Isolation of 35Mycobacteriophagesand Genomic Analysisof the NovelMycobacteriophage,Glass

Thirty-five 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. Purified phage stocks were used to prepare genomic DNA samples for restriction digest analysis. A comparison of those 35 digest results revealed few similarities among the group, further supporting our interpretation that most of the new phage isolates were distinct. One mycobacteriophage, Glass, was chosen for complete genome sequencing using the Illumina MiSeq platform and comparative genomic analysis. The predominant plaque produced by Glass at 32°C was turbid and 0.5-1.0mm in diameter, while plaque produced at 42°C was clear and 1.0-1.5mm in diameter. Genome sequence data for Glass revealed a relationship to a group of 12 mycobacteriophages in Cluster B2. The genome of Glass is 67.5 Kb, 69.0% GC, and contains 91 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.