Structural studies of the O polysaccharides from the lipopolysaccharides of Azospirillum thiophilum BV-ST and Azospirillum griseum L-25-5w-1T.

Diazotrophic bacteria of the genus Azospirillum are known widely, because they are ubiquitous in the rhizosphere and can promote the growth and performance of nonlegume plants. Recently, more Azospirillum species have been isolated from sources other than plants or soil. We report the structures of the O polysaccharides (OPSs) from the lipopolysaccharides of the type strains A. thiophilum BV-ST (1) and A. griseum L-25-5w-1T (2), isolated from aquatic environments. Both structures have a common tetrarhamnan in the repeating-unit, which is decorated with a side xylose in the OPS of A. thiophilum BV-ST.

Structure of the O-polysaccharide from the moderately halophilic bacterium Halomonas fontilapidosi KR26.

Lipopolysaccharide was obtained from the aerobic moderately halophilic bacterium Halomonas fontilapidosi KR26. The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide and was examined by chemical methods and by 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC, and HMBC experiments. The following structure of the linear tetrasaccharide repeating unit was deduced. →2)-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-ß-d-Galp-(1→.

Structure of the O-specific polysaccharide of Ochrobactrum endophyticum KCTC 42485T containing 3-(3-hydroxy-2,3-dimethyl-5-oxoprolyl)amino-3,6-dideoxy-d-galactose.

Ochrobactrum endophyticum (syn. Brucella endophytica) is an aerobic species of Alphaproteobacteria isolated from healthy roots of Glycyrrhiza uralensis. Here we report the structure of the O-specific polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of the type strain KCTC 42485:→3)-α-l-FucpNAc-(1→3)-ß-d-QuipNAc-(1→2)-ß-d-Fucp3NAcyl-(1→ where Acyl is 3-hydroxy-2,3-dimethyl-5-oxoprolyl. The structure was elucidated using chemical analyses along with 1H and 13C NMR spectroscopy (including 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC, HSQC-TOCSY and HSQC-NOESY experiments). To our knowledge the OPS structure is novel and has not been previously published.

Structural characterization and physicochemical properties of the exopolysaccharide produced by the moderately halophilic bacterium Chromohalobacter salexigens, strain 3EQS1.

A strain, 3EQS1, was isolated from a salt sample taken from Lake Qarun (Fayoum Province, Egypt). On the basis of physiological, biochemical, and phylogenetic analyses, the strain was classified as Chromohalobacter salexigens. By 72 h of growth at 25 °C, strain 3EQS1 produced large amounts (15.1 g L-1) of exopolysaccharide (EPS) in a liquid mineral medium (initial pH 8.0) containing 10% sucrose and 10% NaCl. The EPS was precipitated from the cell-free culture medium with chilled ethanol and was purified by gel-permeation and anion-exchange chromatography. The molecular mass of the EPS was 0.9 × 106 Da. Chemical analyses, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy showed that the EPS was a linear ß-D-(2 → 6)-linked fructan (levan). In aqueous solution, the EPS tended to form supramolecular aggregates with a critical aggregation concentration of 240 µg mL-1. The EPS had high emulsifying activity (E24, %) against kerosene (31.2 ± 0.4%), sunflower oil (76.9 ± 1.3%), and crude oil (98.9 ± 0.8%), and it also had surfactant properties. A 0.1% (w/v) aqueous EPS solution reduced the surface tension of water by 11.9%. The levan of C. salexigens 3EQS1 may be useful in various biotechnological processes.

Structure and antiproliferative activity of the polysaccharide from Halomonas aquamarina related to Cobetia pacifica.

Here, the results of the structure and the activity of capsular polysaccharides isolated from the Halomonas aquamarina EG27S8QL and Cobetia pacifica KMM3878 have been described. Both polysaccharides were studied by spectroscopic and chemical methods and were found to be structurally related sulfated galactans differing in the position of the sulfate group: →6)-ß-D-Galp3S-(1 â†’ 4)-ß-D-Galp3S-(1 â†’ 6)-ß-D-Galp3,4(S-Pyr)-(1 â†’ [H. aquamarina EG27S8QL] →6)-ß-D-Gal-(1 â†’ 4)-ß-D-Gal2,3S-(1 â†’ 6)-ß-D-Gal3,4(S-Pyr)-(1 â†’ [C. pacifica KMM3878] Structure of the CPS from H. aquamarina EG27S8QL has not been hitherto reported, whereas the CPS from C. pacifica KMM3878 was identical to the previously studied O-polysaccharide. The CPSs exhibited an antiproliferative effect and suppressed the colony formation of DLD-1 and MCF-7 cells in a different manner.

Lipopolysaccharide and flagellin of Azospirillum brasilense Sp7 influence callus morphogenesis and plant regeneration in wheat.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7 days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro.

Structure of the 4-O-[1-Carboxyethyl]-d-Mannose-Containing O-Specific Polysaccharide of a Halophilic Bacterium Salinivibrio sp. EG9S8QL.

The moderately halophilic strain Salinivibrio sp. EG9S8QL was isolated among 11 halophilic strains from saline mud (Emisal Salt Company, Lake Qarun, Fayoum, Egypt). The lipopolysaccharide was extracted from dried cells of Salinivibrio sp. EG9S8QL by the phenol-water procedure. The OPS was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The OPS was found to be composed of linear tetrasaccharide repeating units of the following structure: →2)-ß-Manp4Lac-(1→3)-α-ManpNAc-(1→3)-ß-Rhap-(1→4)-α-GlcpNAc-(1→, where Manp4Lac is 4-O-[1-carboxyethyl]mannose.

Structure of the O-specific polysaccharide from Azospirillum formosense CC-Nfb-7(T).

The lipopolysaccharide was obtained from the cells of Azospirillum formosense CC-Nfb-7(T), a diazotrophic bacterium isolated from agricultural soil. The O-specific polysaccharide (OPS) was released by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments, and Smith degradation. The following structure of partially methylated OPS composed of trisaccharide repeating units was established.

Structural and genetic characterization of the colitose-containing O-specific polysaccharide from the lipopolysaccharide of Herbaspirillum frisingense GSF30T.

The lipopolysaccharide (LPS) of Herbaspirillum frisingense GSF30T (HfGSF30), a non-pathogenic diazotrophic endobiont, was isolated by phenol-water extraction from bacterial cells and was characterized by chemical analyses and SDS PAGE. The O-specific polysaccharide (OPS, O-antigen), obtained by mild acid hydrolysis of the LPS, was examined by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments. The OPS was found to consist of branched tetrasaccharide repeating units of the following structure: [Formula: see text] This structure is unique among the known bacterial polysaccharide structures. Analysis of the HfGSF30 genome showed that it contained a set of sequentially arranged operons (presumably a cluster of genes) associated with the O-antigen. Amino acid sequence analysis using the BLAST program demonstrated the specificity of this putative cluster for Herbaspirillum spp. The genes responsible for the biosynthesis of the OPS of HfGSF30 were dispersed in the genome, constituting small operons. A putative O-antigen gene cluster of HfGSF30 was identified and found to be consistent with the OPS structure.

Isolation, structure, and potential biotechnological applications of the exopolysaccharide from Paenibacillus polymyxa 92.

Paenibacillus polymyxa 92, isolated from wheat roots, produced large amounts (38.4 g L-1) of exopolysaccharide (EPS) in a liquid nutrient medium containing 10 % (w/v) sucrose. The EPS was precipitated from the culture broth with cold acetone and was purified by gel filtration and anion-exchange chromatography. The molecular mass of the EPS was 2.29-1.10 × 105 Da. Diffuse reflectance infrared Fourier transform and nuclear magnetic resonance spectra showed that the EPS was a linear ß-(2→6)-linked fructan (levan). Aqueous EPS solutions showed pseudoplastic behavior when shear stress was applied at different temperatures. By using the Ostwald-de Waele model, the rheological characteristics of the EPS solution were ascertained. The sorption capacity of the EPS for Zn(II), Cd(II), Pb(II), and Cu(II) was investigated. Sorption was maximal (q = 481 mg g-1) for Cu(II) ions. In model experiments, treatment of wheat seeds with EPS solution significantly increased the length of seedling roots and shoots.

Bioremediation potential of a halophilic Halobacillus sp. strain, EG1HP4QL: exopolysaccharide production, crude oil degradation, and heavy metal tolerance.

A halophilic bacterial strain, EG1HP4QL, was isolated from a salt sample from Lake Qarun, Fayoum Province, Egypt. Morphological, physiological, biochemical, and phylogenetic analyses indicated that the strain belonged to the genus Halobacillus. Strain EG1HP4QL produced an extracellular polysaccharide (EPS), with production peaking (5.9 g L-1) during growth on medium S-G containing 2% (w/v) sucrose at 35 °C (pH 8.0). The EPS had significant emulsifying activity (E24 %) against kerosene (65.7 ± 0.8%), o-xylene (64.0 ± 1%), and sunflower oil (44.7 ± 0.5%). The composition of the EPS included two polymers-a negatively charged and a neutral one (~ 3:1)-in which mannose and glucose were the main neutral monosaccharide constituents. Strain EG1HP4QL was able to utilize crude oil (35.3%) as the sole carbon source within 12 days. The minimum inhibitory concentrations of heavy metals [Zn(II), Cd(II), Pb(II), Ni(II), and Cu(II)] for strain EG1HP4QL were 1.0, 2.0, 2.0, 2.5, and 5 mM, respectively.

Structure, gene cluster of the O antigen and biological activity of the lipopolysaccharide from the rhizospheric bacterium Ochrobactrum cytisi IPA7.2.

Lipopolysaccharide (LPS) of Ochrobactrum cytisi IPA7.2, a bacterium isolated from the roots of Solanum tuberosum L., was extracted from dry bacterial cells and chemically characterized. The O-specific polysaccharide was obtained by mild acid hydrolysis of the LPS and studied by sugar analysis and 1H and 13C NMR spectroscopy, including 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments. The polysaccharide was linear and consisted of trisaccharide repeating units of the following structure: A putative O-antigen gene cluster of O. cytisi IPA7.2 was identified and found to be consistent with the O-specific polysaccharide structure. The LPS of Ochrobactrum cytisi IPA7.2 promoted the growth of potato microplants in vitro.

Structure of the O-specific polysaccharide of Azospirillum doebereinerae type strain GSF71T.

O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of plant-growth-promoting rhizobacteria Azospirillum doebereinerae GSF71T and studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. It was established that the polysaccharide is linear and consists of tetrasaccharide repeating units with the following structure.

Structure of the O-specific polysaccharide from a halophilic bacterium Halomonas ventosae RU5S2EL.

Halomonas ventosae RU5S2EL, a halophilic Gram-negative bacterium isolated from salt sediments of lake Elton (Russia), was cultivated and the lipopolysaccharide was extracted by the Westphal procedure. The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments as well as Smith degradation. The OPS was found to consist of branched pentasaccharide repeating units of the following structure.

Structure of the O-specific polysaccharide from Azospirillum fermentarium CC-LY743T.

O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of nitrogen-fixing bacterium Azospirillum fermentarium CC-LY743T (IBPPM 578) and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments. The polysaccharide was found to be linear and to consist of alterating α-l-fucose and α-d-mannose residues in tetrasaccharide repeating units of the following structure: →2)-α-D-Manp-(1 → 3)-α-L-Fucp-(1 → 3)-α-D-Manp-(1 → 3)-α-L-Fucp-(1→.

Structural studies of the O-specific polysaccharide from detergent degrading bacteria Pseudomonas putida TSh-18.

An O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of bacteria Pseudomonas putida TSh-18, capable of degrading non-ionogenic technical detergents. The polysaccharide was found to contain a rarely occurring sugar derivative 4,6-dideoxy-4-[(R)-3-hydroxybutanoylamino]-d-galactose [d-Fucp4N(RHb)]. Sugar and methylation analyses, Smith degradation, solvolysis with CF3CO2H, and 1H and 13C NMR spectroscopy enabled elucidation of the following structure of the branched trisaccharide repeating units of the polysaccharide.

Elucidation of a masked repeating structure of the O-specific polysaccharide of the halotolerant soil bacteria Azospirillum halopraeferens Au4.

An O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide isolated by the phenol-water extraction from the halotolerant soil bacteria Azospirillum halopraeferens type strain Au4. The polysaccharide was studied by sugar and methylation analyses, selective cleavages by Smith degradation and solvolysis with trifluoroacetic acid, one- and two-dimensional (1)H and (13)C NMR spectroscopy. The following masked repeating structure of the O-specific polysaccharide was established: →3)-α-L-Rhap2Me-(1→3)-[ß-D-Glcp-(1→4)]-α-D-Fucp-(1→2)-ß-D-Xylp-(1→, where non-stoichiometric substituents, an O-methyl group (~45%) and a side-chain glucose residue (~65%), are shown in italics.

Structure of the polysaccharides from the lipopolysaccharide of Azospirillum brasilense Jm125A2.

Two polysaccharides were obtained by mild acid degradation of the lipopolysaccharide of associative nitrogen-fixing bacteria Azospirillum brasilense Jm125A2 isolated from the rhizosphere of a pearl millet. The following structures of the polysaccharides were established by sugar and methylation analyses, Smith degradation, and (1)H and (13)C NMR spectroscopy: [Formula: see text] Structure 1 has been reported earlier for a polysaccharide from A. brasilense S17 (Fedonenko YP, Konnova ON, Zdorovenko EL, Konnova SA, Zatonsky GV, Shaskov AS, Ignatov VV, Knirel YA. Carbohydr Res 2008;343:810-6), whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.

Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14.

Lipopolysaccharides from closely related Azospirillum brasilense strains, Sp246 and SpBr14, were obtained by phenol-water extraction. Mild acid hydrolysis of the lipopolysaccharides followed by GPC on Sephadex G-50 resulted in polysaccharide mixtures. On the basis of sugar and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy data, it was concluded that both bacteria possess the same two distinct polysaccharides having structures 1 and 2: [structure: see text]. Structure 1 has been reported earlier for a polysaccharide of A. brasilense 54 [Fedonenko et al., 2011] whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.

Structural studies of the O-specific polysaccharide(s) from the lipopolysaccharide of Azospirillum brasilense type strain Sp7.

Lipopolysaccharide was obtained by phenol-water extraction from dried bacterial cells of Azospirillum brasilense type strain Sp7. Mild acid hydrolysis of the lipopolysaccharide followed by GPC on Sephadex G-50 resulted in a polysaccharide mixture, which was studied by composition and methylation analyses, Smith degradation and (1)H and (13)C NMR spectroscopy. The following polysaccharide structures were established, where italics indicate a non-stoichiometric (∼40%) 2-O-methylation of l-rhamnose.