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.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Expansion of the global RNA virome reveals diverse clades of bacteriophages

High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Thousands of small, novel genes predicted in global phage genomes

Small genes (<150nucleotides) have been systematically overlooked in phage genomes. We employ a large scale comparative genomics approach to predict >40,000 small-gene families in 2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000families encode predicted anti-CRISPR proteins, and more than500families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.

Fremin, Brayon↗