Structural characterization of lipid A from nontypeable and type f Haemophilus influenzae: variability of fatty acid substitution

Lipid A isolated by mild acid hydrolysis from lipopolysaccharides of 22 nontypeable and 2 type f Haemophilus influenzae strains was investigated using electrospray ionization coupled to quadrupole ion trap mass spectrometry. The lengths, positions, and number of acyl chains in the lipid A molecule were determined using multiple-step tandem mass spectrometry (MSn). All of the analyzed strains showed a major lipid A molecule comprising beta-2-amino-2-deoxy-D-glucopyranose-(1-->6)-alpha-2-amino-2-deoxy-D-glucopyranose phosphorylated at the C4' and C1 positions. The C2/C2' and C3/C3' positions were substituted by amide-linked and ester-linked 3-hydroxytetradecanoic acid chains, respectively. The fatty acid chains on C3' and C2' were further esterified by tetradecanoic acid chains. In all strains, minor amounts of lipid A molecules with different acylation patterns were identified. Thus, structures comprising the hexaacylated lipid A with the C2 or C3 position being substituted by 3-hydroxydecanoic acid, and hexaacylated lipid A with the C3 and C3' positions being substituted by 3-hydroxydodecanoic or dodecanoyloxytetradecanoic acid, respectively, were found. In addition, lipid A with an acetyl group attached to the 3-hydroxytetradecanoic acid groups attached to the C2 or C3 position was detected in two nontypeable H. influenzae strains.     

Complete lipopolysaccharide of Plesiomonas shigelloides O74:H5 (strain CNCTC 144/92). 2. Lipid A, its structural variability, the linkage to the core oligosaccharide, and the biological activity of the lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1-->6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS.     

Structural studies of the core region of Aeromonas salmonicida subsp. salmonicida lipopolysaccharide

The core oligosaccharide structure of the in vivo derived rough phenotype of Aeromonas salmonicida subsp. salmonicida was investigated by a combination of compositional, methylation, CE-MS and one- and two-dimensional NMR analyses and established as the following: [carbohydrate: see text] where R=alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1--> or alpha-D-Galp-(1--> (approx. ratio 4:3). Comparative CE-MS analysis of A. salmonicida subsp. salmonicida core oligosaccharides from strains A449, 80204-1 and an in vivo rough isolate confirmed that the structure of the core oligosaccharide was conserved among different isolates of A. salmonicida.     

Structural analysis of the lipid A component of Campylobacter jejuni CCUG 10936 (serotype O:2) lipopolysaccharide. Description of a lipid A containing a hybrid backbone of 2-amino-2-deoxy-D-glucose and 2,3-diamino-2,3-dideoxy-D-glucose

The chemical structure of Campylobacter jejuni CCUG 10936 lipid A was elucidated. The hydrophilic backbone of the lipid A was shown to consist of three (1----6)-linked bisphosphorylated hexosamine disaccharides. Neglecting the phosphorylation pattern, a D-glucosamine (2-amino-2-deoxy-D-glucose) disaccharide [beta-D-glucosaminyl-(1----6)-D-glucosamine], a hybrid disaccharide of 2,3-diamino-2,3-dideoxy-D-glucose and D-glucosamine [2,3-diamino-2,3-dideoxy-beta-D-glucopyranosyl-(1----6)-D-glucosamine], and a 2,3-diamino-2,3-dideoxy-D-glucose disaccharide were present in a molar ratio of 1:6:1.2. Although the backbones are bisphosphorylated, heterogeneity exists in the substitution of the polar head groups. Phosphorylethanolamine is alpha-glycosidically bound to the reducing sugar residue of the backbone, though C-1 is also non-stoichiometrically substituted by diphosphorylethanolamine. Position 4' of the non-reducing sugar residue carries an ester-bound phosphate group or is non-stoichiometrically substituted by diphosphorylethanolamine. By methylation analysis it was shown that position 6' is the attachment site for the polysaccharide moiety in lipopolysaccharide. These backbone species carry up to six molecules of ester- and amide-bound fatty acids. Four molecules of (R)-3-hydroxytetradecanoic acid are linked directly to the lipid A backbone (at positions 2, 3, 2', and 3'). Laser desorption mass spectrometry showed that both (R)-3-hydroxytetradecanoic acids linked to the non-reducing sugar unit carry, at their 3-hydroxyl group, either two molecules of hexadecanoic acid or one molecule of tetradecanoic and one of hexadecanoic acid. It also suggested that the (R)-3-(tetradecanoyloxy)-tetradecanoic acid was attached at position 2', whereas (R)-3-(hexadecanoyloxy)-tetradecanoic acid was attached at position 3', or at positions 2' and 3'. Therefore, the occurrence of three backbone disaccharides differing in amino sugar composition and presence of a hybrid disaccharide differentiate the lipid A of this C. jejuni strain from enterobacterial and other lipids A described previously.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

Synthesis of the optically active 4-O-phosphono-D-glucosamine derivatives related to the nonreducing-sugar subunit of bacterial lipid A

The optically active lipid A-subunit homologs named GLA-46, GLA-47, GLA-59, and GLA-60 have been synthesized stepwise by successive acylation at N-2 and O-3 of benzyl 2-amino-2-deoxy-4,6-O-isopropylidene-beta-D-glucopyranoside with the 3-9O-(benzyloxy)methyl or 39O-tetradecanoyl derivative of optically active 3-hydroxytetradecanoic acid, and phosphorylation at O-4 of the D-glucosamine residue.     

Structure of the lipid A-inner core region and biological activity of Plesiomonas shigelloides O54 (strain CNCTC 113/92) lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative rod associated with episodes of intestinal infections and outbreaks of diarrhea in humans. The extraintestinal infections caused by this bacterium, for example, endopthalmitis, meningitidis, bacteremia, and septicemia, usually have gastrointestinal origin and serious course. The lipopolysaccharide (LPS, endotoxin) as virulence factor is important in enteropathogenicity of this bacterium. LPSs of P. shigelloides and especially their lipid A part, that is, the immunomodulatory center of LPS, have not been extensively investigated. The structure of P. shigelloides O54 lipid A was determined by chemical analysis combined with MALDI-TOF mass spectrometry, and the intact Kdo-containing core region was investigated by NMR spectroscopy on deacylated LPS. Products from alkaline deacylation of LPS, containing 4-substituted uronic acids, are usually very complex and difficult to separate. Since Kdo residues, like sialic acids, form complexes with serotonin, we used immobilized serotonin for one-step isolation of oligosaccharide containing the intact Kdo region from the reaction mixture by affinity chromatography. The major form of lipid A was built of beta-d-GlcpN4PPEtn-(1-->6)-alpha-d-GlcpN1P disaccharide substituted with 14:0(3-OH), 12:0(3-OH), 14:0(3-O-14:0), and 12:0(3-O-12:0) acyl groups at N-2, O-3, N-2', and O-3', respectively. This is a novel structure among known lipid A molecules. Analysis of intact Kdo-lipid A region, lipid A and its linkage with the core oligosaccharide completes the structural investigation of P. shigelloides O54 LPS, resolving the entire molecule. Biological activities and observed discrepancy between in vitro and in vivo activity of P. shigelloides and Escherichia coli LPS are discussed.     

Lipid a component of Salmonella typhimurium carrying the derepressed Col Ib plasmids

The structure of the lipid A from S. typhimurium harboring the derepressed plasmids Col Ib is very similar: i, 1,4'-bis-phosphorylated-beta-1',6-linked glucosamine disaccharide forms a backbone of the lipid; ii, lipid preparations contain four residues of 3-hydroxytetradecanoic acid at positions C3, C3' and the amide linked at C2, C2' and two free hydroxyl groups at positions C4 and C6'. Differences concern: i, substitution of phosphoryl groups by 4-amino-4-deoxy-L-arabinopyranose and phosphorylethanolamine in S. typhimurium with Col Ib plasmids; ii, the degree of acylation of hydroxyl groups of 3-hydroxytetradecanoic acid by myristic, lauric and palmatic acids; iii, presence of tridecanoic acid bound to hydroxyl of 3-hydroxy-tetradecanate residue in S. typhimurium with Col Ibdrd2 plasmid. Lipopolysaccharides from the plasmid mutant strains express several times higher lethal toxicity in chick embryos compared to lipopolysaccharides from the strain with the wild type Col Ib.     

Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd-/b+. Description of a novel deep-rough chemotype

The chemical structure of the lipopolysaccharide of a deep-rough mutant (strain I-69 Rd-/b+) of Haemophilus influenzae was investigated. The hydrophilic backbone of lipid A was shown to consist of a beta-(1',6)-linked D-glucosamine disaccharide with phosphate groups at C-1 of the reducing D-glucosamine and at C-4' of the non-reducing one. Four molecules of (R)-3-hydroxytetradecanoic acid were found directly linked to the lipid A backbone, two by amide and two by ester linkage (positions 2,2' and 3,3', respectively). Laser-desorption mass spectrometry showed that both 3-hydroxytetradecanoic acids linked to the non-reducing glucosamine carry tetradecanoic acid at their 3-hydroxyl group, so that altogether six molecules of fatty acid are present in lipid A. The lipopolysaccharide was the first described to contain only one sugar unit linked to lipid A. This, sugar in accordance with a previous report [Zamze et al. (1987) Biochem. J. 245, 583-587], was shown to be a dOclA phosphate. The phosphate group was found at position 4, but the analytical procedures employed (permethylation and methanolysis followed by gas-liquid chromatography/mass spectrometry) also revealed dOclA 5-phosphate. Since a cyclic 4,5-phosphate could be ruled out by 31P-NMR, we conclude that, in this lipopolysaccharide, a mixture of dOclA 4- and 5-phosphate is present. By methylation analysis of the dephosphorylated, deacylated and reduced lipopolysaccharide the attachment site of the dOclA was assigned to position C-6' of the non-reducing glucosamine of lipid A. The anomeric linkages present in the lipopolysaccharide were assessed by 1H-NMR and 13C-NMR of deacylated lipopolysaccharide. The saccharide backbone of this Haemophilus influenzae lipopolysaccharide possesses the following structure: (Formula; see text)     

Chemical structure of the lipid A component of lipopolysaccharides from Fusobacterium nucleatum.

The lipid A component of lipopolysaccharides from Fusobacterium nucleatum Fev 1 consists of beta-1',6-linked D-glucosamine disaccharides, which carry two phosphate groups: one in glycosidic and one in ester linkage. The amino groups of the glucosamine disaccharides are substituted by D-3-hydroxyhexadecanoic acid. The hydroxyl groups of the disaccharide backbone are acylated by tetradecanoic, hexadecanoic, and D-3-hydroxytetradecanoic acids. Part of the ester-bound D-3-hydroxytetradecanoic acid is 3-O-substituted by tetradecanoic acid. Whereas a similar pattern of fatty acids was detected in lipopolysaccharides from two other F. nucleatum strains, the amide-bound fatty acid in F. varium and F. mortiferum was D-3-hydroxytetradecanoic acid. The chemical relationships of lipid A from Fusobacteria and other gram-negative bacteria are discussed.     

Structural studies on the R-type lipopolysaccharide of Aeromonas hydrophila

A rough strain of Aeromonas hydrophila, AH-901, has an R-type lipopolysaccharide with the complete core. The following core structure was established by chemical degradations followed by sugar and methylation analyses along with ESIMS and NMR spectroscopy: [formula: see text] where D-alpha-D-Hep and l-alpha-D-Hep stand for D-glycero- and l-glycero-alpha-D-manno-heptose, respectively; Kdo stands for 3-deoxy-D-manno-oct-2-ulosonic acid; all monosaccharides are in the pyranose form; the degree of substitution with beta-D-Gal is approximately 50%. Lipid A of the lipopolysaccharide has a 1,4(')-bisphosphorylated beta-D-GlcN-(1-->6)-alpha-D-GlcN disaccharide backbone with both phosphate groups substituted with 4-amino-4-deoxyarabinose residues.     

Complete structural characterization of the lipid A fraction of a clinical strain of B. cepacia genomovar I lipopolysaccharide

Burkholderia cepacia, a Gram-negative bacterium ubiquitous in the environment, is a plant pathogen causing soft rot of onions. This microorganism has recently emerged as a life-threatening multiresistant pathogen in cystic fibrosis patients. An important virulence factor of B. cepacia is the lipopolysaccharide (LPS) fraction. Clinical isolates and environmental strains possess LPS of high inflammatory nature, which induces a high level production of cytokines. For the first time, the complete structure of the lipid A components isolated from the lipopolysaccharide fraction of a clinical strain of B. cepacia is described. The structural studies carried out by selective chemical degradations, MS, and NMR spectroscopy revealed multiple species differing in the acylation and in the phosphorylation patterns. The highest mass species was identified as a penta-acylated tetrasaccharide backbone containing two phosphoryl-arabinosamine residues in addition to the archetypal glucosamine disaccharide [Arap4N-l-beta-1-P-4-beta-D-GlcpN-(1-6)-alpha-D-GlcpN-1-P-1-beta-L-Arap4N]. Lipid A fatty acids substitution was also deduced, with two 3-hydroxytetradecanoic acids 14:0 (3-OH) in ester linkage, and two 3-hydroxyhexadecanoic acids 16:0 (3-OH) in amide linkage, one of which was substituted by a secondary 14:0 residue at its C-3. Other lipid A species present in the mixture and exhibiting lower molecular weight lacked one or both beta-L-Arap4N residues.     

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.     

Structural studies on the O-antigen of Aeromonas salmonicida

Lipopolysaccharide from a strain of Aeromonas salmonicida salmonicida was isolated from cells by the aqueous phenol method in 2.3% yield (based on dry weight of bacteria). Hydrolysis of the lipopolysaccharide in 1% acetic acid afforded O-polysaccharide (19% by weight), core-oligosaccharide (12.2%) and lipid A (44.6%). Analysis indicated that 3-deoxy-D-manno-2-octulosonic acid was absent from the lipopolysaccharide and that no low-molecular-weight compounds were released by the mild hydrolysis. The O-polysaccharide had the monosaccharide composition of rhamnose, glucose and N-acetylmannosamine in molar ratio of 1.0:1.58:0.83. 75% of the N-acetylmannosamine residues were substituted at position 4 by O-acetyl groups. Hydrolysis of the methylated polysaccharide proved to be both difficult and dependent on the method of hydrolysis chosen, in all cases a partially methylated disaccharide of rhamnose and N-acetylmannosamine was identified in the hydrolysate. Methylation analysis, periodate oxidation and proton magnetic resonance analysis were used to confirm the structure of the repeating unit as: (formula; see text).     

Characterization of a novel lipid A containing D-galacturonic acid that replaces phosphate residues. The structure of the lipid a of the lipopolysaccharide from the hyperthermophilic bacterium Aquifex pyrophilus

According to the 16 S rRNA phylogenetic tree, the hyperthermophilic bacterium Aquifex pyrophilus represents the deepest and shortest branching species of the kingdom Bacteria. We show for the first time that an organism, which is phylogenetically ancient on the basis of its 16 S rRNA and that exists at extreme conditions, may contain lipopolysaccharide (LPS). The LPS was extracted from dried bacteria using a modified phenol/water method. SDS-polyacrylamide gel electrophoresis and silver stain displayed a ladder-like pattern, which is typical for smooth-form LPS (possessing an O-specific polysaccharide). The molecular masses of the LPS populations were determined by matrix-assisted laser-desorption ionization mass spectrometry. Lipid A was precipitated after mild acid hydrolysis of LPS. Its complete structure was determined by chemical analyses, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser-desorption ionization mass spectrometry, and one- and two-dimensional NMR spectroscopy. The lipid A consists of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-D-glucopyranose (DAG) disaccharide carrying two residues each of (R)-3-hydroxytetradecanoic acid and (R)-3-hydroxyhexadecanoic acid in amide linkage and one residue of octadecanoic acid in ester linkage. Each DAG moiety carries one residue of each 3-hydroxytetradecanoic and 3-hydroxyhexadecanoic acid. In the nonreducing DAG, the octadecanoic acid is attached to the 3-hydroxy group of 3-hydroxytetradecanoic acid. Each DAG is substituted by one D-galacturonic acid residue, which is linked to O-1 of the reducing and to O-4 of the nonreducing end. This structure represents a novel type of lipid A.     

Structural characterization of lipid A component of Erwinia carotovora lipopolysaccharide

The chemical structure of the lipid A component of lipopolysaccharide excreted into the liquid medium by the plant pathogenic enterobacterium Erwinia carotovora FERM P-7576 was characterized. It consists of a -1, 6-linked glucosamine disaccharide which carries ester-and amide-bound fatty acids and phosphate similar to the lipid A from other gram-negative bacteria. The lipid A preparation was not uniform in the number and composition of the fatty acids linked to the disaccharide. Four prominent lipids A were involved, they were composed of five to seven residues of fatty acid. Among them the major component was hexa-acyl lipid A, in which the hydroxyl group at position 3 and the amino group of the non-reducing glucosamine unit carry 3-dodecanoyl-oxytetradecanoyl residues. Positions 2 and 3 of the reducing glucosamine unit were substituted by 3-hydroxytetradecanoic acid. In the hepta-acyl lipid A, an additional hexadecanoic acid was linked to the hydroxyl group of the 3-hydroxytetradecanoyl residue at position 2 of the hexa-acyl lipid A. Two penta-acyl lipids A were the homologs of the hexa-acyl lipid A with decreasing acylation. Dodecanoic acid was missing from one, and 3-hydroxytetradecanoic acid from another. 3-Dodecanoyloxytetradecanoyl residue at position 3 differentiates E. carotovora lipid A from that of other gram-negative bacteria.Abbreviations LPS lipopolysaccharide - GlcN glucosamine - KDO 3-deoxy-d-manno-octulosonic acid - FAB-MS fast atom bombardment mass spectrometry - u atomic mass unit     

Structural characterization of the lipid A component of pathogenic Neisseria meningitidis.

The lipid A component of meningococcal lipopolysaccharide was structurally characterized by using chemical modification methods, methylation analysis, 31P nuclear magnetic resonance, and laser desorption mass spectroscopy. It was shown that Neisseria meningitidis lipid A consists of a 1,4'-bisphosphorylated beta(1'----6)-linked D-glucosamine disaccharide (lipid A backbone), both phosphate groups being largely replaced by O-phosphorylethanolamine. This disaccharide harbors two nonsubstituted hydroxyl groups at positions 4 and 6', the latter representing the attachment site of the oligosaccharide portion in lipopolysaccharide. In addition, it is substituted by up to six fatty acid residues. In the major lipid A component, representing a hexaacyl species, the hydroxyl groups at positions 3 and 3' carry (R)-3-hydroxydodecanoic acid [12:0(3-OH)], whereas the amino groups at positions 2 and 2' are substituted by (R)-3-(dodecanoyloxy)tetradecanoic acid [3-O(12:0)-14:0]. A minor portion was present as a tetraacyl lipid A component lacking either dodecanoic acid (12:0) or 12:0 and 12:0(3-OH). N. meningitidis lipid A, therefore, significantly differs from Escherichia coli lipid A by the nature and locations of fatty acids and the substitution of O-phosphorylethanolamine for the nonglycosyl (4'-P) and glycosyl phosphate.     

The structure of the rough-type lipopolysaccharide from Shewanella oneidensis MR-1, containing 8-amino-8-deoxy-Kdo and an open-chain form of 2-acetamido-2-deoxy-D-galactose

The LPS from Shewanella oneidensis strain MR-1 was analysed by chemical methods and by NMR spectroscopy and mass spectrometry. The LPS contained no polysaccharide O-chain, and its carbohydrate backbone had the following structure: (1S)-GalNAco-(1-->4,6)-alpha-Gal-(1-->6)-alpha-Gal-(1-->3)-alpha-Gal-(1-P-3)-alpha-DDHep-(1-->5)-alpha-8-aminoKdo4R-(2-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P, where R is P or EtNPP. There are several novel aspects to this LPS. It contains a novel linking unit between the core polysaccharide and lipid A moieties, namely 8-amino-3,8-dideoxy-D-manno-octulosonic acid (8-aminoKdo) and a residue of 2-acetamido-2-deoxy-D-galactose (N-acetylgalactosamine, GalNAco) in an open-chain form, linked as cyclic acetal to O-4 and O-6 of D-galactopyranose. The structure contains a phosphodiester linkage between the alpha-D-galactopyranose and D-glycero-D-manno-heptose (DDHep) residues.     

The structure of the carbohydrate backbone of the LPS from Shewanella spp. MR-4

The rough type lipopolysaccharide isolated from Shewanella spp. strain MR-4 was analyzed using NMR, mass spectroscopy, and chemical methods. Two structural variants have been found, both contained 8-amino-3,8-dideoxy-d-manno-octulosonic acid and lacked l-glycero-d-manno-heptose. A minor variant of the LPS contained phosphoramide substituent.     

Characterization of a novel lipid A structure isolated from Azospirillum lipoferum lipopolysaccharide

The structure of lipid A from Azospirillum lipoferum, a plant-growth-promoting rhizobacterium, was investigated. It was determined by chemical analysis, mass spectrometric methods, as well as 1D and 2D NMR spectroscopy. Because of the presence of substituents, the investigated lipid A differs from typical enterobacterial lipid A molecules. Its backbone is composed of a beta-(1,6)-linked D-glucosamine disaccharide but lacks phosphate residues. Moreover, the reducing end of the backbone (position C-1) is substituted with alpha-linked d-galacturonic acid. 3-hydroxypalmitoyl residues are exclusively connected to amino groups of the glucosamine disaccharide. Hydroxyls at positions C-3 and C-3' are esterified with 3-hydroxymyristic acids. Primary polar fatty acids are partially substituted by nonpolar fatty acids (namely, 18:0, 18:1 or 16:0), forming acyloxyacyl moieties.