Mechanisms of biosorption of different heavy metals by brown marine macroalgae

The biosorption mechanisms of different heavy metallic cations (Cd, Ni, Pb) to active chemical groups on the cell wall matrix of the nonliving brown marine macroalga, Sargassum vulgaris in its natural form, were examined by the following instrumental and chemical techniques: Fourier-transform infrared (FTIR) analysis, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and extraction of alginic acid and sulfated polysaccharides, which act as metal-binding moieties present in cell wall. From the different techniques used and the known chemical composition of the algal cell wall, it was observed that biosorption of the metallic cations to the algal cell wall component was a surface process. The binding capacities of the different metal cations were between 1 and 1.2 mmol metal/g on a dry weight basis. The main chemical groups involved in the metallic cation biosorption were apparently carboxyl, amino, sulfhydryl, and sulfonate. These groups were part of the algal cell wall structural polymers, namely, polysaccharides (alginic acid, sulfated polysaccharides), proteins, and peptidoglycans. The main cadmium cation sequestration mechanism by the algal biomass was apparently chelation, while the nickel cation sequestration mechanism was mainly ion exchange. Lead cations exhibit higher affinity to the algal biomass, and their binding mechanism included a combination of ion exchange, chelation, and reduction reactions, accompanied by metallic lead precipitation on the cell wall matrix. During the ion exchange process, calcium, magnesium, hydrogen cations, and probably other cations (sodium and potassium) in the algal cell wall matrix were replaced by the tested heavy metals.     

Studies on the Cell Wall of Piricularia oryzae†

The chemical constituents of the cell wall of Piricularia oryzae, the pathogenic fungus of rice blast disease, were studied with the aids of chemical analysis, X-ray diffraction, infra-red absorption and enzymatic degradation. The sugar constituents were identified by chromatography as glucose (62%), mannose (4%), galactose (0.5%), and hexosamine (13%). The acidic amino acid rich protein was comprised 4.6% in the cell wall. The cell wall consists of at least three different polysaccharide complexes: a) α-Heteropolysaccharide protein complex containing mannose, glucose and galactose, b) β-1,3-Glucan containing β-1, 6-linked branch, c) Chitin like substance.     

细叶黄芪叶肉原生质体发育早期细胞壁再生的研究

采用透射电镜术、电镜多糖细胞化学染色、细胞壁荧光染色以及香豆素抑制细胞壁再生等方法,对细叶黄芪（Ａｓｔｒａｇａｌｕｓｍ ｅｌｉｌｏｔｏｉｄｅｓ ｖａｒ．ｔｅｎｕｉｓ）叶肉原生质体细胞壁的再生及其化学特点进行了研究。结果表明,离体培养２４ 小时的原生质体表面产生一些突起小泡,有时可见少量纤维组分的形成。培养３ 天时这种纤维组分明显增多。至５ 天时可清楚看到再生壁是由纤维和颗粒构成。六亚甲四胺银染色证明它们都是由多糖组分组成的。另外,培养３６ 小时的原生质体有相互粘连的现象。电镜观察、荧光染色及香豆素处理的研究表明粘连与再生壁的形成有关。根据上述观察结果,对原生质体再生壁的结构及其化学性质等问题进行了讨论     

Cell Wall Regenration During Early Development of Mesophyll Protoplasts of Astragalus melilotoides var. tenuis

The techniques of transmission electron microscopy (TEM), electron microscopy (EM) cytochemical visualization of polysaccharide, cell wall flourescence labelling of cell wall and inhibition of wall formation by coumarin treatment were used to explore the cell wall regeneration and its chemical characteristics in mesophyll protoplasts of Astragalus melilotoides var. tenuis. The results showed that after 24 h in culture a number of protruding vesicles, as well as a small amount of fibrillar component were formed on the surface of protoplasts. On day 3, the amount of fibrils increased significantly. On day 5, regenerated primary wall composed of fibrils and granules were observed, in which polysacchaides were detected as result of the periodic acid-silver methenamine reaction. In addition, after 36 h in culture, the protoplasts tended to coalesce, flourescence staining and coumarin treatment demonstrated that the protoplast adhesion was the result of cell wall formation. Based on these data, problems such as the structure of regenerated wall and its chemical nature, etc. were discussed.     

Genetic control of a structural polymer of the Neurospora crassa cell wall.

The heteropolysaccharide present in fraction 1 of the Neurospora crassa cell wall has been characterized in wild-type and morphological mutant strains of this fungus. Single and double mutations have been studied to determine possible genetic interactions controlling the chemical composition of such heteropolysaccharides . Single mutations studied were peak-2, scumbo ( FGSC 49), ragged ( FGSC 296), and crisp -1 ( FGSC 488). Double mutations studied were peak-2, scumbo ( FGSC 419), and ragged crisp -1. In all these strains, the main constituents of the heteropolysaccharide were glucose, mannose and galactose. Glycosidic linkages binding these neutral sugars have been identified by gas-liquid chromatography. A chemical structure of fraction I heteropolysaccharide is proposed. The results obtained with double mutants suggest the existence of genetic interactions, such as complementation or additive effects of lesions of different genes, to control the chemical composition and structure of the cell wall and the morphology of N. crassa mycelium.     

Fourier transform infrared-spectroscopic characterisation of isolated endodermal cell walls from plant roots: chemical nature in relation to anatomical development

The chemical nature of enzymatically isolated endodermal cell walls from Cicer arietinum L., Clivia miniata Reg. and Iris germanica L. was studied by FTIR (Fourier transform infrared) spectroscopy. Observed frequencies were assigned to functional groups present in the cell wall and relative amounts of the biopolymers suberin and lignin, cell wall carbohydrates and proteins were determined. Infrared absorption spectra indicated structural characteristics for the three different developmental states of the isolated endodermal cell wall: primary endodermis with Casparian strips (state I), secondary endodermis with suberin lamellae (state II), and tertiary endodermis with U-shaped cell wall depositions (state III). The data obtained from this study are compared with previous results obtained by chemical degradation of isolated endodermal cell walls and subsequent determination of monomeric degradation products by gas chromatography and mass spectrometry. It is concluded that FTIR spectroscopy represents a direct and nondestructive method suitable for the rapid investigation of isolated plant cell walls. Furthermore, the observation that the suberin-assigned absorption bands disappeared after transesterification of the samples with BF₃-methanol confirmed that suberin is completely degraded by this treatment. Received: 20 February 1999 / Accepted: 25 May 1999     

Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells

Plant cell walls serve several functions: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.     

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.     

Cell wall functions in growth and development —a physical and chemical point of view

The plant cell changes its cell wall architecture during growth and development through synthesis and degradation of wall polysaccharides. Changes of chemical components in the cell wall include not only the synthesis and degradation but also the shift of molecular-weight distribution of certain species of the component polysaccharides. The changes in chemical structure, in turn lead to alteration of physical properties of the cell wall. Changes of physical parameters of cell walls obtained by a physical method accord with the biochemical degradation of polysaccharides. The changes in chemical structures of the cell wall are regulated by plant hormones, stress signals and gene expression. The physical and chemical studies of the cell wall have disclosed that degradation and/or depolymerization of wall polysaccahrides causes decrease in viscosity of the cell wall, leading further extension of the cell wall even under the unchanged osmotic relation. Furthermore, cell walls of outer and inner tissues play different regulatory roles in tissue growth and stem strength was governed by the number of cellulose molecules in the cell wall. Recipient of the Botanical Society Award for Young Scientists, 1990.     

Studies of the cell walls of Schizophyllum commune

Mechanically isolated cell wall materials of eight strains of Schizophyllum commune were studied by chemical and enzymatic procedures. Isolated wall material of each strain was separated by chemical methods into three fractions: A (cold alkali-soluble, , amorphous), B (warm alkali-soluble, amorphous), and C (alkali-insoluble, retaining appearance of hyphal fragments). Chemical tests indicated the presence of chitin in Fraction C and the absence of cellulose, lignin and pectic substances from all fractions. Analyses of acid hydrolysates indicated the presence of glucose in Fractions A, B and C, of hexosamine in Fraction C and the absence of galactose, mannose, 6-deoxyhexoses, xylose and other pentoses from all fractions. Unfractionated material, Fraction A and Fraction B were slightly attacked by commercial cellulase whereas Fraction C was heavily attacked. Commercial chitinase by itself did not attack Fraction C or unfractionated material to a significant extent. In the presence of cellulase, it was active upon Fraction C. Qualitative differences in cell wall composition between strains were not detected; however, quantitative differences were observed in the proportion of Fraction A and Fraction C as well as in the amount of the various breakdown products in certain strains. It is visualized that the cell wall of this fungus consists of a core of chitin covered by or intermeshed with glucose-containing polymers.     

Penicillin-induced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurence of subsequent bacteriolysis at different drug concentrations. The results are not consistent with hypotheses explaining penicillin-induced lysis as a result of an overall weakened cell wall structure or an overall activation of autolytic wall enzymes but not conflicting with the model that ascribes penicillin-induced bacteriolysis as the result of a very restricted, local perforation of the peripheral cell wall (murosome-induced bacteriolysis).Abbreviations CL Cross-linking - DNFB 2,4-dinitro-1-fluorobenzole - MIC Minimal inhibitory concentration - OD Optical density at 578 nm - PEN Penicillin G     

Characterization of Solubilized Products from the Cell Wall of Streptococcus pneumoniae during Autoplast Formation

The structure of the cell wall of Streptococcus pneumoniae R36a was investigated by means of chemical analysis of the solubilized products formed during autoplast formation. By autoplast formation, almost all of the cell wall components were solubilized as the end products within several hours. Analysis of the solubilized products revealed that the pneumococcal cell wall consists of three macromolecular components, teichoic acid-glycopeptide I (TA-GP I), teichoic acid-glycopeptide II (TA-GP II), and glycopeptide (GP III). The molecular size of TA-GP I was larger than that of TA-GP II. TA-GP I and TA-GP II were constituted of similar components, galactosamine, 2-acetamido-4-amino-2, 4, 6-trideoxyhexose, glucose, ribitol, choline, phosphate, and peptidoglycan components, but the ratio of teichoic acid to glycopeptide in TA-GP II was higher than that in TA-GP I. TA-GP II was solubilized more slowly than TA-GP I and GP III during autoplast formation. The assembly of the cell wall by TA-GP I and II, and GP III is discussed in connection with the action of autolysin.     

d-canavanine affects peptidoglycan structure,morphogenesis and fitness in Rhizobiales

The bacterial cell wall is made of peptidoglycan (PG), a polymer that is essential for maintenance of cell shape and survival. Many bacteria alter their PG chemistry as a strategy to adapt their cell wall to external challenges. Therefore, identifying these environmental cues is important to better understand the interplay between microbes and their habitat. Here, we used the soil bacterium Pseudomonas putida to uncover cell wall modulators from plant extracts and found canavanine (CAN), a non-proteinogenic amino acid. We demonstrated that cell wall chemical editing by CAN is licensed by P. putida BSAR, a broad-spectrum racemase which catalyses production of dl -CAN from l -CAN, which is produced by many legumes. Importantly, d -CAN diffuses to the extracellular milieu thereby having a potential impact on other organisms inhabiting the same niche. Our results show that d -CAN alters dramatically the PG structure of Rhizobiales (e.g., Agrobacterium tumefaciens, Sinorhizobium meliloti), impairing PG crosslinkage and cell division. Using A. tumefaciens, we demonstrated that the detrimental effect of d -CAN is suppressed by a single amino acid substitution in the cell division PG transpeptidase penicillin binding protein 3a. Collectively, this work highlights the role of amino acid racemization in cell wall chemical editing and fitness.     

Fluorescence and infrared spectrometric study of cell walls from Candida, kluyveromyces, Rhodotorula and schizosaccharomyces yeasts in relation with their chemical composition

Composition, level, and arrangement of the structural polysaccharides determine biophysical properties of fungal cell walls. A small amount of a beta(1-->4) linear homopolymer of GlcNAc in the cell wall forms chitin. To study the components of the cell walls and to estimate the quantity of chitin for different strains, two spectroscopic methods were applied. Because chemical and enzymatic methods are destructive, long, and complex, fluorescence and infrared (IR) spectroscopies were applied on cell walls and on chitin enriched fractions. The results were compared to chemical assays. IR spectra allow identifying the structural types of polysaccharides in yeast walls. Fluorescence spectroscopy was not appropriated for a full and accurate quantitative determination of the polymers but revealed level variations similar to results obtained by chemical analytical methods. The infrared spectra, using a chemometric approach (PLS1), allowed a fairly good estimation of chitin in enriched fractions with respect to the chemical assays.     

Modification of chemical properties of cell walls by silicon and its role in regulation of the cell wall extensibility in oat leaves

Effects of silicon on the mechanical and chemical properties of cell walls in the second leaf of oat (Avena sativa L.) seedlings were investigated. The cell wall extensibility in the basal region of the second leaf was considerably higher than that in the middle and subapical regions. Externally applied silicon increased the cell wall extensibility in the basal region, but it did not affect the extensibility in the middle and subapical regions. The amounts of cell wall polysaccharides and phenolic compounds, such as diferulic acid (DFA) and ferulic acid (FA), per unit length were lower in the basal region than in the middle and subapical regions of the leaf, and silicon altered these amounts in the basal region. In this region, silicon decreased the amounts of matrix polymers and cellulose per unit length and of DFA and FA, both per unit length and unit matrix polymer content. Silicon treatment also lowered the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) in the basal region. In contrast, the amount of silicon in cell walls increased in response to silicon treatment in three regions. These results suggest that in the basal region, silicon reduces the net wall mass and the formation of phenolic acid-mediated cross-linkages between wall polysaccharides. Such modifications of wall architecture may be responsible for the silicon-induced increase in the cell wall extensibility in oat leaves.     

Approximate digestibility of fibre for a graminivorous caterpillar

Final instar Persectania ewingii (Westwood) (Lepidoptera: Noctuidae) were fed seedling Triticum aestivum L. for 2 days to determine the approximate digestibility of the cell wall and cell content fractions. Cell wall content was estimated using a micro-analytical neutral detergent fibre technique. Approximate digestibilites of neutral detergent fibre, neutral detergent solubles and dry matter were calculated for individuals and pooled samples. P. ewingii larvae digested a small but significant proportion of the fibre ingested (13–21%), higher than that previously reported for herbivorous insects. The micro-analytical and previously used macro-analytical techniques produced similar estimates of digestibility although both techniques have inherent shortcomings, the latter requiring the pooling of samplex and the former limiting the number of replicates during chemical analysis. Differences in the amount of larval frass collected during the feeding trial (corrected for consumption) explained much of the variation in digestibility values, while there were no effects of larval mass, overall consumption and total frass produced on digestibility estimates. These results confirm that plant cell contents are the major source of nutrients to larval Lepidoptera although there is some chemical disruption of the plant cell wall.     

Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl

The occurrence and chemical nature of the cross-links between cellulose microfibrils in outer epidermal cell walls in Pisum sativum cv. Alaska was investigated by rapid-freezing and deep-etching techniques coupled with chemical and enzymatic treatments. The cell wall in the elongating region of epidermal cells was characterized by the absence of the cross-links, while in the elongated region, the cell wall was characterized by the presence of cross-links. The cross-links remained in the cell wall of the elongated region after treatment with SDS electrophoresis sample buffer and treatment with 4% potassium hydroxide. After treatment with endo-1,4-beta-glucanase, which fragments xyloglucan, the cross-links were remarkably reduced from the cell wall of the elongated region. The endoglucanase treatment also reduced immunogold labeling of xyloglucan in the cell wall. The endoglucanase hydrolysate from the cell wall fraction of the elongated region gave spots of oligosaccharides in thin layer chromatography, which were identical to the spots of xyloglucan oligosaccharides produced by xyloglucanase from both the cell wall fraction and tamarind xyloglucan. These results indicate that the cross-links are made of xyloglucan. We discussed the possibility of cross-links involved in the control of mechanical properties of the cell wall.     

Induced Autolysis of Aspergillus oryzae (A. niger group): IV. Carbohydrates

The examination of substances formed during induced autolysis by Aspergillus niger was continued in this work, which dealt in particular with carbohydrates. The autolysate contained a large amount of d-glucose (14 to 20% dry wt) and traces of glycolic aldehyde, dihydroxyacetone, ribose, xylose, and fructose. It also contained glycopeptides (about 10% dry wt), which were split from the cell wall during autolysis and which differed from one another in their level of polymerization and their composition. They were constituted by glucose and mannose, glucose and galactose, or mannose, glucose, and galactose (mannose being the most abundant in this case), and amino acids (chiefly alanine, serine, glutamic acid, and aspartic acid). During autolysis, only a part of the cell wall was dissolved, since it retained its shape. Upon further chemical hydrolysis, it produced mostly glucose and glucosamine, and smaller amounts of mannose, galactose, and amino acids. Presumably, glucomannoproteins and glucogalactoproteins were present in the intact cell as a macromolecular complex, constituting, together with chitin, the major part of the cell wall of Aspergillus.     

Cell wall composition of ethambutol susceptible and resistant strains of Mycobacterium smegmatis ATCC 607

Changes in the cell wall that accompany acquisition of ethambutol (EMB) resistance ina single step mutant of Mycobacterium smegmatis ATCC 607 were analysed. Quantitative changes were seen in the chemical constituents of the cell wall of resistant cultures in comparison with EMB-susceptible M. smegmatis . Alterations in the binding of 1-anilinonaphthalene-8-sulphonate (ANS) were suggestive of structural changes in the cell surface.