Functional Characterization of 123 Genes from Mycobacteriophage LeBron Uncovers Cytotoxic Gene Families in Cluster L1

Bacteriophages represent a vast reservoir of genetic diversity however, functional annotation remains a major challenge, as most predicted gene products lack detectable similarity to characterized gene families. Experimental approaches such as systematic overexpression screens provide an avenue for identifying phage genes that influence bacterial physiology. Here, we report an overexpression screen of all 123 predicted protein-coding genes from Cluster L1 mycobacteriophage LeBron, the first representative of this cluster to undergo genome-wide functional analysis. Expression assays in Mycobacterium smegmatis revealed that 39 genes (32%) impaired host growth, with nineteen of these toxic genes (49%) assigned no known function. The proportion of cytotoxic genes observed in LeBron is comparable to findings from Clusters K and F, despite minimal sequence conservation across clusters. Interestingly, a subset of LeBrons toxic genes appear to be functionally analogous to previously identified toxic genes in other clusters, suggesting conserved biological activities carried out by non-homologous proteins. Additionally, this analysis uncovered several novel gene families that elicit strong cytotoxic effects, expanding the known catalog of phage-derived bacterial growth inhibitors. These results provide new insights into phage gene functions and demonstrate the value of genome-wide expression screening for uncovering conserved and cluster-specific interactions between bacteriophages and their hosts.