Approaches and applications for targeted phage discovery

Abstract

Bacteriophages, phages for short, are viruses that infect bacteria. They are the most abundant biological entities on Earth and store a huge amount of genetic diversity. From this great diversity, we have gained insights ranging from fundamental biology to treatments for bacterial infections. Despite their importance across scientific and medical fields, techniques used to isolate phages from the environment have remained largely unchanged for nearly a century. In this work, I present a modification to the classic plaque assay to improve efficiency and information gained during phage screens and show its utility across multiple applications. This method involves co-culturing multiple, genetically distinct bacterial strains to identify phages based on differential lysis patterns. In the first half of the thesis, I demonstrate that the approach can distinguish between previously characterized phages with known receptor or defense system interactions. I then apply the same screening strategy to environmental samples and successfully isolate phages with desired characteristics. In the second half of the thesis, I deploy this screening method across four specific discovery projects. First, I identify phages dependent on diverse bacterial receptors and show that the method is especially useful when phage diversity in the sample is low. Second, I confirm the functionality of restriction enzyme homologs as phage defense systems by screening for phages they inhibit. Third, I identify candidate plasmid-dependent phages that appear to rely on a conjugative pilus encoded by an outbreak-associated IncX3 plasmid. Last, I isolate phages capable of infecting a genetically recoded bacterial strain by supplying the missing tRNAs. In addition to these biological findings, I include a parts list for the custom-built imaging system used to visualize fluorescent plaques. Collectively, this work offers a practical tool for improving phage discovery and contributes to our broader understanding of phage biology.