Reekeekee- and roodoodooviruses, two different Microviridae clades constituted by the smallest DNA phages


 Small circular single-stranded DNA viruses of the Microviridae family are both prevalent and diverse in all ecosystems. They usually harbor a genome between 4.3 and 6.3 kb, with a microvirus recently isolated from a marine Alphaproteobacteria being the smallest known genome of a DNA phage (4.248 kb). A subfamily, the Amoyvirinae, has been proposed to classify this virus and other related small Alphaproteobacteria-infecting phages. Here, we report the discovery, in meta-omics datasets from various aquatic ecosystems, of 16 complete microvirus genomes significantly smaller (from 2.991 to 3.692 kb) than known ones. Phylogenetic analysis reveals that these 16 genomes represent two related, yet distinct and diverse, novel groups of microviruses, amoyviruses being their closest known relatives. We propose that these small microviruses be members of two tentatively named subfamilies Reekeekeevirinae and Roodoodoovirinae. As known microvirus genomes encode many overlapping and overprinted genes that are not identified by gene prediction softwares, we developed a new methodology to identify all genes based on protein conservation, amino-acid composition, and selection pressure estimations. Surprisingly, only 4 to 5 genes could be identified per genome, with a number of overprinted genes lower than in phiX174. These small genomes thus tend to have both a lower number of genes and a shorter length for each gene, leaving no place for variable gene regions that could harbor overprinted genes. Even more surprisingly, these two Microviridae groups had specific and different gene content, and major differences in their conserved protein sequences, highlighting that these two related groups of small genome microviruses use very different strategies to fulfill their lifecycle with such a small numbers of genes. The discovery of these genomes and the detailed prediction and annotation of their genome content expand our understanding of ssDNA phages in nature and is further evidence that these viruses have explored a wide range of possibilities during their long evolution.