Bacteriophages from family Podoviridae
are characteristic by their short tails. Bacteriophage P68 is dsDNA virus
infecting Staphylococcus aureus. To penetrate in the host cell, the
phage tail is equipped with proteins that cleave a pore in the bacterial cell
wall and cell membrane. Subsequently, the phage genome is ejected into the host
cell where the replication process takes place. However, there is limited
information about the native structure and cell penetration mechanism of Podoviridae
phages infecting gram-positive bacteria.
Here we present in situ cryo-EM structure of the full capsid
and tail at resolutions of 3.3 Å and 3.9 Å respectively. The atomic
model reveals unique interconnections between phage structural proteins, and
structural differences between full and empty particle show proteins involved
in membrane penetration. We found two novel proteins (Arstotzka protein and
Acne protein) in capsid, which have not been observed before. Structural
analysis of the Arstotzka protein suggests its participation in dsDNA and
capsid stabilization, while structural and sequential analysis of the Acne
protein has shown its involvement in receptor binding. Native structure of the
tail revealed 72 copies of a unique structural protein forming dodecameric ring
positioned between portal and dsDNA, which is not present in the electron
density map of the empty particle. Sequence analysis and electron microscopy
shows that this protein might be released before dsDNA and penetrate the
bacterial cell membrane. Furthermore, we found out that the structure of
receptor binding protein (RBP) from P68 solved at 2.0 Å resolution by X-ray
crystallography is similar to RBP from family Siphoviridae.
Here we present a function of different structural proteins of P68,
and show how native particle of bacteriophage P68 is assembled. These findings
demonstrate that there is more than one mechanism of the cell membrane
penetration in the family Podoviridae, and that the receptor binding
mechanisms are conserved among different families of bacteriophages infecting
gram-positive bacteria.