Non-cyanobacterial Diazotrophs 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.

Fronts divide diazotroph communities in the Southern Indian Ocean.

Dinitrogen (N2) fixation represents a key source of reactive nitrogen in marine ecosystems. While the process has been rather well-explored in low latitudes of the Atlantic and Pacific Oceans, other higher latitude regions and particularly the Indian Ocean have been chronically overlooked. Here, we characterize N2 fixation and diazotroph community composition across nutrient and trace metals gradients spanning the multifrontal system separating the oligotrophic waters of the Indian Ocean subtropical gyre from the high nutrient low chlorophyll waters of the Southern Ocean. We found a sharp contrasting distribution of diazotroph groups across the frontal system. Notably, cyanobacterial diazotrophs dominated north of fronts, driving high N2 fixation rates (up to 13.96nmol N l-1 d-1) with notable peaks near the South African coast. South of the fronts non-cyanobacterial diazotrophs prevailed without significant N2 fixation activity being detected. Our results provide new crucial insights into high latitude diazotrophy in the Indian Ocean, which should contribute to improved climate model parameterization and enhanced constraints on global net primary productivity projections.

Heterogeneous Diazotroph Communities in the Subtropical‐Subantarctic Transition and Aphotic Zones Off the Coast of Patagonia, Eastern South Pacific Ocean

AbstractSubtropical‐subpolar transition water is a potential domain for N2 fixation, but the understanding of N2 fixation in such waters remains incomplete. We simultaneously examined the N2 fixation activity and community structures of diazotrophs and all prokaryotes from the surface to just above the seafloor off Patagonia in the transitional region of the eastern South Pacific Ocean. N2 fixation activity was not detected in the surface waters, but was observed sporadically and only in subpolar bathypelagic waters (>1,000 m) at very low rates (0.02–0.06 nmol N L−1 d−1). By contrast, the nifH gene, a key gene involved in N2 fixation, was detected widely from the surface to the bottom waters. The majority of diazotrophs were classified as non‐cyanobacterial diazotrophs (NCDs), and the nifH amino acid sequences of major diazotrophs were similar to sequences detected in the Southern Ocean, the aphotic zone and sediment of other oceans, and estuarine waters, suggesting that the NCDs are distributed across diverse marine environments. The overall prokaryotic communities were generally similar to those in other open ocean regions at the phylum level (class level for Proteobacteria) and differed among water depths. Diazotrophs, in contrast, showed vertical and horizontal heterogeneity below the euphotic zone and little association with water depth, indicating a lack of cohesion within the community, which may characterize diazotroph community in the transitional surface water and aphotic zones. Elucidating this community heterogeneity may provide pivotal information about N2 fixation in these waters.

Biofilm formation and cell plasticity drive diazotrophy in an anoxygenic phototrophic bacterium.

The contribution of non-cyanobacterial diazotrophs (NCDs) to total N2 fixation in the marine water column is unknown, but their importance is likely constrained by the limited availability of dissolved organic matter and low O2 conditions. Light could support N2 fixation and growth by NCDs, yet no examples from bacterioplankton exist. In this study, we show that the phototrophic NCD, Rhodopseudomonas sp. BAL398, which is a member of the diazotrophic community in the surface waters of the Baltic Sea, can utilize light. Our study highlights the significance of biofilm formation for utilizing light and fixing N2 under oxic conditions and the role of cell plasticity in regulating these processes. Our findings have implications for the general understanding of the ecology and importance of NCDs in marine waters.

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.

Global oceanic diazotroph database version 2 and elevated estimate of global oceanic N2 fixation

Abstract. Marine diazotrophs convert dinitrogen (N2) gas into bioavailable nitrogen (N), supporting life in the global ocean. In 2012, the first version of the global oceanic diazotroph database (version 1) was published. Here, we present an updated version of the database (version 2), significantly increasing the number of in situ diazotrophic measurements from 13 565 to 55 286. Data points for N2 fixation rates, diazotrophic cell abundance, and nifH gene copy abundance have increased by 184 %, 86 %, and 809 %, respectively. Version 2 includes two new data sheets for the nifH gene copy abundance of non-cyanobacterial diazotrophs and cell-specific N2 fixation rates. The measurements of N2 fixation rates approximately follow a log-normal distribution in both version 1 and version 2. However, version 2 considerably extends both the left and right tails of the distribution. Consequently, when estimating global oceanic N2 fixation rates using the geometric means of different ocean basins, version 1 and version 2 yield similar rates (43–57 versus 45–63 Tg N yr−1; ranges based on one geometric standard error). In contrast, when using arithmetic means, version 2 suggests a significantly higher rate of 223±30 Tg N yr−1 (mean ± standard error; same hereafter) compared to version 1 (74±7 Tg N yr−1). Specifically, substantial rate increases are estimated for the South Pacific Ocean (88±23 versus 20±2 Tg N yr−1), primarily driven by measurements in the southwestern subtropics, and for the North Atlantic Ocean (40±9 versus 10±2 Tg N yr−1). Moreover, version 2 estimates the N2 fixation rate in the Indian Ocean to be 35±14 Tg N yr−1, which could not be estimated using version 1 due to limited data availability. Furthermore, a comparison of N2 fixation rates obtained through different measurement methods at the same months, locations, and depths reveals that the conventional 15N2 bubble method yields lower rates in 69 % cases compared to the new 15N2 dissolution method. This updated version of the database can facilitate future studies in marine ecology and biogeochemistry. The database is stored at the Figshare repository (https://doi.org/10.6084/m9.figshare.21677687; Shao et al., 2022).

Seasonal and spatial patterns in diazotroph community composition at Station ALOHA

Dinitrogen (N2) fixation is carried out by specialized microbes, called diazotrophs, and is a major source of nitrogen supporting primary production in oligotrophic oceans. One of the best-characterized diazotroph habitats is the North Pacific Subtropical Gyre (NPSG), where warm, chronically N-limited surface waters promote year-round N2 fixation. At Station ALOHA (A Long-Term Oligotrophic Habitat Assessment) in the NPSG, N2 fixation is typically ascribed to conspicuous, filamentous cyanobacterial diazotrophs (Trichodesmium and Richelia), unicellular free-living Crocosphaera, and the UCYN-A/haptophyte symbiosis, based on using microscopy and quantitative PCR (qPCR). However, the diazotroph community in this ecosystem is diverse and includes non-cyanobacterial diazotrophs (NCDs). We investigated the diversity, depth distributions, and seasonality of diazotroph communities at Stn. ALOHA using high throughput sequencing (HTS) of nifH gene fragments from samples collected throughout the euphotic zone (0-175 m) at near-monthly intervals from June 2013 to July 2016. The UCYN-A symbioses and Trichodesmium sp. consistently had the highest relative abundances and seasonal patterns that corroborated qPCR-based analyses. Other prevalent community members included a new Crocosphaera-like species, and several NCDs affiliated with γ- and δ-proteobacteria. Notably, some of the NCDs appear to be stable components of the community at Stn. ALOHA, having also been reported in prior studies. Depth and temporal patterns in microdiversity within two major diazotroph groups (Trichodesmium and UCYN-A) suggested that sub-populations are adapted to time- and depth-dependent environmental variation. A network analysis of the upper euphotic (0-75 m) HTS data identified two modules that reflect a diazotroph community structure with seasonal turnover between UCYN-A/Gamma A, and Trichodesmium/Crocosphaera. It also reveals the seasonality of several important cyanobacteria and NCDs about which little is known, including a putative δ-proteobacterial phylotype originally discovered at Stn. ALOHA. Collectively, these results underscore the importance of coupling nifH gene HTS with other molecular techniques to obtain a comprehensive view of diazotroph community composition in the marine environment and reveal several understudied diazotroph groups that may contribute to N2 fixation in the NPSG.

Nitrogen-fixing sulfate reducing bacteria in shallow coastal sediments under simulated resuspension

Nitrogen (N2) fixation by heterotrophic non-cyanobacterial diazotrophs is common in marine deep-sea sediments. However, in shallow coastal areas, where resuspension of sediments is extensive, the magnitude of sediment-associated N2 fixation during resuspension is unknown. We examined nitrogen fixation in dark slurry incubations with sediments (0–5 cm and 0–10 cm depths) under simulated resuspension from five shallow stations (water depth <1 m) and an anoxic site in the Bay of Gdansk (109 m) in the Baltic Sea. Abiotic variables and the composition of nitrogen fixing organisms (diazotrophs) were measured at the study sites. To estimate the contribution of nitrogen fixing sulfate reducing bacteria to total nitrogen fixation, parallel incubations with sodium molybdate as inhibitor were performed. Our data show low but variable nitrogen fixation rates (n.d. - 23.7 nmol N g−1 d−1), promoted in small-grained sediments associated with increased organic carbon content and high nutrient concentrations in pore waters. Highest nitrogen fixation at the shallow sites was encountered in the upper 0–5 cm of the sediments while rates were negligible below. Sulfate reducing bacteria (e.g. Desulfobacterales and Desulfovibrionales) were responsible for most of the heterotrophic nitrogen fixation and appear as key players for pelagic N2 fixation during resuspension. Our study reveals an important sediment – water coupling, which may be accentuated by the increased storms and resuspension events predicted for the Baltic Sea region.

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

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

Cell-specific measurements show nitrogen fixation by particle-attached putative non-cyanobacterial diazotrophs in the North Pacific Subtropical Gyre

Biological nitrogen fixation is a major important source of nitrogen for low-nutrient surface oceanic waters. Nitrogen-fixing (diazotrophic) cyanobacteria are believed to be the primary contributors to this process, but the contribution of non-cyanobacterial diazotrophic organisms in oxygenated surface water, while hypothesized to be important, has yet to be demonstrated. In this study, we used simultaneous 15N-dinitrogen and 13C-bicarbonate incubations combined with nanoscale secondary ion mass spectrometry analysis to screen tens of thousands of mostly particle-associated, cell-like regions of interest collected from the North Pacific Subtropical Gyre. These dual isotope incubations allow us to distinguish between non-cyanobacterial and cyanobacterial nitrogen-fixing microorganisms and to measure putative cell-specific nitrogen fixation rates. With this approach, we detect nitrogen fixation by putative non-cyanobacterial diazotrophs in the oxygenated surface ocean, which are associated with organic-rich particles (<210 µm size fraction) at two out of seven locations sampled. When present, up to 4.1% of the analyzed particles contain at least one active putative non-cyanobacterial diazotroph. The putative non-cyanobacterial diazotroph nitrogen fixation rates (0.76 ± 1.60 fmol N cell−1 d−1) suggest that these organisms are capable of fixing dinitrogen in oxygenated surface water, at least when attached to particles, and may contribute to oceanic nitrogen fixation.

Genetic and physiological insights into the diazotrophic activity of a non-cyanobacterial marine diazotroph.

Nitrogen (N2 ) fixation, or diazotrophy, supports a large part of primary production in oceans. Culture-independent approaches highlighted the presence in abundance of marine non-cyanobacterial diazotrophs (NCD), but their ecophysiology remains elusive, mostly because of the low number of isolated NCD and because of the lack of available genetic tools for these isolates. Here, a dual genetic and functional approach allowed unveiling the ecophysiology of a marine NCD affiliated to the species Vibrio diazotrophicus. Physiological characterization of the first marine NCD mutant obtained so far was performed using a soft-gellan assay, demonstrating that a ΔnifH mutant is not able to grow in nitrogen-free media. Furthermore, we demonstrated that V. diazotrophicus produces a thick biofilm under diazotrophic conditions, suggesting biofilm production as an adaptive response of this NCD to cope with the inhibition of nitrogen fixation by molecular oxygen. Finally, the genomic signature of V. diazotrophicus is essentially absent from metagenomic data of Tara Ocean expeditions, despite having been isolated from various marine environments. We think that the genetically tractable V. diazotrophicus strain used in this study may serve as an ideal model to study the ecophysiology of these overlooked procaryotic group.

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

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

Controlling factors on the global distribution of a representative marine non-cyanobacterial diazotroph phylotype (Gamma A)

Abstract. Non-cyanobacterial diazotrophs may be contributors to global marine N2 fixation, although the factors controlling their distribution are unclear. Here, we explored what controls the distribution of the most sampled non-cyanobacterial diazotroph phylotype, Gamma A, in the global ocean. First, we represented Gamma A abundance by its nifH quantitative polymerase chain reaction (qPCR) copies reported in the literature and analyzed its relationship to climatological biological and environmental conditions. There was a positive correlation between the Gamma A abundance and local net primary production (NPP), and the maximal observed Gamma A abundance increased with NPP and became saturated when NPP reached ∼ 400 mg C m−2 d−1. Additionally, an analysis using a multivariate generalized additive model (GAM) revealed that the Gamma A abundance increased with light intensity but decreased with increasing iron concentration. The GAM also showed a weak but significant positive relationship between Gamma A abundance and silicate concentration, as well as a substantial elevation of Gamma A abundance when the nitrate concentration was very high (≳ 10 µM). Using the GAM, these climatological factors together explained 43 % of the variance in the Gamma A abundance. Second, in addition to the climatological background, we found that Gamma A abundance was elevated in mesoscale cyclonic eddies in high-productivity (climatological NPP &gt; 400 mg m−2 d−1) regions, implying that Gamma A can respond to mesoscale features and benefit from nutrient inputs. Overall, our results suggest that Gamma A tends to inhabit ocean environments with high productivity and low iron concentrations and therefore provide insight into the niche differentiation of Gamma A from cyanobacterial diazotrophs, which are generally most active in oligotrophic ocean regions and need a sufficient iron supply, although both groups prefer well-lit surface waters. More sampling on Gamma A and other non-cyanobacterial diazotroph phylotypes is needed to reveal the controlling mechanisms of heterotrophic N2 fixation in the ocean.

Planktonic Aggregates as Hotspots for Heterotrophic Diazotrophy: The Plot Thickens.

Biological dinitrogen (N2) fixation is performed solely by specialized bacteria and archaea termed diazotrophs, introducing new reactive nitrogen into aquatic environments. Conventionally, phototrophic cyanobacteria are considered the major diazotrophs in aquatic environments. However, accumulating evidence indicates that diverse non-cyanobacterial diazotrophs (NCDs) inhabit a wide range of aquatic ecosystems, including temperate and polar latitudes, coastal environments and the deep ocean. NCDs are thus suspected to impact global nitrogen cycling decisively, yet their ecological and quantitative importance remain unknown. Here we review recent molecular and biogeochemical evidence demonstrating that pelagic NCDs inhabit and thrive especially on aggregates in diverse aquatic ecosystems. Aggregates are characterized by reduced-oxygen microzones, high C:N ratio (above Redfield) and high availability of labile carbon as compared to the ambient water. We argue that planktonic aggregates are important loci for energetically-expensive N2 fixation by NCDs and propose a conceptual framework for aggregate-associated N2 fixation. Future studies on aggregate-associated diazotrophy, using novel methodological approaches, are encouraged to address the ecological relevance of NCDs for nitrogen cycling in aquatic environments.

Long-term warming effects on the microbiome and nifH gene abundance of a common moss species in sub-Arctic tundra.

Bacterial communities form the basis of biogeochemical processes and determine plant growth and health. Mosses harbour diverse bacterial communities that are involved in nitrogen fixation and carbon cycling. Global climate change is causing changes in aboveground plant biomass and shifting species composition in the Arctic, but little is known about the response of moss microbiomes in these environments. Here, we studied the total and potentially active bacterial communities associated with Racomitrium lanuginosum in response to a 20-yr in situ warming in an Icelandic heathland. We evaluated the effect of warming and warming-induced shrub expansion on the moss bacterial community composition and diversity, and nifH gene abundance. Warming changed both the total and the potentially active bacterial community structure, while litter abundance only affected the total bacterial community structure. The abundance of nifH genes was negatively affected by litter abundance. We also found shifts in the potentially nitrogen-fixing community, with Nostoc decreasing and noncyanobacterial diazotrophs increasing in relative abundance. Our data suggest that the moss microbial community and potentially nitrogen fixing taxa will be sensitive to future warming, partly via changes in litter and shrub abundance.

Some light on diazotrophs

Nitrogen Fixation About half of the planet's nitrogen fixation occurs out at sea by prokaryote diazotrophs, yet we still have a poor understanding of which ones do what. Karlusich et al. applied a combination of image capture and nitrogenase gene ( nifH ) sequencing on globally gathered data to map diazotrophs sampled at different depths. Unexpected hotspots of diazotroph abundance were discovered in the Mozambique Channel and the south Pacific Ocean. However, in the Arctic Ocean, nitrogen fixers were found among ultrasmall bacterioplankton, whereas in the tropics, larger cyanobacterial symbionts and colony-dwelling species such as Trichodesmium and Richelia dominated. Although single-cell, free-living, noncyanobacterial diazotrophs were the most abundant overall, Trichodesmium dominated by sheer size; however, several species tended to co-occur in assemblies. Whether or how such assemblages interact will have to await further study. Nat. Commun. 12 , 4160 (2021).

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

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

Gamma4: a genetically versatile Gammaproteobacterial nifH phylotype that is widely distributed in the North Pacific Ocean.

Despite the increasing reports of non-cyanobacterial diazotrophs (NCDs) in pelagic waters, only one NCD (GammaA) has been relatively well described, whose genome and physiology are still unclear. Here we present a comprehensive analysis of the biogeography and ecophysiology of a widely distributed NCD, Gamma4. Gamma4 was the most abundant Gammaproteobacterial NCD along transects across the subtropical North Pacific. Using quantitative PCR, Gamma4 was detectable throughout the surface waters of North Pacific (7°N-55°N, 138°E-80°W), whereas GammaA was detected at <2/3 of the stations. Gamma4 was abundant during autumn-winter and positively correlated with chlorophyll a, while GammaA thrived during spring-summer and was positively correlated with temperature. Environmental clones affiliated with Gamma4 were widely detected in pelagic waters, oxygen minimum zones and even dinoflagellate microbiomes. By analysing the metabolic potential of a genome of Gamma4 reconstructed from the Tara Oceans dataset, we suggest that Gamma4 is a versatile heterotrophic NCD equipped with multiple strategies in scavenging phosphate (and iron) and for respiratory protection of nitrogenase. The transcription of nitrogenase genes is putatively regulated by Fnr-NifL-NifA and GlnD-GlnK systems that respond to intracellular oxygen and glutamate concentration. These results provide important implications for the potential life strategies of pelagic NCDs.

Temperate southern Australian coastal waters are characterised by surprisingly high rates of nitrogen fixation and diversity of diazotrophs.

Biological dinitrogen (N2) fixation is one mechanism by which specific microorganisms (diazotrophs) can ameliorate nitrogen (N) limitation. Historically, rates of N2 fixation were believed to be limited outside of the low nutrient tropical and subtropical open ocean; however, emerging evidence suggests that N2 fixation is also a significant process within temperate coastal waters. Using a combination of amplicon sequencing, targeting the nitrogenase reductase gene (nifH), quantitative nifH PCR, and 15N2 stable isotope tracer experiments, we investigated spatial patterns of diazotroph assemblage structure and N2 fixation rates within the temperate coastal waters of southern Australia during Austral autumn and summer. Relative to previous studies in open ocean environments, including tropical northern Australia, and tropical and temperate estuaries, our results indicate that high rates of N2 fixation (10–64 nmol L−1 d−1) can occur within the large inverse estuary Spencer Gulf, while comparatively low rates of N2 fixation (2 nmol L−1 d−1) were observed in the adjacent continental shelf waters. Across the dataset, low concentrations of NO3/NO2 were significantly correlated with the highest N2 fixation rates, suggesting that N2 fixation could be an important source of new N in the region as dissolved inorganic N concentrations are typically limiting. Overall, the underlying diazotrophic community was dominated by nifH sequences from Cluster 1 unicellular cyanobacteria of the UCYN-A clade, as well as non-cyanobacterial diazotrophs related to Pseudomonas stutzeri, and Cluster 3 sulfate-reducing deltaproteobacteria. Diazotroph community composition was significantly influenced by salinity and SiO4 concentrations, reflecting the transition from UCYN-A-dominated assemblages in the continental shelf waters, to Cluster 3-dominated assemblages in the hypersaline waters of the inverse estuary. Diverse, transitional diazotrophic communities, comprised of a mixture of UCYN-A and putative heterotrophic bacteria, were observed at the mouth and southern edge of Spencer Gulf, where the highest N2 fixation rates were observed. In contrast to observations in other environments, no seasonal patterns in N2 fixation rates and diazotroph community structure were apparent. Collectively, our findings are consistent with the emerging view that N2 fixation within temperate coastal waters is a previously overlooked dynamic and potentially important component of the marine N cycle.

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

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