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 was characterized by the presence of the S- and R-forms of molecules. The following structural portions of the LPS molecule were obtained in the individual state and characterized: lipid A, core oligosaccharide, and O-specific polysaccharide. 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, 2-keto-3-desoxyoctulosonic acid (KDO), as well as 2-amino-2,6-didesoxygalactose (FucN) and 3-amino-3,6-didesoxyglucose (Qui3N), were revealed in the composition of the core oligosaccharide fractions. O-specific polysaccharide chains were established to be composed of repeating trisaccharide units consisting of residues of L-rhamnose (L-Rha), 2-acetamido-2,6-didesoxy-D-galactose (D-FucNAc), and 3-acylamido-3,6-didesoxy-D-glucose (D-Qui3NAcyl), where Acyl = 3-hydroxy-2,3-dimethyl-5-hydroxyprolyl. Neither double immunodiffusion in agar not the immunoenzyme assay revealed serological relations between the strain studied and the P. fluorescens strains studied earlier.     

Comparative characteristics of lipopolysaccharides of various Pseudomonas fluorescens strains (Biovar I)

From the biomass of five Pseudomonas fluorescens biovar I strains, including the P. fluorescens type strain IMV 4125 (ATCC 13525), lipopolysaccharides (LPS) were isolated (by extraction with a phenol-water mixture followed by repeated ultracentrifugation), as well as individual structural components of the LPS macromolecule: lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS). 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, octadecanoic, hexadecenoic, and octadecenoic fatty acids were present in lipid A of the LPS of all the strains studied. Glucosamine, ethanolamine, and phosphoethanolamine were revealed in the lipid A hydrophilic part of all of KDO, a trace amount of heptoses, ethanolamine, phosphoethanolamine, alanine, and phosphorus were identified as the main core components. Interstrain differences in the core oligosaccharide composition were revealed. Structural analysis showed that the O-PS of the type strain, as distinct from that of other strains, is heterogeneous and contains two types of repetitive units, including (1) three L-rhamnose residues (L-Rha), one 3-acetamide-3,6-dideoxy-D-galactose residue (D-Fuc3NAc) as a branching substitute of the L-rhamnan chain and (2) three L-Rha residues and two branching D-Fuc3NAc residues. The type strain is also serologically distinct from other biovar I strains due to the LPS O-chain structure, which is similar to those of the strains of the species Pseudomonas syringae, including the type strain. The data of structural analysis agree well with the results of immunochemical studies of LPS.     

The distinctive features of the structure of the Pseudomonas fluorescens IMV 247 (biovar II) lipopolysaccharide

The results of the study of the Pseudomonas fluorescens IMV 247 (biovar II) lipopolysaccharide (LPS) isolated from the dry bacterial mass by Westphal's method and purified by repeated ultracentrifugation are presented. The macromolecular organization of the LPS is characterized by the presence of S and R forms of LPS molecules in a 1:1 ratio. The structural components of the LPS molecule--lipid A, the core oligosaccharide, and the O-specific polysaccharide--were isolated and characterized. 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, and dodecanoic acids proved to be the main lipid A fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic portion. Glucose, galactose, arabinose, rhamnose, glucosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulonate (KDO) were revealed in the heterogeneous fraction of the core oligosaccharide. The O-specific polysaccharide chain was composed of repeating tetrasaccharide units consisting of L-rhamnose (L-Rha), 3,6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido-2,4,6-trideoxy-4[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb), and 2-acetamido-2-deoxy-D-galacturonic acid (D-GalNAcA) residues. A peculiarity of the O-specific polysaccharide was that it released, upon partial acid hydrolysis, the nonreducing disaccharide GalNAcA-->QuiNAc4NHb with a 3-hydroxybutyryl group glycosylated intramolecularly with a QuiN4N residue. Double immunodiffusion in agar and lipopolysaccharide precipitation reactions revealed no serological interrelationship between the strain studied and the P. fluorescens strains studied earlier.     

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.     

Peculiarities of the structure of thePseudomonas fluorescens IMV 247 (Biovar II) lipopolysaccharide

The results of the study of thePseudomonas fluorescens IMV 247 (biovar II) lipopolysaccharide (LPS) isolated from the dry bacterial mass by Westphal’s method and purified by repeated ultracentrifugation are presented. The macromolecular organization of the LPS is characterized by the presence of S and R forms of LPS molecules in a 1 : 1 ratio. The structural components of the LPS molecule-lipid A, the core oligosaccharide, and the 0-specific polysaccharide-were isolated and characterized. 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, and dodecanoic acids proved to be the main lipid A fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic portion. Glucose, galactose, arabinose, rhamnose, glucosamine, galactosamine alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulonate (KDO) were revealed in the heterogeneous fraction of the core oligosaccharide. The 0-specific polysaccharide chain was composed of repeating tetrasaccharide units consisting of L-rhamnose (L-Rha), 3,6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido-2,4,6-trideoxy4 [(S)-3-hydroxybutyramido]-D-glucose (D-QuiNAc4NHb), and 2-acetamido-2-deoxy-D-galacturonic acid (D-GalNAcA) residues. A peculiarity of the 0-specific polysaccharide was that it released, upon partial acid hydrolysis, the nonreducing disaccharide GalNAcA→ QuiNAc4NHb with a 3-hydroxybutyryl group glycosylated intramolecularly with a QuiN4N residue. Double immunodiffusion in agar and lipopolysaccharide precipitation reactions revealed no serological interrelationship between the strain studied and theP. fluorescens strains studied earlier.     

Comparative Characterization of the Lipopolysaccharides of Different Pseudomonas fluorescensBiovar I Strains

From the biomass of five Pseudomonas fluorescensbiovar I strains, including the P. fluorescenstype strain IMV 4125 (ATCC 13525), lipopolysaccharides (LPS) were isolated (by extraction with a phenol–water mixture followed by repeated ultracentrifugation), as well as individual structural components of the LPS macromolecule: lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS). 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, octadecanoic, hexadecenoic, and octadecenoic fatty acids were present in lipid A of the LPS of all the strains studied. Glucosamine, ethanolamine, and phosphoethanolamine were revealed in the lipid A hydrophilic part of all of the strains. Glucose, rhamnose, mannoze, glucosamine, galactosamine, KDO, a trace amount of heptoses, ethanolamine, phosphoethanolamine, alanine, and phosphorus were identified as the main core components. Interstrain differences in the core oligosaccharide composition were revealed. Structural analysis showed that the O-PS of the type strain, as distinct from that of other strains, is heterogeneous and contains two types of repetitive units, including (1) three L-rhamnose residues (L-Rha), one 3-acetamide-3,6-dideoxy-D-galactose residue (D-Fuc3NAc) as a branching substitute of the L-rhamnan chain and (2) three L-Rha residues and two branching D-Fuc3NAc residues. The type strain is also serologically distinct from other biovar I strains due to the LPS O-chain structure, which is similar to those of the strains of the species Pseudomonas syringae, including the type strain. The data of structural analysis agree well with the results of immunochemical studies of LPS.     

Characterization of lipopolysaccharides from Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) with O-chains represented by alpha-glucan

Results of studies of the structurally unique O-chains of lipopolysaccharides, which were isolated from the dry biomass of Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) by the Westphal technique and purified by repeated ultracentrifugation, are reported. The bulk of the lipopolysaccharide preparations contained S- and R-molecules at an average molar ratio of 1: 2. The main components of the hydrophobic moiety of lipid A were 3-hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, and octadecanoic acids, as well as hexadecenoic and octadecenoic acids. Glucosamine and phosphoethanolamine were identified as components of the hydrophilic moiety of lipid A. The degree of lipid A phosphorylation amounted to 3-4%. Fractions of the core oligosaccharide contained glucose, galactose, mannose, rhamnose, arabinose, glucosamine (only in strain IMB 2108), alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO). Heptose was present in trace amounts. O-specific polysaccharide chains were represented by a linear polymer of D-glucose units, which were linked together via alpha-(1,4) glycoside bonds. The existence of P. fluorescens strains that have alpha-1,4-glucan as the O-chain of their lipopolysaccharides has not been described before.     

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).     

Characterization of Lipopolysaccharides from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> IMB 2108 (Biovar II) and IMB 2111 (Biovar IV) with O-Chains Represented by α-Glucan

Results of studies of the structurally unique O-chains of lipopolysaccharides, which were isolated from the dry biomass of Pseudomonas fluorescens IMB 2108 (biovar II) and IMB 2111 (biovar IV) by the Westphal technique and purified by repeated ultracentrifugation, are reported. The bulk of the lipopolysaccharide preparations contained S- and R-molecules at an average molar ratio of 1 : 2. The main components of the hydrophobic moiety of lipid A were 3-hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, and octadecanoic acids, as well as hexadecenoic and octadecenoic acids. Glucosamine and phosphoethanolamine were identified as components of the hydrophilic moiety of lipid A. The degree of lipid A phosphorylation amounted to 3–4%. Fractions of the core oligosaccharide contained glucose, galactose, mannose, rhamnose, arabinose, glucosamine (only in strain IMB 2108), alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO). Heptose was present in trace amounts. O-specific polysaccharide chains were represented by a linear polymer of D-glucose units, which were linked together via α-(1,4) glycoside bonds. The existence of P. fluorescens strains that have α-1,4-glucan as the O-chain of their lipopolysaccharides has not been described before.     

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).     

Fatty acid composition of lipid A in Pseudomonas fluorescens

The fatty acid composition of lipid A was studied using gas-liquid chromatography (GLC) and GLC-mass spectrometry in Pseudomonas fluorescens strains of biovars A, B, C, i, F and G, the type strain ATCC 13525 (biovar A) inclusive. The following fatty acids were identified as predominant in the composition of lipid A in the strains representing biovars A, B, C, i, F and G: 3-hydroxydecanoic (3-OH C10:0), 2-hydroxydodecanoic (2-OH C12:0), 3-hydroxydodecanoic (3-OH C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoic (C18:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acids. Lipid A of a biovar G strain differed noticeably from other strains in its fatty acid composition. Its main components were as follows: 3-hydroxytetradecanoic (3-OH C14:0), 3-hydroxypentadecanoic (3-OH C15:0) and dodecanoic (C12:0) fatty acids. The coefficients of similarity were determined for lipid A specimens isolated from the studied strains of P. fluorescens by calculating their fatty acid composition with a computer.     

Composition, structure, and biological properties of lipopolysaccharides from different strains of Pseudomonas syringae pv. atrofaciens

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C10:0, C12:0, 2-HO-C12:0, 3-HO-C12:0, C16:0, C16:1, C18:0, and C18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L- (two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substitute. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.     

Studies on the antigenic S-type lipopolysaccharides of Brucella abortus strains 7 and Mustapha

Antigenic phenol phase S-type lipopolysaccharides (LPS) isolated from Brucella abortus (B. abortus) strains 7 and Mustapha were observed to have 13C n.m.r. spectra which were almost identical to the one reported for the Brucella abortus 1119-3. The glycosyl content of the lipid A obtained from the LPS of strain 7 was found to be 2-acetamido-2-deoxyglucose only while strain Mustapha was found to contain both 2-acetamido-2-deoxyglucose and 2-acetamido-2-deoxygalactose. The fatty acid present in the lipid A of both strains was mainly n-hexadecanoic acid. Octadecanoic acid, 3-hydroxytetradecanoic acid as well as small quantities of 3-hydroxydodecanoic acid were also identified. This contrasts with the earlier reports of the absence of 3-OH-14:0 in the LPS of Brucella abortus.     

Genetic locus and structural characterization of the biochemical defect in the O-antigenic polysaccharide of the symbiotically deficient Rhizobium etli mutant, CE166. Replacement of N-acetylquinovosamine with its hexosyl-4-ulose precursor

The O-antigen polysaccharide (OPS) of Rhizobium etli CE3 lipopolysaccharide (LPS) is linked to the core oligosaccharide via an N-acetylquinovosaminosyl (QuiNAc) residue. A mutant of CE3, CE166, produces LPS with reduced amounts of OPS, and a suppressed mutant, CE166 alpha, produces LPS with nearly normal OPS levels. Both mutants are deficient in QuiNAc production. Characterization of OPS from CE166 and CE166 alpha showed that QuiNAc was replaced by its 4-keto derivative, 2-acetamido-2,6-dideoxyhexosyl-4-ulose. The identity of this residue was determined by NMR and mass spectrometry, and by gas chromatography-mass spectrometry analysis of its 2-acetamido-4-deutero-2,6-dideoxyhexosyl derivatives produced by reduction of the 4-keto group using borodeuteride. Mass spectrometric and methylation analyses showed that the 2-acetamido-2,6-dideoxyhexosyl-4-ulosyl residue was 3-linked and attached to the core-region external Kdo III residue of the LPS, the same position as that of QuiNAc in the CE3 LPS. DNA sequencing revealed that the transposon insertion in strain CE166 was located in an open reading frame whose predicted translation product, LpsQ, falls within a large family of predicted open reading frames, which includes biochemically characterized members that are sugar epimerases and/or reductases. A hypothesis to be tested in future work is that lpsQ encodes UDP-2-acetamido-2,6-dideoxyhexosyl-4-ulose reductase, the second step in the synthesis of UDP-QuiNAc from UDP-GlcNAc.     

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient

Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient. Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V. parahaemolyticus serotype O2. A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep). Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone. Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below. The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions. Both similarity and differences are observed between the LPS of V. parahaemolyticus serotype O2 and strain KX-V212. [carbohydrate structure: see text]     

Hafnia alvei lipopolysaccharides: Isolation, sugar composition and SDS-PAGE analysis

Abstract Lipopolysaccharides (LPS) of 33 strains of Hafnia alvei were isolated and purified. LPS content of the dry bacterial mass ranged from 1.2 to 4.5%. All examined lipopolysaccharides contained glucose, glucosamine, heptose, 3-deoxy-octulosonic acid and often galactose. Rhamnose, mannose, galactosamine, mannosamine and unidentified amino sugars were found in some H. alvei strains. Sialic acid was present in LPS of one strain. d -3-Hydroxybutyryl groups also were identified in lipopolysaccharides of 5 strains of this genus.
SDS-PAGE of the lipopolysaccharides was presented in the paper. According to these results two core types exist in H. alvei .     

Chemical characteristics and endotoxic activity of the lipopolysaccharide of Rahnella aquatilis 2-95

The lipopolysaccharide (LPS) from a new Enterobacteriaceae species, Rahnella aquatilis 2-95, was isolated and investigated. The structural components of the LPS molecule, namely, lipid A, core oligosaccharide, and O-specific polysaccharide, were obtained by mild acid hydrolysis. In lipid A, 3-oxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. The major monosaccharides of the core oligosaccharide were galactose, arabinose, fucose, rhamnose, and an unidentified component. The O-specific polysaccharide was found to be assembled of a repeated trisaccharide unit of the following structure: [structure: see text]. The R. aquatilis 2-95 LPS is less toxic and more pyrogenic as compared to the one from the R. aquatilis 1-95 strain studied earlier. Both acyl and phosphate groups are essential for toxic and pyrogenic activity of R. aquatilis 2-95 LPS.     

A Comparative Study of the Sugar Composition of O-antigenic Lipopolysaccharides Isolated from Vibrio alginolyticus and Vibrio parahaemolyticus

A comparative study of the sugar composition of O-antigenic lipopolysaccharides (LPS) isolated from Vibrio alginolyticus and those from V. parahaemolyticus was carried out. 3-Deoxy-d-mannooctulosonic acid, 2-keto-3-deoxy octonate (KDO), a regular sugar constituent of gram-negative bacterial LPS, was totally absent from LPS of all V. alginolyticus strains examined as it was from those of V. parahaemolyticus. Furthermore, a KDO-like thiobarbituric acid test-positive substance, identical with that of either V. parahaemolyticus 07 or 012, was also found in LPS from three strains, 505–78, 905–78, and 1013–79 (designated tentatively as group I), out of the five strains of V. alginolyticus tested. LPS from the members of group I contained, as component sugars, glucose, galactose, l-glycero-d-manno-heptose, glucosamine, galactosamine, the KDO-like substance, and an unidentified amino sugar P1. Thus, LPS of the members of group I possessed a similar sugar composition which is similar to that of LPS from either V. parahaemolyticus 07 or 012. LPS of strain 1027–79, one of the other two strains (designated tentatively as gorup II), contained as component sugars, glucose, l-glycero-d-mannoheptose, glucosamine, galactosamine, and the other unidentified amino sugar P2, while LPS of strain 53–79, the other member of group II, contained galactose as an additional component. The results indicate that LPS of strain 1027–79 has a sugar composition similar to that of V. parahaemolyticus 09 LPS.     

C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella montevideo

We studied the population of LPS molecules on Salmonella montevideo that bind C3 during alternative pathway activation in serum. LPS molecules of Salmonella are composed of lipid A:core oligosaccharide (one copy per molecule), substituted by an O-polysaccharide (O-PS) side chain, which is a linear polymer of 0 to greater than 60 O-antigen repeat units containing mannose. A mutant of S. montevideo called SL5222 that inserts galactose only into core oligosaccharide and mannose only into O-antigen subunits was grown with [3H]mannose and [14C]galactose, so that LPS molecules bearing large numbers of O-antigen subunits have high 3H to 14C ratios, whereas molecules with few O-antigen subunits have lower 3H to 14C ratios. Double-labeled SL5222 was incubated in C8-deficient (C8D) serum or C8D serum with 2 mM Mg++Cl2 and 10 mM ethylene glycoltetraacetic acid (MgEGTA C8D). LPS molecules with covalently attached C3 were identified by binding to anti-C3. LPS molecules that bound C3 under both incubation conditions had O chains seven to eight times longer than the average LPS molecule. SL5222 was then grown in suboptimal concentrations of mannose in order to decrease the number of LPS molecules with long O-PS side chains. C3 attached to progressively shorter chain molecules of LPS as the mannose input was lowered, but still chose the longest available molecules. This finding and recently published observations indicate that C3 can bind to LPS molecules with short O-PS side chains. We postulate that preferential attachment of C3 to long-chain LPS in SL5222 results because long-chain LPS molecules sterically hinder shorter chain LPS molecules from macromolecules. This study provides direct proof that the O-PS of LPS sterically hinders access of large molecules to the outer membrane and indicates that the LPS coat of these bacteria functions as a barrier against large protein molecules.     

Composition,structure, and biological properties of lipopolysaccharides from different strains of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">atrofaciens</Emphasis>

The composition, structure, and certain biological properties of lipopolysaccharides (LPS) isolated from six strains of bacteria Pseudomonas syringae pv. atrofaciens pathogenic for grain-crops (wheat, rye) are presented. The LPS-protein complexes were isolated by a sparing procedure (extraction from microbial cells with a weak salt solution). They reacted with the homologous O sera and contained one to three antigenic determinants. Against the cells of warm-blooded animals (mice, humans) they exhibited the biological activity typical of endotoxins (stimulation of cytokine production, mitogenetic activity, etc.). The LCD of the biovar type strain was highly toxic to mice sensitized with D-galactosamine. The structural components of LPS macromolecules obtained by mild acidic degradation were characterized: lipid A, core oligosaccharide, and O-specific polysaccharide (OPS). Fatty acids 3-HO-C_10:0, C_12:0, 2-HO-C_12:0, 3-HO-C_12:0, C_16:0, C_16:1, C_18:0, and C_18:1 were identified in lipid A of all the strains, as well as the components of the hydrophilic part: glucosamine (GlcN), ethanolamine (EtN), phosphate, and phosphoethanolamine (EtN-P). In the core LPS, glucose (Glc), rhamnose (Rha), L-glycero-D-manno-heptose (Hep), GlcN, galactosamine (GalN), 2-keto-3-deoxy-D-mannooctonoic acid (KDO), alanine (Ala), and phosphate were present. The O chain of all the strains consisted of repeated elements containing a linear chain of three to four L-(two strains) or D-Rha (four strains) residues supplemented with a single residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fucp3Nac), N-acetyl-D-glucosamine (D-GlcpNAc), D-fucose (D-Fucf), or D-Rhap (strain-dependent) as a side substituent. In different strains the substitution position for Rha residues in the repeated components of the major rhamnan chain was also different. One strain exhibited a unique type of O-chain heterogeneity. Immunochemical investigation of the LPS antigenic properties revealed the absence of close serological relations between the strains of one pathovar; this finding correlates with the differences in their OPS structure. Resemblance between the investigated strains and other P. syringae strains with similar LPS structures was revealed. The results of LPS analysis indicate the absence of correlation between the OPS structure and the pathovar affiliation of the strains.