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...

Members of the Bacillus cereus group contain cell wall carbohydrates that vary in their glycosyl compositions. Recent multilocus sequence typing (MLST) refined the relatedness of B. cereus group members by separating them into clades and lineages. Based on MLST, we selected several B. anthracis, B. cereus, and B. thuringiensis strains and compared their cell wall carbohydrates. The cell walls of different B. anthracis strains (clade 1/Anthracis) were composed of glucose (Glc), galactose (Gal), N-acetyl mannosamine (ManNAc), and Nacetylglucosamine (GlcNAc). In contrast, the cell walls from clade 2 strains (B. cereus type strain ATCC 14579 and B. thuringiensis strains) lacked Gal and contained N-acetylgalactosamine (GalNAc). The B. cereus clade 1 strains had cell walls that were similar in composition to B. anthracis in that they all contained Gal. However, the cell walls from some clade 1 strains also contained GalNAc, which was not present in B. anthracis cell walls. Three recently identified clade 1 strains of B. cereus that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241, had cell wall compositions that closely resembled those of the B. anthracis strains. It was also observed that B. anthracis strains cell wall glycosyl compositions differed from one another in a plasmiddependent manner. When plasmid pXO2 was absent, the ManNAc/Gal ratio decreased, while the Glc/Gal ratio increased. Also, deletion of atxA, a global regulatory gene, from a pXO2؊ strain resulted in cell walls with an even greater level of Glc.

Root nodule development is orchestrated by a symbiotic molecular dialogue between Gram-negative Rhizobium bacteria (e.g. Azorhizobium sp., Bradyrhizobium sp., Rhizobium sp., Sinorhizobium sp.) and specific legume host plants. Nodules are newly formed organs consisting of plant cells occupied with bacteroids that provide the host plant with fixed nitrogen. In the best studied symbiotic interactions, bacteria enter the roots via susceptible curled root hairs, and intracellular infection threads guide the bacteria toward de novo nodule primordia, where internalization into plant cells takes place. Initiation of nodule development and invasion require the production of bacterial signal molecules, including fatty acylated chitin oligosaccharides known as Nod factors (1), and structurally complex surface polysaccharides (SPS) 3 (2, 3). The outer surface of rhizobia typically consists of SPS that include extracellular polysaccharides (EPS) that are released into the media, capsular polysaccharides that are tightly associated with the bacterial surface, and lipopolysaccharides (LPS) that are anchored in the outer membrane (4). LPS are composed of lipid A, a core oligosaccharide, and an O-antigen polysaccharide. Accumulating data demonstrate the important role that rhizobial SPS play in invasion and nodule development and their involvement in the initiation of infection and invasion, suppression of plant defense, bacterial release from infection threads, bacteroid development and senescence, induction of plant gene expression, and protection against antimicrobial compounds (2, 3).Various observations suggest that proper LPS synthesis is required for invasion and nodule development in various symbiotic interactions, including the interaction between Rhizobium etli and Phaseolus vulgaris (2, 4). An R. etli mutant that lacks the O-chain polysaccharide portion of its LPS elicited the formation of infection threads on P. vulgaris; however, the bacteria ceased to develop within the root hair that formed thick walls (5, 6). The formation of nodule primordia was normal, but no bacteria were released from infection threads and internalized into plant cells (6). Occasionally, some bacteria were present in intercellular spaces. It was furthermore demonstrated that not only the presence of the O-chain polysaccharide on the LPS but also the abundance of O-chain polysaccharide was important for nodulation. For example, mutant strain R. etli CE166 produced, based on PAGE analysis of the LPS, only 40% LPS containing the O-chain polysaccharide compared with the parent strain, and the symbiotic phenotype of this mutant was

Nonclassical secondary cell wall polysaccharides constitute a major cell wall structure in the Bacillus cereus group of bacteria. The structure of the secondary cell wall polysaccharide from Bacillus cereus ATCC 10987, a strain that is closely related to Bacillus anthracis, was determined. This polysaccharide was released from the cell wall with aqueous hydrogen fluoride (HF) and purified by gel filtration chromatography. The purified polysaccharide, HF-PS, was characterized by glycosyl composition and linkage analyses, mass spectrometry, and one-and twodimensional NMR analysis. The results showed that the B. cereus ATCC 10987 HF-PS has a repeating oligosaccharide consisting of a 36)-␣-GalNAc- (134) The Bacillus cereus is a group of Gram-positive bacteria that includes Bacillus anthracis, B. cereus, and Bacillus thuringiensis strains. Members of this group are very closely related. In fact, on the basis of detailed phylogenetic analysis, it has been suggested that they all may belong to a single species (1). Despite the very close relatedness of B. cereus group members, there is considerable pathogenic variability. Some members of this group are not pathogenic, whereas others are opportunistic pathogens causing a range of conditions on a variety of hosts. Bacillus thuringiensis is an insect pathogen, and B. cereus is normally a soil-dwelling bacterium, which in rare cases, causes, in humans, usually nonfatal cases of food poisoning, sepsis, endophthalmitis, and occasionally severe or fatal pneumonia. One member, B. anthracis, causes anthrax in animals and humans and is considered a high threat bioterrorism agent.This variability in pathogenicity and host range is largely attributed to the plasmid content of the B. cereus group members, which can vary in size and number (2). For example, the crystal toxin genes of B. thuringiensis are carried on a plasmid (3), and plasmids pXO1 and pXO2 contain the genes required for the production of the B. anthracis toxin proteins and ␥-polyglutamate capsule, respectively (4, 5). Further, recent B. cereus isolates from cases of severe and fatal pneumonia were found to have the pXO1 plasmid (6, 7), and another report showed that B. cereus or B. thuringiensis isolates from cases of "anthrax-like" disease in gorillas contain both pXO1 and pXO2 (8, 9).For numerous bacterial pathogens, both Gram-positive and Gram-negative, cell wall polysaccharides are known virulence factors. However, little work has been done on the cell wall polysaccharides from members of the B. cereus group. We recently showed that the polysaccharides released from the cell walls from members of the B. cereus group using aqueous hydrogen fluoride (HF) 3 have carbohydrate compositions that vary qualitatively in a manner that is correlated, at least in part, to phylogenetic relatedness as determined by multilocus

The immunoreactivities of hydrogen fluoride (HF)-released cell wall polysaccharides (HF-PSs) from selected Bacillus anthracis and Bacillus cereus strains were compared using antisera against live and killed B. anthracis spores. These antisera bound to the HF-PSs from B. anthracis and from three clinical B. cereus isolates (G9241, 03BB87, and 03BB102) obtained from cases of severe or fatal human pneumonia but did not bind to the HF-PSs from the closely related B. cereus ATCC 10987 or from B. cereus type strain ATCC 14579. Antiserum against a keyhole limpet hemocyanin conjugate of the B. anthracis HF-PS (HF-PS-KLH) also bound to HF-PSs and cell walls from B. anthracis and the three clinical B. cereus isolates, and B. anthracis spores. These results indicate that the B. anthracis HF-PS is an antigen in both B. anthracis cell walls and spores, and that it shares cross-reactive, and possibly pathogenicity-related, epitopes with three clinical B. cereus isolates that caused severe disease. The anti-HF-PS-KLH antiserum cross-reacted with the bovine serum albumin (BSA)-conjugates of all B. anthracis and all B. cereus HF-PSs tested, including those from nonclinical B. cereus ATCC 10987 and ATCC 14579 strains. Finally, the serum of vaccinated (anthrax vaccine adsorbed (AVA)) Rhesus macaques that survived inhalation anthrax contained IgG antibodies that bound the B. anthracis HF-PS-KLH conjugate. These data indicate that HF-PSs from the cell walls of the bacilli tested here are (i) antigens that contain (ii) a potentially virulence-associated carbohydrate antigen motif, and (iii) another antigenic determinant that is common to B. cereus strains.