In 2019, there were 4.95 million deaths from antibiotic-resistant bacteria. Identifying bacteriophages (phages) that have base-pair (base) modifications which can evade bacterial defenses, such as restriction enzymes, can help researchers select the best phages to treat bacterial infections in humans, livestock, and agriculture, as well as impact ocean nutrient cycles to combat climate change. We are developing a framework for discovering new base modifications in phages and identifying their function within the phage life cycle.

The DNA of eukaryotes, prokaryotes, and their viruses can be modified by adding methyl groups to their nucleotides which can regulate gene expression. Phage DNA modifications are more diverse, for example,
adding amino acids, polyamines, monosaccharides, disaccharides, and methyl groups. These modifications can regulate gene expression, assist in recognizing self-DNA and assist in DNA packaging. It can also protect DNA from degradation by bacteria restriction enzymes (REs), CRISPR-Cas, and environmental damage. Many of these DNA modifications are yet to be discovered.
Most DNA sequencing technologies require PCR amplification which results in the loss of base modifications before sequencing. However, the DNA sequence they provide can be used to obtain a virtual restriction enzyme digest (virtual digest). The proposed framework will use virtual digests to help identify DNA modifications using the following steps.

Step one: screen phage genomes for DNA modifications by comparing phage DNA RE digests to their virtual DNA RE digests. For example, the phages Kharcho and Ottawa show a blockage in some RE digests. In contrast, their virtual RE digest reveals that the DNA should be cut. This inability of the restriction enzyme to cut the phage DNA may indicate a DNA modification in the phage genome.

Step two: Oxford Nanopore Technology (ONT) DNA Sequencing can reveal the presence of base modifications. We are using ONT DNA sequencing to sequence the raw phage DNA in order to identify known base modifications already in the nanopore algorithms or reveal an unknown base modification.

Step three: if the nanopore sequencer detects an unknown base-pair modification, mass spectrometry will be used to identify the unknown modification. New modifications will be added to the nanopore algorithm to recognize the newly identified base modification in other phages. Expanding these algorithms will speed up this workflow, allowing us to use ONT to quickly identify phages with specific DNA modifications without needing Mass Spectrometry or DNA digests.

Step four: Following the discovery of novel base modifications, I will focus on identifying the genes within the phage genome required for adding the modification using a candidate approach. Candidates will be mutated using reverse genetic strategies to test if their mutation impacts the base modification and if the modification is required for phage viability and evasion of host defence systems. The genes involved in base modification will be inserted into phages which lack those genes to see if they can gain the ability to evade bacterial defenses.