Identification of novel inhibitors of Mycobacterium smegmatis growth through genome-wide overexpression of Cluster P3 mycobacteriophage Xavia genes

Summary We examined whether genes encoded by the mycobacteriophage Xavia disrupt growth of Mycobacterium smegmatis , a widely used mycobacterial model. Seventy-one Xavia genes were individually expressed using an inducible plasmid system and assessed for effects on colony formation. Two genes were lethal even without induction, indicating toxicity under basal expression. Induction of sixteen additional genes reduced bacterial growth, spanning structural proteins, lysogeny regulators, DNA-associated enzymes, a lysis protein, and several genes with no known function. These findings expand functional insights into mycobacteriophage gene repertoires and identify candidates for future mechanistic studies. Abstract Bacteriophage genomes encode large numbers of genes with no known function, and many of these genes affect essential host processes when expressed in a heterologous system. For mycobacteriophages, genome-wide overexpression in Mycobacterium smegmatis provides a direct way to identify proteins that impair growth and to determine which mycobacterial pathways are sensitive to phage gene products. To evaluate the cytotoxic potential of the Cluster P3 phage Xavia, a lineage that has not undergone functional screening, we constructed an arrayed pExTra library containing 71 predicted Xavia genes under control of the anhydrotetracycline inducible promoter pTet . All constructs were sequence-verified and transformed into M. smegmatis , and induction allowed measurement of gene-specific effects on growth. Two genes prevented recovery of transformants, suggesting toxicity under basal promoter leakiness. Inducible expression of 16 additional genes impaired growth, and these inhibitory proteins include structural components, regulators of lysogeny, enzymes of DNA metabolism, a lysis factor, and several proteins with no known function. Four of the strongest inhibitors were genes with no known function. These results extend functional screening into the previously untested P3 branch of Actinobacteriophages and identify new proteins that require mechanistic analysis.