Nitrogen Fixation In Rhizobia Research Articles

Effectiveness of nitrogen fixation in rhizobia.

SummaryBiological nitrogen fixation in rhizobia occurs primarily in root or stem nodules and is induced by the bacteria present in legume plants. This symbiotic process has fascinated researchers for over a century, and the positive effects of legumes on soils and their food and feed value have been recognized for thousands of years. Symbiotic nitrogen fixation uses solar energy to reduce the inert N2 gas to ammonia at normal temperature and pressure, and is thus today, especially, important for sustainable food production. Increased productivity through improved effectiveness of the process is seen as a major research and development goal. The interaction between rhizobia and their legume hosts has thus been dissected at agronomic, plant physiological, microbiological and molecular levels to produce ample information about processes involved, but identification of major bottlenecks regarding efficiency of nitrogen fixation has proven to be complex. We review processes and results that contributed to the current understanding of this fascinating system, with focus on effectiveness of nitrogen fixation in rhizobia.

Oxygen regulatory mechanisms of nitrogen fixation in rhizobia.

Rhizobia are α- and β-proteobacteria that form a symbiotic partnership with legumes, fixing atmospheric dinitrogen to ammonia and providing it to the plant. Oxygen regulation is key in this symbiosis. Fixation is performed by an oxygen-intolerant nitrogenase enzyme but requires respiration to meet its high energy demands. To satisfy these opposing constraints the symbiotic partners cooperate intimately, employing a variety of mechanisms to regulate and respond to oxygen concentration. During symbiosis rhizobia undergo significant changes in gene expression to differentiate into nitrogen-fixing bacteroids. Legumes host these bacteroids in specialized root organs called nodules. These generate a near-anoxic environment using an oxygen diffusion barrier, oxygen-binding leghemoglobin and control of mitochondria localization. Rhizobia sense oxygen using multiple interconnected systems which enable a finely-tuned response to the wide range of oxygen concentrations they experience when transitioning from soil to nodules. The oxygen-sensing FixL-FixJ and hybrid FixL-FxkR two-component systems activate at relatively high oxygen concentration and regulate fixK transcription. FixK activates the fixNOQP and fixGHIS operons producing a high-affinity terminal oxidase required for bacterial respiration in the microaerobic nodule. Additionally or alternatively, some rhizobia regulate expression of these operons by FnrN, an FNR-like oxygen-sensing protein. The final stage of symbiotic establishment is activated by the NifA protein, regulated by oxygen at both the transcriptional and protein level. A cross-species comparison of these systems highlights differences in their roles and interconnections but reveals common regulatory patterns and themes. Future work is needed to establish the complete regulon of these systems and identify other regulatory signals.

Genesis of Antibiotic Resistance (AR) XXXVII: Fracking fluid and produced water induced Alteration of commensal microbial genome exacerbate AR pathogen (ARP), consequently Antibiotic Resistance Pandemic (ARP).

“Produced Water” is defined as all water that is returned to the surface through a good borehole made up of water injected during fracture stimulation process as well as natural formation water. Flow back Process allows the well to flow back excess fluids and sand. Once sand and fluid have been removed, gas and/or petroleum liquids begin to flow (the purpose). Flow back process equipment is designed to handle heavy solids so permanent equipment is put in place when the process is complete. Actual duration of the process varies from well to well and play to play. Therefore, all “flow back water (FW) is “produced water” (PW) (FW/PW).FW/PW is more of a concern with respect to drinking water than fracking fluids because flow back water has organic compounds from the formation, as well as heavy metals. Prefracturing fluids, drilling mud, FW/PW and impoundment water has been shown to induce an alteration in the microbial morphology colonizing shale gas reservoirs (reservoir souring, plugging, equipment corrosion, and a decrease in hydrocarbon production volume and quality). As a result of the selection pressure (halotolerant anaerobic microorganisms) leading to Clostridia, a class that includes spore‐forming microorganisms becomes dominant. 250ppm Cu2 in FW/PW has been shown to have a toxic effect on commensal microbial genome while the molecular mechanism remains elusive. Effluent samples collected from facilities treating oil and gas FW/PW were known to contain nickel 8 – 22mg. Metal accumulation due to FW/PW contaminated soil has been shown to enhance antibiotic resistance through co‐selection pressure plausibly via mobile genetic elements in vivo. Heavy metal contamination of soil has been shown to impart a selection pressure on antibiotic resistance in the commensal microbes. An analysis on the spectrum of soil antibiotic resistance genes (ARGs) following 4–5‐year nickel exposure (0–800 mg kg−1) in two long‐term experimental sites showed that a total of 149 unique ARGs were detected, with multidrug and β‐lactam resistance as the most prevailing ARG types. Increase in frequency and abundance of ARGs has been shown to correspond an increase along the gradient of increasing nickel concentrations, with the highest values recorded in the treatments amended with 400 mg nickel kg−1 soil. Correlation of the increase in nickel concentration coinciding with an abundance of the mobile genetic elements (MGEs) along with ARGs, has been implicated in the soil contamination by FW/PW/PM enriched in heavy metal (Nickel). Taken together, FF‐PW‐PM has mutagenic potential in altering the genome of commensal microbes imparting selection pressure for antibiotic resistance. It is suggested that contamination of the water table, agricultural produce, and human exposure to the commensal microbes with an altered ARG's would likely to exacerbate AR thus potentiate ARP. Based on the detailed chemical analysis of produced water/wastewater originating from hydraulic fracking indicates that such epigenetic factors are hypothesized to alter the pattern of methylation/demethylation of a multitude of ARG in soil microbiome. Increased hydraulic fracturing across the globe plausibly alters the soil microbiome (Rhizobium‐Nitrogen fixation in legumes) impinging on microbes that are essential in nitrogen cycle directly entering the food chain thus affecting the “Human Gut Resistome”.Support or Funding InformationSupported by the Professional Development Funds by SWTJC to Subburaj Kannan.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantitative modelling of legume root nodule primordium induction by a diffusive signal of epidermal origin that inhibits auxin efflux.

BackgroundRhizobium nitrogen fixation in legumes takes place in specialized organs called root nodules. The initiation of these symbiotic organs has two important components. First, symbiotic rhizobium bacteria are recognized at the epidermis through specific bacterially secreted lipo-chitooligosaccharides (LCOs). Second, signaling processes culminate in the formation of a local auxin maximum marking the site of cell divisions. Both processes are spatially separated. This separation is most pronounced in legumes forming indeterminate nodules, such as model organism Medicago truncatula, in which the nodule primordium is formed from pericycle to most inner cortical cell layers.ResultsWe used computer simulations of a simplified root of a legume that can form indeterminate nodules. A diffusive signal that inhibits auxin transport is produced in the epidermis, the site of rhizobium contact. In our model, all cells have the same response characteristics to the diffusive signal. Nevertheless, we observed the fastest and strongest auxin accumulation in the pericycle and inner cortex. The location of these auxin maxima correlates with the first dividing cells of future nodule primordia in M. truncatula. The model also predicts a transient reduction of the vascular auxin concentration rootward of the induction site as is experimentally observed. We use our model to investigate how competition for the vascular auxin source could contribute to the regulation of nodule number and spacing.ConclusionOur simulations show that the diffusive signal may invoke the strongest auxin accumulation response in the inner root layers, although the signal itself is strongest close to its production site.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0935-9) contains supplementary material, which is available to authorized users.

Metabolic Reconstruction and Modeling of Nitrogen Fixation in Rhizobium etli

Rhizobiaceas are bacteria that fix nitrogen during symbiosis with plants. This symbiotic relationship is crucial for the nitrogen cycle, and understanding symbiotic mechanisms is a scientific challenge with direct applications in agronomy and plant development. Rhizobium etli is a bacteria which provides legumes with ammonia (among other chemical compounds), thereby stimulating plant growth. A genome-scale approach, integrating the biochemical information available for R. etli, constitutes an important step toward understanding the symbiotic relationship and its possible improvement. In this work we present a genome-scale metabolic reconstruction (iOR363) for R. etli CFN42, which includes 387 metabolic and transport reactions across 26 metabolic pathways. This model was used to analyze the physiological capabilities of R. etli during stages of nitrogen fixation. To study the physiological capacities in silico, an objective function was formulated to simulate symbiotic nitrogen fixation. Flux balance analysis (FBA) was performed, and the predicted active metabolic pathways agreed qualitatively with experimental observations. In addition, predictions for the effects of gene deletions during nitrogen fixation in Rhizobia in silico also agreed with reported experimental data. Overall, we present some evidence supporting that FBA of the reconstructed metabolic network for R. etli provides results that are in agreement with physiological observations. Thus, as for other organisms, the reconstructed genome-scale metabolic network provides an important framework which allows us to compare model predictions with experimental measurements and eventually generate hypotheses on ways to improve nitrogen fixation.

Genetic regulation of nitrogen fixation in rhizobia.

This review presents a comparison between the complex genetic regulatory networks that control nitrogen fixation in three representative rhizobial species, Rhizobium meliloti, Bradyrhizobium japonicum, and Azorhizobium caulinodans. Transcription of nitrogen fixation genes (nif and fix genes) in these bacteria is induced primarily by low-oxygen conditions. Low-oxygen sensing and transmission of this signal to the level of nif and fix gene expression involve at least five regulatory proteins, FixL, FixJ, FixK, NifA, and RpoN (sigma 54). The characteristic features of these proteins and their functions within species-specific regulatory pathways are described. Oxygen interferes with the activities of two transcriptional activators, FixJ and NifA. FixJ activity is modulated via phosphorylation-dephosphorylation by the cognate sensor hemoprotein FixL. In addition to the oxygen responsiveness of the NifA protein, synthesis of NifA is oxygen regulated at the level of transcription. This type of control includes FixLJ in R. meliloti and FixLJ-FixK in A. caulinodans or is brought about by autoregulation in B. japonicum. NifA, in concert with sigma 54 RNA polymerase, activates transcription from -24/-12-type promoters associated with nif and fix genes and additional genes that are not directly involved in nitrogen fixation. The FixK proteins constitute a subgroup of the Crp-Fnr family of bacterial regulators. Although the involvement of FixLJ and FixK in nifA regulation is remarkably different in the three rhizobial species discussed here, they constitute a regulatory cascade that uniformly controls the expression of genes (fixNOQP) encoding a distinct cytochrome oxidase complex probably required for bacterial respiration under low-oxygen conditions. In B. japonicum, the FixLJ-FixK cascade also controls genes for nitrate respiration and for one of two sigma 54 proteins.

Genetic regulation of nitrogen fixation in rhizobia.

This review presents a comparison between the complex genetic regulatory networks that control nitrogen fixation in three representative rhizobial species, Rhizobium meliloti, Bradyrhizobium japonicum, and Azorhizobium caulinodans. Transcription of nitrogen fixation genes (nif and fix genes) in these bacteria is induced primarily by low-oxygen conditions. Low-oxygen sensing and transmission of this signal to the level of nif and fix gene expression involve at least five regulatory proteins, FixL, FixJ, FixK, NifA, and RpoN (sigma 54). The characteristic features of these proteins and their functions within species-specific regulatory pathways are described. Oxygen interferes with the activities of two transcriptional activators, FixJ and NifA. FixJ activity is modulated via phosphorylation-dephosphorylation by the cognate sensor hemoprotein FixL. In addition to the oxygen responsiveness of the NifA protein, synthesis of NifA is oxygen regulated at the level of transcription. This type of control includes FixLJ in R. meliloti and FixLJ-FixK in A. caulinodans or is brought about by autoregulation in B. japonicum. NifA, in concert with sigma 54 RNA polymerase, activates transcription from -24/-12-type promoters associated with nif and fix genes and additional genes that are not directly involved in nitrogen fixation. The FixK proteins constitute a subgroup of the Crp-Fnr family of bacterial regulators. Although the involvement of FixLJ and FixK in nifA regulation is remarkably different in the three rhizobial species discussed here, they constitute a regulatory cascade that uniformly controls the expression of genes (fixNOQP) encoding a distinct cytochrome oxidase complex probably required for bacterial respiration under low-oxygen conditions. In B. japonicum, the FixLJ-FixK cascade also controls genes for nitrate respiration and for one of two sigma 54 proteins.

Stability of plasmids in Rhizobium phaseoli during culture

Genes governing symbiotic nitrogen fixation in rhizobia reside on symbiotic (Sym) plasmids. Additional plasmids with unknown functions (“cryptic” plasmids) usually accompany Sym plasmids in rhizobial cells. These cryptic plasmids are assumed to have beneficial roles in the soil environment. Little is known about the maintenance of cryptic plasmids during culture in the laboratory. We have examined the stabilities of both Sym and cryptic plasmids of three Rhizobium phaseoli strains cultured for 30, 60, 90 and 120 generations. Individual plasmids were labeled with Tn5-Mob, which codes for kanamycin resistance, to provide an initial means of detecting plasmid loss. Agarose gel-electrophoresis and hybridization with Tn5 probe were then used to identify alterations or loss of resistance. Kanamycin-sensitive isolates were infrequent and were due to either losses of TN5-Mob-labeled plasmids or deletions which included Tn5-Mob DNA. The three Sym plasmids and eight cryptic plasmids studied were highly stable.

Response of Rhizobium leguminosarum isolates to different forms of inorganic nitrogen during nodule development in pea ( Pisum sativum L.)

Combined nitrogen inhibits nodule development and nitrogen fixation in Rhizobium -legume symbioses. Isolates of R. leguminosarum which had been shown to vary in symbiotic effectiveness in the presence of NN 4NO 3 were used as inoculum for peas grown with NH 4NO 3 KNO 3 or NH 4Cl and harvested between 14 and 24 days after planting. Plants grown in 2m m NH 4NO 3 showed inhibition of nodule development and function 17 days after seeding but there were no significant differences (SDs) between isolates of high and low effectiveness. In the presence of 5 m M NH 4NO 3 nodule development and C 2H 2 reduction were severely inhibited from 14 days onwards. Plants inoculated with isolates of high effectiveness had more tap-root nodules, higher C 2H 2 reduction rates and higher leghemoglobin content than those inoculated with isolates of low effectiveness. In plants grown with 5 m m KNO 3 or 5 m m NN 4Cl, NO 3 − had the major inhibitory effect on nodulation and nodule development, but NH 4 + began to show inhibitory effects on nodule development after 17 days. There were no SDs between isolates of low and high effectiveness at these concentrations of NO 3 − and NH 4 +. Differences in symbiotic response of R. leguminosarum to combined nitrogen are small and appear during nodule development rather than at nodule initiation.

Hydrogen oxidation and nitrogen fixation in rhizobia, with special attention focused on strain ORS 571.

In this survey we describe the influence of hydrogen oxidation on the physiology of Rhizobium ORS 571. The presence of hydrogen is required for the synthesis of hydrogenase. Carbon substrates do not repress the synthesis of hydrogenase. The respiratory system contains cytrochromes of the b- and c-type. Cytochrome alpha 600 is present after growth at high oxygen tensions. The nature of the terminal oxidases functioning at low oxygen tensions has not been established yet----H+/O values with endogenous substrates are between 6 and 7. The results show the presence of two phosphorylation sites: site 1 (ATP/2e = 1.0) and site 2(ATP/2e = 1.33). By measuring molar growth yields it has been demonstrated that carbon-limited, nitrogen-fixing cultures obtain additional ATP from hydrogen oxidation, and that site 2 of oxidative phosphorylation is passed during hydrogen oxidation. A method is described to calculate ATP/N2 values (the total amount of ATP used by nitrogenase during the fixation of 1 mol N2) and H2/N2 ratios (mol hydrogen formed per mol N2 fixed) in aerobic organisms. For Rhizobium ORS 571 the ATP/N2 value is about 40 and the H2/N2 ratio is between 5 and 7.5. Cells obtained from oxygen-limited nitrogen-fixing cultures contain 30-40% poly-beta-hydroxybutyrate, which explains the high molar growth yields found. Hydrogen has not been detected in the effluent gas of these cultures, which may point to reoxidation of the hydrogen formed at nitrogen fixation. Calculations show that the effect of hydrogen reoxidation on the efficiency of nitrogen fixation (g N fixed X mol-1 substrate converted) is not very large and that the actual H2/N2 ratio is of much more importance. After addition of hydrogen to succinate-limited, ammonia-assimilating cultures, an initial increase of the Ysuccinate value (g dry wt X mol-1 succinate) is followed by a gradual decrease. This is accompanied by a large decrease of the YO2 value, and an increased permeability of the cytoplasmic membrane to protons. The results may be explained by a transition of the culture from an energy-limited state to a carbon-limited state.

Hydrogen oxidation and efficiency of nitrogen fixation in succinate-limited chemostat cultures ofRhizobium ORS 571

Rhizobium ORS 571, isolated from stem nodules of the tropical legumeSesbania rostrata is able to grow in the chemostat with molecular nitrogen as sole nitrogen source at a specific growth rate of 0.1 h-1. Samples from nitrogenfixing cultures showed high acetylene reduction activities: 1,500 nmol ethylene formed per milligram dry weight per hour. Under nitrogen-fixing conditions an uptake hydrogenase is induced. Ammonia-assimilating cultures, without additional hydrogen, did not induce hydrogenase. The addition of hydrogen to succinate-limited nitrogen-fixing cultures resulted in an increase in the molar growth yield on succinate (Ysuccinate) from 27 to 35 and a slight decrease in the molar growth yield on oxygen (\(Y_{O_2 }\)), showing that hydrogen oxidation is less energy-yielding than the oxidation of endogenous substrates. Respiration-driven proton translocation measured with starved cells indicated the functioning of site 1 and 2 of oxidative phosphorylation. Cytochrome spectra showed that cytochromea600, present at high dissolved oxygen tension (d.o.t.) almost completely disappeared at low d.o.t. In flash-photolysis spectra only thea-type cytochrome could be detected as an oxidase in cells both grown at high and low d.o.t. Growth yields in ammonia-assimilating cultures were higher than those measured in nitrogen-fixing cultures. Assuming two sites of oxidative phosphorylation, a molar growth yield on ATP (YATP) of about 3 and 6 was calculated for respecticely nitrogen-fixing and ammonia-assimilating cultures. TheYATP under nitrogen-fixing conditions is dependent on the amount of H2 formed per mol N2 fixed (H2/N2 ratio). A method has been described to calculate the total amount of ATP use by nitrogenase during the fixation of 1 mol N2 (ATP/N2 ratio) and H2/N2 ratios in aerobic nitrogen fixing organisms. This calculation yielded that nitrogen fixation inRhizobium ORS 571 is a high ATP-consuming process. The calculated ATP/N2 and H2/N2 ratios were respectively 42 and 7.5.

Bacterial plasmids of agricultural and environmental importance

Many important characteristics of bacteria are determined by relatively small genetic elements known as bacterial plasmids. Such agriculturally and environmentally important bacterial characteristics as multiple antibiotic resistance in animal pathogens, nodulation and nitrogen fixation in Rhizobium—plant symbioses, crown gall formation by plant pathogenic Agrobacterium species and pesticide degradation by members of the genus Alcaligenes have been shown to be carried as part of transmissible plasmids. The widespread use of antibiotics in human and veterinary medicine and agriculture has resulted in the evolution and spread of multiple drug resistance plasmids. The problem of drug resistance might be overcome by reduction or pronhibition of the use of clinically important antibiotics for non-therapeutic purposes such as growth promotion or prophylaxis. An understanding of the role of plasmid-borne nitrogen fixation/nodulation characteristics of Rhizobium species may provide an alternative to the use of very expensive nitrogenous fertilizers. Studies of the plant pathogenic Agrobacterium have led to successful biological control of crown gall. Finally, the manipulation of pesticide degrading plasmids has already demonstrated their potential for reducing environmental pollution resulting from the widespread use of complex synthetic molecules in both industry and agriculture.

Glutamine synthetase and control of nitrogen fixation in Rhizobium.

Rhizobia have recently been shown to fix nitrogen while living free1,2,3,4. Unfortunately, free-living fixation occurs only at the end of exponential growth, and there is no condition in which bacterial growth is dependent on reduction of dinitrogen by nitrogenase (EC 1.7.99.2). In the unrelated Klebsiella pneumoniae where fixation does occur in culture, nitrogenase expression is regulated by the enzyme glutamine synthetase (GS) (EC 6.3.1.2). Mutants lacking GS activty (glnˉ) fail to induce measurable nitrogenase activity5,6.

Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O 2 tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O 2 carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.