Salmonella enterica Serovar typhimurium infects 100,000 people annually and causes Salmonellosis, which can be potentially fatal due to its dehydrating effects, among other symptoms like fever and diarrhea. An agent of Salmonella enterica Serovar typhimurium eradication, bacteriophage P22, has been studied over the years as a model system phage. P22 is a generalized transducing bacteriophage of the Podoviridae superfamily and the prototype of the P22-like genus.1 P22 capsid is assembled with 415 copies of coat protein (gp5), a triangulation number of 7, and an overall diameter of ∼630 Å in the mature virion (MV). P22-like phages possess a short and noncontractile tail apparatus, too short to span the host cell envelope.2 The P22 tail is a multi-subunit molecular machine3, 4 that provides all structural determinants for host attachment, adsorption, cell envelope penetration, and DNA delivery. Asymmetric cryo-EM reconstructions of the P22 MV proved invaluable in deciphering the organization of the capsid and tail apparatus.5, 6, 7, 8, 9 The tail emanates at a unique 5-fold vertex, interrupting the icosahedral capsid symmetry.10 Cryo-EM reconstructions of the isolated tail apparatus extracted from the infectious P22 virion11, 12 together with crystal structures of P22 tail factors (e.g., the portal protein, gp113, 14; portal protein bound to the head-to-tail adapter protein, gp413; the tail needle, gp2615, 16; the tailspike, gp917, 18, 19, 20 aided in elucidating the tail structure and assembly (Table S1). In vitro studies using purified P22 proteins revealed how the portal protein initiates procapsid assembly and tail factors assemble hierarchically at the end of genome packaging. The scaffolding protein regulates the polymerization of the P22 coat protein (gp5)21 and facilitates the incorporation of portal protein monomers into nascent procapsids by triggering portal ring formation.22, 23 Cryo-EM analysis of P22 precursor capsid (or procapsid) provided evidence that the scaffolding protein interacts with the N-terminus of the coat protein and the portal protein.8 Biochemical studies found that in addition to interaction with portal protein, the scaffolding protein C-terminal helix-turn-helix motif interacts with the coat protein N-terminus. Scaffolding protein is also critical for incorporating the ejection proteins gp7, gp20, and gp16.24, 25 P22 procapsids package concatemeric DNA until the head is full by the action of a powerful motor formed by the large (TerL)26 and small (TerS)27 terminase subunits, possibly assembled into a complex.28 After packaging is complete, the TerL nuclease domain cleaves off the concatemer, releasing the packaging motor and allowing tail factors to assemble onto the portal protein. The gp4 head-to-tail adaptor oligomerizes upon binding to a portal dodecamer,29, 30, 31 providing an assembly platform for the tail hub gp10, which is monomeric in solution but assembles onto gp4,32 generating a 6-fold channel that is sealed by gp26.33

The three ejection proteins, gp7, gp20, and gp16, are essential for infectivity and required for genome ejection into the host.34, 35, 36, 37 Unlike tail components, identifying and characterizing P22 ejection proteins has been challenging.34 These proteins are encapsulated into procapsids during morphogenesis and ejected into the host at infection.36 They play a crucial role in the early stage of transferring P22 DNA into the host,38 as supported by in vitro liposome experiments.39 Cryo-electron tomography (cryo-ET) analysis of P22 virions infecting Salmonella minicells revealed that the ejection proteins form a trans-envelope channel in the host cell envelope, involved in genome ejection.40 However, unlike T7, where the ejection proteins stack symmetrically onto the portal protein,41 or phage ΦcrAss001 that contains cargo proteins poised for ejection around the portal perimeter,42 P22 ejection proteins reside inside the virion, loosely associated with the portal barrel, as suggested by bubblegram studies.43 When the ejection proteins are genetically deleted from the virion, the P22 genome-packaging machinery packages a longer than average DNA molecule, suggesting the ejection proteins occupy volume within the virion that can be filled by packaged DNA when they are absent.44 Finally, in vitro studies revealed that P22 ejection proteins are not ejected in Salmonella unless both lipopolysaccharide (LPS) and the membrane protein OmpA are present.38 In contrast, slow ejection of P22 DNA without release of the ejection proteins can be triggered by the addition of LPS only.45

This paper describes the high-resolution structure of the phage P22 tail machine before genome delivery that we characterized using cryo-EM, genetic, and biochemical analysis. Our work sheds light on the components in the tail and inside the virion responsible for host attachment and genome ejection.