Lipopolysaccharides of Thiobacillus species containing lipid A with 2,3-diamino-2,3-dideoxyglucose

Lipopolysaccharides were isolated from two strains of Thiobacillus ferrooxidans and one strain each of Thiobacillus thiooxidans, Thiobacillus novellus and Thiobacillus sp. IFO 14570. Neutral sugars, 2-keto-3-deoxyoctonate, fatty acids and the rare 2,3-diamino-2,3-dideoxyglucose were detected in all lipopolysaccharides. Lipopolysaccharides of both T. ferrooxidans strains contained l-glycero-d-manno-heptose, whereas that of T. thiooxidans contained both l-glycero-d-manno-heptose and d-glycero-d-manno-heptose. On the other hand, heptoses were absent in lipopolysaccharides of T. novellus and Thiobacillus sp. IFO 14570. Lipid A of T. ferrooxidans and T. thiooxidans contained both glucosamine and 2,3-diamino-2,3-dideoxyglucose, in contrast, lipid A of T. novellus and the Thiobacillus sp. IFO 14570 most likely contain only 2,3-diamino-2,3-dideoxyglucose as backbone sugar. Deoxycholate polyacrylamide gel electrophoresis revealed S-type character for all lipopolysaccharides studied. The significance of the lipopolysaccharide composition for taxonomic and phylogenetic questions with regard to thiobacilli is discussed.Abbreviations DAG 2,3-diamino-2,3-dideoxyglucose - DOC sodium deoxycholate - GC gas-liquid chromatography - GC/MS gas-liquid chromatography/mass spectrometry - d,d-Heptose d-glycero-d-manno-heptose - l,d-Heptose l-glycero-d-manno-heptose - KDO 2-keto-3-deoxyoctonate - LPS lipopolysaccharide - 3-OH-14:0 3-hydroxy-tetradecanoic acid - PAGE polyacrylamide gel electrophoresis - PCP phenol-chloroform-petroleum ether     

Assimilatory sulfur metabolism in Rhodopseudomonas sulfoviridis

Rhodopseudomonas sulfoviridis is unable to grow with sulfate as sole sulfur source. Radioactively labelled sulfate is not incorporated into the cells. Growth only occurs in the presence of reduced sulfur compounds, such as sulfide, thiosulfate, elemental sulfur and cysteine. ATP sulfurylase, adenylylsulfate kinase, O-acetylserine sulfhydrylase and cysteine desulfhydrase are present. Adenylylsulfate sulfotransferase and thiosulfonate reductase are lacking. The enzymes of the sulfate-activating system are not derepressed by O-acetylserine.Non common Abbreviations APS Adenosine 5-phosphosulfate - PAPS 3-phosphoadenosine 5-phosphosulfate     

Chemical characterization of Campylobacter jejuni lipopolysaccharides containing N-acetylneuraminic acid and 2,3-diamino-2,3-dideoxy-D-glucose.

Lipopolysaccharides (LPS) of four nonencapsulated strains of the human enteric pathogen Campylobacter jejuni were chemically characterized. When applied to two of the strains, extraction by a modified phenol-chloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982) gave better yields of LPS than did extraction by the conventional hot phenol-water technique. Constituents common to all LPS were D-glucose, D-galactose, L-glycero-D-manno-heptose, 3-deoxy-D-manno-2-octulosonic acid, D-glucuronic acid, D-galactosamine, and phosphorylethanolamine. Phosphate was present in a relatively high amount. In addition, the LPS of three strains contained N-acetylneuraminic acid, whereas the LPS of the strain lacking this component contained 3-amino-3,6-dideoxy-D-glucose. The lipid A component contained phosphate with D-glucosamine and 2,3-diamino-2,3-dideoxy-D-glucose as the major amino sugars. Ethanolamine-phosphate was present also. The major fatty acids were ester- and amide-bound 3-hydroxytetradecanoic and ester-bound hexadecanoic acids, with a minor amount of ester-bound tetradecanoic acid. This is the first report of N-acetylneuraminic acid in the oligosaccharide moiety and diaminoglucose in the lipid A of C. jejuni LPS.     

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.     

Identification of a 2,3-diamino-2,3-dideoxyhexose in the lipid a component of lipopolysaccharides of Rhodopseudomonas viridis and Rhodopseudomonas palustris

A hitherto unknown amino sugar (Compound A), detected in acid hydrolyzates of lipopolysaccharides of Rhodopsuedomonas viridis and Rhodopseudomonas palustris, is present in the Lipid A component but not in the O-specific part of the lipopolysaccharides. 2-Amino-2-deoxy-D-glucose is lacking in the purified Lipid A of both strains. Compound A, characterized by a very high migration in paper electrophoresis was obtained in a pure state by ion-exchange chromatography and shown by m.s of the alditol acetate to be a 2,3-diamino-2,3-dideoxyhexose. G.I.c. and periodate oxidation excluded all possible stereoisomers with the exception of 2,3-diamino-2,3-dideoxyglucose and 2,3-diamino-2,3-dideoxyidose. G.I.c. of the alditol acetates of Compound A and of the glucose derivative suggests that Compound A is 2,3-diamino-2,3-dideoxyglucose. The significance of the occurrence of this new aminodeoxy sugar in the lipid A component of Rhodopsuedomonas viridis and Rhodopseudomonas palustris O-antigens for the biological properties of the respective lipopolysaccharides and for the taxonomy of the Rhodospirillaceae family is discussed.     

Automated analysis of 2,3-diamino-2,3-dideoxy-D-glucuronic acid by cation exchange chromatography with fluorometric postcolumn derivatization

2,3-Diamino-2,3-dideoxy-D-glucuronic acid (diaminoglucuronic acid) occurs as its di-N-acetyl derivative as a unique constituent of some bacterial cell walls. A sensitive chromatographic method for its determination is described. Diaminoglucuronic acid was well separated from glucosamine and galactosamine in about 80 min on a Dionex DC-6A cation exchange column (0.9 x 18 cm, 50 degrees C) with a sodium citrate buffer (pH 5.28) containing boric acid (0.2 M). The amino sugars in the eluate were monitored fluorometrically by postcolumn derivatization with orthophthalaldehyde detection reagent. This method allowed the automated determination of 50-100 pmol of glucosamine, galactosamine, and diaminoglucuronic acid and was applied successfully to the analysis of diaminoglucuronic acid in Propionibacterium acnes cells.     

Localization and Biological and Physicochemical Properties of the Cell Wall Lipopolysaccharide of Rhodopseudomonas capsulata

Electron micrographs of phenol-water-extracted lipopolysaccharide (LPS) of Rhodopseudomonas capsulata show filamentous and netlike aggregates. Treatment of the LPS with sodium deoxycholate resulted in a reversible splitting into subunits. The LPS represents a cell wall constituent with O-antigenic specificity. In passive hemagglutination tests, high titers were obtained when erythrocytes sensitized with untreated or heat-treated LPS were incubated with antisera obtained by immunization of rabbits with whole cells of R. capsulata. The alkali-treated LPS was not active in this test. Mouse lethality tests have shown that the LPS of R. capsulata is less toxic than LPS of Escherichia coli. Also, the X-ray protection efficacy and the phagocytic activity stimulation of LPS from R. capsulata in mice are small, as compared with LPS of E. coli. Incubation of living bacteria in saline (37 C) resulted in a solubilization of an LPS-protein-lipid complex from the outer layer of the cell wall. The isolated complex contained the components which were found in the LPS. In addition, 20% amino acids and a large amount of palmitic and stearic acids, which are typical phospholipid components, were present.     

Acyl lipid changes in photosynthetic mutants of Rhodopseudomonas sphaeroides correlate with blocks in bacteriochlorophyll synthesis

Abstract In comparison with the wild-type, mutants of Rhodopseudomonas sphaeroides defective in bacteriochlorophyll synthesis fail to alter their lipid composition on shifting from non-photosynthetic to photosynthetic growth conditions. The earlier the lesion in the bacteriochlorophyll synthetic pathway, the more severe the effect on membrane lipid composition, indicating that acyl lipid and pigment syntheses are co-ordinated and linked to pigment-protein complex assembly.     

Radioprotective effects of lipid A, liposomes, and liposomes containing lipid A in mice

Lipid A from Gram-negative bacterial lipopolysaccharide (endotoxin) was incorporated into liposomal membranes and examined as a prophylactic radioprotectant compound in lethally irradiated mice. Splenic hematopoietic activity, resulting in increased numbers of spleen cell colonies, was induced both by lipid A alone or more strongly by liposomal lipid A. Increased survival of lethally irradiated animals was induced to a slight extent by liposomes alone, to a greater extent by lipid A, and at the highest level by liposomes containing lipid A. Under conditions where 100% of untreated or saline-treated animals died of acute radiation syndrome after 20 days, more than 90% of the animals pretreated with liposomal lipid A were still alive 30 days after irradiation. We conclude that lipid A had substantial radioprotectant activity by itself, and the activity was enhanced by incorporation into liposomes. Liposomes alone also exhibited mild radioprotectant effects.     

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.     

Lipopolysaccharide containing L-acofriose in the filamentous blue-green alga Anabaena variabilis

For the first time, an O-antigenic lipopolysaccharide (LPS) has been isolated from a filamentous blue-green alga (Anabaena variabilis). It was extractable with phenol-water, resulting in extraction of the bulk of the LPS into the phenol phase. The polysaccharide moiety of this LPS consists of l-rhamnose, its 3-O-methyl ether l-acofriose, d-mannose, d-glucose, and d-galactose. l-Glycero-d-mannoheptose and 2-keto-3-deoxyoctonate, the two characteristic sugar components of enteric LPS, and phosphate groups are absent from the A. variabilis O antigen. The only amino sugar present is d-glucosamine. Three hydroxy fatty acids were identified, namely, beta-hydroxymyristic, beta-hydroxypalmitic and beta-hydroxystearic acids, in addition to palmitic and unidentified fatty acid. The LPS of A. variabilis is localized in the outermost cell wall layer and behaves like a bacterial O antigen in serological tests. The passive hemagglutination yielded high titers with isolated LPS (pretreated by heat or by alkali) and rabbit antisera prepared against living or heat-killed cells. The position of the precipitation arcs after immunoelectrophoresis of the O antigen indicates the lack of charged groups. The water phase of the phenol-water extract contains, in high yield, a glucose polymer. It is serologically inactive as shown by the passive hemagglutination test and by agar-gel precipitation.     

The structure of lipid A from the lipopolysaccharide of Rhodopseudomonas gelatinosa 29/1

The structure of the lipid A component of Rhodopseudomonas gelatinosa 29/1 lipopolysaccharide was established. It constitutes a -1,6-glucosamine disaccharide substituted on either side by ester-and glycosidically-bound phosphate residues. Both phosphate groups are in turn nonstoichiometrically substituted by ethanolamine. The amino groups of the disaccharide are N-acylated by 3-acyloxyacyl residues: that at the reducing glucosamine by 3-O-(14:0) 10:0, and that at the non-reducing one by 3-O-(12:0)10:0. Hydroxyl groups at C-3 and C-3 are esterified by hydroxycapric acid. Hydroxyl groups at C-4 and C-6 in free hydroxycapric acid. Hydroxyl groups at C-4 and C-6 in free lipid A were shown to be unoccupied by methylation with diazomethane. A similar methylation of the intact lipopolysaccharide revealed a free hydroxyl group only at C-4, indicating that C-6 is the attachment site of 3-deoxy-d-anno-octulosonic acid.By preparative thin-layer chromatography free lipid A could be resolved into at least two major and one minor fractions. Lipid A of R. gelatinosa 29/1 shows high lethal toxicity, comparable to that of Salmonella lipid A.Abbreviations GlcN d-Glucosamine - dOclA 2-keto-3-deoxy-d-manno-octonate - GC-MS combined gas liquid chromatographymass spectrometry - LPS lipopolysaccharide Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

Pigment containing lipid vesicles

Summary Vesicles obtained by sonication of chlorophylla-lecithin mixtures dispersed in an aqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophylla. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophylla molecule per 55 lecithins can be incorporated into the membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102±5 Å and independent of the chlorophylla content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).     

Interaction of photosensitizers with liposomes containing unsaturated lipid

Small unilamellar liposomes were made of dipalmitoyl-phosphatidylcholine and dioleoyl-phosphatidylcholine, and photosensitized by a symmetrically or an asymmetrically substituted glycosilated tetraphenyl-porphyrin derivative. As differential scanning calorimetry and electron paramagnetic resonance spectroscopy (EPR) revealed these porphyrin derivatives were localized in different depth within the lipid bilayer. Both porphyrin derivatives were able to induce photoreaction and consequent structural changes in the membrane. 5-, 12-, or 16-doxyl stearic acid labeled lipid bilayers were applied and the efficiency of photoinduced reaction was followed by the decay of their EPR signal amplitude. Light dose-dependent destruction of nitroxide radical proved to be dependent on the position of spin label. In this process the porphyrin localized in closer connection with the double bond of unsaturated fatty acid was more effective. EPR signal decay was also dependent on the unsaturated fatty acid content of the liposome and the oxygen saturation of the solvent.     

Lipopolysaccharide ofRhodospirillum salinarum 40: structural studies on the core and lipid A region

The structural elucidation of lipid A of the cell wall lipopolysaccharide (LPS) ofRhodospirillum salinarum 40 by chemical methods and laser desorption mass spectrometry revealed the presence of a mixed lipid A composed of three different 1,4 bisphosphorylated β(1→6)-linked backbone hexosaminyl-hexosamine disaccharides, i.e. those composed of GlCN→GlcN, 2,3-diamino-2,3-dideoxy-d-Glc-(DAG)→DAG, and DAG→GlcN. Lipid A ofR. salinarum contained preferentially 3-OH-18:0 and 3-OH-14:0 as amide-linked andcisΔ¹¹-18:1 and c19:0 as ester-linked fatty acids. The mass spectra of the liberated acyl-oxyacyl residues proved the concomitant presence of 3-O-(cisΔ¹¹-18:1)-18:0 and 3-O-(c19:0)-14:0 as the predominating diesters in this mixed lipid A. The glycosidically linked and the ester-linked phosphate groups of the backbone disaccharide were neither substituted by ethanolamine phosphorylethanolamine, nor by 4-amino-4-deoxy-l-arabinose, in contrast to most of the enterobacterial lipid As. In the core oligosaccharide fraction, a HexA (1→4)HexA(1→5)Kdo-trisaccharide was identified by methylation analysis. The terminal HexA (hexuronic acid) is possibly 4-OMe-GalA, a component described here as an LPS constituent for the first time. LPS ofR. salinarum showed a lethality in C57BL/10 ScSN (LPS-responder)-mice) of an order of 10⁻¹–10⁻² of that reported forSalmonella abortus equi LPS, and it was also capable of inducing TNFα and IL6 in macrophages of C57BL/10ScSN mice.     

Diffusion in lipid bilayers containing barriers

In epithelial cells, a barrier or tight junction restricts the diffusion of lipid probes from the apical to the basolateral side of the outer membrane bilayer. This phenomenon is studied theoretically with the diffusion equation on planar and spherical surfaces. Two models for the tight junction are considered: a penetrable barrier embedded in a monolayer and an impenetrable obstacle in the outer membrane of a bilayer than must be bypassed by flip-flopping between inner and outer membranes. The rate of passing from one side of the cell to the other is calculated for each of these models under steady state conditions. The results are compared with recent fluorescent photobleaching recovery experiments. The theoretical interpretation indicates that it would be difficult to distinguish experimentally between the flip-flop case and the barrier crossing case. Assuming a flip-flop model, large differences in the magnitude of the flip-flop rates of probes are necessary to explain the experimental results as suggested by Dragsten et al. (Dragsten, P. R., R. Blumenthal, and J. S. Handler, 1981, Nature [Lond.], 294:718--722).