Discovery of a pentose as a cytosine nucleobase modification in Shewanella phage Thanatos-1 genomic DNA mediating enhanced resistance towards host restriction systems

Co-evolution of bacterial defense systems and phage counter defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. To evade nuclease-based mechanisms targeting the DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits recognition by endonucleases. We found that Shewanella phage Thanatos-1 DNA is insensitive to multiple restriction enzymes and, partially, also to Cas I-Fv and Cas9 cleavage. Furthermore, the phage genome shows strongly impaired basecalling with nanopore sequencing. We characterised the phage adenine methyltransferase TH1_126 in methylase-free E. coli ER3413 and derived and confirmed its recognition motif 5-ATC-3. Moreover, the data pointed to an additional, much more substantial nucleobase modification. Using LC-MS, we identified a deoxypentose of unknown configuration attached to cytosine as a yet undiscovered phage DNA modification, which is present in Thanatos-1 genomic DNA, likely mediates the observed resistance to restriction endonucleases, as well as a strong reduction in Cas nuclease activity. To elucidate the underlying enzyme functions, we determined structural homologs of Thanatos-1 proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of phage protein TH1_063 by in vitro enzyme assays.