Metabolic trade-offs constrain the cell size ratio in a nitrogen-fixing symbiosis.

Biological dinitrogen (N2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N2-fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N2-fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N2-fixing organelle (or nitroplast).

Hopanoid lipids may facilitate aerobic nitrogen fixation in the ocean.

Cyanobacterial diazotrophs are considered to be the most important source of fixed N2 in the open ocean. Biological N2 fixation is catalyzed by the extremely O2-sensitive nitrogenase enzyme. In cyanobacteria without specialized N2-fixing cells (heterocysts), mechanisms such as decoupling photosynthesis from N2 fixation in space or time are involved in protecting nitrogenase from the intracellular O2 evolved by photosynthesis. However, it is not known how cyanobacterial cells limit O2 diffusion across their membranes to protect nitrogenase in ambient O2-saturated surface ocean waters. Here, we explored all known genomes of the major marine cyanobacterial lineages for the presence of hopanoid synthesis genes, since hopanoids are a class of lipids that might act as an O2 diffusion barrier. We found that, whereas all non-heterocyst-forming cyanobacterial diazotrophs had hopanoid synthesis genes, none of the marine Synechococcus, Prochlorococcus (non-N2-fixing), and marine heterocyst-forming (N2-fixing) cyanobacteria did. Finally, we conclude that hopanoid-enriched membranes are a conserved trait in non-heterocyst-forming cyanobacterial diazotrophs that might lower the permeability to extracellular O2 This membrane property coupled with high respiration rates to decrease intracellular O2 concentration may therefore explain how non-heterocyst-forming cyanobacterial diazotrophs can fix N2 in the fully oxic surface ocean.

UCYN-A3, a newly characterized open ocean sublineage of the symbiotic N2 -fixing cyanobacterium Candidatus Atelocyanobacterium thalassa.

The symbiotic unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) is one of the most abundant and widespread nitrogen (N2 )-fixing cyanobacteria in the ocean. Although it remains uncultivated, multiple sublineages have been detected based on partial nitrogenase (nifH) gene sequences, including the four most commonly detected sublineages UCYN-A1, UCYN-A2, UCYN-A3 and UCYN-A4. However, very little is known about UCYN-A3 beyond the nifH sequences from nifH gene diversity surveys. In this study, single cell sorting, DNA sequencing, qPCR and CARD-FISH assays revealed discrepancies involving the identification of sublineages, which led to new information on the diversity of the UCYN-A symbiosis. 16S rRNA and nifH gene sequencing on single sorted cells allowed us to identify the 16S rRNA gene of the uncharacterized UCYN-A3 sublineage. We designed new CARD-FISH probes that allowed us to distinguish and observe UCYN-A2 in a coastal location (SIO Pier; San Diego) and UCYN-A3 in an open ocean location (Station ALOHA; Hawaii). Moreover, we reconstructed about 13% of the UCYN-A3 genome from Tara Oceans metagenomic data. Finally, our findings unveil the UCYN-A3 symbiosis in open ocean waters suggesting that the different UCYN-A sublineages are distributed along different size fractions of the plankton defined by the cell-size ranges of their prymnesiophyte hosts.

Primer Design for an Accurate View of Picocyanobacterial Community Structure by Using High-Throughput Sequencing.

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.

Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria.

Prochlorococcus and Synechococcus are the two most abundant and widespread phytoplankton in the global ocean. To better understand the factors controlling their biogeography, a reference database of the high-resolution taxonomic marker petB, encoding cytochrome b6, was used to recruit reads out of 109 metagenomes from the Tara Oceans expedition. An unsuspected novel genetic diversity was unveiled within both genera, even for the most abundant and well-characterized clades, and 136 divergent petB sequences were successfully assembled from metagenomic reads, significantly enriching the reference database. We then defined Ecologically Significant Taxonomic Units (ESTUs)-that is, organisms belonging to the same clade and occupying a common oceanic niche. Three major ESTU assemblages were identified along the cruise transect for Prochlorococcus and eight for Synechococcus Although Prochlorococcus HLIIIA and HLIVA ESTUs codominated in iron-depleted areas of the Pacific Ocean, CRD1 and the yet-to-be cultured EnvB were the prevalent Synechococcus clades in this area, with three different CRD1 and EnvB ESTUs occupying distinct ecological niches with regard to iron availability and temperature. Sharp community shifts were also observed over short geographic distances-for example, around the Marquesas Islands or between southern Indian and Atlantic Oceans-pointing to a tight correlation between ESTU assemblages and specific physico-chemical parameters. Together, this study demonstrates that there is a previously overlooked, ecologically meaningful, fine-scale diversity within some currently defined picocyanobacterial ecotypes, bringing novel insights into the ecology, diversity, and biology of the two most abundant phototrophs on Earth.

Validation of a new catalysed reporter deposition-fluorescence in situ hybridization probe for the accurate quantification of marine Bacteroidetes populations.

Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) is a powerful approach to quantify bacterial taxa. In this study, we compare the performance of the widely used Bacteroidetes CF319a probe with the new CF968 probe. In silico analyses and tests with isolates demonstrate that CF319a hybridizes with non-Bacteroidetes sequences from the Rhodobacteraceae and Alteromonadaceae families. We test the probes' accuracy in 37 globally distributed marine samples and over two consecutive years at the Blanes Bay Microbial Observatory (NW Mediterranean). We also compared the CARD-FISH data with the Bacteroidetes 16S rRNA gene sequences retrieved from 27 marine metagenomes from the TARA Oceans expedition. We find no significant differences in abundances between both approaches, although CF319a targeted some unspecific sequences and both probes displayed different abundances of specific Bacteroidetes phylotypes. Our results demonstrate that quantitative estimations by using both probes are significantly different in certain oceanographic regions (Mediterranean Sea, Red Sea and Arabian Sea) and that CF968 shows seasonality within marine Bacteroidetes, notably large differences between summer and winter that is overlooked by CF319a. We propose CF968 as an alternative to CF319a for targeting the whole Bacteroidetes phylum since it has better coverage, greater specificity and overall better quantifies marine Bacteroidetes.

Closing the gaps on the viral photosystem-I†psaDCAB gene organization.

Marine photosynthesis is largely driven by cyanobacteria, namely Synechococcus and Prochlorococcus. Genes encoding for photosystem (PS) I and II reaction centre proteins are found in cyanophages and are believed to increase their fitness. Two viral PSI gene arrangements are known, psaJF→C→A→B→K→E→D and psaD→C→A→B. The shared genes between these gene cassettes and their encoded proteins are distinguished by %G + C and protein sequence respectively. The data on the psaD→C→A→B gene organization were reported from only two partial gene cassettes coming from Global Ocean Sampling stations in the Pacific and Indian oceans. Now we have extended our search to 370 marine stations from six metagenomic projects. Genes corresponding to both PSI gene arrangements were detected in the Pacific, Indian and Atlantic oceans, confined to a strip along the equator (30°N and 30°S). In addition, we found that the predicted structure of the viral PsaA protein from the psaD→C→A→B organization contains a lumenal loop conserved in PsaA proteins from Synechococcus, but is completely absent in viral PsaA proteins from the psaJF→C→A→B→K→E→D gene organization and most Prochlorococcus strains. This may indicate a co-evolutionary scenario where cyanophages containing either of these gene organizations infect cyanobacterial ecotypes biogeographically restricted to the 30°N and 30°S equatorial strip.

Sample dilution and bacterial community composition influence empirical leucine-to-carbon conversion factors in surface waters of the world's oceans.

The transformation of leucine incorporation rates to prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world's oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e., removal of grazers [F treatment]) or filtration combined with dilution (i.e., also relieving resource competition [FD treatment]). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu(-1) and were significantly lower in the FD than in the F samples. Also, changes in bacterial community composition during the incubations, as assessed by automated ribosomal intergenic spacer analysis (ARISA), were more pronounced in the FD than in the F treatments, compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCF determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyll a concentration, picocyanobacterial abundance (mostly Prochlorococcus), and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCF variability in the F treatment, supporting the view that both Prochlorococcus and HNA prokaryotes incorporate leucine in substantial amounts, although this results in relatively low carbon production rates in the oligotrophic ocean.

Particle-association lifestyle is a phylogenetically conserved trait in bathypelagic prokaryotes.

The free-living (FL) and particle-attached (PA) marine microbial communities have repeatedly been proved to differ in their diversity and composition in the photic ocean and also recently in the bathypelagic ocean at a global scale. However, although high taxonomic ranks exhibit preferences for a PA or FL mode of life, it remains poorly understood whether two clear lifestyles do exist and how these are distributed across the prokaryotic phylogeny. We studied the FL (<0.8 µm) and PA (0.8-20 µm) prokaryotes at 30 stations distributed worldwide within the bathypelagic oceanic realm (2150-4000 m depth) using high-throughput sequencing of the small subunit ribosomal RNA gene (16S rRNA). A high proportion of the bathypelagic prokaryotes were mostly found either attached to particles or freely in the surrounding water but rarely in both types of environments. In particular, this trait was deeply conserved through their phylogeny, suggesting that the deep-ocean particles and the surrounding water constitute two highly distinct niches and that transitions from one to the other have been rare at an evolutionary timescale. As a consequence, PA and FL communities had clear alpha- and beta-diversity differences that exceeded the global-scale geographical variation. Our study organizes the bathypelagic prokaryotic diversity into a reasonable number of ecologically coherent taxa regarding their association with particles, a first step for understanding which are the microbes responsible for the processing of the dissolved and particulate pools of organic matter that have a very different biogeochemical role in the deep ocean.

Metagenomic 16S rDNA Illumina tags are a powerful alternative to amplicon sequencing to explore diversity and structure of microbial communities.

Sequencing of 16S rDNA polymerase chain reaction (PCR) amplicons is the most common approach for investigating environmental prokaryotic diversity, despite the known biases introduced during PCR. Here we show that 16S rDNA fragments derived from Illumina-sequenced environmental metagenomes (mi tags) are a powerful alternative to 16S rDNA amplicons for investigating the taxonomic diversity and structure of prokaryotic communities. As part of the Tara Oceans global expedition, marine plankton was sampled in three locations, resulting in 29 subsamples for which metagenomes were produced by shotgun Illumina sequencing (ca. 700 Gb). For comparative analyses, a subset of samples was also selected for Roche-454 sequencing using both shotgun (m454 tags; 13 metagenomes, ca. 2.4 Gb) and 16S rDNA amplicon (454 tags; ca. 0.075 Gb) approaches. Our results indicate that by overcoming PCR biases related to amplification and primer mismatch, mi tags may provide more realistic estimates of community richness and evenness than amplicon 454 tags. In addition, mi tags can capture expected beta diversity patterns. Using mi tags is now economically feasible given the dramatic reduction in high-throughput sequencing costs, having the advantage of retrieving simultaneously both taxonomic (Bacteria, Archaea and Eukarya) and functional information from the same microbial community.

Primer design for the amplification of the ammonium transporter genes from the uncultured haptophyte algal species symbiotic with the marine nitrogen-fixing cyanobacterium UCYN-A1.

The multiple symbiotic partnerships between closely related species of the haptophyte algae Braarudosphaera bigelowii and the nitrogen-fixing cyanobacteria Candidatus Atelocyanobacterium thalassa (UCYN-A) contribute importantly to the nitrogen and carbon cycles in vast areas of the ocean. The diversity of the eukaryotic 18S rDNA phylogenetic gene marker has helped to identify some of these symbiotic haptophyte species, yet we still lack a genetic marker to assess its diversity at a finer scale. One of such genes is the ammonium transporter (amt) gene, which encodes the protein that might be involved in the uptake of ammonium from UCYN-A in these symbiotic haptophytes. Here, we designed three specific PCR primer sets targeting the amt gene of the haptophyte species (A1-Host) symbiotic with the open ocean UCYN-A1 sublineage, and tested them in samples collected from open ocean and near-shore environments. Regardless of the primer pair used at Station ALOHA, which is where UCYN-A1 is the pre-dominant UCYN-A sublineage, the most abundant amt amplicon sequence variant (ASV) was taxonomically classified as A1-Host. In addition, two out of the three PCR primer sets revealed the existence of closely-related divergent haptophyte amt ASVs (>95% nucleotide identity). These divergent amt ASVs had higher relative abundances than the haptophyte typically associated with UCYN-A1 in the Bering Sea, or co-occurred with the previously identified A1-Host in the Coral Sea, suggesting the presence of new diversity of closely-related A1-Hosts in polar and temperate waters. Therefore, our study reveals an overlooked diversity of haptophytes species with distinct biogeographic distributions partnering with UCYN-A, and provides new primers that will help to gain new knowledge of the UCYN-A/haptophyte symbiosis.

Non-cyanobacterial diazotrophs: global diversity, distribution, ecophysiology, and activity in marine waters.

Biological dinitrogen (N2) fixation supplies nitrogen to the oceans, supporting primary productivity, and is carried out by some bacteria and archaea referred to as diazotrophs. Cyanobacteria are conventionally considered to be the major contributors to marine N2 fixation, but non-cyanobacterial diazotrophs (NCDs) have been shown to be distributed throughout ocean ecosystems. However, the biogeochemical significance of marine NCDs has not been demonstrated. This review synthesizes multiple datasets, drawing from cultivation-independent molecular techniques and data from extensive oceanic expeditions, to provide a comprehensive view into the diversity, biogeography, ecophysiology, and activity of marine NCDs. A NCD nifH gene catalog was compiled containing sequences from both PCR-based and PCR-free methods, identifying taxa for future studies. NCD abundances from a novel database of NCD nifH-based abundances were colocalized with environmental data, unveiling distinct distributions and environmental drivers of individual taxa. Mechanisms that NCDs may use to fuel and regulate N2 fixation in response to oxygen and fixed nitrogen availability are discussed, based on a metabolic analysis of recently available Tara Oceans expedition data. The integration of multiple datasets provides a new perspective that enhances understanding of the biology, ecology, and biogeography of marine NCDs and provides tools and directions for future research.

Diazotrophs are overlooked contributors to carbon and nitrogen export to the deep ocean.

Diazotrophs are widespread microorganisms that alleviate nitrogen limitation in 60% of our oceans, thereby regulating marine productivity. Yet, the group-specific contribution of diazotrophs to organic matter export has not been quantified, which so far has impeded an accurate assessment of their impact on the biological carbon pump. Here, we examine the fate of five groups of globally-distributed diazotrophs by using an original combination of mesopelagic particle sampling devices across the subtropical South Pacific Ocean. We demonstrate that cyanobacterial and non-cyanobacterial diazotrophs are exported down to 1000 m depth. Surprisingly, group-specific export turnover rates point to a more efficient export of small unicellular cyanobacterial diazotrophs (UCYN) relative to the larger and filamentous Trichodesmium. Phycoerythrin-containing UCYN-B and UCYN-C-like cells were recurrently found embedded in large (>50 µm) organic aggregates or organized into clusters of tens to hundreds of cells linked by an extracellular matrix, presumably facilitating their export. Beyond the South Pacific, our data are supported by analysis of the Tara Oceans metagenomes collected in other ocean basins, extending the scope of our results globally. We show that, when diazotrophs are found in the euphotic zone, they are also systematically present in mesopelagic waters, suggesting their transport to the deep ocean. We thus conclude that diazotrophs are a significant part of the carbon sequestered in the deep ocean and, therefore, they need to be accounted in regional and global estimates of export.

Intriguing size distribution of the uncultured and globally widespread marine non-cyanobacterial diazotroph Gamma-A.

Non-cyanobacterial diazotrophs (NCDs) have recently emerged as potentially important contributors to marine nitrogen fixation. One of the most widely distributed NCDs is Gamma-A, yet information about its autecology is still scarce and solely relies on the PCR-based detection of its nitrogenase (nifH) gene in seawater, since previous metagenomic surveys targeting free-living planktonic size fractions (<3 µm) have not detected it. Here, we explore the diversity, biogeography, size-distribution, and nitrogenase gene expression of Gamma-A across four larger planktonic size-fractions (0.8-5, 5-20, 20-180, and 180-2000 µm) using metagenomes and metatranscriptomes from the Tara Oceans. We detected a single variant of a complete Gamma-A nifH gene along with other nitrogenase-related genes (nifKDT) within a metatranscriptomic-based contig of the Marine Atlas of Tara Ocean Unigenes. Gamma-A was detected in tropical and subtropical oceanic regions across all the size-fractions. However, the highest gene and transcript abundances were found in the 0.8-5 and 5-20 µm size-fractions at the surface, whereas abundances at the deep chlorophyll maximum were lower and similar across all size-fractions. The ubiquitous presence of active Gamma-A in large planktonic size-fractions suggests a filamentous or particle-attached lifestyle and places its potential to fix nitrogen in larger planktonic compartments.

Critical Role of Light in the Growth and Activity of the Marine N2-Fixing UCYN-A Symbiosis.

The unicellular N2-fixing cyanobacteria UCYN-A live in symbiosis with haptophytes in the Braarudosphaera bigelowii lineage. Maintaining N2-fixing symbioses between two unicellular partners requires tight coordination of multiple biological processes including cell growth and division and, in the case of the UCYN-A symbiosis, N2 fixation of the symbiont and photosynthesis of the host. In this system, it is thought that the host photosynthesis supports the high energetic cost of N2 fixation, and both processes occur during the light period. However, information on this coordination is very limited and difficult to obtain because the UCYN-A symbiosis has yet to be available in culture. Natural populations containing the UCYN-A2 symbiosis were manipulated to explore the effects of alterations of regular light and dark periods and inhibition of host photosynthesis on N2 fixation (single cell N2 fixation rates), nifH gene transcription, and UCYN-A2 cell division (fluorescent in situ hybridization and nifH gene abundances). The results showed that the light period is critical for maintenance of regular patterns of gene expression, N2 fixation and symbiont replication and cell division. This study suggests a crucial role for the host as a producer of fixed carbon, rather than light itself, in the regulation and implementation of these cellular processes in UCYN-A.

Global distribution patterns of marine nitrogen-fixers by imaging and molecular methods.

Nitrogen fixation has a critical role in marine primary production, yet our understanding of marine nitrogen-fixers (diazotrophs) is hindered by limited observations. Here, we report a quantitative image analysis pipeline combined with mapping of molecular markers for mining >2,000,000 images and >1300 metagenomes from surface, deep chlorophyll maximum and mesopelagic seawater samples across 6 size fractions (<0.2-2000 µm). We use this approach to characterise the diversity, abundance, biovolume and distribution of symbiotic, colony-forming and particle-associated diazotrophs at a global scale. We show that imaging and PCR-free molecular data are congruent. Sequence reads indicate diazotrophs are detected from the ultrasmall bacterioplankton (<0.2 µm) to mesoplankton (180-2000 µm) communities, while images predict numerous symbiotic and colony-forming diazotrophs (>20 µm). Using imaging and molecular data, we estimate that polyploidy can substantially affect gene abundances of symbiotic versus colony-forming diazotrophs. Our results support the canonical view that larger diazotrophs (>10 µm) dominate the tropical belts, while unicellular cyanobacterial and non-cyanobacterial diazotrophs are globally distributed in surface and mesopelagic layers. We describe co-occurring diazotrophic lineages of different lifestyles and identify high-density regions of diazotrophs in the global ocean. Overall, we provide an update of marine diazotroph biogeographical diversity and present a new bioimaging-bioinformatic workflow.

Deep ocean metagenomes provide insight into the metabolic architecture of bathypelagic microbial communities.

The deep sea, the largest ocean's compartment, drives planetary-scale biogeochemical cycling. Yet, the functional exploration of its microbial communities lags far behind other environments. Here we analyze 58 metagenomes from tropical and subtropical deep oceans to generate the Malaspina Gene Database. Free-living or particle-attached lifestyles drive functional differences in bathypelagic prokaryotic communities, regardless of their biogeography. Ammonia and CO oxidation pathways are enriched in the free-living microbial communities and dissimilatory nitrate reduction to ammonium and H2 oxidation pathways in the particle-attached, while the Calvin Benson-Bassham cycle is the most prevalent inorganic carbon fixation pathway in both size fractions. Reconstruction of the Malaspina Deep Metagenome-Assembled Genomes reveals unique non-cyanobacterial diazotrophic bacteria and chemolithoautotrophic prokaryotes. The widespread potential to grow both autotrophically and heterotrophically suggests that mixotrophy is an ecologically relevant trait in the deep ocean. These results expand our understanding of the functional microbial structure and metabolic capabilities of the largest Earth aquatic ecosystem.

Exploring Microdiversity in Novel Kordia sp. (Bacteroidetes) with Proteorhodopsin from the Tropical Indian Ocean via Single Amplified Genomes.

Marine Bacteroidetes constitute a very abundant bacterioplankton group in the oceans that plays a key role in recycling particulate organic matter and includes several photoheterotrophic members containing proteorhodopsin. Relatively few marine Bacteroidetes species have been described and, moreover, they correspond to cultured isolates, which in most cases do not represent the actual abundant or ecologically relevant microorganisms in the natural environment. In this study, we explored the microdiversity of 98 Single Amplified Genomes (SAGs) retrieved from the surface waters of the underexplored North Indian Ocean, whose most closely related isolate is Kordia algicida OT-1. Using Multi Locus Sequencing Analysis (MLSA) we found no microdiversity in the tested conserved phylogenetic markers (16S rRNA and 23S rRNA genes), the fast-evolving Internal Transcribed Spacer and the functional markers proteorhodopsin and the beta-subunit of RNA polymerase. Furthermore, we carried out a Fragment Recruitment Analysis (FRA) with marine metagenomes to learn about the distribution and dynamics of this microorganism in different locations, depths and size fractions. This analysis indicated that this taxon belongs to the rare biosphere, showing its highest abundance after upwelling-induced phytoplankton blooms and sinking to the deep ocean with large organic matter particles. This uncultured Kordia lineage likely represents a novel Kordia species (Kordia sp. CFSAG39SUR) that contains the proteorhodopsin gene and has a widespread spatial and vertical distribution. The combination of SAGs and MLSA makes a valuable approach to infer putative ecological roles of uncultured abundant microorganisms.

A myovirus encoding both photosystem I and II proteins enhances cyclic electron flow in infected Prochlorococcus cells.

Cyanobacteria are important contributors to primary production in the open oceans. Over the past decade, various photosynthesis-related genes have been found in viruses that infect cyanobacteria (cyanophages). Although photosystem II (PSII) genes are common in both cultured cyanophages and environmental samples 1-4 , viral photosystem I (vPSI) genes have so far only been detected in environmental samples 5,6 . Here, we have used a targeted strategy to isolate a cyanophage from the tropical Pacific Ocean that carries a PSI gene cassette with seven distinct PSI genes (psaJF, C, A, B, K, E, D) as well as two PSII genes (psbA, D). This cyanophage, P-TIM68, belongs to the T4-like myoviruses, has a prolate capsid, a long contractile tail and infects Prochlorococcus sp. strain MIT9515. Phage photosynthesis genes from both photosystems are expressed during infection, and the resultant proteins are incorporated into membranes of the infected host. Moreover, photosynthetic capacity in the cell is maintained throughout the infection cycle with enhancement of cyclic electron flow around PSI. Analysis of metagenomic data from the Tara Oceans expedition 7 shows that phages carrying PSI gene cassettes are abundant in the tropical Pacific Ocean, composing up to 28% of T4-like cyanomyophages. They are also present in the tropical Indian and Atlantic Oceans. P-TIM68 populations, specifically, compose on average 22% of the PSI-gene-cassette carrying phages. Our results suggest that cyanophages carrying PSI and PSII genes are likely to maintain and even manipulate photosynthesis during infection of their Prochlorococcus hosts in the tropical oceans.

Viral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition.

A unique collection of oceanic samples was gathered by the Tara Oceans expeditions (2009-2013), targeting plankton organisms ranging from viruses to metazoans, and providing rich environmental context measurements. Thanks to recent advances in the field of genomics, extensive sequencing has been performed for a deep genomic analysis of this huge collection of samples. A strategy based on different approaches, such as metabarcoding, metagenomics, single-cell genomics and metatranscriptomics, has been chosen for analysis of size-fractionated plankton communities. Here, we provide detailed procedures applied for genomic data generation, from nucleic acids extraction to sequence production, and we describe registries of genomics datasets available at the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena). The association of these metadata to the experimental procedures applied for their generation will help the scientific community to access these data and facilitate their analysis. This paper complements other efforts to provide a full description of experiments and open science resources generated from the Tara Oceans project, further extending their value for the study of the world's planktonic ecosystems.