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The structure of the repeating unit of the O-specific polysaccharide from the lipopolysaccharide of the enterobacterium Raoultella terrigena was determined by means of chemical and spectroscopical methods and was found to be a linear tetrasaccharide containing a cyclic acetal of pyruvic acid (Pyr) as depicted below. Download : Download full-size image

The structure of the O-polysaccharide from the lipopolysaccharide of Pseudomonas stutzeri OX1 was determined by chemical procedures and by 1D and 2D NMR spectroscopy. The analysis revealed the presence of a heterogeneous polymer made by 4-acetamido-4,6-dideoxy-D-mannopyranose (D-Rhap4NAc) and 4-formamido-4,6-dideoxy-D-galactopyranose (d-Fucp4NFo). The combination of chemical and NMR analyses indicates that the heterogeneity of the polymer depends on its non-stoichiometric glycosylation by Fuc4NFo, as shown below: [formula: see text]. The structure of the heterogeneous polymer was confirmed by Smith degradation that significantly simplified the structure of the O-polysaccharide, allowing for the isolation and identification of a linear homopolymer of Rhap4NAc.

Pseudomonas stutzeri OXI is a Gram-negative microorganism able to grow in media containing aromatic hydrocarbons. A novel lipo-oligosaccharide from P. stutzeri OX1 was isolated and characterized. For the first time, the presence of two moieties of 4,6-O-(1-carboxy)-ethylidene residues (pyruvic acid) was identified in a core region; these two residues were found to possess different absolute configuration. The structure of the oligosaccharide backbone was determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de-O-acylation) followed by de-N-acylation using hot KOH. The lipo-oligosaccharide was also analyzed after acid treatment, attained by mild hydrolysis with acetic acid, to obtain information on the nature of the phosphate and acyl groups. The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H-, 31P- and 13C-NMR spectroscopy. These experiments led to the identification of the major oligosaccharide structure representative of core region-lipid A. All sugars are D-pyranoses and alpha-linked, if not stated otherwise. Based on the structure found, the hypothesis can be advanced that pyruvate residues are used to block elongation of the oligosaccharide chain. This would lead to a less hydrophilic cellular surface, indicating an adaptive response of P. sutzeri OX1 to a hydrocarbon-containing environment.

Spectroscopic investigation of the O-antigen from Salmonella enterica subsp. enterica revealed fine details on the acetylation pattern, the biological repeating unit and the polymerization degree. Acetylation at O-2 of the abequose residue, defined both O-factors 4 and 5 in the O-antigen chain of the lipopolysaccharide. NMR observation of the terminal non-reducing end of the polymer confirmed previous data regarding the biological repeating unit and showed an average polymerization degree of 5. The information about these structural elements might contribute to the understanding of key features of the biology of this pathogen, as phase variation and/or adaptation to the external environment.

The Gram-negative bacterium Pseudomonas sp. OX1, previously known as Pseudomonas stutzeri OX1, is endowed with a high metabolic versatility. In fact, it is able to utilize a wide range of toxic organic compounds as the only source of carbon and energy for growth. It has been recently observed that, while growing on a glucose-containing liquid medium, Pseudomonas sp. OX1 can reduce azo dyes, ubiquitous pollutants particularly resistant to chemical and physical degradation, with this azoreduction being a process able to generate enough energy to sustain bacterial survival. We have found that, under these conditions, modifications in the primary structure of the O-specific polysaccharide (OPS) within the lipopolysaccharides occur, leading to remarkable changes both in the monosaccharide composition and in the architecture of the repeating unit, with respect to the polysaccharide produced in the absence of azo dyes. In the present paper, we present the complete structure of this O-specific polysaccharide, whose repeating unit is the following: Download full-size image This structure is totally different from the one determined from Pseudomonas sp. OX1 grown on rich medium.

The novel monosaccharide 8-amino-3,8-dideoxyoct-2-ulosonic acid (Kdo8N) was isolated by methanolysis from the lipooligosaccharide of the marine Gram-negative bacterium Shewanella pacifica. After HPLC purification, the absolute configuration was determined by the Mosher ester method and proven to be 4 R,5 R,6 R,7 R. This established the d- manno- configuration of the monosaccharide.

The complete structure of the lipopolysaccharide isolated from the Gram-negative marine bacterium Alteromonas addita, type strain KMM 3600T = R10SW13T, has been elucidated by means of a combined chemical approach and state-of-the-art NMR and MS analyses. Isolation and characterisation of the lipid A moiety and the core oligosaccharide were pursued separately after either acid or alkaline treatment of the lipopolysaccharide. The structure detected was identified as a novel, highly negatively charged, deep-rough lipopolysaccharide in which a trisaccharide subunit is connected to a typical lipid A glucosamine backbone. Within the core oligosaccharide, a phosphodiester bridge connects a glucose unit to a heptose residue.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Klebsiella oxytoca BAS-10 is a Gram-negative micro-organism capable of growing on high concentrations of heavy metals. This bacterium produces large amounts of an iron-binding exopolysaccharide that, in the presence of metallic cations, precipitates as a dense gel. The primary and secondary structure of the repeating unit of such polysaccharide has been characterised by chemical and spectroscopic methods, resulting in the following heptasaccharide: 2)-α-Rha-(13)-β-Gal-(12)-α-Rha-(14)-β-GlcA-[β-GlcA-(14)]-(13)-α-Rha-(13)-α-Rha-(1. The absolute configurations for the Rha units are L, while those for the GlcA and Gal residues are D. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

The O-chain polysaccharide of the lipopolysaccharide from the endophytic bacterium Burkholderia cepacia strain was characterized. The structure was studied by means of chemical analysis and 2D NMR spectroscopy and shown to be the following: Download : Download full-size image

The structure of the major constituent of the biofilm matrix produced by Pseudomonas sp. OX1, when grown on phenol as the sole carbon source is described. This investigation, carried out by chemical analysis, NMR spectroscopy and MALDI-TOF MS spectrometry, showed the presence of an oligosaccharide blend with the typical alginate structure, namely (1→4) substituted β- d -mannuronic (ManA) and α- l -guluronic acid (GulA). GulA residues were non-acetylated whereas ManA was always O-acetylated at C-2 or C-3.

The structures of two teichoic acid fractions (TA1 and TA2) isolated from the thermophilic gram-positive bacterium Geobacillus thermoleovorans strain Fango were investigated by means of chemical and NMR spectroscopic methods. The most abundant species (TA1) exhibited a rather regular structure comprising two different repeating units of 1,3-glycerol phosphate nonstoichiometrically substituted by terminal-alpha-D-Gal p (t-alpha-D-Gal p). The second molecular species (TA2) presented a higher structural variability and t-alpha-D-Glc p and the disaccharides t-alpha-D-Glc pNAc-(1-->2)-alpha-D-Glc p and t-alpha-D-Glc pNAc-(1-->3)-alpha-D-Glc p were also present as minor substituents at O-2 of the glycerol phosphate residues. Minor substitution by alanine could also be detected.

The structure of the core oligosaccharide of the lipopolysaccharide from an organic solvent tolerant Gram-negative bacterium, Acinetobacter radioresistens S13, was investigated by chemical analysis, NMR spectroscopy and MALDI-TOF mass spectrometry. All the experiments were performed on the oligosaccharides obtained either by alkaline degradation or mild acid hydrolysis. The data showed the presence of two novel oligosaccharide molecules containing a trisaccharide of 3-deoxy-D-manno-octulopyranosonic acid in the inner core region and a glucose rich outer core whose structure is the following: [structure: see text] R=H in the main oligosaccharide and beta-Glc in the minor product. The bacterium was grown on aromatic (phenol and benzoic acid) and nonaromatic carbon sources and the core oligosaccharide resulted to occur always with this novel structure.

Shewanella pacifica is a Gram-negative microrganism that is able to grow in sea water. A novel lipooligosaccharide (LOS) has been isolated from the outer membrane of this bacterium and its primary structure fully characterised. For the first time, the presence of a 2,3-dihydroxypropanoic acid residue (glyceric acid) has been identified in the core region. The complete structure of the LOS was determined by compositional and methylation analyses, by MALDI mass spectrometry, and by 1H, 13C and 31P NMR spectroscopy on the oligosaccharides formed by selective degradation of the LOS. Strong alkaline treatment, aimed at recovering and identifying the complete carbohydrate backbone, was carried out by hydrazinolysis followed by de-N-acylation with hot KOH, whereas mild hydrazinolysis (de-O-acylation) allowed us to gain information about the nature of the phosphate and other non-carbohydrate substituents on the core oligosaccharide. Mild acid hydrolysis was employed to obtain a lipid A moiety with which further degradation and mass spectrometry experiments were carried out in order to determine its primary structure. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

A novel core-lipid A backbone oligosaccharide was isolated and identified from the lipopolysaccharide fraction of the mushrooms pathogen bacterium Pseudomonas tolaasii. The oligosaccharide was obtained by alkaline treatment of the lipopolysaccharide fraction. Since the repeating unit of the O-antigen contained one residue of -->4)-alpha-l-GulpNAcAN, the hydrolysis was accompanied by beta-elimination on this residue and following depolymerization, producing a mixture of oligosaccharides. The complete structural elucidation showed the presence of a single core glycoform and was achieved by chemical analysis and by (1)H, (31)P, and (13)C NMR spectroscopy applying various 1D and 2D experiments. [structure: see text]. All sugars are alpha-d-pyranoses, if not stated otherwise. Hep is l-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid, P is phosphate. QuiN and DeltaGulNA are present in nonstoichiometric amount.

A minor oligosaccharide fraction was isolated after complete de-acylation of the lipooligosaccharide extracted from Pseudomonas stutzeri OX1. The full structure of this oligosaccharide was obtained by chemical degradation, NMR spectroscopy and MALDI-TOF MS spectrometry. These experiments showed the presence of two novel oligosaccharides ( OS1 and OS2 ): where R = ( S )-Pyr(→4,6) in OS1 and α-Rha-(1→3) in OS2 . All sugars are d -pyranoses, except Rha, which is l -pyranose. Hep is l - glycero - d - manno -heptose, Kdo is 3-deoxy- d - manno -oct-2-ulosonic acid, Pyr is pyruvic acid, P is phosphate.