FhuA-mediated phage genome transfer into liposomes: a cryo-electron tomography study.
BACKGROUND
The transfer of phage genomes into host cells is a well established but only dimly understood process. Following the irreversible phage binding to a receptor in the bacterial outer membrane, the DNA is ejected from the viral capsid and transferred across the bacterial cell envelope. In Escherichia coli, the mere interaction of the phage T5 with its outer membrane receptor, the ferrichrome transporter FhuA, is sufficient to trigger the release of the DNA from the phage capsid. Although the structure of FhuA has been determined at atomic resolution, the understanding of the respective roles of phage and bacterial proteins in DNA channeling and the mechanisms by which the transfer of the DNA is mediated remains fragmentary.


RESULTS
We report on the use of cryo-electron tomography to analyze, at a molecular level, the interactions of T5 phages bound to FhuA-containing proteoliposomes. The resolution of the three-dimensional reconstructions allowed us to visualize the phage-proteoliposome interaction before and after release of the genome into the vesicles. After binding to its receptor, the straight fiber of the phage T5 (the "tip" of the viral tail made of pb2 proteins) traverses the lipid bilayer, allowing the transfer of its double-stranded DNA (121,000 bp) into the proteoliposome. Concomitantly, the tip of the tail undergoes a major conformational change; it shrinks in length (from 50 to 23 nm), while its diameter increases (from 2 to 4 nm).


CONCLUSIONS
Taking into account the crystal structure of FhuA, we conclude that FhuA is only used as a docking site for the phage. The tip of the phage tail acts like an "injection needle," creating a passageway at the periphery of FhuA, through which the DNA crosses the membrane. A possible mechanistic scenario for the transfer of the viral genome into bacteria is discussed.
Böhm
J
J
Max-Planck-Institut für Biochemie, Molekulare Strukturbiologie, Am Klopferspitz 18a, 82152 Martinsried, Germany. boehm@biochem.mpg.de
Lambert
O
O
Frangakis
A S
AS
Letellier
L
L
Baumeister
W
W
Rigaud
J L
JL
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D020285:Cryoelectron Microscopy; D004279:DNA, Viral / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D018014:Gene Transfer Techniques; D016679:Genome, Viral; D008567:Membranes, Artificial; D011510:Proteolipids; D011991:Receptors, Virus / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D004279:DNA, Viral; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D008567:Membranes, Artificial; D011510:Proteolipids; D011991:Receptors, Virus; C020485:proteoliposomes