The O-chain structure from the LPS of the bacterium Naxibacter alkalitolerans YIM 31775T

The O-chain polysaccharide of the lipopolysaccharide from the bacterium Naxibacter alkalitolerans strain YIM 31775(T) was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be built up by the following tetrasaccharide repeating unit: -->3)-alpha-D-FucpNAc-(1-->2)-beta-D-Quip3NHBu-(1-->2)-alpha-D-Rhap-(1-->)-beta-D-Galp-(1--> where HBu is hydroxy-butanoyl.     

Structure of the O-polysaccharide of Pseudomonas putida FERM P-18867

The O-polysaccharide of the lipopolysaccharide of Pseudomonas putida FERM P-18867 was found to contain D-mannose and D-rhamnose and have the following structure of the trisaccharide repeating unit:-->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-beta-D-Manp-(1-->     

Isolation and characterization of lectin from the surface of Grifola frondosa (FR.) S.F. Gray mycelium

For the first time, from the surface of the dikaryotic mycelium of the xylotrophic basidiomycete Grifola frondosa 0917 a lectin has been isolated with a molecular mass of 68 +/- 1 kDa, consisting of two subunits of 33-34 kDa each. The lectin is a hydrophilic glycoprotein with the protein : glycan ratio of 3 : 1. It exhibits high affinity to native rabbit erythrocytes and to human erythrocytes of the 0 blood group, but not to trypsin-treated ones. The hemagglutination (HA) caused by lectin was not blocked by any of the 25 tested mono-, di-, and amino sugars; it was also not blocked by some of glyco derivatives. Only 13.9 microg/ml of the homogeneous preparation of a polysaccharide, a linear D-rhamnan with the structure of the repetitive component --> 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 --> blocked hemagglutination completely. The analysis of the amino acid composition of the lectin showed the greatest percentage of amino acids with positively charged R groups, arginine, lysine, and histidine, as well as the complete absence of sulfur-containing amino acids, cysteine, and methionine. D-glucose and D-glucosamine were detected in the carbohydrate part.     

Nitrogen-fixing bacterium Burkholderia brasiliensis produces a novel yersiniose A-containing O-polysaccharide

Burkholderia brasiliensis, a Gram-negative diazotrophic endophytic bacterium, was first isolated from roots, stems, and leaves of rice plant in Brazil. The polysaccharide moiety was released by ammonolysis from the B. brasiliensis lipopolysaccharide (LPS), allowing the unambiguous characterization of a 3,6-dideoxy-4-C-(1-hydroxyethyl)-D-xylo-hexose (yersiniose A), an uncommon feature for Burkholderia LPS. The complete structure of the yersiniose A-containing O-antigen was identified by sugar and methylation analyses and NMR spectroscopy. Our results show that the repeating oligosaccharide motif of LPS O-chain consists of a branched tetrasaccharide with the following structure:-->2-alpha-d-Rhap-(1-->3)-[alpha-YerAp-(1-->2)]-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->.     

A comparison of the lipopolysaccharides and O-specific polysaccharides of Azospirillum brasilense Sp245 and its omegon-Km mutants KM018 and 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.     

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.     

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain L-rhamnose, 2-acetamido-4-[D-3-hydroxybutyramido)]-2,4,6-trideoxy-D-glucose (D-BacNAc4NHbu), and 2-amino-2,6-dideoxy-L-galactose, N-acylated by the 4-carboxyl group of L-malic acid (L-malyl-(4-->2)-alpha-L-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established:-3)-alpha-D-BacNAc4NHbu-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->2)-L-malyl-(4-->2)-alpha-L-FucN-(1-. The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Structure of the O-specific polysaccharide of Proteus vulgaris O15 containing a novel regioisomer of N-acetylmuramic acid, 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O15 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, and H-detected 1H,(13)C HMQC experiments. The polysaccharide was found to contain an ether of GlcNAc with lactic acid, and the following structure of the repeating unit was established:-->3)-alpha-D-GlcpNAc4(R-Lac)6Ac-(1-->2)-beta-D-GlcpA-(1-->3)-alpha-L-6dTalp2Ac-(1-->3)-beta-D-GlcpNAc-(1-->where L-6dTal and D-GlcNAc4(R-Lac) are 6-deoxy-L-talose and 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose, respectively. The latter sugar, which to our knowledge has not been hitherto found in nature, was isolated from the polysaccharide by solvolysis with anhydrous triflic acid and identified by comparison with the authentic synthetic compound. Serological studies with the Smith-degraded polysaccharide showed an importance of 2-substituted GlcA for manifesting of the immunospecificity of P. vulgaris O15.     

Facile synthesis of a pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C

A pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[alpha-D-Galp-(1-->2)-beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->OCH2CH2N3) (1) was synthesized in a regio- and stereoselective manner. The 2-azidoethyl-spacered pentasaccharide mimic 1 can be used to construct a neoglycoconjugate antigen.     

Structure of the O-specific polysaccharide chain of the lipopolysaccharide of Bordetella hinzii

The lipopolysaccharide of Bordetella hinzii was analyzed after various chemical degradations by NMR spectroscopy and MALDI mass spectrometry, and the following structure of the polysaccharide chain was determined: 4-O-Me-alpha-GalpNAc3NAcAN-(1-->[-->4)-beta-GlcpNAc3NAcAN-(1-->4)-beta-GlcpNAc3NAcAN-(1-->4)-alpha-GalpNAc3NAcAN-(1-](n)-where GlcNAc3NAcAN and GalNAc3NAcAN stand for 2,3-diacetamido-2,3-dideoxy-glucuronamide and -galacturonamide, respectively. The polysaccharide chain is terminated with a 4-O-methylated GalNAc3NAcAN residue and is rather short (n < or = 5).     

Structure of the O-specific polysaccharide of Proteus vulgaris O45 containing 3-acetamido-3,6-dideoxy-D-galactose

An O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O45 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established:-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Fucp3NAc4Ac-(1-->where Fuc3NAc4Ac is 3-acetamido-4-O-acetyl-3,6-dideoxygalactose. A cross-reactivity of anti-P. vulgaris O45 serum was observed with several other Proteus lipopolysaccharides, which contains Fuc3N derivatives.     

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T)

An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.     

The structure of the rough-type lipopolysaccharide from Shewanella oneidensis MR-1, containing 8-amino-8-deoxy-Kdo and an open-chain form of 2-acetamido-2-deoxy-D-galactose

The LPS from Shewanella oneidensis strain MR-1 was analysed by chemical methods and by NMR spectroscopy and mass spectrometry. The LPS contained no polysaccharide O-chain, and its carbohydrate backbone had the following structure: (1S)-GalNAco-(1-->4,6)-alpha-Gal-(1-->6)-alpha-Gal-(1-->3)-alpha-Gal-(1-P-3)-alpha-DDHep-(1-->5)-alpha-8-aminoKdo4R-(2-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P, where R is P or EtNPP. There are several novel aspects to this LPS. It contains a novel linking unit between the core polysaccharide and lipid A moieties, namely 8-amino-3,8-dideoxy-D-manno-octulosonic acid (8-aminoKdo) and a residue of 2-acetamido-2-deoxy-D-galactose (N-acetylgalactosamine, GalNAco) in an open-chain form, linked as cyclic acetal to O-4 and O-6 of D-galactopyranose. The structure contains a phosphodiester linkage between the alpha-D-galactopyranose and D-glycero-D-manno-heptose (DDHep) residues.     

Structure of the O-polysaccharide of Providencia stuartii O49

The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->     

Structure of a heteroxylan of gum exudate of the palm Scheelea phalerata (uricuri)

The polysaccharide isolated from the gum exudate of palm Scheelea phalerata (SPN) was water-insoluble and composed of Fuc, Ara, Xyl, and uronic acid moieties in a 5:34:54:7 molar ratio: 12% of phenolics were also present. A soluble polysaccharide (SPNa) was obtained after alkaline treatment, which contained Fuc, Ara, Xyl and uronic acid in a 7:44:42:7 molar ratio, with only 2% phenolics. SPNa had an M(W) approximately 1.04 x 10(5) g mol(-1) and was almost monodisperse (M(W)/M(N) : 1.25 +/-0.22). It had a branched structure with side chains of 2-O-substituted Xylp (approximately 8%) and 3-O-substituted Araf (12%) units, and a large proportion of nonreducing end-units of Araf (15%), Fucp (10%), Xylp (4%), and Arap (6%). The (1 --> 4)-linked beta-Xylp main-chain units were 3-O- (9%), 2-O- (13%), and 2,3-di-O- (13%) substituted. Its (13)C NMR spectrum contained at least 9 C-1 signals, those at delta 108.6 and 107.7 arising from alpha-Araf units. Others were present at delta 175.4 from C-6 of alpha-GlcpA and delta 15.6 from C-6 of Fucp units. The main chain of SPNa was confirmed by analysis of a Smith-degraded polysaccharide (SPDS): methylation analysis provided a 2,3-Me(2)-Xyl (65%) derivative and its (13)C NMR spectrum showed five main signals typical of a (1 --> 4)-linked beta-Xylp units. Methylation analysis of a carboxy-reduced polysaccharide (SPN-CR) revealed a 2,3,4,6-Me(4)-Glc derivative (4%) arising from nonreducing end-units of GlcpA. Alpha-GlcpA-(1 --> 2)-alphabeta-Xy1p and alpha-GlcpA-(1 --> 2)-beta-Xylp-(1 --> 4)-alphabeta-Xylp were obtained via partial acid hydrolysis of SPN, showing the structure of side-chain substituents on O-2 of the main-chain units.     

Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS

Cryptococcus flavescens, a strain originally identified as C. laurentii, was isolated from the cerebrospinal fluid of an AIDS patient, and the soluble capsular polysaccharide of the yeast was investigated. Glucuronoxylomannan (GXM) was obtained from C. flavescens under conditions similar to those used to obtain C. neoformans polysaccharide. However, the GXM differed from C. neoformans polysaccharide in the decreased O-acetyl group content. The structure of GXM was determined by methylation analysis, partial acid hydrolysis, NMR analyses, and controlled Smith degradation. These analyses indicated that GXM has the following structure: an alpha-(1-->3)-D-mannan backbone with side chains of beta-D-glucuronic acid residues bound to the C-2 position of the mannose residue. The C-6 position of the mannose is substituted with D-man-beta-(1-->4)-D-xyl-beta-(1--> disaccharide. Furthermore, the existence of side chains containing more than two xylose residues was suggested. This mannosylxylose side chain is a novel structure in polysaccharides of C. neoformans and other Cryptococcus species.     

O-Specific chain structure from the lipopolysaccharide fraction of Pseudomonas reactans: a pathogen of the cultivated mushrooms

An O-specific polysaccharide containing 2-acetamidino-2-deoxy-beta-D-glucopyranose (Glcp2Am), 2,4-diacetamido-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAc4NAc, bacillosamine) and 2,4-di-(N-acetyl-L-alanylamino)-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAlaAc4NAlaAc) was isolated from the phenol-soluble lipopolysaccharide fraction of the mushroom-associated bacterium Pseudomonas reactans. The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a linear trisaccharide-repeating unit, as shown below:-->3)-beta-D-QuipNAlaAc4NAlaAc-(1-->3)-alpha-D-Glcp2Am-(1-->3)-alpha-D-QuipNAc4NAc(1-->To our knowledge, this is the first complete O-chain structure reported for the lipopolysaccharide of a mushroom-associated bacterium.     

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.