Interactions between Host and Phage Encoded Factors Shape Phage Infection

Evolution of phages and their bacterial hosts are directed by interaction between phage and host-encoded factors. These interactions have resulted in the development of several defense and counter-defense strategies such as DNA restriction and antirestriction systems. Type I restriction-modification (R-M) systems present a barrier to foreign DNA, including phage, entering the bacterial cell, by cleaving inappropriately modified DNA in a sequence-specific manner. Phages have evolved diverse mechanisms to overcome restriction systems. The temperate coliphage P1 encodes virion-associated proteins that protect its DNA from host type I R-M systems. By using genetic and biochemical analysis, it has been established that the P1 Dar (Dar for defense against restriction) system is comprised of at least six virion-associated proteins: DarB, Ulx, Hdf, DarA, DdrA and DdrB. DarB protects P1 DNA from EcoB and EcoK restriction in cis by an unknown mechanism and is incorporated into the virions only in the presence of Hdf, DarA and DdrA. Hdf and DarA have also been found to affect capsid morphogenesis, as their absence results in phage progeny with predominantly aberrant small heads. Examination of purified P1 proheads shows that Dar system proteins are incorporated into the virion before DNA packaging, and an N-terminal signal is required for DarB packaging. Twenty-four additional P1 genes of unknown function were disrupted and none were found to alter the antirestriction phenotype. A purification protocol for the ~250 kDa antirestriction protein DarB has been optimized, which will facilitate biochemical approaches for determining its mechanism of action. While the phage-host interactions of classical phages such as P1 are better understood, relatively little is known about how phages outside of the common paradigm organisms interact with their hosts. A high throughput genetic screen of the T1-like coliphage LL5 and the rV5-like coliphage LL12 was conducted against the E. coli Keio collection. This screen revealed host receptors required for both phages to initiate infection and two chaperones, PpiB and SecB, required for efficient propagation of phage LL5