Going to extremes - a metagenomic journey into the dark matter of life.

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.

Extremophile deep-sea viral communities from hydrothermal vents: Structural and functional analysis.

Ten publicly available metagenomic data sets from hydrothermal vents were analyzed to determine the taxonomic structure of the viral communities present, as well as their potential metabolic functions. The type of natural selection on two auxiliary metabolic genes was also analyzed. The structure of the virome in the hydrothermal vents was quite different in comparison with the viruses present in sediments, with specific populations being present in greater abundance in the plume samples when compared with the sediment samples. ssDNA genomes such as Circoviridae and Microviridae were predominantly present in the sediment samples, with Caudovirales which are dsDNA being present in the vent samples. Genes potentially encoding enzymes that participate in carbon, nitrogen and sulfur metabolic pathways were found in greater abundance, than those involved in the oxygen cycle, in the hydrothermal vents. Functional profiling of the viromes, resulted in the discovery of genes encoding proteins involved in bacteriophage capsids, DNA synthesis, nucleotide synthesis, DNA repair, as well as viral auxiliary metabolic genes such as cytitidyltransferase and ribonucleotide reductase. These auxiliary metabolic genes participate in the synthesis of phospholipids and nucleotides respectively and are likely to contribute to enhancing the fitness of their bacterial hosts within the hydrothermal vent communities. Finally, evolutionary analysis suggested that these auxiliary metabolic genes are highly conserved and evolve under purifying selection, and are thus maintained in their genome.

Deep-Sea Hydrothermal Vent Viruses Compensate for Microbial Metabolism in Virus-Host Interactions.

Viruses are believed to be responsible for the mortality of host organisms. However, some recent investigations reveal that viruses may be essential for host survival. To date, it remains unclear whether viruses are beneficial or harmful to their hosts. To reveal the roles of viruses in the virus-host interactions, viromes and microbiomes of sediment samples from three deep-sea hydrothermal vents were explored in this study. To exclude the influence of exogenous DNAs on viromes, the virus particles were purified with nuclease (DNase I and RNase A) treatments and cesium chloride density gradient centrifugation. The metagenomic analysis of viromes without exogenous DNA contamination and microbiomes of vent samples indicated that viruses had compensation effects on the metabolisms of their host microorganisms. Viral genes not only participated in most of the microbial metabolic pathways but also formed branched pathways in microbial metabolisms, including pyrimidine metabolism; alanine, aspartate, and glutamate metabolism; nitrogen metabolism and assimilation pathways of the two-component system; selenocompound metabolism; aminoacyl-tRNA biosynthesis; and amino sugar and nucleotide sugar metabolism. As is well known, deep-sea hydrothermal vent ecosystems exist in relatively isolated environments which are barely influenced by other ecosystems. The metabolic compensation of hosts mediated by viruses might represent a very important aspect of virus-host interactions.IMPORTANCE Viruses are the most abundant biological entities in the oceans and have very important roles in regulating microbial community structure and biogeochemical cycles. The relationship between virus and host microbes is broadly thought to be that of predator and prey. Viruses can lyse host cells to control microbial population sizes and affect community structures of hosts by killing specific microbes. However, viruses also influence their hosts through manipulation of bacterial metabolism. We found that viral genes not only participated in most microbial metabolic pathways but also formed branched pathways in microbial metabolisms. The metabolic compensation of hosts mediated by viruses may help hosts to adapt to extreme environments and may be essential for host survival.

Genome sequence of an inducible phage in Rhodovulum sp. P5 isolated from the shallow-sea hydrothermal system.

A prophage namely vB_RhkS_P1 was induced by mitomycin C from Rhodovulum sp. P5 in the shallow-sea hydrothermal systems. The vB_RhkS_P1 had siphovirus-like morphology, and the average particle had a head size of approximately 61nm, and the tail length approximately 93nm. The genome of vB_RhkS_P1 was a size of 38.8kbp, 67.5% GC content, and 59 open reading frames. The genome contained Mu-like head structural genes but its genomic content was distinct from Mu or Mu-like phages.

Biogeography of bacteriophages at four hydrothermal vent sites in the Antarctic based on g23 sequence diversity.

In this study, which was carried out within the ChEsSO consortium project (Chemosynthetically driven ecosystems south of the Polar Front), we sampled two hydrothermal vent sites on the East Scotia Ridge, Scotia Sea, one in the Kemp Caldera, South Sandwich Arc and one in the Bransfield Strait, north-west of the Antarctic Peninsula, which exhibit strong differences in their chemical characteristics. We compared a subset of their bacteriophage population by Sanger- and 454-sequencing of g23, which codes for the major capsid protein of T4likeviruses. We found that the sites differ vastly in their bacteriophage diversity, which reflects the differences in the chemical conditions and therefore putatively the differences in microbial hosts living at these sites. Comparing phage diversity in the vent samples to other aquatic samples, the vent samples formed a distinct separate cluster, which also included the non-vent control samples that were taken several hundred meters above the vent chimneys. This indicates that the influence of the vents on the microbial population and therefore also the bacteriophage population extends much further than anticipated.

An abyssal mobilome: viruses, plasmids and vesicles from deep-sea hydrothermal vents.

Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents.

'Ménage à trois': a selfish genetic element uses a virus to propagate within Thermotogales.

Prokaryotic viruses play a major role in the microbial ecology and evolution. However, the virosphere associated with deep-sea hydrothermal ecosystems remains largely unexplored. Numerous instances of lateral gene transfer have contributed to the complex and incongruent evolutionary history of Thermotogales, an order well represented in deep-sea hydrothermal vents. The presence of clustered regularly interspaced short palindromic repeats (CRISPR) loci has been reported in all Thermotogales genomes, suggesting that these bacteria have been exposed to viral infections that could have mediated gene exchange. In this study, we isolated and characterized the first virus infecting bacteria from the order Thermotogales, Marinitoga piezophila virus 1 (MPV1). The host, Marinitoga piezophila is a thermophilic, anaerobic and piezophilic bacterium isolated from a deep-sea hydrothermal chimney. MPV1 is a temperate Siphoviridae-like virus with a 43.7 kb genome. Surprisingly, we found that MPV1 virions carry not only the viral DNA but preferentially package a plasmid of 13.3 kb (pMP1) also carried by M. piezophila. This 'ménage à trois' highlights potential relevance of selfish genetic elements in facilitating lateral gene transfer in the deep-sea biosphere.

Evolutionary strategies of viruses, bacteria and archaea in hydrothermal vent ecosystems revealed through metagenomics.

The deep-sea hydrothermal vent habitat hosts a diverse community of archaea and bacteria that withstand extreme fluctuations in environmental conditions. Abundant viruses in these systems, a high proportion of which are lysogenic, must also withstand these environmental extremes. Here, we explore the evolutionary strategies of both microorganisms and viruses in hydrothermal systems through comparative analysis of a cellular and viral metagenome, collected by size fractionation of high temperature fluids from a diffuse flow hydrothermal vent. We detected a high enrichment of mobile elements and proviruses in the cellular fraction relative to microorganisms in other environments. We observed a relatively high abundance of genes related to energy metabolism as well as cofactors and vitamins in the viral fraction compared to the cellular fraction, which suggest encoding of auxiliary metabolic genes on viral genomes. Moreover, the observation of stronger purifying selection in the viral versus cellular gene pool suggests viral strategies that promote prolonged host integration. Our results demonstrate that there is great potential for hydrothermal vent viruses to integrate into hosts, facilitate horizontal gene transfer, and express or transfer genes that manipulate the hosts' functional capabilities.

Living side by side with a virus: characterization of two novel plasmids from Thermococcus prieurii, a host for the spindle-shaped virus TPV1.

Microbial cells often serve as an evolutionary battlefield for different types of mobile genetic elements, such as viruses and plasmids. Here, we describe the isolation and characterization of two new archaeal plasmids which share the host with the spindle-shaped Thermococcus prieurii virus 1 (TPV1). The two plasmids, pTP1 and pTP2, were isolated from the hyperthermophilic archaeon Thermococcus prieurii (phylum Euryarchaeota), a resident of a deep-sea hydrothermal vent located at the East Pacific Rise at 2,700-m depth (7°25'24 S, 107°47'66 W). pTP1 (3.1 kb) and pTP2 (2.0 kb) are among the smallest known plasmids of hyperthermophilic archaea, and both are predicted to replicate via the rolling-circle mechanism. The two plasmids and the virus TPV1 do not have a single gene in common and stably propagate in infected cells without any apparent antagonistic effect on each other. The compatibility of the three genetic elements and the high copy number of pTP1 and pTP2 plasmids (50 copies/cell) might be useful for developing new genetic tools for studying hyperthermophilic euryarchaea and their viruses.

Genome sequence of a novel deep-sea vent epsilonproteobacterial phage provides new insight into the co-evolution of Epsilonproteobacteria and their phages.

Epsilonproteobacteria are among the predominant primary producers in deep-sea hydrothermal vent ecosystems. However, phages infecting deep-sea vent Epsilonproteobacteria have never been isolated and characterized. Here, we successfully isolated a novel temperate phage, NrS-1, that infected a deep-sea vent chemolithoautotrophic isolate of Epsilonproteobacteria, Nitratiruptor sp. SB155-2, and its entire genome sequence was obtained and analyzed. The NrS-1 genome is linear, circularly permuted, and terminally redundant. The NrS-1 genome is 37,159 bp in length and contains 51 coding sequences. Five major structural proteins including major capsid protein and tape measure protein were identified by SDS-PAGE and mass spectrometry analysis. NrS-1 belongs to the family Siphoviridae, but its sequence and genomic organization are distinct from those of any other previously known Siphoviridae phages. Homologues of genes encoded in the NrS-1 genome were widely distributed among the genomes of diverse Epsilonproteobacteria. The distribution patterns had little relation to the evolutionary traits and ecological and physiological differentiation of the host epsilonproteobacterial species. The widespread occurrence of phage genes in diverse Epsilonproteobacteria supports early co-evolution between temperate phages and Epsilonproteobacteria prior to the divergence of their habitats and physiological adaptation.

The role of interactions between bacterial chaperone, aspartate aminotransferase, and viral protein during virus infection in high temperature environment: the interactions between bacterium and virus proteins.

BACKGROUND: The life cycle of a bacteriophage has tightly programmed steps to help virus infect its host through the interactions between the bacteriophage and its host proteins. However, bacteriophage-host protein interactions in high temperature environment remain poorly understood. To address this issue, the protein interaction between the thermophilic bacteriophage GVE2 and its host thermophilic Geobacillus sp. E263 from a deep-sea hydrothermal vent was characterized. RESULTS: This investigation showed that the host's aspartate aminotransferase (AST), chaperone GroEL, and viral capsid protein VP371 formed a linearly interacted complex. The results indicated that the VP371-GroEL-AST complex were up-regulated and co-localized in the GVE2 infection of Geobacillus sp. E263. CONCLUSIONS: As reported, the VP371 is a capsid protein of GVE2 and the host AST is essential for the GVE2 infection. Therefore, our study revealed that the phage could use the anti-stress system of its host to protect the virus reproduction in a high-temperature environment for the first time.

Finding a needle in the virus metagenome haystack--micro-metagenome analysis captures a snapshot of the diversity of a bacteriophage armoire.

Viruses are ubiquitous in the oceans and critical components of marine microbial communities, regulating nutrient transfer to higher trophic levels or to the dissolved organic pool through lysis of host cells. Hydrothermal vent systems are oases of biological activity in the deep oceans, for which knowledge of biodiversity and its impact on global ocean biogeochemical cycling is still in its infancy. In order to gain biological insight into viral communities present in hydrothermal vent systems, we developed a method based on deep-sequencing of pulsed field gel electrophoretic bands representing key viral fractions present in seawater within and surrounding a hydrothermal plume derived from Loki's Castle vent field at the Arctic Mid-Ocean Ridge. The reduction in virus community complexity afforded by this novel approach enabled the near-complete reconstruction of a lambda-like phage genome from the virus fraction of the plume. Phylogenetic examination of distinct gene regions in this lambdoid phage genome unveiled diversity at loci encoding superinfection exclusion- and integrase-like proteins. This suggests the importance of fine-tuning lyosgenic conversion as a viral survival strategy, and provides insights into the nature of host-virus and virus-virus interactions, within hydrothermal plumes. By reducing the complexity of the viral community through targeted sequencing of prominent dsDNA viral fractions, this method has selectively mimicked virus dominance approaching that hitherto achieved only through culturing, thus enabling bioinformatic analysis to locate a lambdoid viral "needle" within the greater viral community "haystack". Such targeted analyses have great potential for accelerating the extraction of biological knowledge from diverse and poorly understood environmental viral communities.

Spatial distribution of viruses associated with planktonic and attached microbial communities in hydrothermal environments.

Viruses play important roles in marine surface ecosystems, but little is known about viral ecology and virus-mediated processes in deep-sea hydrothermal microbial communities. In this study, we examined virus-like particle (VLP) abundances in planktonic and attached microbial communities, which occur in physical and chemical gradients in both deep and shallow submarine hydrothermal environments (mixing waters between hydrothermal fluids and ambient seawater and dense microbial communities attached to chimney surface areas or macrofaunal bodies and colonies). We found that viruses were widely distributed in a variety of hydrothermal microbial habitats, with the exception of the interior parts of hydrothermal chimney structures. The VLP abundance and VLP-to-prokaryote ratio (VPR) in the planktonic habitats increased as the ratio of hydrothermal fluid to mixing water increased. On the other hand, the VLP abundance in attached microbial communities was significantly and positively correlated with the whole prokaryotic abundance; however, the VPRs were always much lower than those for the surrounding hydrothermal waters. This is the first report to show VLP abundance in the attached microbial communities of submarine hydrothermal environments, which presented VPR values significantly lower than those in planktonic microbial communities reported before. These results suggested that viral lifestyles (e.g., lysogenic prevalence) and virus interactions with prokaryotes are significantly different among the planktonic and attached microbial communities that are developing in the submarine hydrothermal environments.