Rhizobia-diatom symbiosis fixes missing nitrogen in the ocean

Abstract

Nitrogen (N₂) fixation in oligotrophic surface waters is the main source of new nitrogen to the ocean¹ and has a key role in fuelling the biological carbon pump². Oceanic N₂ fixation has been attributed almost exclusively to cyanobacteria, even though genes encoding nitrogenase, the enzyme that fixes N₂ into ammonia, are widespread among marine bacteria and archaea^3-5. Little is known about these non-cyanobacterial N₂ fixers, and direct proof that they can fix nitrogen in the ocean has so far been lacking. Here we report the discovery of a non-cyanobacterial N₂-fixing symbiont, 'Candidatus Tectiglobus diatomicola', which provides its diatom host with fixed nitrogen in return for photosynthetic carbon. The N₂-fixing symbiont belongs to the order Rhizobiales and its association with a unicellular diatom expands the known hosts for this order beyond the well-known N₂-fixing rhizobia-legume symbioses on land⁶. Our results show that the rhizobia-diatom symbioses can contribute as much fixed nitrogen as can cyanobacterial N₂ fixers in the tropical North Atlantic, and that they might be responsible for N₂ fixation in the vast regions of the ocean in which cyanobacteria are too rare to account for the measured rates.

Fig. 1. Phylogeny and visualization of Candidatus Tectiglobus diatomicola and its diatom host.

a, Maximum likelihood phylogenetic tree of concatenated bacterial marker genes from the order Rhizobiales, showing the placement of Ca. T. diatomicola within the Hyphomicrobiaceae family (see Methods). The novel genus Ca. Tectiglobus, comprising Ca. T. diatomicola and its closest relative Ca. T. profundi, is highlighted in pink. Families within the Rhizobiales that contain known N₂-fixing legume symbionts and their exemplary host plants are shown. The order Parvibaculales was used as an outgroup. Black dots indicate more than 95% bootstrap support. Scale bar indicates amino acid substitutions per site. Plant icons were designed by Freepik (Neptunia oleracea) or created with BioRender.com. b,c, False coloured scanning electron microscopy (SEM) image (b) and confocal laser scanning microscopy image (c) of a Haslea diatom. Four Ca. T. diatomicola cells (pink, overlay of Hypho1147 and Hypho734 fluorescence in situ hybridization (FISH) probes; Extended Data Table 2) were detected next to the host nucleus (white; stained with DAPI). Scale bars, 5 µm.

Fig. 2. Genome properties, gene transcription and proposed metabolism of the Candidatus Tectiglobus diatomicola symbiont.

a, Circular representation of the Ca. T. diatomicola genome with 13 encoding contigs (grey), GC content (black) and the average transcription of protein-coding genes as transcripts per million (TPM) (blue; TPM values higher than 800 were cut off). Genes related to N₂ fixation (orange), electron transport chain and ATP generation (blue) and the TCA cycle (red) are highlighted. CDS, coding sequence; comp., completeness; red., redundancy; tmRNA, transfer-messenger RNA. b, Schematic of the proposed metabolic potential of Ca. T. diatomicola (white) and its interactions with Haslea (grey and green), indicating the transfer of fixed nitrogen from the N₂-fixing symbiont in return for diatom-derived C₄-dicarboxylic acids, such as succinate. Proteins and corresponding gene names are: Complex I (NADH–quinone oxidoreductase, nuoBNEF), Complex II (succinate dehydrogenase, sdhABCD), Complex III (cytochrome b/c1, fbcH_1/2, fbcF), Complex IV (cbb₃-type oxidase, ccoNOP), Complex V (ATP synthase, atpABDEGF); fumarate hydratase (fumC); aconitate hydratase (acnB); 2-oxoglutarate dehydrogenase (sucAB, lpd); succinyl-CoA synthetase (sucCD); malate dehydrogenase (mdh); isocitrate dehydrogenase (icd); citrate synthase (gltA); nitrogenase (nifHDK) and its ancillary proteins (nifAENBMQSTUVWXZ) and ferredoxins (fdxABN); rnf complex (rnfBCD); dicarboxylic acid transporter (dctPQM); pyruvate dehydrogenase (aceEF, lpd); pyruvate kinase (pyk); malic enzyme (maeB); and phosphoenolpyruvate carboxykinase (pckA). 2-OG, 2-oxoglutarate; PEP, phosphoenolpyruvate.

Fig. 3. Activity of the Candidatus Tectiglobus diatomicola symbiont and its diatom host.

a,b, NanoSIMS images showing the enrichment in ¹⁵N from ¹⁵N₂ fixation (a) and ¹³C from ¹³CO₂ fixation (b). The inset shows the corresponding fluorescence image after hybridization of Ca. T. diatomicola cells (indicated by white arrowheads) with specific oligonucleotide probes (in pink, overlay of Hypho638–Hypho825 mix in blue and Hypho1147 in red, respectively) (Extended Data Table 2). Scale bars, 5 µm. c, Cellular CO₂ and N₂ fixation rates of Ca. T. diatomicola symbionts (pink triangles, n = 64) and their diatom hosts (blue circles, n = 16). d, Carbon-based growth rates of symbionts (pink triangles, n = 64) and hosts (blue circles, n = 16) (black lines indicate mean; see Methods). Source Data

Fig. 4. Distribution of Candidatus Tectiglobus diatomicola and other N₂ fixers in the world’s oceans.

a, Distribution of Ca. T. diatomicola (pink circles) and Ca. T. profundi (black circles) based on read detection in metagenome datasets from Tara Oceans and our own samples (see Methods and Supplementary Table 4). Black-and-pink circles are metagenomes in which both Ca. Tectiglobus species were detected. The abundance of Ca. T. diatomicola on the basis of gamma-A-specific nifH qPCR data is shown (circles in blue-to-yellow gradient; data from a previous study). Sample locations in which gamma-A nifH qPCR counts were zero are shown in Extended Data Fig. 5b. b, Proportion of heterotrophic (orange) versus cyanobacterial (cyan) N₂ fixers identified in a previous study (0.8–2,000 µm size fraction) in metagenome datasets from Tara Oceans.

Extended Data Fig. 1. Relative abundance of heterotrophic and cyanobacterial N₂ fixers in the tropical North Atlantic.

a, Relative abundance of ‘Ca. T. diatomicola’ (gamma-A), ‘Ca. T. profundi’, as well as selected cosmopolitan heterotrophic and cyanobacterial N₂ fixers based on read detection in metagenome data from different size fractions from the tropical North Atlantic. Note that only few known heterotrophic N₂ fixers were detected in the dataset, and that ‘Ca. T. diatomicola’ had the highest relative abundance. b, Relative abundance of the most abundant known N₂ fixers determined from direct microscopy counts (surface waters of stations 4 and 7 from cruise M161 in the tropical North Atlantic). Note that the metagenome based relative abundances of Trichodesmium in the large size fraction overestimate its microscopy-based abundance by an order of magnitude, most likely due to the polyploidy of this genus. By contrast, metagenome based relative abundances of Crocosphaera in the large size fraction underestimate its microscopy-based abundance, most likely due to DNA extraction biases. Source Data

Extended Data Fig. 2. Organization and phylogeny of nitrogen fixation (nif) genes in ‘Candidatus Tectiglobus diatomicola’.

a, Organization of the nif regulon showing the phylogenetic affiliation and GC content of each gene. All nif genes are affiliated to gammaproteobacteria (yellow), whereas almost all other genes in and around the nif regulon are affiliated to alphaproteobacteria (blue). The presence of nifV indicates that unlike most nodulating rhizobia, ‘Ca. T. diatomicola’ has the capacity to synthesize homocitrate, a ligand of the FeMo cofactor. Genes affiliated to gamma-and alphaproteobacteria were found together on individual PacBio reads (solid black lines). Note that there is no substantial difference in GC content between genes of gamma- and alphaproteobacterial origin. b, Maximum likelihood phylogenetic trees of NifHDKENBS sequences of the Pseudomonadota. Phylogeny is based on the alignment of full-length amino acid sequences retrieved from the GTDB. Cyanobacterial Nif sequences were used as outgroups. All ‘Ca. Tectiglobus diatomicola’/‘Ca. Tectiglobus profundi’ (pink line; 1) Nif protein sequences cluster with gammaproteobacterial sequences, while Nif sequences from other members of the Hyphomicrobiaceae family (purple lines; 2–9; Extended Data Fig. 3) cluster with alphaproteobacteria. These alphaproteobacterial Nif protein sequences form deeply branching sister clades to two of the major nodulating Rhizobiales nif clusters; i.e. Bradyrhizobium (I, solid line) and Allorhizobium-Mesorhizobium-Rhizobium-Sinorhizobium (II, dashed line). Note that some other Rhizobiales (turquoise lines; members of the Rhodobiaceae, Rhizobiaceae, Cohaesibacteraceae and BM303 families) Nif protein sequences also cluster with the gammaproteobacterial sequences indicating that they were also obtained via horizontal gene transfer from a (common) gammaproteobacterial donor. Tree scales indicate amino acid substitutions per site. Note that in panel A, individual nif genes are indicated by capital letters; fd, ferredoxin; hr, hemerythrin; hyp, hypothetical protein; prx, peroxiredoxin; rnd, ribonuclease D.

Extended Data Fig. 3. Maximum likelihood phylogenetic tree of concatenated bacterial marker genes, highlighting genomic features of the Hyphomicrobiaceae family.

Columns from left to right represent genome size (Mb), GC content (%), presence and phylogeny of nifH genes (yellow, gammaproteobacteria; blue, alphaproteobacteria), and presence (green) of ammonium transporter genes (amt). The numbers on the right indicate the nif-containing members of the Hyphomicrobiaceae from Extended Data Fig. 2. ‘Ca. T. diatomicola’ and ‘Ca. T. profundi’ are highlighted in pink. Only genomes with >80% completion are shown. Black dots indicate >95% bootstrap support. Scale bar indicates amino acid substitutions per site. Source Data

Extended Data Fig. 4. Microscopic characterization of ‘Candidatus Tectiglobus diatomicola’–Haslea symbioses.

a–d, Confocal laser scanning microscopy images (a,b) and corresponding scanning electron micrographs (c,d) of dividing diatoms with six (a,c) and eight (b,d) ‘Ca. T. diatomicola’ cells, after hybridization with oligonucleotide probes (in pink, overlay of Hypho1147 and Hypho734 in red and blue, respectively; see Extended Data Table 2) and counterstaining with DAPI (white). Solid and dashed lines indicate assumed outline of the dividing diatoms. e, Confocal laser scanning microscopy image of a Haslea diatom after CARD-FISH with the NON338-probe showing no unspecific binding at the typical symbiont location (autofluorescence of the chloroplasts at 488 nm in green, NON338-probe background signal in light blue, DAPI in white). f, Epifluorescence image of the ‘Ca. T. diatomicola’–Haslea symbiosis, showing ‘Ca. T. diatomicola’ cells after hybridization with a specific oligonucleotide probe (Hypho638 in blue), autofluorescence of the chloroplasts in green and the H-shaped nucleus counterstained with DAPI (white). The bilobed chloroplasts are located on either side of the central valve area, and the lobes of the chloroplasts are connected along the cell’s transapical axis. g–k, Scanning electron micrographs of the diatom host. g, Whole frustule showing the internal raphe structure and an apparent hyaline area around the central raphe region (arrow) as well as sections of the hyaline girdle bands (arrowheads). h, Valve end showing the ridges bordering the central raphe. i, Central valve area. The valve’s normal surface structure of longitudinal ribs overlying the transapical striae appears modified with the longitudinal ribs damaged and only the transapical structures visible. j, Valve pole showing the shape of raphe end (arrow head). k, Central valve area with the central valve area collapsed. Arrowheads point to a chamber-like structure. Scale bars are 5 µm in a–g and 1 µm in h–k.

Extended Data Fig. 5. Detection of ‘Candidatus Tectiglobus’ and Haslea species in metagenomic and nifH qPCR datasets.

a, Relative abundance of Haslea in the 0.8 to 5 µm size fraction determined from V9-18S rRNA amplicon sequencing data from Tara Oceans. b, qPCR datasets in which no ‘Ca. T. diatomicola’ nifH were detected (grey circles; data from Shao et al. ^,). c,d, Identification of ‘Ca. Tectiglobus’ in metagenomes from Tara Oceans (blue), the South Pacific gyre (yellow) and the tropical North Atlantic (green) datasets. Coverage and breadth (fraction of the genome covered by at least one read) of the ‘Ca. T. diatomicola’ (c) and ‘Ca. T. profundi’ (d) genomes within the metagenomic samples. The black line indicates the expected breadth, see Methods. ‘Ca. Tectiglobus’ was only considered to be present in metagenomes where the actual breadth was close to the expected breadth (all metagenomes inside the pink-shaded ellipses). Source Data

Extended Data Fig. 6. Maximum likelihood phylogenetic tree of the ‘Candidatus Tectiglobus diatomicola’ and ‘Candidatus Tectiglobus profundi’ high-affinity cytochrome cbb₃-type terminal oxidase.

The tree is constructed from closely related full-length CcoN amino acid sequences retrieved from the GTDB. The CcoN sequences of the two ‘Ca. Tectiglobus’ species are highlighted in pink. The coloured squares indicate the taxonomic affiliation of the genome. Black dots indicate > 95% bootstrap support. Scale bar indicates amino acid substitutions per site.

Extended Data Fig. 7. Single-cell elemental imaging of the ‘Candidatus Tectiglobus diatomicola’–Haslea symbiosis.

NanoSIMS image of the distribution of carbon (¹²C, red) and nitrogen (¹²C¹⁴N, green) showing the carbon-rich symbionts embedded in nitrogen-rich host biomass. The image shows the same ‘Ca. T. diatomicola’–Haslea symbiosis as in Fig. 3a,b. SE, secondary electrons. Scale bar, 5 µm.

Extended Data Fig. 8. Genome comparison of ‘Candidatus T. diatomicola’ and ‘Candidatus T. profundi’.

a, Whole-genome alignment of ‘Ca. T. diatomicola’ (top) and ‘Ca. T. profundi’ (bottom). Coloured boxes represent colinear regions present in both genomes. Grey vertical lines indicate contig boundaries. Numbers represent genomic positions in kilobase. b, Distribution of genes among the COG functional gene categories for the genomes of ‘Ca. T. diatomicola’, ‘Ca. T. profundi’ and their two closest relatives (GCA_905480435 and GCA_002689605). Source Data