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Escherichia coli includes clonal groups of both commensal and pathogenic strains, with some of the latter causing serious infectious diseases. O antigen variation is current standard in defining strains for taxonomy and epidemiology, providing the basis for many serotyping schemes for Gram-negative bacteria. This review covers the diversity in E. coli O antigen structures and gene clusters, and the genetic basis for the structural diversity. Of the 187 formally defined O antigens, six (O31, O47, O67, O72, O94 and O122) have since been removed and three (O34, O89 and O144) strains do not produce any O antigen. Therefore, structures are presented for 176 of the 181 E. coli O antigens, some of which include subgroups. Most (93%) of these O antigens are synthesized via the Wzx/Wzy pathway, 11 via the ABC transporter pathway, with O20, O57 and O60 still uncharacterized due to failure to find their O antigen gene clusters. Biosynthetic pathways are given for 38 of the 49 sugars found in E. coli O antigens, and several pairs or groups of the E. coli antigens that have related structures show close relationships of the O antigen gene clusters within clades, thereby highlighting the genetic basis of the evolution of diversity., (© The Author(s) 2019. Published by Oxford University Press on behalf of FEMS.)

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)-β-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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 1 H and 13 C NMR spectroscopy applied to both initial and modified O-deacetylated and colitose-free polysaccharides: where β-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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In 2017, we reported a new database on glycosyltransferase (GT) activities, CSDB_GT (http://csdb.glycoscience.ru/gt.html), which was built at the platform of the Carbohydrate Structure Database (CSDB, http://csdb.glycoscience.ru/database/index.html) and contained data on experimentally confirmed GT activities from Arabidopsis thaliana. All entries in CSDB_GT are curated manually upon the analysis of scientific publications, and the key features of the database are accurate structural, genetic, protein and bibliographic references and close-to-complete coverage on experimentally proven GT activities in selected species. In 2018, CSDB_GT was supplemented with data on Escherichia coli GT activities. Now it contains ca. 800 entries on E. coli GTs, including ca. 550 entries with functions predicted in silico. This information was extracted from research papers published up to the year 2018 or was obtained by the authors' efforts on GT annotation. Thus, CSDB_GT was extended to provide not only experimentally confirmed GT activities, but also those predicted on the basis of gene or protein sequence homology that could carry valuable information. Accordingly, a new confirmation status-predicted in silico-was introduced. In addition, the coverage on A. thaliana was extended up to ca. 900 entries, all of which had experimental confirmation. Currently, CSDB_GT provides close-to-complete coverage on experimentally confirmed GT activities from A. thaliana and E. coli presented up to the year 2018., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 CF 3 CO 2 H along with one- and two-dimensional 1 H and 13 C 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 1 H and 13 C 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 β-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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 1 H and 13 C NMR spectroscopy: →3)-α-l-Rhap-(1 → 2)-α-l-Rhap-(1 → 3)-β-l-Rhap-(1 → 4)-β-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., (Copyright © 2018. Published by Elsevier Ltd.)

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 1 H and 13 C NMR spectroscopy. Solvolysis cleaved predominantly the linkage of β-d-Ribf and, to a lesser extent, that of β-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)-β-d-Ribf-(1 → 4)-β-d-Galp-(1 → 3)-β-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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

An O-polysaccharide was isolated from the lipopolysaccharide of Escherichia albertii O2 and studied by chemical methods and 1D and 2D 1 H and 13 C 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 β-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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 1 H and 13 C NMR spectroscopy: The O33-antigen gene cluster was analyzed and found to be essentially consistent with the O-polysaccharide structure., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 1 H and 13 C NMR spectroscopy. Partial solvolysis with anh (anhydrous) CF 3 CO 2 H and hydrolysis with 0.05 M CF 3 CO 2 H cleaved predominantly the glycosidic linkage of β-GalpNAc or β-Galf, respectively, whereas the linkages of α-GlcpNAc and β-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)-β-d-Galp6Ac-(1 → 6)-β-d-Galf-(1 → 3)-β-d-GalpNAc-(1→where the degree of O-acetylation of d-Galp is ∼70%. The O-polysaccharide studied has a β-d-Galp-(1 → 6)-β-d-Galf-(1 → 3)-β-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 β-d-GalpNAc-(1 → 4)-α-d-GlcpNAc linkage., (Copyright © 2017. Published by Elsevier Ltd.)

The O-specific polysaccharide (O-antigen) is a part of the lipopolysaccharide on the cell surface of Gram-negative bacteria. The O-polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O27 and studied by sugar analysis and Smith degradation along with 1 H and 13 C NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established, which is unique among known structures of bacterial polysaccharides:where GlcA is non-stoichiometrically O-acetylated at position 3 (∼22%) or 4 (∼37%). Functions of genes in the O-antigen gene cluster of E. coli O27 were tentatively assigned by comparison with sequences in the available databases and found to be consistent with the O-polysaccharide structure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The O-antigen is a part of the outer membrane of Gram-negative bacteria and is related to bacterial virulence. It is one of the most variable cell constituents, and its structural diversity is almost entirely due to genetic variation of the O-antigen gene cluster. In this study, the O-antigen structure of Escherichia coli O62 was elucidated by chemical analysis and nuclear magnetic resonance spectroscopy, but showing not consistent with the O-antigen gene cluster between conserved genes galF and gnd reported earlier. The complete genome of E. coli O62 was then sequenced and analyzed, and another O-antigen gene cluster was found and characterized that correlated perfectly with the established O-antigen structure. A deletion and complementation experiment confirmed the functionality of the novel gene cluster and demonstrated that the O62-antigen is synthesized by the ABC transporter-dependent system. To our knowledge, this is the first report that the O-antigen gene cluster is positioned at a novel locus in E. coli. Comparative analysis indicated that E. coli O62 likely originated from E. coli O68 via an IS event resulting in the repression of the O68-antigen synthesis, followed by the acquisition of a novel O-antigen gene cluster from Enterobacter aerogenes., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Based on the O-specific polysaccharides of the lipopolysaccharides (O-polysaccharides, O-antigens), strains of a clonal species Escherichia coli are classified into 184 O serogroups. In this work, structures of the O-polysaccharides of E. coli O69 and O146 were elucidated and gene clusters for their biosynthesis were characterized. The O-polysaccharides were released from the lipopolysaccharides by mild acid hydrolysis and studied by sugar analysis and one- and two-dimensional 1 H and 13 C NMR spectroscopy before and after O-deacetylation. The O146 polysaccharide was also studied by Smith degradation. The O69 and O146 polysaccharides were found to contain ether conjugates of monosaccharides with lactic acid called glycolactilic acids: 2-acetamido-2-deoxy-4-O-[(R)-1-carboxyethyl]-D-glucose (D-GlcNAc4Rlac) and 3-O-[(S)-1-carboxyethyl]-D-glucose (D-Glc3Slac), respectively. Structures of the pentasaccharide repeats of the O-polysaccharides were established, and that of E. coli O69 was found to differ in the presence of D-GlcNAc4Rlac from the structure reported for this bacterium earlier (Erbing C, Kenne L, Lindberg B. 1977. Carbohydr Res. 56:371-376). The O-antigen gene clusters of E. coli O69 and O146 between conserved genes galF and gnd were analyzed taking into account the O-polysaccharide structures established, and functions of putative genes for synthesis of D-Glc3Slac and D-GlcNAc4Rlac and for glycosyltransferases were assigned based on homology with O-antigen biosynthesis genes of other enteric bacteria. It was found that in E. coli and Shigella spp. predicted enolpyruvate reductases of the biosynthesis pathway of glycolactilic acids, LarR and LarS, which catalyze formation of conjugates with (R)- or (S)-lactic acid, respectively, are distinguished by sequence homology and size.

The O-polysaccharide (O-antigen) of Escherichia coli O131 was studied by sugar analysis along with 1D and 2D 1 H and 13 C NMR spectroscopy. The following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: →8)-α-Neup5Ac-(2 → 6)-β-D-Galp-(1 → 6)-β-D-Galp-(1 → 3)-β-D-GalpNAc-(1→ The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the E. coli O131-antigen structure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 1 H and 13 C 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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mild alkaline degradation of the lipopolysaccharide of Escherichia coli O80 afforded a polysaccharide, which was studied by sugar analysis, selective cleavage of glycosidic linkages, and (1)H and (13)C NMR spectroscopy. Solvolysis of the polysaccharide with CF3CO2H cleaved the linkages of α-Fuc and β-linked GlcNAc and GalNAc residues to give two disaccharides. The following structure of the hexasaccharide repeating unit of the O-polysaccharide was established: The polysaccharide repeat also contains a minor O-acetyl group but its position was not determined. The O-antigen gene cluster of E. coli O80 between the conserved galF and gnd genes was analyzed and found to be consistent with the O-polysaccharide structure established., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The lipopolysaccharide of Escherichia coli O156 was degraded under mild acidic and alkaline conditions and the resulting polysaccharides were studied by sugar analysis and (1)H and (13)C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established: where Rpyr indicates R-configurated pyruvic acid acetal. Minor O-acetyl groups also were present and tentatively localized on the Gal residues. The gene cluster for biosynthesis of the O-antigen of E. coli O156 was analyzed and shown to be consistent with the O-polysaccharide structure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The O-specific polysaccharide (O-antigen) of Escherichia coli O133 was obtained by mild acid hydrolysis of the lipopolysaccharide of E. coli O133. The structure of the hexasaccharide repeating unit of the polysaccharide was elucidated by (1)H and (13)C NMR spectroscopy, including a two-dimensional (1)H-(1)H ROESY experiment: Functions of genes in the O-antigen gene cluster were putatively identified by comparison with sequences in the available databases and, particularly, an encoded predicted multifunctional glycosyltransferase was assigned to three α-l-rhamnosidic linkages., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fine structure of the O-polysaccharide chain of the lipopolysaccharide (O-antigen) defines the serospecificity of bacterial cells, which is the basis for O-serotyping of medically and agriculturally important gram-negative bacteria including Escherichia coli. In order to obtain the O-polysaccharide for structural analysis, the lipopolysaccharide was isolated from cells of E. coli O84a by phenol/water extraction and degraded with mild acid. However, the O-polysaccharide was cleaved at a highly acid-labile β-l-fucopyranosyl phosphate (β-l-Fucp-1-P) linkage to give mainly a pentasaccharide that corresponded to the O-polysaccharide repeat. Therefore, the lipopolysaccharide and the pentasaccharide as well as their O-deacylated derivatives were studied using sugar analysis, NMR spectroscopy, and (for oligosaccharides) ESI HR MS, and the O84-polysaccharide structure was established. The O-polysaccharide is distinguished by the presence of β-l-Fucp-1-P and randomly di-O-acetylated 6-deoxy-d-talose, which are found for the first time in natural carbohydrates. The gene cluster for the O84-antigen biosynthesis was analysed and its content was found to be consistent with the O-polysaccharide structure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mild acid degradation of the lipopolysaccharide of Escherichia coli O132 released its O-polysaccharide. Analysis by 1D and 2D (1)H and (13)C NMR spectroscopy prior and subsequent to O-deacetylation, in conjunction with sugar analysis, revealed a linear pentasaccharide repeating unit of the O-polysaccharide having the following structure: →2)-α-d-Galf-(1→3)-α-l-Rhap2Ac-(1→4)-α-d-Glcp-(1→2)-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→ Putative functions of genes in the O-antigen gene cluster of E. coli O132 are consistent with the O-polysaccharide structure., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

O-polysaccharides of E. coli O28ab, O37, and O100 were found to contain glycerol 1-phosphate and the following structures of their oligosaccharide repeats were established by sugar analysis, Smith degradation (for O28ab), 1D and 2D (1)H, (13)C, and (13)P NMR spectroscopy: [Formula: see text]. Functions of putative glycosyltransferases genes in the O-antigen gene clusters of the strains studied were tentatively assigned based on similarities to genes of other E. coli O-serogroups available from GenBank and taking into account the O-polysaccharide structures established., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The O-polysaccharide (O-antigen) was isolated from the lipopolysaccharide of Escherichia coli O137 and studied by sugar analysis and NMR spectroscopy. The following structure of the branched tetrasaccharide repeating unit was established: Formula: see text] Both structure and gene cluster of the E. coli O137 polysaccharide are related to those of the E. coli K40 polysaccharide (Amor et al., 1999), which lacks the side-chain glucosylation but contains serine that is amide-linked to GlcA. Functions of genes in the O137-antigen gene cluster were assigned by a comparison with those in K40 and sequences in the available databases. Particularly, predicted glycosyltransferases encoded in the gene cluster were assigned to the formation of three glycosidic linkages in the O-polysaccharide repeating unit., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mild acid degradation of the lipopolysaccharide of Escherichia coli O96 afforded a mixture of two polysaccharides. The following structure of the pentasaccharide repeating unit of the major polymer was established by sugar analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy: [Formula: see text]. The O-antigen gene cluster of E. coli O96 between conserved galF and gnd genes was found to be consistent with this structure, and hence, the major polysaccharide represents the O96-antigen. The O96-antigen structure and gene cluster are similar to those of E. coli O170, and two proteins encoded in the gene clusters of both bacteria were putatively assigned a function of galactofuranosyltransferases. The minor polymer has the same structure as a peptidoglycan-related polysaccharide reported earlier in Providencia alcalifeciens O45 and several other O-serogoups of this species (Ovchinnikova OG, Liu B, Kocharova NA, Shashkov AS, Kondakova AN, Siwinska M, Feng L, Rozalski A, Wang L, Knirel YA. Biochemistry (Moscow) 2012;77:609-15) → 4)-β-D-GlcpNAc-(1 → 4)-β-D-GlcpNAc3(Rlac-lAla)-(1 → where Rlac-lAla indicates (R)-1-[(S)-1-carboxyethylaminocarbonyl]ethyl., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

On mild acid degradation of the lipopolysaccharide of Escherichia coli O160, the O-polysaccharide was cleaved by acid-labile glycosyl phosphate linkages in the main chain. The resultant oligosaccharide and the alkali-treated lipopolysaccharide were studied by sugar analysis along with (1)H and (13)C NMR spectroscopies, and the following structure of the branched pentasaccharide repeating unit of the O-polysaccharide was established: The O-antigen gene cluster of E. coli O160 was found to be consistent with the O-polysaccharide structure established., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The O-polysaccharide (O-antigen) of Escherichia coli O43 was isolated from the lipopolysaccharide and studied by chemical methods, including sugar analyses, Smith degradation, and solvolysis with anhydrous trifluoroacetic acid, along with (1)H and (13)C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established: [Formula: see text] Functions of genes in the O-antigen gene cluster of E. coli O43 were assigned by a comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The O-polysaccharide (O-antigen) of Escherichia coli O169 was studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established: [Formula: see text] The O-polysaccharide of E. coli O169 differs from that of Shigella boydii type 6 only in the presence of a side-chain glucose residue. A comparison of the O-antigen biosynthesis gene clusters between the galF to gnd genes in the genomes of the two bacteria revealed their close relationship. The glycosyltransferase gene responsible for the formation of the β-D-Glcp-(1 → 6)-α-D-Galp linkage in the O-antigen was identified in the gene cluster., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Escherichia coli strains are normally identified by the combination of their O and H (and sometimes K) antigens, and serotyping based on the antigens is believed to be crucial for clinical detection and epidemiological investigation. Two E. coli strains, G5413 and G5287, were isolated from faecal samples of female patients with diarrhoea and were not agglutinated with any antisera that cover the well-known O serogroups of E. coli. We elucidated the O-polysaccharide (OPS) structures and analysed the O-antigen gene clusters of these bacteria. The OPS structure of G5413 established by monosaccharide analysis and NMR spectroscopy was found to be unique amongst known bacterial polysaccharide structures. The O-antigen gene cluster of this strain was sequenced and did not match sequence data with any of the 184 O serogroups that have been recognized internationally. Gene functions were tentatively assigned and were appropriate to the OPS structure. Based on these data, we suggest G5413 as a candidate for a new E. coli O serogroup. Both the OPS structure and O-antigen gene cluster of G5287 were identical to those of E. coli L-19, a candidate for another new O serogroup characterized by us recently. Recognition of these two provisional O serogroups increases the number of known O-antigen forms of E. coli to 186.

The following structure of the O-polysaccharide of Escherichia coli HS1/2 serving as a primary receptor for bacteriophage DT57-12 was elucidated by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy: This structure is shared by E. coli O87 type strain. Putatively assigned functions of genes in the O-antigen gene cluster of E. coli O87 are consistent with the O-polysaccharide structure established., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The O-polysaccharides (O-antigens) were isolated by mild acid degradation of the lipopolysaccharide (LPS) of Escherichia coli O46 and O134. The structures of their linear tetrasaccharide repeating units were established by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy: [Formula: see text], where D-aThr indicates D-allothreonine and R indicates O-acetyl substitution (∼ 70% on aThr and ∼ 15% on GalNAc) in E. coli O46 whereas the O-acetylation is absent in E. coli O134. Functions of genes in the essentially identical O-antigen gene clusters of E. coli O46 and O134 were tentatively assigned by a comparison with sequences in available databases and found to be in agreement with the O-polysaccharide structures established., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An acidic O-polysaccharide (O-antigen) of Escherichia coli O163 was obtained by mild acid hydrolysis of the lipopolysaccharide and studied by sugar analysis and Smith degradation along with 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the linear tetrasaccharide repeating unit was established, which is unique among known structures of bacterial polysaccharides: -->2)-β-D-Manp-(1-->4)-β-D-GlcpA-(1-->3)-α-L-QuipNAc-(1-->3)-α-D-GlcpNAc-(1-->. Functions of genes in the O-antigen gene cluster of E. coli O163 were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure. Relationships between O-antigen structures and gene clusters of E. coli O163 and Salmonella enterica O41 are discussed., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

O-Antigen (O-polysaccharide) variation is the basis for bacterial serotyping and is important in bacterial virulence and niche adaptation. In this work, we present structural and genetic evidences for close relationships between the O-antigens of the Cronobacter spp. and Escherichia coli. Cronobacter sakazakii G2594 (serotype O4) and Cronobacter malonaticus G3864 (serotype O1) are structurally related to those of E. coli O103 and O29, respectively, and some other members of the Enterobacteriaceae family differing in the patterns of lateral glucosylation (C. sakazakii G2594) or O-acetylation (C. malonaticus G3864). The O-antigen gene clusters of the corresponding Cronobacter and E. coli strains contain the same genes with high-level similarity, and the structural differences within both O-antigen pairs were suggested to be due to modification genes carried by prophages., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The O polysaccharide of the lipopolysaccharide (O antigen) of Gram-negative bacteria often serves as a receptor for bacteriophages that can make the phage dependent on a given O-antigen type, thus supporting the concept of the adaptive significance of the O-antigen variability in bacteria. The O-antigen layer also modulates interactions of many bacteriophages with their hosts, limiting the access of the viruses to other cell surface receptors. Here we report variations of O-antigen synthesis and structure in an environmental Escherichia coli isolate, 4s, obtained from horse feces, and its mutants selected for resistance to bacteriophage G7C, isolated from the same fecal sample. The 4s O antigen was found to be serologically, structurally, and genetically related to the O antigen of E. coli O22, differing only in side-chain α-D-glucosylation in the former, mediated by a gtr locus on the chromosome. Spontaneous mutations of E. coli 4s occurring with an unusually high frequency affected either O-antigen synthesis or O-acetylation due to the inactivation of the gene encoding the putative glycosyltransferase WclH or the putative acetyltransferase WclK, respectively, by the insertion of IS1-like elements. These mutations induced resistance to bacteriophage G7C and also modified interactions of E. coli 4s with several other bacteriophages conferring either resistance or sensitivity to the host. These findings suggest that O-antigen synthesis and O-acetylation can both ensure the specific recognition of the O-antigen receptor following infection by some phages and provide protection of the host cells against attack by other phages., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The O-polysaccharide (O-antigen) of Escherichia coli O68 was studied by sugar analysis, partial solvolysis with anhydrous trifluoroacetic acid, and 1D and 2D (1)H and (13)C NMR spectroscopies. The following structure of the branched heptasaccharide repeating unit was established: [structure: see text]. The O-antigen gene cluster of E. coli O68 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the O-antigen structure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Escherichia coli L-19 isolated from a healthy individual did not agglutinate with any of 21 polyvalent antisera that cover 174 E. coli O-serogroups. The strain was studied in respect to the O-antigen (O-specific polysaccharide, OPS) structure and genetics. The LPS was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the OPS. The OPS was studied by sugar and methylation analyses, along with 1D and 2D (1)H and (13)C NMR spectroscopy. The established structure of the linear tetrasaccharide repeating unit was found to be unique among known bacterial polysaccharide structures. A peculiar component of the L-19 OPS was an amide of glucuronic acid with 2-amino-1,3-propanediol (2-amino-2-deoxyglycerol) (GroN). The O-antigen gene cluster of L-19 between the conserved genes galF and gnd was sequenced, and gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in agreement with the OPS structure. Except for putative genes for synthesis and transfer of GroN, the sequences in the L-19 O-antigen gene cluster were little related to those of reference strains of the 174 known E. coli O-serogroups. The data obtained suggest that L-19 can be considered as a candidate for a new E. coli O-serogroup., (© 2014 The Authors.)

The O-polysaccharide (O-antigen) of Escherichia coli O36 was isolated from the lipopolysaccharide and studied by sugar analyses and Smith degradation along with (1)H and (13)C NMR spectroscopy. The following structure of the branched pentasaccharide repeating unit was established, which is unique among the known structures of bacterial polysaccharides: The O-antigen gene cluster of E. coli O36 has been sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the O-polysaccharide structure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

O-Polysaccharide (O-antigen) accompanied by a minor mannan was isolated from the lipopolysaccharide of Escherichia coli O39 and studied by component analyses, methylation, Smith degradation, mass spectrometry, and 1D and 2D NMR spectroscopy. In addition, a new approach, solvolysis with anhydrous trifluoroacetic acid, was applied to cleave selectively the rhamnosidic linkage. The following structure of the O-polysaccharide was established: α--D-Galpl-->3-->3)-β-D-Quip4N(R3Hb)-(1-->2)-α-D-Manp-(l-->4)-α-L-Rhap-(1-->3)-α-D-GlcpNAc-(1--> where D-Qui4N(R3Hb) indicates 4,6-dideoxy-4-[(R)-3-hydroxybutanoylamino]-d-glucose. The O-antigen gene cluster of E. coli O39 has been sequenced. The gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in agreement with the O-polysaccharide structure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The O-polysaccharide (O-antigen) of Escherichia coli O154 was studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the branched pentasaccharide repeating unit was established: [structure: see text]. The O-antigen gene cluster of E. coli O154 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the O-polysaccharide structure., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The O-polysaccharide (O-antigen) of Escherichia coli O102 was studied by sugar analysis along with one- and two-dimensional (1)H and (13)C NMR spectroscopy. The following structure of the branched pentasaccharide repeating unit was established: [formula: see text]. The O-antigen gene cluster of E. coli O102 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the E. coli O102 O-polysaccharide structure., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The O antigen is an essential component of the lipopolysaccharides on the surface of Gram-negative bacteria and its variation provides a major basis for serotyping schemes. The Escherichia coli O-antigen form O180 was first designated in 2004, and O180 strains were found to contain virulence factors and cause diarrhea. Different O-antigen forms are almost entirely due to genetic variations in the O-antigen gene clusters. In this study, the chemical structure and gene cluster of E. coli O180 O antigen were investigated. A tetrasaccharide repeating unit with the following structure: →4)-β-D-ManpNAc3NAcA-(1 → 2)-α-L-Rhap(I)-(1 → 3)-β-L-Rhap(II)-(1 → 4)-α-D-GlcpNAc-(1→was identified in the E. coli O180 O antigen, including the residue D-ManpNAc3NAcA (2,3-diacetamido-2,3-dideoxy-D-mannopyranuronic acid) that had not been hitherto identified in E. coli. Genes in the O-antigen gene cluster were assigned functions based on their similarities with those from available databases, and five genes involved in the synthesis of UDP-D-ManpNAc3NAcA (the nucleotide-activated form of D-ManpNAc3NAcA) were identified. The gnaA gene, encoding the enzyme involved in the initial step of the UDP-D-ManpNAc3NAcA biosynthetic pathway, was cloned and the enzyme product was expressed, purified and assayed for its activity. GnaA was characterized using capillary electrophoresis and electrospray ionization mass spectrometry and identified as a UDP-GlcNAc 6-dehydrogenase. The kinetic and physicochemical parameters of GnaA also were determined.

The presence of cell-bound K1 capsule and K1 polysaccharide in culture supernatants was determined in a series of in-frame nonpolar core biosynthetic mutants from Escherichia coli KT1094 (K1, R1 core lipopolysaccharide [LPS] type) for which the major core oligosaccharide structures were determined. Cell-bound K1 capsule was absent from mutants devoid of phosphoryl modifications on L-glycero-D-manno-heptose residues (HepI and HepII) of the inner-core LPS and reduced in mutants devoid of phosphoryl modification on HepII or devoid of HepIII. In contrast, in all of the mutants, K1 polysaccharide was found in culture supernatants. These results were confirmed by using a mutant with a deletion spanning from the hldD to waaQ genes of the waa gene cluster to which individual genes were reintroduced. A nuclear magnetic resonance (NMR) analysis of core LPS from HepIII-deficient mutants showed an alteration in the pattern of phosphoryl modifications. A cell extract containing both K1 capsule polysaccharide and LPS obtained from an O-antigen-deficient mutant could be resolved into K1 polysaccharide and core LPS by column chromatography only when EDTA and deoxycholate (DOC) buffer were used. These results suggest that the K1 polysaccharide remains cell associated by ionically interacting with the phosphate-negative charges of the core LPS.

The acidic O-polysaccharide (O-antigen) of Escherichia coli O41 was studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy, and the following structure of the branched hexasaccharide repeating unit was established: This structure is unique among the known structures of bacterial polysaccharides. The O-antigen gene cluster of E. coli O41 was sequenced. The gene functions were tentatively assigned by a comparison with sequences in the available databases and found to be in full agreement with the E. coli O41 O-polysaccharide structure., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The O-polysaccharide (O-antigen) of Escherichia coli O19ab was studied by sugar analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy. The following structure of the linear pentasaccharide repeating unit was established: [See formula in text] where the degree of O-acetylation of GlcNAc is ∼33%. The O-antigen gene cluster of E. coli O19ab was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in full agreement with the E. coli O19ab-antigen structure., (Copyright © 2011. Published by Elsevier Ltd.)

A structure of the O-polysaccharide (O-antigen) of Escherichia coli O158 has been reported (Datta, A. K.; Basu, S.; Roy, N. Carbohydr. Res.1999, 322, 219-227). In this work, we reinvestigated the O158 polysaccharide using sugar analyses, Smith degradation, and (1)H and (13)C NMR spectroscopy and established the following structure, which is at variance with the structure established earlier: [see formula in text]. This structure is in agreement with the predicted functions of genes found in the O-antigen gene cluster of E. coli O158., (Copyright © 2011. Published by Elsevier Ltd.)

The O-polysaccharides were released by mild acid hydrolysis from the lipopolysaccharides of Escherichia coli O51 and Salmonella enterica O57 and found to possess the same structure, which was established by sugar analysis and 1D and 2D NMR spectroscopy: [formula in text]. The O-antigen gene clusters of E. coli O51 and S. enterica O57 were sequenced and found to contain the same genes with a high-level similarity. All genes expected for the synthesis of the O-antigen were identified based on their similarity to genes from available databases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

O-Antigen is a part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which confers major antigenic variability to the cell surface. In this study, we report on a previously undefined pair of Escherichia coli and Salmonella enterica with closely related O-antigens. The O-polysaccharides were isolated from the lipopolysaccharides of E. coli O85 and S. enterica O17 by mild acid degradation and studied by sugar analysis and NMR spectroscopy. The following structure was established for the O-unit of the E. coli O85-polysaccharide: The S. enterica O17-polysaccharide has the same carbohydrate backbone and, in addition, contains an O-acetyl group at position 2 of ~80% β-Galf residues. The O-antigen gene cluster of E. coli O85 was found to be closely related to that of S. enterica O17. Screening of type strains of all E. coli and S. enterica O-serogroups revealed two genes specific to the E. coli O85 O-antigen gene cluster, which can be used for development of PCR-based assays for identification and detection of E. coli O85 strains.

The O-antigen is a part of the lipopolysaccharide molecule present in the outer membrane of Gram-negative bacteria, and is essential for the full function of the microorganisms. Salmonella enterica and Escherichia coli are taxonomically closely related species. In this study, the O-antigen structures of S. enterica O16 and O38 and E. coli O11 were determined. Salmonella enterica O38 and E. coli O21 were found to have identical O-antigen structures, whereas S. enterica O16 and E. coli O11 had closely related structures, differing only in the presence of a lateral glucose residue and O-acetylation of a mannose residue in the former. The O-antigen gene clusters of S. enterica O16 and O38 and E. coli O11 were sequenced and analyzed together with that of E. coli O21 retrieved from the GenBank. Each S. enterica/E. coli pair was found to contain the same set of genes organized in the same manner and to share 56-78% overall DNA identity. These data suggest that the O-antigen gene clusters of each pair studied originated from a common ancestor. Thus, it has become evident that in the past, the degree of relatedness between the O-antigens of S. enterica and E. coli was underestimated., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

O-antigen representing the O-polysaccharide chain of the lipopolysaccharide is the most variable constituent on the cell surface of Gram-negative bacteria and a player in their pathogenicity. The O-polysaccharide of Escherichia coli O109 was studied by sugar analysis and nuclear magnetic resonance spectroscopy and found to contain a rarely occurring monosaccharide, 2,3-diacetamido-2,3,6-trideoxy-l-mannose (l-RhaNAc3NAc). The following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established, which is closely related to that of Proteus penneri O66: Ac--4-β-L-RhapNAc3NAc -->4)-α-D-Glcp-(1-->3)-α-L-6dTalp-(1-->3)-β-D-GlcpNAc-(1-->. The O-antigen gene cluster of E. coli O109 was sequenced and all 14 genes found were assigned functions based on their similarity to genes from the available databases. Putative genes for synthesis of l-RhaN3N were found in E. coli O109 and their homologues in E. coli O119, whose O-antigen has been reported earlier to contain 2-acetamido-2,3,6-trideoxy-3-formamido-d-mannose (d-RhaNAc3NFo). Analysis by GLC of the (S)-2-octyl glycosides confirmed that the absolute configuration of RhaN3N in E. coli O119 should be revised from D TO L., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The O-polysaccharide of Salmonella enterica O59 was studied using sugar analysis and 2D (1)H and (13)C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit was established: →2)-β-d-Galp-(1→3)-α-d-GlcpNAc-(1→4)-α-l-Rhap-(1→3)-β-d-GlcpNAc-(1→ Accordingly, the O-antigen gene cluster of S. enterica O59 includes all genes necessary for the synthesis of this O-polysaccharide. Earlier, another structure has been reported for the O-polysaccharide of Salmonella arizonae (S. enterica IIIb) O59, which later was found to be identical to that of Citrobacter (Citrobacter braakii) O35 and, in this work, also to the O-polysaccharide of Escherichia coli O15., (2010 Elsevier Ltd. All rights reserved.)

O-antigen (O-polysaccharide) is a highly variable part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which is used as the basis for bacterial serotyping and is essential for the full function and virulence of bacteria. In this work, the structure and genetics of the O-antigens of Escherichia coli O118 and O151 were investigated. Both O-polysaccharides were found to contain ribitol phosphate and have similar structures, the only difference between their backbones being one linkage mode (β1→3 in E. coli O118 vs. β1→2 in E. coli O151), which, most probably, is the linkage between the oligosaccharide repeats (O-units). The O-antigen gene clusters of the two bacteria are organized in the same manner and share high-level identity (>99%). Analysis of the wzy genes from E. coli O118 and O151 strains, which are responsible for the linkage between O-units, revealed only one nucleotide substitution, resulting in one amino acid residue substitution. The possible genetic events that may lead to the structural difference between two O-antigen structures are discussed. Salmonella O47 has the same O-unit backbone and a similar O-antigen gene cluster (OGC) (the DNA identity ranges from 74% to 83%) as E. coli O118 and O151. It was suggested that the OGCs of the three bacteria studied originated from a common ancestor., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)