Identification and characterization of host-modulating effectors encoded by the Cluster F1 mycobacteriophage NormanBulbieJr

NormanBulbieJr (NBJ) is a temperate siphovirus isolated on the host Mycobacterium smegmatis mc 2 155 that encodes 102 gene products, 60 of which have no known function (NKF). Based on gene content, NBJ is classified as a Cluster F, Subcluster F1 phage and shares 70% of its encoded gene phamilies with Girr, another F1 mycobacteriophage that was recently analyzed in a genome-wide overexpression screen and found to encode 29 diverse gene products capable of inhibiting growth of M. smegmatis . Similar functional screens in other mycobacteriophages have uncovered a growing repertoire of diverse, phage-encoded bacterial growth inhibitors, providing prime candidates for dissecting novel bacterial-phage interactions. An arrayed overexpression library encoding all 102 genes was constructed and systematically screened using a plate-based cytotoxicity assay, identifying 29 genes that inhibit mycobacterial growth. Because mycobacteriophage genomes are also known to encode systems involved in phage-phage competition, we conducted additional phage defense assays for a subset of NBJ genes in our library. This analysis confirmed homotypic immunity by the predicted immunity repressor and identified gp45 as a second defense factor that protects against Cluster F phages and promotes NBJ lysogen stability. Additional mechanistic analyses indicate that gp45 functions in a secretion-dependent manner, acting after phage adsorption to reduce productive infection and early phage transcript accumulation. Escape mutant and bacterial two-hybrid analyses implicate Cluster F tape measure proteins as the likely target of gp45, consistent with disruption of an early step in phage genome delivery. Finally, we extended our analysis to explore the essentiality of all identified host-modulating genes in the NBJ life cycle, using CRISPR-enhanced recombineering to generate phage deletion mutants, revealing two host modulators that are critical for lytic growth. Conducted as part of the SEA-GENES undergraduate research consortium, this study adds to a growing functional genomics resource and provides new insights into the complex interactions between phage gene products and the mycobacterial host cell.