Denitrification by Azospirillum brasilense Sp 7

Nitrous oxide reduction can consistently be demonstrated with high activities in cells of Azospirillum brasilense Sp 7 which are grown anaerobically in the presence of low amounts of nitrite. Azospirillum can even grow anaerobically with nitrous oxide in the absence of any other respiratory electron acceptor. Nitrous oxide reduction by Azospirillum is inhibited by acetylene, amytal and weakly by carbon monoxide. Azospirillum converts nitrous oxide to molecular nitrogen without the formation of ammonia. The cells must, therefore, be supplied with ammonia from nitrogen fixation during anaerobic growth with nitrous oxide. When no other nitrogen compound besides nitrous oxide is available in the medium, the bacteria synthesize nitrogenase from protein reserves in about 2 h. Nitrogenase synthesis is blocked by chloramphenicol under these conditions. In contrast, the addition of nitrate or nitrite to the medium represses the synthesis of nitrogenase. Nitrous oxide reduction by Azospirillum and other microorganisms is possibly of ecological significance, because the reaction performed by the bacteria may remove nitrous oxide from soils.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Effect of inoculation of Zea mays with Azospirillum brasilense strains under temperate conditions

Seven strains of Azospirillum brasilense were compared for their effect on the growth of Zea mays grown under temperate conditions in sand--vermiculite pot cultures. Inoculation with all seven strains tested, including Fix(-) mutant strains, increased dry weight and total nitrogen content of shoots, but nitrogen concentrations were unaffected. Low and variable rates of acetylene reduction activity were observed from excised roots of inoculated plants without preincubation. Estimates of N2-fixing A. brasilense associated with inoculated roots showed differences between strains in establishing themselves in the rhizosphere and endorhizosphere. In some strains enrichment in the endorhizosphere of roots occurred following inoculation, but the relative numbers and location of the strains did not appear to affect the yield response.     

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.     

Fructose catabolism in Azospirillum brasilense and Azospirillum lipoferum.

The pathways for catabolism of fructose were investigated in the type strains of Azospirillum lipoferum and Azospirillum brasilense grown aerobically with (NH4)2SO4 as the nitrogen source. When grown on fructose, the former species possessed a complete Entner-Doudoroff pathway, whereas the latter species lacked activity for glucose-6-phosphate dehydrogenase. Both species possessed a complete catabolic Embden-Meyerhof-Parnas pathway. Neither species possessed the key enzyme of the hexose monophosphate pathway, 6-phosphogluconate dehydrogenase. Both species could phosphorylate fructose to fructose-1-phosphate by means of a phosphoenolpyruvate-phosphotransferase system, and high activities of 1-phosphofructokinase occurred. Both species possessed glucokinase activity, but only A. lipoferum had hexokinase activity; moreover, the cells of A. brasilense were nearly impermeable to glucose, accounting for the inability of this species to grow on glucose. Both species possessed pyruvate dehydrogenase, a complete tricarboxylic acid cycle, a glyoxylate shunt, and malic enzyme. Analysis of the acidic end products for both species indicated the formation of only small amounts of various organic acids, and most of the titratable acidity was due to utilization of the ammonium ions of the medium. Gluconic acid was not formed during growth of either species on fructose but was detected during growth of A. lipoferum on glucose; this species also possessed an NADP-linked glucose dehydrogenase and gluconokinase.     

Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.     

Cell Ultrastructure in Azospirillum brasilense Biofilms

Microbiology - Due to the primary localization of both epiphytic and endophytic plant growth-promoting rhizobacteria on the surface of the plant root system, biofilm formation is an adaptive trait...     

Detection of chemotaxis in Azospirillum brasilense

Azospirillum brasilense strain Cd responded chemotactically to amino acids, sugars and organic acids. Chemotactic rings were observed in semisolid agar plates containing oxidizable substrates. Increasing sodium succinate concentration decreased the velocity of ring expansion. Chemotactic activity of Azospirillum was also examined by a newly developed technique using a channelled chamber. Varying the concentrations of aspartic and glutamic acids affected the chemotactic response of the bacteria. In both assays chemotaxis was obtained under conditions that prevented aerotaxis.     

Metabolism of various carbon sources by Azospirillum brasilense

Azospirillum brasilense Sp7 and two mutants were examined for 19 carbon metabolism enzymes. The results indicate that this nitrogen fixer uses the Entner-Doudoroff pathway for gluconate dissimilation, lacks a catabolic but has an anabolic Embden-Meyerhof-Parnas hexosephosphate pathway, has amphibolic triosephosphate enzymes, lacks a hexose monophosphate shunt, and has lactate dehydrogenase, malate dehydrogenase, and glycerokinase. The mutants are severely deficient in phosphoglycerate and pyruvate kinase and also have somewhat reduced levels of other carbon enzymes.     

Atypical R-S dissociation in Azospirillum brasilense

It was found that atypical R-S dissociation in the type strain A. brasilense Sp7 is not accompanied by drastic changes in the carbohydrate moieties of bacterial lipopolysaccharides but is rather due to different contributions of two O-specific polysaccharides (found in both R and S dissociants) to the age-dependent architectonics of the cell surface.     

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.     

Changes in Azospirillum brasilense motility and the effect of wheat seedling exudates

The rhizobacterium Azospirillum brasilense Sp245 swims, swarms (Swa⁺ phenotype) or, very rarely, migrates with the formation of granular macrocolonies (Gri⁺ phenotype). Our aims were (i) to identify Sp245 mutants that swarm faster than the parent strain or differ from it in the mode of spreading and (ii) to compare the mutants’ responses to wheat seedling exudates. In isotropic liquid media, the swimming speeds of all motile A. brasilense strains were not influenced by the exudates. However, the exudates significantly stimulated the swarming of Sp245. In several Sp245 mutants, the superswarming phenotype was insensitive to local colonial density and to the presence of wheat seedling exudates. An A. brasilense polar-flagellum-defective Gri⁺ mutant BK759.G gave rise to stable Swa⁺⁺ derivatives with restored flagellum production. This transition was concurrent with plasmid rearrangements and was stimulated in the presence of wheat seedling exudates. The swarming rate of the Swa⁺⁺ derivatives of BK759.G was affected by the local density of their colonies but not by the presence of the exudates.     

Production of vitamins by Azospirillum brasilense in chemically-defined media

The production of vitamins by Azospirillum brasilense was studied in chemically-defined media amended with malate, gluconate and fructose. The liberation of vitamins was significantly affected by the presence of different carbon sources and the age of the culture. Thiamine, niacin and pantothenic acid were produced in large amounts. Thiamine and riboflavin were produced only in culture containing malate or fructose. Biotin was not detected in the supernatants of the culture media.     

Production of bacteriocins and siderophore-like activity by Azospirillum brasilense

Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium. Mitomycin C treatment enhanced the proportion to 80%. Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium. These inhibition zones probably result from the activity of siderophores. Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C. Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity. These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.     

Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and EGTA in vitro

Mature plant cell walls lose their ability to expand and become unresponsive to expansin. This phenomenon is believed to be due to cross-linking of hemicellulose, pectin, or phenolic groups in the wall. By screening various hydrolytic enzymes, we found that pretreatment of nongrowing, heat-inactivated, basal cucumber (Cucumis sativus) hypocotyls with pectin lyase (Pel1) from Aspergillus japonicus could restore reconstituted exogenous expansin-induced extension in mature cell walls in vitro. Recombinant pectate lyase A (PelA) and polygalacturonase (PG) from Aspergillus spp. exhibited similar capacity to Pel1. Pel1, PelA, and PG also enhanced the reconstituted expansin-induced extension of the apical (elongating) segments of cucumber hypocotyls. However, the effective concentrations of PelA and PG for enhancing the reconstituted expansin-induced extension were greater in the apical segments than in the basal segments, whereas Pel1 behaved in the opposite manner. These data are consistent with distribution of more methyl-esterified pectin in cell walls of the apical segments and less esterified pectin in the basal segments. Associated with the degree of esterification of pectin, more calcium was found in cell walls of basal segments compared to apical segments. Pretreatment of the calcium chelator EGTA could also restore mature cell walls' susceptibility to expansin by removing calcium from mature cell walls. Because recombinant pectinases do not hydrolyze other wall polysaccharides, and endoglucanase, xylanase, and protease cannot restore the mature wall's extensibility, we can conclude that the pectin network, especially calcium-pectate bridges, may be the primary factor that determines cucumber hypocotyl mature cell walls' unresponsiveness to expansin.     

Changes in motility of the rhizobacterium Azospirillum brasilense in the presence of plant lectins

The plant-beneficial bacterium Azospirillum brasilense can swim in liquids and swarm or migrate with the formation of microcolonies in soft media. To get closer to understanding the influence of natural environments on A. brasilense motility, we studied the individual and social movement of the bacterium in the presence of various plant lectins. The lectins with specificity for N-acetyl-beta-d-glucosamine oligomers (wheat germ, Solanum tuberosum and Ulex europeus agglutinins) decreased A. brasilense swimming speed and induced the formation of branched-granular colonies instead of the swarming rings. These effects seemed to be a consequence of specific interactions between the agglutinins and the lectin-binding polymers present in the A. brasilense cell envelope. Concanavalin A (with an affinity for terminal alpha-d-mannosyl and alpha-d-glucosyl residues) and Phaseolus vulgaris phytohemagglutinin P (with unknown specificity) almost did not affect the motility of A. brasilense.     

Changes in the properties of cell wall pectin methylesterase along the Vigna radiata hypocotyl

The properties of cell wall pectinesterase (EC 3.1.1.11) from Vigna radiata (L.) Wilczek hypocotyl were investigated with immobilized and solubilized wall enzymes. Along the hypocotyl, the decrease of growth potential coincides with significant changes of the characteristics of the pectinesterase activities. As hypocotyl cells grow older, the proportion of ionically bound enzymes decreases and pH profile, sensitivity to cations and affinity for pectin change. The possible significance of these observations is discussed.