The effect of mutations affecting synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.     

Antigenic identity of the capsule lipopolysaccharides,exopolysaccharides, and O-specific polysaccharides in Azospirillum brasilense

The antigenic identity (and close values of electrophoretic mobility) of capsular polysaccharides, exopolysaccharides, and O-specific polysaccharides was revealed in the Azospirillum brasilense strains Sp7 and Sp245 by the immunodiffusion and immunoelectrophoretic methods. Together with the literature data on the identity of the monosaccharides composition of these polymers, this gives evidence of the absence of a specific capsular antigen in the bacteria studied. Thus, extracellular Azospirillum brasilense polysaccharides are likely to represent O-antigenic lipopolysaccharide fragments excreted by the bacteria into the culture medium, and their identification as a capsule or as an exopolysaccharide depends on the strength of the attachment of these polysaccharides to the cell surface.     

Calcofluor- and lectin-binding exocellular polysaccharides of Azospirillum brasilense and Azospirillum lipoferum.

Extracellular polysaccharides synthesized by Azospirillum brasilense and A. lipoferum were shown on agar plates and liquid flocculating cultures. The six strains used in this work expressed a mucoid phenotype, yielding positive calcofluor fluorescence under UV light. The calcofluor-binding polysaccharides were distributed between the capsular and exopolysaccharide fractions, suggesting exocellular localization. No calcofluor fluorescence was observed in residual cells after separation of the capsular and exopolysaccharide fractions. Cellulose content was significantly higher in flocculating than in nonflocculating cultures. Failure to induce flocculation by addition of cellulose (100 mg/ml) to nonflocculating cultures, together with the sensitivity of flocs to cellulase digestion, suggested that cellulose is involved in maintenance of floc stability. Different A. brasilense and A. lipoferum strains bound to a wheat lectin (fluorescein isothiocyanate-wheat germ agglutinin), indicating the occurrence of specific sugar-bearing receptors for wheat germ agglutinin on the cell surface. The biochemical specificity of the reaction was shown by hapten inhibition with N-acetyl-D-glucosamine. All six strains failed to recognize fluorescein isothiocyanate-soybean seed lectin under our experimental conditions. We conclude that azospirilla produce exocellular polysaccharides with calcofluor- and lectin-binding properties.     

The nodPQ genes in Azospirillum brasilense Sp7 are involved in sulfation of lipopolysaccharides

Here we report on the presence of sulfated lipopolysaccharide molecules in Azospirillum brasilense, a plant growth-promoting rhizosphere bacterium. Chemical analysis provided structural data on the O-antigen composition and demonstrated the possible involvement of the nodPQ genes in O-antigen sulfation.     

A comparison of the lipopolysaccharides and O-specific polysaccharides of Azospirillum brasilense Sp245 and its omegon-Km mutants KM018 and Km252

The lipopolysaccharides (LPSs) extracted from the outer membrane of Azospirillum brasilense Sp245 and its Omegon-Km mutants KM018 and KM252 with a hot aqueous solution of phenol were found to differ in the content of carbohydrates, glucosamine, and total phosphorus and in the proportion of octadecenoic and hexadecanoic acids in the lipid moieties of the LPSs. The carbohydrate moieties of the LPSs were heterogeneous in charge. The analysis of the O-specific polysaccharides (O-PSs) of the mutants KM018 and KM252 by gas-liquid chromatography, IR spectroscopy, and NMR spectroscopy showed that they are composed of the same linear pentasugar repeating units-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)- alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->as the O-PSs of the parent strain Sp245. The reported differences in the biological activity of the LPSs of the parent and mutant strains can be due to their different chemical structure.     

Flocculation in Azospirillum brasilense and Azospirillum lipoferum: exopolysaccharides and cyst formation.

The phenomena of flocculation and floc formation by Azospirillum brasilense Sp7 (ATCC 29145) and Azospirillum lipoferum Sp59b (ATCC 29707) were studied in aerobic liquid cultures. Carbon sources representative of various entry pathways in combination with various nitrogen sources induced flocculation in both species of azospirilla. Noticeably, the combination of fructose and nitrate was the most effective in terms of floc yields. Phase-contrast microscopic observations revealed a transition in cell morphology from freely motile, vibrioid cells to nonmotile, highly refractile encysting forms during the formation of flocs. The nonmotile forms in flocs appeared to be entangled within a fibrillar matrix, and the cells were highly resistant to desiccation. Dried flocs kept for almost 6 months still maintained the highly refractile encysting forms, and their viability was confirmed by pellicle formation and acetylene reduction in semisolid malate medium. Electron microscopic observations of the desiccated flocs revealed the presence of cell forms containing abundant poly beta-hydroxybutyrate granules within a central body and surrounded by a thick layer of exopolysaccharides. The latter were characterized by alkali and acid digestion, crude cellulase hydrolysis, and calcofluor staining. It was concluded that the overproduction of exocellular polymers induces the flocculent growth and is associated with the concomitant transformation of vegetative cells to the desiccation-resistant encysting forms under limiting cultural conditions.     

Immunochemical analysis of O-specific polysaccharides from the soil nitrogen-fixing bacteria Azospirillum brasilense

Immunochemical reactivity of O-specific polysaccharide and the monosaccharide composition of O-antigenic determinants of the lipopolysaccharide isolated from the type strain Sp7 of Azospirillum brasilense were studied. An original modification of the method of spectroturbidimetry for disperse biological systems and a nonstandard procedure for the preparation of monospecific antibodies against cell surface antigens were used. The polysaccharide fraction, which contained residues of galactose, rhamnose, and galacturonic acid, was able to bind about 50% of the antibodies raised against whole bacterial cells. Twelve immunodeterminant groups were shown to be present in its molecule. Galactose and, less effectively, rhamnose but not galacturonic acid inhibited the antigen-antibody reactions. It is concluded that the serotype of the strain studied is determined by galactose residues.     

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.     

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria

The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria.     

The random walk of Azospirillum brasilense

The bacterium Azospirillum brasilense has been frequently studied in laboratory experiments. It performs movements in space where long forward and backward runs on a straight line occur simultaneously with slow changes of direction of the line. A model is presented in which a correlated random walk on a line is joined to diffusion on a sphere of directions. For this transport system, a hierarchy of moment approximations is derived, ranging from a hyperbolic system with four dependent variables to a scalar damped wave equation (telegraph equation) and then to a single diffusion equation for particle density. The original parameters are compounded in the diffusion quotient. The effects of these parameters, such as particle speed or turning rate, on the diffusion coefficient are discussed in detail.     

Negative effect of alkylresorcinols on motility of rhizobacteria Azospirillum brasilense

In liquid media, rhizobacteria Azospirillum brasilense are motile by means of a polar flagellum and in semisolid media, also by means of additionally synthesized lateral flagella. In the presence of 4-n-hexylre-sorcinol (0.04–0.10 mM), the swimming rate of A. brasilense Sp245 decreased significantly. At the concentrations over 0.10 mM, motility was completely suppressed. The presence of 4-n-hexylresorcinol in liquid medium also had a negative effect on motility of the cells of Sp245 and other A. brasilense strains (Sp7, Cd, and SR75). The concentrations of 5-methylresorcinol which negatively affected the rate of bacterial spreading in semisolid media were an order of magnitude higher than those of 4-n-hexylresorcinol. Alkylresorcinols, which belong to alkylhydroxybenzene autoregulators, probably affect the cellular mechanisms (systems) responsible for flagella rotation in azospirilla.     

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.     

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.     

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.     

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.     

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.     

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.