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The 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 (Ik) 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. En prensa

Summary Close associations between single‐celled marine organisms can have a central role in biogeochemical processes and are of great interest for understanding the evolution of organisms. The global significance of such associations raises the question of whether unidentified associations are yet to be discovered. In this study, fluorescence‐activated cell sorted photosynthetic picoeukayote (PPE) populations and single cells were analysed by sequencing of 16S rRNA genes in the oligotrophic North Pacific Subtropical Gyre. Samples were collected during two cruises, spanning depths near the deep chlorophyll maximum, where the abundance of PPEs was highest. The association between the widespread and significant nitrogen (N2)‐fixing cyanobacterium, UCYN‐A and its prymnesiophyte host was prevalent in both population and single‐cell sorts. Several bacterial sequences, affiliating with previously described symbiotic taxa were detected but their detection was rare and not well replicated, precluding identification of novel tightly linked species‐specific associations. Similarly, no enrichment of dominant seawater taxa such as Prochlorococcus, SAR11 or Synechococcus was observed suggesting that these were not systematically ingested by the PPE in this study. The results indicate that apart from the UCYN‐A symbiosis, similar tight species‐specific associations with PPEs are unusual in the oligotrophic ocean.

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

Results of recent modelling efforts imply denitrification-influenced waters, such as those in the Eastern Tropical South Pacific (ETSP), may support high rates of biological nitrogen fixation (BNF), yet little is known about the N2 -fixing microbial community in this region. Our characterization of the ETSP diazotrophic community along a gradient from upwelling-influenced to oligotrophic waters did not detect cyanobacterial diazotrophs commonly found in other open ocean regions. Most of the nifH genes amplified by polymerase chain reaction (PCR) from DNA and RNA samples clustered with γ-proteobacterial nifH sequences, although a novel Trichodesmium phylotype was also recovered. Three quantitative PCR assays were developed to target γ-proteobacterial phylotypes, but all were found to be present at low abundances. An analysis of the expected BNF rates based on abundances and plausible cell-specific N2 fixation rates indicates that these γ-proteobacteria are unlikely to be responsible for previously reported BNF rates from corresponding samples. Therefore, the organisms responsible for the measured BNF rates remain poorly understood. Furthermore, there is little direct evidence, at this time, to support the hypothesis that heterotrophic N2 fixation contributes significantly to oceanic BNF rates based on our analysis of heterotrophic cell-specific N2 fixation rates required to explain BNF rates reported in previously published studies.

Summary Synechococcus is a cosmopolitan marine cyanobacterial genus, and is often the most abundant picocyanobacterial genus in coastal waters. Little is known about Synechococcus seasonal dynamics in coastal zones highly impacted by upwelling. This was investigated by collecting seasonal samples from an upwelling-impacted Monterey Bay (MB) monitoring station M0, in parallel with measurements of oceanographic conditions during 2006–2008. Synechococcus abundances were determined using quantitative PCR (qPCR) assays and flow cytometry (FCM). A new qPCR assay was designed to target dominant Synechococcus in MB using the rbcL gene, while previously designed assays targeted distinct phylotypes (called narB subgroups) with the narB gene. The rbcL qPCR assay successfully tracked abundant Synechococcus in MB, accounting for on average 89% (± 57%) of FCM-based counts. Annual spring upwelling caused decreases in Synechococcus and narB subgroup abundances. Differences in narB subgroup abundance maxima and abundance patterns support the view that subgroups differ in their ecologies, including subgroup D_C1, which seems to specifically thrive in coastal waters. Correlations between narB subgroup abundances and measured environmental variables were similar among the subgroups. Therefore, non-measured environmental factors (e.g. metals, mortality) likely had different influences on subgroups, which led to their distinct abundance patterns at M0.

The abundances of six N₂-fixing cyanobacterial phylotypes were profiled at 22 stations across the tropical Atlantic Ocean during June 2006, and used to model the contribution of the diazotrophs to N₂ fixation. Diazotroph abundances were measured by targeting the nifH gene of Trichodesmium, unicellular groups A, B, C (UCYN-A, UCYN-B and UCYN-C), and diatom-cyanobiont symbioses Hemiaulus-Richelia, Rhizosolenia-Richelia and Chaetoceros-Calothrix. West to east gradients in temperature, salinity and nutrients [NO₃⁻ + NO₂⁻, PO₄³⁻, Si(OH)₄] showed the influence of the Amazon River plume and its effect on the distributions of the diazotrophs. Trichodesmium accounted for more than 93% of all nifH genes detected, dominated the warmer waters of the western Atlantic, and was the only diazotroph detected at the equatorial upwelling station. UCYN-A was the next most abundant (> 5% of all nifH genes) and dominated the cooler waters of the eastern Atlantic near the Cape Verde Islands. UCYN-C was found at a single depth (200 m) of high salinity and low temperature and nutrients, whereas UCYN-B cells were widespread but in very low abundance (6.1 × 10¹ ± 4.6 × 10² gene copies l⁻¹). The diatom-cyanobionts were observed primarily in the western Atlantic within or near the high Si(OH)₄ input of the Amazon River plume. Overall, highest diazotroph abundances were observed at the surface and declined with depth, except for some subsurface peaks in Trichodesmium, UCYN-B and UCYN-A. Modelled contributions of Trichodesmium, UCYN-B and UCYN-A to total N₂ fixation suggested that Trichodesmium had the largest input, except for the potential of UCYN-A at the Cape Verde Islands.

Summary Mechanisms of dimethyl sulphide (DMS) and methanethiol (MT) production and consumption were determined in moderately hypersaline mats, Guerrero Negro, Mexico. Biological pathways regulated the net flux of DMS and MT as revealed by increases in flux resulting from decreased salinity, increased temperature and the removal of oxygen. Dimethylsulphoniopropionate (DMSP) was not present in these microbial mats and DMS and MT are probably formed by the reaction of photosynthetically produced low-molecular weight organic carbon and biogenic hydrogen sulphide derived from sulphate reduction. These observations provide an alternative to the notion that DMSP or S-containing amino acids are the dominant precursors of DMS in intertidal sediment systems. The major sink for DMS in the microbial mats was biological consumption, whereas photochemical oxidation to dimethylsulphoxide was the major sink for DMS in the overlying water column. Diel flux measurements demonstrated that significantly more DMS is released from the system during the night than during the day. The major consumers of DMS in the presence of oxygen were monooxygenase-utilizing bacteria, whereas under anoxic conditions, DMS was predominantly consumed by sulphate-reducing bacteria and methanethiol was consumed by methanogenic bacteria. Aerobic and anaerobic consumption rates of DMS were nearly identical. Mass balance estimates suggest that the consumption in the water column is likely to be smaller than net the flux from the mats. Volatile organic sulphur compounds are thus indicators of high rates of carbon fixation and sulphate reduction in these laminated sediment ecosystems, and atmospheric sulphur can be generated as a biogenic signature of the microbial mat community.

Results of recent modelling efforts imply denitrification-influenced waters, such as those in the Eastern Tropical South Pacific (ETSP), may support high rates of biological nitrogen fixation (BNF), yet little is known about the N2 -fixing microbial community in this region. Our characterization of the ETSP diazotrophic community along a gradient from upwelling-influenced to oligotrophic waters did not detect cyanobacterial diazotrophs commonly found in other open ocean regions. Most of the nifH genes amplified by polymerase chain reaction (PCR) from DNA and RNA samples clustered with γ-proteobacterial nifH sequences, although a novel Trichodesmium phylotype was also recovered. Three quantitative PCR assays were developed to target γ-proteobacterial phylotypes, but all were found to be present at low abundances. An analysis of the expected BNF rates based on abundances and plausible cell-specific N2 fixation rates indicates that these γ-proteobacteria are unlikely to be responsible for previously reported BNF rates from corresponding samples. Therefore, the organisms responsible for the measured BNF rates remain poorly understood. Furthermore, there is little direct evidence, at this time, to support the hypothesis that heterotrophic N2 fixation contributes significantly to oceanic BNF rates based on our analysis of heterotrophic cell-specific N2 fixation rates required to explain BNF rates reported in previously published studies.

Synechococcus is a cosmopolitan marine cyanobacterial genus, and is often the most abundant picocyanobacterial genus in coastal waters. Little is known about Synechococcus seasonal dynamics in coastal zones highly impacted by upwelling. This was investigated by collecting seasonal samples from an upwelling-impacted Monterey Bay (MB) monitoring station M0, in parallel with measurements of oceanographic conditions during 2006-2008. Synechococcus abundances were determined using quantitative PCR (qPCR) assays and flow cytometry (FCM). A new qPCR assay was designed to target dominant Synechococcus in MB using the rbcL gene, while previously designed assays targeted distinct phylotypes (called narB subgroups) with the narB gene. The rbcL qPCR assay successfully tracked abundant Synechococcus in MB, accounting for on average 89% (± 57%) of FCM-based counts. Annual spring upwelling caused decreases in Synechococcus and narB subgroup abundances. Differences in narB subgroup abundance maxima and abundance patterns support the view that subgroups differ in their ecologies, including subgroup D_C1, which seems to specifically thrive in coastal waters. Correlations between narB subgroup abundances and measured environmental variables were similar among the subgroups. Therefore, non-measured environmental factors (e.g. metals, mortality) likely had different influences on subgroups, which led to their distinct abundance patterns at M0.

The abundances of six N₂-fixing cyanobacterial phylotypes were profiled at 22 stations across the tropical Atlantic Ocean during June 2006, and used to model the contribution of the diazotrophs to N₂ fixation. Diazotroph abundances were measured by targeting the nifH gene of Trichodesmium, unicellular groups A, B, C (UCYN-A, UCYN-B and UCYN-C), and diatom-cyanobiont symbioses Hemiaulus-Richelia, Rhizosolenia-Richelia and Chaetoceros-Calothrix. West to east gradients in temperature, salinity and nutrients [NO₃⁻ + NO₂⁻, PO₄³⁻, Si(OH)₄] showed the influence of the Amazon River plume and its effect on the distributions of the diazotrophs. Trichodesmium accounted for more than 93% of all nifH genes detected, dominated the warmer waters of the western Atlantic, and was the only diazotroph detected at the equatorial upwelling station. UCYN-A was the next most abundant (5% of all nifH genes) and dominated the cooler waters of the eastern Atlantic near the Cape Verde Islands. UCYN-C was found at a single depth (200 m) of high salinity and low temperature and nutrients, whereas UCYN-B cells were widespread but in very low abundance (6.1 × 10¹ ± 4.6 × 10² gene copies l⁻¹). The diatom-cyanobionts were observed primarily in the western Atlantic within or near the high Si(OH)₄ input of the Amazon River plume. Overall, highest diazotroph abundances were observed at the surface and declined with depth, except for some subsurface peaks in Trichodesmium, UCYN-B and UCYN-A. Modelled contributions of Trichodesmium, UCYN-B and UCYN-A to total N₂ fixation suggested that Trichodesmium had the largest input, except for the potential of UCYN-A at the Cape Verde Islands.

To assess and study the heterogeneity of delta(13)C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the delta(13)C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal delta(13)C values (50 per thousand) in this microbial community. The delta(13)C of ANME-1 rods ranged from -24 per thousand to -87 per thousand. The delta(13)C of ANME-2 sarcina ranged from -18 per thousand to -75 per thousand. Initial measurements of shell aggregates were as heavy as -19.5 per thousand with none observed to be lighter than -57 per thousand. Subsequent measurements on shell aggregates trended lighter reaching values as (13)C-depleted as -73 per thousand. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more (13)C-depleted (with values as light as -56 per thousand). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean delta(13)C = -38 +/- 3 per thousand), suggesting that this bacterial morphotype was capable of rapid colonization and growth.

Methane release from the oceans is controlled in large part by syntrophic interactions between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (DSS), frequently found as organized consortia. An understanding of the specifics of this symbiotic relationship and the metabolic heterogeneity existing between and within individual methane-oxidizing aggregates is currently lacking. Here, we use the microanalytical method FISH-SIMS (fluorescence in situ hybridization-secondary ion mass spectrometry) to describe the physiological traits and anabolic activity of individual methanotrophic consortia, specifically tracking ^(15)N-labelled protein synthesis to examine the effects of organization and size on the metabolic activity of the syntrophic partners. Patterns of 15N distribution within individual aggregates showed enhanced ^(15)N assimilation in ANME-2 cells relative to the co-associated DSS revealing a decoupling in anabolic activity between the partners. Protein synthesis in ANME-2 cells was sustained throughout the core of individual ANME-2/DSS consortia ranging in size range from 4 to 20 μm. This indicates that metabolic activity of the methane-oxidizing archaea is not limited to, or noticeably enhanced at the ANME−2/DSS boundary. Overall, the metabolic activity of both syntrophic partners within consortia was greater than activity measured in representatives of the ANME-2 and DSS observed alone, with smaller ANME-2/DSS aggregates displaying a tendency for greater ^(15)N uptake and doubling times ranging from 3 to 5 months. The combination of ^(15)N-labelling and FISH-SIMS provides an important perspective on the extent of heterogeneity within methanotrophic aggregates and may aid in constraining predictive models of activity and growth by these syntrophic consortia.