SignificanceBacterial viruses (bacteriophages) append a variety of molecules, including sugars, amino acids, and polyamines, to the nucleobases of their genomic DNA to circumvent the endonuclease-based defenses of their hosts. These DNA hypermodifications are formed through bacteriophage-encoded biosynthetic pathways, with steps occurring before and after replication of bacteriophage DNA. We report here the discovery of two thymidine hypermodifications: 5-(2-aminoethoxy)methyluridine replacing 40% of thymidine nucleotides in the Salmonella phage ViI and 5-(2-aminoethyl)uridine replacing 30% of thymidine in the DNA of the Pseudomonas phage M6. Additionally, we show in vitro reconstitution of 5-(2-aminoethyl)uridine biosynthesis from five recombinantly expressed proteins. These findings reveal an expanded diversity in the types of naturally occurring DNA modifications and their biosynthetic pathways.

The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.

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