Bacillusanthracis Surface-Layer Proteins Assemble by Binding to the Secondary Cell Wall Polysaccharide in a Manner that Requires csaB and tagO

Bacillus anthracis, the causative agent of anthrax, requires surface (S)-layer proteins for the pathogenesis of infection. Previous work characterized S-layer protein binding via the surface layer homology domain to a pyruvylated carbohydrate in the envelope of vegetative forms. The molecular identity of this carbohydrate and the mechanism of its display in the bacterial envelope are still unknown. Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, we identify secondary cell wall polysaccharide (SCWP) as the ligand of S-layer proteins. In agreement with the model that surface layer homology domains bind to pyruvylated carbohydrate, SCWP was observed to be linked to pyruvate in a manner requiring csaB, the only structural gene known to be required for S-layer assembly. B. anthracis does not elaborate wall teichoic acids; however, its genome harbors tagO and tagA, genes responsible for the synthesis of the linkage unit that tethers teichoic acids to the peptidoglycan layer. The tagO gene appears essential for B. anthracis growth and complements the tagO mutant phenotypes of staphylococci. Tunicamycin-mediated inhibition of TagO resulted in deformed, S-layer-deficient bacilli. Together, these results suggest that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S-layer assembly and providing for the pathogenesis of anthrax infections.     

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.     

Isolation, Properties, Function, and Regulation of Endo-(1 → 3)-β-Glucanases in Schizosaccharomyces pombe

Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 → 3)-β-, (1 → 3)-α-, and (1 → 6)-β-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 → 3)-β-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 → 3)-β-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37°C. Glucanase activity was higher in vegetative cells held at 37°C than cells held at 25°C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.     

Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051T,Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP

The gram-positive bacterium Paenibacillus alvei CCM 2051^T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051^T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051^T.Bacterial cell surface layer (S-layer) glycoproteins provide a unique self-assembly matrix that has been optimized by nature for regular and periodic display of glycans with nanometer scale accuracy (21, 31). Exploitation of this self-assembly system for surface display of functional, tailor-made glycans is an attractive alternative to the use of common cell surface anchors (7), with potential areas of application relating to any biological phenomenon that is based on carbohydrate recognition, such as receptor-substrate interaction, signaling, or cell-cell communication. A prerequisite for this endeavor is the availability of an S-layer glycoprotein-covered bacterium that is amenable to genetic manipulation. Despite the high application potential offered by the S-layer glycan display system, there are so far only two reports in the literature dealing with the genetic manipulation of S-layer glycoprotein-carrying bacteria. Both reports concern the gram-negative periodontal pathogen Tannerella forsythia ATCC 43037, but neither of them affects S-layer protein glycosylation (12, 24). In archaea, in contrast, molecular studies of S-layer protein glycosylation are quite advanced (1), but with the archaeal system, S-layer glycoprotein self-assembly, which is a prerequisite for the desired glycan display, has not been manageable in vitro so far.Our model organisms and, hence, candidates for S-layer-mediated glycan display enabled by carbohydrate engineering techniques are members of the Bacillaceae family. Currently, the S-layer glycosylation system of the thermophilic bacterium Geobacillus stearothermophilus NRS 2004/3a is best understood (20, 23, 29, 33, 34). However, a drawback of this organism is its resistance to take up foreign DNA. Although described in the literature (13, 14, 37), transformation of thermophilic bacilli seems to be a strain-specific trait. Based on successful transformation experiments in our laboratory, the mesophilic bacterium Paenibacillus alvei CCM 2051^T (ATCC 6344; DSM 29) (formerly Bacillus alvei [4]) was chosen to set up a system for genetic manipulation. The bacterium is completely covered with an oblique S-layer lattice composed of glycoprotein species. Various aspects of its S-layer, including ultrastructural characterization (27), glycosylation analysis (2, 18), and glycan biosynthesis (11), have been investigated so far. The S-layer O-glycans are polymers of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that are linked via the adaptor -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d- Galp-(1→ to specific tyrosine residues (2, 18) of the S-layer protein SpaA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ751775","term_id":"225546017","term_text":"FJ751775"}}FJ751775).Due to the presence of an identical adaptor saccharide backbone in the S-layer glycan of G. stearothermophilus NRS 2004/3a (29), where its biosynthesis is initiated by the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WsaP (33), it was conceivable that a homologous enzyme would initiate S-layer glycosylation in P. alvei CCM 2051^T. Considering that the S-layer protein glycosylation machinery has been found to be encoded by S-layer glycosylation (slg) gene clusters (21), degenerate primers for the rml genes catalyzing the dTDP-l-Rha biosynthesis required for building up the adaptor saccharide of the P. alvei CCM 2051^T S-layer glycan were used to define a point of entry into the glycosylation locus (K. Zarschler, B. Janesch, P. Messner, and C. Schäffer, unpublished data). Chromosome walking revealed the existence of an slg gene cluster of about 24 kb, including an open reading frame (ORF) predicted to encode the initiation enzyme of S-layer protein glycosylation. The corresponding gene, named wsfP, served as a first target for the gene knockout system developed in the course of the present study. This target was chosen because loss of function would be easily screenable, resulting in an S-layer glycosylation-deficient mutant. The gene knockout system constructed for insertional inactivation of the chromosomal wsfP gene of P. alvei CCM 2051^T is based on the commercially available bacterial mobile group II intron Ll.LtrB of Lactococcus lactis, in combination with further components available in our laboratory, including the broad-host-range S-layer gene promoter of sgsE from G. stearothermophilus NRS 2004/3a (22) and the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. Bacterial mobile group II introns are retroelements inserted into specific DNA target sites at high frequency by use of the retrohoming mechanism, by which the excised intron lariat RNA is inserted directly into a DNA target site and is then reverse transcribed by the associated intron-encoded enzyme protein (6, 8, 17). Since the DNA target site is recognized primarily by base pairing of intron RNA, which can be modified, and a few intron-encoded-enzyme-protein recognition positions, these introns can be inserted efficiently into any specific DNA target (9, 15, 35, 40).The aim of this study is the development of a genetic tool for manipulation of S-layer protein glycosylation in P. alvei CCM 2051^T. For proof of concept, we specifically deal with (i) the construction of a broad-host-range gene knockout system based on the L. lactis Ll.LtrB intron; (ii) its modification for specific disruption of the wsfP gene on the P. alvei CCM 2051^T chromosome, encoding the putative initiation enzyme of S-layer glycan biosynthesis; and (iii) the reconstitution of enzyme activity by plasmid-based expression of wsfP and its predicted functional homologue wsaP from G. stearothermophilus NRS 2004/3a.     

Structure of the Type IX Group B Streptococcus Capsular Polysaccharide and Its Evolutionary Relationship with Types V and VII

The Group B Streptococcus capsular polysaccharide type IX was isolated and purified, and the structure of its repeating unit was determined. Type IX capsule →4)[NeupNAc-α-(2→3)-Galp-β-(1→4)-GlcpNAc-β-(1→6)]-β-GlcpNAc-(1→4)-β-Galp-(1→4)-β-Glcp-(1→ appears most similar to types VII and V, although it contains two GlcpNAc residues. Genetic analysis identified differences in cpsM, cpsO, and cpsI gene sequences as responsible for the differentiation between the three capsular polysaccharide types, leading us to hypothesize that type V emerged from a recombination event in a type IX background.     

Hydrolytic Activity and Substrate Specificity of an Endoglucanase from Zea mays Seedling Cell Walls

An endoglucanase was isolated from cell walls of Zea mays seedlings. Characterization of the hydrolytic activity of this glucanase using model substrates indicated a high specificity for molecules containing intramolecular (1→3),(1→4)-β-d-glucosyl sequences. Substrates with (1→4)-β-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1→3)-β-glucans such as laminarin were not hydrolyzed. When (1→3),(1→4)-β-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 × 10⁴). Activity against a water soluble (1→3),(1→4)-β-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1→3),(1→4)-β-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1→3),(1→4)-β-d-glucan that contained blocks of (1→4) linkages and/or more than a single contiguous (1→3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1→3),(1→4)-β-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase.     

Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1-->3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase

Polyclonal antibodies raised against barley (1→3,1→4)-β-d-glucanase, α-amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley. In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding α-amylase or (1→3,1→4)-β-d-glucanase, while in the aleurone α-amylase and (1→3,1→4)-β-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1→3,1→4)-β-d-glucanase, α-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation products were identical for each enzyme.     

Chromosomal Location of Genes Encoding Barley (1-->3, 1-->4)-beta-Glucan 4-Glucanohydrolases

Preparations of DNA from wheat (Triticum aestivum, cv Chinese Spring), barley (Hordeum vulgare, cv Betzes) and six euplasmic wheat-barley addition lines were digested to completion with restriction endonucleases and the products probed by Southern blot analysis using a cDNA-encoding barley (1→3, 1→4)-β-glucanase isoenzyme II. It is shown that one of the barley (1→3, 1→4)-β-glucanase genes is located on chromosome 1.     

Metabolism of the reserve polysaccharide of Streptococcus mitis. Properties of alpha-(1-->6)-glucosidase, its separation from transglucosylase, and the action of the two enzymes on branched oligosaccharides

1. An α-(1→6)-glucosidase has been separated from cell extracts of Streptococcus mitis. The enzyme was freed from transglucosylase by adsorption of the latter on retrograded amylose. 2. The enzyme was detected in five of the six strains of S. mitis that were studied; α-(1→6)-glucosidase was not found in strain RB1633, a strain that did not store polysaccharide. 3. The glucosidase could act on compounds in which α-glucose is joined through an α-(1→6)-bond to either a maltosaccharide or an isomaltosaccharide. 6²-α-Glucosylmaltose (panose) and 6³-α-glucosylmaltotriose were hydrolysed more rapidly and isomaltodextrins more slowly than isomaltose. 4. Transferring activity towards isomaltose and panose was appreciable when the concentration of substrate was 2% or higher. 5. The enzyme had no action on α-(1→4)-glucosidic linkages. 6-α-Maltodextrinylglucoses were hydrolysed only after transglucosylase action had attenuated them to isomaltose.     

Dissection of Hexosyl- and Sialyltransferase Domains in the Bifunctional Capsule Polymerases from Neisseria meningitidis W and Y Defines a New Sialyltransferase Family

Crucial virulence determinants of disease causing Neisseria meningitidis species are their extracellular polysaccharide capsules. In the serogroups W and Y, these are heteropolymers of the repeating units (→6)-α-d-Gal-(1→4)-α-Neu5Ac-(2→)_n in NmW and (→6)-α-d-Glc-(1→4)-α-Neu5Ac-(2→)_n in NmY. The capsule polymerases, SiaD_W and SiaD_Y, which synthesize these highly unusual polymers, are composed of two predicted GT-B fold domains separated by a large stretch of amino acids (aa 399–762). We recently showed that residues critical to the hexosyl- and sialyltransferase activity are found in the predicted N-terminal (aa 1–398) and C-terminal (aa 763–1037) GT-B fold domains, respectively. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain extends well beyond the predicted C-terminal GT-B domain and defines a new glycosyltransferase family, GT97, in CAZy (Carbohydrate-Active enZYmes Database).     

A (1-->3)-beta-d-Glucan Isolated From Zea Shoot Cell Wall Preparations

A small quantity of (1→3)-β-d-glucan was extracted with a (1→3),(1→4)-β-d-glucan by hot water after treatment of the insoluble fraction of a buffer homogenate of Zea shoots with 3 molar LiCl. An ammonium sulfate precipitation procedure effected a separation of the (1→3)-β-d-glucan from the more prevalent (1→3),(1→4)-β-d-glucan. The minor component polysaccharide precipitated at a concentration of 20% ammonium sulfate (w/v) and was, as a consequence of precipitation, rendered insoluble in water. The insoluble products were dissolved in 1 normal NaOH followed by neutralization with CH₃COOH. The purified polysaccharide accounted for approximately 0.3% of total hot water extract. It consisted mostly of glucose and its average mol wt was estimated to be about 7.0 × 10⁴, based on elution from a calibrated Sepharose CL-4B column. Methylation analysis and enzymic hydrolysis or partial acid-hydrolysis of the polysaccharide followed by analysis of the hydrolysate showed that the polysaccharide consisted of (1→3)-β-linked glucose residues.     

The S-layer homology domains of Paenibacillus alvei surface protein SpaA bind to cell wall polysaccharide through the terminal monosaccharide residue

Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaA_SLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaA_SLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaA_SLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.     

The Bacillus anthracis Protein MprF Is Required for Synthesis of Lysylphosphatidylglycerols and for Resistance to Cationic Antimicrobial Peptides

During inhalational anthrax, Bacillus anthracis survives and replicates in alveolar macrophages, followed by rapid invasion into the host's bloodstream, where it multiplies to cause heavy bacteremia. B. anthracis must therefore defend itself from host immune functions encountered during both the intracellular and the extracellular stages of anthrax infection. In both of these niches, cationic antimicrobial peptides are an essential component of the host's innate immune response that targets B. anthracis. However, the genetic determinants of B. anthracis contributing to resistance to these peptides are largely unknown. Here we generated Tn917 transposon mutants in the ΔANR strain (pXO1⁻ pXO2⁻) of B. anthracis and screened them for altered protamine susceptibility. A protamine-sensitive mutant identified carried the transposon inserted in the BA1486 gene encoding a putative membrane protein homologous to MprF known in several gram-positive pathogens. A mutant strain with the BAS1375 gene (the orthologue of BA1486) deleted in the Sterne 34F2 strain (pXO1⁺ pXO2⁻) of B. anthracis exhibited hypersusceptibility not only to protamine but also to α-helical cathelicidin LL-37 and β-sheet defensin human neutrophil peptide 1 compared to the wild-type Sterne strain. Analysis of membrane lipids using isotopic labeling demonstrated that the BAS1375 deletion mutant is unable to synthesize lysinylated phosphatidylglycerols, and this defect is rescued by genetic complementation. Further, we determined the structures of these lysylphosphatidylglycerols by using various mass spectrometric analyses. These results demonstrate that in B. anthracis a functional MprF is required for the biosynthesis of lysylphosphatidylglycerols, which is critical for resistance to cationic antimicrobial peptides.     

Plant O-Hydroxyproline Arabinogalactans Are Composed of Repeating Trigalactosyl Subunits with Short Bifurcated Side Chains

Classical arabinogalactan proteins partially defined by type II O-Hyp-linked arabinogalactans (Hyp-AGs) are structural components of the plant extracellular matrix. Recently we described the structure of a small Hyp-AG putatively based on repetitive trigalactosyl subunits and suggested that AGs are less complex and varied than generally supposed. Here we describe three additional AGs with similar subunits. The Hyp-AGs were isolated from two different arabinogalactan protein fusion glycoproteins expressed in tobacco cells; that is, a 22-residue Hyp-AG and a 20-residue Hyp-AG, both isolated from interferon α2b-(Ser-Hyp)₂₀, and a 14-residue Hyp-AG isolated from (Ala-Hyp)₅₁-green fluorescent protein. We used NMR spectroscopy to establish the molecular structure of these Hyp-AGs, which share common features: (i) a galactan main chain composed of two 1→3 β-linked trigalactosyl blocks linked by a β-1→6 bond; (ii) bifurcated side chains with Ara, Rha, GlcUA, and a Gal 6-linked to Gal-1 and Gal-2 of the main-chain trigalactosyl repeats; (iii) a common side chain structure composed of up to six residues, the largest consisting of an α-l-Araf-(1→5)-α-l-Araf-(1→3)-α-l-Araf-(1→3- unit and an α-l-Rhap-(1→4)-β-d-GlcUAp-(1→6)-unit, both linked to Gal. The conformational ensemble obtained by using nuclear Overhauser effect data in structure calculations revealed a galactan main chain with a reverse turn involving the β-1→6 link between the trigalactosyl blocks, yielding a moderately compact structure stabilized by H-bonds.     

The isolation of oligosaccharides from the cell-wall polysaccharide of Lactobacillus casei, serological group C

1. A number of disaccharides and oligosaccharides have been isolated from the products of mild acid hydrolysis of the specific substance from Lactobacillus casei, serological group C. 2. The major disaccharide is O-β-d-glucopyranosyl-(1→3)-N-acetyl- d-galactosamine (B4) and evidence is presented for the structure of a tetrasaccharide composed of O-β-d-glucopyranosyl-(1→6)-d-galactose (B1) joined through its reducing end group to B4. 3. Disaccharide B1 is also a component of a trisaccharide O-β-d-glucopyranosyl-(1→6)-O-β- d-galactopyranosyl-(1→6)-N-acetyl-d-glucosamine (A7). 4. A number of other oligosaccharides have been shown to be related structurally. 5. The ability of certain of the oligosaccharides to inhibit the precipitin reaction has been studied. The disaccharide B1 is more effective as an inhibitor than gentiobiose and the trisaccharide A7 is considerably more effective than B1. 6. These results have been compared with those obtained previously for the composition of the cell wall.     

Translesion Synthesis across 1,N6-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N6-γ-HMHP-dA) Adducts by Human and Archebacterial DNA Polymerases

The 1,N⁶-(2-Hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (1,N⁶-γ-HMHP-dA) adducts are formed upon bifunctional alkylation of adenine nucleobases in DNA by 1,2,3,4-diepoxybutane, the putative ultimate carcinogenic metabolite of 1,3-butadiene. The presence of a substituted 1,N⁶-propano group on 1,N⁶-γ-HMHP-dA is expected to block the Watson-Crick base pairing of the adducted adenine with thymine, potentially contributing to mutagenesis. In this study, the enzymology of replication past site-specific 1,N⁶-γ-HMHP-dA lesions in the presence of human DNA polymerases (hpols) β, η, κ, and ι and archebacterial polymerase Dpo4 was investigated. Run-on gel analysis with all four dNTPs revealed that hpol η, κ, and Dpo4 were able to copy the modified template. In contrast, hpol ι inserted a single base opposite 1,N⁶-γ-HMHP-dA but was unable to extend beyond the damaged site, and a complete replication block was observed with hpol β. Single nucleotide incorporation experiments indicated that although hpol η, κ, and Dpo4 incorporated the correct nucleotide (dTMP) opposite the lesion, dGMP and dAMP were inserted with a comparable frequency. HPLC-ESI-MS/MS analysis of primer extension products confirmed the ability of bypass polymerases to insert dTMP, dAMP, or dGMP opposite 1,N⁶-γ-HMHP-dA and detected large amounts of −1 and −2 deletion products. Taken together, these results indicate that hpol η and κ enzymes bypass 1,N⁶-γ-HMHP-dA lesions in an error-prone fashion, potentially contributing to A→T and A→C transversions and frameshift mutations observed in cells following treatment with 1,2,3,4-diepoxybutane.     

Identification of the UDP-N-Acetylglucosamine 4-Epimerase Involved in Exosporium Protein Glycosylation in Bacillus anthracis

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation.Bacillus anthracis, the causative agent of anthrax, is a gram-positive, rod-shaped soil bacterium that forms spores when deprived of essential nutrients (15). Spore formation begins with an asymmetric septation that divides the developing cell into a smaller forespore compartment and a larger mother cell compartment, each containing a copy of the genome. The mother cell then engulfs the forespore and surrounds it with three protective layers: a cortex composed of peptidoglycan, a closely apposed proteinaceous coat, and a loosely fitting exosporium (11). Mother cell lysis releases the mature spore, which is dormant and capable of surviving in harsh environments for many years (16). When spores encounter an aqueous environment containing nutrients, they can germinate and grow as vegetative cells (23).Recently, interest in B. anthracis spores has intensified in response to their use as agents of bioterrorism. Of particular interest has been the outermost exosporium layer, which serves as a semipermeable barrier excluding potentially harmful macromolecules (9, 26) and as a vital first point of contact with the immune system of an infected host (14, 18, 32). The exosporium of B. anthracis and closely related species such as Bacillus cereus and Bacillus thuringiensis is a prominent structure comprised of a paracrystalline basal layer and an external hair-like nap (2). The basal layer contains approximately 20 different proteins (22, 25), while the filaments of the nap are formed by trimers of a single collagen-like glycoprotein called BclA (4, 27). The central region of BclA contains a large number of GXX repeats, mostly GTP triplets, and this region varies in length in naturally occurring strains of B. anthracis, resulting in hair-like naps of differing lengths (24, 28). Multiple copies of two O-linked oligosaccharides, a trisaccharide and a pentasaccharide, are attached to the protein component of BclA. The pentasaccharide side chains appear to be attached to threonine residues within the central region, while the trisaccharide side chains are attached to presently undefined residues in the protein (7).The precise structure of the trisaccharide side chain has not been determined, but its sequence is 3-O-methyl-l-rhamnose-l-rhamnose-N-acetylgalactosamine (GalNAc) (7). Except for a single glycosidic linkage, the structure of the pentasaccharide is known. Its structure is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranosyl-(1→?)-N-acetylgalactosamine (7). Both oligosaccharides are attached to the BclA protein backbone through GalNAc residues. The pentasaccharide sugar 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-d-glucose, which was given the trivial name anthrose, has been found only in B. anthracis strains and a limited number of highly pathogenic strains of B. cereus and B. thuringiensis (7, 8). For that reason, anthrose has joined other exosporium components as targets for the detection of B. anthracis spores and as new targets for therapeutic intervention in anthrax (6, 26, 29).In view of the potential importance of the BclA oligosaccharides, especially the anthrose-containing pentasaccharide, we have undertaken a comprehensive study of their biosynthesis. This effort involves identifying the biosynthetic genes for the three component sugars, anthrose, rhamnose, and GalNAc, as well as the genes involved in assembling the oligosaccharides and attaching them to the protein backbone of BclA. We recently reported the identification of a four-gene anthrose biosynthetic operon (8). A four-gene rhamnose biosynthetic operon has also been identified (24). This paper describes the identification of the gene encoding the UDP-N-acetylglucosamine (GlcNAc) 4-epimerase necessary for GalNAc biosynthesis. It also describes a surprising alternative BclA oligosaccharide biosynthetic pathway, which is active only in the absence of the UDP-GlcNAc 4-epimerase. Finally, this paper reports a biphasic pattern of expression of the gene encoding this epimerase during sporulation.     

A Galacturonic Acid–Containing Xyloglucan Is Involved in Arabidopsis Root Hair Tip Growth

Root hairs provide a model system to study plant cell growth, yet little is known about the polysaccharide compositions of their walls or the role of these polysaccharides in wall expansion. We report that Arabidopsis thaliana root hair walls contain a previously unidentified xyloglucan that is composed of both neutral and galacturonic acid–containing subunits, the latter containing the β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→ and/or α-l-fucosyl-(1→2)-β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→) side chains. Arabidopsis mutants lacking root hairs have no acidic xyloglucan. A loss-of-function mutation in At1g63450, a root hair–specific gene encoding a family GT47 glycosyltransferase, results in the synthesis of xyloglucan that lacks galacturonic acid. The root hairs of this mutant are shorter than those of the wild type. This mutant phenotype and the absence of galacturonic acid in the root xyloglucan are complemented by At1g63450. The leaf and stem cell walls of wild-type Arabidopsis contain no acidic xyloglucan. However, overexpression of At1g63450 led to the synthesis of galacturonic acid–containing xyloglucan in these tissues. We propose that At1g63450 encodes XYLOGLUCAN-SPECIFIC GALACTURONOSYLTRANSFERASE1, which catalyzes the formation of the galactosyluronic acid-(1→2)-α-d-xylopyranosyl linkage and that the acidic xyloglucan is present only in root hair cell walls. The role of the acidic xyloglucan in root hair tip growth is discussed.     

Pen and Pal Are Nucleotide-Sugar Dehydratases That Convert UDP-GlcNAc to UDP-6-Deoxy-d-GlcNAc-5,6-ene and Then to UDP-4-Keto-6-deoxy-l-AltNAc for CMP-Pseudaminic Acid Synthesis in Bacillus thuringiensis

CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-d-GlcNAc-5,6-ene. Pen contains strongly bound NADP⁺ and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-d-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-l-AltNAc. Pal is NAD⁺-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-l-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.