Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmission electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCl), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6%) and proteins (19%). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.     

Novel bacterial exopolysaccharides from deep-sea hydrothermal vents

Five bacterial strains recovered from deep-sea hydrothermal vents were studied for their ability to secrete extracellular polymers. A preliminary characterization displayed four different polysaccharides in terms of both chemical composition and rheological properties. One of them was secreted by Alteromonas macleodii subsp. fijiensis and exhibited similarities with xanthan, a commercial polysaccharide. Two of the three Pseudoalteromonas species were shown to produce the same polymer. The last polymer was secreted by a bacterium belonging to the Vibrio genus. They all contained glucose, galactose, mannose, glucuronic and galacturonic acids as the main sugars with the exception of the last one which was only constituted by uronic acids and hexosamines, in that similar to the structure of heparin, a glycosaminoglycan useful in pharmaceutical area. Applications for these polysaccharides could be expected in various biotechnological fields including the food industry, the wastewater treatment and pharmaceutical areas.     

Sugars in hydrolysates of fungal melanins and soil humic acids

Humic acids from four Brazilian topsoils of different origin and four fungal melanins, synthesized under two cultural conditions were subjected to a two step hydrolysis procedure and the released monosaccharides qualitatively and quantitatively determined by gas-liquid chromatography. The neutral sugars, glucose, galactose, mannose, arabinose, xylose, fucose, rhamnose and the alcohol sugar inositol, were detected in most of the soil humic acid samples. The fungal melanins showed the presence of glucose, galactose, mannose and arabinose. Ribose was present in two out of the eight samples tested. Some quantitative differences in the two types of humic polymers were noted and expected considering their origins. However, similarities were more apparent than differences and give further indication that melanic fungi may play a significant role in the formation of soil humic acids.     

Polymeric Beta-Hydroxyalkanoates from Environmental Samples and Bacillus megaterium

The procaryotic endogenous storage polymer known as poly-beta-hydroxybutyrate is actually a mixed polymer of short-chain beta-hydroxy fatty acids. A method for the quantitative recovery of this mixed polymer, called poly-beta-hydroxyalkanoate (PHA), with analysis by capillary gas-liquid chromatography, showed the presence of at least 11 short-chain beta-hydroxy acids in polymers extracted from marine sediments. Polymers extracted from Bacillus megaterium monocultures were also a complex mixture of beta-hydroxy acids with chain lengths between four and eight carbons. Lyophilized sediments were extracted in a modified Soxhlet extractor, and the polymer was purified with ethanol and diethyl ether washes. The purified polymer was treated with ethanol-chloroform-hydrochloric acid (8.5:2.5:1) for 4 h at 100°C, a treatment which resulted in the formation of the ethyl esters of the constituent beta-hydroxy acids. Subsequent assay of the products by gas-liquid chromatography indicated excellent reproducibility and sensitivity (detection limit, 100 fmol). Disturbing sediments mechanically or adding natural chelators increased all major PHA components relative to the bacterial biomass. Gardening of sedimentary microbes by Clymenella sp., an annelid worm, induced decreases in PHA, with changes in the relative proportion of component beta-hydroxy acids. The concentration of PHA relative to the bacterial biomass can reflect the recent metabolic status of the microbiota.     

The extracellular proteoglycan produced by Rhodella grisea

Highly viscous extracellular proteoglycan (EPG) has been isolated from culture medium of the unicellular red alga Rhodella grisea (Rhodophyceae) by ethanol precipitation. EPG was composed of xylose (29.3%), 3-O-methyl-xylose (26.0%), uronic acids (17.1%), rhamnose (14.4%), galactose (7.5%), glucose (3.9%), arabinose (1.4%) and mannose (0.4%), and traces of fucose, 4-O-methyl-xylose and 2,3-di-O-methyl-rhamnose or fucose. In addition, the polymer contained proteins (13.1%), sulphates and 13C-CP MAS spectra indicated the presence of acetyl and succinyl groups. The molecular mass was estimated to be 136,000. Ion-exchange chromatography afforded five fractions differing in composition of neutral sugars, uronic acids, and protein content indicating thus the complex structure of the EPG.     

A gas chromatographic method for the quantitative detemination of hexuronic acids in alginic acid

A method has been developed for the quantitative determination of the relative proportions of d-mannuronic and l-guluronic acids in alginic acid. To obtain homogeneous reaction conditions the viscosity of the alginic acid sample was first decreased by limited hydrolysis with mineral acid. The carboxyl groups were then esterified by reaction with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide, and reduced with sodium borohydride. The resulting hexosans were converted by acid hydrolysis to d-mannose and an equilibrium mixture of l-gulose and 1,6-anhydro-l-gulose. These were treated with sodium borohydride; the 1,6-anhydro-l-gulose was not reduced whereas d-mannose and l-gulose were converted to d-mannitol and d-glucitol. The hexitols were estimated by gas-liquid chromatography as the n-butane boronic acid esters, and the relative proportions of the uronic acids in the alginic acid were calculated by taking into account the equilibrium ratio of l-gulose and 1,6-anhydro-l-gulose. The method can be used to analyze as little as 2 mg of alginic acid.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.     

The identification and quantitation of connective tissue hexosamines using a combination of gas liquid chromatographic and colorimetric procedures.

Due to interactions between amino sugars, amino acids, and/or carbohydrate breakdown products from acid hydrolysis, the quantitation of individual amino sugars from connective tissue hydrolysates, requires a number of indirect steps involving separation and purification of the hexosamines prior to gas-liquid chromatography. In this paper, a method is reported which permits the direct quantitation of galactosamine and glucosamine from connective tissue hydrolysates, utilising a combination of both gas-liquid chromatographic and colorimetric procedures. A two-phase extraction system which selectively eliminates pyridine and amino acids from the T.M.S. ethers of glucosamine and galactosamine is also described.     

Determination of hexuronic acids in glycosaminoglycans by automated ion-exchange chromatography.

This report describes a procedure for analyzing glucuronic and iduronic acids using the Technicon automated sugar chromatography system. Glueronic and iduronic acids of standard samples of glycosaminoglycans have been analyzed after hydrolysis by formic acid. The method has been applied to quantitate uronic acids in chondroitin sulfates and dermatan sulfate mixtures obtained by Dowex 1 Cl^? column fractionation of glycosaminoglycans from aortas of different animal species. The results are in good agreement with those obtained by the gas-liquid chromatographic technique.     

Determination of the gram-positive bacterial content of soils and sediments by analysis of teichoic acid components

Many gram-positive bacteria form substituted polymers of glycerol and ribitol phosphate esters known as teichoic acids. Utilizing the relative specificity of cold concentrated hydroflouric acid in the hydrolysis of polyphosphate esters it proved possible to quantitatively assay the teichoic acid-derived glycerol and ribitol from gram-positive bacteria added to various soils and sediments. The lipids are first removed from the soils or sediments with a one phase chloroform-methanol extraction and the lipid extracted residue is hydrolyzed with cold concentrated hydrofluoric acid. To achieve maximum recovery of the teichoic acid ribitol, a second acid hydrolysis of the aqueous extract is required. The glycerol and ribitol are then acetylated after neutralization and analyzed by capillary gas-liquid chromatography. This technique together with measures of the total phospholipid, the phospholipid fatty acid, the muramic acid and the hydroxy fatty acids of the lipopolysaccharide lipid A of the gram-negative bacteria makes it possible to describe the community structure of environmental samples. The proportion of gram-positive bacteria measured as the teichoic acid glycerol and ribitol is higher in soils than in sediments and increases with depth in both.     

Purification and characterization of an extracellular polysaccharide from haloalkalophilic Bacillus sp. I-450

During the course of investigation of haloalkalophilic bacteria, we screened some heavily polluted soil samples from the mudflats surrounding the city of Inchon, Korea, for their bioflocculant producing ability. Based on the screening, one isolate no. 450 tentatively identified as Bacillus sp. produced an extracellular polysaccharide having flocculation activity. The isolate produced the polysaccharide during the late logarithmic growth phase. The polymer could be recovered from the supernatant of the fermented medium by cold ethanol precipitation and purified by treating with cetylpyridinium chloride (CPC). The polymer was identified as an acidic polysaccharide containing neutral sugars, namely, galactose, fructose, glucose and raffinose, and uronic acids as major and minor components, respectively. The amount of neutral sugars, uronic acid and amino sugars were 52.4, 17.2 and 2.4%, respectively. The molecular weight of the polysaccharide was found to be 2.2×10⁶ Da. The Fourier transform infrared spectrophotometer (FT-IR) revealed typical characteristics of polysaccharides. ¹H NMR spectrum showed that the polymer is a heteroglycan. Thermogravimetric (TGA) analysis indicated the degradation temperature (T_d) at 290 °C. The rheological analysis of the polymer 450 revealed the pseudoplastic property with shear-thinning effect, while the compression test indicated that the polymer had high gel strength, and the S.E.M. studies showed that the polymer has a porous structure with small pore-size distribution indicating the compactness of the polymer.     

Quantitative analysis of total membrane-bound sugars and amino sugars as alditol acetates by combined thin-layer chromatography,gas-liquid chromatography,and radiogas chromatography

A sensitive method (0.4 μg of hexoses routinely detectable) for quantitative determinations of sugars and amino sugars in biological material, particularly in membranes, is described. The method consists of a combination of thin-layer chromatography (tlc), gas-liquid chromatography (glc), and radiogas chromatography (rgc), using a highly thermostable phase (Silar 10 c) for the analysis of the specific alditol acetates. In this method, the losses incurred during hydrolysis and preparation for glc are assessed by comparison with the specific recoveries of added radiolabeled internal standards.     

Two distinct classes of polyuronide from the cell walls of a dimorphic fungus, Mucor rouxii

Polyuronides were extracted from purified yeast and mycelial walls of Mucor rouxii by sequential treatments with lithium chloride and potassium hydroxide and were fractionated by ion-exchange chromatography on DEAE-Sephadex. Two polymers (I and II) of different acidity were found in both wall types. Polymer I contained D-glucuronic acid, L-fucose, D-mannose, and much smaller amounts of D-galactose. Yeast and mycelial polymer I had similar uronic acid contents but differed in their neutral sugar compositions and molecular weights. Polymer II from both cell types contained largely D-glucuronic acid and had similar molecular weights. On partial acid hydrolysis, both polymers I and II gave rise to insoluble glucuronans which appeared to be homopolymeric. One-third of the total uronosyl residues of polymer I, and almost all of the uronosyl residues of polymer II, were present in homopolymeric segments. However, homopolymers derived from polymers I and II may not be identical.     

Sugar constituents of fucose-containing polysaccharides from various Japanese brown algae

Sugar constituents of the fucose-containing polysaccharides (FCPs) from 21 species of brown algae were analyzed. FCPs were extracted with hot water (100 °C, 4 h), separated by precipitation with 20% (v:v) ethanol in the presence of 0.05 M MgCl₂ to remove contaminating soluble alginate, and purified by DEAE-Sephadex column chromatography. The samples were hydrolyzed with HCI, and neutral sugar and uronic acid were separated by anion exchange chromatography. Their amounts were determined by gas-liquid chromatography. The neutral sugars in the FCPs from Ishige okamurae, Laminaria ochotensis, Myelophycus simplex, Padina arborescens and Sargassum thunbergii all contained arabinose, fucose, galactose, glucose, mannose, rhamnose and xylose residues. The FCPs from Ishige okamurae, Padina arborescens, Sargassum hemiphyllum, S. patents and S. sagamianum contained the four uronic acids, galacturonic acid, glucuronic acid, guluronic acid and mannuronic acid.     

Quantitative microanalysis of cell walls of Saprolegnia diclina humphrey and Tremella mesenterica fries

Cell walls of the fungi Saprolegnia diclina Humphrey and Tremella mesenterica Fries were analyzed quantitatively. Particular attention was paid to the hydrolysis and analysis of neutral sugars, amino sugars and amino acids. These components, together with total lipids, total uronic acids and the ashed residue, accounted for more than 90% by weight of the original dry cell wall preparation. There were substantial losses of amino acids during hydrolysis; however, analytical recovery approached 100% when total protein was calculated from the total nitrogen analysis. The analytical procedures were reproducible (±3% for amino acids and amino sugars, and ±5–10% for other components) when applied to individual cell wall preparations. However, even under carefully standardized conditions, different cell wall preparations from the same species showed variable composition.Glucose was the predominant neutral sugar in the cell wall polymers of both species. The amino acid compositions were remarkable in that neither species contained detectable levels of cyst(e)ine. Hydroxyproline was detected in both species. The report from Tremella mesenterica is the first for this imino acid from the cell wall of a Basidiomycete.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected.We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80°C for 1 min, and purified by ultrafiltration and ethanol precipitation.The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem. Biophys. Res. Commun. 63, 875–880).A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB³H₄. The polysaccharide was only slightly degraded (2%) by jack bean α-mannosidase and could precipitate 15 times its own weight of concanavalin A.The acidic polymer was also detected in the cell “periplasm” and was secreted from cells grown in defined media during the period of decelerating growth.     

A gas chromatographic method for the determination of aldose and uronic Acid constituents of plant cell wall polysaccharides

A major problem in determining the composition of plant cell wall polysaccharides has been the lack of a suitable method for accurately determining the amounts of galacturonic and glucuronic acids in such polymers. A gas chromatographic method for aldose analysis has been extended to include uronic acids. Cell wall polysaccharides are depolymerized by acid hydrolysis followed by treatment with a mixture of fungal polysaccharide-degrading enzymes. The aldoses and uronic acids released by this treatment are then reduced with NaBH₄ to alditols and aldonic acids, respectively. The aldonic acids are separated from the alditols with Dowex-1 (acetate form) ion exchange resin, which binds the aldonic acids. The alditols, which do not bind, are washed from the resin and then acetylated with acetic anhydride to form the alditol acetate derivatives. The aldonic acids are eluted from the resin with HCl. After the resin has been removed, the HCl solution of the aldonic acids is evaporated to dryness, converting the aldonic acids to aldonolactones. The aldonolactones are reduced with NaBH₄ to the corresponding alditols, dried and acetylated. The resulting alditol acetate mixtures produced from the aldoses and those from the uronic acids are analyzed separately by gas chromatography. This technique has been used to determine the changes in composition of Red Kidney bean (Phaseolus vulgaris) hypocotyl cell walls during growth, and to compare the cell wall polysaccharide compositions of several parts of bean plants. Galacturonic acid is found to be a major component of all the cell wall polysaccharides examined.     

The composition of β-lactamase I and β-lactamase II from Bacillus cereus 569/H

1. Crystalline beta-lactamase I from Bacillus cereus 569/H yielded only amino acids on acid hydrolysis, but crystalline beta-lactamase II from the same organism yielded also substantial quantities of neutral sugars and amino sugars. 2. Analysis with an amino acid analyser indicated that the two enzymes were similar though not identical in overall amino acid composition. Analysis of neutral and amino sugars as their silyl derivatives by gas-liquid chromatography showed that the carbohydrate moiety of beta-lactamase II contained residues of glucose, galactose, mannose, fucose, glucosamine and galactosamine. 3. After oxidation and hydrolysis both beta-lactamases gave small amounts of cysteic acid. After treatment of inactive Zn(2+)-free beta-lactamase II with N-ethylmaleimide or iodoacetate enzymic activity was not restored by the addition of Zn(2+).