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.