Structural Elucidation of the Nonclassical Secondary Cell Wall Polysaccharide from Bacillus cereus ATCC 10987

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 two-dimensional NMR analysis. The results showed that the B. cereus ATCC 10987 HF-PS has a repeating oligosaccharide consisting of a →6)-α-GalNAc-(1→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→ trisaccharide that is substituted with β-Gal at O3 of the α-GalNAc residue and nonstoichiometrically acetylated at O3 of the N-acetylmannosamine (ManNAc) residue. Comparison of this structure with that of the B. anthracis HF-PS and with structural data obtained for the HF-PS from B. cereus type strain ATCC 14579 revealed that each HF-PS had the same general structural theme consisting of three HexNAc and one Hex residues. A common structural feature in the HF-PSs from B. cereus ATCC 10987 and B. anthracis was the presence of a repeating unit consisting of a HexNAc3 trisaccharide backbone in which two of the three HexNAc residues are GlcNAc and ManNAc and the third can be either GlcNAc or GalNAc. The implications of these results with regard to the possible functions of the HF-PSs are discussed.

sequence typing (10). That work also revealed quantitative glycosyl composition differences that indicated that structural variation can occur in the cell wall carbohydrates among B. cereus strains belonging to the same clade and lineage. Interestingly, it was shown that the B. cereus isolates which caused severe or fatal pneumonia and contained the pXO1 plasmid had HF-released polysaccharide (HF-PS) glycosyl compositions that closely resembled that of B. anthracis (10), indicating that HF-PS structural conservation plays a role in pathogenic function. Further work has shown that antiserum against B. anthracis cross-reacts to some extent with the HF-PS from these B. cereus isolates, indicating that they are structurally related to the B. anthracis HF-PS. 4 Future publications will describe the structures of the HF-PSs from these B. cereus isolates.
Recent structural determination of B. anthracis HF-PS showed that it was composed of a trisaccharide backbone consisting of two GlcNAc residues and one N-acetylmannosamine (ManNAc) residue and that this backbone is variably substituted with terminal Gal residues (10,12). This structure falls into the class of nonclassical secondary cell wall polymers (SCWP) of Gram-positive bacteria, as defined by Schäffer and Messner (13), that are covalently attached to the peptidoglycan through a phosphate or pyrophosphate bond. The S-layer proteins are anchored to the cell wall by binding to the SCWP polysaccharide via a carbohydrate-binding domain known as the S-layer homology (SLH) domain (14). Secondary cell wall polysaccharide structures have been determined for a number of Gram-positive bacteria, including some B. cereus strains (13). Older reports described that B. cereus AHU 1356 produced a neutral carbohydrate composed of GlcNAc, ManNAc, GalNAc, and Glc (15). In addition, an acidic carbohydrate composed of GlcNAc, Gal, rhamnose (Rha), glycerol, and phosphate was also identified in this strain (16). Because of the considerable importance of bacilli with regard to public health, a more complete picture of these carbohydrates is needed. Structural determination of the HF-PSs is necessary as the first step into structurefunction studies of these SCWP polysaccharides as well as to answer questions about their suitability for developing new and/or improved vaccines and diagnostic agents.
In order to determine the relationship of these polysaccharides with pathogenicity, it is necessary to systematically characterize their structures. In this effort, we have initially selected the HF-PS from pathogenic B. anthracis and compared it with the HF-PSs from two normally nonpathogenic strains, the dairy isolate B. cereus ATCC 10987 and the B. cereus type strain ATCC 14579. We selected B. cereus strain ATCC 10987 and the B. cereus type strain ATCC 14579, since the genome of strain B. cereus ATCC 10987 is 93.7%, similar to that of B. anthracis and 90.9% similar to the genome of the B. cereus type strain, ATCC 14579, and since strain ATCC 10987 also contains a plasmid that is similar to the B. anthracis pXO1 virulence plasmid but lacks the pathogenicity island (17). We have already published, in this journal, the structure of the HF-PSs from B. anthracis Ames, Sterne, and Pasteur (12).
Here, we report the structure of the HF-PS from B. cereus strain ATCC 10987 as well as partial structural data for the HF-PS from the B. cereus type strain ATCC 14579 and compare those structures with that of the HF-PS from B. anthracis. We have included the partial structural data of the HF-PS from B. cereus type strain ATCC 14579, since these data, together with the complete structure of the B. cereus ATCC 10987 described here and that reported for B. anthracis (12), reveal that the HF-PSs from these strains contain unique structural features that are present on a possibly B. cereus-conserved structural scaffold. The implications of these results with regard to possible functions are discussed.

Bacterial Strains and Cultural Conditions-B. anthracis
Ames, B. cereus ATCC 10987, and B. cereus ATCC 14579 were provided from the Centers for Disease Control and Prevention culture collection. Cultures were grown were grown as described in our previous report (12).
Preparation of Bacillus Cell Wall Polysaccharides-The bacterial cell walls were prepared using a modified procedure described by Brown (18) as we previously reported (10,12). The polysaccharides were released from the isolated cell walls by treatment with aqueous HF and purified by gel permeation chromatography, as described in our previous report (12).
Composition and Glycosyl Linkage Analysis of the Cell Wall Polysaccharides-Glycosyl composition analysis was done by the preparation and gas chromatography-mass spectrometric analysis of trimethylsilyl methyl glycosides (19). The trimethylsilyl methyl glycosides were analyzed by combined gas chromatography-mass spectrometry, as previously reported (12). The glycosyl linkage analysis was performed according to a modification of the method of Ciucanu and Kerek (20), as described in our previous report (12).
De-O-acetylation of the Cell Wall Polysaccharides-The HF-PS was deacetylated by mild hydrazinolysis. Approximately, 4 mg of sample was treated with 200 l of anhydrous hydrazine (Pierce) at 37°C for 1 h. The sample was cooled on an ice bath, and 2 ml of cold acetone (Ϫ70°C) was added dropwise with mild shaking. The mixture was kept overnight at Ϫ4°C for complete precipitation of de-O-acetylated HF-PS. The de-Oacetylated HF-PS was collected by centrifugation at 10,000 ϫ g for 10 min at 7°C. The supernatant was discarded, and precipitate was washed with cold acetone (Ϫ70°C), followed by centrifugation. (This step was repeated two more times.) Finally, the precipitate was dried under nitrogen flow, dissolved in water, and lyophilized.
NMR Analysis-The HF-PS sample (2-3 mg) was dissolved in 0.5 ml of regular grade deuterium oxide (D 2 O 98.5%) and lyophilized. This process was done twice to exchange the hydroxyl and amide protons with deuterium. The sample was finally dissolved in 0.5 ml of 100% D 2 O (Cambridge Isotopes) and transferred to a 5-mm NMR tube. All one-and two-dimensional NMR spectra were acquired with a 600-MHz Varian Inova instrument using the standard software supplied by Varian. 1 H-1 H homonuclear two-dimensional experiments were done after perfect 90°pulse calibration and 3.5 K spectral width in both dimensions; however, 1 H-13 C HSQC data were acquired taking 3.5 and 12 K in direct and indirect dimensions, respectively.
Mass Spectrometry-Mass spectral analysis of the isolated HF-PS was determined using a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometer from Applied Biosystems. The HF-PS was dissolved in a 1:1 mixture of methanol/water and mixed in equal proportion (v/v) with 0.5 M 2,5-dihydroxybenzoic acid as the matrix. About 0.7 l of the mixture was loaded on each spot on a stainless steel MALDI plate and air-dried. The spectra were acquired in either the linear or reflectron-positive modes using a 337-nm N 2 laser with acceleration voltage of 20 kV.

Isolation and Initial Analysis; Glycosyl Composition and
Linkage Analysis-The HF-PS from the investigated B. cereus strains eluted as a single major peak within the void volume of the Bio-Gel P2 column; the peak fractions were collected, lyophilized, and used for detailed structural analysis. The HF-PS composition from B. anthracis Ames consisted of Gal, GlcNAc, and ManNAc in an approximate 3:2:1 ratio, as previously described for four B. anthracis strains (Ames, Pasteur, Sterne 34F 2 , and UT60) (10,12). The composition of the HF-PS from B. cereus ATCC 10987 consisted of Gal, ManNAc, Glc-NAc, and GalNAc in a 1.6:1.5:1.0:1.4 ratio ( Table 1). A small amount of Glc was present due to contamination of the HF-PS preparation with a Glc-rich polysaccharide, as previously reported (10). The composition of B. cereus ATCC 14579 HF-PS consisted of Glc, ManNAc, GlcNAc, and GalNAc in approximately a 1.8:1.5:3.1:1.0 ratio (Table 1). Thus, the differences in glycosyl compositions of these two B. cereus HF-PSs shows that their structures are not identical to each other and that they also differ from that of the B. anthracis HF-PS, as reported earlier (10,12). Moreover, these two B. cereus HF-PSs contain GalNAc, which is not present in the B. anthracis HF-PS, and the B. cereus ATCC 14579 HF-PS contains Glc, which is not found in either the B. cereus ATCC 10987 or in the B. anthracis HF-PSs. These results are consistent with our earlier report (12), in which proton NMR analysis also showed that these HF-PSs have different structures.
Methylation analysis showed that the B. cereus ATCC 10987 HF-PS consisted of terminally linked Gal, 3,6-linked GalNAc, 4-linked ManNAc, and 4-linked GlcNAc. Minor components included terminally linked GlcNAc, ManNAc, and GalNAc, indicating the possibility of molecular microheterogeneity within the HF-PS preparation. The presence of minor amounts of these terminal HexNAc residues is probably due to variously sized HF-PS molecules that differ in their nonreducing termi-nal glycosyl residues. They are present as minor components, since the HF-PS consists of variously sized molecules of up to 7759 daltons (as shown by MS analysis and described further below) and, therefore, the HexNAc residues at the nonreducing termini of these differently sized molecules found in the HF-PS preparation would be present in small amounts relative to the glycosyl residues in the multiple repeat units.
For  1 tetramer. These results suggest that the m/z ϭ 1583.5 ion is due to an octasaccharide composed of two (HexNAc) 3 (Hex) 1 tetrameric repeating units, and that the ion at m/z ϭ 2354.6 corresponds to a molecule with three tetrameric repeat units. Based on glycosyl composition results described above, probable compositions for these masses are given in Table 2. In addition to these major ions for the two ion clusters, several other ions were observed. The ions at m/z 1625.5 and 1667.5 are 42 and 84 mass units greater, respectively, than the m/z 1583.5 ion, indicating that these ions are due to mono-and di-O-acetylation, respectively, of the ditetrasaccharide repeat structure. The O-acetyl groups, when present, are located on the ManNAc residues, as determined by NMR analysis, which is described below. The ions at m/z 1607.5 and m/z 1649.5 are due to minor amounts of dehydrated (Ϫ18 mass units) versions of these acetylated molecules. We also observe ions at m/z 1786. 5 and 1990.6, which are consistent with one and two additional Hex-NAc residues, respectively, to the m/z 1583.5 ion. The difference between the m/z 1990.6 ion and the m/z 2354.6 ion is 365 mass units. This difference is consistent with the addition of a HexHexNAc disaccharide to the 1990.6 molecule.
Mass spectrometric analysis, in the linear mode, of the HF-PS after de-O-acetylation (Fig. 1B) shows that that there is considerable size heterogeneity in the HF-PS preparation. A series of [M ϩ Na] ϩ ions are observed up to m/z 7759. Each of these ions differs from the previous ion by 772 mass units, which is the mass of the (HexNAc) 3 (Hex) 1 repeat unit. These results show that the HF-PS polysaccharide consists of a mixture of molecules that vary from 2 to at least 10 repeating tetrasaccharide units. It is possible that this size heterogeneity is present in vivo or is produced during the release of the polysaccharide from the cell wall by aqueous HF. De-Oacetylation allows these ions to be observed by removing the considerable microheterogeneity caused by variation in acetylation, microheterogeneity that increases with increasing number of repeat units. The anomeric configuration and the sequence of the glycosyl residues in the B. cereus ATCC 10987 were determined by both one-and two-dimensional NMR analysis of the HF-PS before and after removal of the O-acetyl groups. The complete proton and carbon assignments of this HF-PS were obtained by one-dimensional proton NMR (Fig. 2) and by two-dimensional gCOSY, TOCSY (Fig. 3), and gHSQC (Fig. 4) experiments. The proton spectrum shows four major anomeric H1 signals at ␦ 5.23, ␦ 5.04, ␦ 4.57, and ␦ 4.45 and a minor anomeric H1 signal at ␦ 4.92. Each of these anomeric protons is due to a unique glycosyl ring system in this HF-PS, and the gCOSY, TOCSY (Fig. 3), and gHSQC ( Fig. 4) experiments allowed the proton and carbon assignments of each of these ring systems as explained below and given in Table 3.  Table 2. The spectrum shown in A was obtained in the reflectron mode, whereas the spectrum shown in B was obtained in the linear mode. R 2 , two (HexNAc) 3 (Hex) 1 repeat units; R 3 , three repeat units, etc.

TABLE 2 Proposed structures for the various molecular ions observed during MALDI-TOF MS analysis of the HF-PS from B. cereus ATCC 10987
The proposed structures are consistent with the glycosyl composition, linkage, and NMR data.

Repeat units
Proposed structure Observed ͓M ؉ Na͔ ؉ ion The H1 signal at ␦ 5.23 (1H, s) is the most downfield signal in the spectrum and was assigned to the anomeric proton of an ␣-glycosyl residue (A). The H2 of residue A resonates at ␦ 4.32, as was confirmed from the gCOSY experiment (spectrum not shown). The chemical shift of the carbon to which this proton is attached was determined by gHSQC (Fig. 4) to be ␦ 48.2, indicating it to be nitrogen-bearing carbon consistent with a Hex-NAc residue. In addition, the downfield chemical shifts of C3 and C6 of this residue at ␦ 76.4 and 69.5, respectively, indicate that residue A is substituted at O3 and O6. The gCOSY and TOCSY (Fig. 3) spectra showed that H3 and H4 resonate at ␦ 3.94 and ␦ 4.23, respectively, and that the small overall J 3,4 and J 4,5 coupling constant of H4 (Ͻ9.6 Hz) indicates that residue A has a galacto-configuration. Therefore, residue A was assigned to be O3and O6-substituted ␣-GalNAc, which is consistent with the glycosyl composition and linkage analysis described above showing the presence of a 3,6-linked GalNAc residue.
The next upfield anomeric proton signal at ␦ 5.04 (s, 1H) had a corresponding C1 chemical shift of ␦ 98.56. The characteristic downfield chemical shift of H2 at ␦ 4.66 with small J 1,2 and J 2,3 coupling constants indicated that this residue has a manno-configuration. The chemical shift of C2 is at ␦ 50.8 and shows that C2 is a nitrogen-bearing carbon. Therefore, residue B was assigned as ManNAc. Since both ␣/␤-anomeric configurations of manno-glycosyl residues have low coupling constants, the ␤-anomeric configuration of residue B was confirmed by comparing the TOCSY with NOESY spectrum. The NOESY spectrum (not given) showed strong intraresidue NOE interactions of H1 at ␦ 5.04 to H2, H3, and H5 at ␦ 4.66, ␦ 5.15, and ␦ 3.65, respectively. These NOE interactions are consistent with the axial positions of H1, H3, and H5 of a ␤-anomeric configuration for residue B (the NOESY spectrum for the de-Oacetylated HF-PS is shown in Fig. 5 and also shows the H1/H3/H5 interactions). The downfield chemical shift of H3 at ␦ 5.15, which is attached to a carbon with a chemical shift of ␦ 75.8, is consistent with O-acetylation at O3 of this ␤-ManNAc residue. The presence of O-acetyl groups on this HF-PS is also consistent with the mass spectrometric data as described above.
The minor H1 anomeric signal at ␦ 4.92 was identified to be another ␤-ManNAc residue (B) that does not bear an O-acetyl ester group on O3. The upfield chemical shifts of H2 at ␦ 4.53 (compared with ␦ 4.66 for residue B) and H3 at ␦ 4.08 (com-   Fig. 2. The complete proton assignments were made from the TOCSY and COSY (spectrum not shown) data and are given in Table 3.
pared with ␦ 5.15 for residue B), respectively, are consistent with the lack of an O-acetyl group at O3 of this residue. The relative quantification of HF-PS bearing the O-acetyl group was done by comparing the H1 integral values of residues B and B from the proton NMR spectrum, and it was calculated that about 60% of the HF-PS was O-acetylated at O3 of the ManNAc residue. It is quite possible that the polysaccharide as found in the cell wall is completely O-acetylated and that the 60% value is due to partial removal of O-acetyl groups during its release by aqueous HF.
The next upfield signal at ␦ 4.57 is a doublet with a J 1,2 coupling constant of 7.8 Hz, indicating it to have a ␤-anomeric configuration. The C1 chemical shift of ␦ 101.88 also supports the ␤-anomeric configuration. The H2 of this residue resonates at ␦ 3.65 and is attached to a nitrogen-bearing carbon at ␦ 55.1 (see Fig. 4), showing that this is a ␤-HexNAc residue. The TOCSY spectrum showed relatively large J 2,3 , J 3,4 , and J 4,5 coupling constants, which is consistent with a gluco-configuration; therefore, this residue (C) was assigned as ␤-GlcNAc. The downfield chemical shift of C4 at ␦ 79.3 indicates that this residue is 4-substituted ␤-GlcNAc.
The remaining H1 anomeric signal has a proton chemical shift at ␦ 4.45 (1H, J 1,2 ϭ 7.8 Hz) and a C1 chemical shift at ␦ 104.75. These H1 and C1 chemical shifts and the large J 1,2 coupling constant are consistent with this glycosyl residue (D) having a ␤-anomeric configuration. By comparing gCOSY and TOCSY (Fig. 3) spectra, it was found that the H4 signal resonates at ␦ H 3.91 and has small J 3,4 and J 4,5 coupling constants (Ͻ9.6 Hz), showing that this residue has a galacto-configuration. Therefore, this residue (D) was assigned as Gal.
The NMR and mass spectral data just described indicate that this HF-PS preparation consists of a microheterogeneous mixture of molecules due to nonstoichiometric O-acetyl substitution at O3 of the ManNAc residue, to a varying number of tetrasaccharide repeating units, and to the addition of HexNAc and HexHexNAc saccharides. This microheterogeneity impacts the chemical shift values, which makes determining the glycosyl sequence by NMR analysis difficult. Therefore, in order to reduce the molecular heterogeneity, the sample was treated with anhydrous hydrazine, which removes the O-acetyl groups while leaving the rest of the structure intact. The removal of O-acetyl groups was confirmed in the NMR experiments, and the proton and carbon chemical shifts were determined by one-dimensional proton NMR analysis and two-dimensional gCOSY, TOCSY, and gHSQC NMR experiments (spectra not shown). These assignments are given in Table 3. The inset in Fig. 2 shows the proton spectrum of the anomeric region of the de-O-acetylated HF-PS. After hydrazine treatment, NMR analysis showed the presence of four glycosyl residues between ␦ 4.4 and ␦ 6.0 and three N-acetyl methyl protons near ␦ 2.0, indicating the presence of three N-acetylamino sugars. The H1 (␦ 5.04) and the H3 (␦ 5.15) resonances due to the presence of an acetylated ␤-ManNAc residue are both absent in the de-O-acetylated HF-PS.
Due to spectral simplicity of the de-O-acetylated HF-PS, the glycosyl sequence was determined from a NOESY experiment. The NOE spectrum (Fig. 5) showed a through space interresidue connectivity between H1 of residue A (␦ 5.29) to H4 of residue B (␦ 3.66) along with an intraresidue NOE with H2 at  The assignments are as indicated, and the labeling for the various glycosyl residues is as defined in the legend to Fig. 2. The carbon chemical shift assignments for the various glycosyl residues are given in Table 3.

TABLE 3 1 H and 13 C chemical shifts for the B. cereus ATCC 10987 HF-PS
The values in parentheses are for the de-O-acetylated HF-PS.   (10) and proton NMR spectroscopy (12) as well as the composition and methylation analysis described above show that the HF-PS from the B. cereus type strain, ATCC 14579, has a different structure than the HF-PSs of B. cereus ATCC 10987 and B. anthracis. For example, it contains GalNAc, as is found in B. cereus ATCC 10987 HF-PS but not found in B. anthracis HF-PS, and it contains Glc rather than Gal, which is found in both B. cereus ATCC 10987 and in B. anthracis HF-PSs. The B. cereus ATCC 14579 HF-PS is similar to these latter HF-PSs in that it also contains both ManNAc and GlcNAc. However, despite the obvious structural differences in these HF-PSs, mass spectrometric analysis, using the reflectron mode, of the B. cereus ATCC 14579 HF-PS (Fig. 6) gives a spectrum with a pattern of ions very similar to that obtained for the B. cereus ATCC 10987 HF-PS (Fig. 1A). The ion with the greatest intensity is the [M ϩ Na] ϩ ion at m/z 1583.4, as is the case for the B. cereus ATCC 10987 HF-PS. The ion at m/z 1786.4 is consistent with an added HexNAc residue (i.e. ϩ203 mass   Table 4. units), and the ion at m/z 1989.6 is probably due to the addition of a second HexNAc residue. The ion at m/z 2354.6 is 365 mass units greater than the 1989.6 ion and is consistent with a HexHexNAc disaccharide added to the m/z 1989.6 structure. There are also ions consistent with the loss of a HexHexNAc from the 1583.5 ion (i.e. that at m/z 1218.4) and an ion at m/z 1015.3, which is consistent with the loss of a HexNAc from the 1218.4 ion. The proposed compositions for the pattern of ions are shown in Table 4. This pattern indicates that this HF-PS preparation consists of one, two, and three HexNAc 3 Hex trisaccharides, as observed for the B. cereus ATCC 10987 HF-PS (see Fig. 1A), with heterogeneity due to molecular species with added HexNAc and Hex residues. There is no evidence that the B. cereus ATCC 14579 HF-PS is acetylated, since ions with added increments of 42 mass units are not observed. do not yet know if it contains this same consensus structural feature; however, composition analysis together with the similarity of its mass spectrum with that of B. cereus ATCC 10987 HF-PS supports the possibility that these three HF-PSs share a general structural theme of a polysaccharide with an aminoglycosyl-rich backbone in which at least two of the aminoglycosyl residues are ManNAc and GlcNAc. The complete structure of the B. cereus ATCC 14579 HF-PS is under investigation and will be the subject of a subsequent report.

DISCUSSION
We have previously reported the structure of the HF-PS from B. anthracis, and comparison of NMR spectra showed that its structure varied from the HF-PS of B. cereus ATCC 10987 and ATCC 14579 (12). Here, we report the structure of a nonclassical SCWP polysaccharide isolated from B. cereus ATCC 10987 as well as structural data on this polysaccharide from the B. cereus type strain, ATCC 14579, and we have compared those structural data with the reported B. anthracis HF-PS structure (12). The results are summarized as follows: (i) the HF-PS from B. cereus ATCC 10987 is composed of a tetrasac-charide repeat unit consisting of a 36)-␣-D-GalNAc-(134)-␤-D-ManNAc-(134)-␤-D-GlcNAc-(13 trisaccharide in which the GalNAc residue is substituted at O3 with ␤-D-Gal and the ManNAc residue is 3-O-acetylated; (ii) there is heterogeneity in this HF-PS polysaccharide due to variation in the number of repeating units, the presence of O-acetyl groups, and the addition of ManNAc, GlcNAc, and GalGalNAc, respectively, to the direpeat and, possibly, mono-and trirepeat unit structures; (iii) in B. cereus ATCC 10987 and B. anthracis strains, there is a consensus HF-PS structural feature in that the repeating unit consists of a trisaccharide aminoglycosyl backbone in which two of the three aminoglycosyl residues are ManNAc and GlcNAc, whereas the third is either GlcNAc or GalNAc. Variability between the various HF-PS structures occurs in the substitution pattern of this trisaccharide with regard to both glycosyl and nonglycosyl substituents; (iv) the HF-PS from B. cereus ATCC 14579 is structurally more complex than those of B. cereus ATCC 10987 and B. anthracis. However, the similarities in the MS HF-PS ion patterns of these strains to that of the B. cereus ATCC 14579 HF-PS suggest that they share a general structural theme. Taken together, these data support the conclusion that the HF-PSs from B. anthracis strains, B. cereus ATCC 10987, and B. cereus ATCC 14579 each have unique structural features residing on an overall similar structural theme of a polysaccharide backbone that is It should be emphasized that the last structure has not been unambiguously determined and is currently under investigation. A consensus structure based on these structures is also given, in which X represents substitution of the aminoglycosyl backbone with other glycosyl residues, such as Gal or Glc, or with noncarbohydrate substituents, such as an O-acetyl group.