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At least 19 records

Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism—two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCHN cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation.

59 BASIC BIOLOGICAL SCIENCES↗

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

Biological nitrogen fixation (BNF) is the reduction of N 2 into NH 3 in a group of prokaryotes by an extremely O 2 -sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S 2 V red ) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S 2 V red to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O 2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O 2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O 2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

59 BASIC BIOLOGICAL SCIENCES↗

Characterization of ferredoxins involved in electron transfer pathways for nitrogen fixation implicates differences in electronic structure in tuning 2[4Fe 4S] Fd activity

Ferredoxins (Fds) are small proteins which shuttle electrons to pathways like biological nitrogen fixation. Physical properties tune the reactivity of Fds with different pathways, but knowledge on how these properties can be manipulated to engineer new electron transfer pathways is lacking. Recently, we showed that an evolved strain of Rhodopseudomonas palustris uses a new electron transfer pathway for nitrogen fixation. This pathway involves a variant of the primary Fd of nitrogen fixation in R. palustris, Fer1, in which threonine at position 11 is substituted for isoleucine (Fer1 T11I ). To understand why this substitution in Fer1 enables more efficient electron transfer, we used in vivo and in vitro methods to characterize Fer1 and Fer1 T11I . Electrochemical characterization revealed both Fer1 and Fer1 T11I have similar redox transitions (–480 mV and – 550 mV), indicating the reduction potential was unaffected despite the proximity of T11 to an iron-sulfur (Fe—S) cluster of Fer1. Additionally, disruption of hydrogen bonding around an Fe—S cluster in Fer1 by substituting threonine with alanine (T11A) or valine (T11V) did not increase nitrogenase activity, indicating that disruption of hydrogen bonding does not explain the difference in activity observed for Fer1 T11I . Electron paramagnetic resonance spectroscopy studies revealed key differences in the electronic structure of Fer1 and Fer1 T11I , which indicate changes to the high spin states and/or spin-spin coupling between the Fe—S clusters of Fer1. Finally, our data implicates these electronic structure differences in facilitating electron flow and sets a foundation for further investigations to understand the connection between these properties and intermolecular electron transfer.

59 BASIC BIOLOGICAL SCIENCES↗

A global dataset of terrestrial biological nitrogen fixation

Biological nitrogen fixation (BNF) is the main natural source of new nitrogen inputs in terrestrial ecosystems, supporting terrestrial productivity, carbon uptake, and other Earth system processes. We assembled a comprehensive global dataset of field measurements of BNF in all major N-fixing niches across natural terrestrial biomes derived from the analysis of 376 BNF studies. The dataset comprises 32 variables, including site location, biome type, N-fixing niche, sampling year, quantification method, BNF rate (kg N ha −1 y −1 ), the percentage of nitrogen derived from the atmosphere (%N dfa ), N fixer or N-fixing substrate abundance, BNF rate per unit of N fixer abundance, and species identity. Overall, the dataset combines 1,207 BNF rates for trees, shrubs, herbs, soil, leaf litter, woody litter, dead wood, mosses, lichens, and biocrusts, 152 herb %N dfa values, 1,005 measurements of N fixer or N-fixing substrate abundance, and 762 BNF rates per unit of N fixer abundance for a total of 424 species across 66 countries. This dataset facilitates synthesis, meta-analysis, upscaling, and model benchmarking of BNF fluxes at multiple spatial scales.

Reis Ely, Carla R. [Oregon State Univ., Corvallis,↗

Gel-Derived Amorphous Bismuth–Nickel Alloy Promotes Electrocatalytic Nitrogen Fixation via Optimizing Nitrogen Adsorption and Activation

To achieve the electrochemical nitrogen reduction reaction (NRR) for efficient and sustainable NH 3 production, catalysts should exhibit high selectivity and activity with optimal adsorption energy. Herein we developed a three-dimensional (3D) amorphous BiNi alloy toward a significantly enhanced NRR compared with its crystalline and metal counterparts. Ni alloying enables the chemisorption of nitrogen and the lower fre-energy change for the *NNH formation, and the 3D alloy electrocatalyst exhibits high catalytic activity for NH 3 production with a yield rate of 17.5 μg h -1 mg cat -1 and Faradaic efficiency of 13.8 %. The enhanced electron transfer and increased electrochemical surface area were revealed in the interconnected porous scaffold, affording it sufficiently efficient and stable activity for potential practical applications. Furthermore, this work offers new insights into optimizing the adsorption energy of reactants and intermediates combined with tuning the crystallinity of NRR electrocatalysts.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Gel‐Derived Amorphous Bismuth–Nickel Alloy Promotes Electrocatalytic Nitrogen Fixation via Optimizing Nitrogen Adsorption and Activation

Abstract To achieve the electrochemical nitrogen reduction reaction (NRR) for efficient and sustainable NH 3 production, catalysts should exhibit high selectivity and activity with optimal adsorption energy. Herein we developed a three‐dimensional (3D) amorphous BiNi alloy toward a significantly enhanced NRR compared with its crystalline and metal counterparts. Ni alloying enables the chemisorption of nitrogen and the lower free‐energy change for the *NNH formation, and the 3D alloy electrocatalyst exhibits high catalytic activity for NH 3 production with a yield rate of 17.5 μg h −1 mg cat −1 and Faradaic efficiency of 13.8 %. The enhanced electron transfer and increased electrochemical surface area were revealed in the interconnected porous scaffold, affording it sufficiently efficient and stable activity for potential practical applications. This work offers new insights into optimizing the adsorption energy of reactants and intermediates combined with tuning the crystallinity of NRR electrocatalysts.

Fang, Zhiwei↗

Rnf and Fix Have Specific Roles during Aerobic Nitrogen Fixation in Azotobacter vinelandii

Biological nitrogen fixation requires large amounts of energy in the form of ATP and low potential electrons to overcome the high activation barrier for cleavage of the dinitrogen triple bond. The model aerobic nitrogen-fixing bacteria, Azotobacter vinelandii, generates low potential electrons in the form of reduced ferredoxin (Fd) and flavodoxin (Fld) using two distinct mechanisms via the enzyme complexes Rnf and Fix. Both Rnf and Fix are expressed during nitrogen fixation, but deleting either rnf1 or fix genes has little effect on diazotrophic growth. However, deleting both rnf1 and fix eliminates the ability to grow diazotrophically. Rnf and Fix both use NADH as a source of electrons, but overcoming the energetics of NADH's endergonic reduction of Fd/Fld is accomplished through different mechanisms. Rnf harnesses free energy from the chemiosmotic potential, whereas Fix uses electron bifurcation to effectively couple the endergonic reduction of Fd/Fld to the exergonic reduction of quinone. Different reaction stoichiometries and condition-specific differential gene expression indicate specific roles for the two reactions. This work's complementary physiological studies and thermodynamic modeling reveal how Rnf and Fix balance redox homeostasis in various conditions. Specifically, the Fix complex is required for efficient growth under low oxygen concentrations, while Rnf is presumed to maintain reduced Fd/Fld production for nitrogenase under standard conditions. This work provides a framework for understanding how the production of low potential electrons sustains robust nitrogen fixation in various conditions.

59 BASIC BIOLOGICAL SCIENCES↗

Lightning - Estimates of the rates of energy dissipation and nitrogen fixation

The nitrogen needed by plants can normally not be directly obtained from the nitrogen present in molecular form in the atmosphere. The reason for this situation is related to the great energy required to break the N-N bond. Only a few organisms, such as algae and certain bacteria, can 'fix' nitrogen. An abiological process for breaking the N-N bond is provided by lightning. The present investigation is concerned with this possibility. It is found that lightning produces approximately 2.6 x 10 to the 9th kg N per year. There are, however, uncertainties, which are mainly related to the energy of a lightning flash.

Borucki, W. J.↗

Modeling biological nitrogen fixation in global natural terrestrial ecosystems

Biological nitrogen fixation plays an important role in the global nitrogen cycle. However, the fixation rate has been usually measured or estimated at a particular observational site. To quantify the fixation amount at the global scale, process-based models are needed. This study develops a biological nitrogen fixation model to quantitatively estimate the nitrogen fixation rate by plants in a natural environment. The revised nitrogen module better simulates the nitrogen cycle in comparison with our previous model that has not considered the fixation effects. The new model estimates that tropical forests have the highest fixation rate among all ecosystem types, which decreases from the Equator to the polar region. The estimated nitrogen fixation in global terrestrial ecosystems is 61.5 Tg N yr-1 with a range of 19.8–107.9 Tg N yr-1 in the 1990s. Our estimates are relatively low compared to some early estimates using empirical approaches but comparable to more recent estimates that involve more detailed processes in their modeling. Furthermore, the contribution of nitrogen made by biological nitrogen fixation depends on ecosystem type and climatic conditions. This study highlights that there are relatively large effects of biological nitrogen fixation on ecosystem nitrogen cycling. and the large uncertainty of the estimation calls for more comprehensive understanding of biological nitrogen fixation. More direct observational data for different ecosystems are in need to improve future quantification of fixation and its impacts.

54 ENVIRONMENTAL SCIENCES↗

Dynamic nitrogen fixation in an aerobic endophyte of Populus

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte–poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15 N 2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant–endophyte interactions.

15 N-tracking metabolomics↗

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

Abstract 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 15 N-dinitrogen and 13 C-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.

59 BASIC BIOLOGICAL SCIENCES↗

Bioavailability of molybdenite to support nitrogen fixation on early Earth by an anoxygenic phototroph

Biological nitrogen fixation, which converts atmospheric dinitrogen to ammonia, is catalyzed mostly by Mo-nitrogenase and is a primary contributor to bioavailable nitrogen on early Earth. Mo-nitrogenase is believed to have evolved during the Archean, despite the extremely low concentration of dissolved Mo. However, it remains unclear whether Mo minerals could serve as a source of Mo to support the prevalence of Mo-nitrogenase on early Earth. Here, in this study, we investigated the bioavailability of molybdenite by incubating it with a metabolically ancient anoxygenic phototroph (Rhodopseudomonas palustris) under anoxic conditions. In the laboratory, R. palustris utilized molybdenum from molybdenite as a cofactor for nitrogen fixation. This bacterium extracted Mo from molybdenite by secreting molybdophores rhodopetrobactin A and B and by expressing Mo transport proteins. Surface-sensitive techniques demonstrated significant changes in surface chemistry of molybdenite after its interaction with cells. These findings provide novel explanations for the prevalence of Mo-nitrogenase on early Earth, with significant implications for nitrogen fixation in modern Mo-deficient environments.

54 ENVIRONMENTAL SCIENCES↗

Phylogenomic discovery and engineering of nitrogen fixation into the bioenergy woody crop poplar

Biological nitrogen fixation (BNF) is a key process enabling plants in specific lineages to convert atmospheric dinitrogen (N₂) into bioavailable ammonia through symbioses with diazotrophic microbes. Expanding this capability beyond native nitrogen-fixing clades into non-nodulating crops would reduce synthetic fertilizer use, lowering energy inputs and environmental impacts in agriculture. Supported by DOE Funding Award DE-SC0018247, the NitFix project advanced foundational knowledge required to engineer root-nodule symbioses in new host species. The team generated the most comprehensive phylogenomic analysis to date of all known nodulating lineages, resolving the evolutionary history of nitrogen-fixing symbiosis and identifying core gene suites retained across nodulating taxa. Through multimodal genomics, transcriptomics, and functional analyses in Medicago truncatula and related species, the project mapped regulatory networks underlying nodule organogenesis, bacterial infection, and nitrogen-fixation efficiency. Key discoveries include the identification of conserved signaling modules for rhizobial recognition, transcription factors controlling nodule differentiation, and metabolic pathways integrating fixed nitrogen into plant growth. The project also developed enabling tools—including optimized transformation pipelines, gene-editing workflows, and imaging-based phenotyping—to accelerate engineering efforts in emerging models. Together, these results refine the mechanistic framework of symbiotic nitrogen fixation and highlight transferable components essential for rewiring these traits into non-nodulating crops.

59 BASIC BIOLOGICAL SCIENCES↗

Nitrogen fixation rates in the Guinea Dome and the equatorial upwelling regions in the Atlantic Ocean

Abstract Biological nitrogen fixation is a key process balancing the loss of combined nitrogen in the marine nitrogen cycle. Its relevance in upwelling or high nutrient regions is still unclear, with the few available studies in these regions of the ocean reporting rates that vary widely from below detection limit to > 100 nmol N L −1 d −1 . In the eastern tropical Atlantic Ocean, two open ocean upwelling systems are active in boreal summer. One is the seasonal equatorial upwelling, where the residual phosphorus associated with aged upwelled waters is suggested to enhance nitrogen fixation in this season. The other is the Guinea Dome, a thermal upwelling dome. We conducted two surveys along 23° W across the Guinea Dome and the Equator from 15° N to 5° S in September 2015 and August–September 2016 with high latitudinal resolution (20–60 nm between stations). The abundance of Trichodesmium colonies was characterized by an Underwater Vision Profiler 5 and the total biological nitrogen fixation in the euphotic layer was measured using the 15 N 2 technique. The highest abundances of Trichodesmium colonies were found in the area of the Guinea Dome (9°–15° N) with a maximum of 3 colonies L −1 near the surface. By contrast, colonies were almost absent in the Equatorial band between 2° N and 5° S. The highest nitrogen fixation rate was measured at the northern edge of the Guinea Dome in 2016 (ca. 31 nmol N L −1 d −1 ). In this region, where diazotrophs thrived on a sufficient supply of both phosphorus and iron, a patchy distribution was unveiled by our increased spatial resolution scheme. In the Equatorial band, rates were considerably lower, ranging from below detection limit to ca. 4 nmol N L −1 d −1 , with a clear difference in magnitude between 2015 (rates close to zero) and 2016 (average rates around 2 nmol N L −1 d −1 ). This difference seemed triggered by a contrasting supply of phosphorus between years. Our study stresses the importance of surveys with sampling at fine-scale spatial resolution, and shows unexpected high variability in the rates of nitrogen fixation in the Guinea Dome, a region where diazotrophy is a significant process supplying new nitrogen into the euphotic layer.

Fernández-Carrera, Ana (ORCID:0000000183470996)↗

Modeling of nitrogen fixation and polymer production in the heterotrophic diazotroph Azotobacter vinelandii DJ

Nitrogen fixation is an important metabolic process carried out by microorganisms, which converts molecular nitrogen into inorganic nitrogenous compounds such as ammonia (NH 3 ). These nitrogenous compounds are crucial for biogeochemical cycles and for the synthesis of essential biomolecules, i.e. nucleic acids, amino acids and proteins. Azotobacter vinelandii is a bacterial non-photosynthetic model organism to study aerobic nitrogen fixation (diazotrophy) and hydrogen production. Moreover, the diazotroph can produce biopolymers like alginate and polyhydroxybutyrate (PHB) that have important industrial applications. However, many metabolic processes such as partitioning of carbon and nitrogen metabolism in A. vinelandii remain unknown to date. Genome-scale metabolic models (M-models) represent reliable tools to unravel and optimize metabolic functions at genome-scale. M-models are mathematical representations that contain information about genes, reactions, metabolites and their associations. M-models can simulate optimal reaction fluxes under a wide variety of conditions using experimentally determined constraints. Here we report on the development of a M-model of the wild type bacterium A. vinelandii DJ (iDT1278) which consists of 2,003 metabolites, 2,469 reactions, and 1,278 genes. We validated the model using high-throughput phenotypic and physiological data, testing 180 carbon sources and 95 nitrogen sources. iDT1278 was able to achieve an accuracy of 89% and 91% for growth with carbon sources and nitrogen source, respectively. This comprehensive M-model will help to comprehend metabolic processes associated with nitrogen fixation, ammonium assimilation, and production of organic nitrogen in an environmentally important microorganism.

59 BASIC BIOLOGICAL SCIENCES↗

Enhancing nitrogen fixation efficiency in glow-like discharge by reducing cathode-fall voltage

In plasma nitrogen fixation devices, discharge electrodes are crucial yet susceptible to oxidation and corrosion due to plasma’s high temperatures and oxygen content, which could alter discharge modes. This research evaluates the impact of different electrode materials, including iron, chromium, nickel, copper, and 304 stainless steel, on nitrogen fixation efficiency in glow-like discharges driven by high-voltage DC power. Notably, iron and 304 stainless steel cathodes undergo a mode transition at increased currents, evident from plasma color shifts and significant voltage reductions. Fourier transform infrared spectroscopy analyses reveal that such mode changes minimally affect nitrogen oxide production rates, leading to a notable decrease in energy consumption for nitrogen fixation by up to 40%. OES and SEM-EDS measurements suggest that iron oxide, with its higher secondary electron emission, replaces metal as the cathode material, facilitating mode transitions and maintaining discharge current at lower voltages. Further, this voltage change is largely attributed to the cathode voltage drop, highlighting the minimal role of the cathode fall region in NO x synthesis. These findings underscore the potential for improving plasma nitrogen fixation energy efficiency by choosing suitable cathode materials to lower the cathode-fall voltage.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗

Unbalancing Symbiotic Nitrogen Fixation: Can We Make Effectiveness More Effective?

One of the most critical uses of carbon fuels is in generating nitrogen fertilizer. Atmospheric dinitrogen is too stable to be used directly by plants, so plants need other chemical forms of nitrogen. Nitrogen fertilizer production uses ~4% of the world’s natural gas. Making N fertilizers leverages methane’s energy content by enabling additional energy to be captured into food and fiber through increased photosynthesis. Manufacturing fertilizer is essential—without it there would only be enough combined nitrogen to feed ~3 billion of the world’s ~7 billion people. How to alter this addiction is not obvious, but the best chance to secure substantial and sustainable amounts of future N might be through symbiotic nitrogen fixation (SNF). SNF describes mutualistic interactions where nitrogen-fixing bacteria provide fixed nitrogen to their plant hosts, freeing them from the need for nitrogen fertilizers. SNF already occurs on a large scale; "biological nitrogen” contributes about 5 Tg N/yr to American agriculture, a quarter of the nitrogen needed. Improving SNF and replacing inorganic fertilizers are both important goals in establishing sustainable and more energy-efficient farming. SNF has been used in agriculture for millennia, largely by using legumes like beans or alfalfa in mixed cropping systems. Legume SNF occurs in root nodules, organs that develop after a growing root is infected by bacteria called rhizobia. In the plant cells of a legume nodule, specialized forms of rhizobia fix nitrogen and can produce enough ammonia to supply the growing plant. The exchange of photosynthetically derived plant carbon compounds for nitrogen reduced by the bacteria is the engine that drives the symbiosis. Important details about the organization of development and metabolism in SNF remain to be determined, such as how plant and bacterial metabolism are coordinated as nitrogen is being fixed and what governs the overall level of nitrogen fixation. Most bacterial mutations that affect SNF completely block fixation (e.g. are Fix – ), providing a limited window into the feedback interactions that must be part of the process. We have investigated an unusual mutant of Sinorhizobium meliloti, a symbiotic partner of alfalfa, that fixes dinitrogen at a normal rate (i.e., it is Fix + ) but is not effective in supporting plant growth (i.e., it is ineffective or Eff – ). We have shown that the mutant has a specific mutation in the glnD gene, which codes for a protein that regulates the bacterial response to nitrogen stress, among other key pathways. A Fix + Eff – phenotype contains an unavoidable puzzle—how is it possible for the bacteria to fix nitrogen at a normal rate and without benefiting the nitrogen starved plant host? After eliminating other possibilities, we proposed that the bacteria synthesize a nitrogen-containing compound that the plant can’t use so that acquiring this compound does not relieve plant nitrogen stress. Extracts of nodules made by glnD mutant strains have high concentrations of pyruvate canaline oxime (PCO), a derivative of the plant defensive compound canaline, a toxic analog of ornithine, an amino acid. Many legumes produce canaline and the related arginine mimic canavanine. For reasons we do not yet understand, the mutation in glnD appears to trigger significant overproduction of canaline in a defense response that leads to substantial synthesis of PCO. We expected the bacteria to be able to use PCO, but we have not been able to grow S. meliloti on PCO under several conditions. PCO thus appears to be a metabolic dead end for both the plant and bacteria. We also examined bacterial catabolic pathways that degrade arginine and canavanine and showed that mutation of these did not alter the symbiosis in an obvious way. In related experiments to examine the metabolic development of root nodules, we carried out an ambitious experiment to simultaneously measure metabolites and protein changes during nodule maturation. For these experiments we used a related symbiotic interaction between S. medicae and Medicago truncatula. M. truncatula has a simpler genome than alfalfa so we could identify plant proteins based on predictions from the DNA sequence. Nodules formed on Medicago grow linearly, with the least mature tissues at the tip. Because development of nitrogen fixation is accompanied by the production of the pigmented plant oxygen carrier protein leghemoglobin, we were able to locate and manually separate the tip, transitional, and mature nitrogen-fixing regions from each other. Advances in proteomic and metabolomic techniques allowed us to analyze these tissues from a pool of as few as 10 nodules. We observed correlated transitions of various enzymes in the plant and bacterial proteomes from those characteristic of free-living metabolism to the microaerobic metabolism of the nitrogen-fixing tissues. The metabolome could not be separated into plant and bacterial domains but was consistent with this overall transition. A subsequent paper examined proteolysis in nodule bacteria. Protein turnover in mature regions of the nodule is significant. We were able to show complexes between various proteins and important proteases, using precipitation capture techniques to isolate the proteases and sensitive proteomic analysis to reveal the associated proteins.

09 BIOMASS FUELS↗

Advancing electrocatalytic nitrogen fixation: insights from molecular systems

Nitrogen fixation has a rich history within the inorganic chemistry community. In recent years attention has (re)focused on developing electrocatalytic systems capable of mediating the nitrogen reduction reaction (N 2 RR). Well-defined molecular catalyst systems have much to offer in this context. This personal perspective summarizes recent progress from our laboratory at Caltech, pulling together lessons learned from a number of studies we have conducted, placing them within the broader context of thermodynamic efficiency and selectivity for the N 2 RR. Here, in particular, proton-coupled electron transfer (PCET) provides an attractive strategy to achieve enhanced efficiency for the multi-electron/proton reduction of N 2 to produce NH 3 (or NH 4 + ), and electrocatalytic PCET (ePCET) via an ePCET mediator affords a promising means of mitigating HER such that the N 2 RR can be achieved in a catalytic fashion.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗