Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans

Enzymatic modification of DNA nucleobases can coordinate gene expression, protection from nucleases, or mutagenesis. We recently discovered a new clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX, e.g., TET) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for additional post-replicative enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-dependent glycosyltransferase (GT) enzymes that catalyze linkage of diverse glycans directly onto 5hmC reactive nucleobase substrates. Using targeted bioinformatic mining of the phage metavirome databases, we discovered thousands of new biosynthetic gene clusters (BGCs) containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a pathway reassembly platform for high-throughput functional screening of GT-containing BGCs, relying on the endogenous E. coli metabolome as a substrate pool. We successfully reconstituted a subset of phage BGCs and isolated novel and highly diverse sugar modifications appended to 5hmC, including mono-, di-, or tri-saccharide moieties comprised of hexose, N-acetylhexosamine or heptose sugars. Structural predictions and sugar product analyses suggest that phage GTs are related to host lipopolysaccharide, teichoic acid, and other small molecule biosynthesis enzymes and have been repurposed for DNA substrates. An expanded metagenomic search revealed hypermodification BGCs within gene neighborhoods containing phage structural proteins and putative genome defense systems. These findings enrich our knowledge of secondary modifications on DNA and the origins of corresponding sugar writer enzymes. Post-replicative cytosine hypermodification by virus-encoded GTs is discussed in the context of genome defense, DNA partitioning and virion assembly, and host-pathogen co-evolution.