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.     

Structural studies on the D-arabinose-containing lipid A from Rhodospirillum tenue 2761

Structural studies carried out on the isolated free lipid A of Rhodospirillum tenue 2761 revealed a new type of structure for this lipid. The lipid A backbone of 1',6-linked glucosamine disaccharide (central disaccharide) is substituted by three different sugar residues: the non-reducing end of the disaccharide by 4-amino-4-deoxy-L-arabinose 1-phosphate and its reducing end glycosidically by D-arabinofuranose 1-phosphate; further, the reducing glucosamine of the disaccharide is branched to a third glucosamine residue by a 1',4-glycosidic linkage. The amino and the hydroxyl groups of the central disaccharide are acylated by 3-hydroxydecanoic acid (amide-linked) and palmitic and myristic acids (ester-linked). Neither amino nor hydroxyl groups of the three external sugar residues are acylated. The results suggest the following chemical structure for the lipid A of R. tenue 2761: (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.     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

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.     

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     

In vitro antigenic reactivity of synthetic lipid A analogues as determined by monoclonal and conventional antibodies

Cross-reactivities of synthetic lipid A analogues with monoclonal and conventional antibodies against Salmonella lipid A were studied. It was shown that the in vitro antigenicity of a synthetic compound 506, beta-(1----6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, was practically indistinguishable from that of the natural E. coli lipid A preparation, and that both phosphates in positions 1 and 4' as well as ester- and amide-linked fatty acyl residues, particularly 3-acyloxyacyl group, of the glucosamine disaccharide are involved in the cross-reactivity of lipid A as important antigenic determinants.     

Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acid is conserved in rhizobium and Sinorhizobium sp

A broad-host-range endosymbiont, Sinorhizobium sp. NGR234 is a component of several legume-symbiont model systems; however, there is little structural information on the cell surface glycoconjugates. NGR234 cells in free-living culture produce a major rough lipopolysaccharide (LPS, lacking O-chain) and a minor smooth LPS (containing O-chain), and the structure of the lipid A components was investigated by chemical analyses, mass spectrometry, and NMR spectroscopy of the underivatized lipids A. The lipid A from rough LPS is heterogeneous and consists of six major bisphosphorylated species that differ in acylation. Pentaacyl species (52%) are acylated at positions 2, 3, 2', and 3', and tetraacyl species (46%) lack an acyl group at C-3 of the proximal glucosamine. In contrast to Rhizobium etli and Rhizobium leguminosarum, the NGR234 lipid A contains a bisphosphorylated beta-(1' --> 6)-glucosamine disaccharide, typical of enterobacterial lipid A. However, NGR234 lipid A retains the unusual acylation pattern of R. etli lipid A, including the presence of a distal, amide-linked acyloxyacyl residue containing a long chain fatty acid (LCFA) (e.g. 29-hydroxytriacontanoate) attached as the secondary fatty acid. As in R. etli, a 4-carbon fatty acid, beta-hydroxybutyrate, is esterified to (omega - 1) of the LCFA forming an acyloxyacyl residue at that location. The NGR234 lipid A lacks all other ester-linked acyloxyacyl residues and shows extensive heterogeneity of the amide-linked fatty acids. The N-acyl heterogeneity, including unsaturation, is localized mainly to the proximal glucosamine. The lipid A from smooth LPS contains unique triacyl species (20%) that lack ester-linked fatty acids but retain bisphosphorylation and the LCFA-acyloxyacyl moiety. The unusual structural features shared with R. etli/R. leguminosarum lipid A may be essential for symbiosis.     

Characterization of Mesorhizobium huakuii lipid A containing both D-galacturonic acid and phosphate residues.

The chemical structure of the free lipid A isolated from Mesorhizobium huakuii IFO 15243(T) was elucidated. Lipid A is a mixture of at least six species of molecules whose structures differ both in the phosphorylation of sugar backbone and in fatty acylation. The backbone consists of a beta (1'-->6) linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharide that is partly substituted by phosphate at position 4'. The aglycon of the DAG-disaccharide has been identified as alpha-D-galacturonic acid. All lipid A species carry four amide-linked 3-hydroxyl fatty residues. Two of them have short hydrocarbon chains (i.e. 3-OH-i-13:0) while the other two have longer ones (i.e. 3-OH-20:0). Distribution of 3-hydroxyl fatty acids between the reducing and nonreducing DAG is symmetrical. The nonpolar as well as (omega-1) hydroxyl long chain fatty acids are components of acyloxyacyl moieties. Two acyloxyacyl residues occur exclusively in the nonreducing moiety of the sugar backbone but their distribution has not been established yet. The distal DAG amide-bound fatty acid hydroxyls are not stoichiometrically substituted by ester-linked acyl components.     

Isolation and structural characterization of an R-form lipopolysaccharide from Yersinia enterocolitica serotype O:8.

The lipopolysaccharide (LPS) of strain 8081-c-R2, a spontaneous R-mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/light petroleum. Its compositional analysis indicated the presence of D-GlcN, D-Glc, L-glycero-D-manno- and D-glycero-D-manno-heptose, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1-3) were isolated using high-performance anion-exchange chromatography, the structures of which were determined by compositional analysis and one- and two-dimensional NMR spectroscopy as [carbohydrate structure see text] in which all sugars are pyranoses, and R and R' represent beta-D-Glc (in 1 and 2) and beta-D-GlcN (in 1 only), respectively. D-alpha-D-Hep is D-glycero-alpha-D-manno-heptose, L-alpha-D-Hep is L-glycero-alpha-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI-TOF MS. Its beta-D-GlcN4P-(1-->6)-alpha-D-GlcN-1-->P backbone is mainly tetra-acylated with two amide- and one ester-linked (at O3 of the reducing GlcN) (R)-3-hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3-OH group of the amide-linked (R)-3-hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri- and hexa-acylated lipid A species occur.     

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.     

Structural studies on the immunodominant group of lipid A from lipopolysaccharide of Yersinia pseudotuberculosis.

Lipid A isolated from lipopolysaccharide of Yersinia pseudotuberculosis was used for immunization of rabbits to afford antisera to lipid A with titers of 1:640 in the passive hemolysis test. Exhaustion of immune serume with sheep erythrocytes decreased antibody titers up to 1:160. Authentic samples of 2-(DL-3-hydroxytetradecanoyl)amino-2-deoxy-D-glucose 6-phosphate, 2-tetradecanoylamino-2-deoxy-D-glucose 6-phosphate and 2-acetamido-2-deoxy-D-glucose 6-phosphate have been synthesized in order to carry out a comparative study of inhibitory activity of these compounds and lipid A using a system of lipid A and antiserum to lipid A. As a result, the immunodominant moiety of the lipid A of Y. pseudotuberculosis proved to contain a D-glucosamine residue acylated with 3-hydroxytetradecanoic acid at the amino group. The nature of the fatty acid acylating the amino group of glucosamine does not play an important role in the structure of immunodominant moiety of lipid A.     

Structure of Shigella sonnei lipid A

The detailed chemical structure of lipid A of Shigella sonnei phase II was elucidated. The lipid A backbone consists of a β-1,6-linked glucosamine disaccharide substituted with (mono) phosphates both at C-1 and C-4′. This was shown by selective degradation followed by ³¹P-NMR studies. C-4 and C-6′ were found to contain unsubstituted hydroxyl groups, the latter being the point of attachment of KDO as reported for other enterobacterial lipids A.The amino groups of the glucosamine disaccharide are substituted by 3-hydroxy fatty acids: 3-O-(14:0) 14:0 at the non-reducing glucosamine and 3-O-(12:0) 14:0 at the reducing glucosamine. In contrast to earlier reports, no ethanolamine or phosphodiester linkages were found in lipid A.     

Ornithine lipids and their structural modifications: from A to E and beyond

Ornithine lipids (OLs) are phosphorus-free membrane lipids that are widespread in eubacteria, but absent from archaea and eukaryotes. They contain a 3-hydroxy fatty acyl group attached in amide linkage to the α-amino group of the amino acid ornithine. A second fatty acyl group is ester-linked to the 3-hydroxy position of the first fatty acid. About 25% of the bacterial species whose genomes have been sequenced are predicted to have the capacity to form OLs. Distinct OL hydroxylations have been described in the ester-linked fatty acid, the amide-linked fatty acid, and the ornithine moiety. These modifications often seem to form part of a bacterial stress response to changing environmental conditions, allowing the bacteria to adjust membrane properties by simply modifying already existing membrane lipids without the need to synthesize new lipids.     

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.     

Characterization of lipid A and polysaccharide moieties of the lipopolysaccharides from Vibrio cholerae.

Lipid A and polysaccharide moieties obtained by mild acid hydrolysis of the lipopolysaccharides from Vibrio cholerae 569 B (Inaba) and Vibrio el-tor (Inaba) were characterized. Heterogeneity of lipid A fractions was indicated by t.l.c. and by gel filtration of the de-O-acylated products from mild alkaline methanolysis of the lipids. Presumably lipid A contains a glucosamine backbone, and the fatty acids are probably bound to the hydroxyl and amino groups of glucosamine residues. Approximately equal amounts of fatty acids C16:0, C18:1 and 3-hydroxylauric acid were involved in ester linkages, but 3-hydroxymyristic acid was the only amide-linked fatty acid. Sephadex chromatography of the polysaccharide moiety showed the presence of a high-molecular-weight heptose-free fraction and a low-molecular-weight heptose-containing fraction. Haemagglutination-inhibition assays of these fractions showed the heptose-free fraction to be an O-specific side-chain polysaccharide, whereas the heptose-containing fraction was the core polysaccharide region of the lipopolysaccharides. Identical results were obtained for both organisms.     

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota strain R7 (chemotype Rd1P−)

Abstract Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd₁P⁻ mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd₁P⁻ LPS bound to the β 1 → 6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide l -glycero-α- d -manno-heptopyranose(1 → 3)- l -glycero-α- d -manno-heptopyranose(1 → 5)3-deoxy- d -manno-octulosonic acid. Antibodies against the 1 → 3- and 1 → 7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.     

The structure of the lipid A component of Sphaerotilus natans

The lipopolysaccharide of Sphaerotilus natans afforded a ladder-like pattern of bands in sodium deoxycholate-polyacrylamide gel electrophoresis, indicating the presence of a S-form lipopolysaccharide. The chemical analysis showed neutral sugars (rhamnose, glucose, l-glycero-d-manno-heptose), 3-deoxy-octulosonic acid (Kdo), amino compounds (glucosamine, glucosamine phosphate, ethanolamine and ethanolamine phosphate), and phosphorus. The lipid A fraction contained saturated and unsaturated capric, lauric, and myristic acids, and 3-hydroxy capric acid (3-OH-10:0). Its chemical structure was consisting of a glucosamine disaccharide, glycosidically substituted by a phosphomonoester, and substituted at C-4 by a pyrophosphodiester esterified with ethanolamine. The amino groups of both glucosamines are acylated by 3-hydroxy capric acids and these in turn are substituted by saturated and unsaturated capric, lauric, and myristic acids. Hydroxyl groups of the backbone disaccharide at C-3 and C-3 were also esterified by 3-hydroxy capric acid, those at C-4 and C-6 were unsubstituted. The latter provides the attachment site for Kdo.Abbreviations Kdo 3-deoxy-d-manno-octulosonic acid - 3-OH-10:0 3-hydroxy capric acid - DOC-PAGE deoxycholate-polyacrylamide gel electrophoresis - GC-MS gas chromatography/mass spectrometry - LD-MS laser desorption mass spectrometry - LPS lipopolysaccharide - PS polysaccharide     

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of Gram-negative bacteria.

In the presence of MgCl2, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl2 and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I MgCl2 and detergent were unable to either entrap or retain [14C]-sucrose, [3H=inulin or [3H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [3H]-inulin out of re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl2 to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occurring in their normal intestinal habitat.     

Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety

The chemical structures of six lipid A species (A, B, C, D-1, D-2, and E) purified from Rhizobium etli CE3 were investigated by one- and two-dimensional NMR spectroscopy. The R. etli lipid A subtypes each contain an unusual acyloxyacyl residue at position 2' as part of a conserved distal glucosamine moiety but differ in their proximal units. All R. etli lipid A species lack phosphate groups. However, they are derivatized with an alpha-linked galacturonic acid group at position 4', as shown by nuclear Overhauser effect spectroscopy. Component B, which had been not been reported in previous studies, features a beta, 1'-6 linked disaccharide of glucosamine acylated at positions 2, 3, 2', and 3' in a pattern that is typical of lipid A found in other Gram-negative bacteria. D-1 contains an acylated aminogluconate unit in place of the proximal glucosamine residue of B. C and E lack ester-linked beta-hydroxyacyl chains at position 3, as judged by their H-3 chemical shifts, and may be synthesized from B and D-1, respectively, by the R. etli 3-O-deacylase. D-2 is an isomer of D-1 that forms nonenzymatically by acyl chain migration. A may be an elimination product derived from D-1 during hydrolysis at 100 degrees C (pH 4.5), a step needed to release lipid A from lipopolysaccharide. Based on these findings, we propose a biosynthetic scheme for R. etli lipid A in which B is generated first by a variation of the E. coli pathway. The aminogluconate unit of D-1 could then be made from B by enzymatic oxidation of the proximal glucosamine. As predicted by our hypothesis, enzyme(s) can be demonstrated in extracts of R. etli that convert (14)C-labeled B to D-1.