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1. Unusual marine cyanobacteria/haptophyte symbiosis relies on N2 fixation even in N-rich environments

   https://doi.org/10.1038/s41396-020-0691-6  

   Mills, Matthew M. ; Turk-Kubo, Kendra A. ; van Dijken, Gert L. ; Henke, Britt A. ; Harding, Katie ; Wilson, Samuel T. ; Arrigo, Kevin R. ; Zehr, Jonathan P. ( October 2020 , The ISME Journal)

   null (Ed.)

   Abstract The microbial fixation of N 2 is the largest source of biologically available nitrogen (N) to the oceans. However, it is the most energetically expensive N-acquisition process and is believed inhibited when less energetically expensive forms, like dissolved inorganic N (DIN), are available. Curiously, the cosmopolitan N 2 -fixing UCYN-A/haptophyte symbiosis grows in DIN-replete waters, but the sensitivity of their N 2 fixation to DIN is unknown. We used stable isotope incubations, catalyzed reporter deposition fluorescence in-situ hybridization (CARD-FISH), and nanoscale secondary ion mass spectrometry (nanoSIMS), to investigate the N source used by the haptophyte host and sensitivity of UCYN-A N 2 fixation in DIN-replete waters. We demonstrate that under our experimental conditions, the haptophyte hosts of two UCYN-A sublineages do not assimilate nitrate (NO 3 − ) and meet little of their N demands via ammonium (NH 4 + ) uptake. Instead the UCYN-A/haptophyte symbiosis relies on UCYN-A N 2 fixation to supply large portions of the haptophyte’s N requirements, even under DIN-replete conditions. Furthermore, UCYN-A N 2 fixation rates, and haptophyte host carbon fixation rates, were at times stimulated by NO 3 − additions in N-limited waters suggesting a link between the activities of the bulk phytoplankton assemblage and the UCYN-A/haptophyte symbiosis. The results suggest N 2 fixation may be an evolutionarily viable strategy for diazotroph–eukaryote symbioses, even in N-rich coastal or high latitude waters. 

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2. Seasonal and spatial patterns in diazotroph community composition at Station ALOHA

   https://doi.org/10.3389/fmars.2023.1130158  

   Turk-Kubo, Kendra A. ; Henke, Britt A. ; Gradoville, Mary R. ; Magasin, Jonathan D. ; Church, Matthew J. ; Zehr, Jonathan P. ( May 2023 , Frontiers in Marine Science)

   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 N₂fixation. At Station ALOHA (A Long-Term Oligotrophic Habitat Assessment) in the NPSG, N₂fixation is typically ascribed to conspicuous, filamentous cyanobacterial diazotrophs (TrichodesmiumandRichelia), unicellular free-livingCrocosphaera, 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) ofnifHgene 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 andTrichodesmiumsp. consistently had the highest relative abundances and seasonal patterns that corroborated qPCR-based analyses. Other prevalent community members included a newCrocosphaera-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 (Trichodesmiumand 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, andTrichodesmium/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 couplingnifHgene 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 N₂fixation in the NPSG.

    

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3. Isolation, growth, and nitrogen fixation rates of the Hemiaulus-Richelia (diatom-cyanobacterium) symbiosis in culture

   https://doi.org/10.7717/peerj.10115  

   Pyle, Amy E. ; Johnson, Allison M. ; Villareal, Tracy A. ( January 2020 , PeerJ)

   Nitrogen fixers (diazotrophs) are often an important nitrogen source to phytoplankton nutrient budgets in N-limited marine environments. Diazotrophic symbioses between cyanobacteria and diatoms can dominate nitrogen-fixation regionally, particularly in major river plumes and in open ocean mesoscale blooms. This study reports the successful isolation and growth in monocultures of multiple strains of a diatom-cyanobacteria symbiosis from the Gulf of Mexico using a modified artificial seawater medium. We document the influence of light and nutrients on nitrogen fixation and growth rates of the host diatom Hemiaulus hauckii Grunow together with its diazotrophic endosymbiont Richelia intracellularis Schmidt, as well as less complete results on the Hemiaulus membranaceus - R. intracellularis symbiosis. The symbioses rates reported here are for the joint diatom-cyanobacteria unit. Symbiont diazotrophy was sufficient to support both the host diatom and cyanobacteria symbionts, and the entire symbiosis replicated and grew without added nitrogen. Maximum growth rates of multiple strains of H. hauckii symbioses in N-free medium with N 2 as the sole N source were 0.74–0.93 div d −1 . Growth rates followed light saturation kinetics in H. hauckii symbioses with a growth compensation light intensity (E C ) of 7–16 µmol m −2 s −1 and saturation light level (E K ) of 84–110 µmol m −2 s −1 . Nitrogen fixation rates by the symbiont while within the host followed a diel pattern where rates increased from near-zero in the scotophase to a maximum 4–6 h into the photophase. At the onset of the scotophase, nitrogen-fixation rates declined over several hours to near-zero values. Nitrogen fixation also exhibited light saturation kinetics. Maximum N 2 fixation rates (84 fmol N 2 heterocyst −1 h −1 ) in low light adapted cultures (50 µmol m −2 s − 1) were approximately 40–50% of rates (144–154 fmol N 2 heterocyst −1 h −1 ) in high light (150 and 200 µmol m −2 s −1 ) adapted cultures. Maximum laboratory N 2 fixation rates were ~6 to 8-fold higher than literature-derived field rates of the H. hauckii symbiosis. In contrast to published results on the Rhizosolenia-Richelia symbiosis, the H. hauckii symbiosis did not use nitrate when added, although ammonium was consumed by the H. hauckii symbiosis. Symbiont-free host cell cultures could not be established; however, a symbiont-free H. hauckii strain was isolated directly from the field and grown on a nitrate-based medium that would not support DDA growth. Our observations together with literature reports raise the possibility that the asymbiotic H. hauckii are lines distinct from an obligately symbiotic H. hauckii line. While brief descriptions of successful culture isolation have been published, this report provides the first detailed description of the approaches, handling, and methodologies used for successful culture of this marine symbiosis. These techniques should permit a more widespread laboratory availability of these important marine symbioses. 

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4. The rate and fate of N2 and C fixation by marine diatom-diazotroph symbioses

   https://doi.org/10.1038/s41396-021-01086-7  

   Foster, Rachel A. ; Tienken, Daniela ; Littmann, Sten ; Whitehouse, Martin J. ; Kuypers, Marcel M. M. ; White, Angelicque E. ( August 2021 , The ISME Journal)

   N2 fixation constitutes an important new nitrogen source in the open sea. One group of filamentous N2 fixing cyanobacteria (Richelia intracellularis, hereafter Richelia) form symbiosis with a few genera of diatoms. High rates of N2 fixation and carbon (C) fixation have been measured in the presence of diatom-Richelia symbioses. However, it is unknown how partners coordinate C fixation and how the symbiont sustains high rates of N2 fixation. Here, both the N2 and C fixation in wild diatom-Richelia populations are reported. Inhibitor experiments designed to inhibit host photosynthesis, resulted in lower estimated growth and depressed C and N2 fixation, suggesting that despite the symbionts ability to fix their own C, they must still rely on their respective hosts for C. Single cell analysis indicated that up to 22% of assimilated C in the symbiont is derived from the host, whereas 78–91% of the host N is supplied from their symbionts. A size-dependent relationship is identified where larger cells have higher N2 and C fixation, and only N2 fixation was light dependent. Using the single cell measures, the N-rich phycosphere surrounding these symbioses was estimated and contributes directly and rapidly to the surface ocean rather than the mesopelagic, even at high estimated sinking velocities (<10 m d−1). Several eco-physiological parameters necessary for incorporating symbiotic N2 fixing populations into larger basin scale biogeochemical models (i.e., N and C cycles) are provided.

    

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

   https://doi.org/10.1038/s41467-022-34585-y  

   Harding, Katie J. ; Turk-Kubo, Kendra A. ; Mak, Esther Wing Kwan ; Weber, Peter K. ; Mayali, Xavier ; Zehr, Jonathan P. ( November 2022 , Nature Communications)

   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 simultaneous¹⁵N-dinitrogen and¹³C-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⁻¹d⁻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.

    

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