Bacteriophages from the family Podoviridae use short non-contractile
tails to eject their genomes into bacteria. However, there is limited
high-resolution information about structure and mechanism of genome delivery of
Podoviruses that infect Gram-positive bacteria such as S. aureus.
Here we used cryo-electron microscopy and X-ray crystallography to determine
the structures of S. aureus phage P68 in its native form, genome
ejection intermediate, and empty particle. The structure of the native phage
was solved to 3.3 Å and 3.9 Å for capsid and tail respectively. We
show that residues from N-terminus of the major capsid protein enable
incorporation of P68 portal complex into phage head by forming a special
interface. P68 head contains seventy-two subunits of an inner core protein,
which are positioned between the portal complex and phage genome. Fifteen of
the inner core proteins bind to and alter the structure of adjacent major
capsid proteins and thus specify attachment sites for head fibers. Unlike in
the previously studied phages, head fibers of P68 enable positioning of its
virion at cell surface for genome delivery. P68 genome ejection is triggered by
disruption of interaction of one of the portal protein subunits with phage DNA.
The inner core proteins are released before the DNA and probably enable translocation
of phage DNA across bacterial membrane into cytoplasm. The genome translocation
mechanism and the portal assembly mechanism is likely to be conserved among
bacteriophages infecting gram-positive bacteria.