Document Type
Article
Publication Date
12-2025
Abstract
NormanBulbieJr (NBJ) is a temperate siphovirus isolated on the host Mycobacterium smegmatis mc2155 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 an additional gene involved in host defense against Cluster F phages and which was found to be critical for NBJ lysogen stability. 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 (Heller et al. 2024), 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.
Publication Title
bioRxiv
Recommended Citation
Wise, Bethany M.; Edwards, Kaylia; Jirsa, Cole R.; Abbruzzese, Sabrina; Adebiyi, Amina; Bapat, Sarika; Barnhardt, Tyler; Bastiampillai, Naomi; Begovic, Ella; Berchick, Mary G.; Bocco, Gilda; Bonoris, John; Boos, Elizabeth; Cassady, Margaret; Chehab, Julia; Cooper, Gene; Coyle, Hunter; Davis, Joseph; De la Cruz Vargas, Natalia; Delach, Matthew; Dowiak, Carly; Ferraro, David; Fuller, Madison; Glasser, Bootsie; Gordon, Kyle; Hoch, Hannah; Holleman, Sarah; Hood, Luke; Hurrell, Brett; Jacobs, Nicholas; Jiang, Danny; Kefalos, Steven; Maher, Salma; Martin, Jocelyn; Mengesha, Rohebot; Merenich, Daniel; Nayak, Ashwin; Nesterova, Dasha; Nguyen, Joshua; Okonkwo, Chukwudumebi; Pompan, Taylor; Redwood, Lauren; Scanlon, Christina; Schneider, Lily; Shenai, Aparna; Siatkowski, Colton; Strausner, Lisa; Vizzarri, Joseph; Watson, Jordan; Wilson, Angela; Wynne, Catherine; Zygmunt, Anthony; Kanther, Michelle; Payne, Kimberly M.; Pollenz, Richard S.; Sunnen, C N.; and Heller, Danielle M., "Identification and characterization of host-modulating effectors encoded by the Cluster F1 mycobacteriophage NormanBulbieJr" (2025). PCOM Scholarly Works. 2356.
https://digitalcommons.pcom.edu/scholarly_papers/2356
DOI: https://doi.org/https://doi.org/10.64898/2025.12.12.693979
Comments
This preprint was published in bioRxiv.
The original version is available at http://dx.doi.org/10.64898/2025.12.12.693979.
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. CC BY 4.0.