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Nitrogenase

Nitrogenase is a complex enzyme system responsible for the biological fixation of atmospheric nitrogen, a critical process in the global nitrogen cycle.
This metalloenzyme catalyzes the reduction of atmospheric dinitrogen (N2) to ammonia (NH3), making nitrogen available for incorporation into organic compounds.
Nitrogenase plays a pivotal role in the growth and development of many nitrogen-fixing organisms, including bacteria, archaea, and some eukaryotes.
Understanding the structure, function, and regulation of nitrogenase is an active area of research, with applications in agriculture, bioenergy, and environmental remediation.
Optimizing protocols for nitrogenase studies can help advance our knowledge of this essential biological catalyst and its potential for tackling global challneges.

Most cited protocols related to «Nitrogenase»

1
Modeling Oxygen Management in Nitrogen Fixers
Oxygen management is a key cost for nitrogen fixers that we seek to quantitatively model. First consider the rate of change of the intracellular oxygen, QO2 (mol O2 per cell) in a spherical microbe:

Here, [O2] and [O2]C are the environmental and intracellular oxygen concentrations, respectively (mol O2 m−3). The first term on the right, PO2 (mol O2 per cell per s) represents a source from oxygenic photosynthesis. The second term is a source because of transfer across the membranes of cell with the cytoplasmic radius r (m per cell), governed by the oxygen gradient and the effective diffusivity across the membrane and external molecular boundary layer, κO2 (m2 s−1). The third term, in parentheses, represents consumption of intracellular oxygen by respiration associated with synthesis (RS) including the direct cost of nitrogen fixation, maintenance (Rm) and respiratory protection (RP) (mol O2 per cell per s). RS is related to the growth rate of the population, μ (s−1) by

where QC is the carbon quota (mol C per cell) of the species in question and YSO2:BIO is the growth yield with respect to oxygen (mol O2 consumed per mol C biomass synthesized), which can be evaluated from the overall stoichiometry of the reactions (Heijnen and Roels, 1981 ; Rittmann and McCarty, 2001 ; see Supplementary Material S1).
As reducing intracellular oxygen concentration is critical for nitrogen fixers, consider the solution for the intracellular oxygen concentration [O2]C at steady state (dQO2/dt≈0):

Oxygenic photosynthesis, PO2, always acts to increase intracellular oxygen concentration along with invasion from the environment, if the external concentration is higher. In contrast, there are numerous strategies to reduce intracellular oxygen levels and protect nitrogenase, as mentioned in the introduction: living in a low-oxygen environment, reducing [O2] increasing the efficiency of respiratory oxygen consumption, YSO2:BIO; and creating thick membranes or mucus layers to reduce the effective diffusivity of oxygen, κO2 into the cell. As carbon quota, QC, increases with cell volume (r3), increasing cell radius will increase RS and reduce [O2]C, as will increasing growth rate μ also increase the respiratory oxygen demand. A high maintenance respiration or deliberate respiratory protection, RP, consumes oxygen. The investment in respiratory protection to reduce the intracellular oxygen concentration to very low levels can be estimated by setting [O2]C=0 in Equation (4) and re-arranging:

The required RP is the difference between sources due to oxygenesis and diffusive invasion, and the demand from growth and maintenance.
Inomura K., Bragg J, & Follows M.J. (2016). A quantitative analysis of the direct and indirect costs of nitrogen fixation: a model based on Azotobacter vinelandii. The ISME Journal, 11(1), 166-175.
Publication 2016
Anabolism Carbon Cells Chemical Oxygen Demand Cytoplasm Diffusion M Cells Mucus Nitrogen Nitrogenase Nitrogen Fixation Oxygen Consumption Photosynthesis Plasma Membrane Protoplasm Radius Respiration Respiratory Rate SERPINA3 protein, human Therapies, Oxygen Inhalation Tissue, Membrane
2
Nitrogenase Activity Assay Protocol
For nitrogenase activity assays, Paenibacillus sp.WLY78 and the engineered E. coli strains were grown in 5 ml of LD media (supplemented with antibiotics) in 50 ml flasks shaken at 250 rpm for 16 h at 30°C. The cultures were collected by centrifugation, washed three times with sterilized water and then resuspended in nitrogen-deficient medium containing 2 mM glutamate as nitrogen source (supplemented with antibiotics for the engineered E. coli strains and IPTG when necessary) to a final OD600 of 0.2–0.4. Then, 1 ml of the culture was transferred to a 25-ml test tube and the test tube was sealed with robber stopper. The headspace in the tube was then evacuated and replaced with argon gas [14] . After incubating the cultures for 6–8 h at 30°C with shaking at 250 rpm, C2H2 (10% of the headspace volume) was injected into the test tubes. After incubating the cultures for a further 3 h, 100 µl of culture headspace was withdrawn through the rubber stopper with a gas tight syringe and manually injected into a HP6890 gas chromatograph to quantify ethylene production. All treatments were in three replicates and all the experiments were repeated three or more times.
For measuring the effect of ammonium on nitrogenase activity, nitrogen-deficient medium was supplemented with NH4Cl at the concentrations indicated and the cultures were also grown under anaerobic conditions. For measuring the effect of oxygen on nitrogenase activity, nitrogen-deficient medium containing 2 mM glutamate as nitrogen source was used, and oxygen was adjusted to the initial concentration indicated at the start of the incubation.
Wang L., Zhang L., Liu Z., Zhao D., Liu X., Zhang B., Xie J., Hong Y., Li P., Chen S., Dixon R, & Li J. (2013). A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli. PLoS Genetics, 9(10), e1003865.
Publication 2013
Ammonium Antibiotics, Antitubercular Argon Biological Assay Centrifugation Escherichia coli ethylene Gas Chromatography Glutamate Isopropyl Thiogalactoside Nitrogen Nitrogenase Oxygen Paenibacillus Rubber Strains Syringes
3
Microbial Community Sampling at Nakabusa Hot Springs
Samples were collected from two hot spring sites at Nakabusa hot spring located in Nagano prefecture, Japan (Wall Site [36°23′20″N 137°44′52″E], Stream Site [36°23′33″N, 137°44′52″E]). The linear distance between the two sites was approximately 170 m. Hot spring water was slightly alkaline (pH 8.5–8.9) and contained 0.046–0.138 mM of sulfide, 0.019–0.246 mM of sulfate, and 5.0–6.1 μM of ammonia (32 , 33 (link), 52 (link)). Nitrate and nitrite were not detected (32 , 34 (link)). The microbial communities that developed at 72°C to 75°C were collected from the two sites (Fig. 1) on 22 July, 25 August, 18 November 2016, and 8 and 29 January 2017 using sterilized tweezers: pale tan colored microbial mats developed on a concrete wall that hot spring water runs down (Wall Site, Figs. 1A and 1B); similar pale tan colored filamentous microbial communities (streamers) were observed in a stream of the hot springs (Stream Site, Figs. 1C and 1D). The samples for DNA extraction were placed in 2.0-mL screw cap plastic tubes, frozen immediately in dry ice-ethanol slurry at the site, and stored on dry ice or at –80°C until further use. Samples for nitrogenase activity measurements were appropriately incubated in situ or at the laboratory without freezing (see below).
Nishihara A., Haruta S., McGlynn S.E., Thiel V, & Matsuura K. (2018). Nitrogen Fixation in Thermophilic Chemosynthetic Microbial Communities Depending on Hydrogen, Sulfate, and Carbon Dioxide. Microbes and Environments, 33(1), 10-18.
Publication 2018
Ammonia Cytoskeletal Filaments Dry Ice Ethanol Figs Hot Springs Microbial Community Nitrates Nitrites Nitrogenase Sulfates, Inorganic Sulfides
4
Phylogenetic Analysis of Nitrogenase-like Proteins
An initial list of 75 NifD/E and NifK/N-like sequences belonging to the PFAM family PF00148 were selected manually from the IMG database [33 (link)] (http://img.jgi.doe.gov) and then used as queries in a BLAST [32 (link)] search against the NCBI NR protein database with an e-value cut-off of 10−20. This returned 1117 unique geneIDs, which were then filtered against known NifD/E and NifK/N sequences (Additional file 2: Table S3) to remove hits to conventional nitrogenase. The remaining 900 unique gene IDs were further filtered with a BLAST search against ChlB (accession GenBank:AAT28195.1), BchB (SwissProt:Q3APL0.1), ChlN (GenBank:AAP99591.1) and BchN (SwissProt:Q3APK9.1) to remove homologs of protochlorophylide reductase. Fused protein sequences (NifHD/E) were also filtered out and were not subject to further phylogenetic analysis. Another filtering was done with a preliminary tree built using FastTree 2.1 [34 (link)] to identify very similar sequences; only one member of each set of similar sequences was kept. The final compilation contained 472 unique gene IDs.
Manual inspection of the 472-sequence tree yielded a “core” list of 73 representative sequences. These 73 sequences were then aligned with ClustalW version 2.1 [35 (link)] with the Gonnet 250 protein matrix and default pairwise alignment options. A phylogenetic tree was built with FastTree 2.1 [34 (link)] using the WAG + gamma20 likelihood model; the result is shown in Figure 4.
Dos Santos P.C., Fang Z., Mason S.W., Setubal J.C, & Dixon R. (2012). Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genomics, 13, 162.
Publication 2012
Amino Acid Sequence Genes Nitrogenase Oxidoreductase Proteins Trees
5
Comprehensive Rumen Microbiome Enzymatic Profiling
The protein sequences of the 501 genomes of cultured rumen bacteria (410 from Hungate Collection [21 (link)], 91 from other sources) were retrieved from the Joint Genome Institute (JGI) genome portal. These sequences were then screened against local protein databases for the catalytic subunits of the three classes of hydrogenases (NiFe-hydrogenases, FeFe-hydrogenases, Fe-hydrogenases), nitrogenases (NifH), methyl-CoM reductases (McrA), acetyl-CoA synthases (AcsB), adenylylsulfate reductases (AprA), dissimilatory sulfite reductases (DsrA), alternative sulfite reductases (AsrA), fumarate reductases (FrdA), dissimilatory nitrate reductases (NarG), periplasmic nitrate reductases (NapA), ammonia-forming nitrite reductases (NrfA), DMSO/TMAO reductases (DmsA) and cytochrome bd oxidases (CydA). Hydrogenases were screened using the HydDB data set [66 (link), 67 (link)], targeted searches were used to screen six protein families (AprA, AsrA, NarG, NapA, NrfA, DmsA, CydA) and comprehensive custom databases were constructed to screen five other protein families (NifH, McrA, AcsB, DsrA, FrdA) based on their total reported genetic diversity [70 (link)–74 (link)]. A custom Python script incorporating the Biopython package [75 (link)] was designed to produce and parse BLAST results (https://github.com/woodlaur189/get_flanks_blast/releases). This script was used to batch-submit the protein sequences of the 501 downloaded genomes as queries for BLAST searches against the local databases. Specifically, hits were initially called for alignments with an e-value threshold of 1e-50 and the resultant XML files were parsed. Alignments producing hits were further filtered for those with coverage values exceeding 90% and percent identity values of 30–70%, depending on the target, and hits were subsequently manually curated. Table S1 and S2 provide the FASTA protein sequences, alignment details and distribution summaries of the filtered hits. For hydrogenases, the protein sequences flanking the hydrogenase large subunits were also retrieved; these sequences were used to classify group A [FeFe]-hydrogenases into subtypes (A1–A4), as previously described [66 (link)], and retrieve diaphorase sequences (HydB) associated with the A3 subtype. Partial [FeFe]-hydrogenase protein sequences from six incompletely sequenced rumen ciliates and fungi genomes were retrieved through targeted blastP searches [76 (link)] in NCBI.
Greening C., Geier R., Wang C., Woods L.C., Morales S.E., McDonald M.J., Rushton-Green R., Morgan X.C., Koike S., Leahy S.C., Kelly W.J., Cann I., Attwood G.T., Cook G.M, & Mackie R.I. (2019). Diverse hydrogen production and consumption pathways influence methane production in ruminants. The ISME Journal, 13(10), 2617-2632.
Publication 2019
adenylylsulfate reductase Amino Acid Sequence Ammonia Bacteria Catalytic Domain Ciliata Coenzyme A, Acetyl Dihydrolipoamide Dehydrogenase dimethyl sulfoxide reductase Dissimilatory Sulfite Reductase Genetic Diversity Genome Genome, Bacterial Genome, Fungal Hydrogenase iron hydrogenase Joints methyl coenzyme M reductase nickel-iron hydrogenase Nitrate Reductases Nitrates Nitric Oxide Synthase Nitrite Reductase Nitrogenase Oxidase, Cytochrome-c Patient Discharge Periplasm periplasmic oxidoreductase Proteins Protein Subunits Python Rumen Strains Succimer Succinate Dehydrogenase Sulfite Dehydrogenase Sulfoxide, Dimethyl trimethylamine oxidase trimethyloxamine

Most recents protocols related to «Nitrogenase»

1
Nitrogen Fixation Assay of Bacterial Cells
Bacterial cells were harvested from YEM broth after 5 days of cultivation and washed twice with liquid BNM-B medium without C and N sources. The washed cells were inoculated into 150-ml glass bottles containing 50 ml of BNM-B medium (Wongdee et al., 2018 (link)). After closure of the bottle with an airtight stopper, 15 ml of acetylene (10% final concentration) was injected, and the cultures were incubated at 30°C without shaking. Every 2 days after inoculation, a 1-ml air sample was taken to determine the nitrogenase enzyme activity as described previously (Renier et al., 2011 (link)). The nitrogen fixation activity was calculated from three independent cell cultures. To determine the number of viable cells in the culture at the time of the determination of the nitrogenase enzyme activity, a sample from the culture was taken to determine the colony forming units (CFU on YEM agar plates).
Wongdee J., Piromyou P., Songwattana P., Greetatorn T., Teaumroong N., Boonkerd N., Giraud E., Nouwen N, & Tittabutr P. (2023). Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth. Frontiers in Microbiology, 14, 1131860.
Publication 2023
Acetylene Agar Bacteria Cell Culture Techniques Cells enzyme activity Nitrogenase Nitrogen Fixation Vaccination
2
Symbiotic Interaction Analysis of Bradyrhizobium and A. americana
The symbiotic interaction of wild-type Bradyrhizobium DOA9 and derivatives with A. americana was analyzed as described by Wongdee et al. (2016 (link), 2018) (link). At 20 days post-inoculation (dpi), the numbers of nodules on the roots were determined, and the nitrogenase enzyme activity of the plants was measured using the acetylene reduction assay (ARA). For cytological analysis, fresh nodules were sliced into 30-micron sections using a vibratome (Leica VT1000S Vibrating blade microtome, United States). Nodule sections of plants inoculated with DOA9-PmrpoN strains were stained with X-gluc (Bonaldi et al., 2010 (link)). After staining, the sections were mounted on microscope slides and observed by light microscopy. Nodule section of plants inoculated with rpoN mutants were stained with Live-Dead staining reagent (Invitrogen), after which they were analyzed using confocal microscopy as described by Okazaki et al. (2016) (link).
Wongdee J., Piromyou P., Songwattana P., Greetatorn T., Teaumroong N., Boonkerd N., Giraud E., Nouwen N, & Tittabutr P. (2023). Role of two RpoN in Bradyrhizobium sp. strain DOA9 in symbiosis and free-living growth. Frontiers in Microbiology, 14, 1131860.
Publication 2023
Acetylene Biological Assay Bradyrhizobium derivatives enzyme activity Light Microscopy Microscopy Microscopy, Confocal Microtomy Nitrogenase Plant Roots Plants Strains Symbiosis Vaccination
3
Identifying Nitrogen-fixing Genes via MEGAN5
To identify potential genes encoding nitrogenases and to complement the nifH PCR results, including the classical and alternative enzymes previously described, the reads associated with the nitrogen metabolic pathway were analyzed in MEtaGenome Analyzer 5 (MEGAN5) software26 (link), using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway and NCBI databases as references.
Pinheiro Y., Faria da Mota F., Peixoto R.S., van Elsas J.D., Lins U., Mazza Rodrigues J.L, & Rosado A.S. (2023). A thermophilic chemolithoautotrophic bacterial consortium suggests a mutual relationship between bacteria in extreme oligotrophic environments. Communications Biology, 6, 230.
Publication 2023
Enzymes Genes Genome Metagenome Nitrogen Nitrogenase
4
Nitrogen Fixation Determination via PCR
The presence of nitrogen-fixation systems in the consortia was determined using PCR by targeting the nifH, a gene that encodes the dinitrogenase reductase subunit of the nitrogenase enzyme, which is a biological marker for nitrogen fixation50 . The primers PolF and PolR51 (link) were used according to the following protocol: initial denaturation at 94 °C for 1 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min, and final amplification at 72 °C for 5 min. Herbaspirillum seropedicae ATCC 35892 and Escherichia coli BL21 were used as positive and negative controls in JNFb and SOC media, respectively1 (link). Electrophoresis of the amplicons was performed on 1.5% gels for 40 min at 100 V, after which the amplicons were stained with SYBR Safe and observed under a UV transilluminator.
Pinheiro Y., Faria da Mota F., Peixoto R.S., van Elsas J.D., Lins U., Mazza Rodrigues J.L, & Rosado A.S. (2023). A thermophilic chemolithoautotrophic bacterial consortium suggests a mutual relationship between bacteria in extreme oligotrophic environments. Communications Biology, 6, 230.
Publication 2023
Biological Markers Dinitrogenase Reductase Electrophoresis Enzymes Escherichia coli Gels Genes Herbaspirillum seropedicae Nitrogen Nitrogenase nitrogenase reductase Nitrogen Fixation Oligonucleotide Primers Protein Subunits
5
Protein Structure and Sequence Analysis
Structural homology models of ancestral sequences were generated by MODELLER v10.2 (Webb and Sali, 2016 (link)) using PDB 1M34 as a template for all nitrogenase protein subunits and visualized by ChimeraX v1.3 (Pettersen et al., 2021 (link)).
Extant and ancestral protein sequence space was visualized by machine-learning embeddings, where each protein embedding represents protein features in a fixed-size, multidimensional vector space. The analysis was conducted on concatenated (HDK) nitrogenase protein sequences in our phylogenetic dataset. The embeddings were obtained using the pre-trained language model ESM2 (Lin et al., 2022 (link); Rives et al., 2021 (link)), a transformer architecture trained to reproduce correlations at the sequence level in a dataset containing hundreds of millions of protein sequences. Layer 33 of this transformer was used, as recommended by the authors. The resulting 1024 dimensions were reduced by UMAP (McInnes et al., 2020 ) for visualization in a two-dimensional space.
Protein site-wise conservation analysis was performed using the Consurf server (Ashkenazy et al., 2016 (link)). An input alignment containing only extant, Group I Mo-nitrogenases was submitted for analysis under default parameters. Conserved sites were defined by a Consurf conservation score >7.
Garcia A.K., Harris D.F., Rivier A.J., Carruthers B.M., Pinochet-Barros A., Seefeldt L.C, & Kaçar B. (2023). Nitrogenase resurrection and the evolution of a singular enzymatic mechanism. eLife, 12, e85003.
Publication 2023
Amino Acid Sequence Cloning Vectors Homologous Sequences Nitrogenase Proteins Protein Subunits Spatial Visualization

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More about "Nitrogenase"

Nitrogenase is a critical enzyme system that plays a pivotal role in the global nitrogen cycle.
This metalloenzyme, also known as nitrogen fixation enzyme, is responsible for the biological conversion of atmospheric dinitrogen (N2) into ammonia (NH3), a process essential for the growth and development of many nitrogen-fixing organisms like bacteria, archaea, and certain eukaryotes.
Understanding the structure, function, and regulation of nitrogenase is an active area of research with applications in agriculture, bioenergy, and environmental remediation.
Optimizing research protocols for nitrogenase studies is crucial for advancing our knowledge of this essential biological catalyst and tackling global challenges.
Researchers can leverage tools like GC-2014 gas chromatograph, GC-8A, Agilent 6890N GC, BD Vacutainer, Clarus 480, and DB-1701 column to analyze and quantify nitrogenase activity and nitrogen fixation.
Statistical analysis software like SPSS version 20.0 and HP 6890 Series Gas Chromatograph System can also be employed to interpret experimental data and optimize research protocols.
By understanding the role of nitrogenase in the nitrogen cycle and exploring innovative techniques for its study, scientists can unlock new possibilities for sustainable agriculture, renewable energy production, and environmental remediation, ultimately contributing to the betterment of our planet.