Structural studies on the O-specific side-chains of the cell-wall lipopolysaccharide from Escherichia coli O 75

The structure of the O-specific side-chains of the cell-wall lipopolysaccharide of Escherichia coli O 75 has been investigated, using methylation analysis and Smith degradation as the principal methods. The O-specific side-chain was found to be composed of a tetrasaccharide repeating-unit of the following structure:

     

Structural studies of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide

The structure of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide has been investigated, different 1H- and 13C-n.m.r. techniques being the main methods used. It is concluded that they are composed of pentasaccharide repeating-units having the following structure, in which D-Fuc3NAc is 3-acetamido-3,6-dideoxy-D-galactose. ----4)-beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----2)-alpha-L-Rh ap-(1----3)-beta-L-Rhap-(1----2 increases 1 alpha-D-Fucp3NAc.     

Structural studies of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O 55

The structure of the O-specific side-chains of the lipopolysaccharide from Escherichia coli O 55 has been investigated, methylation analysis, specific degradations, and n.m.r. spectroscopy being the principal methods used. It is concluded that the O-specific side-chains are composed of pentasaccharide repeating-units having the following structure [where Col stands for colitose (3,6-dideoxy-l-xylo-hexose)].

     

Structural analysis of the O-specific polysaccharide isolated from Plesiomonas shigelloides O51 lipopolysaccharide

Plesiomonasshigelloides strain CNCTC 110/92 (O51) was identified as a new example of plesiomonads synthesising lipopolysaccharides (LPSs) that show preference for a non-aqueous surrounding during phenol/water extraction. Chemical analyses combined with ¹H and ¹³C NMR spectroscopy, MALDI-TOF and ESI mass spectrometry showed that the repeating units of the O-specific polysaccharides isolated from phenol and water phase LPSs of P. shigelloides O51 have the same structure: →4)-β-d-GlcpNAc3NRA-(1→4)-α-l-FucpAm3OAc-(1→3)-α-d-QuipNAc-(1→, containing the rare sugar constituent 2,3-diamino-2,3-dideoxyglucuronic acid (GlcpNAc3NRA), and substituents such as d-3-hydroxybutyric acid (R) and acetamidino group (Am). The HR-MAS NMR spectra obtained for the isolated LPSs and directly on bacteria indicated that the O-acetylation pattern was consistent throughout the entire preparation. The ¹H chemical shift values of the structure reporter groups identified in the isolated O-antigens matched those present in bacteria. We have found that the O-antigens recovered from the phenol phase showed a higher degree of polymerisation than those isolated from the water phase.     

Structural studies on the Klebsiella O group 12 lipopolysaccharide

The structure of the O-specific side-chains in the KlebsiellaO group 12 lipopolysaccharide has been investigated. Methylation analysis, and N-deacetylation followed by deamination or acid hydrolysis, with subsequent isolation and characterisation of the resulting oligosaccharides, were the principal methods used. From these studies, it is concluded that the O-specffic side-chains are composed of disaccharide repeating-units having the structure: →3)-β-d-GlcNAcp-(1→3)-α-l-Rhap-(1→.     

Immunochemical studies on shigella dysenteriae type 9 bacterial polysaccharide

On graded hydrolysis and Smith degradation, the O-somatic polysaccharide isolated from Shigella dysenteriae type 9 bacteria, strain NCTC 7919, yielded five oligosaccharides which were characterized. The positions of the O-acetyl and pyruvic acetal groups in the repeating unit were identified. Immunochemical studies indicated that d-galactose is the immunodominant sugar in the polysaccharide, and one of the oligomers, having the structure Gal-(1→3)-GlcNAc-(1→3)-Gal-(1→4)-Man, showed maximum inhibition of the homologous precipitation.     

Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide.

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS, fast atom bombardment (FAB)-MS/MS and 1H- and 13C-NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: The polysaccharide is partially (approximately 10%) substituted with O-acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR-MAS) NMR analysis, which showed both the signals of the O-specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.     

Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7

The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O:7 has been investigated, using n.m.r. spectroscopy, methylation analysis, partial hydrolysis, and Smith degradation as the principal methods. It is concluded that the polysaccharide is constructed of repeating pentasaccharide units having the structure (formula; see text) where D-QuipNAc stands for 4-acetamido-4,6-dideoxy-D-glucopyranose. The 13C-n.m.r. spectrum of the polysaccharide has been interpreted completely.     

Structural studies on the hexose region of the lipopolysaccharide from Escherichia coli C

Escherichia coli C is an R-strain, and hence its lipopolysaccharide consists only of lipid A joined to a basal core. Intact core-polysaccharides have been prepared from this strain, and from mutants of the same strain defective in various stages of core biosynthesis. Using sugar and methylation analyses, and chemical and enzymic degradations, the hexose region of the core of the parent strain has been shown to be a pentasaccharide for which the following structure is proposed:

     

Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg²⁺, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.     

Structural elucidation of the O-specific polysaccharide of the phenol-phase soluble lipopolysaccharide of Vibrio anguillarum

The structure of the O-specific polysaccharide of the phenol-soluble cellular lipopolysaccharide of Vibrio anguillarum has been investigated. The studies involved the use of methylation analysis, partial hydrolysis with 48% hydrogen fluoride, Smith degradation, oxidation with chromium trioxide, and comprehensive proton and carbon-13 nuclear magnetic resonance studies, in which one- and two-dimensional experiments were carried out. As a result of these studies it is proposed that the O-specific polysaccharide of Vibrio anguillarum is composed of a regular heteropolymer, i.e., a main chain of (1----4)-linked 3-acetamido-3,6-dideoxy-beta-L-glucose residues alternately substituted through O-2 with side chain residues of 2-acetamido-2,6-dideoxy-alpha-D-glucose, which seem to be substituted through either O-3 or O-4 with propionyl groups (R), as in the following structure. (Formula: see text)     

Structural studies on a carbohydrate chain isolated from the lipopolysaccharide of Shigella boydii type 8

The lipopolysaccharide isolated from the cells of Shigella boydii type 8 bacteria gave precipitin bands against homologous antisera on Ouchterlony plates, whereas the carbohydrate-containing fractions obtained from it did not. One of the fractions was obtained in major proportion and contained 23.5% of sugars. A structure was assigned to the carbohydrate chain in this material by using the results of methylation, periodate oxidation, and deamination studies.     

Structural and serological studies on Hafnia alvei O-specific polysaccharide of alpha-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223

On the basis of chemical and methylation analyses, one- and two-dimensional (1)H- and (13)C-NMR spectroscopy, including COSY, TOCSY, NOESY and (1)H, (13)C HSQC experiments, a neutral O-specific polysaccharide isolated from Hafnia alvei strain PCM 1223 lipopolysaccharide (LPS) was found to be an alpha-mannan composed of pentasaccharide repeating units having the following structure:-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->. Immunoblotting showed a strong cross-reactivity between anti-H. alvei PCM 1223 serum and LPSs of Escherichia coli O9 and Klebsiella pneumoniae O3. The serological relationship of the LPSs of these bacteria is due to the structural identity of their O-specific polysaccharides, though the LPSs differ in their core regions.     

Structural studies of the O-specific polysaccharide from the lipopolysaccharide of Mesorhizobium huakuii strain S-52, the symbiotic partner of Astragalus sinicus

The O-specific polysaccharide (OPS) obtained by mild-acid degradation of the lipopolysaccharide isolated from Mesorhizobium huakuii strain S-52 was studied by sugar and ethylation analyses along with ¹H and ¹³C NMR spectroscopy. It was concluded that the OPS was composed of trisaccharide repeating units containing two residues of 6-deoxy-l-talose (6dTal) and one l-rhamnose (Rha), whose sequence in the OPS was determined by NOESY and HMBC experiments. The minor 3-O-acetylation (about 10%) of 6-deoxytalose glycosidically substituted at position-2 was judged by relative signal intensities of corresponding O-acetylated and non-acetylated 6dTal residues. Moreover, it was found that the non-reducing end of the OPS repeating unit was occupied by 3-O-methyl-d-fucose, which terminated the O-chain as a cap-residue. These data defined the structure of the OPS as:α-3-OMe-d-Fucp-(1→[2)-α-l-6dTalp-(1→3)-α-l-6dTalp-(1→2)-α-l-Rhap-(1→]_n     

Structural determination of the O-specific chain of the lipopolysaccharide from the mushrooms pathogenic bacterium Pseudomonas tolaasii

The complete structure of the O-specific polysaccharide of the lipopolysaccharide isolated from the cultivated mushrooms pathogen Pseudomonas tolaasii is described. The structural determination, achieved by chemical and spectroscopical analyses, indicates a novel tetrasaccharide repeating unit built up of two units of 2-acetamido-2,6-di-deoxy-glucopyranose (Quinovosamine, QuipNAc) and two units of 2-acetamido-2-deoxy-gulopyranuronamide (GulpNAcAN), one of which is acetylated at C-3 position: