Cofactor maturase NifEN: A prototype ancient nitrogenase?

Nitrogenase plays a key role in the global nitrogen cycle; yet, the evolutionary history of nitrogenase and, particularly, the sequence of appearance between the homologous, yet distinct NifDK (the catalytic component) and NifEN (the cofactor maturase) of the extant molybdenum nitrogenase, remains elusive. Here, we report the ability of NifEN to reduce N2 at its surface-exposed L-cluster ([Fe8S9C]), a structural/functional homolog of the M-cluster (or cofactor; [(R-homocitrate)MoFe7S9C]) of NifDK. Furthermore, we demonstrate the ability of the L-cluster-bound NifDK to mimic its NifEN counterpart and enable N2 reduction. These observations, coupled with phylogenetic, ecological, and mechanistic considerations, lead to the proposal of a NifEN-like, L-cluster-carrying protein as an ancient nitrogenase, the exploration of which could shed crucial light on the evolutionary origin of nitrogenase and related enzymes.

Discovery of Eremiobacterota with nifH homologues in tundra soil.

We describe the genome of an Eremiobacterota population from tundra soil that contains the minimal set of nif genes needed to fix atmospheric N2. This putative diazotroph population, which we name Candidatus Lamibacter sapmiensis, links for the first time Eremiobacterota and N2 fixation. The integrity of the genome and its nif genes are well supported by both environmental and taxonomic signals. Ca. Lamibacter sapmiensis contains three nifH homologues and the complementary set of nifDKENB genes that are needed to assemble a functional nitrogenase. The putative diazotrophic role of Ca. Lamibacter sapmiensis is supported by the presence of genes that regulate N2 fixation and other genes involved in downstream processes such as ammonia assimilation. Similar to other Eremiobacterota, Ca. Lamibacter sapmiensis encodes the potential for atmospheric chemosynthesis via CO2 fixation coupled with H2 and CO oxidation. Interestingly, the presence of a N2O reductase indicates that this population could play a role as a N2O sink in tundra soils. Due to the lack of activity data, it remains uncertain if Ca. Lamibacter sapmiensis is able to assemble a functional nitrogenase and participate in N2 fixation. Confirmation of this ability would be a testament to the great metabolic versatility of Eremiobacterota, which appears to underlie their ecological success in cold and oligotrophic environments.

Deciphering the Nitrogen Fixation Gene Cluster in Vibrio natriegens: A Study on Optimized Expression and Application of Nitrogenase.

Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C2H2/mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.

Degradation of Complex Carbon Sources in Organic Fertilizers Facilitates Nitrogen Fixation.

Biological nitrogen fixation is crucial for agriculture and improving fertilizer efficiency, but organic fertilizers in enhancing this process remain debated. Here, we investigate the impact of organic fertilizers on biological nitrogen fixation through experiments and propose a new model where bacterial interactions with complex carbon sources enhance nitrogen fixation. Field experiments showed that adding organic fertilizers increased the nitrogenase activity by 57.85%. Subculture experiments revealed that organic fertilizer addition enriched genes corresponding to complex carbon and energy metabolism, as well as nifJ involved in electron transfer for nitrogenase. It also enhanced bacterial interactions and enhanced connectors associated with complex carbon degradation. Validation experiments demonstrated that combinations increased nitrogenase activity by 2.98 times compared to the single. Our findings suggest that organic fertilizers promoted nitrogen fixation by enhancing microbial cooperation, improved the degradation of complex carbon sources, and thereby provided utilizable carbon sources, energy, and electrons to N-fixers, thus increasing nitrogenase activity and nitrogen fixation.

Analysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra.

Due to the complexity of the catalytic FeMo cofactor site in nitrogenases that mediates the reduction of molecular nitrogen to ammonium, mechanistic details of this reaction remain under debate. In this study, selenium- and sulfur-incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii are prepared under turnover conditions and investigated by using different EPR methods. Complex signal patterns are observed in the continuous wave EPR spectra of selenium-incorporated samples, which are analyzed by Tikhonov regularization, a method that has not yet been applied to high spin systems of transition metal cofactors, and by an already established grid-of-error approach. Both methods yield similar probability distributions that reveal the presence of at least four other species with different electronic structures in addition to the ground state E0. Two of these species were preliminary assigned to hydrogenated E2 states. In addition, advanced pulsed-EPR experiments are utilized to verify the incorporation of sulfur and selenium into the FeMo cofactor, and to assign hyperfine couplings of 33S and 77Se that directly couple to the FeMo cluster. With this analysis, we report selenium incorporation under turnover conditions as a straightforward approach to stabilize and analyze early intermediate states of the FeMo cofactor.

Genetic characterization of biohydrogen-producing purple non-sulfur bacteria Rhodobacter johrii MAY2 isolate via whole genome analysis.

Purple non-sulfur bacteria (PNSB) are a diverse group of bacteria studied for various possible applications. They are commonly surveyed in bioenergy research as they produce biohydrogen, a candidate for clean alternative energy. This study aimed to assess the biohydrogen production ability and genetically characterize a high biohydrogen-producing PNSB (MAY2) isolated from Los Baños, Laguna, Philippines via whole genome sequencing (WGS). MAY2, when grown in mixed volatile fatty acids, produced biogas with 38% hydrogen. WGS results revealed that the isolate is positively classified under the genus Rhodobacter johrii. Also, 82 genetic hallmarks for biohydrogen production were found in the isolated genome which are involved in the production of key enzymes and proteins relevant to the photofermentative and hydrogen regulation pathways. Its nitrogenase gene cluster is stringently regulated by two genes, nifA and rofN, whose function and expression are easily affected by several environmental factors.

Endogenous clock-mediated regulation of intracellular oxygen dynamics is essential for diazotrophic growth of unicellular cyanobacteria.

The discovery of nitrogen fixation in unicellular cyanobacteria provided the first clues for the existence of a circadian clock in prokaryotes. However, recalcitrance to genetic manipulation barred their use as model systems for deciphering the clock function. Here, we explore the circadian clock in the now genetically amenable Cyanothece 51142, a unicellular, nitrogen-fixing cyanobacterium. Unlike non-diazotrophic clock models, Cyanothece 51142 exhibits conspicuous self-sustained rhythms in various discernable phenotypes, offering a platform to directly study the effects of the clock on the physiology of an organism. Deletion of kaiA, an essential clock component in the cyanobacterial system, impacted the regulation of oxygen cycling and hindered nitrogenase activity. Our findings imply a role for the KaiA component of the clock in regulating the intracellular oxygen dynamics in unicellular diazotrophic cyanobacteria and suggest that its addition to the KaiBC clock was likely an adaptive strategy that ensured optimal nitrogen fixation as microbes evolved from an anaerobic to an aerobic atmosphere under nitrogen constraints.

Light-driven, bias-free nitrogenase-based bioelectrochemical cell for ammonia generation.

Nitrogen fixation is a key process that sustains life on Earth. Nitrogenase is the sole enzyme capable of fixing nitrogen under ambient conditions. Extensive research efforts have been dedicated to elucidating the enzyme mechanism and its artificial activation through high applied voltage, photochemistry, or strong reducing agents. Harnessing light irradiation to minimize the required external bias can lower the process's high energy investment. Herein, we present the development of photo-bioelectrochemical cells (PBECs) utilizing BiVO4/CoP or CdS/NiO photoanodes for nitrogenase activation toward N2 fixation. The constructed PBEC based on BiVO4/CoP photoanode requires minimal external bias (200 mV) and suppresses O2 generation that allows efficient activation of the nitrogenase enzyme, using glucose as an electron donor. In a second developed PBEC configuration, CdS/NiO photoanode was used, enabling bias-free activation of the nitrogenase-based cathode to produce 100 µM of ammonia at a faradaic efficiency (FE) of 12%. The ammonia production was determined by a commonly used fluorescence probe and further validated using 1H-NMR spectroscopy. The presented PBECs lay the foundation for biotic-abiotic systems to directly activate enzymes toward value-added chemicals by light-driven reactions.

Harnessing iron­sulfur enzymes for synthetic biology.

Reactions catalysed by iron-sulfur (Fe-S) enzymes appear in a variety of biosynthetic pathways that produce valuable natural products. Harnessing these biosynthetic pathways by expression in microbial cell factories grown on an industrial scale would yield enormous economic and environmental benefits. However, Fe-S enzymes often become bottlenecks that limits the productivity of engineered pathways. As a consequence, achieving the production metrics required for industrial application remains a distant goal for Fe-S enzyme-dependent pathways. Here, we identify and review three core challenges in harnessing Fe-S enzyme activity, which all stem from the properties of Fe-S clusters: 1) limited Fe-S cluster supply within the host cell, 2) Fe-S cluster instability, and 3) lack of specialized reducing cofactor proteins often required for Fe-S enzyme activity, such as enzyme-specific flavodoxins and ferredoxins. We highlight successful methods developed for a variety of Fe-S enzymes and electron carriers for overcoming these difficulties. We use heterologous nitrogenase expression as a grand case study demonstrating how each of these challenges can be addressed. We predict that recent breakthroughs in protein structure prediction and design will prove well-suited to addressing each of these challenges. A reliable toolkit for harnessing Fe-S enzymes in engineered metabolic pathways will accelerate the development of industry-ready Fe-S enzyme-dependent biosynthesis pathways.

What triggers the coupling of proton transfer and electron transfer at the active site of nitrogenase?

In converting N2 to NH3 the enzyme nitrogenase utilises 8 electrons and 8 protons in the complete catalytic cycle. The source of the electrons is an Fe4S4 reductase protein (Fe-protein) which temporarily docks with the MoFe-protein that contains the catalytic active cofactor, FeMo-co, and an electron transfer cluster called the P cluster. The overall mechanism involves 8 repetitions of a cycle in which reduced Fe-protein docks with the MoFe-protein, one electron transfers to the P-cluster, and then to FeMo-co, followed by dissociation of the two proteins and re-reduction of the Fe-protein. Protons are supplied serially to FeMo-co by a Grotthuss proton translocation mechanism from the protein surface along a conserved chain of water molecules (a proton wire) that terminates near S atoms of the FeMo-co cluster [CFe7S9Mo(homocitrate)] where the multiple steps of the chemical conversions are effected. It is assumed that the chemical mechanisms use proton-coupled electron-transfer (PCET) and that H atoms (e- + H+) are involved in each of the hydrogenation steps. However there is neither evidence for, or mechanism proposed, for this coupling. Here I report calculations of cluster charge distribution upon electron addition, revealing that the added negative charge is on the S atoms of FeMo-co, which thereby become more basic, and able to trigger proton transfer from H3O+ waiting at the near end of the proton wire. This mechanism is supported by calculations of the dynamics of the proton transfer step, in which the barrier is reduced by ca. 3.5 kcal mol-1 and the product stabilised by ca. 7 kcal mol-1 upon electron addition. H tunneling is probable in this step. In nitrogenase it is electron transfer that triggers proton transfer.

ATP-Independent Turnover of Dinitrogen Intermediates Captured on the Nitrogenase Cofactor.

Nitrogenase reduces N2 to NH3 at its active-site cofactor. Previous studies of an N2-bound Mo-nitrogenase from Azotobacter vinelandii suggest binding of three N2 species via asymmetric belt-sulfur displacements in the two cofactors of its catalytic component (designated Av1*), leading to the proposal of stepwise N2 reduction involving all cofactor belt-sulfur sites; yet, the evidence for the existence of multiple N2 species on Av1* remains elusive. Here we report a study of ATP-independent, EuII/SO3 2--driven turnover of Av1* using GC-MS and frequency-selective pulse NMR techniques. Our data demonstrate incorporation of D2-derived D by Av1* into the products of C2H2- and H+-reduction, and decreased formation of NH3 by Av1* concomitant with the release of N2 under H2; moreover, they reveal a strict dependence of these activities on SO3 2-. These observations point to the presence of distinct N2 species on Av1*, thereby providing strong support for our proposed mechanism of stepwise reduction of N2 via belt-sulfur mobilization.

Emergence of an Orphan Nitrogenase Protein Following Atmospheric Oxygenation.

Molecular innovations within key metabolisms can have profound impacts on element cycling and ecological distribution. Yet, much of the molecular foundations of early evolved enzymes and metabolisms are unknown. Here, we bring one such mystery to relief by probing the birth and evolution of the G-subunit protein, an integral component of certain members of the nitrogenase family, the only enzymes capable of biological nitrogen fixation. The G-subunit is a Paleoproterozoic-age orphan protein that appears more than 1 billion years after the origin of nitrogenases. We show that the G-subunit arose with novel nitrogenase metal dependence and the ecological expansion of nitrogen-fixing microbes following the transition in environmental metal availabilities and atmospheric oxygenation that began ∼2.5 billion years ago. We identify molecular features that suggest early G-subunit proteins mediated cofactor or protein interactions required for novel metal dependency, priming ancient nitrogenases and their hosts to exploit these newly diversified geochemical environments. We further examined the degree of functional specialization in G-subunit evolution with extant and ancestral homologs using laboratory reconstruction experiments. Our results indicate that permanent recruitment of the orphan protein depended on the prior establishment of conserved molecular features and showcase how contingent evolutionary novelties might shape ecologically important microbial innovations.

Rice N-biofertilization by inoculation with an engineered photosynthetic diazotroph.

Photosynthetic diazotrophs expressing iron-only (Fe-only) nitrogenase can be developed into a promising biofertilizer, as it is independent on the molybdenum availability in the soil. However, the expression of Fe-only nitrogenase in diazotrophs is repressed by the fixed nitrogen of the soil, limiting the efficiency of nitrogen fixation in farmland with low ammonium concentrations that are inadequate for sustainable crop growth. Here, we succeeded in constitutively expressing the Fe-only nitrogenase even in the presence of ammonium by controlling the transcription of Fe-only nitrogenase gene cluster (anfHDGK) with the transcriptional activator of Mo nitrogenase (NifA*) in several different ways, indicating that the engineered NifA* strains can be used as promising chassis cells for efficient expression of different types of nitrogenases. When applied as a biofertilizer, the engineered Rhodopseudomonas palustris effectively stimulated rice growth, contributing to the reduced use of chemical fertilizer and the development of sustainable agriculture.

Restoration of the Functional nif Gene Cluster by Complex Recombination Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101.

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on a combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, the DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then, the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

Lineage-specific evolution of Aquibium, a close relative of Mesorhizobium, during habitat adaptation.

The novel genus Aquibium that lacks nitrogenase was recently reclassified from the Mesorhizobium genus. The genomes of Aquibium species isolated from water were smaller and had higher GC contents than those of Mesorhizobium species. Six Mesorhizobium species lacking nitrogenase were found to exhibit low similarity in the average nucleotide identity values to the other 24 Mesorhizobium species. Therefore, they were classified as the non-N2-fixing Mesorhizobium lineage (N-ML), an evolutionary intermediate species. The results of our phylogenomic analyses and the loss of Rhizobiales-specific fur/mur indicated that Mesorhizobium species may have evolved from Aquibium species through an ecological transition. Halotolerant and alkali-resistant Aquibium and Mesorhizobium microcysteis belonging to N-ML possessed many tripartite ATP-independent periplasmic transporter and sodium/proton antiporter subunits composed of seven genes (mrpABCDEFG). These genes were not present in the N2-fixing Mesorhizobium lineage (ML), suggesting that genes acquired for adaptation to highly saline and alkaline environments were lost during the evolution of ML as the habitat changed to soil. Land-to-water habitat changes in Aquibium species, close relatives of Mesorhizobium species, could have influenced their genomic evolution by the gain and loss of genes. Our study indicated that lineage-specific evolution could have played a significant role in shaping their genome architecture and conferring their ability to thrive in different habitats.IMPORTANCEPhylogenetic analyses revealed that the Aquibium lineage (AL) and non-N2-fixing Mesorhizobium lineage (N-ML) were monophyletically grouped into distinct clusters separate from the N2-fixing Mesorhizobium lineage (ML). The N-ML, an evolutionary intermediate species having characteristics of both ancestral and descendant species, could provide a genomic snapshot of the genetic changes that occur during adaptation. Genomic analyses of AL, N-ML, and ML revealed that changes in the levels of genes related to transporters, chemotaxis, and nitrogen fixation likely reflect adaptations to different environmental conditions. Our study sheds light on the complex and dynamic nature of the evolution of rhizobia in response to changes in their environment and highlights the crucial role of genomic analysis in understanding these processes.

A CRISPR interference system for engineering biological nitrogen fixation.

A grand challenge for the next century is in facing a changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, Azotobacter vinelandii (A. vinelandii). Here, we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in A. vinelandii that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by ~60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system fills an important gap for engineering microbial nitrogen fixation for desired purposes.IMPORTANCEAll life on Earth requires nitrogen to survive. About 78% of the atmosphere alone is nitrogen, yet humans cannot use it directly. Instead, we obtain the nitrogen we need for our survival through the food we eat. For more than 100 years, a substantial portion of agricultural productivity has relied on industrial methods for nitrogen fertilizer synthesis, which consumes significant amounts of nonrenewable energy resources and exacerbates environmental degradation and human-induced climate change. Promising alternatives to these industrial methods rely on engineering the only biological pathway for generating bioaccessible nitrogen: microbial nitrogen fixation. Bioengineering strategies require an extensive understanding of underlying genetics in nitrogen-fixing microbes, but genetic tools for this critical goal remain lacking. The CRISPRi gene silencing system that we report, developed in the broadly utilized nitrogen-fixing bacterial model, Azotobacter vinelandii, is an important step toward elucidating the complexity of nitrogen fixation genetics and enabling their manipulation.

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus.

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviors of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.IMPORTANCENitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.

Precipitation affects soil nitrogen fixation by regulating active diazotrophs and nitrate nitrogen in an alpine grassland of Qinghai-Tibetan Plateau.

Soil asymbiotic nitrogen (N) fixation provides a critical N source to support plant growth in alpine grasslands, and precipitation change is expected to lead to shifts in soil asymbiotic N fixation. However, large gaps remain in understanding the response of soil asymbiotic N fixation to precipitation gradients. Here we simulated five precipitation gradients (10 % (0.1P), 50 % (0.5P), 70 % (0.7P), 100 % (1.0P) and 150 % (1.5P) of the natural precipitation) in an alpine grassland of Qinghai-Tibetan Plateau and examined the soil nitrogenase activity and N fixation rate for each gradient. Quantitative PCR and high-throughput sequencing were used to measure the abundance and community composition of the soil nifH DNA (total diazotrophs) and nifH RNA reverse transcription (active diazotrophs) gene. Our results showed that the soil diazotrophic abundance, diversity and nifH gene expression rate peaked under the 0.5P. Soil nitrogenase activity and N fixation rate varied in the range 0.032-0.073 nmol·C2H4·g-1·h-1 and 0.008-0.022 nmol·N2·g-1·h-1 respectively, being highest under the 0.5P. The 50 % precipitation reduction enhanced the gene expression rates of Azospirillum and Halorhodospira which were likely responsible for the high N fixation potential. The 0.5P treatment also possessed a larger and more complex active diazotrophic network than the other treatments, which facilitated the resistance of diazotrophic community to environmental stress and thus maintained a high N fixation potential. The active diazotrophic abundance had the largest positive effect on soil N fixation, while nitrate nitrogen had the largest negative effect. Together, our study suggested that appropriate precipitation reduction can enhance soil N fixation through promoting the abundance of the soil active diazotrophs and decreasing soil nitrate nitrogen, and soil active diazotrophs and nitrate nitrogen should be considered in predicting soil N inputs in the alpine grassland of Qinghai-Tibetan Plateau under precipitation change.

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 15N2 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.

Mapping Geological Events and Nitrogen Fixation Evolution Onto the Timetree of the Evolution of Nitrogen-Fixation Genes.

Nitrogen is essential for all organisms, but biological nitrogen fixation (BNF) occurs only in a small fraction of prokaryotes. Previous studies divided nitrogenase-gene-carrying prokaryotes into Groups I to IV and provided evidence that BNF first evolved in bacteria. This study constructed a timetree of the evolution of nitrogen-fixation genes and estimated that archaea evolved BNF much later than bacteria and that nitrogen-fixing cyanobacteria evolved later than 1,900 MYA, considerably younger than the previous estimate of 2,200 MYA. Moreover, Groups III and II/I diverged ∼2,280 MYA, after the Kenorland supercontinent breakup (∼2,500-2,100 MYA) and the Great Oxidation Event (∼2,400-2,100 MYA); Groups III and Vnf/Anf diverged ∼2,086 MYA, after the Yarrabubba impact (∼2,229 MYA); and Groups II and I diverged ∼1,920 MYA, after the Vredefort impact (∼2,023 MYA). In summary, this study provided a timescale of BNF events and discussed the possible effects of geological events on BNF evolution.