FhuA, an Escherichia coli outer membrane protein with a dual function of transporter and channel which mediates the transport of phage DNA.
FhuA (M(r) = 78,900) is an Escherichia coli outer membrane protein which transports the ferric siderophore ferrichrome and is the receptor for phage T5, phi 80 and T1 and for colicin M. FhuA was purified chromatographically in non-ionic detergent (octyl glucoside). The circular dichroism spectrum indicates that FhuA is essentially organized in beta-strands like the majority of proteins of the outer membrane of Gram-negative bacteria. The structural parameters of FhuA were assessed from size exclusion chromatography, sedimention equilibrium and velocity experiments. FhuA is monomeric in solution and functional since binding of phage T5 causes the release of the phage genome, a double-stranded DNA of 121,000 base pairs, into the surrounding medium. Planar lipid bilayer experiments showed that the FhuA transporter is converted into a high conductance channel upon binding of phage T5. FhuA was reconstituted into large unilamellar vesicles (mean diameter 125 nm). Cryo-electron microscopy and fluorescence experiments, using a DNA intercalant YO-PRO 1, showed that binding of T5 to FhuA triggers the transfer of the phage genome into the proteoliposomes without altering their morphology. Two models can account for these observations, which apply both to in vitro and in vivo DNA transport. The simplest model supposes that the naked DNA is transported through the FhuA channel. Alternatively transfer of DNA might be mediated by pb2, the protein forming the phage straight fiber. pb2 would insert either directly in the membrane or inside the FhuA channel.
Laboratoire des Biomembranes, ERS 571 CNRS, Université Paris-Sud, Orsay, France.
M
M
Bonhivers
L
L
Plançon
A
A
Ghazi
P
P
Boulanger
M
M
le Maire
O
O
Lambert
J L
JL
Rigaud
L
L
Letellier
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics*; D001692:Biological Transport; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism* / Q000821:virology; D029968:Escherichia coli Proteins; D007473:Ion Channels / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008954:Models, Biological; D011991:Receptors, Virus / Q000737:chemistry / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D004279:DNA, Viral; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D007473:Ion Channels; D008081:Liposomes; D011991:Receptors, Virus