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At least 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Allosteric control of electron transfer in nitrogenase and nitrogenase-like enzymes (Final Report)

More than a third of all known proteins to date are either oxidoreductases or metalloproteins. The diversity of metal cofactors and redox centers in these proteins make them attractive candidates for engineering protein-based biocatalysts, biosensors, and biofuel cells, among other necessary tools for the bioeconomy. Many of these enzymes function as large multi-subunit complexes arranged as symmetric or asymmetric assemblies, and the purpose of this architecture is unclear. The goal of the project was to address this fundamental question by investigating the mechanism of action of two oxidoreductases: Nitrogenase and DPOR (Dark-Operative Protochlorophyllide Oxidoreductase, a nitrogenase-like oxidoreductase). Nitrogenase and DPOR play key roles in nitrogen fixation and photosynthesis, processes essential to sustaining life. Both proteins are arranged as two symmetrical halves with each half capable of substrate reduction. For more than half a century, substrate reduction by nitrogenase has been presumed to occur independently within the two identical halves. Funded by this DOE grant we have now shown that both nitrogenase and DPOR function asymmetrically. Electron transfer in one half suppresses activity in the other. Thus, we have demonstrated that the two halves do not function in an independent manner; rather, catalytic events in one-half allosterically suppress activity and electron transfer (ET) in the other. This alternating-site mechanism has significant implications on our current understanding of substrate reduction by these enzymes and offers a new mechanistic paradigm for the reactions they catalyze. Our work now provides a mechanistic map of the how the two symmetric catalytic halves work together to establish and maintain the sequential-site mechanism through multiple ET cycles to achieve substrate reduction. There is substantive need for bio-inspired tools that enable reduction of nitrogen and other substrates, as well as photosynthesis, for energy and food production. Understanding the workings of enzymes like nitrogenase and DPOR that mediate multi-electron redox chemistry is vital for designing such synthetic tools. The timing of our research coincides with the biotechnological breakthrough of light-driven artificial N2 reduction by nitrogenase, achieved using the native enzyme with both halves arranged in a mirror-like architecture as catalyst. Together with such discoveries, our research now reveal unknown facets of the reaction mechanism and hold promise for designing more effective catalysts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

How do Hydrological Variability and Human Activities Control the Spatiotemporal Changes of Riverine Nitrogen Export in the Upper Mississippi River Basin?

Excessive nitrogen export from agricultural watersheds remains a critical water quality challenge, with the Upper Mississippi River Basin (UMRB) significantly contributing to downstream eutrophication and hypoxia in the Gulf. This study investigates the spatiotemporal dynamics of riverine nitrate plus nitrite (NO 3 − + NO 2 − -N) export across the UMRB at high spatial resolution (12-digit Hydrologic Unit Codes or HUC12 subwatershed scale) during 2001−2020 and quantifies the effects of anthropogenic activities and hydrological variability on riverine NO 3 − + NO 2 − -N export changes in the region between 2001−2005 and 2016−2020. Our results revealed hotspots of substantial increases in NO 3 − + NO 2 − -N yields across the UMRB, with distinct regional patterns in driving factors. Over the entire UMRB, NO 3 − + NO 2 − -N yields increased by 9.7 kg/ha/yr on average from 2001−2005 to 2016−2020, with anthropogenic activities contributing 4.8 kg/ha/yr and hydrological variability contributing 4.9 kg/ha/yr. The northern and western UMRB had combined influences from both anthropogenic activities and hydrological variability, while the east-central regions had predominantly hydrologically driven changes. Agricultural sources, including fertilizer, manure, and biological nitrogen fixation, collectively contributed over 80% of NO 3 − + NO 2 − -N loading throughout the basin. Furthermore, this framework for disentangling human and hydrological impacts provides critical insights for developing effective and targeted watershed management strategies to reduce nutrient losses and improve water quality.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases

Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which facilitates the cleavage of the relatively inert triple bond of N 2 . Nitrogenase is most commonly associated with the molybdenum–iron cofactor called FeMoco or the M-cluster, and it has been the subject of extensive structural and spectroscopic characterization over the past 60 years. In the late 1980s and early 1990s, two “alternative nitrogenase” systems were discovered, isolated, and found to incorporate V or Fe in place of Mo. These systems are regulated by separate gene clusters; however, there is a high degree of structural and functional similarity between each nitrogenase. Limited studies with the V- and Fe-nitrogenases initially demonstrated that these enzymes were analogously active as the Mo-nitrogenase, but more recent investigations have found capabilities that are unique to the alternative systems. In this review, we will discuss the reactivity, biosynthetic, and mechanistic proposals for the alternative nitrogenases as well as their electronic and structural properties in comparison to the well-characterized Mo-dependent system. Studies over the past 10 years have been particularly fruitful, though key aspects about V- and Fe-nitrogenases remain unexplored.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Electron Redistribution within the Nitrogenase Active Site FeMo-cofactor During Reductive Elimination of H2 to Achieve N=N Triple-Bond Activation

Nitrogen fixation by nitrogenase begins with accumulation of four reducing equivalents at the active-site FeMo-cofactor (FeMo-co), generating a state (denoted E4(4H)) with two [Fe-H-Fe] bridging hydrides. Recently, photolytic reductive elimination (re) of the E4(4H) hydrides showed that enzymatic re of E4(4H) hydride yields an H2-bound complex (E4(H2,2H)), in a process corresponding to a formal 2-electron reduction of the metal-ion core of FeMo-co. The resulting electron-density redistribution from Fe-H bonds to the metal ions themselves enables N2 to bind with concomitant H2 release, a process illuminated here by QM/MM molecular dynamics simulations. What is the nature of this redistribution? Although E4(H2,2H) hasn’t been trapped, cryogenic photolysis of E4(4H) provides a means to address this question. Photolysis of E4(4H) causes hydride-re with release of H2, generating doubly-reduced FeMo-co (denoted E4(2H)*), the extreme limit of the electron-density redistribution upon formation of E4(H2,2H). Here we examine the doubly-reduced FeMo-co core of the E4(2H)* limiting-state by 1H, 57Fe, and 95Mo ENDOR to illuminate the partial electron-density redistribution upon E4(H2,2H) formation during catalysis, complementing these results with corresponding DFT computations. Inferences from the E4(2H)* ENDOR results as extended by DFT computations include: (i) the Mo-site participates negligibly, and overall it is unlikely that Mo changes valency throughout the catalytic cycle; (ii) two distinctive E4(4H) 57Fe signals are suggested as associated with ‘anchors’ of one bridging hydride, two others with anchors of the second, with NBO-analysis identifying one anchor of each hydride as a major recipient of electrons released upon breaking Fe-H bonds.

Lukoyanov, Dmitriy↗
DOE OSTI2025

Electrochemical Observation and pH Dependence of All Three Expected Redox Couples in an Extremophilic Bifurcating Electron Transfer Flavoprotein with Fused Subunits

Bifurcating enzymes employ energy from a favorable electron transfer to drive unfavorable transfer of a second electron, thereby generating a more reactive product. They are therefore highly desirable in catalytic systems, for example, to drive challenging reactions such as nitrogen fixation. While most bifurcating enzymes contain air-sensitive metal centers, bifurcating electron transfer flavoproteins (bETFs) employ flavins. However, they have not been successfully deployed on electrodes. Herein, we demonstrate immobilization and expected thermodynamic reactivity of a bETF from a hyperthermophilic archaeon, Sulfolobus acidocaldarius (SaETF). SaETF differs from previously biochemically characterized bETFs in being a single protein, representing a concatenation of the two subunits of known ETFs. However, SaETF retains the chemical properties of heterodimeric bETFs, including possession of two FADs: one that undergoes sequential 1-electron (1e) reductions at high E° and forms an anionic semiquinone, and another that is amenable to lower-E° 2e reduction, including by NADH. We found homologous monomeric ETF genes in archaeal and bacterial genomes, accompanied by genes that also commonly flank heterodimeric ETFs, and SaETF’s sequence conservation is 50% higher with bETFs than with canonical ETFs. Thus, SaETF is best described as a bETF. Our direct electrochemical trials capture reversible redox couples for all three thermodynamically expected redox events. We document electrochemical activity over a range of pH values and reveal a conformational change coupled to proton acquisition that affects the electrochemical activity of the higher-E° FAD. Thus, this well-behaved monomeric bETF opens the door to bioinspired bifurcating devices or bifurcation on a chip.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Nutrient Dynamics in a Coupled Terrestrial Biosphere and Land Model (ELM-FATES-CNP)

We present a representation of nitrogen and phosphorus cycling in the Functionally Assembled Terrestrial Ecosystem Simulator, a demographic vegetation model within the Energy Exascale Earth System land model. This representation is modular, and designed to allow testing of multiple hypothetical approaches for carbon-nutrient coupling in plants. Novel model hypotheses introduced in this work include, (a) the controls on plant acquisition of aqueous mineralized nutrients in the soil and (b) fairly straight forward methods of allocating nutrients to specific plant organs and their losses through live plant turnover as well as litter fluxes generated through plant mortality. This combines the new with pre-existing hypotheses (such as nitrogen fixation and soil decomposition) into a system that can accommodate plant-soil dynamics for a large number of size- and functional-type-resolved plant cohorts within a time-since-disturbance-resolved ecosystem. Root uptake of nutrients is governed by fine root biomass, and plants vary in their fine root biomass allocation in order to balance carbon and nutrient limitations to growth. We test the sensitivity of the model to a wide range of parameter variations and structural representations, and in the context of observations at Barro Colorado Island, Panama. A key model prediction is that plants in the high-light-availability canopy positions allocate more carbon to fine roots than plants in low-light understory environments, given the widely different carbon versus nutrient constraints of these two niches within a given ecosystem. This model provides a basis for exploring carbon-nutrient coupling with vegetation demography within Earth system models.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Experimentally-validated correlation analysis reveals new anaerobic methane oxidation partnerships with consortium-level heterogeneity in diazotrophy

Abstract Archaeal anaerobic methanotrophs (“ANME”) and sulfate-reducing Deltaproteobacteria (“SRB”) form symbiotic multicellular consortia capable of anaerobic methane oxidation (AOM), and in so doing modulate methane flux from marine sediments. The specificity with which ANME associate with particular SRB partners in situ, however, is poorly understood. To characterize partnership specificity in ANME-SRB consortia, we applied the correlation inference technique SparCC to 310 16S rRNA amplicon libraries prepared from Costa Rica seep sediment samples, uncovering a strong positive correlation between ANME-2b and members of a clade of Deltaproteobacteria we termed SEEP-SRB1g. We confirmed this association by examining 16S rRNA diversity in individual ANME-SRB consortia sorted using flow cytometry and by imaging ANME-SRB consortia with fluorescence in situ hybridization (FISH) microscopy using newly-designed probes targeting the SEEP-SRB1g clade. Analysis of genome bins belonging to SEEP-SRB1g revealed the presence of a complete nifHDK operon required for diazotrophy, unusual in published genomes of ANME-associated SRB. Active expression of nifH in SEEP-SRB1g within ANME-2b—SEEP-SRB1g consortia was then demonstrated by microscopy using hybridization chain reaction (HCR-) FISH targeting nifH transcripts and diazotrophic activity was documented by FISH-nanoSIMS experiments. NanoSIMS analysis of ANME-2b—SEEP-SRB1g consortia incubated with a headspace containing CH4 and 15N2 revealed differences in cellular 15N-enrichment between the two partners that varied between individual consortia, with SEEP-SRB1g cells enriched in 15N relative to ANME-2b in one consortium and the opposite pattern observed in others, indicating both ANME-2b and SEEP-SRB1g are capable of nitrogen fixation, but with consortium-specific variation in whether the archaea or bacterial partner is the dominant diazotroph.

58 GEOSCIENCES↗
DOE OSTI2023

The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

Abstract Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans , which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2 , respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1 . In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Proton transfer during reduction of the catalytic metallo-cofactors of the three nitrogenase isozymes

Nitrogenase catalyzes biological nitrogen fixation, the conversion of atmospheric N 2 into bioavailable ammonia. The three nitrogenase isozymes—Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase—utilize catalytic cofactors distinguished by their metal composition (Fe 7 M, M = Mo, V, or Fe; denoted FeM-co). Their catalytic cycles involve stepwise addition of 8[e−/H+] to FeM-co, generating intermediates designated E n , where n is the number of [e − /H + ] delivered. The electron-transfer has been extensively characterized, but the proton delivery has not. Here, we investigate [e − /H + ] delivery during early-stage conversions, primarily E 0 → E 1 (H), for each of the three nitrogenases, using as reductants γ-ray-generated thermolyzed, mobile electrons at 77 K, and radiation-generated solvent radicals during subsequent annealing to higher temperatures. Our results show E 0 → E 1 (H) conversion differs among the three MFe-proteins. The FeMo-co of MoFe-protein accepts an electron (ET) during 77 K γ-irradiation, but proton transfer (PT) to generate E 1 (H) is only enabled by conformational or thermodynamic activation upon cryoannealing to ∼200 K(ET/PT). For VFe-protein, E 1 (H) forms during annealing at-and-above 210 K by electron-transfer to FeV-co from radicals through proton-coupled electron transfer (PCET), which too is enabled by activated proton transfer. FeFe-protein differs in directly exhibiting delivery of protons at 77 K, which together with the mobile electrons react to form E 1 (H). This could well occur by PCET at 77 K, but does not preclude the possibility of sequential 77 K electron/proton transfer (ET/PT). In addition, 450 nm photolysis reveals the E 1 (H) state of FeV-co, like that of FeFe-co, contains a hydride bound to a formally oxidized cofactor. The mechanistic differences observed here provide a contribution towards understanding the sources of catalytic differences among the three nitrogenase isozymes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

From thermal catalysis to plasma catalysis: a review of surface processes and their characterizations

The use of atmospheric pressure plasma to enhance catalytic chemical reactions involves complex surface processes induced by the interactions of plasma-generated fluxes with catalyst surfaces. Industrial implementation of plasma catalysis necessitates optimizing the design and realization of plasma catalytic reactors that enable chemical reactions that are superior to conventional thermal catalysis approaches. This requires the fundamental understanding of essential plasma-surface interaction mechanisms of plasma catalysis from the aspect of experimental investigation and theoretical analysis or computational modeling. In addition, experimental results are essential to validate the relative theoretical models and hypotheses of plasma catalysis that was rarely understood so far, compared to conventional thermal catalysis. This overview focuses on two important application areas, nitrogen fixation and methane reforming, and presents a comparison of important aspects of the state of knowledge of these applications when performed using either plasma-catalysis or conventional thermal catalysis. We discuss the potential advantage of plasma catalysis over thermal catalysis from the aspects of plasma induced synergistic effect and in situ catalyst regeneration. In-situ/operando surface characterization of catalysts in plasma catalytic reactors is a significant challenge since the high pressure of realistic plasma catalysis systems preclude the application of many standard surface characterization techniques that operate in a low-pressure environment. Here, we present a review of the status of experimental approaches to probe gas-surface interaction mechanisms of plasma catalysis, including an appraisal of demonstrated approaches for integrating surface diagnostic tools into plasma catalytic reactors.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Plasma-catalyst modeling for materials selection: challenges and opportunities in nitrogen oxidation

Reliable kinetic models are essential to rationalize observations and to guide the selection of appropriate plasma-catalyst combinations. Kinetic models coupling plasma- and heterogeneous-catalytic-chemistries, however, are primitive. Here, we combine reduced plasma chemistries and density functional theory parameterized surface reactions to model NO formation from N2 oxidation, an alternative nitrogen fixation process. We first show plasma excited species can enhance turnover frequencies of both Pt and Au. The enhancing potential is a function of plasma species and their densities. We then compare NO production between plasma/Pt and plasma/Au with an integral reactor model and explore the dependence of NO production as a function of plasma conditions, reactor configurations and chemical compositions. Plasma/Pt generates more NO than plasma/Au under most thermal and plasma parametric conditions. Plasma/Au outperforms plasma/Pt under high plasma excitations at low temperatures. Furthermore, these results highlight that selection of plasma-catalyst combinations should consider the properties of plasmas, materials and the coupling of the two.

70 PLASMA PHYSICS AND FUSION TECHNOLOGY↗
DOE OSTI2020

Dual-cone variational calculation of the two-electron reduced density matrix

The computation of strongly correlated quantum systems is challenging because of its potentially exponential scaling in the number of electron configurations. Variational calculation of the two-electron reduced density matrix (2-RDM) without the many-electron wave function exploits the pairwise nature of the electronic Coulomb interaction to compute a lower bound on the ground-state energy with polynomial computational scaling. Recently, a dual-cone formulation of the variational 2-RDM calculation was shown to generate the ground-state energy, albeit not the 2-RDM, at a substantially reduced computational cost, especially for higher $\textit{N}$-representability conditions such as the T2 constraint. Here we generalize the dual-cone variational 2-RDM method to compute not only the ground-state energy but also the 2-RDM. The central result is that we can compute the 2-RDM from a generalization of the Hellmann-Feynman theorem. Specifically, we prove that in the Lagrangian formulation of the dual-cone optimization the 2-RDM is the Lagrange multiplier. Further, we apply the method to computing the energies and properties of strongly correlated electrons—including atomic charges, electron densities, dipole moments, and orbital occupations—in an illustrative hydrogen chain and the nitrogen-fixation catalyst FeMoco. The dual variational computation of the 2-RDM with T2 or higher N -representability conditions provides a polynomially scaling approach to strongly correlated molecules and materials with significant applications in atomic and molecular and condensed-matter chemistry and physics.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2025

Hidden Allies: Decoding the Core Endohyphal Bacteriome of Aspergillus fumigatus

ABSTRACT Bacterial–fungal interactions that influence the behaviour of one or both organisms are common in nature. Well‐studied systems include endosymbiotic relationships that range from transient to long‐term associations. Diverse endohyphal bacteria associate with fungal hosts, emphasising the need to better comprehend the fungal bacteriome. We evaluated the hypothesis thatAspergillus fumigatusharbours an endohyphal community of bacteria that influence the host phenotype. We analysed whether 38A. fumigatusstrains show stable association with diverse endohyphal bacteria; all derived from single‐conidium cultures that were subjected to antibiotic and heat treatments. The fungal bacteriome, inferred through analysis of bacterial diversity within the fungal strains (short‐ and long‐ read sequencing methods), revealed the presence of core endohyphal bacterial genera. Microscopic analysis further confirmed the presence of endohyphal bacteria. The fungal strains exhibited high genetic diversity and phenotypic heterogeneity in drug susceptibility and in vivo virulence. No correlations were observed between genomic or functional traits and bacteriome diversity, but the abundance of some bacterial genera correlated with fungal virulence or posaconazole susceptibility. The observed endobacteriome may play functional roles, for example, nitrogen fixation. Our study emphasises the existence of complex interactions between fungi and endohyphal bacteria, possibly impacting the phenotype of the fungal host, including virulence.

Environmental Sciences & Ecology↗
DOE OSTI2021

The soybean β‐expansin gene GmINS1 contributes to nodule development in response to phosphate starvation

Abstract Legume biological nitrogen fixation (BNF) is the most important N source in agricultural ecosystems. Nodule organogenesis from the primordia to the development of mature nodules with the ability to fix N 2 largely determines BNF capacity. However, nodule growth is often limited by low phosphorus (P) availability, while the mechanisms underlying nodule development responses to P deficiency remain largely unknown. In this study, we found that nodule enlargement is severely inhibited by P deficiency, as reflected by the smaller individual nodule size from a soybean core collection in the field. Wide‐ranging natural diversity in nodule size was further identified in soybeans reared in low P soils, with the FC‐1 genotype outperforming FC‐2 in assessments of nodulation under low P conditions. Among β‐expansin members, GmINS1 expression is most abundantly enhanced by P deficiency in FC‐1 nodules, and its transcript level is further displayed to be tightly associated with nodule enlargement. Four single nucleotide polymorphisms discovered in the GmINS1 promoter distinguished the FC‐1 and FC‐2 genotypes and accounted for the differential expression levels of GmINS1 responses to P deficiency. GmINS1 overexpression led to increases in nodule size, infected cell abundance, and N 2 fixation capacity and subsequently promoted increases in N and P content, soybean biomass, and yield. Our findings provide a candidate gene for optimizing BNF capacity responses to low P stress in soybean molecular breeding programs.

Yang, Zhaojun↗
DOE OSTI2024

Inoculated biocrust cover and functions diverged over a gradient of soil textures and water availability

Restoring biological crust (biocrust) in disturbed drylands is challenging due to the difficult environmental conditions, such as limited soil moisture, low soil nutrients, and extreme temperatures, that impede growth. Understanding how the key components of biocrust—mosses, lichens, and cyanobacteria—react to different environmental factors informs the optimal timing, locations, and species composition for biocrust reintroduction, thereby increasing the likelihood of establishment. Here, we inoculated soils with a diverse range of biocrust organisms, analogous to seeding an area with diverse vascular plant seeds, and varied environmental conditions to observe how these changes influenced the development and functions of reintroduced biocrust. We found that by manipulating soil texture and time spent wet, we can change the proportional cover of biocrust within a restoration‐like setting. Specifically, we found that 4 months after inoculation, finer textured soils that received more water become dominated by moss cover, while coarser textured soils with less water remained dominated by cyanobacteria cover, and the interactions between texture and time spent wet strongly influenced cover. We found biocrust morphological group cover had a small, but detectable, effect on ecosystem functions (soil stability and nitrogenase activity, a proxy for nitrogen fixation), but that environmental conditions had a stronger impact on the functions we measured. Manipulative experiments in controlled environments, like this one, can help elucidate the mechanisms underlying the establishment rate and patterns of biocrusts post‐inoculation, and inform implementation of inoculations in the field.

Environmental Sciences & Ecology↗
DOE OSTI2021

Metagenomic Characterization of Soil Microbial Communities in the Luquillo Experimental Forest (Puerto Rico) and Implications for Nitrogen Cycling

The phylogenetic and functional diversities of microbial communities in tropical rainforests and how these differ from those of temperate communities remain poorly described but are directly related to the increased fluxes of greenhouse gases such as nitrous oxide (N 2 O) from the tropics. Toward closing these knowledge gaps, in this study we analyzed replicated shotgun metagenomes representing distinct life zones and an elevation gradient from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico. These soils had a distinct microbial community composition and lower species diversity compared to those of temperate grasslands or agricultural soils. In contrast to the overall distinct community composition, the relative abundances and nucleotide sequences of N 2 O reductases (nosZ) were highly similar between tropical forest and temperate soils. However, respiratory NO reductase (norB) was 2-fold more abundant in the tropical soils, which might be relatable to their greater N 2 O emissions. Nitrogen fixation (nifH) also showed higher relative abundance in rainforest than in temperate soils, i.e., 20% versus 0.1 to 0.3% of bacterial genomes in each soil type harbored the gene, respectively. Finally, unlike temperate soils, LEF soils showed little stratification with depth in the first 0 to 30 cm, with ~45% of community composition differences explained solely by location. Collectively, these results advance our understanding of spatial diversity and metabolic repertoire of tropical rainforest soil communities and should facilitate future ecological studies of these ecosystems.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Gallionellaceae in rice root plaque: metabolic roles in iron oxidation, nutrient cycling, and plant interactions

On the roots of wetland plants such as rice, Fe(II) oxidation forms Fe(III) oxyhydroxide-rich plaques that modulate plant nutrient and metal uptake. The microbial roles in catalyzing this oxidation have been debated and it is unclear if these iron-oxidizers mediate other important biogeochemical and plant interactions. To investigate this, we studied the microbial communities, metagenomes, and geochemistry of iron plaque on field-grown rice, plus the surrounding rhizosphere and bulk soil. Plaque iron content (per mass root) increased over the growing season, showing continuous deposition. Analysis of 16S rRNA genes showed abundant Fe(II)-oxidizing and Fe(III)-reducing bacteria (FeOB and FeRB) in plaque, rhizosphere, and bulk soil. FeOB were enriched in relative abundance in plaque, suggesting FeOB affinity for the root surface. Gallionellaceae FeOB Sideroxydans were enriched during vegetative and early reproductive rice growth stages, while a Gallionella was enriched during reproduction through grain maturity, suggesting distinct FeOB niches over the rice life cycle. FeRB Anaeromyxobacter and Geobacter increased in plaque later, during reproduction and grain ripening, corresponding to increased plaque iron. Metagenome-assembled genomes revealed that Gallionellaceae may grow mixotrophically using both Fe(II) and organics. The Sideroxydans are facultative, able to use non-Fe substrates, which may allow colonization of rice roots early in the season. FeOB genomes suggest adaptations for interacting with plants, including colonization, plant immunity defense, utilization of plant organics, and nitrogen fixation. Taken together, our results strongly suggest that rhizoplane and rhizosphere FeOB can specifically associate with rice roots, catalyzing iron plaque formation, with the potential to contribute to plant growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Expression of V-nitrogenase and Fe-nitrogenase in Methanosarcina acetivorans is controlled by molybdenum, fixed nitrogen, and the expression of Mo-nitrogenase

ABSTRACT All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N 2 ) to ammonia, with some also containing vanadium (V) and iron-only (Fe) nitrogenases that lack Mo. Among diazotrophs, the regulation and usage of the alternative V-nitrogenase and Fe-nitrogenase in methanogens are largely unknown. Methanosarcina acetivorans contains nif , vnf , and anf gene clusters encoding putative Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. This study investigated nitrogenase expression and growth by M. acetivorans in response to fixed nitrogen, Mo/V availability, and CRISPRi repression of the nif , vnf , and/or anf gene clusters. The availability of Mo and V significantly affected growth of M. acetivorans with N 2 but not with NH 4 Cl. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N 2 in medium containing Mo, and the slowest growth in medium lacking Mo and V. qPCR analysis revealed the transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo, whereas vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being key. Immunoblot analysis revealed Mo-nitrogenase is detected when fixed nitrogen is depleted regardless of Mo availability, while V-nitrogenase and Fe-nitrogenase are detected only in the absence of fixed nitrogen and Mo. CRISPRi repression studies revealed that V-nitrogenase and/or Fe-nitrogenase are required for Mo-independent diazotrophy, and unexpectedly that the expression of Mo-nitrogenase is also required. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and dependent on Mo-nitrogenase expression. IMPORTANCE Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens.

Biotechnology & Applied Microbiology↗