Escherichia albertii EA046 (O9) harbors two polysaccharide gene clusters for synthesis of the O-antigen by the Wzx/Wzy-dependent pathway and a mannan shared by Escherichia coli O8 by the Wzm/Wzt-dependent pathway.

O antigen is a polysaccharide chain of a lipopolysaccharide on the outer membrane of Gram-negative bacteria. O-antigen-based serotyping and molecular typing are widely used for epidemiological and surveillance purposes. Two polysaccharides were isolated by Sephadex G-50 gel-permeation chromatography following mild acid degradation of the lipopolysaccharide of Escherichia albertii EA046 assigned to serotype O9. The polysaccharide eluted first was considered as the O-antigen. It was composed of tetrasaccharide repeating units containing two residues of d-Man and one residue each of d-Gal and d-GlcNAc as well as glycerol phosphate. It had the following unique structure which was established by NMR spectroscopy applied to the initial and dephosphorylated polysaccharides: The polysaccharide eluted from the gel second was identified as a mannan with a → 3)-ß-d-Manp-(1 → 2)-α-d-Manp-(1 → 2)-α-d-Manp-(1 → trisaccharide repeating unit. In E. albertii EA046, two polysaccharide gene clusters were found at a chromosomal locus flanked by the conserved galF gene and the histidine synthesis operon (his). They were suggested to drive the biosynthesis of the O-antigen by the Wzy/Wzy-dependent pathway and the mannan by the Wzm/Wzt-dependent pathway. The mannan shares the structure and gene cluster with a polysaccharide isolated earlier from the lipopolysaccharide of Escherichia coli O8.

Colitose-containing O-polysaccharide structure and O-antigen gene cluster of Escherichia albertii HK18069 related to those of Escherichia coli O55 and E. coli O128.

A 3,6-dideoxy-l-xylo-hexose (colitose)-containing partially O-acetylated branched polysaccharide was obtained by mild acid hydrolysis (2% HOAc, 100 °C, 2 h) of the lipopolysaccharide of Escherichia albertii HK18069 followed by gel-permeation chromatography on Sephadex G-50 Superfine. Part of colitose residues (~40%) was cleaved upon hydrolysis, and the full cleavage was achieved by prolonged hydrolysis (8 h) under the same conditions and resulted in a modified linear polysaccharide. Structure of the O-polysaccharide of E. albertii HK18069 was established by 1D and 2D 1H and 13C NMR spectroscopy applied to both initial and modified O-deacetylated and colitose-free polysaccharides: where ß-d-Galp is mono-O-acetylated at position either 3 (~50%) or 4 (~30%). The O-antigen gene cluster of E. albertii HK18069 between conserved galF and gnd genes together with flanking regions was sequenced, and predicted functions of the genes were found to be consistent with the O-polysaccharide structure established. The O-polysaccharide structure and the O-antigen gene cluster of E. albertii HK18069 are related to those of Esherichia coli O55 and E. coli O128 reported earlier. It is proposed to create for strain HK18069 a new E. albertii O-serogroup, O8.

Structure and gene cluster of the O antigen of Escherichia coli F17, a candidate for a new O-serogroup.

Escherichia coli F17 isolated from horse feces was studied in respect to the O antigen (O polysaccharide) structure and genetics. The lipopolysaccharide was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the O polysaccharide, which was studied by sugar analysis and selective solvolysis with CF3CO2H along with one- and two-dimensional 1H and 13C NMR spectroscopy. The O polysaccharide was found to have a branched pentasaccharide repeat (O-unit) containing one residue each of d-galactose, d-mannose, l-rhamnose, d-glucuronic acid, and N-acetyl-d-glucosamine; about 2/3 units bear a side-chain glucose residue. To our knowledge, the F17 O-polysaccharide structure established is unique among known bacterial polysaccharide structures. The O-antigen gene cluster of E. coli F17 between the conserved genes galF and gnd was sequenced and found to be 99% identical to that of E. coli 102,755 assigned to a novel OgN8 genotype (A. Iguchi, S. Iyoda, K. Seto, H. Nishii, M. Ohnishi, H. Mekata, Y. Ogura, T. Hayashi, Front. Microbiol. 7 (2016) 765). Genes in the cluster were annotated taking into account the F17 O-polysaccharide structure. The data obtained confirm that E. coli F17 and E. coli strains belonging to the OgN8 genotype can be considered as a candidate to a new E. coli O-serogroup. The O antigen of this novel type was demonstrated to make for an effective shield protecting the intimate outer membrane surface of bacteria from direct interaction with bacteriophages.

Structural studies on the O-polysaccharide of Escherichia coli O57.

Mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O57 afforded an O-polysaccharide, which was isolated by gel permeation chromatography (GPC) and studied by sugar analysis, Smith degradation and solvolysis with trifluoroacetic acid, along with 2D 1H and 13C NMR spectroscopy. The O-polysaccharide was found to contain d-Glc, d-Gal, d-GalA, d-GlcNAc, and l-FucNAc, as well as O-acetyl groups. Smith degradation of the O-deacetylated polysaccharide destroyed side-branch ß-Glсp and α-GalpA to give a modified linear polysaccharide. Solvolysis cleaved selectively the linkage of α-l-FucpNAc to give a pentasaccharide corresponding to the O-polysaccharide repeat. A comparison of the NMR spectra of the initial and O-deacetylated polysaccharides showed that α-GalpA is non-stoichiometrically O-acetylated at position either 2 (∼30%) or 3 (∼40%). The following structure of the O-polysaccharide was established, which is unique among known bacterial polysaccharide structures.

Structural and genetic relatedness of the O-antigens of Escherichia coli O50 and O2.

An O-specific polysaccharide (O-antigen) was isolated by mild acid degradation of the lipopolysaccharide of Escherichia coli O50 followed by gel chromatography on Sephadex G-50. The following structure of the tetrasaccharide repeat was established by sugar analysis and 1D and 2D 1H and 13C NMR spectroscopy: →3)-α-l-Rhap-(1 → 2)-α-l-Rhap-(1 → 3)-ß-l-Rhap-(1 → 4)-ß-d-GlcpNAc-(1→ The linear O50 polysaccharide has the same structure as the main chain of the branched O polysaccharide of E. coli O2 studied earlier [Jansson et al., Carbohydr. Res. 161 (1987) 273-279], which differs in the presence of a side-chain α-d-Fucp3NAc residue. In spite of the difference between the O-polysaccharides, the corresponding genes in the O2- and O50-antigen gene cluster are 99-100% identical. The genetic basis for the lack of d-Fucp3NAc from the O50 polysaccharide is evidently a point mutation in the aminotransferase gene fdtB of the d-Fucp3NAc synthesis pathway resulting in a single amino acid change from histidine in O2 to arginine in O50.

Structure and gene cluster of the O-antigen of Escherichia coli O54.

Mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O54 afforded an O-polysaccharide, which was studied by sugar analysis, solvolysis with anhydrous trifluoroacetic acid, and 1H and 13C NMR spectroscopy. Solvolysis cleaved predominantly the linkage of ß-d-Ribf and, to a lesser extent, that of ß-d-GlcpNAc, whereas the other linkages, including the linkage of α-l-Rhap, were stable under selected conditions (40 °C, 5 h). The following structure of the O-polysaccharide was established: →4)-α-d-GalpA-(1 → 2)-α-l-Rhap-(1 → 2)-ß-d-Ribf-(1 → 4)-ß-d-Galp-(1 → 3)-ß-d-GlcpNAc-(1→ The O-antigen gene cluster of E. coli O54 was analyzed and found to be consistent in general with the O-polysaccharide structure established but there were two exceptions: i) in the cluster, there were genes for phosphoserine phosphatase and serine transferase, which have no apparent role in the O-polysaccharide synthesis, and ii) no ribofuranosyltransferase gene was present in the cluster. Both uncommon features are shared by some other enteric bacteria.

Studies on the O-polysaccharide of Escherichia albertii O2 characterized by non-stoichiometric O-acetylation and non-stoichiometric side-chain l-fucosylation.

An O-polysaccharide was isolated from the lipopolysaccharide of Escherichia albertii O2 and studied by chemical methods and 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the O-polysaccharide was established: . The O-polysaccharide is characterized by masked regularity owing to a non-stoichiometric O-acetylation of an l-fucose residue in the main chain and a non-stoichiometric side-chain l-fucosylation of a ß-GlcNAc residue. A regular linear polysaccharide was obtained by sequential Smith degradation and alkaline O-deacetylation of the O-polysaccharide. The content of the O-antigen gene cluster of E. albertii O2 was found to be essentially consistent with the O-polysaccharide structure established.

Structure and genetics of a glycerol 2-phosphate-containing O-specific polysaccharide of Escherichia coli O33.

An O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Escherichia coli O33 followed by gel-permeation chromatography on Sephadex G-50. The polysaccharide was found to contain glycerol 2-phosphate (Gro-2-P), and the following structure of its tetrasaccharide repeat was established by sugar analysis, dephosphorylation, and 1D and 2D 1H and 13C NMR spectroscopy: The O33-antigen gene cluster was analyzed and found to be essentially consistent with the O-polysaccharide structure.

Structure elucidation of the O-specific polysaccharide by NMR spectroscopy and selective cleavage and genetic characterization of the O-antigen of Escherichia albertii O5.

The O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Escherichia albertii O5 (strain T150248) and studied by sugar analysis, selective cleavages of glycosidic linkages, and 1D and 2D 1H and 13C NMR spectroscopy. Partial solvolysis with anh (anhydrous) CF3CO2H and hydrolysis with 0.05 M CF3CO2H cleaved predominantly the glycosidic linkage of ß-GalpNAc or ß-Galf, respectively, whereas the linkages of α-GlcpNAc and ß-Galp were stable. Mixtures of the corresponding tri- and tetra-saccharides thus obtained were studied by NMR spectroscopy and high-resolution ESI MS. The following new structure was established for the tetrasaccharide repeat (O-unit) of the O-polysaccharide: →4)-α-d-GlcpNAc-(1 → 4)-ß-d-Galp6Ac-(1 → 6)-ß-d-Galf-(1 → 3)-ß-d-GalpNAc-(1→where the degree of O-acetylation of d-Galp is ∼70%. The O-polysaccharide studied has a ß-d-Galp-(1 → 6)-ß-d-Galf-(1 → 3)-ß-d-GalpNAc trisaccharide fragment in common with the O-polysaccharides of E. albertii O7, Escherichia coli O124 and O164, and Shigella dysenteriae type 3 studied earlier. The orf5-7 in the O-antigen gene cluster of E. albertii O5 are 47%, 78%, and 75% identical on the amino acid level to genes for predicted enzymes of E. albertii O7, including Galp-transferase wfeS, UDP-d-Galp mutase glf, and Galf-transferase wfeT, respectively, which are putatively involved with the synthesis of the shared trisaccharide fragment of the O-polysaccharides. The occurrence upstream of the O-antigen gene cluster of a 4-epimerase gene gnu for conversion of undecaprenyl diphosphate-linked d-GlcNAc (UndPP-d-GlcNAc) into UndPP-d-GalNAc indicates that d-GalNAc is the first monosaccharide of the O-unit, and hence the O-units are interlinked in the O-polysaccharide of E. albertii O5 by the ß-d-GalpNAc-(1 → 4)-α-d-GlcpNAc linkage.

Structures and gene clusters of the O-antigens of Escherichia albertii O3, O4, O6, and O7.

The O-specific polysaccharides (OPSs) called O-antigens were obtained by mild acid degradation of the lipopolysaccharides of Escherichia albertii serotypes O3, O4, O6, and O7 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure was established for the OPS of E. albertii O4, which, to our knowledge, is unique among known bacterial polysaccharide structures: →2)-α-l-Rhap-(1 â†’ 2)-α-l-Fucp-(1 â†’ 2)-ß-d-Galp-(1 â†’ 3)-α-d-GalpNAc-(1 â†’ 3)-ß-d-GlcpNAc-(1→ The OPS structure of the strain of E. albertii O7 studied was identical to that of strain LMG 20973 (= Albert 10457), whose structure has been reported earlier (R. Eserstam et al. Eur. J. Biochem. 269 (2002) 3289-3295). E. albertii O3 and O6 shared the OPS structures with Escherichia coli O181 and O3, respectively, except for the lack of O-acetylation in E. albertii O3, which is present in E. coli O181. The gene clusters driving the O-antigen biosynthesis of the E. albertii strains were sequenced, the genes were annotated by comparison with sequences in the available databases, and the predicted functions of the encoded proteins were found to be consistent with the OPS structures established. In accordance with the relatedness of the OPS structures, the O-antigen gene clusters of E. albertii O3 and O6 contain the same genes and have the same organization as those of E. coli O181 and O3, the entire gene clusters being 83% and 98% identical, respectively.

Structure and gene cluster of the O-antigen of Escherichia albertii O1 resembling the O-antigen of Pseudomonas aeruginosa O5.

The O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Escherichia albertii serotype O1 strain SP20140089 and studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure was established for the trisaccharide repeating unit of the O-polysaccharide: →4)-ß-d-ManpNAc3NAcA-(1 â†’ 4)-ß-d-GlcpNAm3NAcA-(1 â†’ 3)-α-d-GlcpNAc-(1→ where ManNAc3NAcA and GlcNAm3NAcA indicate 2,3-diacetamido-2,3-dideoxymannuronic acid and 2-acetimidoylamino-3-acetamido-2,3-dideoxyglucuronic acid, respectively. While showing some similarity with O-polysaccharide structures of a group of Pseudomonas aeruginosa serotypes (O2, O5, O16, O18, and O20), that of E. albertii O1 is unique among known bacterial polysaccharide structures. The gene cluster for biosynthesis of the O1-antigen was sequenced and functions of the genes were predicted by comparison with sequences in the available databases, including those involved in the synthesis of nucleotide precursors of 2,3-diamino-2,3-dideoxyhexuronic acid derivatives in P. aeruginosa O5.

Structure and genetics of the O-antigens of Escherichia coli O182-O187.

O-polysaccharides (OPSs) were obtained by mild acid degradation of the lipopolysaccharides of Escherichia coli O182-O187, and their structures were established by sugar analysis, Smith degradation, and 1H and 13C NMR spectroscopy. In addition to the monosaccharides that occur often in E. coli OPSs (d-Glc, d-Gal, d-Man, d-GlcNAc, d-GalNAc, d-GlcA, l-Fuc, d-Rib), a number of less common components were identified as the OPS constituents, including 2-acetamido-2-deoxy-l-quinovose and 4-deoxy-4-[(S)-3-hydroxybutanoyl-l-alanyl]-d-quinovose (O186), 3-acetamido-3-deoxy-d-fucose (O187), 3-deoxy-3-[(R)-3-hydroxybutanoyl]-d-fucose (O184), and 2,3-diacetamido-2,3-dideoxy-l-rhamnose (O182). The OPS structures of E. coli O183 and O182 are identical to those of the OPS of Shigella boydii type 10 and the capsular polysaccharide of E. coli K48, respectively. The OPSs of E. coli O186 and O123 are closely related differing in the presence of a Glc residue in the former in place of a GlcNAc residue in the latter. The O-antigen gene clusters of the bacteria studied were analyzed and their contents were found to be consistent with the OPS structures. Predicted glycosyltransferases encoded in the gene clusters were tentatively assigned to glycosidic linkages based on similarities to sequences of other E. coli O-serogroups available from GenBank and taking into account the OPS structures established.