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Soft rot caused by numerous species of Pectobacterium and Dickeya is a serious threat to the world production of potatoes. The application of bacteriophages to combat bacterial infections in medicine, agriculture, and the food industry requires the selection of comprehensively studied lytic phages and the knowledge of their infection mechanism for more rational composition of therapeutic cocktails. We present the study of two bacteriophages, infective for the Pectobacterium brasiliense strain F152. Podoviridae PP99 is a representative of the genus Zindervirus , and Myoviridae PP101 belongs to the still unclassified genomic group. The structure of O-polysaccharide of F152 was established by sugar analysis and 1D and 2D NMR spectroscopy: → 4)-α-D-Man p 6Ac-(1→ 2)-α-D-Man p -(1→ 3)-β-D-Gal p -(1→ 3 ↑ 1 α -l- 6 dTal p Ac 0 − 2 The recombinant tail spike protein of phage PP99, gp55, was shown to deacetylate the side chain talose residue of bacterial O-polysaccharide, thus providing the selective attachment of the phage to the cell surface. Both phages demonstrate lytic behavior, thus being prospective for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Background: Citrobacter strains are opportunistic pathogens often responsible for serious enteric as well as extraintestinal diseases, and therefore the O-antigenic scheme, still in use in diagnostic identification, should be set for proper serotyping. The structures of more than 30 different Citrobacter O-antigens (O-polysaccharide chains of the lipopolysaccharides) of 43 Citrobacter O-serogroups have been elucidated so far. However, relationships between strains in several heterogeneous serogroups still need to be clarified by immunochemical studies. These include complex serogroups O3 and O8, represented by 20 and 7 strains, respectively, which are the subject of the present work. Earlier, the O-polysaccharide structures have been determined for Citrobacter O3 strain Be35/57 (PCM 1508) and Citrobacter O8 strain Be64/57 (PCM 1536). Results: Serological studies (immunoblotting) carried out on Citrobacter lipopolysaccharides from different strains ascribed to serogroups O3 and O8 showed that each of these serogroups should be divided into non-cross-reacting subgroups. Based on the results of chemical analyses and ¹H and 13C NMR spectroscopy the structure of Citrobacter O-antigens from strains PCM 1504 (O6) and PCM 1573 (O2) have been established. Chemical data combined with serological analyses showed that several Citrobacter strains should be reclassified into other serogroups. Conclusions: Immunochemical studies carried out on Citrobacter LPS, described in this paper, showed the expediency of reclassification of: 1) strains PCM 1504 and PCM 1573 from serogroups O6 and O2 to serogroups O3 and O8, respectively, 2) strains PCM 1503 and PCM 1505 from serogroups O3 and O8 to new serogroups O3a and O8a, respectively. [ABSTRACT FROM AUTHOR]

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

Capsular polysaccharides (CPSs), from Acinetobacter baumannii isolates 1432, 4190 and NIPH 70, which have related gene content at the K locus, were examined, and the chemical structures established using 2D 1H and 13C NMR spectroscopy. The three isolates produce the same pentasaccharide repeat unit, which consists of 5-N-acetyl-7-N-[(S)-3-hydroxybutanoyl] (major) or 5,7-di-N-acetyl (minor) derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid (Leg5Ac7R), D-galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. However, the linkage between repeat units in NIPH 70 was different to that in 1432 and 4190, and this significantly alters the CPS structure. The KL27 gene cluster in 4190 and KL44 gene cluster in NIPH 70 are organized identically and contain lga genes for Leg5Ac7R synthesis, genes for the synthesis of the common sugars, as well as an itrA2 initiating transferase and four glycosyltransferases genes. They share high-level nucleotide sequence identity for corresponding genes, but differ in the wzy gene encoding the Wzy polymerase. The Wzy proteins, which have different lengths and share no similarity, would form the unrelated linkages in the K27 and K44 structures. The linkages formed by the four shared glycosyltransferaseswere predicted by comparison with gene clusters that synthesize related structures. These findings unambiguously identify the linkages formed by WzyK27 andWzyK44, and show that the presence of different wzy genes in otherwise closely related K gene clusters changes the structure of the CPS. This may affect its capacity as a protective barrier for A. baumannii. [ABSTRACT FROM AUTHOR]

Upon mild acid degradation of the lipopolysaccharide of Escherichia coli O165, the O-polysaccharide chain was cleaved at the glycosidic linkage of 5-N-acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic acid (Pse5Hb7Ac). Analysis of the resulting linear tetrasaccharide and alkali-treated lipopolysaccharide by ¹H/13C 1D and 2D nuclear magnetic resonance spectroscopy enabled elucidation of the following structure of the O-polysaccharide: →8)-α-Psep5Hb7Ac-(2→6)-β-D-Galp-(1→4)-β-D-Gl?p-(1→3)-α-DGl?pNAc-(1→. The β-D-Galp-(1→4)-β-D-Gl?p-(1→3)-D-Gl?pNAc structural element is also present in the O-polysaccharide of E. coli O82. The content of the O-antigen gene cluster of E. coli O165 was found to be consistent with the O-polysaccharide structure established. Functions of proteins encoded in the gene cluster, including enzymes involved in the Pse5Hb7Ac biosynthesis and glycosyltransferases, were putatively assigned by comparison with sequences in available databases. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides on the cell surface of Gram-negative bacteria are an important factor in pathogenicity, and the O-specific polysaccharide chain ( O-polysaccharide, O-antigen) defines the immunospecificity of different bacterial strains. Cronobacter turicensis is an emerging foodborne pathogen which causes severe invasive infections in neonates. In this study, a new O serotype, C. turicensis O2, was established, the structure and genetics of the O-antigen were investigated, and a serotype-specific gene was identified. Sugar and methylation analyses, and nuclear magnetic resonance spectroscopy indicated that the O-polysaccharide contains d-galactose ( d- Gal), N-acetyl- d-glucosamine ( d- Glc NAc), l-rhamnose ( l- Rha) and 5,7-diacetamido-3,5,7,9-tetradeoxy- d- glycero- d- galacto-non-2-ulosonic acid (di- N-acetyllegionaminic acid, Leg5 Ac7 Ac). The structure of the tetrasaccharide repeat of the O-polysaccharide was established as: [ABSTRACT FROM AUTHOR]

Shigella flexneri is an important human pathogen causing shigellosis. Strains of S. flexneri are serologically heterogeneous and, based on O-antigens, are currently classified into 14 types. Structures of the O-antigens ( O-polysaccharides) of S. flexneri have been under study since 1960s but some gaps still remained. In this work, using one- and two-dimensional 1H- and 13C- NMR spectroscopy, the O-polysaccharides of several S. flexneri types were reinvestigated, and their structures were either confirmed (types 2b, 3b, 3c, 5b, X) or amended in respect to the O-acetylation pattern (types 3a, Y, 6, 6a). As a result, the O-acetylation sites were defined in all O-polysaccharides that had not been studied in detail earlier, and the long story of S. flexneri type strain O-antigen structure elucidation is thus completed. New and published data on the S. flexneri O-antigen structures are summarized and discussed in view of serological and genetic relationships of the O-antigens within the Shigella group and between S. flexneri and Escherichia coli. [ABSTRACT FROM AUTHOR]

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

O-antigen representing the O-polysaccharide chain of the lipopolysaccharide is the most variable constituent on the cell surface of Gram-negative bacteria and a player in their pathogenicity. The O-polysaccharide of Escherichia coli O109 was studied by sugar analysis and nuclear magnetic resonance spectroscopy and found to contain a rarely occurring monosaccharide, 2,3-diacetamido-2,3,6-trideoxy--mannose (-RhaNAc3NAc). The following structure of the tetrasaccharide repeating unit of the O-polysaccharide was established, which is closely related to that of Proteus penneri O66: The O-antigen gene cluster of E. coli O109 was sequenced and all 14 genes found were assigned functions based on their similarity to genes from the available databases. Putative genes for synthesis of -RhaN3N were found in E. coli O109 and their homologues in E. coli O119, whose O-antigen has been reported earlier to contain 2-acetamido-2,3,6-trideoxy-3-formamido--mannose (-RhaNAc3NFo). Analysis by GLC of the ( S)-2-octyl glycosides confirmed that the absolute configuration of RhaN3N in E. coli O119 should be revised from to . [ABSTRACT FROM AUTHOR]

Abstract: A polysaccharide was isolated by GPC after mild acid treatment of the lipopolysaccharide of Vibrio vulnificus CECT4602 and found to contain l-Rha, d-GlcpNAc and 2-acetamido-2,3,6-trideoxy-3-(3-hydroxybutanoylamino)-l-mannose (l-RhaNAc3NHb). GLC analysis of the trifluoroacetylated (S)-2-octyl esters derived by full acid hydrolysis of the polysaccharide showed that ∼80% of the 3-hydroxybutanoic acid has the S configuration and ∼20% the R configuration. The following structure of the polysaccharide was established by 1H and 13C NMR spectroscopies, including 2D ROESY and 1H/13C HMBC experiments: ▪ [Copyright &y& Elsevier]

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]

Lipopolysaccharide was extracted from cells of Citrobacter freundii PCM 1443 from serogroup O39 and degraded by mild acid hydrolysis to give an O-polysaccharide. Based on enzymatic and methylation analyses, along with 1H and 13C nuclear magnetic resonance spectroscopy, it was found that the lipopolysaccharide studied has two different linear polysaccharide chains ofd-galactan type containing 3-substituted galactose residues. One of the galactans has the disaccharide repeating units of α-d-galactopyranose and β-d-galactofuranose and the other is comprised of α-d-galactopyranose and β-d-galactopyranose, the latter being substituted in 25% repeats with PEtN at O-6. An immunoblotting assay demonstrated that the lipopolysaccharide of C. freundii PCM 1443 is serologically related to that of Klebsiella pneumoniae O1, which contains the same galactan chains but is devoid of phosphoethanolamine. [ABSTRACT FROM AUTHOR]

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]

Earlier, the structures of the O-chain polysaccharides of the lipopolysaccharides (LPS) of a number of Hafnia alvei strains have been established. However, it remained unknown, which is the first and the last monosaccharide of the O-chain. This is defined by the structure of the so-called biological repeating unit (O-unit), which is pre-assembled and then polymerised in the course of biosynthesis of bacterial polysaccharides by the Wzy-dependent pathway. Now we report on the structures of the O-units in 10 H. alvei strains. The LPS were cleaved by mild acid hydrolysis and oligosaccharide fractions IIIa and IIIb were isolated by gel chromatography subsequently on Sephadex G-50 and BioGel P-2 and studied by methylation analysis and NMR spectroscopy. Fraction IIIb was found to represent the core oligosaccharide containing a terminal upstream α-D-Glc-(1→3)-α-D-Glc or α-DGal-(1→3)-α-D-Glc disaccharide in the outer region that is typical of H. alvei. Fraction IIIa consists of the LPS core with one Ounit linked by a 3-substituted β-D-GalNAc residue (in strains PCM 1189 and PCM 1546) or a 3-substituted βD-GlcNAc residue (in the other strains studied). In most strains examined the βconfiguration of the D-GlcNAc linkage in the first O-unit attached to the core is the same and in some strains is opposite to that found in the interior O-units of the O-chain polysaccharide. Various monosaccharides, including D-Glc, D-Gal, D-GlcA and acyl derivatives of 3-amino-3,6-dideoxy-D-glucose or 4-amino-4,6-dideoxy-Dglucose, occupy the non-reducing end of the O-unit. [ABSTRACT FROM AUTHOR]

The core structure of the lipopolysaccharide (LPS) isolated from a rough strain of the phytopathogenic bacteriumPseudomonas syringaepv.phaseolicola, GSPB 711, was investigated by sugar and methylation analyses, Fourier transform ion-cyclotron resonance ESI MS, and one- and two-dimensional1H-,13C- and31P-NMR spectroscopy. Strong alkaline deacylation of the LPS resulted in two core-lipid A backbone undecasaccharide pentakisphosphates in the ratio≈ 2.5 : 1, which corresponded to outer core glycoforms 1 and 2 terminated with eitherl-rhamnose or 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), respectively. Mild acid degradation of the LPS gave the major glycoform 1 core octasaccharide and a minor truncated glycoform 2 core heptasaccharide, which resulted from the cleavage of the terminal Kdo residues. The inner core ofP. syringaeis distinguished by a high degree of phosphorylation ofl-glycero-d-manno-heptose residues with phosphate, diphosphate and ethanolamine diphosphate groups. The glycoform 1 core is structurally similar but not identical to one of the core glycoforms of the human pathogenic bacteriumPseudomonas aeruginosa.The outer core composition and structure may be useful as a chemotaxonomic marker for theP. syringaegroup of bacteria, whereas a more conserved inner core structure appears to be representative for the whole genusPseudomonas. [ABSTRACT FROM AUTHOR]

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]

Studies the structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 8 from the serogroup O67. Process used for obtaining bacterial strains for the experiment; Method used for the preparation of O-antiserum and serological assays; Structure of the polysaccharide observed by chemical and nuclear magnetic resonance spectroscopic studies.

Analyzes the structure of the O-specific polysaccharide of Proteus penneri and classification of cross-reactive strains to a proposed serogroup O64. Description of the methods used for the isolation of lipopolysaccharides; Procedure used for the preparation of polyclonal O-antisera; Methylation analysis of the polysaccharide; Structure of the polysaccharide observed by nuclear magnetic resonance spectroscopy.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated. [ABSTRACT FROM AUTHOR]

On the basis of sugar analysis and NMR spectroscopy, including selective spin-decoupling, one-dimensional NOE, two-dimensional homonuclear and 13C,¹H-heteronuclear correlation spectroscopy, the following structure of the acidic O-specific polysaccharide of Proteus mirabilis O43 was established: →4)-α-D-GalpA-(1→3)-α-D-GalpA-(1→3)-α-D-Gl cpNAc-(1→4)-α - D-Glcp-(1→, where GalA is galacturonic acid and Galp is galactopyranose. No serological cross-reactivity was observed between lipopolysaccharides of P. mirabilis O43 and other studied Proteus strains, except for P. mirabilis O10. The O-specific polysaccharide of P. mirabilis O43 was serologically active in precipitation and inhibition tests but the activity was lost after periodate oxidation. These data suggest that the O43 specificity is determined by a wide epitope with the immunodominant role of 4-substituted D-Glc or/and D-GalA, which are destroyed by periodate oxidation. [ABSTRACT FROM AUTHOR]

The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O-specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography. The CPS contained D-galactose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), D-galacturonic acid (D-GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galactose 4,6-cyclophosphate and having the following structure [This structure can not be represented in to (ASCII.txt)] These data basically confirm the structure of the V. cholerae CPS proposed on the basis of an NMR study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835-838] and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate. [ABSTRACT FROM AUTHOR]

O-specific polysaccharides, obtained on mild acid degradation of lipopolysacchrides of the serologically related strains Pseudomonas aeruginosa O3 (L´nyi classification), O25 (Wokatsch classification) and immunotypes 3 and 7 (Fisher classification), are built up of trisaccharide repeating units involving 2-acetamido-2,6-dideoxy-D-galactose (N-acetyl-D-fucosamine), 2,3-diacetarnido-2,3-dideoxy-D-mannuronic acid or 2,3-diacetamido-2,3-dideoxy-L-guluronic acid and 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid or 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic acid. Lányi O3(a),3d,3f and Wokatsch O25 polysaccharides contain also O-acetyl groups. On the basis of solvolysis with anhydrous hydrogen fluoride, resulting in trisaccharide fragments with N-acetylfucosamine residue at the reducing terminus, chemical modifications of the acetamidino group (alkaline hydrolysis to the acetamido group or reductive deamination to the ethylarnino group), as well as analysis by ¹H-NMR (including nuclear Overhauser effect experiments) and 13C-NMR spectroscopy, and fast-atom bombardment mass spectrometry, it was concluded that the repeating units of the polysaccharides have the following structures: [This equation cannot be converted into ASCII text.] where HexNAcAmA = α-L-GulNAcAmA (≈70%) or β-D-ManNAcAmA (≈30%). Lányi O3(a),3d,3f polysaccharide involves two types of repeating units, which differ from each other only in the configuration at C-5 of the 3-acetamidino-2-acetamido-2,3-dideoxyuronic acid residue. Lányi O3(a),3c, O3a,3d,3e and Fisher immunotypes 3 and 7 polysaccharides contain, together with the major repeating units shown above, a small proportion of units in which the derivative of &aalpha;-L-guluronic acid is replaced by the corresponding β-D-manno isomer. The data obtained provide the opportunity to substantiate the serological interrelations between these strains of P. aeruginosa by the presence in the O-specific polysaccharides of common monosaccharides or disaccharide fragments. The distinctions between them stem from the presence or absence of the O-acetyl group, a different configuration of the glycosidic linkage of the N-acetylfucosamine residue and/or a different configuration at C-5 of one or both derivatives of diaminouronic acids. [ABSTRACT FROM AUTHOR]

On mild acid degradation of Pseudomonas aeruginosa O:3(a),c, O:3a,d,e and O:3(a),d,f lipopolysaccharides O-specific polysaccharides were isolated. The trisaccharide repeating units of O:3(a0,c and O:3a,d,e polysaccharides contained 2-acetamido-2,6-dideoxy-D-galactose and 2,3-(l-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, which were identified previously as the constituents of P. aeruginosa O:3a,b and O:3a,d O-specific polysaccharides, as well as 2,3-diacetamido-2,3-dideoxy-L-guluronic acid, which has never before been found in nature. the last monosaccharide was identified without being isolated in the free state, by means of 13 C nuclear magnetic resonance spectroscopy. The structures of O:3(a),c and O:3a,d,e polysaccharides were established by selective hydrogen fluoride solvolysis followed by modification of the trisaccharides obtained and analysis of the 13C nuclear magnetic resonance spectra at each modification stage. The polysaccharides possessed similar structures of repeating units differing from each other in the anomeric configuration of the N-acetylfucosamine residue only: →4)DManIm-(β1→4)LGul(NAc)2U(α1→3)DFucNAc(β1-and → 4)DManImU(β1→4)LGul(NAc)2U(α1→3)DFucNAc(α1-, where DManImU = 2,3-(l-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, LGul(NAc)2U = 2,3-diacetamido-2,3-dideoxy-L-guluronic acid, DFucNAc = 2-acetamido-2,6-dideoxy-D-galactose. The same components were detected in the O:3(a),d,f polysaccharide but not in the same stoichiometric ratio; this polysaccharide possessed no regular structure. The immunochemical study of lipopolysaccharides of all five P. aeruginosa O:3 serotypes showed a definite interrelation between their serological properties and structures of the corresponding O-specific polysaccharides. [ABSTRACT FROM AUTHOR]

Structural and immunochemical studies have been performed on the O-specific antigens of two serologically related types of Pseudomonas aeruginosa O:2a,b and O:2a,c (Lanyi's classification). The O-specific polysaccharide chains of the two serotypes were shown to be acidic polysaccharides composed of repeating trisaccharide units consisting of L-rhamnose, N-acetyl-D-quinovosamine and N-acetyl-L-galactosaminuronic acid residues. Based on 1H and 13C nuclear magnetic resonance spectroscopy, data of methylation analysis and selective solvolysis with hydrogen fluoride, the repeating unit of the O:2a,c O-specific polysaccharide was assigned the following structure: →4)LGalNAcA(α1 → 3)DQuiNAc(α1 » 3)LRha(α1, where GalNAcA = N-acetylgalactosaminuronic acid, QuiNAc = N-acetylquinovosamine and Rha = rhamnose. The O:2a,b O-specific polysaccharide had the same structure of the trisaccharide repeating unit but was distinguished by the presence of O-acetyl groups on 70–80% of the rhamnose residues in position 2. The O-acetyl groups were located both by methylation with methyltrifluoromethane sulphonate and by comparison of the 13C nuclear magnetic resonance spectra of the native and O-deacetylated polysaccharides. Serological studies revealed the determinant role of the O-acetyl groups in the O-antigen O:2a,b and suggested the O-factor 2b to be related to the 2-O-acetyl-L-rhamnose residue, whereas the O-factor 2c was probably determined by the nonacetylated rhamnose residue. The dominant moiety of the determinant 2a common to the two antigens was obviously presented by the N-acetyl-L-galactosaminuronic acid residue. [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]

Mild acid hydrolysis of the smooth-type lipopolysaccharide (LPS) of Pectinatus frisingensis afforded no polysaccharide but monomeric 6-deoxy-L-altrose (L-6dAlt) which was identified by anion-exchange chromatography in borate buffer, GLC/MS, 1H-NMR spectroscopy, and optical rotation. LPS was degraded with alkali under reductive conditions to give a completely O-deacylated polysaccharide, which was studied by methylation analysis, 1H-NMR and 13C-NMR spectroscopy, including sequential, selective spin-decoupling, two-dimensional correlation spectroscopy (COSY), COSY with relayed coherence transfer, two-dimensional heteronuclear 13C, 1H-COSY, one-dimensional NOE and two-dimensional rotating-frame NOE spectroscopy. It was found that the O-specific polysaccharide chain of P. frisingensis LPS is a homopolymer of 6-deoxy-L-altrofuranose built up of tetrasaccharide-repeating units having the following structure: [This text does not exist in ASCII] Similarly, mild acid degradation of smooth-type LPS of Pectinatus cerevisiiphilus resulted in depolymerisation of the polysaccharide chain to give a disaccharide consisting of D-glucose and D-fucose. Study of the disaccharide by methylation analysis and alkali-degraded LPS by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy showed that the O-specific polysaccharide of P. cerevisiiphilus has the following structure: →2)-β-D-Fucf-(1→2)-α-D-Glcp-(1→. [ABSTRACT FROM AUTHOR]

The O-specific polysaccharide, obtained by mild acid degradation of Citrobacter O16 lipopolysaccharide, consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose, glycerol and phosphate in the ratios 2:2:2: 1:1. Selective cleavage of the polysaccharide was caried out by Smith degradation, N-deacetylation-deamination and dephosphorylation with 48% hydrofluoric acid, which was accompanied by unexpected splitting of one of the glycosidic linkages, The structures of the oligosaccharides thus obtained were established using ¹H- and 13C-NMR spectroscopy, including one-dimensional NOE, two-dimensional rotating-frame NOE, homonuclear and heteronuclear 12C,¹H correlation spectroscopy, and, for the Smith degradation product, positive- and negative-ion-mode fast-atom-bombardment MS and MS/MS with collision-induced dissociation. On the basis of these data and the results of methylation analysis, it was concluded that the O-specific polysaccharide has the following repeating unit structure: [ABSTRACT FROM AUTHOR]

O-Specific polysaccharide was obtained by mild acid degradation of Proteus penneri strain 16 lipopolysaccharide and found to contain D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-galactose in the ratio of 2:1:1:1 as well as a small proportion of O-acetyl groups. On the basis of one-dimensional ¹H-NMR13C-NMR and NOE spectroscopy, two-dimensional homonuclear-shift-correlated spectroscopy with one-step and two-step relayed coherence transfer and heteronuclear ¹H/13C NMR shift-correlated spectroscopy, it was concluded that the O-specific polysaccharide of P. penneri strain 16 has the following structure: ["Multiple line equation(s) cannot be represented in ASCII text"] This structure was confirmed by methylation analysis and structural analysis of a linear tetrasaccharide fragment prepared by cleavage of the polysaccharide with anhydrous hydrogen fluoride followed by conversion of the α-tetrosyl fluoride obtained in to the corresponding free oligosaccharide and alditol. O-Acetyl groups were tentatively located at position 3 of the glucuronic acid residue and at position 4 of the 6-substituted glucose residue, the degree of acetylation being less than 20% of the total. Cross-reactions of P. penneri strain 16 anti-(O-specific polysaccharide) antiserum with lipopolysaccharides from several other Proteus strains and the role of 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-galactose in the serological specificity of P. penneri strain 16 are discussed. [ABSTRACT FROM AUTHOR]

The following structure of the repeating unit of the O-specific polysaccharide of Proteus mirabilis O27, containing amides of D-glucuronic and D-galacturonic adds with L-lysine and L-alanine, respectively, N-acetyl-Dglucosamine and phosphoethanolamine, has been determined: ... The main methods used for structural analysis of the polysaccharide were selective solvolysis with anhydrous hydrogen fluoride and Smith degradation, as well as NMR spectroscopy and mass spectrometry. The immunodominant role of the lateral N-acetyl-D-glucosamine and phosphoethanolamine in manifesting the serological specificity of P. mirabilis O27 has been established. [ABSTRACT FROM AUTHOR]

Two acylated forms of the higher sugar, 5,7-diamino-3,5,7,9-tetradeoxy- l - glycero - l - manno -non-2-ulosonic acid called pseudaminic acid, Pse5Ac7Ac and Pse5Ac7RHb where R indicates (R)-3-hydroxybutanoyl, have been found to occur in many capsular polysaccharide (CPS) types produced by isolates of an important human pathogen, Acinetobacter baumannii. The presence of either a psaABCEDF or psaABCGHF gene module at the K locus (KL) for CPS biosynthesis determines the type of the variant produced. Here, an A. baumannii clinical isolate 52-249, recovered in 2015 in Moscow, Russia, was found to include a novel psaABCIJF gene module in the KL218 sequence at the K locus. The CPS from 52‐249 was extracted and studied by sugar analysis and partial acid hydrolysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. A branched tetrasaccharide repeating unit was identified, which included a →3)-α- d -Gal p -(1→6)-α- d -Glc p NAc-(1→3)-β- d -Gal p NAc-(1→ main chain and Pse5Ac7Ac attached as a side branch, indicating that the psaABCIJF gene module is associated with synthesis of this variant. The K218 CPS was found to be structurally related to the K46 CPS of A. baumannii , and a comparison of the two structures enabled the assignment of glycosyltransferases. A KpsS3 protein for the α-(2→6) linkage of the Pse5Ac7Ac residue to D-Gal p in K218 was identified. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii isolate LUH5552 carries the KL89 capsule biosynthesis gene cluster. Capsular polysaccharide (CPS) isolated from LUH5552 was analyzed by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K89 CPS structure has not been seen before in A. baumannii CPS structures resolved to date and includes a 3-acetamido-3,6-dideoxy- d -galactose (d -Fuc p 3NAc) residue which is rare amongst A. baumannii CPS. The K89 CPS has a →3)-α- d -Gal p NAc-(1→3)-β- d -Glc p NAc-(1→ main chain with a β- d -Glc p -(1→2)-β- d -Fuc p 3NAc-(1→6)- d -Glc p side branch that is α-(1→4) linked to d -Gal p NAc. The roles of the Wzy polymerase and the four glycosyltransferases encoded by the KL89 gene cluster in the biosynthesis of the K89 CPS were assigned. Two glycosyltransferases, Gtr121 and Gtr122, link the d -Fuc p 3NAc to its neighboring sugars. [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]

Abstract: A polysaccharide isolated after mild acid degradation of the lipopolysaccharide of Vibrio vulnificus clinical isolate YJ016 was found to contain l-Fuc, d-GlcpNAc, 2,4-diacetamido-2,4,6-trideoxy-d-glucose (di-N-acetylbacillosamine, d-QuiNAc4NAc), and 2-acetimidoylamino-2-deoxy-l-galacturonic acid (l-GalNAmA). The last sugar derivative was confirmed by correlations for nitrogen-linked protons in 2D TOCSY and ROESY spectra measured in a H2O–D2O mixture. The following structure of the polysaccharide was established by 1H and 13C NMR spectroscopy, including 2D ROESY and 1H,13C HMBC experiments: Display Omitted where the degree of 6-O-acetylation of the lateral β-GlcNAc residue is ∼70%. [Copyright &y& Elsevier]

Abstract: The structure of the phenol-soluble polysaccharide from Pseudoalteromonas rubra type strain ATCC 29570T has been elucidated using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, gNOESY, ROESY, 1H,13C gHMQC and gHMBC experiments. It is concluded that the trisaccharide repeating unit of the polysaccharide has the following structure: ▪ where Sug is 2-acetamido-2,6-dideoxy-d-xylo-hexos-4-ulose, Am is acetimidoyl and Acyl is a malic acid residue, which is O-acetylated in ∼70% of the units. [Copyright &y& Elsevier]

Abstract: A neutral O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis OC (CCUG 10702) and studied by sugar and methylation analyses and 1H and 13C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: Based on the unique structure of the O-polysaccharide and serological data, we propose classifying P. mirabilis OC (CCUG 10702) into a new separate Proteus serogroup O75. A weak cross-reaction of O-antiserum against P. mirabilis OC with the lipopolysaccharide of P. mirabilis O49 was accounted for by a similarity in the O-polysaccharide structures. [Copyright &y& Elsevier]

The facultative intracellular pathogen Bartonella henselae is responsible for a broad range of clinical manifestations, including the formation of vascular tumors as a result of increased proliferation and survival of colonized endothelial cells. This remarkable interaction with endotoxin-sensitive endothelial cells and the apparent lack of septic shock are considered to be due to a reduced endotoxic activity of the B. henselae lipopalysaccharide. Here, we show that B. henselae ATCC 49882T produces a deep-rough-type lipopolysaccharide devoid of O-chain and report on its complete structure and Toll-like receptordependent biological activity. The major short-chain lipopolysaccharide was studied by chemical analyses, electrospray ionization, and matrix-assisted laser desorption/ ionization mass spectrometry, as well as by NMR spectroscopy after alkaline deacylation. The carbohydrate portion of the lipopolysaccharide consists of a branched trisaccharide containing a glucose residue attached to position 5 of an α-(2→4)-linked 3-deoxy-D-manno-oct-2-ulosonic acid disaccharide. Lipid A is a pentaacylated β-(1′→6)-linked 2,3-diamino-2,3-dideoxyglucose disaccharide 1,4′-bisphosphate with two amidelinked residues each of 3-hydroxydodecanoic and 3-hydroxyhexadecanoic acids and one residue of either 25-hydroxyhexacosanoic or 27-hydroxyoctacosanoic acid that is O-linked to the acyl group at position 2′. The lipopolysaccharide studied activated Toll-like receptor 4 signaling only to a low extent (1,000-10,000-fold lower compared with that of Salmonella enterica sv. Friedenau) and did not activate Toll-like receptor 2. Some unusual structural features of the B. henselae lipopolysaccharide, including the presence of a long-chain fatty acid, which are shared by the lipopolysaccharides of other bacteria causing chronic intracellular infections (e.g. Legionella and Chlamydia), may provide the molecular basis for low endotoxic potency. [ABSTRACT FROM AUTHOR]

The following structure of the O-polysaccharide of the phytopathogenic bacterium Xanthomonas cassavae GSPB 2437 was determined by sugar analysis along with 1H and 13C NMR spectroscopy: [Copyright &y& Elsevier]

Capsular polysaccharide (CPS) is a key target for bacteriophage and vaccine therapies currently being developed for treatment of infections caused by the extensively antibiotic resistant bacterial species, Acinetobacter baumannii. Identification of new CPS structures and the genetics that drive their synthesis underpins tailored treatment strategies. A novel CPS biosynthesis gene cluster, designated KL139, was identified in the whole genome sequence of a multiply antibiotic resistant clinical isolate, A. baumannii MAR-17-1041, recovered in Russia in 2017. CPS material extracted from A. baumannii MAR-17-1041 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy, and the structure was found to include a branched pentasaccharide repeating unit containing neutral carbohydrates. This structure closely resembles the topology of the A. baumannii K14 CPS but differs in the presence of d -Glc p in place of a d -Gal p sugar in the repeat-unit main chain. The difference was attributed to a change in the sequence for two glycosyltransferases. These two proteins are also encoded by the A. baumannii KL37 gene cluster, and a multiple sequence alignment of KL139 with KL14 and KL37 revealed a hybrid relationship. The global distribution of KL139 was also assessed by probing 9065 A. baumannii genomes available in the NCBI non-redundant and WGS databases for the KL139 gene cluster. KL139 was found in 16 genomes from four different countries. Eleven of these isolates belong to the multidrug resistant global lineage, ST25. [ABSTRACT FROM AUTHOR]

Whole genome sequence from Acinetobacter baumannii isolate Ab-46-1632 reveals a novel KL144 capsular polysaccharide (CPS) biosynthesis gene cluster, which carries genes for d -glucuronic acid (D -GlcA) and l -rhamnose (l -Rha) synthesis. The CPS was extracted from Ab-46-1632 and studied by 1H and 13C NMR spectroscopy, including a two-dimensional 1H,13C HMBC experiment and Smith degradation. The CPS was found to have a hexasaccharide repeat unit composed of four l -Rha p residues and one residue each of d -Glc p A and N -acetyl- d -glucosamine (D -Glc p NAc) consistent with sugar synthesis genes present in KL144. The K144 CPS structure was established and found to be related to those of A. baumannii K55, K74, K85, and K86. A comparison of the corresponding gene clusters to KL144 revealed a number of shared glycosyltransferase genes correlating to shared glycosidic linkages in the structures. One from the enzymes, encoded by only KL144 and KL86, is proposed to be a novel multifunctional rhamnosyltransfaerase likely responsible for synthesis of a shared α- l -Rha p -(1 → 2)-α-L-Rha p -(1 → 3)-L-Rha p trisaccharide fragment in the K144 and K86 structures. [ABSTRACT FROM AUTHOR]

The KL26 gene cluster responsible for the synthesis of the K26 capsular polysaccharide (CPS) of Acinetobacter baumannii includes rmlBDAC genes for l -rhamnose (l -Rha p) synthesis, tle to generate 6-deoxy- l -talose (l -6dTal p) from l -Rha p , and a manC gene for D -mannose (D -Man p) that is rare in Acinetobacter CPS. K26 CPS material was isolated from A. baumannii isolate KZ-1098, and studied by sugar analysis, Smith degradation, and one and two-dimensional 1H and 13C NMR spectroscopy before and after O -deacetylation with aqueous ammonia. The following structure of the branched hexasaccharide repeating unit of the CPS was established: → 2) − β − D − Man p − 1 → 4 − β − D − Glc p − 1 → 3 − α − L − 6 dTal p − 1 → 3 − β − D − Glc p NAc − (1 → 3 ↑ 1 4 │ Ac α − L − Rha p − 2 ← 1 − α − D − Glc p The structural depolymerase of phage vB_AbaP_APK26 cleaved selectively the β-Glc p NAc-(1 → 2)-α-Man p linkage in the K26 CPS formed by Wzy K26 to give monomer, dimer, and trimer of the CPS repeating unit, which were characterized by high-resolution electrospray ionization mass spectrometry as well as 1H and 13C NMR spectroscopy. The wzy K26 gene responsible for this linkage and the manC gene were only found in six A. baumannii genomes carrying KL26 and one carrying the novel KL148 gene cluster, indicating the rare occurrence of β-Glc p NAc-(1 → 2)-α-Man p in A. baumannii CPS structures. However, K26 shares a β- d -Glc p -(1 → 3)-α- l -6dTal p -(1 → 3)-β- d -Glc p NAc trisaccharide fragment with a group of related A. baumannii CPSs that have varying patterns of acetylation of l -6dTal p. [ABSTRACT FROM AUTHOR]

The K239 type capsular polysaccharide (CPS) isolated from Acinetobacter baumannii isolate MAR19-4435 was studied by sugar analysis, one- and two-dimensional 1H and 13C NMR spectroscopy. K239 consists of branched heptasaccharide repeats (K-units) comprised of five residues of l -rhamnose (l -Rha p), and one residue each of d -glucuronic acid (d -Glc p A) and N -acetyl- d -glucosamine (d -Glc p NAc). The structure of K239 is closely related to that of the A. baumannii K86 CPS type, though the two differ in the 2,3-substitution patterns on the l -Rha p residue that is involved in the linkage between K-units in the CPS polymer. This structural difference was attributed to the presence of a gtr221 glycosyltransferase gene and a wzy KL239 polymerase gene in KL239 that replaces the gtr80 and wzy KL86 genes in the KL86 CPS biosynthesis gene cluster. Comparison of the two structures established the role of a novel Wzy KL239 polymerase encoded by KL239 that forms the β-d-GlcpNAc-(1→2)-l-Rha p linkage between K239 units. A. baumannii MAR19-4435 was found to be non-susceptible to infection by the APK86 bacteriophage, which encodes a depolymerase that specifically cleaves the linkage between K-units in the K86 CPS, indicating that the difference in 2,3-substitution of l -Rha p influences the susceptibility of this isolate to bacteriophage activity. [ABSTRACT FROM AUTHOR]

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 -Rha p and d -Glc p A 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 -Glc p A and d -Glc p NAc and six residues of l -Rha p. The K55 unit differs from the K74 unit in the linkage between D-Glc p A and an l -Rha p 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 -Rha p -(1 → 3)-α- l -Rha p 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 -Rha p 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. [ABSTRACT FROM AUTHOR]

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]

Lipopolysaccharide (LPS) of Ochrobactrum cytisi IPA7.2, a bacterium isolated from the roots of Solanum tuberosum L., was extracted from dry bacterial cells and chemically characterized. The O-specific polysaccharide was obtained by mild acid hydrolysis of the LPS and studied by sugar analysis and 1H and 13C NMR spectroscopy, including 1H,1H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments. The polysaccharide was linear and consisted of trisaccharide repeating units of the following structure: A putative O-antigen gene cluster of O. cytisi IPA7.2 was identified and found to be consistent with the O-specific polysaccharide structure. The LPS of Ochrobactrum cytisi IPA7.2 promoted the growth of potato microplants in vitro. [ABSTRACT FROM AUTHOR]

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 β-elimination (NaOH-NaBH 4) 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 -Gal p NAcA6 d Ala-(1 → 4)- α -d -Gal p NAcA-(1 → 3)- β- d -Qui p NAc4NAc-(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 -Qui p NAc4NAc. 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 -Gal p NAcA 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 (al anine t ransferase). Alt1 represents a novel family that amidate the carboxyl group of d -Gal p NAcA or d -Gal p A. [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]

K63 capsular polysaccharide produced by Acinetobacter baumannii isolate LUH5551 (previously designated isolate O24) was re-examined using sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. Though previously reported as O24 consisting of linear tetrasaccharide units that include a 7-acetamido-5-acylamino form of 8-epilegionaminic acid [8eLeg5R7Ac, acylated at C5 with (S)-3-hydroxybutanoyl or acetyl (1:1)], the elucidated structure of the K63 type capsule was found to include a derivative of 5,7-diamino-3,5,7,9-tetradeoxy- d - glycero - d - galacto -non-2-ulosonic (legionaminic) acid, Leg5Ac7R, where R is either (S)-3-hydroxybutanoyl or an acetyl group (∼1:1 ratio). This finding is consistent with the presence of the lgaABCHIFG gene module for Leg5Ac7R biosynthesis in the KL63 gene cluster at the capsular polysaccharide (CPS) biosynthesis K locus in the LUH5551 genome. The glycosyltransferases (Gtrs) and Wzy polymerase encoded by KL63 were assigned to linkages in the linear K63 tetrasaccharide unit and linkage of the K63 units. [Display omitted] • The structure of the A. baumannii K63 capsule has been determined. • This structure revises that reported for O24. • K63 includes 5,7-diamino-3,5,7,9-tetradeoxy- d - glycero - d - galacto -non-2-ulosonic acid. • Roles for the glycosyltransferases and Wzy polymerase encoded by KL63 are assigned. [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]

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