Polysaccharides of the cellular slime mold II. Change of intra- and extracellular polysaccharides during growth phase of Dictyostelium discoideum NC-4

The three intra- and extracellular polysaccharide fractions were isolated during the growth phase of Dictyostelium discoideum NC-4, and the change in content of component sugars of four fractions during the culture period was examined. Myxamoebae most extensively contain a polysaccharide fraction extracted with phenol-water (polysaccharide fraction I) in a quantity of about 15–23% per dry cell. After 15 h the uronic acid formed in the polysaccharide fraction I, and the cell, could be aggregated. The glucosamine content in the polysaccharide fraction I reached a maximum as the myxamoebae entered the exponential phase, and a large amount of galactose was produced as the cell entered the stationary phase. The phenol-water extract from the cells of the stationary phase was reacted with concanavalin-A.     

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.     

Mitogenic andadjuvant activities of polysaccharides from the cellular slime mold, Dictyostelium discoideum NC-4.

Cell surface and extracellular polysaccharide fractions obtained from Dictyostelium discoideum NC-4 cultured in bacteria-free medium showed strong B-cell mitogenic activities. Upon periodate treatment of the extra-cellular polysaccharide fraction this activity completely disappeared. The extracellular polysaccharide fraction could also enhance the antibody response in vitro against sheep red blood cells.     

Structural investigations of the extracellular polysaccharides elaborated by Beijerinckia mobilis

The extracellular mucilage from Beijerinckia mobilis, a member of the Azotobacteriaceae, after removal of contaminating protein, was separated into a neutral polysaccharide (N-2, 10%); a neutral, dialysable fraction (N-1, 5%), consisting of glucose and oligosaccharides containing glucose, arabinose, and rhamnose; and an acidic polysaccharide (85%). N-2 (mol. wt, 1900) was highly branched and comprised glucopyranose, mannopyranose, and arabinofuranose residues (1:1:1). The various linkages were determined. The acid fraction was a polymer of high molecular weight composed of L-guluronic acid (65%), D-glucose (15%), and D-glycero-D-mannoheptose (20%), together with acetic and pyruvic acids. From the results of methylation, periodate oxidation, and partial hydrolysis, a branched molecule with a backbone of guluronic acid and heptose, and side chains of glucose and guluronic acid is proposed. Pyruvic acid was found to be acetal-linked to 2?5% of the heptose residues. The similarities between this polysaccharide and that from the related species Azotobacter indicum are discussed.     

Emergence of Novel Streptococcus iniae Exopolysaccharide-Producing Strains following Vaccination with Nonproducing Strains

Streptococcus iniae is a major pathogen of fish, producing fatal disease among fish species living in very diverse environments. Recently, reoccurrences of disease outbreaks were recorded in rainbow trout (Oncorhynchus mykiss, Walbaum) farms where the entire fish population was routinely vaccinated. New strains are distinguished from previous strains by their ability to produce large amounts of extracellular polysaccharide that is released into the medium. Present findings indicate that the extracellular polysaccharide is a major antigenic factor, suggesting an evolutionary selection of strains capable of extracellular polysaccharide production.     

SUBCELLUAR SITES FOR SYNTHESIS OF CHONDROMUCOPROTEIN OF CARTILAGE

Microsomes from embryonic cartilage have been subfractionated to yield smooth microsomes and rough microsomes. The in vitro enzymic activities involved in chondroitin sulfate biosynthesis have been assayed in these subfractions. The results demonstrate that all of the activities necessary for linkage to protein as well as for completion of the polysaccharide chain are present in both the rough and smooth fractions. Only in the case of the polymerization of N-acetylgalactosamine and glucuronic acid could enzyme assays be done independent of endogenous acceptor. This enzyme(s) was equally distributed between the rough and smooth fractions. The activities for the addition of xylose and galactose to protein were highest in the rough fraction while that for sulfation was highest in the smooth fraction. These findings suggest that polysaccharide chain-initiation occurs in the rough endoplasmic reticulum and that chain completion occurs in the smooth reticulum. This pattern is consistent with modern theories of synthesis, transfer, and export of extracellular macromolecules.     

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.     

Characterization of a Sinorhizobium Isolate and Its Extracellular Polymer Implicated in Pollutant Transport in Soil

A bacterium isolated from soil (designated 9702-M4) synthesizes an extracellular polymer that facilitates the transport of such hydrophobic pollutants as polynuclear aromatic hydrocarbons, as well as the toxic metals lead and cadmium in soil. Biolog analysis, growth rate determinations, and percent G+C content identify 9702-M4 as a strain of Sinorhizobium meliloti. Sequence analysis of a 16S rDNA fragment gives 9702-M4 a phylogenetic designation most closely related to Sinorhizobium fredii. The extracellular polymer of isolate 9702-M4 is composed of both an extracellular polysaccharide (EPS) and a rough lipopolysaccharide. The EPS component is composed mainly of 4-glucose linkages with monomers of galactose, mannose, and glucuronic acid and has pyruval and acetyl constituents. The lipid fraction and the negative charge associated with carbonyl groups of the exopolymer are thought to account for the binding of polynuclear aromatic hydrocarbons and cationic metals.     

Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[¹⁴C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)-α- and (1 → 6)-α-d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.     

Production of carbohydrates by the marine diatom Chaetoceros affinis var. willei (Gran) Hustedt. II. Preliminary investigation of the extracellular polysaccharide

The isolation of an extracellular polysaccharide from cultures of Chaetoceros affinis var. willei (Gran) Hustedt is described. The polysaccharide behaved as a homogeneous, polyanionic compound in free-boundary electrophoresis at both pH 2 and 7. It contained sulphur, presumably as sulphate half ester groups (8.7% of SO₂Na), and the following monosaccharides were tentatively identified: rhamnose, fucose, arabinose, and galactose, with the two former constituting 63% of the polysaccharide preparation. The main cellular polysaccharide was a glucan and could be extracted from the cells by dilute acid. The remaining material gave, after hydrolysis, a complex mixture of monosaccharides with rhamnose as the major component. It is concluded that the extracellular polysaccharide is probably excreted from healthy cells.     

Fractionation of the β-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were β-linked as shown by ¹H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Structure of a pectic polysaccharide fraction from zea shoots

A pectic fraction, accounting for about 0.3% of the total cell wall polysaccharide, was derived from the hot water extract of an insoluble fraction of the buffer-homogenate of Zea shoots. The pectic polysaccharide fraction was characterized by fragmentation analysis after hydrolysis with acid and Erwinia carotovora pectate lyase. The results suggest that the fraction consists of mostly a linear homopolygalacturonan with neutral sugar components or a homogalacturonan and a rhamnogalacturonan with neutral sugar components.     

Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.     

Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein.

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.     

Polysaccharide Produced by Anacystis nidulans: Its Ecological Implication

An extracellular polysaccharide from Anacystis nidulans was extracted from cell-free medium. Analysis showed that the polysaccharide consisted of glucose, galactose, and mannose in the ratio of 60:14:20. The production of polysaccharide depends on the age of culture, the growth temperature, and the form of nitrogen available.     

A glimpse of the diversity of complex polysaccharide-degrading culturable bacteria from Kongsfjorden,Arctic Ocean

Cold-adapted, complex polysaccharide-degrading marine bacteria have important implications in biogeochemical processes and biotechnological applications. Bacteria capable of degrading complex polysaccharide substrates, mainly starch, have been isolated from various cold environments, such as sea ice, glaciers, subglacial lakes, and marine sediments. However, the total diversity of polysaccharide-degrading culturable bacteria in Kongsfjorden, Arctic Ocean, remains unexplored. In the study reported here, we tested 215 cold-adapted heterotrophic bacterial cultures (incubated at 4 and 20 °C, respectively) isolated from Kongsfjorden, for the production of cold-active extracellular polysaccharide-degrading enzymes, including amylase, pectinase, alginase, xylanase, and carboxymethyl (CM)-cellulase. Our results show that 52 and 41% of the bacterial isolates tested positive for extracellular enzyme activities at 4 and 20 °C, respectively. A large fraction of the bacterial isolates (37% of the positive isolates) showed multiple extracellular enzyme activities. Alginase and pectinase were the most predominantly active enzymes, followed by amylase, xylanase, and CM-cellulase. All isolates which tested positive for extracellular enzyme activities were affiliated to microbial class Gammaproteobacteria. The four genera with the highest number of isolates were Pseudomonas, followed by Psychrobacter, Pseudoalteromonas, and Shewanella. The prevalence of complex polysaccharide-degrading enzymes among the isolates indicates the availability of complex polysaccharide substrates in the Kongsfjorden, likely as a result of glacial melting and/or macroalgal load. In addition, the observed high functional/phenotypic diversity in terms of extracellular enzyme activities within the bacterial genera indicates a role in regulating carbon/carbohydrate turnover in the Kongsfjorden, especially by reducing recalcitrance.     

Fine Structure of Extracellular Polysaccharide of Erwinia amylovora

Virulent E₉ and avirulent E₈ strains of Erwinia amylovora were shown by means of light, transmission, and scanning microscopy to be, respectively, encapsulated and unencapsulated. Difficulty was encountered in stabilizing the fibrillar-appearing capsular extracellular polysaccharide. We suggest that the ephemeral nature of extracellular polysaccharide is due to the collapse of its extended structure upon dehydration. This occurs when bacteria are prepared for either transmission or scanning electron microscopy. The electron micrographs support our previous biochemical and immunological studies contending that the capsule is composed of tightly bound and loosely held components. The preparation of bacteria in freeze-dried colonies has permitted us to observe and explain the fluidity of the encapsulated strain. We suggest that this fluidity is a reflection of the loosely held extracellular polysaccharide or slime.     

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.     

Structural studies on a new polysaccharide,containing d-riburonic acid,from Rhizobium meliloti IFO 13336

The structure of an extracellular, acidic polysaccharide from Rhizobium meliloti IFO 13336 was studied by a method involving successive fragmentation with specific β-d-glycanases of Flavobacterium M64. The polysaccharide is composed of repeating units of the octasaccharide shown. An acidic component was identified as d-riburonic acid.     

Chemistry of the polysaccharides of the diatom Coscinodiscus nobilis

The extracellular polysaccharide of Coscinodiscus nobilis, a member of the Coscinodiscaceae, contains a highly branched heteropolysaccharide(s) containing fucose, rhamnose, mannose, d-glucose, xylose, d-glucuronic acid, galactose (trace) and half ester sulphate. The positions of linkages between the monosaccharides have been established and evidence for the linkages between d-glucuronic acid and monosaccharides was obtained. The extracellular polysaccharide contained also a chrysolaminaran, but this may have been derived from dead cells. Fucose and mannose occur also in a separate polymer. The diatom contained polysaccharide material consisting of glucose, mannose, fucose and uronic acid residues.