Cellulophaga phage phi40:2 is a Baltic Sea phage that infects Cellulophaga baltica and displays a large Myovirus morphology. However, sequencing its genome has proven challenging using classical methods. Previous attempts utilizing 454, Ion Torrent, MinIon, Illumina sequencing, or phi29 genome amplification followed by Illumina sequencing were unsuccessful primarily due to difficulties in library preparation. Nevertheless, successful sequencing was achieved using the Neoprep library system followed by Illumina sequencing, resulting in a closed genome of approximately 142.5 kb. This was consistent with the size of the phage capsid but differed largely from the 242 kb genome size determined by Pulsed Field Gel Electrophoresis.

PCR analysis targeting specific 400 bp fragments dispersed across phi40:2 genome, employing three different sets of primers designed to be specific to the phage and not its host DNA, revealed difficulties in successfully amplifying the phage genome depending on the choice of polymerase and experimental conditions. The Phusion High Fidelity DNA polymerase exhibited the highest success rate, which was further enhanced by optimizing the concentration of MgCl₂, as well as the addition of BSA and DMSO. AmpliTaq Gold DNA polymerase did not yield any positive results, while Qiagen Taq DNA polymerase produced weak bands that showed slight improvement under optimized conditions. Invitrogen Platinum Taq DNA polymerase did not yield visible results under standard conditions, but faint bands were observed when using the optimized conditions.

Mass spectrometry analysis of phi40:2 DNA unveiled a unique 346 Da nucleoside modification, likely corresponding to a thymidine modification. This modification differs by only 16 Da from a modification observed in Bacillus phage SP15, known as 5-dihydroxypentyluracyl, which suggests a similar modification with the addition of a hydroxyl group (-OH). A collision- induced dissociation (CID) MS/MS spectra analysis supported the multiple hydroxyl groups in the unknown modification nucleobase. Intriguingly, both phages share a flavin-dependent thymidylate synthase, likely to have diverged into a 5-hydroxymethyluracil (5hmU) synthase, suggesting a conserved mechanism involved in the synthesis of this modified base. We propose that this modification synthesis begins with the insertion of a 5hmU into the DNA during polymerization, followed by further modifications. Alternatively, this protein may have evolved to insert a dihydropentyl group, or similar, from an unknown source. This mechanism introduces the possibility of an unanticipated class of DNA modifications.

Potentially, this modified nucleotide is the reason behind the problems of sequencing or conducting PCR on the phage genome. Given the widespread nature of phages related to other Cellulophaga phages, ranging from the marine environments to the human gut, the geographical distribution and ecological importance of this, as yet, unsequenceable phage is yet to be discovered.