scholarly journals Low potential electron generating enzymes serve different functions during aerobic nitrogen fixation in Azotobacter vinelandii

2021 ◽  
Author(s):  
Alexander B Alleman ◽  
Florence Mus ◽  
John W Peters
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Activation Barrier ◽  
Redox Homeostasis ◽  
Low Oxygen ◽  
High Activation Barrier ◽  
Biological Nitrogen ◽  
Diazotrophic Growth ◽  
Enzyme Complexes ◽  
Fix Genes

Biological nitrogen fixation requires large amounts of energy in the form of ATP and low potential electrons to overcome the high activation barrier for cleavage of the dinitrogen triple bond. The model aerobic nitrogen-fixing bacteria, Azotobacter vinelandii, generates low potential electrons in the form of reduced ferredoxin (Fd) and flavodoxin (Fld) using two distinct mechanisms via the enzyme complexes Rnf and Fix. Both Rnf and Fix are expressed during nitrogen fixation, and deleting either rnf1 or fix genes has little effect on diazotrophic growth. However, deleting both rnf1 and fix eliminates the ability to grow diazotrophically. Rnf and Fix both use NADH as a source of electrons, but overcoming the energetics of NADH's endergonic reduction of Fd/Fld is accomplished through different mechanisms. Rnf harnesses free energy from the proton motive force, whereas Fix uses electron bifurcation to effectively couple the endergonic reduction of Fd/Fld to the exergonic reduction of quinone. Different stoichiometries and gene expression analyses indicate specific roles for the two reactions under different conditions. In this work, complementary physiological studies and thermodynamic modeling reveal how Rnf and Fix simultaneously balance redox homeostasis in various conditions. Specifically, the Fix complex is required for efficient growth under low oxygen concentrations, while Rnf sustains homeostasis and delivers sufficient reduced Fd to nitrogenase under standard conditions. This work provides a framework for understanding how the production of low potential electrons sustains robust nitrogen fixation in various conditions.

Gene Fitness of Azotobacter vinelandii Under Diazotrophic Growth

2021 ◽  
Author(s):  
Carolann M. Knutson ◽  
Meghan N. Pieper ◽  
Brett M. Barney
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Genetic Manipulation ◽  
Nitrogen Fixing ◽  
Obligate Aerobe ◽  
Dependent Growth ◽  
Metal Cofactors ◽  
Biological Nitrogen ◽  
Diazotrophic Growth ◽  
Reference Tool

Azotobacter vinelandii is a nitrogen-fixing free-living soil microbe that has been studied for decades in relation to biological nitrogen fixation (BNF). It is highly amenable to genetic manipulation, helping to unravel the intricate importance of different proteins involved in the process of BNF, including the biosynthesis of cofactors that are essential to assembling the complex metal cofactors that catalyze the difficult reaction of nitrogen fixation. Additionally, A. vinelandii accomplishes this feat while growing as an obligate aerobe, differentiating it from many of the nitrogen-fixing bacteria that are associated with plant roots. The ability to function in the presence of oxygen makes A. vinelandii suitable for application in various potential biotechnological schemes. In this study, we employed transposon sequencing (Tn-seq) to measure the fitness defects associated with disruptions of various genes under nitrogen-fixing dependent growth, versus growth with extraneously provided urea as a nitrogen source. The results allowed us to probe the importance of more than 3800 genes, revealing that many genes previously believed to be important, can be successfully disrupted without impacting cellular fitness. Importance These results provide insights into the functional redundancy in A. vinelandii , while also providing a direct measure of fitness for specific genes associated with the process of BNF. These results will serve as a valuable reference tool in future studies to uncover the mechanisms that govern this process.

THE PRODUCTION OF 13N2 BY 50-MeV PROTONS FOR USE IN BIOLOGICAL NITROGEN FIXATION

1967 ◽  
Vol 13 (5) ◽  
pp. 587-599 ◽  
Author(s):  
N. E. R. Campbell ◽  
Ram Dular ◽  
H. Lees ◽  
K. G. Standing
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Continuous Production ◽  
Target Material ◽  
Experimental System ◽  
Beam Current ◽  
Reaction Products ◽  
Arctic Soils ◽  
Number Of Microorganisms ◽  
Biological Nitrogen

An experimental system for the continuous production of radioisotopic nitrogen, 13N2, has been developed using the sector-focused cyclotron at the University of Manitoba. The radioisotope is produced by 50 MeV proton bombardment of 14N2 with powdered melamine as the nitrogen-containing target material. A trap system necessary for the removal of unwanted reaction products is described and details of experimental procedures involving changes in proton beam current and in state of beam focus are presented.Using the radioisotope, a number of microorganisms isolated from sub-Arctic soils of the Fort Churchill region have been examined for their nitrogen fixation potential. Several of these, including a species of Rhodotorula and a species of Pullularia in addition to bacterial forms, have demonstrated nitrogen fixation at a rate comparable with that shown by Azotobacter vinelandii.

scholarly journals Heterotrophic bacterial diazotrophs are more abundant than their cyanobacterial counterparts in metagenomes covering most of the sunlit ocean

2021 ◽  
Author(s):  
Tom O. Delmont ◽  
Juan Jose Pierella Karlusich ◽  
Iva Veseli ◽  
Jessika Fuessel ◽  
A. Murat Eren ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Nitrogen Balance ◽  
Large Scale ◽  
Current View ◽  
Low Oxygen ◽  
Nitrogen Fixers ◽  
Large Size ◽  
Genomic Characterization ◽  
Biological Nitrogen ◽  
Nifh Genes

Biological nitrogen fixation is a major factor contributing to microbial primary productivity in the open ocean. The current view depicts a few cyanobacterial diazotrophs as the most relevant marine nitrogen fixers, whereas heterotrophic diazotrophs are more diverse and considered to have lower impacts on the nitrogen balance. Here, we used 891 Tara Oceans metagenomes to create a manually curated, non-redundant genomic database corresponding to free-living, as well as filamentous, colony-forming, particle-attached and symbiotic bacterial and archaeal populations occurring in the surface of five oceans and two seas. Notably, the database provided the genomic content of eight cyanobacterial diazotrophs including Trichodesmium populations and a newly discovered population similar to Richelia, as well as 40 heterotrophic bacterial diazotrophs organized into three main functional groups that considerably expand the known diversity of abundant marine nitrogen fixers compared to previous genomic surveys. Critically, these 48 populations may account for more than 90% of cells containing known nifH genes and occurring in the sunlit ocean, suggesting that the genomic characterization of the most abundant marine diazotrophs may be nearing completion. The newly identified heterotrophic bacterial diazotrophs are widespread, express their nifH genes in situ, and co-occur under nitrate-depleted conditions in large size fractions where they might form aggregates providing the low-oxygen microenvironments required for nitrogen fixation. Most significantly, we found heterotrophic bacterial diazotrophs to be more abundant than cyanobacterial diazotrophs in most metagenomes from the open oceans and seas. This large-scale environmental genomic survey emphasizes the considerable potential of heterotrophs in the marine nitrogen balance.

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.

scholarly journals Genetic determinants of ammonia excretion in nifL mutants of Azotobacter vinelandii

2021 ◽  
Author(s):  
Florence Mus ◽  
Devanshi Khokhani ◽  
Esther Rugoli ◽  
Ray Dixon ◽  
Jean-Michel Ané ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Model Organism ◽  
Ammonia Excretion ◽  
Promoter Sequence ◽  
Genetic Determinants ◽  
Two Component System ◽  
Opposite Orientation ◽  
Fixed Nitrogen ◽  
Biological Nitrogen

Abstract The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an anti-activator that tightly controls that activity of the nitrogen fixation specific transcriptional activator, NifA, in response to redox, nitrogen, and carbon status. While several studies reported mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonia, knowledge about the specific determinants for this ammonia-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonia accumulated in liquid culture (~ 12 mM). The ammonia excretion phenotype is solely associated with deletions of NifL domains combined with the insertion of a promoter sequence in the opposite orientation to nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonia excreted by affecting rnf1 gene expression as an additional requirement for ammonia excretion. These ammonia-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines the discreet determinants that bring about A. vinelandii ammonia excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

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.

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.

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