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

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

Diversity, Genomics, and Distribution of Phytoplankton-Cyanobacterium Single-Cell Symbiotic Associations.

Cyanobacteria are common in symbiotic relationships with diverse multicellular organisms (animals, plants, fungi) in terrestrial environments and with single-celled heterotrophic, mixotrophic, and autotrophic protists in aquatic environments. In the sunlit zones of aquatic environments, diverse cyanobacterial symbioses exist with autotrophic taxa in phytoplankton, including dinoflagellates, diatoms, and haptophytes (prymnesiophytes). Phototrophic unicellular cyanobacteria related to Synechococcus and Prochlorococcus are associated with a number of groups. N2-fixing cyanobacteria are symbiotic with diatoms and haptophytes. Extensive genome reduction is involved in the N2-fixing endosymbionts, most dramatically in the unicellular cyanobacteria associated with haptophytes, which have lost most of the photosynthetic apparatus, the ability to fix C, and the tricarboxylic acid cycle. The mechanisms involved in N2-fixing symbioses may involve more interactions beyond simple exchange of fixed C for N. N2-fixing cyanobacterial symbioses are widespread in the oceans, even more widely distributed than the best-known free-living N2-fixing cyanobacteria, suggesting they may be equally or more important in the global ocean biogeochemical cycle of N.Despite their ubiquitous nature and significance in biogeochemical cycles, cyanobacterium-phytoplankton symbioses remain understudied and poorly understood.

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.

Empirical relationship between nifH gene abundance and diazotroph cell concentration in the North Pacific Subtropical Gyre.

Cyanobacterial N2 -fixing microorganisms (diazotrophs) play a critical role in nitrogen and carbon cycling in the oceans; hence, accurate measurements of diazotroph abundance are imperative for understanding ocean biogeochemistry. Marine diazotroph abundances are often assessed using qPCR of the nifH gene, a sensitive, taxa-specific, and time/cost-efficient method. However, the validity of nifH abundance as a proxy for cell concentration has recently been questioned. Here, we compare nifH gene abundances to cell counts for four diazotroph taxa (Trichodesmium, Crocosphaera, Richelia, and Calothrix) on two cruises to the North Pacific Subtropical Gyre, one of the largest habitats for marine diazotrophs. nifH:cell relationships were strong and significant for Crocosphaera, Richelia, and Calothrix (nifH:cell 1.51-2.58; R2  = 0.89-0.96) but were not significant for Trichodesmium, despite previous studies reporting significant nifH:cell relationships for this organism. Limited available data suggest that empirical nifH:cell can vary among studies but that relationships are usually significantly linear and >1:1. Our study indicates that nifH gene abundance, while not a direct measure of cells, is a useful quantitative proxy for diazotroph abundance.

Hopanoid lipids may facilitate aerobic nitrogen fixation in the ocean.

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

Cell sorting reveals few novel prokaryote and photosynthetic picoeukaryote associations in the oligotrophic ocean.

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.

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.

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

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.

Trends in Free-access Genomic Data Accelerate Advances in Cyanobacteria Taxonomy.

Free access databases of DNA sequences containing microbial genetic information have changed the way scientists look at the microbial world. Currently, the NCBI database includes about 516 distinct search results for Cyanobacterial genomes distributed in a taxonomy based on a polyphasic approach. While their classification and taxonomic relationships are widely used as is, recent proposals to alter their grouping include further exploring the relationship between Cyanobacteria and Melainabacteria. Nowadays, most cyanobacteria still are named under the Botanical Code; however, there is a proposal made by the Genome Taxonomy Database (GTDB) to harmonize cyanobacteria nomenclature with the other bacteria, an initiative to standardize microbial taxonomy based on genome phylogeny, in order to contribute to an overall better phylogenetic resolution of microbiota. Furthermore, the assembly level of the genomes and their geographical origin demonstrates some trends of cyanobacteria genomics on the scientific community, such as low availability of complete genomes and underexplored sampling locations. By describing how available cyanobacterial genomes from free-access databases fit within different taxonomic classifications, this mini-review provides a holistic view of the current knowledge of cyanobacteria and indicates some steps towards improving our efforts to create a more cohesive and inclusive classifying system, which can be greatly improved by using large-scale sequencing and metagenomic techniques.

Symbiotic unicellular cyanobacteria fix nitrogen in the Arctic Ocean.

Biological dinitrogen (N2) fixation is an important source of nitrogen (N) in low-latitude open oceans. The unusual N2-fixing unicellular cyanobacteria (UCYN-A)/haptophyte symbiosis has been found in an increasing number of unexpected environments, including northern waters of the Danish Straight and Bering and Chukchi Seas. We used nanoscale secondary ion mass spectrometry (nanoSIMS) to measure 15N2 uptake into UCYN-A/haptophyte symbiosis and found that UCYN-A strains identical to low-latitude strains are fixing N2 in the Bering and Chukchi Seas, at rates comparable to subtropical waters. These results show definitively that cyanobacterial N2 fixation is not constrained to subtropical waters, challenging paradigms and models of global N2 fixation. The Arctic is particularly sensitive to climate change, and N2 fixation may increase in Arctic waters under future climate scenarios.

What's in a name? The case of cyanobacteria.

A redefinition of the cyanobacterial lineage has been proposed based on phylogenomic analysis of distantly related nonphototrophic lineages. We define Cyanobacteria here as "Organisms in the domain bacteria able to carry out oxygenic photosynthesis with water as an electron donor and to reduce carbon dioxide as a source of carbon, or those secondarily evolved from such organisms."

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.

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.

Use of the high-affinity phosphate transporter gene, pstS, as an indicator for phosphorus stress in the marine diazotroph Crocosphaera watsonii (Chroococcales, Cyanobacteria).

The marine diazotroph Crocosphaera watsonii provides fixed carbon (C) and nitrogen (N) to open-ocean regimes, where nutrient deficiency controls productivity. The growth of Crocosphaera can be limited by low phosphorus (P) concentrations in these oligotrophic environments. Biomarkers such as the high-affinity ABC transporter phosphate-binding gene, pstS, are commonly used to monitor when such organisms are under P stress; however, transcriptional regulation of these markers is often complex and not well-understood. In this study, we interrogated changes in pstS transcript levels in C. watsonii cells under P starvation, and in response to added dissolved inorganic phosphorus (DIP), dissolved organic phosphorus (DOP), and changing light levels. We observed elevated relative pstS transcript levels in C. watsonii WH8501 at DIP concentrations below 60 and above 20 nmol · L-1 . Transcript levels were suppressed by both inorganic and bioavailable organic phosphorus; however, the P stress response was more sensitive to DIP than DOP sources. Increasing light intensity resulted in increased relative pstS transcript abundances independently of low external P, and seemed to exacerbate the physiological effects of P stress. The variable response to different P compounds and rapid and transient influence of high light on pstS transcript abundances suggests that pstS is an indicator of internal P status in Crocosphaera.

Distinct ecological niches of marine symbiotic N2 -fixing cyanobacterium Candidatus Atelocyanobacterium thalassa sublineages.

A recently described symbiosis between the metabolically streamlined nitrogen-fixing cyanobacterium UCYN-A and a single-celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN-A sublineages have been defined based on UCYN-A nitrogenase (nifH) sequences. Due to the low abundances of UCYN-A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN-A sublineages based on high throughput sequencing of UCYN-A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN-A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN-A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN-A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN-A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN-A distributions and the ecological importance of the different sublineages.

Two subpopulations of Crocosphaera watsonii have distinct distributions in the North and South Pacific.

Crocosphaera watsonii is a unicellular nitrogen (N2)-fixing cyanobacterium with ecological importance in oligotrophic oceans. In cultivated strains there are two phenotypes of C. watsonii (large and small cells) with differences that could differentially impact biogeochemical processes. Recent work has shown the phenotypes diverged through loss or addition of type-specific genes in a fraction of their genomes, whereas the rest of the genomes were maintained at 99-100% DNA identity. Previous molecular assays for C. watsonii abundances targeted the conserved regions and therefore could not differentiate between phenotypes, so their relative distributions in natural communities were unknown. To determine phenotype distributions, this study developed and applied type-specific quantitative polymerase chain reaction assays to samples from the North and South Pacific. Abundances of both Crocosphaera types declined sharply with depth between 45 and 75 m in both sites. In surface water small cells were 10-100 times more abundant than large cells in the N. Pacific, whereas in the S. Pacific the two phenotypes were nearly equal. Evidence for large cell aggregation was only found in N. Pacific samples. The differences in C. watsonii sub-populations in the North and South Pacific have direct implications for biogeochemistry and carbon export in oligotrophic gyres.

Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium.

Nitrogen (N(2))-fixing marine cyanobacteria are an important source of fixed inorganic nitrogen that supports oceanic primary productivity and carbon dioxide removal from the atmosphere. A globally distributed, periodically abundant N(2)-fixing marine cyanobacterium, UCYN-A, was recently found to lack the oxygen-producing photosystem II complex of the photosynthetic apparatus, indicating a novel metabolism, but remains uncultivated. Here we show, from metabolic reconstructions inferred from the assembly of the complete UCYN-A genome using massively parallel pyrosequencing of paired-end reads, that UCYN-A has a photofermentative metabolism and is dependent on other organisms for essential compounds. We found that UCYN-A lacks a number of major metabolic pathways including the tricarboxylic acid cycle, but retains sufficient electron transport capacity to generate energy and reducing power from light. Unexpectedly, UCYN-A has a reduced genome (1.44 megabases) that is structurally similar to many chloroplasts and some bacteria, in that it contains inverted repeats of ribosomal RNA operons. The lack of biosynthetic pathways for several amino acids and purines suggests that this organism depends on other organisms, either in close association or in symbiosis, for critical nutrients. However, size fractionation experiments using natural populations have so far not provided evidence of a symbiotic association with another microorganism. The UCYN-A cyanobacterium is a paradox in evolution and adaptation to the marine environment, and is an example of the tight metabolic coupling between microorganisms in oligotrophic oceanic microbial communities.

Molecular markers define progressing stages of phosphorus limitation in the nitrogen-fixing cyanobacterium, Crocosphaera.

Crocosphaera watsonii is a marine cyanobacterium that frequently inhabits low phosphate environments in oligotrophic oceans. While C. watsonii has the ability to fix atmospheric nitrogen, its growth may be limited by availability of phosphorus. Biomarkers that indicate cellular phosphorus status give insight into how P-limitation can affect the distribution of nitrogen-fixing cyanobacterial populations. However, adaptation to phosphorus stress is complex and one marker may not be sufficient to determine when an organism is P-limited. In this study, we characterized the transcription of key genes, activated during phosphorus stress in C. watsonii WH8501, to determine how transcription changed during the phosphorus stress response. Transcription of pstS, which encodes a high-affinity phosphate binding protein, was discovered to be quickly up-regulated in phosphorus-depleted cells as an immediate stress response; however, its transcription declined after a period of phosphorus starvation. In addition, diel regulation of pstS in C. watsonii WH8501 complicates the interpretation of this marker in field applications. Transcription of the gene coding for the arsenite efflux protein, arsB, was upregulated after pstS in phosphorus limited cells, but it remained upregulated at later stages of phosphorus limitation. These results demonstrate that a single molecular marker does not adequately represent the entire phosphorus stress response in C. watsonii WH8501. Using both markers, the variations in transcriptional response over a range of degrees of phosphorus limitation may be a better approach for defining cellular phosphorus status.

ARBitrator: a software pipeline for on-demand retrieval of auto-curated nifH sequences from GenBank.

MOTIVATION: Studies of the biochemical functions and activities of uncultivated microorganisms in the environment require analysis of DNA sequences for phylogenetic characterization and for the development of sequence-based assays for the detection of microorganisms. The numbers of sequences for genes that are indicators of environmentally important functions such as nitrogen (N2) fixation have been rapidly growing over the past few decades. Obtaining these sequences from the National Center for Biotechnology Information's GenBank database is problematic because of annotation errors, nomenclature variation and paralogues; moreover, GenBank's structure and tools are not conducive to searching solely by function. For some genes, such as the nifH gene commonly used to assess community potential for N2 fixation, manual collection and curation are becoming intractable because of the large number of sequences in GenBank and the large number of highly similar paralogues. If analysis is to keep pace with sequence discovery, an automated retrieval and curation system is necessary. RESULTS: ARBitrator uses a two-step process composed of a broad collection of potential homologues followed by screening with a best hit strategy to conserved domains. 34 420 nifH sequences were identified in GenBank as of November 20, 2012. The false-positive rate is ∼0.033%. ARBitrator rapidly updates a public nifH sequence database, and we show that it can be adapted for other genes. AVAILABILITY AND IMPLEMENTATION: Java source and executable code are freely available to non-commercial users at http://pmc.ucsc.edu/∼wwwzehr/research/database/. CONTACT: zehrj@ucsc.edu SUPPLEMENTARY INFORMATION: SUPPLEMENTARY INFORMATION is available at Bioinformatics online.