Identification and biosynthesis of thymidine hypermodifications in the genomic DNA of widespread bacterial viruses

Significance Bacterial 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.