Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction

For the first time, the complete structure of the lipid A from the lipopolysaccharide of an Agrobacterium species is here reported. In particular, the structure of the lipid A from A. tumefaciens strain C58, a soil pathogen bacterium strictly related to Rhizobiaceae, was determined. The structural study, carried out by chemical analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy, revealed that lipid A fraction consisted of a mixture of species all sharing the bis-phosphorylated glucosamine disaccharide backbone that could be designated in two main structural motifs, according to the acylation pattern. The main species was a penta-acylated lipid A bearing two unsubstituted 14:0 (3-OH) fatty acids in ester linkage and two 16:0 (3-OH) in amide linkage; the one on GlcN II was O-acylated by a long chain fatty acid, 28:0 (27-OH). This in turn was esterified by a 3-hydroxy-butyroyl residue at its hydroxy group. The second species, in lesser amounts, was identified as a tetra-acylated lipid A and lacked the 14:0 (3-OH) residue on GlcN I. Other species deriving from these two lacked a phosphate group or 3-hydroxy-butyroyl residue or otherwise carried a 26:0 (25-OH) as long chain fatty acid. The lipid A structure of phytopathogen A. tumefaciens strain C58 presents deep structural analogies with lipid A of symbiotic Rhizobium, and the hypothesis is advanced that it can be a strategy of the bacterium to escape or attenuate the plant response.     

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.     

Structural determination of lipid A of the lipopolysaccharide from Pseudomonas reactans. A pathogen of cultivated mushrooms.

The chemical structure of lipid A from the lipopolysaccharide of the mushroom-associated bacterium Pseudomonas reactans, a pathogen of cultivated mushroom, was elucidated by compositional analysis and spectroscopic methods (MALDI-TOF and two-dimensional NMR). The sugar backbone was composed of the beta-(1'-->6)-linked d-glucosamine disaccharide 1-phosphate. The lipid A fraction showed remarkable heterogeneity with respect to the fatty acid and phosphate composition. The major species are hexacylated and pentacylated lipid A, bearing the (R)-3-hydroxydodecanoic acid [C12:0 (3OH)] in amide linkage and a (R)-3-hydroxydecanoic [C10:0 (3OH)] in ester linkage while the secondary fatty acids are present as C12:0 and/or C12:0 (2-OH). A nonstoichiometric phosphate substitution at position C-4' of the distal 2-deoxy-2-amino-glucose was detected. Interestingly, the pentacyl lipid A is lacking a primary fatty acid, namely the C10:0 (3-OH) at position C-3'. The potential biological meaning of this peculiar lipid A is also discussed.     

Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human Toll-like receptor 4 complexes

Burkholderia mallei, the aetiologic agent of glanders, causes a variety of illnesses in animals and humans ranging from occult infections to acute fulminating septicaemias. To better understand the role of lipopolysaccharide (LPS) in the pathogenesis of these diseases, studies were initiated to characterize the structural and biological properties of lipid A moieties expressed by this organism. Using a combination of chemical analyses and MALDI-TOF mass spectrometry, B. mallei was shown to express a heterogeneous mixture of tetra- and penta-acylated lipid A species that were non-stoichiometrically substituted with 4-amino-4-deoxy-arabinose residues. The major penta-acylated species consisted of bisphosphorylated d-glucosamine disaccharide backbones possessing two amide linked 3-hydroxyhexadecanoic acids, two ester linked 3-hydroxytetradecanoic acids [C14:0(3-OH)] and an acyloxyacyl linked tetradecanoic acid, whereas, the major tetra-acylated species possessed all but the 3'-linked C14:0(3-OH) residues. In addition, although devoid of hexa-acylated species, B. mallei LPS was shown to be a potent activator of human Toll-like receptor 4 complexes and stimulated human macrophage-like cells (THP-1 and U-937), monocyte-derived macrophages and dendritic cells to produce high levels of TNF-alpha, IL-6 and RANTES. Based upon these results, it appears that B. mallei LPS is likely to play a significant role in the pathogenesis of human disease.     

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.     

Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides

The structure of the lipid A component of lipopolysaccharides isolated from two wild-type strains (Fisher 2 and 7) and one rough mutant (PAC 605) of Pseudomonas aeruginosa was investigated using chemical analysis, methylation analysis, combined gas-liquid chromatography/mass spectrometry, laser-desorption mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of a pyranosidic beta 1,6-linked D-glucosamine disaccharide [beta-D-GlcpN-(1----6)-D-GlcpN], phosphorylated in positions 4' and 1. Position 6' of the beta-D-GlcpN-(1----6)-D-GlcpN disaccharide was identified as the attachment site of the core oligosaccharide and the hydroxyl group at C-4 was not substituted. Lipid A of the three P. aeruginosa strains expressed heterogeneity with regard to the degree of acylation: a hexaacyl as well as a pentaacyl component were structurally characterized. The hexaacyl lipid A contains two amide-bound 3-O-acylated (R)-3-hydroxydodecanoic acid groups [12:0(3-OH)] at positions 2 and 2' of the GlcN dissacharide and two ester-bound (R)-3-hydroxydecanoic acid groups [10:0(3-OH)] at positions 3 and 3'. The pentaacyl species, which represents the major lipid A component, lacks one 10:0(3-OH) residue, the hydroxyl group in position 3 of the reducing GlcN residue being free. In both hexa- and pentaacyl lipid A the 3-hydroxyl group of the two amide-linked 12:0(3-OH) residues are acylated by either dodecanoic (12:0) or (S)-2-hydroxydodecanoic acid [12:0(2-OH)], the lipid A species with two 12:0(2-OH) residues, however, being absent. The presence of only five acyl residues in the major lipid A fraction may account for the low endotoxic activity observed with P. aeruginosa lipopolysaccharide.     

Construction of a deep-rough mutant of Burkholderia cepacia ATCC 25416 and characterization of its chemical and biological properties

Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I. Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1 --> 8) D-glycero-D-talo-oct-2-ulosonic acid (Ko)-(2 --> 4)3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection. Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor alpha from macrophages to the same extent. Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.     

Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine

The lipopolysaccharide (LPS) of the Gram-negative Acidiphilium strain GS18h/ATCC55963, a new soil isolate, exhibited very low endotoxic activity as determined by Limulus gelation activity, lethal toxicity in galactosamine (GalN) sensitised mice, and level of tumor necrosis factor alpha (TNFalpha) in the blood serum of BALB/c mice. Analysis of the LPS, specially of lipid A which usually accounts for the toxicity, revealed the latter to contain glucosamine and phosphate besides fatty acids, of which 14:0(3-OH), 18:0(3-OH), 18:1 and 19:0(cyclo) are the major components, while 12:0, 16:0, 19:1, 20:0(3-OH) and 20:1(3-OH) are present in small amounts. The 14:0(3-OH) and 18:0(3-OH) fatty acids are amide-linked, whereas the rest are ester bound. Glucose, galactose, mannose, rhamnose, heptose, galacturonic acid and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) were present in the polysaccharide part of this LPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the LPS showed a macromolecular heterogeneity distinctly different from those of Escherichia coli or Salmonella. The toxicity of this LPS being extremely low attributed to fatty acid composition of its lipid A, promises potential therapeutic application.     

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

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

The lipid A from Vibrio fischeri lipopolysaccharide: a unique structure bearing a phosphoglycerol moiety

Vibrio fischeri, a bioluminescent marine bacterium, exists in an exclusive symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, whose light organ it colonizes. Previously, it has been shown that the lipopolysaccharide (LPS) or free lipid A of V. fischeri can trigger morphological changes in the juvenile squid's light organ that occur upon colonization. To investigate the structural features that might be responsible for this phenomenon, the lipid A from V. fischeri ES114 LPS was isolated and characterized by multistage mass spectrometry (MS(n)). A microheterogeneous mixture of mono- and diphosphorylated diglucosamine disaccharides was observed with variable states of acylation ranging from tetra- to octaacylated forms. All lipid A species, however, contained a set of conserved primary acyl chains consisting of an N-linked C14:0(3-OH) at the 2-position, an unusual N-linked C14:1(3-OH) at the 2'-position, and two O-linked C12:0(3-OH) fatty acids at the 3- and 3'-positions. The fatty acids found in secondary acylation were considerably more variable, with either a C12:0 or C16:1 at the 2-position, C14:0 or C14:0(3-OH) at the 2'-position, and C12:0 or no substituent at the 3'-position. Most surprising was the presence of an unusual set of modifications at the secondary acylation site of the 3-position consisting of phosphoglycerol (GroP), lysophosphatidic acid (GroP bearing C12:0, C16:0, or C16:1), or phosphatidic acid (GroP bearing either C16:0 + C12:0 or C16:0 + C16:1). Given their unusual nature, it is possible that these features of the V. fischeri lipid A may underlie the ability of E. scolopes to recognize its symbiotic partner.     

Differential inductions of TNF-alpha and IGTP, IIGP by structurally diverse classic and non-classic lipopolysaccharides

The innate immune system recognizes microbes by characteristic molecules like the Gram-negative lipopolysaccharide (LPS). Lipid A (the LPS bioactive moiety) signals through toll-like receptors (TLRs) to induce pro-inflammatory molecules and small GTPases of the p47 family involved in intracellular pathogen control. We tested TNF-alpha and p47-GTPase induction in macrophages using classical LPSs [lipid As with glucosamine backbones, ester- and amide-linked C14:0(3-OH) and C12 to C16 in acyloxyacyl groups] of wild type and mutant Escherichia coli and Yersinia species and non-classical LPSs [lipid As with diaminoglucose, ester-linked 3-OH-fatty acids and C28:0(27-OH) and C23:0(29-OH) in acyloxyacyl groups] of plant endosymbionts (Rhizobium), intracellular pathogens (Brucella and Legionella) and phylogenetically related opportunistic bacteria (Ochrobactrum). Classical but not non-classical LPSs efficiently induced TNF-alpha, IIGP and IGTP p47-GTPase expression. Remarkably, the acyloxyacyl groups in classical LPSs necessary to efficiently induce TNF-alpha were not necessary to induce p47-GTPases, suggesting that different aspects of lipid A are involved in this differential induction. This was confirmed by using PPDM2, a non-endotoxic lipid A-structurally related synthetic glycolipid. Despite their different bioactivity, all types of LPSs signalled through TLR-4 and not through TLR-2. However, whereas TNF-alpha induction was myeloid differentiation factor 88 (MyD88)-dependent, that of p47-GTPases occurred via a MyD88-independent pathway. These observations show that different aspects of the LPS pathogen-associated molecular pattern may be triggering different signalling pathways linked to the same TLR. They also reinforce the hypothesis that non-classical lipid As act as virulence factors by favouring the escape from the innate immune system.     

Structure of a novel lipid A obtained from the lipopolysaccharide of Caulobacter crescentus

Caulobacter crescentus CB15 is a dimorphic bacterium that is best known as a prokaryotic model for cell development. However, it is also being exploited in biotechnology, where the crystalline surface (S-layer) protein secretion system has been adapted for heterologous protein display or secretion. Because the S-layer attaches to the cell surface via lipopolysaccharide (LPS) and since the LPS represents a potential endotoxin contaminant of recombinant proteins, the lipid A component was examined in detail. LPS was acid hydrolyzed to obtain crude lipid A, which was methylated and purified by HPLC. HPLC peak fractions were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy. The structure of the major lipid A of C. crescentus comprised the tetrasaccharide backbone alpha-D-GalpA-(1-->4)-beta-D-DAG-(1-->6)-alpha-D-DAG-(1-->1)-alpha-D-GalpA substituted with six fatty acids, and a molecular mass of 1875 (GalpA, galactopyranuronic acid; DAG, 2,3-diamino-2,3-dideoxyglucopyranose). No phosphate residues were detected. The major lipid A component had 12:0[3-O[Delta(5)-12:1(3-OH)]] and 12:0[3-O(Delta(5)-12:1)] fatty acyl chains at either the 3'- or the 2' positions of the distal subunit DAG B, and 12:0(3-OH) and 12:0[3,6-(OH)( 2)] fatty acyl chains at 3- and 2- positions of the reducing end subunit DAG A, respectively. In addition, several other variations in the structure were observed. The LPS was evaluated for TNF-alpha inducing activity and consistent with its unusual lipid A structure (relative to that of enteric bacteria), the activity was reduced by greater than 100-fold as compared to Escherichia coli ReLPS. This and other evidence suggests the potential application of this lipid A as a vaccine adjuvant or the suitability of Caulobacter displaying antigens for formulation of whole cell vaccines.     

Chemical characterization of effective and ineffective strains of Rhizobium leguminosarum bv. viciae

Chemical composition of lipopolysaccharide (LPS) isolated from an effective (97) and ineffective (87) strains of R. l. viciae has been determined. LPS preparations from the two strains contained: glucose, galactose, mannose, fucose, arabinose, heptose, glucosamine, galactosamine, quinovosamine, and 3-N-methyl-3,6-dideoxyhexose, as well as glucuronic, galacturonic and 3-deoxyoctulosonic acid. The following fatty acids were identified: 3-OH 14:0, 3-OH 15:0, 3-OH 16:0, 3-OH 18:0 and 27-OH 28:0. The ratio of 3-OH 14:0 to other major fatty acids in LPS 87 was higher that in LPS 97. SDS/PAGE profiles of LPS indicated that, in lipopolysaccharides, relative content of S form LPS I to that of lower molecular mass (LPS II) was much higher in the effective strain 97 than in 87. All types of polysaccharides exo-, capsular-, lipo, (EPS, CPS, LPS, respectively) examined possessed the ability to bind faba bean lectin. The degree of affinity of the host lectin to LPS 87 was half that to LPS 97. Fatty acids (FA) composition from bacteroids and peribacteroid membrane (PBM) was determined. Palmitic, stearic and hexadecenoic acids were common components found in both strains. There was a high content of unsaturated fatty acids in bacteroids as well as in PBM lipids. The unsaturation index in the PBM formed by strain 87 was lower than in the case of strain 97. Higher ratio of 16:0 to 18:1 fatty acids was characteristic for PMB of the ineffective strain.     

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.     

Occurrence of an Unusual Hopanoid-containing Lipid A Among Lipopolysaccharides from Bradyrhizobium Species

The chemical structures of the unusual hopanoid-containing lipid A samples of the lipopolysaccharides (LPS) from three strains of Bradyrhizobium (slow-growing rhizobia) have been established. They differed considerably from other Gram-negative bacteria in regards to the backbone structure, the number of ester-linked long chain hydroxylated fatty acids, as well as the presence of a tertiary residue that consisted of at least one molecule of carboxyl-bacteriohopanediol or its 2-methyl derivative. The structural details of this type of lipid A were established using one- and two-dimensional NMR spectroscopy, chemical composition analyses, and mass spectrometry techniques (electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry and MALDI-TOF-MS). In these lipid A samples the glucosamine disaccharide characteristic for enterobacterial lipid A was replaced by a 2,3-diamino-2,3-dideoxy-d-glucopyranosyl-(GlcpN3N) disaccharide, deprived of phosphate residues, and substituted by an α-d-Manp-(1→6)-α-d-Manp disaccharide substituting C-4′ of the non-reducing (distal) GlcpN3N, and one residue of galacturonic acid (d-GalpA) α-(1→1)-linked to the reducing (proximal) amino sugar residue. Amide-linked 12:0(3-OH) and 14:0(3-OH) were identified. Some hydroxy groups of these fatty acids were further esterified by long (ω-1)-hydroxylated fatty acids comprising 26–34 carbon atoms. As confirmed by mass spectrometry techniques, these long chain fatty acids could form two or three acyloxyacyl residues. The triterpenoid derivatives were identified as 34-carboxyl-bacteriohopane-32,33-diol and 34-carboxyl-2β-methyl-bacteriohopane-32,33-diol and were covalently linked to the (ω-1)-hydroxy group of very long chain fatty acid in bradyrhizobial lipid A. Bradyrhizobium japonicum possessed lipid A species with two hopanoid residues.     

Lipid A precursor from Pseudomonas aeruginosa is completely acylated prior to addition of 3-deoxy-D-manno-octulosonate

Inhibition of lipopolysaccharide (LPS) synthesis in Pseudomonas aeruginosa at the stage of incorporation of 3-deoxy-D-manno-octulosonate (KDO) caused accumulation of a lipid A precursor which contained all of the fatty acids present on the lipid A of mature LPS. The enzyme CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from P. aeruginosa is inhibited by the KDO analog alpha-C-[1,5-anhydro-8-amino-2,7,8-trideoxy-D-manno-octopyranosyl] carboxylate (I), and I is effectively delivered to P. aeruginosa following attachment by amide linkage to the carboxyl terminus of alanylalanine. Intracellular hydrolysis releases the free inhibitor (I) which then inhibits activation of KDO by CMP-KDO synthetase causing accumulation of lipid A precursor and subsequent growth stasis. The major lipid A precursor species accumulated was purified and found to contain glucosamine, phosphate, C12:O, 2OH-C12:O and 3OH-C10:0 (in ester linkage), and 3OH-C12:0 (in amide linkage) in molar ratios of 1:1:0.5:0.5:1:1. Analysis of precursor by fast atom bombardment mass spectroscopy yielded a major ion (M - H)- of mass 1616 and fragments which were consistent with the structure of lipid A from P. aeruginosa. In contrast, Salmonella typhimurium, Escherichia coli, Citrobacter sp., Serratia marcescens, Enterobacter aerogenes, and Enterobacter cloacae all accumulated underacylated lipid A precursors which only contained 3-OH-C14:0, glucosamine, and phosphate. This difference and species-specific patterns of major and minor precursor species show that early steps in the assembly of lipid A are similar, but not identical in enteric and nonenteric Gram-negative bacteria.     

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

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

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.     

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