Carbohydrate engineering of the recognition motifs in streptococcal co-aggregation receptor polysaccharides

The cell wall polysaccharides of certain oral streptococci function as receptors for the lectin-like surface adhesins on other members of the oral biofilm community. Recognition of these receptor polysaccharides (RPS) depends on the presence of a host-like motif, either GalNAcbeta1-3Gal (Gn) or Galbeta1-3GalNAc (G), within the oligosaccharide repeating units of different RPS structural types. Type 2Gn RPS of Streptococcus gordonii 38 and type 2G RPS of Streptococcus oralis J22 are composed of heptasaccharide repeats that are identical except for their host-like motifs. In the current investigation, the genes for the glycosyltransferases that synthesize these motifs were identified by high-resolution nuclear magnetic resonance (NMR) analysis of genetically altered polysaccharides. RPS production was switched from type 2Gn to 2G by replacing wefC and wefD in the type 2Gn gene cluster of S. gordonii 38 with wefF and wefG from the type 2G cluster of S. oralis J22. Disruption of either wefC or wefF abolished cell surface RPS production. In contrast, disruption of wefD in the type 2Gn cluster or wefG in the type 2G cluster eliminated beta-GalNAc from the Gn motif or beta-Gal from the G motif, resulting in mutant polysaccharides with hexa- rather than heptasaccharide subunits. The mutant polysaccharides reacted like wild-type RPS with rabbit antibodies against type 2Gn or 2G RPS but were inactive as co-aggregation receptors. Additional mutant polysaccharides with GalNAcbeta1-3GalNAc or Galbeta1-3Gal recognition motifs were engineered by replacing wefC in the type 2Gn cluster with wefF or wefF in the type 2G cluster with wefC respectively. The reactions of these genetically modified polysaccharides as antigens and receptors provide further insight into the structural basis of RPS function.     

Lectin microarray reveals binding profiles of Lactobacillus casei strains in a comprehensive analysis of bacterial cell wall polysaccharides

We previously showed a pivotal role of the polysaccharide (PS) moiety in the cell wall of the Lactobacillus casei strain Shirota (YIT 9029) as a possible immune modulator (E. Yasuda M. Serata, and T. Sako, Appl. Environ. Microbiol. 74:4746-4755, 2008). To distinguish PS structures on the bacterial cell surface of individual strains in relation to their activities, it would be useful to have a rapid and high-throughput methodology. Recently, a new technique called lectin microarray was developed for rapid profiling of glycosylation in eukaryotic polymers and cell surfaces. Here, we report on the development of a simple and sensitive method based on this technology for direct analysis of intact bacterial cell surface glycomes. The method involves labeling bacterial cells with SYTOX Orange before incubation with the lectin microarray. After washing, bound cells are directly detected using an evanescent-field fluorescence scanner in a liquid phase. Using this method, we compared the cell surface glycomes from 16 different strains of L. casei. The patterns of lectin-binding affinity of most strains were found to be unique. There appears to be two types of lectin-binding profiles: the first is characterized by a few lectins, and the other is characterized by multiple lectins with different specificities. We also showed a dramatic change in the lectin-binding profile of a YIT 9029 derivative with a mutation in the cps1C gene, encoding a putative glycosyltransferase. In conclusion, the developed technique provided a novel strategy for rapid profiling and, more importantly, differentiating numerous bacterial strains with relevance to the biological functions of PS.     

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.     

Glycerolphosphate-containing cell wall polysaccharides from Streptococcus sanguis

Six glycerolphosphate-containing tetraheteroglycans, a, b-1, b-2, b-3, b-4, and b-5, have been purified from the formamide extracts of Streptococcus sanguis by alcohol and acetone precipitations, Sephadex G-75, and diethylaminoethyl-cellulose column chromatography. The polysaccharides were judged as at least 95% pure by analytical disc gel electrophoresis and immune double diffusion against rabbit antiserum. They were shown to be cell wall polysaccharides, since they formed a single band of identity in immune double diffusion with partially purified polysaccharide extracted from a purified cell wall preparation of S. sanguis. The polysaccharides were composed of l-rhamnose, d-glucose, and N-acetyl d-glucosamine in a similar molar ratio, but varied in their glycerol and phosphate contents. They exhibited four different mobilities in polyacrylamide disc gel electrophoresis at pH 8.9. When they were treated with formamide at 170 C for 20 min, the faster moving polysaccharide(s) yielded polysaccharides with mobilities corresponding to the other slower moving polysaccharides. These results indicate that the polysaccharides originated from the same cell wall polysaccharide and were produced as a result of breakage in the phosphodiester bonds during the formamide extraction procedure. A preliminary structural study shows that the terminal reducing sugar is l-rhamnose and that the glycerol moiety is probably linked to the polysaccharide through a phosphodiester bond.     

Changes in cell wall polysaccharides of germinating barley grains

Decorticated barley grains were germinated at 25° for 6 days, until the endosperm reserves were nearly exhausted. The neutral monosaccharide components of the hydrolysates of the cell walls and gums from the embryo, aleurone layer and starchy endosperm and the endospermic starch were determined at daily intervals. The amount of embryo cell wall polysaccharide increased 40 times and glucose became the major component, followed in abundance by xylose and arabinose. The cell wall and gum polysaccharides of the aleurone layer (plus testa) and the starchy endosperm declined during germination and their compositions altered. The endospermic starch also decreased. In the early stages of germination the apparent composition of the cell walls of the aleurone layer and starchy endosperm depended upon how they had been prepared. After 6 days the cell walls and gums had provided a significant carbohydrate supply to the living tissues, equivalent to 18.5% of the endospermic polysaccharide degraded during growth, starch having provided the remaining 81.5%.     

Secretion of cell wall polysaccharides in Vicia root hairs

Root hairs of hairy winter vetch ( Vicia villosa Roth) synthesize and secrete abundant cell wall matrix polysaccharides, making this an excellent system for the study of secretion during tip growth. Roots with newly formed hairs were preserved by cryofixation and freeze substitution. Cryofixed root hairs showed excellent structural and antigenic preservation. Ultrastructural analyses showed numerous vesicles near the tip and a concentration of Golgi bodies in the subapical region of the hair. The distribution of polygalacturonic acid and xyloglucan in the endomembrane system and cell wall were revealed by immunolabeling, using previously characterized monoclonal antibodies. De-esterified polygalacturonic acid was present on the external surface of the cell wall, but was not detected within the cell, although chemical de-esterification revealed abundant antigen in Golgi bodies and secretory vesicles. Methyl-esterified polygalacturonic acid epitopes were detected within the medial and trans cisternae of Golgi bodies, in secretory vesicles, and throughout the wall, indicating that pectin is secreted in a neutral form and may then be de-esterified in muro . Xyloglucan was also detected within the trans cisternae of Golgi bodies, secretory vesicles and throughout the cell wall. Double labeling experiments demonstrated that both polysaccharides occur simultaneously in the same Golgi bodies, and that secretory vesicles containing both polygalacturonic acid and xyloglucan deliver the polysaccharides to the cell wall at the growing tip.     

The specific nature of plant cell wall polysaccharides

Polysaccharide compositions of cell walls were assessed by quantitative analyses of the component sugars. Cell walls were hydrolyzed in 2 n trifluoroacetic acid and the liberated sugars reduced to their respective alditols. The alditols were acetylated and the resulting alditol acetates separated by gas chromatography. Quantitative assay of the alditol acetates was accomplished by electronically integrating the detector output of the gas chromatograph. Myo-inositol, introduced into the sample prior to hydrolysis, served as an internal standard.     

Modification of cell wall polysaccharides during cell elongation in Phaseolus vulgaris hypocotyls

The effect of gibberellic acid (GA) and naphthylacetic acid (NAA) on hypocotyl elongation and cell wall polysaccharides was studied using Phaseolus vulgaris seedlings grown in light condition. The hypocotyl was demarcated into two segments — one near the root was called lower and the one near the cotyledon was called upper. The upper segment showed a typical sigmoidal growth curve while lower segment did not show any growth at all. GA promoted the growth of upper segment while NAA showed clear inhibition in both the segments. Xyloglucan content showed a clear inverse correlation with growth. Pectic polysaccharides did not show a clear trend, though showed an initial inverse correlation with growth. It is concluded that degradation of low and high molecular weight xyloglucans are involved in cell wall loosening which in turn may be responsible for the elongation growth of Phaseolus hypocotyls in light.     

Differences in cell wall polysaccharides of several species of Eupenicillium

Abstract A β-(1–5)-galactofuran was isolated and characterized from fraction F1S (alkali- and water-soluble) of the cell wall of most of the species of Eupenicillium . In E. cryptum, E. euglaucum and E. nepalense the galactan contained galactofuranose with different linkages in addition to β-(1–5). Fraction F1I (alkali-soluble, water-insoluble) was an α-glucan in certain species while in other it was a =gb-glucan. Xylose was detected in some species in F1I or in F3 (alkali-soluble at 70°C). The most abundant fraction (F4), resistant to the alkali treatment, was a β-glucan-chitin complex. Excepting this component, the β-(1–5)-galactofuran was the polysaccharide which appeared more frequently in the cell wall of species of Eupencillium and it may have chemotaxonomic relevance.     

Turnover of cell wall polysaccharides in elongating pea stem segments

Turnover of cell wall polysaccharides and effects of auxin thereon were examined after prelabeling polysaccharides by feeding pea (Pisum sativum var. Alaska) stem segments ¹⁴C-glucose, then keeping the tissue 7 hours in unlabeled glucose with or without indoleacetic acid. There followed an extraction, hydrolysis, and chromatography procedure by which labeled monosaccharides and uronic acids were released and separated with consistently high recovery. Most wall polymers, including galacturonan and cellulose, did not undergo appreciable turnover. About 20% turnover of starch, which normally contaminates cell wall preparations but which was removed by a preliminary step in this procedure, occurred in 7 hours. Quantitatively, the principal wall polymer turnover process observed was a 50% decrease in galactose in the pectinase-extractable fraction, including galactose attached to a pectinase-resistant rhamnogalacturonan. Other pectinase-resistant galactan(s) did not undergo turnover. No turnover was observed in arabinans, but a doubling of radioactivity in arabinose of the pectinase-resistant, hot-acid-degradable fraction occurred in 7 hours, possibly indicating conversion of galactan into arabinan. None of the above changes was affected by indoleacetic acid, but a quantitatively minor turnover of a pectinase-degradable xyloglucan was found to be consistently promoted by indole-acetic acid. This was accompanied by a reciprocal increase in water-soluble xyloglucan, suggesting that indoleacetic acid induces conversion of wall xyloglucan from insoluble to water-soluble form. The results indicate a highly selective pattern of wall turnover processes with an even more specific influence of auxin.     

Rice cell wall polysaccharides: Structure and biosynthesis

Rice is the most widely consumed staple food, and is cultivated worldwide to satisfy our daily caloric needs. Thus, extensive efforts on rice breeding and biotechnology have substantially focused on the development of elite cultivars with high yields and better grain quality, as well as enhanced resistance against biotic and abiotic stresses. Recently, it has been observed that rice is more than a just grain-producing crop. Carbon-rich materials of the rice cell wall polysaccharides from post-harvest wastes, including the straw and husk, have been converted into bioethanol and other invaluable, renewable materials. In order to maximize the utilization of cell wall-derived resources, it is imperative to understand cell wall chemistry and molecular mechanism underlying cell wall biosynthesis in rice. In the last decade, several approaches, including mutational genetics and the functional characterization of candidate genes, have been successful in isolating some of cell wall biosynthetic genes in rice, marking the first step forward in obtaining a complete understanding of rice cell wall biosynthesis, although the exact biochemical functions have not been conclusive. In this paper, we focus on integrating old and new information to provide an updated perspective in the cell wall formation of rice, highlighting the chemical structures and biosynthesis of rice cell wall polysaccharides.     

Composition of the cell wall polysaccharides in some geophilic dermatophytes

The main polysaccharide fractions from cell wall material of several geophilic dermatophyte species were characterized as a glucomannan (F1S) which amounted to 4.0–6.5% and a glucan-chitin complex representing 44.2–71.0%. The neutral sugar content of fraction F1S in these species was mannose (38.7–78.2%), galactose (0.3–7.3%) and glucose (3.2–8.2%) except inM. fulvum (21.9%) andE. stockdaleae (12.5%). Small proportions of xylose, about 1%, were found in this fraction except inM. fulvum which reached 7.8% and inM. nanum which lacked xylose. The main products detected after Smith degradation were glycerol and glucose. From fraction F1S ofM. fulvum a glucan (18.3%) and a mannan (41.5%) were obtained. These two polysaccharides could be used as chemotaxonomic characters for the definition of this group of fungi.     

Enzymatic degradation of cell wall and related plant polysaccharides

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.     

Genetic engineering of grass cell wall polysaccharides for biorefining

Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass‐derived lignocellulose processing industry.     

Enzymatic degradation of cell wall polysaccharides from soybean meal

Soybean meal, soybean water unextractable solids (WUS) and extracts thereof, which contain particular cell wall polysaccharides, were incubated with a number of cell wall degrading enzymes. The intact cell wall polysaccharides in the meal and WUS were hardly degradable, while the extracts from WUS were well degraded. The arabinogalactan side chains in the pectin-rich ChSS fraction (Chelating agent Soluble Solids) could to a large extent be removed from the pectins by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase and arabinofuranosidase B. The remaining polymer was isolated and represented 30% of the polysaccharides in the ChSS fraction. Determination of the sugar composition showed these polymers to be very highly substituted pectic structures. It still contained 5 mol% of arabinose and 12 mol% of galactose, representing 7% and 12%, respectively, of the arabinose and galactose present in the ChSS fraction before degradation. Further, the presence of uronic acid (50 mol%) and of xylose (18 mol%) indicated the presence of a xylogalacturonan.     

Stimulation of TNF-alpha release by fungal cell wall polysaccharides

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.     

Dynamic changes in cell wall polysaccharides during wheat seedling development

Changes in arabinoxylan content and composition during development of wheat seedlings were investigated. The cell walls isolated from the seedlings showed an increasing content of arabinoxylan during development, which could be correlated to increased activity of xylan synthase and arabinoxylan arabinosyltransferase. Arabinoxylan changed from initially having a high degree of arabinose substitution to a much lower degree of substitution. beta-Glucan was present in the walls at the early stages of development, but was actively degraded after day 4. Increased deposition of arabinoxylan did not take place until beta-glucan had been fully degraded. Ferulic and p-coumaric acid esters were present at all points but increased significantly from day 3 to 6, where lignification began. Ferulic acid dimers did not appear in the cell wall until day three and the different ferulic acid dimers varied in the course of accumulation. The ratio of ferulic acid dimers to free ferulic acid was maximal at the time when the wall had been depleted for beta-glucan, which had not yet been fully replaced by arabinoxylan. This pattern suggests a role for ferulic acid dimers in stabilizing the wall during the transition from a flexible to a more rigid structure. To investigate if the same changes could be observed within a single seedling, 7 day old seedlings were divided into four sections and the walls were analyzed. Some of the changes observed during the seedling development could also be observed within a single seedling, when analyzing the segments from the elongation zone at the base to the top of the leaf. However, the expanding region of older seedlings was much richer in hydroxycinnamates than the expanding region of younger seedlings. Diferulic acids are stabilizing the wall in the transition phase from an expanding to a mature wall. This transition can take place in different manners depending on the cell and tissue type.