Identification of a Cytotoxic Gene in Mycobacteriophage Vix

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Dr. Joseph Stukey, Hope College

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The genomes of more than 75 mycobacteriophages, all of which are capable of infecting Mycobacterium smegmatis, have now been sequenced and are available in GenBank. Despite the ability of those phages to infect a single host, their genomes exhibit great genetic diversity. In fact, a comparative genomic analysis of all sequenced mycobacteriophages indicates that there are more than 20 genetically distinct versions of genomes represented in this collection. This raises the interesting question of how the different phages wrest control from the host cell during lytic infection and whether they target the same or different points of the host cell metabolism. We hypothesize that mycobacteriophages assume control over their host by making proteins that directly interact and interfere with the functioning of sensitive host cell proteins. We further hypothesize that isolated expression of the genes that encode those phage proteins in M. smegmatis will be cytotoxic to the bacterium. The only mycobacteriophage genes previously shown to be cytotoxic to M. smegmatis are residents of the L5 genome. A new mycobacteriophage Vix, recently isolated at Hope College, contains a gene (Vix75) that has homology to one of the cytotoxic L5 genes. The Vix75 gene was amplified by PCR and ligated to the promoter region of the inducible M. smegmatis acetamidase gene on plasmid pLAM12. The pVIX75 recombinant plasmid was transferred into M. smegmatis and transformed cells were tested for growth on medium containing acetamide to induce expression of the Vix75 gene. Our results show that Vix75 is cytotoxic to M. smegmatis. We are currently investigating the mechanism of this cytotoxic activity and the relevant host cell target.

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