AbstractMarine 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 culturing of important hosts and the lack of a ‘universal’ gene 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. However, short-read 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. Assembly across genomic islands which likely contain niche-defining genes that drive ecological speciation is also challenging. While such populations and features are successfully captured by single-virus genomics and fosmid-based approaches, both techniques require considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative method for improving assembly of viral metagenomics using long read technology. Named ‘VirION’ (Viral, long-read metagenomics via MinION sequencing), our sequencing approach and complementary bioinformatics pipeline (i) increased number and completeness of assembled viral genomes compared to short-read sequencing methods; (ii) captured populations of abundant viruses with high microdiversity missed by short-read methods and (iii) captured more and longer genomic islands than short-read methods. Thus, VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

Microbes drive most ecosystems and are modulated by viruses that impact their lifespan, gene flow and metabolic outputs. However, the influence of viral community diversity
at the ecosystem level remains difficult to assess due to classification issues and few reference genomes. Here we establish a ~12-fold expanded global ocean virome dataset of 195,728 viral populations, now including the Arctic Ocean, and validate that these populations form discrete genotypic clusters. Meta-community analyses revealed just five ecological zones throughout the global ocean, and established local and global patterns and drivers in viral community diversity at levels of both macrodiversity (inter-population diversity) and microdiversity (intra-population genetic variation). These patterns sometimes, but not always, paralleled those from macro- organisms and revealed temperate and tropical surface waters and the Arctic as biodiversity hotspots and mechanistic hypotheses to explain them. With this further understanding of viral
populations and ecology in the ocean, viruses can be more broadly included in ecosystem models.

Even before the coining of the term “great plate count anomaly” in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the “most wanted” bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress toward answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.

Marine regions that have seasonal to long-term low dissolved oxygen (DO) concentrations, sometimes called "dead zones," are increasing in number and severity around the globe with deleterious effects on ecology and economics. One of the largest of these coastal dead zones occurs on the continental shelf of the northern Gulf of Mexico (nGOM), which results from eutrophication-enhanced bacterioplankton respiration and strong seasonal stratification. Previous research in this dead zone revealed the presence of multiple cosmopolitan bacterioplankton lineages that have eluded cultivation, and thus their metabolic roles in this ecosystem remain unknown. We used a coupled shotgun metagenomic and metatranscriptomic approach to determine the metabolic potential of Marine Group II Euryarchaeota, SAR406, and SAR202. We recovered multiple high-quality, nearly complete genomes from all three groups as well as candidate phyla usually associated with anoxic environments-Parcubacteria (OD1) and Peregrinibacteria Two additional groups with putative assignments to ACD39 and PAUC34f supplement the metabolic contributions by uncultivated taxa. Our results indicate active metabolism in all groups, including prevalent aerobic respiration, with concurrent expression of genes for nitrate reduction in SAR406 and SAR202, and dissimilatory nitrite reduction to ammonia and sulfur reduction by SAR406. We also report a variety of active heterotrophic carbon processing mechanisms, including degradation of complex carbohydrate compounds by SAR406, SAR202, ACD39, and PAUC34f. Together, these data help constrain the metabolic contributions from uncultivated groups in the nGOM during periods of low DO and suggest roles for these organisms in the breakdown of complex organic matter.IMPORTANCE Dead zones receive their name primarily from the reduction of eukaryotic macrobiota (demersal fish, shrimp, etc.) that are also key coastal fisheries. Excess nutrients contributed from anthropogenic activity such as fertilizer runoff result in algal blooms and therefore ample new carbon for aerobic microbial metabolism. Combined with strong stratification, microbial respiration reduces oxygen in shelf bottom waters to levels unfit for many animals (termed hypoxia). The nGOM shelf remains one of the largest eutrophication-driven hypoxic zones in the world, yet despite its potential as a model study system, the microbial metabolisms underlying and resulting from this phenomenon-many of which occur in bacterioplankton from poorly understood lineages-have received only preliminary study. Our work details the metabolic potential and gene expression activity for uncultivated lineages across several low DO sites in the nGOM, improving our understanding of the active biogeochemical cycling mediated by these "microbial dark matter" taxa during hypoxia.

Western Antarctica, one of the fastest warming locations on Earth, is a unique environment that is underexplored with regards to biodiversity. Although pelagic microbial communities in the Southern Ocean and coastal Antarctic waters have been well studied, there are fewer investigations of benthic communities and most have a focused geographic range. We sampled surface sediment from 24 sites across a 5,500 km region of Western Antarctica (covering the Ross Sea to the Weddell Sea) to examine relationships between microbial communities and sediment geochemistry. Sequencing of the 16S and 18S rRNA genes showed microbial communities in sediments from the Antarctic Peninsula (AP) and Western Antarctica (WA), including the Ross, Amundsen, and Bellingshausen Seas, could be distinguished by correlations with organic matter concentrations and stable isotope fractionation (total organic carbon; TOC, nitrogen, and δ13C). Overall, samples from the AP were higher in nutrient content (TOC, nitrogen, and NH4+) and communities in these samples had higher relative abundances of operational taxonomic units (OTUs) classified as the diatom, Chaetoceros, a marine cercozoan and four OTUs classified as Cytophaga or Flavobacteria. As these OTUs were strongly correlated with TOC, the data suggests the diatoms could be a source of organic matter and the Bacteroidetes and cercozoan are grazers that consume the organic matter. Additionally, samples from WA have lower nutrients and were dominated by Thaumarchaeota, which could be related to their known ability to thrive as lithotrophs. This study documents the largest analysis of benthic microbial communities to date in the Southern Ocean, representing almost half the continental shoreline of Antarctica, and documents trophic interactions and coupling of pelagic and benthic communities. Our results indicate potential modifications in carbon sequestration processes related to change in community composition, identifying a prospective mechanism that links climate change to carbon availability.

AbstractMicrobes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus-host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated one new member of the globally dominant bacterial SAR11 clade and three new members of the methylotrophic bacterial clade OM43. We used these as bait to isolate 117 new phages including the first known siphophage infecting SAR11, and the first isolated phage for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that the viruses infecting SAR11 have ecotype-specific host-ranges. Similar to the abundant human-associated phage ΦCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny or chronic infection, despite a lack of associated genes; or host phenotypic bistability with lysis putatively maintained within a susceptible subpopulation. Broader representation of important virus-host systems in culture collections and genomic databases will improve both our understanding of virus-host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.

ABSTRACTThe methylotrophic OM43 clade are Gammaproteobacteria that comprise some of the smallest free-living cells known and have highly streamlined genomes. OM43 represents an important microbial link 0between marine primary production and remineralisation of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of microbial mortality and global marine biogeochemistry. Recent cultivation efforts have brought the first viruses infecting members of the OM43 clade into culture. Here we characterize a novel myophage infecting OM43 called Melnitz. Melnitz was isolated independently on three separate occasions (with isolates sharing >99.95% average nucleotide identity) from water samples from a subtropical ocean gyre (Sargasso Sea) and temperate coastal (Western English Channel) systems. Metagenomic recruitment from global ocean viromes confirmed that Melnitz is globally ubiquitous, congruent with patterns of host abundance. Bacteria with streamlined genomes such as OM43 and the globally dominant SAR11 clade use riboswitches as an efficient method to regulate metabolism. Melnitz encodes a two-piece tmRNA (ssrA), controlled by a glutamine riboswitch, providing evidence that riboswitch use also occurs for regulation during phage infection of streamlined heterotrophs. Virally encoded tRNAs and ssrA found in Melnitz were phylogenetically more closely related to those found within the alphaproteobacterial SAR11 clade and their associated myophages than those within their gammaproteobacterial hosts. This suggests the possibility of an ancestral inter-class host transition event between SAR11 and OM43. Melnitz and a related myophage that infects SAR11 were unable to infect hosts of the SAR11 and OM43, respectively, suggesting host transition rather than a broadening of host range.IMPORTANCEIsolation and cultivation of viruses is the foundation on which the mechanistic understanding of virus-host interactions and ground-truthing is based. This study isolated and characterised the first myophage known to infect the OM43 clade, expanding our knowledge of this understudied group of microbes. The near-identical genomes of four strains of Melnitz isolated from different marine provinces and global abundance estimations from metagenomic data suggest that this viral population is globally ubiquitous. Genome analysis revealed several unusual features in Melnitz and related genomes recovered from viromes, such as a curli operon and virally encoded tmRNA controlled by a glutamine riboswitch, neither of which are found in the host. Further phylogenetic analysis of shared genes indicates that this group of viruses infecting the gammaproteobacterial OM43 shares a recent common ancestor with viruses infecting the abundant alphaproteobacterial SAR11 clade. Host ranges are affected by compatible cell surface receptors, successful circumvention of superinfection exclusion systems and the presence of required accessory proteins, which typically limits phages to singular narrow groups of closely related bacterial hosts. This study provides intriguing evidence that for streamlined heterotrophic bacteria, virus-host transitioning is not necessarily restricted to phylogenetically related hosts, but is a function of shared physical and biochemical properties of the cell.

AbstractMarine 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 culturing of important hosts and the lack of a ‘universal’ gene 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. However, short-read 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. Assembly across genomic islands which likely contain niche-defining genes that drive ecological speciation is also challenging. While such populations and features are successfully captured by single-virus genomics and fosmid-based approaches, both techniques require considerable cost and technical expertise. Here we established a low-cost, low-input, high throughput alternative method for improving assembly of viral metagenomics using long read technology. Named ‘VirION’ (Viral, long-read metagenomics via MinION sequencing), our sequencing approach and complementary bioinformatics pipeline (i) increased number and completeness of assembled viral genomes compared to short-read sequencing methods; (ii) captured populations of abundant viruses with high microdiversity missed by short-read methods and (iii) captured more and longer genomic islands than short-read methods. Thus, VirION provides a high throughput and cost-effective alternative to fosmid and single-virus genomic approaches to more comprehensively explore viral communities in nature.

Microbes drive most ecosystems and are modulated by viruses that impact their lifespan, gene flow and metabolic outputs. However, the influence of viral community diversity
at the ecosystem level remains difficult to assess due to classification issues and few reference genomes. Here we establish a ~12-fold expanded global ocean virome dataset of 195,728 viral populations, now including the Arctic Ocean, and validate that these populations form discrete genotypic clusters. Meta-community analyses revealed just five ecological zones throughout the global ocean, and established local and global patterns and drivers in viral community diversity at levels of both macrodiversity (inter-population diversity) and microdiversity (intra-population genetic variation). These patterns sometimes, but not always, paralleled those from macro- organisms and revealed temperate and tropical surface waters and the Arctic as biodiversity hotspots and mechanistic hypotheses to explain them. With this further understanding of viral
populations and ecology in the ocean, viruses can be more broadly included in ecosystem models.

AbstractMarine regions that have seasonal to long-term low dissolved oxygen (DO) concentrations, sometimes called ‘dead zones,’ are increasing in number and severity around the globe with deleterious effects on ecology and economics. One of the largest of these coastal dead zones occurs on the continental shelf of the northern Gulf of Mexico (nGOM), which results from eutrophication-enhanced bacterioplankton respiration and strong seasonal stratification. Previous research in this dead zone revealed the presence of multiple cosmopolitan bacterioplankton lineages that have eluded cultivation, and thus their metabolic roles in this ecosystem remain unknown. We used a coupled shotgun metagenomic and metatranscriptomic approach to determine the metabolic potential of Marine Group II Euryarchaeota, SAR406, and SAR202. We recovered multiple high-quality, nearly complete genomes from all three groups as well as those belonging to Candidate Phyla usually associated with anoxic environments-Parcubacteria (OD1) and Peregrinibacteria. Two additional groups with putative assignments to ACD39 and PAUC34f supplement the metabolic contributions by uncultivated taxa. Our results indicate active metabolism in all groups, including prevalent aerobic respiration, with concurrent expression of genes for nitrate reduction in SAR406 and SAR202, and dissimilatory nitrite reduction to ammonia and sulfur reduction by SAR406. We also report a variety of active heterotrophic carbon processing mechanisms, including degradation of complex carbohydrate compounds by SAR406, SAR202, ACD39, and PAUC34f. Together, these data help constrain the metabolic contributions from uncultivated groups in the nGOM during periods of low DO and suggest roles for these organisms in the breakdown of complex organic matter.ImportanceDead zones receive their name primarily from the reduction of eukaryotic macrobiota (demersal fish, shrimp, etc.) that are also key coastal fisheries. Excess nutrients contributed from anthropogenic activity such as fertilizer runoff result in algal blooms and therefore ample new carbon for aerobic microbial metabolism. Combined with strong stratification, microbial respiration reduces oxygen in shelf bottom waters to levels unfit for many animals (termed hypoxia). The nGOM shelf remains one of the largest eutrophication-driven hypoxic zones in the world, yet despite its potential as a model study system, the microbial metabolisms underlying and resulting from this phenomenon—many of which occur in bacterioplankton from poorly understood lineages—have received only preliminary study. Our work details the metabolic potential and gene expression activity for uncultivated lineages across several low DO sites in the nGOM, improving our understanding of the active biogeochemical cycling mediated by these “microbial dark matter” taxa during hypoxia.

ABSTRACTMicrobes 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, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow (updated at protocols.io) 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. Taken together, the addition of long-reads uniquely enabled the recovery of 22% of the most abundant viruses—that would have been missed in short-read only assemblies.

AbstractBackgroundWe have successfully used whole-genome sequencing to provide additional information for transmission pathways in infectious spread. We report and interpret genomic sequencing results in clinical context from a large outbreak of COVID-19 with 46 cases across staff and patients in a community hospital in the UK.MethodsFollowing multiple symptomatic cases within a two-week period, all staff and patients were screened by RT-PCR and staff subsequently had serology tests.ResultsThirty staff (25%) and 16 patients (62%) tested positive for COVID-19. Genomic sequencing data showed significant overlap of viral haplotypes in staff who had overlapping shift patterns. Patient haplotypes were more distinct from each other but had overlap with staff haplotypes.ConclusionsThis study includes clinical and genomic epidemiological detail that demonstrates the value of a combined approach. Viral genetic sequencing has identified that staff transmission of COVID-19 was important in this community hospital outbreak.Key pointsDetailed analysis of a large community hospital outbreak in older adults and staff with concurrent clinical and genomic data, including working patterns.Staff transmission was important in this community hospital outbreak.We found plausible associations between staff and patient cases.

70% of the world’s surface is covered by oceans; its impact on the global carbon cycle, climate change, and acid-base biochemistry remain crucial to our understanding of the natural world. The oceans act as important buffers against climate change, absorbing 25% of anthropogenic carbon and over 90% of rising temperatures. 90% of the ocean’s biomass is composed of marine microorganisms and their impact on global systems, particularly in the face of anthropogenic climate change, remains an active area of research. Marine microorganisms are critical in the energy cycle and are the foundation for marine life. Warmer waters have led to increasingly stratified and nutrient-depleted water masses at the ocean surface, favouring low-nutrient microbial specialists. One group of these, known as the SAR11 clade, comprise up to 40% of the microbial community and are estimated to convert up to 20% of all global primary production back to atmospheric CO2 as well as being an important biological source of methane. Increasing SAR11 abundance in warming oceans and concomitant increases in remineralisation of CO 2 and methane may create a positive feedback loop for global warming.

A potential brake on the influence of SAR11 carbon remineralisation is their associated viruses, which are predicted to lyse up to 20% of cellular biomass daily. These viruses also encode an enormous array of genetic diversity and its relationship with both physical and biological factors is key to understanding the marine biome’s population dynamics. Predation of cells by viruses is a major driver of carbon export to the deep ocean, but our knowledge of these interactions in the SAR11 clade is limited, in part due to the paucity of host-virus model systems for this clade.

However, studying these microorganisms remains challenging since only a few SAR11 strains have been isolated and cultured for in vitro experimentation. Alternative study methods include obtaining genomes via metagenomics studies and Single-cell Amplified Genomes (SAGs). Therefore, the goal of this project is to extract and explore SAR11 host and associated phage genomes from metagenomic and SAG data. Here, I present a study of 451 SAGs collected from the Tara Ocean expeditions and twelve prokaryotic metagenomic samples from the Bermuda Atlantic Time Series (BATS).

Overall, I summarise the difficulty of obtaining contiguous and high-quality SAR11 genomes from metagenomic data. I conclude possible reasons why existing bioinformatics tools are ineffective at recovering such sequences and suggest improvements through long-read technology. Through SAG data, I identified and evaluated genomic regions associated with phage defence to improve our understanding of SAR11-associated viral dynamics in the oceans. Additionally, I characterised two previously undescribed clades of SAR11, both phylogenetically and ecologically. Our 451 SAGS contained fewer phage sequences than SAGs from other taxa, indicating the SAR11 clade does not conform to the expected statement that 20% of all marine microorganisms are infected at any given time. Lastly, I confirmed that a hypervariable region identified as a putative site for host-virus Red Queen dynamics is present within all clades of SAR11, and concluded these regions are enriched in genes related to cell wall biosynthesis. I hypothesise that these genes are related to phage defence, altering the cell wall receptors and preventing recognition of a host by SAR11 phages, therefore resisting infection. These findings together increase our understanding of additional host-phage interactions SAR11 has and impact current models when calculating SAR11 phage carbon-sequestering via the viral shunt.

AbstractThe evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus leads to new variants that warrant timely epidemiological characterization. Here we use the dense genomic surveillance data generated by the COVID-19 Genomics UK Consortium to reconstruct the dynamics of 71 different lineages in each of 315 English local authorities between September 2020 and June 2021. This analysis reveals a series of subepidemics that peaked in early autumn 2020, followed by a jump in transmissibility of the B.1.1.7/Alpha lineage. The Alpha variant grew when other lineages declined during the second national lockdown and regionally tiered restrictions between November and December 2020. A third more stringent national lockdown suppressed the Alpha variant and eliminated nearly all other lineages in early 2021. Yet a series of variants (most of which contained the spike E484K mutation) defied these trends and persisted at moderately increasing proportions. However, by accounting for sustained introductions, we found that the transmissibility of these variants is unlikely to have exceeded the transmissibility of the Alpha variant. Finally, B.1.617.2/Delta was repeatedly introduced in England and grew rapidly in early summer 2021, constituting approximately 98% of sampled SARS-CoV-2 genomes on 26 June 2021.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

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 shortand 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 longand 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.

Aquaculture is of major and increasing importance to global food security, particularly in Low Income, Food Deficit Countries (LIFDCs), where it also serves as a significant contribution to poverty alleviation. Disease is widely acknowledged as the prominent bottleneck to achieving global food security and poverty alleviation targets relating to aquaculture, with annual losses exceeding >$6bn (Food and Agriculture Organization 2014). Outbreaks of disease caused by endemic and emerging pathogens impact directly on farmer income and their nutritional security. Avoidance of yield-limiting disease outbreaks is a fundamental requirement for future growth and resilience of aquaculture in LIFDCs. Advances in molecular techniques coupled with next-generation sequencing have provided a step-change in understanding the role of host-associated bacteria, archaea, protists and viruses (the microbiome) in host homeostasis. Shifts in microbiome communities under stressful conditions can contribute to disease states. However, the role of microbiomes in the emergence of diseases in aquaculture, where stressors include feeding, antibiotic and disinfectant use and over-stocking, is poorly studied.
Here our study presents an evaluation of the microbiomes (bacteria and viruses) associated with tilapia and their pond environments in aquaculture, using 16S rRNA community profiling techniques and viral amplicon sequencing. Samples investigated in this project were collected from Malawi tilapia fish farms; their skin community composition and diversity were examined across geographical scales. The high variability observed of the microbial communities in small geographic regions, showed that future sampling to detect shifts due to dysbiosis will require time-resolved sampling of ponds under study. Nanopore sequencing of full length 16S rRNA genes, using MinION, allowed us to examine the microbial communities at higher taxonomic resolution than short read sequencing techniques. Its success lays the foundation for in-situ microbial profiling of aquaculture ponds for disease, and offers independence to farmers to monitor their own ponds. Successful amplification of the T4-like Myoviridae phylogenetic markers from one rearing water sample was achieved, although the required degeneracy of the primers inhibited multiplexing. Therefore, our findings suggest that inclusion of bacteriophages in microbiome studies is better served using shotgun metagenomic methods, rather than amplicon sequencing.

Finally, we investigated the use of skin swabbing as an alternative to bucket incubations to minimise animal stress when categorising the fish skin microbiome. Skin swabbing successfully captured similar microbial communities in comparison to bucket incubations, with greater diversity and variance between fish.

. Here, we present the complete genome sequence of
. Marinobacterium
. sp. strain LSUCC0821, isolated from the coastal Gulf of Mexico with artificial seawater using high-throughput dilution-to-extinction (DTE) cultivation. The 2.36-Mbp circularized genome sequence has 2,231 predicted genes, a 91.5% coding density, and a GC content of 47.8%.
.

Typically, pathogens infect multiple host species. Such multihost pathogens can show considerable variation in their degree of infection and transmission specificity, which has important implications for potential disease emergence. Transmission of multihost pathogens can be driven by key host species and changes in such transmission networks can lead to disease emergence. We study two viruses that show contrasting patterns of prevalence and specificity in managed honeybees and wild bumblebees, black queen cell virus (BQCV) and slow bee paralysis virus (SBPV), in the context of the novel transmission route provided by the virus-vectoring Varroa destructor. Our key result is that viral communities and RNA virus genetic variation are structured by location, not host species or V. destructor presence. Interspecific transmission is pervasive with the same viral variants circulating between pollinator hosts in each location; yet, we found virus-specific host differences in prevalence and viral load. Importantly, V. destructor presence increases the prevalence in honeybees and, indirectly, in wild bumblebees, but in contrast to its impact on deformed wing virus (DWV), BQCV and SBPV viral loads are not increased by Varroa presence, and do not show genetic evidence of recent emergence. Effective control of Varroa in managed honeybee colonies is necessary to mitigate further disease emergence, and alleviate disease pressure on our vital wild bee populations. More generally, our results highlight the over-riding importance of geographical location to the epidemiological outcome despite the complexity of multihost-parasite interactions.

Even before the coining of the term “great plate count anomaly” in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the “most wanted” bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress toward answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.

With our global population expected to increase to as much as 9.8 billion by 2050, strategies for obtaining worldwide food security become increasingly important. The oceans act as a generous resource for reaching our global nutrition targets, yet overfishing in recent decades has caused great harm, including localised population extinction, to fish and shellfish stocks. Aquaculture, the act of maintaining and farming marine or freshwater animal organisms, has become a popular alternative to wild fisheries. However, with a high demand for food sources, comes a move towards more intensive farming practices, whereby denser communities of farmed animals are kept in waters with high nutrient input. Such farming practices can favour pathogenic bacterial communities, which can cause disease in farmed animals and consequently lead to reduced stock numbers. Not only does this affect yield, but there can be further economic impacts to the detriment of those whose livelihoods depend on the aquaculture sector.

Traditionally, antibiotics have been used with abundance to treat disease in aquaculture, however overuse and misuse has led to a global rise in antibiotic resistance. This is particularly apparent in aquaculture, where antibiotics can be directly applied to organisms and also, easily accumulate within the water column. Therefore, as the global antibiotic crisis worsens, it has become ever more important to develop novel therapeutic alternatives. One such promising alternative is the use of bacteriophages (phages) – viruses which kill bacteria. However, application of phages requires more research to become commercially viable. Encapsulation of such phages may improve their therapeutic use through increased concentrations during application, improved stability and increased protection. Long term storage of encapsulated phages, e.g. after lyophilisation (freeze drying), would facilitate development of a robust phage library and enable rapid construction of bespoke phage cocktails, whereby distinct phages are combined to combat bacterial resistance. Droplet microfluidics is an emerging field, which can be used for the high-throughput encapsulation of bacteriophages, for use against bacterial infections, not only in aquaculture, but also in clinical settings.

Vibrio parahaemolyticus is a highly pathogenic bacterium, capable of infecting shellfish and subsequently cause gastroenteritis in humans. V. parahaemolyticus is commonly isolated from oysters, for example Crassostrea gigas, which is the most commonly farmed species of oyster in the UK. and there is increasing evidence of antibiotic resistance in V. parahaemolyticus.

This project aimed to evaluate the use of droplet microfluidics and subsequent freeze-drying in order to encapsulate bacteriophages specific for V. parahaemolyticus, a highly pathogenic bacterium, capable of infecting shellfish and subsequently cause gastroenteritis in humans. V. parahaemolyticus is commonly isolated from oysters, for example Crassostrea gigas, which is the most commonly farmed species of oyster in the UK and furthermore, there is increasing evidence of antibiotic resistance in V. parahaemolyticus. In order to develop of an encapsulated viral library for phage therapy of V. parahaemolyticus, the following four challenges needed to be addressed: (1) isolate novel vibriophages specific for V. parahaemolyticus, (2) develop a novel protocol for the synthesis of monodisperse sodium alginate microcapsules, using microfluidics, (3) encapsulate vibriophages in alginate droplets and (4) use such encapsulated phages to treat V. parahaemolyticus infection of C. gigas.

The genomes of four strains of V. parahaemolyticus (EXE V18/004, V12/024, V05/313 and V05/027) were sequenced. In total, 10 dsDNA high quality (category 5) prophage and 5 Inoviruses were detected and manually curated across the four genomes. Furthermore, in this instance the isolation of novel vibriophages was unsuccessful. Despite this, a novel protocol was developed for the synthesis of monodisperse alginate droplets, using a glass microfluidic device. Droplets were synthesised with an alginate concentration of 1 % (w/w) and collected in calcium chloride (CaCl2) solution with a concentration of 2 % (w/w). Bacteriophage T4, vibriophage sm030 and vibriophage sm031 were successfully encapsulated within alginate microcapsules and later lyophilised. Lyophilised droplets containing bacteriophage T4, vibriophage sm030 or vibriophage sm031 were able to cause infection and reduce cell growth in broth cultures of Escherichia coli and V. parahaemolyticus, respectively. More research is needed into phage encapsulation in bacteriophage therapy before its widespread use.

In many regions of the world oceans, phytoplankton face the problem of discriminating between phosphate, an essential nutrient, and arsenate, a toxic analogue. Many phytoplankton, including the most abundant phytoplankton group known, Prochlorococcus, detoxify arsenate (AsV) by reduction to arsenite (AsIII), followed by methylation and excretion of the methylated arsenic products. We synthesized [14C]dimethyl arsenate (DMA) and used it to show that cultured Pelagibacter strain HTCC7211 (SAR11) cells oxidize the methyl group carbons of DMA, producing 14CO2 and ATP. We measured [14C]DMA oxidation rates in the P-depleted surface waters of the Sargasso Sea, a subtropical ocean gyre. [14C]DMA was oxidized to 14CO2 by Sargasso Sea plankton communities at a rate that would cause turnover of the estimated DMA standing stock every 8.1 days. SAR11 strain HTCC7211, which was isolated from the Sargasso Sea, has a pair of arsenate resistance genes and was resistant to arsenate, showing no growth inhibition at As/P ratios of >65:1. Across the global oceans, there was a strong inverse relationship between the frequency of the arsenate reductase (LMWPc_ArsC) in Pelagibacter genomes and phosphate concentrations. We propose that the demethylation of methylated arsenic compounds by Pelagibacter and possibly other bacterioplankton, coupled with arsenate resistance, results in the transfer of energy from phytoplankton to bacteria. We dub this a parasitic cycle because the release of arsenate by Pelagibacter in principle creates a positive-feedback loop that forces phytoplankton to continually regenerate arsenate detoxification products, producing a flow of energy to P-limited ocean regions.IMPORTANCE in vast, warm regions of the oceans, phytoplankton face the problem of arsenic poisoning. Arsenate is toxic because it is chemically similar to phosphate, a scarce nutrient that phytoplankton cells need for growth. Many phytoplankton, including the commonest phytoplankton type in warm oceans, Prochlorococcus, detoxify arsenate by adding methyl groups. Here we show that the most abundant non-photosynthetic plankton in the oceans, SAR11 bacteria, remove the methyl groups, releasing poisonous forms of arsenic back into the water. We postulate that the methylation and demethylation of arsenic compounds creates a cycle in which the phytoplankton can never get ahead and must continually transfer energy to the SAR11 bacteria. We dub this a parasitic process and suggest that it might help explain why SAR11 bacteria are so successful, surpassing all other plankton in their numbers. Field experiments were done in the Sargasso Sea, a subtropical ocean gyre that is sometimes called an ocean desert because, throughout much of the year, there is not enough phosphorous in the water to support large blooms of phytoplankton. Ocean deserts are expanding as the oceans absorb heat and grow warmer.

The phylum Caldiserica was identified from the hot spring 16S rRNA gene lineage ‘OP5’ and named for the sole isolate Caldisericum exile, a hot spring sulfur-reducing chemoheterotroph. Here we characterize 7 Caldiserica metagenome-assembled genomes (MAGs) from a thawing permafrost site in Stordalen Mire, Arctic Sweden. By 16S rRNA and marker gene phylogenies, and average nucleotide and amino acid identities, these Stordalen Mire Caldiserica (SMC) MAGs form part of a divergent clade from C. exile. Genome and meta-transcriptome and proteome analyses suggest that unlike Caldisericum, the SMCs (i) are carbohydrate- and possibly amino acid fermenters that can use labile plant compounds and peptides, and (ii) encode adaptations to low temperature. The SMC clade rose to community dominance within permafrost, with a peak metagenome-based relative abundance of ∼60%. It was also physiologically active in the upper seasonally-thawed soil. Beyond Stordalen Mire, analysis of 16S rRNA gene surveys indicated a global distribution of this clade, predominantly in anaerobic, carbon-rich and cold environments. These findings establish the SMCs as four novel phenotypically and ecologically distinct species within a single novel genus, distinct from C. exile clade at the phylum level. The SMCs are thus part of a novel cold-habitat phylum for an understudied, globally-distributed superphylum encompassing the Caldiserica. We propose the names Candidatus Cryosericota phylum nov. Ca. Cryosericia class nov. Ca. Cryosericales ord. nov. Ca. Cryosericaceae fam. nov. Ca. Cryosericum gen. nov. Ca. Cryosericum septentrionale sp. nov. Ca. C. hinesii sp. nov. Ca. C. odellii sp. nov. and Ca. C. terrychapinii sp. nov.

Novel transmission routes can directly impact the evolutionary ecology of infectious diseases, with potentially dramatic effect on host populations and knock-on effects on the wider host community. The invasion of Varroa destructor, an ectoparasitic viral vector in Western honeybees, provides a unique opportunity to examine how a novel vector affects disease epidemiology in a host community. This specialist honeybee mite vectors deformed wing virus (DWV), an important re-emerging honeybee pathogen that also infects wild bumblebees. Comparing island honeybee and wild bumblebee populations with and without V. destructor, we show that V. destructor drives DWV prevalence and titre in honeybees and sympatric bumblebees. Viral genotypes are shared across hosts, with the potentially more virulent DWV-B overtaking DWV-A in prevalence in a current epidemic. This demonstrates disease emergence across a host community driven by the acquisition of a specialist novel transmission route in one host, with dramatic community level knock-on effects.

We 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.

The bio-aerosol is an important medium for the potential dispersal of biological warfare agents within the battlefield space. In order to better protect the military personnel who work within this environment it is imperative that we increase our understanding of this matrix, especially the naturally occurring variation and its causes. Understanding the naturally occurring variation within the bio-aerosol will enable future and current biological detection platforms to be put through better test and evaluation processes, thus reducing the potential for false alarms and false negatives. Analysing bio-aerosol samples collected across a temporal gradient through a metagenomics approach will enable the natural variation to be better understood. However, metagenomic analysis tools have been shown to have contradictory reviews within the literature, it is therefore essential to identify the most suitable analysis approach.

Here I developed a metagenomic analysis pipeline which delivers high confidence taxonomic identification to species level, as well as accurate measures of diversity and homogeneity. The analysis pipeline that was developed takes the output from multiple tools thus reducing the number of false positives, delivering high confidence taxonomic identification. The analysis pipeline also gives a more accurate measure of diversity and homogeneity compared to any of the tools being used individually. This improved accuracy will deliver superior results when measuring the change in abundance of species identified within the bio-aerosol in sampling regimes carried out at Dstl. These improvements will lead to more accurate test bio-aerosols being developed for biological detection platform evaluation. Fundamentally this will improve the UK military’s capability to detect biological warfare releases within the battlespace.

Coastal regions experiencing declining dissolved oxygen are increasing in number and severity around the world. However, despite the importance of microbial metabolism in coastal hypoxia, few metagenomic surveys exist. Our data set from within the second largest human-caused hypoxic region provides opportunities to more deeply explore the microbiology of these systems.

Marine regions that have seasonal to long-term low dissolved oxygen (DO) concentrations, sometimes called "dead zones," are increasing in number and severity around the globe with deleterious effects on ecology and economics. One of the largest of these coastal dead zones occurs on the continental shelf of the northern Gulf of Mexico (nGOM), which results from eutrophication-enhanced bacterioplankton respiration and strong seasonal stratification. Previous research in this dead zone revealed the presence of multiple cosmopolitan bacterioplankton lineages that have eluded cultivation, and thus their metabolic roles in this ecosystem remain unknown. We used a coupled shotgun metagenomic and metatranscriptomic approach to determine the metabolic potential of Marine Group II Euryarchaeota, SAR406, and SAR202. We recovered multiple high-quality, nearly complete genomes from all three groups as well as candidate phyla usually associated with anoxic environments-Parcubacteria (OD1) and Peregrinibacteria Two additional groups with putative assignments to ACD39 and PAUC34f supplement the metabolic contributions by uncultivated taxa. Our results indicate active metabolism in all groups, including prevalent aerobic respiration, with concurrent expression of genes for nitrate reduction in SAR406 and SAR202, and dissimilatory nitrite reduction to ammonia and sulfur reduction by SAR406. We also report a variety of active heterotrophic carbon processing mechanisms, including degradation of complex carbohydrate compounds by SAR406, SAR202, ACD39, and PAUC34f. Together, these data help constrain the metabolic contributions from uncultivated groups in the nGOM during periods of low DO and suggest roles for these organisms in the breakdown of complex organic matter.IMPORTANCE Dead zones receive their name primarily from the reduction of eukaryotic macrobiota (demersal fish, shrimp, etc.) that are also key coastal fisheries. Excess nutrients contributed from anthropogenic activity such as fertilizer runoff result in algal blooms and therefore ample new carbon for aerobic microbial metabolism. Combined with strong stratification, microbial respiration reduces oxygen in shelf bottom waters to levels unfit for many animals (termed hypoxia). The nGOM shelf remains one of the largest eutrophication-driven hypoxic zones in the world, yet despite its potential as a model study system, the microbial metabolisms underlying and resulting from this phenomenon-many of which occur in bacterioplankton from poorly understood lineages-have received only preliminary study. Our work details the metabolic potential and gene expression activity for uncultivated lineages across several low DO sites in the nGOM, improving our understanding of the active biogeochemical cycling mediated by these "microbial dark matter" taxa during hypoxia.

Western Antarctica, one of the fastest warming locations on Earth, is a unique environment that is underexplored with regards to biodiversity. Although pelagic microbial communities in the Southern Ocean and coastal Antarctic waters have been well studied, there are fewer investigations of benthic communities and most have a focused geographic range. We sampled surface sediment from 24 sites across a 5,500 km region of Western Antarctica (covering the Ross Sea to the Weddell Sea) to examine relationships between microbial communities and sediment geochemistry. Sequencing of the 16S and 18S rRNA genes showed microbial communities in sediments from the Antarctic Peninsula (AP) and Western Antarctica (WA), including the Ross, Amundsen, and Bellingshausen Seas, could be distinguished by correlations with organic matter concentrations and stable isotope fractionation (total organic carbon; TOC, nitrogen, and δ13C). Overall, samples from the AP were higher in nutrient content (TOC, nitrogen, and NH4+) and communities in these samples had higher relative abundances of operational taxonomic units (OTUs) classified as the diatom, Chaetoceros, a marine cercozoan and four OTUs classified as Cytophaga or Flavobacteria. As these OTUs were strongly correlated with TOC, the data suggests the diatoms could be a source of organic matter and the Bacteroidetes and cercozoan are grazers that consume the organic matter. Additionally, samples from WA have lower nutrients and were dominated by Thaumarchaeota, which could be related to their known ability to thrive as lithotrophs. This study documents the largest analysis of benthic microbial communities to date in the Southern Ocean, representing almost half the continental shoreline of Antarctica, and documents trophic interactions and coupling of pelagic and benthic communities. Our results indicate potential modifications in carbon sequestration processes related to change in community composition, identifying a prospective mechanism that links climate change to carbon availability.

Abstract We report production of chlorophyll f and chlorophyll d in the cyanobacterium Chlorogloeopsis fritschii cultured under near-infrared and natural light conditions. C. fritschii produced chlorophyll f and chlorophyll d when cultured under natural light to a high culture density in a 20 L bubble column photobioreactor. In the laboratory, the ratio of chlorophyll f to chlorophyll a changed from 1:15 under near-infrared, to an undetectable level of chlorophyll f under artificial white light. The results provide support that chlorophylls f and d are both red-light inducible chlorophylls in C. fritschii.

Metagenomics has evolved over the last 3 decades from the analysis of single genes and their apparent diversity in an ecosystem to the provision of complex genetic information relating to whole ecosystems. Metagenomics is a vast subject area in terms of methodology, which encompasses a suite of molecular technologies employed to investigate genomic information from all members of a microbial community. However, the relatively recent developments in high-throughput sequencing platforms have meant that metagenomic can be performed simply by extracting DNA and sequencing it. Here, we outline explicit methodologies for the extraction of metagenomic DNA from marine and sediments/soil environmental samples, the subsequent production and sequencing of large-insert metagenomic libraries, and also shotgun pyrosequencing considerations. We also provide relevant advice on bioinformatic analyses of the complex metagenomic datasets. We hope that the information provided here will be useful to establish the techniques in most reasonably equipped molecular biology laboratories.

Vibrio tubiashii NCIMB 1337 is a major and increasingly prevalent pathogen of bivalve mol-lusks, and shares a close phylogenetic relationship with both V. orientalis and V. coralliilyti-cus. It is a Gram-negative, curved rod-shaped bacterium, originally isolated from a moribund juvenile oyster, and is both oxidase and catalase positive. It is capable of growth under both aerobic and anaerobic conditions. Here we describe the features of this organism, together with the draft genome and annotation. The genome is 5,353,266 bp long, consisting of two chromosomes, and contains 4,864 protein-coding and 86 RNA genes.