Effect of lectins from <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> 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 of lectins 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 of lectin in under 10 min in a concentration of 10 μg/ml. The obtained results show that lectins from Azospirillum are capable of inducing the adaptation processes in the roots of wheat seedlings.     

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 of the functions of lectins remain insufficiently understood.     

The Role of Cell-Surface Lectins in the Aggregation of Azospirilla

The mutant strain Azospirillum brasilenseSp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilenseSp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilenseSp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense75, A. brasilenseSp7, and A. lipoferum59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilenseSp7 cells with Bacillus polymyxa1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Stabilizing effect of <Emphasis Type="Italic">Azospirillum</Emphasis> lectins on β-glucosidase activity

Lectins from the surface of Azospirillum brasilense Sp7 and Azospirillum brasilense Sp7.2.3 (a mutant with impaired lectin activity) were shown to induce a stabilizing effect on the activity of almond β-glucosidase under conditions of thermoinactivation and proteolytic enzyme treatment. Differences were revealed in the influence of lectins with various antigenic properties. Our results indicate that the effects of lectins on the catalytic activity of the enzyme are mainly associated with conformational changes in lectin molecules during mutagenesis, but not with carbohydrate specificity (general property). These data should be taken into account in evaluating the role of lectins in the formation of nitrogen-fixing associations.     

Effect of Azospirillum Lectins on the Activities of Wheat-root Hydrolytic Enzymes

This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast fractions of wheat-seedling roots. Lectin (40 μg mL⁻¹) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase. Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained by change in the antigenic properties of the mutant lectin.     

Wheat root colonization by Azospirillum brasilense strains with different motility

Migration of associative bacteria Azospirillum brasilense in semisolid media is performed mainly by swarming (Swa⁺ phenotype), which depends on the flagellar functioning and intercellular contacts. Non-swarming mutants of A. brasilense Sp245 lacking a polar flagellum migrate in semisolid media with microcolony formation using a unrevealed mechanism (Gri⁺ phenotype). The study of wheat root colonization dynamics demonstrated that A. brasilense Sp245 Gri⁺ mutants exhibited lower capacity for wheat root adsorption. However, after “anchoring” has occurred, both A. brasilense Sp245 and its Swa-Gri⁺ mutants colonized the growing roots with virtually the same efficiency. All strains under study formed microcolonies on the surface of roots, stimulated root branching, and exhibited changes in the composition of protein antigens exposed on the bacterial cell surface. Indirect evidence was obtained for enhanced production of genus-specific protein antigens in the process of A. brasilense Sp245 adaptation to growth on plant roots.     

Effect of Azospirilla Lectins on Germination Capacity of Seeds

We studied the effect of lectins of Azospirillum brasilense Sp 7 on germination capacity of tomato, cucumber, pea, wheat, and onion seeds. The lectins proved to inhibit seed germination. We studied the effect of different concentrations of lectins from four azospirilla strains on the rate of wheat seed germination. Both inhibiting and stimulating effects were demonstrated. Lectin-induced changes in the mitotic state of plant cells were demonstrated with histological sections of the root growing point in onion.     

Maize Response to Salicylic Acid and Fusarium moniliforme

Effects of salicylic acid and Fusarium moniliformeon trypsin inhibitor activity, lectin activity, lectin carbohydrate specificity, and salicylic acid content in maize seedlings were studied. Changes in trypsin inhibitor activity, lectin activity, and the content of endogenous salicylic acid after administration of exogenous salicylic acid or a pathogen were shown to depend on the resistance of maize strains to Fusarium. The data suggest that salicylic acid is involved in the induction of trypsin and lectin inhibitors that are important in the formation of defenses against abiotic and biotic factors in maize sprouts.     

The participation of the cell wall hydrolytic enzymes in the initial colonization of Azospirillum brasilense on wheat roots

The effect of cellulase and pectinase on bacterial colonization of wheat was studied by three different experiments. In the first experiment, the root colonization of 3 wheat cultivars (Ghods, Roshan and Omid) by two A. brasilense strains (Sp7 and Dol) was compared using pre-treated roots with cellulase and pectinase, and non-treated with these enzymes (control). Although the root colonization varied greatly among strain-plant combinations in controls, the pre-treatment of roots with polysaccharide degrading enzymes significantly increased the bacterial count in roots, regardless of the strain-plant combination. This might be an indication that cell wall may act as an important factor in plant-Azospirillum interaction. In the second experiment, the root cellulase activity of the same wheat cultivars treated with and without the two Azospirillum brasilense, strains (Sp7 and Dol) was compared. The pre-treatment of wheat roots with Azospirillum enhanced the cellulase activity of wheat root extracts. Thus, the cellulase activity might participate in the initial colonization of wheat roots by Azospirillum. The comparison of the cellulase activity of root extracts within inoculated and non-inoculated seedlings showed that the inoculation had enhanced the cellulase activity in root extracts, but this effect was directly dependent on the strain-plant combination. Strain Sp7 stimulated the highest cellulase activity in cv. Roshan, but strain Dol induced the highest enzyme activity in cv. Ghods. In the third experiment, several growth parameters of those 3 wheat cultivars treated with and without those two bacterial strains (Sp7 and Dol) were compared. The highest magnitude of growth responses caused by Sp7 strain was in the cv Roshan, but Dol strain stimulated the highest growth in cv Ghods. Therefore, effective colonization may contribute to more growth responses.     

Serological relationships of azospirilla revealed by their motility patterns in the presence of antibodies to lipopolysaccharides

Motility of the serologically different Azospirillum brasilense strains Sp245 (serogroup I) and Sp7 (serogroup II) was studied in the presence of antibodies to their lipopolysaccharides (LPS). A procedure was proposed in order to determine the motility patterns indicating the specificity of the interaction between the anti-LPS antibodies and bacteria. Analysis of the effect of such antibodies on motility of 25 strains (A. brasilense, A. lipoferum, A. irakense, and Azospirillum sp.) revealed bacteria exhibiting antigenic cross reactions with A. brasilense Sp7 or Sp245. The effect of anti-LPS antibodies on motility of azospirilla was in agreement with the results of immune agglutination analysis of bacterial cells and of immunodiffusion analysis of the LPS preparations. According to our results, strains Azospirillum sp. SR81 and A. brasilense SR14 should be included into serogroups I and II, respectively.     

Establishment of inoculatedAzospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum

Summary Four field experiments were carried out with wheat or sorghum in different regions of Brazil. The aim was to study the establishment of inoculatedAzospirillum strains, marked with resistance to various antibiotics, in the rhizosphere and in roots. The levels of the various antibiotics were chosen according to the resistance of the indigenousAzospirillum population.Azospirillum brasilense strains Sp 107 and Sp 245 could be established in all three wheat experiments and predominated within theAzospirillum population in washed, and especially in surface sterilized, roots. Strains Sp 7 and Cd established poorly in wheat roots.Azospirillum lipoferum Sp S82 represented 72% of the root isolates from sorghum inoculated with this strain. This strain and naturalAzospirillum infection became concentrated in the upper parts of the root system. Improved methods for root surface sterilization in which the absence ofAzospirillum on the root surface was established by pre-incubating roots with paraffin-capped ends in NFb medium confirmed the establishment of inoculatedAzospirillum strains within sorghum roots in the field.     

Wheat lectin as a factor in plant-microbial communication and a stress response protein

Wheat lectin (wheat germ agglutinin, WGA), a representative of a broad group of cereal lectins, is excreted by plant roots into the surrounding medium and interacts with both pathogenic microflora and growth-stimulating rhizobacteria. WGA was found to serve as a molecular signal for the rhizobacterium Azospirillum brasilense, which forms endophytic and associative symbioses with wheat plants. The bacterial response to the lectin was pleiotropic: WGA at concentrations from 10^?10 to 10^?6 M exerted a dose-dependent effect on a range of processes in the bacterium that are important for the establishment and functioning of symbiosis. Plants with different WGA content differed in their responses to severe nitrogen starvation and to seed treatment with Azospirillum.     

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 nanoparticles. Extracellular phenoloxidizing enzymes (laccases and Mn peroxidases) were shown to participate in reduction of Au⁺³ (HAuCl₄) to Au⁰. 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.     

Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.     

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

Improved Potential for Nitrogen Fixation in Azospirillum brasilense Sp7‐S Associated with Wheat nifH Expression as a Function of Oxygen Pressure

The use of a nifH‐lacZ fusion as an indicator of nitrogen fixing conditions is investigated in relation to two strains of Azospirillum brasilense with contrasting patterns of colonization on wheat roots. The degree of expression of nifH‐lacZ of Azospirillum brasilense could be manipulated by controlling the oxygen pressure. A strong correlation between nitrogenase activity and nifH expression was found in pure cultures. nifH expression was maximal at 0.5% oxygen in pure cultures of both the wild type Sp7 and spontaneous mutant Sp7‐S. Differentiation of the maximal expression was observed when the two strains were in association with para‐nodulated wheat, resulting in greater expression by Sp7‐S over a broader range of external oxygen concentrations than by Sp7. This result was observed when expressed as activity per mg of plant protein as well as per bacterium. An increase in nifH expression was also noted with para‐nodulated (2,4‐D treated) wheat inoculated with Sp7‐S when compared with untreated wheat. No significant difference was found between treated and untreated wheat inoculated with Sp7. The results indicate that the majority of the Azospirillum brasilense Sp7‐S cells occupy a more protected niche when in association with wheat roots, resulting in conditions that support a greater potential for nitrogen fixation as judged by nifH expression.     

Lipopolysaccharides in four strains of the unicellular cyanobacterium Synechocystis

The results of the cross reactions of the 27 strains of Azospirillum spp. with 4 fluorescent antibodies (FA) show a neat differentiation between the two species. A. lipoferum represents a more homogenous group in respect to FA reactions and highly fluorescent preparations were obtained with strains from a large scope origin against Sp59 FA, the type strain. In contrast A. brasilense contains at least three sub groups in respect to FA reactions. The first includes all denitrifing strains (nir⁺) which react with FA from Sp7 the type strain. None of the nir^- strains reacted strongly with Sp7 FA. One part of the A. brasilense nir^- group which includes the strains isolated from well sterilized rice and wheat roots (Sp 107, 107 st, 106 and 109 st) reacts with FA of their reference strain Sp107 but not with that of Sp28 FA. The strains isolated from unsterilized roots and soils reacted with SP28 FA and not with that of Sp107 FA. In addition there were 3 strains (Sp A4, 34 and 67) which reacted with neither of the FAs.Abbreviations Fa fluorescent antibody - FITC fluorescein isothiocyanate - Rh ITC gelatin-rhodamine isothiocyanate - nir⁺ nitrite reductase positive - nir^- nitrite reductase negative     

Extracellular Proteolytic Enzymes of Azospirillum brasilense Strain Sp7 and Regulation of Their Activity by a Homologous Lectin

It was found that Azospirillum brasilense strain Sp7 is able to produce extracellular proteolytic enzymes. The enzymes were active within a broad range of pH values, with two peaks of activity being located in the acid and alkaline pH areas; required calcium ions; and exhibited substrate specificity with respect to azogelatin. Zymography allowed at least four proteolytic enzymes with molecular weights of 32, 45, 52, and 174 kDa to be detected in A. brasilense Sp7 culture liquid. It was shown that the lectin from A. brasilense Sp7 can inhibit proteolytic enzymes.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 444–448.Original Russian Text Copyright © 2005 by Chernyshova, Alen’kina, Nikitina, Ignatov.     

Changes in cell surface properties and biofilm formation efficiency in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 mutants in the putative genes of lipid metabolism <Emphasis Type="Italic">mmsB1</Emphasis> and <Emphasis Type="Italic">fabG1</Emphasis>

The previously obtained insertion mutants of Azospirillum brasilense Sp245 in the genes mmsB1 and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense strains Sp245, SK039, and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide preparations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.     

Enhanced Growth of Wheat and Soybean Plants Inoculated with Azospirillum brasilense Is Not Necessarily Due to General Enhancement of Mineral Uptake

The capacity of Azospirillum brasilense to enhance the accumulation of K⁺, P, Ca²⁺, Mg²⁺, S, Na⁺, Mn²⁺, Fe²⁺, B, Cu²⁺, and Zn²⁺ in inoculated wheat and soybean plants was evaluated by using two different analytical methods with five A. brasilense strains originating from four distinct geographical regions. A Pseudomonas isolate from the rhizosphere of Zea mays seedlings was included as a control. All A. brasilense strains significantly improved wheat and soybean growth by increasing root and shoot dry weight and root surface area. The degree of plant response to inoculation varied among the different strains of A. brasilense. All strains were capable of colonizing roots, but the best root colonizer, Pseudomonas sp., had no effect on plant growth. The numbers of organisms of Brazilian strains Sp-245 and Sp-246 colonizing roots were similar regardless of the host plant. Numbers of organisms for the other strains were directly dependent on the host plant. The main feature characterizing mineral accumulation in inoculated plants was that all inoculation treatments changed the mineral balance of the plants, but in an inconsistent manner. Enhancement of mineral uptake by plants also varied among strains to a great extent and was directly dependent on the strain-plant combination; i.e., a strain capable of increasing accumulation of a particular ion in one plant species or cultivar often lacked the ability to do so in another. Minerals in inoculated plants were not evenly distributed in different plant tissues, and the changes varied among groups of plants within each bacterial strain inoculation treatment. We suggest that, although A. brasilense strains are capable of changing the mineral balance and content of plants, it is unlikely that this ability is a general mechanism responsible for plant improvement by A. brasilense.