Biogenesis of a Bacteriophage Long Non-Contractile Tail.

Siphoviruses are main killers of bacteria. They use a long non-contractile tail to recognize the host cell and to deliver the genome from the viral capsid to the bacterial cytoplasm. Here, we define the molecular organization of the Bacillus subtilis bacteriophage SPP1 ~ 6.8 MDa tail and uncover its biogenesis mechanisms. A complex between gp21 and the tail distal protein (Dit) gp19.1 is assembled first to build the tail cap (gp19.1-gp21Nter) connected by a flexible hinge to the tail fiber (gp21Cter). The tip of the gp21Cter fiber is loosely associated to gp22. The cap provides a platform where tail tube proteins (TTPs) initiate polymerization around the tape measure protein gp18 (TMP), a reaction dependent on the non-structural tail assembly chaperones gp17.5 and gp17.5* (TACs). Gp17.5 is essential for stability of gp18 in the cell. Helical polymerization stops at a precise tube length followed by binding of proteins gp16.1 (TCP) and gp17 (THJP) to build the tail interface for attachment to the capsid portal system. This finding uncovers the function of the extensively conserved gp16.1-homologs in assembly of long tails. All SPP1 tail components, apart from gp22, share homology to conserved proteins whose coding genes' synteny is broadly maintained in siphoviruses. They conceivably represent the minimal essential protein set necessary to build functional long tails. Proteins homologous to SPP1 tail building blocks feature a variety of add-on modules that diversify extensively the tail core structure, expanding its capability to bind host cells and to deliver the viral genome to the bacterial cytoplasm.

Bacteriophage P22 tailspike: structure of the complete protein and function of the interdomain linker.

Attachment of phages to host cells, followed by phage DNA ejection, represents the first stage of viral infection of bacteria. Salmonella phage P22 has been extensively studied, serving as an experimental model for bacterial infection by phages. P22 engages bacteria by binding to the sugar moiety of lipopolysaccharides using the viral tailspike protein for attachment. While the structures of the N-terminal particle-binding domain and the major receptor-binding domain of the tailspike have been analyzed individually, the three-dimensional organization of the intact protein, including the highly conserved linker region between the two domains, remained unknown. A single amino-acid exchange in the linker sequence made it possible to crystallize the full-length protein. Two crystal structures of the linker region are presented: one attached to the N-terminal domain and the other present within the complete tailspike protein. Both retain their biological function, but the mutated full-length tailspike displays a retarded folding pathway. Fitting of the full-length tailspike into a published cryo-electron microscopy map of the P22 virion requires an elastic distortion of the crystal structure. The conservation of the linker suggests a role in signal transmission from the distal tip of the molecule to the phage head, eventually leading to DNA ejection.

Tailspike interactions with lipopolysaccharide effect DNA ejection from phage P22 particles in vitro.

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.