Regulation Of Nitrogenase Activity Research Articles

Is N‐feedback involved in the regulation of nitrogenase activity in Medicago truncatula?

AbstractTo determine whether senescing leaves provoke an active nitrogen (N) remobilization that results in the reduction of nitrogenase activity, 60% of Medicago truncatula lower leaves were either darkened or individually excised for two weeks. Although a considerable amount of N was remobilized, N2 fixation activity was found to be increased to maintain the N source/sink balance, indicating an absence of the negative N‐feedback regulation of nitrogenase activity in the senescing M. truncatula.

Role of acid phosphatase in the tolerance of the rhizobial symbiosis with legumes to phosphorus deficiency

Phosphorus (P) deficiency initiates a myriad of transcriptional, biochemical and physiological responses stimulating either the root’s extracellular abilities to acquire soil P in the rhizosphere or optimize its intracellular use efficiency and allocation through all plant organs. Enhancing activity of acid phosphatase (APase) to acquire and remobilize Pi from organic P compounds is one important strategy for improving plant P nutrition. The release of APase to the rhizosphere is a typical and almost universal P-starvation response in higher plants. However, relatively little is known about the functions of intracellular APase in legume nodules. The aim of this review was to track the enzyme activity along with the intra-nodular localization of APase, and its contribution in the rhizobial symbiosis tolerance to P-deficiency. Our findings have revealed that expression of APase and phytases genes and activities of the corresponding enzymes were positively correlated with increases both of the P use efficiency for N2 fixation and nodule O2 permeability in the rhizobial symbiosis with legumes. The induced enzyme activity and the marked transcripts localization of APase and phytase in nodule cortex would control nodule respiration and contribute to adaptation of nodulated legumes to low-P availability. Thus, the increase of APase and phytase activities in legume nodules supports a physiological role of these enzymes in the regulation of nitrogenase activity in connection with the nodule-P status, and opens up a new scenario for a better understanding of the regulation of N2 fixation in legumes.

Phylogeny of 16S rRNA and nifH genes and regulation of nitrogenase activity by oxygen and ammonium in the genus Paenibacillus

All Paenibacillus 16S rDNA sequences, except for that of Paenibacillus massiliensis T7, formed a coherent cluster, distinct from Gram-positive nitrogen-fixing Clostridium pasteurianum and Heliobacterium chlorum. All Paenibacillus NifH sequences formed two main clusters. Cluster I encompassing the NifH sequences from most of members of Paenibacillus spp., such as Paenibacillus azotofixans NifH1 and NifH2, Paenibacillus polymyxa and Paenibacillus macerans. Cluster II including only P. azotofixans NifH3. Curiously, three copies of nifH genes of Paenibacillus sabine T27 clustered within P. azotofixans cluster I (NifH1 and NifH2). The effect of O2 and ammonium on nitrogenase activity was studied with 14 different nitrogen-fixing Paenibacillus strains. The optimal oxygen concentration level for all Paenibacillus strains is in the 0 to 0.05% range, similar to that for Klebsiella pneumoniae. In all Paenibacillus strains, the highest nitrogenase activity is obtained in the condition of 0-0.1 mM NH4Cl and the increase of NH4CI from 0.1 to 5 mM caused a rapid inhibition of nitrogenase activity. However, the inhibition was reversible in the presence of 200 mM NH4Cl in some Paenibacillus strains. It is the first time to use almost all of the recognized nitrogen-fixing Paenibacilus spp. to investigate the phylogeny of 16S rRNA and nifH genes. The data that the inhibition of O2 and ammonium on nitrogenase acitivity will provide a base for studying the molecular regulatory mechanism of nitrogen fixation in the genus Paenibacillus.

Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to P(II) protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.

Ammonia-Induced Formation of an AmtB-GlnK Complex Is Not Sufficient for Nitrogenase Regulation in the Photosynthetic BacteriumRhodobacter capsulatus

A series of Rhodobacter capsulatus AmtB variants were created and assessed for effects on ammonia transport, formation of AmtB-GlnK complexes, and regulation of nitrogenase activity and NifH ADP-ribosylation. Confirming previous reports, H193 and H342 were essential for ammonia transport and the replacement of aspartate 185 with glutamate reduced ammonia transport. Several amino acid residues, F131, D334, and D335, predicted to be critical for AmtB activity, are shown here for the first time by mutational analysis to be essential for transport. Alterations of the C-terminal tail reduced methylamine transport, prevented AmtB-GlnK complex formation, and abolished nitrogenase switch-off and NifH ADP-ribosylation. On the other hand, D185E, with a reduced level of transport, was capable of forming an ammonium-induced complex with GlnK and regulating nitrogenase. This reinforces the notions that ammonia transport is not sufficient for nitrogenase regulation and that formation of an AmtB-GlnK complex is necessary for these processes. However, some transport-incompetent AmtB variants, i.e., F131A, H193A, and H342A, form ammonium-induced complexes with GlnK but fail to properly regulate nitrogenase. These results show that formation of an AmtB-GlnK complex is insufficient in itself for nitrogenase regulation and suggest that partial ammonia transport or occupation of the pore by ammonia is essential for this function.

Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1

In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is regulated by reversible ADP-ribosylation of dinitrogenase reductase in response to external so called “switch-off” effectors. Activation of the modified, inactive form is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the ADP-ribose moiety. This study addresses the signal transduction between external effectors and DRAG. R. rubrum, wild-type and P II mutant strains, were studied with respect to DRAG localization. We conclude that GlnJ clearly has an effect on the association of DRAG to the membrane in agreement with the effect on regulation of nitrogenase activity. Furthermore, we have generated a R. rubrum mutant lacking the putative ammonium transporter AmtB1 which was shown not to respond to “switch-off” effectors; no loss of nitrogenase activity and no ADP-ribosylation. Interestingly, DRAG was mainly localized to the cytosol in this mutant. Overall the results support our model in which association to the membrane is part of the mechanism regulating DRAG activity.

Regulation of nitrogenase activity in Rhodobacter capsulatus under dark microoxic conditions.

Rhodobacter capsulatus modulates its in vivo nitrogenase activity in the light in response to the addition of NH4+ in a variety of ways: with ADP-ribosylation of the Fe-protein of nitrogenase, with a switch-off response that is independent of ADP-ribosylation, and with a "magnitude response." In the light, these responses are differentially shown by cultures that differ in the degree of their nitrogen limitation. Here we examined the response of these culture types to the addition of NH4+ under dark, microoxic conditions and found that all three responses can be observed under these conditions. However, the magnitude response was much more sensitive to the ammonium concentration, and the ADP-ribosylation response correlated only poorly with activity changes, similar to results obtained in the light. In contrast to previous reports, Fe-protein was not ADP-ribosylated in response to the presence of oxygen.

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl.

Dinitrogenase reductase-activating glycohydrolase can be released from chromatophores of Rhodospirillum rubrum by treatment with MgGDP.

Dinitrogenase reductase-activating glycohydrolase (DRAG), involved in the regulation of nitrogenase activity in Rhodospirillum rubrum, is associated with chromatophore membranes in cell extracts. We show that DRAG can be specifically released by treatment with MgGDP; other nucleotides studied had no effect. The DRAG activity released corresponds to the release of DRAG protein.

Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio

Regulation of nitrogenase is not sufficiently understood to engineer symbioses that achieve a high N2 fixation rate under high levels of soil N. In the present hydroponic growth chamber study we evaluated the hypothesis that nitrogenase activity and the extent of its inhibition by NO3− may be related to both N and carbohydrate levels in plant tissues. A wide range of C:N ratios in various plant tissues (8.5 to 41.0, 1.9 to 3.7, and 0.8 to 1.8, respectively, in shoots, roots, and nodules) was generated through a combination of light and CO2 levels, using two soybean genotypes differing in C and N acquisition rates. For both genotypes, N concentration in shoots was negatively correlated to nitrogenase activity and positively correlated to the extent of nitrogenase inhibition by NO3−. Furthermore, nitrogenase activity was positively correlated to total nonstructural carbohydrates (TNC) and C:N ratio in shoot and nodules for both genotypes. Nitrogenase inhibition by NO3− was negatively correlated to TNC and C:N ratio in shoots, but not in nodules for both genotypes. At the onset of nitrogenase inhibition by NO3−, C:N ratio declined in shoots but not in nodules. These results indicate that both C and N levels in plant tissues are involved in regulation of nitrogenase activity. We suggest that the level of nitrogenase activity may be determined by (1) N needs (as determined by shoot C:N) and (2) availability of carbohydrates in nodules. Modulation of the nitrogenase activity may occur through sensing changes in plant N, i.e. changes in shoot C:N ratio, possibly through some phloem translocatable compound(s).

Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio

Regulation of nitrogenase activity in Bradyrhizobium japonicum/soybean symbiosis by plant N status as determined by shoot C:N ratio

The nitrogen-sink is involved in the regulation of nitrogenase activity in white clover after defoliation

The nitrogen-sink is involved in the regulation of nitrogenase activity in white clover after defoliation

The Role of Oxygen in the Regulation of Nitrogenase Activity in Drought-Stressed Soybean Nodules.

The aim of this study was to investigate the mechanism of nitrogenase inhibition in drought-stressed soybean (Glycine max L.) nodules to determine whether this stress was similar to other inhibitory treatments (e.g. detopping) known to cause an O2 limitation of nodule metabolism. Nodulated soybean plants were either detopped or subjected to mild, moderate, or severe drought stress by growth in different media and by withholding water for different periods. All treatments caused a decline in nitrogenase activity, and in the drought-stressed nodules, the decline was correlated with more negative nodule water potentials. Increases in rhizosphere O2 concentration stimulated nitrogenase activity much more in detopped plants than in drought-stressed plants, reflecting a greater degree of O2 limitation with the detopped treatment than with the drought-stressed treatment. These results indicated that drought stress differs from many other inhibitory treatments, such as detopping, in that its primary cause is not a decrease in nodule permeability and a greater O2 limitation of nodule metabolism. Rather, drought stress seems to cause a decrease in the maximum O2-sufficient rate of nodule respiration or nitrogenase activity, and the changes in nodule permeability reported to occur in drought-stressed nodules may be a response to elevated O2 concentrations in the infected cell that may occur as nodule respiration declines.

Nitrogenase activity in marine sediments from a temperate saltmarsh lagoon: modulation by complex polysaccharides, ammonium and oxygen

Nitrogenase activity (acetylene reduction) was examined in marine sediments associated with seagrass beds and thalassinidean prawn burrows, in a temperate saltmarsh lagoon. Nitrogenase activity in unamended sediments from different microhabitats ranged from undetectable values to ≈0.75 nmol C 2H 4 (g dry sediment) −1·hour −1. The bulk of organic material available to heterotrophic bacteria in saltmarsh lagoons is probably derived from primary production. Amendment of sediments with structural plant polysaccharides showed that xylan and alginate stimulated nitrogenase activity on average by 5 to 18-fold relative to unamended sediments, whereas cellulose and carrageenan were less effective. Amendment of sediments with storage plant polysaccharides produced the greatest stimulation of nitrogenase activity. Addition of laminarin and glycogen (amylopectin) significantly ( p <0.05) stimulated nitrogenase activity by 19 to 92-fold. In contrast to these polysaccharides of plant origin, chitin (a polymer of prawn exoskeletons) did not significantly enhance nitrogenase activity in these sediments. Aerobic conditions stimulated nitrogenase activity by 2 to 20-fold in surface sediments, but not in sub-surface sediments, indicating that aerobic or microaerophilic respiration was a significant source of energy for nitrogenase activity in surface sediments. Oxygen-stimulation of nitrogenase activity was less marked in aerobic sediments around the rim of prawn burrow openings where subsurface sediments had been displaced to the surface by bioturbation. Ammonium did not appear to play a major role in the regulation of nitrogenase activity in these sediments as in situ concentrations were ≈20-fold lower than the concentration (50 μM) which resulted in a slight (0–15%) reduction in nitrogenase activity.

The nitrogen‐sink is involved in the regulation of nitrogenase activity in white clover after defoliation

In an attempt to manipulate plant nitrogen‐sink strength, various defoliation treatments were applied to white clover (Trifolium repent L. cv. Ladino) grown in a controlled environment. Nitrogenase activity and its oxygen limitation were measured as H; evolution in Ar:O2 using a flow‐through gas exchange system. An experiment to monitor the response of nitrogenase activity to various degrees of defoliation showed that the removal of up to 50% of the leaf are a had no effect on nitrogenase activity within 6 h. If more than 50% of the leaf area was removed. the nitrogenase activity decreased in relation to the loss of leaf area. This was accompanied by a corresponding increase in the O2 limitation of nitrogenase activity. In the experiment to determine the N‐sink strength after defoliation, the dry weight increase w as initially unaffected by the removal of 4% of the leaf area, whereas removal of 85% or 100% of the leaf area resulted in a dry weight loss for several days. The time course of nitrogen assimilation was similar to that of dry weight increase. This study provides substantial evidence that after a severe defoliation the nitrogen demand is temporarily restricted due to the lack of dry weight increase. Since the plant's ability to store organic nitrogen is very limited. it seems plausible that nitrogen assimilation in the still fully intact symbiotic system had to be down‐regulated. Consequently, to avoid ammonia toxicity. nitrogenase activity had to be reduced. Such an interpretation is supported by the fact that complete defoliation of nitrogen‐Starved plants caused a much milder decrease in nitrogenase activity compared to the decrease in plants well supplied with nitrogen. The present data are consistent with the hypothesis that after defoliation nitrogenase activity is adjusted in response to the reduced demand for symbiotically fixed nitrogen (nitrogen‐sink strength), It is proposed that such an adjustment could he made by a nitrogen feedback mechanism that regulates a variable oxygen‐diffusion barrier in the nodules.

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

The relationship between nodule adenylates and the regulation of nitrogenase activity by O2 in soybean

Nodulated soybeans (Glycine max L. Merr, cv. Maple Arrow) were exposed to various physiological and environmental treatments to determine the relationship between nodule adenylate pools and the degree of O2 limitation of nitrogenase. Adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) and ATP/ADP ratios declined under conditions of decreased (10%) external pO2 but increased in nodules exposed to elevated (30%) external pO2. Nitrogenase activity was inhibited by both pO2 treatments, but recovered towards initial levels within 45 min. AEC also returned to initial levels during this period. To account for these and related data in the literature, it was hypothesized that 1) legume nodules regulate infected cell O2 concentration (Oi) to maintain adenylate pools at levels which limit respiratory metabolism: 2) treatments which decrease Oi alter the adenylate pools and further limit nodule metabolism; 3) treatments which increase Oi to levels in excess of a narrow range alter the adenylate pools and activate biochemical pathways which are not conducive to nitrogenase activity. In a preliminary test of these hypotheses, changes in AEC and ATP/ADP ratio were studied in nodules in which nitrogenase activity was inhibited by stem girdling, nitrate fertilization and exposure to an Ar:O2 atmosphere. All three treatments caused an increased O2 limitation of nodule respiration and nitrogenase activity. However, decreases in AEC were observed only in the stem girdling and nitrate fertilization treatment: Ar:O2 exposure had no effect on whole nodule AEC. While this result challenged the hypotheses suggesting a central role for adenylates in the regulation of O2‐limited metabolism, it was noted that the Ar:O2 treatment would differ from the other treatments in that it would have a specific effect on the ATP demands for NH3 assimilation in the plant fraction. Since AEC and ATP/ADP ratio would be affected by both the rate of ATP synthesis (potentially an O2‐limited process) and the demand for ATP, changes in these parameters in the whole nodule may not be a reliable indicator of adenylate‐mediated O2 limitation. Futher studies are needed to examine in vivo changes in adenylate pools in the plant and bacteroid fractions in nodules which vary in their degree of O2‐limited metabolism.

What triggers the regulation of nitrogenase activity in forage legume nodules after defoliation?

The dramatic decrease in nitrogenase activity after the defoliation of forage legumes has been recognized for a long time; however, the underlying mechanisms are not understood yet. The impact of current photosynthesis can be excluded. The precise role of carbohydrate availability is still unclear and remains to be established. From current knowledge we can conclude that, after defoliation, nitrogenase activity in legume nodules is down-regulated by a variable oxygen diffusion resistance. The triggering elements are not known; there is, however, increasing evidence that the plant's demand for symbiotically fixed nitrogen plays an important role. The possibility is here discussed that, after defoliation, a nitrogen feedback mechanism regulates nitrogenase activity through a variable oxygen diffusion resistance in the nodules.

Current Nitrogen Fixation Is Involved in the Regulation of Nitrogenase Activity in White Clover (Trifolium repens L.).

Previous studies have shown that nitrogenase activity decreases dramatically after defoliation, presumably because of an increase in the O2 diffusion resistance in the infected nodules. It is not known how this O2 diffusion resistance is regulated. The aim of this study was to test the hypothesis that current N2 fixation (ongoing flux of N2 through nitrogenase) is involved in the regulation of nitrogenase activity in white clover (Trifolium repens L. cv Ladino) nodules. We compared the nitrogenase activity of plants that were prevented from fixing N2 (by continuous exposure of their nodulated root system to an Ar:O2 [80:20] atmosphere) with that of plants allowed to fix N2 (those exposed to N2:O2, 80:20). Nitrogenase activity was determined as the amount of H2 evolved under Ar:O2. An open flow system was used. In experiment I, 6 h after complete defoliation and the continuous prevention of N2 fixation, nitrogenase activity was higher by a factor of 2 compared with that in plants allowed to fix N2 after leaf removal. This higher nitrogenase activity was associated with a lower O2 limitation (measured as the partial pressure of O2 required for highest nitrogenase activity). In experiment II, the nitrogenase activity of plants prevented from fixing N2 for 2 h before leaf removal showed no response to defoliation. The extent to which nitrogenase activity responded to defoliation was different in plants allowed to fix N2 and those that were prevented from doing so in both experiments. This leads to the conclusion that current N2 fixation is directly involved in the regulation of nitrogenase activity. It is suggested that an N feedback mechanism triggers such a response as a result of the loss of the plant's N sink strength after defoliation. This concept offers an alternative to other hypotheses (e.g. interruption of current photosynthesis, carbohydrate deprivation) that have been proposed to explain the immediate decrease in nitrogenase activity after defoliation.