The role of cell surface bacterial lectins in the aggregation of Azospirilla

The mutant strain Azospirillum brasilense Sp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilense Sp7(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. brasilense Sp7(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. brasilense 75, A. brasilense Sp7, and A. lipoferum 59b 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. brasilense Sp7 cells with Bacillus polymyxa 1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Relationship between lectin, alpha-, beta-glucosidase, and beta-galactosidase activities of Azospirillum]

The activities of alpha-glucosidase, beta-glucosidase, and beta-galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol-acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied among the azospirilla studied. The cofunction of the A. brasilense Sp7 lectin and beta-galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum 59b lectin and glucosidases was revealed. The lectin from A. lipoferum 59b may possess saccharolytic activity.     

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.     

Impact of bacterial lectin on functional activity of lymphocytes

Impact of Azospirillum brasilense Sp7 lectin with carbohydrate specificity to L-fucose on T- and B-lymphocytes production, activity of IL-1alpha, and TNF-alpha was studied in vitro and compared with influence of red kidney bean phytohemagglutinin (PHA) on the same parameters. Increase of growth of T- and B-cells subpopulations has been shown that point to stimulatory effect of the bacterial lectin. Bacterial lectin of A. brasilense also increased synthesis of IL-1alpha and TNF-alpha. It has been shown that the bacterial lectin has more pronounced stimulatory effect on functional activity of lymphocytes compared with PHA.     

Extracellular proteolytic enzymes of Azospirillum brasilensis strain Sp7 and regulation of their activity by a homologous lectin

It was found that Azospirillum brasilensis 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.     

The bactericidal activity of lectins from nitrogen-fixing bacilli

Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 micrograms/ml). Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp. citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A. brasilense 245. The bacillar lectins incubated with Rhizobium and Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function. We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.     

Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.     

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.     

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.     

Intermediary carbon metabolism of Azospirillum brasilense.

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.     

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.     

Effect of Azospirillum brasilense lectin on the kinetics of lymph nodes cell populations and cytokine status in experimental animals

The complex study of the influence of A. brasilense Sp 7 lectin with carbohydrate specificity to L-fucose and D-galactose on the dynamics of cell populations in different structural functional zones of the white mice mesentery, as well as on the capacity of immunocompetent cells of mesenterial lymph nodes for synthesizing cytokines (interleukin-1, interleukin-6 and tumor necrosis factor), was carried out. A single oral administration of bacterial lectin in a dose of 4.5 micrograms produced a pronounced immunostimulating effect.     

Enhanced micropropagation response and biocontrol effect of Azospirillum brasilense Sp245 on Prunus cerasifera L. clone Mr.S 2/5 plants

Inoculation with Azospirillum brasilense Sp245 exerts beneficial effects on micropropagated plants of Prunus cerasifera L. clone Mr.S 2/5, as seen in the results of a comparative analysis of inoculated and non-inoculated explants, during both the rooting and acclimatation phases. The presence of Azospirillum brasilense Sp245 increased root system, root hair biomass production and apical activity. Although the presence of the bacteria had a positive effect on rooting, the addition of indolebutyric acid (IBA) to Murashige and Skoog (MS) medium was seen as indispensable in order to promote the rooting of explants. Aside from the promotion of plant growth, A. brasilense Sp245 protects plants against pathogen attacks, such as Rhizoctonia spp., with a plant survival rate of nearly 100% vs. 0% as seen in the negative control. The biocontrol effect of A. brasilense Sp245 on the fungal rhizospheric community has been confirmed by denaturing gradient gel electrophoresis (DGGE) profiles of the rhizospheric microbial community. This study indicates that A. brasilense Sp245 could be employed as a tool in plant biotechnology.     

The chemotaxis-like Che1 pathway has an indirect role in adhesive cell properties of Azospirillum brasilense

The Azospirillum brasilense chemotaxis-like Che1 signal transduction pathway was recently shown to modulate changes in adhesive cell surface properties that, in turn, affect cell-to-cell aggregation and flocculation behaviors rather than flagellar-mediated chemotaxis. Attachment to surfaces and root colonization may be functions related to flocculation. Here, the conditions under which A. brasilense wild-type Sp7 and che1 mutant strains attach to abiotic and biotic surfaces were examined using in vitro attachment and biofilm assays combined with atomic force microscopy and confocal microscopy. The nitrogen source available for growth is found to be a major modulator of surface attachment by A. brasilense and could be promoted in vitro by lectins, suggesting that it depends on interaction with surface-exposed residues within the extracellular matrix of cells. However, Che1-dependent signaling is shown to contribute indirectly to surface attachment, indicating that distinct mechanisms are likely underlying flocculation and attachment to surfaces in A. brasilense.     

Change in the content of salicylic acid and activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the influence of Azospirilium lectins

The time course of changes in the endogenous content of salicylic acid, the ratio between the acid’s free and bound forms, and changes in the activities of phenylalanine ammonia-lyase and catalase in wheat seedling roots under the effect of lectins of two strains of the associative nitrogen-fixing bacterium Azospirillum (A. brasilense Sp7 and its mutant defective in lectin activity, A. brasilense Sp7.2.3) is investigated. Differences in plant response to the action of the lectins from these two strains are established. On the basis of the obtained data, a model is proposed for lectin-assisted induction of resistance, according to which the lectin effect on the roots of seedlings results in the accumulation of free salicylic acid, which inhibits catalase activity, ultimately leading to accumulation of hydrogen peroxide and formation of induced resistance.     

A mutant of Azospirillum brasilense Sp7 impaired in flocculation with a modified colonization pattern and superior nitrogen fixation in association with wheat.

We report here significant phenotypic and genetic differences between Azospirillum brasilense Sp7 and spontaneous mutant Sp7-S and their related properties in association with wheat. In contrast to the wild-type strain of Sp7, colonies of Sp7-S stained weakly with Congo red when grown on agar media containing the dye and did not flocculate in the presence of fructose and nitrate. Scanning and transmission electron micrographs showed clearly that the Sp7-S strain lacked surface materials present as a thick layer on the surface of the wild-type Sp7 strain. Different patterns of colonization on wheat roots between Sp7 and Sp7-S, revealed by in situ studies using nifA-lacZ as a reporter gene, were related to a large increase in nitrogenase activity (acetylene reduction) with Sp7-S in association with normal and 2,4-dichlorophenoxyacetic acid-treated wheat for assays conducted under conditions in which the nitrogenase activity of free-living Azospirillum organisms was inhibited by an excess of oxygen. Randomly amplified polymorphic DNA analysis indicated the close genetic relationship of Sp7-S to several other sources of Sp7, by comparison to other recognized strains of A. brasilense. Genetic complementation of Sp7-S was achieved with a 9.4-kb fragment of DNA cloned from wild-type Sp7, restoring Congo red staining and flocculation.     

Nature of the lectin-induced activation of plasma membrane Mg2+ATPase.

The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.     

Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat

Auxin production by Azospirillum is believed to play a major role in the observed plant growth promoting effect. By using different genetically modified strains, the contribution of auxin biosynthesis by A. brasilense in altering root morphology was evaluated in a plate assay. Inoculation with the wild type strains A. brasilense Sp245 and Sp7 resulted in a strong decrease in root length and increase in root hair formation. This effect was abolished when inoculating with an ipdC mutant of A. brasilense. The ipdC gene encodes a key enzyme in the IPyA pathway of IAA synthesis by A. brasilense. On the other hand, the observed auxin effect was further enhanced by adding tryptophan, a precursor of IAA, to the plates and could be mimicked by replacing the Azospirillum cells by a particular concentration of IAA. Furthermore, particular mutants (rpoN, scrp) and transconjugants (extra copy of ipdC) of A. brasilense were tested in the plate assay. Together, these results confirm the important role of IAA produced by Azospirillum in altering root morphology and illustrate the power of combining genetic tools and bioassays to elucidate the mechanism of a beneficial Azospirillum-plant interaction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation and purification of Mn-peroxidase from Azospirillum brasilense Sp245

Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3.     

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.