Microbiomes from every environment contain a myriad of uncultivated archaeal and bacterial viruses, but studying these viruses is hampered by the lack of a universal, scalable taxonomic framework. We ...present vConTACT v.2.0, a network-based application utilizing whole genome gene-sharing profiles for virus taxonomy that integrates distance-based hierarchical clustering and confidence scores for all taxonomic predictions. We report near-identical (96%) replication of existing genus-level viral taxonomy assignments from the International Committee on Taxonomy of Viruses for National Center for Biotechnology Information virus RefSeq. Application of vConTACT v.2.0 to 1,364 previously unclassified viruses deposited in virus RefSeq as reference genomes produced automatic, high-confidence genus assignments for 820 of the 1,364. We applied vConTACT v.2.0 to analyze 15,280 Global Ocean Virome genome fragments and were able to provide taxonomic assignments for 31% of these data, which shows that our algorithm is scalable to very large metagenomic datasets. Our taxonomy tool can be automated and applied to metagenomes from any environment for virus classification.
The gut microbiome profoundly affects human health and disease, and their infecting viruses are likely as important, but often missed because of reference database limitations. Here, we (1) built a ...human Gut Virome Database (GVD) from 2,697 viral particle or microbial metagenomes from 1,986 individuals representing 16 countries, (2) assess its effectiveness, and (3) report a meta-analysis that reveals age-dependent patterns across healthy Westerners. The GVD contains 33,242 unique viral populations (approximately species-level taxa) and improves average viral detection rates over viral RefSeq and IMG/VR nearly 182-fold and 2.6-fold, respectively. GVD meta-analyses show highly personalized viromes, reveal that inter-study variability from technical artifacts is larger than any “disease” effect at the population level, and document how viral diversity changes from human infancy into senescence. Together, this compact foundational resource, these standardization guidelines, and these meta-analysis findings provide a systematic toolkit to help maximize our understanding of viral roles in health and disease.
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•Assembly of 2,697 gut metagenomes from 32 studies exposed 33,242 viral populations•Inter-study analyses reveal strong study biases at the viral population-level•Viral population detection was higher in bulk versus VLP-enriched metagenomes•Gut viral diversity is age-dependent across healthy, Western people
At least 32 studies to date have looked at the human gut virome but with limited consistency. Gregory and Zablocki et al. curate and aggregate these data to provide a systematic virome database; use it to assess study biases, global ecological patterns; and show how viromes evolve throughout the human lifespan.
Soils contain a tangle of minerals, water, nutrients, gases, plant roots, decaying organic matter, and microorganisms which work together to cycle nutrients and support terrestrial plant growth. Most ...soil microorganisms live in periodically interconnected communities closely associated with soil aggregates, i.e., small (<2 mm), strongly bound clusters of minerals and organic carbon that persist through mechanical disruptions and wetting events. Their spatial structure is important for biogeochemical cycling, and we cannot reliably predict soil biological activities and variability by studying bulk soils alone. To fully understand the biogeochemical processes at work in soils, it is necessary to understand the micrometer-scale interactions that occur between soil particles and their microbial inhabitants. Here, we review the current state of knowledge regarding soil aggregate microbial communities and identify areas of opportunity to study soil ecosystems at a scale relevant to individual cells. We present a framework for understanding aggregate communities as "microbial villages" that are periodically connected through wetting events, allowing for the transfer of genetic material, metabolites, and viruses. We describe both top-down (whole community) and bottom-up (reductionist) strategies for studying these communities. Understanding this requires combining "model system" approaches (e.g., developing mock community artificial aggregates), field observations of natural communities, and broader study of community interactions to include understudied community members, like viruses. Initial studies suggest that aggregate-based approaches are a critical next step for developing a predictive understanding of how geochemical and community interactions govern microbial community structure and nutrient cycling in soil.
Bacterial viruses (“phages”) play important roles in the regulation and evolution of microbial communities in most ecosystems. Terrestrial hot springs typically contain thermophilic bacterial ...communities, but the diversity and impacts of its associated viruses (“thermophilic phages”) are largely unexplored. Here, we provide a taxonomic overview of phages that have been isolated strictly from terrestrial hot springs around the world. In addition, we placed 17 thermophilic phage genomes in a global phylogenomic context to detect evolutionary patterns. Thermophilic phages have diverse morphologies (e.g., tailed, filamentous), unique virion structures (e.g., extremely long tailed siphoviruses), and span five taxonomic families encompassing strictly thermophilic phage genera. Within the phage proteomic tree, six thermophilic phage-related clades were identified, with evident genomic relatedness between thermophilic phages and archaeal viruses. Moreover, whole proteome analyses showed clustering between phages that infect distinct host phyla, such as Firmicutes and
Deinococcus
–
Thermus
. The potential for discovery of novel phage-host systems in terrestrial hot springs remain mostly untapped, thus additional emphasis on thermophilic phages in ecological prospecting is encouraged to gain insights into the microbial population dynamics of these environments.
Viruses impact microbial diversity, gene flow and function through virus-host interactions. Although metagenomics surveys are rapidly cataloguing viral diversity, methods are needed to capture ...specific virus-host interactions in situ. Here, we leveraged metagenomics and repurposed emulsion paired isolation-concatenation PCR (epicPCR) to investigate viral diversity and virus-host interactions in situ over time in an estuarine environment. The method fuses a phage marker, the ribonucleotide reductase gene, with the host 16S rRNA gene of infected bacterial cells within emulsion droplets providing single-cell resolution for dozens of samples. EpicPCR captured in situ virus-host interactions for viral clades with no closely related database representatives. Abundant freshwater Actinobacteria lineages, in particular Rhodoluna sp., were the most common hosts for these poorly characterized viruses, with interactions correlated with environmental factors. Multiple methods used to identify virus-host interactions, including epicPCR, identified different and largely non-overlapping interactions within the vast virus-host interaction space. Tracking virus-host interaction dynamics also revealed that multi-host viruses had significantly longer periods with observed virus-host interactions, whereas single-host viruses were observed interacting with hosts at lower minimum abundances, suggesting more efficient interactions. Capturing in situ interactions with epicPCR revealed environmental and ecological factors shaping virus-host interactions, highlighting epicPCR as a valuable technique in viral ecology.
The current view of virus diversity in terrestrial hot springs is limited to a few sampling sites. To expand our current understanding of hot spring viral community diversity, this study aimed to ...investigate the first African hot spring (Brandvlei hot spring; 60 °C, pH 5.7) by means of electron microscopy and sequencing of the virus fraction. Microscopy analysis revealed a mixture of regular- and 'jumbo'-sized tailed morphotypes (
), lemon-shaped virions (
like; salterprovirus-like) and pleiomorphic virus-like particles. Metavirome analysis corroborated the presence of His1-like viruses and has expanded the current clade of salterproviruses using a polymerase B gene phylogeny. The most represented viral contig was to a cyanophage genome fragment, which may underline basic ecosystem functioning provided by these viruses. Furthermore, a putative
-related phage was assembled with high coverage, a previously undocumented phage-host association. This study demonstrated that a moderately thermophilic spring environment contained a highly novel pool of viruses and should encourage future characterization of a wider temperature range of hot springs throughout the world.
Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be ...challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit-and revealed many critical ecosystem roles for viruses-microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data ('enhanced' viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a 'living document' to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology.
Climate change threatens Earth's ice-based ecosystems which currently offer archives and eco-evolutionary experiments in the extreme. Arctic cryopeg brine (marine-derived, within permafrost) and sea ...ice brine, similar in subzero temperature and high salinity but different in temporal stability, are inhabited by microbes adapted to these extreme conditions. However, little is known about their viruses (community composition, diversity, interaction with hosts, or evolution) or how they might respond to geologically stable cryopeg versus fluctuating sea ice conditions.
We used long- and short-read viromics and metatranscriptomics to study viruses in Arctic cryopeg brine, sea ice brine, and underlying seawater, recovering 11,088 vOTUs (~species-level taxonomic unit), a 4.4-fold increase of known viruses in these brines. More specifically, the long-read-powered viromes doubled the number of longer (≥25 kb) vOTUs generated and recovered more hypervariable regions by >5-fold compared to short-read viromes. Distribution assessment, by comparing to known viruses in public databases, supported that cryopeg brine viruses were of marine origin yet distinct from either sea ice brine or seawater viruses, while 94% of sea ice brine viruses were also present in seawater. A virus-encoded, ecologically important exopolysaccharide biosynthesis gene was identified, and many viruses (~half of metatranscriptome-inferred "active" vOTUs) were predicted as actively infecting the dominant microbial genera Marinobacter and Polaribacter in cryopeg and sea ice brines, respectively. Evolutionarily, microdiversity (intra-species genetic variations) analyses suggested that viruses within the stable cryopeg brine were under significantly lower evolutionary pressures than those in the fluctuating sea ice environment, while many sea ice brine virus-tail genes were under positive selection, indicating virus-host co-evolutionary arms races.
Our results confirmed the benefits of long-read-powered viromics in understanding the environmental virosphere through significantly improved genomic recovery, expanding viral discovery and the potential for biological inference. Evidence of viruses actively infecting the dominant microbes in subzero brines and modulating host metabolism underscored the potential impact of viruses on these remote and underexplored extreme ecosystems. Microdiversity results shed light on different strategies viruses use to evolve and adapt when extreme conditions are stable versus fluctuating. Together, these findings verify the value of long-read-powered viromics and provide foundational data on viral evolution and virus-microbe interactions in Earth's destabilized and rapidly disappearing cryosphere. Video Abstract.
The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are shaped by both current and historical soil environmental conditions. Here, we developed and ...applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) with an H
O labeling experiment to identify actively growing soil microorganisms and their genomic capacities. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve the active subset of soil community members and identify their characteristic ecophysiological traits. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to heavily isotopically labeled bacteria, we identified abundant isotope-labeled phages, suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capacity to decompose the diverse complex carbohydrates common in soil organic matter or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation.
Soil moisture is a critical factor that strongly shapes the lifestyle of soil organisms by changing access to nutrients, controlling oxygen diffusion, and regulating the potential for mobility. We identified active microorganisms in three grassland soils with similar mineral contexts, yet different historic rainfall inputs, by adding water labeled with a stable isotope and tracking that isotope in DNA of growing microbes. By examining the genomes of active and inactive microorganisms, we identified functions that are enriched in growing organisms, and showed that different functions were selected for in different soils. Wetter soil had higher activity of motile organisms, but activity of pathways for degradation of soil organic carbon compounds, including simple carbon substrates, were comparable for all three soils. We identified many labeled, and thus active bacteriophages (viruses that infect bacteria), implying that the cells they killed contributed to soil organic matter. The activity of these bacteriophages was significantly correlated with activity of their hosts.
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