The Structure of the Major Cell Wall Polysaccharide of Bacillus anthracis Is Species-specific

Abstract

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...

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

“…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).…”

“…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).…”

“…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.…”

“…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.…”

Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

“…Insertional lesions in bslO, sap, and csaB, as well as patA1 and patA2, increase the chain lengths of B. anthracis vegetative forms without affecting the size of bacterial cells (15)(16)(17)(18). bslO encodes a secreted cell wall hydrolase with S-layer homology (SLH) domains that bind to pyruvylated secondary cell wall polysaccharide (SCWP) (15), comprised of the repeating unit [¡4)-␤-ManNAc-(1¡4)-␤-GlcNAc-(1¡6)-␣-GlcNAc-(1¡)] n (19,20). BslO functions as a murein hydrolase when deposited at the division septa of vegetative cells and promotes cell separation, thereby reducing chain length (15).…”