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Bacteriophages or simply “phages” are the most abundant biological entities in the biosphere. They represent a rich source of noncanonical nucleotides and are in fact the organisms with the greatest diversity of DNA modifications. The continues evolutionary arms race with their bacterial hosts have acquired both microorganisms with a myriad of defence and counter-defence mechanisms. These systems do not only provide protection against one another, but also evolutionary benefits for both. So far, more than 21 distinct DNA modifications have been found in the genomes of phages, including the more complex “hypermodifications” like glucosylations, arabinosylations, and deazapurines. Even so, many more DNA modifications are expected to exist. In this study, a novel suspected rhamnose-hypermodification in phapecoctaviruses and its potential role against host encoded antiviral defence systems was investigated. Phages from the two genera, Justugliebigvirus and Phapecoctavirus of the Stephanstirmvirinae subfamily are all unique in encoding a complete or nearly complete dTDP-L-rhamnose biosynthesis pathway. The study includes two phage species: Phapecoctavirus ukendt which encodes the entire rhamnose biosynthesis pathway (rmlABCD) and Phapecoctavirus anhysbys which encodes two out of the four genes (rmlC, rmlD) of the rhamnose biosynthesis pathway. The phages were investigated for novel suspected DNA modifications in their genomes and whether these modifications provided protection towards degradation of their DNA. The phages’ ability to infect various strains including those from the ECOR collection and 33 E. coli strains individually equipped with novel antiviral defence systems was also investigated. Results showed that the genomes of both phages are modified with an unknown suspected hypermodification positioned in the GTAC motif and strongly suggested that thymines of the motif are the ones modified. Nanopore sequencing showed that all GTAC sites in both phages modified

Bacteriophages or simply “phages” are the most abundant biological entities in the biosphere. They represent a rich source of noncanonical nucleotides and are in fact the organisms with the greatest diversity of DNA modifications. The continues evolutionary arms race with their bacterial hosts have acquired both microorganisms with a myriad of defence and counter-defence mechanisms. These systems do not only provide protection against one another, but also evolutionary benefits for both. So far, more than 21 distinct DNA modifications have been found in the genomes of phages, including the more complex “hypermodifications” like glucosylations, arabinosylations, and deazapurines. Even so, many more DNA modifications are expected to exist. In this study, a novel suspected rhamnose-hypermodification in phapecoctaviruses and its potential role against host encoded antiviral defence systems was investigated. Phages from the two genera, Justugliebigvirus and Phapecoctavirus of the Stephanstirmvirinae subfamily are all unique in encoding a complete or nearly complete dTDP-L-rhamnose biosynthesis pathway. The study includes two phage species: Phapecoctavirus ukendt which encodes the entire rhamnose biosynthesis pathway (rmlABCD) and Phapecoctavirus anhysbys which encodes two out of the four genes (rmlC, rmlD) of the rhamnose biosynthesis pathway. The phages were investigated for novel suspected DNA modifications in their genomes and whether these modifications provided protection towards degradation of their DNA. The phages’ ability to infect various strains including those from the ECOR collection and 33 E. coli strains individually equipped with novel antiviral defence systems was also investigated. Results showed that the genomes of both phages are modified with an unknown suspected hypermodification positioned in the GTAC motif and strongly suggested that thymines of the motif are the ones modified. Nanopore sequencing showed that all GTAC sites in both phages modified