Chemotypes of Fusobacterium nucleatum lipopolysaccharides

Lipopolysaccharides (LPS) were isolated from 20 strains of Fusobacterium nucleatum and examined by paper chromatography, gas liquid chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-dexoxy-octonate (KDO). The LPS had in common glucosamine, L-glycero-D-manno-heptose, glucose and KDO. The KDO content was low. Galatose, rhamnose and D-glycero-D-manno-heptose were found in some strains. Based on the sugar composition of the LPS, the F. nucleatum strains could be classified into six chemotypes.     

Characterization and taxonomic significance of lipopolysaccharides of Leptospira interrogans serovar hardjo

Lipopolysaccharides (LPSs) from Leptospira interrogans serovar hardjo (reference strain hardjoprajitno and strain hardjobovis) were prepared by the hot phenol-water procedure. High yields of LPSs were found in the phenol phase. Gel electrophoresis of the phenol phase LPSs showed similar patterns for all strains in contrast to the different patterns found in the water phase LPSs. Sugar composition was also similar among all strains with rhamnose as the predominant sugar. Mannosamine was detected by high performance thin layer and gas-liquid chromatography. 2-Keto-3-deoxyoctonic acid (KDO) was comparable with authentic KDO by paper chromatography. Periodate oxidation at near neutral pH with or without prior hydrolysis showed that most of the KDO was substituted. The fatty acid composition of strain hardjobovis LPS was slightly different from that of the reference strain hardjoprajitno. Myristic and 3-hydroxymyristic acid were not detected in any of the LPS preparations. In conjunction with genetic and other data, the two strains are sufficiently different to be regarded as members of two separate species sharing common antigens. There is sufficient evidence to rename the hardjoprajitno strain type L. interrogans hardjo-p, and the hardjobovis strain type L. borgpeterseni hardjo-b.     

Occurrence of 2-keto-3-deoxyoctonate (KDO) and KDO phosphate in lipopolysaccharides ofBacteriodes species

Occurrence of 2-keto-3-deoxyoctonate (KDO) in lipopolysaccharides (LPS) of genusBacteroides (some strains have recently been reclassified asPorphyromonas orPrevotella) was examined. Strong-acid treatment of LPS isolated fromBacteroides fragilis, Bacteroides (Porphyromonas) gingivalis andBacteroides intermedius, (Prevotella intermedia) released periodate/thiobarbituric acid reaction-positive substances that were not detectable under conventional hydrolysis conditions. These substances were demonstrated to be KDO phosphate by high voltage paper electrophoresis before and after alkaline phosphatase treatment. KDO phosphate was also identified in these LPS by gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. KDO was identified as well in both mild and strong-acid hydrolysates of LPS isolated fromBacteriodes melaninogenicus (Prevotella melaninogenica). Neither KDO nor KDO phosphate was detectable in LPS ofBacteriodes asaccharolyticus (Porphyromonas asaccharolytica) even after the strong-acid treatment of LPS. These findings indicate that there are possible structural variations in the inner core region ofBacteroides LPS.     

Sugar Composition of Lipopolysaccharides of Family Vibrionaceae

A comparative study was carried out on the sugar composition of lipopolysaccharides (LPS) isolated from representative strains of members of the family Vibrionaceae including all of the constituting genera, i.e., Vibrio, Aeromonas, Photobacterium, Plesiomonas, and Lucibacterium. More than 100 strains were examined. It was found that, with the exception of Vibrio parahaemolyticus 06, 2-keto-3-deoxyoctonate (KDO), known generally as a component sugar in the core region of usual gram-negative bacterial LPS, is virtually absent from LPS of the Vibrionaceae strains so far examined. Furthermore, mannose was also lacking in LPS of Vibrionaceae strains with the exception of only one strain, A. anaerogenes (ATCC 15467). Instead, some KDO-like substances were found in LPS from Vibrio (“Beneckea”) nereida (ATCC 25917) and Plesiomonas shigelloides including the type strain (ATCC 14029), the same as those found in LPS from V. parahaemolyticus O7 and O12, and three strains of V. alginolyticus. These substances were strongly positive in the periodate-thiobarbituric acid test, yielding a color with maximum absorption at 549 nm. The spectra were identical to that of KDO, whereas substances differed from KDO at least in behavior in high-voltage paper electrophoresis and thin-layer chromatography. A particularly interesting feature from the chemotaxonomical point of view was found in the sugar composition of LPS isolated from V. cholerae. Fructose was present exclusively in LPS of V. cholerae (both O1 and non-O1 groups and classical and eltor biotypes) with the exception of one strain of Photobacterium phosphoreum (NCMB 844). In addition, a pair of rarely occurring amino sugars, perosamine and quinovosamine, was found in LPS from O1 group V. cholerae regardless of either the biotype (classical or eltor) or the serotype (Inaba or Ogawa), whereas this pair was not present in non-O1 group V. cholerae (the so-called NAG vibrios). This feature was confirmed with LPS from more than 30 additional strains of O1 group V. cholerae isolated from patients. The virtual absence of KDO in LPS of the family Vibrionaceae was demonstrated for the first time in this study. These results are compatible with the interpretation that the absence of KDO in LPS can be used as one of the taxonomical characteristics of Vibrionaceae in addition to (G+C) content, DNA (or RNA) homology, and numerical analysis data.     

Occurrence of 2-keto-3-deoxy-D-manno-octonic acid in lipopolysaccharides isolated from Vibrio parahaemolyticus.

The occurrence of 2-keto-3-deoxy-D-manno-octonic acid (KDO) in lipopolysaccharides (LPS) of Vibrio parahaemolyticus was demonstrated for the first time by gas chromatography-mass spectrometry after dephosphorylation, reduction, and methylation. KDO was virtually completely phosphorylated, since no KDO was detected by either gas chromatography or thiobarbituric acid assay before dephosphorylation. The level of KDO in all six strains of V. parahaemolyticus investigated ranged from 0.37 to 0.69%, which was considerably lower than that in enterobacterial LPS.     

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.     

Characterization of the lipopolysaccharide from the nod mutant of Rhizobium trifolii

Lipopolysaccharides (LPS) from the non-nodulating Rhizobium trifolii 24SM 15 and from the nodulating R. trifolii 24SM 13 were isolated and examined by means of gas-liquid chromatography and mass spectrometry. Analysis of LPS showed these preparations from both strains examined contained Lipid A, 2-keto-3-deoxyoctonate, neutral sugars, amino sugars, and trace amounts of amino acids. In 24SM 13 LPS prevailed glucose and rhamnose whereas LPS from the non-nodulating strain SM 15 contained mainly mannose, galactose and heptose. Quinovosamine and mannosamine were detected only in the nodulating strain. The ratio of glucosamine phosphate to glucosamine was higher in the LPS of the non-nodulating strain SM 15 than in the corresponding material of the nodulating one. An unknown component producing a peak at the position of glyceryl-S-cysteine on amino acid analysis profiles was detected in SM 15 LPS. The differences in LPS composition were associated with the alterations in the sensitivity to phage 3H, and nodulation ability.     

Chemical characterization of the lipopolysaccharides from enteropathogenic Escherichia coli O142 and O158.

Lipopolysaccharides (LPS) from two enteropathogenic strains of E. coli O142 and O158 were isolated by hot phenol-water extraction procedure. Polyacrylamide gel electrophoretic pattern of the LPS showed the typical ladder like pattern of smooth type of LPS. The LPS of E. coli O158 was found to contain L-rhamnose, D-glucose and N-acetyl-D-galactosamine as major constituents together with D-galactose, N-acetyl-D-glucosamine, L-glycero-D-manno-heptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) whereas LPS from E. coli O142 contained L-rhamnose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as major constituents together with D-glucose, D-galactose, N-acetyl-D-glucosamine, L-glycero-D-mannoheptose and 2-keto-3-deoxy-D-manno-octulosonic acid (KDO). LPS was degraded by mild acid hydrolysis to yield a degraded polysaccharide fraction and an insoluble lipid-A fraction. The main fatty acids of the lipid-A fraction of the LPS were C12:O, C14:O, and 3-OH C14:O for O158 strain whereas E. coli O142 lipid-A consisted of C12:O, C14:O, 3-OH C14:O, and C16:O. The degraded polysaccharide fraction on gel permeation chromatography gave a high moleculer weight O-chain fraction and a core oligosaccharide and a fraction containing degraded sugars. The chemical composition of LPS and its fragmented products are reported in this communication.     

Antitumor Lipopolysaccharide from Heptoseless Mutant of Proteus mirabilis

Studies on lipopolysaccharide (LPS) from the cells of Proteus mirabilis RMS-203 were focused upon reduction of lethal toxicity and of pyrogenicity by biological and chemical modification. A heptoseless mutant, strain N-434, was isolated by the use of phage resistancy as a tool. LPS from that heptoseless mutant was completely deficient in neutral sugars and mainly composed of 2-keto-deoxy-octonic acid (KDO), glucosamine and fatty acids. It revealed almost the same antitumor activity as LPS of the wild type but it was less toxic and less pyrogenic.

Hydroxylaminolysis and reduction with LiAlH₄ resulted in removal of fatty acids from LPS accompanied with decrease in lethal toxicity and antitumor acitivity but not in pyrogenicity.

Lipid A fractions showed almost the same antitumor activity as intact LPS but less lethality and less pyrogenicity.     

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.     

Lack of specific binding of labeledRhizobium japonicum lipopolysaccharides to wild soybean (Glycine soja) roots

Summary Lipopolysaccharides (LPS) were extracted from two strains ofRhizobium japonicum (61A76NS and 3I1b110-I). The extracted LPS was purified by gel filtration column chromatography and the amount of 2-keto-3-deoxyoctonate (KDO) was determined. Column purified LPS from both strains were conjugated to rhodamine isothiocyanate on celite to examine binding of this purified, labeled surface component to aseptically grownGlycine soja (wild soybean) seedlings as a basis for symbiotic specificity using fluorescent microscopy. Rhodamine conjugated LPS from both strains ofRhizobium japonicum did not exhibit specific binding to wild soybean seedling roots.Paper no. 8130 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Characterization of the lipopolysaccharide ofVeillonella parvula ATN and growth conditions affecting its yield

The lipopolysaccharide (LPS) ofVeillonella parvula ATN, a human oral isolate, was characterized. Lipid A accounted for, approximately 55% of the weight of the LPS complex. Major fatty acids included tridecanoic acid, 3-hydroxytridecanoic acid, and 3-hydroxypentadecanoic acid. Glucose was the only hexose detected. Amino compounds included glucosamine, galactosamine, and glycine. Heptose and 2-keto-3-deoxyoctonic acid (KDO) were also present. Alterations in the culture medium formulation affected LPS extraction yields. Furthermore, evidence was presented for the existence of a heterogeneous LPS component, with changes in growth conditions giving rise to more or less hydrophilic types of LPS.     

Chemical Analysis of Lipopolysaccharides of Shigella sonnei Form II Strains Expressed by Cloned Form I Antigen Genes

A compositional sugar analysis was carried out on lipopolysaccharide (LPS) from Shigella sonnei form II in which a plasmid with cloned form I antigen genes had been introduced. The recipient form II strains contained galactose, glucose, heptose, glucosamine, and 2-keto-3-deoxyoctonic acid (KDO) (2: 3: 1: 2: 2) in its LPS, while the transformant form I LPS contained, besides these sugars, N-acetyl-L -altrosaminouronic acid as an additional sugar constituent, which is known to be one of the antigenic determinants of form I antigen.     

Characterization of gonidial zone of Cycas revoluta coralloid roots by means of microelectrodes

Abstract Structural analysis of the 2-keto-3-deoxyoctonate region of lipopolysaccharide (LPS) isolated from Porphyromonas (Bacteroides) gingivalis was carried out. The substitution of the polysaccharide portion on the KDO was determined by gas chromatography/mass spectrometry of the product obtained by sequential derivatization of the LPS, including dephosphorylation, permethylation, carboxyl reduction, partial hydrolysis, carbonyl reduction, complete hydrolysis and O -acetylation. It was revealed that the KDO carries the polysaccharide on its position C5 and is phosphorylated on either position C7 or C8, although its exact position is not determined. The structure of the KDO region of P. gingivalis LPS in Gram-negative bacterial LPS had not hitherto been elucidated.     

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.     

Degradation of bacterial lipopolysaccharide by the slime mould Physarum polycephalum.

A strain of the acellular slime mould Physarum polycephalum degraded lipopolysaccharides (LPS) from a variety of bacteria. The anticomplementary (AC) activity of LPS was greatly reduced, as was the content of lauric, myristic, and palmitic acids, and the ability to sensitize erythrocytes to agglutination by antibody. These results indicate that Physarum has enzymes which reduce the lipid A moiety of LPS. In contrast, 2-keto-3-deoxy-D-manno-actanoic acid (KDO), immunodominant sugars, and beta-hydroxymyristic acid were scarcely affected. Both supernates and plasmodial extracts of Physarum had LPS-degradative activity and were able to attack both purified LPS and LPS in killed bacteria.     

Rabbit polymorphonuclear leukocyte migration in vivo in response to lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella

Subcutaneously implanted chambers in rabbits were used for testing the migration of polymorphonuclear leukocytes in response to injected LPS isolated from strains of Bacteroides, Fusobacterium and Veillonella. A salmonella LPS was used as reference endotoxin. No differnece in chemotactic activity between the Veillonella LPS and LPS from Salmoneila was found. Fusobacterium LPS whoed insignificantly lower chemotactic capacity than the Salmonella LPS. The Bacteroides LPS were all significantly less chemotactic than the reference endotoxin. An insignificant correlation between the amount of exudate aspirated from the chambers 5 h after injection of the different LPS preparations and the number of leukocytes per microliter of exudate was found.     

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 SKHM 1-188 and two its LPS-mutants (Th29 and Ts22) with sharply decreased nodulation competitiveness was conducted. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all the three strains: a higher molecular-weight LPS1, containing O-polysaccharide (O-PS), and a and 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--X1 (TGlc 0.53), X2 (TGlc 0.47), and X3 (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 fatty acids, such as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T3-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 a lower X2, X3, and 3-OH C 14:0 content and a higher KDO, C18:0, and hydroxy X content. 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 X1 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. This supposedly contributes to their nonspecific adhesion on the roots of the host plant, thus decreasing their nodulation competitiveness.     

Hydrolytic release, and identification by g.l.c.-m.s., of 3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharides isolated from bacteria of the Vibrionaceae

The identification of the peracetylated methyl glycosides of 3-deoxy-D-manno-2-octulosonic acid (KDO) methyl esters was achieved by g.l.c.-m.s. These peracetylated methyl glycoside methyl esters were obtained from fully acetylated lipopolysaccharides and core oligosaccharides of representative strains of the Vibrionaceae family by the following sequence of mild reactions: acetolysis, methanolysis, and acetylation. KDO was shown to be present in all of the lipopolysaccharides (LPS), a result in direct contrast to the generally accepted view of the absence of this compound in LPS from this family of bacteria.     

Lipopolysaccharide O-chain microheterogeneity of Salmonella serotypes Enteritidis and Typhimurium

Variability in the lipopolysaccharide (LPS) of the two most prevalent Salmonella serotypes causing food-borne salmonellosis was assessed using gas chromatography analysis of neutral sugars from 43 Salmonella enterica serovar Enteritidis ( S . Enteritidis) and 20 Salmonella enterica serovar Typhimurium ( S . Typhimurium) isolates . Four substantially different types of O-chain chemotypes were detected using cluster analysis of sugar compositions; these were low-molecular-mass (LMM) LPS, glucosylated LMM LPS, high-molecular-mass (HMM) LPS and glucosylated HMM LPS. Nineteen out of 20 S . Typhimurium isolates yielded glucosylated LMM . In contrast, S . Enteritidis produced a more diverse structure, which varied according to the source and history of the isolate: 45.5% of egg isolates yielded glucosylated HMM LPS; 100% of stored strains lacked glucosylation but retained chain length in some cases; and 83.3% of fresh isolates from the naturally infected house mouse Mus musculus produced glucosylated LMM LPS. A chain length determinant ( wzz ) mutant of S . Enteritidis produced a structure similar to that of S . Typhimurium and was used to define what constituted significant differences in structure using cluster analysis. Fine mapping of the S . Enteritidis chromosome by means of a two-restriction enzyme-ribotyping technique suggested that mouse isolates producing glucosylated LMM LPS were closely related to orally invasive strains obtained from eggs, and that stored strains were accumulating genetic changes that correlated with suppression of LPS O-chain glucosylation. These results suggest that the determination of LPS chemotype is a useful tool for epidemiological monitoring of S . Enteritidis , which displays an unusual degree of diversity in its LPS O-chain.