficient for TonB‐dependent FhuA activities of Escherichia coli

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Abstract
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 φ80. The previously determined crystal structure reveals that residues 160–714 of the mature protein form a β‐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, FhuAΔ5‐160, that was incorporated in the outer membrane. Cells that synthesized FhuAΔ5‐160 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 FhuAΔ5‐160. These results demonstrate non‐specific diffusion of compounds across the outer membrane of cells that synthesize FhuAΔ5‐160. However, growth of a FhuAΔ5‐160 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. FhuAΔ5‐160 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 FhuAΔ5‐160 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 FhuAΔ5‐160. FhuAΔ5‐160 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 β‐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 FhuAΔ5‐160, interaction of TonB with the β‐barrel is sufficient to release ferrichrome from the residual binding sites at the β‐barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB‐dependent phages T1 and φ80.