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.

Erratum to: "Structural Relationships Between Genetically Closely Related O-Antigens of Escherichia coli and Shigella spp." [Biochemistry (Moscow), 81, 600 (2016)].

This corrects the article DOI: 10.1134/S0006297916060067.

O-Antigens of Escherichia coli Strains O81 and HS3-104 Are Structurally and Genetically Related, Except O-Antigen Glucosylation in E. coli HS3-104.

Glycerophosphate-containing O-specific polysaccharides (OPSs) were obtained by mild acidic degradation of lipopolysaccharides isolated from Escherichia coli type strain O81 and E. coli strain HS3-104 from horse feces. The structures of both OPSs and of the oligosaccharide derived from the strain O81 OPS by treatment with 48% HF were studied by monosaccharide analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy. Both OPSs had similar structures and differed only in the presence of a side-chain glucose residue in the strain HS3-104 OPS. The genes and the organization of the O-antigen biosynthesis gene cluster in both strains are almost identical with the exception of the gtr gene cluster responsible for glucosylations in the strain HS3-104, which is located elsewhere in the genome.

Structural Relationships Between Genetically Closely Related O-Antigens of Escherichia coli and Shigella spp.

Gene clusters for biosynthesis of 24 of 34 basic O-antigen forms of Shigella spp. are identical or similar to those of the genetically closely related bacterium Escherichia coli. For 18 of these relatedness was confirmed chemically by elucidation of the O-antigen (O-polysaccharide) structures. In this work, structures of the six remaining O-antigens of E. coli O32, O53, O79, O105, O183 (all related to S. boydii serotypes), and O38 (related to S. dysenteriae type 8) were established using (1)H and (13)C NMR spectroscopy. They were found to be identical to the Shigella counterparts, except for the O32- and O38-polysaccharides, which differ in the presence of O-acetyl groups. The structure of the E. coli O105-related O-polysaccharide of S. boydii type 11 proposed earlier is revised. The contents of the O-antigen gene clusters of the related strains of E. coli and Shigella spp. and different mechanisms of O-antigen diversification in these bacteria are discussed in view of the O-polysaccharide structures established. These data illustrate the value of the O-antigen chemistry and genetics for elucidation of evolutionary relationships of bacteria.

[Characterization of the Lipopolysaccharides of Pseudomonas chlororaphis].

Lipopolysaccharides (LPS) from two strains ot Pseudomonas chlororaphis subsp. aureofaciens,UCM B-111 and UCM B-306, were isolated and characterized. The LPS preparations exhibited low toxicity, high pyrogenicity and high antiviral activity. Mild acid hydrolysis was used to obtain the O-specific polysaccharides. Their structures were established by monosaccharide analysis and determination of absolute configurations, as well as by 1D and 2D NMR spectroscopy. The O-polysaccharides were shown to contain the linear tri- or tetrasaccharide repeating units. Both O-polysaccharides were structurally heterogeneous: P. chlororaphis subsp. aureofaciens UCM B-111--> 4)-αD-GalpNAc6Ac-(1 --> 3)-ß-D-QuipNAc-(1 --> 6)-αD-GlcpNAc-(l --> ßD-GlcpNAc-(l --> 3)] GalNAc -60%; degree of the non-stoichiometric 6-O-acetylation of GalNAc -60%; P. chlorophis subsp. aureofaciens UCM B-306 --> 3)-α-D-Rhap-(1 --> 4)-α-D-GalpNAcAN-(1 --> 3)-αD-QuipNAc4NAc-(1 -->, where GalNAcAN is 2-acetamido-2-deoxy-D-galacturonamide, the degree of non-stoichiometric amidation of the GalNAcA residue -60%.

[Characterization of the lipopolysaccharide from Budvicia aquatica DLR 20186].

A lipopolysaccharide (LPS) from Budvicia aquatica DRL 20186 was isolated, studied, and chemically identified. It was shown to be lowly toxic, but highly pyrogenic. Its fatty acid composition was similar to that of the LPS from other Enterobacteriaceae, with predominance of tetradecanoic (32.7%) and 3-hydroxytetradecanoic acids (23.8%). Hexadecenoic (20.4%), hexadecanoic (11.8%), and dodecanoic acids (8.4%) were also revealed. Double immunodiffusion in agar by the Ouchterlony method revealed antigenic activity of the B. aquatica DLR 20186 LPS in a homologous system. In cross reactions, however, it did not interact with the antisera to other @B. aquatica@ strains.

[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.

Isolation and structural identification of glycopolymers of Bifidobacterium bifidum BIM B-733D as putative players in pathogenesis of autoimmune thyroid diseases.

Bifidobacterium bifidum 791 (commercially available as B. bifidum BIM B-733D) cell-surface biopolymers (BPs) interact selectively with human serum thyroid peroxidase (TPO) and thyroglobulin (Tg) autoantibodies (anti TPO and anti Tg, respectively). BPanti-TPO and BPanti-Tg were isolated from the soluble fraction of B. bifidum BIM B-733D by affinity chromatography with anti-TPO or anti-Tg, respectively. Homogeneity of affinity eluates (AEanti-TPO and AEanti-Tg) was tested by size exclusion chromatography. For each AE, the elution profiles generated on the basis of absorbance at 280 nm do not conform to ELISA data for functional activity characteristic of BPs. Moreover, high functional activity was detected in chromatographic fractions that had significantly different molecular weights and no absorbance at 280 nm, which suggests a non-protein (carbohydrate) nature of BPanti-TPO and BPanti-Tg. The semi-preparative size exclusion chromatography of AEanti-TPO and AEanti-Tg with detection by refractometer gave 5,000-7,000 Da fractions containing substances that interact selectively with either anti TPO (BPanti-TPO) or anti-Tg (BPanti-Tg) according to ELISA data. Analysis by two-dimensional NMR spectroscopy including a 1H, 13C-heteronuclear single-quantum coherence experiment indicated that both substances are linear α-1,6-glucans. For the first time, an immunological similarity (molecular mimicry) of glycopolymers of B. bifidum BIM B-733D and human thyroid proteins, TPO and Tg, was shown. On the whole, our data point to a possible role of bifidobacteria in the pathogenesis of autoimmune thyroid diseases (ATD). The main requirements for triggering/acceleration or prevention/abrogation of ATD by bifidobacteria through molecular mimicry mechanism are hypothesised to be (1) genetic predisposition to ATD and (2) intestinal epithelium penetration by α-1,6-glucan.

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.

[The structure of the O-specific polysaccharide from a mutant of nitrogen-fixing rhizobacterium Azospirillum brasilense Sp245 with an altered plasmid content].

The rhizobacteria Azospirillum brasilense Sp245 produce antigenically different lipopolysaccharides LPSI and LPSII, both containing identical pentasaccharides built from D-rhamnose residues as the repeated chains of O-specific oligosaccharides (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 A. brasilense mutant Sp245.5 appeared to be a disaccharide consisting of residues of N-acetyl-D-galactosamine and N-acetyl-D-mannosaminuronic acid: [Formula: see text].

[Chemical characteristics and endotoxic activity of the lipopolysaccharide of Rahnella aquatilis 2-95].

The lipopolysaccharide (LPS) from a new Enterobacteriaceae species, Rahnella aquatilis 2-95, was isolated and investigated. The structural components of the LPS molecule, namely, lipid A, core oligosaccharide, and O-specific polysaccharide, were obtained by mild acid hydrolysis. In lipid A, 3-oxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. The major monosaccharides of the core oligosaccharide were galactose, arabinose, fucose, rhamnose, and an unidentified component. The O-specific polysaccharide was found to be assembled of a repeated trisaccharide unit of the following structure: [structure: see text]. The R. aquatilis 2-95 LPS is less toxic and more pyrogenic as compared to the one from the R. aquatilis 1-95 strain studied earlier. Both acyl and phosphate groups are essential for toxic and pyrogenic activity of R. aquatilis 2-95 LPS.

[Composition, structure, and biological properties of lipopolysaccharides from different strains of Pseudomonas syringae pv. atrofaciens].

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C10:0, C12:0, 2-HO-C12:0, 3-HO-C12:0, C16:0, C16:1, C18:0, and C18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L- (two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substitute. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.

[Characterization of lipopolysaccharides from Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) with O-chains represented by alpha-glucan].

Results of studies of the structurally unique O-chains of lipopolysaccharides, which were isolated from the dry biomass of Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) by the Westphal technique and purified by repeated ultracentrifugation, are reported. The bulk of the lipopolysaccharide preparations contained S- and R-molecules at an average molar ratio of 1: 2. The main components of the hydrophobic moiety of lipid A were 3-hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, and octadecanoic acids, as well as hexadecenoic and octadecenoic acids. Glucosamine and phosphoethanolamine were identified as components of the hydrophilic moiety of lipid A. The degree of lipid A phosphorylation amounted to 3-4%. Fractions of the core oligosaccharide contained glucose, galactose, mannose, rhamnose, arabinose, glucosamine (only in strain IMB 2108), alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO). Heptose was present in trace amounts. O-specific polysaccharide chains were represented by a linear polymer of D-glucose units, which were linked together via alpha-(1,4) glycoside bonds. The existence of P. fluorescens strains that have alpha-1,4-glucan as the O-chain of their lipopolysaccharides has not been described before.

[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.

[Characterization of the lipopolysaccharide from Rahnella aquatilis 1-95].

The lipopolysaccharide from the freshwater bacterium Rahnella aquatilis 1-95 has been isolated and investigated for the first time. The structural components of the lipopolysaccharide molecule: lipid A, core oligosaccharide, and O-specific polysaccharide were isolated by mild acidic hydrolysis. In lipid A, 3-hydroxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. In the core oligosaccharide, galactose, arabinose, fucose, and an unidentified component were shown to be the major monosaccharides. The O-specific polysaccharide consists of a regularly repeating trisaccharide unit with the acyl and phosphate following structure: [structure: see text] groups have been shown to be responsible for the toxic and pyrogenic properties of the lipopolysaccharide of R. aquatilis.

[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.