FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

Abstract

The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(‐)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the ‘gating loop’ (delta 322–355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322–355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

“…4); the FhuA-T5 interaction was described in ref. 16]. The ability of colicin Ia to halt the Cir-induced conductance rise across the membrane (Fig.…”

“…Given, as indicated above, that these channels are plugged, it is not surprising that, with the exception of two FhuA deletion mutants that displayed aberrant properties (12,13), no one has thus far reconstituted them in an ion-conducting state in lipid bilayer membranes (14)(15)(16). [The binding of T5 bacteriophage to FhuA generated approximately 1 nS channels (16), but it is not clear that these resulted from opening of the FhuA channel (17,18)].…”

“…[The binding of T5 bacteriophage to FhuA generated approximately 1 nS channels (16), but it is not clear that these resulted from opening of the FhuA channel (17,18)]. Our own initial attempts with several of these proteins were equally unsuccessful.…”

“…Bonhivers et al (16) have reported that the binding of the phage to FhuA in planar phospholipid bilayers resulted in the appearance of approximately 1 nS channels in 0.1 M KCl. We have not been able to reproduce this finding under the conditions described by these authors, but as we shall see below, we have also established conditions for observing phage-induced channels.…”

An internal affinity‐tag for purification and crystallization of the siderophore receptor fhua, integral outer membrane protein from escherichia coli K‐12

“…This model is further supported by a neural network designed for topology predictions of outer membrane proteins and available at http://strucbio.biologie.uni-konstanz.de/-kay/om-topopredict.html . FhuA can be induced to form a channel by the electrochemical potential of the CM via the TonB-ExbB-ExbD complex (Postle, 1993) and by the binding of the bacteriophage T5 (Bonhivers et al, 1996;Plaqon et al, 1997). Ligand binding sites on FhuA have been deduced by competitive peptide mapping (Killmann et al.…”