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Journal of Bacteriology, January 2008, p. 112-121, Vol. 190, No. 1
0021-9193/08/$08.00+0     doi:10.1128/JB.01292-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cell Wall Carbohydrate Compositions of Strains from the Bacillus cereus Group of Species Correlate with Phylogenetic Relatedness

Christine Leoff,^1,3, Elke Saile,^1,2, David Sue,² Patricia Wilkins,² Conrad P. Quinn,² Russell W. Carlson,^1* and Elmar L. Kannenberg^1,3

Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd., Athens, Georgia 30602,¹ Centers for Disease Control and Prevention, 1600 Clifton Rd., MS D-11, Atlanta, Georgia 30333,² Departments of Microbiology and Biotechnology, University of Tübingen, D72076 Tübingen, Germany³

Received 9 August 2007/ Accepted 17 October 2007

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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 N-acetylglucosamine (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 plasmid-dependent 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.

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The Bacillus cereus group of bacteria contains the closely related species B. cereus, B. anthracis, and B. thuringiensis. B. cereus can be a potent opportunistic pathogen, while B. thuringiensis is an insect pathogen, and B. anthracis is the causative organism of anthrax. The distribution of B. anthracis spores in the U.S. postal system in 2001 demonstrated their potential as a bioterrorist weapon. Because of the risks these strains pose for public health, rapid differentiation and identification of the members the B. cereus group of strains through molecular means is an important and ongoing endeavor in many laboratories.

To date, the cell wall carbohydrates in the B. cereus group of strains have, nonetheless, not been investigated systematically with regard to occurrence, structural peculiarities, and their usefulness for taxonomic classification and strain identification. Carbohydrates are a common feature of bacterial cell walls, e.g., as capsules, as S-layer protein components, or as various other cell wall glycoconjugates. In the B. cereus group of strains, these cell wall components can vary from strain to strain and display heterogeneity. For example, while B. cereus and B. thuringiensis are typically not encapsulated, in a number of pathogenic B. cereus strains the occurrence of as yet not fully characterized capsules was indicated, which seems to be, at least in one case, carbohydrate in nature (45). In contrast, pathogenic B. anthracis cells are surrounded by capsules that are comprised of poly--D-glutamic acid (17). In a survey on S-layer distribution in strains of the B. cereus group, ca. 40% of all strains had S-layers. Of the strains that originated from clinical isolates, the vast majority (ca. 70%) contained S-layers in their cell walls (34). The carbohydrate components of S-layers in B. cereus group of strains have thus far not been characterized systematically.

In many bacterial genera, the cell walls are well established as diagnostic targets (1, 33, 48, 49), carbohydrate-based vaccine antigens (29, 49), and virulence factors (35). Therefore, the characterization of cell walls of B. anthracis and other strains of the B. cereus group could be important for identifying potential vaccine antigens, for diagnostics, and for elucidating the molecular basis for their virulence and pathogenicity. Infection by pathogenic strains of the B. cereus group likely involves multiple components of the cell wall, including the cell wall carbohydrate-containing components, that interact with the host. During an infection, these cell wall components may function in bacterial adhesion to host cells and also as barriers to the host defense mechanism, thereby acting as virulence factors. Should the cell wall carbohydrate of B. anthracis prove to have such functions, as is the case with many other bacteria, this would ensure its structural conservation, making this carbohydrate a potentially good candidate for the identification and classification of Bacillus species, as well as for development into a vaccine antigen.

Traditionally, Bacillus species have been differentiated based on their phenotypic and biochemical characteristics. Recently, molecular methods of classification have become more prevalent (2, 3, 18, 19, 22, 24, 25). These molecular classification methods have been used to regroup Bacillus strains. The phylogenetic picture that is emerging from these studies for strains of the B. cereus group is only partially in accordance with the more traditional classification scheme and is, to a degree, still in flux. For example, B. cereus group strains have traditionally been classified as three species: B. cereus, B. thuringiensis, and B. anthracis. In contrast, fluorescent heteroduplex analysis placed these species in only two subgroups (31). Similarly, albeit on a different set of bacterial strains, the analysis of small acid-soluble proteins in the B. cereus group by mass spectrometry also led to only two subgroups (6, 7). These recent findings, as well as those based on other methods including comparative Bacillus species genome analyses, will alter the more traditional Bacillus taxonomic groupings (39, 41). In particular, among these molecular approaches, multilocus sequence typing (MLST) analysis (2, 19, 25, 38) is widely used because of its power to resolve the relatedness of even closely related strains because its findings are unambiguous and because the method is truly portable among laboratories (19).

A study using MLST that was published in 2004 reported that a collection of B. cereus group strains representing 59 sequence types could be assigned to three clades and nine lineages (38). The same laboratory evaluated, also by using MLST, the phylogeny of invasive B. cereus isolated from clinical infections (2). Interestingly, the study showed that pathogenic strains were not restricted to a single clonal group or lineage but were genomically diverse and related to strains traditionally grouped as B. anthracis, B. cereus, or B. thuringiensis. These findings were particularly interesting since it showed that all B. cereus group strains obtained from human or animal infections, including anthrax and bacterial pneumonia, are closely related to each other (2, 38).

Little is known about the carbohydrates that comprise the cell walls of pathogenic B. cereus group strains. However, recent insights into the relatedness of these strains raise the intriguing question of whether function or phylogenetic relatedness governs the occurrence of their cell wall carbohydrates. Previous studies have established a precedent for distinctive glycosyl compositions of the total cell walls of representative strains from B. anthracis, B. cereus, and B. thuringiensis. For example, galactose (Gal) was found only in B. anthracis cell walls, whereas glucose (Glc) and N-acetylgalactosamine (GalNAc) were present in B. cereus cell walls (16, 50). These published data suggest that there could be cell wall carbohydrates that are specific to each of these three Bacillus species. However, a systematic comparison of the cell wall compositions and/or structures from members of the B. cereus group of bacteria as a function of the more detailed MLST phylogenetic classification has not been reported.

In the present study we investigated the glycosyl compositions of the cell walls from a collection of strains of the B. cereus group (Table 1). The strains described here were picked on the basis of their MLST phylogenetic relatedness, as put forward by Priest et al. (38), with a suggested modification adding the Cereus IV lineage to clade 1 (36). In addition, since recent sequencing projects of whole genomes from B. cereus group strains showed that genes involved in carbohydrate biosynthesis and metabolism not only are localized on the chromosome but also can be encoded on plasmids (40), we investigated whether cell wall composition is influenced by the virulence plasmid content in selected B. anthracis strains. The data demonstrate that there is variation in the glycosyl compositions of cell walls among even closely related B. cereus group strains and that this compositional variation correlates with differences in phylogenetic relatedness. Further, we show that under standard laboratory growth conditions the types of carbohydrates found in the cell walls of B. anthracis strains may depend, to some extent, on their virulence plasmid content.

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TABLE 1. MLST groupings, clinical manifestations, and sources of strains in this study

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Bacterial strains and culture conditions. Most B. anthracis strains were obtained from the Centers for Disease Control and Prevention culture collection. The strains B. anthracis 7702 and UT60 were obtained from T. Koehler, University of Texas-Houston Health Science Center. A list of bacterial strains used in the present study and their sources are given in Table 1. Cells cultured overnight in brain heart infusion medium (BHI; BD BBL, Sparks, MD) containing 0.5% glycerol were used to inoculate four 250-ml volumes of BHI medium in 1-liter Erlenmeyer flasks the following morning. Cultures were grown at their optimum growth temperatures: B. anthracis at 37°C and B. cereus and B. thuringiensis at 30°C with shaking at 200 rpm. Growth was monitored by measuring the optical density of the cultures at 600 nm. In mid-log phase (i.e., at optical densities at 600 nm of approximately 2.3 to 2.7 for B. anthracis and 4.0 to 4.6 for B. cereus and B. thuringiensis), the cells were harvested by centrifugation (8,000 x g, 4°C, 15 min), washed two times in sterile saline, enumerated by dilution plating on BHI agar plates, and then autoclaved for 1 h at 121°C before further processing.

Preparation of bacterial cell walls. The bacterial cell walls were prepared by modification of a previously described procedure (5). The autoclaved bacterial cells (3 x 10⁸ to 3 x 10⁹ CFU/ml) were disrupted in 40 ml of sterile saline on ice by four 10-min sonication cycles. The complete or nearly complete disruption of cells was checked microscopically. Unbroken cells were removed by low-speed centrifugation (8,000 x g, 4°C, 15 min). The separated pellet and supernatant fractions were stored at –70°C. The cell walls were separated from the low-speed supernatants by ultracentrifugation at 100,000 x g at 4°C for 4 h. The resulting cell wall pellets were washed by suspension in cold, deionized water, followed by an additional ultracentrifugation at 100,000 x g at 4°C for 4 h and then lyophilized.

Release of phosphate-bound polysaccharides from the cell wall. Phosphate-bound polysaccharides were released from the cell walls by treatment with aqueous hydrogen fluoride (HF) according to a modification of the procedure described by Ekwunife et al. (14). Briefly, the cell walls are subjected to 47% HF under stirring at 4°C for 48 h. The reaction mixture was neutralized with NH₄OH and subjected to a 10-min low-speed centrifugation, and the supernatant with the released polysaccharides was lyophilized, redissolved in deionized water, and subjected to a chromatographic size separation on a BioGel P2 column (Bio-Rad). The fractions eluting from the BioGel P2 column were monitored by using a refractive index detector. Polysaccharide-containing peaks were pooled, lyophilized, and analyzed by gas chromatography-mass spectrometry (GC-MS) as described below. These HF-released polysaccharides are referred to as HF-PSs.

Glycosyl composition analysis. The carbohydrate profiles were determined by GC-MS analysis of the trimethylsilyl (TMS) methylglycosides as previously described by York et al. (51). The cell walls and HF-PSs were subjected to methanolysis at 80°C for 18 h in methanolic HCl (1 M). The resulting methyl glycosides were N acetylated, trimethylsilylated, and then analyzed by GC-MS analysis (5890A GC-MS; Agilent Technologies, Palo Alto, CA) using a 30-m DB-1 fused silica capillary column (J&W Scientific, Folsom, CA). Inositol was used as an internal standard, and retention times were compared to authentic standards. Composition analysis was done on samples obtained from at least two independent cultures of each strain, and each sample was also analyzed at least two times.

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Glycosyl composition analysis for members of the B. cereus group. The strains investigated in the present study and their classifications are given in Table 1. Examples of GC profiles comparing a B. anthracis Sterne 34F₂ cell wall sample with those of B. cereus ATCC 10987 and with B. cereus ATCC 14579 (the B. cereus type strain) are given in Fig. 1. The compiled glycosyl compositions for the cell wall samples from each strain are shown in Table 2. The glycosyl composition of the cell walls from all of the B. anthracis strains contained Glc, Gal, N-acetyl mannosamine (ManNAc), and GlcNAc. Qualitatively, B. cereus strains belonging to clade 1, lineage Cereus IV (clade 1/Cereus IV), had the same cell wall glycosyl components as strains belonging to clade 1/Anthracis. The cell walls from strains of Clade 1/Cereus III differed from those of clade 1/Anthracis in that they contained additional mannose (Man), while strains belonging to clade 1/Cereus I differed in that their cell walls also contained GalNAc, and the cell walls from all strains belonging to clade 2 lacked Gal and contained GalNAc.

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FIG. 1. GC-MS sugar profiles obtained from B. anthracis Sterne 34F2, B. cereus ATCC 10987, and type strain B. cereus ATCC 14579 vegetative cell walls after hydrolysis of the total cell wall preparations and derivatization into TMS methylglycosides. The sample origin is indicated in the profiles. Inos, inositol (internal standard); MurNAc, N-acetylmuramic acid. *, Noncarbohydrate components (not further characterized).

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TABLE 2. Sugar composition of cell walls from members of the B. cereus group

There were some notable differences with regard to the relative amounts of certain glycosyl residues even among strains belonging to the same clade and lineage. For example, strain B. anthracis Sterne 34F₂ had cell walls with notably decreased levels of ManNAc compared to B. anthracis Ames and B. anthracis Pasteur 4229, while B. anthracis 7702 and its atxA deletion mutant UT60 showed an increase in cell wall Glc levels compared to the other B. anthracis strains. Quantitative differences in various glycosyl components were also present between B. cereus clade 1/Cereus I strains F666 and ATCC 10987. Relative to strain B. cereus ATCC 10987, strain B. cereus F666 contained significantly increased amounts of Glc and decreased amounts of Gal. Differences were also noticeable in strains B. cereus B5780 and 03BB102 cell walls, both belonging to clade 1/Cereus III. Strain B5780 had a much higher level of Glc and lower levels of both Gal and ManNAc than strain 03BB102.

Effects of plasmid content on the glycosyl composition in B. anthracis cell walls. In order to determine whether the plasmid content has an effect on the glycosyl composition of cell walls in the different B. anthracis strains, we normalized the glycosyl residue percentages shown in Table 2 to the amount of Gal for each sample. The reason for normalizing to Gal is that, as described below, Gal is the major glycosyl residue found in the HF-PSs for each of the B. anthracis strains, and the HF-PSs of these strains all have the same structure, as reported by Choudhury et al. (9). These Gal-normalized values are given in Table 3, together with the plasmid content in the different strains. Qualitatively, the sugar profiles of the different cell walls were not affected by the plasmid content. Quantitatively, the glycosyl composition of the cell wall from B. anthracis Pasteur that lacks pXO1 (pXO1^–) was the same as that of B. anthracis Ames that contains both pXO1 and pXO2. This finding suggests that the plasmid pXO1 has no impact on sugar composition of the cell wall. In contrast, pXO2^– B. anthracis strains (Sterne 34F₂ and 7702) have cell walls with reduced amounts of ManNAc and increases in Glc relative to Gal, suggesting that the absence of the pXO2 plasmid impacts cell wall glycosyl composition. While the increase in Glc was modest for B. anthracis Sterne 34F2 cell walls, B. anthracis 7702 cell walls displayed a threefold increase (relative to Gal) in Glc levels compared to the cell wall from B. anthracis Ames. The increase in the amounts of cell wall Glc was even more pronounced in B. anthracis UT60, a derivative of B. anthracis 7702 that has a deletion mutation in the atxA regulatory gene on pXO1 in addition to lacking pXO2 (10). In this strain we observed a 5-fold increase in Glc compared to the cell wall from B. anthracis Ames and an 60% increase in Glc compared to the amounts in the parent strain B. anthracis 7702. These data indicate that the absence of pXO2 in combination with the deletion of the regulatory gene atxA from pXO1 result in detectable changes in the cell wall glycosyl composition of B. anthracis.

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TABLE 3. Effect of different plasmid combinations on the sugar composition (normalized to the amount of Gal) of the B. anthracis cell walls

Composition of HF-PSs. Polysaccharides that are attached to the bacterial cell walls through phosphate bonds can be released through HF treatment (26). This procedure was used in other studies to obtain the cell wall polysaccharide from B. anthracis that is thought to anchor the S-layer protein to the peptidoglycan (14, 32). The glycosyl residue compositions of the HF-PSs from the investigated strains of B. anthracis (clade 1/Anthracis), and B. cereus (clade 1/Cereus I, III, and IV and clade 2/Tolworthi) are presented in Table 4, and GC-MS profiles of the cell wall compositions compared to the HF-PSs for one preparation each from pXO2^– B. anthracis Sterne 34F₂ and B. cereus ATCC 10987 are shown in Fig. 2. These results show that the HF-PSs from B. anthracis Ames, B. anthracis Pasteur, B. anthracis Sterne 34F₂, and B. anthracis UT60 all have the same glycosyl residue composition, both qualitatively and quantitatively; they all contain Gal, ManNAc, and GlcNAc in approximately a 3:1:2 ratio as previously reported by Choudhury et al. (9). Each of these polysaccharides has a small amount of Glc, but further structural analysis has shown (9) that this is due to contamination by a Glc-rich component that is not part of this polysaccharide. The presence of a Glc-rich polysaccharide in B. anthracis cell walls that is not part of the HF-PS was most obvious for the preparation of one culture of B. anthracis Sterne 34F₂ (Fig. 1). In that preparation, the cell wall had a relatively large Glc content (Fig. 1), while the HF-PS was greatly reduced in Glc. The "missing" Glc was found in the cell wall debris after HF treatment and, therefore, B. anthracis Sterne 34F₂ apparently has a Glc-rich component in the cell wall that is not released by HF treatment. This relatively large amount of Glc-rich cell wall component was only observed in one of three B. anthracis Sterne 34F₂ cultures. Because of the variability of the increased level of Glc from different cultures, it is not clear what actually governs the different levels of the possible Glc-rich cell wall component observed in the various Sterne 34F₂ cell wall preparations. However, the increased cell wall Glc level, even though variable from different Sterne 34F₂, cultures, was observed only in the pXO2^– B. anthracis strains. The relationship of this possible Glc-rich component with the loss of the pXO2 plasmid requires further investigation.

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TABLE 4. Sugar composition of isolated polysaccharides released from the Bacillus cell walls through HF treatment

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FIG. 2. GC-MS sugar profiles obtained from B. anthracis Sterne 34F2 (A) and B. cereus ATCC 10987 (B) vegetative cell walls after hydrolysis of the total cell wall preparations and derivatization into TMS methylglycosides. Sample origins are indicated in the profiles. HF-PS samples were released from cell walls through HF treatment and purified on BioGel P2 columns. Inos, inositol (internal standard); MurNAc, N-acetylmuramic acid. *, Noncarbohydrate component (not further investigated).

The finding that the cell walls of several pXO2^– B. anthracis strains may contain a Glc-rich component that is not released by treatment with aqueous HF was also observed in one cell wall preparation for B. cereus ATCC 10987 (Fig. 2). This result, as with results for extracts from pXO2^– B. anthracis strains, indicates that B. cereus ATCC 10987 contains a Glc-rich polysaccharide that is not released by HF treatment. As with the pXO2^– B. anthracis strains, the Glc-rich cell wall component was found in the cell wall debris after HF treatment. Further structural analysis (unpublished data) shows that the relatively small amount of Glc found in the B. cereus 10987 HF-PS is due to residual contamination by a four-linked Gl-containing component. Thus, we conclude that the minor amount of Glc found in the B. cereus ATCC 10987 HF-PS is not part of this polysaccharide and, therefore, this HF-PS consists of Gal, ManNAc, GlcNAc, and GalNAc in a 1:1:1:1 ratio. It clearly has a different structure than the B. anthracis HF-PS. This structural difference was also supported by a comparison of the proton nuclear magnetic resonance spectra of these HF-PSs in the report by Choudhury et al. (9). The HF-PS isolated from strain B. cereus F666 (this strain is in the same clade 1/Cereus I lineage as strain B. cereus ATCC 10987) has a glycosyl composition that resembles the HF-PS of strain B. cereus ATCC 10987 but with a significantly increased amount of Glc. In fact, the HF-PS from strain F666 showed three times the amounts of Glc compared to the HF-PS from B. cereus ATCC 10987. This result suggests that Glc is a part of the F666 HF-PS and, therefore, this HF-PS likely consists of Glc, Gal, ManNAc, GlcNAc, and GalNAc in a 1:1:1:1:1 ratio.

The strains that belong to the B. cereus group clade 1/Cereus III, strains B5780 and 03BB102, showed more pronounced differences from one another in their HF-PS sugar compositions. Both strains contained a small amount of Man in their isolated HF-PS fractions, which was not observed in the other HF-PSs examined. In addition, strain B5780 HF-PS contained larger amounts of Glc and lower amounts of Gal and ManNAc than the strain 03BB102 HF-PS, which contained a small amount of Glc and larger amounts of Gal and ManNAc (Table 4). It is possible that the glycosyl residues present in small amounts are due to low levels of contaminating carbohydrates that are not part of the HF-PS structures. If this were the case, then the HF-PS of strain B. cereus B5780 would be composed of Glc and GlcNAc in a 2:1 ratio, and the HF-PS of B. cereus 03BB102 would be composed of Gal, ManNAc, and GlcNAc in a 6:1:2 ratio. Further structural investigation of these HF-PSs is in progress.

The HF-PS preparations that were most similar to the B. anthracis HF-PSs were from the B. cereus group strains that belong to the clade 1/Cereus IV lineage: G9241 and 03BB87. These strains, as with strain 03BB102, are pathogens that caused severe pneumonia (20). The HF-PSs from strains G9241 and 03BB87 consisted of Gal, ManNAc, and GlcNAc in a 3:1:1 (or 3:1:2) ratio. The ratio of these glycosyl residues in the B. anthracis HF-PS is 3:1:2. We are currently characterizing the structures of the 03BB87 and G9241 HF-PSs to determine whether they are the same or different from the B. anthracis HF-PS structure.

The HF-PS from the type strain B. cereus ATCC 14579 (clade 2/Tolworthi) also showed a consistent small decrease in Glc content compared to its cell wall (compare Tables 2 and 4). This result indicates that the B. cereus ATCC 14579 cell wall contained a Glc-rich component that was not released from the cell wall by HF treatment. Unlike the HF-PSs from the B. anthracis strains and from B. cereus ATCC 10987, Glc is also a major glycosyl residue in the B. cereus ATCC 14579 HF-PS and, it is therefore likely that Glc is a component of this polysaccharide. This was verified by further structural analysis of this HF-PS (unpublished data). The components of the HF-PS from B. cereus ATCC 14579 are Glc, ManNAc, GlcNAc, and GalNAc in approximately a 1:1:2:1 ratio.

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We investigated cell wall compositions from a selection of strains belonging to the B. cereus group species B. anthracis, B. cereus, and B. thuringiensis. Recent investigations into the phylogenetic relatedness of these B. cereus group strains, e.g., MLST, offer a more differentiated picture than previous classification schemes and resulted in separating these strains into two clades and several lineages (Table 1) (2, 38). Here we showed that the glycosyl residue composition of the cell walls varied significantly both qualitatively and quantitatively among the investigated strains in a manner that reveals possible correlations with their phylogenetic relatedness. In summary, we observed the following. (i) B. cereus strains that are closely related had cell wall glycosyl compositions that qualitatively varied from one another in a clade/lineage-specific manner. (ii) Quantitative glycosyl analysis showed that strains belonging to the same lineage vary from one another in the amounts of various glycosyl residues, indicating the presence of strain-specific cell wall carbohydrates. (iii) Analysis of the cell walls from recently discovered pathogenic B. cereus strains that caused severe pneumonia, i.e., strains 03BB102, 03BB87, and G9241 (20, 21), showed that they have glycosyl compositions that were most similar to the cell walls of the B. anthracis strains. (iv) The plasmid content of B. anthracis strains appeared to affect cell wall glycosyl compositions, i.e., the amounts of ManNAc and Glc were lower and higher, respectively, in the cell walls from strains that lacked the pXO2 virulence plasmid, and the amount of a possible Glc-rich non-HF-PS cell wall was particularly increased in an atxA mutant of a pXO2-minus B. anthracis strain. (v) The HF-PSs released from the cell walls of the different B. anthracis strains all had the same Gal/ManNAc/GlcNAc ratio, i.e., 3:1:2, as previously reported (9), a finding consistent with the fact that they have the same structure (9). (vi) The HF-PSs from strains of the B. cereus group clade 1/Cereus I (i.e., B. cereus ATCC 10987 and F666), clade 1/Cereus III (i.e., B. cereus B5780 and 03BB102), and clade 2/Tolworthi (i.e., the type strain B. cereus ATCC 14579), each had a unique glycosyl composition that was different from the B. anthracis HF-PSs, indicating that they had structures different from one another and from the B. anthracis HF-PS structure.

To our knowledge, this is the first report that compares, in a systematic manner, the cell wall carbohydrates of several pathogenic and nonpathogenic members of the B. cereus with known phylogenetic grouping based on MLST analysis (2) (38). Earlier studies by Fox et al. (15, 50) determined carbohydrate profiles from vegetative cells and spores of a number of B. cereus and B. anthracis strains that had less clearly defined relationships. As expected, our findings corroborate some of those reported by Fox and coworkers (15, 50). These researchers showed that, in addition to the major glycosyl residues, minor amounts of rhamnose, ribose, and methylated sugars were present in the cell wall preparations; however, these glycosyl residues were attributed to contamination from spore components and RNA (15, 50). It is known that the exosporium BclA protein is glycosylated by a rhamnose-containing oligosaccharide (11).

Our comparative analyses of the cell walls from MLST-defined Bacillus strains provide new information that correlates with their phylogenetic relatedness. Even though our study involved a limited number of strains, the qualitative glycosyl residue differences suggest that cell wall compositions qualitatively varied in a clade/lineage-specific manner. In addition, comparison of two B. cereus strains, B5780 and 03BB102, both belonging to lineage Cereus III of clade 1 showed that, although they contain the same glycosyl residues, these residues are present at very different levels (Table 2). This result suggests the possibility of strain-specific quantitative differences that could, in some cases, allow identification of strains within a single B. cereus lineage. However, a larger sample of Bacillus strains is needed to determine breadth and consistency of these qualitative and quantitative differences.

Glycosyl compositions of the cell walls of B. anthracis strains before and after treatment with HF revealed that the absence of plasmid pXO2 may have some impact on cell wall glycosyl composition. Although the plasmid effects on cell wall carbohydrates are preliminary and in need of confirmation by examining genetically better defined strains, it is worth noting that we observed consistently decreased relative amounts of ManNAc and variably increased levels of Glc (relative to the amounts of Gal) in the cell walls of all B. anthracis strains lacking pXO2. The fact that the HF-PS from all of the pXO2^– B. anthracis strains had the same glycosyl composition and structure (9) as the HF-PSs from B. anthracis Ames and Pasteur suggests that the lower level of ManNAc and increased level of Glc in the cell walls reflect changes in carbohydrate structures that are not part of the HF-PS. An additional effect on cell wall glycosyl composition was detected in B. anthracis UT60; namely, the deletion of atxA from pXO1 results in higher levels of Glc in the cell wall (compared to its parent strain, B. anthracis 7702), presumably due to larger amounts of the Glc-rich non-HF-PS component in its cell wall. Taken together, these results indicate that the pXO1 and pXO2 plasmids may have a role in determining the presence or absence of a Glc-rich component in some cell walls even though there are no known carbohydrate synthesis-related genes on pXO1 or pXO2 that could easily explain the observed glycosyl changes. The gene products of the majority of open reading frames predicted on the pXO1 and pXO2 virulence plasmids are still unidentified (40). It may well be that there are open reading frames that encode as-yet-unidentified carbohydrate synthesis-related genes. In the case of B. anthracis UT60, the deleted atxA gene located on virulence plasmid pXO1 encodes a global regulator and the major transcriptional activator of the pXO1-borne anthrax toxin genes (4). In a genetically complete strain containing both pXO1 and pXO2, atxA has also been shown to be indirectly involved in the regulation of the capsule biosynthesis operon capBCAD located on pXO2 (13). The cap genes are essential for the encapsulation of B. anthracis cells by a poly--D-glutamic acid, one of the identified B. anthracis virulence factors necessary for the protection of B. anthracis cells inside the host (23, 30). The stimulating effect on the Glc level and the relatively lower amount of ManNAc in B. anthracis UT60 (and the other pXO2^– B. anthracis strains) may indicate additional and previously unknown regulatory roles of atxA and of pXO2-encoded genes in cell wall polysaccharide biosynthesis. Further work using isogenic strains of B. anthracis is needed to determine the significance and role of these plasmid effects on the cell wall carbohydrates.

As a first approach to determining the cell wall polysaccharide structures underlying the observed sugar composition profiles, phosphate-bound cell wall polysaccharides were released by HF treatment of the cell walls and purified. This procedure was used to purify the cell wall from B. anthracis that is thought to anchor the S-layer protein to the peptidoglycan (32). Composition analysis of these HF-PSs from the different B. anthracis strains revealed that all had the same 3:1:2 Gal/ManNAc/GlcNAc ratio, reflecting the identical structures of these polysaccharides (9). Since the HF-PS compositions and structures from all of the B. anthracis strains were the same, it is likely that their structures are independent of the presence or absence of the virulence plasmids pXO1 or pXO2. The B. anthracis HF-PSs were clearly different in glycosyl composition from the HF-PSs from the cell walls of other B. cereus group members which differed from one another. Although it is possible that the HF-PSs from the B. cereus strains vary in a manner that correlates with clade or lineage, further work on more strains would be required to determine the validity of this possibility.

An interesting observation is the similarity of glycosyl compositions among the cell walls of B. cereus strains that have recently been shown to cause severe pneumonia in humans (20, 21) to those of B. anthracis (Table 2). These clinical strains, namely, B. cereus G9241, 03BB102, and 03BB87, belong to clade 1, lineage Cereus III or IV (36). Comparing small acid-soluble proteins in B. anthracis to those in strain B. cereus G9241, a recent report noticed that the acid-soluble proteins of G9241 fell into a more distantly related protein cluster and stated that, on the basis of this criterion, pathogenicity and phylogenicity are not necessarily correlated features (7). Our result indicates that the cell walls of these pathogenic B. cereus strains may contain carbohydrates that have structural features in common with each other and with those of B. anthracis. The HF-PS preparations of these B. cereus strains displayed glycosyl compositions that were relatively similar to one another and to the HF-PSs from the B. anthracis strains (Table 4). This finding could be taken as an indication for the functional importance of the HF-PS (and the S-layer anchoring mechanism) in virulence and, possibly, of its relative independence from phylogenetic strain relatedness in pathogenic B. cereus strains.

B. cereus strains G9241, 03BB87, and 03BB102 all contain at least considerable numbers of genes with high similarity to genes of the virulence plasmid pXO1 of B. anthracis (e.g., B. cereus G9241 carries a plasmid that is almost identical to B. anthracis pXO1) (20, 21). Recently, "Bacillus anthracis-like" isolates were obtained from chimpanzees and gorillas from Cote d'Ivoire and Cameroon that were thought to have died from anthrax-like disease (24a, 28). Interestingly, based on molecular analyses (MLST and others), these strains fell outside the well-supported cluster of classic B. anthracis strains and instead clustered with B. cereus and B. thuringiensis strains, most closely with a recently described atypical and pathogenic B. thuringiensis (24a, 28). These B. anthracis-like isolates from great apes reportedly contain both pXO1 and pXO2 plasmids, while the pathogenic B. cereus strains 03BB102, G9241, and 03BB87 all contain a pXO1-like plasmid but not pXO2 (20). The similarity of the HF-PS compositions for strains 03BB87, 03BB102, and G9241 to those observed for the B. anthracis HF-PSs suggests that the underlying HF-PSs in these strains may be structurally related. Perhaps, the HF-PS structure found in B. anthracis and, possibly, the related HF-PS structures of the pathogenic B. cereus strains are necessary for virulence and/or are a characteristic of B. cereus strains that were able to acquire one or both of the B. anthracis virulence plasmids. It is not known whether the African gorilla isolates contain HF-PSs that corroborate these suspicions. To date, these strains have not yet been characterized with regard to their cell wall carbohydrates. The HF-PS structures of the B. cereus strains causing severe pneumonias are currently being investigated in our laboratory.

Much remains to be determined regarding the genetic basis for the synthesis of cell wall carbohydrates, the exact location of these molecules in the cell wall, and their functions. However, the results described here suggest that description of the of cell wall carbohydrates of the B. cereus group strains will eventually be useful for strain classification and, therefore, for the development of diagnostic and vaccine applications. In addition, the functional importance of these molecules with regard to virulence and pathogenicity requires further structural analysis, which is currently under way in our laboratory.

 
We thank Theresa Koehler, University of Texas, Houston Health Science Center, Houston, TX, for kindly providing the strains B. anthracis 7702 and UT60.

This study was supported by NIAID grant R21 AI059577 (R.W.C.) and also, in part, by DOE grant DE-FG02-93ER20097 (to the Complex Carbohydrate Research Center).

 
* Corresponding author. Mailing address: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602. Phone: (706) 542-4439. Fax: (706) 542-4412. E-mail: rcarlson{at}ccrc.uga.edu

Published ahead of print on 2 November 2007.

C.L. and E.S. contributed equally to this study.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, January 2008, p. 112-121, Vol. 190, No. 1
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