Structure of the O-polysaccharide from the Azospirillum lipoferum Sp59b lipopolysaccharide

A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant-growth-promoting bacterium Azospirillum lipoferum Sp59b. On the basis of sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy, including a NOESY experiment, the following structure of the branched hexasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text].     

Chemical composition of lipopolysaccharide from Azospirillum lipoferum

Abstract The lipopolysaccharide isolated from Azospirillum lipoferum strain SpBr17 (ATCC29709) was proven to be composed from 7 neutral sugars, two of which, rhamnose and glucose, were the major constituents. Two heptoses, l -glycero- d -mannoheptose and d -glycero- l -mannoheptose were identified. Among 8 fatty acids isolated from the lipopolysaccharide only 3-hydroxypalmitic acid was amide-bound. The approximate molar ratios of the constituents 3-deoxy- l -mannooctulosonic acid : glucosamine : amide-linked fatty acids : ester-linked fatty acids : phosphate were 0.8 : 4 : 2 : 4 : 2.5.     

Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation

M ichiels , K., V erreth , C. & V anderleyden , J. 1990. Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation. Journal of Applied Bacteriology 69 , 705–711.
Surface polysaccharide production by Azospirillum is demonstrated by fluorescence of colonies grown on media containing the fluorescent dye Calcofluor, which binds to β-linked polysaccharides. Mutants showing decreased and increased levels of fluorescence are obtained from Azospirillum lipoferum strain Sp59b by chemical mutagenesis, and from A. brasilense strain 7030 by Tn5 mutagenesis.
The A. brasilense 7030 fluorescence mutants produce wild-type levels of exo-polysaccharide in their culture supernatant fluids, but are affected in flocculation in liquid culture. On the basis of these observations, we postulate that an A. brasilense surface polysaccharide, different from the exopolysaccharide, is involved in both Calcofluor staining and flocculation.
It is shown by DNA hybridization that the genetic loci affected in the A. brasilense 7030 fluorescence mutants are different from the A. brasilense exoB and exoC loci, which are involved in exopolysaccharide production.     

Floc Formation by Azospirillum lipoferum Grown on Poly-β-Hydroxybutyrate

Azospirillum lipoferum RG6xx was grown under conditions similar to those resulting in encystment of Azotobacter spp. A. lipoferum produced cells of uniform shape when grown on nitrogen-free β-hydroxybutyrate agar. Cells accumulated poly-β-hydroxybutyrate and often grew as chains or filaments that eventually lost motility and formed capsules. Within 1 week, vegetative A. lipoferum inocula were converted into microflocs arising from filaments or chains. Cells within microflocs were pleomorphic, contained much poly-β-hydroxybutyrate, and were encapsulated. Some cells had a cystlike morphology. Up to 57% of the dry weight of encapsulated flocs was poly-β-hydroxybutyrate, whereas vegetative cells grown in broth with combined nitrogen had only 3% of their dry weight as poly-β-hydroxybutyrate. Neither encapsulated cells in flocs nor nonencapsulated vegetative cells were significantly desiccation resistant. Under starvation conditions (9 days) only 25% of encapsulated cells remained viable, whereas vegetative cells multiplied severalfold. In short-term germination experiments with encapsulated flocs, nitrate, ammonium, and soil extract promoted formation of motile vegetative cells. Most cells in treatments lacking combined nitrogen eventually depleted their visible poly-β-hydroxybutyrate reserves without germinating. The remaining cells retained the reserve polymer and underwent size reduction.     

Floc Formation by Azospirillum lipoferum Grown on Poly-beta-Hydroxybutyrate

Azospirillum lipoferum RG6xx was grown under conditions similar to those resulting in encystment of Azotobacter spp. A. lipoferum produced cells of uniform shape when grown on nitrogen-free beta-hydroxybutyrate agar. Cells accumulated poly-beta-hydroxybutyrate and often grew as chains or filaments that eventually lost motility and formed capsules. Within 1 week, vegetative A. lipoferum inocula were converted into microflocs arising from filaments or chains. Cells within microflocs were pleomorphic, contained much poly-beta-hydroxybutyrate, and were encapsulated. Some cells had a cystlike morphology. Up to 57% of the dry weight of encapsulated flocs was poly-beta-hydroxybutyrate, whereas vegetative cells grown in broth with combined nitrogen had only 3% of their dry weight as poly-beta-hydroxybutyrate. Neither encapsulated cells in flocs nor nonencapsulated vegetative cells were significantly desiccation resistant. Under starvation conditions (9 days) only 25% of encapsulated cells remained viable, whereas vegetative cells multiplied severalfold. In short-term germination experiments with encapsulated flocs, nitrate, ammonium, and soil extract promoted formation of motile vegetative cells. Most cells in treatments lacking combined nitrogen eventually depleted their visible poly-beta-hydroxybutyrate reserves without germinating. The remaining cells retained the reserve polymer and underwent size reduction.     

Floc Formation by Azospirillum lipoferum Grown on Beta-Hydroxybutyrate

[This corrects the article on p. 2986 in vol. 54.].     

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.     

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.     

Cloning and expression of draTG genes from Azospirillum lipoferum

A genomic library of Azospirillum lipoferum was constructed with phage lambda EMBL4 as vector. From this library, the genes encoding dinitrogenase reductase ADP-ribosyltransferase (DRAT), draT, and dinitrogenase reductase-activating glycohydrolase (DRAG), draG, were cloned by hybridization with the heterologous probes of Rhodospirillum rubrum. As in R. rubrum, draT is located between draG and nifH, the gene encoding dinitrogenase reductase (a substrate for the DRAG/DRAT system). In the crude extract of Escherichia coli harboring the expression vector for this region, DRAT and DRAG enzyme activities were detected, confirming the identity of the cloned genes. Southern hybridization with genomic DNA from different Azospirillum spp., demonstrated a correlation between observable draTG hybridization and the biochemical demonstration of this covalent modification system.     

Effect of Endosulfan on Azospirillum lipoferum Growth, Morphology, Nitrogenase Activity, and Protein Binding

The organochlorine Thiodan CE inhibited growth and nitrogenase activity of Azospirillum lipoferum. The active ingredient, Endosulfan, was nonspecifically bound to proteins and mainly adsorbed to the cell envelope with small amounts transported into cytosol. The involvement of the external membrane and cyst formation in protection against hazardous substances is discussed.     

Metabolic activities in Azospirillum lipoferum grown in the presence of NH4+

The utilization of some agro-industrial wastes as soil conditioners to provide free-living nitrogen-fixing bacterial populations (e.g. Azospirillum spp.) with carbon and energy sources, may be an interesting perspective for agriculture. However, the presence of ammonium nitrogen in cultivated soils and/or various wastes could inhibit the growth of the nitrogen-fixing populations. The present investigation shows that growth of Azospirillum lipoferum was restricted at a dissolved oxygen (DO) concentration equal to 135 microM, when the initial NH4Cl concentration increased from 0.5 to 0.9 g/l. The activities of both citrate synthase (CS) and isocitrate dehydrogenase were significantly decreased in the presence of 0.9 g/l NH4Cl (e.g., 40% and 66%, respectively, in cells incubated for 95 h), while ammonium assimilation occurred via the glutamate dehydrogenase reaction. Furthermore, growth limitation occurred even in the presence of 0.5 g/l NH4Cl, when the DO concentration decreased from 135 to 30 microM. The activities of both CS and succinate dehydrogenase were dramatically decreased in cells grown at the lower DO concentration (e.g., 90% and 93% respectively, in a 95 h incubation), while ammonium assimilation was limited due to the low activities of both glutamate dehydrogenase and glutamate synthase. It is concluded that the threshold of ammonium concentration at which growth of A. lipoferum is limited, depends on the DO concentration in the medium.     

Effect of Azospirillum brasilense Sp245 lipopolysaccharide on the functional activity of wheat root meristematic cells

We studied changes in the physiological and biochemical parameters of wheat (Triticum aestivum L. ??Saratovskaya 29??) seedlings treated with lipopolysaccharide isolated from the outer membrane of the associative bacterium Azospirillum brasilense Sp245. The obtained data were compared with (i) the results of plant inoculation with whole Sp245 cells and (ii) the effects exerted by the lipopolysaccharide and whole cells of the enterobacterium Escherichia coli K12 and the specific legume symbiont Rhizobium leguminosarum 249. The functional activity of meristematic cells was judged by their mitotic index and by the results of immunochemical determination of the proliferative antigen of initials, a molecular marker for wheat meristem cells. Treatment of the seedling root system with 10 ??g mL^?1 of Sp245 lipopolysaccharide increased the mitotic index (1.8-fold) and the antigen content (approximately 1.4-fold). These increases were comparable to the effects produced by whole cell inoculation (2- and 1.4-fold, respectively). Our findings give grounds to consider lipopolysaccharide as an active component of the Azospirillum cell surface that not only determines bacterial contact interactions with wheat roots but also participates in the induction of plant responses to these interactions. We finally discuss the linkage between the proliferative antigen of initials and the transduction of a hormonal signal to the cell, as well as the informational value of this antigen as an indicator of effectiveness of plant?Cbacterial interactions.     

Characterization of a novel lipid A structure isolated from Azospirillum lipoferum lipopolysaccharide

The structure of lipid A from Azospirillum lipoferum, a plant-growth-promoting rhizobacterium, was investigated. It was determined by chemical analysis, mass spectrometric methods, as well as 1D and 2D NMR spectroscopy. Because of the presence of substituents, the investigated lipid A differs from typical enterobacterial lipid A molecules. Its backbone is composed of a beta-(1,6)-linked D-glucosamine disaccharide but lacks phosphate residues. Moreover, the reducing end of the backbone (position C-1) is substituted with alpha-linked d-galacturonic acid. 3-hydroxypalmitoyl residues are exclusively connected to amino groups of the glucosamine disaccharide. Hydroxyls at positions C-3 and C-3' are esterified with 3-hydroxymyristic acids. Primary polar fatty acids are partially substituted by nonpolar fatty acids (namely, 18:0, 18:1 or 16:0), forming acyloxyacyl moieties.     

Transformation studies of Bacillus thuringiensis cryIC gene into a nitrogen-fixing Azospirillum lipoferum

A lepidopteran toxin gene, cryIC (pSB607) from entomopathogenic Bacillus thuringiensis subsp. aizawai was introduced into nitrogen-fixing Azospirillum lipoferum by transformation. Regeneration of spheroplasts was achieved at 99% with 39% frequency of regeneration. Transformants were screened on NB kanamycin with ampicillin plates and 4 transformants were selected after ten generations. SDS-PAGE and Western blot analysis confirmed the presence of a 68 kDa protein in the transformants. Studies on utilization of carbon sources indicate that glucose and sucrose are the most favorable carbon sources and 2% molasses is the cheap alternate carbon source for the better growth of parent A. lipoferum and transformants.     

Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml⁻¹, whereas HPLC revealed the presence of only 0.5 μg of IAA ml⁻¹. Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml⁻¹, respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml⁻¹ should be viewed with particular caution. Metabolism of [2′-¹⁴C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize (Zea mays L.) seedlings gave rise to [¹⁴C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′-¹⁴C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.