Molecular structure of lipid A, the endotoxic center of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides—Themes and variations

Bacteria which utilize the xenobiotic compounds chloridazon, antipyrin, and pyramidon as sole carbon sources were isolated from various soil samples. The 22 strains isolated are similar with respect to morphological, physiological, biochemical, serological, and genetic properties. These bacteria are aerobic gram-negative rods or coccal rods (0.7 to 1.0 by 1.0 to 2.0 μm) that occur singly, in pairs, or in short chains and are nonmotile and nonsporeforming. Physiological and biochemical characteristics and susceptibility to antibiotics were determined. The strains need vitamin B12 as a growth factor; they are catalase positive and weakly oxidase positive and show slight H2S production. All of the other tests which we performed were negative. The nutritional spectrum is extraordinarily limited, with optimal growth on chloridazon, antipyrin, pyramidon, and l-phenylalanine. Most sugars, alcohols, amino and carboxylic acids, and ordinary complex media are not utilized. The bacteria are osmotically sensitive. They are a serologically uniform group of organisms, which are harmless to rats and rabbits. Their guanine-plus-cytosine contents range between 65 and 68.5 mol%. The chloridazon-degrading bacteria are characterized as a new genus, Phenylobacterium, with a single species, Phenylobacterium immobile. The type strain Phenylobacterium immobile strain E (= DSM 1986), is not closely related to any other gram-negative bacterium, as shown by a 16S ribosomal ribonucleic acid partial sequence analysis. This organism is a member of group I of the purple nonsulfur bacteria, but is phylogenetically isolated in this group. Phenylobacterium immobile is remotely related to Pseudomonas diminuta, Rhizobium leguminosarum, rhodopseudomonads, and Aquaspirillum itersonii. Like other members of this group, Phenylobacterium immobile contains 2,3-diamino-2,3-dideoxy-d-glucose in its lipopolysaccharide. The murein equals a normal murein from a gram-negative bacterium. All citric acid cycle enzymes are detectable in Phenylobacterium immobile.

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Phenylobacterium immobile gen. nov., sp. nov., a Gram-Negative Bacterium That Degrades the Herbicide Chloridazon

Publisher Summary This chapter discusses the analysis of lipopolysaccharides (LPS) of gram-negative bacteria. LPS are characteristic components of the cell wall of all Gram-negative bacteria and of at least some cyanobacteria. They are localized in the outer layer of the outer membrane, and are, in noncapsulated strains, exposed on the cell surface. They contribute to the integrity of the outer membrane and, as has been shown for enteric bacteria, protect the cell against the action of bile salts and (lipophilic) antibiotics. Isolated lipopolysaccharide, upon injection into higher organisms, exhibits a variety of biological (“endotoxic”) activities, such as the induction of fever, changes in the white blood cell count, diarrhoea, or even shock and death. Since the toxic activities are because of substances released from disintegrating bacteria, and not to an excreted exotoxin, lipopolysaccharide has been termed an endotoxin. Thus, the terms lipopolysaccharide, O-antigen, and endotoxin are used synonymously and describe either the chemical structure or discrete biological functions of the same substance.

6 Analysis of Lipopolysaccharides of Gram-Negative Bacteria

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 [β-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-β-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 α-glycosidically bound to the reducing sugar residue of the backbone, though C-1 is also non-stoichiometrically substituted by diphos-phorylethanolamine. 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.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1991.tb16036.x

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.

Lipopolysaccharide of a chloridazon-degrading bacterium was obtained by a two-stage extraction procedure with phenol/EDTA in a yield of 0.3% of dried bacteria. The carbohydrate moiety consisted of heptose, 3-deoxyoctulosonic acid and D-glucose in a molar ratio of 1:2:2 X 3. Lipid A was composed of 1 mol 2,3-diamino-2,3-dideoxy-D-glucose, 2 mol amide-bound and 2.6 mol ester-bound fatty acids/mol. Amide-bound fatty acids were 3-hydroxydodecanoic acid and 3-hydroxyhexadecanoic acid; dodecanoic acid and R-(-)-3-hydroxydodec-5-cis-enoic acid were found to be present in ester linkage. Under conditions of acidic hydrolysis, the latter was converted into the cis and trans isomers of 5-hexyltetrahydrofuran-2-acetic acid. Dodecanoic acid was demonstrated to be linked with the hydroxy groups of the amide-bound fatty acids. The taxonomic significance of these results, especially the demonstration of 2,3-diamino-2, 3-dideoxy-D-glucose, is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07809.x

Rüdiger Weisshaar

Papers

The lipopolysaccharide of a chloridazon-degrading bacterium.