Journey to the center of the phage; revealing the ejectosome of Pectobacterium bacteriophage ΦM1

Podophages that infect gram-negative bacteria, such as Pectobacterium pathogen PhiM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here we describe the ejectosome of bacteriophage PhiM1 to a resolution of 3.32 angstroms by single particle cryo-EM. The core consists of tetrameric and octameric ejection proteins which form a ~1.5 MDa ejectosome that must transition through the ~30 angstrom aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 angstroms and 3.23 angstroms, and note an uncommon fold adopted by the dimerised decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the PhiM1 particle, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages.