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1.
J Colloid Interface Sci ; 607(Pt 2): 1625-1632, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34592549

RESUMO

The "FeMo cofactors" in biological nitrogenase play a decisive role in nitrogen reduction. Herein, a novel bionic Fe/Mo bimetallene was applied in photocatalytic nitrogen reduction. The surface coating Fe/Mo bimetallene of Bi2Mo0.3W0.7O6 (BMWO) nanocrystals could effectively promote the separation and transportation of photogenerated carriers by multi-electron redox reactions and deliver 2.8 times longer photo-carrier lifetime. Consequently, the nitrogen fixation activity of Fe/Mo bimetallene-coated BMWO nanocrystal photocatalyst was obviously enhanced (218.93 µmol g-1h-1), which was about 4.8 times that of unmodified BMWO nanocrystals. This work provides a novel approach to design bionic Fe/Mo bimetallene-modified inorganic semiconductor photocatalysts for nitrogen reduction.


Assuntos
Biônica , Nitrogênio , Molibdoferredoxina/metabolismo , Fixação de Nitrogênio , Nitrogenase/metabolismo
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.
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
4.
Microb Cell Fact ; 20(1): 139, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34281551

RESUMO

BACKGROUND: Biological nitrogen fixation is catalyzed by Mo-, V- and Fe-nitrogenases that are encoded by nif, vnf and anf genes, respectively. NifB is the key protein in synthesis of the cofactors of all nitrogenases. Most diazotrophic Paenibacillus strains have only one nifB gene located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV). But some Paenibacillus strains have multiple nifB genes and their functions are not known. RESULTS: A total of 138 nifB genes are found in the 116 diazotrophic Paenibacillus strains. Phylogeny analysis shows that these nifB genes fall into 4 classes: nifBI class including the genes (named as nifB1 genes) that are the first gene within the compact nif gene cluster, nifBII class including the genes (named as nifB2 genes) that are adjacent to anf or vnf genes, nifBIII class whose members are designated as nifB3 genes and nifBIV class whose members are named as nifB4 genes are scattered on genomes. Functional analysis by complementation of the ∆nifB mutant of P. polymyxa which has only one nifB gene has shown that both nifB1 and nifB2 are active in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Deletion analysis also has revealed that nifB1 of Paenibacillus sabinae T27 is involved in synthesis of Mo-nitrogenase, while nifB3 and nifB4 genes are not. Complementation of the P. polymyxa ∆nifBHDK mutant with the four reconstituted operons: nifB1anfHDGK, nifB2anfHDGK, nifB1vnfHDGK and nifB2vnfHDGK, has shown both that nifB1 and nifB2 were able to support synthesis of Fe- or V-nitrogenases. Transcriptional results obtained in the original Paenibacillus strains are consistent with the complementation results. CONCLUSIONS: The multiple nifB genes of the diazotrophic Paenibacillus strains are divided into 4 classes. The nifB1 located in a compact nif gene cluster (nifBHDKENX(orf1)hesAnifV) and the nifB2 genes being adjacent to nif or anf or vnf genes are active in synthesis of Mo-, Fe and V-nitrogenases, but nifB3 and nifB4 are not. The reconstituted anf system comprising 8 genes (nifBanfHDGK and nifXhesAnifV) and vnf system comprising 10 genes (nifBvnfHDGKEN and nifXhesAnifV) support synthesis of Fe-nitrogenase and V-nitrogenase in Paenibacillus background, respectively.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fixação de Nitrogênio/genética , Nitrogenase/biossíntese , Paenibacillus/genética , Proteínas de Bactérias/classificação , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Filogenia
5.
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
6.
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
7.
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
8.
Chem Soc Rev ; 50(15): 8743-8761, 2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34159992

RESUMO

The reduction of dinitrogen (N2) is essential for its incorporation into nucleic acids and amino acids, which are vital to life on earth. Nitrogenases convert atmospheric dinitrogen to two ammonia molecules (NH3) under ambient conditions. The catalytic active sites of these enzymes (known as FeM-cofactor clusters, where M = Mo, V, Fe) are the sites of N2 binding and activation and have been a source of great interest for chemists for decades. In this review, recent studies on nitrogenase-related synthetic molecular complexes and biological clusters are discussed, with a focus on their reactivity and spectroscopic characterization. The molecular models that are discussed span from simple mononuclear iron complexes to multinuclear iron complexes and heterometallic iron complexes. In addition, recent work on the extracted biological cofactors is discussed. An emphasis is placed on how these studies have contributed towards our understanding of the electronic structure and mechanism of nitrogenases.


Assuntos
Nitrogenase/química , Nitrogenase/metabolismo , Análise Espectral , Amônia/química , Amônia/metabolismo , Biocatálise , Ferro/química , Ferro/metabolismo , Nitrogênio/química , Nitrogênio/metabolismo , Oxirredução
9.
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
10.
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
11.
Nat Chem ; 13(7): 666-670, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34045715

RESUMO

The Fe-S clusters of nitrogenases carry out the life-sustaining conversion of N2 to NH3. Although progress continues to be made in modelling the structural features of nitrogenase cofactors, no synthetic Fe-S cluster has been shown to form a well-defined coordination complex with N2. Here we report that embedding an [MoFe3S4] cluster in a protective ligand environment enables N2 binding at Fe. The bridging [MoFe3S4]2(µ-η1:η1-N2) complex thus prepared features a substantially weakened N-N bond despite the relatively high formal oxidation states of the metal centres. Substitution of one of the [MoFe3S4] cubanes with an electropositive Ti metalloradical induces additional charge transfer to the N2 ligand with generation of Fe-N multiple-bond character. Structural and spectroscopic analyses demonstrate that N2 activation is accompanied by shortened Fe-S distances and charge transfer from each Fe site, including those not directly bound to N2. These findings indicate that covalent interactions within the cluster play a critical role in N2 binding and activation.


Assuntos
Complexos de Coordenação/química , Ferro/química , Molibdênio/química , Nitrogênio/química , Enxofre/química , Biomimética , Complexos de Coordenação/síntese química , Nitrogenase/química , Oxirredução
12.
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
13.
Int J Mol Sci ; 22(7)2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916504

RESUMO

NifS and NifU (encoded by nifS and nifU) are generally dedicated to biogenesis of the nitrogenase Fe-S cluster in diazotrophs. However, nifS and nifU are not found in N2-fixing Paenibacillus strains, and the mechanisms involved in Fe-S cluster biosynthesis of nitrogenase is not clear. Here, we found that the genome of Paenibacillus polymyxa WLY78 contains the complete sufCDSUB operon, a partial sufC2D2B2 operon, a nifS-like gene, two nifU-like genes (nfuA-like and yutI), and two iscS genes. Deletion and complementation studies showed that the sufC, sufD, and sufB genes of the sufCDSUB operon, and nifS-like and yutI genes were involved in the Fe-S cluster biosynthesis of nitrogenase. Heterologous complementation studies demonstrated that the nifS-like gene of P. polymyxa WLY78 is interchangeable with Klebsiella oxytoca nifS, but P. polymyxa WLY78 SufCDB cannot be functionally replaced by K. oxytoca NifU. In addition, K. oxytoca nifU and Escherichia coli nfuA are able to complement the P. polymyxa WLY78 yutI mutant. Our findings thus indicate that the NifS-like and SufCDB proteins are the specific sulfur donor and the molecular scaffold, respectively, for the Fe-S cluster formation of nitrogenase in P. polymyxa WLY78. YutI can be an Fe-S cluster carrier involved in nitrogenase maturation in P. polymyxa WLY78.


Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Proteínas Ferro-Enxofre/genética , Nitrogenase/genética , Paenibacillus polymyxa/genética , Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Nitrogenase/biossíntese , Paenibacillus polymyxa/metabolismo
14.
Bioresour Technol ; 333: 125165, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33894451

RESUMO

This study investigated the effects of garbage enzyme (GE), pelelith (PL), and biochar (BC) on nitrogen (N) conservation, nitrogenase (Nase) and N-fixing bacteria during the composting of sewage sludge. Results showed that the addition of GE, PL, and BC reduced NH3 emissions by 40.9%, 29.3%, and 67.4%, and increased the NO3-N contents of the end compost by 161.4, 88.2, and 105.8% relative to control, respectively, thus increasing the TN content. Three additives improved Nase, cellulase, and fluorescein diacetate hydrolase (FDA) activities and the abundances of nifH gene, and the largest increase was BC, followed by PL and GE. In addition, the additives also markedly influenced the succession of N-fixing bacteria, and significantly increased the abundance of Proteobacteria during the whole process. The BC and PL additions strengthened the sensitivity of N-fixing bacteria to environmental variables, and FDA, TN, moisture content, and NO3-N significantly affected the N-fixing bacteria at genus level.


Assuntos
Compostagem , Bactérias Fixadoras de Nitrogênio , Carvão Vegetal , Nitrogênio/análise , Nitrogenase , Esgotos , Solo
15.
Cells ; 10(4)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924023

RESUMO

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiaceae/enzimologia , Fabaceae/microbiologia , Nitrogenase/metabolismo , Oxo-Ácido-Liases/metabolismo , Simbiose , Burkholderiaceae/genética , Genoma Bacteriano , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Funções Verossimilhança , Metaboloma , Filogenia , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia
16.
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
17.
Environ Microbiol ; 23(6): 3164-3181, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33876566

RESUMO

Sulfate-reducing bacteria (SRB) are widespread in human guts, yet their expansion has been linked to colonic diseases. We report the isolation, sequencing and physiological characterization of strain QI0027T , a novel SRB species belonging to the class Desulfovibrionia. Metagenomic sequencing of stool samples from 45 Chinese individuals, and comparison with 1690 Desulfovibrionaceae metagenome-assembled genomes recovered from humans of diverse geographic locations, revealed the presence of QI0027T in 22 further individuals. QI0027T encoded nitrogen fixation genes and based on the acetylene reduction assay, actively fixed nitrogen. Transcriptomics revealed that QI0027T overexpressed 42 genes in nitrogen-limiting conditions compared to cultures supplemented with ammonia, including genes encoding nitrogenases, a urea uptake system and the urease complex. Reanalyses of 835 public stool metatranscriptomes showed that nitrogenase genes from Desulfovibrio bacteria were expressed in six samples suggesting that nitrogen fixation might be active in the gut environment. Although frequently thought of as a nutrient-rich environment, nitrogen fixation can occur in the human gut. Animals are often nitrogen limited and have evolved diverse strategies to capture biologically active nitrogen, ranging from amino acid transporters to stable associations with beneficial microbes that provide fixed nitrogen. QI0027T is the first Desulfovibrio human isolate for which nitrogen fixation has been demonstrated, suggesting that some sulfate-reducing bacteria could also play a role in the availability of nitrogen in the gut.


Assuntos
Desulfovibrio , Fixação de Nitrogênio , Animais , Bactérias/metabolismo , Desulfovibrio/genética , Desulfovibrio/metabolismo , Humanos , Nitrogenase/metabolismo , Oxirredução , Filogenia , Sulfatos
18.
Sci Rep ; 11(1): 9081, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907268

RESUMO

Phosphorus-solubilizing microorganisms is a microbial fertilizer with broad application potential. In this study, 7 endophytic phosphate solubilizing bacteria were screened out from Chinese fir, and were characterized for plant growth-promoting traits. Based on morphological and 16S rRNA sequence analysis, the endophytes were distributed into 5 genera of which belong to Pseudomonas, Burkholderia, Paraburkholderia, Novosphingobium, and Ochrobactrum. HRP2, SSP2 and JRP22 were selected based on their plant growth-promoting traits for evaluation of Chinese fir growth enhancement. The growth parameters of Chinese fir seedlings after inoculation were significantly greater than those of the uninoculated control group. The results showed that PSBs HRP2, SSP2 and JRP22 increased plant height (up to 1.26 times), stem diameter (up to 40.69%) and the biomass of roots, stems and leaves (up to 21.28%, 29.09% and 20.78%) compared to the control. Total N (TN), total P (TP), total K (TK), Mg and Fe contents in leaf were positively affected by PSBs while showed a significant relationship with strain and dilution ratio. The content of TN, TP, TK, available phosphorus (AP) and available potassium (AK) in the soil increased by 0.23-1.12 mg g-1, 0.14-0.26 mg g-1, 0.33-1.92 mg g-1, 5.31-20.56 mg kg-1, 15.37-54.68 mg kg-1, respectively. Treatment with both HRP2, SSP2 and JRP22 increased leaf and root biomass as well as their N, P, K uptake by affecting soil urease and acid phosphatase activities, and the content of available nutrients in soil. In conclusion, PSB could be used as biological agents instead of chemical fertilizers for agroforestry production to reduce environmental pollution and increase the yield of Chinese fir.


Assuntos
Inoculantes Agrícolas/fisiologia , Cunninghamia/crescimento & desenvolvimento , Cunninghamia/microbiologia , Fosfatos/metabolismo , Plântula/crescimento & desenvolvimento , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Carbono-Carbono Liases/metabolismo , Cunninghamia/metabolismo , Endófitos/fisiologia , Ácidos Indolacéticos/metabolismo , Nitrogenase/metabolismo , Fósforo/metabolismo , RNA Ribossômico 16S , Plântula/metabolismo , Plântula/microbiologia , Sideróforos/metabolismo , Solo/química
19.
Angew Chem Int Ed Engl ; 60(29): 15792-15797, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33928749

RESUMO

Constructing synthetic models of the nitrogenase PN -cluster has been a long-standing synthetic challenge. Here, we report an optimal nitrogenase PN -cluster model [{(TbtS)(OEt2 )Fe4 S3 }2 (µ-STbt)2 (µ6 -S)] (2) [Tbt=2,4,6-tris{bis(trimethylsilyl)methyl}phenyl] that is the closest synthetic mimic constructed to date. Of note is that two thiolate ligands and one hexacoordinated sulfide are connecting the two Fe4 S3 incomplete cubanes similar to the native PN -cluster, which has never been achieved. Cluster 2 has been characterized by X-ray crystallography and relevant physico-chemical methods. The variable temperature magnetic moments of 2 indicate a singlet ground state (S=0). The Mössbauer spectrum of 2 exhibits two doublets with an intensity ratio of 3:1, which suggests the presence of two types of iron sites. The synthetic pathway of the cluster 2 could indicate the native PN -cluster maturation process as it has been achieved from the Fe4 S4 cubane Fe4 S4 (STbt)4 (1).


Assuntos
Compostos Férricos/química , Compostos Férricos/síntese química , Ferro/química , Nitrogenase/química , Enxofre/química , Técnicas de Química Sintética , Ligantes , Modelos Moleculares , Conformação Molecular
20.
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
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