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 Manipulating nitrogen regulation in diazotrophic bacteria for agronomic benefit

2019 ◽  
Vol 47 (2) ◽  
pp. 603-614 ◽  
Author(s):  
Marcelo Bueno Batista ◽  
Ray Dixon
Keyword(s):  
Nitrogen Fixation ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Ammonia Excretion ◽  
Ammonium Assimilation ◽  
Regulatory Mechanisms ◽  
Diazotrophic Bacteria ◽  
Fixed Nitrogen ◽  
Regulatory Circuits ◽  
Biological Nitrogen

AbstractBiological nitrogen fixation (BNF) is controlled by intricate regulatory mechanisms to ensure that fixed nitrogen is readily assimilated into biomass and not released to the environment. Understanding the complex regulatory circuits that couple nitrogen fixation to ammonium assimilation is a prerequisite for engineering diazotrophic strains that can potentially supply fixed nitrogen to non-legume crops. In this review, we explore how the current knowledge of nitrogen metabolism and BNF regulation may allow strategies for genetic manipulation of diazotrophs for ammonia excretion and provide a contribution towards solving the nitrogen crisis.

The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii

2021 ◽  
Author(s):  
Yulia V Bertsova ◽  
Marina V Serebryakova ◽  
Alexander A Baykov ◽  
Alexander V Bogachev
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Growth Defect ◽  
Deficient Mutant ◽  
Fixed Nitrogen ◽  
Oxidoreductase Activity ◽  
Threonine Residue ◽  
Ferredoxin Reductase ◽  
Relative Contribution ◽  
Fixation System

Abstract Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin) acting as an electron donor for nitrogenase. However, the relative contribution of Rnf into nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely unknown. We have constructed A. vinelandii cells with impaired activities of FixABCX and/or putative flavin transferase ApbE. The ApbE-deficient mutant could not produce covalently flavinylated membrane proteins and demonstrated a markedly decreased flavodoxin:NAD+ oxidoreductase activity and significant growth defect under diazotrophic conditions. The double ΔFix/ΔApbE mutation abolished the flavodoxin:NAD+ oxidoreductase activity and the ability of A. vinelandii to grow in the absence of fixed nitrogen source. ApbE flavinylated a truncated RnfG subunit of Rnf1 by forming a phosphoester bond between FMN and a threonine residue. These findings indicate that Rnf (presumably its Rnf1 form) is the major ferredoxin-reducing enzyme in the nitrogen fixation system and that the activity of Rnf depends on its covalent flavinylation by the flavin transferase ApbE.

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 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.

Symbiosomes: temporary moonlighting organelles

2014 ◽  
Vol 460 (1) ◽  
pp. 1-11 ◽  
Author(s):  
David W. Emerich ◽  
Hari B. Krishnan
Keyword(s):  
Nitrogen Fixation ◽  
Symbiotic Nitrogen Fixation ◽  
Infected Plant ◽  
Plant Cells ◽  
Fixed Nitrogen ◽  
Moonlighting Proteins ◽  
Biological Nitrogen ◽  
Structural Entity ◽  
Leguminous Host

Symbiosomes are a unique structural entity that performs the role of biological nitrogen fixation, an energy-demanding process that is the primary entryway of fixed nitrogen into the biosphere. Symbiosomes result from the infection of specific rhizobial strains into the roots of an appropriate leguminous host plant forming an organ referred to as a nodule. Within the infected plant cells of the nodule, the rhizobia are encased within membrane-bounded structures that develop into symbiosomes. Mature symbiosomes create an environment that allows the rhizobia to differentiate into a nitrogen-fixing form called bacteroids. The bacteroids are surrounded by the symbiosome space, which is populated by proteins from both eukaryotic and prokaryotic symbionts, suggesting this space is the quintessential component of symbiosis: an inter-kingdom environment with the single purpose of symbiotic nitrogen fixation. Proteins associated with the symbiosome membrane are largely plant-derived proteins and are non-metabolic in nature. The proteins of the symbiosome space are mostly derived from the bacteroid with annotated functions of carbon metabolism, whereas relatively few are involved in nitrogen metabolism. An appreciable portion of both the eukaryotic and prokaryotic proteins in the symbiosome are also ‘moonlighting’ proteins, which are defined as proteins that perform roles unrelated to their annotated activities when found in an unexpected physiological environment. The essential functions of symbiotic nitrogen fixation of the symbiosome are performed by co-operative interactions of proteins from both symbionts some of which may be performing unexpected roles.

Nitrogen fixation in the high arctic tundra at Sarcpa Lake, Northwest Territories

1985 ◽  
Vol 63 (5) ◽  
pp. 974-979 ◽  
Author(s):  
Jim D. Karagatzides ◽  
Martin C. Lewis ◽  
Herbert M. Schulman
Keyword(s):  
Nitrogen Fixation ◽  
Northwest Territories ◽  
Acetylene Reduction ◽  
Biological Nitrogen Fixation ◽  
Arctic Tundra ◽  
High Arctic ◽  
Fixed Nitrogen ◽  
Free Living ◽  
Blue Green Algae ◽  
Biological Nitrogen

The acetylene reduction assay was used to examine biological nitrogen fixation in the high arctic tundra at Sarcpa Lake, Northwest Territories (68°32′ N, 83°19′ W). The highest rates of acetylene reduction (9.37 ± 3.19 μmol C2H4 m−2 h−1) were in habitats that had a high density of the legumes Oxytropis maydelliana, O. arctobia, and Astragalus alpinus. Nitrogen fixation in the wet soils along the shore of a small lake was similar (8.87 ± 4.35 μmol C2H4 m−2 h−1) because of the blue-green alga Nostoc, which associates with mosses. Free-living blue-green algae and lichens made insignificant contributions to the total nitrogen fixation budget because they were uncommon and fixed nitrogen at a slower rate. Nitrogen-fixing lichens in the area included Stereocaulon arenarium and S. rivulorum. It is concluded that legumes have a significant input to the biological nitrogen fixation budget at Sarcpa Lake.

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.

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.

INFLUENCE OF THE ELEMENTS OF GROWING TECHNOLOGY ON THE ACTIVATION OF PLANT-MICROBIAL SYMBIOSIS AND THE PROCESSES OF NITROGEN LUCOSENOSIS AGROCENOSIS

2020 ◽  
pp. 61-72
Author(s):  
Viacheslav Tsyhanskyi ◽  
Olena Tsyhanska
Keyword(s):  
Nitrogen Fixation ◽  
Plant Growth ◽  
Plant Growth Regulators ◽  
Growth Regulators ◽  
Seed Treatment ◽  
Mineral Fertilizers ◽  
Agricultural Crops ◽  
Fixed Nitrogen ◽  
Sowing Seed ◽  
Biological Nitrogen

In order to improve soil fertility, increase the productivity of agricultural crops, including alfalfa, and to make the best use of mineral fertilizers it is expedient to carry out liming in the range of 1.0-1.5 under hydrolytic acidity. It provides an increase in the amount of 1.2-1.8 tons of feed units per hectare of rotation area. The mobility of toxic elements of aluminum and manganese decreases in the limed soil. These processes contribute to nodule formation and an increase in the nitrogen fixation of alfalfa. The intensified activity of nitrogen fixation processes occurring in the root zone of plants can be achieved with the use of physiologically active substances characterized by auxin-cytokine activity. Both associative and symbiotic nitrogen fixation processes are enhanced by virtue of plant growth stimulants. These preparations are the analogs of exogenous phytohormones of cytokine, gibberellin and auxin effect, and unsaturated fatty acids, carbohydrates, amino acids. They are used for pre-sowing seed treatment and top dressing of plants. The conceptual direction of the development of biotechnologies and biological nitrogen fixation in agriculture is the development of preparations in order to stimulate growth, increase the production of legumes. Creation is important the original complex compositions of multifactorial action, which combine the properties of plant growth regulators, fertilizer elements, means of plant resistance to stress and diseases. In light of this, the study of agrobiological methods of growing forage crops. Including alfalfa, with the use of plant growth regulators, as well as their combination with biological preparations based on active strains of nodule bacteria. It aims to improve the nitrogen nutrition of plants in the monoculture. Important is also a significant increase in the level of the biological conversion of atmospheric nitrogen into organic nitrogen-containing compounds. Is particularly relevant for the creation of sustainable production of feed raw materials and maximization of the biological potential of plants under the unstable soil and climatic conditions. When growing agricultural crops, biologically fixed nitrogen is extremely important for legumes since this reduces the use of mineral fertilizers and the cost of production. Perennial legumes, in particular, alfalfa, are not only a source of high-protein feed, but they are the best precursors, especially for grains, cereals, and industrial crops. They not only increase their yields but also improve the soil structure and fertility. According to the results of our research, under the conditions of Right-Bank Forest-Steppe, the best conditions for the symbiotic activity of alfalfa seedlings were created under the conditions: Non-perishable growing method. The introduction of the herbicide in the year of sowing. Conduct calcining the full norm. Use of pre-sowing seed treatment with bacterial preparation risobofit with plant growth regulator Emistim S. Under these conditions, the maximum indicator of biologically fixed nitrogen is formed-236.1 kg/ha. Key words: alfalfa sowing, liming of soil, inoculation, growth promoter, biological nitrogen.

Sign in / Sign up

Export Citation Format

Share Document