Showcase of Undergraduate Research and Creative Endeavors (SOURCE)

Investigations of Nucleotide Modifications in Winthrop’s Bacteriophage Collection

Bacteriophages (phages) are viruses that replicate in bacteria and have co-existed with their host in a complicated, evolutionary arms race for approximately three billion years. Both continuously evolve attack and defense mechanisms to ensure their own survival. Bacteria employ an array of defense mechanisms, including a Restriction-Modification System that utilizes restriction endonucleases (REs) to destroy infecting phage genomes. Phages can utilize methylation, or more commonly, noncanonical nucleobase substitutions. These substitutions are incorporated into phage genomes during replication, and “camouflage” their RE sites. The objective of this study was to reveal possible phage DNA modifications by comparing patterns of endonuclease cleavage of Winthrop’s collection of phage genomes, to virtual profiles of predicted endonuclease activity. Our protocols utilized methylation-insensitive enzymes, to reveal possible RE site protection in the phage genomes. Simultaneously, an in-silico tool, available at New England Biolabs (NEB), was used to generate virtual digestion patterns using the same suite of enzymes. Our results highlighted that several of our phages’ DNA was blocked from digestion, especially those grouped in the EA cluster. These EA phages show resistance to similar REs, suggesting that phages within the same cluster may display similar nucleobase substitutions. Further investigations are underway to identify modification similarities between phages within related or unrelated clusters. Biochemical analysis to reveal the presence and molecular structure of noncanonical nucleobase substitutions is a future goal of these investigations. Detailed knowledge of phage protective mechanisms is important, since it may contribute a competitive advantage to the phage when used in a therapeutic setting.