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

Abstract

SummaryFhuA 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 f80. The previously determined crystal structure reveals that residues 160±714 of the mature protein form a b-barrel that is closed from the periplasmic side by the globular Nproximal fragment, residues 1±159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuAD5-160, that was incorporated in the outer membrane. Cells that synthesized FhuAD5-160 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 FhuAD5-160. These results demonstrate non-speci®c diffusion of compounds across the outer membrane of cells that synthesize FhuAD5-160. However, growth of a FhuAD5-160 tonB wildtype 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. FhuAD5-160 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 FhuAD5-160 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 FhuAD5-160. FhuAD5-160 still functions as a speci®c 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

Differential substrate-induced signaling through the TonB-dependent transporter BtuB

“…TonB may also interact with other parts of the transporters, as implied from genetic suppression patterns (15). Claims that FepA and FhuA variants lacking the hatch domain and Ton box (32,33) still exhibit TonB-dependent activity have been challenged by the recognition that the empty barrels can be complemented by the hatch domains encoded by the mutated chromosomal alleles (34). The N-terminal regions of FhuA and FecA move extensively after substrate binding, but the Ton box regions themselves are disordered.…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…The structure of wild-type FhuA suggests that deletion of the plug would produce a wide-open channel. However, the deletion made E. coli only very slightly more susceptible to various antibiotics (92), and the mutant protein did not produce stable channels after reconstitution into planar lipid bilayers (93). These observations suggest that the channel might be still closed by the external loops.…”

“…A surprising observation, in view of the general consensus about the role of the TonB box, was that the deletion of the entire "plug" from FhuA still produced a TonB-dependent transport of ferrichrome (92,336). (A much earlier study of the deletion between residues 21 and 128 showed a similar, unimpaired transport phenotype [116], but the mutant protein still contained the Ton box.)…”

Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli

“…Higher resolution shells were omitted from the refinement process because of very high R values (Ͼ50%). The space group was determined to be P6 4 22 with the following unit cell parameters: a ϭ 61.58 Å, b ϭ 61.58 Å, c ϭ 121.95 Å, ␣ ϭ 90°, ␤ ϭ 90°, and ␥ ϭ 120°. The structure of TonB-77 was solved using molecular replacement with the program MOLREP (40) and REFMAC5 (41) from the program package CCP4 (42).…”