Location of fatty acids in lipid A obtained from lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023

Monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was initially purified by silicic acid column chromatography to yield a single major pentaacyl MLA fraction. This fraction was methylated and further purified by reverse-phase high performance liquid chromatography to yield three prominent peak fractions. Laser desorption mass spectrometry of these three fractions allowed us to complete the important structural analysis of lipid A from this source. Three structurally distinct forms of dimethyl MLA were identified where Mr = 1447, 1449, and 1451 atomic mass units. These forms differed only by the presence or absence of unsaturation and keto group in the fatty acids. We established that the acyloxyacyl group (either delta 7-tetradecenoyloxytetradecanoate or tetradecanoyloxytetradecanoate) and the 3-ketotetradecanoate or hydroxytetradecanoate occupied the 2'- and 2-positions of the glucosamine disaccharide, respectively. Analysis of several minor fractions suggests that there is considerable structural heterogeneity in the MLA. With this new knowledge, the study of the structure-to-function relationship of the reported lack of toxicity of lipopolysaccharide from R. sphaeroides can be completed.     

Human low-molecular-mass kininogen. Amino-acid sequence of the light chain; homology with other protein sequences

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.     

Chemical reduction of 3-oxo and unsaturated groups in fatty acids of diphosphoryl lipid A from the lipopolysaccharide of Rhodopseudomonas sphaeroides. Comparison of biological properties before and after reduction

Unlike the diphosphoryl lipid A (DPLA) derived from toxic lipopolysaccharide of Escherichia coli and Salmonella strains, the DPLA from nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 is biologically inactive. This could be due to the presence of 3-oxotetradecanoic and delta 7-tetradecenoic acids. These two fatty acids in R. sphaeroides DPLA were catalytically reduced in platinum oxide/H2 to the 3-hydroxy and saturated fatty acids, respectively. The biologically active E. coli DPLA was also treated with platinum oxide/H2, but as expected, the reduction step did not change the structure. These two preparations were then compared with the untreated samples for biological activity in three select in vitro assays. Over a range of 0.01-100 ng/ml, both normal and reduced DPLA from R. sphaeroides were inactive in priming phorbol myristate acetate-stimulated superoxide anion release in human alveolar macrophages. Over a range of 10-10(3) ng/ml, both samples failed to induce tumor necrosis factor in the RAW 264.7 murine macrophage cell line. The reduced DPLA marginally activated 70Z/3 pre-B cells at concentrations of 0.1-30 micrograms/ml. In every case, both normal and platinum oxide/H2-treated E. coli DPLA were biologically active. These results indicate that the lack of biological activity of R. sphaeroides DPLA is not due to the presence of 3-oxo and unsaturated fatty acids, but rather to one or more of the following: (i) presence of only five fatty acyl groups (compared to six in active lipid A); (ii) presence of 3-hydroxydecanoic acids (rather than 3-hydroxytetradecanoic, in active lipid A); (iii) greater variation in size of the fatty acids.     

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).     

Lipopolysaccharides of two strains of the phototrophic bacterium Rhodopseudomonas capsulata

The lipopolysaccharides of Rhodopseudomonas capsulata strains St. Louis (ATCC 23782) and Sp 11 both contain L-acofriose, rhamnose, glucose and glucosamine as the main sugar constituents. 2-Keto-3-deoxyoctonate and neuraminic acid were tentatively identified. The fatty acid spectrum found with both strains comprises 3-OH-C10 and C12:1 (ester-linked) and 3-oxo-C14 (amide-linked). Isolated lipid A from strain Sp 11 contains glucosamine, glucosamine-phosphate and the total of the fatty acids of the lipopolysaccharide. Methylation analysis of the degraded polysaccharide of this lipopolysaccharide shows L-acofriose in both terminal and 1 leads to 2 chain-linked positions in a 1:4 molar ratio. Rhamnose is exclusively chain-linked (1 leads to 2), glucose is both terminally and chain-linked (1 leads to 6) in a 1:1 molar ratio. The serological activity of the lipopolysaccharide of both the R. capsulata strains is low in antisera against living or heat-killed cells when tested by passive hemagglutination, Ouchterlony immunoprecipitation or gel-immunoelectrophoresis. No crossreaction was observed among the lipopolysaccharides of R. capsulata strains St. Louis, Sp 11 and 37b4 in immunoprecipitation. Lipopolysaccharide of strain Sp 11 was found to lack lethal toxicity in galactosamine-sensitized mice.     

Different lipid A types in lipopolysaccharides of phototrophic and related non-phototrophic bacteria

Lipid A analyses confirm not only the present taxa of the purple nonsulfur bacteria (formerly Rhodospirillaceae), but also phylogenetical relatedness of distinct phototrophic to distinct non-phototrophic bacteria, as was suggested by cataloguing 16S rRNA. For example, lipid A with ester-bound 3-OH-10:0 and the rare amide-linked 3-oxo-14:0 is common to the phototrophic Rhodobacter capsulatus and Rhodobacter sphaeroides and also to Paracoccus denitrificans and Thiobacillus versutus. 'Lipid ADAG' (lipid A with 2,3-diamino-D-glucose (DAG)) occurs in the phototrophic Rhodopseudomonas viridis and Rhodopseudomonas palustris and also in the related non-phototrophic species, e.g., Nitrobacter winogradskyi, Pseudomonas diminuta, or Thiobacillus ferrooxidans. The phylogenetically more coherent purple sulfur bacteria (Chromatiaceae) uniformly contain D-mannose in their phosphate-free lipid A. Among the green bacteria, only the Chlorobiaceae but not the likewise chlorosome-containing Chloroflexaceae contain lipopolysaccharide. Lipid ADAG from R. viridis is a structural analogue of a biosynthetic precursor (lipid X) of enterobacterial lipid A. Lipid A synthase from Salmonella accepts not only lipid X but also the synthetic di-N-acyl-2,3-diamino-D-glucose analogue as substrate (Raetz, C.R.H., unpublished results). More and more naturally occurring lipid A's with both, 2,3-diaminoglucose and glucosamine ('mixed' lipid A, with 2,3-diaminoglucose or glucosamine dominating) are being found. Newly recognized lipid A and lipid ADAG types might offer the possibility of differentially stimulating desired biological activities in animals without also having the undesired endotoxic activities. The non-toxic lipid A from Rhodopseudomonas viridis for example is able to stimulate prostaglandin secretion in peritoneal macrophages and can be used as an antagonist to the endotoxic shock caused by Salmonella lipopolysaccharide.     

The purification and chemical composition of the lipopolysaccharide of Pseudomonas alcaligenes

1. A method for obtaining lipopolysaccharide free from glycosaminopeptide from isolated cell walls of Pseudomonas alcaligenes is discussed. 2. About 70-75% of the lipopolysaccharide and 86-90% of the isolated lipid A have been accounted for in terms of identifiable components. 3. Hydrolysates of lipid A contain mainly inorganic phosphate, glucosamine, O-phosphorylglucosamine and fatty acids (dodecanoic acid, dodec-2-enoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid), of which the last is the main N-acylating acid of the glucosamine backbone. 4. Material corresponding to the polysaccharide moiety of the lipopolysaccharide is extensively degraded. 5. Solubilization of the lipopolysaccharide by using sodium deoxycholate appreciably affects the chemical composition of the material.     

Elucidation of structure of lipid A from the marine Gram-negative bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A from the marine gamma-proteobacterium Pseudoalteromonas haloplanktis ATCC 14393T, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be beta-1,6-glucosaminobiose 1,4'-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3' and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2', respectively.     

Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells

Abstract Pentaacyl diphosphoryllipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 (RsDPLA) did not induce tumour necrosis factor-α nor interleukin-6 release in the murine macrophage-like cell line J774.1. However, it effectively inhibited the induction of these two cytokines by LPS of Salmonella minnesota Re mutant R595 (ReLPA) in a concentration-dependent manner. Maximal inhibition and half-maximal inhibition occured when the ReLPS to RsDPLA mass ratio was 1:30 and 1:1, respectively. A binding study was performed in the presence of serum to determine whether RsDPLA is competing with ReLPS for LPS binding sites on J774.1 cells. This assay allows the determination of LPS binding to J774.1 cells via a mechanism involving CD14, a receptor for complexes of LPS with LPS binding protein (LBP), and its possible inhibition. The results show that RsDPLA strongly inhibits the binding of ¹²⁵I-labelled ReLPS to J774.1 cells. Maximal and one-half maximal inhibition of binding occured when the ReLPS to RsDPLA mass ratios were 1:2.5 and 1:0.5, respectively. It was found that the inhibition of binding by RsDPLA was much stronger than that by unlabelled ReLPS. These results suggest that RsDPLA is competing with ReLPS for CD14-dependent recognition of LPS on J774.1 cells.     

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.     

Structural analysis of the nontoxic lipid A of Rhodobacter capsulatus 37b4

Lipid A from Rhodobacter capsulatus 37b4 consists of a D-glucosaminyl-(beta 1-6)-D-glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C-1 of the reducing glucosamine and phosphorylethanolamine at C-4' of the nonreducing glucosamine. 1,4'-Bisphosphorylated lipid A, lacking the polar head groups, was also encountered and contributed to the observed microheterogeneity in the phosphate substitution. The amino functions of both glucosamines are substituted almost entirely by the rare 3-oxotetradecanoic acid, which is a characteristic constituent of lipid A in the genus Rhodobacter. 3-Hydroxydecanoic acid is ester-bound at C-3 and C-3' of the glucosamine disaccharide and the one at the nonreducing glucosamine (C-3') is partially substituted by dodecenoic acid to form an ester-bound diester. In free lipid A, hydroxy groups at C-4 and C-6' of the glucosamine disaccharide are unsubstituted. C-6' being the putative attachment point of the lipopolysaccharide core. The nontoxic Rhodobacter capsulatus lipid A shows extensive serological cross-reaction with the toxic Salmonella lipid A. Structural similarities in the hydrophilic part of both types of lipid A, dissimilarities in the hydrophobic part and their impacts on serologic properties are discussed.     

The extraction of cell walls of Pseudomonas aeruginosa with aqueous phenol. The insoluble residue and material from the aqueous layers

1. The insoluble residue and material present in the aqueous layers resulting from treatment of cell walls of Pseudomonas aeruginosa with aqueous phenol were examined. 2. The products (fractions AqI and AqII) isolated from the aqueous layers from the first and second extractions respectively account for approx. 25% and 12% of the cell wall and consist of both lipopolysaccharide and muropeptide. 3. The lipid part of the lipopolysaccharide is qualitatively similar to the corresponding material (lipid A) from other Gram-negative organisms, as is the polysaccharide part. 4. The insoluble residue (fraction R) contains sacculi, which also occur in fraction AqII. On hydrolysis, the sacculi yield glucosamine, muramic acid, alanine, glutamic acid and 2,6-diaminopimelic acid, together with small amounts of lysine, and they are therefore similar to the murein sacculi of other Gram-negative organisms. Fraction R also contains substantial amounts of protein, which differs from that obtained from the phenol layer. 5. The possible association or aggregation of lipopolysaccharide, murein and murein sacculi is discussed.     

Studies on the chemical composition of lipopolysaccharide from Neisseria meningitidis group B.

A lipopolysaccharide was isolated from Neisseria meningitidis group B by phenol/water extraction and purified by differential ultracentrifugation. This preparation exhibited endotoxic properties as shown by the limulus-lysate assay. Mild acid hydrolysis of the lipopolysaccharides yielded a lipid A fraction and a polysaccharide fraction. The lipid A fraction contained fatty acids, phosphorus and glucosamine. Analysis of the polysaccharide fraction revealed the presence of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid and phosphorus. There was no heptose.     

The structure of uncommon lipid A from the marine bacterium Marinomonas communis ATCC 27118T

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118T with 1% AcOH. Using chemical analysis and ID and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be beta-(1',6)-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-[(R)-3-hydroxydecanoyloxy)]decanoic acid, and (R)-3-hydroxydecanoic acid at the C2, C2' and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M. communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.     

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.     

Fermentation of pyruvate by 7 species of phototrophic purple bacteria]

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.     

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.     

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.     

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

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)