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:     

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).     

Extracellular polysaccharides of a bacterium associated with a fungal canker disease of Eucalyptus sp

Extracellular polysaccharides (EPSs) produced by an Erwinia sp associated with a fungal canker disease of Eucalyptus were fractionated into one polysaccharide that was identified with that produced by Erwinia chrysanthemi strains SR260, Ech1, and Ech9, and the other distinctively different from any other EPS produced by E. chrysanthemi strains so far studied. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, low pressure gel-filtration, and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharide, identified as EPS Teranera, has the following structure: [structure: see text] The molecular weights of the polysaccharides range from 3.2-6.2 x 10(5) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.     

Structure of a polysaccharide from a Rhizobium species containing 2-deoxy-beta-D-arabino-hexuronic acid.

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.     

Structural studies of an emulsion-stabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain.

The primary structure of an emulsion-stabilizing exopolysaccharide from the adhesive, hydrophobic Rhodococcus strain No. 33 was elucidated by NMR spectroscopy, methylation analyses, periodate oxidation and oligosaccharide analyses. The polysaccharide PS-33 consisted of rhamnose, galactose, glucose and glucuronic acid in molar ratios of 2:1:1:1. The main chain contained 3-substituted alpha-D-glucuronic acid linked to the 3-position at alpha-L-rhamnose, in addition to 3-substituted residues of beta-D-galactose and alpha-D-glucose. The alpha-L-rhamnose of the side chain was linked to position 4 of the galactose. In addition, the polysaccharide was O-acetylated, corresponding to one acetyl group per repeating unit. From the results two structural possibilities could be suggested. As the polysaccharide carries hydrophobic groups (methyl of rhamnose/O-acetyl), it is very likely that these are of general significance for the emulsifying activity of polysaccharides. It also seems to be possible that this polysaccharide is at least partially responsible for the hydrophobic cell surface properties of the Rhodococcus strain No. 33 and it may be involved in hydrophobic interactions when adhering to hydrophobic interfaces.     

Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D (1)H and (13)C NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure: [structure: see text].     

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

Isolation and biochemical characterization of extracellular polymeric secretions (EPS) from modern soft marine stromatolites (Bahamas) and its inhibitory effect on CaCO3 precipitation

Bahamian soft marine stromatolites consist of cyanobacterial biofilms and carbonate sand grains (ooids) embedded in their extracellular polymeric secretions (EPS). EPS were isolated from natural marine stromatolites and the laboratory cultured stromatolite forming cyanobacterium isolate Schizothix sp. Laboratory investigations were conducted to examine biochemical characteristics and the role of EPS in the inhibition of CaCO3 precipitation. EPS consisted of acid polysaccharides and proteins. SDS-PAGE and amino acid analysis suggested that EPS from both soft marine stromatolite and Schizothrix sp. mat contained small proteins (38 kD and 45 kD) enriched in aspartic acid and glutamic acid. Also, immuno blotting suggested that natural EPS contain high molecular weight acid polysaccharide (500 k) which may represent cross-linked products of laboratory cultured Schizothrix sp. acid polysaccharide (300 k). EPS from both soft marine stromatolite and laboratory cultured Schizothrix sp. inhibited CaCO3 precipitation in vitro, as determined using pH drift assays examining pH decrease which occur in response to CaCO3 precipitation. PH drift assays of enzymatically and chemically modified EPS isolated from soft marine stromatolite and laboratory cultured Schizothrix sp. indicated that both uronic acids and protein fractions may be involved in the inhibition of CaCO3 precipitation.     

Structural analysis of mucoidan, an acidic extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides (EPS), which are assumed to play an important role in the hydrocarbon tolerance of R. erythropolis PR4. The strain produced an acidic EPS, mucoidan, together with a fatty acid-containing EPS, PR4 FACEPS. The chemical structure of the mucoidan was determined using (1)H and (13)C NMR spectroscopy and by conducting 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The mucoidan was shown to consist of a pentasaccharide repeating unit with the following structure: [structure: see text].     

Relationships between colony morphotypes and oil tolerance in Rhodococcus rhodochrous

A mucoidal strain of Rhodococcus rhodochrous was resistant to 10% (vol/vol) n-hexadecane, while its rough derivatives were sensitive. When the extracellular polysaccharide (EPS) produced by the mucoidal strain was added to cultures of the rough strains, the rough strains gained resistance to n-hexadecane. Thus, EPS confer tolerance to n-hexadecane in members of the genus Rhodococcus.     

Structure of an acylated exopolysaccharide synthesized by Acinetobacter sp

The complex preparation ethapolan synthesized by Acinetobacter sp. consists of neutral (minor component) and two acidic exopolysaccharides (EPS) one of which is acylated. On the basis of chemical modification of EPS, solvolysis with anhydrous hydrogen fluoride resulting in a penta- and octasaccharide fragments, Smith degradation, 1H- and 13C NMR analysis the following structure of the acylated polysaccharide repeating unit has been established (scheme): It is suggested that in the acylated EPS at least one glucose residue and the galactose residue are O-acylated.     

一株高温产粘菌株的筛选及其产胞外聚合物分析

从油井采出水中分离到一株高温产胞外聚合物的细菌MS-1,经16S rDNA基因序列分析属于芽孢杆菌属(Bacillus sp.).该菌能在60℃生长并产生胞外聚合物,其中胞外多糖含量为48.3%～54.5%.主要由甘露糖、葡萄糖、半乳糖组成,摩尔比分别是2.04:1.00:0.89.胞外聚合物中蛋白含量为37.2%～42.4%,主要由甲硫氨酸、亮氨酸、天门冬氨酸、丙氨酸、组氨酸和丝氨酸组成.利用透射电镜和环境扫描电镜对胞外聚合物的形成进行了观察.该菌的分离和研究为高温油藏的微生物调剖和驱油奠定了生物学基础.     

Structure and hydrodynamic properties of the extracellular polysaccharide from a mutant strain (RA3W) of Erwinia chrysanthemi RA3

The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain RA3W, a mutant strain of E. chrysanthemi RA3, has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl linkage analysis, and 1D 1H NMR spectroscopy. The polysaccharide is structurally similar, if not identical, to the family of EPS produced by such as E. chrysanthemi strains Ech9, Ech9Sm6, and SR260. The molecular weight of EPS RA3W by ultracentrifugation (sedimentation equilibrium) and light scattering is compared with those of other E. chrysanthami EPSs, as are the viscometric properties.     

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.     

The structure of the O-specific polysaccharide from the lipopolysaccharide of Pseudomonas sp. OX1 cultivated in the presence of the azo dye Orange II

The Gram-negative bacterium Pseudomonas sp. OX1, previously known as Pseudomonas stutzeri OX1, is endowed with a high metabolic versatility. In fact, it is able to utilize a wide range of toxic organic compounds as the only source of carbon and energy for growth. It has been recently observed that, while growing on a glucose-containing liquid medium, Pseudomonas sp. OX1 can reduce azo dyes, ubiquitous pollutants particularly resistant to chemical and physical degradation, with this azoreduction being a process able to generate enough energy to sustain bacterial survival. We have found that, under these conditions, modifications in the primary structure of the O-specific polysaccharide (OPS) within the lipopolysaccharides occur, leading to remarkable changes both in the monosaccharide composition and in the architecture of the repeating unit, with respect to the polysaccharide produced in the absence of azo dyes. In the present paper, we present the complete structure of this O-specific polysaccharide, whose repeating unit is the following: [Formula: see text] This structure is totally different from the one determined from Pseudomonas sp. OX1 grown on rich medium.     

Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below. -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.     

Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs

AIMS: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. CONCLUSIONS: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. Significance and Impact of the Study: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.     

Free radical scavenging and antioxidant activities of EPS2, an exopolysaccharide produced by a marine filamentous fungus Keissleriella sp. YS 4108

The reactive oxygen species (ROS) and free radical-initiated reactions are ascertained to play multiple roles in degenerative or pathological events such as aging, cancer, heart dysfunction and Alzheimer's disease. EPS2 with a mean molecular weight of 1.3 x 10(5) was characterized as an antioxidant exopolysaccharide from the broth of a marine filamentous fungus Keissleriella sp. YS 4108. Compositionally, it is composed of galactose, glucose, rhamnose, mannose and glucuronic acid in an approximate proportion of 50:8:1:1:0.4. The radical eliminating and antioxidant actions of the glycan was assessed in different in vitro systems showing that EPS2 exhibited profound scavenging activities in superoxide radical. As a reinforcement of the action, similar radical scavenging effects of EPS2 were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. Moreover, EPS2 effectively blocked as well the non site-specific strand-breaking of DNA induced by the Fenton reaction at concentrations of 0.1 and 1 mg/mL. Further investigation of the effect of EPS2 on human low density lipoprotein (LDL) system demonstrated that it significantly inhibited copper-mediated oxidation of LDL in a dose-dependent manner. These results suggest that EPS2, possessing pronounced free radical scavenging and antioxidant activities, could be of considerable preventive and therapeutic significance to some life-threatening health problems such as cancer, atherogenesis and Alzheimer's disease which pathologically initiated by the presence of free radicals leading to the inevitable peroxidation of important biomolecules.     

Structure of an acidic polysaccharide from the agar-decomposing marine bacterium Pseudoalteromonas atlantica strain IAM 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid

The structure of an acidic polysaccharide from Pseudoalteromonas atlantica strain 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac) has been elucidated. The polysaccharide was studied by 1H and 13C NMR spectroscopy, including 2D experiments, along with sugar and methylation analyses. After a selective hydrolysis a modified polysaccharide devoid of its side chain could be isolated. It was found that the polysaccharide has pentasaccharide repeating units with following structure: [structure: see text].     

Optimization, purification, characterization and antioxidant activity of an extracellular polysaccharide produced by Paenibacillus polymyxa SQR-21

The optimization, purification and characterization of an extracellular polysaccharide (EPS) from a bacterium Paenibacillus polymyxa SQR-21 (SQR-21) were investigated. The results showed that SQR-21 produced one kind of EPS having molecular weight of 8.96 × 10⁵ Da. The EPS was comprised of mannose, galactose and glucose in a ratio of 1.23:1.14:1. The ratio of monosaccharides and glucuronic acid was 7.5:1. The preferable culture conditions for EPS production were pH 6.5, temperature 30 °C for 96 h with yeast extract and galactose as best N and C sources, respectively. The maximum EPS production (3.44 g L⁻¹) was achieved with galactose 48.5 g L⁻¹, Fe³⁺ 242 μM and Ca²⁺ 441 μM. In addition, the EPS showed good superoxide scavenging, flocculating and metal chelating activities while moderate inhibition of lipid peroxidation and reducing activities were determined. These results showed the great potential of EPS produced by SQR-21 to be used in industry in place of synthetic compounds.