Composition of O-antigenic lipopolysaccharides from Enterobacter cloacae

Analyses have been carried out on lipopolysaccharides (LPS) from 14 strains of Enterobacter cloacae representing different O serotypes. All of the products appeared to have a composition and architecture typical of enterobacterial LPS, but points of interest include the absence of phosphate residues from the core oligosaccharide, the presence of both L-glycero-D-mannoheptose and D-glycero-D-mannoheptose (ratio usually about 4:1), and the presence in lipid A of small amounts of fatty acids with odd numbers of carbon atoms (mainly C13) in addition to tetradecanoic acid and 3-hydroxytetradecanoic acid. Monosaccharides identified as components of polymeric fractions from the LPS were glucose, galactose, mannose, rhamnose, glucosamine, galactosamine, fucosamine, and galacturonic acid. Most polymeric fractions also probably contained an O-acetyl substituent. Closely similar chemotypes found for the polymeric fractions from the LPS of cross-reacting serotypes support the view that these fractions contain the O-antigenic determinants and represent the side chains of the LPS.     

Characterization of Exopolysaccharides Produced by Plant-Associated Fluorescent Pseudomonads

A total of 214 strains of plant-associated fluorescent pseudomonads were screened for the ability to produce the acidic exopolysaccharide (EPS) alginate on various solid media. The fluorescent pseudomonads studied were saprophytic, saprophytic with known biocontrol potential, or plant pathogenic. Approximately 10% of these strains exhibited mucoid growth under the conditions used. The EPSs produced by 20 strains were isolated, purified, and characterized. Of the 20 strains examined, 6 produced acetylated alginate as an acidic EPS. These strains included a Pseudomonas aeruginosa strain reported to cause a dry rot of onion, a strain of P. viridiflava with soft-rotting ability, and four strains of P. fluorescens. However, 12 strains of P. fluorescens produced a novel acidic EPS (marginalan) composed of glucose and galactose (1:1 molar ratio) substituted with pyruvate and succinate. Three of these strains were soft-rotting agents. Two additional soft-rotting strains of P. fluorescens produced a third acidic novel EPS composed of rhamnose, mannose, and glucose (1:1:1 molar ratio) substituted with pyruvate and acetate. When sucrose was present as the primary carbon source, certain strains produced the neutral polymer levan (a fructan) rather than an acidic EPS. Levan was produced by most strains capable of synthesizing alginate or the novel acidic EPS containing rhamnose, mannose, and glucose but not by strains capable of marginalan production. It is now evident that the group of bacteria belonging to the fluorescent pseudomonads is capable of elaborating a diverse array of acidic EPSs rather than solely alginate.     

Characterization of the lipopolysaccharide from the nod mutant of Rhizobium trifolii

Lipopolysaccharides (LPS) from the non-nodulating Rhizobium trifolii 24SM 15 and from the nodulating R. trifolii 24SM 13 were isolated and examined by means of gas-liquid chromatography and mass spectrometry. Analysis of LPS showed these preparations from both strains examined contained Lipid A, 2-keto-3-deoxyoctonate, neutral sugars, amino sugars, and trace amounts of amino acids. In 24SM 13 LPS prevailed glucose and rhamnose whereas LPS from the non-nodulating strain SM 15 contained mainly mannose, galactose and heptose. Quinovosamine and mannosamine were detected only in the nodulating strain. The ratio of glucosamine phosphate to glucosamine was higher in the LPS of the non-nodulating strain SM 15 than in the corresponding material of the nodulating one. An unknown component producing a peak at the position of glyceryl-S-cysteine on amino acid analysis profiles was detected in SM 15 LPS. The differences in LPS composition were associated with the alterations in the sensitivity to phage 3H, and nodulation ability.     

RELATIVE INCREASE OF DEOXY SUGARS DURING MICROBIAL DEGRADATION OF AN EXTRACELLULAR POLYSACCHARIDE RELEASED BY A TROPICAL FRESHWATER THALASSIOSIRA SP. (BACILLARIOPHYCEAE)1

The aim of this study was to characterize the extracellular polysaccharides (EPS) released by a freshwater Thalassiosira sp. (Bacillariophyceae) and evaluate their degradation by heterotrophic microbial populations from the same habitat of Thalassiosira sp., a tropical eutrophic reservoir. The EPS were purified by anion exchange column chromatography, the monosaccharide composition was determined by GC, and the linkages of the monosaccharides by GC‐MS. The EPS is a mannose‐rich heteropolysaccharide composed of two different acidic fractions. Both of these fractions are composed of mannose, rhamnose, fucose, xylose, galactose, glucose, glucuronic acid, and N‐acetyl glucosamine but with different proportions. N‐acetyl galactosamine occurs only in fraction 1 and galacturonic acid only in fraction 2. We monitored the concentrations of the monosaccharides in the EPS during its degradation using pulse amperometric detection in an HPLC. The decay patterns of the monosaccharides were varied and the deoxy sugars, fucose and rhamnose, were degraded at a slower rate than the other components, increasing their relative concentrations and the hydrophobic feature of the EPS. The possibility of a selective degradation, which enhances the stickiness of the EPS, promoting transparent exopolymeric particles and aggregate formation, is discussed based on the literature data.     

Chemotypes of Veillonella lipopolysaccharides

Lipopolysaccharides (LPS) were isolated by phenol-water extraction from 34 strains of Veillonella, and examined by paper chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-deoxy-octonate (KDO). Several preparations were also examined for neutral sugars by gas liquid chromatography. The LPS had in common glucosamine, galactosamine, L-glycero-D-manno-heptose glucose and KDO. Most LPS contained galactose, and a few rhamnose. D-glycero-D-manno-heptose was found in LPS from one of the strains. Based on the sugar composition of the LPS, the Veillonella strains could be classified into four chemotypes.     

Characterization of lipopolysaccharides from Ralstonia solanacearum

Lipopolysaccharides (LPSs) from four strains of Ralstonia solanacearum belonging to biovar I (ICMP 6524, 8115, 5712, and 8169) were isolated and investigated. The structural components of the LPS molecule, such as lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS), were obtained after mild acid hydrolysis of the LPS preparations. In lipid A from all the LPS samples studied, 3-hydroxyhexadecanoic, 2-hydroxyhexadecanoic, tetradecanoic, and hexadecanoic fatty acids prevailed. The dominant monosaccharides of the core oligosaccharides of all of the strains studied were rhamnose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and heptose. However, individual strains varied in the content of galactose, ribose, xylose, and arabinose. Three types of the O-PS structure were established, which differed in their configuration (alpha or beta), as well as in the type of the bond between glucosamine and rhamnose residues (1-->2 or 1-->3).     

Production of exopolysaccharides from a thermophilic microorganism isolated from a marine hot spring in flegrean areas

A thermophilic strain isolated from sea sand at Maronti, near Sant' Angelo (Ischia), is described. The organism grows well at an optimal temperature of 60 °C at pH 7.0. The thermophilic bacterium, named strain 4004, produces an exocellular polysaccharide (EPS) in yields of 90 mg/l. The EPS fraction was produced with all substrates tested, although a higher yield was obtained with sucrose or trehalose as sole carbon source. During growth, the EPS content was proportional to the biomass. Three fractions (EPS1, EPS2, EPS3) were obtained after purification. Quantitative monosaccharide analysis of the EPSs revealed the presence of mannose:glucose:galactose in a relative ratio of 0.5:1.0:0.3 in EPS1, mannose:glucose:galactose in a relative ratio of 1.0:0.3:trace in EPS2, and galactose:mannose:glucosamine:arabinose in a relative ratio of 1.0:0.8:0.4:0.2 in EPS3. The average molecular mass of EPS3 was determined to be 1×10⁶ Da. From comparison of the chemical shift values in ¹H and ¹³C spectra, we conclude that EPS3 presents a pentasaccharide repeating unit. Electronic Publication     

Sugar composition and FT-IR analysis of exopolysaccharides produced by microbial isolates from paper mill slime deposits

Thirty exopolysaccharides (EPS) produced by bacteria isolated from biofilms or slimelayers from different paper and board mills in Finland, France and Spain were subjected to size exclusion chromatography and sugar compositional analysis. High performance size exclusion chromatography (HPSEC) analysis revealed that some samples were composed of several molecular weight populations. These samples were fractionated by size exclusion chromatography and pooled accordingly. Principal components analysis (PCA) of the sugar compositions of the different pools indicated the presence of glucans and mannans caused by insufficient removal of the carbon or nitrogen source (yeast extract) from the bacteria growth medium leading to an overestimation of the glucose and mannose level in the sample, respectively. From the point of view of slime problems the EPS populations are the most important for multivariate analysis. Four groups of EPSs have been recognized by PCA analysis: a group of EPSs produced by Enterobacter and related genera similar to the regularly reported colanic acid; a group of Methylobacterium EPSs having high galactose and pyruvate levels and two groups that showed less dense clusters produced by Bacillus and related genera, showing high mannose and/or glucose levels and Klebsiella EPSs that showed galactose with rhamnose as major characteristic sugar moieties. Fourier transform infrared spectroscopy (FT-IR) of the same samples followed by discriminant partial least squares regression (DPLS) and linear discriminant analysis (LDA) showed that, when used with a well-defined training set, FT-IR could be used clustering instead of time-consuming sugar composition analysis. The Enterobacter and Methylobacetrium EPS groups could be recognized clearly. However the fact that this could hardly be done for the other two groups in the dataset indicates the importance of a larger and well-defined training or calibration set. The potential to use FT-IR, as a tool for pattern recognition and clustering with respect to EPS structures produced by micro organisms isolated from a paper mill environment is discussed.     

Composition and Role of Extracellular Polymers in Methanogenic Granules

Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaerobic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H(inf2)-CO(inf2)), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobacterium formicicum.     

Screening fungi for the production of an inhibitor of HMG CoA reductase: Production of mevinolin by the fungi of the genus Pleurotus

Abstract Two thermophilic archaea belonging to the genus Sulfolobus , grown on glucose, produced an extracellular polysaccharide (EPS). The production of EPS conducted both in 90 1 fermentor and in batch culture reached the maximum during the stationary phase of growth. The chemical analysis (FT-IR, HPAE-PAD, UV, Optical Rotation) of the biopolymer and of its acid hydrolizate suggested glucose, mannose, glucosamine and galactose as major components of the sulfated heteropolysaccharide.     

Chemical Composition of Two Exopolysaccharides from Bacillus thermoantarcticus

The thermophilic bacterium Bacillus thermoantarcticus produces two exocellular polysaccharides (EPS 1 and EPS 2), which can be obtained from the supernatant of liquid cultures by cold-ethanol precipitation, in yields as high as 400 mg liter(sup-1). The EPS fraction was produced with all substrates tested, although a higher yield was obtained with mannose as the carbon and energy source. The EPS content was proportional to the total biomass. On a weight basis, EPS 1 and EPS 2 represented about 27 and 71%, respectively, of the total carbohydrate fraction. EPS 1 is a sulfate heteropolysaccharide containing mannose and glucose in a relative molar proportion of 1.0 and 0.7, respectively. EPS 2 is a sulfate homopolysaccharide containing mannose as the major component. The absolute configurations of hexoses were shown to be d for both EPSs. Nuclear magnetic resonance spectra confirmed the presence of (alpha)-d-mannose and (beta)-d-glucose in EPS 1 and only (alpha)-d-mannose in EPS 2. In addition, (sup1)H nuclear magnetic resonance analysis and chemical analysis indicated the presence of pyruvic acid in EPS 2.     

Herbaspirillum seropedicae rfbB and rfbC genes are required for maize colonization

In this study we disrupted two Herbaspirillum seropedicae genes, rfbB and rfbC, responsible for rhamnose biosynthesis and its incoporation into LPS. GC-MS analysis of the H. seropedicae wild-type strain LPS oligosaccharide chain showed that rhamnose, glucose and N-acetyl glucosamine are the predominant monosaccharides, whereas rhamnose and N-acetyl glucosamine were not found in the rfbB and rfbC strains. The electrophoretic pattern of the mutants LPS was drastically altered when compared with the wild type. Knockout of rfbB or rfbC increased the sensitivity towards SDS, polymyxin B sulfate and salicylic acid. The mutants attachment capacity to maize root surface plantlets was 100-fold lower than the wild type. Interestingly, the wild-type capacity to attach to maize roots was reduced to a level similar to that of the mutants when the assay was performed in the presence of isolated wild-type LPS, glucosamine or N-acetyl glucosamine. The mutant strains were also significantly less efficient in endophytic colonization of maize. Expression analysis indicated that the rfbB gene is upregulated by naringenin, apigenin and CaCl(2). Together, the results suggest that intact LPS is required for H. seropedicae attachment to maize root and internal colonization of plant tissues.     

Chemical and biological properties of lipopolysaccharide, lipid A and degraded polysaccharide from Wolinella recta ATCC 33238

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-D-manno-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0, C14:0, 3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.     

Exopolysaccharides of Pseudomonas mendocina P2d

Pseudomonas mendocina P₂d cells grown at room temperature in sodium benzoate as sole source of carbon, followed by storage on ice, form a viscous pellet on centrifugation. Such viscosity is not produced by cells grown on glucose or any other carbohydrates. Viscosity was found to be associated with the extracellular polysaccharide (EPS) of cells and not released into the supernatant fluid. A combination of sodium dodecyl sulphate-citrate buffer and homogenization was effective in releasing the EPS. The EPS is a heteropolysaccharide, consisting of rhamnose, fucose, glucose, ribose, arabinose and mannose, which has good emulsifying activity.     

Composition of the lipopolysaccharide from different capsular serotype strains of Haemophilus influenzae

Lipopolysaccharide (LPS) from all six serotype strains of Haemophilus influenzae was similar in composition. The oligosaccharide, of each LPS, was composed of glucose, galactose, heptose and 2-keto-3-deoxyoctonic acid. The lipid A was composed of glucosamine, phosphate and the fatty acids 14:0 and 3-OH 14:0. Each LPS also contained ethanolamine and ethanolamine phosphate, and the oligosaccharides from two strains additionally contained small amounts of glucosamine. Although the LPS was similar in composition, different serotypes had quantitative differences, especially in the galactose content, which correlated with the antigenic specificity of their homologous antisera and with their mobility on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A survey by SDS-PAGE showed that LPS from strains of the serotypes a, c and d was characteristically of lower Mr than the LPS from most (80%) serotype b strains.     

Chemotypes of Fusobacterium nucleatum lipopolysaccharides

Lipopolysaccharides (LPS) were isolated from 20 strains of Fusobacterium nucleatum and examined by paper chromatography, gas liquid chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-dexoxy-octonate (KDO). The LPS had in common glucosamine, L-glycero-D-manno-heptose, glucose and KDO. The KDO content was low. Galatose, rhamnose and D-glycero-D-manno-heptose were found in some strains. Based on the sugar composition of the LPS, the F. nucleatum strains could be classified into six chemotypes.     

Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

Diverse Exopolysaccharide Producing Bacteria Isolated from Milled Sugarcane: Implications for Cane Spoilage and Sucrose Yield

Bacterial deterioration of sugarcane during harvesting and processing is correlated with significant loss of sucrose yield and the accumulation of bacterial polysaccharides. Dextran, a homoglucan produced by Leuconostoc mesenteroides, has been cited as the primary polysaccharide associated with sugarcane deterioration. A culture-based approach was used to isolate extracellular polysaccharide (EPS) producing bacterial strains from milled sugarcane stalks. Ribosomal RNA sequencing analysis grouped 25 isolates into 4 genera. This study identified 2 bacterial genera not previously associated with EPS production or sucrose degradation. All isolates produced polysaccharide when grown in the presence of sucrose. Monosaccharide analysis of purified polymers by Gas Chromatography revealed 17 EPSs consisting solely of glucose (homoglucans), while the remainder contained traces of mannose or fructose. Dextranase treatment of polysaccharides yielded full digestion profiles for only 11 extracts. Incomplete hydrolysis profiles of the remaining polysaccharides suggest the release of longer oligosaccharides which may interfere with sucrose crystal formation.     

Cell surface polysaccharides from Bradyrhizobium japonicum and a nonnodulating mutant.

The cell surface polysaccharides of wild-type Bradyrhizobium japonicum USDA 110 and a nonnodulating mutant, strain HS123, were analyzed. The capsular polysaccharide (CPS) and exopolysaccharide (EPS) of the wild type and the mutant strain do not differ in their sugar composition. CPS and EPS are composed of mannose, 4-O-methylgalactose/galactose, glucose, and galacturonic acid in a ratio of 1:1:2:1, respectively. H nuclear magnetic resonance spectra of the EPS and CPS of the wild type and mutant strain are very similar, but not identical, suggesting minor structural variation in these polysaccharides. The lipopolysaccharides (LPS) of the above two strains were purified, and their compositions were determined. Gross differences in the chemical compositions of the two LPS were observed. Chemical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that strain HS123 is a rough-type mutant lacking a complete LPS. The LPS of mutant strain HS123 is composed of mannose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, and lipid A. The wild-type LPS is composed of fucose, xylose, arabinose, mannose, glucose, fucosamine, quinovosamine, glucosamine, uronic acid, 2-keto-3-deoxyoctulosonic acid, and lipid A. Preliminary sugar analysis of lipid A from B. japonicum identified mannose, while traces of glucosamine were detected. 3-Hydroxydodecanoic and 3-hydroxytetradecanoic acids formed a major portion of the fatty acids in lipid A. Lesser quantities of nonhydroxylated 16:0, 18:0, 22:0, and 24:0 acids also were detected.     

Composition and biological properties of lipopolysaccharides isolated from Schizothrix calcicola (Ag.) Gomont (Cyanobacteria).

The most common cyanobacterium contaminating drinking water systems in southwestern Pennsylvania is Schizothrix calcicola. Lipoplysaccharides (LPS) were isolated from this species by hot phenol-water extraction. The polysaccharide moiety was composed of glucosamine, galactose, glucose, mannose, xylose and rhamnose. The lipid A part contained beta-hydroxylauric, myristic, pentadecanoic, palmitic, beta-hydroxypalmitic, stearic, oleic, and linoleic acids. In contrast to many LPS isolated from Enterobacteriaceae, the dominant component was not beta-hydroxymyristic but beta-hydroxypalmitic acid. The LPS induced Limulus lysate gelation and Schwartzman reaction but was nontoxic to mice. The identity of LPS was verified by alkali and lysozyme treatment. The results suggest that S. calcicola is one of the principal sources of endotoxins in water systems using open finished-water reservoirs.