PROPERTIES OF THE FHUA CHANNEL IN THE ESCHERICHIA-COLI OUTER-MEMBRANEAFTER 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 pro tein. A FhuA deletion derivative lacking residues 322 to 355 (FhuA Del ta 322-355) 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 e lectrochemical 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 Delta 3 22-336 bound ferrichrome and displayed a residual Ton-dependent ferric hrome transport activity. FhuA Delta 335-355 no longer bound ferrichro me but supported ferrichrome diffusion through the outer membrane in t he absence of the Ton system. FhuA Delta 335-355 rendered cells sensit ive 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 Delta 70-223, whi ch has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only w eak ferrichrome uptake, suggesting formation of a nonspecific pore thr ough the outer membrane. FhuA Delta 457-479 supported Ton-dependent up take of ferrichrome. None of these FhuA deletion derivatives formed po res in black lipid membranes with a stable single-channel conductance. Rather, the conductance displayed a high degree of current noise, ind icating a substantial influence of the deletions on the conformation o f the FhuA protein. FhuA also supports infection by the phages T1, T5, and phi 80 and renders cells sensitive to albomycin and colicin M. Ce lls expressing FhuA Delta 322-336 were sensitive to albomycin and coli cin M but were only weakly sensitive to T5 and phi 80 and insensitive to T1. Cells expressing FhuA Delta 335-355 were resistant to all FhuA ligands. These results indicate different structural requirements with in the gating loop for the various FhuA ligands. Cells expressing FhuA Delta 457-479 displayed a strongly reduced sensitivity to all FhuA li gands, while cells expressing FhuA Delta 70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resist ant. It is concluded that residues 335 to 355 mainly determine the pro perties of the gate with regard to FhuA permeability and ligand bindin g.