Abstract
Bacterial Type I restriction-modification (R-M) systems present a major barrier to foreign DNA entering the bacterial cell. The temperate phage P1 packages several proteins into the virion that protect the phage DNA from host restriction. Isogenic P1 deletion mutants were used to reconstitute the previously described restriction phenotypes associated with darA and darB. While P1ΔdarA and P1ΔdarB produced the expected phenotypes, deletions of adjacent genes hdf and ddrA also produced darA-like phenotypes and deletion of ulx produced a darB-like phenotype, implicating several new proteins of previously unknown function in the P1 dar antirestriction system. Interestingly, disruption of ddrB decreased P1's sensitivity to EcoB and EcoK restriction. Proteomic analysis of purified virions suggests that packaging of antirestriction components into P1 virions follows a distinct pathway that begins with the incorporation of DarA and Hdf and concludes with DarB and Ulx. Electron microscopy analysis showed that hdf and darA mutants also produce abnormally high proportions of virions with aberrant small heads, which suggests Hdf and DarA play a role in capsid morphogenesis. The P1 antirestriction system is more complex than previously realized and is comprised of multiple proteins including DdrA, DdrB, Hdf, and Ulx in addition to DarA and DarB.