Anushka Tennakoon, Iresha K Edirisingha, Cole R Jirsa, Ethan Rutledge, Ankita Bhattacharyya, Ella King, Madelyn Moresi, Amara Lovings, Parker R Yarborough, Anna Hoben, Garrett Manns, Toney Ray Gibson, Syed Mohammed Ashfaque Uddin, Yeseul Bae, Natalie Taylor, Taylor L Johnson, Madelyn Futral, Paige Wilson, Jalyn Brown, Zackari L Arbogast, Abigail Brooks, Carson Ward, Courtney Foxworth, Kacey Nguyen, Daphne Fairchild, Deisy Lemus, Hayley White, Holly Craft, Justin Adonis, Kamiya Givan, Sergio Valdivia, Kayci Beth Wallace, Madeline Bent, Shikelya D Chambliss, Dmitri Mavrodi, Danielle M Heller, Ramesh Rijal
Bacteriophages encode numerous genes with no known function, many of which can affect essential cellular processes when expressed in the bacterial host. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis can be used to identify proteins that impair growth. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, we constructed a plasmid library containing 71 predicted Xavia genes under the anhydrotetracycline inducible promoter pTet and screened this library in a plate-based cytotoxicity assay to measure impacts on M. smegmatis growth. Two genes prevented transformants recovery, consistent with toxicity under basal promoter leakiness, and inducible expression of 18 additional genes impaired growth. These inhibitory proteins include structural components; factors involved in DNA metabolism, lysogeny, and lysis; and several proteins with no known function. These results extend functional screening into a lineage of actinobacteriophages that has not previously been characterized, and identify new proteins that warrant further mechanistic analysis.