Isolation and Genome Sequencing of Two Novel Mycobacteriophages, Optimus and Sassafras

Faculty Mentor(s)

Dr. Joseph Stukey, Hope College

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This research was supported by the Howard Hughes Medical Institute, Science Education Alliance.


Twenty new mycobacteriophages, capable of infecting Mycobacterium smegmatis, were isolated from soil samples collected on or nearby Hope College in Holland, Michigan. Collectively, the group displayed a variety of plaque morphologies indicating 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. Of 20 samples analyzed, a total of 13 phages produced just 4 types of restriction digest patterns indicating some degree of relatedness among some of our new phage isolates. Interestingly, one group of 4 phages (Optimus, Lynx, Aurora and TheCube14) that yielded a similar restriction digest pattern, were all isolated from mulch-covered soil at a depth of 4-8 cm. Two phages (Optimus and Sassafras) were chosen for complete genome sequencing and comparative genomic analyses. Both phages produced plaques of between 1-2 mm in diameter at 24 hours that enlarged to about 4 mm in diameter after 48 hours of incubation at 37°C. Whereas continued incubation of phage Optimus resulted in cessation of plaque growth by 72 hours, plaques produced by Sassafras continued to enlarge beyond 8 days, reaching a diameter of greater than 10 mm. Phage Optimus produced plaques that displayed a clear center surrounded by turbid rings. Phage Sassafras produced clear plaques with defined edges at 24 hours, but all subsequent growth was progressively more turbid in nature, resulting in plaques with a turbid ring around a center clear zone. Comparison of the restriction digest patterns for Optimus and Sassafras with more than 60 existing mycobacteriophage genomes indicates that Optimus may be a new representative of cluster H, while Sassafras shows some similarity to the F cluster of mycobacteriophages. Results of our analyses of both genomes are reported.

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