My research focuses on the interactions between bacteriophages (viruses that infect bacteria) and their bacterial hosts, with a particular emphasis on the epigenetic marks in phages that enable them to circumvent bacterial defenses. Specifically, I investigate how these epigenetic marks shield phage DNA from bacterial restriction systems, which are enzymes that cleave foreign DNA. Phages can evade recognition and destruction by these systems by modifying their DNA with epigenetic marks (e.g., 7-deazaguanine), ensuring their survival and propagation. Understanding these intricate phage-bacteria interactions is crucial for developing effective phage-based therapies to combat bacterial infections, offering a potential solution to the growing problem of antibiotic resistance.
Current Research Projects
- Elucidating the insertion of deazaguanine by DpdA (Biochemistry)
DpdA, a phage-encoded protein, swaps a guanine in DNA with a modified guanine base from the 7-deazaguanine family via a process called transglycosylation. Collaboratively, we have identified several DpdA proteins with slightly different substrates or efficiencies. This project aims to assay various DpdA proteins in vitro to validate in vivo predictions and observations.
- Techniques: Protein purification using Escherichia coli, expression, lysis, and purification conditions, DNA binding and base swapping activity assays.
- Classification of DpdA (Bioinformatics)
DpdA is a paralog of the TGT protein family, which modifies tRNA. Both DpdA and TGT exhibit varying substrate specificity. This project aims to classify TGT and DpdA proteins solely based on their amino acid sequences.
- Techniques: Protein sequence alignment and clustering, clustering tree and network generation.
- Investigating DpdA type 3 (Molecular Biology)
DpdA3 modifies DNA at a 100% rate, which is uncommon for post-replication modification. We hypothesize that it utilizes nucleosides instead of a deazaguanine base to facilitate the base swap. We have also identified a potential deoxyribose loader that we plan to clone and assay.
- Techniques: Gene cloning in plasmid (PCR, restriction digestion, DNA ligation, Escherichia coli transformation, plasmid extraction, and sequencing), restriction activity assays.
- Investigating phage FDTS (Molecular Biology)
Thymidylate synthase (TS) produces dTMP from dUMP, which involves methylation of dUMP. Flavin-dependent thymidylate synthase (FDTS) also produces dTMP from dUMP but uses flavin as a cofactor. We identified a phage FDTS that incorporates a large chain of oxidized carbon instead of a simple methyl group. We aim to clone and assay this FDTS.
- Techniques: Gene cloning in plasmid (PCR, restriction digestion, DNA ligation, Escherichia coli transformation, plasmid extraction, and sequencing), restriction activity assays.
- Classify phage FDTS (Bioinformatics)
Some bacteriophage TS enzymes also perform hydroxymethylation instead of methylation and act on dCMP instead of dUMP, contributing to epigenetic marks in phage DNA. With the discovery of this FDTS, we hypothesize that there might be more unknown modifications facilitated by FDTS. This project aims to identify existing groups of phage FDTS to discover new epigenetic marks.
- Techniques: Protein sequence alignment and clustering, clustering tree and network generation.
- Identifying orthologs in genomes (Coding Bioinformatics)
Identifying orthologs in genomes is challenging because each protein family has a unique evolutionary history, requiring different cutoff thresholds to determine if two homologs share the same function. We developed software that addresses this issue using similarity networks.
Techniques: Python coding, SnakeMake pipeline implementation, software optimization, and efficient threshold detection.