Dual cytosine modification through hydroxylation and arabinofuranosylation in phages

Bacteriophages use diverse genomic modifications, including substitutions on the cytosine pyrimidine ring through C-C and C-N bonds. Here, we report that coliphage HY126 encodes an Afh system with enzymatic activities not previously implicated in DNA modification. This multienzyme cascade comprises the following: AfhB, a reduced flavin-dependent hydroxylase that catalyzes deoxycytidine monophosphate (dCMP) hydroxylation through C-O bond formation to yield 5-hydroxy-dCMP; AfhE and AfhF, which synthesize the sugar donor uridine diphospho-ᴅ-arabinose for AfhC; AfhC, a phage-encoded nucleotide arabinosyltransferase that arabinofuranosylates 5-hydroxy-dCMP, priming it for DNA incorporation; AfhG, a DNA arabinosyltransferase that adds a second arabinofuranose moiety through a β-1,3 linkage to 5-hydroxyarabinofuranosylated cytosine embedded in DNA, forming diarabinofuranosyl-5-hydroxycytosine. This work unveils a unique two-step arabinofuranosylation mechanism-involving distinct nucleotide priming followed by DNA modification-thereby elucidating an enzymatic strategy in the phage-host evolutionary arms race.