Restriction and modification of deoxyarchaeosine (dG+)-containing phage 9 g DNA

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.

Isolation and Characterization of the Novel Bacteriophage AXL3 against Stenotrophomonas maltophilia

“…It is possible that phage-encoded Cas4-like proteins may play an uncharacterized role in defense against host restriction-modification systems; the Campylobacter phages encoding Cas4 nuclease homologs are also predicted to contain modified guanosine nucleotides that provide resistance to gDNA digestion with restriction enzymes [ 47 ], as observed in AXL3. A Cas4-like nuclease has also been identified in close proximity to deoxyarchaeosine (dG+) synthesis genes in the restriction-resistant genome of E. coli phage 9 g, suggesting a possible role for Cas4 in a restriction system of unmodified DNA, or degradation of host DNA for nucleotide recycling [ 48 ]. Further characterization of these phage-encoded Cas4 proteins will likely reveal uncharacterized defense and anti-defense systems.…”

Identification of the minimal bacterial 2′‐deoxy‐7‐amido‐7‐deazaguanine synthesis machinery

“…Results from the transformation assays with both unmodified DNA and DNA containing preQ 0 or ADG show that only ADG confers resistance to the predicted restriction activity encoded by the dpd cluster. Unlike dG + in 9g phage (Tsai et al, 2017), the presence ADG in pUC19 does not confer resistance to the Type II restriction enzymes we tested (Fig. S7).…”

“…[Note that the standard nomenclature uses ‘Q’ to represent the ribonucleoside, ‘preQ 0’ and ‘ADG’ to represent the corresponding base, ‘dPreQ 0 ’ and ‘dADG’ to represent the corresponding 2′‐deoxyribonucleosides.] In phages such as the Escherichia coli phage 9g, the presence of dG + confers resistance to many restriction enzymes (Kulikov et al ., ; Tsai et al ., ). The 11‐gene deazapurine‐in‐DNA ( dpd ) cluster of Salmonella enterica serovar Montevideo ATCC BAA‐710 ( S .…”

“…[Note that the standard nomenclature uses 'Q' to represent the ribonucleoside, 'preQ 0' and 'ADG' to represent the corresponding base, 'dPreQ 0 ' and 'dADG' to represent the corresponding 2′-deoxyribonucleosides. ] In phages such as the Escherichia coli phage 9g, the presence of dG + confers resistance to many restriction enzymes (Kulikov et al, 2014;Tsai et al, 2017). The 11-gene deazapurine-in-DNA (dpd) cluster of Salmonella enterica serovar Montevideo ATCC BAA-710 (S. Montevideo) was shown to be involved in modification of genomic DNA with dPreQ 0 and dADG, and in restriction-like activity that caused low transformation efficiency when using unmodified DNA compared with modified DNA.…”

Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

“…Modified bases in phage or other mobile DNA provide protection against ‘conventional’ host restriction. For example, 2′-deoxyguanosine replacement by 2′-deoxyarchaeosine (dG + ) in the Escherichia coli phage 9g DNA renders it resistant to over 70% of commercially available Type II restriction endonucleases (REases) ( 7 ). Similarly, α-putrescinylthymine (putT) in phi W-14 DNA has been found to block DNA cleavage by more than half of all tested Type II REases ( 8 ).…”