Structure of the O-specific polysaccharide isolated from the lipopolysaccharide of Citrobacter gillenii serotype O12a, 12b strain PCM 1544.

A neutral O-specific polysaccharide was isolated from the lipopolysaccharide of Citrobacter gillenii strain PCM 1544, representing serotype O12a,12b. The polysaccharide was studied by sugar and methylation analyses and Smith degradation along with 1H and 13C NMR spectroscopy, including a ROESY experiment. The following structure of the tetrasaccharide repeating unit was established, in which substitution with terminal GlcNAc is approximately 60%. [structure: see text]

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-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-->.

Structure of the lipopolysaccharide core region of Hafnia alvei strains 1185 and 1204.

Sugar and methylation analyses using gas chromatography/mass spectrometry and NMR spectroscopy proved that the core oligosaccharides of Hafnia alvei strains 1185 and 1204 have the following formula: carbohydrate sequence [see text] where Kdo = 3-deoxy-oct-2-ulosonic acid and P-PEtN = diphosphorylethanolamine. The structure shown above is a slight modification of the typical core region of H. alvei lipopolysaccharides. The difference refers to one sugar only: terminal galactose is present in the core of strains of 1185 and 1204, while terminal glucose in the typical core.

Non-typical lipopolysaccharide core regions of some Hafnia alvei strains: structural and serological studies.

The lipopolysaccharides of Hafnia alvei strains 23, 1222 and 39 were found to have non-typical core region. On the basis of sugar and methylation analyses, 1H-nuclear magnetic resonance spectra and matrix-assisted laser-desorption ionization-time of flight mass spectrometry, it was concluded that the core oligosaccharide of strains 23 and 1222 has the same structure as Escherichia coli R4 core region, and the core oligosaccharide of strain 39 has the structure of Salmonella Ra core. Using the serological methods (passive hemagglutination, enzyme-linked immunosorbent assay and immunoblotting) and the anti-conjugate sera directed against E. coli R4 and Salmonella Ra core oligosaccharides we have confirmed the structural results presented above.

Reinvestigation of the O-specific polysaccharides of Hafnia alvei lipopolysaccharides isolated from strains ATCC 13337 and 1187.

The structure of the O-specific polysaccharides of the lipopolysaccharides produced by Hafnia alvei strains ATCC 13337 and 1187 was reinvestigated. The position of phosphate group in the repeating units of the polysaccharides was established with the aid of 1H detected, 31P edited NMR spectra. According to the results obtained, the polysaccharides are teichoic acid-like polymers with the repeating units of the following structure: [formula: see text] where Acyl = D-3-hydroxylbutyryl, and 3-O-acetylation was approximately 30%.

Immunochemical studies of the lipopolysaccharide O-specific polysaccharide of Hafnia alvei PCM 1199 related to H. alvei PCM 1205.

On the basis of chemical analyses and NMR spectroscopic studies, it was found that the O-specific polysaccharide (O-PS) isolated from the Hafnia alvei PCM 1199 lipopolysaccharide (LPS) is a glycerol teichoic-acid-like polymer having a repeating unit of the following structure: [structure in text] where Qui4NAc is 4-acetamido-4,6-dideoxyglucose and O-acetylation at both positions is non-stoichiometric. The glycosidic linkage of the lateral beta-D-GlcpNAc residue is acid-labile and cleaved from the O-PS during mild acid hydrolysis or dephosphorylation with 48% hydrofluoric acid. Comparative analysis revealed that the structure of the H. alvei PCM 1199 O-PS is similar to that of H. alvei PCM 1205, which differs in the presence of an additional lateral alpha-D-Glcp residue and the position of one of the O-acetyl groups only. Accordingly, serological tests revealed a high degree of serological similarity between LPSs and O-PSs of the two H. alvei strains.

NMR spectroscopic elucidation of the structure of a glycerol teichoic acid-like O-specific polysaccharide of the bacterium Hafnia alvei strain PCM 1199.

The structure of the acidic O-specific polysaccharide of a Gram-negative bacterium, H. alvei strain PCM 1199, was studied by NMR spectroscopy including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), 1H, 13C heteronuclear single-quantum coherence (HSQC), 1H, 13C heteronuclear multiple-bond correlation (HMBC), and one-dimensional 1H, 31P heteronuclear multiple-quantum coherence (HMQC) experiments. It was found that the polysaccharide contains D-galactose, 2-acetamido-2-deoxy-D-glucose, 4-acetamido-4,6-dideoxy-D-glucose, glycerol, and phosphate in the ratios 1:2:1:1:1, as well as O-acetyl groups in non-stoichiometric amounts. The polysaccharide is similar in structure to teichoic acids of Gram-positive bacteria and has the following structure of the repeating unit: 3)-beta-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->3)-beta-D-Quip4NAc-(1- ->1)-Gro- 3-P-(O--> [formula: see text] beta-D-GlcpNAc [formula: see text] The O-specific polysaccharide of H. alvei PCM 1199 is structurally related to another teichoic acid-like O-specific polysaccharide of H. alvei PCM 1205 studied by us earlier.

Structure of the O-specific polysaccharide of Hafnia alvei PCM 1222 containing 2-aminoethyl phosphate.

The O-specific polysaccharide of H. alvei strain PCM 1222 has a branched hexasaccharide repeating unit containing D-galactose, L-rhamnose, D-ribose, D-galacturonic acid, and 2-acetamido-2-deoxy-D-glucose in the ratios 1:2:1:1:1, as well as 2-aminoethyl phosphate (EtNP) and O-acetyl groups in nonstoichiometric amounts. The polysaccharide was modified by carboxyl reduction, O-deacetylation, and dephosphorylation with 48% hydrofluoric acid, the last reaction being accompanied by removal of the lateral residue of beta-galactofuranose. The modified polysaccharides were studied by methylation analysis and 1H and 13C NMR spectroscopy, including 2D correlation spectroscopy (COSY), H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), 1D NOE, 2D rotating-frame NOE spectroscopy (ROESY), and 2D combined total correlation spectroscopy (TOCSY) and ROESY (TORO). The following structure of the O-deacetylated polysaccharide was established: [formula: see text] In different batches of the polysaccharide, the content of EtNP varied from 0.35 to 0.55 and that of the O-acetyl groups from 0.05 to 0.4 per repeating unit. It was tentatively suggested that the O-acetyl group is located at position 4 of a rhamnosyl residue.

Structural study and serological characterisation of the O-specific polysaccharide of Hafnia alvei PCM 1185, another Hafnia O-antigen that contains 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-glucose.

The O-specific polysaccharide of H. alvei strain PCM 1185 contains D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-glucose (Qui3NAcyl) in the ratios 2:1:1:1 as well as O-acetyl groups. On the basis of sugar and methylation analyses of the polysaccharide before and after chemical modifications (O-deacetylation, carboxyl reduction, Smith degradation), as well as 1H and 13C NMR spectroscopy, including 1D sequential, selective spin-decoupling, 2D homonuclear and 13C,1H heteronuclear correlation spectroscopy (COSY), and 2D rotating-frame NOE spectroscopy, it was found that the polysaccharide has a pentasaccharide repeating unit with the following structure: [formula: see text] with O-acetyl groups present in nonstoichiometric amounts, mainly at position 2 of GlcA and position 6 of GlcNAc or lateral Glc. Serological study showed that H. alvei strain PCM 1185 can be placed in a new serotype D and that an O-acetyl group can be a part of its epitope.

Structure of the O-specific polysaccharide, containing a glycerol phosphate substituent, of Hafnia alvei strain 1220 lipopolysaccharide.

The O-specific polysaccharide of the lipopolysaccharide produced by Hafnia alvei strain 1220 contained D-glucose, D-galactose, N-acetyl-D-glucosamine, N-acetyl-L-fucosamine (2-acetamido-2,6-dideoxy-L-galactose), glycerol, and phosphate. It was proved by composition and methylation analyses, Smith degradation, dephosphorylation, and one- and two-dimensional 1H NMR spectroscopy to be a teichoic acid-like polymer with a branched hexasaccharide repeating unit of the following structure. [sequence: see text]

The occurrence of glycine in bacterial lipopolysaccharides.

The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [14C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100 degrees C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.

[Structural studies of carbohydrate lipopolysaccharide sections in Hafnia alvei]. / Badania strukturalne czesci cukrowej lipopolisacharydów Hafnia alvei.

The structures of 14 O-specific polysaccharides isolated from Hafnia alvei lipopolysaccharides have been described. The structures of the common core existing in most Hafnia alvei strains examined so far and the core-like trisaccharide found in some Hafnia alvei strains have been also presented.

[Structural and serologic characteristics of O-specific polysaccharides of Hafnia alvei PCM 1185 and 1199]. / Charakterystyka strukturalna i serologiczna polisacharydów O-swoistych Hafnia alvei PCM 1185 I 1199.

This work describes results of studies of O-specific oligosaccharide repeating units from lipopolysaccharides of two related Hafnia alvei serotypes. The linkage between O-antigen and the core region in PCM 1199 LPS has been also established. The O-acetyl residues present in the polysaccharides are involved in formation of epitopes.

Structure of the Hafnia alvei strain PCM 1188 O-specific polysaccharide.

The lipopolysaccharide was extracted from cells of Hafnia alvei PCM 1188 strain and, after mild acid hydrolysis, the O-specific polysaccharide isolated and characterized. On the basis of sugar and methylation analysis, FAB mass spectrometry and NMR spectroscopy of the polysaccharide and oligosaccharides obtained after Smith degradation, or solvolysis with anhydrous hydrogen fluoride, the repeating unit of the O-specific polysaccharide was shown to be the pentasaccharide: [formula: see text]

Structure of the O-specific polysaccharide of Hafnia alvei 1204 containing 3,6-dideoxy-3-formamido-D-glucose.

The O-specific polysaccharide of Hafnia alvei strain 1204 has a hexasaccharide repeating unit containing D-mannose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, and 3,6-dideoxy-3-formamido-D-glucose (Qui3NFo) in the ratios 2:1:1:1:1 as well as O-acetyl groups. On the basis of methylation analysis of the intact, carboxyl-reduced, and Smith-degraded polysaccharide as well as 1D and 2D NMR spectroscopy, including 1D total correlation spectroscopy, 1D NOE spectroscopy, 2D homonuclear shift-correlated spectroscopy (COSY), and 13C,1H heteronuclear COSY, the following structure of the O-deacetylated polysaccharide was established: -->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-beta-D-GlcpN Ac-(1--> -->2)-beta-D-Quip3NFo-(1-->3)-alpha-D-GalpNAc-(1-->4)-alpha-D-G lcpA-(1--> Location of the N-formyl group, occurring as two stereoisomers in the ratio approximately 3:1, was determined by an NOE on H-3 Qui3N arising on pre-irradiation of HCO of the minor (E) isomer. The O-acetyl groups are attached in nonstoichiometric amounts at position 3 of GlcA and position 6 of a mannose residue or GlcNAc.

Lipopolysaccharide core region of Hafnia alvei: structure elucidation using chemical methods, gas chromatography-mass spectrometry, and NMR spectroscopy.

Sugar and methylation analysis with the use of gas chromatography-mass spectrometry and 1H NMR spectroscopy proved that the core oligosaccharides isolated from lipopolysaccharides of eight Hafnia alvei strains have the identical hexasaccharide skeleton. However, 1H, 31P heterocorrelated spectra showed that the phosphorylation pattern is not the same. The branched heptose for the ATCC 13337, 1187, 2, 1191, 1196, 1220, and 481L strains is phosphorylated as in the following formula, where P = -O-P(O)(O-)2 and P-PEtN = [-O-P(O)(O-)]2-O(CH2)2NH3+ [formula: see text] A different phosphorylation pattern was found for the 1211 strain, where the branched heptose residue is 6-substituted by a monophosphorylethanolamine group, ...-->3(-->7)(PEtN-->6)-alpha-LD-Hepp-(1-->3)..., where PEtN = -O-P(O)(O-)-O(CH2)2NH3+.

The structure of the O-specific polysaccharide of Hafnia alvei strain 1216.

The O-specific polysaccharide of Hafnia alvei strain 1216 is composed of D-galactose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-glucose, and O-acetyl groups in the ratios 1:1:2:1:1. On the basis of sugar and methylation analyses of the intact and chemically degraded (O-deacetylated, carboxyl-reduced, Smith-degraded) polysaccharide and 1H and 13C NMR spectroscopy, including 2D shift-correlated (COSY, relayed COSY, 13C, 1H-COSY) and 1D NOE spectroscopy, it was concluded that the O-antigen is built up of linear pentasaccharide units having the following structure: [formula: see text]

Serological and structural features of Hafnia alvei lipopolysaccharides containing D-3-hydroxybutyric acid.

The serological heterogeneity of Hafnia alvei lipopolysaccharides from strains ATCC 13337, 1187, 1221, 114/60, 1211 and 1216, that contain D-3-hydroxybutyric acid, was analyzed by rocket immunoelectrophoresis, immunoblotting and passive hemagglutination. The significance of D-3-hydroxybutyric acid component for their cross-reactivity has been discussed. The results obtained allowed us to place four H. alvei strains (ATCC 13337, 1187, 1221 and 114/60) in one serotype (A) and to consider two other strains (1211 and 1216) as separate serotypes (B and C, respectively).

3-Deoxy-octulosonic acid-containing trisaccharide fragment of an unusual core type of some Hafnia alvei lipopolysaccharides.

The 3-deoxy-octulosonic acid-containing trisaccharide was isolated from the carbohydrate material obtained after the acid hydrolysis of lipopolysaccharides of Hafnia alvei strains 2 and 1211 using gel filtration. For purification the trisaccharide fraction was rechromatographed on a Bio-Gel P2 column. On the basis of sugar and methylation analyses and 1H NMR spectroscopy the trisaccharide was identified as 3-deoxy-7-O-(6-O-acetyl-alpha-D-galactopyranosyl)-4-O-(alpha-L-glycero-D - mannoheptopyranosyl)-D-manno-2-octulopyranosic acid. The occurrence of such bifurcate Kdo-containing structure has not been previously reported in Enterobacteriaceae.