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.     

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).     

Structural heterogeneity in the core oligosaccharide of the S-layer glycoprotein from Aneurinibacillus thermoaerophilus DSM 10155.

The surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 has a total carbohydrate content of 15% (by mass), consisting of O-linked oligosaccharide chains. After proteolytic digestion of the S-layer glycoprotein byPronase E and subsequent purification of the digestion products by gel permeation chromatography, chromatofocusing and high-performance liquid chromatography two glycopeptide pools A and B with identical glycans and the repeating unit structure -->4)-alpha-l-Rha p -(1-->3)-beta-d- glycero -d- manno -Hep p -(1--> (Kosma et al., 1995b, Glycobiology, 5, 791-796) were obtained. Combined evidence from modified Edman-degradation in combination with liquid chromatography electrospray mass-spectrometry and nuclear magnetic resonance spectroscopy revealed that both glycopeptides contain equal amounts of the complete core structure alpha-l-Rha p -(1-->3)-alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and the truncated forms alpha-l-Rha p -(1-->3)-beta-d-Gal p NAc-(1-->O)-Thr/Ser and beta-d-Gal p NAc-(1-->O)-Thr/Ser. All glycopeptides possessed the novel linkage types beta-d-Gal p NAc-(1-->O)-Thr/Ser. The different cores were substituted with varying numbers of disaccharide repeating units. By 300 MHz proton nuclear magnetic resonance spectroscopy the complete carbohydrate core structure of the fluorescently labeled glyco-peptide B was determined after Smith-degradation of its glycan chain. The NMR data confirmed and complemented the results of the mass spectroscopy experiments. Based on the S-layer glycopeptide structure, a pathway for its biosynthesis is suggested.     

Molecular basis of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus

The Gram-positive bacterium Geobacillus stearothermophilus NRS 2004/3a possesses a cell wall containing an oblique surface layer (S-layer) composed of glycoprotein subunits. O-Glycans with the structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n) (= 13-18), a2-O-methyl group capping the terminal repeating unit at the nonreducing end and a -->2)-alpha-L-Rhap-[(1-->3)-alpha-L-Rhap](n) (= 1-2)(1-->3)- adaptor are linked via a beta-D-Galp residue to distinct sites of the S-layer protein SgsE. S-layer glycan biosynthesis is encoded by a polycistronic slg (surface layer glycosylation) gene cluster. Four assigned glycosyltransferases named WsaC-WsaF, were investigated by a combined biochemical and NMR approach, starting from synthetic octyl-linked saccharide precursors. We demonstrate that three of the enzymes are rhamnosyltransferases that are responsible for the transfer of L-rhamnose from a dTDP-beta-L-Rha precursor to the nascent S-layer glycan, catalyzing the formation of the alpha1,3- (WsaC and WsaD) and beta1,2-linkages (WsaF) present in the adaptor saccharide and in the repeating units of the mature S-layer glycan, respectively. These enzymes work in concert with a multifunctional methylrhamnosyltransferase (WsaE). The N-terminal portion of WsaE is responsible for the S-adenosylmethionine-dependent methylation reaction of the terminal alpha1,3-linked L-rhamnose residue, and the central and C-terminal portions are involved in the transfer of L-rhamnose from dTDP-beta-L-rhamnose to the adaptor saccharide to form the alpha1,2- and alpha1,3-linkages during S-layer glycan chain elongation, with the methylation and the glycosylation reactions occurring independently. Characterization of these enzymes thus reveals the complete molecular basis for S-layer glycan biosynthesis.     

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.     

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.     

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).     

Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide

The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units.     

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.     

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.     

Synthesis of two heptasaccharide analogues of the lentinan repeating unit

beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (18) and the allyl glycoside of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-beta-D-Glcp-(1-->6)]-alpha-D-Glcp (29) were synthesized as the analogues of the lentinan repeating heptaose by building the pentasaccharide backbones first, followed by attaching the side chains. 4,6-O-benzylidenated mono-13 or disaccharide 8 were used as the acceptor to ensure the beta linkage in the synthesis of 18, while 4,6-O-benzylidenated disaccharides 21 and 23 were used as the donor and acceptor, respectively, to ensure the beta linkage in the synthesis of 29.     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Elucidation of two O-chain structures from the lipopolysaccharide fraction of Agrobacterium tumefaciens F/1

Agrobacterium tumefaciens F/1 produces two different O-chains, both are constituted of rhamnose and glucosamine: the less abundant has a linear disaccharidic repeating unit 3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1--> and the second one 4)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->. The two intact antigenic moieties were studied in mixture by 2D NMR. Additional supporting data were obtained by periodate degradation, the major component was cleaved selectively, leading to a glucosamine glycoside, whereas the minor one was recovered unaffected.     

Glycoprotein-bound large carbohydrates of early embryonic cells: structural characteristic of the glycan isolated from F9 embryonal carcinoma cells

The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.     

Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70.

The surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 was shown to contain a new type of glycan chain. Different from all known eubacterial glycoproteins, the saccharide moiety consists only of six sugar residues without any repeat sequences. Proteolytic digestion of purified S-layer glycoprotein resulted in isolation of several glycopeptide fractions. These are composed of the same hexasaccharide portion but are linked to oligopeptides of different length. One of them contains only a single amino acid. As concluded from chemical analyses and proton and carbon nuclear magnetic resonance spectroscopy of this preparation, the hexasaccharide moiety is linked via a novel O-glycosidic linkage. This is a beta-D-glucose residue linked to the phenolic hydroxyl group of tyrosine in intact S-layer glycoprotein.     

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.     

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-->     

Glucuronic acid directly linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins.

Sugar-beet pulp was de-esterified and submitted to 72 h hydrolysis by 0.1 M HCl at 80 degrees C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n(>/= 2) repeats of the dimer -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1--> were isolated from the hydrolysate by ion-exchange and gel-permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O-3 of a galacturonic acid (GalA) residue, as shown for the two pentamers beta-D-GlcpA-(1-->3)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-L-Rhap and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-[beta-D-GlcpA-(1-->3)]-alpha-D-GalpA-(1-->2)-L-Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.     

Structure of the glycan chain from the surface layer glycoprotein of Bacillus alvei CCM 2051

The cell surface of the mesophilic eubacterium Bacillus alvei CCM 2051 is covered by an oblique arranged surface layer glycoprotein. The subunits revealed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis were distinct bands of molecular masses 140,000, 128,000, and 127,000. Proteolytic degradation of the purified S-layer glycoprotein yielded a single glycopeptide fraction with an apparent molecular mass of ca. 25,000. Methylation analysis in conjunction with two-dimensional nuclear magnetic resonance experiments at 500 MHz established the branched trisaccharide (formula; see text) as the repeating unit for this glycan chain.     

Structural characterization of the O-antigenic polysaccharide of the lipopolysaccharide from Rhizobium etli strain CE3. A unique O-acetylated glycan of discrete size, containing 3-O-methyl-6-deoxy-L-talose and 2,3,4-tri-O-,methyl-l fucose

The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.