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, Amoyvirinae, has been proposed to classify this virus and other related small Alphaproteobacteria-infecting phages. Here, we report the discovery, in meta-omics data sets from various aquatic ecosystems, of sixteen complete microvirus genomes significantly smaller (2.991-3.692 kb) than known ones. Phylogenetic analysis reveals that these sixteen genomes represent two related, yet distinct and diverse, novel groups of microviruses-amoyviruses being their closest known relatives. We propose that these small microviruses are 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 software, we developed a new methodology to identify all genes based on protein conservation, amino acid composition, and selection pressure estimations. Surprisingly, only four to five genes could be identified per genome, with the number of overprinted genes lower than that 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 number 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 are further evidence that these viruses have explored a wide range of possibilities during their long evolution.

Microbial communities in lakes can profoundly impact biogeochemical processes through their individual activities and collective interactions. However, the complexity of these communities poses challenges, particularly for studying rare organisms such as Candidate Phyla Radiation bacteria (CPR) and enigmatic entities such as aster-like nanoparticles (ALNs). Here, a reactor was inoculated with water from Lake Fargette, France, and maintained under dark conditions at 4°C for 31 months and enriched for ALNs, diverse Planctomycetes, and CPR bacteria. We reconstructed draft genomes and predicted metabolic traits for 12 diverse Planctomycetes and 9 CPR bacteria, some of which are likely representatives of undescribed families or genera. One CPR genome representing the little-studied lineage "Candidatus Peribacter" was curated to completion (1.239 Mbp) and unexpectedly encodes the full gluconeogenesis pathway. Metatranscriptomic data indicate that some planctomycetes and CPR bacteria were active under the culture conditions, accounting for ∼30% and ∼1% of RNA reads mapping to the genome set, respectively. We also reconstructed genomes and obtained transmission electron microscope images for numerous viruses, including one with a >300-kbp genome and several predicted to infect Planctomycetes. Together, our analyses suggest that freshwater Planctomycetes are central players in a subsystem that includes ALNs, symbiotic CPR bacteria, and viruses. IMPORTANCE Laboratory incubations of natural microbial communities can aid in the study of member organisms and their networks of interaction. This is particularly important for understudied lineages for which key elements of basic biology are still emerging. Using genomics and microscopy, we found that members of the bacterial lineage Planctomycetes may be central players in a subset of a freshwater lake microbiome that includes other bacteria, archaea, viruses, and mysterious entities, called aster-like nanoparticles (ALNs), whose origin is unknown. Our results help constrain the possible origins of ALNs and provide insight into possible interactions within a complex lake ecosystem.

Microbial communities from hypersaline ponds, dominated by halophilic archaea, are considered specific of such extreme conditions. The associated viral communities have accordingly been shown to display specific features, such as similar morphologies among different sites. However, little is known about the genetic diversity of these halophilic viral communities across the Earth. Here, we studied viral communities in hypersaline ponds sampled on the coast of Senegal (8-36% of salinity) using metagenomics approach, and compared them with hypersaline viromes from Australia and Spain. The specificity of hyperhalophilic viruses could first be demonstrated at a community scale, salinity being a strong discriminating factor between communities. For the major viral group detected in all samples (Caudovirales), only a limited number of halophilic Caudovirales clades were highlighted. These clades gather viruses from different continents and display consistent genetic composition, indicating that they represent related lineages with a worldwide distribution. Non-tailed hyperhalophilic viruses display a greater rate of gene transfer and recombination, with uncharacterized genes conserved across different kind of viruses and plasmids. Thus, hypersaline viral communities around the world appear to form a genetically consistent community that are likely to harbour new genes coding for enzymes specifically adapted to these environments.