Chemical structure of the 2-keto-3-deoxyoctonate (KDO) region of the lipopolysaccharide isolated from O1 Vibrio cholerae NIH 4IR (Ogawa).

The chemical structure of the 2-keto-3-deoxyoctonate (KDO) region of the lipopolysaccharide (LPS) isolated from O1 V. cholerae NIH 41R (Ogawa) was elucidated by dephosphorylation, periodate oxidation and methylation analysis. Methylation analysis of KDO in the dephosphorylated LPS revealed the presence of 5-O-acetyl-1,2,4,6,7,8-hexa-O-methyl-3-deoxy-octitol and 2-keto-3-deoxy-heptulosonic acid was detected in the methanolysate of the periodate-oxidized and dephosphorylated LPS. These results indicated that the site of binding of KDO to the core oligosaccharide is position C5 as in enteric gram-negative bacterial LPS, while only one molecule of the KDO residue carrying phosphate on position C4 is present in the inner core region of the LPS in contrast to enteric gram-negative bacterial LPS in which one molecule of KDO carrying KDO or KDO2----4KDO disaccharide instead of the phosphate group at position C4 is present in its main chain.     

Effect of miconazole on the structure and function of plasma membrane of Candida albicans

Abstract Fructose, a rarely occurring sugar constituent of Gram-negative bacterial lipopolysaccharides (LPS), is distributed ubiquitously in LPS of 01 Vibrio cholerae so far examined, but its location in LPS has not hitherto been elucidated. It was found that hydrazinolysis of LPS successfully affords a derivative retaining virtually all the fructose of intact LPS, but no ester-bound phosphate. Structural analysis carried out on the LPS derivative prepared by the hydrazinolysis of R-type LPS isolated from a rough mutant strain (NIH 41R) of 01 V. cholerae NIH 41 (Ogawa) revealed that the fructose is present as a non-reducing terminal residue bound to position C-6 of a glucose residue in the core region. This finding is considered to exclude the possibility that, in the LPS of 01 V. cholerae , the fructose is present in the region of the inner core in place of 2-keto-3-deoxyoctonate.     

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.     

Selective detection of 3-deoxymannooctulosonic acid in intact lipopolysaccharides by spin-echo 13C NMR

The 3-deoxy-D-mannooctulosonic acid (KDO) region of lipopolysaccharides (LPS) from the heptoseless mutant Salmonella minnesota R595 and inner core and heptoseless mutants derived from Escherichia coli K12 was studied by 13C NMR spectroscopy. A spin-echo spectral editing technique was employed for the selective detection of the quaternary anomeric carbon of ketosidically linked KDO. Only two quaternary carbon resonances attributable to KDO were detected in the anomeric carbon spectral region of each LPS from heptoseless mutants E. coli D31m4 (99.7 and 100.8 ppm) and S. minnesota R595 (100.0 and 100.9 ppm). Integrated signal intensities from fully relaxed normal 13C spectra showed that equivalent molar quantities of KDO and glucosamine (i.e. 2 mol of each) were present in each of these samples. Similarly, only two KDO anomeric carbon resonances were detected in the LPS from the inner core mutants E. coli D21f1 (100.8 and 101.2 ppm) and E. coli D21e7 (100.8 and 101.2 ppm). These data confirm the presence of a KDO disaccharide structure rather than a trisaccharide as determined by others using thiobarbituric acid-based assays. The LPS of E. coli D21 (complete inner core oligosaccharide) exhibited four quaternary anomeric carbon resonances (99.4, 100.7, 101.8, and 102.7 ppm). The unequal intensities of these resonances, however, demonstrated that significant heterogeneity exists with respect to KDO substitution in this LPS. A third KDO moiety present in substoichiometric amounts could be consistent with this observation. However, this possibility could not be distinguished from other modes of substitutional heterogeneity involving only 2 KDO residues.     

Immunological properties of Vibrio cholerae lipopolysaccharides.

Immunological effects of wall lipopolysaccharide (LPS) preparations obtained from Vibrio cholerae Inaba 569B, Ogawa NIH 41 and NAG 4715 strains by the hot phenol-water procedure were examined in mice. Although these LPS lack KDO, which are basic components of the core region of most gram-negative LPS, they still have potencies as B-cell mitogens, adjuvants, immunosuppressants, polyclonal B-cell activators and phagocytic stimulants for macrophages. The activities of these V. cholerae LPS on murine immune system seemed to be weaker than those of Salmonella typhimurium LT2-LPS. Among these V. cholerae LPS, NAG 4715-LPS showed the strongest mitogenic activity and phagocytic stimulation, while the potencies of this NAG 4715-LPS for the induction of polyclonal B cell activation, adjuvant effects and immunosuppression did not seem to be greater to those of the other LPS.     

Methylation analysis of the heptose/3-deoxy-D-manno-2-octulosonic acid region (inner core) of the lipopolysaccharide from Salmonella minnesota rough mutants

A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy-D-manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd2P-, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di-O-acetyl and 2,4,6-tri-O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd2P- LPS, the hydrolysis step involved 0.1M trifluoroacetic acid at 100 degrees for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:02 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave 5-O-acetyl-3-deoxy-1,7,8-tri-O-methyl-2,4,6-tri-O-trideuteriomethyl++ +-D- glycero-D-talo/galacto-octitol and 1,5-di-O-acetyl-2,3,4,6,7-penta-O-methyl-L-glycero-D-manno-heptitol++ +. These results prove the presence of a (2----4)-linked KDO disaccharide in Re LPS and show that the core region of Rd2P- LPS contains a terminal alpha-L-glycero-D-manno-heptopyranosyl group and a non-substituted, a 4-O-, and a 4,5-di-O-substituted pyranosidic KDO residue in the molar ratios 1:1:0.2:1.     

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.     

Chemical properties of lipopolysaccharide (LPS) isolated from Vibrio anguillarum PT514

Vibrio anguillarum, one of the causative agents of fish vibriosis, is serologically and biochemically divided into three groups (A, B and C). The chemical composition and molecular architecture of lipopolysaccharide (LPS) isolated from V. anguillarum PT 514, which belongs to serogroup B, were investigated. The LPS contained glucose (Glc), fructose (Fru), L-glycero-D-mannoheptose (L-D Hep), glucosamine (GlcN) and 4-amino-4,6-dideoxyglucose as sugar constituents in molar ratios of 8.9:0.7:3.0:1.1:1.6. Sephadex G-50 gel-chromatography of a degraded polysaccharide fraction separated from the LPS by 5% acetic acid hydrolysis suggested that the O-specific polysaccharide region consists of, in average, as much as 29 moles of Glc per 3 moles of L-D Hep, while the core polysaccharide contains at least Glc, L-D Hep and GlcN in molar ratios of 3.2 : 3.0 : 0.2. Fru and 4-amino-4,6-dideoxyglucose components were released from LPS on weak-acid hydrolysis, indicating that PT 514 LPS is distinguishable from those of Vibrio anguillarum belonging to the other serogroups. 2-Keto-3-deoxyoctonate (KDO), a common sugar constituent of gram-negative bacterial LPS, was not detected by Weissbach's color reaction under the conventional hydrolysis condition, but O-phosphoryl KDO was found in the strong-acid hydrolysate (4 M HCl, 100 C, 45 min). This substance was identical, at least in high-voltage paper electrophoresis, to 5-O-phosphoryl KDO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) are potent endotoxins that are thought to be involved in the pathogenesis of Gram-negative septicemia. The liver is known to be the primary organ responsible for the clearance of LPS from the systemic circulation in mammals. In this work, 125I-labeled LPS have been used in a filtration assay for the specific binding of LPS to intact rat hepatocytes. Eight S-form (smooth) LPS with complete O-specific polysaccharide chains isolated from different O-serotypes of Salmonella and Escherichia coli as well as nine R-form (rough) LPS isolated from Salmonella mutants deficient in synthesis of their core oligosaccharides were used in this study. All 125I-labeled S-form LPS and R-form LPS, except Re, show specific binding to isolated hepatocytes. The binding is saturable, is inhibited with excess unlabeled homologous or heterologous LPS but not lipid A, and is trypsin sensitive. L-Glycero-D-mannoheptose (heptose), a constituent of the inner core region of almost all LPS, is a potent inhibitor of the specific binding of 125I-labeled Rb2 LPS, whereas other monosaccharides, including 3-deoxy-D-manno-2-octulosonic acid (KDO), have weak or negligible inhibitor activity. These results strongly suggest the presence of a lectin-like receptor for the LPS inner core region (heptose-KDO region) on the plasma membrane of rat hepatocytes.     

Sugar composition of the polysaccharide portion of lipopolysaccharides isolated from non-O1 Vibrio cholerae O2 to O41, O44, and O68

The sugar composition of the polysaccharide portion of lipopolysaccharides (LPS) was determined for 42 serovars of non-O1 Vibrio cholerae, i.e., from serogroups O2 to O41, O44, and O68. On the basis of their compositional sugar pattern, they were classified into 24 chemotypes. 2-Keto-3-deoxyoctonate (KDO) was totally undetectable by the conventional color test (Weissbach's reaction) under the conventional hydrolysis conditions. Instead, a kind of KDO-like substance, which was positive in the reaction but not identical to KDO, was found in serogroup O19. Fructose, a characteristic sugar constituent of O1 V. cholerae LPS, was found in 33 serogroups but was absent from nine serogroups, approximately 20% of the members of this group so far examined.     

The core lipopolysaccharide of Escherichia coli is a ligand for the dendritic-cell-specific intercellular adhesion molecule nonintegrin CD209 receptor

The dendritic-cell-specific intercellular adhesion molecule nonintegrin (DC-SIGN) CD209 is a receptor for Escherichia coli K-12 that promotes bacterial adherence and phagocytosis. However, the ligand of E. coli for DC-SIGN has not yet been identified. In this study, we found that DC-SIGN did not mediate the phagocytosis of several pathogenic strains of E. coli, including enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, and uropathogenic E. coli, in dendritic cells or HeLa cells expressing human DC-SIGN antigen. However, we showed that an outer core lipopolysaccharide (LPS) (rough) mutant, unlike an inner core LPS (deep rough) mutant or O-antigen-expressing recombinant of E. coli K-12 was phagocytosed. These results demonstrate that the host cells expressing DC-SIGN can phagocytose E. coli in part by interacting with the complete core region of the LPS molecule. These results provide a mechanism for how O antigen acts as an antiphagocytic factor.     

Isolation of Salmonella typhimurium strains that utilize exogenous 3-deoxy-D-manno-octulosonate for synthesis of lipopolysaccharide.

Spontaneous mutants of Salmonella typhimurium LT2 were selected for the ability to accumulate exogenous 3-deoxy-D-manno-octulosonate (KDO). Bacteria containing a gene (kdsA) which codes for a temperature-sensitive KDO-8-phosphate synthetase were plated at the restrictive temperature of 42 degrees C on medium containing 5 mM KDO. Since bacteria containing the kdsA lesion are unable to grow at 42 degrees C due to inhibition of lipopolysaccharide (LPS) synthesis and accumulation of lipid A precursor, this method allowed direct, positive selection of mutants capable of utilizing exogenous KDO for LPS synthesis. Spontaneous mutants, selected at a frequency of about 10(-6), required exogenous KDO for growth at 42 degrees C. The growth rate at 42 degrees C was nearly normal in the presence of 20 mM KDO and was directly proportional to KDO concentrations below 20 mM. Exogenous KDO also suppressed accumulation of lipid A precursor. The apparent Km for KDO accumulation was 23 mM, and the maximum rate of transport was calculated to be 505 pmol of KDO per min per 10(8) cells. Bacteria incorporated exogenous [3H]KDO exclusively into LPS, with less than 10% dilution in specific activity due to residual endogenous KDO synthesis. The mutation giving rise to the ability to accumulate exogenous KDO was extremely useful in the direct screening for new mutations in the kdsA gene after localized mutagenesis. Five mutations in kdsA were isolated, four of which were new alleles as determined by on fine-structure analysis. The ability to introduce labeled (3H, 13C, and 14C) KDO in vivo should simplify and extend the analysis of this critical metabolic pathway in gram-negative bacteria.     

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.     

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.     

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.     

Differences between the LPS cores in adherent and non-adherent strains of enteropathogenic Escherichia coli 0119.

Adherent enteropathogenic Escherichia coli 0119 strains had a larger lipopolysaccharide core than non-adherent strains, although the O-chains were identical. The core from the non-adherent strain 19392 contained five hexose residues in the outer region, with three L-glycero-D-manno-heptose residues and 3-deoxy-D-manno-octulosonic acid (KDO) in the inner region. The core of adherent strain JCP88 had an atypical structure consisting of six hexose residues, KDO, and equimolar amounts of L-glycero-D-manno-heptose and D-glycero-D-manno-heptose. The core of a rough JCP88 mutant resembled an incomplete 19392 core.     

Effect of lipopolysaccharide core synthesis mutations on the production of Vibrio cholerae O-antigen in Escherixhia coli K-12

The rfb genes of Vibrio cholerae O1 (Ogawa serotype) were subcloned into a derivative of pBR322. This plasmid was transformed into several Escherichia coli K-12 mutant strains which produce an incomplete lipopolysaccharide (LPS)-core-oligosaccharide region. The data indicate that the V. cholerae O-antigen is assembled onto the E. coli LPS and that at least two glucoses are needed in the core in order to achieve a high level of production. These data are consistent with the reported presence of glucose in the V. cholerae LPS-core-oligosaccharide region.     

A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide)

Invasion of host cells is essential for the pathogenicity of Salmonella. The author's group has recently reported the cloning of the rfaE gene of Salmonella typhimurium, previously implicated in biosynthesis of the lipopolysaccharide (LPS)-inner core [Jin et al. (2001); Kim (2002)]. The product of the rfaE gene is involved in ADP-L-glycero-D-manno-heptose biosynthesis. rfaE mutants synthesize heptose-deficient LPS (Re-LPS) consisting only of lipid A and 3-deoxy-D-manno-octulosonic acid (KDO). Mutants that make incomplete LPS are rough mutants and "deep-rough" mutants affected in the heptose region of the inner core have reduced growth rate and increased sensitivity to high temperature. Complementation of S. typhimurium rfaE mutant strain SL1102 (rfaE543) with rfaE demonstrated conclusively that this gene restored the smooth phenotype, and the LPS produced by the complemented strain was indistinguishable from that of wild type smooth strains. In vitro infection experiments showed that complementation with rfaE permitted invasion of human Chang epithelial cells, larynx epidermal carcinoma HEp-2 cells and intestinal epithelial Henle-407 cells. These data imply that the structure of the LPS that is synthesized is critical for Salmonella invasiveness.     

Identification of an ATP-binding cassette transporter for export of the O-antigen across the inner membrane in Rhizobium etli based on the genetic, functional, and structural analysis of an lps mutant deficient in O-antigen

For O-antigen lipopolysaccharide (LPS) synthesis in bacteria, transmembrane migration of undecaprenyl pyrophosphate-bound O-antigen oligosaccharide subunits or polysaccharide occurs before ligation to the core region of the LPS molecule. In this study, we identified by mutagenesis an ATP-binding cassette transporter in Rhizobium etli CE3 that is likely responsible for the translocation of the O-antigen across the inner plasma membrane. Mutant FAJ1200 LPS lacks largely the O-antigen, as shown by SDS-polyacrylamide gel electrophoresis and confirmed by immunoblot analysis. Furthermore, LPS isolated from FAJ1200 is totally devoid of any O-chain glycosyl residues and contains only those glycosyl residues that can be expected for the inner core region. The membrane component and the cytoplasmic ATP-binding component of the ATP-binding cassette transporter are encoded by wzm and wzt, respectively. The Tn5 transposon in mutant FAJ1200 is inserted in the wzm gene. This mutation resulted in an Inf- phenotype in bean plants.     

Isolation and characterisation of the lipopolysaccharide from Xanthomonas hortorum pv. vitians

Xanthomonas hortorum pv. vitians is a Gram-negative bacterium that acts as the causative agent of bacterial leaf spot and headrot in lettuce. The lipopolysaccharide (LPS) of this bacterium is suspected to be an important molecule for adhesion to the plants. We have isolated the LPS, prepared the lipid A and the polysaccharide moieties thereof, and characterised all preparations by compositional analysis. Main sugar components are rhamnose and 3-acetamido-3,6-dideoxy-galactose which presumably furnish the O-specific polysaccharide. Other sugars are mannose, glucose, 6-deoxygalactose (fucose), and galacturonic acid, which should be core region constituents, and glucosamine, which builds up the carbohydrate backbone of lipid A. The LPS contains several phosphate groups, most of which are present in the core region. The main fatty acids in the lipid A are C10:0, 3-OH-C10:0 and 3-OH-C12:0. The latter is the only amide-linked fatty acid. Two fatty acids present in small amounts were identified, C8:0 and C11:0.