The synthesis of exopolysaccharide by Klebsiella aerogenes membrane preparations and the involvement of lipid intermediates

1. Membrane preparations from Klebsiella aerogenes type 8 were shown to transfer glucose and galactose from their uridine diphosphate derivatives to a lipid and to polymer. The ratio of glucose to galactose transfer in both cases was 1:2. This is the same ratio in which these sugars occur in native polysaccharide. Galactose transfer was dependent on prior glucosylation of the lipid. Mutants were obtained lacking (a) glucosyltransferase and (b) galactosyltransferase. The transferase activities in a number of non-mucoid mutants was examined. 2. Glucose transfer was partially inhibited by uridine monophosphate, and incorporation of either glucose or galactose into lipid was decreased in the presence of uridine diphosphate. The sugars are thought to be linked to a lipid through a pyrophosphate bond, and treatment of the lipid intermediates with phenol yielded water-soluble compounds. These could be dephosphorylated with alkaline phosphatase. Transfer of glucuronic acid to lipid or polymer from uridine diphosphate glucuronic acid was much lower than that of the other two sugars. 3. The fate of sugars incorporated into polymer was also followed. Some conversion of glucose into galactose and glucuronic acid occurred. Mutants unable to transfer glucose or galactose to lipid were unable to form polymer. Other mutants capable of lipid glycosylation were in some cases unable to form polymer. A model for capsular polysaccharide synthesis is proposed and its similarity to the formation of other polymers outside the cell membrane is discussed.     

The utilisation of galactose by an Aspergillus nidulans mutant lacking galactose phosphate-UDP glucose transferase and its relation to cell wall synthesis

Summary A mutant of Aspergillus nidulans lacking galactose phosphate-UDP glucose transferase could not grow on galactose but incorporated this sugar into cell constituents when supplied with another carbon source. 75% of the radioactivity taken up was found in the galactose and glucose monomers of the hyphal wall. Most of the remaining label was in a cytoplasmic polysaccharide and in free galactose and galactose phosphate. The composition of the cytoplasmic polysaccharide resembled that of the wall polymers. These findings are taken to indicate that enzymes not connected with the Leloir pathway can activate and epimerise galactose and that polymeric wall precursors may be present in the cytoplasm. The specific labelling obtained with galactose was combined with radioautography to show that glucose and galactose containing polymers are incorporated into the hyphal wall at the growing tip.     

On the Steric Configuration of the Urushi Polysaccharide

This paper presents the steric configuration of a polysaccharide obtained from the latex of the Japanese lacquer tree. Thus, the polysaccharide consisted of galactose and galacturonic acid, and the main chain of polysaccharide was proved to be linked with 1 → 3 linkage of galactose residue of 6-O-galacturonosyl galactose unit by the periodate oxidation method. The molecular weight was calculated to be 6 × 10⁴ by Archibald’s ultracentrifugal method. The configuration of the polysaccharide was discussed to be α helical structure of which one turn of the helices consists of 6 to 7 6-O-galacturonosyl galactose units and the diameter of the helix is 21 Å and one pitch is 9.5 Å on the basis of viscosity measurements, color reaction with iodine, crystallization with capronic acid and consideration of molecular models.     

Polymer-Producing Species of Arthrobacter.

Gasdorf, Helen J. (Northern Regional Research Laboratory, Peoria, Ill.), R. G. Benedict, M. C. Cadmus, R. F. Anderson, and R. W. Jackson. Polymer-producing species of Arthrobacter. J. Bacteriol. 90:147-150.1965.-Two slime-producing microorganisms, designated as NRRL B-1973 and NRRL B-1797, were isolated from a Guatemalan soil sample. Their morphological and physiological characteristics permit their assignment to the genus Arthrobacter. Both cultures produce a large amount of extracellular polysaccharide, the maximal amount being 1.4 g per 3 g of glucose. The carbohydrate constituents of B-1973 polysaccharide are galactose, glucose, and mannuronic acid; those of B-1797 are galactose, glucose, and glucuronic acid. The organisms are morphologically and physiologically alike. The differences between these two cultures and previously described species of Arthrobacter appear sufficient to designate a new species. The name Arthrobacter viscosus sp. n. is proposed.     

Irreversible glucuronyl C5-epimerization in the biosynthesis of heparan sulfate

Glucuronyl C5-epimerase catalyzes the conversion of d-glucuronic acid to l-iduronic acid units in heparan sulfate biosynthesis. Substrate recognition depends on the N-substituent pattern of the heparan sulfate precursor polysaccharide and requires the adjacent glucosamine residue toward the non-reducing end to be N-sulfated. Epimerization of an appropriately N-sulfated substrate is freely reversible in a soluble system, with equilibrium favoring retention of d-gluco configuration (Hagner-McWhirter, A., Lindahl, U., and Li, J.-P. (2000) Biochem. J. 347, 69-75). We studied the reversibility of the epimerase reaction in a cellular system, by incubating human embryonic kidney 293 cells with d-[5-(3)H]galactose. The label was incorporated with glucuronic acid units into the heparan sulfate precursor polysaccharide and was lost upon subsequent C5-epimerization to iduronic acid. However, analysis of oligosaccharides obtained by deaminative cleavage of the mature heparan sulfate chains indicated that all glucuronic acid units retained their C5-(3)H label, irrespective of whether they had occurred in sequences susceptible or resistant to the epimerase. All (3)H-labels of the final products resisted incubation with epimerase in a soluble system, apparently due to blocking O-sulfate groups. These results indicate that glucuronic acid C5-epimerization is effectively irreversible in vivo and argue for a stringent organization of the biosynthetic machinery.     

Extracellular acidic polysaccharide production by a two-membered bacterial coculture

A two-membered coculture of strains KYM-7 and KYM-8, identified as Cellulomonas cellulans and Agrobacterium tumefaciens, respectively, produced a large amount of an extracellular polysaccharide, designated APK-78, from starch. Each strain in pure culture produced only very little amount of polysaccharide from starch; the coexistence of the two strains from the early stage of cultivation was indispensable for a large amount of polysaccharide to be produced. The polysaccharide APK-78 was acidic and composed of glucose, galactose, succinic acid, and pyruvic acid with a molar ratio of 8.1:1.0:1.7:1.0, indicating that it is a succinoglycan type of polysaccharide.     

Patterns of polysaccharide biosynthesis in differentiating cells of maize root-tips

1. The patterns of incorporation of radioactivity from d-[6-(3)H]-, d-[1-(14)C]-, d-[U-(14)C]- and d-[6-(14)C]-glucose and [U-(14)C]myoinositol into the neutral sugars and uronic acids of the polysaccharides synthesized in different regions of the root-tip of maize were determined. 2. The root-cap tissue synthesized a slime in which a polysaccharide that contained a high proportion of fucose (32%) and galactose (21%) was found. This polysaccharide is synthesized only by the root-cap cells, and very little polysaccharide containing fucose is synthesized in adjacent tissues. Part of the meristematic tissue of the root is surrounded by the cap cells. A section of the root that contains both these tissues can be analysed, and the polysaccharide synthesized by the meristematic region can be obtained since the contribution of the root-cap cells can be found by the amount of fucose formed. 3. It was shown that there is very little difference in the polysaccharide synthesis of the meristematic region from that of the cells immediately behind it. In the more mature cells, however, the amount of xylose synthesized relative to that of arabinose is increased, and the proportion of xylose and arabinose formed in the matrix polysaccharides is increased whereas that of galactose is decreased. 4. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on polysaccharide synthesis was to bring about a decrease in the relative amount of galactose synthesized in the matrix polysaccharides of cells immediately adjacent to the meristematic region and also in the more mature tissue. The growth factor also increased the amount of xylose synthesized relative to that of arabinose in the more mature tissue. These metabolic effects were related to a very obvious change in the morphological appearance of the root-tips. 5. Radioactivity from [U-(14)C]myoinositol was incorporated mainly into xylose, arabinose and galacturonic acid rather than into the hexoses, although small amounts of these sugars were formed.     

Acidic exopolysaccharide from Bradyrhizobium (Chamaecytisus proliferus)

M. LEON-BARRIOS, A.M. GUTIERREZ-NAVARRO, R. PEREZ-GALDONA, J. DIAZ-SIVERIO. J. TRUJILLO AND J. CORZO. 1992. The exopolysaccharide from a strain of Bradyrhizobium isolated in the Canary Islands was studied. The polysaccharide was found to be acidic and composed of glucose, galactose, mannose and galacturonic acid in a relation 3: 1: 1: 1, respectively. Acetyl was the only acyl substituent detected. Polyacrylamide gel electrophoresis showed that it had a polymeric structure with a variable degree of polymerization. At low ionic strength it aggregated but in EDTA solutions it was resolved as a polydisperse sample by gel exclusion chromatography.     

Water-soluble polysaccharides from Ginkgo biloba leaves.

The water-soluble polysaccharides from dried Ginkgo biloba leaves were isolated after exhaustive extraction with organic solvents. The polysaccharide mixture could be separated into a neutral (GF1) and two acidic (GF2 and GF3) polysaccharide fractions by ion exchange chromatography. According to the Mr distribution GF1 and GF3 seemed to be homogenous, whereas GF2 could be further fractionated into two subfractions (GF2a and GF2b) by gel permeation chromatography. GF1 (Mr 23,000) showed the structural features of a branched arabinan. The main chain was composed of 1,5-linked arabinose residues and three in 12 arabinose molecules were branched via C-2 or C-3. GF2a (Mr 500,000) consisted mainly of 1,2,4-branched mannose (29%), 1,4-linked glucuronic (32%) and galacturonic (8%) acid as well as terminal rhamnose (25%). After removal of ca 70% of the terminal rhamnose the remaining polysaccharide showed a decrease in 1,2,4-branched mannose and an increase in 1,2-linked mannose indicating that at least half of the rhamnose residues were linked to mannose via C-4. GF3 (Mr 40,000) consisted of 1,4-linked galacturonic (30%) and glucuronic (16) acid, 1,3,6-branched galactose (15%), 1,2-linked (5%) and 1,2,4-branched (3.5%) rhamnose as well as 1,5-linked arabinose (11%). Rhamnose (5%) and arabinose (10%) were present as terminal groups. Mild acid hydrolysis selectively cleaved arabinose and the remaining polysaccharide showed an increased amount of 1,6-linked and terminal galactose and a decreased quantity of 1,3,6-branched galactose. These results indicated that the terminal as well as the 1,5-linked arabinose were mainly connected to galactose via C-3. The GF3 polysaccharide appeared to be a rhamnogalacturonan with arabinogalactan side chains.     

Compositional studies on succinoglycan-like extracellular water-soluble Rhizobium polysaccharides

This study reports structural information on extracellular, water-soluble polysaccharides from 5 different strains of Rhizobium, viz. R. trifolii J60, R. meliloti J1017, 202, 204 and 207. All the 5 polysaccharides had glucose and galactose in approximate molar ratio of 7:1. Methylation analysis revealed that the polysaccharides contained (1 leads to 3), (1 leads to 6), (1 leads to 4), (1 leads to 4, 1 leads to 6)-linked D-glucose residues, (1 leads to 3)-linked D-galactose and non-reducing terminal D-glucose attached to pyruvate. This structure was found to be exactly the same as that of succinoglycan, a succinic acid containing water-soluble polysaccharide elaborated by Alcaligenes faecalis var. myxogenes 10C3. The similarity of the structure of polysaccharides of two different Rhizobium species and also to the polysaccharide produced by Alcaligenes are discussed in terms of host specificity.     

Studies on the Non-starchy Polysaccharides of the Endosperm of Buckwheat

Water soluble polysaccharides from the buckwheat endosperm was fractionated by salting out and a DEAE-cellulose column (phosphate form) chromatography and the main component (polysaccharide A₁) was isolated as an ultracentrifugally and electrophoretically pure preparation.

The content of polysaccharide A₁ in the buckwheat endosperm was 0.1~0.2%.

Its water solution showed high viscosity and [α]_d was +39.4°. The molecular weight was 240,000~260,000.

Polysaccharide A₁ consisted of xylose, mannose, galactose and glucuronic acid. The hydrolysis of methylated polysaccharide A₁ gave 2,3,4-tri-O-methyl-xylose, 2,3,4,6-tetra-O-methyl-galactose, 2,4,6-tri-O-methyl-galactose, di-O-methyl-mannose and 4-O-methyl- and 5-O-methyl-glucuronic acid. These results suggested that the main chain of this polysaccharide consisted of glucuronic acid, mannose and galactose and the former two occupied branching position with xylose and galactose residues as nonreducing end.     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

Endocytic membrane traffic to the Golgi apparatus in a regulated secretory cell line

We have established a ricin-resistant glycosylation-defective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells. The strategy employed in this study is a modification of that used previously (Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628) to demonstrate transport of the cation-independent and -dependent mannose 6-phosphate receptors from the cell surface to the trans-Golgi network in nonsecretory cell types. In ricin-resistant PC12 cells, radiolabeled galactose was incorporated enzymatically into surface glycoconjugates, primarily glycoproteins. Resistance to beta-galactosidase was acquired upon reculture at 37 degrees C due to further terminal glycosylation of the galactose residues. Treatment of N-linked oligosaccharides isolated from recultured cells with a variety of glycosidases in conjunction with beta-galactosidase demonstrated the addition of sialic acid N-acetylglucosamine and fucose residues to the galactose residues in recultured cells. Resistance to beta-galactosidase was not acquired in cells recultured at 19 degrees C, indicating that subsequent glycosylation of galactose residues did not occur at the cell surface or in endosomes. While glycosylation of galactose incorporated into asparagine oligosaccharides in Chinese hamster ovary clone 13 cells was not significant (less than 1%) after 6 h of reculture, approximately 10% of the galactose incorporated into surface oligosaccharides was further glycosylated in PC12 cells in this time. Analysis of total labeled versus beta-galactosidase-resistant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that endocytic traffic to the site of glycosylation activity in mutant PC12 cells was highly selective, but included a much greater number of proteins than were detected in Chinese hamster ovary clone 13 fibroblasts.     

STRUCTURE OF EXTRACELLULAR POLYSACCHARIDES PRODUCED BY A SOIL CRYPTOMONAS SP. (CRYPTOPHYCEAE)1

The Hindak strain of a Cryptomonas species (Cryptophyceae) produces extracellular polysaccharides. Because there is no information on the structure of these compounds in the Cryptophyceae we conducted structural studies. Gas–liquid chromatographic analyses showed that the polysaccharide is composed of fucose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, and traces of 3-O-methyl galactose. The polysaccharide was separated into two subtractions by ion-exchange chromatography. Fraction A consisted mainly of 1,3-linked galactose units and 1,4-linked galacturonic acid. Unlike fraction B, fraction A did not have xylose, 3-O-methyl galactose, or glucuronic acid. Also, its degree of branching was low compared to that of fraction B. Only traces of sulfate were present infraction A, but fraction B was 10–15% sulfated. Protein was approximately 1% in both fractions. These polysaccharides appear to be a novel type of polymer in algae.     

Isolation and analysis of a novel acidic polysaccharide from the case of squid pen.

A new non-sulphated acidic polysaccharide with an average molecular mass of 55 kDa was isolated from squid pen case after papain digestion and beta-elimination. This polysaccharide contains mainly L-iduronic acid, D-glucuronic acid, D-galactosamine, D-glucosamine and significant amounts of neutral sugars as glucose, galactose and fucose. The polysaccharide was not degraded to the relative disaccharides by chondroitinases ABC, AC and B, hyaluronidase and keratanase or by treatment with heparinases, suggesting a structure different from those of known glycosaminoglycans. The polysaccharide cannot form self aggregates.     

Structure of the cell wall of lactobacilli. Role of muramic acid phosphate in Lactobacillus fermenti

1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus fermenti, serological group F, were separated by mild conditions of acid hydrolysis; the polysaccharide was composed of glucose and galactose. 2. Soluble cell-wall products were isolated from cell wall lysed by lysozyme and a Streptomyces enzyme preparation. The lysozyme-dissolved fraction contained a greater proportion of mucopeptide. 3. The soluble preparations were heated in dilute acid to hydrolyse the linkage between the polysaccharide and mucopeptide components and then incubated with acid phosphatase. 4. Inorganic phosphate was released from products of Streptomyces enzyme action but not from products of lysozyme action. 5. The phosphate was shown to be present in the mucopeptide as muramic acid phosphate. It is concluded that in the intact wall polysaccharide is joined to muramic acid by a phosphodiester linkage.     

Characterization of three capsular polysacharides produced by Burkholderia pseudomallei

Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.     

The presence of polysaccharide in normal human gastric mucus.

Polysaccharide material was found in the proteolysis glycopolypeptide fraction from normal human gastric mucus. The polysaccharide was identified by carbohydrate and amino acid analyses, by elemental analysis and from its behaviour on density-gradient ultracentrifugation. The polysaccharide is polydisperse with a weight-average molecular mass of 300 000 Da. Over 85% of the polysaccharide consists of galactose, and this represents 26% of all the galactose present in the fractions after beta-elimination with reduction of the glycopolypeptide material.     

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.     

Utilization of Waste Products of Dehydrated Onion Industry for Production of Fodder Yeast

An organism producing extracellular polysaccharide was isolated from soil and identified as Aeromonas hydrophila (Chester) Stanier. The effects of medium components and cultural conditions on production of the polysaccharide were studied. The optimal concentrations of carbon and nitrogen sources were 5% and 0.3%, respectively, for production of the polysaccharide. The optimal initial pH was 7~9. The maximum polysaccharide yield was obtained at 4~8 days of fermentation. From sucrose and raffinose as carbon source, the organism produced levan and acidic polysac-charide in the ratio of 7:3 and 4:6, respectively. From glucose, galactose, fructose, mannose, maltose and lactose, mainly acidic polysaccharide was produced. The acidic polysaccharide was found to contain galactose, mannose and glucuronic acid in a ratio of 5:4:2. The acidic polysaccharides obtained from sucrose and lactose seemed to be the same polysaccharide.