Abstract
E. coli phage 9 g contains the modified base deoxyarchaeosine (dG+) in its genome. The phage encodes its own primase, DNA ligase, DNA polymerase, and enzymes necessary to synthesize and incorporate dG+. Here we report phage 9 g DNA sensitivity to >200 Type II restriction endonucleases (REases). Among the REases tested approximately 29% generated complete or partial digestions, while the remaining 71% displayed resistance to restriction. Phage 9 g restriction fragments can be degraded by DNA exonucleases or ligated by T3 and T4 DNA ligases. In addition, we examined a number of cytosine and adenine methyltransferases to generate double base modifications. M.AluI, M.CviPI, M.HhaI, and M.EcoGII were able to introduce 5mC or N6mA into 9 g DNA as confirmed by partial resistance to restriction and by liquid chromatography-mass spectrometry. A number of wild-type E. coli bacteria restricted phage 9 g, indicating natural restriction barriers exist in some strains. A BlastP search of GenBank sequences revealed five glutamine amidotransferase-QueC homologs in Enterobacteria and Pseudomonas phage, and distant homologs in other phage and bacterial genomes, suggesting that dG+ is not a rare modification. We also mapped phage 9 g DNA packaging (pac) site containing two 21-bp direct repeats and a major terminase cleavage site in the phage genome.
Highlights
In the restriction and anti-restriction arms race between bacteria and bacteriophage, phage have evolved elaborate DNA modifications on their genomes to evade host-encoded restriction endonucleases (REases) that attack phage DNA during infection[1]
A ~20 kb gene cluster encoding bacterial tRNA-guanine transglycosylase (i.e. TgtA5 encoded by tgtA5 gene), 7-cyano-7-deazaguanine synthase, and other DNA metabolic enzymes, including ATPase, DNA helicase, and a PLD-family endonuclease was found in Samonella enterica subsp. serovar Montevideo, Kineococcus radiotolerans, and other bacteria[14]
If phage 9 g is digested by Type II REases into small fragments, which DNA ligase can be used to ligate the sticky or blunt ends? Another interesting question is whether E. coli, Enterobacteria, and Pseudomonas hosts have evolved modification-dependent REases that target the modified base dG+ in an analogous fashion to the host-encoded restriction enzyme GmrSD, which targets the modified bases5hmC or glc-5hmC on phage T4gt and T4 DNA17–19, the plasmid-encoded restriction system PvuRts1I, which prefers to attack 5hmC-containing DNA20, the host encoded SauUSI endonuclease that restricted 5mC-modified DNA21, and the ScoMcrA restricting phosphorothioated and m5C-modified DNA22
Summary
In the restriction and anti-restriction arms race between bacteria and bacteriophage, phage have evolved elaborate DNA modifications on their genomes to evade host-encoded restriction endonucleases (REases) that attack phage DNA during infection[1]. Only ~15% of N6-(1-acetamido)-adenine in phage Mu renders its gDNA resistant to many Type I and II restriction systems of the host[8] Another example of phage DNA modification is 2′-deoxyarchaeosine (abbreviated as dG+) in E. coli phage 9 g genome[13, 14]. Another interesting question is whether E. coli, Enterobacteria, and Pseudomonas hosts have evolved modification-dependent REases that target the modified base dG+ in an analogous fashion to the host-encoded restriction enzyme GmrSD, which targets the modified bases5hmC or glc-5hmC on phage T4gt and T4 DNA17–19, the plasmid-encoded restriction system PvuRts1I, which prefers to attack 5hmC-containing DNA20, the host encoded SauUSI endonuclease that restricted 5mC-modified DNA21, and the ScoMcrA restricting phosphorothioated and m5C-modified DNA22 If phage 9 g is digested by Type II REases into small fragments, which DNA ligase can be used to ligate the sticky or blunt ends? Another interesting question is whether E. coli, Enterobacteria, and Pseudomonas hosts have evolved modification-dependent REases that target the modified base dG+ in an analogous fashion to the host-encoded restriction enzyme GmrSD, which targets the modified bases5hmC or glc-5hmC on phage T4gt and T4 DNA17–19, the plasmid-encoded restriction system PvuRts1I, which prefers to attack 5hmC-containing DNA20, the host encoded SauUSI endonuclease that restricted 5mC-modified DNA21, and the ScoMcrA restricting phosphorothioated and m5C-modified DNA22
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