Secondary Cell Wall Polysaccharide Research Articles

Contribution of TagA-Like Glycosyltransferases to the Assembly of the Secondary Cell Wall Polysaccharide in Bacillus anthracis.

Bacillus anthracis elaborates a secondary cell wall polysaccharide (SCWP) made of 6 to 12 trisaccharide units. Pyruvyl and acetyl substitutions of the distal unit are prerequisites for the noncovalent retention of 22 secreted Bacillus S-layer (Bsl)-associated proteins bearing an S-layer homology (SLH) domain. Surface display of Bsl proteins contributes to cell separation as well as virulence. Earlier work suggested that TagO initiates the synthesis of SCWP while GneY and GneZ, two UDP-GlcNAc 2-epimerases, synthesize ManNAc that is later incorporated in the repeat unit (→4)-ManNAc-(β1→4)-GlcNAc-(β1→6)-GlcNAc-(α1→). In organisms that synthesize wall teichoic acid, TagA catalysts have been shown to form the glycosidic bond ManNAc-(β1→4)-GlcNAc. Here, we show that genes bas2675 and bas5272, predicted to encode glycosyltransferases of the WecB/TagA/CpsF family (PFAM03808; CAZy GT26), are required for B. anthracis SCWP synthesis and S-layer assembly. Similar to tagO or gneY gneZ mutants, B. anthracis strains depleted of tagA1 (bas5272) cannot maintain cell shape, support vegetative growth, or synthesize SCWP. Expression of tagA2 (bas2675), or Staphylococcus aureus tagA on a plasmid, rescues the nonviable tagA1 mutant. We propose that TagA1 and TagA2 fulfill overlapping and key glycosyltransferase functions for the synthesis of repeat units of the SCWP of B. anthracis. IMPORTANCE Glycosyltransferases (GTs) catalyze the transfer of sugar moieties from activated donor molecules to acceptor molecules to form glycosidic bonds using a retaining or inverting mechanism. Based on the structural relatedness of their catalytic and carbohydrate-binding modules, GTs have been grouped into 115 families in the Carbohydrate-Active EnZyme (CAZy) database. For complex products, the functional assignment of GTs remains highly challenging without the knowledge of the chemical structure of the assembled polymer. Here, we propose that two uncharacterized GTs of B. anthracis belonging to the WecB/TagA/CpsF family incorporate ManNAc in repeat units of the secondary cell wall polymer of bacilli species.

Importance of Water in Maintaining Softwood Secondary Cell Wall Nanostructure.

Water is one of the principal constituents by mass of living plant cell walls. However, its role and interactions with secondary cell wall polysaccharides and the impact of dehydration and subsequent rehydration on the molecular architecture are still to be elucidated. This work combines multidimensional solid-state 13C magic-angle-spinning (MAS) nuclear magnetic resonance (NMR) with molecular dynamics modeling to decipher the role of water in the molecular architecture of softwood secondary cell walls. The proximities between all main polymers, their molecular conformations, and interaction energies are compared in never-dried, oven-dried, and rehydrated states. Water is shown to play a critical role at the hemicellulose–cellulose interface. After significant molecular shrinkage caused by dehydration, the original molecular conformation is not fully recovered after rehydration. The changes include xylan becoming more closely and irreversibly associated with cellulose and some mannan becoming more mobile and changing conformation. These irreversible nanostructural changes provide a basis for explaining and improving the properties of wood-based materials.

Cell-surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis.

Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2nd generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.

Distinct Pathways Carry Out α and β Galactosylation of Secondary Cell Wall Polysaccharide in Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is required for the retention of surface layer (S-layer) and S-layer homology (SLH) domain proteins. Genetic disruption of the SCWP biosynthetic pathway impairs growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats composed of one ManNAc and two GlcNAc residues with O-3-α-Gal and O-4-β-Gal substitutions. UDP-Gal, synthesized by GalE1, is the substrate of galactosyltransferases that modify the SCWP repeat. Here, we show that the gtsE gene, which encodes a predicted glycosyltransferase with a GT-A fold, is required for O-4-β-Gal modification of trisaccharide repeats. We identify a DXD motif critical for GtsE activity. Three distinct genes, gtsA, gtsB, and gtsC, are required for O-3-α-Gal modification of trisaccharide repeats. Based on the similarity with other three-component glycosyltransferase systems, we propose that GtsA transfers Gal from cytosolic UDP-Gal to undecaprenyl phosphate (C55-P), GtsB flips the C55-P-Gal intermediate to the trans side of the membrane, and GtsC transfers Gal onto trisaccharide repeats. The deletion of galE1 does not affect growth in vitro, suggesting that galactosyl modifications are dispensable for the function of SCWP. The deletion of gtsA, gtsB, or gtsC leads to a loss of viability, yet gtsA and gtsC can be deleted in strains lacking galE1 or gtsE We propose that the loss of viability is caused by the accumulation of undecaprenol-bound precursors and present an updated model for SCWP assembly in B. anthracis to account for the galactosylation of repeat units.IMPORTANCE Peptidoglycan is a conserved extracellular macromolecule that protects bacterial cells from turgor pressure. Peptidoglycan of Gram-positive bacteria serves as a scaffold for the attachment of polymers that provide defined bacterial interactions with their environment. One such polymer, B. anthracis SCWP, is pyruvylated at its distal end to serve as a receptor for secreted proteins bearing the S-layer homology domain. Repeat units of SCWP carry three galactoses in B. anthracis Glycosylation is a recurring theme in nature and often represents a means to mask or alter conserved molecular signatures from intruders such as bacteriophages. Several glycosyltransferase families have been described based on bioinformatics prediction, but few have been studied. Here, we describe the glycosyltransferases that mediate the galactosylation of B. anthracis SCWP.

Extraction and Purification of Wall-Bound Polymers of Gram-Positive Bacteria.

The envelope of gram-positive bacteria encompasses the cell wall, a rigid exoskeleton comprised of peptidoglycan that provides protection against lysis and governs bacterial cell shapes. Peptidoglycan also serves as the site of attachment for proteins and nonproteinaceous polymers that interact with the bacterial environment. Nonproteinaceous molecules include teichoic acids, capsular polysaccharides, and secondary cell wall polysaccharides (SCWP). Treatment of gram-positive bacterial cells with proteases, nucleases, and detergents results in the isolation of "murein sacculi" (i.e., peptidoglycan with bound carbohydrate polymers). Incubation of sacculi with acid or base releases carbohydrate polymers that can be purified for further biochemical characterization. This protocol describes the hydrofluoric acid extraction and purification of the secondary cell wall polymer of Bacillus anthracis that is also found in the envelope of the other members of the Bacillus cereus sensu lato group of bacteria.

4,6- O-Pyruvyl Ketal Modified N-Acetylmannosamine of the Secondary Cell Wall Polysaccharide of Bacillus anthracis Is the Anchoring Residue for Its Surface Layer Proteins.

The secondary cell wall polysaccharide (SCWP) of Bacillus anthracis plays a key role in the organization of the cell envelope of vegetative cells and is intimately involved in host-guest interactions. Genetic studies have indicated that it anchors S-layer and S-layer-associated proteins, which are involved in multiple vital biological functions, to the cell surface of B. anthracis. Phenotypic observations indicate that specific functional groups of the terminal unit of SCWP, including 4,6- O-pyruvyl ketal and acetyl esters, are important for binding of these proteins. These observations are based on genetic manipulations and have not been corroborated by direct binding studies. To address this issue, a synthetic strategy was developed that could provide a range of pyruvylated oligosaccharides derived from B. anthracis SCWP bearing base-labile acetyl esters and free amino groups. The resulting oligosaccharides were used in binding studies with a panel of S-layer and S-layer-associated proteins, which identified structural features of SCWP important for binding. A single pyruvylated ManNAc monosaccharide exhibited strong binding to all proteins, making it a promising structure for S-layer protein manipulation. The acetyl esters and free amine of SCWP did not significantly impact binding, and this observation is contrary to a proposed model in which SCWP acetylation is a prerequisite for association of some but not all S-layer and S-layer-associated proteins.

Molecular Basis for the Attachment of S-Layer Proteins to the Cell Wall of Bacillus anthracis.

Bacterial surface (S) layers are paracrystalline arrays of protein assembled on the bacterial cell wall that serve as protective barriers and scaffolds for housekeeping enzymes and virulence factors. The attachment of S-layer proteins to the cell walls of the Bacillus cereus sensu lato, which includes the pathogen Bacillus anthracis, occurs through noncovalent interactions between their S-layer homology domains and secondary cell wall polysaccharides. To promote these interactions, it is presumed that the terminal N-acetylmannosamine (ManNAc) residues of the secondary cell wall polysaccharides must be ketal-pyruvylated. For a few specific S-layer proteins, the O-acetylation of the penultimate N-acetylglucosamine (GlcNAc) is also required. Herein, we present the X-ray crystal structure of the SLH domain of the major surface array protein Sap from B. anthracis in complex with 4,6- O-ketal-pyruvyl-β-ManNAc-(1,4)-β-GlcNAc-(1,6)-α-GlcN. This structure reveals for the first time that the conserved terminal SCWP unit is the direct ligand for the SLH domain. Furthermore, we identify key binding interactions that account for the requirement of 4,6- O-ketal-pyruvyl-ManNAc while revealing the insignificance of the O-acetylation on the GlcNAc residue for recognition by Sap.

Galactosylation of the Secondary Cell Wall Polysaccharide of Bacillus anthracis and Its Contribution to Anthrax Pathogenesis.

Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats with the structure, [→4)-β-ManNAc-(1→4)-β-GlcNAc(O3-α-Gal)-(1→6)-α-GlcNAc(O3-α-Gal, O4-β-Gal)-(1→]6-12 The genes whose products promote the galactosylation of B. anthracis SCWP are not yet known. We show here that the expression of galE1, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for B. anthracis SCWP galactosylation. The galE1 mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-type B. anthracis, but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of galE1 diminishes the capsulation of B. anthracis with poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of the B. anthracis cell wall envelope and for the pathogenesis of anthrax disease.IMPORTANCE Unlike virulent Bacillus anthracis isolates, B. anthracis strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth in vitro in broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants of B. anthracis strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes galE1 and galE2 We identified galE1 as necessary for SCWP galactosylation. Deletion of galE1 decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of B. anthracis and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.

PatB1 is an O-acetyltransferase that decorates secondary cell wall polysaccharides.

O-Acetylation of the secondary cell wall polysaccharides (SCWP) of the Bacillus cereus group of pathogens, which includes Bacillus anthracis, is essential for the proper attachment of surface-layer (S-layer) proteins to their cell walls. Using a variety of pseudosubstrates and a chemically synthesized analog of SCWP, we report here the identification of PatB1 as a SCWP O-acetyltransferase in Bacillus cereus. Additionally, we report the crystal structure of PatB1, which provides detailed insights into the mechanism of this enzyme and defines a novel subfamily of the SGNH family of esterases and lipases. We propose a model for the O-acetylation of SCWP requiring the translocation of acetyl groups from a cytoplasmic source across the plasma membrane by PatA1 and PatA2 for their transfer to SCWP by PatB1.

Assembly and Function of the Bacillus anthracis S-Layer.

Bacillus anthracis, the anthrax agent, is a member of the Bacillus cereus sensu lato group, which includes invasive pathogens of mammals or insects as well as nonpathogenic environmental strains. The genes for anthrax pathogenesis are located on two large virulence plasmids. Similar virulence plasmids have been acquired by other B. cereus strains and enable the pathogenesis of anthrax-like diseases. Among the virulence factors of B. anthracis is the S-layer-associated protein BslA, which endows bacilli with invasive attributes for mammalian hosts. BslA surface display and function are dependent on the bacterial S-layer, whose constituents assemble by binding to the secondary cell wall polysaccharide (SCWP) via S-layer homology (SLH) domains. B. anthracis and other pathogenic B. cereus isolates harbor genes for the secretion of S-layer proteins, for S-layer assembly, and for synthesis of the SCWP. We review here recent insights into the assembly and function of the S-layer and the SCWP.

Structural and immunochemical relatedness suggests a conserved pathogenicity motif for secondary cell wall polysaccharides in Bacillus anthracis and infection-associated Bacillus cereus.

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying “anthrax-like” symptoms in Cameroon (Bc CA) and Côte d’Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10–20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains.

Integrated analysis and transcript abundance modelling of H3K4me3 and H3K27me3 in developing secondary xylem

Despite the considerable contribution of xylem development (xylogenesis) to plant biomass accumulation, its epigenetic regulation is poorly understood. Furthermore, the relative contributions of histone modifications to transcriptional regulation is not well studied in plants. We investigated the biological relevance of H3K4me3 and H3K27me3 in secondary xylem development using ChIP-seq and their association with transcript levels among other histone modifications in woody and herbaceous models. In developing secondary xylem of the woody model Eucalyptus grandis, H3K4me3 and H3K27me3 genomic spans were distinctly associated with xylogenesis-related processes, with (late) lignification pathways enriched for putative bivalent domains, but not early secondary cell wall polysaccharide deposition. H3K27me3-occupied genes, of which 753 (~31%) are novel targets, were enriched for transcriptional regulation and flower development and had significant preferential expression in roots. Linear regression models of the ChIP-seq profiles predicted ~50% of transcript abundance measured with strand-specific RNA-seq, confirmed in a parallel analysis in Arabidopsis where integration of seven additional histone modifications each contributed smaller proportions of unique information to the predictive models. This study uncovers the biological importance of histone modification antagonism and genomic span in xylogenesis and quantifies for the first time the relative correlations of histone modifications with transcript abundance in plants.

Corrigendum: An Overview of Structural Features of Antibacterial Glycoconjugate Vaccines That Influence Their Immunogenicity.

On page 4248, the structure of α-d-galactose depicted by the symbol D was drawn incorrectly in Figure 11. The correct structure has been illustrated below: Figure 11 Secondary cell wall polysaccharide structure of B. anthracis (a) and two synthetic trisaccharide epitopes, antigen I (b) and antigen II (c) where, A = N-acetyl-β-d-glucosamine, B = N-acetyl-β-d-mannosamine, C = N-acetyl-α-d-glucosamine, D = α-d-galactose, E = β-d-galactose. On page 4254, the following contribution was missed from Reference [98]: M. Vasan, J. Rauvolfova, M. A. Wolfert, C. Leoff, E. L. Kannenberg, C. P. Quinn, R. W. Carlson, G. J. Boons, ChemBioChem 2008, 9, 1716–1720. The authors sincerely apologize for the oversights and any inconvenience that this may have caused.

Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis.

The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→) with α-Gal and β-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan.

Increased toxin expression in a Clostridium difficile mfd mutant.

BackgroundThe symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY.ResultsDuring attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations.ConclusionsThis study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.

Bacillus anthracis lcp Genes Support Vegetative Growth, Envelope Assembly, and Spore Formation.

Bacillus anthracis, a spore-forming pathogen, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and B. anthracis S-layer-associated proteins (BSLs) function as chain length determinants and are assembled in the envelope by binding to the secondary cell wall polysaccharide (SCWP). B. anthracis expresses six different genes encoding LytR-CpsA-Psr (LCP) enzymes (lcpB1 to -4, lcpC, and lcpD), which when expressed in Staphylococcus aureus promote attachment of wall teichoic acid to peptidoglycan. Mutations in B. anthracis lcpB3 and lcpD cause aberrations in cell size and chain length that can be explained as discrete defects in SCWP assembly; however, the function of the other lcp genes is not known. By deleting combinations of lcp genes from the B. anthracis genome, we generated variants with single lcp genes. B. anthracis expressing lcpB3 alone displayed physiological cell size, vegetative growth, spore formation, and S-layer assembly. Strains expressing lcpB1 or lcpB4 displayed defects in cell size and shape, S-layer assembly, and spore formation yet sustained vegetative growth. In contrast, the lcpB2 strain was unable to grow unless the gene was expressed from a multicopy plasmid (lcpB2(++)), and variants expressing lcpC or lcpD displayed severe defects in growth and cell shape. The lcpB2(++), lcpC, or lcpD strains supported neither S-layer assembly nor spore formation. We propose a model whereby LCP enzymes fulfill partially overlapping functions in transferring SCWP molecules to discrete sites within the bacterial envelope. Products of genes essential for bacterial envelope assembly represent targets for antibiotic development. The LytR-CpsA-Psr (LCP) enzymes tether bactoprenol-linked intermediates of secondary cell wall polymers to the C6 hydroxyl of N-acetylmuramic acid in peptidoglycan; however, the role of LCPs as a target for antibiotic therapy is not defined. We show here that LCP enzymes are essential for the cell cycle, vegetative growth, and spore formation of Bacillus anthracis, the causative agent of anthrax disease. Furthermore, we assign functions for each of the six LCP enzymes, including cell size and shape, vegetative growth and sporulation, and S-layer and S-layer-associated protein assembly.

Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis.

Bacillus anthracis elaborates a linear secondary cell wall polysaccharide (SCWP) that retains surface (S)-layer and associated proteins via their S-layer homology (SLH) domains. The SCWP is comprised of trisaccharide repeats [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→] and tethered via acid-labile phosphodiester bonds to peptidoglycan. Earlier work identified UDP-GlcNAc 2-epimerases GneY (BAS5048) and GneZ (BAS5117), which act as catalysts of ManNAc synthesis, as well as a polysaccharide deacetylase (BAS5051), as factors contributing to SCWP synthesis. Here, we show that tagO (BAS5050), which encodes a UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme that initiates the synthesis of murein linkage units, is required for B. anthracis SCWP synthesis and S-layer assembly. Similar to gneY-gneZ mutants, B. anthracis strains lacking tagO cannot maintain cell shape or support vegetative growth. In contrast, mutations in BAS5051 do not affect B. anthracis cell shape, vegetative growth, SCWP synthesis, or S-layer assembly. These data suggest that TagO-mediated murein linkage unit assembly supports SCWP synthesis and attachment to the peptidoglycan via acid-labile phosphodiester bonds. Further, B. anthracis variants unable to synthesize SCWP trisaccharide repeats cannot sustain cell shape and vegetative growth. Bacillus anthracis elaborates an SCWP to support vegetative growth and envelope assembly. Here, we show that some, but not all, SCWP synthesis is dependent on tagO-derived murein linkage units and subsequent attachment of SCWP to peptidoglycan. The data implicate secondary polymer modifications of peptidoglycan and subcellular distributions as a key feature of the cell cycle in Gram-positive bacteria and establish foundations for work on the molecular functions of the SCWP and on inhibitors with antibiotic attributes.

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly.

Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes the in vitro formation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly in B. anthracis. S-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identified Bacillus anthracis SlaQ, a small cytoplasmic protein that facilitates S-layer protein assembly in vivo and in vitro.

Restricting lignin and enhancing sugar deposition in secondary cell walls enhances monomeric sugar release after low temperature ionic liquid pretreatment.

BackgroundLignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype.ResultsEngineered Arabidopsis plants were treated with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate 1-ethyl-3-methylimidazolium acetate ([C2C1im][OAc]) at 10 % wt biomass loading at either 70 °C for 5 h or 140 °C for 3 h. After pretreatment at 140 °C and subsequent saccharification, the relative peak sugar recovery of ~26.7 g sugar per 100 g biomass was not statistically different for the wild type than the peak recovery of ~25.8 g sugar per 100 g biomass for the engineered plants (84 versus 86 % glucose from the starting biomass). Reducing the pretreatment temperature to 70 °C for 5 h resulted in a significant reduction in the peak sugar recovery obtained from the wild type to 16.2 g sugar per 100 g biomass, whereas the engineered lines with reduced lignin content exhibit a higher peak sugar recovery of 27.3 g sugar per 100 g biomass and 79 % glucose recoveries.ConclusionsThe engineered Arabidopsis lines generate high sugar yields after pretreatment at 70 °C for 5 h and subsequent saccharification, while the wild type exhibits a reduced sugar yield relative to those obtained after pretreatment at 140 °C. Our results demonstrate that employing cell wall engineering efforts to decrease the recalcitrance of lignocellulosic biomass has the potential to drastically reduce the energy required for effective pretreatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0275-2) contains supplementary material, which is available to authorized users.

LytR-CpsA-Psr enzymes as determinants of Bacillus anthracis secondary cell wall polysaccharide assembly.

Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified the B. anthracis lcpD mutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, the B. anthracis lcpB3 variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan. B. anthracis does not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed in S. aureus, promote WTA attachment. We propose a model whereby B. anthracis LCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.