Nitrogen-fixing Bacterium Azospirillum Brasilense Research Articles

2nd Workshop in Microbial Molecular Biology

2nd Workshop in Microbial Molecular Biology

Vertical migration of nutrients and water-soluble organic matter in the soil profile under pre-sowing seed treatment with plant growth promoting rhizobacteria

Studies conducted in a stationary lysimeter experiment in the conditions of the washing water regime have shown that the use of PGPR for pre-sowing seed inoculation of agricultural crops reduces vertical migration of biogenic nutrients and water-soluble organic matter down the soil profile. The effect of seed inoculation with PGPR on the reduction of nutrient losses was not specific to the type of rhizobacteria and was similar for crops grown on different mineral fertilizers backgrounds (spring barley and winter rye seeds were inoculated with the nitrogen-fixing bacteria—Azospirillum brasilense 410 and A. brasilense 18-2, respectively, while maize seeds were inoculated with the phosphate-mobilizing Paenibacillus polymyxa KB). Seed inoculation has decreased nitrogen leaching down the soil profile by 4–9 kg/ha, phosphorus compounds—by 0.5–3.0 kg/ha, potassium—by 0.6–3.0 kg/ha, calcium—by 6–42 kg/ha, magnesium—by 3.0–6.0 kg/ha, water-soluble organic matter—by 0.8–8.0 kg/ha, subject to crop and norms of mineral fertilizers. Maize seeds inoculated with phosphorous-mobilizing P. polymyxa KB under crop cultivation on the cattle manure background did not affect the intensity of nutrient migration. On the other hand, the combination of green manure (narrow-leaved lupine as an intermediate crop) with pre-sowing seed inoculation had significantly reduced nutrient losses beyond the root zone soil layer. It is concluded that the use of PGPR in crop production on mineral and green manure backgrounds contributes to the preservation of soil fertility by limiting biogenic nutrients and water-soluble organic matter leaching with the water drainage down the soil profile. Pre-sowing seed inoculation had no significant effect on the vertical migration of nutrients in the soil on the background of cattle manure, due to the highly competitive environment created with the introduction of microorganisms from organic fertilizer, preventing the establishment of close interactions between PGPR and plants.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense.

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense.

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status.IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

Biochemical and Structural Characterization of l-2-Keto-3-deoxyarabinonate Dehydratase: A Unique Catalytic Mechanism in the Class I Aldolase Protein Superfamily.

l-2-Keto-3-deoxyarabinonate (l-KDA) dehydratase (AraD) catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde in the nonphosphorylative l-arabinose pathway from bacteria and belongs to the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein superfamily. All members of this superfamily, including several aldolases for l-KDA, share a common catalytic mechanism of retro-aldol fission, in which a lysine residue forms a Schiff base with the carbonyl C2 atom of the substrate, followed by proton abstraction of the substrate by a tyrosine residue as the base catalyst. Only AraD possesses a glutamine residue instead of this active site tyrosine, suggesting its involvement in catalysis. We herein determined the crystal structures of AraD from the nitrogen-fixing bacterium Azospirillum brasilense and AraD in complex with β-hydroxypyruvate and 2-oxobutyrate, two substrate analogues, at resolutions of 1.9, 1.6, and 2.2 Å, respectively. In both of the complexed structures, the ε-nitrogen of the conserved Lys171 was covalently linked to the carbonyl C2 atom of the ligand, which was consistent with the Schiff base intermediate form, similar to other DHDPS/NAL members. A site-directed mutagenic study revealed that Glu173 and Glu200 played important roles as base catalysts, whereas Gln143 was not absolutely essential. The abstraction of one of the C3 protons of the substrate (but not the O4 hydroxyl) by Glu173 was similar to that by the (conserved) tyrosine residues in the two DHDPS/NAL members that produce α-ketoglutaric semialdehyde (d-5-keto-4-deoxygalactarate dehydratase and Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase), indicating that these enzymes evolved convergently despite similarities in the overall reaction.

Ефективність використання асоціативних діазотрофів для підвищення урожайності тритикале ярого

Goal. To study the effectiveness of the use of associative Azospirillum brasilense diazotrophs 10/1 to increase the yield and improve grain quality of spring triticale. Methods. Microbiology, gas chromatography, field experiment, and statistical. The objects of study — plants of spring triticale of varieties Oberig Kharkivskii, the strain of associative nitrogen-fixing bacteria Azospirillum brasilense 10/1. Field experiments were carried out in conditions of Ukrainian Polissia (Institute of Agricultural Microbiology and Agroindustrial Manufacture, NAAS, Chernihiv). The inoculation of seeds of triticale was carried out with 3-days culture of bacteria A. brasilense 10/1 for 2 h before seeding at the rate of 200–300 thousand bacterial cells per seed. Processing of experimental data was performed using the computer program Microsoft Office Excel. Results. It is established that the strain A. brasilense 10/1 positively influenced the growth and development of spring triticale due to improved nitrogen nutrition of plants and the ability to produce growth-stimulation substances. Use of the strain A. brasilense 10/1 for preplant bacterization of seeds triticale provided the increase in crop yield by 0.5 t/ha, or of 16.39% compared to control. Positive influence is revealed on a mass of grains in one ear and weight of 1000 grains. These indicators increased in the variant with inoculation on average on 16.8 and 11.9%, respectively. Also by inoculation of spring triticale with a promising strain of A. brasilense 10/1 the protein content in the grain increased to 14.06%, wet gluten – to the 35, 17, full-scale mass of grain increased by 7.3%. Conclusions. Taking into account the positive effect of associative nitrogen-fixing bacteria A. brasilense 10/1 on the growth and development of triticale plants, perspective variant is the creation of a biological product based on the strain for increasing the yield and improving the quality of the resulting products.

The NADP-dependent malic enzyme MaeB is a central metabolic hub controlled by the acetyl-CoA to CoASH ratio

Malic enzymes participate in key metabolic processes, the MaeB-like malic enzymes carry a catalytic inactive phosphotransacetylase domain whose function remains elusive. Here we show that acetyl-CoA directly binds and inhibits MaeB-like enzymes with a saturable profile under physiological relevant acetyl-CoA concentrations. A MaeB-like enzyme from the nitrogen-fixing bacterium Azospirillum brasilense, namely AbMaeB1, binds both acetyl-CoA and unesterified CoASH in a way that inhibition of AbMaeB1 by acetyl-CoA is relieved by increasing CoASH concentrations. Hence, AbMaeB1 senses the acetyl-CoA/CoASH ratio. We revisited E. coli MaeB regulation to determine the inhibitory constant for acetyl-CoA. Our data support that the phosphotransacetylase domain of MaeB-like enzymes senses acetyl-CoA to dictate the fate of carbon distribution at the phosphoenol-pyruvate / pyruvate / oxaloacetate metabolic node.

Responses of Bruguiera sexangula propagules to beneficial microbes in the nursery

Bruguiera sexangula (Lour) Poir., a threatened mangrove tree, was inoculated with beneficial microbes in a nursery to assess any improvements in growth and biomass. From soil samples from the rhizosphere of B. sexangula in a mangrove forest in Panangadu of Kerala India, nitrogen-fixing bacteria Azotobacter chroococcum and Azospirillum brasilense were isolated. The phosphate-solubilising bacterium Bacillus megaterium and potassium-mobilizing bacteria Frateruria aurantia were also isolated and cultured on suitable media. Later, ripe propagules of B. sexangula were collected from matured trees and raised in sterilized soil bags (13 × 25 cm) containing sterilized soil and sand (2:1 ratio). The cultured beneficial microbes were propagated and used to inoculate the ripe propagules of B. sexangula and maintained in the nursery for 6 months. After 6 months, growth and biomass of the inoculated propagules were greater than for the uninoculated control propagules. Shoot length, number of leaves, stem girth and root length were also significantly greater than in the controls. This study showed that the mangrove-specific beneficial microbes influenced the growth of B. sexangula in the nursery and will help in the establishment of B. sexangula in degraded mangrove forests.

АДИТИВНИЙ ПРОЯВ БІОЦЕНОТИЧНИХ ЗВЯЗКІВ У СИСТЕМІ «МІКРООРГАНІЗМИ РИЗОСФЕРИ ҐРУНТУ-РОСЛИНИ ТРИБИ TRITICEAЕ»

The purpose and objectives of the study. In terms of Polissia, Polissia-Forest-steppe and Forest-steppe ecotopes therewere found out the the possibility of enhance relationships and achievements of biocenotical additive effect in the case of introduction of complex breeding phosphate mobilizing and nitrogen-fixing microorganisms (by means of inoculation) in system "rhizosphere microorganisms- plants tribe Triticeae."Methods. Field, laboratory, statistical methods and method of the ecosystem approach.Results. Sensitivity cultivars of triticale, wheat, rye winter in pre-sowing seed inoculation of nitrogen-fixing bacteria Azospirillum brasilense (diazobacteryn) and Agrobacterium radiobacter 204 (diazofit) and phosphate mobilizing microorganisms of Achromobacter album 1122 (albobacteryn) and Bacillus polymyxa KB (polimiksobacteryn) in a Polissia, Polissia-Forest-steppe and Forest-steppe ecotopes. It was found that the sensitivity of wheat genotypes under conditions of mild winter Polissia ecotypes depends on belonging to particular genotype specific ecotypes. There were established that genotype of wheat and triticale Л 3946/96, Chaian (P = 0.95) respond to polimiksobacteryn effect only in terms Polissia and Polissia-Forest-steppe ecotopes. All tested genotypes of winter triticale and winter rye sensitive to microbial agents as Polissia, Polissia-Forest-steppe and Forest-steppe ecotopes. The impact of bio-agents microbial preparations causes additive effect, which is the fuller disclosure of the tribe Triticeae plant capacity and increase their productivity by improving plant nutrition necessary nutrients (nitrogen, phosphorus).Conclusions. In conditions of Polissia, Forest-Steppe and Polissia-Forest-Steppe ecotopes – there were established the possibility of amplification relations and achieving of biocenotic additive effect in the case of the introduction of comprehensive and phosphate mobilizing there have been accumulated nitrogen-fixing microorganisms (by inoculation) in the "micro rhizosphere soil-plant tribe Triticeaе». It was found that the additive effect is to increase the productivity phytocenoses representatives of the tribe Triticeaе.

Structure of the polysaccharides from the lipopolysaccharide of Azospirillum brasilense Jm125A2

Two polysaccharides were obtained by mild acid degradation of the lipopolysaccharide of associative nitrogen-fixing bacteria Azospirillum brasilense Jm125A2 isolated from the rhizosphere of a pearl millet. The following structures of the polysaccharides were established by sugar and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy: [Display omitted] Structure 1 has been reported earlier for a polysaccharide from A. brasilense S17 (Fedonenko YP, Konnova ON, Zdorovenko EL, Konnova SA, Zatonsky GV, Shaskov AS, Ignatov VV, Knirel YA. Carbohydr Res 2008;343:810–6), whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.

THE INFLUENCE OF BRADYRHIZOBIUM JAPONICUM AND AZOSPIRILLUM BRASILENSE ON THE VERTICAL MIGRATION OF NUTRIENTS AT SOYBEAN CULTIVATION

The paper presents the research results of pre-sowing seeds bacterization with nitrogen fixing bacteria Bradyrhizobium japonicum and Azospirillum brasilense influence on the loss of moisture, water soluble humus and nutrients. It was shown that seeds bacterization reduces the leaching intensity of nitrates, phosphorus, potassium, calcium and magnesium. Moreover, the least losses of nutrients were observed in a variants with joint application of both studied nitrogen fixing microorganisms. It was noted that seeds bacterization with B. japonicum and A. brasilense had promoted increase of chlorophyll content in the leaves of soybean plants. The highest yield was observed in the variant with the joint use of rhizobia and azospirillum.

Immunochemical detection of Azospirilla in soil with genus-specific antibodies

Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

Signal effects of the lectin from the associative nitrogen-fixing bacterium Azospirillum brasilense Sp7 in bacterial–plant root interactions

Azospirillum brasilense, which has the potential to stimulate plant growth, belongs to the group of plant growth-promoting bacteria. The lectin found on the surface of A. brasilense strain Sp7 has the ability to bind specific carbohydrates and ensures adhesion of the bacteria to the root surface. The aim of this work was to investigate possible inductive effects of the Sp7 lectin on the plant cell signal systems. Enzyme-linked immunosorbent assay, spectrophotometry, and thin-layer and gas–liquid chromatography were used to determine the content of signal intermediates in the cells of wheat root seedlings. Laser scanning confocal microscopy was used to examine the localization of fluorescently labeled lectin on the plant cell. The Sp7 lectin acted on the signal system components in wheat seedling roots by regulating the contents of cAMP, nitric oxide, diacylglycerol, and salicylic acid, as well as by modifying the activities of superoxide dismutase and lipoxygenase. The revealed cell membrane localization of the lectin is of deciding importance for its signal function. The results of the study suggest that the A. brasilense Sp7 lectin acts as a signal molecule involved in the interaction of growth-promoting rhizobacteria with plant roots.

Comparative assessment of inductive effects of Azospirillum lectins with different antigenic properties on the signal systems of wheat seedling roots

The lectins of associative nitrogen-fixing bacteria Azospirillum brasilense Sp7 and its mutant A. brasilense Sp7.2.3 were shown to have different effects on the components of the wheat seedling root signal system, namely to regulate the levels of cAMP, nitric oxide, diacylglycerol, and salicylic acid, as well as to induce the activities of superoxide dismutase and lipoxygenase. Our results make it possible to consider azospirilla lectins as inducers of the signal systems in wheat seedling roots, since they cause development of several flows of primary signals. These data are of general biological importance, since lectins are present in all living organisms and most ot the functions of lectins remain insufficiently understood.

Gold(III) Reduction by the Rhizobacterium Azospirillum brasilense with the Formation of Gold Nanoparticles

For the soil nitrogen-fixing bacterium Azospirillum brasilense, the ability to reduce [AuCl4](-) and to form gold nanoparticles (GNPs) has been demonstrated, with the appearance of a mauve tint of the culture. To validate the shapes and chemical nature of nanoparticles, transmission electron microscopy (TEM) and X-ray fluorescence analysis were used. For the widely studied agriculturally important wild-type strains A. brasilense Sp7 and Sp245, GNPs formed after 10days of incubation of cell biomass with 0.25mM [AuCl4](-) were shown (using TEM) to be mainly of spherical form (5 to 20nm in diameter), with rare occasional triangles. In the course of cultivation with [AuCl4](-), after 5days, a mauve tint was already visible for cells of strain Sp245.5, after 6days for Sp245 and after 10days for Sp7. Thus, for the mutant strain Sp245.5 (which has significant differences in the structure and composition of cell-surface polysaccharides as compared with Sp245), a more rapid formation of GNPs was observed. Moreover, their TEM images (also obtained after 10days) showed different shapes: nano-sized spheres, triangles, hexagons and rods, as well as larger round-shaped flower-like nanoparticles about 100nm in size. Since by the time of GNP formation in our experiments the cells were found to be already not viable, this confirms the dominating role of cell surface structure and chemical composition in shaping the GNPs formed in the course of [AuCl4](-) reduction to Au(0). This finding may be useful for understanding the natural biogeochemical mechanisms of gold reduction and formation of GNPs involving microorganisms. The data obtained may also help in developing protocols for environmentally friendly synthesis of nanoparticles and possible use of bacterial cells with modified surface structure and composition for their fabrication.

Biosynthesis of gold nanoparticles by Azospirillum brasilense

Plant-associated nitrogen-fixing soil bacteria Azospirillum brasilense were shown to reduce the gold of chloroauric acid to elemental gold, resulting in formation of gold nanoparicles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au+3 (HAuCl4) to Au(0). Transmission electron microscopy revealed accumulation of colloidal gold nanoparticles of diverse shape in the culture liquid of A. brasilense strains Sp245 and Sp7. The size of the electron-dense nanospheres was 5 to 50 nm, and the size of nanoprisms varied from 5 to 300 nm. The tentative mechanism responsible for formation of gold nanoparticles is discussed.

Agro-process intensification: soilborne micro-bioreactors with nitrogen fixing bacterium Azospirillum brasilense as self-sustaining biofertiliser source for enhanced nitrogen uptake by plants

A new application of agro-process intensification is described for nitrogen fixation by Azospirillum brasilense supported within the pores of sulphonated-neutralised polyHIPE polymers (PHPs) which are highly hydrophilic, elastic, crosslinked and ionic with nano-structured pore walls. These bioactive macroscopic polymer particles, when used as soil additives act as micro-bioreactors within the soil and facilitate the interactions between plant roots, root exudates, water, nutrients and bacteria (reactive components), because plant roots penetrate into these micro-bioreactors which simultaneously absorb water and nutrients while generating biofertiliser through the nitrogen fixing bacteria within them. Hence, these soil additives act as synthetic rhizosphere (SRS). In greenhouse experiments, it is shown that the presence of the bioactive SRS at 0.5 wt% level in the soil without any fertiliser addition increases the dry grass shoots by 9.6%, 9.5%, 40% and 145% after 3, 6, 9 and 12 weeks of growth, respectively, compared to grass grown with no SRS or bacteria. Progressive yield enhancement with Azospirillum brasilense supported on PHPs is due to reduction of soil nutrients thus switching nitrogen fixing bacterium from consumption to production of nitrogen. The environmental impact and sustainability of SRS media are also considered and compared with other soil additives: super absorbent polymers and biochar.

Изучение влияния лектинов азоспирилл на образование перекиси водорода в корнях проростков пшеницы

We show that the lectins isolated from the surface of the nitrogenfixing soil bacterium Azospirillum brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3., can regulate the production of hydrogen peroxide in wheat seedling roots, which is associated with the activation of superoxide dismutase, peroxidase and oxalate oxidase, as well as with the inhibition of catalase activity. We show that activation of oxalate oxidase is the most rapidly inducible pathway for the formation of hydrogen peroxide in wheat seedling roots under the effect of lectins. The obtained data indicate that the Azospirillum lectins can act as inducers of adaptation processes in wheat seedling roots.

Synthesis of Two Tetrasaccharides Related to the O-Antigen from Azospirillum brasilense S17 and Azospirillum lipoferum SR65

Synthesis of two isomeric tetrasaccharides, β-D-Glup-(1→2)-α-L-Rhap-(1→3)-α-L- Rhap-(1→2)-α-L-Rhap (I) and β-D-Glup-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap (II), the repeating units from the lipopolysaccharides of the nitrogen-fixing bacterium Azospirillum brasilense S17 and Azospirillum lipoferum SR65, was achieved via assembly of the building blocks 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl trichloroacetimidate (2), p-methoxyphenyl 3,4-di-O-benzoyl-α-L-rhamnopyranoside (3), 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-α-L-rhamnopyranosyl trichloroacetimidate (6), 2,3,4,6-tetra-O-benzoyl-β-D-glucopyranosyl trichloroacetimidate (8), and p-methoxy phenyl 2,4-di-O-benzoyl-α-L-rhamnopyranoside (14). Condensation of 3 with 6 or 8 provided the disaccharides 9 or 11, respectively. Deallyloxycarbonylation of 11 gave the disaccharide aceptor 12, while removal of the p-methoxyphenyl group in 9 followed by trichloroacetimidation of the anomeric hydroxyl group afforded the disaccharide donor 10. Meanwhile, disaccharide donor 16 and acceptor 18 were prepared from 6, 8, and 14 similarly. Finally, condensation of 10 with 12 or 16 with 18, followed by deprotection, gave the target tetrasaccharides I or II, respectively.

Effect of lectins from Azospirillum brasilense to peroxidase and oxalate oxidase activity regulation in wheat roots

Lectins were extracted from the surface of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 and from its mutant A. brasilense Sp7.2.3 defective in lectin activity. The ability oflectins to stimulate the rapid formation of hydrogen peroxide related to increase of oxalate oxidase and peroxidase activity in the roots of wheat seedlings has been demonstrated. The most rapid induced pathway of hydrogen peroxide formation in the roots of wheat seedlings was the oxalic acid oxidation by oxalate oxidase which is the effect oflectin in under 10 min in a concentration of 10 microg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.