Unraveling the functional dark matter through global metagenomics

30 pages, 4 figures, 1 table, supplementary information https://doi.org/10.1038/s41586-023-06583-7.-- Data availability: All of the analysed datasets along with their corresponding sequences are available from the IMG system (http://img.jgi.doe.gov/). A list of the datasets used in this study is provided in Supplementary Data 8. All data from the protein clusters, including sequences, multiple alignments, HMM profiles, 3D structure models, and taxonomic and ecosystem annotation, are available through NMPFamsDB, publicly accessible at www.nmpfamsdb.org. The 3D models are also available at ModelArchive under accession code ma-nmpfamsdb.-- Code availability: Sequence analysis was performed using Tantan (https://gitlab.com/mcfrith/tantan), BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), LAST (https://gitlab.com/mcfrith/last), HMMER (http://hmmer.org/) and HH-suite3 (https://github.com/soedinglab/hh-suite). Clustering was performed using HipMCL (https://bitbucket.org/azadcse/hipmcl/src/master/). Additional taxonomic annotation was performed using Whokaryote (https://github.com/LottePronk/whokaryote), EukRep (https://github.com/patrickwest/EukRep), DeepVirFinder (https://github.com/jessieren/DeepVirFinder) and MMseqs2 (https://github.com/soedinglab/MMseqs2). 3D modelling was performed using AlphaFold2 (https://github.com/deepmind/alphafold) and TrRosetta2 (https://github.com/RosettaCommons/trRosetta2). Structural alignments were performed using TMalign (https://zhanggroup.org/TM-align/) and MMalign (https://zhanggroup.org/MM-align/). All custom scripts used for the generation and analysis of the data are available at Zenodo (https://doi.org/10.5281/zenodo.8097349)Metagenomes encode an enormous diversity of proteins, reflecting a multiplicity of functions and activities1,2. Exploration of this vast sequence space has been limited to a comparative analysis against reference microbial genomes and protein families derived from those genomes. Here, to examine the scale of yet untapped functional diversity beyond what is currently possible through the lens of reference genomes, we develop a computational approach to generate reference-free protein families from the sequence space in metagenomes. We analyse 26,931 metagenomes and identify 1.17 billion protein sequences longer than 35 amino acids with no similarity to any sequences from 102,491 reference genomes or the Pfam database3. Using massively parallel graph-based clustering, we group these proteins into 106,198 novel sequence clusters with more than 100 members, doubling the number of protein families obtained from the reference genomes clustered using the same approach. We annotate these families on the basis of their taxonomic, habitat, geographical and gene neighbourhood distributions and, where sufficient sequence diversity is available, predict protein three-dimensional models, revealing novel structures. Overall, our results uncover an enormously diverse functional space, highlighting the importance of further exploring the microbial functional dark matterWith the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria

Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome.

Bacteriophages have important roles in the ecology of the human gut microbiome but are under-represented in reference databases. To address this problem, we assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Over 75% of genomes represent double-stranded DNA phages that infect members of the Bacteroidia and Clostridia classes. Based on sequence clustering we identified 54,118 candidate viral species, 92% of which were not found in existing databases. The Metagenomic Gut Virus catalogue improves detection of viruses in stool metagenomes and accounts for nearly 40% of CRISPR spacers found in human gut Bacteria and Archaea. We also produced a catalogue of 459,375 viral protein clusters to explore the functional potential of the gut virome. This revealed tens of thousands of diversity-generating retroelements, which use error-prone reverse transcription to mutate target genes and may be involved in the molecular arms race between phages and their bacterial hosts

The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes.

The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria

IMG/VR v3: an integrated ecological and evolutionary framework for interrogating genomes of uncultivated viruses.

Viruses are integral components of all ecosystems and microbiomes on Earth. Through pervasive infections of their cellular hosts, viruses can reshape microbial community structure and drive global nutrient cycling. Over the past decade, viral sequences identified from genomes and metagenomes have provided an unprecedented view of viral genome diversity in nature. Since 2016, the IMG/VR database has provided access to the largest collection of viral sequences obtained from (meta)genomes. Here, we present the third version of IMG/VR, composed of 18 373 cultivated and 2 314 329 uncultivated viral genomes (UViGs), nearly tripling the total number of sequences compared to the previous version. These clustered into 935 362 viral Operational Taxonomic Units (vOTUs), including 188 930 with two or more members. UViGs in IMG/VR are now reported as single viral contigs, integrated proviruses or genome bins, and are annotated with a new standardized pipeline including genome quality estimation using CheckV, taxonomic classification reflecting the latest ICTV update, and expanded host taxonomy prediction. The new IMG/VR interface enables users to efficiently browse, search, and select UViGs based on genome features and/or sequence similarity. IMG/VR v3 is available at https://img.jgi.doe.gov/vr, and the underlying data are available to download at https://genome.jgi.doe.gov/portal/IMG_VR

Influence of the polar light cycle on seasonal dynamics of an Antarctic lake microbial community.

BackgroundCold environments dominate the Earth's biosphere and microbial activity drives ecosystem processes thereby contributing greatly to global biogeochemical cycles. Polar environments differ to all other cold environments by experiencing 24-h sunlight in summer and no sunlight in winter. The Vestfold Hills in East Antarctica contains hundreds of lakes that have evolved from a marine origin only 3000-7000 years ago. Ace Lake is a meromictic (stratified) lake from this region that has been intensively studied since the 1970s. Here, a total of 120 metagenomes representing a seasonal cycle and four summers spanning a 10-year period were analyzed to determine the effects of the polar light cycle on microbial-driven nutrient cycles.ResultsThe lake system is characterized by complex sulfur and hydrogen cycling, especially in the anoxic layers, with multiple mechanisms for the breakdown of biopolymers present throughout the water column. The two most abundant taxa are phototrophs (green sulfur bacteria and cyanobacteria) that are highly influenced by the seasonal availability of sunlight. The extent of the Chlorobium biomass thriving at the interface in summer was captured in underwater video footage. The Chlorobium abundance dropped from up to 83% in summer to 6% in winter and 1% in spring, before rebounding to high levels. Predicted Chlorobium viruses and cyanophage were also abundant, but their levels did not negatively correlate with their hosts.ConclusionOver-wintering expeditions in Antarctica are logistically challenging, meaning insight into winter processes has been inferred from limited data. Here, we found that in contrast to chemolithoautotrophic carbon fixation potential of Southern Ocean Thaumarchaeota, this marine-derived lake evolved a reliance on photosynthesis. While viruses associated with phototrophs also have high seasonal abundance, the negative impact of viral infection on host growth appeared to be limited. The microbial community as a whole appears to have developed a capacity to generate biomass and remineralize nutrients, sufficient to sustain itself between two rounds of sunlight-driven summer-activity. In addition, this unique metagenome dataset provides considerable opportunity for future interrogation of eukaryotes and their viruses, abundant uncharacterized taxa (i.e. dark matter), and for testing hypotheses about endemic species in polar aquatic ecosystems. Video Abstract

A genomic catalog of Earth's microbiomes.

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth's continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes

Minimum Information about an Uncultivated Virus Genome (MIUViG)

International audienceWe present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere

A genomic catalog of Earth’s microbiomes

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.ISSN:1546-1696ISSN:1087-015