scholarly journals Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph

2018 ◽  
Author(s):  
Deng Liu ◽  
Michelle Liberton ◽  
Jingjie Yu ◽  
Himadri B. Pakrasi ◽  
Maitrayee Bhattacharyya-Pakrasi
Keyword(s):  
Nitrogen Fixation ◽  
Energy Demand ◽  
Nitrogenase Activity ◽  
Crop Plants ◽  
Nitrogen Fertilizers ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Photosynthetic Organism ◽  
Biological Nitrogen ◽  
Oxygenic Phototroph

ABSTRACTBiological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N2fixation is energetically highly expensive, and an energy generating process such as photosynthesis can meet the energy demand of N2fixation. However, synthesis and expression of nitrogenase is exquisitely sensitive to oxygen. Thus, engineering nitrogen fixation activity in photosynthetic organisms that produce oxygen is challenging. Cyanobacteria are oxygenic photosynthetic prokaryotes, and some of them also fix N2. Here, we demonstrate a feasible way to engineer nitrogenase activity in the non-diazotrophic cyanobacteriumSynechocystissp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacteriumCyanothecesp. ATCC 51142. In addition, we have identified the minimalnifcluster required for such activity inSynechocystis6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels ofnifgenes. Importantly, the O2tolerance of nitrogenase was enhanced by introduction of uptake hydrogenase genes, showing this to be a functional way to improve nitrogenase enzyme activity under micro-oxic conditions. To date, our efforts have resulted in engineeredSynechocystis6803 strains that remarkably have more than 30% N2-fixation activity compared to that inCyanothece51142, the highest such activity established in any non-diazotrophic oxygenic photosynthetic organism. This study establishes a baseline towards the ultimate goal of engineering nitrogen fixation ability in crop plants.IMPORTANCEApplication of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes as well as the wide spread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow crop plants the ability to fix atmospheric N2in situ. One long-term approach is to transfer allnifgenes from a prokaryote to plant cells, and express nitrogenase in an energy-producing organelle, chloroplast or mitochondrion. In this context,Synechocystis6803, the non-diazotrophic cyanobacterium utilized in this study, provides a model chassis for rapid investigation of the necessary requirements to establish diazotrophy in an oxygenic phototroph.

scholarly journals Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Deng Liu ◽  
Michelle Liberton ◽  
Jingjie Yu ◽  
Himadri B. Pakrasi ◽  
Maitrayee Bhattacharyya-Pakrasi
Keyword(s):  
Nitrogen Fixation ◽  
Energy Demand ◽  
Nitrogenase Activity ◽  
Crop Plants ◽  
Nitrogen Fertilizers ◽  
Widespread Application ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Photosynthetic Organism ◽  
Oxygenic Phototroph

ABSTRACTBiological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N2fixation is energetically highly expensive, and an energy-generating process such as photosynthesis can meet the energy demand of N2fixation. However, synthesis and expression of nitrogenase are exquisitely sensitive to the presence of oxygen. Thus, engineering nitrogen fixation activity in photosynthetic organisms that produce oxygen is challenging. Cyanobacteria are oxygenic photosynthetic prokaryotes, and some of them also fix N2. Here, we demonstrate a feasible way to engineer nitrogenase activity in the nondiazotrophic cyanobacteriumSynechocystissp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacteriumCyanothecesp. ATCC 51142. In addition, we have identified the minimalnifcluster required for such activity inSynechocystis6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels ofnifgenes. Importantly, the O2tolerance of nitrogenase was enhanced by introduction of uptake hydrogenase genes, showing this to be a functional way to improve nitrogenase enzyme activity under micro-oxic conditions. To date, our efforts have resulted in engineeredSynechocystis6803 strains that, remarkably, have more than 30% of the N2fixation activity ofCyanothece51142, the highest such activity established in any nondiazotrophic oxygenic photosynthetic organism. This report establishes a baseline for the ultimate goal of engineering nitrogen fixation ability in crop plants.IMPORTANCEApplication of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes and the widespread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow to crop plants the ability to fix atmospheric N2in situ. One long-term approach is to transfer allnifgenes from a prokaryote to plant cells and to express nitrogenase in an energy-producing organelle, chloroplast, or mitochondrion. In this context,Synechocystis6803, the nondiazotrophic cyanobacterium utilized in this study, provides a model chassis for rapid investigation of the necessary requirements to establish diazotrophy in an oxygenic phototroph.

scholarly journals Root Distribution and Nitrogen Fixation Activity of Tropical Forage Legume American Jointvetch (Aeschynomene americanaL.) cv. Glenn under Waterlogging Conditions

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Manabu Tobisa ◽  
Masataka Shimojo ◽  
Yasuhisa Masuda
Keyword(s):  
Nitrogen Fixation ◽  
Root Distribution ◽  
Water Surface ◽  
Nitrogenase Activity ◽  
Soil Surface ◽  
Nodule Formation ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Net Assimilation ◽  
Waterlogging Treatment

We investigated the root distribution and nitrogen fixation activity of American jointvetch (Aeschynomene americanaL.) cv. Glenn, under waterlogging treatment. The plants were grown in pots under three different treatments: no waterlogging (control), 30 days of waterlogging (experiment 1), and 40 days of waterlogging (experiment 2). The plants were subjected to the treatments on day 14 after germination. Root dry matter (DM) weight distribution of waterlogged plants was shallower than controls after day 20 of waterlogging. Throughout the study period, the total root DM weight in waterlogged plants was similar to that in the controls. Enhanced rooting (adventitious roots) and nodule formation at the stem base were observed in waterlogged plants after day 20 of waterlogging. The average DM weight of individual nodules on the region of the stem between the soil surface and water surface of waterlogged plants was similar to that of individual taproot nodules in the controls. Waterlogged plants had slightly greater plant DM weight than the controls after 40 days of treatment. The total nitrogenase activity (TNA) of nodules and nodule DM weight were higher in waterlogged plants than in the controls. Waterlogged American jointvetch had roots with nodules both around the soil surface and in the area between the soil surface and water surface after 20 days of waterlogging, and they maintained high nitrogenase activity and net assimilation rate that resulted in an increased growth rate.

scholarly journals Biological Nitrogen Fixation and Denitrification in Rhizosphere of Potato Plants in Response to the Fertilization and Inoculation

2021 ◽  
Vol 5 ◽  
Author(s):  
Vitaliy V. Volkogon ◽  
Svitlana B. Dimova ◽  
Kateryna I. Volkogon ◽  
Vasyl P. Sidorenko ◽  
Mykola V. Volkogon
Keyword(s):  
Nitrogen Fixation ◽  
Green Manure ◽  
Crop Yields ◽  
Nitrogenase Activity ◽  
Farmyard Manure ◽  
Mineral Fertilizers ◽  
Potato Plants ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Fertilizer Rates

The study aim was to evaluate the potential nitrogen fixation and denitrification in the rhizosphere soil of potato plants, crop yield and output quality in response to the different fertilization systems and the inoculation with Azospirillum brasilense 410. Field stationary experiment was conducted between 2016 and 2019 with potato in a crop rotation system on leached chernozem soil. Farmyard manure, 40 t/ha, applied prior to potatoes planting promotes nitrogen fixation (0.8–2.0 times compared to control). However, it has also affected denitrification (in 1.4–2.2 times higher compared to control). The lowest rate of mineral fertilizers used in the experiment, N40P40K40, was shown as most environmentally feasible. Under its use the increase of soil nitrogenase activity and low denitrification levels were observed. Same trends were also noted for the medium fertilizer rate, N80P80K80. The highest doses of mineral fertilizers, N120P120K120, substantially affected the denitrification process and reduced the nitrogen fixation activity (in 1.9–2.2 times). The combination of manure with the medium fertilizers rate has also resulted in high denitrification levels, while the soil nitrogen fixation activity has restored only at flowering stage. Crop inoculation with A. brasilense combined with the manure application, has not affected studied processes. However, crop inoculation after the green manure intercropping has shown the growth of nitrogenase activity. Used on the mineral fertilizers background inoculation has activated nitrogen fixation and has ensured the decrease of denitrification levels, subject to the fertilization background. High fertilizer rates have hampered the inoculation efficiency. Inoculation has promoted crop yields on unfertilized and mineral backgrounds or following green manure. Crop inoculation following organic and the organo-mineral backgrounds had no significant effect, probably due to the competition for A. brasilense from microorganisms that have created a competitive environment for A. brasilense. Despite its environmental expediency, inoculation combined with the low fertilizer doses underperforms the action of inoculation combined with the medium fertilizer rates showing the latter as the compromise between the environmental requirements and crop productivity. The use of inoculation has promoted the accumulation of starch and ascorbic acid and has contributed to the reduction of nitrate contents in the tubers of inoculated plants.

scholarly journals Rhamnolipid Enhances the Nitrogen Fixation Activity of Azotobacter chroococcum by Influencing Lysine Succinylation

2021 ◽  
Vol 12 ◽  
Author(s):  
Jin Li ◽  
Hu Pan ◽  
Hui Yang ◽  
Chong Wang ◽  
Huhu Liu ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Crop Production ◽  
Nitrogenase Activity ◽  
Tricarboxylic Acid Cycle ◽  
Azotobacter Chroococcum ◽  
Mechanistic Study ◽  
Nitrogen Fixation Activity ◽  
Lysine Succinylation ◽  
Fixation Activity ◽  
Proteomic Approach

The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.

scholarly journals THE ORIENTATION OF BIOLOGICAL NITROGEN TRANSFORMATION PROCESSES IN SOIL UNDER THE ORGANIC PRODUCTION OF AGRICULTURAL PRODUCTS

2017 ◽  
Vol 25 ◽  
pp. 18-24
Author(s):  
V. V. Volkohon ◽  
A. M. Moskalenko ◽  
S. B. Dimova ◽  
M. A. Zhurba ◽  
K. I. Volkohon ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Field Experiments ◽  
Root Zone ◽  
Spring Barley ◽  
Organic Fertilizer ◽  
Nitrogen Transformation ◽  
Organic Production ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity ◽  
Biological Nitrogen

The paper covers the study of direct impact and after-effect of 40 t/ha of cattle manure on theorientation of nitrogen fixation and biological denitrification processes in the root zone of potatoes,spring barley, pea, and winter wheat plants in rotation in a stationary field experiments on leachedblack soil. Application of manure had significantly increased the nitrogen fixation activity, whilepromoting a high level of N2O emission. The use of microbial preparations for pre-seeding bacterization of seeds optimizes the course of biological nitrogen transformation process — through theenhancement of nitrogen fixation activity and reduction of gaseous nitrogen losses (with the exception of Biogran use on potatoes in the year of manure application). Introduction with manure of alarge number of microorganisms to the soil offsets the positive effect of biopreparations use. Yieldrecords and estimation of grain output per hectare within the crop rotation cycle indicates the practicability of combined application of manure and microbial preparations (excluding the year of direct effect of organic fertilizer) in organic agriculture.

scholarly journals Effect of the Nitrification Inhibitor 3,4-Dimethylpyrazole Phosphate on the Deep Placement of Nitrogen Fertilizers for Soybean Cultivation

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Soshi Hatano ◽  
Yoichi Fujita ◽  
Yoshifumi Nagumo ◽  
Norikuni Ohtake ◽  
Kuni Sueyoshi ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Absorption Rate ◽  
Nitrification Inhibitor ◽  
Nitrogen Fertilizers ◽  
Control Treatment ◽  
Nitrogen Absorption ◽  
Deep Placement ◽  
Urea Fertilizer ◽  
Nitrogen Fixation Activity ◽  
Fixation Activity

The deep placement of urea fertilizer (DMU) containing 1% (W/W) of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on soybean growth and seed yield was as effective as those of the coated urea (CU) and lime nitrogen (LN) in a field research. The average seed yields were high in LN (464 g·m−2) and DMU (461 g·m−2) and relatively low in CU (405 g·m−2), U (396 g·m−2), and Cont (373 g·m−2) treatments. The accumulations of dry matter and nitrogen in soybean shoots were higher in the plants with deep placement of CU, LN, and DMU than U and Cont. The daily nitrogen fixation activity and daily nitrogen absorption rate were calculated based on the relative ureide method. Both nitrogen fixation activity and nitrogen absorption rate were higher in DMU, CU, and LN compared with control treatment, suggesting that the deep placement of DMU did not repress nitrogen fixation. Soil incubation test was performed using the same field soil with DMU, U, LN, and urea with DMPP 1%, 2%, and 4%. DMU inhibits nitrification similar to the pattern of LN until 8 weeks. The increasing DMPP concentration did not markedly increase the nitrification inhibition. From these results, it was concluded that urea fertilizer with 1% DMPP is efficient for deep placement of N fertilizer for soybean cultivation due to its lower price compared with CU and LN.

Novel rhizobia exhibit superior nodulation and biological nitrogen fixation even under high nitrate concentrations

2019 ◽  
Vol 96 (2) ◽  
Author(s):  
Hien P Nguyen ◽  
Hiroki Miwa ◽  
Jennifer Obirih-Opareh ◽  
Takuya Suzaki ◽  
Michiko Yasuda ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Bradyrhizobium Japonicum ◽  
Root Nodules ◽  
Nitrogen Fertilizers ◽  
Nodule Formation ◽  
Nitrogen Fixing ◽  
Nitrogen Fixation Activity ◽  
Nitrate Concentrations ◽  
Nodulation Competitiveness ◽  
Biological Nitrogen

ABSTRACT Legume–rhizobium symbiosis leads to the formation of nitrogen-fixing root nodules. However, externally applied chemical nitrogen fertilizers (nitrate and ammonia) strongly inhibit nodule formation and nitrogen fixation. Here, we isolated several rhizobial strains exhibiting a superior nodulation and nitrogen fixation with soybean at high nitrate concentrations. The nodulation of soybean symbiont Bradyrhizobium diazoefficiens USDA110 was significantly inhibited at 12.5 mM nitrate; however, three isolates (NKS4, NKM2 and NKTG2) were capable of forming nitrogen-fixing nodules, even at 20 mM nitrate. These isolates exhibited higher nodulation competitiveness and induced larger nodules with higher nitrogen-fixation activity than USDA110 at 5 mM nitrate. Furthermore, these isolates induced more nodules than USDA110 even in nitrate-free conditions. These isolates had a distant lineage within the Bradyrhizobium genus; though they were relatively phylogenetically close to Bradyrhizobium japonicum, their morphological and growth characteristics were significantly different. Notably, in the presence of nitrate, expression of the soybean symbiosis-related genes (GmENOD40 and GmNIN) was significantly higher and expression of GmNIC1 that is involved in nitrate-dependent nodulation inhibition was lower in the roots inoculated with these isolates in contrast with inoculation of USDA110. These novel rhizobia serve as promising inoculants for soybeans cultivated in diverse agroecosystems, particularly on nitrate-applied soils.

scholarly journals Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes

2016 ◽  
Vol 82 (13) ◽  
pp. 3698-3710 ◽  
Author(s):  
Florence Mus ◽  
Matthew B. Crook ◽  
Kevin Garcia ◽  
Amaya Garcia Costas ◽  
Barney A. Geddes ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Synthetic Biology ◽  
Biological Nitrogen Fixation ◽  
Symbiotic Nitrogen Fixation ◽  
Crop Plants ◽  
Symbiotic Relationships ◽  
Mutualistic Relationship ◽  
Interest In Research ◽  
Biological Nitrogen ◽  
Developed World

ABSTRACTAccess to fixed or available forms of nitrogen limits the productivity of crop plants and thus food production. Nitrogenous fertilizer production currently represents a significant expense for the efficient growth of various crops in the developed world. There are significant potential gains to be had from reducing dependence on nitrogenous fertilizers in agriculture in the developed world and in developing countries, and there is significant interest in research on biological nitrogen fixation and prospects for increasing its importance in an agricultural setting. Biological nitrogen fixation is the conversion of atmospheric N2to NH3, a form that can be used by plants. However, the process is restricted to bacteria and archaea and does not occur in eukaryotes. Symbiotic nitrogen fixation is part of a mutualistic relationship in which plants provide a niche and fixed carbon to bacteria in exchange for fixed nitrogen. This process is restricted mainly to legumes in agricultural systems, and there is considerable interest in exploring whether similar symbioses can be developed in nonlegumes, which produce the bulk of human food. We are at a juncture at which the fundamental understanding of biological nitrogen fixation has matured to a level that we can think about engineering symbiotic relationships using synthetic biology approaches. This minireview highlights the fundamental advances in our understanding of biological nitrogen fixation in the context of a blueprint for expanding symbiotic nitrogen fixation to a greater diversity of crop plants through synthetic biology.

scholarly journals Potential of Phototrophic Purple Nonsulfur Bacteria to Fix Nitrogen in Rice Fields

2021 ◽  
Vol 10 (1) ◽  
pp. 28
Author(s):  
Isamu Maeda
Keyword(s):  
Nitrogen Fixation ◽  
Regulatory Networks ◽  
Nitrogenase Activity ◽  
Field Studies ◽  
Rice Fields ◽  
Purple Nonsulfur Bacteria ◽  
Available Nitrogen ◽  
Nitrogenase Activities ◽  
Plant Available Nitrogen ◽  
Biological Nitrogen

Biological nitrogen fixation catalyzed by Mo-nitrogenase of symbiotic diazotrophs has attracted interest because its potential to supply plant-available nitrogen offers an alternative way of using chemical fertilizers for sustainable agriculture. Phototrophic purple nonsulfur bacteria (PNSB) diazotrophically grow under light anaerobic conditions and can be isolated from photic and microaerobic zones of rice fields. Therefore, PNSB as asymbiotic diazotrophs contribute to nitrogen fixation in rice fields. An attempt to measure nitrogen in the oxidized surface layer of paddy soil estimates that approximately 6–8 kg N/ha/year might be accumulated by phototrophic microorganisms. Species of PNSB possess one of or both alternative nitrogenases, V-nitrogenase and Fe-nitrogenase, which are found in asymbiotic diazotrophs, in addition to Mo-nitrogenase. The regulatory networks control nitrogenase activity in response to ammonium, molecular oxygen, and light irradiation. Laboratory and field studies have revealed effectiveness of PNSB inoculation to rice cultures on increases of nitrogen gain, plant growth, and/or grain yield. In this review, properties of the nitrogenase isozymes and regulation of nitrogenase activities in PNSB are described, and research challenges and potential of PNSB inoculation to rice cultures are discussed from a viewpoint of their applications as nitrogen biofertilizer.

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