A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution

Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1, 2, 3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria(4)(,)(5) that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6, 7, 8, 9, 10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11, 12, 13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.(14) In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family (“Naomiviridae”). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment.