Lysogeny and Use of Mycobacteriophage Pita2

The CDC has classified antibiotic resistance as the biggest health challenge of our era; every year 2 million lives are impacted and even lost due to resistant bacteria. Bacteriophages provide an alternative route to fighting infections that does not further the development of antibiotic resistance among bacterial species. A bacteriophage replicates inside a bacterial cell and then causes that cell to lyse, an event that kills the bacterial host. However, some phage can integrate their genomes into the host chromosome without causing lysis. The HHMI SEA-PHAGES program has generated a collection of bacteriophage that infect Actinobacteria species. Over 13,000 phages have been collected thus far, but fewer than 3,000 have been sequenced and genetically analyzed. The purpose of research into the lysogeny of discovered, but unsequenced, bacteriophage is to classify them by immunity range; that is, closely related bacteriophage are unable to infect the lysogenic host, while more distantly related phage are. Initially, a lysogen had to be isolated. For Pita2, a phage isolated and analyzed at Bowling Green State University, the host is Mycobacterium smegmatis. A purified lysogen of Pita2 was analyzed against bacteriophage with known DNA sequences to confirm that it is immune to infection by closely related phage. The lysogeny was then examined for its ability to identify close vs. more distantly related phage among a set unknown bacteriophage. Another virus, namely SARS-CoV2, prevented the completion of this study. However, it was possible to generate preliminary data that established the ability to use immunity as a means to classify newly isolated phage based upon the degree to which they can successfully infect the Pita2 lysogen