Bacteriophage Diversity and Applications: Molecular Omics and Evolutionary Analysis

Bacteriophages or phages are highly abundant viruses that infect specific strains of bacteria. They have many diverse applications, including phage therapy, food biocontrol, and wastewater treatment. The goal of this research is to enhance overall understanding of phages through a series of case studies to inform future research into potential phage applications in medicine, biotechnology, and agriculture. The first case study, conducted through the SEA-PHAGES program, isolated and characterized 48 novel Arthrobacter globiformis phages using methodologies like plaque assays, DNA extraction, restriction enzyme digests, and transmission electron microscopy imaging, significantly enriching the Actinobacteriophage database. The findings demonstrate the effectiveness of SEA-PHAGES methodologies in advancing phage discovery and enhancing understanding of phage diversity. In the second case study, four novel Arthrobacter globiformis phage genomes were annotated using SEA-PHAGES methodologies, bioinformatics tools, and global databases, addressing challenges like hypothetical genes while identifying specialized features such as programmed translational frameshifts and tRNAs. The annotations, once approved and published in GenBank, further enrich the Actinobacteriophage database and highlight the importance of standardized workflows in advancing phage diversity research and applications. In the third case study, a PCR-based protocol was developed for clustering phages that infect Arthrobacter globiformis. This study suggested that phages can be classified on a single gene basis via an evolutionary cluster without the need for sequencing, which is typically required for clustering and can be time and resource intensive. The implementation of this study will help alleviate the bottleneck seen from phage discovery to cluster assignment by eliminating the need for sequencing and thereby help further define phage diversity. In the fourth and final case study, mass spectrometry data was used to analyze lipid profile changes for three phages that infect Mycobacterium smegmatis over time. The results implied that phages require their bacterial host to enhance energy production, metabolic activity, and cell signaling early in the infection process, likely due to the host’s initial recognition of the phage attempting to infect and its early efforts to divert resources to resist this infection. The results also implied that phages require their bacterial host to enhance structural support later in the infection process, likely due to the extensive stress and damage caused by the phage. These results provide insights into the phage-host interaction and the defense mechanisms utilized by bacterial hosts in response to phage infection. Together, these case studies contribute to a deeper understanding of phage biology by enhancing understanding of phage diversity and phage-host interactions while also providing valuable tools to advance phage research. By further contributing to the Actinobacteriophage database, streamlining phage classification, and improving methods for analyzing phage infection dynamics, this research paves the way for future phage-based applications in a variety of fields.