Mycobacteriophage Cluster Identification Using The Minor Tail Protein Gene

Abstract

The HHMI SEA-PHAGES Program, where students isolate a phage from an environmental soil sample, currently consists of 136 colleges and universities. However, due to cost, only two phages from each university are sequenced per annum. There is a high demand for standardization and classification of phages discovered by phage hunting. Although academic laboratories have isolated and purified many novel phages, there is little to no phage-specific information documented other than plaque morphology and phage host used. At the time of writing, PhagesDB.org had records of 10,654 phages isolated on the host Mycobacterium smegmatis. Of these, only 1,782 have been sequenced and their cluster identifications have been denoted. These mycobacteriophages belong to clusters A, AA, AB, AC, B, C, D, E, F, G, H, I, J, K, L. M, N, O, P, Q, R, S, Singleton, T, U, V, W, X, Y, Z, all of which infect M. smegmatis. In this study, we used the Minor Tail Protein (MTP) Gene targeting primers to identify cluster and subcluster designations for novel, not yet sequenced mycobacteriophages. We developed a kit that will enable the students in the SEA-PHAGES Program to characterize their mycobacteriophages. Primer sets were designed computationally in Geneious software according to a set of aligned templates representing sequenced mycobacteriophage genomes. These primer sets were tested using PCR and gel electrophoresis on control phages representing each cluster, then cross tested against each control to verify their specificity. Control phages were then tested against Smith’s Tape Measure Protein (TMP) primers. We show that MTP primers determine the cluster designations of the phages better than TMP. Both MTP and TMP primer sets were tested on novel mycobacteriophages isolated during the 2019 George Mason University Phage Discovery Course (BIOL 401). All positive PCR products were sequenced to verify the MTP sequence. We conclude that MTP primers may be used in combination with Tape Measure Protein (TMP) primers as well as the Phage Enzyme Tool (PET) to positively identify cluster designations prior to sequencing. MTP primers are a cost-effective tool to classify mycobacteriophages prior to sequencing and can help to prioritize which genomes to sequence as certain (sub)clusters have been somewhat saturated. This would make it more efficient to help researchers identify phages that could be used in phage therapy. These primers will aid the SEA-PHAGES program in identifying potential phages to be used in the treatment of Mycobacterium tuberculosis and drug-resistant bacterial infections.