University Archives

Bacteriophages encode diverse proteins capable of manipulating or disrupting bacterial processes, including toxic gene products that may serve as a new antibacterial agent. The mycobacteriophage Fred313, isolated at the University of Detroit Mercy, contains several genes previously identified as toxic to Mycobacterium smegmatis, yet the structural basis for this toxicity remains unclear. In this study, the Fred313 gene library was refined through verifying gene-specific primers using PCR amplification and gel electrophoresis, confirming correct base-pair lengths for the majority of genes that were tested. Three previously identified toxic genes—19, 51, and 54—were examined further using toxicity dot assay using anhydrotetracycline induction varying in concentration levels. All three genes demonstrated lethality in M. smegmatis, confirming their toxic phenotypes. To investigate the mechanism of toxicity, AlphaFold structural predictions were generated for genes 19, 51, and 54. Gene 19 formed a β-rich stopper fold similar to known protein SPP1 gp16. Gene 51 displayed a large, multi-domain enzyme that was consistent with nucleotide-modifying reductase, that disrupts essential metabolic pathways. Gene 54 consisted of α-helical folds relating to interaction based toxins in TA systems. Together, the structural and phenotypic evidence presented explores the functional characteristics of Fred313 and the potential therapeutic advances of phage-derived toxins as an antibacterial.