nitrogenase and malic-acid

Two local hydrogen-evolving strains of purple nonsulfur bacteria have been isolated, characterized, and identified as Rhodopseudomonas sp. TUT (strains Rh1 and Rh2). Lactate followed by succinate and malate supported the highest amounts of H2 production, growth (O.D.660nm, proteins and bacteriochlorphyll contents), nitrogenase activity, and uptake hydrogenase; the least of which was acetate. Alginate-immobilized cells evolved higher hydrogen amounts than free cell counterparts. Rh1 was more productive than Rh2 at all circumstances. Lactate-dependent hydrogen evolution was more than twice that of acetate, due to ATP productivity (2/-1, respectively), which is limiting to the nitrogenase activity. The preference of lactate over other acids indicates the feasibility of using these two strains in hydrogen production from dairy wastewater.

Topics: Acetic Acid; Adenosine Triphosphate; Alginates; Bacteriochlorophylls; Cells, Immobilized; Energy Metabolism; Glucuronic Acid; Hexuronic Acids; Hydrogen; Hydrogenase; Kinetics; Lactic Acid; Malates; Nitrogenase; Photosynthesis; Rhodopseudomonas; Succinic Acid

Chickpea (Cicer arietinum) is an important pulse crop in many countries in the world. The symbioses between chickpea and Mesorhizobia, which fix N₂ inside the root nodules, are of particular importance for chickpea's productivity. With the aim of enhancing symbiotic efficiency in chickpea, we compared the symbiotic efficiency of C-15, Ch-191 and CP-36 strains of Mesorhizobium ciceri in association with the local elite chickpea cultivar 'Bivanij' as well as studied the mechanism underlying the improvement of N₂ fixation efficiency. Our data revealed that C-15 strain manifested the most efficient N₂ fixation in comparison with Ch-191 or CP-36. This finding was supported by higher plant productivity and expression levels of the nifHDK genes in C-15 nodules. Nodule specific activity was significantly higher in C-15 combination, partially as a result of higher electron allocation to N₂ versus H⁺. Interestingly, a striking difference in nodule carbon and nitrogen composition was observed. Sucrose cleavage enzymes displayed comparatively lower activity in nodules established by either Ch-191 or CP-36. Organic acid formation, particularly that of malate, was remarkably higher in nodules induced by C-15 strain. As a result, the best symbiotic efficiency observed with C-15-induced nodules was reflected in a higher concentration of the total and several major amino metabolites, namely asparagine, glutamine, glutamate and aspartate. Collectively, our findings demonstrated that the improved efficiency in chickpea symbiotic system, established with C-15, was associated with the enhanced capacity of organic acid formation and the activities of the key enzymes connected to the nodule carbon and nitrogen metabolism.

Topics: Biomass; Carbon; Cicer; Malates; Mesorhizobium; Models, Biological; Nitrogen; Nitrogen Fixation; Nitrogenase; Plant Roots; Plant Shoots; Root Nodules, Plant; Species Specificity; Sucrose; Symbiosis

Drought negatively impacts symbiotic nitrogen fixation (SNF) in Cicer arietinum L. (chickpea), thereby limiting yield potential. Understanding how drought affects chickpea nodulation will enable the development of strategies to biotechnologically engineer chickpea varieties with enhanced SNF under drought conditions. By analyzing carbon and nitrogen metabolism, we studied the mechanisms of physiological adjustment of nitrogen fixation in chickpea plants nodulated with Mesorhizobium ciceri during both drought stress and subsequent recovery. The nitrogenase activity, levels of several key carbon (in nodules) and nitrogen (in both nodules and leaves) metabolites and antioxidant compounds, as well as the activity of related nodule enzymes were examined in M. ciceri-inoculated chickpea plants under early drought stress and subsequent recovery. Results indicated that drought reduced nitrogenase activity, and that this was associated with a reduced expression of the nifK gene. Furthermore, drought stress promoted an accumulation of amino acids, mainly asparagine in nodules (but not in leaves), and caused a cell redox imbalance in nodules. An accumulation of organic acids, especially malate, in nodules, which coincided with the decline of nodulated root respiration, was also observed under drought stress. Taken together, our findings indicate that reduced nitrogenase activity occurring at early stages of drought stress involves, at least, the inhibition of respiration, nitrogen accumulation and an imbalance in cell redox status in nodules. The results of this study demonstrate the potential that the genetic engineering-based improvement of SNF efficiency could be applied to reduce the impact of drought on the productivity of chickpea, and perhaps other legume crops.

Topics: Carbon; Cell Respiration; Cicer; Droughts; Gene Expression Regulation, Plant; Malates; Mesorhizobium; Models, Biological; Nitrogen; Nitrogen Fixation; Nitrogenase; Oxidation-Reduction; Oxidative Stress; Plant Leaves; Plant Proteins; Plant Roots; Root Nodules, Plant; Symbiosis; Water

Unlike the most of α-alkoxy coordination in α-hydroxycarboxylates to vanadium, novel α-hydroxy coordination to vanadium(IV) has been observed for a series of chiral and achiral monomeric α-hydroxycarboxylato vanadyl complexes [VO(H2cit)(bpy)]·2H2O (1), [VO(Hmal)(bpy)]·H2O (2), [VO(H2cit)(phen)]·1.5H2O (3), [VO(Hmal)(phen)]·H2O (4), and [(Δ)VO(S-Hcitmal)(bpy)]·2H2O (5), [VO(H2cit)(phen)]2·6.5H2O (6), which were isolated from the reactions of vanadyl sulfate with α-hydroxycarboxylates and N-heterocycle ligands in acidic solution. The complexes feature a tridentate citrate, malate or citramalate that chelates to vanadium atom through their α-hydroxy, α-carboxy and β-carboxy groups; while the other β-carboxylic acidic group of citrate is free to participate strong hydrogen bonds with lattice water molecule. The neutral α-hydroxy group also forms strong intermolecular hydrogen bonds with water molecule and the negatively-charged α-carboxy group in the environment. The inclusion of a hydrogen ion in α-alkoxy group results in the formation of a series of neutral complexes with one less positive charge. There are two different configurations of citrate with respect to the trans-position of axial oxo group, where the complex with trans-hydroxy configuration seems more stable with less hindrance. The average bond distances of V-Ohydroxy and V-Oα-carboxy are 2.196 and 2.003Å respectively, which are comparable to the VO distance (2.15Å) of homocitrate in FeV-cofactor of V-nitrogenase. A new structural model is suggested for R-homocitrato iron vanadium cofactor as VFe7S9C(R-Hhomocit) (H4homocit=homocitric acid) with one more proton in homocitrate ligand.

Topics: Algal Proteins; Bacterial Proteins; Carboxylic Acids; Coordination Complexes; Crystallography, X-Ray; Hydrogen Bonding; Hydrogen-Ion Concentration; Ligands; Malates; Metalloproteins; Molecular Structure; Nitrogenase; Organometallic Compounds; Protons; Solutions; Static Electricity; Stereoisomerism

Reaction of sodium or potassium molybdate and excess malic acid in a wide range of pH values (pH 4.0-7.0) resulted in the isolation of two cis-dioxo-bis(malato)-Mo(VI) complexes, viz. Na(3)[MoO(2)H(S-mal)(2)] and K(3)[MoO(2)H(S-mal)(2)].H(2)O (H(3)mal=malic acid). The sodium complex is also characterized by an X-ray structure analysis, showing that the mononuclear Mo units are linked together via very strong symmetric CO(2)...H... O(2)C-hydrogen bond [2.432(5) A], forming a polymeric chain. The molybdenum atoms are quasi-octahedrally coordinated by two cis-oxo groups and two bidentate malate ligands via its alkoxy and alpha-carboxyl groups, while the beta-carboxylic and carboxylate groups remain uncomplexed, as the coordination of vicinal carboxylate and alkoxide of homocitrate in FeMo cofactor of nitrogenase. The absolute configuration of the metal center in this S-malato complex is assigned as Lambda and the homochirality within the chain is established as a homochiral form ...Lambda(S)-Lambda(S)-Lambda(S)-Lambda(S)... . It is proposed that the chiral configuration of the metal center in wild-type FeMo-co biosynthesis might be induced by the early coordination of the chiral R-homocitric acid, while a mixture of raceme might be obtained in the biosynthesis of NifV(-) FeMo-cofactor. The absolute configuration of wild-type FeMo-cofactor is assigned as Delta(R).

Topics: Crystallography, X-Ray; Magnetic Resonance Spectroscopy; Malates; Molybdenum; Molybdoferredoxin; Nitrogenase; Organometallic Compounds; Polymers; Spectroscopy, Fourier Transform Infrared; Stereoisomerism

Analysis of the content and distribution of organic acids in chickpea plants (Cicer arietinum L.) showed that malonate was the most abundant acid in roots and nodules, whereas malate was the main acid in leaves and stems. The highest concentration of malonate in roots was in the apices. Malonate metabolism did not appear to be directly related to abiotic stress. We suggest that malonate has a role as a defensive chemical in roots and nodules of chickpeas.

Topics: Fabaceae; Fumarates; Malates; Malonates; Nitrogenase; Plant Roots; Plant Shoots; Plants, Medicinal; Succinates

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Kinetics; Malates; Nitrogen Fixation; Nitrogenase; Oxidation-Reduction; Oxygen Consumption; Pisum sativum; Protons; Rhizobium leguminosarum; Uncoupling Agents

Rhodospirillum rubrum grown in continuous culture with glutamate as the sole fixed C and N source produced no nitrogenase, and the cultures were characterized by high extracellular ammonium concentrations. Addition of organic acids derepressed nitrogenase. Glutamate dehydrogenase, glutamine synthetase, glutamate synthase, malate dehydrogenase, nitrogenase, and ammonium were assayed before and after malate addition.

Topics: Ammonia; Enzyme Repression; Glutamate-Ammonia Ligase; Glutamates; Glutamic Acid; Kinetics; Malate Dehydrogenase; Malates; Nitrogen; Nitrogenase; Rhodospirillum rubrum