Analyses of amber mutants of Salmonella phage SPN3US

The giant Salmonella enterica phage SPN3US belongs to the &phis;KZ-related phages which are amongst the most complex virions of prokaryotic viruses, even more complex than most eukaryotic viruses. SPN3US for example assigns 46% of its 240kbp genome to code for virion proteins. We have limited knowledge about these giant phages as they encode extremely diverged genes, from other phages and even to one another. The &phis;KZ-related phages possess large genomes and correspondingly large and complex capsids, two sets of fragmented RNAP beta and beta'-like proteins and they also exhibit an unusual replication having a DNA polymerase split into two polypeptides.;The complete genome of phage SPN3US was previously sequenced by Lee et al. (2011). As is typical for a newly sequenced phage, 80% of its ORFs were annotated as hypothetical. To address this problem of functionally unassigned genes, a novel strategy for phages is to target the essential genes and work to determine their functions using genetic approaches. In this work amber mutants of a giant phage were sequenced for the first time. Once the amber mutation sites were determined the sequence similarity of those genes in other &phis;KZ-related phages was analyzed. This has enabled us to identify fourteen essential genes in SPN3US, ten of which code for the components of the phage virion. Previously we knew the function of three genes, now we can infer the function of four more genes. The mutants encoding these essential genes will now form the basis of further studies to determine specific functions for these novel genes. Ten of these genes are well conserved among the &phis;KZ-related phages being classified as core genes for this subfamily.;These studies have demonstrated that SPN3US is an excellent model system to study the &phis;KZ-related phages.