The nature of the interaction Azospirillum-Arabidopsis determine the molecular and morphological changes in root and plant growth promotion.

Plant growth promoting rhizobacteria influence host functional and adaptive traits via complex mechanisms that are just started to be clarified. Azospirillum brasilense acts as a probiotic bacterium, but detailed information about its molecular mechanisms of phytostimulation is scarce. Three interaction systems were established to analyze the impact of A. brasilense Sp245 on the phenotype of Arabidopsis seedlings, and underlying molecular responses were assessed under the following growth conditions: (1) direct contact of roots with the bacterium, (2) chemical communication via diffusible compounds produced by the bacterium, (3) signaling via volatiles. A. brasilense Sp245 improved shoot and root biomass and lateral root production in the three interaction systems assayed. Cell division, quiescent center, and differentiation protein reporters pCYCB1;1::GUS, WOX5::GFP, and pAtEXP7::GUS had a variable expression in roots depending of the nature of interaction. pCYCB1;1::GUS and WOX5::GFP increased with volatile compounds, whereas pAtEXP7::GUS expression was enhanced towards the root tip in plants with direct contact with the bacterium. The auxin reporter DR5::GUS was highly expressed with diffusible and volatile compounds, and accordingly, auxin signaling mutants pin3, slr1, arf7arf19, and tir1afb2afb3 showed differential phytostimulant responses when compared with the wild type. By contrast, ethylene signaling was not determinant to mediate root changes in response to the different interactions, as observed using the ethylene-related mutants etr1, ein2, and ein3. Our data highlight the diverse effects by which A. brasilense Sp245 improves plant growth and root architectural traits and define a critical role of auxin but not ethylene in mediating root response to bacterization.

Azospirillum brasilense Sp245 lipopolysaccharides induce target of rapamycin signaling and growth in Arabidopsis thaliana.

The Target of Rapamycin (TOR) protein kinase plays a pivotal role in metabolism and gene expression, which enables cell proliferation, growth and development. Lipopolysaccharides (LPS) are a class of complex glycolipids present in the cell surface of Gram-negative bacteria and mediate plant-bacteria interactions. In this study, we examined whether LPS from Azospirillum brasilense Sp245 affect Arabidopsis thaliana growth via a mechanism involving TOR. A. thaliana plants were treated with LPS and plant growth and development were analyzed in mature plants. Morphological and molecular changes as well as TOR expression and activity were analyzed in root tissues. LPS increased total fresh weight, root length and TOR::GUS expression in the root meristem. Phosphorylation of S6k protein, a downstream target of TOR, increased following LPS treatment, which correlated with increased or decreased expression of CycB1;1::GUS protein upon treatment with LPS or TOR inhibitor AZD-8055, respectively. Long term LPS treatment further increased the rosette size as well as the number of stems and siliques per plant, indicating an overall phytostimulant effect for these signaling molecules. Taken together, the results suggest that A. brasilense LPS play probiotic roles in plants influencing TOR-mediated processes.

Crystal structure of bacterial L-arabinose 1-dehydrogenase in complex with L-arabinose and NADP.

L-Arabinose 1-dehydrogenase (AraDH) is responsible for the first step of the non-phosphorylative L-arabinose pathway from bacteria, and catalyzes the NAD(P)+-dependent oxidation of L-arabinose to L-arabinonolactone. This enzyme belongs to the so-called Gfo/Idh/MocA protein superfamily, but has a very poor phylogenetic relationship with other functional members. We previously reported the crystal structures of AraDH without a ligand and in complex with NADP+. To clarify the underlying catalytic mechanisms in more detail, we herein elucidated the crystal structure in complex with L-arabinose and NADP+. In addition to the previously reported five amino acid residues (Lys91, Glu147, His153, Asp169, and Asn173), His119, Trp152, and Trp231 interacted with L-arabinose, which were not found in substrate recognition by other Gfo/Idh/MocA members. Structure-based site-directed mutagenic analyses suggested that Asn173 plays an important role in catalysis, whereas Trp152, Trp231, and His119 contribute to substrate binding. The preference of NADP+ over NAD+ was significantly subjected by a pair of Ser37 and Arg38, whose manners were similar to other Gfo/Idh/MocA members.

Flagellin of polar flagellum from Azospirillum brasilense Sp245: Isolation, structure, and biological activity.

Glycosylated flagellin of the polar flagellum of the plant growth-promoting rhizobacteria Azospirillum brasilense Sp245 was for the first time isolated and characterized by biochemical and bioinformatics methods. Using the amino acid sequence taken from the NCBI database of bacterial whole-genome DNA sequencing, the secondary and tertiary structures of the protein part of this glycoprotein were determined by template-based molecular modeling. With the use of a set of predictors, regions of its intrinsic structural disorder were identified, and binding sites of carbohydrate fragments to the surface of the molecule were determined. A positive effect of the polar flagellum flagellin on the root meristem of wheat seedlings was for the first time revealed for associative bacteria. The effect was manifested in an increase in the division rate of plant cells - a significant increase in the mitotic index. Thus, the induction of specific responses of plants to their interactions with flagellin of the associative bacteria may probably be considered as a demonstration of its elicitor properties.

Biochemical and molecular characterization of arsenic response from Azospirillum brasilense Cd, a bacterial strain used as plant inoculant.

Azospirillum brasilense Cd is a bacterial strain widely used as an inoculant of several crops due to its plant growth promoting properties. However, its beneficial effects depend on its viability and functionality under adverse environmental conditions, including the presence of arsenic (As) in agricultural soils. Therefore, the aim of this work was to evaluate the response of A. brasilense Cd to arsenate (AsV) and arsenite (AsIII). This bacterium was tolerant to As concentrations frequently found in soils. Moreover, properties related to roots colonization (motility, biofilm, and exopolymers) and plant growth promotion (auxin, siderophore production, and N2 fixation) were not significantly affected by the metalloid. In order to deepen the understanding on As responses of A. brasilense Cd, As resistance genes were sequenced and characterized for the first time in this work. These genes could mediate the redox As transformation and its extrusion outside the cell, so they could have direct association with the As tolerance observed. In addition, its As oxidation/reduction capacity could contribute to change the AsV/AsIII ratio in the environment. In conclusion, the results allowed to elucidate the As response of A. brasilense Cd and generate interest for its potential use in polluted environments.

Structure elucidation and gene cluster characterization of the O-antigen of Vibrio cholerae O14.

The O-polysaccharide (O-antigen) of Vibrio cholerae O14 was studied using chemical analyses and 1D and 2D NMR spectroscopy. The following structure of the repeating unit of the O-antigen was established: where GlcpN(SHb) indicates 2-deoxy-2-[(S)-3-hydroxybutanoylamino]-d-glucose. We found that Vibrio cholerae O14 is similar to that of O-polysaccharide of Azospirillum brasilense S17, which has been reported earlier. Moreover, we predicted functions of all the genes in the O-antigen gene cluster according to the structure established. Our study enriches the existing O-antigen database of Vibrio cholerae, and further facilitates the bacterial serotype identification.

Azospirillum brasilense inoculation counteracts the induction of nitrate uptake in maize plants.

Nitrogen (N) represents one of the limiting factors for crop growth and productivity and to date has been widely supplied via external application of fertilizers. However, the use of plant growth-promoting rhizobacteria (PGPR) might represent a valuable tool to further improve plant nutrition. This study examines the influence of Azospirillum brasilense strain Cd on nitrate uptake in maize (Zea mays) plants, focusing on the high-affinity transport system (HATS). Plants were induced with nitrate (500 µM) and either inoculated or not with Azospirillum. Inoculation decreased the nitrate uptake rate in induced plants, suggesting that Azospirillum may negatively affect HATS in the short term. The expression dynamics of ZmNF-YA and ZmLBD37 suggested that Azospirillum affected the N balance in the plants, most probably by supplying them with reduced N, i.e. NH4+. This was further corroborated by measurements of total N and the expression of ammonium transporter genes. Overall, our data demonstrate that Azospirillum can counteract the plant response to nitrate induction, albeit without compromising N nutrition. This suggests that the agricultural application of microbial inoculants requires fine-tuning of external fertilizer inputs.

Structural studies of O-specific polysaccharide(s) and biological activity toward plants of the lipopolysaccharide from Azospirillum brasilense SR8.

Lipopolysaccharide (LPS) was extracted from dry bacterial cells of plant-growth-promoting bacterium Azospirillum brasilense SR8 (IBPPM 5). The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis, 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments, computational NMR-based structure analysis, and Smith degradation. The OPS was shown to contain two types of repeating units of the following structure: Both OPS structures are present in A. brasilense 54, from which structure 1 has been reported earlier (Fedonenko et al., 2011), whereas to our knowledge structure 2 has not been hitherto found in bacterial saccharides. Treatment of wheat seedling roots with LPS of A. brasilense SR8 increased the number of root hair deformations as compared to seedlings grown without LPS, but had no effect on adsorption of the bacteria to the root surface. A. brasilense SR8 was able to utilize LPS of several structurally related Azospirillum strains.

Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside.

Total chemical synthesis of the linear tetrasaccharide repeating unit ß-D-Glc-(1 → 2)-α-L-Rha-(1 → 3)-α-L-Rha-(1 → 2)-α-L-Rha-CH2CH2NH2 of the O-antigen from Azospirillum brasilense Jm125A2 is accomplished through rational protecting group manipulations of commercially available monosaccharides and stereoselective glycosylations. The target tetrasaccharide in the form of its 2-aminoethyl glycoside is obtained in ∼24% yield over 10 steps following a linear strategy. The structure is particularly suitable for further glycoconjugate formation through the terminal free amine without hampering the reducing end stereochemistry.

Application of a Janus aglycon with dual function in benzyl-free synthesis of spacer-armed oligosaccharide fragments of polysaccharides from rhizobacterium Azospirillum brasilense sp7.

Both protective and pre-spacer features of 4-(2-chloroethoxy)phenyl (CEP) aglycon, which belong to the class of Janus aglycons, were engaged in a benzyl-free synthesis of oligosaccharide fragments of polysaccharides from rhizobacterium Azospirillum brasilense sp7. Introduction of α-1,4-linked L-fucose residue was performed using 3,4-di-O-benzoyl-2-O-triisopropylsilyl-α-L-fucopyranosyl N-phenyltrifluoroacetimidate in excellent stereoselectivity and high yields. The obtained deprotected di-, tri- and tetrasaccharides contain 4-(2-azidoethoxy)phenyl (AEP) spacer aglycon, which allows straightforward preparation of neoglycoconjugates that will be used for the study of the role of lipopolysaccharide of rhizobacterium A. brasilense sp7 in plant-microbe symbiosis. The intermediate protected oligosaccharide building blocks with cleavable CEP/AEP aglycons have a strong potential for further application in the synthesis of more complex oligosaccharides.

Early plant growth and biochemical responses induced by Azospirillum brasilense Sp245 lipopolysaccharides in wheat (Triticum aestivum L.) seedlings are attenuated by procyanidin B2.

This study analyzes the effects of procyanidin B2 on early wheat plant growth and plant biochemical responses promoted by lipopolysaccharides (LPS) derived from the rhizobacteria Azospirillum brasilense Sp245. Measurements of leaf, root length, fresh weight, and dry weight showed in vitro plant growth stimulation 4 days after treatment with A. brasilense as well as LPS. Superoxide anion (O2·-) and hydrogen peroxide (H2O2) levels increased in seedling roots treated with LPS (100 µg mL-1). The chlorophyll content in leaf decreased while the starch content increased 24 h after treatment in seedling roots. The LPS treatment induced a high increase in total peroxidase (POX) (EC 1.11.1.7) activity and ionically bound cell wall POX content in roots, when compared to respective controls. Early plant growth and biochemical responses observed in wheat seedlings treated with LPS were inhibited by the addition of procyanidin B2 (5 µg mL-1), a B type proanthocyanidin (PAC), plant-derived polyphenolic compound with binding properties of LPS. All results suggest first that the ionically bound cell wall POX enzymes could be a molecular target of A. brasilense LPS, and second that the recognition or association of LPS by plant cells is required to activate plant responses. This last event could play a critical role during plant growth regulation by A. brasilense LPS.

[A STUDY OF THE ISOLATED BACTERIOPHAGE ΦAB-SP7 ADSORPTION ON THE CELL SURFACE OF THE AZOSPIRILLUM BRASILENSE SP7].

The bacteriophage ΦAb-Sp7 was isolated from the cells of the Azospirillum brasilense Sp7. The morphology, size of the gram-negative colonies, and range of lytic activity against other strains and species of the genus Azospirillum was tested. The isolated phage DNA was examined using electrophoretic and restriction analysis, and the size of the genome were established. The electron microscopy. resuIts show that the phage (capsid) has a strand-like form. The electron microscopy study of the bacteriophage ΦAb-Sp7 adsorption on the A. brasilense Sp7 bacterial surface was performed.

Evidence for ferritin as dominant iron-bearing species in the rhizobacterium Azospirillum brasilense Sp7 provided by low-temperature/in-field Mössbauer spectroscopy.

For the ubiquitous diazotrophic rhizobacterium Azospirillum brasilense, which has been attracting the attention of researchers worldwide for the last 35 years owing to its significant agrobiotechnological and phytostimulating potential, the data on iron acquisition and its chemical speciation in cells are scarce. In this work, for the first time for azospirilla, low-temperature (at 80 K, 5 K, as well as at 2 K without and with an external magnetic field of 5 T) transmission Mössbauer spectroscopic studies were performed for lyophilised biomass of A. brasilense (wild-type strain Sp7 grown with (57)Fe(III) nitrilotriacetate complex as the sole source of iron) to enable quantitative chemical speciation analysis of the intracellular iron. In the Mössbauer spectrum at 80 K, a broadened quadrupole doublet of high-spin iron(III) was observed with a few percent of a high-spin iron(II) contribution. In the spectrum measured at 5 K, a dominant magnetically split component appeared with the parameters typical of ferritin species from other bacteria, together with a quadrupole doublet of a superparamagnetic iron(III) component and a similarly small contribution from the high-spin iron(II) component. The Mössbauer spectra recorded at 2 K (with or without a 5 T external field) confirmed the assignment of ferritin species. About 20% of total Fe in the dry cells of A. brasilense strain Sp7 were present in iron(III) forms superparamagnetic at both 5 and 2 K, i.e. either different from ferritin cores or as ferritin components with very small particle sizes.

Reorganization of Azospirillum brasilense cell membrane is mediated by lipid composition adjustment to maintain optimal fluidity during water deficit.

AIMS: We study the Azospirillum brasilense tolerance to water deficit and the dynamics of adaptive process at the level of the membrane. METHODS AND RESULTS: Azospirillum brasilense was exposed to polyethylene glycol (PEG) growth and PEG shock. Tolerance, phospholipids and fatty acid (FA) composition and membrane fluidity were determined. Azospirillum brasilense was able to grow in the presence of PEG; however, its viability was reduced. Cells grown with PEG showed membrane fluidity similar to those grown without, the lipid composition was modified, increasing phosphatidylcholine and decreasing phosphatidylethanolamine amounts. The unsaturation FAs degree was reduced. The dynamics of the adaptive response revealed a decrease in fluidity 20 min after the addition of PEG, indicating that the PEG has a fluidizing effect on the hydrophobic region of the cell membrane. Fluidity returned to initial values after 60 min of PEG exposure. CONCLUSION: Azospirillum brasilense is able to perceive osmotic changes by changing the membrane fluidity. This effect is offset by changes in the composition of membrane phospholipid and FA, contributing to the homeostasis of membrane fluidity under water deficit. SIGNIFICANCE AND IMPACT OF THE STUDY: This knowledge can be used to develop new Azospirillum brasilense formulations showing an adapted membrane to water deficit.

ProClaT, a new bioinformatics tool for in silico protein reclassification: case study of DraB, a protein coded from the draTGB operon in Azospirillum brasilense.

BACKGROUND: Azopirillum brasilense is a plant-growth promoting nitrogen-fixing bacteria that is used as bio-fertilizer in agriculture. Since nitrogen fixation has a high-energy demand, the reduction of N2 to NH4+ by nitrogenase occurs only under limiting conditions of NH4+ and O2. Moreover, the synthesis and activity of nitrogenase is highly regulated to prevent energy waste. In A. brasilense nitrogenase activity is regulated by the products of draG and draT. The product of the draB gene, located downstream in the draTGB operon, may be involved in the regulation of nitrogenase activity by an, as yet, unknown mechanism. RESULTS: A deep in silico analysis of the product of draB was undertaken aiming at suggesting its possible function and involvement with DraT and DraG in the regulation of nitrogenase activity in A. brasilense. In this work, we present a new artificial intelligence strategy for protein classification, named ProClaT. The features used by the pattern recognition model were derived from the primary structure of the DraB homologous proteins, calculated by a ProClaT internal algorithm. ProClaT was applied to this case study and the results revealed that the A. brasilense draB gene codes for a protein highly similar to the nitrogenase associated NifO protein of Azotobacter vinelandii. CONCLUSIONS: This tool allowed the reclassification of DraB/NifO homologous proteins, hypothetical, conserved hypothetical and those annotated as putative arsenate reductase, ArsC, as NifO-like. An analysis of co-occurrence of draB, draT, draG and of other nif genes was performed, suggesting the involvement of draB (nifO) in nitrogen fixation, however, without the definition of a specific function.

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 (1)H and (13)C NMR spectroscopy: [Formula: see text] 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.

Chemical synthesis of the tetrasaccharide repeating unit of the O-polysaccharide isolated from Azospirillum brasilense SR80.

A linear strategy has been developed for the synthesis of the tetrasaccharide repeating unit of the O-polysaccharide from Azospirillum brasilense SR80. Stepwise glycosylation of the rationally protected thioglycoside donors activated by NIS in the presence of La(OTf)3 furnished the target tetrasaccharide. The glycosylation reactions resulted in the formation of the desired linkage with absolute stereoselectivity and afforded the required derivatives in good to excellent yields. The phthalimido group has been used as the precursor of the desired acetamido group to meet the requirement of 1,2-trans glycosidic linkage.

Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris.

Synthesis of the tetrasaccharide repeating unit of the O-glycan from the polar flagellum flagellin of Azospirillum brasilense Sp7.

Chemical synthesis of the tetrasaccharide repeating unit of the O-glycan from the polar flagellum flagellin of Azospirillum brasilense Sp7 in the form of its p-methoxyphenyl glycoside is reported. The required glycosidic linkages have been accomplished by activation of thioglycosides with N-iodosuccinimide in the presence of H2SO4-silica. H2SO4-silica was found to be an effective alternative to the classical acid promoters like TfOH or TMSOTf and it can lead to the formation of both 1,2-cis and 1,2-trans glycosidic linkages depending on the protecting group manipulation and control of the reaction condition.

Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14.

Lipopolysaccharides from closely related Azospirillum brasilense strains, Sp246 and SpBr14, were obtained by phenol-water extraction. Mild acid hydrolysis of the lipopolysaccharides followed by GPC on Sephadex G-50 resulted in polysaccharide mixtures. On the basis of sugar and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy data, it was concluded that both bacteria possess the same two distinct polysaccharides having structures 1 and 2: [structure: see text]. Structure 1 has been reported earlier for a polysaccharide of A. brasilense 54 [Fedonenko et al., 2011] whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.