Finding Functions for Unfamiliar Phage Genes
Drs. Virginia McDonough Stukey and Joseph Stukey
Mycobacteriophages are viruses that infect bacterial cells of the genus Mycobacterium. With more than 330 mycobacteriophage genomes sequenced and available in GenBank, they represent the largest collection of sequenced phages that infect a single host (Mycobacterium smegmatis). Comparative genomic analyses show the mycobacteriophages are highly diverse, representing more than 25 distinct genetic “types”. They possess a multitude of unfamiliar or novel genes –genes encoding protein sequences that do not resemble any previously studied proteins. Consequently, the functions of the proteins encoded in those novel phage genes cannot be predicted but need to be discovered. Some of the novel phage proteins are predicted to have roles in subverting host cell systems following phage infection, to benefit phage reproduction. To identify those critical phage gene functions, we expressed individual phage genes or small clusters of phage genes in the host cell and examined for effects on host cell growth (cytotoxicity). We are using this approach on a mycobacteriophage called Pumpkin, which was isolated at Hope College in 2008. We have identified three phage genes, Pumpkin_115, Pumpkin_119, and Pumpkin_143 and at least two other small genomic regions, encompassing genes 121-127 and genes 130-133 that are cytotoxic to M. smegmatis. Future work will include dissecting those genomic regions to identify the relevant cytotoxic genes, testing all cytotoxic phage gene products for interactions with host cell proteins to identify the host systems targeted by mycobacteriophage Pumpkin, and deleting each cytotoxic gene from the phage Pumpkin genome to confirm a role in the phage infection process.
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