Isolation of 32 Mycobacteriophages and Genomic Analysis of the Novel Mycobacteriophage, Roscoe
Drs. Aaron Best and Joseph Stukey
Thirty-two new mycobacteriophages were isolated from soil samples collected on or nearby Hope College in Holland, Michigan. All were capable of infecting Mycobacterium smegmatis and produced a variety of plaque morphologies based on size, shape, and clarity, consistent with the isolation of an assortment of different phages. Both lytic and temperate phages appear represented in this collection. Thirty-two purified phage stocks were used to prepare genomic DNA samples for restriction digest analysis. A comparison of those 32 digest results revealed few similarities among the group, further supporting our interpretation that most of the new phage isolates were distinct. One mycobacteriophage, Roscoe, was chosen for complete genome sequencing using the Ion Torrent Personal Genome Machine platform and comparative genomic analysis. The predominant plaque produced by Roscoe was 2-4 mm in diameter and displayed a clear center surrounded by a wide turbid comet-tailed ring after 24 hours of growth at 37°C. Comparison of the restriction digest pattern for Roscoe with more than 200 known mycobacteriophage genomes did not yield an exact match, suggesting Roscoe was a novel mycobacteriophage. Genome sequence data for Roscoe supported that prediction but also revealed a relationship to a large group of mycobacteriophages in Cluster B1. The genome of Roscoe is 68 Kb, 66.5% GC, and contains 103 genes in agreement with the genome characteristics of closely related phage. A detailed analysis of the complete genome sequence and comparison with sequenced members of this small and unique group of mycobacteriophages is the subject of the second semester of this yearlong course and is presented.
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