Atelocyanobacterium Research Articles

Eddy-driven diazotroph distribution in the subtropical North Atlantic: horizontal variability prevails over particle sinking speed

Mesoscale eddies influence the distribution of diazotrophic (nitrogen-fixing) cyanobacteria, impacting marine productivity and carbon export. Non-cyanobacterial diazotrophs (NCDs) are emerging as potential contributors to marine nitrogen fixation, relying on organic matter particles for resources, impacting nitrogen and carbon cycling. However, their diversity and biogeochemical importance remain poorly understood. In the subtropical North Atlantic along a single transect, this study explored the horizontal and vertical spatial variability of NCDs associated with suspended, slow-sinking, and fast-sinking particles collected with a marine snow catcher. The investigation combined amplicon sequencing with hydrographic and biogeochemical data. Cyanobacterial diazotrophs and NCDs were equally abundant, and their diversity was explained by the structure of the eddy. The unicellular symbiotic cyanobacterium UCYN-A was widespread across the eddy, whereas Trichodesmium and Crocosphaera accumulated at outer fronts. The diversity of particle-associated NCDs varied more horizontally than vertically. NCDs constituted most reads in the fast-sinking fractions, mainly comprising Alphaproteobacteria, whose abundance significantly differed from the suspended and slow-sinking fractions. Horizontally, Gammaproteobacteria and Betaproteobacteria exhibited inverse distributions, influenced by physicochemical characteristics of water intrusions at the eddy periphery. Niche differentiations across the anticyclonic eddy underscored NCD-particle associations and mesoscale dynamics, deepening our understanding of their ecological role and impact on ocean biogeochemistry.

Nitrogen-fixing organelle in a marine alga.

Symbiotic interactions were key to the evolution of chloroplast and mitochondria organelles, which mediate carbon and energy metabolism in eukaryotes. Biological nitrogen fixation, the reduction of abundant atmospheric nitrogen gas (N2) to biologically available ammonia, is a key metabolic process performed exclusively by prokaryotes. Candidatus Atelocyanobacterium thalassa, or UCYN-A, is a metabolically streamlined N2-fixing cyanobacterium previously reported to be an endosymbiont of a marine unicellular alga. Here we show that UCYN-A has been tightly integrated into algal cell architecture and organellar division and that it imports proteins encoded by the algal genome. These are characteristics of organelles and show that UCYN-A has evolved beyond endosymbiosis and functions as an early evolutionary stage N2-fixing organelle, or "nitroplast."

Distribution of the N2 -fixing cyanobacterium Candidatus Atelocyanobacterium thalassa in the Mexican Pacific upwelling system under two contrasting El Niño Southern Oscillation conditions.

The unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) is a key diazotroph in the global ocean owing to its high N2 fixation rates and wide distribution in marine environments. Nevertheless, little is known about UCYN-A in oxygen-deficient zones (ODZs), which may be optimal environments for marine diazotrophy. Therefore, the distribution and diversity of UCYN-A were studied in two consecutive years under contrasting phases (La Niña vs. El Niño) of El Niño Southern Oscillation (ENSO) along a transect in the ODZ of the Mexican Pacific upwelling system. Of the three UCYN-A sublineages found, UCYN-A1 and UCYN-A3 were barely detected, whereas UCYN-A2 was dominant in all the stations and showed a wide distribution in both ENSO phases. The presence of UCYN-A was associated with well-oxygenated waters, but it was also found for the first time under suboxic conditions (<20 μM) at the bottom of a shallow coastal station, within the oxygen-poor and nutrient-rich Subsurface Subtropical water mass. This study contributes to the understanding of UCYN-A distribution under different oceanographic conditions associated with ENSO phases in upwelling systems, especially because of the current climate change and increasing deoxygenation in many areas of the world's oceans.

Composition and distribution of diazotrophs in the Baltic Sea

Nitrogen (N2) fixation rates in the brackish Baltic Sea are among the highest per unit of area in the world. However, beyond the filamentous heterocyst-forming cyanobacteria, knowledge about the composition and distribution of N2-fixing microbes (diazotrophs) is limited. To address this, we investigated nitrogenase gene (nifH) composition and expression at coastal (<10 km offshore) and offshore (>10 km offshore) stations, at surface (avg. 1.8 m) and at depth (avg. 24 m) and in free-living (0.2–3.0 μm) and particle-associated size fractions (>3 μm). Surprisingly, nifH genes affiliated with Pseudanabaena and non-cyanobacterial diazotrophs (NCDs) dominated the composition whereas filamentous heterocyst-forming cyanobacteria accounted for almost 80% of the nifH transcripts. Salinity had a minor influence on the composition, but Aphanizomenon and Nodularia showed increased relative nifH gene expression at low and higher salinity, respectively. Pseudanabaena only accounted for up to 5% of the nifH transcripts and nifH gene expression by Candidatus Atelocyanobacterium thalassa (sublineage UCYN-A2) was mainly observed in the most saline western part of the Baltic. The only notable expression by NCDs (up to 15% of nifH transcripts at a given station) coincided with an upwelling event at the southern coast and was largely accounted for by a Pseudomonas-like nifH phylotype, recurrently found in the Baltic Sea. NCD relative abundances were dominant in coastal stations, presumably driven by sediment resuspension as evidenced by higher turbidity and DOC levels and the recovery of sediment diazotrophs in the pelagic zone. This study reveals the heterogeneity of the composition and activity of diazotrophs in the Baltic Sea, and underscores the need for future N2 fixation studies that include coastal and offshore Baltic waters.

Microevolutionary patterns in ecotypes of the symbiotic cyanobacterium UCYN-A revealed from a Northwest Atlantic coastal time series.

UCYN-A is a globally important nitrogen-fixing symbiotic microbe often found in colder regions and coastal areas where nitrogen fixation has been overlooked. We present a 3-year coastal Northwest Atlantic time series of UCYN-A by integrating oceanographic data with weekly nifH and16S rRNA gene sequencing and quantitative PCR assays for UCYN-A ecotypes. High UCYN-A relative abundances dominated by A1 to A4 ecotypes reoccurred annually in the coastal Northwest Atlantic. Although UCYN-A was detected every summer/fall, the ability to observe separate ecotypes may be highly dependent on sampling time given intense interannual and weekly variability of ecotype-specific occurrences. Additionally, much of UCYN-A's rarer diversity was populated by short-lived neutral mutational variants, therefore providing insight into UCYN-A's microevolutionary patterns. For instance, rare ASVs exhibited community composition restructuring annually, while also sharing a common connection to a dominant ASV within each ecotype. Our study provides additional perspectives for interpreting UCYN-A intraspecific diversity and underscores the need for high-resolution datasets when deciphering spatiotemporal ecologies within UCYN-A.

Symbiotic UCYN-A strains co-occurred with El Niño, relaxed upwelling, and varied eukaryotes over 10 years off Southern California

Biological nitrogen fixation, the conversion of N2 gas into a bioavailable form, is vital to sustaining marine primary production. Studies have shifted beyond traditionally studied tropical diazotrophs. Candidatus Atelocyanobacterium thalassa (or UCYN-A) has emerged as a focal point due to its streamlined metabolism, intimate partnership with a haptophyte host, and broad distribution. Here, we explore the environmental parameters that govern UCYN-A’s presence at the San Pedro Ocean Time-series (SPOT), its host specificity, and statistically significant interactions with non-host eukaryotes from 2008-2018. 16S and 18S rRNA gene sequences were amplified by “universal primers” from monthly samples and resolved into Amplicon Sequence Variants, allowing us to observe multiple UCYN-A symbioses. UCYN-A1 relative abundances increased following the 2015-2016 El Niño event. This “open ocean ecotype” was present when coastal upwelling declined, and Ekman transport brought tropical waters into the region. Network analyses reveal all strains of UCYN-A co-occur with dinoflagellates including Lepidodinium, a potential predator, and parasitic Syndiniales. UCYN-A2 appeared to pair with multiple hosts and was not tightly coupled to its predominant host, while UCYN-A1 maintained a strong host-symbiont relationship. These biological relationships are particularly important to study in the context of climate change, which will alter UCYN-A distribution at regional and global scales.

Distribution and survival strategies of endemic and cosmopolitan diazotrophs in the Arctic Ocean

Dinitrogen (N2) fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent studies have shown that N2 fixation also occurs in the polar regions and thus is a global process, although the physiological and ecological characteristics of polar diazotrophs are not yet known. Here, we successfully reconstructed diazotroph genomes, including that of cyanobacterium UCYN-A (Candidatus ‘Atelocyanobacterium thalassa’), from metagenome data corresponding to 111 samples isolated from the Arctic Ocean. These diazotrophs were highly abundant in the Arctic Ocean (max., 1.28% of the total microbial community), suggesting that they have important roles in the Arctic ecosystem and biogeochemical cycles. Further, we show that diazotrophs within genera Arcobacter, Psychromonas, and Oceanobacter are prevalent in the <0.2 µm fraction in the Arctic Ocean, indicating that current methods cannot capture their N2 fixation. Diazotrophs in the Arctic Ocean were either Arctic-endemic or cosmopolitan species from their global distribution patterns. Arctic-endemic diazotrophs, including Arctic UCYN-A, were similar to low-latitude-endemic and cosmopolitan diazotrophs in genome-wide function, however, they had unique gene sets (e.g., diverse aromatics degradation genes), suggesting adaptations to Arctic-specific conditions. Cosmopolitan diazotrophs were generally non-cyanobacteria and commonly had the gene that encodes the cold-inducible RNA chaperone, which presumably makes their survival possible even in deep, cold waters of global ocean and polar surface waters. This study shows global distribution pattern of diazotrophs with their genomes and provides clues to answering the question of how diazotrophs can inhabit polar waters.

Open ocean and coastal strains of the N2-fixing cyanobacterium UCYN-A have distinct transcriptomes.

Decades of research on marine N2 fixation focused on Trichodesmium, which are generally free-living cyanobacteria, but in recent years the endosymbiotic cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) has received increasing attention. However, few studies have shed light on the influence of the host versus the habitat on UCYN-A N2 fixation and overall metabolism. Here we compared transcriptomes from natural populations of UCYN-A from oligotrophic open-ocean versus nutrient-rich coastal waters, using a microarray that targets the full genomes of UCYN-A1 and UCYN-A2 and known genes for UCYN-A3. We found that UCYN-A2, usually regarded as adapted to coastal environments, was transcriptionally very active in the open ocean and appeared to be less impacted by habitat change than UCYN-A1. Moreover, for genes with 24 h periodic expression we observed strong but inverse correlations among UCYN-A1, A2, and A3 to oxygen and chlorophyll, which suggests distinct host-symbiont relationships. Across habitats and sublineages, genes for N2 fixation and energy production had high transcript levels, and, intriguingly, were among the minority of genes that kept the same schedule of diel expression. This might indicate different regulatory mechanisms for genes that are critical to the symbiosis for the exchange of nitrogen for carbon from the host. Our results underscore the importance of N2 fixation in UCYN-A symbioses across habitats, with consequences for community interactions and global biogeochemical cycles.

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.

Coastal upwelling enhances abundance of a symbiotic diazotroph (UCYN-A) and its haptophyte host in the Arctic Ocean

The apparently obligate symbiosis between the diazotroph Candidatus Atelocyanobacterium thalassa (UCYN-A) and its haptophyte host, Braarudosphaera bigelowii, has recently been found to fix dinitrogen (N2) in polar waters at rates (per cell) comparable to those observed in the tropical/subtropical oligotrophic ocean basins. This study presents the novel observation that this symbiosis increased in abundance during a wind-driven upwelling event along the Alaskan Beaufort shelfbreak. As upwelling relaxed, the relative abundance of B. bigelowii among eukaryotic phytoplankton increased most significantly in waters over the upper slope. As the host’s nitrogen demands are believed to be supplied primarily by UCYN-A, this response suggests that upwelling may enhance N2 fixation as displaced coastal waters are advected offshore, potentially extending the duration of upwelling-induced phytoplankton blooms. Given that such events are projected to increase in intensity and number with ocean warming, upwelling-driven N2 fixation as a feedback on climate merits investigation.

Salinity as a key control on the diazotrophic community composition in the southern Baltic Sea

Abstract. Over the next decade, the Baltic Sea is predicted to undergo severe changes including decreased salinity due to altered precipitation related to climate changes. This will likely impact the distribution and community composition of Baltic Sea dinitrogen-fixing (N2-fixing) microbes, among which heterocystous cyanobacteria are especially adapted to low salinities and may expand to waters with currently higher salinity, including the Danish Strait and Kattegat, while other high-salinity-adapted N2 fixers might decrease in abundance. In order to explore the impact of salinity on the distribution and activity of different diazotrophic clades, we followed the natural salinity gradient from the eastern Gotland and Bornholm basins through the Arkona Basin to the Kiel Bight and combined N2 fixation rate measurements with a molecular analysis of the diazotrophic community using the key functional marker gene for N2 fixation nifH, as well as the key functional marker genes anfD and vnfD, encoding for the two alternative nitrogenases. We detected N2 fixation rates between 0.7 and 6 nmol N L−1 d−1, and the diazotrophic community was dominated by the cyanobacterium related to Nodularia spumigena and the small unicellular, cosmopolitan cyanobacterium UCYN-A. Nodularia was present in gene abundances between 8.07 × 105 and 1.6 × 107 copies L−1 in waters with salinities of 10 and below, while UCYN-A reached gene abundances of up to 4.5 × 107 copies L−1 in waters with salinity above 10. Besides those two cyanobacterial diazotrophs, we found several clades of proteobacterial N2 fixers and alternative nitrogenase genes associated with Rhodopseudomonas palustris, a purple non-sulfur bacterium. Based on principal component analysis (PCA), salinity was identified as the primary parameter describing the diazotrophic distribution, while pH and temperature did not have a significant influence on the diazotrophic distribution. While this statistical analysis will need to be explored in direct experiments, it gives an indication for the future development of diazotrophy in a freshening Baltic Sea with UCYN-A retracting to more saline North Sea waters and heterocystous cyanobacteria expanding as salinity decreases.

Integrating Stochastic and Deterministic Process in the Biogeography of N2-Fixing Cyanobacterium Candidatus Atelocyanobacterium Thalassa.

UCYN-A is one of the most widespread and important marine diazotrophs. Its unusual distribution in both cold/warm and coastal/oceanic waters challenges current understanding about what drives the biogeography of diazotrophs. This study assessed the community assembly processes of the nitrogen-fixing cyanobacterium UCYN-A, developing a framework of assembly processes underpinning the microbial biogeography and diversity. High-throughput sequencing and a qPCR approach targeting the nifH gene were used to investigate three tropical seas: the Bay of Bengal, the Western Pacific Ocean, and the South China Sea. Based on the neutral community model and two types of null models calculating the β-nearest taxon index and the normalized stochasticity ratio, we found that stochastic assembly processes could explain 66–92% of the community assembly; thus, they exert overwhelming influence on UCYN-A biogeography and diversity. Among the deterministic processes, temperature and coastal/oceanic position appeared to be the principal environmental factors driving UCYN-A diversity. In addition, a close linkage between assembly processes and UCYN-A abundance/diversity/drivers can provide clues for the unusual global distribution of UCYN-A.

Light and depth dependency of nitrogen fixation by the non-photosynthetic, symbiotic cyanobacterium UCYN-A.

SummaryThe symbiotic cyanobacterium UCYN‐A is one of the most globally abundant marine dinitrogen (N2)‐fixers, but cultures have not been available and its biology and ecology are poorly understood. We used cultivation‐independent approaches to investigate how UCYN‐A single‐cell N2 fixation rates (NFRs) and nifH gene expression vary as a function of depth and photoperiod. Twelve‐hour day/night incubations showed that UCYN‐A only fixed N2 during the day. Experiments conducted using in situ arrays showed a light‐dependence of NFRs by the UCYN‐A symbiosis, with the highest rates in surface waters (5–45 m) and lower rates at depth (≥ 75 m). Analysis of NFRs versus in situ light intensity yielded a light saturation parameter (I k ) for UCYN‐A of 44 μmol quanta m−2 s−1. This is low compared with other marine diazotrophs, suggesting an ecological advantage for the UCYN‐A symbiosis under low‐light conditions. In contrast to cell‐specific NFRs, nifH gene‐specific expression levels did not vary with depth, indicating that light regulates N2 fixation by UCYN‐A through processes other than transcription, likely including host–symbiont interactions. These results offer new insights into the physiology of the UCYN‐A symbiosis in the subtropical North Pacific Ocean and provide clues to the environmental drivers of its global distributions.

Elucidation of trophic interactions in an unusual single-cell nitrogen-fixing symbiosis using metabolic modeling.

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A (Candidatus Atelocyanobacterium thalassa). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems.

Biological nitrogen fixation detected under Antarctic sea ice

Nitrogen fixation is the primary source of reactive nitrogen in the ocean. Most ecological models do not predict nitrogen fixation in the Antarctic Ocean because of the low availability of iron and high abundance of nitrogen. Here we extensively examined nitrogen fixation in the Antarctic Ocean, and found substantial nitrogen fixation (maximum: 44.4 nmol N l−1 d−1) near the Antarctic coast, especially around ice-covered regions. The nitrogenase gene (nifH) was detected at all coastal stations, including stations where no nitrogen fixation was found. At the stations where nitrogen fixation was detected, the nitrogen-fixing cyanobacterium UCYN-A (Candidatus ‘Atelocyanobacterium thalassa’) dominated nifH gene expression, and the nifH sequence was identical to that of the major oligotype in tropical and subtropical oceans. Our results suggest that marine nitrogen fixation is a ubiquitous process in the global ocean, and that UCYN-A is the keystone species for making it possible. Observational evidence of cyanobacterial activity in the Antarctic Ocean suggests that nitrogen fixation could be a ubiquitous process in the global ocean.

Unexpected presence of the nitrogen-fixing symbiotic cyanobacterium UCYN-A in Monterey Bay, California.

In the last decade, the known biogeography of nitrogen fixation in the ocean has been expanded to colder and nitrogen‐rich coastal environments. The symbiotic nitrogen‐fixing cyanobacteria group A (UCYN‐A) has been revealed as one of the most abundant and widespread nitrogen‐fixers, and includes several sublineages that live associated with genetically distinct but closely related prymnesiophyte hosts. The UCYN‐A1 sublineage is associated with an open ocean picoplanktonic prymnesiophyte, whereas UCYN‐A2 is associated with the coastal nanoplanktonic coccolithophore Braarudosphaera bigelowii, suggesting that different sublineages may be adapted to different environments. Here, we study the diversity of nifH genes present at the Santa Cruz Municipal Wharf in the Monterey Bay (MB), California, and report for the first time the presence of multiple UCYN‐A sublineages, unexpectedly dominated by the UCYN‐A2 sublineage. Sequence and quantitative PCR data over an 8‐year time‐series (2011–2018) showed a shift toward increasing UCYN‐A2 abundances after 2013, and a marked seasonality for this sublineage which was present during summer‐fall months, coinciding with the upwelling‐relaxation period in the MB. Increased abundances corresponded to positive temperature anomalies in MB, and we discuss the possibility of a benthic life stage of the associated coccolithophore host to explain the seasonal pattern. The dominance of UCYN‐A2 in coastal waters of the MB underscores the need to further explore the habitat preference of the different sublineages in order to provide additional support for the hypothesis that UCYN‐A1 and UCYN‐A2 sublineages are different ecotypes.

Transcriptional Patterns of Biogeochemically Relevant Marker Genes by Temperate Marine Bacteria.

Environmental microbial gene expression patterns remain largely unexplored, particularly at interannual time scales. We analyzed the variability in the expression of marker genes involved in ecologically relevant biogeochemical processes at a temperate Atlantic site over two consecutive years. Most of nifH transcripts, involved in nitrogen (N) fixation, were affiliated with the symbiotic cyanobacterium Candidatus Atelocyanobacterium thalassa, suggesting a key role as N providers in this system. The expression of nifH and amoA (i.e., marker for ammonia oxidation) showed consistent maxima in summer and autumn, respectively, suggesting a temporal succession of these important N cycling processes. The patterns of expression of genes related to the oxidation of carbon monoxide (coxL) and reduced sulfur (soxB) were different from that of amoA, indicating alternate timings for these energy conservation strategies. We detected expression of alkaline phosphatases, induced under phosphorus limitation, in agreement with the reported co-limitation by this nutrient at the study site. In contrast, low-affinity phosphate membrane transporters (pit) typically expressed under phosphorus luxury conditions, were mainly detected in post-bloom conditions. Rhodobacteraceae dominated the expression of soxB, coxL and ureases, while Pelagibacteraceae dominated the expression of proteorhodopsins. Bacteroidetes and Gammaproteobacteria were major contributors to the uptake of inorganic nutrients (pit and amt transporters). Yet, in autumn, Thauma- and Euryarchaeota unexpectedly contributed importantly to the uptake of ammonia and phosphate, respectively. We provide new hints on the active players and potential dynamics of ecologically relevant functions in situ, highlighting the potential of metatranscriptomics to provide significant input to future omics-driven marine ecosystem assessment.

Evidence of high N&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; fixation rates in the temperate northeast Atlantic

Abstract. Diazotrophic activity and primary production (PP) were investigated along two transects (Belgica BG2014/14 and GEOVIDE cruises) off the western Iberian Margin and the Bay of Biscay in May 2014. Substantial N2 fixation activity was observed at 8 of the 10 stations sampled, ranging overall from 81 to 384 µmol N m−2 d−1 (0.7 to 8.2 nmol N L−1 d−1), with two sites close to the Iberian Margin situated between 38.8 and 40.7∘ N yielding rates reaching up to 1355 and 1533 µmol N m−2 d−1. Primary production was relatively lower along the Iberian Margin, with rates ranging from 33 to 59 mmol C m−2 d−1, while it increased towards the northwest away from the peninsula, reaching as high as 135 mmol C m−2 d−1. In agreement with the area-averaged Chl a satellite data contemporaneous with our study period, our results revealed that post-bloom conditions prevailed at most sites, while at the northwesternmost station the bloom was still ongoing. When converted to carbon uptake using Redfield stoichiometry, N2 fixation could support 1 % to 3 % of daily PP in the euphotic layer at most sites, except at the two most active sites where this contribution to daily PP could reach up to 25 %. At the two sites where N2 fixation activity was the highest, the prymnesiophyte–symbiont Candidatus Atelocyanobacterium thalassa (UCYN-A) dominated the nifH sequence pool, while the remaining recovered sequences belonged to non-cyanobacterial phylotypes. At all the other sites, however, the recovered nifH sequences were exclusively assigned phylogenetically to non-cyanobacterial phylotypes. The intense N2 fixation activities recorded at the time of our study were likely promoted by the availability of phytoplankton-derived organic matter produced during the spring bloom, as evidenced by the significant surface particulate organic carbon concentrations. Also, the presence of excess phosphorus signature in surface waters seemed to contribute to sustaining N2 fixation, particularly at the sites with extreme activities. These results provide a mechanistic understanding of the unexpectedly high N2 fixation in productive waters of the temperate North Atlantic and highlight the importance of N2 fixation for future assessment of the global N inventory.

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.

Dynamics and interactions of highly resolved marine plankton via automated high-frequency sampling.

Short timescale observations are valuable for understanding microbial ecological processes. We assessed dynamics in relative abundance and potential activities by sequencing the small sub-unit ribosomal RNA gene (rRNA gene) and rRNA molecules (rRNA) of Bacteria, Archaea, and Eukaryota once to twice daily between March 2014 and May 2014 from the surface ocean off Catalina Island, California. Typically Ostreococcus, Braarudosphaera, Teleaulax, and Synechococcus dominated phytoplankton sequences (including chloroplasts) while SAR11, Sulfitobacter, and Fluviicola dominated non-phytoplankton Bacteria and Archaea. We observed short-lived increases of diatoms, mostly Pseudo-nitzschia and Chaetoceros, with quickly responding Bacteria and Archaea including Flavobacteriaceae (Polaribacter & Formosa), Roseovarius, and Euryarchaeota (MGII), notably the exact amplicon sequence variants we observed responding similarly to another diatom bloom nearby, 3 years prior. We observed correlations representing known interactions among abundant phytoplankton rRNA sequences, demonstrating the biogeochemical and ecological relevance of such interactions: (1) The kleptochloroplastidic ciliate Mesodinium 18S rRNA gene sequences and a single Teleaulax taxon (via 16S rRNA gene sequences) were correlated (Spearman r = 0.83) yet uncorrelated to a Teleaulax 18S rRNA gene OTU, or any other taxon (consistent with a kleptochloroplastidic or karyokleptic relationship) and (2) the photosynthetic prymnesiophyte Braarudosphaera bigelowii and two strains of diazotrophic cyanobacterium UCYN-A were correlated and each taxon was also correlated to other taxa, including B. bigelowii to a verrucomicrobium and a dictyochophyte phytoplankter (all r > 0.8). We also report strong correlations (r > 0.7) between various ciliates, bacteria, and phytoplankton, suggesting interactions via currently unknown mechanisms. These data reiterate the utility of high-frequency time series to show rapid microbial reactions to stimuli, and provide new information about in situ dynamics of previously recognized and hypothesized interactions.