Characterization of 2-amino-2,6-dideoxy-D-glucose as a constituent of the lipopolysaccharide antigen of Pseudomonas aeruginosa immunotype 4.

The title lipopolysaccharide was freed from its lipid A component by mild, acid hydrolysis, to give a polysaccharide fraction that was subsequently hydrolyzed completely to afford a mixture of neutral sugars and amino sugars. The amino sugars were separated, and identified as 2-amino-2-deoxy-D-galactose, 2-amino-2,6-dideoxy-galactose as a 2:1 mixture of the D and L enantiomers, and 2-amino-2,6-dideoxy-D-glucose. A reference sample of 2-amino-2,6-dideoxy-D-glucose was synthesized by an improved preparative route. Among the lipopolysaccharide antigens of the seven recognized immunotypes of Pseudomonas aeruginosa, 2-amino-2,6-dideoxyglucose is also characterized as a constituent of two others, types 3 and 5.     

Carbohydrate composition of the phenol-soluble lipopolysaccharides of Citrobacter freundii

Phenol-soluble lipopolysaccharides were obtained from the interphase and phenol phase fractions of 44% aqueous phenol-extracted Citrobacter species. Upon detailed investigation of C. freundii 8090, the two lipopolysaccharide fractions were found to contain different amounts of lipid A, although qualitative composition was similar. Both contained lipid A, 2-keto-deoxyoctonic acid, heptose, phosphate, d-glucose, galactose, rhamnose, 2-acetamido-2 deoxy-d-glucose, 3-acetamido-3,6-dideoxy-d-glucose, O-acetyl, and trace amino acids. Partially purified phenol-phase lipopolysaccharide partitioned into the phenol-soluble phase when refractionated with 44% aqueous phenol, and was further found to be soluble in 88% phenol, 95% ethyl alcohol, and chloroform-methanol (2:1).     

Immunochemical characteristics of a lipopolysaccharide from Pseudomonas aurantiaca

A lipopolysaccharide was isolated from Pseudomonas aurantiaca IMB 31 by extraction with aqueous phenol and purified by ultracentrifugation. The lipopolysaccharide was confined to the phenol phase. Fucosamine (2-amino-2,6-dideoxygalactose) (36%) and bacillosamine (2,4-diamino-3,4,6-trideoxyglucose) (23%) were identified as hypothetic components of the O-chain in the carbohydrate moiety of the macromolecule using the techniques of paper chromatography, gas-liquid chromatography and ion-exchange chromatography on an amino acid analyser. Rhamnose, glucose, galactose, glucosamine and galactosamine were detected as hypothetical components of the core in the lipopolysaccharide composition, as well as 2-keto-3-deoxyoctonic acid, heptose, alpha-alanine and phosphorus, usual components of the core in Pseudomonas. The following predominant fatty acids were identified in the composition of lipid A using the techniques of gas-liquid chromatography with standard compounds and gas-liquid mass spectrometry: 3-OH C10:0 (14.4%), C12:0 (30.5%), 2-OH C12:0 (14.9%), 3-OH C12:0 (17.4%), C16:0 (9.9%). The serological relationship between P. aurantiaca strains was studied, and their phylogenetic relationship with P. fluorescens is discussed.     

Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3-diamino-2,3-dideoxyglucose

Lipopolysaccharides were isolated from two strains of Thiobacillus ferrooxidans and one strain each of Thiobacillus thiooxidans, Thiobacillus novellus and Thiobacillus sp. IFO 14570. Neutral sugars, 2-keto-3-deoxyoctonate, fatty acids and the rare 2,3-diamino-2,3-dideoxyglucose were detected in all lipopolysaccharides. Lipopolysaccharides of both T. ferrooxidans strains contained l-glycero-d-manno-heptose, whereas that of T. thiooxidans contained both l-glycero-d-manno-heptose and d-glycero-d-manno-heptose. On the other hand, heptoses were absent in lipopolysaccharides of T. novellus and Thiobacillus sp. IFO 14570. Lipid A of T. ferrooxidans and T. thiooxidans contained both glucosamine and 2,3-diamino-2,3-dideoxyglucose, in contrast, lipid A of T. novellus and the Thiobacillus sp. IFO 14570 most likely contain only 2,3-diamino-2,3-dideoxyglucose as backbone sugar. Deoxycholate polyacrylamide gel electrophoresis revealed S-type character for all lipopolysaccharides studied. The significance of the lipopolysaccharide composition for taxonomic and phylogenetic questions with regard to thiobacilli is discussed.Abbreviations DAG 2,3-diamino-2,3-dideoxyglucose - DOC sodium deoxycholate - GC gas-liquid chromatography - GC/MS gas-liquid chromatography/mass spectrometry - d,d-Heptose d-glycero-d-manno-heptose - l,d-Heptose l-glycero-d-manno-heptose - KDO 2-keto-3-deoxyoctonate - LPS lipopolysaccharide - 3-OH-14:0 3-hydroxy-tetradecanoic acid - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether     

O-Methyl sugars in lipopolysaccharides of Rhodospirillacea. Identification of 3-O-methyl-d-mannose in Rhodopseudomonas viridis and of 4-O-methyl-d-xylose and 3-O-methyl-6-deoxy-d-talose in Rhodopseudomonas palustris respectively

1. This paper deals with the identification of three O-methyl sugars in lipopolysaccharides isolated from strains of the Gram-negative photosynthetic family Rhodospirillaceae. In addition to the previously described 3-O-methyl-l-xylose, a second O-methyl sugar was encountered in the lipopolysaccharide of Rhodopseudomonas viridis F, namely 3-O-methyl-d-mannose. The lipopolysaccharides of two strains of Rhodopseudomonas palustris (strain 1e5 and 8/1) contain two O-methylsugars, 4-O-methyl-d-xylose and 3-O-methyl-6-deoxy-d-talose (d-acovenose). 4-O-Methyl-d-xylose, but not 3-O-methyl-6-deoxy-d-talose, could be identified in the lipopolysaccharides of the strains K/1 and 2/2 of the same species. 2. The O-methyl sugars described in this communication were isolated by paper chromatography and identified by g.l.c., paper chromatography, high-voltage electrophoresis and mass spectrometry. Besides the genuine sugars, their alditol acetates and their demethylated (parental) forms were investigated. Optical rotation measurements and, in one case, enzymic reactions were used to establish the optical configuration of the sugars under investigation.     

Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293.

Glycoconjugates are distributed on the cell surfaces of some small-sized treponemes and have been reported to be completely different from lipopolysaccharides. We separated a glycoconjugate fraction from Treponema medium ATCC 700293, a medium-sized oral spirochete, to assess its immunobiological activities and elucidate the chemical structure of its polysaccharide part using phenol/water extraction, hydrophobic chromatography, and gel filtration. The glycoconjugate showed negligible or weak endotoxic and immunobiological properties. The chemical structure of the polysaccharide part was shown by two-dimensional NMR and MALDI-TOF-MS to be a tetrasaccharide backbone with two amino acids: [-->4)beta-d-GlcpNAc3NAcA(1-->4)beta-d-ManpNAc3NAOrn(1-->3)beta-d-GlcpNAc(1-->3)alpha-D-Fucp4NAsp(1-->] where GlcNAc3NAcA is 2,3-diacetamido-2,3-dideoxyglucuronic acid, ManNAc3NAOrn is Ndelta-(2-acetamido-3-amino-2,3-dideoxymannuronyl)ornithine, and Fuc4NAsp is 4-(alpha-aspartyl)amino-4,6-dideoxygalactose.     

Structural analysis of the core region of lipopolysaccharides from Proteus mirabilis serotypes O6, O48 and O57.

The structure of lipid A core region of the lipopolysaccharides (LPS) from Proteus mirabilis serotypes O6, O57 and O48 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of LPS: [see text for structure]. Incomplete substitutions are indicated by bold italic type. All sugars are present in pyranose form, alpha-Hep is the residue of L-glycero-alpha-D-manno-Hep, alpha-DD-Hep is the residue of D-glycero-alpha-D-manno-Hep, L-Ara4N is 4-amino-4-deoxy-L-arabinose, Qui4NAlaAla is the residue of 4-N-(L-alanyl-L-alanyl)-4-amino-4,6-dideoxyglucose. All sugars except L-Ara4N have D-configuration. beta-GalA* is partially present in the form of amide with 1,4-diaminobutane (putrescine)-HN(CH2)4NH2 or spermidine-HN(CH2)3NH(CH2)4NH2.     

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.     

Analysis of the lipopolysaccharide of Pseudomonas maltophilia 555

The phenol phase soluble lipopolysaccharide of Pseudomonas maltophilia strain 555, obtained from cells by the hot aqueous phenol method, was of the smooth type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hydrolysis, methylation, and 13C and 1H nuclear magnetic resonance analyses showed that this lipopolysaccharide has an O-chain polysaccharide composed of a repeating pentasaccharide unit, containing D-rhamnose (D-Rha, one part), 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc, one part), and 4-acetamido-4,6-dideoxy-D-mannose (D-Rha4NAc, three parts) and having the structure (formula; see text) The serological cross-reactions between P. maltophilia 555 and Brucella species can now be related to the occurrence of N-acyl derivatives of 4-amino-4,6-dideoxy-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharide components.     

Structure and Properties of the Lipopolysaccharide of Pseudomonas fluorescens IMV 2366 (Biovar III)

The lipopolysaccharide (LPS) preparation isolated from the bacterial mass of Pseudomonas fluorescens IMV 2366 (biovar III) by Westphal's method and purified by repeated ultracentrifugation contained S- and R-forms of molecules. The structural components of the LPS molecule—lipid A, core oligosaccharide, and O-specific polysaccharide—were obtained in the individual state and characterized. The main components of the lipid A hydrophobic moiety were 3-hydoxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, and hexadecanoic fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic moiety. Rhamnose, glucose, galactose, glucosamine, galactosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as 2-amino-2,6-dideoxygalactose (FucN) and 3-amino-3,6-dideoxyglucose (Qui3N), were revealed in the composition of the core oligosaccharide fractions. O-specific polysaccharide chains were composed of repeating trisaccharide units consisting of residues of L-rhamnose (L-Rha), 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc), and 3-acylamido-3,6-dideoxy-D-glucose (D-Qui3NAcyl), where Acyl = 3-hydroxy-2,3-dimethyl-5-hydroxyprolyl. Neither double immunodiffusion in agar not the immunoenzymatic assay revealed serological relations between the strain studied and the P. fluorescens strains studied earlier.     

Capsular polysaccharides and lipopolysaccharides from two Bacteroides fragilis reference strains: chemical and immunochemical characterization.

Fermentor growth of Bacteroides fragilis under controlled conditions in a complex medium containing 1% glucose and 10% fetal calf serum resulted in high yields of bacteria. After hot phenol-water extraction of the organisms, capsular polysaccharide was isolated from the aqueous phase and purified by Sephacryl S-300 chromatography in a buffer with 3% sodium deoxycholate. Lipopolysaccharide was isolated by phenol-chloroform-light petroleum ether extraction. The capsular polysaccharide from B. fragilis strain NCTC 9343 contained six sugars: L-fucose, D-galactose, D- and L-quinovosamine, D-glucosamine, and galacturonic acid. The capsule of strain ATCC 23745 also contained D-glucose, L-fucosamine, L-rhamnosamine, and a 3-amino-3,6-dideoxyhexose but lacked D-quinovosamine. The latter capsule also contained alanine (4%). The capsular polysaccharides were different immunochemically by ELISA inhibition. The lipopolysaccharide of both strains contained the same sugars (L-rhamnose, D-glucose, D-galactose, and D-glucosamine) and fatty acids (13-methyl-tetradecanoic and 3-hydroxy-hexadecanoic and 3-hydroxy-15 methyl-hexadecanoic as major constituents) and were identical by ELISA inhibition.     

Antigenic bacterial polysaccharides. 24. The structure of the O-specific polysaccharide chain of Salmonella arizonae 063 (Arizona 08) lipopolysaccharide

The O-specific polysaccharide chain of the Salmonella arizonae O63 lipopolysaccharide is composed of D-glucose, D-galactose, N-acetyl-D-galactosamine, and 3-acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) residues in the ratio 1:1:2:1. On the basis of methylation analysis and calculations of 13C-NMR-spectra of the polysaccharide and of the product of its selective cleavage with anhydrous hydrogen fluoride, the linear polymer lacking 3-acetamido-3,6-dideoxygalactose, it was concluded that the polysaccharide has the following structure: (Formula: see text).     

Homogeneity of lipopolysaccharides from Acholeplasma.

Five methods were employed to determine the heterogeneity or homogeneity of lipopolysaccharides from four acholeplasmal species, Acholeplasma axanthum, A. granularum, A. laidlawii, and A. modicum. A axanthum lipopolysaccharide behaved as a single component in all tests. A. granularum exhibited two components of identical composition and antigenic specificity. A. modicum lipopolysaccharide behaved as three components in two tests, but all three were similar in composition and identical serologically. The separable components of lipopolysaccharides from A. granularum and A. modicum probably represent size differences only. A. laidlwii lipopolysaccharide contained two distinct components by all methods. One was identified as the previously reported amino sugar polymer, whereas the other was a lipopolysaccharide containing both neutral and amino sugars.     

New Structures of the O-Specific Polysaccharides of Proteus. 4. Polysaccharides Containing Unusual Acidic N-Acyl Derivatives of 4-Amino-4,6-dideoxy-D-glucose

The structures of the O-polysaccharides of the lipopolysaccharides of Proteus mirabilis O7 and O49 were determined by chemical methods, mass spectrometry, including MS/MS, and NMR spectroscopy, including experiments run in an H2O/D2O mixture to reveal correlations for NH protons. The O-polysaccharides were found to contain N-carboxyacetyl (malonyl) and N-(3-carboxypropanoyl) (succinyl) derivatives of 4-amino-4,6-dideoxyglucose (4-amino-4-deoxyquinovose, Qui4N), respectively. The behavior of Qui4N derivatives with the dicarboxylic acids under conditions of acid hydrolysis and methanolysis was studied using GLC-MS.     

Distribution and composition of lipopolysaccharides from mycoplasmas.

Polymeric carbohydrates containing glycerol and fatty acids were isolated from whole cells and membranes of mycoplasmas by hot aqueous phenol extraction and gel filtration. Lipopolysaccharides were found to occur in four species of Acholeplasma, two of Anaeroplasma, and in Mycoplasma neurolyticum. None were detected in Spiroplasma citri or in five species of Mycoplasma. All lipopolysaccharides contained both neutral and N-acylated amino sugars in ratios varying from 1:1 to 3:1. The neutral sugars found in varying distribution were glucose, galactose, and mannose. The amino sugars included fucosamine, an unidentified deoxyhexosamine, galactosamine, and glucosamine. Fucosamine and glucose were the only sugars common to all lipopolysaccharides. The fatty acids were similar to those found in the lipids of each organism.     

Identification of D-mannosamine and quinovosamine in Salmonella and related bacteria

Lipopolysaccharides of several strains of Salmonella, Arizona, and Proteus vulgaris, have been found to contain two unusual amino sugars not previously reported. These amino sugars were identified as d-mannosamine and quinovosamine (2-amino-2,6-dideoxy glucose).     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 1. Structural analysis of the highly hydrophobic lipopolysaccharide, including the O-antigen, its biological repeating unit, the core oligosaccharide, and the linkage between them

The lipopolysaccharide of Plesiomonas shigelloides serotype O74:H5 (strain CNCTC 144/92) was obtained with the hot phenol/water method, but unlike most of the S-type enterobacterial lipopolysaccharides, the O-antigens were preferentially extracted into the phenol phase. The poly- and oligosaccharides released by mild acidic hydrolysis of the lipopolysaccharide from both phenol and water phases were separated and investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF mass spectrometry, and sugar and methylation analysis. The O-specific polysaccharide and oligosaccharides consisting of the core, the core with one repeating unit, and the core with two repeating units were isolated. It was concluded that the O-specific polysaccharide is composed of a trisaccharide repeating unit with the [-->2)-beta-d-Quip3NAcyl-(1-->3)-alpha-l-Rhap2OAc-(1-->3)-alpha-d-FucpNAc-(1-->] structure, in which d-Qui3NAcyl is 3-amino-3,6-dideoxy-d-glucose acylated with 3-hydroxy-2,3-dimethyl-5-oxopyrrolidine-2-carboxylic acid. The major oligosaccharide consisted of a single repeating unit and a core oligosaccharide. This undecasaccharide contains information about the biological repeating unit and the type and position of the linkage between the O-specific chain and core. The presence of a terminal beta-d-Quip3NAcyl-(1--> residue and the -->3)-beta-d-FucpNAc-(1-->4)-alpha-d-GalpA element showed the structure of the biological repeating unit of the O-antigen and the substitution position to the core. The -->3)-beta-d-FucpNAc-(1--> residue has the anomeric configuration inverted compared to the same residue in the repeating unit. The core oligosaccharide was composed of a nonphosphorylated octasaccharide, which represents a novel core type of P. shigelloides LPS characteristic of serotype O74. The similarity between the isolated O-specific polysaccharide and that found on intact bacterial cells and lipopolysaccharide was confirmed by HR-MAS NMR experiments.     

Structure of the O-polysaccharide of Providencia alcalifaciens O19

Studies of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) of Providencia alcalifaciens O19 by sugar and methylation analyses along with NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments, showed that the pentasaccharide repeating unit of the polysaccharide has the following structure: [structure: see text] where Fuc3NAc is 3-acetamido-3,6-dideoxygalactose. The unique structure of the O-antigen and serological data are in consistence with classification of this bacterium in a separate Providencia serogroup.     

The structure of the O-polysaccharide isolated from the lipopolysaccharide of Salmonella Dakar (serogroup O:28)

The structure of the O-antigenic part of the lipopolysaccharide (LPS) of Salmonella Dakar was analysed using chemical methods, NMR spectroscopy and mass spectrometry. The following structure for the repeating unit of the O-polysaccharide was determined: [see text] where Quip3NAc is 3-acetamido-3,6-dideoxyglucose. This is the first published structure of the O-polysaccharides from 101 serotypes of Salmonella bacteria belonging to serogroup O:28 (formerly M) in the Kauffmann-White scheme.     

Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9

The phenol-phase soluble cellular lipopolysaccharide isolated by the phenol/water extraction method from Yersinia enterocolitica serotype O:9 cells was shown by hydrolytic, periodate oxidation, methylation and nuclear magnetic resonance studies to be an S-type lipopolysaccharide with a linear O-antigenic polysaccharide of 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The serological cross-reactivity between Y. enterocolitica serotype O:9 and the lipopolysaccharides of Vibrio cholerae and Brucella species can now be related to the presence of N-acylated 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residues in their respective O-antigenic chains.