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Viruses infecting marine prokaryotes have a large impact on the diversity and dynamics of their hosts. Model systems suggest that viral infection is frequency dependent and constrained by the virus-host encounter rate. However, it is unclear whether frequency-dependent infection is pervasive among the abundant prokaryotic populations with different temporal dynamics. To address this question, we performed a comparison of prokaryotic and viral communities using 16S rRNA amplicon and virome sequencing based on samples collected monthly for 2 years at a Japanese coastal site, Osaka Bay. Concurrent seasonal shifts observed in prokaryotic and viral community dynamics indicated that the abundance of viruses correlated with that of their predicted host phyla (or classes). Cooccurrence network analysis between abundant prokaryotes and viruses revealed 6, 423 cooccurring pairs, suggesting a tight coupling of host and viral abundances and their “one-to-many” correspondence. Although stable dominant species, such as SAR11, showed few cooccurring viruses, a fast succession of their viruses suggests that viruses infecting these populations changed continuously. Our results suggest that frequency-dependent viral infection prevails in coastal marine prokaryotes regardless of host taxa and temporal dynamics.

Marine viruses play a major role in the energy and nutrient cycle and affect the evolution of their hosts. Despite their importance, there is still little knowledge about RNA viruses. Here, we have explored the Atlantic Ocean, from surface to deep (4.296 m), and used viromics and quantitative methods to unveil the genomics, biogeography, and the mass contribution of RNA viruses to the total viroplankton. A total of 2481 putative RNA viral contigs (>500 bp) and 107 larger bona fide RNA viral genomes (>2.5 kb) were identified; 88 of them representing novel viruses belonging mostly to two clades: Yangshan assemblage (sister clade to the class Alsuviricetes) and Nodaviridae. These viruses were highly endemic and locally abundant, with little or no presence in other oceans since only ≈15% of them were found in at least one of the Tara sampling metatranscriptomes. Quantitative data indicated that the abundance of RNA viruses in the surface and deep chlorophyll maximum zone was within ≈106 VLP/mL representing a potential contribution of 5.2%–24.4% to the total viroplankton community (DNA and RNA viruses), with DNA viruses being the predominant members (≈107 VLP/mL). However, for the deep sample, the observed trend was the opposite, although as further discussed, several biases should be considered. Together these results contribute to our understanding of the diversity, abundance, and distribution of RNA viruses in the oceans and provide a basis for further investigation into their ecological roles and biogeography.

Single-virus genomics (SVGs) has been successfully applied to ocean surface samples allowing the discovery of widespread dominant viruses overlooked for years by metagenomics, such as the uncultured virus vSAG 37-F6 infecting the ubiquitous Pelagibacter spp. In SVGs, one uncultured virus at a time is sorted from the environmental sample, whole-genome amplified, and sequenced. Here, we have applied SVGs to deep-ocean samples (200–4000 m depth) from global Malaspina and MEDIMAX expeditions, demonstrating the feasibility of this method in deep-ocean samples. A total of 1328 virus-like particles were sorted from the North Atlantic Ocean, the deep Mediterranean Sea, and the Pacific Ocean oxygen minimum zone (OMZ). For this proof of concept, sixty single viruses were selected at random for sequencing. Genome annotation identified 27 of these genomes as bona fide viruses, and detected three auxiliary metabolic genes involved in nucleotide biosynthesis and sugar metabolism. Massive protein profile analysis confirmed that these viruses represented novel viral groups not present in databases. Although they were not previously assembled by viromics, global fragment recruitment analysis showed a conserved profile of relative abundance of these viruses in all analyzed samples spanning different oceans. Altogether, these results reveal the feasibility in using SVGs in this vast environment to unveil the genomes of relevant viruses.

Large double-stranded DNA viruses of the phylum Nucleocytoviricota, often referred to as "giant viruses," are ubiquitous members of marine ecosystems that are important agents of mortality for eukaryotic plankton. Although giant viruses are known to be prevalent in marine systems, their activities in oligotrophic ocean waters remain unclear. Oligotrophic gyres constitute the majority of the ocean and assessing viral activities in these regions is therefore critical for understanding overall marine microbial processes. In this study, we generated 11 metagenome-assembled genomes (MAGs) of giant viruses from samples previously collected from Station ALOHA in the North Pacific Subtropical Gyre. Phylogenetic analyses revealed that they belong to the orders Imitervirales (n =6), Algavirales (n =4), and Pimascovirales (n =1). Genome sizes ranged from similar to 119-574 kbp, and several of the genomes encoded predicted TCA cycle components, cytoskeletal proteins, collagen, rhodopsins, and proteins potentially involved in other cellular processes. Comparison with other marine metagenomes revealed that several have broad distribution across ocean basins and represent abundant viral constituents of pelagic surface waters. Our work sheds light on the diversity of giant viruses present in oligotrophic ocean waters across the globe.

Large double-stranded DNA viruses of the phylum Nucleocytoviricota, often referred to as "giant viruses," are ubiquitous members of marine ecosystems that are important agents of mortality for eukaryotic plankton. Although giant viruses are known to be prevalent in marine systems, their activities in oligotrophic ocean waters remain unclear. Oligotrophic gyres constitute the majority of the ocean and assessing viral activities in these regions is therefore critical for understanding overall marine microbial processes. In this study, we generated 11 metagenome-assembled genomes (MAGs) of giant viruses from samples previously collected from Station ALOHA in the North Pacific Subtropical Gyre. Phylogenetic analyses revealed that they belong to the orders Imitervirales (n =6), Algavirales (n =4), and Pimascovirales (n =1). Genome sizes ranged from similar to 119-574 kbp, and several of the genomes encoded predicted TCA cycle components, cytoskeletal proteins, collagen, rhodopsins, and proteins potentially involved in other cellular processes. Comparison with other marine metagenomes revealed that several have broad distribution across ocean basins and represent abundant viral constituents of pelagic surface waters. Our work sheds light on the diversity of giant viruses present in oligotrophic ocean waters across the globe.

Single-virus genomics (SVGs) has been successfully applied to ocean surface samples allowing the discovery of widespread dominant viruses overlooked for years by metagenomics, such as the uncultured virus vSAG 37-F6 infecting the ubiquitous Pelagibacter spp. In SVGs, one uncultured virus at a time is sorted from the environmental sample, whole-genome amplified, and sequenced. Here, we have applied SVGs to deep-ocean samples (200–4000 m depth) from global Malaspina and MEDIMAX expeditions, demonstrating the feasibility of this method in deep-ocean samples. A total of 1328 virus-like particles were sorted from the North Atlantic Ocean, the deep Mediterranean Sea, and the Pacific Ocean oxygen minimum zone (OMZ). For this proof of concept, sixty single viruses were selected at random for sequencing. Genome annotation identified 27 of these genomes as bona fide viruses, and detected three auxiliary metabolic genes involved in nucleotide biosynthesis and sugar metabolism. Massive protein profile analysis confirmed that these viruses represented novel viral groups not present in databases. Although they were not previously assembled by viromics, global fragment recruitment analysis showed a conserved profile of relative abundance of these viruses in all analyzed samples spanning different oceans. Altogether, these results reveal the feasibility in using SVGs in this vast environment to unveil the genomes of relevant viruses.

Background Bacteriophages, the viruses infecting bacteria, are biological entities that can control their host populations. The ecological relevance of phages for microbial systems has been widely explored in aquatic environments, but the current understanding of the role of phages in terrestrial ecosystems remains limited. Here, our objective was to quantify the extent to which phages drive the assembly and functioning of soil bacterial communities. We performed a reciprocal transplant experiment using natural and sterilized soil incubated with different combinations of two soil microbial communities, challenged against native and non-native phage suspensions as well as against a cocktail of phage isolates. We tested three different community assembly scenarios by adding phages: (a) during soil colonization, (b) after colonization, and (c) in natural soil communities. One month after inoculation with phage suspensions, bacterial communities were assessed by 16S rRNA amplicon gene sequencing. Results By comparing the treatments inoculated with active versus autoclaved phages, our results show that changes in phage pressure have the potential to impact soil bacterial community composition and diversity. We also found a positive effect of active phages on the soil ammonium concentration in a few treatments, which indicates that increased phage pressure may also be important for soil functions. Conclusions Overall, the present work contributes to expand the current knowledge about soil phages and provide some empirical evidence supporting their relevance for soil bacterial community assembly and functioning.

Background Bacteriophages, the viruses infecting bacteria, are biological entities that can control their host populations. The ecological relevance of phages for microbial systems has been widely explored in aquatic environments, but the current understanding of the role of phages in terrestrial ecosystems remains limited. Here, our objective was to quantify the extent to which phages drive the assembly and functioning of soil bacterial communities. We performed a reciprocal transplant experiment using natural and sterilized soil incubated with different combinations of two soil microbial communities, challenged against native and non-native phage suspensions as well as against a cocktail of phage isolates. We tested three different community assembly scenarios by adding phages: (a) during soil colonization, (b) after colonization, and (c) in natural soil communities. One month after inoculation with phage suspensions, bacterial communities were assessed by 16S rRNA amplicon gene sequencing. Results By comparing the treatments inoculated with active versus autoclaved phages, our results show that changes in phage pressure have the potential to impact soil bacterial community composition and diversity. We also found a positive effect of active phages on the soil ammonium concentration in a few treatments, which indicates that increased phage pressure may also be important for soil functions. Conclusions Overall, the present work contributes to expand the current knowledge about soil phages and provide some empirical evidence supporting their relevance for soil bacterial community assembly and functioning.

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 9 (2018): 358, doi:10.3389/fmicb.2018.00358., Viruses are key players in ocean ecology and biogeochemistry, not only because of their functional roles but also partially due to their sheer abundance (Fuhrman, 1999; Wilhelm and Suttle, 1999). Because viruses cannot replicate without their hosts' machinery, their abundance is inextricably related to that of their (mostly microbial) hosts. The relationship between viral and microbial abundances is thus of great interest.

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 9 (2018): 358, doi:10.3389/fmicb.2018.00358., Viruses are key players in ocean ecology and biogeochemistry, not only because of their functional roles but also partially due to their sheer abundance (Fuhrman, 1999; Wilhelm and Suttle, 1999). Because viruses cannot replicate without their hosts' machinery, their abundance is inextricably related to that of their (mostly microbial) hosts. The relationship between viral and microbial abundances is thus of great interest.

About a quarter of anthropogenic CO2 emissions are currently taken up by the oceans, decreasing seawater pH. We performed a mesocosm experiment in the Baltic Sea in order to investigate the consequences of increasing CO2 levels on pelagic carbon fluxes. A gradient of different CO2 scenarios, ranging from ambient (similar to 370 mu atm) to high (similar to 1200 mu atm), were set up in mesocosm bags (similar to 55m(3)). We determined standing stocks and temporal changes of total particulate carbon (TPC), dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and particulate organic carbon (POC) of specific plankton groups. We also measured carbon flux via CO2 exchange with the atmosphere and sedimentation (export), and biological rate measurements of primary production, bacterial production, and total respiration. The experiment lasted for 44 days and was divided into three different phases (I: t0-t16; II: t17-t30; III: t31-t43). Pools of TPC, DOC, and DIC were approximately 420, 7200, and 25 200 mmol Cm-2 at the start of the experiment, and the initial CO2 additions increased the DIC pool by similar to 7% in the highest CO2 treatment. Overall, there was a decrease in TPC and increase of DOC over the course of the experiment. The decrease in TPC was lower, and increase in DOC higher, in treatments with added CO2. During phase I the estimated gross primary production (GPP) was similar to 100 mmol C m(-2) day(-1), from which 75-95% was respired, similar to 1% ended up in the TPC (including export), and 5-25% was added to the DOC pool. During phase II, the respiration loss increased to similar to 100% of GPP at the ambient CO2 concentration, whereas respiration was lower (85-95% of GPP) in the highest CO2 treatment. Bacterial production was similar to 30% lower, on average, at the highest CO2 concentration than in the controls during phases II and III. This resulted in a higher accumulation of DOC and lower reduction in the TPC pool in the elevated CO2 treatme

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ~20% transfer of organic matter derived from the infected 13C- and 15N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate 13C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.

Marine viruses have only relatively recently come to the attention of molecular biologists, and the extraordinary diversity of potential host organisms suggests a new wealth of genetic and structural forms. A promising technology for characterizing and describing the viruses structurally is atomic force microscopy (AFM). We provide examples here of some of the different architectures and novel structural features that emerge from even a very limited investigation, one focused on cyanophages, viruses that infect cyanobacteria (blue-green algae). These were isolated by phage selection of viruses collected from California coastal waters. We present AFM images of tailed, spherical, filamentous, rod shaped viruses, and others of eccentric form. Among the tailed phages numerous myoviruses were observed, some having long tail fibers, some other none, and some having no visible baseplate. Syphoviruses and a podovirus were also seen. We also describe a unique structural features found on some tailed marine phages that appear to have no terrestrial homolog. These are long, 450 nm, complex helical tail fibers terminating in a unique pattern of 3+1 globular units made up of about 20 small proteins.

Marine viruses have only relatively recently come to the attention of molecular biologists, and the extraordinary diversity of potential host organisms suggests a new wealth of genetic and structural forms. A promising technology for characterizing and describing the viruses structurally is atomic force microscopy (AFM). We provide examples here of some of the different architectures and novel structural features that emerge from even a very limited investigation, one focused on cyanophages, viruses that infect cyanobacteria (blue-green algae). These were isolated by phage selection of viruses collected from California coastal waters. We present AFM images of tailed, spherical, filamentous, rod shaped viruses, and others of eccentric form. Among the tailed phages numerous myoviruses were observed, some having long tail fibers, some other none, and some having no visible baseplate. Syphoviruses and a podovirus were also seen. We also describe a unique structural features found on some tailed marine phages that appear to have no terrestrial homolog. These are long, 450 nm, complex helical tail fibers terminating in a unique pattern of 3+1 globular units made up of about 20 small proteins.

The South China Sea is one of the largest marginal seas in the world. A cruise was conducted during September 2005 to investigate the spatial distribution of viral and bacterial abundance as well as nutrient and chlorophyll a (chl a) concentrations in the northern South China Sea (SCS). The northern SCS was divided into 3 regions: the estuarine coastal plume, continental shelf, and open ocean. Except for the estuarine coastal waters, the northern SCS is oligotrophic. The distribution of chl a and viral and bacterial abundances were closely related to the water mass since higher chl a and viral and bacterial abundances occurred in the upwelling region and the cold eddy. Viral and bacterial abundances decreased from the estuarine waters to offshore waters. On average, viral abundance was significantly higher (p < 0.05) in the estuarine coastal plume (25.2 +/- 3.1 x 10(6) ml(-1)), than on the continental shelf (14.1 +/- 6.5 x 10(6) ml(-1)) or the open ocean (11.7 +/- 5.3 x 10(6) ml(-1)). Bacterial abundance followed a similar spatial distribution, ranging from 4.6 +/- 1.1 x 10(6) ml(-1) in the estuary to 1.6 +/- 0.8 x 10(6) ml(-1) in the open ocean. Ratios of viral to bacterial abundance (VBR) increased from near-shore to the coast and open ocean (5.6 +/- 0.7, 6.1 +/- 2.2 and 8.4 +/- 4.7, respectively). This is due to the fact that viral abundance decreased slower than bacterial abundance from the estuarine plume to the open ocean. Viral abundance was more significantly correlated with bacterial abundance (p < 0.0001) than with chl a concentration (p = 0.001), suggesting that bacteria were the major host members for marine viruses in the oligotrophic northern SCS. The highest abundance of viruses highlighted the significant influence of the relatively nutrient- and organic-rich Pearl River outflow on the northern SCS and indicated that viruses also responded to anthropogenic inputs in marine ecosystems.

The South China Sea is one of the largest marginal seas in the world. A cruise was conducted during September 2005 to investigate the spatial distribution of viral and bacterial abundance as well as nutrient and chlorophyll a (chl a) concentrations in the northern South China Sea (SCS). The northern SCS was divided into 3 regions: the estuarine coastal plume, continental shelf, and open ocean. Except for the estuarine coastal waters, the northern SCS is oligotrophic. The distribution of chl a and viral and bacterial abundances were closely related to the water mass since higher chl a and viral and bacterial abundances occurred in the upwelling region and the cold eddy. Viral and bacterial abundances decreased from the estuarine waters to offshore waters. On average, viral abundance was significantly higher (p < 0.05) in the estuarine coastal plume (25.2 +/- 3.1 x 10(6) ml(-1)), than on the continental shelf (14.1 +/- 6.5 x 10(6) ml(-1)) or the open ocean (11.7 +/- 5.3 x 10(6) ml(-1)). Bacterial abundance followed a similar spatial distribution, ranging from 4.6 +/- 1.1 x 10(6) ml(-1) in the estuary to 1.6 +/- 0.8 x 10(6) ml(-1) in the open ocean. Ratios of viral to bacterial abundance (VBR) increased from near-shore to the coast and open ocean (5.6 +/- 0.7, 6.1 +/- 2.2 and 8.4 +/- 4.7, respectively). This is due to the fact that viral abundance decreased slower than bacterial abundance from the estuarine plume to the open ocean. Viral abundance was more significantly correlated with bacterial abundance (p < 0.0001) than with chl a concentration (p = 0.001), suggesting that bacteria were the major host members for marine viruses in the oligotrophic northern SCS. The highest abundance of viruses highlighted the significant influence of the relatively nutrient- and organic-rich Pearl River outflow on the northern SCS and indicated that viruses also responded to anthropogenic inputs in marine ecosystems.

The South China Sea is one of the largest marginal seas in the world. A cruise was conducted during September 2005 to investigate the spatial distribution of viral and bacterial abundance as well as nutrient and chlorophyll a (chl a) concentrations in the northern South China Sea (SCS). The northern SCS was divided into 3 regions: the estuarine coastal plume, continental shelf, and open ocean. Except for the estuarine coastal waters, the northern SCS is oligotrophic. The distribution of chl a and viral and bacterial abundances were closely related to the water mass since higher chl a and viral and bacterial abundances occurred in the upwelling region and the cold eddy. Viral and bacterial abundances decreased from the estuarine waters to offshore waters. On average, viral abundance was significantly higher (p < 0.05) in the estuarine coastal plume (25.2 +/- 3.1 x 10(6) ml(-1)), than on the continental shelf (14.1 +/- 6.5 x 10(6) ml(-1)) or the open ocean (11.7 +/- 5.3 x 10(6) ml(-1)). Bacterial abundance followed a similar spatial distribution, ranging from 4.6 +/- 1.1 x 10(6) ml(-1) in the estuary to 1.6 +/- 0.8 x 10(6) ml(-1) in the open ocean. Ratios of viral to bacterial abundance (VBR) increased from near-shore to the coast and open ocean (5.6 +/- 0.7, 6.1 +/- 2.2 and 8.4 +/- 4.7, respectively). This is due to the fact that viral abundance decreased slower than bacterial abundance from the estuarine plume to the open ocean. Viral abundance was more significantly correlated with bacterial abundance (p < 0.0001) than with chl a concentration (p = 0.001), suggesting that bacteria were the major host members for marine viruses in the oligotrophic northern SCS. The highest abundance of viruses highlighted the significant influence of the relatively nutrient- and organic-rich Pearl River outflow on the northern SCS and indicated that viruses also responded to anthropogenic inputs in marine ecosystems.

The research funded by this award, sponsored investigations on novel marine viruses that were isolated in British Columbia coastal waters and the Gulf of Mexico. It was a continuation of Grant NOOOl4-92-J-l676 awarded at The University of Texas. The results included the isolation of a viral pathogen that infects a eukaryotic toxin-producing phytoplankton. The phytoplankton that is infected has been responsible for extensive fish kills in North American and Asia. The ultimate goal is to use these and other viral isolates as vectors for transforming and studying genetics in eukaryotic microalgae. As part of the research an unknown viral pathogen that infects microzooplankton has begun to be characterized. Ultimately, this research may lead to the development of in situ PCR-based methods to detect the presence of viral genes within infected cells, and for detection of novel and rare virus types in seawater. This will allow us to more effectively screen for the presence of potential lysogens, as well as allow us to determine the presence of infected cells in natural populations. We have also continued work on the development of fluorescently labeled virus technology as a tool to localize and isolate viral receptors on cell surfaces.

The report is to isolate representative marine virus systems; to develop taxon-specific probes for marine viruses; to determine spatial and temporal distributions of specific viral taxa; to establish a library of natural marine virus communities and a culture collection of marine viruses and their hosts, and to characterize the viruses that we isolate.

This report documents R and D performed by Principal Investigators under the sponsorship of the ONR Biological Sciences Division during fiscal year 1991. This report includes the following topics: Biocatalysis in Non Aqueous Solvents, Biodeterioration, Biomimetics, Biopolymeric Materials, Education, High Temperature, Pressure Biology, Marine Symbiosis, Marine Viruses, Molecular Biology, Marine Organisms, Water Biological Interfaces, Immunophysiology, Cell Biology, Pressure Biology, Sensory Biophysics, Membrane Biology I, Membrane Biology, Single Neuron Computation.