Localization and partial characterization of soybean lectin-binding polysaccharide of Rhizobium japonicum.

Immunoelectron microscopy was combined with partial characterization of isolated exopolysaccharide to study binding of soybean lectin by Rhizobium japonicum strain USDA 138. Lectin-binding activity resided in two forms of exopolysaccharide produced during growth: an apparently very high-molecular-weight capsular form and a lower-molecular-weight diffusible form. At low-speed centrifugation, the capsular form cosedimented with cells to form a viscous, white, cell-gel complex which was not diffusible in 1% agar, and the diffusible form remained in the cell-free supernatant. Electron microscopic observation of the cell-gel complex after labeling with soybean lectin-ferritin conjugate revealed that capsular polysaccharides, frequently attached to one end of the cells, were receptors for lectin. The outer membrane of the cell bound no lectin. Various preparations of exopolysaccharide isolated from the culture supernatant were tested for lectin binding, interaction with homologous somatic antigen, and the presence of 2-keto-3-deoxyoctonate and were chromatographed in Sepharose 4B and 6B gel beds. Lectin binding was restricted to a polysaccharide component designated as lectin-binding polysaccharide. This polysaccharide, as present in the cell-free culture supernatant, was a diffusible acidic polysaccharide devoid of 2-keto-3-deoxyoctonate, with a molecular weight of 2 X 10(6) to 5 X 10(6). It was concluded that the soybean lectin-binding component of R. japonicum is an extracellular polysaccharide and not a lipopolysaccharide and that the diffusible lectin-binding polysaccharide probably differs from the very high-molecular-weight lectin-binding polysaccharide of the loose capsule (slime) only in the degree of polymerization.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

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.     

Production of Extracellular Polysaccharide by Zoogloea ramigera

In batch cultures of Zoogloea ramigera the maximum rate of exopolysaccharide synthesis occurred in a partly growth-linked process. The exopolysaccharide was attached to the cells as a capsule. The capsules were released from the cell walls after 150 h of cultivation, which caused the fermentation broth to be highly viscous. Ultrasonication could be used to release capsular polysaccharide from the microbial cell walls. Treatment performed after 48 to 66 h of cultivation revealed exopolysaccharide concentration and apparent viscosity values in accordance with values of untreated samples withdrawn after 161 h of cultivation. The yield coefficient of exopolysaccharide on the basis of consumed glucose was in the range of 55 to 60% for batch cultivations with an initial glucose concentration of 25 g liter⁻¹. An exopolysaccharide concentration of up to 38 g liter⁻¹ could be attained if glucose, nitrogen, and growth factors were fed into the batch culture. The oxygen consumption rate in batch fermentations reached 25 mmol of O₂ liter⁻¹ h⁻¹ during the exopolysaccharide synthesis phase and then decreased to values below 5 mmol of O₂ liter⁻¹ h⁻¹ during the release phase. The fermentation broth showed pseudoplastic flow behavior, and the polysaccharide was not degraded when growth had ceased.     

Exopolysaccharide production by Pseudomonas NCIB11264 grown in batch culture.

Fermentation studies using batch culture indicated that exopolysaccharide production by Pseudomonas NCIBI1264 in a chemically defined medium increased under conditions of nitrogen limitation and excess carbon substrate at pH values above 6. The polysaccharide was formed from a variety of carbon substrates and its composition was not affected by the nature of the carbohydrate source. Polysacharide formation did not increase in media containing small amounts of phosphate, and, as in secondary metabolite production, it started late in the exponential growth phase continuing maximally after growth had ceased. The efficiency of glucose conversion into exopolysaccharide was low. Colorimetric, viscometric, and total carbon estimation techniques are described for determining exopolysaccharide levels in cell-free culture supernatants.     

A natural terrestrial biofilm

Material recovered from an extensive and viscous biofilm found on areas of rough hill pasture in Southern Scotland proved to consist of a thick mucilaginous deposit of polysaccharide in which species of green algae, withGleocystis spp as the dominant microorganism and lesser numbers ofOocystis spp. Cyanobacteria were also entrapped. On laboratory culture other green algal and cyanobacterial species were detected. Analysis of the native polysaccharide and of the exopolysaccharide from a mixed culture of the dominant algal species derived from the original material, revealed the major components as glucose, galactose, mannose and rhamnose. The content of uronic acids was very low. The viscosity of the polysaccharide preparations was determined and compared with bacterial biofilm material; viscosity was lost following phenol extraction indicating that the original material was probably closely associated with proteins.     

Synthesis of a Low-Molecular-Weight Form of Exopolysaccharide by Bradyrhizobium japonicum USDA 110

A novel extracellular low-molecular-weight polysaccharide was detected as a contaminant within extracellular cyclic β-1,6-β-1,3-glucan preparations from Bradyrhizobium japonicum USDA 110 cultures. Compositional analysis, methylation analysis, and nuclear magnetic resonance analysis revealed that this low-molecular-weight polysaccharide was composed of the same pentasaccharide repeating unit previously described for the high-molecular-weight form of the exopolysaccharide (EPS) synthesized by B. japonicum strains. Mass spectrometry analysis indicated that the size of this low-molecular-weight form of EPS was consistent with a dimeric form of the pentasaccharide repeating unit.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal⁻ ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Exopolysaccharide Distribution of and Bioemulsifier Production by Acinetobacter calcoaceticus BD4 and BD413

The heavily encapsulated Acinetobacter calcoaceticus BD4 and the “miniencapsulated” single-step mutant A. calcoaceticus BD413 produced extracellular polysaccharides in addition to the capsular material. The molar ratio of rhamnose to glucose (3:1) in the extracellular BD413 polysaccharide fraction was similar to the composition of the capsular material. In both strains, the increase in capsular polysaccharide was parallel to cell growth and remained constant in stationary phase. The extracellular polysaccharides were detected starting from mid-logarithmic phase and continued to accumulate in the growth medium for 5 to 8 h after the onset of stationary phase. Strain BD413 produced one-fourth the total rhamnose exopolysaccharide per cell that strain BD4 did. Depending on the growth medium, 32 to 63% of the rhamnose polysaccharide produced by strain BD413 was extracellular, whereas in strain BD4 only 7 to 14% was extracellular. In all cases, strain BD413 produced more extracellular rhamnose polysaccharide than strain BD4 did. In glucose medium, strain BD413 also produced approximately 10 times more extracellular emulsifying activity than strain BD4 did. The isolated capsular polysaccharide obtained after shearing of BD4 cells showed no emulsifying activity. Thus, strain BD413 either produces a modified extracellular polysaccharide or excretes an additional substance(s) that is responsible for the emulsifying activity. Emulsions induced by the ammonium sulfate-precipitated BD413 extracellular emulsifier require the presence of magnesium ion and a mixture of an aliphatic and an aromatic hydrocarbon.     

The red microalga <Emphasis Type="Italic">Flintiella sanguinaria</Emphasis> as a new exopolysaccharide producer

Flintiella sanguinaria is a red unicellular microalgae belonging to Porphyridiophyceae with a high potential as an exopolysaccharide producer. A new culture medium was developed by a stoichiometry approach based on elemental analysis of microalgae, and culture conditions were improved after measurements of photosynthetic activity as a function of irradiance, temperature and pH. These optimal conditions were thus validated in a photobioreactor for the production of biomass and exopolysaccharide (EPS). After extraction and purification of the EPS, it was characterised by HPAEC-PAD, HPLC and FT-IR as a methylated and acetylated galactoxylan including a significant amount of rhamnose and glucuronic acid in its structure. Macromolecular conformation in dilute solution of native, deproteinised and desubstituted EPS from F. sanguinaria revealed that this polysaccharide had a strong associative behaviour in which hydrophobic interactions or hydrogen bonding but also proteins were implicated.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal- ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Pseudomonas aeruginosa Psl is a galactose- and mannose-rich exopolysaccharide

The Pseudomonas aeruginosa polysaccharide synthesis locus (psl) is predicted to encode an exopolysaccharide which is critical for biofilm formation. Here we used chemical composition analyses and mannose- or galactose-specific lectin staining, followed by confocal laser scanning microscopy and electron microscopy, to show that Psl is a galactose-rich and mannose-rich exopolysaccharide.     

Production and Characterization of an Exopolysaccharide by Yeast

Antarctic yeast strains were investigated for exopolysaccharide biosynthesis and the Sporobolomyces salmonicolor AL₁ strain was selected. It was studied for exopolysaccharide biosynthesis on different carbon and nitrogen sources. The investigations showed that sucrose and ammonium sulphate were suitable culture medium components for polymer biosynthesis. Exopolysaccharide formation by the yeast strain was accompanied by a decrease in the culture medium pH value from the initial pH 5.3 to pH 1.7–2.0. During the biosynthetic process, the dynamic viscosity of the culture broth increased to the maximum value of 15.37 mPas and the polysaccharide yield reached 5.63 g/l on a culture medium containing 5.00% sucrose and 0.25% ammonium sulphate at a temperature of 22 °C for 120 h. The crude polysaccharide obtained from Sp. salmonicolor AL₁ featured high purity (90.16% of carbon content) and consisted of glucose (54.1%), mannose (42.6%) and fucose (3.3%). Pure mannan containing 98.6% of mannose was isolated from it.     

Effect of the polysaccharide ofAgrobacterium radiobacter on the growth of plants and occurrence of damping-off in sugar beet

Agrobacterium radiobacter, strain B6 (a strain isolated in this laboratory, which limited the occurrence of damping-off of sugar beet and influenced growth of plants in hot-house and field experiments) was found to produce an acidic exopolysaccharide in a mineral medium with various carbon sources. Hydrolyzates of the polysaccharide contained glucose, galactose, glycerol, succinic acid and pyruvic acid, whose quantitative content varied according to the carbon source used. The polysaccharide isolated from the medium containing glucose exhibited the highest physiological activity. Seeds germinated best and sugar beet roots were found to grow most rapidly in a medium containing 0.2 % (W/W) of the polysaccharide. The roots exposed for 3 d in this medium grew 2.7-fold as compared with non-treated plants. Higher sumbers of microorganisms were detected on the surface of roots treated with the polysaccharide. Growth of roots was also stimulated when immersing the seeds (30 min) in a 0.2 –0.4 % solution of this polysaccharide. After a two-fold treatment the roots were less damaged by the fungusPythium ultimum. Plants from seeds treated with the polysaccharide grew in the field soil more rapidly than the non-treated plants but worse than after bacterization of the seeds byA. radiobacter B6 and were only partially protected against the damping-off of sugar beet.     

Mixotrophic production of phycoerythrin and exopolysaccharide by the microalga Porphyridium cruentum

The ability of the unicellular rhodophyte Porphyridium cruentum to grow mixotrophically on the soluble fraction of Solarium tuberosum meal was tested. At the beginning of stationary phase Porphyridium cruentum produced 7 μg ml⁻¹ of phycoerythrin and 129 μg ml⁻¹ of total soluble exopolysaccharide when cultured autotrophically. When cultured mixotrophically with the soluble fraction of Solanum tuberosum meal, the productivity increased to 10 μg ml⁻¹ of phycoerythrin and 330 μg ml⁻¹ of total soluble exopolysaccharide. When the soluble fraction of S. tuberosum meal was supplied together with nitrate and phosphate, the productivity of phycoerythrin increased to 21 μg ml⁻¹ while the production of total soluble exopolysaccharide decreased to 195 μg ml⁻¹. Results demonstrate that the soluble fraction of S. tuberosum meal can be used as substrate for the production of phycoerythrin and exopolysaccharide by P. cruentum improving the results obtained with the autotrophic culture medium.     

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 °C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium. Received: 11 April 1997 / Received revision: 19 June 1997 / Accepted: 23 June 1997     

Purification and chemical characterization of an exopolysaccharide isolated from Capnocytophaga ochracea

Purification and chemical characterization of an immunosuppressive exopolysaccharide from Capnocytophaga ochracea strain 25 are described. This polysaccharide was extracted from spent culture medium by cold ethanol precipitation. Purification was accomplished by trichloroacetic acid and pronase treatments in combination with diethylaminoethyl-Sepharose and concanavalin A-Sepharose chromatography. Purity of the exopolysaccharide was ascertained by polyacrylamide gel electrophoresis using periodic acid--Schiff staining. The exopolysaccharide was free of protein, nucleic acid, and lipopolysaccharide, but contained large amounts of mannose with lesser quantities of glucose, galactose, glucuronic acid, and glucosamine.     

Structural characterization of the exopolysaccharide PS-EDIV from <Emphasis Type="Italic">Sphingomonas pituitosa</Emphasis> strain DSM 13101

Members of the bacterial genus Sphingomonas are known to produce highly viscous polysaccharides in solution. The exopolysaccharide PS-EDIV was produced by Sphingomonas pituitosa strain DSM 13101, purified using centrifugation, and precipitation and its structure was elucidated by 1D and 2D NMR techniques and chemical microderivatization combined with various mass spectrometric techniques. The following repeating unit of the polysaccharide could be identified: In addition, the polysaccharide also contains acetyl and glyceryl groups whose exact positions were not determined. PS-EDIV is similar in structure to a known exopolysaccharide but differs in being the first bacterial polysaccharide in which two different glucuronic acids are combined. It caused a high viscosity of the culture broth after cultivation for 48 h, although a gelation was not observed.     

Peanut agglutinin induced alterations in capsular and extracellular polysaccharide synthesis andex -planta nitrogenase activity of cowpea rhizobia

The root exudate ofArachis hypogea (groundnut) and its seed lectin peanut agglutinin were found to stimulate the synthesis of exopolysaccharide and capsular polysaccharide of the microsymbiont cowpeaRhizobium strain JLn (c). The synthesis of capsular polysaccharide was enhanced 1.5-fold and 2-fold in the presence of peanut agglutinin and root exudate, respectively. The synthesis of capsular polysaccharide was suppressed in the presence of different forms of combined nitrogen. Quantitative differences were also detected between the exopolysaccharide of cells grown in the presence and absence of root exudate. Electron microscopic examination of negatively stained lectin-treated JLn (c) cells showed an increased deposition of capsular polysaccharide surrounding the cells. Hurthermore,ex planta nitrogenase activity of JLn(c) cells in the presence of lectin was found to be enhanced by 63% in correlation with the increased synthesis of polysaccharides. Part of this work was presented at the colloquium session of the 4th Hederation of Asian and Oceanian Biochemists Congress, held at Singapore, in November 1986.     

Experimental analysis of the oil addition effect on mycelia and polysaccharide productions in Ganoderma lucidum submerged culture

The effect of corn oil addition on mycelium growth and polysaccharide productions in the medicinal mushroom Ganoderma lucidum was studied. The results showed that when a level of 2% corn oil was added at the beginning of culture, the biomass and polysaccharide productions reached a maximum of 12.9 and 1.038 g/L, respectively, during 13-day cultivation. The pH variation along with morphology observation in culture provided an indirect inference to the promotional effect of oil addition. Moreover, a curve fitting analysis was carried out to assay the elevated effect on biomass and exopolysaccharide productions in oil added culture. The experimental data of substrates consumption and products formation in culture with oil addition were predicted through the fitting equations obtained in single carbon source culture. The numerical results further clarified the stimulatory effects of oil addition in G. lucidum culture.