New lactate- and pyruvate-containing polysaccharide and rhamnomannan with xylose residues from the cell wall of Rathayibacter oskolensis VKM Ac-2121T.

The cell wall of endophytic strain Rathayibacter oskolensis VKM Ac-2121T (family Microbacteriaceae, class Actinomycetes) was found to contain neutral and acidic glycopolymers. The neutral polymer is a block-type rhamnomannan partially should be substitutied by xylose residues, [→2)-α-[ß-D-Xylp-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼45. The acidic polymer has branched chain, bearing lactate and pyruvate residues, →4)-α-D-[S-Lac-(2-3)-α-L-Rhap-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-[4,6-R-Pyr]-D-Galp-(1 â†’ 3)-ß-D-Glcp-(1 â†’. The structures of both glycopolymers were not described in the Gram-positive bacteria to date. The glycopolymers were studied by chemical and NMR spectroscopic methods. The results of this study provide new data on diversity of bacterial glycopolymers and may prove useful in the taxonomy of the genus Rathayibacter and for understanding the molecular mechanisms of interaction between plants and plant endophytes.

Glycoengineering directs de novo biomanufacturing of UPEC O21 O-antigen polysaccharide based glycoprotein.

Glycoproteins, in which polysaccharides are usually attached to proteins, are an important class of biomolecules that are widely used as therapeutic agents in clinical treatments for decades. Uropathogenic Escherichia coli (UPEC) O21 has been identified as a serogroup that induces urinary tract infections, with a global increasing number among women and young children. Therefore, there is an urgent need to establish protective vaccines against UPEC infection. Herein, we engineered non-pathogenic E. coli MG1655 to achieve robust, cost-effective de novo biosynthesis of O21 O-antigen polysaccharide-based glycoprotein against UPEC O21. Specifically, this glycoengineered E. coli MG1655 was manipulated for high-efficient glucose-glycerol co-utilization and for the gene cluster installation and O-glycosylation machinery assembly. The key pathways of UDP-sugar precursors were also strengthened to enforce more carbon flux towards the glycosyl donors, which enhanced the glycoprotein titer by 5.6-fold. Further optimization of culture conditions yielded glycoproteins of up to 35.34 mg/L. Glycopeptide MS confirmed the preciset biosynthesis of glycoprotein. This glycoprotein elicited antigen-specific IgG immune responses and significantly reduced kidney and bladder colonization. This bacterial cell-based glyco-platform and optimized strategies can provide a guideline for the biosynthesis of other value-added glycoproteins.

A highly branched novel galactofuranan in the cell wall of Clavibacter tesselarius VKM Ac-1406T.

The structures of two cell wall glycopolymers were studied in the plant pathogenic bacterium Clavibacter tesselarius VKM Ac-1406T (family Microbacteriaceae, order Micrococcales, class Actinomycetes). The predominant polymer was a novel (1 â†’ 6)-linked ß-d-galactofuranan with a highly branched repeating unit, α-L-Rhap-(1 â†’ 3)-α-D-Galp-(1 â†’ 2)-[α-L-Rhap-(1 â†’ 3)]-α-D-Fucp-(1 →, at O-2 on every second galactofuranose residue. The second polymer present in small amounts was acidic with the repeating unit, →3)-α-D-Galp-(1 â†’ 3)-α-D-[4,6-S-Pyr]-Manp-(1 â†’ 3)-α-D-Manp-[2OAc]0.2-(1→, and was reported in all Clavibacter species investigated to date. The presented results expand our knowledges of structural diversity of phosphate-free cell wall glycopolymers and provide evidence in support of their taxonomic specificity for bacterial species and genera.

Structural and Serological Characterization of the O Antigen of Proteus mirabilis Clinical Isolates Classified into a New Proteus Serogroup, O84.

Two closely related Proteus mirabilis smooth strains, Kr1 and Ks20, were isolated from wound and skin samples, respectively, of two infected patients in central Poland. Serological tests, using the rabbit Kr1-specific antiserum, revealed that both strains presented the same O serotype. Their O antigens are unique among the Proteus O serotypes, which had been described earlier, as they were not recognized in an enzyme-linked immunosorbent assay (ELISA) by a set of Proteus O1-O83 antisera. Additionally, the Kr1 antiserum did not react with O1-O83 lipopolysaccharides (LPSs). The O-specific polysaccharide (OPS, O antigen) of P. mirabilis Kr1 was obtained via the mild acid degradation of the LPSs, and its structure was established via a chemical analysis and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy applied to both initial and O-deacetylated polysaccharides, where most ß-2-acetamido-2-deoxyglucose (N-acetylglucosamine) (GlcNAc) residues are non-stoichiometrically O-acetylated at positions 3, 4, and 6 or 3 and 6, and a minority of α-GlcNAc residues are 6-O-acetylated. Based on the serological features and chemical data, P. mirabilis Kr1 and Ks20 were proposed as candidates to a new successive O-serogroup in the genus Proteus, O84, which is another example of new Proteus O serotypes identified lately among serologically differentiated Proteus bacilli infecting patients in central Poland.

A novel cell wall galactofuranan in Clavibacter phaseoli VKM Ac-2641T.

A glycopolymer of novel structure was found in the cell wall of plant pathogen Clavibacter phaseoli VKM Ac-2641T (family Microbacteriaceae, class Actinomycetes). The glycopolymer was (1 â†’ 6)-linked ß-d-galactofuranan with side branched trisaccharide, α-D-Manp-(1 â†’ 2)-[α-D-Manp-(1 â†’ 3)]-α-D-Ribf-(1→ at O-2 on every second galactofuranose residue. The galactofuranan structure was established by chemical and NMR spectroscopic methods using one- and two-dimensional techniques 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC. The results of this study provide new data on diversity of bacterial glycopolymers, may prove useful for bacterial taxonomy and contribute to the understanding of the host plant-microbiota interaction mechanisms.

Complete chemical structure of the K135 capsular polysaccharide produced by Acinetobacter baumannii RES-546 that contains 5,7-di-N-acetyl-8-epipseudaminic acid.

A structurally diverse capsular polysaccharide (CPS) in the outer cell envelope plays an important role in the virulence of the important bacterial pathogen, Acinetobacter baumannii. More than 75 different CPS structures have been determined for the species to date, and many CPSs include isomers of a higher sugar, namely 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid. Recently, a novel isomer having the d-glycero-l-manno configuration (5,7-di-N-acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac) has been identified in the CPS from A. baumannii clinical isolate RES-546 [Carbohydr. Res. 513 (2022) 108,531]. Here, the complete chemical structure of this CPS, designated K135, was elucidated. The CPS was found to have a branched tetrasaccharide K unit and to include the higher sugar as part of a 8ePse5Ac7Ac-(2 â†’ 6)-α-Gal disaccharide branching from a →3)-α-D-GlcpNAc-(1 â†’ 3)-ß-D-GlcpNAc-(1→ main chain. Assignment of glycosyltransferases encoded by the CPS biosynthesis gene cluster in the RES-546 genome enabled the first sugar of the K unit, and hence the topology of the K135 CPS, to be determined.

NoteIdentification of 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-l-manno-non-2-ulosonic acid (di-N-acetyl-8-epipseudaminic acid) in the capsular polysaccharide of Acinetobacter baumannii Res546.

A structurally diverse capsular polysaccharide that surrounds the bacterial cell plays an important role in virulence of Acinetobacter baumannii, a cause of nosocomial infections worldwide. Various isomers of 5,7-diacylamido-3,5,7,9-tetradeoxynon-2-ulosonic acid have been identified as components of bacterial polysaccharides. In this work, we report on the identification of a new isomer having the d-glycero-l-manno configuration (8-epipseudaminic acid) in the capsular polysaccharide of A. baumannii Res546. The higher sugar was isolated by Smith degradation of the polysaccharide followed by mild acid hydrolysis and identified by a comparison with all isomers using NMR spectroscopy and optical rotation.

Cell wall galactofuranan and pyruvate-containing galactomannan in the cell walls of Clavibacter strains.

The cell wall glycopolymer structures of plant-associated strains Clavibacter sp. VKM Ac-1371, Clavibacter sp. VKM Ac-1372 and Clavibacter sp. VKM Ac-1374, members of three putative new species (family Microbacteriaceae, class Actinobacteria) were studied. Each strain was found to contain two glycopolymers, neutral and acidic ones. The main chain of neutral polymer, identical in all three strains, is (1 â†’ 6)-linked ß-d-galactofuranan with every second galactofuranose residue substituted at position 2 by side disaccharide, α-d-Manp-(1 â†’ 2)-α-d-Ribf-(1 â†’ . The second, acidic polymer, is pyruvate-containing galactomannan with the repeating unit, →3)-α-d-Galp-(1 â†’ 3)-α-d-[4,6-S-Pyr]-Manp-(1 â†’ 3)-α-d-Manp-(1 â†’ . Reducing mannopyranose residues of the acidic polysaccharides repeating unit from strains VKM Ac-1372 and VKM Ac-1374 bear O-acetyl residues additionally. The cell wall glycopolymer structures were established by chemical and NMR spectroscopic methods with using one- and two-dimensional techniques 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC. The results obtained provide new data on diversity of the bacterial cell wall glycopolymers and may prove valuable for microbial taxonomy and insight into the molecular mechanisms of interactions between bacteria and plants and also of bacterial adaptation to survival in desert systems.

A novel ItrA4 d-galactosyl 1-phosphate transferase is predicted to initiate synthesis of an amino sugar-lacking K92 capsular polysaccharide of Acinetobacter baumannii B8300.

The K92 capsular polysaccharide (CPS) from Acinetobacter baumannii B8300 was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The elucidated CPS includes a branched pentasaccharide repeat unit containing one d-Galp and four l-Rhap residues; an atypical composition given that all A. baumannii CPS structures determined to date contain at least one amino sugar. Accordingly, biosynthesis of A. baumannii CPS types are initiated by initiating transferases (Itrs) that transfer 1-phosphate of either a 2-acetamido-2-deoxy-d-hexose, a 2-acetamido-2,6-dideoxy-d-hexose or a 2-acetamido-4-acylamino-2,4,6-trideoxy-d-hexose to an undecaprenyl phosphate (UndP) carrier. However, the KL92 capsule biosynthesis gene cluster in the B8300 genome sequence includes a gene for a novel Itr type, ItrA4, which is predicted to begin synthesis of the K92 CPS by transferring D-Galp 1-phosphate to the UndP lipid carrier. The itrA4 gene was found in a module transcribed in the opposite direction to the majority of the K locus. This module also includes an unknown open reading frame (orfKL92), a gtr166 glycosyltransferase gene, and a wzi gene predicted to be involved in the attachment of CPS to the cell surface. Investigation into the origins of orfKL92-gtr166-itrA4-wziKL92 revealed it might have originated from Acinetobacter junii.

Novel galactofuranan and pyruvylated galactomannan in the cell wall of Clavibacter michiganensis subsp. michiganensis VKM Ac-1403T.

The cell wall of Clavibacter michiganensis subsp. michiganensis VKM Ас-1403Т (family Microbacteriaceae, class Actinobacteria) contains two polysaccharides. The first one is neutral (1 â†’ 6) linked galactofuranan in which every second galactofuranose residue in the main chain substituted at position 3 by side trisaccharide, ß-D-GlcpNAc-(1 â†’ 3)-α-L-Rhap-(1 â†’ 2)-α-D-Fucp-(1 â†’. The second polymer is pyruvylated galactomannan with the repeating unit, →3)-α-D-Galp-(1 â†’ 3)-α-D-[4,6-S-Pyr]-Manp-(1 â†’ 3)-α-D-Manp-(1 â†’. The cell wall glycopolymer structures were established by chemical and NMR spectroscopic methods. The obtained results provide new data on the cell wall composition of plant pathogenic species of the genus Clavibacter and can promote understanding the molecular mechanisms involved in colonization and infection of plants.

D-rhamnan and teichuronic acid from the cell wall of Rathayibacter caricis VKM Ac-1799T.

The cell wall of Rathayibacter caricis VKM Ac-1799T (family Microbacteriaceae, class Actinobacteria) was found to contain both neutral and acidic glycopolymers. The first one is D-rhamnopyranan with main chain →2)-α-D-Rhap-(1 â†’ 3)-α-D-Rhap-(1→, where a part of 2-substituted residues bears as a side-chain at position 3 α-D-Manp residues or disaccharides α-D-Araf-(1→2)-α-D-Manp-(1 â†’ . The second polymer is a teichuronic acid with a branched repeating units composed of seven monosaccharides →4)-α-[ß-D-Manp-(1 â†’ 3)]-D-Glcp-(1 â†’ 4)-ß-D-GlcpA-(1 â†’ 2)-ß-[4,6Pyr]-D-Manp-(1 â†’ 4)-ß-L-Rhap-(1 â†’ 4)-ß-D-Glcp-(1 â†’ 4)-ß-D-Glcp-(1 â†’ . The structures of the polymers were determined by chemical and NMR spectroscopic methods.

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.

K17 capsular polysaccharide produced by Acinetobacter baumannii isolate G7 contains an amide of 2-acetamido-2-deoxy-d-galacturonic acid with d-alanine.

The K17 capsular polysaccharide (CPS) produced by Acinetobacter baumannii G7, which carries the KL17 configuration at the capsule biosynthesis locus, was isolated and studied by chemical methods along with one- and two-dimensional 1H and 13C NMR spectroscopy. Selective cleavage of the glycosidic linkage of a 2,4-diacetamido-2,4,6-trideoxy-d-glucose (d-QuiNAc4NAc) residue by (i) trifluoroacetic acid solvolysis or (ii) alkaline ß-elimination (NaOH-NaBH4) of the 4-linked D-alanine amide of a 2-acetamido-2-deoxy-d-galacturonic acid residue (d-GalNAcA6DAla) yielded trisaccharides that were isolated by Fractogel TSK HW-40 gel-permeation chromatography and identified by using NMR spectroscopy and high-resolution electrospray ionization mass spectrometry. The following structure was established for the trisaccharide repeat (K unit) of the CPS: →4)-α-d-GalpNAcA6dAla-(1→4)-α-d-GalpNAcA-(1→3)-ß-d-QuipNAc4NAc-(1→ . The presence of the itrA1 gene coding for the initial glycosylphosphotransferase in the KL17 gene cluster established the first sugar of the K unit as d-QuipNAc4NAc. KL17 includes genes for three transferases that had been annotated previously as glycosyltransferases (Gtrs). As only two Gtrs are required for the K17 structure and one d-GalpNAcA residue is modified by a d-alanine amide, these assignments were re-assessed. One transferase was found to belong to the ATPgrasp_TupA protein family that includes d-alanine-d-alanine ligases, and thus was renamed Alt1 (alanine transferase). Alt1 represents a novel family that amidate the carboxyl group of d-GalpNAcA or d-GalpA.

New glycopolymers containing both D- and L-rhamnopyranoses from Rathayibacter iranicus VKM Ac-1602T cell wall.

The cell wall of Rathayibacter iranicus VKM Ac-1602T (family Microbacteriaceae, class Actinobacteria) is characterised by the absence of phosphate-containing and by the presence of two rhamnose-containing glycopolymers. The first is a branched rhamnomannan, in which 60% of mannose residues of the main chain are glycosylated by terminal mannose residues: →2)-α-D-Rhap-(1 → 3)-α-[α-D-Manp-(1 → 6)]-D-Manp-(1 → . The second is a branched teichuronic acid, in which all the rhamnose residues of the main chain are glycosylated by glucose residues:→3)-α-[α-D-Glcp-(1 → 2)]-L-Rhap-(1 → 4)-ß-D-GlcpA-(1 → 2)-α-D-Manp-(1 → 3)-α-D-Galp-(1 → 3)-ß-D-Glcp-(1 → . Both glycopolymers have the unique structures and described in the cell walls of Gram-positive bacteria for the first time. The obtained data allow for a more complete characterisation of the cell wall of the microorganism under investigation and can serve as a phenotypic characterisation of this bacterium. The glycopolymer structures were established using chemical and nuclear magnetic resonance (NMR) spectroscopy methods.

The K90 capsular polysaccharide produced by Acinetobacter baumannii LUH5553 contains di-N-acetylpseudaminic acid and is structurally related to the K7 polysaccharide from A. baumannii LUH5533.

Acinetobacter baumannii isolate LUH5553 carries the KL90 capsule gene cluster, which includes genes for three glycosyltransferases (Gtrs) and the ItrA3 initiating transferase, as well as a set of genes for synthesis of a higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid (Pse5Ac7Ac). The K90 capsular polysaccharide (CPS) has a tetrasaccharide repeat (K90 unit), which begins with d-GlcpNAc and contains Pse5Ac7Ac. The higher sugar was cleaved by mild acid hydrolysis of the CPS, and structures of the initial and modified polysaccharides were established by 1D and 2D 1H and 13C NMR spectroscopy. K90 contains α-d-Galp-(1 → 6)-d-GlcpNAc and α-d-GlcpNAc-(1 → 3)-d-GlcpNAc fragments, and formation of these glycosidic linkages is catalysed respectively by Gtr14 and Gtr15. The gtr14 and gtr15 genes occur in several A. baumannii KL gene clusters, including KL5 and KL7 that carry itrA2 rather than itrA3. As ItrA2 introduces d-GalpNAc rather than d-GlcpNAc as the first monosaccharide, Gtr15 can transfer d-GlcpNAc to either of these amino sugars, suggesting that this enzyme has relaxed specificity. Consequently, the third, novel glycosyltransferase, Gtr163, forms the ß-(2 → 3) linkage between Pse5Ac7Ac and d-Galp. Wzy polymerases encoded by KL90 and KL7 are 54% identical and form the same linkage between the K units to give branched polysaccharides with the same main chain but different disaccharide side chains, ß-Pse5Ac7Ac-(2 → 3)-d-Galp in K90 and α-Leg5Ac7Ac-(2 → 6)-d-Galp in K7.

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.

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 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.

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.