Studies of the polysaccharide fraction from the lipopolysaccharide of Pseudomonas alcaligenes

Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2-3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5-6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, P(i) and PP(i). The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.     

An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension

A water-soluble arabinogalactan, isolated from the extracellular medium of suspension-cultured cells of Rubus fruticosus, contained arabinose, rhamnose, galactose, and also protein (6.5%) and uronic acid (2.5%). Methylation analysis of the arabinogalactan and the arabinose-free product obtained by mild acid hydrolysis showed that the polysaccharide was a typical arabino-3,6-galactan in which rhamnose and glucuronic acid occupied non-reducing terminal positions. Successive Smith-degradations combined with methylation analysis and ¹³C-n.m.r. spectroscopy revealed that the arabinogalactan contained a main chain of (→3)-linked β-d-galactopyranosyl residues with a high degree of branching at positions 6 by (1→6)-linked d-galactopyranosyl side-chains of various lengths, in which several contiguous residues were substituted at positions 3. The polymer is thus an arabinogalactan-protein belonging to the galactans of Type II.     

Characterization of lipopolysaccharides from Ralstonia solanacearum

Lipopolysaccharides (LPSs) from four strains of Ralstonia solanacearum belonging to biovar I (ICMP 6524, 8115, 5712, and 8169) were isolated and investigated. The structural components of the LPS molecule, such as lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS), were obtained after mild acid hydrolysis of the LPS preparations. In lipid A from all the LPS samples studied, 3-hydroxyhexadecanoic, 2-hydroxyhexadecanoic, tetradecanoic, and hexadecanoic fatty acids prevailed. The dominant monosaccharides of the core oligosaccharides of all of the strains studied were rhamnose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and heptose. However, individual strains varied in the content of galactose, ribose, xylose, and arabinose. Three types of the O-PS structure were established, which differed in their configuration (alpha or beta), as well as in the type of the bond between glucosamine and rhamnose residues (1-->2 or 1-->3).     

Immunochemical analysis of O-specific polysaccharides from the soil nitrogen-fixing bacteria Azospirillum brasilense

Immunochemical reactivity of O-specific polysaccharide and the monosaccharide composition of O-antigenic determinants of the lipopolysaccharide isolated from the type strain Sp7 of Azospirillum brasilense were studied. An original modification of the method of spectroturbidimetry for disperse biological systems and a nonstandard procedure for the preparation of monospecific antibodies against cell surface antigens were used. The polysaccharide fraction, which contained residues of galactose, rhamnose, and galacturonic acid, was able to bind about 50% of the antibodies raised against whole bacterial cells. Twelve immunodeterminant groups were shown to be present in its molecule. Galactose and, less effectively, rhamnose but not galacturonic acid inhibited the antigen-antibody reactions. It is concluded that the serotype of the strain studied is determined by galactose residues.     

Structural features of an arabinogalactan gum exudates from Spondias dulsis (Anacardiaceae)

The tree Spondias dulcis, located in Venezuela, exudes a light-brown gum. The polysaccharide, isolated from the original gum, contains galactose, arabinose, mannose, rhamnose, glucuronic acid, and its 4-O-methyl derivative. Application of chemical methods, in combination with 1D and 2D NMR spectroscopy afforded interesting structural features of the gum polysaccharide. The unequivocal presence of rhamnose in the polymer structure was confirmed by chemical and spectral data [1H (1.03 ppm); 13C (16.92 ppm)]. Also confirmed was the existence of 3-O- and 6-O-substitutes galactose residues by the spectral data correlations observed in Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Correlation (HMBC). Also observed were unequivocal resonances for beta-D-glucuronic acid and its 4-O-methyl derivative, and the presence of 3-O-alpha-L-arabinofuranose and 3-O-beta-L-arabinopyranose residues.     

Isolation and characterization of a succinylated polysaccharide from the cell wall of Micrococcus agilis

A polysaccharide consisting of rhamnose, galactose, glucosamine and ester-linked succinic acid was extracted from the isolated cell walls of Micrococcus agilis by the hot water-phenol and 5% trichloroacetic acid (TCA) extraction methods. The hot water-phenol extractable polysaccharide accounted for 30% of the weight of the wall, with 23% by the TCA method. Phosphorus contents were less than 0.01% of the polysaccharide. Succinyl residues released by alkali treatment (0.1 N NaOH, 30 min, 37 degrees C) were identified by gas-liquid chromatography, and accounted for 6.3% and 5.1% of the polysaccharide purified from the hot water-phenol and TCA extracts, respectively. The polysaccharide was not bound when chromatography on Concanavalin A-Sepharose 4B (Con A/Sepharose 4B) columns was performed and it could thus be separated from any residual membrane lipomannan. The purified polysaccharide behaved as a negatively-charged polymer on electrophoresis in 1% agarose (at pH 8.6). A strong cross-reaction, unaffected by removal of the succinyl groups, was observed with type XXIII pneumococcal polysaccharide antiserum indicating the presence of L-rhamnose, linked through non-reducing, lateral end groups.     

Isolation and structural study of polysaccharides from campion Silene vulgaris

Silenan SV, a pectic polysaccharide, was isolated from the aerial part of Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn.), widespread through the European North of Russia. The polysaccharide was found to contain residues of galacturonic acid (63%), arabinose, galactose, and rhamnose as the main constituents. The results of a partial acidic hydrolysis, pectinase digestion, and NMR studies of silenan SV indicated that its molecule contains a linear alpha-1,4-D-galacturonan backbone and ramified regions. The core of the ramified regions is composed of residues of alpha-1,4-D-galacturonic acid along with 2-substituted alpha-rhamnopyranose residues. The NMR data showed that the silenan SV side chains are composed of the blocks built from the terminal alpha-1,5-linked arabinofuranose and beta-1,4-linked galactopyranose residues; these most likely are the side chains of rhamnogalacturonan, characteristic of other pectic polysaccharides. The nonreducing ends of these side chains contain alpha-arabinofuranose residues.     

Isolation and characterization of polysaccharides from Tansy

Using extraction with 0.75% aqueous ammonium oxalate, the following polysaccharide fractions were isolated: tanacetans TVF, TVS, and TVR from floscules, sprouts, and roots, respectively, of Tanacetum vulgare L., spread throughout the European North of Russia. The sugar chain of tanacetan TVF consists of D-galacturonic acid (61.4%), arabinose (14.7%), galactose (10.2%), and rhamnose (3.7%) as the main constituents as well as xylose, glucose, mannose, apiose, and 2-O-methylxylose in trace amounts. Tanacetans TVS and TVR were shown to differ in the sugar quantitative composition. They contain 67 and 28% galacturonic acid, respectively. A partial acid hydrolysis of the tanacetan TVF gave a polysaccharide fragment TVF1, alpha-1,4-D-galacturonan (GalA 98.2%). Digestion with pectinase (alpha-1,4-D-polygalacturonase) resulted in fragment TVF3, containing residues of arabinose (27.1%) and galactose (17.3%). NMR spectroscopy allowed detection of the terminal residues of alpha-Araf and beta-Galp as well as of the residues of alpha-Araf substituted in 3,5- and 5-positions. Thus, tanacetan TVF was proved to be a pectic polysaccharide.     

Partial structure of a polysaccharide from leaves of Welwitschia mirabilis

Gum-tears from the leaves of Welwitschia mirabilis contain a polysaccharide composed of arabinose, galactose and glucuronic acid as main constituents with xylose, fucose and rhamnose in smaller quantities. Periodate oxidation and permethylation studies indicated that the gum could consist of a framework of glucuronic acid residues linked 1 → 4 and galactose residues linked 1 → 6 and of short chains of arabinose, xylose, fucose and rhamnose linked 1 → 3 to both residues. All rhamnose and fucose and part of arabinose were found as non-reducing terminal units.     

Structural studies on a polysaccharide obtained from the cambium layer of a bael (Aegle marmelos) tree

The purified polysaccharide isolated from the cambium layer of a young bael (Aegle marmelos) tree contains galactose, arabinose, rhamnose, xylose, and glucose in the molar ratios of 10.0:9.8:1.4:1.9:1. Methylation analysis and Smith degradation studies established the linkages of the different monosaccharide residues. The anomeric configurations of the various sugar units were determined by oxidation of the acetylated polysaccharide with chromium(VI) trioxide. The oligosaccharides isolated from the polysaccharide by graded hydrolysis were characterized. The structural significance of these results is discussed.     

Water-soluble polysaccharides from Ginkgo biloba leaves.

The water-soluble polysaccharides from dried Ginkgo biloba leaves were isolated after exhaustive extraction with organic solvents. The polysaccharide mixture could be separated into a neutral (GF1) and two acidic (GF2 and GF3) polysaccharide fractions by ion exchange chromatography. According to the Mr distribution GF1 and GF3 seemed to be homogenous, whereas GF2 could be further fractionated into two subfractions (GF2a and GF2b) by gel permeation chromatography. GF1 (Mr 23,000) showed the structural features of a branched arabinan. The main chain was composed of 1,5-linked arabinose residues and three in 12 arabinose molecules were branched via C-2 or C-3. GF2a (Mr 500,000) consisted mainly of 1,2,4-branched mannose (29%), 1,4-linked glucuronic (32%) and galacturonic (8%) acid as well as terminal rhamnose (25%). After removal of ca 70% of the terminal rhamnose the remaining polysaccharide showed a decrease in 1,2,4-branched mannose and an increase in 1,2-linked mannose indicating that at least half of the rhamnose residues were linked to mannose via C-4. GF3 (Mr 40,000) consisted of 1,4-linked galacturonic (30%) and glucuronic (16) acid, 1,3,6-branched galactose (15%), 1,2-linked (5%) and 1,2,4-branched (3.5%) rhamnose as well as 1,5-linked arabinose (11%). Rhamnose (5%) and arabinose (10%) were present as terminal groups. Mild acid hydrolysis selectively cleaved arabinose and the remaining polysaccharide showed an increased amount of 1,6-linked and terminal galactose and a decreased quantity of 1,3,6-branched galactose. These results indicated that the terminal as well as the 1,5-linked arabinose were mainly connected to galactose via C-3. The GF3 polysaccharide appeared to be a rhamnogalacturonan with arabinogalactan side chains.     

Localization of mucilaginous polysaccharides in mulberry leaves

Mulberry tree leaves were shown to have mucilaginous polysaccharides. The extracted water-soluble mucilage was separated into three fractions via a cetylpyridinum chloride complex and purified by anion-exchange chromatography. Five acidic polysaccharides were separated from these fractions, one of which was a major polysaccharide (Mp-3) that was structurally analyzed and used for antibody preparation. The Mp-3 polysaccharide contained rhamnose, galactose, glucose, galacturonic acid, and glucuronic acid in a molar ratio of 1 : 0.2 : 0.5 : 2.3 : 1.5 as constituent monosaccharides. Methylation and gas chromatography-mass spectrometry analysis indicated that the polysaccharide was a rhamnogalacturonan mainly consisting of 1,2,3-linked rhamnose residues, 1,3,4- and 1,4-linked uronic acid residues, and terminal uronic acid residues. Its molecular weight was estimated to be 5.5 x 10(5). Immunohistological observation revealed that the Mp-3 polysaccharide is specifically localized in inner epidermal cells situated in adaxial leaves, and electron microscopy showed that its subcellular location is between the plasma membrane and the cell wall. In young leaves, numerous secretory vesicles were present in a shrunken cytoplasm that was surrounded by fibers. In mature leaves, more than 20% of total epidermal cells were these inner cells in which polysaccharide deposition was significantly increased. The deposits appeared as a rounded electron-dense mass throughout the inner cells by electron microscopy.     

Characterization of the group-specific polysaccharide of group B Streptococcus

The group-specific polysaccharide of the group B Streptococcus was isolated by nitrous acid extraction followed by gel filtration on Sepharose 6B and chromatography on DEAE-Bio-Gel A. It was composed of rhamnose, galactose, N-acetylglucosamine, and glucitol phosphate. Mild periodate oxidation of the polysaccharide resulted in a rapid reduction in molecular weight, indicating that the glucitol was located in the backbone of the polymer. High-resolution 31P NMR showed the presence of a single type of phosphodiester bond in the molecule. Methylation analysis and several specific chemical degradations were done to determine sugar linkages. The basic structure of the group B polysaccharide consists of a backbone of 2-linked rhamnose, 2,4-linked rhamnose, and glucitol phosphate, and side chains of rhamnose(1----3)galactose(1----3)N-acetylglucosamine linked to the 4-position of a rhamnose in the backbone.     

The capsule and slime polysaccharides of the wild type and a phage resistant mutant ofRhodopseudomonas capsulata St. Louis

A rhamnose, galactose and pyruvic acid containing polysaccharide (capsule) together with the peptidoglycan was isolated fromRhodopseudomonas capsulata St. Louis as the insoluble sediment after sodium dodecyl sulfate extraction of cell envelope fractions. Treatment with pronase E separated the soluble polysaccharide from the insoluble peptidoglycan. After lysozyme-digestion, both the capsule polysaccharide and peptidoglycan were soluble.The capsule was also accumulated in the combined interphase/phenol-phase of hot phenol-water extracts of whole cells. Again, the capsule and peptidoglycan were sedimented together as long as no pronase E-treatment was performed. With the phage-resistant mutant (R. capsulata St. Louis RC1^-), no capsule polysaccharide was obtained in the combined interphase/phenol phase.An acidic polysaccharide (slime) different from the capsule in composition and serology was obtained by Cetavlon fractionation of hot phenol/water extracts of cells of both the wild-type and the mutant cells. It was shown to consist mainly of rhamnose, glucosamine and galacturonic acid.The use of O/K-antisera and of capsule polysaccharideantisera allowed a separate visualization of the capsule and slime layers.This paper is dedicated to Professor Hans G. Schoegel on the occasion of his 60th birthday     

Structural study of bergenan, a pectin from Bergenia crassifolia

A pectin polysaccharide named bergenan was isolated from the freshly collected leaves of the leather bergenia Bergenia crassifolia by extraction with an aqueous solution of ammonium oxalate. The main component of its carbohydrate chain was shown to be the residues of D-galacturonic acid (about 80%). In addition, the polysaccharide contains residues of galactose, arabinose, and rhamnose; their total content is less than 15%. It was shown that the bergenan samples from bergenia leaves collected at different vegetation periods (from July to September) do not substantially differ either in monosaccharide composition or in the viscosity of aqueous solutions they form. The results of enzymatic hydrolysis by alpha-1,4-galacturonase (pectinase), partial acidic hydrolysis, NMR spectroscopy, and methylation with subsequent analysis of the results by GC-MS indicate that the bergenan macromolecule contains the regions of a linear alpha--1,4-D-galactopyranosyluronan and rhamnogalacturonan-I (RG-1). Galacturonan responds for a greater part of the macromolecule. A considerable amount of its constituent galacturonic acid residues are present as methyl esters. The side chains in RG-I are attached to the rhamnopyranose residues of the main carbohydrate chain by 1,4-link and are composed of the residues of terminal arabinofuranose and galactopyranose, 1,5-linked (-arabinofuranose, and 1,4-and 1,6-linked beta-galactopyranose. The branching points of the side chains of the RG-I molecule are 3,4- and 3,6-di-O-substituted galactose residues.     

Pectin substances of the callus culture of Silene vulgaris (M.) G

Pectin-protein fraction SVC was isolated from the callus culture of the bladder campion (Silene vulgaris). The main components in it were residues of D-galacturonic acid, galactose, arabinose, rhamnose, and protein. Using ion-exchange chromatography, ultrafiltration, and acid and enzymatic hydrolysis, it was shown that SVC contained a mixture of molecules of linear pectin, branched pectin polysaccharide, and pectin-protein polymer. A fragment of the linear chain of galacturonan amounted to more than half of the entire carbohydrate silenan chain. The branched area of the macromolecule is represented by rhamnogalacturonan I. The pectin-protein polymer consisted mainly of protein and weakly branched pectin fragments with molecular mass of more than 300 kDa.     

Characterization of Lipopolysaccharides from Ralstonia solanacearum

Lipopolysaccharides (LPSs) from four strains of Ralstonia solanacearum belonging to biovar I (ICMP 6524, 8115, 5712, and 8169) were isolated and investigated. The structural components of the LPS molecule, such as lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS), were obtained after mild acid hydrolysis of the LPS preparations. In lipid A from all the LPS samples studied, 3-hydroxytetradecanoic, 2-hydroxyhexadecanoic, tetradecanoic, and hexadecanoic fatty acids prevailed. The dominant monosaccharides of the core oligosaccharides of all of the strains studied were rhamnose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and heptose. However, individual strains varied in the content of galactose, ribose, xylose, and arabinose. Three types of the O-PS structure were established, which differed in their configuration ( or ), as well as in the type of the bond between glucosamine and rhamnose residues (1 2 or 1 3).     

Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.     

An arabinogalactan from the fibres of cotton (Gossypium arboreum L.)

An acidic arabinogalactan has been isolated from fibres of the cotton plant (Gossypium arboreum L.) at the stage of intensive secondary-wall formation. The polysaccharide contains arabinose, galactose, rhamnose, and glucuronic acid residues in the molar ratios 1:1.2:0.1:0.2. Periodate oxidation and methylation studies showed that there is a main chain of (1→3)-linked galactopyranosyl residues to which side chains are attached at O-6. The side chains consist of (1→6)-linked galactopyranosyl residues substituted at O-3 by (1→5)-linked arabinofuranosyl chains. Terminal galactopyranosyl, rhamnopyranosyl, and glucopyranuronosyl groups are also present. Enzymic hydrolysis showed that the configurations of the galactose and arabinose residues are d and l, respectively.     

Structural studies on the O-antigen of Aeromonas salmonicida

Lipopolysaccharide from a strain of Aeromonas salmonicida salmonicida was isolated from cells by the aqueous phenol method in 2.3% yield (based on dry weight of bacteria). Hydrolysis of the lipopolysaccharide in 1% acetic acid afforded O-polysaccharide (19% by weight), core-oligosaccharide (12.2%) and lipid A (44.6%). Analysis indicated that 3-deoxy-D-manno-2-octulosonic acid was absent from the lipopolysaccharide and that no low-molecular-weight compounds were released by the mild hydrolysis. The O-polysaccharide had the monosaccharide composition of rhamnose, glucose and N-acetylmannosamine in molar ratio of 1.0:1.58:0.83. 75% of the N-acetylmannosamine residues were substituted at position 4 by O-acetyl groups. Hydrolysis of the methylated polysaccharide proved to be both difficult and dependent on the method of hydrolysis chosen, in all cases a partially methylated disaccharide of rhamnose and N-acetylmannosamine was identified in the hydrolysate. Methylation analysis, periodate oxidation and proton magnetic resonance analysis were used to confirm the structure of the repeating unit as: (formula; see text).