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.     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Chemical composition and ultrastructure of cellular and extracellular Moraxella glucidolytica lipopolysaccharides

A cellular (LPS I) and extracellular (LPS II) lipopolysaccharide were isolated from Moraxella glucidolytica cells grown on ethanol and from the culture fluid, respectively. Both LPS were toxic when injected to mice and chick embryos. These LPS contained glucose, galactose, glucosamine, galactosamine, 2-keto-3-deoxyoctonate and lipids. By permethylation studies, glucose was found to be linked (16) and (13) in LPS I and only (16) in LPS II. Galactose was the terminal non-reducing sugar. Branching occurred at positions 3 and 4 of galactose residues. LPS I was rich in - and -hydroxylauric and -hydroxymyristic acids and LPS II contained mainly stearic and -hydroxymyristic acids. LPS I was detoxified by mild acid and alkaline treatments. It was also dissociated by sodium deoxycholate and chromatographed on Sephadex G-75. The main fraction was reassociated by removing the surfactant by dialysis. The morphology of LPS I and LPS II was examined by electron microscopy. LPS I (original and reassociated fractions) consisted exclusively of ribbons while LPS II contained ribbons and vesicles.Non-Standard Abbreviations KDO 2-Keto-3-deoxyoctonic acids - LPS Lipopolysaccharide - NaD Sodium deoxycholate     

Chemical Characterization of the Lipopolysaccharide of Pseudomonas solanacearum

The carbohydrates present in lipopolysaccharide (LPS) from Pseudomonas solanacearum are rhamnose, xylose, 2-amino-2-deoxyglucose, glucose, heptose, and 2-keto-3-deoxyoctonate. LPS extracted from cultures grown on either glycerol or glucose (as the major source of carbon) and extracted after various incubation periods had similar compositions. The LPS from several strains of the bacterium contained the same component sugars, but the amounts of each sugar varied considerably. It was observed, however, that xylose and 2-amino-2-deoxyglucose increased proportionately with rhamnose, the major component. Phenol-water-extracted LPS contained measurable amounts of nucleic acid, protein, and arabinan, but none of these polymers were detected in LPS extracted with phenol-chloroform-petroleum ether. Polysaccharides liberated from LPS by mild acid hydrolysis were purified by gel filtration. Carbohydrate analysis of the LPS from a virulent, fluidal strain (K60) showed that the O-specific antigen consisted of rhamnose, xylose, and 2-amino-2-deoxyglucose in the proportions 4:1:1. The LPS of an avirulent, afluidal strain (B1) lacked the O-specific antigen; the R-core region consisted of rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate. Methylation analysis indicated that the K60 O-specific antigen was composed of a hexasaccharide repeating unit containing 3-, 2-, and 3,4-substituted rhamnopyranosyl residues, 3-substituted 2-amino-2-deoxyglucose, and terminal xylopyranose in the molar ratios 2:1:1:1:1.     

Cleavage of the O antigen 4,5,12 of Salmonella typhimurium by hydrofluoric acid

The effect of hydrofluoric acid (aqueous 48% HF) upon different lipopolysaccharides (LPS) was studied, employing conditions (48 h at + 4°C) that are commonly used to dephosphorylate LPS. From the LPS of Salmonella typhimurium having the O antigen 4,5,12 almost all of the O-antigenic sugars (Abe, Gal, Glc, Man, Rha) were liberated in dialysable form, whereas the saccharide chains of Salmonella LPS with O antigen 6,7 (Man, Glc, GlcNAc) were resistant to HF. The lability towards HF was shown to be due to the presence of the deoxysugar L-rhamnose in the saccharide backbone of the O antigen 4,5,12, since only Rha was found as the terminal sugar in the corresponding dialysable material. Hydrofluoric acid can thus be used to specifically cleave Rha-containing polysaccharides.     

Chemical analysis of Azospirillum lipopolysaccharides

Lipopolysaccharides (LPS) were extracted by hot phenol-water from five strains each of Azospirillum lipoferum and Azospirillum brasilense. Rhamnose, glucose, glucosamine and 3-deoxy-d-mannooctulosonic acid were comon sugar constituents of all LPS preparations. 2-O-Mefucose, 3-O-Me-fucose, 3-O-Me-rhamnose and 2-O-Megalactose were found in LPSs of some A. brasilense strains. Fatty acid spectra from all LPSs studied were almost identical with predominance of 3-hydroxymyristic and 3-hydroxypalmitic acids. 3-Hydroxypalmitic acid was the only amide-linked fatty acid. Lipopolysaccharides isolated from A. brasilense showed higher heterogeneity in sugar composition than those from A. lipoferum.Abbreviations glc gas liquid chromatography - ms mass spectrometry - LPS lipopolysaccharide - dOclA 3-deoxy-d-mannooctulosonic acid - 3-OH-16:0 3-hydroxypalmitic acid - nir^- nitrite reductase negative - nir⁺ nitrite reductase positive     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.     

2-Keto-l-Gulonic Acid Fermentation

Fractionation of sorbitol metabolites in the culture liquid of Gluconobacter melanogenus IFO 3292 was examined by column chromatographic techniques. Ion exchange column chromatography of the culture supernatant allowed to divide the components of the metabolites into Fractions I, II, III and IV. Paperelectrophoretic and paperchromatographic analyses of these fractions revealed that Fractions I, II, III and IV contained neutral sugar, hexonic acids, 5-ketohexonic acid and 2-ketohexonic acids, respectively.

The neutral sugar in Fraction I, the 5-ketohexonic acid in Fraction III and the 2-ketohexonic acids in Fraction IV were isolated and determined to be l-sorbose, 5-keto-d- mannonic, 2-keto-d-gluconic and 2-keto-l-gulonic acids, respectively, from their physical properties. In Fraction II were contained two different hexonic acids, one of which was identified to be l-idonic acid by the aid of substrate specificity of a hexonic acid dehydrogenase of Pseudomonas aeruginosa, and the other was determined to be d-mannonic acid as the phenylhydrazide derivative.     

Sugar Composition of O-Antigenic Lipopolysaccharides Isolated from Vibrio parahaemolyticus

Vibrio parahaemolyticus, a causative bacterium of food poisoning unique for its particular primary association with sea products, is now divided serologically into 11 or 12 O-forms based on agglutination and agglutinin-absorption tests. We determined the sugar composition of the somatic O-antigens, i.e., lipopolysaccharides (LPS), of representative strains of each O-form. Of particular interest is the absence of evidence for the presence of 2-keto-3-deoxy-octonic acid (KDO), a regular sugar component of gram-negative bacterial LPS, in any LPS examined, with the exception of 06. Furthermore, 07 and 012 LPS contained a KDO-like compound that is, however, not identical with KDO. Glucose, glucosamine, and L-glycero-D-mannoheptose were found as common sugar constituents. Three unidentified amino sugars, designated here as P1, P2, and P3, were found. Various combinations of each of these unidentified amino sugars, and of galactose, fucose, arabinose, D-glycero-D-mannoheptose, galactosamine, KDO, and the KDO-like substance were detected in accordance with the O-form of LPS. On the basis of the sugar composition, LPS of the 12 O-forms of V. parahaemolyticus can be classified into nine chemotypes, because 03, 05, and 011 LPS belong to the same chemotype and 07 and 012 to another chemotype.     

Chemical composition of lipopolysaccharides from Legionella bozemanii and Legionella longbeachae

Lipopolysaccharides (LPS) from Legionella bozemanii serogroup 1 and Legionella longbeachae serogroup 1 were subjected to chemical analyses. The lipid A part of both LPSs contained 2,3-dideoxy-2,3-diamino-d-glucose as major constituents and d-glucosamine and glycerol as minor constituents of the sugar backbone structure. Both LPSs exhibited a very complex fatty acid composition. Twenty amide-linked 3-hydroxy fatty acids were detected in LPS of L. longbeachae, whereas seventeen were encountered in LPS of L. bozemanii. Both LPSs contained nine ester-linked nonhydroxy fatty acids and the unique long-chain fatty acids 27-oxo-octacosanoic acid, 29-oxotriacontanoic acid, heptacosane-1,27-dioic acid and nonacosane-1,29-dioic acid. SDS-PAGE showed that L. bozemanii produced smooth-form LPS, whereas L. longbeachae LPS was mainly of the R-type. Composition analyses were in accordance with these electrophoretic patterns. d-Quinovosamine and l-fucosamine constituted 80 mol% of the polysaccharide part of L. bozemanii LPS. Other sugars identified were d-glucosamine, d-mannose, d-glucose, l-rhamnose, d-glycero-d-manno-heptose, l-glycero-d-mannoheptose, 2-keto-3-deoxy-octonic acid and glycerol. The polysaccharide chain from LPS of L. longbeachae appeared to be shorter, but composed of the same sugars except l-fucosamine. Both LPSs contained glycerol phosphate and glucosamine phosphate and L. longbeachae LPS contained in addition glucose phosphate.Abbreviations EI Electron impact - GlcN3N 2,3-Diamino-2,3-dideoxy-d-glucose - HPAEC High pH anion-exchange chromatography - Kdo 2-Keto-3-deoxy-octonic acid - LPS Lipopolysaccharide - PCP Phenol/chloroform/petroleum ether solvent - PED Pulsed electrochemical detection - PS Polysaccharide - TFA Trifluoroacetyl - TMS Trimethylsilyl     

New variants of polar glycopeptidolipids detected in Mycobacterium simiae, including 'habana' strains, as evidenced by electrospray ionization-ion trap-mass spectrometry

Aims: To determine the composition of polar glycopeptidolipids (pGPLs) of Mycobacterium simiae and, particularly, those of ‘habana’ strains, in a search for specific markers given the immunogenic potential of ‘habana’ TMC 5135 in experimental tuberculosis. Methods and Results: pGPLs were determined in free lipid extracts using electrospray ionization‐ion trap‐mass spectrometry (ESI‐IT‐MS), working in both negative‐ and positive‐ion mode. In the case of TMC 5135, the presence of the previously characterized GPL‐II (containing 2,4‐di‐O‐CH₃ glucuronic acid as distal sugar in the oligosaccharide antigenic moiety) and GPL‐III (containing 4‐O‐CH₃ glucuronic acid as distal sugar) was confirmed using MS/MS and MS/MS/MS approaches. Interestingly, some ‘habana’ strains presented variants of GPL‐II, designated GPL‐II′‐A and GPL‐II′‐B. A di‐O‐CH₃‐deoxy‐hexose (tentatively, 2,3‐di‐O‐CH₃‐fucose) was identified as the penultimate sugar in the oligosaccharide moiety of GPL‐II′‐A, whereas in GPL‐II′‐B the penultimate sugar was fucose (tentative identification). On the contrary, the distal sugar of the oligosaccharide chain of pGPLs of Myco. simiae ATCC 25275^T was identified as tri‐O‐CH₃‐glucuronic acid (designated GPL‐sim^T‐I, with two variants: GPL‐sim^T‐I‐A and GPL‐sim^T‐I‐B), O‐CH₃‐glucuronic acid (designated GPL‐sim^T‐II) and di‐O‐CH₃‐glucuronic acid (GPL‐II′‐A and GPL‐II′‐B). The penultimate sugar of the oligosaccharide chain of GPL‐sim^T‐I‐A and GPL‐sim^T‐II was identified as di‐O‐CH₃‐deoxy‐hexose (tentatively, 2,3‐di‐O‐CH₃ fucose), and that of GPL‐sim^T‐I‐B as deoxy‐hexose (tentatively, fucose). In all strains studied, each [M‐H]^? and [M+Na]⁺ ion was revealed as a mixture of homologous compounds varying in the number of –O‐CH₃ groups present in the oligosaccharide moiety and in the length of the fatty acyl linked to the peptide. Conclusions: The present work indicates that, within a similar general pattern of pGPLs, different strains of Myco. simiae present some variations, so that new compounds (GPL‐II′‐A, GPL‐II′‐B, GPL‐sim^T‐I‐A, GPL‐sim^T‐I‐B and GPL‐sim^T‐II) were defined. Noteworthy was the fact that the ‘habana’ strains clearly differed from the type strain of Myco. simiae. Significance and Impact of the Study: The data obtained can be used in the delineation of the ‘habana’ group of Myco. simiae, including the quality control of the immunogenic strain ‘habana’ TMC 5135.     

Investigation of Lipopolysaccharides from Sinorhizobium meliloti SKHM1-188 and Two of Its Mutants with Decreased Nodulation Competitiveness

A comparative study of the lipopolysaccharides (LPS) isolated from Sinorhizobium meliloti SKHM1-188 and two of its LPS mutants (Tb29 and Ts22) with sharply decreased nodulation competitiveness was conducted. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all three strains: a higher molecular weight LPS1, containing O-polysaccharide (O-PS), and a lower molecular weight LPS2, without O-PS. However, the LPS1 content in mutants was significantly smaller than in the parent strain. The LPS of the strains studied contained glucose, galactose, mannose, xylose, three nonidentified sugars (X ₁ (TGlc 0.53), X ₂ (TGlc 0.47), and X ₃ (TGlc 0.43)), glucosamine, and ethanolamine, while the LPS of S. meliloti SKHM1-188 additionally contained galactosamine, glucuronic and galacturonic acids, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as such fatty acids as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T_{3-OH C14:0} 1.33), C18:0, and unsaturated C18:1 fatty acids. The LPS of both mutants were similar in the component composition but differed from the LPS of the parent strain by lower X ₂, X ₃, and 3-OH C14:0 contents and higher KDO, C18:0, and hydroxy X contents. The LPS of all the strains were subjected to mild hydrolysis with 1% acetic acid and fractionated on a column with Sephadex G-25. The higher molecular weight fractions (2500–4000 Da) contained a set of sugars typical of intact LPS and, supposedly, corresponded to the LPS polysaccharide portion (PS1). In the lower molecular weight fractions (600–770 Da, PS2), glucose and uronic acids were the major components; galactose, mannose, and X ₁ were present in smaller amounts. The PS1/PS2 ratio for the two mutants was significantly lower than for strain SKHM1-188. The data obtained show that the amount of O-PS–containing molecules (LPS1) in the heterogeneous lipopolysaccharide complex of the mutants was smaller than in the SKHM1-188 LPS; this increases the hydrophobicity of the cell surface of the mutant bacteria, which supposedly contributes to their nonspecific adhesion to the roots of the host plant, thus decreasing their nodulation competitiveness.     

Incorporation of Substrate Cell Lipid A Components into the Lipopolysaccharide of Intraperiplasmically Grown Bdellovibrio bacteriovorus

The composition of Bdellovibrio bacteriovorus lipopolysaccharide (LPS) was determined for cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas putida. The LPS of axenically grown bdellovibrios contained glucose and fucosamine as the only detectable neutral sugar and amino sugar, and nonadecenoic acid (19:1) as the predominant fatty acid. Additional fatty acids, heptose, ketodeoxyoctoic acid, and phosphate were also detected. LPS from bdellovibrios grown intraperiplasmically contained components characteristic of both axenically grown bdellovibrios and the substrate cells. Substrate cell-derived LPS fatty acids made up the majority of the bdellovibrio LPS fatty acids and were present in about the same proportions as in the substrate cell LPS. Glucosamine derived from E. coli LPS amounted to about one-third of the hexosamine residues in intraperiplasmically grown bdellovibrio LPS. However, galactose, characteristic of the E. coli outer core and O antigen, was not detected in the bdellovibrio LPS, suggesting that only lipid A components of the substrate cell were incorporated. Substrate cell-derived and bdellovibrio-synthesized LPS materials were conserved in the B. bacteriovorus outer membrane for at least two cycles of intraperiplasmic growth. When bdellovibrios were grown on two different substrate cells successively, lipid A components were taken up from the second while the components incorporated from the lipid A of the first were conserved in the bdellovibrio LPS. The data show that substrate cell lipid A components were incorporated into B. bacteriovorus lipid A during intraperiplasmic growth with little or no change, and that these components, fatty acids and hexosamines, comprised a substantial portion of bdellovibrio lipid A.     

Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii.

Lipopolysaccharides (LPSs) isolated from phase I and phase II Coxiella burnetii (LPS I and LPS II, respectively) were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The yields of crude phenol-water extracts from phase I cells were roughly three to six times higher than those from phase II cells. Purification of LPSs by ultracentrifugation gave similar yields for both LPS I and LPS II. Purified LPS I and LPS II contained roughly 0.8 and 0.6% protein, respectively. The fatty acid constituents of the LPSs were different in composition and content, with branched-chain fatty acids representing about 15% of the total. beta-Hydroxymyristic acid was not detected in either LPS I or LPS II. A thiobarbituric acid-periodate-positive compound was evident in the LPSs; however, this component was not identified as 3-deoxy-D-mannooctulosonic acid by gas and paper chromatographies. LPS II contained D-mannose, D-glucose, D-glyceromannoheptose, glucosamine, ethanolamine, 3-deoxy-D-mannooctulosonic acid-like material, phosphate, and fatty acids. LPS I contained the unique disaccharide galactosaminuronyl glucosamine and nine unidentified components in addition to the components of LPS II. The hydrophobic, putative lipid A fraction of LPS I and LPS II contained the above constituents, but the hydrophilic fraction was devoid of ethanolamine. The LPS I disaccharide galactosaminuronyl glucosamine was found in both fractions of the acetic acid hydrolysates. Analysis of LPSs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that LPS II was composed of only one band, whereas LPS I consisted of six or more bands with irregular spacing. Ouchterlony immunodiffusion tests demonstrated that LPS I reacted with phase I but not with phase II whole-cell hyperimmune antibody, and LPS II reacted neither with phase I nor phase II hyperimmune antibody. From these results, it was concluded that the chemical structures of LPSs from C. burnetii were different from those of the LPSs of gram-negative bacteria; however, the LPS structural variation in C. burnetii may be similar to the smooth-to-rough mutational variation of saccharide chain length in gram-negative bacteria.     

A Chemotaxonomic Study of Vibrio fluvialis Based on the Sugar Composition of the Polysaccharide Portion of the Lipopolysaccharides

A chemotaxonomic study was carried out with a new serotyping scheme comprising 35 O-antigen groups of Vibrio fluvialis on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharide (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. The 35 O-antigen groups were divided into 21 chemotypes. It is noted that a rarely occurring component sugar of gram-negative bacterial LPS, D -glycero-D -manno-heptose, and two kinds of uronic acids, i.e., galacturonic acid of a weakly bound type and glucuronic acid of a strongly bound type, were found in common in all the 21 chemotypes. A characteristic sugar component of gram-negative bacterial LPS, 2-keto-3-deoxyoctonate (KDO), was not detectable in any of the 21 chemotypes. Instead, three kinds of “KDO-like substances” were found, one in each of three chemotypes (III, XI and XVII). They were strongly positive in Weissbach's periodate-thiobarbituric acid test for KDO, but definitely not identical to it in high-voltage paper electrophoresis (HVPE); the substance present in chemotype XI was indicated by HVPE to be 3-deoxy-D -threo-hexulosonic acid which is a sugar constituent of Vibrio parahaemolyticus O7 and O12 LPS.     

Isolation of lipopolysaccharides and the effect of Polymyxin B on the outer membrane of Corynebacterium autotrophicum

Lipopolysaccharides (LPS) from Corynebacterium autotrophicum were isolated and analyzed. Autotrophically grown cells contained 2–5 mg of partly purified LPS per g dry weight of lyophilized cells. Serological cross reaction with Lipid A antigen of Salmonella minnesota confirmed the presence of LPS in C. autotrophicum. Electron microscopy of negatively stained Polymyxin B-treated cells showed formation of blebs on the Outer Membrane indicating an interaction of Polymyxin B specifically with LPS. Up to now, no Gram-positive organisms are known which contain any LPS. Thus, C. autotrophicum, though giving opposite results when the Gram-staining reaction was applied by several authors, has to be classified into the group of Gram-negative bacteria.Non-Common Abbreviations LPS lipopolysaccharide - KDO 2-keto-3-deoxyoctonate     

Immunochemical investigations of antigens isolated fromBacteroides ovatus strain ATCC 8483

A saline extract (SE) and a phenol/water extract (WL) were prepared fromBacteroides ovatus strain ATCC 8483. A fraction CS was isolated from the culture supernatant. WL was further split by ultracentrifugation into lipopolysaccharide (LPS) and supernatant (L₁). Fractions SE, WL, LPS and L₁ reacted serologically with homologous antiserum but did not cross-react with antisera against heterologousBacteroides serotypes. Fraction CS was inactive in haemagglutination, haemagglutination inhibition and immunoelectrophoresis tests. SE, WL, LPS and L₁ proved to be serologically heterogeneous. A distinct serological specificity for SE was demonstrated. The serological reactivity in SE and WL was not altered after treatment with proteolytic enzymes yet completely destroyed in WL and partially in SE by sodium metaperiodate. SE, WL, LPS and L₁ contained the sugar components rhamnose, fucose, ribose, mannose, galactose, glucose and glucosamine in different molar ratios for each fraction. Galactosamine was found in WL and LPS, uronic acid in WL and L₁. Two unidentified aminohexoses were detected in WL, one of which was also detectable in L₁ and SE. 2-Keto-3-deoxyaldonic acid was demonstrated in LPS and L₁ after strong acid hydrolysis.     

Biological activity of (Lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. trifolii strain TA1

Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssB-pssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and ¹H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major ¹H NMR signals for O-acetyl and N-acetyl groups were 1: 0.8 and 1: 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1β and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.     

Isolation of two different polysaccharides from halophilic Zoogloea sp.

Summary An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from marine environments. This strain could produce two different polysaccharides. One (water-soluble polysaccharide : WSP) was from cell-free liquid medium, the other (cell-bound polysaccharide : CBP) was obtained from cell surface. Both polysaccharides contained glucose, galactose and mannose as sugar components, but their molar ratios were different (WSP : 2:2:3, CBP : 1:2:2) and half of the sugar components existed as uronic acid form. Both polysaccharide productions started at the early stage of the logarithmic growth phase. The amount of WSP and CBP was influenced by culture conditions such as additional carbon and nitrogen sources. Isolated Zoogloea sp. showed a high product yield without the increase of cell mass.