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

Abstract

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.

CodingDiv: analyzing SNP-level microdiversity to discriminate between coding and noncoding regions in viral genomes

“…The remaining gene is C4, nested within the replication protein on an alternate reading frame, and is the only region with ratio lower to 1.65 (ratio of 1.35). Selection pressures on the ancestral gene constrain the nature of substitutions of an overprinted gene and lead to pNeg/pS ratios greater than 1 for overlapping genes ( Pavesi 2006 , Olo Ndela et al 2023 ). Thus, ORFs with values up to 1.5 should be considered for overprinted regions.…”

“…Yet, nonsynonymous mutations with positive substitution scores in the BLOSUM62 matrix can be abundant in coding regions and also occur frequently in random noncoding regions. A ratio was thus computed considering only negative score mutations and synonymous ones (pNeg/pS, see Olo Ndela et al 2023 ). Results are provided as two files and are used to generate a plot in SVG format.…”

Bacterial isolation and genome analysis of a novel Klebsiella quasipneumoniae phage in southwest China’s karst area

“…Through NCBI research, it is found that the genome size of the known Klebsiella phage is generally between ~17 kb and 269 kb, while the genome size of the phage KL01 is 45.9 kb, which is one of the smaller sequenced Klebsiella phage genome. The GC content of different phages is also very different, and the difference of GC content can lead to great differences in the protein and amino acid content of phages [ 68 ]. KL01 encodes holin, which is an essential membrane protein that participates in host cell lysis.…”

Microviruses: A World Beyond phiX174

“…Most coding sequences beyond the three core genes are small and therefore less easily recognizable across lineages via homology searches (11). Based on the biology of isolated microviruses, members of the group also need to encode scaffolding and DNA-binding/packaging proteins for virion assembly (39), but not all MAGs appear to encode such proteins (21). Other, presumably nonessential coding sequences are also predicted to be involved in lysis (peptidases, amidases, and other cell wall degrading enzymes), overcoming phage defenses (methyltransferases, anti-CRISPR proteins), or containing secretion signals, although the vast majority are of unknown functions (40).…”