Construction of recombinant Escherichia coli producing nitrogenase-related proteins from Azotobacter vinelandii

2021 ◽  
Author(s):  
Yuki Tatemichi ◽  
Takeharu Nakahara ◽  
Mitsuyoshi Ueda ◽  
Kouichi Kuroda
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Fossil Fuels ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Gene Clusters ◽  
Fixation Method ◽  
Overlap Extension ◽  
Biological Nitrogen

Abstract Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.

Evaluation of Biological Nitrogen Fixation Capacity in Arachis Species and the Possible Role of Polyploidy1

1994 ◽  
Vol 21 (1) ◽  
pp. 55-60 ◽  
Author(s):  
H. T. Stalker ◽  
M. L. Nickum ◽  
J. C. Wynne ◽  
G. H. Elkan ◽  
T. J. Schneeweis
Keyword(s):  
Nitrogen Fixation ◽  
Arachis Hypogaea ◽  
Cover Crops ◽  
Biological Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Diploid Species ◽  
Dry Matter Accumulation ◽  
Arachis Species ◽  
Fixation Capacity ◽  
Biological Nitrogen

Abstract Arachis species have potential for enhancing cultivated peanut (Arachis hypogaea L.) germplasm as forages and cover crops. This study's objective was to evaluate a range of Arachis species for biological nitrogen fixation capacity. Several Arachis species are tetraploids, and it has been shown that tetraploidy may play an important role in nodule initiation. Species were first tested under natural field conditions and then in the greenhouse using three Bradyrhizobium strains that had been previously shown to be effective on peanut. Nodule number, nodule weight, nitrogenase activity determined by acetylene reduction, and shoot dry weight were measured as indicators of nitrogen fixation capacity. In the field, tetraploid species produced significantly more nodules than the diploids, but total dry matter accumulation was independent of the number of nodules or rate of fixation. In the greenhouse, no significant differences were observed among the bradyrhizobial strains. Arachis hypogaea and A. monticola showed significantly higher measures of nitrogen fixation capacity for all measured traits than the diploid species. However, autotetraploid plants of A. villosa did not have significantly more nodules than diploids of the same accession; the autotetraploids consistently had higher nitrogenase activity. Arachis pusilla never formed a symbiotic relationship with the bradyrhizobial strains used.

scholarly journals Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase

2015 ◽  
Vol 198 (4) ◽  
pp. 633-643 ◽  
Author(s):  
Marie-Christine Hoffmann ◽  
Eva Wagner ◽  
Sina Langklotz ◽  
Yvonne Pfänder ◽  
Sina Hött ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Central Process ◽  
Content Type ◽  
Proteome Profiling ◽  
Biological Nitrogen ◽  
Diazotrophic Growth

ABSTRACTRhodobacter capsulatusis capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N2) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, andlacZreporter fusions. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed.IMPORTANCEBiological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, includingRhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome inR. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions.

scholarly journals Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Brett M. Barney ◽  
Mary H. Plunkett ◽  
Velmurugan Natarajan ◽  
Florence Mus ◽  
Carolann M. Knutson ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stationary Phase ◽  
Gene Transcription ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Content Type ◽  
Ammonium Production ◽  
Biological Nitrogen

ABSTRACT Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

scholarly journals Metabolic model of nitrogen-fixing obligate aerobe Azotobacter vinelandii demonstrates adaptation to oxygen concentration and metal availability

2021 ◽  
Author(s):  
Alexander B Alleman ◽  
Florence Mus ◽  
John W Peters
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Metabolic Model ◽  
Metal Availability ◽  
Respiratory Protection ◽  
Nitrogen Fixing ◽  
A Genome ◽  
Biological Nitrogen ◽  
Genome Scale

There is considerable interest in promoting biological nitrogen fixation as a mechanism to reduce the inputs of nitrogenous fertilizers in agriculture, a problem of agronomic, economic, and environmental importance. For the potential impact of biological nitrogen fixation in agriculture to be realized, there are considerable fundamental knowledge gaps that need to be addressed. Biological nitrogen fixation or the reduction of N2 to NH3 is catalyzed by nitrogenase which requires a large amount of energy in the form of ATP and low potential electrons. Nitrogen-fixing organisms that respire aerobically have an advantage in meeting the energy demands of biological nitrogen fixation but face challenges of protecting nitrogenase from inactivation in the presence of oxygen. Here, we have constructed a genome-scale metabolic model of the aerobic metabolism of nitrogen-fixing bacteria Azotobacter vinelandii, which uses a complex electron transport system, termed respiratory protection, to consume oxygen at a high rate keeping intracellular conditions microaerobic. Our model accurately determines growth rate under high oxygen and high substrate concentration conditions, demonstrating the large flux of energy directed to respiratory protection. While respiratory protection mechanisms compensate the energy balance in high oxygen conditions, it does not account for all substrate intake, leading to increased maintenance rates. We have also shown how A. vinelandii can adapt under different oxygen concentrations and metal availability by rearranging flux through the electron transport system. Accurately determining the energy balance in a genome-scale metabolic model is required for future engineering approaches.

The fixed nitrogen sensitivity of biological nitrogen fixation in salt marshes sediments from the northeastern United States

2020 ◽  
Author(s):  
Romain Darnajoux ◽  
Rei Zhang ◽  
Katja Luxem ◽  
Xinning Zhang
Keyword(s):  
Nitrogen Fixation ◽  
Salt Marshes ◽  
Biological Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Inorganic Nitrogen ◽  
Ammonium Concentration ◽  
Desulfovibrio Vulgaris ◽  
Fixed Nitrogen ◽  
Sulfate Reducing ◽  
Biological Nitrogen

<p>Biological nitrogen fixation, the main input of fixed N into ecosystems, converts inert N<sub>2</sub> gas into bioavailable ammonium in an energetically costly reaction catalyzed by the prokaryotic metalloenzyme nitrogenase.  The high ATP and reductant requirements of N<sub>2</sub> fixation explain why this process is highly regulated in diazotrophs, with the presence of ammonium inhibiting nitrogenase expression and activity. Yet, several reports of N<sub>2</sub> fixation in ammonium- and nitrate-rich (10 to 300 µM) benthic environments challenge our understanding of a key environmental sensitivity of N<sub>2</sub> fixation. Field studies point to heterotrophic sulfate reducers as the likely diazotrophs in these benthic settings, but the fixed N sensitivity of sulfate-reducing diazotrophs is not well understood due to a dearth of culture studies. Additionally, assays of N<sub>2</sub> fixation in incubations rarely involve parallel measurements of dissolved inorganic nitrogen, possibly leading to experimental bias in favor of detecting activity under ammonium-replete initial conditions.</p><p>To help reconcile the environmental results, we investigate the ammonium sensitivity of N<sub>2</sub> fixation using the acetylene reduction assay and <sup>15</sup>N<sub>2</sub> tracer methods in i) the model sulfate-reducing diazotroph, <em>Desulfovibrio vulgaris</em> str. Hildenborough (DvH), ii) four enrichment cultures from salt marsh sediments of New Jersey, and iii) slurry incubations of sediments collected from three northeastern salt marshes. In all instances, we found that ammonium strongly inhibits biological nitrogen fixation, with nitrogenase activity only detectable when ammonium concentration is below a threshold of 10 µM (slurry incubation) or 2 µM (pure cultures, enrichments). Amendment of ammonium quickly inhibits nitrogen fixation and nitrogenase activity only resumes  once ammonium is depleted to the threshold level. Ammonium additions to actively fixing samples show complete inhibition of N<sub>2</sub> fixation within several hours post-addition. </p><p>Our measurements of the ammonium sensitivity of benthic N<sub>2</sub> fixation are consistent with the traditional understanding of nitrogen fixer metabolism and with early findings of Postgate et al. (1984) demonstrating that N<sub>2</sub> fixation by the sulfate reducer <em>Desulfovibrio gigas</em> is inhibited by ammonium levels that exceed 10 µM. These results help clarify a long-standing paradox in benthic nitrogen cycling. We suggest that prior observations of N<sub>2</sub> fixation at elevated ammonium levels could reflect methodological artifacts due to very fast depletion of ammonium during activity assays, legacy N<sub>2</sub> fixation activity associated with incomplete inhibition by ammonium, or spatial heterogeneity. Further work to standardize fixed N sensitivity assays could help with cross-study comparisons and with clarifying inconsistencies in our understanding of how environmental fixed nitrogen levels control nitrogen fixation.</p>

Biological nitrogen fixation associated with tropical pasture grasses

2001 ◽  
Vol 28 (9) ◽  
pp. 837 ◽  
Author(s):  
Veronica M. Reis ◽  
Fábio B. dos Reis Jr ◽  
Diego M. Quesada ◽  
Octávio C. A. de Oliveira ◽  
Bruno J. R. Alves ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Fossil Fuels ◽  
Pennisetum Purpureum ◽  
N Accumulation ◽  
Fast Growing ◽  
15N Enrichment ◽  
Pasture Grasses ◽  
High Yields ◽  
Biological Nitrogen

This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 The semi-humid or humid tropics are ideal for the production of large quantities of biomass from fast-growing C4 grasses, but high yields normally require large quantities of fertiliser, especially N, which has a very high input from fossil fuels (natural gas). A program has been started recently to use elephant grass (Pennisetum purpureum Schum.) to substitute firewood as a fuel and also to make charcoal for iron production. In this case, any large N fertiliser additions would mean that the yield of bio fuel per unit of fossil fuel invested would be detrimentally affected. In this study, we report on the potential for the selection of genotypes of fast-growing C4 tropical grasses of the genera Pennisetum and Brachiaria for their capacity to obtain N inputs from plant-associated biological nitrogen fixation (BNF). Fourteen genotypes each of Brachiaria and Pennisetum were screened for BNF contributions by growing them in 15N-labelled soil. In the case of the Pennisetum, after a suitable cutting height for the crop had been selected, there were large differences in dry matter production, N accumulation and 15N enrichment. The differences in 15N enrichment between genotypes were statistically significant and BNF inputs were estimated as high as 41% of accumulated N. In the study on Brachiaria genotypes, potential inputs of BNF seemed lower. Only one or two genotypes of B. brizantha and B. ruziziensis obtained more then 20% of their N from BNF. The N2-fixing bacteria that were most commonly associated with shoots and roots the Pennisetum genotypes were of the genus Herbaspirillum, but predominantly of a recently described new species. The Brachiaria spp. from three different sites (Rio de Janeiro, Goânia, Bahia) were predominately colonised by Azospirillum spp., most of the isolates being of the species Azospirillum amazonense. Very few Herbaspirilla were isolated from these plants.

scholarly journals Specificity of NifEN and VnfEN for the Assembly of Nitrogenase Active Site Cofactors in Azotobacter vinelandii

mBio ◽  
2021 ◽  
Author(s):  
Ana Pérez-González ◽  
Emilio Jimenez-Vicente ◽  
Jakob Gies-Elterlein ◽  
Alvaro Salinero-Lanzarote ◽  
Zhi-Yong Yang ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Active Site ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Complex Process ◽  
Number Of Genes ◽  
Biological Nitrogen

Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products.

Helping plants get more nitrogen from the air

2000 ◽  
Vol 8 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Edward C. Cocking
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Fossil Fuels ◽  
Crop Yields ◽  
Atmospheric Nitrogen ◽  
Fixed Nitrogen ◽  
Free Living ◽  
World Agriculture ◽  
Biological Nitrogen ◽  
Inoculation Technology

Plants cannot themselves obtain their nitrogen from the air but rely mainly on the supply of combined nitrogen in the form of ammonia, or nitrates, resulting from nitrogen fixation by free-living bacteria in the soil or bacteria living symbiotically in nodules on the roots of legumes. Increased crop yields in the twentieth century required this biological nitrogen fixation to be supplemented increasingly by the use of fixed nitrogen from chemical fertilizers. The development of the Haber–Bosch process for catalytically combining atmospheric nitrogen with hydrogen from fossil fuels to produce ammonia enabled increased crop yields. However, energy and environmental concerns arising from the overuse of nitrogenous fertilizers have highlighted the need for plants to obtain more of their nitrogen from the air by biological nitrogen fixation. New systems are being developed for increased biological nitrogen fixation with cereals and other non-legumes by establishing nitrogen-fixing bacteria within their roots. This new inoculation technology is aimed at significantly reducing the use of synthetic nitrogenous fertilizers in world agriculture.

Sign in / Sign up

Export Citation Format

Share Document