A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein-and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose-and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

In this report we describe the structure of the polysaccharide released from Bacillus anthracis vegetative cell walls by aqueous hydrogen fluoride (HF). This HF-released polysaccharide (HF-PS) was isolated and structurally characterized from the Ames, Sterne, and Pasteur strains of B. anthracis. The HF-PSs were also isolated from the closely related Bacillus cereus ATCC 10987 strain, and from the B. cereus ATCC 14579 type strain and compared with those of B. anthracis. The structure of the B. anthracis HF-PS was determined by glycosyl composition and linkage analyses, matrix-assisted laser desorption-time of flight mass spectrometry, and one-and two-dimensional nuclear magnetic resonance spectroscopy. The HF-PSs from all of the B. anthracis isolates had an identical structure consisting of an amino sugar backbone of 36)-␣-GlcNAc- (134) Generally, the carbohydrate-containing components of the vegetative cell walls of Gram-positive bacteria consist of the extensive peptidoglycan layer, teichoic acids, lipoteichoic acids, capsular polysaccharides, and crystalline cell surface proteins known as S-layer proteins that are often glycosylated (2). However, the B. anthracis cell wall differs in several aspects from this generalized description. First, B. anthracis cells are surrounded by a poly-␥-D-glutamate capsule and not by a polysaccharide capsule. Second, their cell walls do not contain teichoic acid (3), and last, their S-layer proteins are not glycosylated (1, 4). However, glycosyl composition comparisons of the cell walls of B. anthracis, Bacillus cereus, and Bacillus thuringiensis show that they do contain glycosyl residues and that they differ from one another in their glycosyl compositions (5).To date, cell wall carbohydrates from the vegetative cells of members of the B. cereus group have been addressed only to a limited extent (6 -8). All of these carbohydrates are rich in amino glycosyl residues but have variations in the type and amounts of these residues. The study of Ekwunife et al. (6) focused on the glycosyl composition of a carbohydrate polymer released from the cell wall through hydrogen fluoride (HF) treatment (HF treatment releases wall polysaccharides covalently bound via a phosphate bond to the peptidoglycan) of B. anthracis (⌬ Sterne) and found that the HF-released polysaccharide (HF-PS) 3 contained Gal, GlcNAc, and ManNAc in an approximate ratio of 3:2:1. This HF-PS was also further investigated by Mesnage et al. (4). They reported the importance of a pyruvyl substituent with regard to the function of this polysaccharide in anchoring the S-layer proteins to the cell wall.Fox et al. (7) investigated a number of B. anthracis and B. cereus strains for their total cell glycosyl compositions, which showed interesting differences between the different strains. For example, in contrast to the B. anthracis strains, all B. cereus strains investigated contained GalNAc, suggesting possible differences in cell wall architecture in the different Bacillus species cell walls and, possibly, the occurrence of...

Secondary cell wall polysaccharides (SCWPs) are important structural components of the Bacillus cell wall and contribute to the array of antigens presented by these organisms in both spore and vegetative forms. We previously found that antisera raised to Bacillus anthracis spore preparations cross-reacted with SCWPs isolated from several strains of pathogenic B. cereus, but did not react with other phylogenetically related but nonpathogenic Bacilli, suggesting that the SCWP from B. anthracis and pathogenic B. cereus strains share specific structural features. In this study, SCWPs from three strains of B. cereus causing severe or fatal pneumonia (G9241, 03BB87 and 03BB102) were isolated and subjected to structural analysis and their structures were compared to SCWPs from B. anthracis. Complete structural analysis was performed for the B. cereus G9241 SCWP using NMR spectroscopy, mass spectrometry and derivatization methods. The analyses show that SCWPs from B. cereus G9241 has a glycosyl backbone identical to that of B. anthracis SCWP, consisting of multiple trisaccharide repeats of: →6)-α-d-GlcpNAc-(1 → 4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1→. Both the B. anthracis and pathogenic B. cereus SCWPs are highly substituted at all GlcNAc residues with α- and β-Gal residues, however, only the SCWPs from B. cereus G9241 and 03BB87 carry an additional α-Gal substitution at O-3 of ManNAc residues, a feature lacking in the B. anthracis SCWPs. Both the B. anthracis and B. cereus SCWPs are pyruvylated, with an approximate molecular mass of ≈12,000 Da. The implications of these findings regarding pathogenicity and cell wall structure are discussed.

L-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-b-L-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-b-L-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 308C. The apparent K m for NADPH is 90 mM and 16.9 mM for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-b-L-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.L-Rhamnose is a component of the plant cell wall pectic polysaccharides rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II; Ridley et al., 2001) and is also present in diverse secondary metabolites including anthocyanins, flavonoids, and triterpenoids (Das et al., 1987;Bar-Peled et al., 1991;van Setten et al., 1995;Shinozaki et al., 1996;Markham et al., 2000), in certain types of plant glycoproteins (Haruko and Haruko, 1999), and in arabinogalactan proteins (Pellerin et al., 1995). The specific enzymes that attach rhamnose to each molecule are known as rhamnosyltransferases (RhaTs). To date, only a small number of RhaTs have been studied, and those were involved in flavonoid rhamnosylation. The characterized RhaTs utilize UDP-b-L-rhamnose (UDP-b-L-Rha) as the donor substrate (Kamsteeg et al., 1978;Feingold, 1982;Bar-Peled et al., 1991), although in mung bean (Vigna radiata), both dTDP-b-L-rhamnose (dTDP-b-L-Rha) and UDP-b-L-Rha were reported to act as sugar donors for the rhamnosylation of flavonoids (Barber and Neufeld, 1961).We are studying the enzymes involved in the synthesis of the nucleotide-rhamnose as part of our effort to understand the synthesis of pectic polysaccharides. To date, the rhamnosylation of plant polysaccharides and glycoproteins has not been studied. Thus, the identity of the activated form(s) of rhamnose needed for the synthesis of these macromolecules is not known with certainty. The enzymes required for the synthesis of the activated form(s) of rhamnose in plants have also not been purified.In contrast, much more is known about the synthesis of rhamnose in...