Acidic Exopolysaccharide Research Articles

Acidic exopolysaccharide produced by Enterobacter sp.

An Enterobacter sp. capable of efficiently producing an acidic exopolysaccharide was recently isolated. The bacterium was cultivated in a synthetic medium for production of a homogeneous polysaccharide. The polysaccharide isolated by anion exchange chromatography gave a peak on gel filtration in the presence of 1 M NaCl. The concentration of the polysaccharide in the medium was maximum on the third day of cultivation, by which time 13.5% (w/w) of the sucrose which had been supplied as the sole carbon source was converted to the polysaccharide. The polysaccharide consists of glucose, mannose, rhamnose, fucose, galacturonic acid and glucuronic acid, and was concluded to be an acidic exopolysaccharide with novel chemical properties on the basis of its monomeric sugar composition. A rheological comparison of the polysaccharide with xanthan gum revealed they are rheologically similar but that the former is stiffer than the latter. In addition, strong gelation of the polysaccharide with Cu 2+ suggests that the polysaccharide would be useful for copper removal from wastewater.

Structural characterization and solution properties of an acidic branched (1→3)- β-D-glucan from Aureobasidium pullulans

An acidic exopolysaccharide was isolated from a selected strain of Aureobasidium pullulans. On the basis of spectroscopic and chromatographic techniques, the polymer was identified as a β-D-glucan containing a main chain of (1 → 3)-linked β-D-glucopyranosyl units substituted at the O-6 position by single β-D-glucopyranosyl side chains. The ratio of units in the main chain to units in the side chain was found to be 1.4:1. The ionic character of this exopolysaccharide is due to the presence of malate residues which are linked to the polymer through ester bonds. The degree of substitution was estimated to be very low (0.05). In aqueous solution no signals are present in the NMR spectra strongly suggesting that the polymer adopts a rigid ordered conformation as further confirmed by rheological data. A solvent-induced conformational transition was observed in DMSO in which NMR spectra with good signal-to-noise ratio were obtained. The solution behaviour of the polymer is similar to that of other branched (1 → 3)- β-D-glucans in spite of both the degree of branching and the substitution with malate groups.

Presence of acetate and succinate in the exopolysaccharide produced by Zoogloea ramigera 115SLR

The extracellular acidic polysaccharide produced by Zoogloea ramigera 115SLR contains O-acetyl and O-succinyl groups, in addition to glucose, galactose and pyruvate already reported. Methylation analysis of both the native and the chemically modified zooglan showed that these O-acyl groups are attached to position 6 of a 4-substituted glucosyl residue and of a terminal glucose.

Structure of stewartan, the capsular exopolysaccharide from the corn pathogen Erwinia stewartii

Stewartan, the acidic exopolysaccharide of Erwinia stewartii, was purified from agar grown cells. To facilitate its structural analysis, chemical and enzymatic depolymerizations were carried out using hydrofluoric acid and E. amylovora phage /gf-Ealh, respectively. Structural characterization of the resulting oligosaccharides was performed by a combination of mass spectrometric and one- and two-dimensional (1D/2D) NMR spectroscopic techniques. A branched repeating unit with seven monosaccharide residues, all in their pyranose forms, was found: ▪.

Structure of amylovoran, the capsular exopolysaccharide from the fire blight pathogen Erwinia amylovora

The acidic exopolysaccharide (EPS) of Erwinia amylovora, amylovoran, was purified from culture supernatants of bacteria in minimal medium and cleaved chemically either by treatment with trifluoroacetic acid or hydrofluoric acid, and enzymatically by digestion with depolymerase from E. amylovora phage φ-Ea1h. Structural characterization of the resulting oligosaccharides was performed by a combination of mass spectrometric and NMR [one- and two-dimensional (1D and 2D)] spectroscopic techniques. A branched repeating unit with five monosaccharide residues and various substituents was determined: ▪ The terminal monosaccharide of the side branch, which bears a 4,6-bound pyruvate residue in the R-configuration, was found to be modified with 2-linked (26%), 3-linked (24%), 2-,3-linked (40%) O-acetyl groups, or these were absent (10%). An additional glucose residue is linked to approximately 10% of the core α-galactose of the repeating unit.

Structural studies of the acidic exopolysaccharide produced by a mucoid strain of Burkholderia cepacia, isolated from cystic fibrosis

Structural studies of the acidic exopolysaccharide produced by a mucoid strain of Burkholderia cepacia, isolated from cystic fibrosis

Structural studies of the acidic exopolysaccharide produced by a mucoid strain of Burkholderia cepacia, isolated from cystic fibrosis

The acidic exopolysaccharide produced by a mucoid strain of Burkholderia cepacia isolated from a cystic fibrosis patient, was purified by cetyltrimethylammonium bromide precipitation and/or anion-exchange chromatography. Based on the sugar composition and permethylation analyses, supported by GLC-MS and NMR spectroscopy analyses, the repeating-unit of the polysaccharide was established as → 3)- β-d-Glc p-(1 → 3)-[4,6- O-(1-carboxyethylidene)]-α-d-Gal p-(1 →.

ChemInform Abstract: Structure of the Acidic Exopolysaccharide Produced by Pseudomonas “ gingeri” Strain Pf9.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Defective Infection and Nodulation of Clovers by Exopolysaccharide Mutants of Rhizobium leguminosarum bv. trifolii

The biological and symbiotic properties of two types of exopolysaccharide deficient (Exo- phenotype) mutants of Rhizobium leguminosarum were investigated. One mutant, a pss1 derivative (ANU437) of R. l. bv. trifolii, produces very low levels of acidic exopolysaccharide (EPS) and of the acidic oligosaccharide (AOS) repeating unit lacking its O-acetyl substituent. The second strain, originally a R. I. bv. viciae derivative, but containing a clover pSym plasmid (Exo-1 mutant ANU54(pBRIAN)), did not produce any EPS polymer or detectable oligosaccharide repeating unit. The Exo- mutants formed no capsules and their growth in laboratory media was comparable to their parent strains but was inhibited by the addition of the phytoalexins medicarpin and kievitone, but not by the phytoalexin pisatin. Both Exo- mutants were slow to induce clover root hair curling (Hac+), and formed growing infection threads (Inf-) only on white clovers. However, the extent of these early symbiotic defects depended on the plant growth conditions used. Both mutants induced small non-nitrogen-fixing (Fix-) nodules on white and subterranean clovers. Within the white clover nodules induced by mutant ANU437, bacteria were released into plant cells. On white clovers at elevated light and temperature conditions, the Exo-1 mutant ANU54(pBRIAN) usually failed to initiate infection threads and thus formed empty nodules which do not contain any bacteria. In addition, this mutant formed only empty nodules without infection threads on subterranean clovers under all growth conditions tested. The outermost layers of plant cells in these empty nodules had thickened cell walls and deposited materials. These experiments show that EPS synthesis is essential for the induction of rapid root hair curling, proper infection thread formation and the ability to sustain the growth of induced nodules on various clovers.

The structure of the acidic exopolysaccharide produced by Pseudomonas “gingeri” strain Pf9

The structure of the acidic exopolysaccharide produced by the mushroom pathogen Pseudomonas “gingeri” strain Pf9, a bacterium which causes ginger blotch, was investigated by chemical analysis, mass spectrometry and 1D and 2D NMR spectroscopy. The polysaccharide consists of the linear trisaccharide repeating unit ▪ where the cyclic pyruvic acetal groups at O-4 and O-6 of the mannopyranosyl residues have the S-configuration. Methylation analysis under neutral conditions and NMR data showed that the mannose residues are acetylated at O-2. This exopolysaccharide has the same structure as the E. coli K55 capsular polysaccharide and differs from the Klebsiella K5 capsular polysaccharide only in the position of acetylation (C-2 of the glucopyranose residue).

Nodulation response of exopolysaccharide deficient mutants of Rhizobium leguminosarum bv. trifolii to addition of acidic exopolysaccharide

Several transposon Tn5-induced mutants of Rhizobium leguminosarum bv. trifolii 24.1 synthesized no detectable acidic exopolysaccharide (EPS) and were unable to induce nitrogen-fixing nodules on clover plants. Clover (Trifolium protense) co-inoculated with R. leguminosarum bv. trifolii 2407 Nod - mutant and the mutants deficient in exopolysaccharide production (Exo -) formed mixed infected nodules that did not fix nitrogen. The ability some of these Exo - mutants to induce nitrogen-fixing nodules on clover was restored in the presence of added homologous EPS from R. leguminosarum bv. trifolii 24.1 wild-type strain.

Exopolysaccharide production by Bifidobacterium longum BB‐79

Bifidobacterium longum BB‐79 produced an acidic extracellular polysaccharide (EPS), especially when grown on solid medium. The EPS was isolated by ethanol precipitation followed by dialysis and lyophilization. Anion exchange and gel‐filtration chromatography were used to further purify and characterize the EPS. The average molecular weight was greater than 200 kDa as estimated by chromatography. Based on gas‐liquid chromatography (GLC) and GLC‐mass spectrometry analyses, the EPS appears to be composed of galactose and an unidentified hexose (possibly glucose) with a carboxyethyl (lactic acid) substituent. Lactose, when used as the primary carbon source in liquid media, gave the highest yield of EPS. Incubation times longer than 24 h and the initial culture pH (pH 6·0–9·0) had little effect on the amount of EPS produced.

Surface polysaccharide mutants of Rhizobium sp. (Acacia) strain GRH2: major requirement of lipopolysaccharide for successful invasion of Acacia nodules and host range determination

Two transposon Tn5-induced mutants of wild-type broad-host-range Rhizobium sp. GRH2 were isolated and found to harbour different alterations in surface polysaccharides. These mutants, designated GRH2-14 and GRH2-50, induced a few, empty nodules on Acacia and lost the ability to nodulate most host herbaceous legumes. Whereas mutant GRH2-14 produces an acidic exopolysaccharide (EPS) similar to the wild-type, the acidic EPS of mutant GRH2-50 lacks galactose and the pyruvyl and 3-hydroxybutyryl substituents attached to this sugar moiety. In addition, both mutants GRH2-50 and GRH2-14 were altered in smooth lipopolysaccharides (LPS). DNA sequence analyses of the corresponding Tn5 insertions revealed that strain GRH2-50 was mutated in a DNA locus homologous to galE, and in vitro enzyme assays indicated that the UDPglucose 4-epimerase (GalE) activity was missing in this mutant strain. DNA hybridization studies showed that the GRH2-50 mutant DNA has homologous sequences within the different biovars of Rhizobium leguminosarum. However, no DNA homology to GRH2-14 altered DNA was found in those rhizobial strains, indicating that it represents a new chromosomal lps locus in Rhizobium sp. (Acacia) involved in symbiotic development.

Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora

A 16 kb transcript of the ams region, which is essential for biosynthesis of amylovoran, the acidic exopolysaccharide of Erwinia amylovora, was detected by Northern hybridization analysis. The positive regulator RcsA enhanced transcription of the large mRNA from the ams operon. The nucleotide sequence of this area revealed 12 open reading frames (ORFs), which are all transcribed in the same direction. Five ORFs corresponded to the previously mapped genes amsA to amsE. Sequence analysis of the insertion sites of several Tn5 mutations confirmed these data. Tn5 or site-directed mutagenesis of the ORFs 477, 377, 144, and 743 revealed an amylovoran-deficient phenotype, and the newly identified genes were named amsG, amsH, amsI, and amsF, respectively. The predicted amino acid sequence of AmsG is highly homologous to galactosyl-1-phosphate undecaprenylphosphate transferases. AmsB and AmsD are similar to other glycosyl transferases, and AmsH may be related to BexD. A significant homology to mammalian phosphatases was observed for AmsI. AmsA shows characteristic motifs for membrane association and ATP binding. AmsF carries a secretory signal sequence in the N-terminus and could be involved in periplasmic processing of the repeating units. Complementation experiments located a promoter region required for gene expression as far as 500 bp upstream of amsG. It is preceded by a typical transcriptional termination sequence. A mutation upstream of the terminator did not affect amylovoran synthesis. Partial nucleotide sequences further upstream of the ams region showed homology to genes mapped at 45 min on the Escherichia coli chromosome. A termination sequence was also found downstream of the ams operon at a distance of 16 kb from the promoter. Between amsF and this terminator, three additional ORFs were detected.

Identification of exopolysaccharides produced by fluorescent pseudomonads associated with commercial mushroom (Agaricus bisporus) production.

The acidic exopolysaccharides (EPSs) from 63 strains of mushroom production-associated fluorescent pseudomonads which were mucoid on Pseudomonas agar F medium (PAF) were isolated, partially purified, and characterized. The strains were originally isolated from discolored lesion which developed postharvest on mushroom (Agaricus bisporus) caps or from commercial lots of mushroom casing medium. An acidic galactoglucan, previously named marginalan, was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens (biovars III and V) isolated from casing medium. One biovar II strain (J1) of P. fluorescens produced alginate, a copolymer of mannuronic and guluronic acids, and one strain (H13) produced an apparently unique EPS containing neutral and amino sugars. Of 10 strains of the pathogen "P. gingeri," the causal agent of mushroom ginger blotch, 8 gave mucoid growth on PAF. The "P. gingeri" EPS also was unique in containing both neutral sugar and glucuronic acid. Mucoid, weakly virulent strains of "P. reactans" produced either alginate or marginalan. All 10 strains of the pathogen P. tolaasii, the causal agent of brown blotch of mushrooms were nonnmucoid on PAF. Production of EPS by these 10 strains plus the 2 nonmucoid strains of "P. gingeri" also was negative on several additional solid media as well as in two broth media tested. The results support our previous studies indicating that fluorescent pseudomonads are a rich source of novel EPSs.

Pleiotropic effects of purine auxotrophy inRhizobium meliloti on cell surface molecules

Rhizobial purine auxotrophs have earlier been shown to be defective in symbiosis, though the exact reason for this failure is not clear. Using various dyes that specifically bind different cell surface molecules, we show that there are multiple changes in the cell surface molecules associated with different purine auxotrophs. Affected molecules in different purine auxotrophs that were tested include (i) acidic exopolysaccharides, (ii) cellulose fibrils, and (iii) beta (1–3) glucans. Our results show that the symbiotic deficiency of purine auxotrophs is likely to be a result of these associated changes on the cell surface

Structure of simusan, a new acidic exopolysaccharide from Arthrobacter sp.

Simusan, a major exopolysaccharide produced by an ethanol-utilizing Arthrobacter sp. strain CE-17, contains d-glucose, d-mannose, d-galactose, l-rhamnose, d-glucuronic acid, and pyruvic acid in the ratios ∼ 3:2:1:1:1:1 as well as O-acyl groups (presumably residues of acetic and palmitic acid). On the basis of chemical modifications of the polysaccharide, solvolysis with anhydrous hydrogen fluoride resulting in a penta- and an octa-saccharide fragment, Smith degradation, and 1H and 13C NMR analysis, the following structure of the repeating unit was established: ▪ It is suggested that at least one of the glucose residues and the galactose residue are O-acetylated.

Pleiotropic effects of regulatory ros mutants of Agrobacterium radiobacter and their interaction with Fe and glucose.

Four exo mutants of Agrobacterium radiobacter, defective in the synthesis of acidic exopolysaccharide were complemented by a gene from that species, which is similar to the transcriptional regulator, ros, of A. tumefaciens. It was confirmed that this A. radiobacter gene, which we term rosAR, like ros, repressed its own transcription as well as that of virC and virD, two loci involved in tumorigenesis. The sequence of RosAR suggested that it might bind to a transition metal and its repressor abilities were shown to require Fe in the medium; repression was also enhanced with increasing levels of glucose. Certain rosAR mutants, in which its 3' end was removed were dominant; i.e., when plasmids containing such mutant forms of the gene were introduced into wild-type A. radiobacter, the transconjugants were nonmucoid. Such effects were also seen in a wide range of bacteria, including Escherichia coli and Xanthomonas. Several mutants that were complementd by rosAR also accumulated protoporphyrin, suggesting a defect in haem synthesis.

Two Types of Nodules Induced on Trifolium pratense by Mutants of Rhizobium leguminosarum bv. trifolii deficient in Exopolysaccharide Production

Summary Several Tn5 mutants of the symbiotic bacterium Rhizobium leguminosarum bv. trifolii 24.1 that fail to synthesize acidic exopolysaccharide, are able to induce non-nitrogen-fixing nodules on Trifolium pratense . Hybridization analysis indicated that Tn5 insertions were located on megaplasmids pRtb and pRtc and on the chromosome of R. leguminosarum bv. trifolii 24.1. Microscopic analysis of nodules induced by these non-mucoid mutants revealed two types of nodules: infected nodules with infection threads and bacteroids, and non-infected nodules which neither contained infection threads nor bacteroids. Ultrastructural data on bacteroid development and plant cell differentiation in nodules induced by the mutants indicate a decrease in bacteroid and plant metabolism. Starch accumulation in the plant plastids and presence of osmiophilic droplets in the plant cells of the peripheral tissue was observed.

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 Fe but not of Fe 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 Fe reduction or chelation. Deacetylation of B. japonicum polysaccharide increased its precipitation by Fe. At pH near neutrality, the polysaccharide from Bradyrhizobium (Chamaecytisus) strain BGA-1 stabilized Fe 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 Al chelation with EDTA. Precipitation was inhibited by increases in the ionic strength over 10 mM. Bradyrhizobium (Chamaecytisus) polysaccharide was also precipitated by Th, Sn, Mn, and Co. The presence of Fe increased the exopolysaccharide precipitation by aluminum. No precipitation, gelation, or increase in turbidity of polysaccharide solutions occurred when K, Na, Ca, Mg, Cu, Cd, Pb, Zn, Hg, or U 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 Fe, Al, Th, and Sn.