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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445505

RESUMO

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme's sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle's genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an 'Fe-only' nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.


Assuntos
Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloroplastos/metabolismo , Engenharia Genética/métodos , Nitrogenase/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Genoma de Cloroplastos , Fixação de Nitrogênio , Nitrogenase/metabolismo , Fotossíntese , Biologia Sintética
2.
Nat Commun ; 12(1): 4774, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362886

RESUMO

Biological N2 fixation was key to the expansion of life on early Earth. The N2-fixing microorganisms and the nitrogenase type used in the Proterozoic are unknown, although it has been proposed that the canonical molybdenum-nitrogenase was not used due to low molybdenum availability. We investigate N2 fixation in Lake Cadagno, an analogue system to the sulfidic Proterozoic continental margins, using a combination of biogeochemical, molecular and single cell techniques. In Lake Cadagno, purple sulfur bacteria (PSB) are responsible for high N2 fixation rates, to our knowledge providing the first direct evidence for PSB in situ N2 fixation. Surprisingly, no alternative nitrogenases are detectable, and N2 fixation is exclusively catalyzed by molybdenum-nitrogenase. Our results show that molybdenum-nitrogenase is functional at low molybdenum conditions in situ and that in contrast to previous beliefs, PSB may have driven N2 fixation in the Proterozoic ocean.


Assuntos
Chromatiaceae/metabolismo , Molibdênio/metabolismo , Fixação de Nitrogênio , Nitrogênio/metabolismo , Biomassa , Ciclo do Carbono , Dióxido de Carbono , Tamanho Celular , Chromatiaceae/genética , Metagenoma , Modelos Teóricos , Nitrogenase/metabolismo , Oceanos e Mares , Análise de Célula Única
3.
NPJ Biofilms Microbiomes ; 7(1): 54, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210981

RESUMO

Biofilm and nitrogen fixation are two competitive strategies used by many plant-associated bacteria; however, the mechanisms underlying the formation of nitrogen-fixing biofilms remain largely unknown. Here, we examined the roles of multiple signalling systems in the regulation of biofilm formation by root-associated diazotrophic P. stutzeri A1501. Physiological analysis, construction of mutant strains and microscale thermophoresis experiments showed that RpoN is a regulatory hub coupling nitrogen fixation and biofilm formation by directly activating the transcription of pslA, a major gene involved in the synthesis of the Psl exopolysaccharide component of the biofilm matrix and nifA, the transcriptional activator of nif gene expression. Genetic complementation studies and determination of the copy number of transcripts by droplet digital PCR confirmed that the regulatory ncRNA RsmZ serves as a signal amplifier to trigger biofilm formation by sequestering the translational repressor protein RsmA away from pslA and sadC mRNAs, the latter of which encodes a diguanylate cyclase that synthesises c-di-GMP. Moreover, RpoS exerts a braking effect on biofilm formation by transcriptionally downregulating RsmZ expression, while RpoS expression is repressed posttranscriptionally by RsmA. These findings provide mechanistic insights into how the Rpo/Gac/Rsm regulatory networks fine-tune nitrogen-fixing biofilm formation in response to the availability of nutrients.


Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Fixação de Nitrogênio , Pseudomonas stutzeri/fisiologia , Proteínas de Bactérias/metabolismo , Ordem dos Genes , Nitrogenase/genética , Nitrogenase/metabolismo , Sequências Repetitivas de Ácido Nucleico , Ativação Transcricional
4.
Arch Microbiol ; 203(7): 4367-4383, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34120198

RESUMO

The present study was undertaken to investigate the arsenite (As III)-induced changes in the diazotrophic cyanobacterium Anabaena PCC 7120. It was observed that the growth of cyanobacterial decreased with increase in As (III) concentration. The cells exposed to As (III) showed morphological variation (deformity) due to the formation of deeper constrictions in vegetative cells. Strain showed increased heterocyst differentiation (1.6-fold higher) whereas decreased nitrogenase activity at the concentration of 40 ppm As (III). The activities of NR, NiR, urease and GS decreased with increase in As (III) concentrations and attained their minimum levels at 40 ppm of As (III). The Ca2+-dependent ATPase activity increased with increase in As (III) concentration and attained its about 2.72-fold higher level at 40 ppm of As (III). In contrast, sharp decline in Mg2+-dependent ATPase activity (28%) was recorded at 1 ppm of As (III) over untreated control. The rates of photosynthetic O2 evolution and respiration decreased with increase in As (III) concentration and attained its minimal level at 40 ppm of As (III). Therefore, this study highlighted arsenite regimes efficiently correlated with behavioral changes in consort with strain.


Assuntos
Anabaena , Arsenitos , Anabaena/efeitos dos fármacos , Anabaena/metabolismo , Arsenitos/farmacologia , Proteínas de Bactérias/metabolismo , Poluentes Ambientais/farmacologia , Ativação Enzimática/efeitos dos fármacos , Nitrogênio/metabolismo , Fixação de Nitrogênio/efeitos dos fármacos , Nitrogenase/metabolismo
5.
PLoS Genet ; 17(6): e1009617, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34111137

RESUMO

The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation (nif) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii. The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A. vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family.


Assuntos
Amônia/metabolismo , Azotobacter vinelandii/genética , Proteínas de Bactérias/genética , Glutamato-Amônia Ligase/genética , Nitrogênio/metabolismo , Nitrogenase/genética , Fatores de Transcrição/genética , Substituição de Aminoácidos , Azotobacter vinelandii/enzimologia , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Glutamato-Amônia Ligase/metabolismo , Mutação , Fixação de Nitrogênio , Nitrogenase/metabolismo , Oxigênio/metabolismo , Solo/química , Microbiologia do Solo , Fatores de Transcrição/metabolismo , Transcrição Genética
6.
Microbes Environ ; 36(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34108360

RESUMO

Fermentative nitrogen-fixing bacteria have not yet been examined in detail in thermal environments. In the present study, we isolated the thermophilic fermentative bacterium, strain YA01 from a hot spring. This strain grew at temperatures up to 78°C. A phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain YA01 belonged to the genus Caldicellulosiruptor, which are fermentative bacteria in the phylum Firmicutes, with 97.7-98.0% sequence identity to its closest relatives. Strain YA01 clearly exhibited N2-dependent growth at 70°C. We also confirmed N2-dependent growth in the relatives of strain YA01, Caldicellulosiruptor hydrothermalis 108 and Caldicellulosiruptor kronotskyensis 2002. The nitrogenase activities of these three strains were examined using the acetylene reduction assay. Similar activities were detected for all tested strains, and were slightly suppressed by the addition of ammonium. A genome analysis revealed that strain YA01, as well as other Caldicellulosiruptor, possessed a gene set for nitrogen fixation, but lacked the nifN gene, which encodes a nitrogenase iron-molybdenum cofactor biosynthesis protein that is commonly detected in nitrogen-fixing bacteria. The amino acid sequences of nitrogenase encoded by nifH, nifD, and nifK shared 92-98% similarity in Caldicellulosiruptor. A phylogenetic tree of concatenated NifHDK sequences showed that NifHDK of Caldicellulosiruptor was in the deepest clade. To the best of our knowledge, this is the first study to demonstrate the nitrogen-fixing ability of fermentative bacteria at 70°C. Caldicellulosiruptor may have retained an ancient nitrogen-fixing enzyme system.


Assuntos
Caldicellulosiruptor/isolamento & purificação , Caldicellulosiruptor/fisiologia , Fixação de Nitrogênio , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caldicellulosiruptor/classificação , Caldicellulosiruptor/genética , Fermentação , Genoma Bacteriano , Fontes Termais/química , Fontes Termais/microbiologia , Temperatura Alta , Nitrogênio/metabolismo , Nitrogenase/química , Nitrogenase/genética , Nitrogenase/metabolismo , Filogenia
7.
Toxins (Basel) ; 13(5)2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946501

RESUMO

Non-proteinogenic neurotoxic amino acid ß-N-methylamino-L-alanine (BMAA) is synthesized by cyanobacteria, diatoms, and dinoflagellates, and is known to be a causative agent of human neurodegenerative diseases. Different phytoplankton organisms' ability to synthesize BMAA could indicate the importance of this molecule in the interactions between microalgae in nature. We were interested in the following: what kinds of mechanisms underline BMAA's action on cyanobacterial cells in different nitrogen supply conditions. Herein, we present a proteomic analysis of filamentous cyanobacteria Nostoc sp. PCC 7120 cells that underwent BMAA treatment in diazotrophic conditions. In diazotrophic growth conditions, to survive, cyanobacteria can use only biological nitrogen fixation to obtain nitrogen for life. Note that nitrogen fixation is an energy-consuming process. In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by using LC-MS/MS spectrometry. Among them, 123 proteins belonging to different functional categories were selected-due to their notable expression differences-for further functional analysis and discussion. The presented proteomic data evidences that BMAA treatment leads to very strong (up to 80%) downregulation of α (NifD) and ß (NifK) subunits of molybdenum-iron protein, which is known to be a part of nitrogenase. This enzyme is responsible for catalyzing nitrogen fixation. The genes nifD and nifK are under transcriptional control of a global nitrogen regulator NtcA. In this study, we have found that BMAA impacts in a total of 22 proteins that are under the control of NtcA. Moreover, BMAA downregulates 18 proteins that belong to photosystems I or II and light-harvesting complexes; BMAA treatment under diazotrophic conditions also downregulates five subunits of ATP synthase and enzyme NAD(P)H-quinone oxidoreductase. Therefore, we can conclude that the disbalance in energy and metabolite amounts leads to severe intracellular stress that induces the upregulation of stress-activated proteins, such as starvation-inducible DNA-binding protein, four SOS-response enzymes, and DNA repair enzymes, nine stress-response enzymes, and four proteases. The presented data provide new leads into the ecological impact of BMAA on microalgal communities that can be used in future investigations.


Assuntos
Diamino Aminoácidos/farmacologia , Fixação de Nitrogênio/efeitos dos fármacos , Nostoc/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Bicarbonatos/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Regulação para Baixo/efeitos dos fármacos , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Nostoc/metabolismo , Nostoc/fisiologia , Fosforilação/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Proteômica , Estresse Fisiológico/efeitos dos fármacos
8.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836573

RESUMO

Nitrogenases utilize Fe-S clusters to reduce N2 to NH3 The large number of Fe sites in their catalytic cofactors has hampered spectroscopic investigations into their electronic structures, mechanisms, and biosyntheses. To facilitate their spectroscopic analysis, we are developing methods for incorporating 57Fe into specific sites of nitrogenase cofactors, and we report herein site-selective 57Fe labeling of the L-cluster-a carbide-containing, [Fe8S9C] precursor to the Mo nitrogenase catalytic cofactor. Treatment of the isolated L-cluster with the chelator ethylenediaminetetraacetate followed by reconstitution with 57Fe2+ results in 57Fe labeling of the terminal Fe sites in high yield and with high selectivity. This protocol enables the generation of L-cluster samples in which either the two terminal or the six belt Fe sites are selectively labeled with 57Fe. Mössbauer spectroscopic analysis of these samples bound to the nitrogenase maturase Azotobacter vinelandii NifX reveals differences in the primary coordination sphere of the terminal Fe sites and that one of the terminal sites of the L-cluster binds to H35 of Av NifX. This work provides molecular-level insights into the electronic structure and biosynthesis of the L-cluster and introduces postbiosynthetic modification as a promising strategy for studies of nitrogenase cofactors.


Assuntos
Azotobacter vinelandii/metabolismo , Molibdoferredoxina/metabolismo , Nitrogenase/metabolismo , Precursores de Proteínas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Espectroscopia de Mossbauer
9.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809732

RESUMO

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.


Assuntos
Paenibacillus polymyxa/metabolismo , Riboswitch/genética , Serina/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Códon/genética , Sequência Conservada , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Nitrogenase/metabolismo , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Paenibacillus polymyxa/efeitos dos fármacos , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/crescimento & desenvolvimento , RNA Bacteriano/química , RNA Bacteriano/genética , Serina/farmacologia
10.
J Biol Inorg Chem ; 26(2-3): 341-353, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33713183

RESUMO

Recently, a 1.83 Å crystallographic structure of nitrogenase was suggested to show N2-derived ligands at three sites in the catalytic FeMo cluster, replacing the three [Formula: see text] bridging sulfide ligands (two in one subunit and the third in the other subunit) (Kang et al. in Science 368: 1381-1385, 2020). Naturally, such a structure is sensational, having strong bearings on the reaction mechanism of the enzyme. Therefore, it is highly important to ensure that the interpretation of the structure is correct. Here, we use standard crystallographic refinement and quantum refinement to evaluate the structure. We show that the original crystallographic raw data are strongly anisotropic, with a much lower resolution in certain directions than others. This, together with the questionable use of anisotropic B factors, give atoms an elongated shape, which may look like diatomic atoms. In terms of standard electron-density maps and real-space Z scores, a resting-state structure with no dissociated sulfide ligands fits the raw data better than the interpretation suggested by the crystallographers. The anomalous electron density at 7100 eV is weaker for the putative N2 ligands, but not lower than for several of the [Formula: see text] bridging sulfide ions and not lower than what can be expected from a statistical analysis of the densities. Therefore, we find no convincing evidence for any N2 binding to the FeMo cluster. Instead, a standard resting state without any dissociated ligands seems to be the most likely interpretation of the structure. Likewise, we find no support that the homocitrate ligand should show monodentate binding.


Assuntos
Nitrogênio/metabolismo , Nitrogenase/química , Nitrogenase/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ligantes , Modelos Moleculares
11.
J Biol Chem ; 296: 100501, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33667548

RESUMO

Nitrogenase is the only enzyme capable of catalyzing nitrogen fixation, the reduction of dinitrogen gas (N2) to ammonia (NH3). Nitrogenase is tightly inhibited by the environmental gas carbon monoxide (CO). Nitrogen-fixing bacteria rely on the protein CowN to grow in the presence of CO. However, the mechanism by which CowN operates is unknown. Here, we present the biochemical characterization of CowN and examine how CowN protects nitrogenase from CO. We determine that CowN interacts directly with nitrogenase and that CowN protection observes hyperbolic kinetics with respect to CowN concentration. At a CO concentration of 0.001 atm, CowN restores nearly full nitrogenase activity. Our results further indicate that CowN's protection mechanism involves decreasing the binding affinity of CO to nitrogenase's active site approximately tenfold without interrupting substrate turnover. Taken together, our work suggests CowN is an important auxiliary protein in nitrogen fixation that engenders CO tolerance to nitrogenase.


Assuntos
Proteínas de Bactérias/metabolismo , Monóxido de Carbono/farmacologia , Gluconacetobacter/metabolismo , Fixação de Nitrogênio , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Proteínas de Bactérias/química , Catálise , Gluconacetobacter/efeitos dos fármacos , Gluconacetobacter/genética , Cinética , Modelos Moleculares , Nitrogenase/química , Oxirredução , Domínios e Motivos de Interação entre Proteínas
12.
Curr Protoc ; 1(3): e57, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33656286

RESUMO

Azotobacter vinelandii (A. vinelandii) is a commonly used model organism for the study of aerobic respiration, the bacterial production of several industrially relevant compounds, and, perhaps most significantly, the genetics and biochemistry of biological nitrogen fixation. Laboratory growth assessments of A. vinelandii are useful for evaluating the impact of environmental and genetic modifications on physiological properties, including diazotrophy. However, researchers typically rely on manual growth methods that are oftentimes laborious and inefficient. We present a protocol for the automated growth assessment of A. vinelandii on a microplate reader, particularly well-suited for studies of diazotrophic growth. We discuss common pitfalls and strategies for protocol optimization, and demonstrate the protocol's application toward growth evaluation of strains carrying modifications to nitrogen-fixation genes. © 2021 The Authors. Basic Protocol 1: Preparation of A. vinelandii plate cultures from frozen stock Basic Protocol 2: Preparation of A. vinelandii liquid precultures Basic Protocol 3: Automated growth rate experiment of A. vinelandii on a microplate reader.


Assuntos
Azotobacter vinelandii , Azotobacter vinelandii/genética , Laboratórios , Fixação de Nitrogênio , Nitrogenase/metabolismo
13.
Appl Microbiol Biotechnol ; 105(7): 2889-2899, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33745008

RESUMO

Biological nitrogen fixation is usually inhibited by fixed nitrogen. Paenibacillus sabinae T27, a Gram-positive, spore-forming diazotroph, possesses high nitrogenase activity and has 3 copies of nifH (nifH, nifH2, nifH3), a copy of nifDK, and multiple nifHDK-like genes. In this study, we found that P. sabinae T27 showed nitrogenase activities not only in low (0-3 mM) concentrations of NH4+ but also in high (30-300 mM) concentrations of NH4+, no matter whether this bacterium was grown in a flask or in a fermenter on scale cultivation. qRT-PCR and western blotting analyses supported that Fe protein and MoFe protein were synthesized under both low (0-3 mM) and high (30-300 mM) concentrations of NH4+. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that MoFe protein was encoded by nifDK and Fe protein was encoded by both nifH and nifH2. The cross-reaction suggested the purified Fe and MoFe components from P. sabinae T27 grown in both nitrogen-limited and nitrogen-excess conditions were active. This is the first time to report that diazotrophs show nitrogenase activity in presence of high (30-300 mM) concentrations of NH4+. Our study will provide a clue for studying the mechanisms of nitrogen fixation in presence of the high concentration of NH4+. KEY POINTS: • P. sabinae T27 can synthesize active nitrogenase in presence of high levels of ammonia. •Fe and MoFe proteins of nitrogenase purified in absence of ammonia are the same as those purified from the high concentration of ammonia. • Fe protein is encoded by nifH and nifH2, and MoFe protein is encoded by nifDK.


Assuntos
Amônia , Nitrogenase , Anaerobiose , Fermentação , Fixação de Nitrogênio , Nitrogenase/metabolismo , Paenibacillus
14.
Science ; 371(6530)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33574183

RESUMO

Kang et al (Reports, 19 June 2020, p. 1381) report a structure of the nitrogenase MoFe protein that is interpreted to indicate binding of N2 or an N2-derived species to the active-site FeMo cofactor. Independent refinement of the structure and consideration of biochemical evidence do not support this claim.


Assuntos
Azotobacter vinelandii , Molibdoferredoxina , Domínio Catalítico , Nitrogenase/metabolismo
15.
Science ; 371(6530)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33574184

RESUMO

Peters et al comment on our report of the dynamic structure of the nitrogenase metallocofactor during N2 reduction. Their claim that their independent structural refinement and consideration of biochemical data contradict our finding is incorrect and is strongly refuted by our biochemical and structural data that collectively and conclusively point to the binding of dinitrogen species to the nitrogenase cofactor.


Assuntos
Nitrogenase , Nitrogenase/metabolismo , Oxirredução
16.
J Phycol ; 57(3): 863-873, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33450056

RESUMO

The production of dihydrogen (H2 ) is an enigmatic yet obligate component of biological dinitrogen (N2 ) fixation. This study investigates the effect on H2 production by N2 fixing cyanobacteria when they are exposed to either air or a gas mixture consisting of argon, oxygen, and carbon dioxide (Ar:O2 :CO2 ). In the absence of N2 , nitrogenase diverts the flow of electrons to the production of H2 , which becomes a measure of Total Nitrogenase Activity (TNA). This method of argon-induced hydrogen production (AIHP) is much less commonly used to infer rates of N2 fixation than the acetylene reduction (AR) assay. We provide here a full evaluation of the AIHP method and demonstrate its ability to achieve high-resolution measurements of TNA in a gas exchange flow-through system. Complete diel profiles of H2 production were obtained for N2 fixing cyanobacteria despite the absence of N2 that broadly reproduced the temporal patterns observed by the AR assay. Comparison of H2 production under air versus Ar:O2 :CO2 revealed the efficiency of electron usage during N2 fixation and place these findings in the broader context of cell metabolism. Ultimately, AIHP is demonstrated to be a viable alternative to the AR assay with several additional merits that provide an insight into cell physiology and promise for successful field application.


Assuntos
Cianobactérias , Fixação de Nitrogênio , Argônio , Cianobactérias/metabolismo , Hidrogênio , Nitrogênio , Nitrogenase/metabolismo
17.
Commun Biol ; 4(1): 4, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33398015

RESUMO

Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.


Assuntos
Bactérias/enzimologia , Engenharia Genética/métodos , Fixação de Nitrogênio/genética , Nitrogenase/genética , Biblioteca Gênica , Ferro/metabolismo , Mitocôndrias/enzimologia , Nitrogenase/isolamento & purificação , Nitrogenase/metabolismo , Saccharomyces cerevisiae/enzimologia , Tabaco/metabolismo
18.
Angew Chem Int Ed Engl ; 60(18): 10112-10121, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33497500

RESUMO

The ability of resonant X-ray emission spectroscopy (XES) to recover physical oxidation state information, which may often be ambiguous in conventional X-ray spectroscopy, is demonstrated. By combining Kß XES with resonant excitation in the XAS pre-edge region, resonant Kß XES (or 1s3p RXES) data are obtained, which probe the 3dn+1 final-state configuration. Comparison of the non-resonant and resonant XES for a series of high-spin ferrous and ferric complexes shows that oxidation state assignments that were previously unclear are now easily made. The present study spans iron tetrachlorides, iron sulfur clusters, and the MoFe protein of nitrogenase. While 1s3p RXES studies have previously been reported, to our knowledge, 1s3p RXES has not been previously utilized to resolve questions of metal valency in highly covalent systems. As such, the approach presented herein provides chemists with means to more rigorously and quantitatively address challenging electronic-structure questions.


Assuntos
Compostos de Ferro/química , Nitrogenase/química , Compostos de Ferro/metabolismo , Conformação Molecular , Nitrogenase/metabolismo , Oxirredução , Espectrometria por Raios X
19.
Microb Biotechnol ; 14(3): 1073-1083, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33507628

RESUMO

Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe-only nitrogenase component proteins in the low-oxygen mitochondria matrix of S. cerevisiae. As-isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo-AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo-AnfDK could be activated for acetylene reduction by the simple addition of FeMo-co in vitro, indicating preexistence of the P-clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe-only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.


Assuntos
Nitrogenase , Saccharomyces cerevisiae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ferro , Fixação de Nitrogênio , Nitrogenase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
20.
ISME J ; 15(1): 124-128, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32918066

RESUMO

Non-cyanobacterial diazotrophs (NCDs) have recently emerged as potentially important contributors to marine nitrogen fixation. One of the most widely distributed NCDs is Gamma-A, yet information about its autecology is still scarce and solely relies on the PCR-based detection of its nitrogenase (nifH) gene in seawater, since previous metagenomic surveys targeting free-living planktonic size fractions (<3 µm) have not detected it. Here, we explore the diversity, biogeography, size-distribution, and nitrogenase gene expression of Gamma-A across four larger planktonic size-fractions (0.8-5, 5-20, 20-180, and 180-2000 µm) using metagenomes and metatranscriptomes from the Tara Oceans. We detected a single variant of a complete Gamma-A nifH gene along with other nitrogenase-related genes (nifKDT) within a metatranscriptomic-based contig of the Marine Atlas of Tara Ocean Unigenes. Gamma-A was detected in tropical and subtropical oceanic regions across all the size-fractions. However, the highest gene and transcript abundances were found in the 0.8-5 and 5-20 µm size-fractions at the surface, whereas abundances at the deep chlorophyll maximum were lower and similar across all size-fractions. The ubiquitous presence of active Gamma-A in large planktonic size-fractions suggests a filamentous or particle-attached lifestyle and places its potential to fix nitrogen in larger planktonic compartments.


Assuntos
Cianobactérias , Cianobactérias/metabolismo , Imunoglobulina A , Fixação de Nitrogênio , Nitrogenase/genética , Nitrogenase/metabolismo , Oceanos e Mares , Filogenia , Água do Mar , gama-Globulinas
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