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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. [ABSTRACT FROM AUTHOR]

Background O-antigen (O-polysaccharide) of the lipopolysaccharide is a highly variable cell component of the outer membrane in Shigella flexneri. It defines the serospecificity and plays an important role in the pathogenesis of shigellosis. There are two distinct O-antigen forms for the 19 serotypes of S. flexneri: one for serotypes 1-5, X, Y, 7 (and their subtypes), and the other for serotype 6. Although having different basal O-polysaccharide structures, the two forms share a common disaccharide fragment [→2)-α-L-RhapIII-(1 → 2)-α-L-RhapII]. In serotype 6 and some non-6 serotypes, RhaIII is O-acetylated at position either 3 or 4 (3/4-Oacetylation), conferring to the hosts a novel antigenic determinant named O-factor 9. An acyltransferase gene (oacB) responsible for this modification has been identified in serotypes 1a, 1b, 2a, 5a, and Y, but not in serotype 6. Results Using genetic, serological, and chemical approaches, another acyltransferase gene named oacC was demonstrated to be responsible for the 3/4-O-acetylation on RhaIII in the O-antigen of S. flexneri serotype 6. Inactivation of the oacC gene resulted in the loss of the 3/4-Oacetyltion, and the cloned oacC gene restored this modification upon transformation. In accordance with the similarity in the acceptor substrate structure and high sequence homology (72% identity) between oacC and oacB, oacC has the interchangeable function with the oacB gene in mediation of the 3/4-O-acetylation. The oacC gene is located in a prophage on the chromosome and presented in all 77 serotype 6 strains tested. Conclusions Identification and functional characterization of the O-acetyltransferase encoding gene, oacC, shows that it is involved in O-antigen modification by 3/4-O-acetylation on RhaIII specific to serotype 6. [ABSTRACT FROM AUTHOR]

The O-antigens of all Shigella flexneri serotypes, except serotype 6, share a linear tetrasaccharide repeat composed of one N-acetylglucosamine and three l-rhamnose residues, and differences between the serotypes are due to modification of various monosaccharide residues with glucosyl and/or O-acetyl and/or phosphoethanolamine (PEtN) groups. Plasmid-borne opt (formerly lpt-O) gene encoding a PEtN transferase which modifies the O-antigens of S. flexneri serotype X, 4a and Y strains and converts the hosts into MASF IV-1 (E1037) positive “variant” (v) Xv, 4av and Yv serotypes, respectively. In this study, we showed that the opt-carrying plasmid pSFxv_2 can transform strains of all S. flexneri serotypes (1–6) to confer them with the MASF IV-1 epitope recognized by monoclonal antibody MASF IV-1 and typing antiserum IV. The transformants possessed modified O-antigens with a PEtN group(s) at position 3 of one or two rhamnose residues. In some serotypes, the PEtN modification competed or/and interfered with glucosylation and O-acetylation at the same or its neighboring sugar residue. We also showed that the plasmid pSFxv_2 is mobilizable to other S. flexneri strains by conjugation. Although pSFxv_2-harboring S. flexneri strains found in clinical infections are restricted to serotypes Xv, 4av, Yv and, possibly, 6v, our results demonstrate a high potential of dissemination of this plasmid in S. flexneri and emergence of new S. flexneri serotypes. [ABSTRACT FROM AUTHOR]

Shigella flexneri is the major human pathogen causing shigellosis. O-antigens of all S. flexneri serotypes (except for serotype 6) share the →2)-α-l-RhapIII-(1 → 2)-α-l-RhapII-(1 → 3)-α-l-RhapI-(1 → 3)-β-d-GlcpNAc-(1→ basic O-unit, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation at various positions. Recently, in serotype X and 4a variants called Xv and 4av, respectively, O-antigen modification with phosphoethanolamine (PEtN) has been identified, which is encoded by a plasmid-borne gene (lpt-O) for a PEtN-transferase and confers the monoclonal antibody IV-1(MASF IV-1) determinant to the bacteria. In this study, we elucidated the O-antigen structure of serotype Yv, another MASF IV-1-positive novel variant of S. flexneri. The serotype Yv O-antigen has the same basic carbohydrate backbone structure as that of the “classical” serotype Y, but differs in the presence of PEtN at position 3 of RhaIII (major) or both RhaII and RhaIII (minor). This pattern is similar to that of serotype 4av, but different from the pattern of serotype Xv, which is characterized by major PEtN modification on RhaII. In serotype Yv, mono- and bisphosphorylated O-units generate a block-copolymeric structure, the former being partially O-acetylated at position 6 of GlcNAc and the latter lacking O-acetylation. Functional analysis revealed a correlation between the serotype-specific PEtN modification pattern and the lpt-O variation in different serotypes: lpt-ORII in serotype Xv is better tuned for phosphorylation of RhaII and lpt-ORIII in serotypes Yv and 4av for phosphorylation of RhaIII. These data enhance our knowledge of S. flexneri serotype conversion mechanisms and help to understand the biosynthesis process of the new O-antigen variants. [ABSTRACT FROM AUTHOR]

The O-antigen is one of the most variable Gram-negative cell constituents, and its specificity is important for bacterial niche adaptation. The observed diversity of O-antigen forms is mainly due to genetic variations in O-antigen gene clusters. Less common is a change of gene function due to nucleotide substitution; a new instance of which is reported here. The O-antigens of E. coli O107 and O117 have similar structures differing only in a single sugar residue (GlcNAc in O107 substituted for Glc in O117). These O-antigen gene clusters contain the same set of 11 genes and share 98.6% overall DNA identity. The function of the genes in the gene clusters have been proposed previously, and a glycosyltransferase gene (wclY) with nucleotide polymorphism in each strain was proposed to transfer different sugars in different strains. To identify the gene responsible for the transfer of different sugars, wclY mutants of E. coli O107 and O117 were constructed, and each mutant was complemented with the wclY genes cloned from both O107 and O117. Structural analysis of the O-antigens of the four recombinant strains identified wclY as a Glc-transferase in O117 and a GlcNAc-transferase in O107. The evolutionary relationship of E. coli O107 and O117 O-antigens is also discussed. [ABSTRACT FROM PUBLISHER]

This review is devoted to structural and serological characteristics of the O-antigens (O-polysaccharides) of the lipopolysaccharides of various Proteus species, which provide the basis for classifying Proteus strains to Oserogroups. The antigenic relationships of Proteus strains within and beyond the genus as well as their O-antigenrelated bioactivities are also discussed. [ABSTRACT FROM AUTHOR]

O-antigen (O-polysaccharide), a part of the outer membrane of Gram-negative bacteria, is one of the most variable cell constituents and is related to bacterial virulence. O-antigen diversity is almost entirely due to genetic variations in O-antigen gene clusters. In this study, the O-polysaccharide structures of Salmonella O55 and Escherichia coli O103 were elucidated by chemical analysis and nuclear magnetic resonance spectroscopy. It was found that the O-polysaccharides have similar pentasaccharide O-units, which differ only in one sugar (glucose versus N-acetylglucosamine) and in the N-acyl group (acetyl versus 3-hydroxybutanoyl) on 3-amino-3,6-dideoxy-d-galactose (d-Fuc3N). The Salmonella O55 antigen gene cluster was sequenced and compared with the E. coli O103 antigen gene cluster reported previously. The two gene clusters were found to share high-level similarity (DNA identity ranges from 53% to 76%), except for two putative acyl transferase genes (fdtC in Salmonella O55 and fdhC in E. coli O103) which show no similarity. Replacement of the fdtC gene in Salmonella O55 with the fdhC gene from E. coli O103 resulted in production of a modified O-antigen, which contains a 3-hydroxybutanoyl derivative of Fuc3N in place of 3-acetamido-3,6-dideoxygalactose. This finding strongly suggests that fdhC is a 3-hydroxybutanoyltransferase gene. The sequence similarity level suggested that the O-antigen gene clusters of Salmonella O55 and E. coli O103 originate from a common ancestor, and this evolutionary relationship is discussed. [ABSTRACT FROM PUBLISHER]

O-antigen is part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, and contributes the major antigenic variability to the cell surface. Screening for the Escherichia coli O-serogroup is the conventional method for identifying E. coli clones. In this study, we investigated the structural characteristics of the E. coli O99 O-antigen and the organization of the genes involved in its synthesis. On the basis of sugar and methylation analysis and nuclear magnetic resonance spectroscopy data, we established the structure of the branched hexasaccharide repeat unit of the O-polysaccharide. This unit consists of fourd-rhamnose (d-Rha) moieties in the backbone and twod-glucose (d-Glc) moieties in the side chain, as shown below: The O-antigen gene cluster of E. coli O99, which was located between galF and gnd, was found to contain putative genes for the synthesis ofd-Rha, genes encoding sugar transferases, and ATP-binding cassette (ABC) transporter genes ( wzm and wzt). Our findings indicate that in E. coli O99, the synthesis and translocation of the O-antigen occurs by an ABC transporter-dependent process. [ABSTRACT FROM AUTHOR]

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β-d-Gal pNAc-(1→3)-α-l-Rha p-(1→4)-α-d-Glc pNAc-(1→4)-β-d-Gal p-(1→3)-α-d-Gal pNAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution ofd-GlcNAc ford-Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 (d-GlcNAc) and O117 (d-Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations. [ABSTRACT FROM AUTHOR]

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to containd-glucose,d-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a β configuration, except for the side-chain Glc, which is α. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti- C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue. [ABSTRACT FROM AUTHOR]

The review is devoted to recent progress in the structural elucidation of the lipopolysaccharide of the bacterium Pseudomonas aeruginosa, including O-antigen biological repeats, core oligosaccharide, and lipid A. Data on biosynthesis, genetics and serology of the lipopolysaccharide isolated from various P. aeruginosa O-serogroups are discussed in relation to the chemical structures. [ABSTRACT FROM AUTHOR]

The review is devoted to recent progress in the structural elucidation of the lipopolysaccharide of the bacterium Pseudomonas aeruginosa, including O-antigen biological repeats, core oligosaccharide, and lipid A. Data on biosynthesis, genetics and serology of the lipopolysaccharide isolated from various P. aeruginosa O-serogroups are discussed in relation to the chemical structures. [ABSTRACT FROM PUBLISHER]

O-Polysaccharides were obtained from the lipopolysaccharides of Proteus mirabilis CCUG 10704 (OE) and Proteus vulgaris TG 103 and studied by chemical analyses and one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy, including rotating-frame nuclear Overhauser effect spectroscopy, H-detected 1H,13C heteronuclear single-quantum spectroscopy and 1H,31P heteronuclear multiple-quantum spectroscopy experiments. The Proteus mirabilis OE polysaccharide was found to have a trisaccharide repeating unit with a lateral glycerol phosphate group. The Proteus vulgaris TG 103 produces a similar O-polysaccharide, which differs in incomplete substitution with glycerol phosphate ( c. 50% of the stoichiometric amount) and the presence of an O-acetyl group at position 6 of the 2-acetamido-2-deoxygalactose (GalNAc) residue. These structures are unique among the known bacterial polysaccharide structures. Based on the structural and serological data of the lipopolysaccharides, it is proposed to classify both strains studied into a new Proteus serogroup, O54, as two subgroups, O54a,54b and O54a,54c. The serological relatedness of the Proteus O54 and some other Proteus lipopolysaccharides is discussed. [ABSTRACT FROM AUTHOR]

Highly phosphorylated core oligosaccharides and those substituted with one O-antigen repeating unit were obtained by mild acid degradation or strong alkaline hydrolysis of lipopolysaccharide samples from 23 reference strains representing all Pseudomonas aeruginosa O-serogroups. Studies by high-resolution electrospray ionization mass spectrometry and two-dimensional NMR spectroscopy revealed both conserved and variable structural features of the lipopolysaccharides of various O-serogroups. The upstream terminal saccharide of the O-antigen, which contributes most to the immunospecificity of the bacteria, was defined in 11 from a total of 13 O-serogroups. The data obtained link together the known biosynthesis pathways, genetics and serology of the P. aeruginosa lipopolysaccharide. [ABSTRACT FROM AUTHOR]

An acidic branched O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide (LPS) of Proteus genomospecies 4 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected 1H,13C HSQC experiments. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established, which is unique among Proteus polysaccharide structures: where Qui3NAc stands for 3-acetamido-3,6-dideoxyglucose. Based on the O-polysaccharide structure and serological data, we propose classifying Proteus genomospecies 4 into a new, separate Proteus serogroup, O56. A weak cross-reactivity of Proteus genomospecies 4 antiserum with LPS of Providencia stuartii O18 and Proteus vulgaris OX2 was observed and is discussed in view of a similarity of the O-polysaccharide structures. Structural and serological investigations showed that Proteus genomospecies 5 and 6 should be classified into the existing Proteus serogroups O8 and O69, respectively. [ABSTRACT FROM AUTHOR]

Abstract: An alkali-treated lipopolysaccharide of Proteus penneri strain 60 was studied by chemical analyses and 1H, 13C and 31P NMR spectroscopy, and the following structure of the linear pentasaccharide–phosphate repeating unit of the O-polysaccharide was established:Rabbit polyclonal O-antiserum against P. penneri 60 reacted with both core and O-polysaccharide moieties of the homologous LPS. Based on the unique O-polysaccharide structure and serological data, we propose to classify P. penneri 60 into a new, separate Proteus serogroup O70. A weak cross-reactivity of P. penneri 60 O-antiserum with the lipopolysaccharide of Proteus vulgaris O8, O15 and O19 was observed and discussed in view of the chemical structures of the O-polysaccharides. [Copyright &y& Elsevier]

Abstract: The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 75 consists of tetrasaccharide-ribitol phosphate repeating units and resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was elucidated by chemical methods and 1H and 13C NMR spectroscopy: where Rib-ol is ribitol. Serological studies with polyclonal antisera showed that the same structure of the O-polysaccharide occurred in two strains: P. penneri 75 and 128. A similar structure has been established earlier for the O-polysaccharide of P. penneri 103 [Drzewiecka, D., et al., Carbohydr. Res. 337 (2002) 1535–1540]. On the basis of complex serological investigations with use of two polyclonal P. penneri 75 and 103 O-antisera, five strains could be classified into Proteus O73 serogroup: P. penneri 48, 75, 90, 103 and 128, two of which (P. penneri 75 and 128) should be subdivided into subgroup 73a, 73b and three others (P. penneri 48, 90 and 103) into subgroup 73a, 73c. Epitopes responsible for the cross-reactivity of P. penneri O73 strains and a related strain of P. mirabilis O20 were tentatively defined. [Copyright &y& Elsevier]

The O-polysaccharide of Providencia stuartii O33 was obtained by mild acid degradation of the lipopolysaccharide and the following structure of the tetrasaccharide repeating unit was established:→ 6)-α-d-GlcpNAc-(1 → 4)-α-d-GalpA-(1 → 3)-α-d-GlcpNAc-(1 → 3)-β-d-Quip4N(Ac-d-Asp)-(1 →, where d-Qui4N(Ac-d-Asp) is 4-(N-acetyl-d-aspart-4-yl)amino-4,6-dideoxy-d-glucose. Structural studies were performed using sugar and methylation analyses and NMR spectroscopy, including conventional 2D 1H, 1H COSY, TOCSY, NOESY and 1H, 13C HSQC experiments as well as COSY and NOESY experiments in an H2O–D2O mixture to reveal correlations for NH protons. The O-polysaccharide of P. stuartii O33 shares an α-d-Glcp NAc-(1 → 3)-β-d-Quip4N(Ac-d-Asp) epitope with that of Proteus mirabilis O38, which seems to be responsible for a marked serological cross-reactivity of anti-P. stuartii O33 serum with the lipopolysaccharide of the latter bacterium. P. stuartii O33 is serologically related also to P. stuartii O4, whose O-polysaccharide contains a lateral β-d-Qui4N(Ac-l-Asp) residue. [Copyright &y& Elsevier]

The lipopolysaccharides (LPS) of Proteus penneri 28 and Proteus vulgaris O31 (PrK 55/57) were degraded with dilute acetic acid and structurally identical high-molecular-mass O-polysaccharides were isolated by gel-permeation chromatography. Sugar analysis and nuclear magnetic resonance (NMR) spectroscopic studies showed that both polysaccharides contain D-GlcNAc, 2-acetamido-2,6-dideoxy-L-glucose (L-2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine)) and 2-acetamido-3-O-[(S)-1-carboxyethyl]-2-deoxy-D-glucose (N-acetylisomuramic acid) and have the following structure: where (S)-1-carboxyethyl [a residue of (S)-lactic acid] (S-Lac) is an ether-linked residue of (S)-lactic acid. The O-polysaccharide studied is structurally similar to that of P. penneri 26, which differs only in the absence of S-Lac from the GlcNAc residue. Based on the O-polysaccharide structures and serological data of the LPS, it was suggested classifying these strains in one Proteus serogroup, O31, as two subgroups: O(31a), 31b for P. penneri 28 and P. vulgaris PrK 55/57 and O31a for P. penneri 26. A serological relatedness of the LPS of Proteus O(31a), 31b and P. penneri 62 was revealed and substantiated by sharing epitope O31b, which is associated with N-acetylisomuramic acid. It was suggested that a cross-reactivity of P. penneri 28 O-antiserum with the LPS of several other P. penneri strains is due to a common epitope(s) on the LPS core. [Copyright &y& Elsevier]

O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide (LPS) of Proteus penneri strain 31. Sugar and methylation analyses along with NMR spectroscopic studies, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C and 1H,31P HMQC experiments, demonstrated the following structure of the polysaccharide: where FucNAc is 2-acetamido-2,6-dideoxygalactose and EtnP is 2-aminoethyl phosphate. The polysaccharide studied has the same carbohydrate backbone as the O-polysaccharide of Proteus vulgaris O19. Based on this finding and close serological relatedness of the LPS of the two strains, it is proposed to classify P. penneri 31 in Proteus serogroup O19 as an additional subgroup. In contrast, D-GlcNAc6PEtn and α-L-FucNAc-(1→3)-D-GlcNAc shared with a number of other Proteus O-polysaccharides could not provide any significant cross-reactivity of the corresponding LPS with rabbit polyclonal O-antiserum against P. penneri 31. [Copyright &y& Elsevier]

Mild acid degradation of the lipopolysaccharide of Citro- bacter gillenii O9a,9b released a polysaccharide (PS), which was found to consist of a single monosaccharide, 4-acetamido-4,6-dideoxy-d-mannose (d-Rha4NAc, N -acetyl-d-perosamine). PS was studied by methylation analysis␣and 1 H-NMR and 13 C-NMR spectroscopy, using two-dimensional 1 H,1 H COSY, TOCSY, NOESY, and H-detected 1 H,13 C heteronuclear correlation experiments. It was␣found that PS includes two structurally different polysaccharides: an α1→2-linked homopolymer of N -acetyl-d-perosamine [→2)-α-d-Rhap 4NAc-(1→, PS2] and a␣polysaccharide composed of tetrasaccharide repeating units (PS1) with the␣following structure: →3)-α-d-Rhap 4NAc-(1→2)-α-d-Rhap 4NAc-(1→2)-α-d-Rhap 4NAc-(1→3)-α-d-Rhap 4N Ac2Ac-(1→ where the degree of O-acetylation of a 3-substituted Rha4NAc residue at position 2 is ≈ 70%. PS could be fractionated into PS1 and PS2 by gel-permeation chromatography on TSK HW-50S. Matrix-assisted laser desorption ionization MS data indicate sequential chain elongation of both PS1 and PS2 by a single sugar unit, with O-acetylation in PS1 beginning at a certain chain length. Anti-(C. gillenii O9a,9b) serum reacted with PS1 in double immunodiffusion and immunoblotting, whereas neither PS2 nor the lipopolysaccharide of Vibrio cholerae O1 with a structurally related O-chain polysaccharide were reactive. [ABSTRACT FROM AUTHOR]

The following structure of the O‐specific polysaccharide (OPS) of Proteus vulgaris O45 lipopolysaccharide (LPS) was established using 1H‐ and 13C‐NMR spectroscopy, including two‐dimensional NOESY and H‐detected 1H, 13C heteronuclear multiple‐quantum coherence (HMQC) experiments: &figfu1; Immunochemical studies, using rabbit polyclonal anti‐P. vulgaris O45 serum and LPS, OPS and Smith‐degraded OPS of P. vulgaris O45, showed the importance of β‐d‐GlcA in manifesting the serological specificity of the O‐antigen studied. [ABSTRACT FROM AUTHOR]

The following structure of the O-specific polysaccharide chain (O-antigen) of the Proteus vulgaris O32 lipopolysaccharide (LPS) was established by 1H-NMR and 13C-NMR spectroscopy, including two-dimensional NOESY and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments : ←2)-α-L-RhapI-(1←2)-α-L-RhapII-(1←4)-β-D-GalpAI-(1←3)-β-D-GlcpNAc-(1←4)-α-D-GalpAII-(1←. In addition, an O-acetyl group was detected, which, most probably, is located at position 3 of a part of RhapI residues. Serological studies, using rabbit polyclonal anti-(P. vulgaris O32) serum, homologous and heterologous Proteus O-antigens and related artificial antigens, revealed the importance of an α-D-GalA-associated epitope in manifesting the immunospecificity of P. vulgaris O32 and substantiated serological relationships between the O-antigen studied and those of some other Proteus strains. [ABSTRACT FROM AUTHOR]

A neutral O-specific polysaccharide obtained from the lipopolysaccharide of Proteus penneri strain 26 was studied using sugar analysis and 1H and 13C NMR spectroscopy, including two-dimensional NMR techniques. The following structure of the trisaccharide repeating unit was established : ←6)-α-D-GlcpNAc-(1←3)-α-L-QuipNAc-(1←3)-α-D-GlcpNAc-(1← where L-QuiNAc is 2-acetamido-2,6-dideoxy-L-glucose (N-acetyl-L-quinovosamine). Cross-reactivity of the Proteus penneri 26 anti-O serum with other strains of P. penneri isolated in Poland and USA and one strain of P. vulgaris is discussed. [ABSTRACT FROM AUTHOR]

On the basis of chemical analyses and NMR spectroscopic studies, it was found that the O-specific polysaccharide (O-PS) isolated from the Hafnia alvei PCM 1199 lipopolysaccharide (LPS) is a glycerol teichoic-acid-like polymer having a repeating unit of the following structure : 3)-β-D-Quip4NAc-(1←1)-Gro3P-(O←3)-β-D-Galp-(1←3)-α-D-GlcpNAc-(1← 2 6 ↑ ∣ 1 OAc β-D-GlcpNAc 6 ∣ OAc where Qui4NAc is 4-acetamido-4,6-dideoxyglucose and O-acetylation at both positions is non-stoichiometric. The glycosidic linkage of the lateral β-D-GlcpNAc residue is acid-labile and cleaved from the OPS during mild acid hydrolysis or dephosphorylation with 48 % hydrofluoric acid. Comparative analysis revealed that the structure of the H. alvei PCM 1199 O-PS is similar to that of H. alvei PCM 1205, which differs in the presence of an additional lateral α-D-Glcp residue and the position of one of the O-acetyl groups only. Accordingly, serological tests revealed a high degree of serological similarity between LPSs and O-PSs of the two H. alvei strains. [ABSTRACT FROM AUTHOR]

O-specific polysaccharide was isolated from Proteus penneri strain 12 (ATCC 33519) lipopolysaccharide (LPS) and studied using NMR spectroscopy, including selective spin-decoupling, one-dimensional NOE, two-dimensional homonuclear correlation spectroscopy, 13C,¹H heteronuclear correlation spectroscopy and chemical methods (O-deacetylation, Smith degradation, partial acid hydrolysis followed by borohydride reduction and methylation). The amide of D-galacturonic acid with L-threonine [D-GalA(L-Thr)] was identified as a constituent of the polysaccharide and the following structure of the tetrasaccharide repeating unit was established: [This equation cannot be represented into ASCII Text] where the degree of O-acetylation at either position varies over 20&ndash40%. Serological study with LPS, its degradation products and related synthetic glyeoconjugates (2-acrylamidoethyl glycosides of amides of α-D-GalA with L-amino acids copolymerised with acrylamide) showed that D-GalA(L-Thr) plays an important role in manifesting the serological specificity of the penneri 12 O-antigen. Serological cross reactions between LPSs of P. penneri 12 and Proteus mirabilis S1959, R14/S1959 (transient-like form), O23 and O28 are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The O-polysaccharide of Vibrio cholerae O43 was studied using chemical analyses, triflic acid solvolysis and 2D NMR spectroscopy, including 1H/1H COSY, TOCSY, NOESY and 1H/13C gradient-selected HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: →3)-β-d-Quip4NAcyl-(1→3)-α-d-GalpNAcA-(1→4)-α-d-GalpNAc-(1→3)-α-d-QuipNAc-(1→ where d-QuiNAc stands for 2-acetamido-2,6-dideoxy-d-glucose, d-Qui4NAcyl for 4-(N-acetyl-l-allothreonyl)amino-4,6-dideoxy-d-glucose and d-GalNAcA for 2-acetamido-2-deoxy-d-galacturonic acid. [Copyright &y& Elsevier]

The serological classification scheme of the opportunistic Proteus bacilli includes a number of Proteus penneri strains. The tested P. penneri 4034-85 strain turned out to be serologically distinguished in ELISA and Western blotting. The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of this strain and studied by sugar and methylation analyses and dephosphorylation along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, HMBC, and HSQC-TOCSY experiments, The O-polysaccharide was found to have a linear repeating unit containing glycerol 1-phosphate and two residues each of Gal and GlcNAc. The following O-polysaccharide structure was established, which, to our knowledge, is unique among known bacterial polysaccharide structures: →4)-β- d -Glc p NAc-(1→3)-α- d -Gal p -(1→3)-β- d -Glc p NAc-(1→2)-β- d -Gal p -(1→3)-Gro-1- P -(O→. We identified a unique O-specific polysaccharide from a clinical Proteus penneri strain, creating a new O83 serogroup in the genus Proteus →4)-β- d -Glc p NAc-(1→3)-α- d -Gal p -(1→3)-β- d -Glc p NAc-(1→2)-β- d -Gal p -(1→3)-Gro-1- P -(O→. Unlabelled Image [ABSTRACT FROM AUTHOR]

• The lipopolysaccharide of Escherichia albertii O9 contains two polysaccharides. • One is composed of tetrasaccharide repeating units. • The other is identified as a mannan. • Two polysaccharide gene clusters flanked by galF gene and the histidine synthesis operon. 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. [ABSTRACT FROM AUTHOR]

Mild acid degradation of the lipopolysaccharide of Yersinia kristensenii C-134 afforded a glycerol teichoic acid-like O-polysaccharide, which was studied by sugar analysis, O-deacetylation and dephosphorylation along with 1D and 2D NMR spectroscopy. The following structure of the O-polysaccharide was established: This structure is related to those of other Y. kristensenii O-polysaccharides studied earlier. The O-antigen gene cluster of Y. kristensenii С-134 was analyzed and found to be consistent with the O-polysaccharide structure established. Image 1 • Structure of the O-polysaccharide of Yersinia kristensenii C-134 was established. • The O-polysaccharide has a glycerol teichoic acid-like structure. • Dephosphorylation of the O-polysaccharide afforded a glycerol-linked hexasaccharide. • The O-antigen gene cluster was found to be consistent with the O-polysaccharide structure. [ABSTRACT FROM AUTHOR]

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 -Rha p -(1 → 2)-α- l -Rha p -(1 → 3)-β- l -Rha p -(1 → 4)-β- d -Glc p NAc-(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 -Fuc p 3NAc 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 -Fuc p 3NAc from the O50 polysaccharide is evidently a point mutation in the aminotransferase gene fdtB of the d -Fuc p 3NAc synthesis pathway resulting in a single amino acid change from histidine in O2 to arginine in O50. [ABSTRACT FROM AUTHOR]

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 -Rib f and, to a lesser extent, that of β- d -Glc p NAc, whereas the other linkages, including the linkage of α- l -Rha p, were stable under selected conditions (40 °C, 5 h). The following structure of the O-polysaccharide was established: →4)-α- d -Gal p A-(1 → 2)-α- l -Rha p -(1 → 2)-β- d -Rib f -(1 → 4)-β- d -Gal p -(1 → 3)-β- d -Glc p NAc-(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. [ABSTRACT FROM AUTHOR]

Lipopolysaccharide was isolated from bacteria Yersinia intermedia H9-36/83 (O:17) and degraded with mild acid to give an O-specific polysaccharide, which was isolated by GPC on Sephadex G-50 and studied by sugar analysis and 1D and 2D NMR spectroscopy. The polysaccharide was found to contain 3-deoxy-3-[( R )-3-hydroxybutanoylamino]- d -fucose ( d -Fuc3N R 3Hb) and the following structure of the heptasaccharide repeating unit was established: Image 2 The structure established is consistent with the gene content of the O-antigen gene cluster. The O-polysaccharide structure and gene cluster of Y. intermedia are related to those of Hafnia alvei 1211 and Escherichia coli O:103. [ABSTRACT FROM AUTHOR]

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: Image 2 The O33-antigen gene cluster was analyzed and found to be essentially consistent with the O-polysaccharide structure. [ABSTRACT FROM AUTHOR]

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: Image 2 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. [ABSTRACT FROM AUTHOR]

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Enterobacter cloacae C4115 and 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 of the O-polysaccharide was established: →2)-α- l -Rha p -(1 → 2)-α- l -Rha p -(1 → 2)-α- l -Rha p -(1 → 2)-α- d -Gal p -(1 → 3)-α- d -Fuc p NAc-(1→ The O-antigen gene cluster of E. cloacae C4115 was sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in consistence with the O-polysaccharide structure. The O-antigen structure and gene cluster of E. cloacae C4115 are similar to those of E. cloacae G3421 studied by us earlier (Perepelov A.V. et al. Carbohydr. Res. 427 (2016) 55–59). [ABSTRACT FROM AUTHOR]

On mild acid degradation of the lipopolysaccharide of Enterobacter cloacae C5529, the O-polysaccharide chain was cleaved at the linkages of 5,7-diacetamido-3,5,7,9-tetradeoxy- l - glycero - l - manno -non-2-ulosonic acid (di- N -acetylpseudaminic acid, Pse p 5Ac7Ac). The resultant oligosaccharide and an alkali-treated lipopolysaccharide were studied by sugar analysis along with 1 H and 13 C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established: →4)-β-Pse p 5Ac7Ac-(2 → 3)-β- d -Gal p -(1 → 6)-β- d -Gal f -(1 → 3)-α- d -Gal p -(1→ It differs from a structurally related O-polysaccharide of E. cloacae G3045 studied early (Perepelov, A. V.; Wang, M.; Filatov, A. V.; Guo, X.; Shashkov, A. S.; Wang, L.; Knirel, Y. A. Carbohydr. Res. 2015; 407:59–62) in positions of substitution of β-Pse p 5Ac7Ac (O-4 vs. O-8) and β-Gal p (O-3 vs. O-6) and the absence of a side-chain α-Gal p residue. The O-antigen gene clusters of E. cloacae C5529 and G3045 are organized identically and include genes with the same putative functions in the O-polysaccharide synthesis. Based on these and serological data, it is suggested to combine E. cloacae C5529 and G3054 in one O-serogroup as two subgroups. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 CF 3 CO 2 H 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. [ABSTRACT FROM AUTHOR]

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 R pyr 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. [ABSTRACT FROM AUTHOR]

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: 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. [ABSTRACT FROM AUTHOR]

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: 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 -Glc p NAc-(1→4)-β- d -Glc p NAc3( R lac- l Ala)-(1→ where R lac- l Ala indicates ( R )-1-[( S )-1-carboxyethylaminocarbonyl]ethyl. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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: 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. [ABSTRACT FROM AUTHOR]

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: 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 -Glc p -(1→6)-α- d -Gal p linkage in the O-antigen was identified in the gene cluster. [ABSTRACT FROM AUTHOR]

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-Glс p and α-Gal p A to give a modified linear polysaccharide. Solvolysis cleaved selectively the linkage of α- l -Fuc p NAc 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-Gal p A 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: Image 2 [ABSTRACT FROM AUTHOR]

Structure of the O-specific polysaccharide from Pseudomonas chlororaphis subsp. aureofaciens UCM B-306 was elucidated by sugar analysis along with 1D and 2D 1 H and 13 C NMR spectroscopy. The polysaccharide is built up of trisaccharide repeats containing d -rhamnose, 2,4-diacetamido-2,4,6-trideoxy- d -glucose ( d -QuiNAc4NAc), and 2-acetamido-2-deoxy- d -galacturonic acid ( d -GalNAcA), which is amidated in ∼40% repeats. It was suggested that the O-polysaccharide has a blockwise structure, which can be presented as follows: -[→3)-α- d -Rha p -(1→4)-α- d -Gal p NAcA-(1→3)-α- d -Qui p NAc4NAc-(1-] n → and -[→3)-α- d -Rha p -(1→4)-α- d -Gal p NAcAN-(1→3)-α- d -Qui p NAc4NAc-(1-] m →, where GalNAcAN indicates 2-acetamido-2-deoxy- d -galacturonamide, n: m =∼3:2. [ABSTRACT FROM AUTHOR]