Rathayibacter festucae VKM Ac-1390T (family Microbacteriaceae, class Actinomycetes) contains three rhamnose-containing glycopolymers in the cell wall, the structures of which were established by chemical and NMR spectroscopy methods. The first polymer, a rhamnomannan, consists of repeating tetrasaccharide units with xylopyranose side residues, →2)-α-[β-D-Xylp-(1→3)]-D-Rhap-(1→3)-α-D-Manp-(1→2)-α-D-Rhap-(1→3)-α-D-Manp-(1→. The second polymer found in minor amounts, is a rhamnan, →2)-α-D-Rhap-(1→3)-α-D-Rhap-(1→. The third polymer is a teichuronic acid acetalated with pyruvic acid, →2)-α-[4,6-S-Pyr]-D-Manp-(1→4)-α-L-Rhap-(1→4)-β-D-Glcp-(1→4)-α-D-Glcp-(1→4)-β-D-GlcpA-(1→. The structures of rhamnomannan and teichuronic acid are new for Ratayibacter and prokaryotes in a whole. The results of this study expand our understanding of the structural diversity of microbial glycopolymers and are consistent with previous reports on the species specificity of glycopolymer structures and composition in the genus Rathayibacter.

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-[β-D-Xylp-(1→3)]-D-Manp-(1→3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1→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→3)]-D-Manp-(1→3)-α-D-[4,6-R-Pyr]-D-Galp-(1→3)-β-D-Glcp-(1→. 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.

ABSTRACT The carbapenem-resistant Acinetobacter baumannii isolate BAL062 is a clinical reference isolate used in several recent experimental studies. It is from a ventilator-associated pneumonia (VAP) patient in an intensive care unit at the Hospital for Tropical Diseases (HTD), Ho Chi Minh City, Vietnam in 2009. Here, BAL062 was found to belong to the B sub-lineage of global clone 2 (GC2) isolates in the previously reported outbreak (2008 and 2012) of carbapenem-resistant VAP A. baumannii at the HTD. While related sub-lineage B outbreak isolates were extensively antibiotic-resistant and carry GC2-associated genomic resistance islands, AbGRI1, AbGRI2, and AbGRI3, BAL062 has lost AbGRI3 and three aminoglycoside resistance genes, armA, aacA4, and aphA1 , leading to amikacin, tobramycin and kanamycin susceptibility. The location of Tn 2008 VAR found in the chromosome of this sub-lineage was also corrected. Like many of the outbreak isolates, BAL062 carries the KL58 gene cluster at the capsular polysaccharide (CPS) synthesis locus and an annotation key is provided. As information about K type is important for the development of novel CPS-targeting therapies, the BAL062 K58-type CPS structure was established using NMR spectroscopy. It is most closely related to K2 and K93, sharing similar configurations and linkages between K units, and contains the rare higher monosaccharide, 5,7-diacetamido-3,5,7,9-tetradeoxy- d - glycero - l - manno -non-2-ulosonic acid (5,7-di- N -acetyl-8-epipseudaminic acid; 8ePse5Ac7Ac), the 8-epimer of Pse5Ac7Ac (5,7-di- N -acetylpseudaminic acid). Inspection of publicly available A. baumannii genomes revealed a wide distribution of the KL58 locus in geographically diverse isolates belonging to several sequence types that were recovered over two decades from clinical, animal, and environmental sources. IMPORTANCE Many published experimental studies aimed at developing a clearer understanding of the pathogenicity of carbapenem-resistant Acinetobacter baumannii strains currently causing treatment failure due to extensive antibiotic resistance are undertaken using historic, laboratory-adapted isolates. However, it is ideal if not imperative that recent clinical isolates are used in such studies. The clinical reference isolate characterized here belongs to the dominant A. baumannii GC2 clone causing extensively resistant infections and has been used in various recent studies. The correlation of resistance profiles and resistance gene data is key to identifying genes available for gene knockout and complementation analyses, and we have mapped the antibiotic resistance genes to find candidates. Novel therapies, such as bacteriophage or monoclonal antibody therapies, currently under investigation as alternatives or adjuncts to antibiotic treatment to combat difficult-to-treat CRAb infections often exhibit specificity for specific structural epitopes of the capsular polysaccharide (CPS), the outer-most polysaccharide layer. Here, we have solved the structure of the CPS type found in BAL062 and other extensively resistant isolates. As consistent gene naming and annotation are important for locus identification and interpretation of experimental studies, we also have correlated automatic annotations to the standard gene names.

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 → 6)-linked β-d-galactofuranan with side branched trisaccharide, α-D-Manp-(1 → 2)-[α-D-Manp-(1 → 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.

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 glycoplatform and optimized strategies can provide a guideline for the biosynthesis of other value-added glycoproteins.

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 β-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 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 → 6)-α-Gal disaccharide branching from a →3)-α-D-GlcpNAc-(1 → 3)-β-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.

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 → 6)-linked β-d-galactofuranan with a highly branched repeating unit, α-L-Rhap-(1 → 3)-α-D-Galp-(1 → 2)-[α-L-Rhap-(1 → 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 → 3)-α-D-[4,6-S-Pyr]-Manp-(1 → 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.

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.

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.

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 → 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 → . The second polymer is a teichuronic acid with a branched repeating units composed of seven monosaccharides →4)-α-[β-D-Manp-(1 → 3)]-D-Glcp-(1 → 4)-β-D-GlcpA-(1 → 2)-β-[4,6Pyr]-D-Manp-(1 → 4)-β-L-Rhap-(1 → 4)-β-D-Glcp-(1 → 4)-β-D-Glcp-(1 → . The structures of the polymers were determined by chemical and NMR spectroscopic methods.

The cell wall of Clavibacter michiganensis subsp. michiganensis VKM Ас-1403Т (family Microbacteriaceae, class Actinobacteria) contains two polysaccharides. The first one is neutral (1 → 6) linked galactofuranan in which every second galactofuranose residue in the main chain substituted at position 3 by side trisaccharide, β-D-GlcpNAc-(1 → 3)-α-L-Rhap-(1 → 2)-α-D-Fucp-(1 →. The second polymer is pyruvylated galactomannan with the repeating unit, →3)-α-D-Galp-(1 → 3)-α-D-[4,6-S-Pyr]-Manp-(1 → 3)-α-D-Manp-(1 →. 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.

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 → 6)-linked β-d-galactofuranan with every second galactofuranose residue substituted at position 2 by side disaccharide, α-d-Manp-(1 → 2)-α-d-Ribf-(1 → . The second, acidic polymer, is pyruvate-containing galactomannan with the repeating unit, →3)-α-d-Galp-(1 → 3)-α-d-[4,6-S-Pyr]-Manp-(1 → 3)-α-d-Manp-(1 → . 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.

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 09. 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-GIcNAc as well as glycerol phosphate. It had the following unique structure which was established by NMR spectroscopy applied to the initial and dephosphorylated polysaccharides: [GRAPHICS] The polysaccharide eluted from the gel second was identified as a mannan with a -> 3)-beta-D-Manp- (1 -> 2)-alpha-D-Manp-(1 -> 2)-alpha-D-Manp-(1 -> trisaccharide repeating unit. In E. albertii EA046, two polysac- charide 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. (C) 2019 Elsevier B.V. All rights reserved.

KL55, KL74, and KL85 capsular polysaccharide (CPS) biosynthesis loci in Acinetobacter baumannii BAL_204, BAL_309, and LUH5543 genomes, respectively, are related and each contains genes for l-Rhap and d-GlcpA synthesis. The CPSs were isolated and studied by sugar analysis, Smith degradation, and 1H and 13C NMR spectroscopy. The K55 and K74 CPSs are built up of branched octasaccharide repeats (K units) containing one residue each of d-GlcpA and d-GlcpNAc and six residues of l-Rhap. The K55 unit differs from the K74 unit in the linkage between D-GlcpA and an l-Rhap residue in the K unit (1 → 3 versus 1 → 2) and linkage between K units. However, most K units in the isolated K74 CPS were modified by β-elimination of a side-chain α-l-Rhap-(1 → 3)-α-l-Rhap disaccharide from position 4 of GlcA to give 4-deoxy-l-threo-hex-4-enuronic acid (1:~3 ratio of intact and modified units). The K85 CPS has a branched heptasaccharide K unit similar to the K74 unit but with one fewer α-l-Rhap residue in the side chain. In contrast to previous findings on A. baumannii CPSs, each K locus includes fewer glycosyltransferase (Gtr) genes than the number required to form all linkages in the K units. Hence, one Gtr appears to be multifunctional catalysing formation of two 1 → 2 and one 1 → 3 linkages between the l-Rha residues.

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 β-(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, β-Pse5Ac7Ac-(2 → 3)-d-Galp in K90 and α-Leg5Ac7Ac-(2 → 6)-d-Galp in K7.

This review is devoted to methods for the selective cleavage of glycosidic bonds. The mechanisms of reactions underlying these methods are considered and examples of their practical application in the structural analysis of bacterial polysaccharides are given. Specific methods for the selective cleavage of polysaccharides, remaining relevant for researchers, include the Smith degradation based on destruction of monosaccharides containing vicinal diol groups, dephosphorylation of phosphate-containing polysaccharides with hydrofluoric acid and the hydrolytic cleavage of glycosyl phosphate bonds in the latter compounds. Non-specific methods, including partial acid hydrolysis, acetolysis and solvolysis with anhydrous organic (CF3SO3H, MeSO3H, CF3CO2H) and inorganic (HF) acids do not make any specific demands on the composition and structure of the polysaccharide and are sensitive to its fine structural features. The review addesses the issue of stability of glycosidic bonds in various monosaccharides to reagents used for non-specific selective cleavage. The bibliography includes 58 references.

This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.

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)-β-D-GlcpA-(1 → 2)-α-D-Manp-(1 → 3)-α-D-Galp-(1 → 3)-β-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 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 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.