Structural and Genomic Characterization of a Novel B. Subtilis Phage

Bacteriophages (phages) are viruses that infect specific bacterial hosts. They are found everywhere their hosts are found, making them incredibly abundant in almost every habitat. This project aims to characterize novel bacteriophages that infect the soil bacteria Bacillus subtilis from the environment in Nashville, TN. After successfully isolating a B. subtilis phage from the environment, we optimized culturing conditions for isolation of high-titer purified phage. With this high-titer phage, we developed a protocol to structurally characterize the bacteriophage by scanning electron microscopy in collaboration with the electron microscopy core facility at Vanderbilt University. We then developed a protocol to isolate and purify phage genetic material. This sample was sequenced by Next Generation Sequencing at Yale University. The resulting DNA reads were computationally aligned to generate a whole genome sequence ~145kb in length. The genome was was compared against a sequence database, which confirmed it to be related to other B. subtilis-infecting phages, and which and supports that the phage isolated is a unique species. We then designed primers to generate amplicons of predicted sizes, and resulting PCR and gel electrophoresis confirmed the sequencing results and a linear phage genome, as predicted. Future analysis aims to annotate the genome in order to identify genes and predict function. Therefore, these results demonstrate that we generated successful protocols to characterize novel bacteriophages at Belmont, which can be used to characterize additional environmentally-derived samples