Structural analysis of a novel sialic-acid-containing trisaccharide from Rhodobacter capsulatus 37b4 lipopolysaccharide.

Sialic-acid-containing lipopolysaccharides from Rhodobacter capsulatus 37b4 (S-form lipopolysaccharide), KB-1 (R-type lipopolysaccharide) and Sp 18 (deep R-type lipopolysaccharide) were investigated for the linkage and substitution of sialic acids. Methylation analysis and behaviour towards acid and enzymic hydrolysis indicated a non-reducing terminal location of sialic acids in the R-type lipopolysaccharide of strain Sp 18, whereas an internal, chain-linked location of sialic acids was found in the lipopolysaccharides of strains 37b4 and KB-1. For these latter strains, methylation analysis revealed a substitution of sialic acids by other sugars at position 7 for strain 37b4 and positions 4 and 7 for strain KB-1. In accordance with the chain-linked position of sialic acids, mild hydrolysis of R. capsulatus 37b4 lipopolysaccharide with acetic acid released a trisaccharide with sialic acid at the reducing terminus. Structural investigation of this trisaccharide by methylation analysis, 1H- and 13C-NMR spectroscopy revealed the presence of the disaccharide Gal1-6Glc at the non-reducing end, probably with an alpha-anomeric configuration of the galactose residue, i.e. melibiose, beta-glycosidically linked to position 7 of sialic acid. Therefore the structure Gal alpha 1-6Glc beta 1-7Neu5Ac is proposed for this core oligosaccharide from R. capsulatus 37b4 lipopolysaccharide.

Cytokine induction by lipopolysaccharide (LPS) corresponds to lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS.

Many pathological effects of gram-negative bacteria are produced by their cell wall-derived lipopolysaccharides (LPSs). Differing pathogenicity of gram-negative LPSs, however, may depend on their capacities to induce cytokines. Thus, we studied the lethal toxicity of four nonenterobacterial LPSs and compared it with their capacity to induce mononuclear cell (MNC)-derived interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). Unstimulated MNC did not release these cytokines. LPS from the phototrophic strain Rhodobacter capsulatus 37b4 elaborated little toxicity in galactosamine-treated mice (10 micrograms of LPS per mouse was the 100% lethal dose [LD100]) and induced IL-1 and IL-6 release only at high concentrations (10 to 50 micrograms of LPS per ml). R. capsulatus LPS failed to induce TNF activity even at the highest concentration tested (100 micrograms of LPS per ml). In contrast, LPS derived from Pseudomonas diminuta NCTC 8545 or the nodulating species Bradyrhizobium lupini DSM 30140 and Rhizobium meliloti 10406 expressed lethal toxicity (LD100, 1,000, 100, and 10 ng per mouse, respectively) and induced IL-1 or IL-6 (10 to 100, 10, and 1 ng of LPS per ml, respectively) at concentrations 1,000- to 10,000-fold lower than effective levels of R. capsulatus LPS. LPSs from P. diminuta, B. lupini, and R. meliloti also stimulated TNF production and release. MNC accumulated cell-associated IL-1 activities under circumstances in which released activity was readily detected. The cells contained only scant IL-6 activity, indicating release of this mediator rather than intracellular accumulation. Antisera to the respective cytokines inactivated biological activities of the samples selectively. The R. capsulatus LPS inhibited cytokine induction by LPS from P. diminuta, B. lupini, and R. meliloti in coincubation experiments. These results show that the in vivo lethality of the LPSs tested correlates with the induction of monocyte-derived cytokines in vitro. The results of this study suggest that the different lethality of various LPSs from gram-negative bacteria may be due to the differential ability of these LPSs to induce cytokine production.

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.

Lipid A with 2,3-diamino-2,3-dideoxy-glucose in lipopolysaccharides from slow-growing members of Rhizobiaceae and from "Pseudomonas carboxydovorans".

Lipid A's from two Bradyrhizobium species and from the phylogenetically closely related species "Pseudomonas carboxydovorans" were found to contain 2,3-diamino-2,3-dideoxy-glucose as lipid A backbone sugar. In contrast, three representatives of the genus Rhizobium, as well as the phylogenetically related species Agrobacterium tumefaciens, contain solely glucosamine as lipid A backbone sugar. These findings support independent studies on the phylogenetical relatedness based on 16S rRNA-data of the genus Bradyrhizobium with "Pseudomonas carboxydovorans" and Rhodopseudomonas palustris, which form a tight phylogenetical cluster and which all contain the 2,3-diamino-2,3-dideoxy-glucose-containing lipid A. The relatedness of these species to the glucosamine-containing species of the genus Rhizobium and to Agrobacterium tumefaciens is rather distant as documented by 16S rRNA studies.

Absence of a characteristic cell wall lipopolysaccharide in the phototrophic bacterium Chloroflexus aurantiacus.

Two strains of the gliding phototrophic bacterium Chloroflexus aurantiacus were investigated for the presence of lipopolysaccharide (LPS). With both strains, all fractions of hot phenol-water extracts and the extracted cell residues from whole cells or cell homogenates were found to be free from characteristic LPS constituents, such as 3-hydroxy fatty acids, 2-keto-3-deoxyoctonate, heptoses, or O-chain sugars. Phenolchloroform-petroleum ether extracts were also free from precipitable LPS. A lipid A fraction could not be obtained, and there was no hint for glucosamine as a possible lipid A backbone amino sugar. Absence of LPS was confirmed by sodium deoxycholate gel electrophoresis.

Lipopolysaccharides of Thiocystis violacea, Thiocapsa pfennigii, and Chromatium tepidum, species of the family Chromatiaceae.

The lipopolysaccharides (LPS) of three species of purple sulfur bacteria (Chromatiaceae), Thiocystis violacea, Thiocapsa pfennigii, and the moderately thermophilic bacterium Chromatium tepidum, were isolated. The LPS of Thiocystis violacea and Chromatium tepidum contained typical O-specific sugars, indicating O-chains. Long O-chains were confirmed for these species by sodium deoxycholate gel electrophoresis of their LPS. Thiocapsa pfennigii, however, had short or no O-chains. The core region of the LPS of all three species comprised D-glycero-D-mannoheptose as the only heptose and 2-keto-3-deoxyoctonate. The lipid A, obtained from the LPS by mild acid hydrolysis, contained glucosamine as the main amino sugar. Amide-bound 3-hydroxymyristic acid was the only hydroxy fatty acid. The main ester-bound fatty acid in all lipid A fractions was 12:0. Mannose and small amounts of 2,3-diamino-2,3-dideoxy-D-glucose were common constituents of the lipid A of the three Chromatiaceae species investigated. All lipid A fractions were essentially free of phosphate.

Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop.

The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH). The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline. When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E. coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA. Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit. This material was judged to be in the P site by the criterion of puromycin reactivity. As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit. The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.