The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Analysis of its glycosylation.

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n = 13-18), with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core -->3)-alpha-l-Rhap-(1-->3)-alpha-L-Rhap-(L--> and the linkage glycose beta-D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.     

New insights into the glycosylation of the surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a

The surface of Geobacillus stearothermophilus NRS 2004/3a cells is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. To this S-layer glycoprotein, elongated glycan chains are attached that are composed of [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-L-Rhap-(1-->] repeating units, with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain and a core saccharide as linker to the S-layer protein. On sodium dodecyl sulfate-polyacrylamide gels, four bands appear, of which three represent glycosylated S-layer proteins. In the present study, nanoelectrospray ionization time-of-flight mass spectrometry (MS) and infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry were adapted for analysis of this high-molecular-mass and water-insoluble S-layer glycoprotein to refine insights into its glycosylation pattern. This is a prerequisite for artificial fine-tuning of S-layer glycans for nanobiotechnological applications. Optimized MS techniques allowed (i) determination of the average masses of three glycoprotein species to be 101.66 kDa, 108.68 kDa, and 115.73 kDa, (ii) assignment of nanoheterogeneity to the S-layer glycans, with the most prevalent variation between 12 and 18 trisaccharide repeating units, and the possibility of extension of the already-known -->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1--> core by one additional rhamnose residue, and (iii) identification of a third glycosylation site on the S-layer protein, at position threonine-590, in addition to the known sites threonine-620 and serine-794. The current interpretation of the S-layer glycoprotein banding pattern is that in the 101.66-kDa glycoprotein species only one glycosylation site is occupied, in the 108.68-kDa glycoprotein species two glycosylation sites are occupied, and in the 115.73-kDa glycoprotein species three glycosylation sites are occupied, while the 94.46-kDa band represents nonglycosylated S-layer protein.     

A novel type of carbohydrate-protein linkage region in the tyrosine-bound S-layer glycan of Thermoanaerobacterium thermosaccharolyticum D120-70.

The surface-layer (S-layer) protein of Thermoanaerobacterium thermosaccharolyticum D120-70 contains glycosidically linked glycan chains with the repeating unit structure -->4)[alpha-D-Galp-(1-->2)]-alpha-L-Rhap-(1-->3)[beta-D-Glcp-(1--> 6)] -beta-D-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Glcp-(1--> . After proteolytic degradation of the S-layer glycoprotein, three glycopeptide pools were isolated, which were analyzed for their carbohydrate and amino-acid compositions. In all three pools, tyrosine was identified as the amino-acid constituent, and the carbohydrate compositions corresponded to the above structure. Native polysaccharide PAGE showed the specific heterogeneity of each pool. For examination of the carbohydrate-protein linkage region, the S-layer glycan chain was partially hydrolyzed with trifluoroacetic acid. 1D and 2D NMR spectroscopy, including a novel diffusion-edited difference experiment, showed the O-glycosidic linkage region beta-D-glucopyranose-->O-tyrosine. No evidence was found of additional sugars originating from a putative core region between the glycan repeating units and the S-layer polypeptide. For the determination of chain-length variability in the S-layer glycan, the different glycopeptide pools were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, revealing that the degree of polymerization of the S-layer glycan repeats varied between three and 10. All masses were assigned to multiples of the repeating units plus the peptide portion. This result implies that no core structure is present and thus supports the data from the NMR spectroscopy analyses. This is the first observation of a bacterial S-layer glycan without a core region connecting the carbohydrate moiety with the polypeptide portion.     

N-acetylmuramic acid as capping element of alpha-D-fucose-containing S-layer glycoprotein glycans from Geobacillus tepidamans GS5-97T

Geobacillus tepidamans GS5-97(T) is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106-166 kDa. The three high molecular mass bands gave a positive periodic acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Fucp-(1-->](n=approximately 20), with D-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-acetylmuramic acid, the key component of prokaryotic peptidoglycan, was found in an alpha-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a beta-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core -->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1--> to carbon 3 of beta-D-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5-97(T).     

Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.     

The Shigella flexneri O-antigenic polysaccharide chain. Nature of the biological repeating unit

The sequence of monosaccharides in the biological repeating tetrasaccharide unit of Shigella flexneri variant Y O-antigenic polysaccharide chain was determined by subjecting three oligosaccharides of the polysaccharide, obtained by phage-Sf6-mediated enzymatic hydrolysis, to methylation analysis and proton nuclear magnetic resonance spectroscopy. The smallest saccharide was shown to be a tetrasaccharide with the structure alpha-L-Rhap-(1-2)-L-Rha. The next saccharide, an octasaccharide, was shown to be a dimer of the tetrasaccharide with the L-Rha residues linked alpha 1.3. The longest saccharide was shown to be a decasaccharide with the following structure: alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-beta-D-GlcpNAc-(1-2)-alpha-L-Rhap-(1-2)-alpha-L-Rhap++ + +-(1-3)-alpha-L-Rhap-(1-3)-beta-D-GlcpNAc-(1-2)-alpha-L-R hap-(1-2)-L-Rha. Thus the decasaccharide differed from the octasaccharide and tetrasaccharide by having the alpha-L-Rhap-(1-2)-L-Rhap disaccharide added in the terminal non-reducing end of the saccharide chain. This shows that the alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-D-GlcpNAc tetrasaccharide is the biological repeating unit of the O chain and that the repeating units are joined through a beta-D-GlcpNAc-(1-2)-L-Rhap linkage. Inhibition experiments utilizing the enzyme-linked immunosorbent assay (ELISA) with S. flexneri Y lipopolysaccharide/S. flexneri Y rabbit antiserum showed that the decasaccharide was the best inhibitor (threefold as active as the octasaccharide and sixtyfold as active as the tetrasaccharide); this supports the postulated structure of the biological repeating unit.     

Homologs of the Rml enzymes from Salmonella enterica are responsible for dTDP-beta-L-rhamnose biosynthesis in the gram-positive thermophile Aneurinibacillus thermoaerophilus DSM 10155

The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus) thermoaerophilus strain DSM 10155 are composed of L-rhamnose- and D-glycero-D-manno-heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho-L-rhamnose (dTDP-L-rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP-L-rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP-D-glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli. Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91(T) produces D-rhamnose- and 3-acetamido-3,6-dideoxy-D-galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91(T) for L-rhamnose and D-rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91(T) is not able to synthesize dTDP-L-rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.     

Efficient synthesis of a heptasaccharide,the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642

alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-Xylp-(1-->2)-][beta-D-Xylp-(1-->4)-]alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642 was synthesized as its methyl glycoside via 3-O-selective glycosylation of methyl alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (9) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (8), followed by dixylosylation with 2,3,4-tri-O-benzoyl-alpha,beta-D-xylopyranosyl trichloroacetimidate (12) and subsequent deacylation.     

The dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene is part of the surface layer glycoprotein glycosylation gene cluster of Geobacillus tepidamans GS5-97T

The glycan chain of the S-layer protein of Geobacillus tepidamans GS5-97(T) consists of disaccharide repeating units composed of L-rhamnose and D-fucose, the latter being a rare constituent of prokaryotic glycoconjugates. Although biosynthesis of nucleotide-activated L-rhamnose is well established, D-fucose biosynthesis is less investigated. The conversion of alpha-D-glucose-1-phosphate into thymidine diphosphate (dTDP)-4-dehydro-6-deoxyglucose by the sequential action of RmlA (glucose-1-phosphate thymidylyltransferase) and RmlB (dTDP-glucose-4,6-dehydratase) is shared between the dTDP-D-fucose and the dTDP-L-rhamnose biosynthesis pathway. This key intermediate is processed by the dTDP-4-dehydro-6-deoxyglucose reductase Fcd to form dTDP-alpha-D-fucose. We identified the fcd gene in G. tepidamans GS5-97(T) by chromosome walking and performed functional characterization of the recombinant 308-amino acid enzyme. The in vitro activity of the enzymatic cascade (RmlB and Fcd) was monitored by high-performance liquid chromatography and the reaction product was confirmed by (1)H and (13)C nuclear magnetic resonance spectroscopy. This is the first characterization of the dTDP-alpha-D-fucopyranose biosynthesis pathway in a Gram-positive organism. fcd was identified as 1 of 20 open reading frames contained in a 17471-bp S-layer glycosylation (slg) gene cluster on the chromosome of G. tepidamans GS5-97(T). The sgtA structural gene is located immediately upstream of the slg gene cluster with an intergenic region of 247 nucleotides. By comparison of the SgtA amino acid sequence with the known glycosylation pattern of the S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a, two out of the proposed three glycosylation sites on SgtA could be identified by electrospray ionization quadrupole-time-of-flight mass spectrometry to be at positions Ser-792 and Thr-583.     

Structure of the O-specific polysaccharide of Vibrio fluvialis serovar 3]

O-Specific polysaccharide composed of L-rhamnose and 2-acetamido-2-deoxy-D-mannose was obtained on mild acid degradation of the V. fluvialis lipopolysaccharide. On the basis of the 13C-NMR data and methylation studies, the following structure was suggested for the polysaccharide repeating unit: ----4)-alpha-L-Rhap-(1----3)-beta-D-ManpNAc-(1---- This structure was confirmed by calculations using known glycosidation effects on 13C chemical shifts.     

The structure of the O-specific polysaccharide of Escherichia coli O117:K98:H4

The primary structure of the O-antigen of Escherichia coli O117 was shown by monosaccharide analysis, methylation analysis, and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of linear pentasaccharide repeating units with the structure: -->3)-alpha-D-GalpNAc-(1-->4)-beta-D-GalpNAc-(1-->3)-alpha-L-Rhap- (1-->4)- alpha-D-Glcp-(1-->4)-beta-D-Galp-(1-->     

Structural Basis of Substrate Binding in WsaF, a Rhamnosyltransferase from Geobacillus stearothermophilus

Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by α-1,3-, α-1,2-, and β-1,2-linkages. The formation of the β-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-β-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-β-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition.     

Structure of the O-specific polysaccharide of Mesorhizobium huakuii IFO15243T

The structure of the O-specific polysaccharide isolated by mild acid hydrolysis of the lipopolysaccharide of Mesorhizobium huakuii IFO15243T was studied using methylation analysis and various one- and two-dimensional 1H and 13C NMR experiments. The O-antigen polysaccharide was found to be linear polymer constituted by a trisaccharide repeating unit of the following structure: --> 2)-alpha-L-6dTalp-(1 --> 3)-alpha-L-6dTalp-(1 --> 2)-alpha-L-Rhap-(1 -->.     

Synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-[beta-L-Xylp-(1-->4)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap,the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942

An efficient synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-[beta-L-Xylp-(1-->4)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942 and its isomer beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->4)-[beta-L-Xylp-(1-->2)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap was achieved via sequential assembly of the building blocks, allyl 2,3-O-isopropylidene-alpha-L-rhamnopyranoside (2), allyl 3,4-O-isopropylidene-alpha-L-rhamnopyranoside (3), allyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate (7), and 2,3,4-tri-O-benzoyl-beta-L-xylopyranosyl trichloroacetimidate (12). The process was carried out in a regio- and stereoselective manner using glycosyl trichloroacetimidates as donors and unprotected or partially protected rhamnopyranosides as acceptors in the presence of a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf).     

Structural studies on the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide of Klebsiella serotype K40 contained D-mannose, D-glucuronic acid, D-galactose, and L-rhamnose in the approximate molar ratios 1:1:1:2. The primary structure of the capsular polysaccharide has been investigated mainly by methylation analysis, periodate oxidation, characterization of oligosaccharides, base degradation reaction, and 1H and 13CNMR spectroscopy. The polysaccharide does not contain any pyruvic acetal or O-acetyl substitution. It has a pentasaccharide repeating unit of the following primary structure: alpha-D-Manp 1----4 ----4)-beta-D-GlcpA-(1----2)-alpha-L-Rhap-(1----3)-beta-D-Ga lp-(1----2)-alpha- L-Rhap-(1----.     

Analysis of structural components and molecular construction of soybean soluble polysaccharides by stepwise enzymatic degradation.

Soybean soluble polysaccharides (SSPS) extracted from soybean cotyledons have a pectin-like structure. The core polysaccharides after treatments with four kinds of hemicellulases and a pectinase contained approximately equal numbers of L-rhamnose and D-galacturonate residues, suggesting the presence of the rhamnogalacturonan (RG) I structure consisting of the diglycosyl repeating unit, -4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-. The lengths of RG chains were calculated as approximately 15, 28, and 100 diglycosyl repeats. The RG components linked to each other by intervention of galacturonan (GN) chains, constituting the backbone of SSPS. All arabinose residues, which constitute 21% of total SSPS sugars, were found to be in side chains from RG regions, and this was also true for galactose residues, which constitute 50% of total sugars. Of arabinose residues, 94% are present as alpha-1,3- or alpha-1,5-arabinans, and 89% of galactose residues were present as beta-1,4-galactans. Galactan chains are modified with arabinose, xylose, fucose, and glucose at the sites close to the RG regions.     

Structure of the O-polysaccharide of Pseudomonas syringae pv. delphinii NCPPB 1879(T) having side chains of 3-acetamido-3,6-dideoxy-D-galactose residues

The O-polysaccharide (OPS) was obtained from the lipopolysaccharide of Pseudomonas syringae pv. delphinii NCPPB 1879(T) and studied by sugar and methylation analyses, Smith degradation, and (1)H- and (13)C-NMR spectroscopy. The OPS was found to contain residues of L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), and the following structure of the major (n = 2) and minor (n = 3) heptasaccharide repeating units of the OPS was established: [carbohydrate structure: see text]. The OPS is distinguished by the presence of oligosaccharide side chains consisting of three D-Fuc3NAc residues that are connected to each other by the (alpha 1-->2)-linkage. The OPS is characterized by a structural heterogeneity due to a different position of substitution of one of the four L-rhamnose residues in the main chain of the repeating unit as well as to the presence of oligosaccharide units with an incomplete side chain.     

Antigenic bacterial polysaccharides. 13. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from Pseudomonas cepacia strain IMV 4137

On mild acid degradation of a lipopolysaccharide from Pseudomonas cepacia strain IMV 4137, a serologically active O-specific polysaccharide was obtained and shown to contain L-rhamnose and D-galactose. According to 1H- and 13C-NMR data as well as methylation analysis, the polysaccharide is made up of disaccharide repeating units of the following structure:----2)-alpha-L-Rhap-(1----4)-alpha-D-Galp-(1----.     

Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50.

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro-inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg-gingipains of P. gingivalis are post-translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O-polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF-COSY, TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-31P HXTOCSY) and GC-MS data. These data allowed us to conclude that the O-polysaccharide is built up of the tetrasaccharide repeating sequence: -->6)-alpha-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Galp-(1--> and carries a monophosphoethanolamine residue at position C-2 of the alpha-rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O-polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.     

Structure of a capsular polysaccharide isolated from Salmonella enteritidis

Salmonella enteritidis is a food-borne enteric human pathogen that can form a complex protective extracellular matrix. We describe here a component of this matrix which is distinct from other known salmonella extracellular polysaccharides such as cellulose and colanic acid. We have used glycosyl composition and linkage analysis, as well as 1D and 2D NMR spectroscopy to determine the structure of this polysaccharide. We propose that the primary saccharide in the S. enteritidis capsule has a branched tetrasaccharide repeating unit having the following structure: -->3)-alpha-D-Galp-(1-->2)-[alpha-Tyvp-(1-->3)]-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->. This structure is partially substituted on both tyvelose and galactose with a glucose-containing side chain. It further bears considerable similarity to the O antigen from this organism, a feature found in a number of other capsules from Gram-negative bacteria. In addition, we have detected fatty acids at levels that indicate the presence of a lipid anchor.