An inexpensive, accurate, and precise wet-mount method for enumerating aquatic viruses.

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the "filter mount" method, in which samples are filtered onto costly 0.02-µm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5×10(7) viruses ml(-1). The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17×10(6) to 1.37×10(8) viruses ml(-1) when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1×10(6) viruses ml(-1)) encountered in field and laboratory samples.

Mixotrophic growth of a ubiquitous marine diatom.

Diatoms are major players in the global carbon cycle, and their metabolism is affected by ocean conditions. Understanding the impact of changing inorganic nutrients in the oceans on diatoms is crucial, given the changes in global carbon dioxide levels. Here, we present a genome-scale metabolic model (iMK1961) for Cylindrotheca closterium, an in silico resource to understand uncharacterized metabolic functions in this ubiquitous diatom. iMK1961 represents the largest diatom metabolic model to date, comprising 1961 open reading frames and 6718 reactions. With iMK1961, we identified the metabolic response signature to cope with drastic changes in growth conditions. Comparing model predictions with Tara Oceans transcriptomics data unraveled C. closterium's metabolism in situ. Unexpectedly, the diatom only grows photoautotrophically in 21% of the sunlit ocean samples, while the majority of the samples indicate a mixotrophic (71%) or, in some cases, even a heterotrophic (8%) lifestyle in the light. Our findings highlight C. closterium's metabolic flexibility and its potential role in global carbon cycling.

Viral community analysis in a marine oxygen minimum zone indicates increased potential for viral manipulation of microbial physiological state.

Microbial communities in oxygen minimum zones (OMZs) are known to have significant impacts on global biogeochemical cycles, but viral influence on microbial processes in these regions are much less studied. Here we provide baseline ecological patterns using microscopy and viral metagenomics from the Eastern Tropical North Pacific (ETNP) OMZ region that enhance our understanding of viruses in these climate-critical systems. While extracellular viral abundance decreased below the oxycline, viral diversity and lytic infection frequency remained high within the OMZ, demonstrating that viral influences on microbial communities were still substantial without the detectable presence of oxygen. Viral community composition was strongly related to oxygen concentration, with viral populations in low-oxygen portions of the water column being distinct from their surface layer counterparts. However, this divergence was not accompanied by the expected differences in viral-encoded auxiliary metabolic genes (AMGs) relating to nitrogen and sulfur metabolisms that are known to be performed by microbial communities in these low-oxygen and anoxic regions. Instead, several abundant AMGs were identified in the oxycline and OMZ that may modulate host responses to low-oxygen stress. We hypothesize that this is due to selection for viral-encoded genes that influence host survivability rather than modulating host metabolic reactions within the ETNP OMZ. Together, this study shows that viruses are not only diverse throughout the water column in the ETNP, including the OMZ, but their infection of microorganisms has the potential to alter host physiological state within these biogeochemically important regions of the ocean.

Protist impacts on marine cyanovirocell metabolism.

The fate of oceanic carbon and nutrients depends on interactions between viruses, prokaryotes, and unicellular eukaryotes (protists) in a highly interconnected planktonic food web. To date, few controlled mechanistic studies of these interactions exist, and where they do, they are largely pairwise, focusing either on viral infection (i.e., virocells) or protist predation. Here we studied population-level responses of Synechococcus cyanobacterial virocells (i.e., cyanovirocells) to the protist Oxyrrhis marina using transcriptomics, endo- and exo-metabolomics, photosynthetic efficiency measurements, and microscopy. Protist presence had no measurable impact on Synechococcus transcripts or endometabolites. The cyanovirocells alone had a smaller intracellular transcriptional and metabolic response than cyanovirocells co-cultured with protists, displaying known patterns of virus-mediated metabolic reprogramming while releasing diverse exometabolites during infection. When protists were added, several exometabolites disappeared, suggesting microbial consumption. In addition, the intracellular cyanovirocell impact was largest, with 4.5- and 10-fold more host transcripts and endometabolites, respectively, responding to protists, especially those involved in resource and energy production. Physiologically, photosynthetic efficiency also increased, and together with the transcriptomics and metabolomics findings suggest that cyanovirocell metabolic demand is highest when protists are present. These data illustrate cyanovirocell responses to protist presence that are not yet considered when linking microbial physiology to global-scale biogeochemical processes.

Genetic Manipulation of Competition for Nitrate between Heterotrophic Bacteria and Diatoms.

Diatoms are a dominant group of eukaryotic phytoplankton that contribute substantially to global primary production and the cycling of important elements such as carbon and nitrogen. Heterotrophic bacteria, including members of the gammaproteobacteria, are commonly associated with diatom populations and may rely on them for organic carbon while potentially competing with them for other essential nutrients. Considering that bacterioplankton drive oceanic release of CO 2 (i.e., bacterial respiration) while diatoms drive ocean carbon sequestration vial the biological pump, the outcome of such competition could influence the direction and magnitude of carbon flux in the upper ocean. Nitrate availability is commonly a determining factor for the growth of diatom populations, particularly in coastal and upwelling regions. Diatoms as well as many bacterial species can utilize nitrate, however the ability of bacteria to compete for nitrate may be hindered by carbon limitation. Here we have developed a genetically tractable model system using the pennate diatom Phaeodactylum tricornutum and the widespread heterotrophic bacteria Alteromonas macleodii to examine carbon-nitrogen dynamics. While subsisting solely on P. tricornutum derived carbon, A. macleodii does not appear to be an effective competitor for nitrate, and may in fact benefit the diatom; particularly in stationary phase. However, allochthonous dissolved organic carbon addition in the form of pyruvate triggers A. macleodii proliferation and nitrate uptake, leading to reduced P. tricornutum growth. Nitrate reductase deficient mutants of A. macleodii (ΔnasA) do not exhibit such explosive growth and associated competitive ability in response to allochthonous carbon when nitrate is the sole nitrogen source, but could survive by utilizing solely P. tricornutum-derived nitrogen. Furthermore, allocthonous carbon addition enables wild-type A. macleodii to rescue nitrate reductase deficient P. tricornutum populations from nitrogen starvation, and RNA-seq transcriptomic evidence supports nitrogen-based interactions between diatoms and bacteria at the molecular level. This study provides key insights into the roles of carbon and nitrogen in phytoplankton-bacteria dynamics and lays the foundation for developing a mechanistic understanding of these interactions using co-culturing and genetic manipulation.

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

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

Ocean plankton. Patterns and ecological drivers of ocean viral communities.

Viruses influence ecosystems by modulating microbial population size, diversity, metabolic outputs, and gene flow. Here, we use quantitative double-stranded DNA (dsDNA) viral-fraction metagenomes (viromes) and whole viral community morphological data sets from 43 Tara Oceans expedition samples to assess viral community patterns and structure in the upper ocean. Protein cluster cataloging defined pelagic upper-ocean viral community pan and core gene sets and suggested that this sequence space is well-sampled. Analyses of viral protein clusters, populations, and morphology revealed biogeographic patterns whereby viral communities were passively transported on oceanic currents and locally structured by environmental conditions that affect host community structure. Together, these investigations establish a global ocean dsDNA viromic data set with analyses supporting the seed-bank hypothesis to explain how oceanic viral communities maintain high local diversity.

Ocean plankton. Environmental characteristics of Agulhas rings affect interocean plankton transport.

Agulhas rings provide the principal route for ocean waters to circulate from the Indo-Pacific to the Atlantic basin. Their influence on global ocean circulation is well known, but their role in plankton transport is largely unexplored. We show that, although the coarse taxonomic structure of plankton communities is continuous across the Agulhas choke point, South Atlantic plankton diversity is altered compared with Indian Ocean source populations. Modeling and in situ sampling of a young Agulhas ring indicate that strong vertical mixing drives complex nitrogen cycling, shaping community metabolism and biogeochemical signatures as the ring and associated plankton transit westward. The peculiar local environment inside Agulhas rings may provide a selective mechanism contributing to the limited dispersal of Indian Ocean plankton populations into the Atlantic.