Extracellular polysaccharides of a bacterium associated with a fungal canker disease of Eucalyptus sp

Extracellular polysaccharides (EPSs) produced by an Erwinia sp associated with a fungal canker disease of Eucalyptus were fractionated into one polysaccharide that was identified with that produced by Erwinia chrysanthemi strains SR260, Ech1, and Ech9, and the other distinctively different from any other EPS produced by E. chrysanthemi strains so far studied. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, low pressure gel-filtration, and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharide, identified as EPS Teranera, has the following structure: [structure: see text] The molecular weights of the polysaccharides range from 3.2-6.2 x 10(5) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.     

Structure and hydrodynamic properties of the extracellular polysaccharide from a mutant strain (RA3W) of Erwinia chrysanthemi RA3

The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain RA3W, a mutant strain of E. chrysanthemi RA3, has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl linkage analysis, and 1D 1H NMR spectroscopy. The polysaccharide is structurally similar, if not identical, to the family of EPS produced by such as E. chrysanthemi strains Ech9, Ech9Sm6, and SR260. The molecular weight of EPS RA3W by ultracentrifugation (sedimentation equilibrium) and light scattering is compared with those of other E. chrysanthami EPSs, as are the viscometric properties.     

Structural features of immunologically active polysaccharides from Ganoderma lucidum.

Three polysaccharides, two heteroglycans (PL-1 and PL-4) and one glucan (PL-3), were solubilized from the fruit bodies of Ganoderma lucidum and isolated by anion-exchange and gel-filtration chromatography. Their structural features were elucidated by glycosyl residue and glycosyl linkage composition analyses, partial acid hydrolysis, acetolysis, periodate oxidation, 1D and 2D NMR spectroscopy, and ESI-MS experiments. The data obtained indicated that PL-1 had a backbone consisting of 1,4-linked alpha-D-glucopyranosyl residues and 1,6-linked beta-D-galactopyranosyl residues with branches at O-6 of glucose residues and O-2 of galactose residues, composed of terminal glucose, 1,6-linked glucosyl residues and terminal rhamnose. PL-3 was a highly branched glucan composed of 1,3-linked beta-D-glucopyranosyl residues substituted at O-6 with 1,6-linked glucosyl residues. PL-4 was comprised of 1,3-, 1,4-, 1,6-linked beta-D-glucopyranosyl residues and 1,6-linked beta-D-mannopyranosyl residues. These polysaccharides enhanced the proliferation of T- and B-lymphocytes in vitro to varying contents and PL-1 exhibited an immune-stimulating activity in mice.     

Extracellular polysaccharide of Erwinia chrysanthemi A350 and ribotyping of Erwinia chrysanthemi spp

Erwinia chrysanthemi spp. are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by the E. chrysanthemi strain A350, which is a lacZ- mutant of the wild type strain 3937, pathogenic to Saintpaulia, has been determined using a combination of chemical and physical techniques including methylation analysis, low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry and 1- and 2D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain A350 contains D-GalA, together with L-Rhap and D-Galp in a 1:4:1 ratio. Evidence is presented for the following hexasaccharide repeat unit: [structure: see text] All the Erwinia chrysanthemi spp. studied to date have been analyzed by ribotyping and collated into families, which are consistent with the related structures of their EPS.     

Structural characterisation of novel lichen heteroglycans by NMR spectroscopy and methylation analysis

Two galactofuranomannans, Ths-4 and Ths-5, were isolated from the lichen, Thamnolia vermicularis var. subuliformis, using ethanol fractionation and anion-exchange and size-exclusion chromatography. The average molecular weights of Ths-4 and Ths-5 were estimated to be 19 and 200 kDa, respectively. Structural characterisation of Ths-4, Ths-5 and their partially hydrolysed derivatives was performed by methanolysis and methylation analysis. The intact and partially hydrolysed Ths-4 was further analysed using NMR spectroscopy (1D, COSY, NOESY, TOCSY, HSQC and HMBC). According to the data obtained, the heteroglycans Ths-4 and Ths-5 have similar structures, but have large differences in molecular weight. The structure is composed of 3-O-linked and 5-O-linked galactofuranosyl chains linked to a mannan core. The mannan core consists of a main chain of alpha-(1-->6)-linked mannopyranosyl residues, substituted at O-2 with either a single alpha-mannopyranosyl unit or an alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp group in the ratio of approximately 1:3, respectively. The polysaccharides have idealised repeating blocks as is shown.     

Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1.23:0.36 was isolated from the Pacific brown seaweed Fucus evanescens. The structures of its desulfated and de-O-acetylated derivatives were investigated by 1D and 2D (1)H and (13)C NMR spectroscopy, and the data obtained were confirmed by methylation analysis of the native and desulfated polysaccharides. The fucoidan was shown to contain a linear backbone of alternating 3- and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp(2SO(3)(-))-(1-->4)-alpha-L-Fucp(2SO(3)(-))-(1-->. Additional sulfate occupies position 4 in a part of 3-linked fucose residues, whereas a part of the remaining hydroxyl groups is randomly acetylated.     

In vitro antioxidant activities of sulfated polysaccharide fractions extracted from Corallina officinalis

Sulfated polysaccharides (F1, F2) from seaweed Corallina officinalis were isolated through anion-exchange column chromatography. Their chemical characteristics were determined by GC, HPLC, FT-IR and UV spectra. F1 and F2 contained only two monosaccharides, namely galactose and xylose. The antioxidant activities of F1, F2 and the de-sulfated polysaccharides (DF-1, DF-2) in vitro were investigated, including hydroxyl radicals scavenging effect, superoxide radical scavenging capacity, DPPH radical activity and reducing power. As expected, antioxidant assay showed that the two sulfated polysaccharide fractions (F1, F2) possessed considerable antioxidant properties and had more excellent abilities than de-sulfated polysaccharides (DF-1, DF-2).     

New oligosaccharides prepared by acid hydrolysis of the polysaccharides from Nerium indicum Mill and their anti-angiogenesis activities

To discover drug candidates with anti-angiogenesis activity for cancer therapeutics, three galactooligosaccharides (OJ1-OJ3) were prepared by acid hydrolysis of the polysaccharides from Nerium indicum Mill. Their structures were characterized using sugar analysis, methylation analysis, and both 1D and 2D NMR spectroscopy, complemented by mass spectrometry. They were hexasaccharide (OJ1), a pentasaccharide (OJ2), and an undecasaccharide (OJ3), which was a new linear galactan. Bioactivity testing in vitro showed that OJ2 and OJ3 significantly inhibited the HMEC-1 (human microvascular endothelial cell) cell tube formation.     

Furostanol saponins from Yucca gloriosa L. rhizomes

Two new and one known furostanol saponins were isolated from the rhizomes of Yucca gloriosa. The structures of the isolated compounds were established by detailed spectroscopic analysis (1D-, 2D NMR, ESI-MS, HR-MALDI-MS). The glycosidic profile of Y. gloriosa in comparison to that of Yucca schidigera, the well known commercial source of saponins, is briefly discussed. The similarity between the two species regarding the high steroidal content suggests that it might be possible to use Y. gloriosa for the same applications as Y. schidigera.     

Structural elucidation of a cell wall fungal polysaccharide isolated from Ustilaginoidea virens, a pathogenic fungus of Oriza sativa and Zea mays

The alkali-extractable water-soluble polysaccharides (F1SS) isolated from the outer cell wall of two strains of Ustilaginoidea virens have been studied by chemical and methylation analyses, and 1D and 2D ¹H and ¹³C NMR spectroscopy. The structures of these polysaccharides are very similar, and can be described by the following idealized repeating unit: where n and m are approximately 1 and 2, respectively.     

E/Z-Thesinine-O-4'-alpha-rhamnoside, pyrrolizidine conjugates produced by grasses (Poaceae)

Based on direct infusion mass spectrometry we identified a novel alkaloid as a major component of perennial ryegrass (Lolium perenne). Initial mass spectral data suggested it to be a pyrrolizidine conjugate. As this class of alkaloids has not been described before from grasses, we isolated it to elucidate its structure. The isolated alkaloid proved to be a mixture of two stereoisomers. The structures of the two compounds as determined by 1D and 2D NMR spectroscopy, were E-thesinine-O-4'-alpha-rhamnoside (1) and Z-thesinine-O-4'-alpha-rhamnoside (2). These identifications were supported by the characterisation by GC-MS and optical rotation of (+)-isoretronecanol as the necine base released on alkaline hydrolysis of these alkaloids. 1 and 2 together with the aglycone and a hexoside were also detected in tall fescue (Festuca arundinacea). This is the first report of pyrrolizidine alkaloids produced by grasses (Poaceae).     

Steroids from the soft coral Dendronephthya sp

Fifteen steroids were isolated from the soft coral Dendronephthya sp., of which five are determined as new compounds, namely (22E)-3-O-beta-formylcholest-5,22-diene (1), (22E)-3-O-beta-formyl-24-methyl-cholest-5,22-diene (2), 2-ethoxycarbonyl-2-beta-hydroxy-A-nor-cholest-5-ene-4-one (3), (22E)-2-ethoxycarbonyl-2-beta-hydroxy-A-nor-cholest-5,22-diene-4-one (4), and (22E)-2-ethoxycarbonyl-2-beta-hydroxy-24-mthyl-A-nor-cholest-5,22-diene-4- one (5). 1 and 2 belonged to 3-O-formylated cholesterol analogues, and 3 to 5 are unique ring A-contracted steroids. Their structures were elucidated by extensive 2D NMR in association with IR, MS analysis.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy

Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, a (1-->6)-beta-d-glucan, and a alpha-galactomannan. To refine the structures of these polysaccharides, cell-wall glucans of S. pombe were extracted, fractionated, and analyzed by NMR spectroscopy. S. pombe cells were treated with 3% NaOH, and alkali-soluble and insoluble fractions were prepared. The alkali-insoluble fraction was treated with 0.5M acetic acid or Zymolyase 100T to yield an alkali-insoluble, acetic acid-insoluble fraction, an alkali-insoluble, Zymolyase-insoluble fraction, and an alkali-insoluble, Zymolyase-soluble fraction. (13)C NMR and 2D-NMR spectra disclosed that the cell wall of S. pombe is composed of three types of glucans, specifically, a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, which may either be linear or slightly branched, and a highly branched (1-->6)-beta-d-glucan, in addition to alpha-galactomannan. The highly branched (1-->6)-beta-d-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1-->3)-beta-branched (1-->6)-beta-d-glucan with more branches than that of Saccharomyces cerevisiae. Flexibility of these polysaccharides in the cell wall was analyzed by (13)C NMR spectra in D(2)O. The data collectively indicate that (1-->3)-alpha- and (1-->3)-beta-d-glucans are rigid and contribute to the cell shape, while the highly branched (1-->6)-beta-d-glucan and alpha-galactomannan are flexible.     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.     

Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.     

Structural investigation of the O-specific polysaccharides of Morganella morganii consisting of two higher sugars

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,(13)C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-2,6-dideoxy-D-galactose (shewanellose, She). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2.     

Structural characterization of a 2-O-acetylglucomannan from Dendrobium officinale stem

A heteropolysaccharide obtained from an aqueous extract of dried stem of Dendrobium officinale Kimura and Migo by anion-exchange chromatography and gel-permeation chromatography, was investigated by chemical techniques and NMR spectroscopy, and is demonstrated to be a 2-O-acetylglucomannan, composed of mannose, glucose, and arabinose in 40.2:8.4:1 molar ratios. It has a backbone of (1-->4)-linked beta-d-mannopyranosyl residues and beta-d-glucopyranosyl residues, with branches at O-6 consisting of terminal and (1-->3)-linked Manp, (1-->3)-linked Glcp, and a small proportion of arabinofuranosyl residues at the terminal position. The acetyl groups are substituted at O-2 of (1-->4)-linked Manp and Glcp. The main repeating unit of the polysaccharides is reported.     

Environmental and biofilm-dependent changes in a Bacillus cereus secondary cell wall polysaccharide

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(β1-3)]GlcNAc(β1-6)[Glc(β1-3)][ManNAc(α1-4)]GalNAc(α1-4)ManNAc(β1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm.