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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 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.

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 (

15 pages, 7 figures, supplementary information https://doi.org/10.1038/s42003-021-02112-2.-- All data generated or analyzed during this study are included in this published article (and its supplementary information files). All raw sequences are publicly available at both DOE’s JGI Integrated Microbial Genomes and Microbiomes (IMG/MER) and the European Nucleotide Archive (ENA). Individual metagenome assemblies, annotation files, and alignment files can be accessed at IMG/MER. All accession numbers are listed in Supplementary Data 1. The co-assembly for the MAG dataset construction can be found through ENA at https://www.ebi.ac.uk/ena with accession number PRJEB40454, the nucleotide sequence for each MAG and their annotation files can be found through BioStudies at https://www.ebi.ac.uk/biostudies with accession S-BSST457 and also in the companion website to this manuscript at https://malaspina-public.gitlab.io/malaspina-deep-ocean-microbiome/.-- All software used in this work is publicly available distributed by their respective developers, and it is described in “Methods”, including the versions and options used. Additional custom scripts to assign taxonomy to the M-geneDB genes and to filter and format FRA results are available through BioStudies at https://www.ebi.ac.uk/biostudies with accession S-BSST457, 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, This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) through the Consolider-Ingenio program (Malaspina 2010 Expedition, ref. CSD2008-00077). The sequencing of 58 bathypelagic metagenomes was done by the U.S. Department of Energy Joint Genome Institute, supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02 05CH11231 to SGA (CSP 612 “Microbial metagenomics and transcriptomics from a global deep-ocean expedition”). Additional funding was provided by the project MAGGY (CTM2017-87736-R) to S.G.A. from the Spanish Ministry of Economy and Competitiveness, Grup de Recerca 2017SGR/1568 from Generalitat de Catalunya, and King Abdullah University of Science and Technology (KAUST) under contract OSR #3362 and by funding of the EMFF Program of the European Union (MERCLUB project, Grant Agreement 863584). The ICM researchers have had the institutional support of the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S). High-Performance computing analyses were run at the Marine Bioinformatics Service (MARBITS, https://marbits.icm.csic.es) of the Institut de Ciències del Mar (ICM-CSIC), Barcelona, Supercomputing Center (Grant BCV-2013-2-0001) and KAUST’s Ibex HPC

Diazotrophs are widespread microorganisms that regulate marine productivity in 60% of our oceans by alleviating nitrogen limitation. Yet, their contribution to organic carbon and nitrogen export fluxes has never been quantified, making an assessment of their impact on the biological carbon pump impossible. Here, we examine species-specific fates of several groups of globally-distributed unicellular (UCYN) and filamentous diazotrophs in the mesopelagic ocean. We used an innovative approach consisting of the combined deployment of surface-tethered drifting sediment traps, Marine Snow Catcher, and Bottle-net, in which we performed nifH sequencing and quantitative PCR on major diazotroph groups across the subtropical South Pacific Ocean. nifH sequencing data from sediment traps deployed at 170 m, 270 m and 1000 m provide clear evidence that cyanobacterial and non-cyanobacterial diazotrophs are systematically present in sinking particles down to 1000 m, with export fluxes being the highest for the UCYN-A1 symbiosis, followed by UCYN-B or Trichodesmium (depending on station and depth), Gamma A and UCYN-C. Specific export turnover rates (a metric similar to the export efficiency adapted to organisms) point to a more efficient export of UCYN groups relative to the filamentous Trichodesmium. This is further confirmed by Marine Snow catcher data showing that the proportion of sinking cells was significantly higher for UCYN compared to Trichodesmium. Phycoerythrin-containing UCYN-B and UCYN-C-like cells were indeed recurrently found embedded in large (> 50 micrometers) seemingly organic aggregates, or organized into clusters of tens to hundreds of cells linked by an extracellular matrix, facilitating the export. Overall, diazotrophs accounted for 6-13% (170 m) to 45-100% (1000 m) of the total particulate nitrogen export fluxes in our study. We thus conclude that diazotrophs are important contributors to carbon sequestration in the subtropical South Pacific Ocean and need to be considered in future studies to improve the accuracy of current regional and global estimates of export.

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.

The unicellular N2-fixing cyanobacteria UCYN-A live in symbiosis with haptophytes in theBraarudosphaera bigelowiilineage. 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, N2fixation of the symbiont and photosynthesis of the host. In this system, it is thought that the host photosynthesis supports the high energetic cost of N2fixation, 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 N2fixation (single cell N2fixation rates),nifHgene transcription, and UCYN-A2 cell division (fluorescentin situhybridization andnifHgene abundances). The results showed that the light period is critical for maintenance of regular patterns of gene expression, N2fixation 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.

This article is contribution number 118 of Tara Oceans.-- 18 pages, 10 figures, supplementary information https://doi.org/10.1038/s41467-021-24299-y.-- Data availability: The authors declare that all the data supporting the findings of this study are publicly available in the following repositories and in the supplementary information files of this paper. The contextual data are available in Pangaea (www.pangaea.de)21 with the identifier https://doi.org/10.1594/PANGAEA.875582. Flow cytometry data23,108 are available in Mendeley Data (https://data.mendeley.com) with the identifier https://doi.org/10.17632/p9r9wttjkm.2. The confocal microscopy30 and UVP533 images annotated as diazotrophs in the current work and their corresponding metadata were submitted to the EMBL-EBI repository BioStudies (www.ebi.ac.uk/biostudies) under accession S-BSST529; while the whole image databases and their metadata are available at Ecotaxa (confocal microscopy of 5–20 µm sized-fractionated samples: https://ecotaxa.obs-vlfr.fr/prj/3365; confocal microscopy of 20–180 μm sized-fractionated samples: https://ecotaxa.obs-vlfr.fr/prj/2274; UVP5: https://ecotaxa.obs-vlfr.fr/prj/579). Tara Oceans metagenomes22,24,25 are archived at ENA under the accession numbers: PRJEB1787, PRJEB1788, PRJEB4352, PRJEB4419, PRJEB9691, PRJEB9740, and PRJEB9742. The nifH and recA catalogs were compiled from Integrated Microbial Genomes (IMG, https://img.jgi.doe.gov/); Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP; https://github.com/dib-lab/dib-MMETSP); OM-Reference Gene Catalog version 2 (OM-RGC-v2, https://www.ocean-microbiome.org/); Tara Oceans assemblies (Supplementary Table 8 from ref. 16); and the nifH database (version April 2014) curated and hosted at Zehr Lab, University of California, Santa Cruz, CA, USA (https://www.jzehrlab.com/nifh). The 10 nifH sequences generated in the clone libraries of the current work were submitted to ENA under the accession numbers: MW590317–MW590326. Source data are provided with this paper, 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 (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, This work has been supported by the FFEM - French Facility for Global Environment, French Government ‘Investissements d’Avenir’ programs OCEANOMICS (ANR-11-BTBR-0008), FRANCE GENOMIQUE (ANR-10-INBS-09-08), MEMO LIFE (ANR-10-LABX-54), and PSL Research University (ANR-11-IDEX-0001-02). R.A.F. acknowledges funding from Knut and Alice Wallenberg foundation. C.B. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (Diatomic; grant agreement No. 835067) and Agence Nationale de la Recherche “Phytomet” (ANR-16-CE01-0008) projects. F.M.C.-C. acknowledges funding from the European Union’s Horizon 2020 research program under the Marie Sklodowska-Curie grant agreement No. 749380 (UCYN2PLAST). S.G.A. acknowledges funding from the project “MAGGY” (CTM2017-87736-R) from the Spanish Ministry of Economy and Competitiveness and the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S). J.J.P.K. acknowledges postdoctoral funding from the Fonds Français pour l’Environnement Mondial

The deep sea, the largest compartment of the ocean, is an essential component of the Earth system, but the functional exploration of its microbial communities lags far behind that of other marine realms. Here we analyze 58 bathypelagic microbial metagenomes from the Atlantic, Indian, and Pacific Oceans in an unprecedented sampling effort from the Malaspina Global Expedition, to resolve the metabolic architecture of the deep ocean microbiome. The Malaspina Deep-Sea Gene Collection, 71% of which consists of novel genes, reveals a strong dichotomy between the functional traits of free-living and particle-attached microorganisms, and shows relatively patchy composition challenging the paradigm of a uniform dark ocean ecosystem. Metagenome Assembled Genomes uncovered 11 potential new phyla, establishing references for deep ocean microbial taxa, and revealed mixotrophy to be a widespread trophic strategy in the deep ocean. These results expand our understanding of the functional diversity, metabolic versatility, and carbon cycling in the largest ecosystem on Earth.One Sentence SummaryA whole community genomic survey of the deep microbiome sheds light on the microbial and functional diversity of the dark ocean.

17 pages, 5 figures, 3 tables, supplemental material https://doi.org/10.1128/AEM.02659-18.-- Data availability.All sequences generated in this study can be accessed through European Nucleotide Archive (ENA) under the study accession number PRJEB27291 and accession numbers ERR2639355 to ERR2639364, 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, This study was supported by the Argentinean Network for the Assessment and Monitoring of Pampean Shallow Lakes (PAMPA2–CONICET), the ANPCyT (PICT-2014-1290 and PICT-2016-1079), and by MAGGY (grant CTM2017-87736-R) to S. G. Acinas and REMEI (grant CTM2015-70340-R) to J. M. Gasol from the Spanish Ministry of Economy and Competitiveness (MINECO)

Teira, Eva ... et. al.-- 9 pages, 6 figures, 2 tables, 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 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, This study was supported by the by the Spanish Ministry of Science and Innovation through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077) and project DIFUNCAR (CTM2008-03790/MAR)

This protocol is part of Nucleic acids preparationsforViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition.

This protocol describes the sequencing and data quality control for theTaraOceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition. Figure 3:Overview of experimental pipeline from nucleic acids to sequences. (Red crosses highlight QC steps where experiments can be stopped.)

This protocol describes the sequencing library preparation for theTaraOceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.

This protocol describes the handling of genomics samples for the Tara Oceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from the Tara Oceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.

The protocols in this collection are from the Alberti A., et al manuscript (Alberti A. 2017, Scientific Data). These protocols 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). This collection complements other efforts to provide a full description of experiments and open science resources generated from theTaraOceans project, further extending their value for the study of the world’s planktonic ecosystems.' From the Methods section: 'The generation of information-rich data from marine plankton samples presents unique challenges that are inherent to the particular sampling conditions at sea and the wide spectrum of organisms included in that environment. All processing steps, including biomass collection, sample preservation, nucleic acids extractions and sequencing library preparation, are critical and require specific protocols and robust methods in order to ensure comparability of results and limit potential biases. Our methods were either developed specifically forTaraOceans samples or carefully selected among existing ones in order to meet the requirements of our sequencing strategy and to produce optimized datasets for downstream bioinformatics analyses, as for example the production of overlapping reads from metagenomics libraries to facilitate assembly. They are presented in five sub-sections, starting with a brief description of how samples were handled between the research vessel and the processing laboratories (protocol 1). Protocol 2reports on DNA and RNA extractions procedures for -omics analyses, including the generation of amplified genomic DNA from uncultured isolated unicellular eukaryotes. The generation of 18S and 16S rRNA amplicons from DNA of specific size-fractions is described in protocol 3 and Illumina libraries preparation in protocol 4. Sequencing procedures and post-sequencing data processing are described in protocol 5. For details on the onboard sampling protocols, see Pesantet al.'

This protocol describes thenucleic acids preparations for theTaraOceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.

This protocol describes the 18S and 16S rRNA genes amplicon generation for eukaryotic and prokaryotic metabarcoding for theTaraOceans expedition and is part ofViral to metazoan marine plankton nucleotide sequences from theTaraOceans expedition. Figure 1: Overview of -omics analysis strategy applied on Tara Oceans samples.

13 pages, 6 figures, 1 table, supplementary Information http://www.nature.com/ismej/journal/v10/n3/suppinfo/ismej2015137s1.html, The deep-sea is the largest biome of the biosphere, and contains more than half of the whole ocean’s microbes. Uncovering their general patterns of diversity and community structure at a global scale remains a great challenge, as only fragmentary information of deep-sea microbial diversity exists based on regional-scale studies. Here we report the first globally comprehensive survey of the prokaryotic communities inhabiting the bathypelagic ocean using high-throughput sequencing of the 16S rRNA gene. This work identifies the dominant prokaryotes in the pelagic deep ocean and reveals that 50% of the operational taxonomic units (OTUs) belong to previously unknown prokaryotic taxa, most of which are rare and appear in just a few samples. We show that whereas the local richness of communities is comparable to that observed in previous regional studies, the global pool of prokaryotic taxa detected is modest (~3600 OTUs), as a high proportion of OTUs are shared among samples. The water masses appear to act as clear drivers of the geographical distribution of both particle-attached and free-living prokaryotes. In addition, we show that the deep-oceanic basins in which the bathypelagic realm is divided contain different particle-attached (but not free-living) microbial communities. The combination of the aging of the water masses and a lack of complete dispersal are identified as the main drivers for this biogeographical pattern. All together, we identify the potential of the deep ocean as a reservoir of still unknown biological diversity with a higher degree of spatial complexity than hitherto considered, This work was supported by the Spanish Ministry of Science and Innovation through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077). [...] Sequencing at the Joint Genome Intitute was supported by US Department of Energy Joint Genome Intitute 2011 Microbes Program grant CSP 602 grant to SGA. The work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract No. DE-AC02–05CH11231. [...] Additional funding was provided by Spanish Ministry of Science and Innovation grant CGL2011–26848/BOS MicroOcean PANGENOMICS and the Spanish Ministry of Economy and Competitivity grant MALASPINOMICS (CTM2011–15461-E). GS and FMCC held PhD JAE-Predoc (CSIC) and FPI (MICINN) fellowships respectively and SGA was supported by FP7-OCEAN-2011 project ‘Micro B3’

The symbiotic unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) is one of the most abundant and widespread nitrogen (N

Royo-Llonch, Marta ... et al.-- 14 pages, 6 figures, supplementary material http://journal.frontiersin.org/article/10.3389/fmicb.2017.01317/full#supplementary-material, 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, Funding was provided by the Spanish Ministry of Economy and Competitivity grants CTM2013-48292-C3 “EcoBGM” to CP-A and JG, United States NSF grants OCE-1232982 and OCE-1335810 to RS and CGL2011-26848/BOS MicroOcean PANGENOMICS to SGA, as well as grant BIOSENSOMICS through “Ayudas Fundación BBVA a investigadores y creadores culturales” to SGA. MR-L held a Ph.D. Fellowship FPI (BES-2014-068285) funded by the Spanish Ministry of Economy and Competitivity.

Fridman, Svetlana ... et al.-- This is contribution number 54 of Tara Oceans.-- 8 pages, 4 figures, supplementary material https://dx.doi.org/10.1038/s41564-017-0002-9, 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 , viral photosystem I (vPSI) genes have so far only been detected in environmental samples . 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 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, This work was funded by a European Commission ERC Advanced Grant (no. 321647), the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA Grant Agreement No. 317184, an Israel Science Foundation grant (no. 580/10) and the Louis and Lyra Richmond Memorial Chair in Life Sciences to O.B., a European Commission ERC starting grant (no. 203406) to D.L. and the Technion’s Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering and the Russell Berrie Nanotechnology Institute.

Special issue: Marine Microbes.-- This is Contribution No. 16 of the TARA Oceans expedition 2009–2012.-- 13 pages, 5 figures, additional supporting information http://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12517/suppinfo

14 pages, 6 figures, 1 table, A marine symbiosis has been recently discovered between prymnesiophyte species and the unicellular diazotrophic cyanobacterium UCYN-A. At least two different UCYN-A phylotypes exist, the clade UCYN-A1 in symbiosis with an uncultured small prymnesiophyte and the clade UCYN-A2 in symbiosis with the larger Braarudosphaera bigelowii. We targeted the prymnesiophyte–UCYN-A1 symbiosis by double CARD-FISH (catalyzed reporter deposition-fluorescence in situ hybridization) and analyzed its abundance in surface samples from the MALASPINA circumnavigation expedition. Our use of a specific probe for the prymnesiophyte partner allowed us to verify that this algal species virtually always carried the UCYN-A symbiont, indicating that the association was also obligate for the host. The prymnesiophyte–UCYN-A1 symbiosis was detected in all ocean basins, displaying a patchy distribution with abundances (up to 500 cells ml− 1) that could vary orders of magnitude. Additional vertical profiles taken at the NE Atlantic showed that this symbiosis occupied the upper water column and disappeared towards the Deep Chlorophyll Maximum, where the biomass of the prymnesiophyte assemblage peaked. Moreover, sequences of both prymnesiophyte partners were searched within a large 18S rDNA metabarcoding data set from the Tara-Oceans expedition around the world. This sequence-based analysis supported the patchy distribution of the UCYN-A1 host observed by CARD-FISH and highlighted an unexpected homogeneous distribution (at low relative abundance) of B. bigelowii in the open ocean. Our results demonstrate that partners are always in symbiosis in nature and show contrasted ecological patterns of the two related lineages, Financial support has been provided by the Spanish Ministry of Economy and Competitivity through project Consolider-Ingenio Malaspina 2010 (CSD2008-00077) to CMD, FLAME (CGL2010-16304) to RM, and PANGENOMICS (CGL2011-26848/BOS) to SGA. AMC was recipient of a Spanish FPI grant (BES-2009-027194)

Farrant, Gregory K. ... et al.-- 10 pages, 5 figures.-- Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. KU377785-990, KU670814-6, KU705397-460, and KU937818-30).-- This article is contribution number 41 of Tara Oceans, 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, This work was supported by the French “Agence Nationale de la Recherche” Programs SAMOSA (ANR-13-ADAP-0010) and France Génomique (ANR-10-INBS-09), the French Government “Investissements d’Avenir ”Program OCEANOMICS (ANR-11-BTBR-0008), UK Natural Environment Research Council Grants NE/I00985X/1 and NE/J02273X/1, and the European Union’s Seventh Framework Programs FP7 MicroB3 (Grant 287589) and MaCuMBA (Grant 311975)

Roitman, Sheila ... et. al.-- 9 pages, 4 figures, supporting information http://dx.doi.org/10.1111/1462-2920.13036.-- This is contribution number 30 of the Tara Oceans, 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, This work was supported by funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA Grant Agreement No. 317184, European Commission ERC Advanced Grant No. 321647, and Israel Science Foundation Grant No. 580/10

Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic zone interactome using environmental factors and organismal abundance profiles and found that environmental factors are incomplete predictors of community structure. We found associations across plankton functional types and phylogenetic groups to be nonrandomly distributed on the network and driven by both local and global patterns. We identified interactions among grazers, primary producers, viruses, and (mainly parasitic) symbionts and validated network-generated hypotheses using microscopy to confirm symbiotic relationships. We have thus provided a resource to support further research on ocean food webs and integrating biological components into ocean models.

Lima-Mendez, Gipsi ... et. al.-- 9 pages, 4 figures, 1 table, supplementary materials www.sciencemag.org/content/348/6237/1262073/suppl/DC1, Species interaction networks are shaped by abiotic and biotic factors. Here, as part of the Tara Oceans project, we studied the photic zone interactome using environmental factors and organismal abundance profiles and found that environmental factors are incomplete predictors of community structure. We found associations across plankton functional types and phylogenetic groups to be nonrandomly distributed on the network and driven by both local and global patterns. We identified interactions among grazers, primary producers, viruses, and (mainly parasitic) symbionts and validated networkgenerated hypotheses using microscopy to confirm symbiotic relationships. We have thus provided a resource to support further research on ocean food webs and integrating biological components into ocean models. © 2015, American Association for the Advancement of Science. All rights reserved, We thank the commitment of the following people and sponsors:Centre National de la Recherche Scientifique (CNRS) (in particular, Groupement de Recherche GDR3280), European Molecular Biology Laboratory (EMBL), Genoscope/CEA, VIB, Stazione Zoologica Anton Dohrn, UNIMIB, Fund for Scientific Research – Flanders (G.L.M., K.F., S.C., and J.R.), Rega Institute (J.R.), KU Leuven(J.R.), The French Ministry of Research, the French Government “Investissements d’Avenir” programmes OCEANOMICS (ANR-11-BTBR-0008), FRANCE GENOMIQUE (ANR-10-INBS-09-08), MEMO LIFE (ANR-10-LABX-54), PSL* Research University (ANR-11-IDEX-0001-02), ANR (projects POSEIDON/ANR-09-BLAN-0348, PHYTBACK/ANR-2010-1709-01, PROMETHEUS/ANR-09-PCSGENM- 217, TARA GIRUS/ANR-09-PCS-GENM-218, European Union FP7 (MicroB3/No.287589, IHMS/HEALTH-F4-2010-261376, ERC Advanced Grant Awards to CB (Diatomite: 294823), Gordon and Betty Moore Foundation grant (3790) to M.B.S., Spanish Ministry of Science and Innovation grant CGL2011-26848/BOS MicroOcean PANGENOMICS to S.G.A., TANIT (CONES 2010-0036) from the Agència de Gestió d´Ajusts Universitaris i Reserca funded to S.G.A., JSPS KAKENHI grant number 26430184 to H.O., FWO, BIO5, Biosphere 2, Agnès b., the Veolia Environment Foundation, Region Bretagne, Lorient Agglomeration, World Courier, Illumina, the EDF Foundation, FRB, the Prince Albert II de Monaco Foundation, Etienne Bourgois, the Tara schooner, and its captain and crew. We are also grateful to the French Ministry of Foreign Affairs for supporting the expedition and to the countries that graciously granted sampling permissions. Tara Oceans would not exist without continuous support from 23 institutes (http://oceans.taraexpeditions.org)

15 pages, 5 figures, data accessibility, supporting information http://dx.doi.org/10.1111/mec.13419, 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 (, This research was funded by the Spanish Ministry of Economy and Competitiveness Science and Innovation through the Consolider-Ingenio programme (project Malaspina 2010 Expedition, ref. CSD2008–00077. [...] Sequencing at the JGI was supported by U.S. Department of Energy (DOE) JGI 2011 Microbes Program grant CSP 387 grant to S.G.A. The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. [...] Additional funding was provided by the Spanish Ministry of Science and Innovation grant CGL2011-26848/BOS MicroOcean PANGENOMICS and the Spanish Ministry of Economy and Competitivity grant MALASPINOMICS (CTM2011-15461-E). GS and FMCC held a Ph.D. JAE-Predoc (CSIC) and an FPI (MICINN) fellowships, respectively. SGA was supported by a project FP7-OCEAN-2011 ‘Micro B3’

Lara, Elena ... et. al.-- 26 pages, 5 figures, 1 table, supporting information https://doi.org/10.1371/journal.pone.0114829, Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9%16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new ‘rare virosphere’ phage–host model system, EL was a recipient of the FPI-predoc fellowship from the Spanish Ministry of Science and Innovation (MICINN). SGA was supported by a Ramon y Cajal contract from MICINN and by MicroB3 (FP7-OCEAN-2011). KH was supported by a postdoctoral fellowship from the Swedish Research Council (623-2010-6548). This work was supported by grants MICROVIS (CTM2007-62140) and MEFISTO (CTM2013-43767-P) to DV, MICRODIVERSITY (CGL2008-00762/BOS) and PANGENOMICS CGL2011-26848/BOS) to SGA from the MICINN, a Gordon and Betty Moore Foundation (grant #2631) to MBS

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.

Summary 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 (mitags) 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 (m454tags; 13 metagenomes, ca. 2.4 Gb) and 16S rDNA amplicon (454tags; ca. 0.075 Gb) approaches. Our results indicate that by overcoming PCR biases related to amplification and primer mismatch, mitags may provide more realistic estimates of community richness and evenness than amplicon 454tags. In addition, mitags can capture expected beta diversity patterns. Using mitags 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.

Eric Karsenti et al.-- 5 pages, 2 figures, With biology becoming quantitative, systems-level studies can now be performed at spatial scales ranging from molecules to ecosystems. Biological data generated consistently across scales can be integrated with physico-chemical contextual data for a truly holistic approach, with a profound impact on our understanding of life [1]–[5]. Marine ecosystems are crucial in the regulation of Earth's biogeochemical cycles and climate [6],[7]. Yet their organization, evolution, and dynamics remain poorly understood [8],[9]. The Tara Oceans project was launched in September 2009 for a 3-year study of the global ocean ecosystem aboard the ship Tara. A unique sampling programme encompassing optical and genomic methods to describe viruses, bacteria, archaea, protists, and metazoans in their physico-chemical environment has been implemented. Starting as a grassroots initiative of a few scientists, the project has grown into a global consortium of over 100 specialists from diverse disciplines, including oceanography, microbial ecology, genomics, molecular, cellular, and systems biology, taxonomy, bioinformatics, data management, and ecosystem modeling. This multidisciplinary community aims to generate systematic, open access datasets usable for probing the morphological and molecular makeup, diversity, evolution, ecology, and global impacts of plankton on the Earth system, We are keen to thank the commitment of the following people and sponsors who made this singular expedition possible: CNRS, EMBL, Genoscope/CEA, VIB, Stazione Zoologica Anton Dohrn, UNIMIB, ANR (projects POSEIDON, BIOMARKS, PROMETHEUS, and TARA-GIRUS), FWO, BIO5, Biosphere 2, agnès b., the Veolia Environment Foundation, Region Bretagne, World Courier, Illumina, Cap L'Orient, the EDF Foundation EDF Diversiterre, FRB, the Prince Albert II de Monaco Foundation, Etienne Bourgois, the Tara schooner and its captain and crew. Tara Oceans would not exist without the continuous support of the participating 23 institutes (see http://oceans.taraexpeditions.org). Special thanks also goes to Fabrice Not and Noan Le Bescot for creating the figures of this manuscript. This is contribution number 0001 of the Tara Oceans Expedition 2009–2012

Cyanobacterial diazotrophs are considered to be the most important source of fixed N 2 in the open ocean. Biological N 2 fixation is catalyzed by the extremely O 2 -sensitive nitrogenase enzyme. In cyanobacteria without specialized N 2 -fixing cells (heterocysts), mechanisms such as decoupling photosynthesis from N 2 fixation in space or time are involved in protecting nitrogenase from the intracellular O 2 evolved by photosynthesis. However, it is not known how cyanobacterial cells limit O 2 diffusion across their membranes to protect nitrogenase in ambient O 2 -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 O 2 diffusion barrier. We found that, whereas all non−heterocyst-forming cyanobacterial diazotrophs had hopanoid synthesis genes, none of the marine Synechococcus , Prochlorococcus (non−N 2 -fixing), and marine heterocyst-forming (N 2 -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 O 2 . This membrane property coupled with high respiration rates to decrease intracellular O 2 concentration may therefore explain how non−heterocyst-forming cyanobacterial diazotrophs can fix N 2 in the fully oxic surface ocean.