Phage antibodies for the immunochemical characterization of Herbaspirillum seropedicae Z78 glycopolymers

Microbial carbohydrate antigens are targets of the immune systems of hosts. In this context, it is of interest to obtain data that will permit judgment of the degree of heterogeneity, chemical makeup, and localization of the antigenic determinants of the Herbaspirillum surface glycopolymers. A sheep single-chain antibody-fragment phage library (Griffin.1, UK) was used to obtain miniantibodies to the exopolysaccharides (EPS-I and EPS-II), capsular polysaccharides (CPS-I and CPS-II) and lipopolysaccharide (LPS) of Herbaspirillum seropedicae Z78. To infer about the presence or absence of common antigenic determinants in the cell-surface polysaccharides of H. seropedicae Z78, we ran a comparative immunoassay using rabbit polyclonal and phage recombinant antibodies to the surface glycopolymers of H. seropedicae Z78. We isolated and purified the exopolysaccharides (EPS-I and EPS-II), capsular polysaccharides (CPS-I and CPS-II), and lipopolysaccharide (LPS) of Herbaspirillum seropedicae Z78. Using rabbit polyclonal antibodies, we found that these cell-surface polysaccharides were of a complex nature. EPS-I, EPS-II, CPS-I, CPS-II, and LPS contained common antigenic determinants. CPS-I, CPS-II, and LPS also contained individual antigenic determinants composed of rhamnose, N-acetyl-d-glucosamine, and N-acetyl-d-galactosamine—sugars responsible for cross-reactions with miniantibodies. The anti-LPS miniantibodies were more specific for the core region of the LPS, in which rhamnose was the most abundant sugar, than they were specific for its O portion. The miniantibodies we isolated can be useful reagents not only in basic biochemical research but also in clinical diagnostic and therapeutic applications.     

Structural analysis of the O-polysaccharide from the lipopolysaccharide of Azospirillum brasilense S17

A mixture of two structurally distinct neutral O-polysaccharides was obtained by mild acid degradation of the lipopolysaccharide isolated by the phenol/water extraction from the asymbiotic diazotrophic rhizobacterium Azospirillum brasilense S17. The following structures of the O-polysaccharides were established by composition and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy, including a 2D NOESY experiment: [Formula: see text] where L-Rha2Me stands for 2-O-methyl-L-rhamnose and SHb for the (S)-3-hydroxybutanoyl group. The occurrence of two distinct polysaccharides is reported for the first time in Azospirillum spp.     

Role of the Polysaccharide Components of Azospirillum brasilense Capsules in Bacterial Adsorption on Wheat Seedling Roots

Azospirillum brasilense cells deprived of capsular exopolysaccharides completely lost their ability to bind wheat germ agglutinin (WGA) and much of their ability to attach to wheat seedling roots. The decapsulation of bacterial cells by washing them with a NaCl solution led to an increase in the relative hydrophobicity of the cell surface. The pretreatment of wheat seedling roots with N-acetyl-D-glucosamine (GlcNAc) or the GlcNAc-containing polysaccharide complexes stripped from Azospirillum cells reduced their attachment to the roots. Under the experimental conditions used (3-h incubation of wheat seedling roots with exponential-phase azospirilla), bacterial adsorption is mainly driven by the specific mechanisms attachment of the cells to the roots, whose operation is due to the capsular polysaccharide components and the WGA present on the wheat seedling roots.     

Structural and functional peculiarities of the lipopolysaccharide of Azospirillum brasilense SR55, isolated from the roots of Triticum durum

Azospirillum brasilense SR55, isolated from the rhizosphere of Triticum durum, was classified as serogroup II on the basis of serological tests. Such serogroup affiliation is uncharacteristic of wheat-associated Azospirillum species. The lipid A of A. brasilense SR55 lipopolysaccharide contained 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, hexadecanoic and octadecenoic fatty acids. The structure of the lipopolysaccharide's O polysaccharide was established, with the branched octasaccharide repeating unit being represented by l-rhamnose, l-3-O-Me-rhamnose, d-galactose and d-glucuronic acid. The SR55 lipopolysaccharide induced deformations of wheat root hairs. The lipopolysaccharide was not involved in bacterial cell aggregation, but its use to pretreat wheat roots was conducive to cell adsorption. This study shows that Azospirillum bacteria can utilise their own lipopolysaccharide as a carbon source, which may give them an advantage in competitive natural environments.     

The use and development of the dynamic light-scattering method to investigate supramolecular structures in aqueous solutions of bacterial lipopolysaccharides

The temperature dependence of the scattering intensity, average size, and size distribution for supramolecular particles in aqueous solutions of lipopolysaccharides from Azospirillum bacteria was investigated by dynamic light scattering. Relationships were obtained that made it possible to comparatively estimate the mass–volume concentration of the biopolymeric substance in suspensions and the number concentration of supramolecular particles with their size and degree of polydispersity taken into account. In the range from 0 to 60°C, two types of the temperature dependence of scattering intensity were found: (a) with an irregular spasmodic change in scattering intensity and with considerable heterogeneity of the systems with respect to particle size and (b) with a smoother character of this dependence with considerably decreased heterogeneity of the suspensions. In the ranges of the latter type, whose location depended on what strain was used to isolate lipopolysaccharides, it proved to be possible to correctly determine the parameters of the supramolecular particles (of the supposedly formed micellar phase) by dynamic light scattering. The revealed statistically significant differences in the size and the concentration of the micellar particles are explained by their dependence on the peculiarities of the chemical structure of lipopolysaccharides. Atomic-force microscopy was used for an independent morphological estimation of the preparations, yielding good agreement with the dynamic light-scattering results.     

Immunochemical Characterization of the Capsular Polysaccharide of Azospirillum irakense KBC1

The repeating unit structure of Azospirillum irakense KBC1 capsular polysaccharide (CPS) was established and was found to be identical to that of the O polysaccharide of A. irakense KBC1 lipopolysaccharide (LPS). The antigenic heterogeneity of the LPS and the CPS was shown to be related to differences in the macromolecular organization of these glycopolymers. After an immune response activation, R-form CPS molecules were found to be predominant.     

Structural peculiarities of the O-specific polysaccharides of <Emphasis Type="Italic">Azospirillum</Emphasis> bacteria of serogroup III

Lipopolysaccharides and O-specific polysaccharides were isolated from the outer membrane of bacterial cells of three strains belonging to two Azospirillum species, and their structures were established by monosaccharide analysis including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy. It was shown that while having the identical composition, the O-polysaccharides have different branched tetrasaccharide repeating units. Two neutral polysaccharides were found in the lipopolysaccharide of A. brasilense 54, and the structure for the predominant O-polysaccharide was determined. The structural data, together with results of serological studies, enabled assignment of strains examined to a novel serogroup, III. The chemical basis for the serological relatedness among the azospirilla of this serogroup is presumably the presence of a common →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→oligosaccharide motif in their O-polysaccharides.     

The structure of the O-specific polysaccharide from a mutant of nitrogen-fixing rhizobacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 with an altered plasmid content

The rhizobacteria Azospirillum brasilense Sp245 produce immunochemically different lipopolysaccharides LPSI and LPSII, both containing identical pentasaccharides built from D-rhamnose residues as the repeating units of O-specific polysaccharides (OPS). In this study, we report the structure of the OPS from A. brasilense LPSI⁻LPSII⁻ mutant Sp245.5, which spontaneously lost the p85 and p120 plasmids upon the formation of a new 300-MDa megaplasmid after the long-term storage of the bacteria in a rich medium. The repeating unit of the OPS of A. brasilense Sp245.5 appeared to be a disaccharide consisting of residues of N-acetyl-D-galactosamine and N-acetyl-D-mannosaminuronic acid:

The role of polysaccharide-containing components of the Azospirillum brasilense capsule in adsorbing bacteria on wheat seedling roots

Azospirillum brasilense cells deprived of capsular exopolysaccharides completely lost their ability to bind wheat germ agglutinin (WGA) and much of their ability to attach to wheat seedling roots. The decapsulation of bacterial cells by washing them with a NaCl solution led to an increase in the relative hydrophobicity of the cell surface. The pretreatment of wheat seedling roots with N-acetyl-D-glucosamine (GlcNAc) or the GlcNAc-containing polysaccharide complexes stripped from Azospirillum cells reduced their attachment to the roots. Under the experimental conditions, 3-h incubation of wheat seedling roots with exponential-phase azospirilla, bacterial adsorption is mainly driven by the attachment of the cells to the roots, whose operation is due to the capsular polysaccharide components and the WGA present on the wheat seedling roots.