Nitrogen-fixing Conditions Research Articles

Influence of Microbiological Preparations and Microelements on Indicators of Photosynthetic and Symbiotic Activity of Soybean Crops

The article presents the results of studies on the effect of the combined use of microbiological fertilizers and microelements on the formation of the photosynthetic and symbiotic apparatus, the size and structure of the yield, the chemical composition and quality of soybean seeds, carried out on leached chernozems in the Kabardino-Balkarian Republic foothills zone. The use of microbiological fertilizers and microelements as preparations for pre-sowing treatment of soybean seeds increases the weight of active nodules by 46.7–61.0 kg / ha, the active symbiotic potential – 2.4–2.8 thousand units, the amount of fixed atmospheric nitrogen by the symbiotic soybean system – 36.1–40.1 kg / ha. In 2022, the maximum number of nodules, including active ones, was formed in the Fon + Ultrastim® variant, the smallest – in the control. In 2023 which was less moisture-rich for nodule formation, the maximum number of nodules was in the Background + ATUVA® variant. Creating optimal conditions for biological nitrogen fixation (Background + ATUVA®) increases the area of soybean leaves by 14.1 thousand m²/ha, photosynthetic potential is 72.7 thousand units, dry matter accumulation is 1.84 t/ha, seed yield – 1.33 t/ha. The microbiological fertilizers and microelements application (Background + ATUVA®) increases the nitrogen content in the vegetative and generative organs of soybeans throughout the growing season, increases the nitrogen accumulation by seeds by 36.1–40.1 kg/ha, the share of atmospheric nitrogen in plant nutrition – 42–50%; the protein content in seeds is 1.1%, while the collection of protein with soybean seeds increases by 479 kg.

Identification of Yarrowia lipolytica as a platform for designed consortia that incorporate in situ nitrogen fixation to enable ammonia-free bioconversion

Bioconversion processes require nitrogen for growth and production of intracellular enzymes to produce biofuels and bioproducts. Typically, this is supplied as reduced nitrogen in the form of ammonia, which is produced offsite from N2 and H2 via the Haber-Bosch process. While this has revolutionized industries dependent on fixed nitrogen (e.g., modern agriculture), it is highly energy-intensive and its reliance on natural gas combustion results in substantial global CO2 emissions. Here we investigated the feasibility of in situ biological nitrogen fixation from N2 gas as a strategy to reduce greenhouse gas impacts of aerobic bioconversion processes. We developed an efficient and cost-effective method to screen fungal bioconversion hosts for compatibility with the free-living diazotrophic bacterium Azotobacter vinelandii under nitrogen fixing conditions. Our screening revealed that the genus Yarrowia is particularly enriched during co-culture experiments. Follow-up experiments identified four Y. lipolytica strains (NRRL Y-11853, NRRL Y-7208, NRRL Y-7317, and NRRL YB-618) capable of growth in co-culture with A. vinelandii. These strains utilize ammonium secreted during diazotrophic fixation of N2, which is provided as a component of the air input stream during aerobic fermentation. This demonstrates the feasibly of in situ biological nitrogen fixation to support heterotrophic fermentation processes for production of fuels and chemicals.

Proteomic changes orchestrate metabolic acclimation of a unicellular diazotrophic cyanobacterium during light-dark cycle and nitrogen fixation states.

Cyanobacteria have developed an impressive array of proteins and pathways, each tailored for specific metabolic attributes, to execute photosynthesis and biological nitrogen (N2)-fixation. An understanding of these biologically incompatible processes provides important insights into how they can be optimized for renewable energy. To expand upon our current knowledge, we performed label-free quantitative proteomic analysis of the unicellular diazotrophic cyanobacterium Crocosphaera subtropica ATCC 51142 grown with and without nitrate under 12-hour light-dark cycles. Results showed significant shift in metabolic activities including photosynthesis, respiration, biological nitrogen fixation (BNF), and proteostasis to different growth conditions. We identified 14 nitrogenase enzymes which were among the most highly expressed proteins in the dark under nitrogen-fixing conditions, emphasizing their importance in BNF. Nitrogenase enzymes were not expressed under non nitrogen fixing conditions, suggesting a regulatory mechanism based on nitrogen availability. The synthesis of key respiratory enzymes and uptake hydrogenase (HupSL) synchronized with the synthesis of nitrogenase indicating a coordinated regulation of processes involved in energy production and BNF. Data suggests alternative pathways that cells utilize, such as oxidative pentose phosphate (OPP) and 2-oxoglutarate (2-OG) pathways, to produce ATP and support bioenergetic BNF. Data also indicates the important role of uptake hydrogenase for the removal of O2 to support BNF. Overall, this study expands upon our knowledge regarding molecular responses of Crocosphaera 51142 to nitrogen and light-dark phases, shedding light on potential applications and optimization for renewable energy.

An Azospirillum brasilense chemoreceptor that mediates nitrate chemotaxis has conditional roles in the colonization of plant roots.

Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacterium Azospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter of tlp2 is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.IMPORTANCEBacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere.

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.

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.

Nitrogen fixation by rotational gliding arc plasma at surrounding conditions

Nitrogen fixation (NF) was investigated in a rotational gliding arc plasma based on various energy inputs, flow rates, and N2:O2 ratios under atmospheric conditions. For practical application, the effect of water content in the feed, self-heating during plasma discharge, prolonged operation, and comparison between plasma and thermal process for N2 oxidation were also conducted. The comparison results reveal that NF by the gliding arc plasma is superior to the thermal process in terms of NOx yield and energy consumption at low temperatures and atmospheric pressure. Herein, energy input and flow rate are two controlled factors that significantly influence NOx production rate. Simultaneously, O2 content played a crucial role in determining NOx yield, selectivity of NO/NO2, and energy consumption. Observations during plasma discharge indicated an initial rise in gas and reactor body temperatures, coinciding with decrease in total NOx formation. Moreover, the presence of water had a negative effect on NOx yield. Accordingly, the optimized conditions for NF by gliding arc plasma for NOx formation was identified at a N2 to O2 ratio of 3:2 without water vapor in the feed gas. Furthermore, a 14-h continuous operational test validated the robustness of our reactor configuration, affirming its long-term viability. The study concludes by performing oxidation of the NOx gas followed by dissolution in water, successfully generating nitric acid. These findings underscore the potential of gliding arc plasma for efficient NF processes, offering insights into optimal operational conditions and downstream applications for nitric acid generation.

How Can Plants Help Restore Degraded Tropical Soils?

In the tropics, anthropogenic activities can lead to water and wind erosion, a loss of biodiversity, and a reduction in sequestered carbon, fertility, and organic matter content in the soils concerned, potentially resulting in their degradation. This study therefore aims to identify the mechanisms used by plant species to restore degraded tropical soils and plant species characteristics that are best suited to achieve this through a critical scoping review of the peer-reviewed literature. Soil restoration leads to the re-establishment of ecosystem services and an increase in soil production potential, the regeneration of biodiversity, the stopping of organic matter losses, and the creation of favorable conditions for carbon sequestration and nitrogen fixation. The choice of appropriate plant species depends on the restoration objectives to be achieved. Five key mechanisms by which plant species contribute to restore degraded tropical soils include: (1) nitrogen fixation, (2) carbon sequestration, (3) organic matter addition, (4) structure stabilization, and (5) erosion control. The main characteristics of plant species and vegetation involved in these mechanisms are (a) the capacity to form symbiotic associations with N-fixing bacteria and mycorrhizae, (b) the production of abundant root biomass releasing litter and exudates, (c) roots having a high length density, branching intensity, and depth distribution, (d) the production of an abundant and easily decomposed above ground litter, (e) the production of a vast canopy, and (f) the presence of different vegetation strata. Targeting these characteristics will contribute to acting on several mechanisms simultaneously, which will increase the chance of success in tropical soil restoration.

Nitrogen fixation and transcriptome of a new diazotrophic Geomonas from paddy soils.

The ability of Geomonas species to fix nitrogen gas (N2) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria. We demonstrated N2-dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas.

Continuous Bioproduction of Alginate Bacterial under Nitrogen Fixation and Nonfixation Conditions

Alginate is a biomaterial produced by Azotobacter vinelandii, a diazotroph that, under nitrogen-fixing conditions, can fix nitrogen under high oxygen levels. In A. vinelandii, alginate is synthesized from fructose-6P via synthesis of precursor, polymerization, and modification/exportation. Due to its viscosifying, gelling, and thickening characteristics, alginate is widely used in food, pharmaceutical, and cosmetical industries. This study aimed to develop a continuous bioprocess and a comparative analysis of alginate production under diazotrophic and nondiazotrophic conditions. Continuous cultures were developed at three dilution rates (0.06, 0.08 and 0.10 h−1). In steady state, the respiratory activity, alginate production, alginate molecular weight and the genes encoding alginate polymerase were determined. Under the conditions studied, the specific oxygen uptake rate and respiratory quotient were similar. The diazotrophic conditions improved the conversion of sucrose to alginate and the specific productivity rate, which was 0.24 ± 0.03 g g−1 h−1. A higher alginate molecular weight (725 ± 20 kDa) was also achieved under diazotrophic conditions, which can be explained by an increase in the gene expression of genes alg8 and alg44 (encoding polymerase). The results of this work show the feasibility of enhancing alginate production (yields and specific productivity rates) and quality (molecular weight) under nitrogen-fixing conditions, opening the possibility of developing a continuous bioprocess to produce alginate with specific characteristics under conditions of diazotrophy.

Overexpression of LjPLT3 Enhances Salt Tolerance in Lotus japonicus.

Intracellular polyols are used as osmoprotectants by many plants under environmental stress. However, few studies have shown the role of polyol transporters in the tolerance of plants to abiotic stresses. Here, we describe the expression characteristics and potential functions of Lotus japonicus polyol transporter LjPLT3 under salt stress. Using LjPLT3 promoter-reporter gene plants showed that LjPLT3 was expressed in the vascular tissue of L. japonicus leaf, stem, root, and nodule. The expression was also induced by NaCl treatment. Overexpression of LjPLT3 in L. japonicus modified the growth rate and saline tolerance of the transgenic plants. The OELjPLT3 seedlings displayed reduced plant height under both nitrogen-sufficient and symbiotic nitrogen fixation conditions when 4 weeks old. The nodule number of OELjPLT3 plants was reduced by 6.7-27.4% when 4 weeks old. After exposure to a NaCl treatment in Petri dishes for 10 days, OELjPLT3 seedlings had a higher chlorophyll concentration, fresh weight, and survival rate than those in the wild type. For symbiotic nitrogen fixation conditions, the decrease in nitrogenase activity of OELjPLT3 plants was slower than that of the wild type after salt treatment. Compared to the wild type, both the accumulation of small organic molecules and the activity of antioxidant enzymes were higher under salt stress. Considering the concentration of lower reactive oxygen species (ROS) in transgenic lines, we speculate that overexpression of LjPLT3 in L. japonicus might improve the ROS scavenging system to alleviate the oxidative damage caused by salt stress, thereby increasing plant salinity tolerance. Our results will direct the breeding of forage legumes in saline land and also provide an opportunity for the improvement of poor and saline soils.

Evolving a New Electron Transfer Pathway for Nitrogen Fixation Uncovers an Electron Bifurcating-Like Enzyme Involved in Anaerobic Aromatic Compound Degradation.

Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin (Fd) or flavodoxin (Fld) to reduce dinitrogen gas (N2) to produce ammonia, generating hydrogen gas (H2) as an obligate product of this activity. Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes multiple proteins that can reduce Fd, the FixABCX complex is the only one shown to support nitrogen fixation, and R. palustris Fix- mutants grow poorly under nitrogen-fixing conditions. To investigate how native electron transfer chains (ETCs) can be redirected toward nitrogen fixation, we leveraged the strong selective pressure of nitrogen limitation to isolate a suppressor of an R. palustris ΔfixC strain that grows under nitrogen-fixing conditions. We found two mutations were required to restore growth under nitrogen-fixing conditions in the absence of functional FixABCX. One mutation was in the gene encoding the primary Fd involved in nitrogen fixation, fer1, and the other mutation was in aadN, which encodes a homolog of NAD+-dependent Fd:NADPH oxidoreductase (Nfn). We present evidence that AadN plays a role in electron transfer to benzoyl coenzyme A reductase, the key enzyme involved in anaerobic aromatic compound degradation. Our data support a model where the ETC for anaerobic aromatic compound degradation was repurposed to support nitrogen fixation in the ΔfixC suppressor strain. IMPORTANCE There is increasing evidence that protein electron carriers like Fd evolved to form specific partnerships with select electron donors and acceptors to keep native electron transfer pathways insulated from one another. This makes it challenging to integrate a Fd-dependent pathway such as biological nitrogen fixation into non-nitrogen-fixing organisms and provide the high-energy reducing power needed to fix nitrogen. Here, we show that amino acid substitutions in an electron donor for anaerobic aromatic compound degradation and an Fd involved in nitrogen fixation enabled electron transfer to nitrogenase. This study provides a model system to understand electron transfer chain specificity and how new electron transfer pathways can be evolved for biotechnologically valuable pathways like nitrogen fixation.

NtcA, LexA and heptamer repeats involved in the multifaceted regulation of DNA repair genes recF, recO and recR in the cyanobacterium Nostoc PCC7120

Regulation of DNA repair genes in cyanobacteria is an unexplored field despite some of them exhibiting high radio-resistance. With RecF pathway speculated to be the major double strand break repair pathway in Nostoc sp. strain PCC7120, regulation of recF, recO and recR genes was investigated. Bioinformatic approach-based identification of promoter and regulatory elements was validated using qRT-PCR analysis, reporter gene and DNA binding assays. Different deletion constructs of the upstream regulatory regions of these genes were analysed in host Nostoc as well as heterologous system Escherichia coli. Studies revealed: (1) Positive regulation of all three genes by NtcA, (2) Negative regulation by LexA, (3) Involvement of contiguous heptamer repeats with/without its yet to be identified interacting partner in regulating (i) binding of NtcA and LexA to recO promoter and its translation, (ii) transcription or translation of recF, (4) Translational regulation of recF and recO through non-canonical and distant S.D. sequence and of recR through a rare initiation codon. Presence of NtcA either precludes binding of LexA to AnLexA-Box or negates its repressive action resulting in higher expression of these genes under nitrogen-fixing conditions in Nostoc. Thus, in Nostoc, expression of recF, recO and recR genes is intricately regulated through multiple regulatory elements/proteins. Contiguous heptamer repeats present across the Nostoc genome in the vicinity of start codon or promoter is likely to have a global regulatory role. This is the first report detailing regulation of DSB repair genes in any algae.

Fixing N2 into cyanophycin: continuous cultivation of Nostoc sp. PCC 7120

Two diazotrophic cyanobacteria (Anabaena cylindrica PCC 7122 and Nostoc sp. PCC 7120) were cultivated to produce cyanophycin, a nitrogen reserve compound, under nitrogen fixing conditions. In preliminary continuous experiments, Nostoc sp. was shown to be more efficient, accumulating a higher amount of cyanophycin and showing a greater capability to fix atmospheric nitrogen in the biomass (67 mgN d−1 of fixed nitrogen per liter of culture). The operating conditions were then optimized to maximize the cyanophycin productivity: the effect of incident light intensity, residence time and nitrogen availability were investigated. Nitrogen availability and/or pH played a major role with respect to biomass production, whereas phosphorus limitation was the main variable to maximize cyanophycin accumulation. In this way, it was possible to achieve a stable and continuous production of cyanophycin (CGP) under diazotrophic conditions, obtaining a maximum cyanophycin productivity of 15 mgCGP L−1 d−1.Key points• Diazotrophic cyanobacteria produce stable amount of cyanophycin in continuous PBR.• Nostoc sp. proved to be more efficient in producing cyanophycin than Anabaena sp.• P deprivation is the major variable to increase cyanophycin productivity in continuous.

Elucidation of Essential Genes and Mutant Fitness during Adaptation toward Nitrogen Fixation Conditions in the Endophyte Azoarcus olearius BH72 Revealed by Tn-Seq.

Azoarcus olearius BH72 is a diazotrophic model endophyte that contributes fixed nitrogen to its host plant, Kallar grass, and expresses nitrogenase genes endophytically. Despite extensive studies on biological nitrogen fixation (BNF) of diazotrophic endophytes, little is known about global genetic players involved in survival under respective physiological conditions. Here, we report a global genomic screen for putatively essential genes of A. olearius employing Tn5 transposon mutagenesis with a modified transposon combined with high-throughput sequencing (Tn-Seq). A large Tn5 master library of ~6 × 105 insertion mutants of strain BH72 was obtained. Next-generation sequencing identified 183,437 unique insertion sites into the 4,376,040-bp genome, displaying one insertion every 24 bp on average. Applying stringent criteria, we describe 616 genes as putatively essential for growth on rich medium. COG (Clusters of Orthologous Groups) assignment of the 564 identified protein-coding genes revealed enrichment of genes related to core cellular functions and cell viability. To mimic gradual adaptations toward BNF conditions, the Tn5 mutant library was grown aerobically in synthetic medium or microaerobically on either combined or atmospheric nitrogen. Enrichment and depletion analysis of Tn5 mutants not only demonstrated the role of BNF- and metabolism-related proteins but also revealed that, strikingly, many genes relevant for plant-microbe interactions decrease bacterial competitiveness in pure culture, such type IV pilus- and bacterial envelope-associated genes. IMPORTANCE A constantly growing world population and the daunting challenge of climate change demand new strategies in agricultural crop production. Intensive usage of chemical fertilizers, overloading the world's fields with organic input, threaten terrestrial and marine ecosystems as well as human health. Long overlooked, the beneficial interaction of endophytic bacteria and grasses has attracted ever-growing interest in research in the last decade. Capable of biological nitrogen fixation, diazotrophic endophytes not only provide a valuable source of combined nitrogen but also are known for diverse plant growth-promoting effects, thereby contributing to plant productivity. Elucidation of an essential gene set for a prominent model endophyte such as A. olearius BH72 provides us with powerful insights into its basic lifestyle. Knowledge about genes detrimental or advantageous under defined physiological conditions may point out a way of manipulating key steps in the bacterium's lifestyle and plant interaction toward a more sustainable agriculture.

Photosynthetic modulation during the diurnal cycle in a unicellular diazotrophic cyanobacterium grown under nitrogen-replete and nitrogen-fixing conditions

Cyanobacteria are the only oxygenic photosynthetic organisms that can fix nitrogen. In diazotrophic cyanobacteria, the regulation of photosynthesis during the diurnal cycle is hypothesized to be linked with nitrogen fixation and involve the D1 protein isoform PsbA4. The amount of bioavailable nitrogen has a major impact on productivity in aqueous environments. In contrast to low- or nitrogen-fixing (−N) conditions, little data on photosynthetic regulation under nitrogen-replete (+ N) conditions are available. We compared the regulation of photosynthesis under −N and + N conditions during the diurnal cycle in wild type and a psbA4 deletion strain of the unicellular diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. We observed common changes to light harvesting and photosynthetic electron transport during the dark in + N and −N conditions and found that these modifications occur in both diazotrophic and non-diazotrophic cyanobacteria. Nitrogen availability increased PSII titer when cells transitioned from dark to light and promoted growth. Under −N conditions, deletion of PsbA4 modified charge recombination in dark and regulation of PSII titer during dark to light transition. We conclude that darkness impacts the acceptor-side modifications to PSII and photosynthetic electron transport in cyanobacteria independently of the nitrogen-fixing status and the presence of PsbA4.

Efficient sunlight promoted nitrogen fixation from air under room temperature and ambient pressure via Ti/Mo composites

Photocatalytic nitrogen fixation is an important pathway for carbon neutralization and sustainable development. Inspired by nitrogenase, the participation of molybdenum can effectively activate nitrogen. A novel Ti/Mo composites photocatalyst is designed by sintering the molybdenum acetylacetonate precursor with TiO2. The special carbon-coated hexagonal photocatalyst is obtained which photocatalytic nitrogen fixation performance is enhanced 16 times compared to pure TiO2 at room temperature and ambient pressure. The abundant surface defects in this composite were confirmed to be the key factor for nitrogen fixation. The 15N2 isotope labeling experiment was used to demonstrate the feasibility of nitrogen to ammonia conversion. Also, modelling on the interactions between light and the synthesized photocatalyst particle was examined for the light absorption. The optimum nitrogen fixation conditions have been examined, and the nitrogen fixation performance can reach up to 432 μg·gcat-1·h−1. Numerical simulations via the field-only surface integral method were also carried out to study the interactions between light and the photocatalytic particles to further confirm that it can be a useful material for photocatalyst. This newly developed Ti/Mo composites provide a simple and effective strategy for photocatalytic nitrogen fixation from air directly under ambient conditions.

Efficient photocatalytic nitrogen fixation from air under sunlight via iron-doped WO3

Photocatalytic nitrogen fixation from air directly under sunlight can contribute significantly to carbon neutralization. It is an ideal pathway to replace the industrial Haber Bosch process in future. A Fe-doped layered WO3 photocatalyst containing oxygen vacancies was developed which can fix nitrogen from air directly under sunlight at atmospheric pressure. The iron doping enhances the transport efficiency of photogenerated electrons. The photocatalytic efficiency is around 4 times higher than that of pure WO3. The optium nitrogen fixation conditions were examined by orthogonal experiments and its nitrogen fixation performance could reach up to 477 μg·gcat−1·h−1 under sunlight. In addition, the process of nitrogen fixation was detected by situ infrared, which confirmed the reliability of nitrogen fixation. Also, modeling on the interactions between light and the photocatalyst was carried out to study the distribution of surface charge and validate the light absorption of the photocatalyst. This work provides a simple and cheap strategy for photocatalytic nitrogen fixation from air under mild conditions.

A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli

Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense.

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.