The wbnH gene of Escherichia coli O86:H2 encodes an alpha-1,3-N-acetylgalactosaminyl transferase involved in the O-repeating unit biosynthesis

O-repeating unit biosynthesis is the first committed step in lipopolysaccharide (LPS) biosynthesis in a variety of gram-negative bacteria. The wbnH gene was previously proposed to encode a glycosyltransferase involved in O-repeating unit synthesis in Escherichia coli O86:H2 strain. In this work, we provide biochemical evidence to show that wbnH encodes a N-acetylgalactosaminyl transferase (GalNAcT) that catalyzes the transfer of GalNAc from UDP-GalNAc to the GalNAc-pyrophosphate-lipid acceptor. WbnH activity was characterized using a synthetic acceptor substrate GalNAc alpha-PP-O(CH2)11-OPh. The resulting disaccharide product GalNAc-alpha-1,3-GalNAc alpha-PP-O(CH2)11-OPh was analyzed by LC-MS and NMR spectroscopy. Substrate specificity study indicates that pyrophosphate and hydrophobic lipid moiety are structural requirements for WbnH activity. Divalent metal cations are not required for enzyme catalysis, suggesting WbnH belongs to glycosyltransferase GT-B superfamily. Our results complete the characterization of O86 O-unit assembly pathway, and provide the access of chemically defined O-unit substrates for the further investigation of O-antigen biosynthetic mechanism.     

Structure of a teichoic acid-like O-polysaccharide of Escherichia coli O29

A teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O29. The O-polysaccharide and an oligosaccharide obtained by dephosphorylation of the O-polysaccharide were studied by sugar analysis along with 1H and 13C NMR spectroscopy. The following structure of the branched oligosaccharide repeating unit, containing five monosaccharide residues and glycerol 1-phosphate (D-Gro-1-P), was established: [carbohydrate structure: see text].     

Structural determination of the O-antigenic polysaccharide from Escherichia coli O166

The O-antigen of the lipopolysaccharide from Escherichia coli O166 has been determined by component analysis together with 1D and 2D NMR spectroscopy techniques. The polysaccharide has pentasaccharide repeating units consisting of D-glucose (1), D-galactose (2) and N-acetyl-D-galactosamine (2) with the following structure: [STRUCTURE: SEE TEXT]. In the 1H NMR, spectrum resonances of low intensity were observed. Further analysis of these showed that they originate from the terminal part of the polysaccharide, thereby revealing that the repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.     

Novel glycosyltransferase genes involved in the acetan biosynthesis of Acetobacter xylinum

Novel aceQ and aceR genes involved in the acetan biosynthesis of Acetobacter xylinum were newly isolated. The homology search with DNA Data Bank of Japan indicated that aceQ and aceR were glycosyltransferases. Their gene-disrupted mutants were obtained by homologous recombination using the tetracycline resistance gene and the electroporation method. By NMR and ESI-MS analyses, aceQ-disrupted mutant DQ was found to secrete a water-soluble polysaccharide harboring the -Man-GlcUA side chain and the aceR-disrupted mutant DR was found to secrete an acetan analog, lacking the terminal Rha residue. These results suggested that aceQ and aceR encode a glucosyltransferase and a rhamnosyltransferase, respectively. It was indicated that acetan analogs harboring various side chains can be generated easily by genetic engineering.     

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).     

Structure of a highly phosphorylated O-polysaccharide of Proteus mirabilis O41

A highly phosphorylated O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O41 followed by GPC. The initial and dephosphorylated polysaccharides and phosphorylated products from two sequential Smith degradations were studied by (1)H, (13)C and (31)P NMR spectroscopy and ESI-MS. The O-polysaccharide was found to have a tetrasaccharide repeating unit containing one ribitol phosphate (presumably d-Rib-ol-5-P) and two ethanolamine phosphate (Etn-P) groups, one of which is present in the stoichiometric amount and the other in a nonstoichiometric amount. The following structure of the O-polysaccharide was established:     

Close relation of the O-polysaccharide structure of Escherichia coli O168 and revised structure of the O-polysaccharide of Shigella dysenteriae type 4

The O-polysaccharide was isolated from the lipopolysaccharide of Escherichia coli O168 and studied by chemical analyses and Smith degradation along with (1)H and (13)C NMR spectroscopies. The following structure of the branched pentasaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text] where 6-O-acetylation of GlcNAc is partial. Reinvestigation of the O-polysaccharide of Shigella dysenteriae type 4 established earlier showed it to have the same structure except for that the lateral Fuc residue is nonstoichiometrically O-acetylated at each position.     

New structure for the O-polysaccharide of Providencia alcalifaciens O27 and revised structure for the O-polysaccharide of Providencia stuartii O43

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide from Providencia alcalifaciens O27 and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, H-detected (1)H,(13)C HSQC, and HMBC experiments. It was found that the polysaccharide is built up of linear partially O-acetylated tetrasaccharide repeating units and has the following structure: [structure: see text] where Qui4NFo stands for 4-formamido-4,6-dideoxyglucose (4-formamido-4-deoxyquinovose). The O-polysaccharide structure of Providencia stuartii O43 established earlier was revised with respect to the configuration of the constituent 4-amino-4,6-dideoxyhexose (from Rha4N to Qui4N).     

Structure of the O-polysaccharide of Providencia alcalifaciens O19

Studies of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) of Providencia alcalifaciens O19 by sugar and methylation analyses along with NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments, showed that the pentasaccharide repeating unit of the polysaccharide has the following structure: [structure: see text] where Fuc3NAc is 3-acetamido-3,6-dideoxygalactose. The unique structure of the O-antigen and serological data are in consistence with classification of this bacterium in a separate Providencia serogroup.     

Structure of the O-polysaccharide of Providencia stuartii O49

The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->     

Structure of the N-acetyl-L-rhamnosamine-containing O-polysaccharide of Proteus vulgaris TG 155 from a new Proteus serogroup, O55

The O-polysaccharide of the lipopolysaccharide (LPS) of Proteus vulgaris TG 155 was found to contain 2-acetamido-2,6-dideoxy-L-mannose (N-acetyl-L-rhamnosamine, L-RhaNAc), a monosaccharide that occurs rarely in Nature. The following structure of the O-polysaccharide was established by NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H,13C HSQC experiments, along with chemical methods: [carbohydrate structure in text] Rabbit polyclonal O-antiserum against P. vulgaris TG 155 reacted with both core and O-polysaccharide moieties of the homologous LPS but showed no cross-reactivity with other LPS from the complete set of serologically different Proteus strains. Based on the unique O-polysaccharide structure and the serological data, we propose classifying P. vulgaris TG 155 into a new, separate Proteus O-serogroup, O55.     

Structure of the O-polysaccharide of Proteus mirabilis OC (CCUG 10702) from a new proposed Proteus serogroup O75

A neutral O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis OC (CCUG 10702) and studied by sugar and methylation analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: [structure: see text]. Based on the unique structure of the O-polysaccharide and serological data, we propose classifying P. mirabilis OC (CCUG 10702) into a new separate Proteus serogroup O75. A weak cross-reaction of O-antiserum against P. mirabilis OC with the lipopolysaccharide of P. mirabilis O49 was accounted for by a similarity in the O-polysaccharide structures.     

Structure of a colitose-containing O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O6

The O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O6 and studied by sugar and methylation analysis, selective hydrolytic removal of 3,6-dideoxy-L-xylo-hexose (colitose, Col), (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY and H-detected (1)H,(13)C HSQC and HMBC experiments. The polysaccharide was found to have a branched pentasaccharide repeating unit with the following structure: [see text] Remarkably, the trisaccharide side chain of the O6-polysaccharide represents a colitose ('3-deoxy-L-fucose') analogue of the H type 1 (precursor) antigenic determinant.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10701 (OB) classified into a new Proteus serogroup, O74

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus mirabilis CCUG 10701 (OB) and studied by chemical analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: --> 3)-beta-D-GlcpNAc6Ac-(1 --> 2)-beta-D-GalpA4Ac-(1--> 3)-alpha-D-GalpNAc-(1 --> 4)-alpha-D-GalpA-(1 -->, where the degree of O-acetylation at position 6 of GlcNAc is approximately 50% and at position 4 of beta-GalA approximately 60%. Based on the unique structure of the O-polysaccharide and serological data, it is proposed to classify P. mirabilis CCUG 10701 (OB) into a new Proteus serogroup, O74.     

Structure of the O-polysaccharide of Escherichia coli O112ab containing L-iduronic acid

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Escherichia coli O112ab and studied by sugar analysis along with (1)H and (13)C NMR spectroscopy. The O-polysaccharide was found to contain a rarely occurring sugar component, L-iduronic acid (L-IdoA), and the following structure of the branched pentasaccharide repeating unit was established: [structure: see text].     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O29

The O-polysaccharide was obtained by a mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O29. Structural studies were performed using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional 1H, 1H COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. On the basis of the data obtained, the following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: [structure: see text].     

Structure of a glucosyl phosphate-containing O-polysaccharide of Proteus vulgaris O42

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O42 and studied by sugar and methylation analyses along with 1H, 13C and 31P NMR spectroscopy. The following structure of the polysaccharide having a linear pentasaccharide phosphate repeating unit was established: -->3)-alpha-L-FucpNAc4Ac-(1-->4)-alpha-D-Glcp-1-P-(O-->4)-alpha-D-GlcpNAc-(1-->3)-alpha-L-FucpNAc4Ac-(1-->3))-alpha-D-GlcpNAc6Ac-(1--> where the degree of O-acetylation is approximately 80% on GlcNAc and approximately 40% on each of the FucNAc residues. A weak serological cross-reaction of anti-P. vulgaris O42 serum with the lipopolysaccharide of P. vulgaris O39 was observed and accounted for by the sharing of a disaccharide fragment of the O-polysaccharides.     

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O47

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O47:H4, strain 3646/51. Studies by sugar and methylation analyses along with Smith degradation and 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, ROESY and H-detected 1H,13C HSQC and HMBC experiments, showed that the polysaccharide has a branched hexasaccharide repeating unit with the following structure: [carbohydrate structure: see text]     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Structure of the O-polysaccharide from the lipopolysaccharide from Vibrio cholerae O6

The O-polysaccharide from Vibrio cholerae O6 was isolated from the LPS by mild-acid hydrolysis and has been investigated by sugar and methylation analysis and NMR spectroscopy. The polysaccharide was also depolymerized with aqueous hydrofluoric acid to give the repeating unit and multiples thereof. The O-polysaccharide had the following tetrasaccharide repeating unit. Two O-acetyl groups are present, one of them making the GlcNAc residue fully substituted and the steric crowding considerable at the branching residue.