The Salmonella-infecting bacteriophage P22 packages its genome from concatemers of dsDNA. This packaging machinery consists of a large (L-) and small (S-) terminase complex. The small terminase functions to properly position the large terminase for packaging initiation via the pac site. The large terminase uses ATP hydrolysis to translocate a single copy of the genome into the bacteriophage procapsid. The motor activity of L-terminase results in dsDNA packaging at a rate of up to 2000 bp/sec. Although poorly understood, termination and cleavage are induced upon reaching a filled procapsid. Upon cleavage, the L-/S-terminase complex quickly disassociates from the capsid enabling tail and associated proteins to bind, sealing the genome inside the procapsid. Upon binding to surface receptors of Salmonella, dsDNA along with ejection proteins (gp7, gp16, and gp20) are injected to host cytoplasm. These ejection proteins are required for correct delivery of dsDNA to host cytoplasm.
Aim 1 will [use single particle electron microscopy] elucidate the 3D structure and stoichiometry of L-/S- terminase holoenzyme;a state just prior to binding to procapsid. [Current proposed mechanisms for terminase mechanism is based on inconclusive, low-resolution, 34 angstrom data. Resolution better than 7A will resolve mechanism controversies in the field.] Aim 2 will characterize the 3D structure of P22 with its terminase complex bound;a state just prior to genome packaging.
Aim 3 will capture the structure of genome and gp26 (tail needle) deficient P22;a state just after genome ejection. We have preliminary [single particle] data of a gp26 minus phage that shows extra density where the tail needle used to be. It is our hypothesis that an ejection (gp7, gp16, or gp20) contributes to this density. Together, the aims will enable new hypotheses to be developed which will elucidate the mechanism of genome packaging and injection into the host
P22 serves as a model for more complex human pathogens such as Herpes simplex virus (HSV). The goals of this proposal are to understand structural mechanisms of genome packaging and host cell injection.