Copper and cobalt alter the cell wall composition of Cunninghamella blakesleeana

Cunninghamella blakesleeana was highly sensitive to Cu and Co on a medium containing NaNO3 as the sole nitrogen source. The nitrate reductive pathway was altered by Cu and Co, and NO-2 accumulated in the medium. Under conditions of Cu toxicity, the mycelium and the cell walls acquired a blue color, and most of the Cu was located in the cell walls, which differed in several aspects from cell walls derived from Co-containing or control cultures. At half-maximal growth inhibition by Cu (2.5 micrograms/mL or 39.3 microM) or Co (3.5 micrograms/mL or 59.4 microM), the mycelia contained 1.5 micrograms Cu or 1.0 microgram Co/mg dry tissue, respectively, but the isolated cell walls contained 33.5 micrograms Cu or 1.8 micrograms Co/mg dry cell wall. The phosphorous content of mycelia from Co-containing cultures was the same as that from control cultures, whereas that of mycelia from Cu-containing cultures contained 36% less. However, the phosphorous content of the cell walls from mycelia cultured in the presence of Cu or Co was two- and three-fold higher, respectively, than that of cell walls from control cultures. The cell walls of Cu-containing cultures contained significantly less hexosamine than the control cell walls, and chitin and chitosan were present in equal quantities. The cell walls of Co-containing cultures had the same amount of hexosamine as the control cell walls, but 88% of the hexosamine was present as chitosan and bound very little Co. The control cell walls contained approximately 60% chitosan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular weight distribution of chitosan isolated from Mucor rouxii under different culture and processing conditions

The isolation of chitosan from a fungal source offers the potential of a product with controlled physicochemical properties not obtainable by the commercial chemical conversion of crustacean chitin. A variety of culture and processing protocols using Mucor rouxii were studied for their effects on biomass yield and chitosan molecular weight. Weight-averaged molecular weight determined by gel permeation chromotography ranged from 2.0 x 10(5) to approximately 1.4 x 10(6) daltons. The chitosan yield ranged from 5% to 10% of total biomass dry weight and from 30% to 40% of the cell wall. Of the culture parameters studied, length of incubation and medium composition effected biomass production and molecular weight. Modification of the processing protocol, including the type and strength of acid, and cell wall disruption in acid prior to refluxing were used to optimize the efficiency of chitosan extraction.The degree of deacetylation of fungal and commercial chitosans was compared using infrared spectrometry, titration, and first derivative of UV absorbance spectrometry. The chitosan obtained directly from the fungal cell wall had a higher degree of deacetylation than commercial chitosan from the chemical conversion process.     

Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan

In a preceding paper (Briza, P., Winkler, G., Kalchhauser, H., and Breitenbach, M. (1986) J. Biol. Chem. 261, 4288-4294), we reported the presence of dityrosine in the outer layers of yeast ascospore walls. Both outer layers seen in electron micrographs of yeast ascospore walls are sporulation-specific. Here we show that the second of these two outer layers consists of chitosan. In intact spores, it is shielded from staining with primulin by the outermost layer. However, in purified spore walls, the second layer is brightly stained by primulin, and hydrolysates of such preparations contain about 10% glucosamine relative to spore wall dry weight. The spore wall material staining with primulin is resistant to chitinase, but readily degraded by treatment with HNO2. Acetylation prior to HNO2 treatment completely prevents its degradation. A partial acid hydrolysate of spore walls contains predominantly soluble poly-beta-(1,4)-glucosamine as determined by 13C NMR spectroscopy. By these criteria, the glucosamine polymer of yeast ascospore walls is chitosan. As spore walls treated with alkali lack the inner layers but contain chitosan and as chitosan is not exposed at the surface of the spore, we conclude that it is localized in the second outer layer of the spore wall.     

Composition of the Cellular Envelopes of Anabaena cylindrica

Comparative chemical analyses were made of the walls of vegetative cells, heterocysts, and spores, and of the mucilage of Anabaena cylindrica. The wall of the vegetative cell is composed predominantly of amino compounds, with a mannose-rich carbohydrate component comprising only 18% of the dry weight. In contrast, 62% of the heterocyst wall and 41% of the spore wall is carbohydrate. The carbohydrate moieties of the heterocyst wall and spore wall are similar in that the ratio of glucose, mannose, galactose, and xylose is approximately 75:20:3:4 in both walls. It appears that, during the differentiation of a vegetative cell into either a spore or a heterocyst, a glucose-rich wall polysaccharide is produced that is different from the polysaccharide component of the wall of the vegetative cell and of the sheath. In the case of the heterocyst, the wall was estimated to account for approximately 52% of the dry weight of the whole cell.     

Tumor induction by agrobacterium involves attachment of the bacterium to a site on the host plant cell wall

Cell wall preparations from primary bean leaves were found to inhibit tumor initiation by Agrobacterium tumefaciens strain B6 when inoculated with the bacteria on bean leaves. Membrane fractions from these same leaves were noninhibitory. The cell walls were effective when applied prior to or with bacteria, but application of cell walls about 15 minutes after bacteria did not affect the number of tumors initiated. Much of the inhibitory activity of the plant cell walls was eliminated by pretreatment with dead site-attaching bacteria or with lipopolysaccharide from these bacteria. Cells and lipopolysaccharide from non-site-attaching agrobacteria had no effect on the activity of the plant cell walls. About 30% inhibition of tumor initiation was obtained with plant cell walls at 50 μg/ml dry weight, and at 10 mg/ml dry weight about 70% inhibition was typical. Both early and late appearing tumors were affected by the cell walls, indicating that they do not exclusively affect tumors arising from either small or large wounds. These data show that plant cell walls but not membranes contain surfaces to which A. tumefaciens adheres and these exhibit the specificity typical of the host site to which virulent agrobacteria must attach to induce tumors. It is concluded that some portion of wound-exposed plant cell wall constitutes the host adherence site in Agrobacterium infections.     

Peptides isolated from cell walls of Medicago truncatula nodules and uninfected root

The hydroxyproline-rich root nodules of legumes provide a microaerobic niche for symbiotic nitrogen-fixing Rhizobacteria. The contributions of the cell wall and associated structural proteins, particularly the hydroxyproline-rich glycoproteins (HRGPs), are therefore of interest. Our approach involved identification of the protein components by direct chemical analysis of the insoluble wall. Chymotryptic peptide mapping showed a "P3-type" extensin containing the highly arabinosylated Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Tyr3-Lys motif as a major component. Cell wall amino acid analyses and quantitative hydroxyproline arabinoside profiles, predominantly of tri- and tetraarabinosides, confirmed this extensin as the major structural protein in the cell walls of both root nodules and uninfected roots. On the other hand, judging from the Pro, Glu and non-glycosylated Hyp content, the nodule-specific proline-rich glycoproteins, such as the early nodulins (ENOD-PRPs), are present in much lesser amounts. Although we isolated no PRP peptides from nodule cell walls, a single PRP peptide from root cell walls confirmed the presence of a PRP in roots and represented the first direct evidence for a crosslinked PRP in muro. Compared with root cell walls (approximately 7% protein dry weight) nodule cell walls contained significantly more protein (approximately 13% dry weight) with an overall amino acid and peptide composition indicating the presence of structural protein unrelated to the HRGPs.     

Degradation of barley straw, ryegrass, and alfalfa cell walls by Clostridium longisporum and Ruminococcus albus

The recently isolated ruminal sporeforming cellulolytic anaerobe Clostridium longisporum B6405 was examined for its ability to degrade barley straw, nonlignified cell walls (mesophyll and epidermis) and lignified cell walls (fiber) of ryegrass, and alfalfa cell walls in comparison with strains of Ruminococcus albus. R. albus strains degraded 20 to 28% of the dry matter in barley straw in 10 days, while the clostridium degraded less than 2%. A combined inoculum of R. albus SY3 and strain B6405 was no more efficient than SY3 alone, and the presence of Methanobacterium smithii PS did not increase the amount of dry matter degraded. In contrast, with alfalfa cell walls as the substrate, the clostridium was twice as active (28% weight loss) as R. albus SY3 (15%). The percentages of dry matter degraded from ryegrass cell walls of mesophyll, epidermis, and fiber for the clostridium were 50, 47, and 32%, respectively, and for R. albus SY3 they were 77, 73, and 63%, respectively. Analyses of the predominant neutral sugars (arabinose, xylose, and glucose) in the plant residues after bacterial attack were consistent with the values for dry matter weight loss. Measurements of the amount of carbon appearing in the fermentation products indicated that R. albus SY3 degraded ryegrass mesophyll cell walls most rapidly, with epidermis and fiber cell walls being degraded at similar rates. Strain B6405 attacked alfalfa cell walls at a rate greater than that of any of the ryegrass substrates. These results indicate an unexpected degree of substrate specificity in the ability of C. longisporum to degrade plant cell wall material.     

Degradation of barley straw, ryegrass, and alfalfa cell walls by Clostridium longisporum and Ruminococcus albus.

The recently isolated ruminal sporeforming cellulolytic anaerobe Clostridium longisporum B6405 was examined for its ability to degrade barley straw, nonlignified cell walls (mesophyll and epidermis) and lignified cell walls (fiber) of ryegrass, and alfalfa cell walls in comparison with strains of Ruminococcus albus. R. albus strains degraded 20 to 28% of the dry matter in barley straw in 10 days, while the clostridium degraded less than 2%. A combined inoculum of R. albus SY3 and strain B6405 was no more efficient than SY3 alone, and the presence of Methanobacterium smithii PS did not increase the amount of dry matter degraded. In contrast, with alfalfa cell walls as the substrate, the clostridium was twice as active (28% weight loss) as R. albus SY3 (15%). The percentages of dry matter degraded from ryegrass cell walls of mesophyll, epidermis, and fiber for the clostridium were 50, 47, and 32%, respectively, and for R. albus SY3 they were 77, 73, and 63%, respectively. Analyses of the predominant neutral sugars (arabinose, xylose, and glucose) in the plant residues after bacterial attack were consistent with the values for dry matter weight loss. Measurements of the amount of carbon appearing in the fermentation products indicated that R. albus SY3 degraded ryegrass mesophyll cell walls most rapidly, with epidermis and fiber cell walls being degraded at similar rates. Strain B6405 attacked alfalfa cell walls at a rate greater than that of any of the ryegrass substrates. These results indicate an unexpected degree of substrate specificity in the ability of C. longisporum to degrade plant cell wall material.     

Cell wall composition of Streptomyces roseoflavus var. roseofungini and its Nocardia-like variant

The composition of cell walls was comparatively studied in Streptomyces roseoflavus var. roseofungini 1128 and in its variant 1-68. In the logarithmic phase of growth, the content of teichoic acid in the cell wall of the parent culture was four times as high as in the cell wall of the variant. The cell walls of the parent culture contained 5 to 7 times more O-lysyl residues not only due to a higher content of teichoic acid in the walls but also owing to a lower content of lysyl groups in the teichoic acid of the variant. An additional polysaccharide comprising galactose and glucosamine was found in the cell wall of the variant but not in the parent strain. The peptidoglycan of the both cultures had a structure typical of Streptomyces spp.; its content in the cell walls of the two cultures was identical (ca. 50% of the dry cell wall biomass weight). The results are discussed in connection with the peculiarities of the variant hyphal septation.     

Changes in cell wall polysaccharides associated with growth

Changes in the polysaccharide composition of Phaseolus vulgaris, P. aureus, and Zea mays cell walls were studied during the first 28 days of seedling development using a gas chromatographic method for the analysis of neutral sugars. Acid hydrolysis of cell wall material from young tissues liberates rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose which collectively can account for as much as 70% of the dry weight of the wall. Mature walls in fully expanded tissues of these same plants contain less of these constituents (10%-20% of dry wt). Gross differences are observed between developmental patterns of the cell wall in the various parts of a seedling, such as root, stem, and leaf. The general patterns of wall polysaccharide composition change, however, are similar for analogous organs among the varieties of a species. Small but significant differences in the rates of change in sugar composition were detected between varieties of the same species which exhibited different growth patterns. The cell walls of species which are further removed phylogenetically exhibit even more dissimilar developmental patterns. The results demonstrate the dynamic nature of the cell wall during growth as well as the quantitative and qualitative exactness with which the biosynthesis of plant cell walls is regulated.     

Degradation of isolated grass mesophyll, epidermis and fibre cell walls in the rumen and by cellulolytic rumen bacteria in axenic culture

The degradation of cell walls of mesophyll, epidermis and fibre cells isolated from leaves of perennial and Italian ryegrass within the sheep rumen or by selected strains of rumen bacteria in vitro , was followed by estimation of dry matter loss, or loss of neutral sugar residues. Primary cell walls (mesophyll and epidermis) were fully degraded within 12 h in the rumen, while the more heavily lignified fibre cell walls showed only a 40% loss of dry matter over the same period. Neutral sugar residues were lost at a common rate from walls of all three cell types. Incubation of cell walls with cellulolytic bacteria showed that the extent to which cell walls were attacked was constantly ordered (epidermis > mesophyll > fibre). The rate of degradation of cell walls was less in axenic culture than within the rumen. Greatest weight losses were produced by Ruminococcus albus , followed by Bacteroides succinogenes , with Ruminococcus flavefaciens effecting the least change, regardless of the nature of the cell wall provided as a substrate. Xylose was more readily lost from primary cell walls than glucose during the early stages of attack, but both were lost at a common rate from fibre cell walls. Dry matter losses produced by the hemicellulolytic strain, Bacteroides ruminocola , were limited even after extended incubation. Electron microscopy indicated that R. albus was less commonly attached to cell walls than were the other cellulolytic strains, although evidence of capsular material was present. Bacteroides succinogenes was seen with an extensive capsule which enveloped clusters of cells, forming micro-colonies in association with the plant cell wall. Vesicle-like structures, commonly associated with the cellulolytic bacteria R. albus and B. succinogenes , were found on comparatively few occasions in this study.     

Changes in composition of harvested mushrooms (Agaricus bisporus)

Post-harvest changes in the biochemical composition of the mushroom were studied. Non-structural polysaccharide was found at levels greater than 10% dry wt in the fresh mushroom. After 4 days storage, the level had decreased to below 5% dry weight. The polysaccharide appeared to contain only glucose residues joined by α-1,4 and α-1,6 linkages. The chitin content of cell walls increased by ca 50% during 4 days storage, while cell wall glucan decreased. There was a large increase in urea content.     

A comparison of the polysaccharides extracted from dried and non-dried walls of suspension-cultured sycamore cells

Suspension-cultured sycamore cells (Acer pseudoplatanus) were disrupted in aqueous K-Pi buffer and the insoluble residue (the cell wall) purified by extraction with organic solvents and air-dried (dry cell walls) or by washing with aqueous sodium dodecyl sulphate and stored frozen (wet cell walls). Polysaccharides solubilized from the purified wet and dry cell walls by enzymatic digestion and chemical extraction were isolated and their glycosyl-residue compositions compared. No significant differences were found in the types or yields of the polysaccharides solubilized by enzymatic digestion and chemical extraction of the wet and dry cell wall preparations. Moreover, the glycosyl-residue compositions of the so-called ‘-cellulose’ fraction that remains after extraction of the wet and dry cell wall preparations with alkali was indistinguishable from the glycosyl-residue compositions of the walls prior to extraction.     

Changes in Microsporum gypseum Mycelial Wall and Spore Coat Glycoproteins During Sporulation and Spore Germination

Ethylenediamine-soluble glycoproteins were extracted from isolated Microsporum gypseum hyphal walls during sporulation and from spore coats before and after germination. This study was carried out to identify a sporulation-specific cell wall protein that possibly served as a substrate for the alkaline protease which initiated the macroconidial germination of this fungus. Analyses revealed that water-insoluble glycoprotein accounted for 10% of the ungerminated spore coat but only for 4 to 5% of the mycelial wall dry weight. This fraction was modified in its amino acid composition during sporulation, and it decreased in protein content during spore germination. Water-soluble glycoprotein, which accounted for approximately 3 to 3.5% of either the spore coat or mycelial wall dry weight, was of similar amino acid composition from both sources and did not decrease in protein content upon spore germination. The water-insoluble glycoprotein was found to be rich in leucine, aspartic acid, glycine, glutamic acid, and phenylalanine residues. The water-soluble glycoprotein was rich in proline, threonine, glycine, serine, glutamic acid, and alanine.     

The lipid content of cell walls obtained from juvenile,yeast-like and filamentous cells ofCandida albicans

Purified cell walls ofCandida albicans obtained from juvenile cells, mature yeast-like cells and filamentous cells were analyzed for their lipid components. Chloroform: methanol (2:1 v v) extraction of the acetone-treated dried cell walls indicated the total lipid content to be 2.1% of the dry weight of the juvenile cell walls, 1.8% of the mature yeast-like cell walls and 4.5% of the filamentous cell walls. Separation of the chloroform: methanol extractable fraction through a silicie acid column and quantitative determination of the fractions showed significant amounts of sterol esters, triglycerides, sterols, free fatty acids, and phospholipids in these extracts. Following acetone extraction sterols were shown to constitute a greater percentage of the cell wall of juvenile cells than mature cells. Thin-layer chromatography separated the acetone-extractable lipids into at least four components. Diethyl ether extracts of the cell walls indicated the presence of small amounts of glycerol phospholipids in the cell walls of juvenile and mature yeast cells. Boiling 95% ethanol also removed a small lipid fraction from the cell walls of both juvenile and mature yeast which could include sphingosine phosphatides or glycosides.     

A HISTOCHEMICAL STUDY OF ABSCISSION LAYER FORMATION IN THE BEAN

In debladed bean petioles calcium and dry weight increased in the abscission zone during an induction period of 14 hr. Before the microscopic appearance of the abscission layer calcium decreased in the abscission zone and increased in the petiole. Dry matter began to decrease in both the abscission zone and the petiole 24 hr after deblading. The first visual change in the cells of the abscission zone was a swelling of the pectic materials of the cell walls. This was followed by breakdown of other cell wall components, i.e., non-cellulosic polysaccharides and cellulose. The cellulose of the cell walls adjacent and distal to the abscission layer was found to be altered; however, no lignin was present during abscission layer development. The alteration of pectic materials, coupled with breakdown of cell wall components, resulted in the collapse of cells of the abscission layer just prior to separation. Auxin delayed abscission and also delayed the initial increase in calcium, the movement of calcium from the abscission zone to the petiole, and the decrease in dry weight.     

Autolytic activities of the cell wall in rice coleoptiles. Effects of nojirimycin

Oryza sativa L. var. bahia coleoptile cell walls show sufficient autolytic activity for the release into the surrounding medium of amounts up to 60 μg of sugars per mg of dry weight of cell wall. The products released elute in Bio-gel P.2 as mono- and polysaccharides with glucose as the sole component. The polysaccharide component releases tri- and tetrasaccharides on treatment with a glucanase specific for β (1–3) (1–4) linkages in the same proportion as that of the mixed glucan of the cell wall. This supports the hypothesis that the polysaccharide component originates from the cell wall glucan and that autolysis is therefore related to the processes of the loss of rigidity of the cell wall. Nojirimycin (a specific glucanase inhibitor and inhibitor of auxin-induced elongation) decreases autolytic activity of the cell walls, reducing it to 30% of its normal value. Bio-gel P. 2 elution of the products released in autolysis in the presence of nojirimycin shows that only the monosaccharide fraction was affected.     

Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls.

A strain of Bacillus sp (Bacillus R-4) produces a protease and a carbohydrolase both of which have the ability to lyse Rhizopus cell walls. Of the enzymes, the carbohydrolase has been purified to an ultracentrifugally and electrophoretically homogeneous state, and identified as a chitosanase. The enzyme was active on glycol chitosan as well as chitosan. Molecular weight of the purified enzyme was estimated as 31 000 and isoelectric point as pH 8.30. The enzyme was most active at pH 5.6 and at 40 degrees C with either Rhizopus cell wall or glycol chitosan as substrate, and was stable over a range of pH 4.5 to 7.5 at 40 degrees C for 3 h. The activity was lost by sulfhydryl reagents and restored by either reduced glutathione of L-cysteine. An abrupt decrease in viscosity of the reaction mixture suggested an endowise cleavage of chitosan by this enzyme.     

Hyphal wall composition inApodachlyella completa

Mechanically isolated hyphal walls of the oomyceteApodachlyella completa (Humphrey) Indoh. (Leptomitales) were investigated by means of biochemical, cytochemical, and X-ray analyses. Microscopic examination of the cell wall preparations revealed them to be free of cytoplasmic contaminants β-Glucans account for 88% of wall weight and contain predominantly I→3 and I→6 linkages. Cellulose was demonstrated cytochemically and by X-ray diffraction and accounts for approximately 6% of wall dry weight. Additionally, walls contain 5% insoluble hexosamine, 4.8% protein, 1.1% lipid, and 0.5% ash. Chitin was qualitatively detected by means of cytochemical and X-ray analysis. The cell wall composition ofA. completa is similar to that reported for other oomycetes, in that β-glucans are the primary wall constituents. However, this fungus shows the unusual feature of chitin occurring simultaneously with cellulose, as do the closely related speciesApodachlya sp. andLeptomitus lacteus. *** DIRECT SUPPORT *** A01R4022 00007     

CELL WALL AND PEPTIDOGLYCAN FROM Lactobacillus fermenti

Cell walls from Lactobacillus fermenti were prepared by differential centrifugation of disrupted cells, with and without trypsin treatment. Approximately 16% of the dry weight of walls was found in a crude trichloroacetic acid extract of the walls; half of this amount remained upon further purification. The purufied extract lacked alanine, but contained substantial amounts of glucosamine. The walls constituted 23 to 33% of the dry weight of the cell. The chemical composition of the various types of wall preparations and of the peptidoglycan from them was studied. The peptidoglycan contained equimolar proportions of glucosamine, muramic acid, l-alanine, d-glutamic acid, and lysine, with somewhat lower proportions of d-aspartic acid and d-alanine. The chemical composition of the peptidoglycan is similar to that reported for three other lactobacilli. In addition to the major constituents of walls and peptidoglycan, there were several minor amino acids. The protein and the amounts of the minor amino acids decreased, and among these threonine and arginine were completely absent from preparations obtained with trypsin. Such preparations contained higher proportions of the d-isomers of alanine, glutamic acid, and aspartic acid as compared to walls and peptidoglycan prepared without trypsin. In addition, walls isolated with the use of trypsin were susceptible to lysozyme, whereas those prepared without trypsin were not. However, the trypsin treatment did not result in any change of the ultrastructure as revealed by electron microscope studies.