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.     

Structural analysis of the alpha-D-glucan (EPS180) produced by the Lactobacillus reuteri strain 180 glucansucrase GTF180 enzyme

The neutral exopolysaccharide EPS180 produced from sucrose by the glucansucrase GTF180 enzyme from Lactobacillus reuteri 180 was found to be a (1-->3,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis, periodate oxidation, and 1D/2D 1H and 13C NMR spectroscopy of the intact EPS180, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of EPS180, a composite model, that includes all identified structural features, was formulated as follows: [Formula: see text].     

Structural analysis of the alpha-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme

The neutral exopolysaccharide EPS35-5 (reuteran) produced from sucrose by the glucansucrase GTFA enzyme from Lactobacillus reuteri 35-5 was found to be a (1-->4,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis and 1D/2D 1H and 13C NMR spectroscopy of intact EPS35-5, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis and enzymatic hydrolysis, using pullulanase M1 (Klebsiella planticola), of EPS35-5, a composite model, that includes all identified structural elements, was formulated as follows: [Formula: see text].     

Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodochrous strain S-2

A possibility has been suggested of applying the EPS produced by Rhodococcus rhodochrous strain S-2 (S-2 EPS) to the bioremediation of oil-contaminated environments, because its addition, together with minerals, to oil-contaminated seawater resulted in emulsification of the oil, increased the degradation of polyaromatic hydrocarbons (PAH) of the oil, and led to the dominance of PAH-degrading marine bacteria. To understand the underlying principles of these phenomena, we determined the chemical structure of the sugar chain of S-2 EPS. The EPS was found to be composed of D-galactose, D-mannose, D-glucose, and D-glucuronic acid, in a molar ratio of 1:1:1:1. In addition, 0.8% (w/w) of octadecanoic acid and 2.7% (w/w) of hexadecanoic acid were also contained in its structure. By 1H and 13C NMR spectroscopy, including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments, as well as chemical and enzymatic analyses, the polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (see formula in text).     

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain L-rhamnose, 2-acetamido-4-[D-3-hydroxybutyramido)]-2,4,6-trideoxy-D-glucose (D-BacNAc4NHbu), and 2-amino-2,6-dideoxy-L-galactose, N-acylated by the 4-carboxyl group of L-malic acid (L-malyl-(4-->2)-alpha-L-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established:-3)-alpha-D-BacNAc4NHbu-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->2)-L-malyl-(4-->2)-alpha-L-FucN-(1-. The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.     

Structure of the high molecular weight exopolysaccharide produced by Bifidobacterium animalis subsp. lactis IPLA-R1 and sequence analysis of its putative eps cluster

The bile adapted strain Bifidobacterium animalis subsp. lactis IPLA-R1 secretes a high molecular weight exopolysaccharide (HMW-EPS) when grown on the surface of agar-MRSC. This EPS is composed of l-rhamnopyranosyl, d-glucopyranosyl, d-galactopyranosyl and d-galactofuranosyl residues in the ratio of 3:1:1:1. Linkage analysis and 1D and 2D NMR spectroscopy were used to show that the EPS has a hexasaccharide repeating unit with the following structure:Treatment of the EPS with mild acid cleanly removed the terminal d-galactofuranosyl residue. The eps cluster sequenced for strain IPLA-R1 showed high genetic homology with putative eps clusters annotated in the genomes of strains from the same species. It is of note that several genes coding for rhamnose-precursors are present in the eps cluster, which could be correlated with the high percentage of rhamnose detected in its EPS repeated unit.     

Structure of the O-specific polysaccharide of Providencia rustigianii O14 containing N(epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine

The O-specific polysaccharide of Providencia rustigianii O14 was obtained by mild acid degradation of the LPS and studied by chemical methods and NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, NOESY, and 1H,(13)C HSQC experiments. The polysaccharide was found to contain N (epsilon)-[(S)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine ('alaninolysine', 2S,8S-AlaLys). The amino acid component was isolated by acid hydrolysis and identified by 13C NMR spectroscopy and specific optical rotation, using synthetic diastereomers for comparison. The following structure of the trisaccharide repeating unit of the polysaccharide was established:Anti-P. rustigianii O14 serum was found to cross-react with O-specific polysaccharides of Providencia and Proteus strains that contains amides of uronic acid with N(epsilon)-[(R)-1-carboxyethyl]-L-lysine and L-lysine.     

Structural analysis of an extracellular polysaccharide produced by a benzene tolerant bacterium, Rhodococcus sp. 33

Rhodococcus sp. 33 can tolerate and efficiently degrade various concentrations of benzene, one of the most toxic and prevailing environmental pollutants. This strain produces a large quantity of extracellular polysaccharide (33 EPS), which plays an important role in the benzene tolerance in Rhodococcus sp. 33, especially by helping the cells to survive an initial challenge with benzene. This EPS has been reported to be composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1. To understand the protective effect of 33 EPS, we determined its chemical structure by using 1H and 13C NMR spectroscopy including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: [structure: see text].     

Structural elucidation of the O-antigenic polysaccharide from enterohemorrhagic (EHEC) Escherichia coli O48:H21

The structure of the antigenic O-polysaccharide (O-PS) of the lipopolysaccharide (LPS) produced by the enterohemorrhagic strain of Escherichia coli O48:H21 (EHEC) has been elucidated. The O-PS obtained by mild acid hydrolysis of the LPS had [alpha]D +95 (water) and was composed of L-rhamnose (L-Rha), D-galactose (D-Gal), 2-amino-2-deoxy-D-glucose (D-GlcN), 2-amino-2-deoxy-D-galactose (D-GalN), and D-galacturonic acid (D-GalA) (1:1:1:1:1). From the results of methylation analysis, mass spectrometry, 2D NMR, and DOC-PAGE, the O-PS was shown to be a high molecular mass polymer of a repeating pentasaccharide unit having the structure: [structure: see text]. The D-Gal pA non-reducing end groups in the O-PS were partially O-acetylated (approximately 30%) at the O-2 and O-3 positions and the degree of acetylation was variable from batch to batch cell production.     

Structural studies on exopolysaccharides produced by three different propionibacteria strains

The exopolysaccharides produced by three propionibacteria strains, Propionibacterium freudenreichii 109, Propionibacterium freudenreichii 111, and Propionibacterium thoenii 126, grown on whey-based media, were found to be charged heteropolymers, composed of D-glucose, D-mannose, and D-glucuronic acid in molar ratios of 2:2:1. By means of methylation analysis, mass spectrometry, partial acid hydrolysis, and 1D/2D NMR (1H and 13C) studies, it was determined that all three exopolysaccharides contain the same branched, pentasaccharide repeating unit: [Formula: see text].     

Chemical structure of aeromonas gum--extracellular polysaccharide from Aeromonas nichidenii 5797

Aeromonas (A) gum, an extracellular heteropolysaccharide produced by the bacterium Aeromonas nichidenii strain 5797, was studied by 1H and 13C NMR spectroscopy including 2D COSY, TOCSY, 1H, 13C HMQC, HMBC and ROESY experiments after O-deacetylation and Smith degradation. These investigations revealed the presence of an O-acetylated pentasaccharide repeating unit composed of mannose, glucose, xylose and glucuronic acid, and it has the following structure: [Image: see text]     

Structure of the O-polysaccharide of Providencia alcalifaciens O8 containing (2S,4R)-2,4-dihydroxypentanoic acid, a new non-sugar component of bacterial glycans

A glycerol teichoic acid-like O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O8 and studied by chemical methods and NMR spectroscopy, including 2D ROESY, {(1)H,(13)C} HSQC, and HMQC-TOCSY experiments. It was found that the compound contains a new component of bacterial lipopolysaccharides: ether-linked (2S,4R)-2,4-dihydroxypentanoic acid (Dhpa), which was identified by NMR spectroscopy. The following structure of the repeating unit of the polysaccharide was established:     

The O-polysaccharide of Escherichia coli O112ac has the same structure as that of Shigella dysenteriae type 2 but is devoid of O-acetylation: a revision of the S. dysenteriae type 2 O-polysaccharide structure

The O-antigen structure of Shigella dysenteriae type 2 was reinvestigated using chemical modifications along with high-resolution 2D (1)H and (13)C NMR spectroscopy. The O-antigen was found to contain a pyruvic acid acetal, which was overlooked in an early study, and the following revised structure of the pentasaccharide repeating unit was established: where approximately 70% GlcNAc residues bear an O-acetyl group at position 3. The O-antigen of Escherichia coli O112ac was found to have the same carbohydrate structure but to lack O-acetylation.     

The complete structure of the core of the LPS from Plesiomonas shigelloides 302-73 and the identification of its O-antigen biological repeating unit

Plesiomonas shigelloides is a Gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal infections, which especially invades immunocompromised patients and neonates. The lipopolysaccharides are one of the major virulence determinants in Gram-negative bacteria and are structurally composed of three different domains: the lipid A, the core oligosaccharide and the O-antigen polysaccharide.In the last few years we elucidated the structures of the O-chain and the core oligosaccharide from the P. shigelloides strain 302-73. In this paper we now report the characterization of the linkage between the core and the O-chain. The LPS obtained after PCP extraction contained a small number of O-chain repeating units. The product obtained by hydrazinolysis was analysed by FTICR-ESIMS and suggested the presence of an additional Kdo in the core oligosaccharide. Furthermore, the LPS was hydrolysed under mild acid conditions and a fraction that contained one O-chain repeating unit linked to a Kdo residue was isolated and characterized by FTICR-ESIMS and NMR spectroscopy. Moreover, after an alkaline reductive hydrolysis, a disaccharide α-Kdo-(2→6)-GlcNol was isolated and characterized. The data obtained proved the presence of an α-Kdo in the outer core and allowed the identification of the O-antigen biological repeating unit as well as its linkage with the core oligosaccharide.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

The structure of the exocellular polysaccharide produced by Rhodococcus sp. RHA1

Rhodococcus sp. RHA1 is a Gram-positive actinomycete capable of metabolizing a wide spectrum of organic compounds whose survival in chemically hostile environments is believed to be in part due to the production of an exocellular polysaccharide (EPS). In order to investigate the functional nature of the EPS, its structure was determined using a combinatory approach including hydrolysis, composition, and methylation, analysis methods, as well as 2D (1)H and (13)C NMR spectroscopy. The EPS was found to be a high-molecular-mass polymer of a repeating tetrasaccharide unit composed of D-glucuronic acid, D-glucose, D-galactose, L-fucose and O-acetyl (1:1:1:1:1), and has the structure:     

The O-polysaccharide from the lipopolysaccharide of Providencia stuartii O44 contains L-quinovose, a 6-deoxy sugar rarely occurring in bacterial polysaccharides

The O-polysaccharide (O-antigen) of Providencia stuartii O44:H4 (strain 3768/51) was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC, and HMQC-TOCSY experiments. The O-polysaccharide was found to have a branched hexasaccharide repeating unit of the following structure: [Formula: see text].     

Structural assignment of a heteropolysaccharide isolated from the gum of Cochlospermum religiosum (Katira gum)

A heteropolysaccharide isolated from the gum (Katira) of Cochlospermum religiosum was found to consist of D-galactose, D-galacturonic acid and L-rhamnose in a molar ratio 2:1:3. Structural assignment of the polysaccharide was carried out using total acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation and NMR studies (1H, 13C, DQF-COSY, TOCSY, NOESY, HMBC and HSQC) and the repeating unit of the polysaccharide was established as [Formula: see text].     

Structure and serological analysis of the Hafnia alvei 481-L O-specific polysaccharide containing phosphate in the backbone chain

The lipopolysaccharide was extracted from cells of Hafnia alvei 481-L bacterial strain and, after mild acid hydrolysis, the O-specific polysaccharide was isolated and characterised. On the basis of chemical analyses and NMR spectroscopic studies of the polysaccharide and oligosaccharides obtained after Smith degradation, or hydrogen fluoride treatment, it was found that the repeating unit of the O-specific polysaccharide is a phosphorylated hexasaccharide: [see text]. The biological repeating unit of the H. alvei 481-L O-antigen has galactose phosphate at the nonreducing terminus. Serological tests indicate that this strain represents an individual serotype in the H. alvei genus.     

Structural determination of the O-antigenic polysaccharide from the verocytotoxin-producing Escherichia coli O176

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O176 has been determined. Component analysis together with 1H and 13C NMR spectroscopy was employed to elucidate the structure. Inter-residue correlations were determined by 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [Formula: see text] Cross-peaks of low intensity from alpha-linked mannopyranosyl residues were present in the 1H, 1H TOCSY NMR spectra and further analysis of these showed that they originate from the terminal part of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-galactosamine residue at its reducing end. The repeating unit of the E. coli O176 O-antigen is similar to those from E. coli O17 and O77, thereby explaining the reported cross-reactivities between the strains, and identical to that of Salmonella cerro (O:6, 14, 18).