Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth’s biomes

Abstract

Bacteriophages from the Inoviridae family (inoviruses) are characterized by their unique morphology, genome content and infection cycle. One of the most striking features of inoviruses is their ability to establish a chronic infection whereby the viral genome resides within the cell in either an exclusively episomal state or integrated into the host chromosome and virions are continuously released without killing the host. To date, a relatively small number of inovirus isolates have been extensively studied, either for biotechnological applications, such as phage display, or because of their effect on the toxicity of known bacterial pathogens including Vibrio cholerae and Neisseria meningitidis. Here, we show that the current 56 members of the Inoviridae family represent a minute fraction of a highly diverse group of inoviruses. Using a machine learning approach leveraging a combination of marker gene and genome features, we identified 10,295 inovirus-like sequences from microbial genomes and metagenomes. Collectively, our results call for reclassification of the current Inoviridae family into a viral order including six distinct proposed families associated with nearly all bacterial phyla across virtually every ecosystem. Putative inoviruses were also detected in several archaeal genomes, suggesting that, collectively, members of this supergroup infect hosts across the domains Bacteria and Archaea. Finally, we identified an expansive diversity of inovirus-encoded toxin–antitoxin and gene expression modulation systems, alongside evidence of both synergistic (CRISPR evasion) and antagonistic (superinfection exclusion) interactions with co-infecting viruses, which we experimentally validated in a Pseudomonas model. Capturing this previously obscured component of the global virosphere may spark new avenues for microbial manipulation approaches and innovative biotechnological applications.

Ecology and molecular targets of hypermutation in the global microbiome

“…For contigs <3kb, taxonomic classification was set as "unclassified" because there are not enough predicted genes beyond the RT and DGR target on these contigs for a robust classification using the majority rule approach. Viral origin was predicted using a combination of VirSorter 45 v1.0.5 (as for genomes), the Earth's Virome pipeline 46 , and the inovirus detector pipeline 47 . All sequences identified as viral with the Earth's Virome pipeline, the inovirus detector pipeline, or VirSorter categories 1, 2, 4, and 5 were considered "viral", while sequences only predicted with VirSorter as categories 3 or 6 were listed as "putative viral" for the RT OTU-level aggregation (see below).…”

“…These high-quality target genes were clustered at 99% AAI using cd-hit 41 v4.8.1, then clustered in a two-step process as in ref. 47 . Briefly, sequences are first clustered using MCL 52 from an all-vs-all blastp, using blast score as edge weight and an inflation value of 2.0, then HMM profiles were built for these clusters and hhsearch 55 v3.1.0 was used to identify similarities between clusters.…”

“…Notably, while many giant viruses have been recently identified from metagenome assemblies 79 , no DGR were detected in these genomes or co-localized with an NCLDV marker gene, suggesting that DGRs are rare or absent from these large eukaryotic viruses despite their ability to exchange genes with bacteriophages. The only exceptions were two sequences (Meta_3300029305_Ga0307249_1003718615 and Meta_3300018430_Ga0187902_100048955) identified as putative inoviruses because of the presence of an inovirus ATPase marker nearby 47 . However, in both cases, the metagenome contig was too short to distinguish whether the DGR was encoded by the inovirus genome or by a neighboring Caudovirales prophage, as previously observed 47 .…”

Advantages and limits of metagenomic assembly and binning of a giant virus

“…Substantial advances in metagenomics and single-cell genomics have rapidly expanded known biodiversity by recovering sequences of 100s of thousands of uncultured bacteria and archaea from the environment and from the human microbiome ( Brown et al, 2015;Parks et al, 2017;Rinke et al, 2013;Schulz et al, 2017a) . Metagenomics has also recently proven to be a powerful method for assessing diversity and coding potential of environmental viruses (Paez-Espino et al, 2016;Roux et al, 2019) . Most viral genomes are small in size and when found in metagenomic data they are readily present on a single contig and thus often considered complete or near complete (Roux et al, 2018) .…”

Methods for Extraction and Detection of Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis

“…We and others have recently reported that Pf phage influence the pathogenesis of P. aeruginosa infections in the lung and other environments by inducing liquid crystal biofilm and promoting chronic infection. [11,12,[15][16][17][20][21][22] For all of these reasons there is a pressing need for effective and reliable protocols for assessments of both phage and bacteria in CF sputum. [23,24] Respiratory secretions are primarily composed of polymeric mucins (MUC5AC and MUC5B) that form a gel like material with complex rheological properties that form the basis for its function as an innate defense barrier, including viscoelasticity, cohesiveness, adhesiveness and wetness.…”