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1.
World J Microbiol Biotechnol ; 40(5): 136, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38499730

RESUMO

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.


Assuntos
Compostos de Amônio , Oryza , Fixação de Nitrogênio , Nitrogenase/genética , Nitrogenase/metabolismo , Nitrogênio/metabolismo , Solo
2.
mBio ; 15(3): e0331423, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38377621

RESUMO

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.


Assuntos
Nitrogenase , Rhodobacter capsulatus , Nitrogenase/metabolismo , Fixação de Nitrogênio/genética , Ferredoxinas/metabolismo , Proteoma/metabolismo , Ferro/metabolismo , Amônia/metabolismo , Nitrogênio/metabolismo
3.
Sci Total Environ ; 919: 170648, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38336078

RESUMO

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.


Assuntos
Fixação de Nitrogênio , Solo , Pradaria , Tibet , Nitratos/análise , Nitrogênio/análise , Microbiologia do Solo , Nitrogenase/metabolismo
4.
ISME J ; 18(1)2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38365250

RESUMO

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.


Assuntos
Fixação de Nitrogênio , Populus , Fixação de Nitrogênio/fisiologia , Populus/genética , Populus/metabolismo , Endófitos/genética , Oxirredutases/genética , Hibridização in Situ Fluorescente , Nitrogenase/genética , Nitrogenase/metabolismo , Nitrogênio
5.
Appl Environ Microbiol ; 90(3): e0209123, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38412007

RESUMO

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.


Assuntos
Mesorhizobium , Mesorhizobium/genética , Fixação de Nitrogênio , Nitrogenase/genética , Ecossistema , Água , Simbiose , Filogenia
6.
Mol Biol Evol ; 41(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38319744

RESUMO

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.


Assuntos
Cianobactérias , Fixação de Nitrogênio , Fixação de Nitrogênio/genética , Nitrogenase/genética , Nitrogenase/metabolismo , Cianobactérias/genética , Archaea/metabolismo , Nitrogênio
7.
mSystems ; 9(3): e0015524, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38376168

RESUMO

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.


Assuntos
Azotobacter vinelandii , Fixação de Nitrogênio , Humanos , Fixação de Nitrogênio/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Nitrogenase/genética , Nitrogênio/metabolismo , Sequência de Bases , Azotobacter vinelandii/genética
8.
J Phys Chem B ; 128(4): 985-989, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38237063

RESUMO

The mechanism for N2 activation in the E4 state of nitrogenase was investigated by model calculations. In the experimentally suggested mechanism, the E4 state is obtained after four reductions to the ground state. In a recent theoretical study, results for a different mechanism have been found in excellent agreement with available Electron Paramagnetic Resonance (EPR) experiments for E4. The two hydrides in E4 leave as H2 concertedly with the binding of N2. The mechanism suggested differs from the experimentally suggested one by a requirement for four activation steps prior to catalysis. In the present study, the experimentally suggested mechanism is studied using the same methods as those used in the previous study on the theoretical mechanism. The computed results make it very unlikely that a structure obtained after four reductions from the ground state has two hydrides, and the experimentally suggested mechanism does therefore not agree with the EPR experiments for E4. Another structure with only one hydride is here suggested to be the one that has been observed to bind N2 after only four reductions of the ground state.


Assuntos
Nitrogenase , Nitrogenase/química , Oxirredução , Espectroscopia de Ressonância de Spin Eletrônica , Catálise
9.
FEMS Microbiol Lett ; 3712024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-38168702

RESUMO

The characterization of cyanobacteria communities remains challenging, as taxonomy of several cyanobacterial genera is still unresolved, especially within Nostocales taxa. Nostocales cyanobacteria are capable of nitrogen fixation; nitrogenase genes are grouped into operons and are located in the same genetic locus. Structural nitrogenase genes (nifH, nifK and nifD) as well as 16S rRNA have been shown to be adequate genetic markers for distinguishing cyanobacterial genera. However, there is no available information regarding the phylogeny of regulatory genes of the nitrogenase cluster. Aiming to provide a more accurate overview of the evolution of nitrogen fixation, this study analyzed for the first time nifE and nifN genes, which regulate the production of nitrogenase, alongside nifH. Specific primers were designed to amplify nifE and nifN genes, previously not available in literature and phylogenetic analysis was carried out in 13 and 14 TAU-MAC culture collection strains, respectively, of ten Nostocales genera along with other sequences retrieved from cyanobacteria genomes. Phylogenetic analysis showed that these genes seem to follow a common evolutionary pattern with nitrogenase structural genes and 16S rRNA. The classification of cyanobacteria based on these molecular markers seems to distinguish Nostocales strains with common morphological, ecological, and physiological characteristics.


Assuntos
Cianobactérias , Nitrogenase , Nitrogenase/genética , Filogenia , RNA Ribossômico 16S/genética , Fixação de Nitrogênio/genética , Cianobactérias/genética
10.
J Inorg Biochem ; 253: 112484, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38219407

RESUMO

The light-driven reduction of dinitrogen (N2) to ammonia (NH3) catalyzed by a cadmium sulfide (CdS) nanocrystal­nitrogenase MoFe protein biohybrid is dependent on a range of different factors, including an appropriate hole-scavenging sacrificial electron donor (SED). Here, the impact of different SEDs on the overall rate of N2 reduction catalyzed by a CdS quantum dot (QD)-MoFe protein system was determined. The selection of SED was guided by several goals: (i) molecules with standard reduction potentials sufficient to reduce the oxidized CdS QD, (ii) molecules that do not absorb the excitation wavelength of the CdS QD, and (iii) molecules that could be readily reduced by sustainable processes. Earlier studies utilized buffer molecules or ascorbic acid as the SED. The effectiveness of ascorbic acid as SED was compared to dithionite (DT), triethanolamine (TEOA), and hydroquinone (HQ) across a range of concentrations in supporting N2 reduction to NH3 in a CdS QD-MoFe protein photocatalytic system. It was found that TEOA supported N2 reduction rates comparable to those observed for dithionite and ascorbic acid. HQ was found to support significantly higher rates of N2 reduction compared to the other SEDs at a concentration of 50 mM. A comparison of the rates of N2 reduction by the biohybrid complex to the standard reduction potential (Eo) of the SEDs reveals that Eo is not the only factor impacting the efficiency of hole-scavenging. These findings reveal the importance of the SED properties for improving the efficiency of hole-scavenging in the light-driven N2 reduction reaction catalyzed by a CdS QD-MoFe protein hybrid.


Assuntos
Azotobacter vinelandii , Compostos de Cádmio , Nitrogenase , Sulfetos , Nitrogenase/metabolismo , Molibdoferredoxina/metabolismo , Oxirredução , Ditionita/metabolismo , Catálise , Ácido Ascórbico/metabolismo , Azotobacter vinelandii/metabolismo
11.
Plant Physiol Biochem ; 207: 108362, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38266561

RESUMO

Nodule symbiosis is an energetic process that demands a tremendous carbon (C) cost, which massively increases in responses to environmental stresses. Notably, most common respiratory pathways (e.g., glycolysis and Krebs cycle) that sustain nitrogenase activity and subsequent nitrogen (N) assimilation (amino acid formation) display a noncyclic mode of C flux. In such circumstances, the nodule's energy charge could markedly decrease, leading to a lower symbiotic activity under stresses. The host plant then attempts to induce alternative robust metabolic pathways to minimize the C expenditure and compensate for the loss in respiratory substrates. GABA (γ-aminobutyric acid) shunt appears to be among the highly conserved metabolic bypass induced in responses to stresses. Thus, it can be suggested that GABA, via its primary biosynthetic pathway (GABA shunt), is simultaneously induced to circumvent stress-susceptible decarboxylating portion of the Krebs cycle and to replenish symbiosome with energy and C skeletons for enhancing nitrogenase activity and N assimilation besides the additional C costs expended in the metabolic stress acclimations (e.g., biosynthesis of secondary metabolites and excretion of anions). The GABA-mediated C/N balance is strongly associated with interrelated processes, including pH regulation, oxygen (O2) protection, osmoregulation, cellular redox control, and N storage. Furthermore, it has been anticipated that GABA could be implicated in other functions beyond its metabolic role (i.e., signaling and transport). GABA helps plants possess remarkable metabolic plasticity, which might thus assist nodules in attenuating stressful events.


Assuntos
Fabaceae , Fabaceae/metabolismo , Simbiose/fisiologia , Nitrogênio/metabolismo , Carbono/metabolismo , Ácido gama-Aminobutírico/metabolismo , Verduras , Plantas/metabolismo , Homeostase , Nitrogenase/metabolismo , Fixação de Nitrogênio/fisiologia , Nódulos Radiculares de Plantas
12.
Nat Struct Mol Biol ; 31(1): 150-158, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38062208

RESUMO

Nitrogenases are best known for catalyzing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO2) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the three nitrogenase isozymes, the iron nitrogenase has the highest wild-type activity for the reduction of CO2, but the molecular architecture facilitating these activities has remained unknown. Here, we report a 2.35-Å cryogenic electron microscopy structure of the ADP·AlF3-stabilized iron nitrogenase complex from Rhodobacter capsulatus, revealing an [Fe8S9C-(R)-homocitrate] cluster in the active site. The enzyme complex suggests that the iron nitrogenase G subunit is involved in cluster stabilization and substrate channeling and confers specificity between nitrogenase reductase and catalytic component proteins. Moreover, the structure highlights a different interface between the two catalytic halves of the iron and the molybdenum nitrogenase, potentially influencing the intrasubunit 'communication' and thus the nitrogenase mechanism.


Assuntos
Dióxido de Carbono , Ferro , Ferro/metabolismo , Dióxido de Carbono/química , Oxirredução , Nitrogenase/química , Nitrogenase/metabolismo , Hidrocarbonetos/metabolismo
13.
Sci Total Environ ; 912: 169263, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38092216

RESUMO

Biochar is an efficient and inexpensive carrier for bacteria that stimulate plant development and growth. In this study, different biopolymer additives (cellulose, xanthan gum, chitin and tryptone) were tested with different addition ratios (1:0.1, 1:0.5 and 1:1) on further enhancing biochar capacity for supporting the growth and activity of Bradyrhizobium japonicum (CB1809). We utilized pine wood biochar (PWBC) pyrolyzed at 400 °C as the base inoculum carrier. The shelf life and survival rate of CB1809 were counted using the colony-forming unit (CFU) method for up to 120 days. Peat served as a standard reference material against which all treatments were compared. Subsequent experiments evaluated the ability of carrier inoculants to promote Glycine max L. (soybean) plant growth and nodulation under different watering regimes, i.e., 55 % water holding capacity (WHC) (D0), 30 % WHC (D1) and, 15 % WHC (D2) using sandy loam soil. Results revealed that among different additives; xanthan gum with 1:0.5 to PWBC [PWBC-xanthan gum(1:0.5)] was observed as a superior formulation in supporting rhizobial shelf life and survival rate of CB1809. In pot experiments, plants with PWBC-xanthan gum(1:0.5) formulation showed significant increase in various physiological characteristics (nitrogenase activity, chlorophyll pigments, membrane stability index, and relative water content), root architecture (root surface area, root average diameter, root volume, root tips, root forks and root crossings), and plant growth attributes (shoot/root dry biomass, shoot/root length, and number of nodules). Additionally, a reduced enrichment of isotopic signatures (δ13C, δ15N) was observed in plants treated with PWBC-xanthan gum(1:0.5), less enrichment of δ15N indicates an inverse link to nodulation and nitrogenase activity, while lower δ13C values indicates effective water use efficiency by plants during drought stress. These results suggest that biopolymers supplementation of the PWBC is useful in promoting shelf life or survival rate of CB1809.


Assuntos
Carvão Vegetal , Rhizobium , Água , Solo , Biopolímeros , Nitrogenase
14.
Biochemistry ; 63(1): 152-158, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38091601

RESUMO

Nitrogenase is the only enzyme that catalyzes the reduction of nitrogen gas into ammonia. Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Many nitrogen fixing bacteria protect nitrogenase from CO inhibition using the protective protein CowN. This work demonstrates that a conserved glutamic acid residue near the C-terminus of Gluconacetobacter diazotrophicus CowN is necessary for its function. Mutation of the glutamic acid residue abolishes both CowN's protection against CO inhibition and the ability of CowN to bind to nitrogenase. In contrast, a conserved C-terminal cysteine residue is not important for CO protection by CowN. Overall, this work uncovers structural features in CowN that are required for its function and provides new insights into its nitrogenase binding and CO protection mechanism.


Assuntos
Ácido Glutâmico , Nitrogenase , Nitrogenase/química , Ácido Glutâmico/genética , Monóxido de Carbono/metabolismo
15.
Sci Total Environ ; 912: 169005, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38065494

RESUMO

Biological nitrogen fixation and nitrification inhibitor applications contribute to improving soil nitrogen (N) availability, however, free-living N fixation affected by nitrification inhibitors has not been effectively evaluated in soils under different weed management methods. In this study, the effects of the nitrification inhibitors dicyandiamide (DCD) and 3, 4-dimethylpyrazole phosphate (DMPP) on the nitrogenase, nifH gene,and diazotrophic communities in soils under different weed management methods (AMB, weeds growth without mowing or glyphosate spraying; GS, glyphosate spraying; MSG, mowing and removing weeds and glyphosate spraying; and WM, mowing aboveground weeds) were investigated. Compared to the control counterparts, the DCD application decreased soil nitrogenase activity and nifH gene abundance by 4.5 % and 37.9 %, respectively, under the GS management method, and the DMPP application reduced soil nitrogenase activity by 20.4 % and reduced the nifH gene abundance by 83.4 % under the MSG management method. The application of nitrification inhibitors significantly elevated soil NH4+-N contents but decreased NO3--N contents, which had adverse impacts on soil nifH gene abundance and nitrogenase activity. The nifH gene abundances were also negatively impacted by dissolved organic N and Geobacter but were positively affected by available phosphorus and diazotrophic community structures. Nitrification inhibitors significantly inhibited Methylocella but stimulated Rhizobiales and affected soil diazotrophic communities. The nitrification inhibitors DCD and DMPP significantly altered soil diazotrophic community structures, but weed management outweighed nitrification inhibitors in reshaping soil diazotrophic community structures. The non-targeted effects of the nitrification inhibitors DMPP and DCD on soil free-living N fixation were substantially influenced by the weed management methods.


Assuntos
Fixação de Nitrogênio , Solo , Solo/química , Nitrificação , Iodeto de Dimetilfenilpiperazina/farmacologia , Nitrogenase , Fosfatos , Microbiologia do Solo , Nitrogênio/análise , Fertilizantes
16.
J Proteomics ; 294: 105061, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38154550

RESUMO

Paenibacillus sonchi SBR5T is a Gram-positive, endospore-forming facultative aerobic diazotrophic bacterium that can fix nitrogen via an alternative Fe-only nitrogenase (AnfHDGK). In several bacteria, this alternative system is expressed under molybdenum (Mo)-limiting conditions when the conventional Mo-dependent nitrogenase (NifHDK) production is impaired. The regulatory mechanisms, metabolic processes, and cellular functions of N2 fixation by alternative and/or conventional systems are poorly understood in the Paenibacillus genus. We conducted a comparative proteomic profiling study of P. sonchi SBR5T grown under N2-fixing conditions with and without Mo supply through an LC-MS/MS and label-free quantification analysis to address this gap. Protein abundances revealed overrepresented processes related to anaerobiosis growth adaption, Fe-S cluster biosynthesis, ammonia assimilation, electron transfer, and sporulation under N2-fixing conditions compared to non-fixing control. Under Mo limitation, the Fe-only nitrogenase components were overrepresented together with the Mo-transporter system, while the dinitrogenase component (NifDK) of Mo­nitrogenase was underrepresented. The dinitrogenase reductase component (NifH) and accessory proteins encoded by the nif operon had no significant differential expression, suggesting post-transcriptional regulation of nif gene products in this strain. Overall, this was the first comprehensive proteomic analysis of a diazotrophic strain from the Paenibacillaceae family, and it provided insights related to alternative N2-fixation by Fe-only nitrogenase. SIGNIFICANCE: In this work, we try to understand how the alternative nitrogen fixation system, presented by some diazotrophic bacteria, works. For this, we used the SBR5 lineage of P. sonchi, which presents the alternative system in which the nitrogenase cofactor is composed only of iron. In addition, we tried to unravel the proteome of this strain in different situations of nitrogen fixation, since, for Gram-positive bacteria, these systems are little known. The results achieved, through LC-MS/MS and label-free quantitative analysis, showed an overrepresentation of proteins related to different processes involved with growth under stressful conditions in situations of nitrogen deficiency, in addition to suggesting that some encoded proteins by the nif operon may be regulated at post-transcriptional levels. Our findings represent important steps toward the elucidation of nitrogen fixation systems in Gram-positive diazotrophic bacteria.


Assuntos
Fixação de Nitrogênio , Paenibacillus , Proteoma/metabolismo , Proteômica , Cromatografia Líquida , Espectrometria de Massas em Tandem , Nitrogenase/metabolismo , Paenibacillus/genética , Paenibacillus/metabolismo , Molibdênio/metabolismo , Ferro/metabolismo , Nitrogênio/metabolismo
17.
Phys Chem Chem Phys ; 26(3): 1684-1695, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38126534

RESUMO

The main class of nitrogenases has a molybdenum in its cofactor. A mechanism for Mo-nitrogenase has recently been described. In the present study, another class of nitrogenases has been studied, the one with a vanadium instead of a molybdenum in its cofactor. It is generally believed that these classes use the same general mechanism to activate nitrogen. The same methodology has been used here as the one used for Mo-nitrogenase. N2 activation is known to occur after four reductions in the catalytic cycle, in the E4 state. The main features of the mechanism for Mo-nitrogenase found in the previous study are an activation process in four steps prior to catalysis, the release of a sulfide during the activation steps and the formation of H2 from two hydrides in E4, just before N2 is activated. The same features have been found here for V-nitrogenase. A difference is that five steps are needed in the activation process, which explains why the ground state of V-nitrogenase is a triplet (even number) and the one for Mo-nitrogenase is a quartet (odd number). The reason an additional step is needed for V-nitrogenase is that V3+ can be reduced to V2+, in contrast to the case for Mo3+ in Mo-nitrogenase. The fact that V3+ is Jahn-Teller active has important consequences. N2H2 is formed in E4 with reasonably small barriers.


Assuntos
Nitrogenase , Vanádio , Nitrogenase/metabolismo , Molibdênio , Oxirredução , Nitrogênio
18.
mBio ; 15(2): e0308823, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38126768

RESUMO

Biological nitrogen fixation, the conversion of inert N2 to metabolically tractable NH3, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo-R-homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N2 reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as Saccharomyces cerevisiae, represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation. A central player in FeMo-co assembly is the scaffold protein NifEN upon which processing of NifB-co, an [8Fe-9S-C] precursor produced by NifB, occurs. Prior work established that NifB-co can be produced in S. cerevisiae mitochondria. In the present work, a library of nifEN genes from diverse diazotrophs was expressed in S. cerevisiae, targeted to mitochondria, and surveyed for their ability to produce soluble NifEN protein complexes. Many such NifEN variants supported FeMo-co formation when heterologously produced in the diazotroph A. vinelandii. However, only three of them accumulated in soluble forms in mitochondria of aerobically cultured S. cerevisiae. Of these, two variants were active in the in vitro FeMo-co synthesis assay. NifEN, NifB, and NifH proteins from different species, all of them produced in and purified from S. cerevisiae mitochondria, were combined to establish successful FeMo-co biosynthetic pathways. These findings demonstrate that combining diverse interspecies nitrogenase FeMo-co assembly components could be an effective and, perhaps, the only approach to achieve and optimize nitrogen fixation in a eukaryotic organism.IMPORTANCEBiological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo-R-homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae. NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae. FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.


Assuntos
Compostos de Ferro , Nitrogenase , Saccharomyces cerevisiae , Ácidos Tricarboxílicos , Nitrogenase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Molibdoferredoxina/metabolismo , Proteínas de Bactérias/metabolismo , Mitocôndrias/metabolismo , Nitrogênio/metabolismo
19.
Phys Chem Chem Phys ; 26(2): 1364-1375, 2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38108422

RESUMO

Nitrogenase is the only enzyme that can cleave the strong triple bond in N2, making nitrogen available for biological lifeforms. The active site is a MoFe7S9C cluster (the FeMo cluster) that binds eight electrons and protons during one catalytic cycle, giving rise to eight intermediate states E0-E7. It is experimentally known that N2 binds to the E4 state and that H2 is a compulsory byproduct of the reaction. However, formation of H2 is also an unproductive side reaction that should be avoided, especially in the early steps of the reaction mechanism (E2 and E3). Here, we study the formation of H2 for various structural interpretations of the E2-E4 states using combined quantum mechanical and molecular mechanical (QM/MM) calculations and four different density-functional theory methods. We find large differences in the predictions of the different methods. B3LYP strongly favours protonation of the central carbide ion and H2 cannot form from such structures. On the other hand, with TPSS, r2SCAN and TPSSh, H2 formation is strongly exothermic for all structures and En and therefore need strict kinetic control to be avoided. For the E2 state, the kinetic barriers for the low-energy structures are high enough to avoid H2 formation. However, for both the E3 and E4 states, all three methods predict that the best structure has two hydride ions bridging the same pair of Fe ions (Fe2 and Fe6) and these two ions can combine to form H2 with an activation barrier of only 29-57 kJ mol-1, corresponding to rates of 7 × 102 to 5 × 107 s-1, i.e. much faster than the turnover rate of the enzyme (1-5 s-1). We have also studied H-atom movements within the FeMo cluster, showing that the various protonation states can quite freely be interconverted (activation barriers of 12-69 kJ mol-1).


Assuntos
Nitrogenase , Prótons , Nitrogenase/química , Oxirredução , Nitrogênio/química , Catálise
20.
Mol Cells ; 46(12): 736-742, 2023 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-38052488

RESUMO

NifB, a radical S-adenosylmethionine (SAM) enzyme, is pivotal in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), commonly referred to as the M-cluster. This cofactor, located within the active site of nitrogenase, is essential for the conversion of dinitrogen (N2) to NH3. Recognized as the most intricate metallocluster in nature, FeMo-co biosynthesis involves multiple proteins and a sequence of steps. Of particular significance, NifB directs the fusion of two [Fe4S4] clusters to assemble the 8Fe core, while also incorporating an interstitial carbide. Although NifB has been extensively studied, its molecular mechanisms remain elusive. In this review, we explore recent structural analyses of NifB and provide a comprehensive overview of the established catalytic mechanisms. We propose prospective directions for future research, emphasizing the relevance to biochemistry, agriculture, and environmental science. The goal of this review is to lay a solid foundation for future endeavors aimed at elucidating the atomic details of FeMo-co biosynthesis.


Assuntos
Compostos de Ferro , Nitrogenase , Nitrogenase/química , Nitrogenase/metabolismo , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Compostos de Ferro/química , Compostos de Ferro/metabolismo , Estudos Prospectivos , Domínio Catalítico , Proteínas de Bactérias/metabolismo
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