The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion‐exchange chromatography, could be determined accurately since, xylem‐specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.     

Environmental and biofilm-dependent changes in a Bacillus cereus secondary cell wall polysaccharide

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(β1-3)]GlcNAc(β1-6)[Glc(β1-3)][ManNAc(α1-4)]GalNAc(α1-4)ManNAc(β1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm.     

Cell wall proteomics of sugarcane cell suspension cultures

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl₂ and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second‐generation ethanol production.     

Xylogenesis in tissue culture II: Microtubules,cell shape and secondary wall patterns

Summary InZinnia suspension cultures, two general categories of tracheary element (TE) secondary wall patterns can be distinguished: bands and webs. Band patterns are found in elongated cells or regions of cells, web patterns in isodiametric cells or regions of cells. Interphase cortical microtubule arrays, organized before overt differentiation occurs, determine both the shape of the cell and whether band or web patterns will be deposited at the time of TE formation. By altering cell shape and consequently also altering the interphase microtubule array, it is possible to control the type of wall pattern which is deposited.These results provide support for the hypothesis which states that the organization of interphase cortical microtubule arrays (i.e., random or parallel), which laterally associate during tracheary element differentiation, determines the pattern in which secondary walls will be deposited.     

Role of net charge on catalytic domain and influence of cell wall binding domain on bactericidal activity, specificity, and host range of phage lysins

The recombinant lysins of lytic phages, when applied externally to Gram-positive bacteria, can be efficient bactericidal agents, typically retaining high specificity. Their development as novel antibacterial agents offers many potential advantages over conventional antibiotics. Protein engineering could exploit this potential further by generating novel lysins fit for distinct target populations and environments. However, access to the peptidoglycan layer is controlled by a variety of secondary cell wall polymers, chemical modifications, and (in some cases) S-layers and capsules. Classical lysins require a cell wall-binding domain (CBD) that targets the catalytic domain to the peptidoglycan layer via binding to a secondary cell wall polymer component. The cell walls of Gram-positive bacteria generally have a negative charge, and we noticed a correlation between (positive) charge on the catalytic domain and bacteriolytic activity in the absence of the CBD (nonclassical behavior). We investigated a physical basis for this correlation by comparing the structures and activities of pairs of lysins where the lytic activity of one of each pair was CBD-independent. We found that by engineering a reversal of sign of the net charge of the catalytic domain, we could either eliminate or create CBD dependence. We also provide evidence that the S-layer of Bacillus anthracis acts as a molecular sieve that is chiefly size-dependent, favoring catalytic domains over full-length lysins. Our work suggests a number of facile approaches for fine-tuning lysin activity, either to enhance or reduce specificity/host range and/or bactericidal potential, as required.     

Ethanol yields and cell wall properties in divergently bred switchgrass genotypes

Genetic modification of herbaceous plant cell walls to increase biofuels yields is a primary bioenergy research goal. Using two switchgrass populations developed by divergent breeding for ruminant digestibility, the contributions of several wall-related factors to ethanol yields was evaluated. Field grown low lignin plants significantly out yielded high lignin plants for conversion to ethanol by 39.1% and extraction of xylans by 12%. However, across all plants analyzed, greater than 50% of the variation in ethanol yields was attributable to changes in tissue and cell wall architecture, and responses of stem biomass to dilute-acid pretreatment. Although lignin levels were lower in the most efficiently converted genotypes, no apparent correlation were seen in the lignin monomer G/S ratios. Plants with higher ethanol yields were associated with an apparent decrease in the lignification of the cortical sclerenchyma, and a marked decrease in the granularity of the cell walls following dilute-acid pretreatment.     

Buckling of inner cell wall layers after manipulations to reduce tensile stress: observations and interpretations for stress transmission

The inner layer of the cell wall in tissues that are under tensile stress in situ, e.g. epidermis and collenchyma of etiolated sunflower hypocotyls, shows a pattern of transverse folds when the tissues are detached and plasmolysed. This can be observed by Nomarski imaging of inner surfaces of the outer cell walls and electron microscopy of longitudinal sections after peeling the epidermis and bathing it in plasmolysing solutions. The folds are apparently caused by buckling of the inner layer due to the longitudinal compressive force exerted on this layer by the outer wall layer, when it shrinks after the removal of the longitudinal tensile stresses. In these stresses, two components can be distinguished: the tissue stress, disappearing on peeling, and that caused directly by turgor pressure, disappearing in hyperosmotic solution. Investigation of the buckling indicates that the outer layer of the cell wall transmits in situ most of the longitudinal tensile stress in the wall. The common concept that the inner layer of the wall is the region bearing most stress and therefore regulating growth can still be valid with respect to the transverse stress component.     

Unique N-glycan moieties of the 66-kDa cell wall glycoprotein from the red microalga Porphyridium sp

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man(8-9)Xyl(1-2)Me(3)GlcNAc(2). The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins.     

Yeast cell attachment: a tool modulating wall composition and resistance to 5-bromo-6-azauracil

The attachment of Candida utilis, Kluyveromyces lactis, and Saccharomyces cerevisiae cells stimulates an increase in the content of cell wall polysaccharides and mannoproteins, accompanied by increased resistance to the inhibitory effect of 5-bromo-6-azauracil. The covalent attachment of viable yeasts was accomplished (via dialdehyde-amino spacers) by reaction of aldehyde groups of the carrier with reactive amino groups in accessible cell surface proteins. The employed technique enables the optimization of yeast sources of β-1,3-, β-1,6- glucans, mannan, and mannoprotein. The modulatory effect of the cell attachment is discussed.     

The cell wall proteins of Clostridium difficile

Abstract The proteins which can be released by 6 M urea treatment from the cell walls of Clostridium difficile represent the major cell surface proteins. In the 5 strains examined there are one to three of these major proteins. They appear to be strain-specific antigens being detected in immunoblots only with homologous antiserum. A common cell-surface protein of M _r 73 kDa has been identified as a minor component of the urea extract.     

Microtubules do not control orientation of secondary cell wall microfibril deposition inMicrasterias

Summary The influence of the microtubule disorganizing substances amiprophos-methyl (APM) and colchicine on secondary wall formation inMicrasterias denticulata was investigated by the freezeetch technique. The results reveal that neither microtubule inhibitor changes the pattern of microfibril deposition. The application of APM or colchicine also does not cause any structural alterations of the microfibrils or of the protoplasmic (Pf) and the exoplasmic (Ef) fracture face of the plasma membrane, thus indicating that microtubules are not involved in secondary wall formation inM. denticulata. However, since areas of the plasma membrane which collapsed upon freeze-etching are restricted to the Pf-face of cells treated with microtubule inhibitors, cortical microtubules may function as mechanical support during secondary wall formation. In the cortical cytoplasm filamentous structures are found in close spatial relationship and an almost parallel alignment to rosettes of the plasma membrane.     

The effect of strain rate on the failure stress and toughness of bone of different mineral densities

The risk of low energy fracture of the bone increases with age and osteoporosis. This paper investigates the effect of strain rate and mineral level on the peak stress and toughness of whole ovine bones.     

Scaffoldin Conformation and Dynamics Revealed by a Ternary Complex from the Clostridium thermocellum Cellulosome

Cellulosomes are multienzyme complexes responsible for efficient degradation of plant cell wall polysaccharides. The nonenzymatic scaffoldin subunit provides a platform for cellulolytic enzyme binding that enhances the overall activity of the bound enzymes. Understanding the unique quaternary structural elements responsible for the enzymatic synergy of the cellulosome is hindered by the large size and inherent flexibility of these multiprotein complexes. Herein, we have used x-ray crystallography and small angle x-ray scattering to structurally characterize a ternary protein complex from the Clostridium thermocellum cellulosome that comprises a C-terminal trimodular fragment of the CipA scaffoldin bound to the SdbA type II cohesin module and the type I dockerin module from the Cel9D glycoside hydrolase. This complex represents the largest fragment of the cellulosome solved by x-ray crystallography to date and reveals two rigid domains formed by the type I cohesin·dockerin complex and by the X module-type II cohesin·dockerin complex, which are separated by a 13-residue linker in an extended conformation. The type I dockerin modules of the four structural models found in the asymmetric unit are in an alternate orientation to that previously observed that provides further direct support for the dual mode of binding. Conserved intermolecular contacts between symmetry-related complexes were also observed and may play a role in higher order cellulosome structure. SAXS analysis of the ternary complex revealed that the 13-residue intermodular linker of the scaffoldin subunit is highly dynamic in solution. These studies provide fundamental insights into modular positioning, linker flexibility, and higher order organization of the cellulosome.     

Relationships between spatial configuration of tropical forest patches and woody plant diversity in northeastern Puerto Rico

The destruction and fragmentation of tropical forests are major sources of global biodiversity loss. A better understanding of anthropogenically altered landscapes and their relationships with species diversity and composition is needed in order to protect biodiversity in these environments. The spatial patterns of a landscape may control the ecological processes that shape species diversity and composition. However, there is little information about how plant diversity varies with the spatial configuration of forest patches especially in fragmented tropical habitats. The northeastern part of Puerto Rico provides the opportunity to study the relationships between species richness and composition of woody plants (shrubs and trees) and spatial variables [i.e., patch area and shape, patch isolation, connectivity, and distance to the Luquillo Experimental Forest (LEF)] in tropical forest patches that have regenerated from pasturelands. The spatial data were obtained from aerial color photographs from year 2000. Each photo interpretation was digitized into a GIS package, and 12 forest patches (24–34 years old) were selected within a study area of 28 km². The woody plant species composition of the patches was determined by a systematic floristic survey. The species diversity (Shannon index) and species richness of woody plants correlated positively with the area and the shape of the forest patch. Larger patches, and patches with more habitat edge or convolution, provided conditions for a higher diversity of woody plants. Moreover, the distance of the forest patches to the LEF, which is a source of propagules, correlated negatively with species richness. Plant species composition was also related to patch size and shape and distance to the LEF. These results indicate that there is a link between landscape structure and species diversity and composition and that patches that have similar area, shape, and distance to the LEF provide similar conditions for the existence of a particular plant community. In addition, forest patches that were closer together had more similarity in woody plant species composition than patches that were farther apart, suggesting that seed dispersal for some species is limited at the scale of 10 km.     

Structure and conservation of the periplasmic targeting factor Tic22 protein from plants and cyanobacteria

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.