Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly

Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" β' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution.