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 Leeuwenhoek Lecture, 1992 Bacterial evolution and the nitrogen-fixing plant

1992 ◽  
Vol 338 (1286) ◽  
pp. 409-416 ◽  
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Bacterial Species ◽  
Nitrogen Fixing ◽  
Bacterial Evolution ◽  
Late Emergence ◽  
Biological Nitrogen

Biological nitrogen fixation is fundamental to the economy of the biosphere, yet it is restricted to a few dozen bacterial species. Why have plants not acquired it during evolution? No serious physiological or genetic obstacles seem to exist. Has a relatively late emergence, among genomically flexible prokaryotes, effectively precluded appropriate seletion pressure?

scholarly journals Biological Nitrogen Fixation in the Context of Global Change

2013 ◽  
Vol 26 (5) ◽  
pp. 486-494 ◽  
Author(s):  
José Olivares ◽  
Eulogio J. Bedmar ◽  
Juan Sanjuán
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Food Production ◽  
Agricultural Practices ◽  
N Fertilization ◽  
Agricultural Crops ◽  
Nitrogen Fixing ◽  
Sustainable Technologies ◽  
Use Efficiency ◽  
Biological Nitrogen

The intensive application of fertilizers during agricultural practices has led to an unprecedented perturbation of the nitrogen cycle, illustrated by the growing accumulation of nitrates in soils and waters and of nitrogen oxides in the atmosphere. Besides increasing use efficiency of current N fertilizers, priority should be given to value the process of biological nitrogen fixation (BNF) through more sustainable technologies that reduce the undesired effects of chemical N fertilization of agricultural crops. Wider legume adoption, supported by coordinated legume breeding and inoculation programs are approaches at hand. Also available are biofertilizers based on microbes that help to reduce the needs of N fertilization in important crops like cereals. Engineering the capacity to fix nitrogen in cereals, either by themselves or in symbiosis with nitrogen-fixing microbes, are attractive future options that, nevertheless, require more intensive and internationally coordinated research efforts. Although nitrogen-fixing plants may be less productive, at some point, agriculture must significantly reduce the use of warming (chemically synthesized) N and give priority to BNF if it is to sustain both food production and environmental health for a continuously growing human population.

Understanding plant-root interactions with rhizobacteria to improve biological nitrogen fixation in crops

2021 ◽  
pp. 163-194
Author(s):  
Ulrike Mathesius ◽  
◽  
Jian Jin ◽  
Yansheng Li ◽  
Michelle Watt ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Plant Root ◽  
Current Understanding ◽  
Plant Roots ◽  
Nitrogen Fixing ◽  
Root Interactions ◽  
Molecular Determinants ◽  
Abiotic Constraints ◽  
Biological Nitrogen

Plant roots have evolved with the presence of rhizobacteria that can colonise the surface or interior of the plant. Some of these rhizobacteria are actively recruited by the plant and carry out particular functions, in particular in nutrient acquisition. Nitrogen-fixing bacteria form associations with many plant species, either as external associations or as symbiotic endophytes. The symbiosis between legumes and nitrogen-fixing rhizobia has been studied in most detail and is the most important contributor to nitrogen fixation in agriculture. This chapter highlights our current understanding of the molecular determinants of legume nodulation as well as challenges for improvements of biological nitrogen fixation in legumes and non-legumes. There is a need for connecting out knowledge of the molecular regulation of nodulation with field-based studies that take into account the interaction of nodulation with biotic and abiotic constraints. In addition, current approaches for engineering new symbioses are discussed.

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 Diazotrophic Growth Allows Azotobacter vinelandii To Overcome the Deleterious Effects of a glnE Deletion

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Florence Mus ◽  
Alex Tseng ◽  
Ray Dixon ◽  
John W. Peters
Keyword(s):  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Ammonium Assimilation ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Posttranslational Regulation ◽  
Knockout Mutant ◽  
Content Type ◽  
Carbon And Nitrogen ◽  
Ammonium Production

ABSTRACT Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity and preventing ammonium assimilation have been considered strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the posttranslational regulation of ammonium assimilation that is critical for the appropriate coordination of carbon and nitrogen assimilation. Since GS is key to the sole ammonium assimilation pathway of Azotobacter vinelandii, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain, thus preventing posttranslational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium, as previously observed in other organisms, and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of downregulation of GS activity, resulting in high intracellular glutamine levels and severe perturbation of the ratio of glutamine to 2-oxoglutarate under excess-nitrogen conditions. Interestingly, the mutant can grow diazotrophically at rates comparable to those of the wild type. This observation suggests that the control of nitrogen fixation-specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at levels appropriate for optimal carbon and nitrogen balance. IMPORTANCE In this study, the characterization of the glnE knockout mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation is of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.

scholarly journals Nitrogen utilization and transformation ofStenotrophomonas maltophiliaW-6 with nitrogen-fixing ability

2018 ◽  
Author(s):  
Shutong Wang ◽  
Yi Xu ◽  
Zhenlun Li
Keyword(s):  
Nitrogen Fixation ◽  
Nitrogen Cycle ◽  
Biological Nitrogen Fixation ◽  
Stenotrophomonas Maltophilia ◽  
Inorganic Nitrogen ◽  
Nitrate Removal ◽  
Nitrogen Utilization ◽  
Purple Soil ◽  
Nitrogen Fixing ◽  
Biological Nitrogen

AbstractStrain W-6 was isolated from the purple soil and successfully identifed asStenotrophomonas maltophiliaand used for the investigation on nitrogen utilization. Strain W-6 was monitored with the ability of biological nitrogen fixation when N2was used for the sole nitrogen source, and yet nitrogenase activity would be inhibited in the presence of extra nitrogen. Moreover, Strain W-6 could utilize NO3−, NO2−and NH4+for cell growth through assimilation, but unable to convert them to atmospheric nitrogen. Meantime, accumulation of nitrite was observed during the nitrate removal process, and the optimal conditions for nitrate removal were temperature of 20°C, shaking speed of 150 rpm, sodium succinate as the carbon source and C/N of 12. The experimental results indicate thatStenotrophomonas maltophiliautilize W-6 could utilize not only N2but also other nitrogen sources directly as its N substance. Therefore, heterotrophicAzotobactermay possess a great significance to nitrogen cycle except in biological nitrogen fixation.ImportanceAzotobacterspp. are found in soils worldwide, with features not simply for the nitrogen fixation, but for the energy metabolism relevant to agriculture. However, the role ofAzotobacterpotential in the function of nitrogen cycle except in biological nitrogen fixation is largely unknown. As such, whether bacteria utilize either inorganic nitrogen or organic nitrogen has remained obscure. The present studies indicate thatStenotrophomonas maltophiliaW-6 could highly efficient utilize nitrate, nitrite and ammonium etc. N substance and detect NH4+as final product. The transport velocities of nitrate-N to nitrite-N was quickly without gaseous nitrogen was produced. We probed the relationship between biological nitrogen fixation and N cycle via N conversion processes byS. maltophiliaW-6 with nitrogen-fixing ability

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.

Biological Nitrogen Fixation and Nitrogen Fixing Trees

2021 ◽  
pp. 413-443
Author(s):  
P. K. Ramachandran Nair ◽  
B. Mohan Kumar ◽  
Vimala D. Nair
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Nitrogen Fixing ◽  
Biological Nitrogen ◽  
Nitrogen Fixing Trees
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