scholarly journals Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Mary H. Plunkett ◽  
Carolann M. Knutson ◽  
Brett M. Barney
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Key Factors ◽  
Factors Affecting ◽  
Ammonium Production ◽  
Biological Nitrogen

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 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.

Nitrogen Special Issue: summing up of papers and recommendations for future research

2001 ◽  
Vol 41 (3) ◽  
pp. 459 ◽  
Author(s):  
E. C. Wolfe
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Supply And Demand ◽  
Crop Rotations ◽  
Future Research ◽  
Special Issue ◽  
Factors Affecting ◽  
Slow Progress ◽  
Dryland Crops ◽  
Biological Nitrogen

The 12th Australian Nitrogen Fixation Conference was the third in a series of national workshops that began in 1991 and dealt with aspects of the nitrogen dynamics of Australian pastures and croplands. The conference and the papers published in the Special Issue addressed, at least in part, the slow progress that is evident in improving the rate of biological nitrogen fixation by enhancing inoculating techniques and Rhizobium strains. An important output from the conference was an analysis of nitrogen supply and demand in Australian dryland crops, indicating less reliance on biological nitrogen fixation due to higher wheat yields, the increased use of canola in crop rotations and problems with pulses. In 3 keynote reviews, the factors affecting the fixation and release of biologically fixed nitrogen to non-legume crops were comprehensively detailed. These factors included the frequency of legumes in rotations and their individual biomass rather than the efficiency of nitrogen fixation itself. The further development and use of models is a way of predicting outcomes for various combinations of management and crop rotations. The present trend towards fewer years of legumes in phase farming in Australia may reverse, resulting in renewed interest in ley pastures and pulse crops.

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