Precipitation of Metallic Cations by the Acidic Exopolysaccharides from Bradyrhizobium japonicum and Bradyrhizobium (Chamaecytisus) Strain BGA-1

The interaction between the acidic exopolysaccharides produced by two Bradyrhizobium strains and several metal cations has been studied. Aqueous solutions in the millimolar range of Fe³⁺ but not of Fe²⁺ precipitated the exopolysaccharides from Bradyrhizobium (Chamaecytisus) strain BGA-1 and, to a lesser extent, Bradyrhizobium japonicum USDA 110. The precipitation was pH dependent, with a maximum around pH 3. The precipitate was redissolved by changing the pH and by Fe³⁺ reduction or chelation. Deacetylation of B. japonicum polysaccharide increased its precipitation by Fe³⁺. At pH near neutrality, the polysaccharide from Bradyrhizobium (Chamaecytisus) strain BGA-1 stabilized Fe³⁺ solutions, despite the insolubility of Fe(OH)₃. Aluminum precipitated Bradyrhizobium (Chamaecytisus) polysaccharide but not the polysaccharide produced by B. japonicum. The precipitation showed a maximum at about pH 4.8, and the precipitate was redissolved after Al³⁺ chelation with EDTA. Precipitation was inhibited by increases in the ionic strength over 10 mM. Bradyrhizobium (Chamaecytisus) polysaccharide was also precipitated by Th⁴⁺, Sn²⁺, Mn²⁺, and Co²⁺. The presence of Fe³⁺ increased the exopolysaccharide precipitation by aluminum. No precipitation, gelation, or increase in turbidity of polysaccharide solutions occurred when K⁺, Na⁺, Ca²⁺, Mg²⁺, Cu²⁺, Cd²⁺, Pb²⁺, Zn²⁺, Hg²⁺, or U⁶⁺ was added at several pH values. The results suggest that the precipitation is based on the interaction between carboxylate groups from different polysaccharide chains and the partially hydrolyzed aquoions of Fe³⁺, Al³⁺, Th⁴⁺, and Sn²⁺.     

Effects of amino acids on polysaccharide synthesis ofEscherichia coli K-12lon + andlon − strains

Ultraviolet-sensitivelon ^? mutant ofEscherichia coli K-12 produced abundant polysaccharide when grown in a minimal medium at 37 C, but not when grown in a broth medium. The repression of polysaccharide synthesis in the broth-grownlon ^? andlon ⁺ cells was studied. The effects were largely dependent on the amino acid concentrations and on the requirements of the strain used. At 200 μg per ml of each of the essential amino acids, histidine, proline, and threonine, there was complete inhibition of polysaccharide synthesis. At 200 μg per ml the required amino acids, tryptophane and tyrosine promoted polysaccharide synthesis. Most amino acids inhibited cell growth at 200 μg per ml but the inhibiting effect was smaller at 400 μg per ml. Polysaccharide synthesis of cells was not correlated with the growth rate, and occurred even under non-growing conditions.     

Glycosylation by d-fructofuranose thio-orthoesters

The capsular polysaccharide from Klebsiella type K54, containing both O-formyl and O-acetyl groups, has been investigated by using the techniques of methylation analysis (by gas-liquid chromatography), periodate oxidation-Smith degradation, and both ¹H- and ¹³C-n.m.r. spectroscopy. Degradation of the native polysaccharide with a bacteriophage-induced glucosidase generated a formylated, as well as a formylated and acetylated, tetrasaccharide, whereas similar depolymerization of the deacetylated polysaccharide yielded a single tetrasaccharide; the corresponding, O-acylated octasaccharides were also isolated and characterized. These oligosaccharides, utilized in chemical and spectroscopic studies in order to determine the location of the O-acyl substituents in the repeating sequence, indicated formylation at O-4 of each lateral d-glucosyl group and acetylation at O-2 of alternate l-fucosyl residues. A new structure for the repeating unit in the polysaccharide is proposed.     

Structure of the capsular polysaccharide of Klebsiella K- type 63

Structural investigation of the capsular polysaccharide from Klebsiella K type 63 by methylation analysis, periodate oxidation, and uronic acid degradation showed the repeating unit to consist of →3)-α-D-Galp-(1→3)-α-D-GalpA-(1→3)-α-L-Fucp(1→. This structure is identical to that of Escherichia coli serotype K-42 capsular polysaccharide. The ¹H- and¹³C-n.m.r. spectra of the original and modified polysaccharide are consistent with the foregoing structure.     

A comparison of some hydrolytic and gas chromatographic procedures used in methylation analysis of the carbohydrate units of glycopeptides

The polysaccharide secreted by Klebsiella aerogenes type 54 strain A3 was isolated, methylated, the ester carboxyl-reduced, and the product partially hydrolyzed. The resulting, partially O-methylated oligosaccharides were reduced and ethylated, and the mixture of products was fractionated by l.c. The l.c. fractions containing per-O-alkylated oligosaccharide-alditols were analyzed by e.i.-m.s. Pure per-O-alkylated oligosaccharide-alditols were also analyzed by ¹H-n.m.r. spectroscopy. The products obtained by base-catalyzed degradation and subsequent ethylation of the per-O-methylated polysaccharide were fractionated by l.c. The main product isolated was analyzed by e.i.-m.s., c.i.-m.s., and ¹H-n.m.r. spectroscopy. The results of these studies, in conjunction with results of analytical methods commonly used in the elucidation of polysaccharide structures, unambiguously characterized the primary glycosyl structure of the polysaccharide. Base-labile substituents, previously reported to be present in the polysaccharide, were not studied. Structure 1 revises, and complements, previously reported structures.

     

Utilization of unhydrolyzed cheese whey for the production of extracellular polysaccharide by Xanthomonas cucurbitae PCSIR B-52

A strain of Xanthomonas cucurbitae PCSIR B-52 produced extracellular polysaccharide using partially deproteinized cheese whey without hydrolysis. A synthetic lactose-salt medium was also utilized to determine the optiomum level of lactose desirable for successfull fermentation. The amount of extracellular polysaccharide was maximised at 7.8 gl⁻¹ in the presence of 40 gl⁻¹ lactose. The bacterium efficiently consumed cheese whey, particularly in the presence of corn steep liquor and penicillin waste mycelium in shaken flasks. The polysaccharide, bacterial cell mass and viscosity gradients were improved as a result of efficient oxygen transfer in a mechanically agitated fermentor. A depletion in dissolved oxygen tension resulted during the exponential growth phase. The fermentation pattern of extracellular polysaccharide was also studied by repeated batch process.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype 55

The structure of the capsular polysaccharide from Klebsiella type 55 has been investigated by using the techniques of methylation, Smith periodate oxidation, and partial, acid hydrolysis. The anomeric configurations of the glycosidic linkages were determined by performing ¹H-n.m.r. and ¹³C-n.m.r.spectroscopy on the polysaccharide and derived poly- and oligo-saccharides obtained through degradative procedures. The position of the O-acetyl group was located by devising an improved method for its replacement by a methyl ether group. The structure was shown to consist of the following tetrasaccharide repeating unit.     

Structure of a polysaccharide from the lipopolysaccharide of Vibrio vulnificus CECT4602 containing 2-acetamido-2,3,6-trideoxy-3-[(S)- and (R)-3-hydroxybutanoylamino]-l-mannose

A polysaccharide was isolated by GPC after mild acid treatment of the lipopolysaccharide of Vibrio vulnificus CECT4602 and found to contain l-Rha, d-GlcpNAc and 2-acetamido-2,3,6-trideoxy-3-(3-hydroxybutanoylamino)-l-mannose (l-RhaNAc3NHb). GLC analysis of the trifluoroacetylated (S)-2-octyl esters derived by full acid hydrolysis of the polysaccharide showed that ∼80% of the 3-hydroxybutanoic acid has the S configuration and ∼20% the R configuration. The following structure of the polysaccharide was established by ¹H and ¹³C NMR spectroscopies, including 2D ROESY and ¹H/¹³C HMBC experiments:      

INCORPORATION OF SULFATE INTO THE CAPSULAR POLYSACCHARIDE OF THE RED ALGA PORPHYRIDIUM

The accumulation of sulfate-³⁵S by Porphyridium aerugineum cells and subsequent appearance of solubilized capsular polysaccharide-³⁵S in the growth medium were examined The uptake of label by the cells was largely light dependent. Pulse-chase experiments using log phase cells revealed a rapid labeling of solubilized capsular polysaccharide, recovered from the medium as the cetylpyridinium chloride precipitate Polyacrylamide gel electrophoresis of the polysaccharide-³⁵S showed the sulfate to be firmly bound to an immobile fraction. Sephadex chromatography revealed the molecular weight of the polysaccharide to be in excess of 2 x 10⁵. Acid hydrolysis of the polysaccharide-³⁵S released sulfate-³⁵S ion as evidenced by radioautography of thin layer chromatographs Preliminary electron microscope evidence suggests that the synthesis, movement, and deposition of the capsular polysaccharide on the cell surface are Golgi complex-mediated processes     

Stimulatory Effects of Polysaccharide Fraction from Solanum nigrum on RAW 264.7 Murine Macrophage Cells

The polysaccharide fraction from Solanum nigrum Linne has been shown to have antitumor activity by enhancing the CD4⁺/CD8⁺ ratio of the T-lymphocyte subpopulation. In this study, we analyzed a polysaccharide extract of S. nigrum to determine its modulating effects on RAW 264.7 murine macrophage cells since macrophages play a key role in inducing both innate and adaptive immune responses. Crude polysaccharide was extracted from the stem of S. nigrum and subjected to ion-exchange chromatography to partially purify the extract. Five polysaccharide fractions were then subjected to a cytotoxicity assay and a nitric oxide production assay. To further analyze the ability of the fractionated polysaccharide extract to activate macrophages, the phagocytosis activity and cytokine production were also measured. The polysaccharide fractions were not cytotoxic, but all of the fractions induced nitric oxide in RAW 264.7 cells. Of the five fractions tested, SN-ppF3 was the least toxic and also induced the greatest amount of nitric oxide, which was comparable to the inducible nitric oxide synthase expression detected in the cell lysate. This fraction also significantly induced phagocytosis activity and stimulated the production of tumor necrosis factor-α and interleukin-6. Our study showed that fraction SN-ppF3 could classically activate macrophages. Macrophage induction may be the manner in which polysaccharides from S. nigrum are able to prevent tumor growth.     

Novel O-antigen of Hafnia alvei PCM 1195 lipopolysaccharide with a teichoic acid-like structure

The lipopolysaccharide (LPS) of Hafnia alvei strain PCM 1195 was obtained by the hot phenol/water method. The O-specific polysaccharide was released by mild acidic hydrolysis and isolated by gel filtration. The structure of the O-specific polysaccharide was investigated by ¹H, ¹³C, and ³¹P NMR spectroscopy, MALDI-TOF MS, and GC-MS, accompanied by monosaccharide and methylation analysis. It was concluded that the O-specific polysaccharide is composed of a hexasaccharide repeating units interlinked with a phosphate group: {→4-α-d-Glcp-(1→3)-α-l-FucpNAc-(1→3)-[α-d-Glcp-(1→4)]-α-d-GlcpNAc-(1→3)-α-l-FucpNAc-(1→4)-α-d-Glcp-(1→P}_n.     

Polysaccharide Fraction from Higher Plants which Strongly Interacts with the Cytosolic Phosphorylase Isozyme : I. Isolation and Characterization

From leaves of Spinacia oleracea L. or from Pisum sativum L. and from cotyledons of germinating pea seeds a high molecular weight polysaccharide fraction was isolated. The apparent size of the fraction, as determined by gel filtration, was similar to that of dextran blue. Following acid hydrolysis the monomer content of the polysaccharide preparation was studied using high pressure liquid and thin layer chromatography. Glucose, galactose, arabinose, and ribose were the main monosaccharide compounds. The native polysaccharide preparation interacted strongly with the cytosolic isozyme of phosphorylase (EC 2.4.1.1). Interaction with the plastidic phosphorylase isozyme(s) was by far weaker. Interaction with the cytosolic isozyme was demonstrated by affinity electrophoresis, kinetic measurements, and by ¹⁴C-labeling experiments in which the glucosyl transfer from [¹⁴C]glucose 1-phosphate to the polysaccharide preparation was monitored.     

Decreased Exopolysaccharide Synthesis by Anaerobic and Symbiotic Cells of Bradyrhizobium japonicum

Experiments were conducted to determine whether symbiotic bacteroids of Bradyrhizobium japonicum produce exopolysaccharide within soybean (Glycine max [L.] Merr. cv `Lee 74') nodules. B. japonicum strains RT2, a derivative of USDA 110 with resistance to streptomycin and rifampicin, and RT176-1, a mutant deficient in exopolysaccharide synthesis, were used. Although aerobically cultured RT2 produced 1550 micrograms of exopolysaccharide per 10¹⁰ cells, root nodules formed by RT2 contained only 55.7 micrograms of polysaccharide per 10¹⁰ bacteroids, indicating that little exopolysaccharide synthesis occurred within the nodules. The polysaccharide level of RT2 nodules was about equal to that of nodules containing the exopolysaccharide mutant RT176-1 (61.0 micrograms per 10¹⁰ bacteroids). Gas chromatographic analysis showed that the sugar composition of polysaccharide from nodules of RT2 or RT176-1 was almost the same as that of polysaccharide from unnodulated root tissue, but differed strikingly from that of rhizobial exopolysaccharide from aerobic cultures. Thus, the host plant and not the bacteroids was probably the source of most or all of the polysaccharide in the nodule extracts. Also, bacteroids from nodules failed to bind soybean lectin, confirming the absence of an exopolysaccharide capsule.     

Structure of the 3-deoxy-d-manno-octulosonic acid-containing polysaccharide (K6 antigen) from escherichia coli LP 1092

The acidic polysaccharide (K6) antigen from Escherichia coli LP 1092 contains d-ribose and 3-deoxy-d-manno-octulosonic acid in the molar ratio of 2:1, respectively. Spectroscopic data (¹³C- and ¹H-n.m.r.), methylation analyses, and periodate oxidation indicate that the polysaccharide is composed of the foregoing components essentially in the following trisaccharide sequence: →2)-β-d-Ribf-(1→2)-β-d-Ribf-(1→7)-α-d-KDO-(2→The polysaccharide also contains O-acetyl substituents (～0.2–0.3 mol per KDO residue).     

Production and Characterization of an Exopolysaccharide Excreted by a Deep-Sea Hydrothermal Vent Bacterium Isolated from the Polychaete Annelid Alvinella pompejana

The heterotrophic and mesophilic marine bacterium HYD-1545 was isolated on a metal-amended medium from the dorsal integument of the hydrothermal vent polychaete Alvinella pompejana. This strain, which can be assigned to the genus Alteromonas on the basis of its G+C content and phenotypical features, produced large amounts of an acidic polysaccharide in batch cultures. The polysaccharide was excreted during the stationary phase of growth and contained glucose, galactose, glucuronic acid, galacturonic acid, and 4,6-O-(1-carboxyethilidene)-galactose as major components. This polysaccharide was a polyelectrolyte, and the viscosity of its solutions depended on the ionic strength. The decrease in viscosity with increasing NaCl concentrations and the effect of Ca²⁺ in decreasing the viscosity at low Ca²⁺ concentrations support a model in which the polysaccharide carries anionic groups. However, an unusual behavior was observed at higher concentrations and could be related to intermolecular interactions involving Ca²⁺ ions.     

Glucogalactan: A polysaccharide isolated from the cell-wall of Verticillium Lecanii

A water-soluble polysaccharide was extracted with alkali from the cell wall of Verticillium lecanii (also called Lecanicillium lecanii). After freezing and thawing, the water-soluble fraction was purified by gel filtration chromatography on Sepharose CL-6B and eluted as one peak by HPSEC/RID. Monosaccharide analysis showed galactose and glucose (1.1:1), with traces of mannose (<1%). The structural characteristics were determined by spectroscopic analysis, FT-IR and 1D and 2D ¹H and ¹³C NMR, and methylation results. On the basis of the data obtained, the following structure of the polysaccharide (E₃S_IV fraction) was established:     

Relative Contributions of Bacteria and Fungi to Rates of Degradation of Lignocellulosic Detritus in Salt-Marsh Sediments

Specifically radiolabeled [¹⁴C-lignin]lignocellulose and [¹⁴C-polysaccharide]lignocellulose from the salt-marsh cordgrass Spartina alterniflora were incubated with an intact salt-marsh sediment microbial assemblage, with a mixed (size-fractionated) bacterial assemblage, and with each of three marine fungi, Buergenerula spartinae, Phaeosphaeria typharum, and Leptosphaeria obiones, isolated from decaying S. alterniflora. The bacterial assemblage alone mineralized the lignin and polysaccharide components of S. alterniflora lignocellulose at approximately the same rate as did intact salt-marsh sediment inocula. The polysaccharide component was mineralized twice as fast as the lignin component; after 23 days of incubation, ca. 10% of the lignin component and 20% of the polysaccharide component of S. alterniflora lignocellulose were mineralized. Relative to the total sediment and bacterial inocula, the three species of fungi mediated only very slow mineralization of the lignin and polysaccharide components of S. alterniflora lignocellulose. Experiments with uniformly ¹⁴C-labeled S. alterniflora material indicated that the three fungi and the bacterial assemblage were capable of degrading the non-lignocellulosic fraction of S. alterniflora material, but only the bacterial assemblage significantly degraded the lignocellulosic fraction. Our results suggest that bacteria are the predominant degraders of lignocellulosic detritus in salt-marsh sediments.     

Structure of a peptidoglycan-related polysaccharide from <Emphasis Type="Italic">Providencia alcalifaciens</Emphasis> O45

A polysaccharide was isolated from the opportunistic human pathogen Providencia alcalifaciens O45:H26 by extraction with aqueous phenol and studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional ROESY and H-detected ¹H,¹³C HSQC experiments. The polysaccharide contains N-acetylglu-cosamine and N-acetylmuramic acid (D-GlcpNAc3Rlac) amidated with L-alanine and has the following structure:

$\to 4) - \beta - D - GlcpNAc - (1 \to 4) - \beta - D - GlcpNAc3(Rlac - L - Ala) - (1 \to .$

The polysaccharide possesses a remarkable structural similarity to the bacterial cell wall peptidoglycan. It is not unique to the strain studied but is common to strains of at least four P. alcalifaciens O-serogroups (O3, O24, O38, and O45). No evidence was obtained that the polysaccharide is associated with the LPS, and hence it might represent a bacterial capsule component.

     

Heparan sulfate biosynthesis by embryonic tissues and primary fibroblast populations.

Fibroblasts from cornea, heart, and skin of day 14 embryonic chicks demonstrate the ability to make heparan sulfate-like polysaccharide when examined during the 10 hr period immediately following their removal from the embryo. Both the whole tissues from which these fibroblasts are isolated and the fibroblasts grown for 2–5 weeks in vitro also synthesize heparan sulfate. During their first few days in vitro, the three fibroblast populations display increasing rates of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporation into glycosaminoglycans and sharp fluctuations of those rates, yet the percentage of total [³⁵S]-sulfate incorporated into heparan sulfate-like polysaccharide and the distribution of this polysaccharide between cells and nutrient medium do not change significantly. During their first 48 hr in vitro, skin fibroblasts, but not those from cornea or heart, show steadily decreasing discrepancies between the proportions of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporated into heparan sulfate, suggesting a sharp decline in the synthesis of nonsulfated glycosaminoglycans. These data support the hypothesis of Kraemer than many cell-types in vivo may normally make heparan sulfate. The data largely eliminate the hypothesis that the biosynthesis of this polysaccharide is selectively stimulated as embryonic cells adapt to growth in vitro.     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.