Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1546

An acidic O-polysaccharide isolated by mild acid hydrolysis from the lipopolysaccharide of Hafnia alvei PCM 1546 is composed of D-Gal, D-Glc, D-GlcA, D-GalNAc and O-acetyl groups in the ratios 1:1:1:2:1.6. On the basis of sugar and methylation analyses along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the pentasaccharide repeating unit of the polysaccharide was established: [see equation in text].     

Structure of the O-polysaccharide of Hafnia alvei strain PCM 1189 that has hexa- to octasaccharide repeating units owing to incomplete glucosylation

The O-polysaccharide of Hafnia alvei PCM 1189 consists of D-glucose, D-galactose, D-GalNAc and D-GlcA and lacks the strict regularity. The intact and carboxyl-reduced polysaccharides as well as oligosaccharides obtained by partial acid hydrolysis were studied by chemical and enzymatic analyses, methylation and NMR spectroscopy. The following structure was established for the O-polysaccharide, which is built up of branched hexa- to octasaccharide repeating units differing in the number of lateral glucose residues: [structure: see text] where the glucose residues shown in italics are nonstoichiometric substituents. The repeating units include also a minor O-acetyl group, whose position was not determined.     

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established: -->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Galp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-GlcpNAc-(1-->.     

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.     

Structure of the O-polysaccharide of the lipopolysaccharide of Pragia fontium 97U116

The O-polysaccharide of Pragia fontium 97U116 was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar analysis along with 1D and 2D ¹H and ¹³C NMR spectroscopy. The following structure of the pentasaccharide-repeating unit was established: →2)-α-d-Galf-(1→3)-α-l-Rhap2Ac^I-(1→4)-α-d-GlcpNAc^I-(1→2)-α-l-Rhap^II-(1→3)-β-d-GlcpNAc^II-(1→     

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].     

The O-acetylation patterns in the O-antigens of Hafnia alvei strains PCM 1200 and 1203, serologically closely related to PCM 1205

Serological tests revealed immunochemical similarities between the lipopolysaccharides of Hafnia alvei strains PCM 1200, 1203 and 1205. Immunoblotting and ELISA showed cross-reactions between the strains. NMR spectroscopy showed that the O-deacetylated O-specific polysaccharides isolated from lipopolysaccharides of H. alvei strains PCM 1200 and 1203 possessed the same composition and sequence as the O-deacetylated O-specific polysaccharide of H. alvei strain PCM 1205, that is a glycerol teichoic-acid-like polymer with a repeating unit of the following structure: [carbohydrate structure: see text] NMR spectroscopic studies of the polysaccharides concluded that O-3 of the side chain beta-D-GlcpNAc is partially O-acetylated (50-80%) in both investigated strains. In strain PCM 1203 an additional O-acetyl group (50-80%) is linked to O-6 of the chain -->3)-alpha-D-GlcpNAc-(1--> residue. The structural features of the isolated O-specific polysaccharides were also the same as those of the O-specific polysaccharides on the bacterial cells directly observed by the HR-MAS NMR technique.     

Structure of the O-polysaccharide of the lipopolysaccharide of Azospirillum irakense KBC1

The O-polysaccharide was isolated from the lipopolysaccharide of the plant-growth-promoting bacterium Azospirillum irakense KBC1 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 1H, 13C HSQC and NOESY experiments for linkage and sequence analysis. The following structure of the branched hexasaccharide repeating unit of the O-polysaccharide with an unusually long side chain was established: [carbohydrate structure: see text].     

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-specific polysaccharide of Hafnia alvei PCM 1196

An acidic O-specific polysaccharide was isolated from Hafnia alvei PCM 1196 lipopolysaccharide and studied by sugar and methylation analyses along with one- and two-dimensional ¹H and ¹³C NMR spectroscopy, including NOESY and HMBC experiments. The following structure of the pentasaccharide repeating unit was established:

     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O60

An O-polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O60 and studied by sugar and methylation analyses as well as ¹H and ¹³C NMR spectroscopy, including 2D ROESY and ¹H,¹³C HMBC experiments in D₂O and a ROESY experiment in a 9:1 H₂O–D₂O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear pentasaccharide repeating units containing an amide of d-glucuronic acid with l-serine and has the following structure:The O-antigen studied is structurally and serologically closely related to the O-antigen of Proteus vulgaris O44.     

Structure of the O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii O11, strain PCM 1540

The O-specific polysaccharide of the lipopolysaccharide of Citrobacter gillenii PCM 1540 (serogroup O11) consists of D-Glc, D-Man, D-GalNAc, D-GlcNAc, 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) and O-acetyl groups in the ratios 2:1:1:1:1:1. On the basis of sugar and methylation analyses and Smith-degradation along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched hexasaccharide repeating unit was established: [structure: see text]. Citrobacter werkmanii PCM 1541 belonging to the same serogroup O11 was found to have an R-form lipopolysaccharide devoid of the O-specific polysaccharide.     

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.     

Structures of the biological repeating units in the O-chain polysaccharides of Hafnia alvei strains having a typical lipopolysaccharide outer core region

Earlier, the structures of the O-chain polysaccharides of the lipopolysaccharides (LPS) of a number of Hafnia alvei strains have been established. However, it remained unknown, which is the first and the last monosaccharide of the O-chain. This is defined by the structure of the so-called biological repeating unit (O-unit), which is pre-assembled and then polymerised in the course of biosynthesis of bacterial polysaccharides by the Wzy-dependent pathway. Now we report on the structures of the O-units in 10 H. alvei strains. The LPS were cleaved by mild acid hydrolysis and oligosaccharide fractions IIIa and IIIb were isolated by gel chromatography subsequently on Sephadex G-50 and BioGel P-2 and studied by methylation analysis and NMR spectroscopy. Fraction IIIb was found to represent the core oligosaccharide containing a terminal upstream alpha-d-Glc-(1-->3)-alpha-d-Glc or alpha-d-Gal-(1-->3)-alpha-d-Glc disaccharide in the outer region that is typical of H. alvei. Fraction IIIa consists of the LPS core with one O-unit linked by a 3-substituted beta-d-GalNAc residue (in strains PCM 1189 and PCM 1546) or a 3-substituted beta-d-GlcNAc residue (in the other strains studied). In most strains examined the beta-configuration of the d-GlcNAc linkage in the first O-unit attached to the core is the same and in some strains is opposite to that found in the interior O-units of the O-chain polysaccharide. Various monosaccharides, including d-Glc, d-Gal, d-GlcA and acyl derivatives of 3-amino-3,6-dideoxy-d-glucose or 4-amino-4,6-dideoxy-d-glucose, occupy the non-reducing end of the O-unit.     

Structure of the O-polysaccharide of the lipopolysaccharide of Rahnella aquatilis 1-95

The O-polysaccharide was isolated by mild acid hydrolysis of the lipopolysaccharide of Rahnella aquatilis 1-95 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including NOESY and 1H,13C HSQC experiments for linkage and sequence analysis. The following structure of the branched trisaccharide repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text].     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O28

An O-polysaccharide and oligosaccharides were isolated by GPC following mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O28. The O-polysaccharide was studied by sugar and methylation analyses, ¹H and ¹³C NMR spectroscopy, including 2D ROESY and H-detected ¹H,¹³C HSQC and HMBC experiments, and the following structure of the branched pentasaccharide repeating unit was established:This structure was confirmed by ESI MS of the isolated tridecasaccharide consisting of the lipopolysaccharide core and one O-polysaccharide repeat. The ESI mass spectrum also enabled inferring the composition of the core oligosaccharide.     

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.     

Structure of an abequose-containing O-polysaccharide from Citrobacter freundii O22 strain PCM 1555

The lipopolysaccharide of Citrobacter freundii O22 (strain PCM 1555) was degraded under mild acidic conditions and the O-polysaccharide released was isolated by gel chromatography. Sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ¹H,¹H ROESY and ¹H,¹³C HMBC experiments, showed that the repeating unit of the O-polysaccharide has the following structure:

Full-size image (4K)

where Abe is abequose (3,6-dideoxy-d-xylo-hexose). SDS–PAGE and immunoblotting revealed that the O-antigen of C. freundii O22 is serologically indistinguishable from those of Salmonella group B serovars (Typhimurium, Brandenburg, Sandiego, Paratyphi B) but not related to other abequose-containing O-antigens tested (Citrobacter werkmanii O38 and Salmonella Kentucky) or colitose (l enantiomer of abequose)-containing O-antigen of Escherichia coli O111.