Long-read powered viral metagenomics in the oligotrophic Sargasso Sea.

Dominant microorganisms of the Sargasso Sea are key drivers of the global carbon cycle. However, associated viruses that shape microbial community structure and function are not well characterised. Here, we combined short and long read sequencing to survey Sargasso Sea phage communities in virus- and cellular fractions at viral maximum (80 m) and mesopelagic (200 m) depths. We identified 2,301 Sargasso Sea phage populations from 186 genera. Over half of the phage populations identified here lacked representation in global ocean viral metagenomes, whilst 177 of the 186 identified genera lacked representation in genomic databases of phage isolates. Viral fraction and cell-associated viral communities were decoupled, indicating viral turnover occurred across periods longer than the sampling period of three days. Inclusion of long-read data was critical for capturing the breadth of viral diversity. Phage isolates that infect the dominant bacterial taxa Prochlorococcus and Pelagibacter, usually regarded as cosmopolitan and abundant, were poorly represented.

The Citizen Phage Library: Rapid Isolation of Phages for the Treatment of Antibiotic Resistant Infections in the UK.

Antimicrobial resistance poses one of the greatest threats to global health and there is an urgent need for new therapeutic options. Phages are viruses that infect and kill bacteria and phage therapy could provide a valuable tool for the treatment of multidrug-resistant infections. In this study, water samples collected by citizen scientists as part of the Citizen Phage Library (CPL) project, and wastewater samples from the Environment Agency yielded phages with activity against clinical strains Klebsiella pneumoniae BPRG1484 and Enterobacter cloacae BPRG1482. A total of 169 and 163 phages were found for K. pneumoniae and E. cloacae, respectively, within four days of receiving the strains. A third strain (Escherichia coli BPRG1486) demonstrated cross-reactivity with 42 E. coli phages already held in the CPL collection. Seed lots were prepared for four K. pneumoniae phages and a cocktail combining these phages was found to reduce melanisation in a Galleria mellonella infection model. The resources and protocols utilised by the Citizen Phage Library enabled the rapid isolation and characterisation of phages targeted against multiple strains. In the future, within a clearly defined regulatory framework, phage therapy could be made available on a named-patient basis within the UK.

Novel pelagiphage isolate Polarivirus skadi is a polar specialist that dominates SAR11-associated bacteriophage communities at high latitudes.

The SAR11 clade are the most abundant members of surface marine bacterioplankton and a critical component of global biogeochemical cycles. Similarly, pelagiphages that infect SAR11 are ubiquitous and highly abundant in the oceans. Pelagiphages are predicted to shape SAR11 community structures and increase carbon turnover throughout the oceans. Yet, ecological drivers of host and niche specificity of pelagiphage populations are poorly understood. Here we report the global distribution of a novel pelagiphage called "Polarivirus skadi", which is the sole representative of a novel genus. P. skadi was isolated from the Western English Channel using a cold-water ecotype of SAR11 as bait. P. skadi is closely related to the globally dominant pelagiphage HTVC010P. Along with other HTVC010P-type viruses, P. skadi belongs to a distinct viral family within the order Caudovirales, for which we propose the name Ubiqueviridae. Metagenomic read recruitment identified P. skadi as one of the most abundant pelagiphages on Earth. P. skadi is a polar specialist, replacing HTVC010P at high latitudes. Experimental evaluation of P. skadi host range against cold- and warm-water SAR11 ecotypes supported cold-water specialism. Relative abundance of P. skadi in marine metagenomes correlated negatively with temperature, and positively with nutrients, available oxygen, and chlorophyll concentrations. In contrast, relative abundance of HTVC010P correlated negatively with oxygen and positively with salinity, with no significant correlation to temperature. The majority of other pelagiphages were scarce in most marine provinces, with a few representatives constrained to discrete ecological niches. Our results suggest that pelagiphage populations persist within a global viral seed bank, with environmental parameters and host availability selecting for a few ecotypes that dominate ocean viromes.

A Novel and Ubiquitous Marine Methylophage Provides Insights into Viral-Host Coevolution and Possible Host-Range Expansion in Streamlined Marine Heterotrophic Bacteria.

The 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 between marine primary production and remineralization of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of 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 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 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. IMPORTANCE Isolation and cultivation of viruses are the foundations on which the mechanistic understanding of virus-host interactions and parameterization of bioinformatic tools for viral ecology are based. This study isolated and characterized the first myophage known to infect the OM43 clade, expanding our knowledge of this understudied group of microbes. The nearly identical genomes of four strains of Melnitz isolated from different marine provinces and the 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 may not be necessarily restricted to phylogenetically related hosts but is a function of shared physical and biochemical properties of the cell.

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.

Draft Genome Sequences of Pelagimyophage Mosig EXVC030M and Pelagipodophage Lederberg EXVC029P, Isolated from Devil's Hole, Bermuda.

We present the genomes of two isolated bacteriophages infecting Pelagibacter ubique HTCC1062. Pelagibacter phage Mosig EXVC030M (Myoviridae) and Pelagibacter phage Lederberg EXVC029P (Podoviridae) were isolated by dilution-to-extinction culturing from the oxygen minimum zone at Devil's Hole (Harrington Sound, Bermuda).

Efficient dilution-to-extinction isolation of novel virus-host model systems for fastidious heterotrophic bacteria.

Microbes 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 ɸCrAss001, 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.

Pangenomics Analysis Reveals Diversification of Enzyme Families and Niche Specialization in Globally Abundant SAR202 Bacteria.

It has been hypothesized that the abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolisms for the oxidation of organic compounds that are resistant to microbial degradation via common metabolic pathways. Expansions of paralogous enzymes were reported and implicated in hypothetical metabolism involving monooxygenase and dioxygenase enzymes. In the proposed metabolic schemes, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore SAR202 evolution and metabolism, we reconstructed single amplified genomes and metagenome-assembled genomes from locations around the world that included the deepest ocean trenches. In an analysis of 122 SAR202 genomes that included seven subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, as well as further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the group III SAR202 genomes, and expansions of dioxygenase enzymes were prevalent in those of group VII. We found that group I SAR202 genomes encode expansions of racemases in the enolase superfamily, which we propose evolved for the degradation of compounds that resist biological oxidation because of chiral complexity. Supporting the conclusion that the paralog expansions indicate metabolic specialization, fragment recruitment and fluorescent in situ hybridization (FISH) with phylogenetic probes showed that SAR202 subclades are indigenous to different ocean depths and geographical regions. Surprisingly, some of the subclades were abundant in surface waters and contained rhodopsin genes, altering our understanding of the ecological role of SAR202 species in stratified water columns.IMPORTANCE The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Information about microbial interactions with this vast resource is limited, despite broad recognition that DOM turnover has a major impact on the global carbon cycle. To explain patterns in the genomes of marine bacteria, we propose hypothetical metabolic pathways for the oxidation of organic molecules that are resistant to oxidation via common pathways. The hypothetical schemes we propose suggest new metabolic pathways and classes of compounds that could be important for understanding the distribution of organic carbon throughout the biosphere. These genome-based schemes will remain hypothetical until evidence from experimental cell biology can be gathered to test them. Our findings also fundamentally change our understanding of the ecology of SAR202 bacteria, showing that metabolically diverse variants of these cells occupy niches spanning all depths and are not relegated to the dark ocean.