Environment-specific virocell metabolic reprogramming.

Viruses impact microbial systems through killing hosts, horizontal gene transfer, and altering cellular metabolism, consequently impacting nutrient cycles. A virus-infected cell, a "virocell", is distinct from its uninfected sister cell as the virus commandeers cellular machinery to produce viruses rather than replicate cells. Problematically, virocell responses to the nutrient-limited conditions that abound in nature are poorly understood. Here we used a systems biology approach to investigate virocell metabolic reprogramming under nutrient limitation. Using transcriptomics, proteomics, lipidomics, and endo- and exo-metabolomics, we assessed how low phosphate (low-P) conditions impacted virocells of a marine Pseudoalteromonas host when independently infected by two unrelated phages (HP1 and HS2). With the combined stresses of infection and nutrient limitation, a set of nested responses were observed. First, low-P imposed common cellular responses on all cells (virocells and uninfected cells), including activating the canonical P-stress response, and decreasing transcription, translation, and extracellular organic matter consumption. Second, low-P imposed infection-specific responses (for both virocells), including enhancing nitrogen assimilation and fatty acid degradation, and decreasing extracellular lipid relative abundance. Third, low-P suggested virocell-specific strategies. Specifically, HS2-virocells regulated gene expression by increasing transcription and ribosomal protein production, whereas HP1-virocells accumulated host proteins, decreased extracellular peptide relative abundance, and invested in broader energy and resource acquisition. These results suggest that although environmental conditions shape metabolism in common ways regardless of infection, virocell-specific strategies exist to support viral replication during nutrient limitation, and a framework now exists for identifying metabolic strategies of nutrient-limited virocells in nature.

Four additional natural 7-deazaguanine derivatives in phages and how to make them.

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

Glacier ice archives nearly 15,000-year-old microbes and phages.

BACKGROUND: Glacier ice archives information, including microbiology, that helps reveal paleoclimate histories and predict future climate change. Though glacier-ice microbes are studied using culture or amplicon approaches, more challenging metagenomic approaches, which provide access to functional, genome-resolved information and viruses, are under-utilized, partly due to low biomass and potential contamination. RESULTS: We expand existing clean sampling procedures using controlled artificial ice-core experiments and adapted previously established low-biomass metagenomic approaches to study glacier-ice viruses. Controlled sampling experiments drastically reduced mock contaminants including bacteria, viruses, and free DNA to background levels. Amplicon sequencing from eight depths of two Tibetan Plateau ice cores revealed common glacier-ice lineages including Janthinobacterium, Polaromonas, Herminiimonas, Flavobacterium, Sphingomonas, and Methylobacterium as the dominant genera, while microbial communities were significantly different between two ice cores, associating with different climate conditions during deposition. Separately, ~355- and ~14,400-year-old ice were subject to viral enrichment and low-input quantitative sequencing, yielding genomic sequences for 33 vOTUs. These were virtually all unique to this study, representing 28 novel genera and not a single species shared with 225 environmentally diverse viromes. Further, 42.4% of the vOTUs were identifiable temperate, which is significantly higher than that in gut, soil, and marine viromes, and indicates that temperate phages are possibly favored in glacier-ice environments before being frozen. In silico host predictions linked 18 vOTUs to co-occurring abundant bacteria (Methylobacterium, Sphingomonas, and Janthinobacterium), indicating that these phages infected ice-abundant bacterial groups before being archived. Functional genome annotation revealed four virus-encoded auxiliary metabolic genes, particularly two motility genes suggest viruses potentially facilitate nutrient acquisition for their hosts. Finally, given their possible importance to methane cycling in ice, we focused on Methylobacterium viruses by contextualizing our ice-observed viruses against 123 viromes and prophages extracted from 131 Methylobacterium genomes, revealing that the archived viruses might originate from soil or plants. CONCLUSIONS: Together, these efforts further microbial and viral sampling procedures for glacier ice and provide a first window into viral communities and functions in ancient glacier environments. Such methods and datasets can potentially enable researchers to contextualize new discoveries and begin to incorporate glacier-ice microbes and their viruses relative to past and present climate change in geographically diverse regions globally. Video Abstract.

VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature.

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.

Viral Ecogenomics of Arctic Cryopeg Brine and Sea Ice.

Arctic regions, which are changing rapidly as they warm 2 to 3 times faster than the global average, still retain microbial habitats that serve as natural laboratories for understanding mechanisms of microbial adaptation to extreme conditions. Seawater-derived brines within both sea ice (sea-ice brine) and ancient layers of permafrost (cryopeg brine) support diverse microbes adapted to subzero temperatures and high salinities, yet little is known about viruses in these extreme environments, which, if analogous to other systems, could play important evolutionary and ecosystem roles. Here, we characterized viral communities and their functions in samples of cryopeg brine, sea-ice brine, and melted sea ice. Viral abundance was high in cryopeg brine (1.2 × 108 ml-1) and much lower in sea-ice brine (1.3 × 105 to 2.1 × 105 ml-1), which roughly paralleled the differences in cell concentrations in these samples. Five low-input, quantitative viral metagenomes were sequenced to yield 476 viral populations (i.e., species level; ≥10 kb), only 12% of which could be assigned taxonomy by traditional database approaches, indicating a high degree of novelty. Additional analyses revealed that these viruses: (i) formed communities that differed between sample type and vertically with sea-ice depth; (ii) infected hosts that dominated these extreme ecosystems, including Marinobacter, Glaciecola, and Colwellia; and (iii) encoded fatty acid desaturase (FAD) genes that likely helped their hosts overcome cold and salt stress during infection, as well as mediated horizontal gene transfer of FAD genes between microbes. Together, these findings contribute to understanding viral abundances and communities and how viruses impact their microbial hosts in subzero brines and sea ice.IMPORTANCE This study explores viral community structure and function in remote and extreme Arctic environments, including subzero brines within marine layers of permafrost and sea ice, using a modern viral ecogenomics toolkit for the first time. In addition to providing foundational data sets for these climate-threatened habitats, we found evidence that the viruses had habitat specificity, infected dominant microbial hosts, encoded host-derived metabolic genes, and mediated horizontal gene transfer among hosts. These results advance our understanding of the virosphere and how viruses influence extreme ecosystems. More broadly, the evidence that virally mediated gene transfers may be limited by host range in these extreme habitats contributes to a mechanistic understanding of genetic exchange among microbes under stressful conditions in other systems.

Phage-specific metabolic reprogramming of virocells.

Ocean viruses are abundant and infect 20-40% of surface microbes. Infected cells, termed virocells, are thus a predominant microbial state. Yet, virocells and their ecosystem impacts are understudied, thus precluding their incorporation into ecosystem models. Here we investigated how unrelated bacterial viruses (phages) reprogram one host into contrasting virocells with different potential ecosystem footprints. We independently infected the marine Pseudoalteromonas bacterium with siphovirus PSA-HS2 and podovirus PSA-HP1. Time-resolved multi-omics unveiled drastically different metabolic reprogramming and resource requirements by each virocell, which were related to phage-host genomic complementarity and viral fitness. Namely, HS2 was more complementary to the host in nucleotides and amino acids, and fitter during infection than HP1. Functionally, HS2 virocells hardly differed from uninfected cells, with minimal host metabolism impacts. HS2 virocells repressed energy-consuming metabolisms, including motility and translation. Contrastingly, HP1 virocells substantially differed from uninfected cells. They repressed host transcription, responded to infection continuously, and drastically reprogrammed resource acquisition, central carbon and energy metabolisms. Ecologically, this work suggests that one cell, infected versus uninfected, can have immensely different metabolisms that affect the ecosystem differently. Finally, we relate phage-host genome complementarity, virocell metabolic reprogramming, and viral fitness in a conceptual model to guide incorporating viruses into ecosystem models.

Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils.

Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ∼1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1-2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ∼4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates 'living' protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.

Long-read viral metagenomics captures abundant and microdiverse viral populations and their niche-defining genomic islands.

Marine viruses impact global biogeochemical cycles via their influence on host community structure and function, yet our understanding of viral ecology is constrained by limitations in host culturing and a lack of reference genomes and 'universal' gene markers to facilitate community surveys. Short-read viral metagenomic studies have provided clues to viral function and first estimates of global viral gene abundance and distribution, but their assemblies are confounded by populations with high levels of strain evenness and nucleotide diversity (microdiversity), limiting assembly of some of the most abundant viruses on Earth. Such features also challenge assembly across genomic islands containing niche-defining genes that drive ecological speciation. These populations and features may be successfully captured by single-virus genomics and fosmid-based approaches, at least in abundant taxa, but at considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative sequencing and informatics workflow to improve viral metagenomic assemblies using short-read and long-read technology. The 'VirION' (Viral, long-read metagenomics via MinION sequencing) approach was first validated using mock communities where it was found to be as relatively quantitative as short-read methods and provided significant improvements in recovery of viral genomes. We then then applied VirION to the first metagenome from a natural viral community from the Western English Channel. In comparison to a short-read only approach, VirION: (i) increased number and completeness of assembled viral genomes; (ii) captured abundant, highly microdiverse virus populations, and (iii) captured more and longer genomic islands. Together, these findings suggest that VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

Whole-Genome Sequences of Phages p000v and p000y, Which Infect the Bacterial Pathogen Shiga-Toxigenic Escherichia coli.

We report here the genome sequences and morphological characterizations of phages p000v and p000y, which infect the bacterial pathogen Shiga-toxigenic Escherichia coli O157:H7 and which are potential candidates for phage therapy against such pathogens.

Fighting Fire with Fire: Phage Potential for the Treatment of E. coli O157 Infection.

Hemolytic⁻uremic syndrome is a life-threating disease most often associated with Shiga toxin-producing microorganisms like Escherichia coli (STEC), including E. coli O157:H7. Shiga toxin is encoded by resident prophages present within this bacterium, and both its production and release depend on the induction of Shiga toxin-encoding prophages. Consequently, treatment of STEC infections tend to be largely supportive rather than antibacterial, in part due to concerns about exacerbating such prophage induction. Here we explore STEC O157:H7 prophage induction in vitro as it pertains to phage therapy-the application of bacteriophages as antibacterial agents to treat bacterial infections-to curtail prophage induction events, while also reducing STEC O157:H7 presence. We observed that cultures treated with strictly lytic phages, despite being lysed, produce substantially fewer Shiga toxin-encoding temperate-phage virions than untreated STEC controls. We therefore suggest that phage therapy could have utility as a prophylactic treatment of individuals suspected of having been recently exposed to STEC, especially if prophage induction and by extension Shiga toxin production is not exacerbated.

Soil Viruses Are Underexplored Players in Ecosystem Carbon Processing.

Rapidly thawing permafrost harbors ∼30 to 50% of global soil carbon, and the fate of this carbon remains unknown. Microorganisms will play a central role in its fate, and their viruses could modulate that impact via induced mortality and metabolic controls. Because of the challenges of recovering viruses from soils, little is known about soil viruses or their role(s) in microbial biogeochemical cycling. Here, we describe 53 viral populations (viral operational taxonomic units [vOTUs]) recovered from seven quantitatively derived (i.e., not multiple-displacement-amplified) viral-particle metagenomes (viromes) along a permafrost thaw gradient at the Stordalen Mire field site in northern Sweden. Only 15% of these vOTUs had genetic similarity to publicly available viruses in the RefSeq database, and ∼30% of the genes could be annotated, supporting the concept of soils as reservoirs of substantial undescribed viral genetic diversity. The vOTUs exhibited distinct ecology, with different distributions along the thaw gradient habitats, and a shift from soil-virus-like assemblages in the dry palsas to aquatic-virus-like assemblages in the inundated fen. Seventeen vOTUs were linked to microbial hosts (in silico), implicating viruses in infecting abundant microbial lineages from Acidobacteria, Verrucomicrobia, and Deltaproteobacteria, including those encoding key biogeochemical functions such as organic matter degradation. Thirty auxiliary metabolic genes (AMGs) were identified and suggested virus-mediated modulation of central carbon metabolism, soil organic matter degradation, polysaccharide binding, and regulation of sporulation. Together, these findings suggest that these soil viruses have distinct ecology, impact host-mediated biogeochemistry, and likely impact ecosystem function in the rapidly changing Arctic. IMPORTANCE This work is part of a 10-year project to examine thawing permafrost peatlands and is the first virome-particle-based approach to characterize viruses in these systems. This method yielded >2-fold-more viral populations (vOTUs) per gigabase of metagenome than vOTUs derived from bulk-soil metagenomes from the same site (J. B. Emerson, S. Roux, J. R. Brum, B. Bolduc, et al., Nat Microbiol 3:870-880, 2018, https://doi.org/10.1038/s41564-018-0190-y). We compared the ecology of the recovered vOTUs along a permafrost thaw gradient and found (i) habitat specificity, (ii) a shift in viral community identity from soil-like to aquatic-like viruses, (iii) infection of dominant microbial hosts, and (iv) carriage of host metabolic genes. These vOTUs can impact ecosystem carbon processing via top-down (inferred from lysing dominant microbial hosts) and bottom-up (inferred from carriage of auxiliary metabolic genes) controls. This work serves as a foundation which future studies can build upon to increase our understanding of the soil virosphere and how viruses affect soil ecosystem services.

Clean Low-Biomass Procedures and Their Application to Ancient Ice Core Microorganisms.

Microorganisms in glacier ice provide tens to hundreds of thousands of years archive for a changing climate and microbial responses to it. Analyzing ancient ice is impeded by technical issues, including limited ice, low biomass, and contamination. While many approaches have been evaluated and advanced to remove contaminants on ice core surfaces, few studies leverage modern sequencing to establish in silico decontamination protocols for glacier ice. Here we sought to apply such "clean" sampling techniques with in silico decontamination approaches used elsewhere to investigate microorganisms archived in ice at ∼41 (D41, ∼20,000 years) and ∼49 m (D49, ∼30,000 years) depth in an ice core (GS3) from the summit of the Guliya ice cap in the northwestern Tibetan Plateau. Four "background" controls were established - a co-processed sterile water artificial ice core, two air samples collected from the ice processing laboratories, and a blank, sterile water sample - and used to assess contaminant microbial diversity and abundances. Amplicon sequencing revealed 29 microbial genera in these controls, but quantitative PCR showed that the controls contained about 50-100-times less 16S DNA than the glacial ice samples. As in prior work, we interpreted these low-abundance taxa in controls as "contaminants" and proportionally removed them in silico from the GS3 ice amplicon data. Because of the low biomass in the controls, we also compared prokaryotic 16S DNA amplicons from pre-amplified (by re-conditioning PCR) and standard amplicon sequencing, and found the resulting microbial profiles to be repeatable and nearly identical. Ecologically, the contaminant-controlled ice microbial profiles revealed significantly different microorganisms across the two depths in the GS3 ice core, which is consistent with changing climate, as reported for other glacier ice samples. Many GS3 ice core genera, including Methylobacterium, Sphingomonas, Flavobacterium, Janthinobacterium, Polaromonas, and Rhodobacter, were also abundant in previously studied ice cores, which suggests wide distribution across glacier environments. Together these findings help further establish "clean" procedures for studying low-biomass ice microbial communities and contribute to a baseline understanding of microorganisms archived in glacier ice.

Multiple mechanisms drive phage infection efficiency in nearly identical hosts.

Phage-host interactions are critical to ecology, evolution, and biotechnology. Central to those is infection efficiency, which remains poorly understood, particularly in nature. Here we apply genome-wide transcriptomics and proteomics to investigate infection efficiency in nature's own experiment: two nearly identical (genetically and physiologically) Bacteroidetes bacterial strains (host18 and host38) that are genetically intractable, but environmentally important, where phage infection efficiency varies. On host18, specialist phage phi18:3 infects efficiently, whereas generalist phi38:1 infects inefficiently. On host38, only phi38:1 infects, and efficiently. Overall, phi18:3 globally repressed host18's transcriptome and proteome, expressed genes that likely evaded host restriction/modification (R/M) defenses and controlled its metabolism, and synchronized phage transcription with translation. In contrast, phi38:1 failed to repress host18's transcriptome and proteome, did not evade host R/M defenses or express genes for metabolism control, did not synchronize transcripts with proteins and its protein abundances were likely targeted by host proteases. However, on host38, phi38:1 globally repressed host transcriptome and proteome, synchronized phage transcription with translation, and infected host38 efficiently. Together these findings reveal multiple infection inefficiencies. While this contrasts the single mechanisms often revealed in laboratory mutant studies, it likely better reflects the phage-host interaction dynamics that occur in nature.

Comparative Omics and Trait Analyses of Marine Pseudoalteromonas Phages Advance the Phage OTU Concept.

Viruses influence the ecology and evolutionary trajectory of microbial communities. Yet our understanding of their roles in ecosystems is limited by the paucity of model systems available for hypothesis generation and testing. Further, virology is limited by the lack of a broadly accepted conceptual framework to classify viral diversity into evolutionary and ecologically cohesive units. Here, we introduce genomes, structural proteomes, and quantitative host range data for eight Pseudoalteromonas phages isolated from Helgoland (North Sea, Germany) and use these data to advance a genome-based viral operational taxonomic unit (OTU) definition. These viruses represent five new genera and inform 498 unaffiliated or unannotated protein clusters (PCs) from global virus metagenomes. In a comparison of previously sequenced Pseudoalteromonas phage isolates (n = 7) and predicted prophages (n = 31), the eight phages are unique. They share a genus with only one other isolate, Pseudoalteromonas podophage RIO-1 (East Sea, South Korea) and two Pseudoalteromonas prophages. Mass-spectrometry of purified viral particles identified 12-20 structural proteins per phage. When combined with 3-D structural predictions, these data led to the functional characterization of five previously unidentified major capsid proteins. Protein functional predictions revealed mechanisms for hijacking host metabolism and resources. Further, they uncovered a hybrid sipho-myovirus that encodes genes for Mu-like infection rarely described in ocean systems. Finally, we used these data to evaluate a recently introduced definition for virus populations that requires members of the same population to have >95% average nucleotide identity across at least 80% of their genes. Using physiological traits and genomics, we proposed a conceptual model for a viral OTU definition that captures evolutionarily cohesive and ecologically distinct units. In this trait-based framework, sensitive hosts are considered viral niches, while host ranges and infection efficiencies are tracked as viral traits. Quantitative host range assays revealed conserved traits within virus OTUs that break down between OTUs, suggesting the defined units capture niche and fitness differentiation. Together these analyses provide a foundation for model system-based hypothesis testing that will improve our understanding of marine copiotrophs, as well as phage-host interactions on the ocean particles and aggregates where Pseudoalteromonas thrive.

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus.

This corrects the article DOI: 10.1038/ismej.2016.81.

Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus.

Bacteria impact humans, industry and nature, but do so under viral constraints. Problematically, knowledge of viral infection efficiencies and outcomes derives from few model systems that over-represent efficient lytic infections and under-represent virus-host natural diversity. Here we sought to understand infection efficiency regulation in an emerging environmental Bacteroidetes-virus model system with markedly different outcomes on two genetically and physiologically nearly identical host strains. For this, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout both infections. While phage transcriptomes were similar, transcriptional differences between hosts suggested host-derived regulation of infection efficiency. Specifically, the alternative host overexpressed DNA degradation genes and underexpressed translation genes, which seemingly targeted phage DNA particle production, as experiments revealed they were both significantly delayed (by >30 min) and reduced (by >50%) in the inefficient infection. This suggests phage failure to repress early alternative host expression and stress response allowed the host to respond against infection by delaying phage DNA replication and protein translation. Given that this phage type is ubiquitous and abundant in the global oceans and that variable viral infection efficiencies are central to dynamic ecosystems, these data provide a critically needed foundation for understanding and modeling viral infections in nature.

Towards quantitative viromics for both double-stranded and single-stranded DNA viruses.

BACKGROUND: Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). METHODS: Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. RESULTS: Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. DISCUSSION: Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

Ecogenomics and potential biogeochemical impacts of globally abundant ocean viruses.

Ocean microbes drive biogeochemical cycling on a global scale. However, this cycling is constrained by viruses that affect community composition, metabolic activity, and evolutionary trajectories. Owing to challenges with the sampling and cultivation of viruses, genome-level viral diversity remains poorly described and grossly understudied, with less than 1% of observed surface-ocean viruses known. Here we assemble complete genomes and large genomic fragments from both surface- and deep-ocean viruses sampled during the Tara Oceans and Malaspina research expeditions, and analyse the resulting 'global ocean virome' dataset to present a global map of abundant, double-stranded DNA viruses complete with genomic and ecological contexts. A total of 15,222 epipelagic and mesopelagic viral populations were identified, comprising 867 viral clusters (defined as approximately genus-level groups). This roughly triples the number of known ocean viral populations and doubles the number of candidate bacterial and archaeal virus genera, providing a near-complete sampling of epipelagic communities at both the population and viral-cluster level. We found that 38 of the 867 viral clusters were locally or globally abundant, together accounting for nearly half of the viral populations in any global ocean virome sample. While two-thirds of these clusters represent newly described viruses lacking any cultivated representative, most could be computationally linked to dominant, ecologically relevant microbial hosts. Moreover, we identified 243 viral-encoded auxiliary metabolic genes, of which only 95 were previously known. Deeper analyses of four of these auxiliary metabolic genes (dsrC, soxYZ, P-II (also known as glnB) and amoC) revealed that abundant viruses may directly manipulate sulfur and nitrogen cycling throughout the epipelagic ocean. This viral catalog and functional analyses provide a necessary foundation for the meaningful integration of viruses into ecosystem models where they act as key players in nutrient cycling and trophic networks.

Optimization of viral resuspension methods for carbon-rich soils along a permafrost thaw gradient.

Permafrost stores approximately 50% of global soil carbon (C) in a frozen form; it is thawing rapidly under climate change, and little is known about viral communities in these soils or their roles in C cycling. In permafrost soils, microorganisms contribute significantly to C cycling, and characterizing them has recently been shown to improve prediction of ecosystem function. In other ecosystems, viruses have broad ecosystem and community impacts ranging from host cell mortality and organic matter cycling to horizontal gene transfer and reprogramming of core microbial metabolisms. Here we developed an optimized protocol to extract viruses from three types of high organic-matter peatland soils across a permafrost thaw gradient (palsa, moss-dominated bog, and sedge-dominated fen). Three separate experiments were used to evaluate the impact of chemical buffers, physical dispersion, storage conditions, and concentration and purification methods on viral yields. The most successful protocol, amended potassium citrate buffer with bead-beating or vortexing and BSA, yielded on average as much as 2-fold more virus-like particles (VLPs) g(-1) of soil than other methods tested. All method combinations yielded VLPs g(-1) of soil on the 10(8) order of magnitude across all three soil types. The different storage and concentration methods did not yield significantly more VLPs g(-1) of soil among the soil types. This research provides much-needed guidelines for resuspending viruses from soils, specifically carbon-rich soils, paving the way for incorporating viruses into soil ecology studies.

Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems.

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. Here, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.