Structural studies of O-specific polysaccharide(s) and biological activity toward plants of the lipopolysaccharide from Azospirillum brasilense SR8.

Lipopolysaccharide (LPS) was extracted from dry bacterial cells of plant-growth-promoting bacterium Azospirillum brasilense SR8 (IBPPM 5). The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis, 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments, computational NMR-based structure analysis, and Smith degradation. The OPS was shown to contain two types of repeating units of the following structure: Both OPS structures are present in A. brasilense 54, from which structure 1 has been reported earlier (Fedonenko et al., 2011), whereas to our knowledge structure 2 has not been hitherto found in bacterial saccharides. Treatment of wheat seedling roots with LPS of A. brasilense SR8 increased the number of root hair deformations as compared to seedlings grown without LPS, but had no effect on adsorption of the bacteria to the root surface. A. brasilense SR8 was able to utilize LPS of several structurally related Azospirillum strains.

[Effect of flavonoids on the composition of surface glycopolymers in Azospirillum lipoferum Sp59b].

Cultivation of the type strain Azospirillum lipoferum Sp59b in the presence of the flavonoid quercetin induced modification of the structure of the bacterial lipopolysaccharide. Cultivation in the presence of the flavonoid was shown to result in altered serological characteristics of the bacteria, increased heterogeneity of the outer membrane lipopolysaccharide pool, as well as in modified composition and fatty acid ratio of lipid A. The flavonoid was shown to induce the synthesis of the O-specific polysaccharide with the repeating structure represented by a tetrasaccharide consisting of a linear trisaccharide fragment of α-L-Rhap residues in the main chain and the terminal ß-D-Glcp residue. The structure of this O-specific polysaccharide was identical to the previously determined structure of the capsular polysaccharide of these bacteria grown without quercetin. Modifications in the structural composition of the capsular polysaccharide induced by cultivation in the presence of quercetin were revealed.

[Characterization of the lipopolysaccharides of serogroup II Azospirillum].

Lipopolysaccharides of six Azospirillum strains (A. brasilense SR50, SR80, SR88, SR109, SR111, SR115, and A. lipoferum SR 42) isolated from the rhizosphere of cereal plants of Saratov oblast, Russia and assigned to serogroup II by serological analysis were studied. In the lipid A fatty acid composition, the lipopolysaccharides under study were similar to those of other Azospirillum strains and were characterized by predominance of 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, and octadecenoic acids. Monosaccharide analysis of the O-specific polysaccharides (including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy) revealed the presence of two types of repeating units in varying ratios. High degree of serological similarity between the strains under study was shown to result from the presence of repeating units with identical structure in their O antigens.

Structural peculiarities of the O-specific polysaccharides of Azospirillum 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.

Capsular polysaccharide of the bacterium Azospirillum lipoferum Sp59b: structure and antigenic specificity.

Antigenic differences were revealed between the cell wall outer membrane lipopolysaccharides and the capsular high molecular weight bioglycans for a typical strain of the nitrogen-fixing rhizobacterium Azospirillum lipoferum Sp59b using antibodies prepared against the homologous lipopolysaccharide and lipopolysaccharide-protein complex. From the capsular lipopolysaccharide-protein and polysaccharide-lipid complexes of A. lipoferum Sp59b, polysaccharides were isolated and their structure was for the first time established in Azospirillum by monosaccharide analysis which included determination of the absolute configurations, methylation, O-deacetylation, and one- and two-dimensional NMR spectroscopy. The polysaccharides of the capsular complexes were shown to have identical structure of the branched tetrasaccharide repeating unit, which differs from the structure of the O-specific polysaccharide within the outer membrane lipopolysaccharide of this strain.

[Chemical and serological studies of liposaccharides of bacteria of the genus Azospirillum].

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS(Cd), LPS(Sp59b), LPS(Sp7), LPS(S17), and LPS(KBC1) preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab(Sp59b) and Ab(Cd) were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.

[Role of the clasping-leaved pondweed (Potamogeton perfoliatus) polysaccharides in formation of its bacterial environment].

The effect of the polysaccharides of clasping-leaved pondweed (Potamogeton perfioliatus) on the formation of a bacteriocenosis of this plant was demonstrated by research on chemoreception, relative surface hydrophobicity, and the growth characteristics of the members of five bacterial genera abundant in this microbiocenosis. The plant heteropolysaccharides of anionic and cationic nature were found to participate in selective stimulation or inhibition of growth of some microbial groups in surrounding water. These findings improve our understanding of the spectrum of physiological activity of glycopolymers of diverse origin.

[Chemical composition and immunochemical characteristics of the lipopolysaccharide of nitrogen-fixing rhizobacterium Azospirillum brasilense CD].

The chemical composition of the lipopolysaccharide of the associative diazotrophic rhizobacterium Azospirillum brasilense Cd has been studied. Among the main components of the hydrophobic part of the lipopolysaccharide, we identified 3-hydroxytetradecanoic, hexadecenoic, 3-hydroxyhexadecanoic, hexadecanoic, octadecenoic, and nanodecanoic fatty acids; the carbohydrate part contained rhamnose, galactose, and mannose. Polyclonal antibodies against the preparation under study were raised in rabbits. Serological relations between A. brasilense Cd and other strains of Azospirillum spp. were studied using double radial immunodiffusion and enzyme-linked immunosorbent assay.

[Determination of the structure of the repeated unit of the Azospirillum brasilense SR75 O-specific polysaccharide and homology of the lps loci in the plasmids of Azospirillum brasilense strains SR75 and Sp245].

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the OPSs of previously studied A. brasilense strain Sp245, isolated from surface-sterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains was localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects.

[A comparison of the lipopolysaccharides and O-specific polysaccharides of Azospirillum brasilense Sp245 and its omegon-Km mutants KM018 and Km252]. / Sravnitel'naia kharakteristika lipopolisakharidov i O-spetsificheskikh polisakharidov Azospirillum brasilense Sp245 i ego omegon-Km mutantov KM018 i 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.

[Effect of cultivation parameters on the composition of extracellular polysaccharide containing substances in bacterium Azospirillum brasilense]. / Issledovanie vliianiia uslovii vyrashchivaniia bakterii Azospirillum brasilense na sostav vnekletochnykh polisakharidsoderzhashchikh materialov.

Maintenance of pH 7.0 during the fermentation period favors accumulation of high-molecular polysaccharide-containing components, the so-called lipopolysaccharide-protein and polysaccharide-lipid complexes, in the capsules and culture medium. Increased pH of the culture medium to 8.0 reduced the period of exponential growth and the yield of polysaccharide-containing complexes as compared to the optimal conditions. Maintenance of pH 5.5 suppressed the culture growth and polysaccharide production. The polysaccharide-lipid complexes obtained when pH was stabilized at the level of 7.0-8.0 had a relatively low molecular weight and contained only acidic polysaccharides. The use of potassium gluconate instead of sodium malate as a source of carbon in the culture medium changed the polysaccharide composition and increased the content of glucosamine, which increased the attraction of polysaccharides to wheat germ agglutinin. Prolongation of Azospirillum cultivation to five days introduced new glucose-containing polysaccharide components in the capsule.

[Biological role of lectins from Bacillus polymyxa during interaction with surface carbohydrates of wheat roots ]. / Izuchenie biologicheskoi roli lektinov Bacillus polymyxa pri vzaimodeistvii s uglevodami fraktsii ékzokomponentov kornei pshenitsy.

The data obtained demonstrate the capacity of lectins LI and LII from the soil nitrogen-fixing bacteria Bacillus polymyxa 1460 to change their proteolytic activity after interaction with surface carbohydrates of wheat seedling roots.

[The role of cell surface bacterial lectins in the aggregation of Azospirilla]. / Uchastie bakterial'nykh lektinov kletochnoi poverkhnosti v agregatsii Azospirill.

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.

[Study of protective role of the polysaccharide-containing components of capsules of Azospirillum brasilense]. / Issledovanie zashchitnoi roli polisakharidsoderzhashchikh komponentov kapsuly bakterii Azospirillum brasilense.

The involvement of the carbohydrate components of the Azospirillum brasilense Sp245 capsules in bacterial protection from the action of extreme factors was investigated. The survival of encapsulated and non-encapsulated azospirilla exposed to elevated (46-48 degrees C) and below-freezing (-20 and -70 degrees C) temperatures, extreme pH values (2 and 10), and to drying was studied. High-molecular-weight carbohydrate-containing complexes (lipopolysaccharide-protein complex and polysaccharide-lipid complex) were isolated from the capsular material of azospirilla. It was shown that the addition of these complexes to the suspension of decapsulated cells before exposing them to extreme factors enhanced their survival rates by 15 to 51%.

[Role of the agglutinating proteins of bacilli and rhizobia in bacterial interaction]. / Rol' aggliutiniruiushchikh belkov batsill i rizobii v mezhbakterial'nykh vzaimodeistviiakh.

The surface agglutinating proteins of the soil nitrogen-fixing bacteria Bacillus polymyxa 1460 and Rhizobium leguminosarum 252 were found to be able to interact with the polysaccharide complexes of certain azospirilla and rhizobia. Such interactions are most likely involved in the formation of nitrogen-fixing plant-bacterial associations in the rhizosphere.

[Participation of azospirillium polysaccharides in interaction with wheat root surface]. / Uchastie lipopolisakharidov azospirill vo vzaimodeistvii s poverkhnost'iu kornei pshenitsy.

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilense Sp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillum cell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilense Sp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillum cells with plants is discussed.

[Role of the surface and extracellular substances of the phytopathogenic bacterium Xanthomonas campestris in its interactions with cabbage plants]. / Rol' poverkhnostnykh i vnekletochnykh soedinenii bakterii Xanthomonas campestris v patogennykh vzaimootnosheniiakh s rasteniiami kapusty.

Changes in some physiological and biochemical characteristics of cabbage (cv. Slava) seedling roots in response to inoculation with the phytopathogen Xanthomonas campestris and its surface and extracellular substances were evaluated. Seven days after the inoculation, the growth of the roots was slightly suppressed and they contained increased amounts of peroxidase. The effect of the lipopolysaccharides stripped from the cell surface or isolated from the culture liquid of X. campestris was similar to that of the whole cells of the phytopathogen. The bacterial lectin isolated from the cell surface material did not induce any defense response in cabbage plants but, presumably, could play a role in the contact interactions between bacteria and plants.

[The role of polysaccharide-containing components of the Azospirillum brasilense capsule in adsorbing bacteria on wheat seedling roots]. / Rol' polisakharidsoderzhashchikh komponentov kapsuly Azospirillum brasilense v adsorbtsii bakterii na korniakh prorostkov pshenitsy.

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.

[Initial stages of interaction of Azospirillum brasilense bacteria with wheat germ roots: adsorption, deformation of root hairs]. / Issledovanie nachal'nykh étapov vzaimodeistviia bakterii Azospirillum brasilense s korniami prorostkov pshenitsy: adsorbtsii, deformatsii kornevykh voloskov.

The initial stages of colonization of wheat roots by cells of Azospirillum brasilense strains 75 and 80 isolated from soils of the Saratov oblast were studied. The adsorption of azospirilla on root hairs of soft spring wheats rapidly increased in the first hours of incubation, going then to a plateau phase. Within the first 15 h of incubation, exponential-phase cells were adsorbed more intensively than stationary-phase cells. Conversely, stationary-phase cells were adsorbed more intensively than exponential-phase cells, if the period of azospirilla incubation with the wheat roots was extended. As the time of incubation increased, the attachment of azospirilla to the wheat roots became stronger. The effect of cell attachment to root hairs was strain-dependent; the number of adsorbed cells of a given strain of azospirilla was greater in the case of host wheat cultivars. The deformation of wheat root hairs was affected by the polysaccharide-containing complexes isolated from the capsular material of azospirilla. The suggestion is made that common receptor systems are involved in the adsorption of azospirilla on roots and in root hair deformation.

[Properties of polysaccharide complexes produced by Azospirillum brasilense and polysaccharides isolated from these complexes]. / Svoistv polisakharidnykh kompleksov, produtsiruemykh Azospirillum brasilense, i poluchaemykh iz nikh polisakharidov.

Azospirillum brasilense strains Sp7, Sp7 (S-form), Sp107, Sp245, Sp246, S17, and 15 when cultivated in a liquid synthetic malate medium up to the end of the logarithmic phase of growth were shown to produce at least two complex polysaccharide-containing components. The components were arbitrarily called lipopolysaccharide-protein complex (LPPC) and polysaccharide-lipid complex (PSLC). The LPPCs and PSLCs from the strains Sp7, Sp107, Sp245, Sp246, and S17 were shown to interact with a wheat germ agglutinin (WGA). From PSLCs, acidic polysaccharides (PS) were isolated and their specific rotation, molecular masses, affinity for WGA, and monosaccharide composition were determined. The PSs of all strains contained rhamnose, galacturonic acid, and galactose (except the strain Sp246), and glucosamine (except the strain Sp7 (S-form)), while the PSs of the strains Sp7 and S17 also contained fucose and mannose, respectively. It is suggested that LPPCs and PSLCs may be involved in the process of interaction of azospirilla with wheat root surfaces, and that PSsare probably active parts of the LPPCs and PSLCs. The ability of LPPCs and PSs to interact with Congo red in aqueous solutions was investigated.