Structural studies of the side chain of outer membrane lipopolysaccharide from Pseudomonas syringae pv. coriandricola W-43.

The lipopolysaccharide (LPS) was isolated from Pseudomonas syringae pv. coriandricola W-43 by hot phenol-water extraction. Rhamnose and 3-N-acetyl-3-deoxyfucose were found to be the major sugar constituents of the LPS together with N-acetylglucosamine, N-acetylgalactosamine, heptose, and 3-deoxy-D-manno-octulosonic acid (Kdo). The main fatty acids of lipid A of the LPS were 3-OH-C:10, C12:0, 2-OH-C12:0, and 3-OH-C12:0. The O-specific polysaccharide liberated from the LPS by mild-acid hydrolysis was purified by gel permeation chromatography. The compositional analysis of the O-specific polysaccharide revealed the presence of L-rhamnose and 3-N-acetyl-3-deoxy-D-fucose in a molar ratio of 4:1. The primary structure of the O-specific polysaccharide was established by methylation analysis together with 1H and 13C nuclear magnetic resonance spectroscopy, including two-dimensional shift-correlated and one-dimensional nuclear Overhauser effect spectroscopy. The polysaccharide moiety was found to consist of a tetrasaccharide rhamnan backbone, and 3-N-acetyl-3-deoxy-D-fucose constitutes the side chain of the branched pentasaccharide repeating unit of the polysaccharide.     

Antigenic bacterial polysaccharides. 28. The structure of the O-specific lipopolysaccharide chain of Pseudomonas syringae pv. atrofaciens K-1025 and Pseudomonas holci 90a (serogroup II)

Lipopolysaccharides of serologically related strains of Pseudomonas syringae pv. atrofaciens K-1025 and Pseudomonas holci 90a possess the identical O-specific polysaccharide chains, representing a homopolymer of D-rhamnose. On the basis of methylation, partial and complete Smith degradation, and analysis by 1H- and 13C-NMR-spectroscopy, it was concluded that the repeating unit of the polysaccharide is a branched pentasaccharide of the following structure: (formula; see text)     

Antigenic bacterial polysaccharides. 13. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from Pseudomonas cepacia strain IMV 4137

On mild acid degradation of a lipopolysaccharide from Pseudomonas cepacia strain IMV 4137, a serologically active O-specific polysaccharide was obtained and shown to contain L-rhamnose and D-galactose. According to 1H- and 13C-NMR data as well as methylation analysis, the polysaccharide is made up of disaccharide repeating units of the following structure:----2)-alpha-L-Rhap-(1----4)-alpha-D-Galp-(1----.     

Antigenic polysaccharides of bacteria. 31. The structure of the O-specific polysaccharide chain of Pseudomonas aurantiaca IMB 31 lipopolysaccharide

The O-specific polysaccharide chain of the Pseudomonas aurantiaca IMV 31 lipopolysaccharide contains N-acetyl-L-fucosamine (FucNAc) and di-N-acetyl-D-bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose, Bac(NAc)2) in the ratio 2:1. On the basis of methylation, solvolysis with anhydrous hydrogen fluoride, and computer-assisted analysis of 13C-NMR spectrum, it was concluded that the trisaccharide repeating unit of the polysaccharide possesses the following structure: structure: ----3)-beta-D-Bac(NAc)2-(1----3)-alpha-L-FucNAc-(1----3)-alpha-L- FucNAc-(1----.     

Antigenic polysaccharides of bacteria. 25. Structure of the O-specific polysaccharide chain of Pseudomonas cepacia 673/2 lipopolysaccharide containing L-glycero-D-manno-heptose

O-Specific polysaccharide, consisting of D-rhamnose and L-glycero-D-manno-heptose (LD-Hep) in a 2 : 1 ratio, was obtained on the mild acid degradation of the Pseudomonas cepacia IMV 673/2 lipopolysaccharide; monosaccharide LD-Hep has not previously been found in O-specific chains of lipopolysaccharides. On the basis of methylation and 13C-NMR data, it was concluded that the polysaccharide is composed of trisaccharide repeating units having the following structure: ----3)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1----2)-alpha-LD-Hep-(1----     

Antigenic bacterial polysaccharides. 36. A study of the structure of the O-specific polysaccharide chain of the lipopolysaccharide of Pseudomonas fluorescens IMV 2763 (biostrain G)

Chemical and serological characterization of the Pseudomonas fluorescens IMV 2763 (biovar G) lipopolysaccharide was carried out. The O-specific polysaccharide chain of the lipopolysaccharide is composed of D-mannose, 6-deoxy-L-talose, N-acetyl-D-galactosamine and O-acetyl groups in the ratio of approximately 2:1:1:1. The polysaccharide is branched and a half of residues of 6-deoxytalose and monosubstituted mannose carry O-acetyl groups. On the basis of methylation, partial acid hydrolysis and 13C NMR analysis it was concluded that the repeating unit of the polysaccharide has the following structure: (formula; see text)     

The structure of O-specific polysaccharide chain of Pseudomonas fluorescens lipopolysaccharide

An O-specific polysaccharide, containing 6-deoxy-L-talose (6dTal), N-acetyl-D-fucosamine (FucNAc), 3-amino-3,6-dideoxy-D-glucose with an unidentified N-acyl substituent (Qui3NR), and O-acetyl groups, was obtained on mild acid degradation of a Pseudomonas fluorescens strain 361 lipopolysaccharide. On the basis of O-deacetylation, acid hydrolysis, methylation, selective solvolysis with anhydrous hydrogen fluoride, and 13C NMR analysis, the polysaccharide is built up of trisaccharide repeating units of the following structure: (Formula: see text).     

Antigenic polysaccharides of bacteria. 26. Structure of O-specific polysaccharides from Pseudomonas cerasi 467 and Pseudomonas syringae pv. syringae strains 218 and P-55 belonging to serogroups II and III

Serologically active O-specific polysaccharides were obtained on mild acid hydrolysis of lipopolysaccharides from Pseudomonas cerasi 467 and Pseudomonas syringae pv. syringae strains 218 and P-55. On the basis of 1H- and 13C-NMR analysis, it was concluded that the P. cerasi polysaccharide has the following structure: ----3)-alpha-D-Rhap-(1----3)-alpha-D-Rhap-(1----2)-alpha-D-+ ++Rhap-(1---- which is identical to that of O-specific polysaccharide from P. syringae pv. morsprunorum C28 (Smith A. R. W. et al. Eur. J. Biochem., 1985, V. 149, No 1, p. 73-78). The polysaccharides from P. syringae pv. syringae strains possess the same backbone but differ by the presence of D-fucose as monosaccharide branches. Methylation and 1H- and 13C-NMR analysis revealed the following structure of these polysaccharides: (Formula: see text). The degree of substitution of the backbone trisaccharide units by the fucofuranose residues is about 35% for the strain 218 and about 85% for the strain P-55.     

The structure of the O-specific polysaccharide from the lipopolysaccharide of Pseudomonas sp. OX1 cultivated in the presence of the azo dye Orange II

The Gram-negative bacterium Pseudomonas sp. OX1, previously known as Pseudomonas stutzeri OX1, is endowed with a high metabolic versatility. In fact, it is able to utilize a wide range of toxic organic compounds as the only source of carbon and energy for growth. It has been recently observed that, while growing on a glucose-containing liquid medium, Pseudomonas sp. OX1 can reduce azo dyes, ubiquitous pollutants particularly resistant to chemical and physical degradation, with this azoreduction being a process able to generate enough energy to sustain bacterial survival. We have found that, under these conditions, modifications in the primary structure of the O-specific polysaccharide (OPS) within the lipopolysaccharides occur, leading to remarkable changes both in the monosaccharide composition and in the architecture of the repeating unit, with respect to the polysaccharide produced in the absence of azo dyes. In the present paper, we present the complete structure of this O-specific polysaccharide, whose repeating unit is the following: [Formula: see text] This structure is totally different from the one determined from Pseudomonas sp. OX1 grown on rich medium.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the polysaccharide chain of Ps. aeruginosa O-serogroup 7 (Lanyi) lipopolysacharide

A loosely bound lipopolysaccharide-protein complex was extracted from cells of Pseudomonas aeruginosa strain 170015 (O:7ab; Lanyi classification) by saline solution and purified from contaminant nucleic acid by Cetavlon treatment followed by precipitation in an ultracentrifuge. The saline-treated cells were re-extracted with hot aqueous phenol to give firmly bound lipopolysaccharide which was isolated from the phenol layer and purified by ultracentrifugaiton. The identity of both lipopolysaccharide preparations was proved by serological and chemical evidence. Mild acid degradation of the lipopolysaccharide resulted in the splitting off of a lipid component and led to polysaccharide which was purified by gel-filtration on a Sephadex G-50 column. The polysaccharide consisted of N-acetyl-D-fucosamine, N-acetyl-L-fucosamine and D-glucose in the ratio 1:1:1. On the basis of nuclear magnetic resonance spectra, results of methylation analysis and two sequential Smith degradations, the following structure can be assigned to the repeating unit of the polysaccharide: -3)LFucNAc(alpha 1-3)DFucNAc(beta 1-2)DGlc(beta 1-. The polysaccharide did not show serological activity whereas alkali-treated lipopolysaccharide readily sensitised sheep erythrocytes and inhibited the passive haemagglutination reaction with anti-(O:7a,b)serum. Evidence is presented that the oligosaccharide repeating units of the polysaccharide and alkali-treated lipopolysaccharide are indistinguishable. Ps. aeruginosa strain 170016 (O:7a,c) was shown to have the O-specific lipopolysaccharide identical with that from strain 170015. The presented data show that subfactors 7b and 7c in the Lanyi classification of Ps. aeruginosa O-antigens seem to relate to components of the bacterial surface other than lipopolysaccharides.     

Antigenic bacterial polysaccharides. 14. Structure of the O-specific polysaccharide chain of a lipopolysaccharide from Pseudomonas aeruginosa (Lányi)

The lipopolysaccharide from Pseudomonas aeruginosa O12 (Lányi classification) gave on mild acid hydrolysis an O-specific polysaccharide built of D-ribose and N-acetyl-D-galactosamine. The disaccharide structure----4)-alpha-GalNAcp-(1----2)-beta-Ribf-(1----for the repeating unit of the polysaccharide was established by nondestructive way involving full interpretation of its 1H- and 13C-NMR-spectra, using homonuclear and selective heteronuclear 13C[1H] double resonances.     

Antigenic polysaccharides of bacteria. 35. Establishment of the structure of polysaccharide chains of lipopolysaccharides of Pseudomonas cepacia IMV 4176 and IMV 4202 (Serotype 3)

On mild acid degradation of the Pseudomonas cepacia strain IMV 4176 lipopolysaccharide, two polysaccharides were obtained, one of which is a homopolymer of N-acetyl-D-galactosamine and the other is composed of equal amounts of N-acetyl-D-galactosamine and D-ribose. Partial hydrolysis with aqueous oxalic acid caused depolymerization of the heteropolysaccharide, and the homopolysaccharide was isolated in the individual state. On the basis of methylation and 13C NMR analysis, it was concluded that both polysaccharides are built up of disaccharide repeating units having the following structures: ----4)-alpha-D-GalpNAc-(1----4)-beta-D-GalpNAc-(1---- and ----4)-alpha-D-GalpNAc-(1----2)-beta-D-Ribf-(1----. The heteropolysaccharide from P. cepacia strain 4176 is identical by the structure of the repeating unit to the O-specific polysaccharide of P. cepacia strain IMV 4202 (serotype 3), Pseudomonas aeruginosa O12 and Serratia marcescens O14.     

Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D-rhamnan

Structural studies were carried out on a rhamnose-rich polysaccharide isolated from the O-polysaccharide fraction of lipopolysaccharide in Pseudomonas aeruginosa IID 1008 (ATCC 27584) after destruction of the major O-specific chain by alkaline treatment. The isolated polysaccharide contained rhamnose, 3-O-methyl-6-deoxyhexose, glucose, xylose, alanine, galactosamine and phosphorus in a molar ratio of 67:6.9:4.3:2.1:1.1:1.0:4.1. Data from analysis involving Smith degradation, methylation, 1H-NMR spectroscopy and optical rotation measurement showed that the polysaccharide was built up of three moieties, a rhamnan chain composed of about 70 D-rhamnose residues, the core chain and an oligosaccharide chain comprising 3-O-methyl-6-deoxyhexose, xylose, rhamnose and probably glucose. The repeating unit of the rhamnan chain was indicated to have the following structure:----3)D-Rha(alpha 1----3)D-Rha(alpha 1----2)D-Rha(alpha 1----. This structure is identical with that proposed previously for the repeating unit of the side chain of lipopolysaccharide from plant pathogenic bacteria Pseudomonas syringae pv. morsprunorum C28 [Smith, A.R.W., Zamze, S.E., Munro, S.M., Carter, K. J. and Hignett, R.C. (1985) Eur. J. Biochem. 149, 73-78].     

Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C.     

Structural determination of the O-specific chain of the lipopolysaccharide from the mushrooms pathogenic bacterium Pseudomonas tolaasii

The complete structure of the O-specific polysaccharide of the lipopolysaccharide isolated from the cultivated mushrooms pathogen Pseudomonas tolaasii is described. The structural determination, achieved by chemical and spectroscopical analyses, indicates a novel tetrasaccharide repeating unit built up of two units of 2-acetamido-2,6-di-deoxy-glucopyranose (Quinovosamine, QuipNAc) and two units of 2-acetamido-2-deoxy-gulopyranuronamide (GulpNAcAN), one of which is acetylated at C-3 position:     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from P. aeruginosa O13 (Lányi)

The O-specific polysaccharide, obtained on mild acid degradation of lipopolysaccharide of Pseudomonas aeruginosa O13 (Lányi classification), is built up of trisaccharide repeating units involving 2-acetamidino-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), 2-acetamidino-2,6-dideoxy-L-galactose (L-fucosacetamidine, L-FucAm), and a new sialic-acid-like sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso n ic acid (Sug), and thus contains simultaneously both acidic and basic functions. Cleavage of the polysaccharide with hydrogen fluoride in methanol revealed the high stability of the glycosidic linkage of the ulosonic acid and afforded methyl glycosides of a disaccharide and a trisaccharide. The structures of the new ulosonic acid and acetamidino group were established by analysing the oligosaccharide fragments by 1H, 13C nuclear magnetic resonance spectrometry, as well as on the basis of their chemical conversions: alkaline hydrolysis of the acetamidino group into acetamido group, reductive deamination with lithium borohydride into the ethylamino group and acetylation with acetic anhydride in pyridine accompanied by intramolecular acylation of the acetamidino function by the ulosonic acid to form a six-membered lactam ring. Identification of the oligosaccharide fragments and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and polysaccharide revealed the following structure of the repeating unit: ----3)D-QuiNAcp(alpha 1----3)Sugp(alpha 2----3)L-FucAmp(alpha 1----.     

Structure of the O-polysaccharide of the lipopolysaccharide of Pseudomonas syringae pv. garcae ICMP 8047.

The composition and structure of the O-polysaccharide of the lipopolysaccharide of Pseudomonas syringae pathovar garcae ICMP 8047 were studied using methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The polysaccharide was found to contain L-rhamnose and 3-acetamido-3, 6-dideoxy-D-galactose (D-Fuc3NAc) in the ratio 4:1 and to consist of two types of pentasaccharide repeating units. The major (1) and minor (2) repeating units differ from each other only in the position of substitution of one of the rhamnose residues in the main chain. Similar structural heterogeneity has been reported formerly in O-polysaccharides of some other P. syringae strains having a similar monosaccharide composition. A Fuc3NAc residue is attached to the main rhamnan chain as a side chain by a (alpha1-->4) glycosidic linkage; this has not hitherto been described in P. syringae: [figure].     

Structural diversity of O-polysaccharides and serological classification of Pseudomonas syringae pv. garcae and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I: --> 3)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 -->; II: --> 3)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 --> 2)-alpha-L-Rha-(1 --> 3)-alpha-L-Rha-(1 -->. The branched polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588 have the same L-rhamnan backbone with repeating units I and II and a lateral chain of (alpha1 --> 4)- or (alpha1 --> 3)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral alpha-D-Fuc3NAc residues were characterized.     

Structure of O-specific polysaccharide chains of Pseudomonas aeruginosa II (Sandwick) and V (Verder-Evans) lipopolysaccharides containing N-acetyl-D-galactosaminuronic acid

Acidic polysaccharides were isolated from the Pseudomonas aeruginosa II (Sandvik) and V (Verder-Evans) lipopolysaccharides on mild acid hydrolysis followed by gel filtration on Sephadex G-50. The Sandvik II polysaccharide consists of 2-acetamido-2-deoxy-D-galacturonic acid, 2-acetamido-2,6-dideoxy-D-glucose, and L-rhamnose in the ratio 1:1:2. The Verder-Evans V polysaccharide contained the same monosaccharides and, in addition, a D-glucose residue. On the basis of 13C NMR data, methylation analysis, Smith degradation and solvolysis with hydrogen fluoride, the following structures were determined for the repeating units of the polysaccharides: (Formula: see text).     

Structure of the repeating chain of the O-specific polysaccharide of Vibrio fluvialis

O-Specific polysaccharide chain of the Vibrio fluvialis lipopolysaccharide is built up of pentasaccharide repeating units, containing one N-acetyl-D-glucosamine and four L-rhamnose residues. The structure of the polysaccharide was elucidated using two-dimensional correlation 1H-NMR-spectroscopy, 13C-NMR-spectroscopy and nuclear Overhauser effect and confirmed by methylation analysis and selective cleavage of N-acetylglucosamine residues by the N-deacetylation-deamination method which yielded linear L-rhamnan representing the backbone of the polysaccharide. Thus, the repeating unit of the O-specific polysaccharide has the following structure: (formula; see text)