Characterization of Miscanthus cell wall polymers

Efficient utilization of lignocellulosic Miscanthus biomass for the production of biochemicals, such as ethanol, is challenging due to its recalcitrance, which is influenced by the individual plant cell wall polymers and their interactions. Lignocellulosic biomass composition differs depending on several factors, such as plant age, harvest date, organ type, and genotype. Here, four selected Miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus, Miscanthus × giganteus, Miscanthus sinensis × Miscanthus sacchariflorus hybrid) were grown and harvested, separated into stems and leaves, and characterized for their non‐starch polysaccharide composition and structures, lignin contents and structures, and hydroxycinnamate profiles (monomers and ferulic acid dehydrodimers). Polysaccharides of all genotypes are mainly composed of cellulose and low‐substituted arabinoxylans. Ratios of hemicelluloses to cellulose were comparable, with the exception of Miscanthus sinensis that showed a higher hemicellulose/cellulose ratio. Lignin contents of Miscanthus stems were higher than those of Miscanthus leaves. Considering the same organs, the four genotypes did not differ in their Klason lignin contents, but Miscanthus × giganteus showed the highest acetylbromide soluble lignin content. Lignin polymers isolated from stems varied in their S/G ratios and linkage type distributions across genotypes. p‐Coumaric acid was the most abundant ester‐bound hydroxycinnamte monomer in all samples. Ferulic acid dehydrodimers were analyzed as cell wall cross‐links, with 8‐5‐coupled diferulic acid being the main dimer, followed by 8‐O‐4‐, and 5‐5‐diferulic acid. Contents of p‐coumaric acid, ferulic acid, and ferulic acid dimers varied depending on genotype and organ type. The largest amount of cell wall cross‐links was analyzed for Miscanthus sinensis.     

Mode of cell wall synthesis in gram-positive bacilli.

Ultrastructural experiments on plasmolyzed cells suggested that the information for the position and orderly synthesis of septa is not determined by the attachment of cell membrane to previously formed wall. These experiments, in conjunction with others on cells disrupted by the freeze-fracture technique, are most consistent with wall growth over the entire surface of the rods, with wall material gradually moving from a position next to the cell membrane to a position at the outer surface of the cell.     

Cross wall synthesis and the arrangement of the wall polymers in the cell wall of Staphylococcus spp

The growing process and the fine structure of the cross wall of Staphylococcus were investigated by electron microscopy. Examination of the tangentially sectioned cross wall revealed that it was initially synthesized as a thin cell wall layer by an invaginated cytoplasmic membrane. The wall thickness soon increased by additional synthesis of the wall from the cytoplasmic membrane located at the side region of the cross wall. Scanning electron microscopic observation of sodium dodecyl sulfate-treated and mechanically separated cross walls revealed that the outer surface of the cross wall exhibits regular circular structures and the inner surface showed has an irregular surface. This indicates that cell wall materials were arranged in a regular circular manner in the initially synthesized thin layer. It is conceivable that in Staphylococcus spp. two cell wall synthesizing systems are present: wall-elongation synthesis in which wall materials are arranged in a regular circular manner and wall-thickening synthesis in which wall materials are arranged in an irregular manner.     

Compositional changes in cell wall polymers during mango fruit ripening

Softening of mango fruit has been investigated by analysis of ripening related changes in the composition of the fruit cell walls. There is an apparent overall loss of galactosyl and deoxyhexosyl residues during ripening, the latter indicating degradation of the pectin component of the wall. The loss of galactose appears to be restricted to the chelator soluble fraction of the wall pectin, whilst loss of deoxyhexose seems to be more evenly distributed amongst the pectin. The chelator soluble pectin fraction is progressively depolymerised and becomes more polydisperse during ripening. These changes are similar to those occurring in other fruit and are related to the action of wall hydrolases during ripening.     

Anionic carbohydrate-containing cell wall polymers of Streptomyces melanosporofaciens and related species

The structures of cell wall anionic carbohydrate-containing polymers in Streptomyces melanosporofaciens VKM Ac-1864T and phylogenetically close organisms-S. hygroscopicus subsp. hygroscopicus VKM Ac-831T, S. violaceusniger VKM Ac-583T, S. endus VKM Ac-1331, S. endus VKM Ac-129, and S. rutgersensis subsp. castelarensis VKM Ac-832T--have been comparatively studied by chemical and NMR spectroscopic methods. The natural polymer of a new, previously unknown structure, Kdn (3-deoxy-D-glycero-Dgalacto-non-2-ulopyranosonic acid) with beta-galactose residues at C-9, has been found in the cell walls of all the strains under study. The cell walls of all the studied organisms contain three teichoic acids (TA): a predominant TA (1,3-poly(glycerol phosphate) with N-acetylated alpha-glucosaminyl substitutes by C-2 of glycerol, and minor TAs, 1,3- and 2,3-poly(glycerol phosphate) polymers without substitution. Their chains have O-acetyl and O-lysyl groups. Microorganisms of the above-mentioned species differ in the number of alpha-glucosaminyl substitutes and in the degree of their acetylation in the predominant teichoic acid.     

Raman imaging of cell wall polymers in Arabidopsis thaliana

We present chemical images of Arabidopsis thaliana stem cross-sections acquired by confocal Raman microscopy. Using green light (532 nm) from a continuous wave laser, the spatial distributions of cell wall polymers in Arabidopsis are visualized for the first time with lateral resolution that is sub-μm. Our results facilitate the label-free in situ characterization and screening of cell wall composition in this plant biology and genetics model organism, contributing ultimately towards an understanding of the molecular biology of many plant traits.     

Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis.

The biosynthesis of peptidoglycan and teichoic acid by reverting protoplasts of Bacillus licheniformis 6346 His-, in cubated at 35 C on medium containing 2.5% agar, is detectable after 40 min. The amount of N-acetyl-[1-14C]glucosamine incorporated into peptidoglycan and teichoic acid on continued incubation doubles at the same rate as the incorporation of [3H]tryptophan into protein. At the early stages of reversion the average glycan chain length, measured by the ratio of free reducing groups of muramic acid and glucosamine to total muramic acid present, is very short. As reversion proceeds, the average chain length increases to a value similar to the found in the wall of the parent bacillus. The extent of cross-linkage found in the peptide side chains of the peptidoglycan also increases as reversion proceeds. At the completion of reversion the wall material synthesized has similar characteristics to those of the walls of the parent bacilli, containing peptidoglycan and teichoic and teichuronic acids in about the same proportions. Soluble peptidoglycan can be isolated from the reversion medium, amounting to 30% of the total formed after 3 h of incubation and 8% after 12 h. This amount was reduced by the presence in the medium of the walls of an autolysin-deficient mutant; they were not formed at all by reverting protoplasts of the autolysin-deficient mutant itself. Analysis of the soluble material provided additional evidence for their being autolytic products rather than small unchanged molecules. When protoplasts were incubated on medium containing only 0.8% agar, 53 to 67% of the peptidoglycan formed after 3 h of incubation was soluble, and 21% after 12 h. Fibers that appeared to be sheared from the protoplasts at intermediate stages of reversion on medium containing 2.5% agar were similar in composition to the bacillary walls.     

Anionic polymers of the cell wall of Streptomyces sp. VKM An-2534

The cell wall of Streptomyces sp. VKM An-2534, the causative agent of common scab in potato tubers, which does not synthesize thaxtomin and is phylogenetically close to phytopathogen Streptomyces setonii sp. ATCC 25497, contains two anionic carbohydrate-containing polymers. The major polymer is teichuronic acid, whose repeating unit is disaccharide --> 4)-beta-D-ManpNAc3NAcyA-(1 --> 3)-alpha-D-GalpNAc-(1-->, where Acy is a residue of acetic or L-glutamic acid. The polymer of such structure has been found in Gram-positive bacteria for the first time. The minor polymer is teichoic acid [1,5-poly(ribitol phosphate)], in which a part of the ribitol residues are glycosylated at C4 with beta-D-Glcp and, probably, with beta-D-GlcpNAc and some residues are O-acylated with Lys residues. The structures were proved by chemical and NMR spectroscopic methods. It is likely that the presence of acidic polysaccharides on the surface of the phytopathogenic streptomycete is necessary for its attachment to the host plant.     

Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriae

Many surface proteins in Gram-positive bacteria are covalently linked to the cell wall through a transpeptidation reaction catalysed by the enzyme sortase. Corynebacterium diphtheriae encodes six sortases, five of which are devoted to the assembly of three distinct types of pilus fibres--SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. We demonstrate here the function of SrtF, the so-called housekeeping sortase, in the cell wall anchoring of pili. We show that a multiple deletion mutant strain expressing only SrtA secretes a large portion of SpaA polymers into the culture medium, with concomitant decrease in the cell wall-linked pili. The same phenotype is observed with the mutant that is missing SrtF alone. By contrast, a strain that expresses only SrtF displays surface-linked pilins but no polymers. Therefore, SrtF can catalyse the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins. We show that SrtA and SrtF together generate wild-type levels of the SpaA-type pilus on the bacterial surface. Furthermore, by regulating the expression of SpaA in the cell, we demonstrate that the SrtF function becomes critical when the SpaA level is sufficiently high. Together, these findings provide key evidence for a two-stage model of pilus assembly: pilins are first polymerized by a pilus-specific sortase, and the resulting fibre is then attached to the cell wall by either the cognate sortase or the housekeeping sortase.     

Modifications of cell wall polymers in Gram-positive bacteria by multi-component transmembrane glycosylation systems

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Anionic polymers of Bacillus subtilis cell wall modulate the folding rate of secreted proteins

In order to characterize the dynamics of the interaction between the emergent membrane translocated exoprotein and the components of Bacillus subtilis cell wall, we examined the kinetics of the in vitro refolding of levansucrase and alpha-amylase, at pH 7 and 37 degrees C, in the presence of polyphosphates (polyP) of various chain lengths (2/=16. In contrast, polyP modulate indirectly the rate of alpha-amylase refolding via their affinity for calcium. These differential effects might explain that the rate of the cell wall translocation of alpha-amylase secretion was found to be half that of levansucrase.     

Structure of anionic carbohydrate-containing cell wall polymers in several representatives of the order actinomycetales

A new teichoic acid was identified in the cell walls of Streptomyces griseoviridis VKM Ac-622T, Streptomyces sp. VKM Ac-2091, and Actinoplanes campanulata VKM Ac-1319T. The polymer is poly(glycosylglycerol phosphate). The repeating units of the polymer, alpha-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-galactopyran+ ++ osyl-(1-->1)-glycerols, are in phosphodiester linkage at C-3 of glycerol and C-6 of galactose. The structures of cell wall teichoic acids in the strains Streptomyces chryseus VKM Ac-200T and "Streptomyces subflavus" VKM Ac-484 similar in morphology and growth characteristics are also identical: 1,5-poly(ribitol phosphate) substituted at C-4(2) by 2-acetamido-2-deoxy-beta-glucopyranosyl residues and 1,3-poly(glycerol phosphate). The taxonomic aspects of these results are discussed.     

Regular arrangement of wall polymers in staphylococci

Concentric circular structures were observed on the newly exposed surface of the wall of Staphylococcus epidermidis and on its isolated cross-wall. These structures were removed by treatment with trichloroacetic acid. Chemical analysis revealed that after treatment with trichloroacetic acid most of the wall phosphorus was extracted but more than half of the N-acetylglucosamine remained associated with the wall. These observations suggest that polysaccharides are likely to be arranged circularly on the surface of the wall.     

Effect of nitrogen deficiency on the ion-exchange properties of cell wall polymers from wheat roots

The ion-exchange properties of cell wall polymers isolated from the roots of wheat (Triticum aestivum L.) plants grown on either nitrate-free (N-deficient) or nitrate-containing (+N) hydroponic nutrient medium have been investigated. Irrespective of the nitrogen nutrition regimen, the studied cell walls contained four types of ion-exchange groups: primary amino groups of structural proteins (pK_a < 3), carboxyl groups of polygalacturonic acid in pectin (pK _a ~4.7), carboxyl groups of hydroxycinnamic acids (pK _a ~7.3), and phenolic OH-groups of lignin (pK_a ~10.2). The quantitative ratio between these types of ion-exchange groups, the mass fraction of cell walls in the dry weight of roots, and the swelling coefficient of cell walls depended on the nitrate presence in the growing medium. Compared to the +N variant, the N-deficient variant was characterized by a 2.4 times higher content of phenolic OH-groups in cell walls and 1.24 times higher mass fraction of cell walls; at the same time, the swelling coefficient for this variant was lower by 10%. The obtained data indicate that nitrogen deficiency results in a formation of thicker root cell walls with a higher degree of polymer cross-linking that may be caused by the increased lignin content.     

The carbohydrate-containing cell wall polymers of some species from the cluster "Streptomyces lavendulae"

The type strains of the cluster "Streptomyces lavendulae" species with a low level of DNA-DNA relatedness were found to contain different cell-wall carbohydrate polymers, whereas the species of this cluster with a level of DNA-DNA relatedness of about 60% contain similar or identical carbohydrate polymers. The type strains Streptomyces katrae VKM Ac-1220T and S. polychromogenes VKM Ac-1207T synthesize mannan with different amounts of alpha-1,2- and alpha-1,3-substituted mannopyranose units and a small number of 1,3-poly(glycerophosphate) chains. The cell walls of S. lavendulocolor VKM Ac-215T and Streptomyces sp. VKM Ac-2117 were found to contain a hitherto unknown teichuronic acid, whose repeating unit is a disaccharide consisting of diaminomannuronic acid and N-acetylgalactosamine:-->4)-beta-D-ManpNAc3NAcA-(1-->3)-alpha-D- GalpNAc-(1-->. In addition, the cell walls of these two streptomycetes contain beta-glucosylated 1,5-poly(ribitol phosphate). The cell walls of S. virginiae VKM Ac-1218T and S. flavotricini VKM Ac-1277T contain the same poly(glucosyl-glycerophosphate). The results presented in this paper are in accordance with the DNA-DNA relatedness data published earlier and indicate a taxonomic significance of the structure of the cell-wall polysaccharides for the delineation of pheneticall/related Streptomyces species.