Document Type

Honors Thesis

Publication Date

Spring 2015

Abstract

Mycobacteriophages (phages) are diverse and abundant viruses that infect species of the genus Mycobacterium. Mycobacteriophages are categorized into clusters based primarily on nucleotide similarity (18). Some clusters are well-characterized, while others, such as Cluster E, are poorly characterized (20). There are 54 members of Cluster E (39) including the phage Ukulele that was isolated at the University of Maine in 2011. This thesis is aimed towards characterizing Cluster E phages using Ukulele as a model. Cluster E phages have long tails of approximately 300 nm and they produce slightly turbid plaques on a lawn of Mycobacterium smegmatis. Putative simple terminators and two repeat sequences potentially involved in regulating gene transcription or translation were identified, as well as an HNH endonuclease at the right end of the genome that may functionally associate with the terminase. Cluster E phages are temperate but genes that encode the repressor and excise are not obvious. Gp88 was identified as a potential repressor gene and gp52 was identified as a potential excise, repressor, or Cro-like gene. Each of these genes was deleted independently from the Ukulele genome using the Bacteriophage Recombineering of Electroporated DNA system (35) to learn more about its function. Gp88Δ mutants are not viable and have not yet been isolated using complementation. Therefore, gp88 does not encode the repressor. Gp52Δ mutants have been detected but a pure mutant has not yet been isolated and further experiments are needed to determine the function of gp52.

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