Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201, USA.
Gosnell School of Life Sciences, Rochester Institute of Technology, 85 Lomb Memorial Drive, Rochester, NY 14623, USA.
Viruses. 2018 Jun 8;10(6):313. doi: 10.3390/v10060313.
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 phage JS09 and 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.
在与 T4 相关的噬菌体中，羟甲基胞嘧啶 (hmC) 在 DNA 复制过程中被掺入基因组 DNA 中，然后由噬菌体编码的葡糖基转移酶进一步修饰为葡糖基-hmC。先前的研究表明，RB69 与 T4 和相关噬菌体共享一组核心基因。然而，与其他“RB”噬菌体不同，RB69 无法将其 DNA 与 T4 或其他“RB”分离株进行重组。此外，尽管 RB69 具有用于合成 hmC 的 T4 酶的同源物，但它没有鉴定出与已知葡糖基转移酶基因的同源物。在这项研究中，我们使用高 pH 阴离子交换色谱 (HPAEC) 和质谱法来了解 RB69 行为的基础。我们的分析在 RB69 DNA 中鉴定出一种新型噬菌体表观遗传 DNA 糖修饰，我们将其命名为阿拉伯糖基-hmC (ara-hmC)。我们寻找负责这种新型修饰的假定葡糖基转移酶，并确定 RB69 还具有一种新型转移酶基因 ORF003c，它可能负责阿拉伯糖基特异性修饰。我们提出，ara-hmC 是 RB69 无法与其他含有 hmC 的 T-偶数噬菌体进行遗传交换以及其描述的初生种化的原因。RB69 的 ara-hmC 还可能使其 DNA 免受某些抗噬菌体类型 IV 限制内切酶的影响。一些与 T4 相关的噬菌体，如 phage JS09 和 phage Shf125875，与 RB69 ORF003c 具有同源物，这表明 ara-hmC 修饰在 T4 相关噬菌体中可能较为常见，这突显了进一步研究了解该修饰的作用及其产生的生化途径的重要性。
