Abstract
Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G+), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2′-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2′-deoxy-7-amido-7-deazaguanine (dADG), 2′-deoxy-7-cyano-7-deazaguanine (dPreQ0) and 2′-deoxy-7- aminomethyl-7-deazaguanine (dPreQ1). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2′-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.
Highlights
wild type (WT) ΔqueC ΔqueDΔtgt pBAD24 0 A 0 A C 0 CACACAC pBAD33 0 0 C C 0 AACACACA Lane # 1 2 3 4 5 6 7 8 9 10 11 12 13 kbp 10 A = 9g dpdA C = 9g gat-queC0 = empty plasmid dG+, in E. coli DNA
The expression of folE, queD, and queE from Enterobacteria phage 9g in trans in E. coli MG1655 ΔfolE, ΔqueD, and ΔqueE strains, respectively, successfully re-established the production of Q, demonstrating the isofunctionality of the tested pairs (Fig. 2a). This complementation was not observed when the viral gat-queC and dpdA genes were expressed in E. coli ΔqueC and Δtgt, respectively
This result was unexpected for gat-queC, as we had previously shown that expression of an archaeal gat-queC homolog in E. coli could lead to G+ in tRNA and the formation of a preQ0 intermediate[16]
Summary
Because the presence of dG+ confers resistance to EcoRI digestion[29], we used restriction profiles as a first indication for the presence of modifications in plasmid DNA. EcoRI cuts pBAD24 once and pBAD33 twice, as shown in the digestion profiles of plasmids extracted from E. coli cotransformed with the two empty plasmids (Fig. 2b, c, lane 1). Because the gat-queC and dpdA genes of phage 9g lack EcoRI sites, the restriction profiles of plasmids extracted from E. coli derivatives cotransformed with an empty plasmid and a plasmid containing one of the two genes are shifted by the insert sizes (Fig. 2c, lanes 2, 3, 5, and 6). The single digestion by PsiI linearized all these plasmids, and the plasmids encoding both dpdA and gat-queC of phage 9g were again partially resistant to EcoRI digestion (red arrows in Supplementary Fig. 1)
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