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The Odd "RB" Phage-Identification of Arabinosylation as a New Epigenetic Modification of DNA in T4-Like Phage RB69
Julie A Thomas1,2, Jared Orwenyo3,4, Lai-Xi Wang5,6
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201, USA. jatsbi@rit.edu.
Abstract:
In bacteriophages related to T4, hydroxymethylcytosine (hmC) is incorporated into the genomic DNA during DNA replication and is then further modified to glucosyl-hmC by phage-encoded glucosyltransferases. Previous studies have shown that RB69 shares a core set of genes with T4 and relatives. However, unlike the other “RB” phages, RB69 is unable to recombine its DNA with T4 or with the other “RB” isolates. In addition, despite having homologs to the T4 enzymes used to synthesize hmC, RB69 has no identified homolog to known glucosyltransferase genes. In this study we sought to understand the basis for RB69’s behavior using high-pH anion exchange chromatography (HPAEC) and mass spectrometry. Our analyses identified a novel phage epigenetic DNA sugar modification in RB69 DNA, which we have designated arabinosyl-hmC (ara-hmC). We sought a putative glucosyltranserase responsible for this novel modification and determined that RB69 also has a novel transferase gene, ORF003c, that is likely responsible for the arabinosyl-specific modification. We propose that ara-hmC was responsible for RB69 being unable to participate in genetic exchange with other hmC-containing T-even phages, and for its described incipient speciation. The RB69 ara-hmC also likely protects its DNA from some anti-phage type-IV restriction endonucleases. Several T4-related phages, such as E. coli phage JS09 and Shigella phage Shf125875 have homologs to RB69 ORF003c, suggesting the ara-hmC modification may be relatively common in T4-related phages, highlighting the importance of further work to understand the role of this modification and the biochemical pathway responsible for its production.
Insights
Researchers discovered a new DNA modification in RB69 phage, arabinosyl-hydroxymethylcytosine (ara-hmC). This modification likely explains RB69
Area of Science:
- * Molecular Biology
- * Virology
- * Genetics
Background:
- * T4-related bacteriophages utilize hydroxymethylcytosine (hmC) in their DNA, often further modified by glucosyltransferases.
- * The RB69 phage, despite sharing genes with T4, exhibits unique behaviors like failed DNA recombination and lacks known glucosyltransferase genes.
- * Previous research indicates RB69's inability to genetically exchange DNA with T4 and other related phages.
Purpose of the Study:
- * To investigate the molecular basis for RB69's distinct DNA modification and recombination-defective phenotype.
- * To identify novel epigenetic DNA modifications and responsible enzymes in RB69 bacteriophage.
- * To understand the implications of these findings for phage evolution and host interactions.
Main Methods:
- * High-pH anion exchange chromatography (HPAEC) was employed to analyze RB69 DNA.
- * Mass spectrometry was utilized to identify and characterize DNA modifications.
- * Bioinformatic analysis was performed to identify potential enzyme homologs.
Main Results:
- * A novel DNA sugar modification, arabinosyl-hmC (ara-hmC), was identified in RB69 genomic DNA.
- * A previously uncharacterized gene, RB69 ORF003c, was identified as the likely transferase responsible for the ara-hmC modification.
- * The ara-hmC modification is proposed to be responsible for RB69's inability to recombine with T4 phages and its incipient speciation.
Conclusions:
- * RB69 bacteriophage possesses a unique DNA modification, ara-hmC, mediated by a novel transferase (ORF003c).
- * This modification likely prevents genetic exchange with other T-even phages and offers protection against certain restriction enzymes.
- * The presence of homologs to ORF003c in other phages suggests ara-hmC modification may be widespread in the T4 phage family.