From High Street to Bishop Woods: The “Lives” of Two Phages Mary Owen, Andrew Schaff, Kayla Cartwright, Angel Chen, Jessica Clark, Bradley Davis, Paige Etchison, Shelby Frieszell, Jacquelyn Gross, Steph Smith, Elsa Clenny, Sammi Podolyan, Stephanie Swedik, Gabrielle Lopez, Tzvia Springer, Luis Actis, Mitchell Balish Miami University, College of Arts and Science: Department of Microbiology Abstract Methods Background Results Acknowledgments Conclusions Purpose: To study the data collected from mycobacteriophages HighStump and Mortcellus to gain new insights on the properties and characteristics of bacteriophages To broaden our current understanding of how we categorize and analyze diverse samples of bacteriophages Project Goal of this research To collect, isolate, and purify bacteriophage samples which infect the bacteria Mycobacterium smegmatis from various areas in Oxford, OH To study phage characteristics and assign them to specific clusters Plaque morphology Restriction digests Transmission electron microscopy images The genomes of HighStump and Mortcellus were annotated to analyze possible gene composition and function The Basic Local Alignment Search Tool (BLAST) was used to search protein databases for known protein sequences in other bacteriophages similar to those of HighStump and Mortcellus HHpred was used to detect homology between the predicted bacteriophage protein sequences and proteins with known structure First-year students in Miami University’s Microbiology 223/224 classes isolated and characterized the growth, appearance, and gene content of two Mycobacterium smegmatis bacteriophages isolated at or near Miami University’s main campus in Oxford, Ohio. The bacteriophage HighStump was found in soil close to a tree stump not far from a construction site on High Street, the main road connecting campus with the commercial center of Oxford. HighStump exhibited lytic growth and large, bullseye plaques on M. smegmatis, and was characterized visually by a long tail, exceeding a length of 250 nm. Analysis of its 68,821 bp-long genome placed HighStump in the B1 subcluster of mycobacteriophages, consistent with these phenotypes. With 103 genes (preliminarily), HighStump was found to be a fairly typical member of this well- characterized cluster. It had a very long tapemeasure protein, 1,991 amino acids in length, consistent with the length of its tail. The class did not identify any genes that were unique to this bacteriophage, and overall, as determined by examination of best BLAST hits, the predicted amino acid sequences of its genes had particularly strong phylogenetic relatedness to those of B1 subcluster bacteriophages Newman, Oline, and Vivaldi. Interestingly, despite the lytic growth of this bacteriophage in our laboratory conditions, the genome of HighStump included a gene potentially encoding an excisionase, suggesting that it could be lysogenic under some as-yet-unestablished set of conditions. The bacteriophage Mortcellus was cultured from soil beneath decomposing leaves in Bishop Woods, a wooded area on the Miami University main campus that was recently subjected to substantial thinning of arboreal growth and then reopened to public traffic. Mortcellus grew as pinprick plaques on M. smegmatis and exhibited lysogenic behavior. Like those of HighStump, Mortcellus virions had long tails, but not as long. The genome of Mortcellus was 69,800 bp in length and its nucleotide sequence clustered with the B3 subcluster of bacteriophages, despite an early prediction from DNA analysis that it was a member of cluster D. Though not currently as well-populated as the B1 subcluster, subcluster B3 is fairly replete with characterized species. BLAST analysis indicated close phylogenetic relatedness of a substantial portion of its predicted 103 genes to bacteriophages Pipefish, Phlyer, and Phaedrus. Like HighStump, there were no surprises among the Mortcellus genes in comparison with other members of its subcluster. The value of studying these organisms, aside from the educational component, is in broadening understanding of the genes common to members of the respective subclusters at the sequence level, enabling greater insight when the products of these genes are understood at the biochemical, genetic, and cellular levels in the future. Phages were isolated from warm, moist environments in Oxford, Ohio: HighStump: a decaying tree stump on High Street Mortcellus: mulchy soil underneath decaying leaves by the side walk in Bishop Woods Phages were both isolated through enrichment of culture and plated using the host Mycobacterium smegmatis (1) Phages were purified through serial dilution: HighStump: 4 rounds of purification Mortcellus: 3 rounds of purification Stock solution of each phage was prepared with a titer of: HighStump: 9.4 ✕ pfu/mL Mortcellus: 5.56 ✕ 10 9 pfu/mL Transmission electron microscopy (TEM) was used to view the two phages In preparation for sequencing (1): The phage solutions were first treated with nucleases Then, the phage particles were precipitated with polyethylene glycol and the capsids were denatured Finally, the phage DNA were isolated by a DNA-binding column and eluted with distilled water Phage DNA was sequenced at the Pittsburgh Bacteriophage Institute The genomes of the two phages were annotated using the Artemis and DNAMaster computer program interfaces Functions of the proteins coded by these genes were predicted using BLAST and HHpred 1. Science Education Alliance (2011). NGRI Phage Laboratory Manual. Chevy Chase, Maryland: Howard Hughes Medical Institute. 2. Belcaid, M., Bergeron, A., & Poisson, G. (2011). The evolution of the tape measure protein: Units, duplications and losses. BMC Bioinformatics, 12. References Figure 1. HighStump plaques on M. smegmatis. Plaques exhibited a bullseye, lytic morphology with a radius of 4.7 mm. Figure 2. Mortcellus plaques on M. smegmatis. Plaques exhibited a pinprick, lysogenic morphology with a radius of 0.35 mm. This work was supported by the Howard Hughes Medical Institute, the Miami University College of Arts and Science, and the Miami University Department of Microbiology. We thank Dr. Richard E. Edelmann of the Miami University Center for Microscopy and Imaging and the staff of the Pittsburgh Bacteriophage Institute. Figure 5. Organization of HighStump genome. HighStump contained 103 genes spanning its 68,821-bp chromosome. Gene content and organization were typical of Subcluster B1 phages, as predicted from restriction analysis. In agreement with the long tail (Fig. 3), the tape measure protein (2) was predicted to be a very long 1,991 amino acids. Figure 6. Organization of Mortcellus genome. Mortcellus contained 103 genes spanning its 69,800-bp chromosome. Gene content and organization were typical of Subcluster B3 phages, despite prediction from restriction analysis that it would be in Cluster D. Interestingly, the block of genes from exhibited 100% amino acid conservation with 14 other completely annotated phages, suggesting strong conservation of this unit, which contains no genes of known function. HighStump and Mortcellus are newly described members of cluster B isolated in Oxford, Ohio HighStump: subcluster B1 Mortcellus: subcluster B3 Both have 103 genes Their gene content is very similar to other members of their subclusters No new genes Some highly conserved genes In the interest of exploring diversity of mycobacteriophages, it might be worth taking steps to avoid genome sequencing of phages from well-explored clusters and subclusters The question of what makes cluster B so well-represented is interesting Do they have a highly successful set of genes that makes them well- adapted? Are they particularly easily cultured compared to some other clusters? Figure 3. TEM of HighStump. HighStump had a tail length of 290 nm with distinct tail fiber proteins visible. Figure 4. TEM of Mortcellus. Mortcellus had a tail length of 250 nm. Tail fiber proteins are less distinct than in Fig. 3.