Nitrogenase Activity Of Azospirillum Brasilense Research Articles

The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillumbrasilense.

The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillumbrasilense requires dinitrogenase reductase ADP-ribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A.brasilense DraG invitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex invitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZΔ42-54). However, although the GlnZΔ42-54 variant was more effective in inhibiting DraG invitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either invitro or invivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG.

English

The association of Cymbopogon winterianus (Citronella), a member of the Poaceae family, with the N2-fixing bacteria, Azospirillum was examined to evaluate possible benefits for agriculture. The nodules on citronella were induced using plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 ppm in nitrogen free Hoagland’s solution. Concentration of 2,4-D greater than 1.0 ppm caused stunning and death, while concentration of 2,4-D less than 1.0 ppm has no obvious effect on seedling growth. The presence of nitrogenase activity (acetylene reduction assay) and leghemoglobin (hemoprotein found in the nitrogen-fixing root nodules) was noticed in the plants treated with Azospirillum either alone or with nodulated plants, but higher activity was noticed in nodulated Azospirillum treated plants. These results confirmed the nitrogenase activity of Azospirillum brasilense in association with 2,4-D induced nodules of citronella. After transplanting to pots, it was noticed that nodulated and Azospirillum - treated plants showed higher chlorophyll content in leaf, enhanced nitrate reductase (NR) activity, leading to higher yield as compared to the control plants (non-nodulated). The nodulated plants treated with Azospirillum also had higher physiological activities as compared to plants treated only with Azospirillum. The results provide evidence that the plants belonging to the family Poaceae are potentially able to create a symbiosis with diazotropic bacteria, which colonize para-nodule tissue intracellularly, promoting a higher level of nitrogen (N2) fixation leading to better growth and plant development.   Key words: Cymbopogon, nodulation, 2,4-D, Azospirillum brasilense, nitrogen fixation, oil yield.

Effect of the over-expression of PII and PZ proteins on the nitrogenase activity of Azospirillum brasilense

The Azospirillum brasilense PII and PZ proteins, encoded by the glnB and glnZ genes respectively, are intracellular transducers of nitrogen levels with distinct functions. The PII protein participates in nif regulation by controlling the activity of the transcriptional regulator NifA. PII is also involved in transducing the prevailing nitrogen levels to the Fe-protein ADP-ribosylation system. PZ regulates negatively ammonium transport and is involved in nitrogenase reactivation. To further investigate the role of PII and PZ in the regulation of nitrogen fixation, broad-host-range plasmids capable of over-expressing the glnB and glnZ genes under control of the ptac promoter were constructed and introduced into A. brasilense. The nitrogenase activity and nitrate-dependent growth was impaired in A. brasilense cells over-expressing the PII protein. Using immunoblot analysis we observed that the reduction of nitrogenase activity in cells over-expressing PII was due to partial ADP-ribosylation of the Fe-protein under derepressing conditions and a reduction in the amount of Fe-protein. These results support the hypothesis that the unmodified PII protein act as a signal to the DraT enzyme to ADP-ribosylate the Fe-protein in response to ammonium shock, and that it also inhibits nif gene expression. In cells over-expressing the PZ protein the nitrogenase reactivation after an ammonium shock was delayed indicating that the PZ protein is involved in regulation of DraG activity.

Nodule-like Structures Induced on the Roots of Maize Seedlings by the Addition of Synthetic Auxin 2,4-D and its Effects on Growth and Yield

Induction of para-nodules on maize contributed to increase in yield. The nodules on maize (Zea mays L.) cv. Kiran were induced using plant growth regulator 2,4-D (lppm) in nitrogen free Hoagland solution. Concentrations of 2,4-D greater than lppm caused stunting and death, while concentrations less than lppm has no obvious effect on seedling growth. The associative symbiosis (using Azospirillum) was noticed in the nodulated and non-nodulated maize plants. The presence of nitrogenase activity (acetylene reduction assay) and leghemoglobin was noticed in the plants treated with Azospirillum either alone or with nodulated plants, but higher activity was noticed in the nodulated Azospirillum treated plants. The 2,4-D induced nodule like structures provided niche for the establishment of the bacteria, thereby enhancing nitrogen fixation in the nodulated roots. These results confirmed the nitrogenase activity of Azospirillum brasilense in association with the 2,4-D induced nodules of maize. After transplanting in the pots, it was noticed that nodulated and Azospirillum treated plants showed higher chlorophyll content in leaf, enhanced NR activity leading to higher yield as compared to controlled plants (non-nodulated). The nodulated plants treated with Azospirillum had higher physiological activities as compared to only Azospirillum treated plants. Thus, the gramineous plants are potentially able to create a symbiosis with diazotropic bacteria, which colonize para-nodule tissue intracellularly, promoting a higher level of N2 fixation for the better growth and development of the plants.

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.

Posttranslational regulatory system for nitrogenase activity in Azospirillum spp

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.

Plant, Microbial and Soil Factors, Determining Nitrogen Fixation in the Rhizosphere

AbstractA genotype effect on associative (rhizosphere) N2‐fixation was observed with two cultivars of Sorghum bicolor (nutans) with a maximum rate of 8 μmol C2H4 · h−1 · plant−1 in one genotype compared to 0.9 μmol in the other. Characteristics of the high fixing genotype were a reduced transpiration rate, a lower number of stomata and increased root exudate production per gram root dry weight with higher concentration of dicarboxylic acids. The bacterial rhizosphere composition revealed a three times higher number of N2‐fixing bacteria, a tenfold reduction of actinomycetes and a threefold reduction of Arthrobacter associated with the high fixing cultivar compared to the low fixing genotype. From these and other plant rhizospheres two new nitrogen fixing bacteria, Pseudomonas stutzeri and Erwinia herbicola, were characterized. With the N2‐fixing bacteria Azospirillum brasilense and Klebsiella pneumoniae an enhancement of specific nitrogenase activity by aromatic compounds, for example phenolics, the herbicide alachlor and the insecticide carbofuran was demonstrated. An oscillating nitrogenase activity in Azospirillum brasilense under microaerobic conditions was found, resulting from an encystation and deencystation under those conditions. Experiments with wheat roots demonstrated that reduced oxygen tensions, essential for a maximum rhizosphere N2‐fixation, reduced root growth significantly and altered the N‐metabolism of the roots.

Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp.

Ammonium chloride (greater than or equal to 0.05 mM) effectively and reversibly inhibited the nitrogenase activity of Azospirillum brasilense, Azospirillum lipoferum and Azospirillum amazonense. The glutamine synthetase inhibitor L-methionine-DL- sulfoximine abolished this "switch-off" in A. lipoferum and A. brasilense, but not in A. amazonense. Azaserine, an inhibitor of glutamate synthase, inhibited nitrogenase activity itself. This provides further evidence for glutamine as a metabolite of regulatory importance in the NH4+ switch-off phenomenon. In A. brasilense and A. lipoferum, a transition period before the complete inhibition of nitrogenase activity after the addition of 1 mM ammonium chloride was observed. The in vitro nitrogenase activity also was decreased after treatment with ammonium. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a second dinitrogenase reductase (Fe protein) subunit appeared, which migrated in coincidence with the modified subunit of the inactive Fe protein of the nitrogenase of Rhodospirillum rubrum. After the addition of ammonium 32P was incorporated into this subunit of the Fe protein of A. brasilense. In A. amazonense, the inhibition of nitrogenase activity by ammonium was only partial, and no transition period could be observed. The in vitro nitrogenase activity of ammonium-treated cells was not decreased, and no evidence for a modified Fe protein subunit was found. Nitrogenase extracts of A. amazonense were active and had an Fe protein that migrated as a close double band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense

Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense

Organic acid utilization, succinate excretion, encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium.

Induction of Nitrogenase Activity in Azospirillum brasilense by Conditioned Medium from Cell Suspension Cultures of Pennisetum americanum (Pearl Millet) and Panicum maximum (Guinea Grass)

Summary Nitrogenase activity ( = acetylene reduction activity) was observed in Azospirillum brasilense cultures grown on conditioned medium obtained from embryogenic pearl millet ( Pennisetum americanum ) and Guinea grass ( Panicum maximum ) cell suspension cultures, but no bacterial growth or activity was observed on unconditioned medium. A peak of maximum activity was obtained with medium harvested mid-way through a 13 d culture period. Non-embryogenic suspension cultures of pearl millet and Guinea grass supported little or no bacterial growth or nitrogenase activity.

The dependence of hydrogen recycling upon nitrogenase activity in Azospirillum brasilense Sp. 7

The dependence of hydrogen recycling upon nitrogenase activity in Azospirillum brasilense Sp. 7