The goal of these studies is to characterize the large and complex virion of Salmonella phage SPN3US and toestablish a genetic system to facilitate studies of the fundamental features of giant phages and how theyparasitize their hosts. This research is significant as recent studies have shown that giant phages abound inthe environment. However, the long genomes of giant phages (>200 kb) encode many genes of whichtypically 80% are functionally uncharacterized. That is, essentially we have extremely limitedunderstanding as to the molecular mechanisms of giant phage infection within the host cell. This is aproblem because most giant phages were isolated with the goal of using them for phage therapy to treat multi-drug resistant pathogens or other novel biocontrol applications. The lack of knowledge regarding giant phagesrepresents a significant hurdle to obtaining regulatory approval for therapeutic phages. Additionally, withoutinsight into the processes of host takeover and infection, there can be no rational optimization of the mostappropriate phage(s) to select for therapeutics or identification of phage proteins to target for further researchas novel biotechnological tools. This research will address the lack of fundamental knowledge aboutgiant phages by the characterization of Salmonella phage SPN3US as a model for an expanding groupof phages that infect human pathogens. Preliminary studies have shown SPN3US is a suitable model phage sharing a core set of genes withother giant phages. In addition, SPN3US infects the genetically tractable host Salmonella Typhimurium LT2which has facilitated the isolation of SPN3US amber mutants, the first such collection for any giant phage. Ourstudies have shown SPN3US is an extraordinarily large tailed phage with novel structural features and iscomprised of >70 different proteins. As with all tailed phages, the role of the SPN3US capsid or head is toprotect the phage genome while in the environment to enable its delivery to a new bacterium. Wehypothesize that the SPN3US head has evolved an additional role to transport a large cargo of ejectionproteins that enter the Salmonella cell with the dsDNA and ensure its subordination to the goal of viralprogeny production. Consequently, we also hypothesize that giant phages have a set of essentialvirion proteins to achieve both these roles. This research aims to test these hypotheses with the followingthree aims: (1) An in-depth characterization of the SPN3US virion and head to identify all component proteinsand the copy numbers of each per phage particle. This will be achieved via structural analyses using Cryo-electron microscopy and proteomic analyses using mass spectrometry. (2) Identification of the essentialproteins in the SPN3US virion. This aim will be achieved via the isolation and genome sequencing of ambermutant phages, and (3) Delineation and characterization of SPN3US head proteins that are ejection proteinsvia biochemical, structural and proteomic analyses of mutant phage particles.