In this report we describe the structure of the polysaccharide released from B. 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 B. cereus ATCC 10987 strain, and from the B. cereus ATCC 14579 type strain and compared to 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 (MALDI-TOF MS), and 1- and 2-D nuclear magnetic resonance (NMR) spectroscopy. The HF-PSs from all of the B. anthracis isolates had an identical structure consisting of an amino sugar backbone of (R)6)--GlcNAc-(1(R)4)--ManNAc-(1(R)4)--GlcNAc-(1(R) in which the -GlcNAc residue is substituted with -Gal and -Gal at O3 and O4, respectively, and the -GlcNAc substituted with -Gal at O3. There is some variability in the presence of two of these three Gal substitutions. Comparison with the HF-PSs from B. cereus ATCC 10987 and B. cereus ATCC 14579 showed that the B. anthracis structure was clearly different from each of these HF-PSs and, further, that the B. cereus ATCC 10987 HF-PS structure was different from that of B. cereus ATCC 14579. The presence of a B. anthracis-specific polysaccharide structure in its vegetative cell wall is discussed with regard to its relationship to those of other Bacilli

NMR, structure, isolate, strain, polysaccharide, carbohydrate, cell, hydrogen, Research, type, complex, specific, spectrometry, cell wall, linkage, sugar, mass spectrometry, nuclear, nuclear magnetic resonance, resonance, spectroscopy, backbone, MALDI-TOF, polysaccharide structure, composition, MS, time, amino, aqueous, relationship, glycosyl, comparison, species, Bacillus, cell wall polysaccharide, PDF, MALDI-TOF MS, amino sugar, substitution, variability, matrix, hydrogen fluoride, matrix-assisted, HF, laser desorption, species-specific

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,4,2	Ac
4,4,3	aDGalp	100.3	70.3	70.6	70.3	72.2	61.9
4,4,4	bDGalp	104.1	72.5	73.8	70.0	73.8	61.9
4,4	aDGlcpN	99.7	54.1	76.3	77.5	71.9	68.1
4,2	Ac
4	bDManpN	99.7	55.0	73.8	75.3	76.3	62.2
2	Ac
3	aDGalp	98.8	70.0	70.6	72.2	72.2	61.9
	bDGlcpN	101.9	55.3	76.6	78.1	76.3	61.6


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,4,2	Ac
4,4,3	aDGalp	5.53	4.77	3.72	3.98	3.87	3.73
4,4,4	bDGalp	4.44	3.54	3.64	3.94	3.63	3.77-3.84
4,4	aDGlcpN	5.22	4.09	4.02	4.03	3.94	4.07-4.12
4,2	Ac
4	bDManpN	4.91	4.51	4.10	3.74	3.51	3.77-3.84
2	Ac
3	aDGalp	5.64	3.82	3.74	4.00	3.84	3.76
	bDGlcpN	4.67	3.92	3.92	4.10	3.54	3.77-3.84


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,4,2	Ac
4,4,3	aDGalp	100.3/5.53	70.3/4.77	70.6/3.72	70.3/3.98	72.2/3.87	61.9/3.73
4,4,4	bDGalp	104.1/4.44	72.5/3.54	73.8/3.64	70.0/3.94	73.8/3.63	61.9/3.77-3.84
4,4	aDGlcpN	99.7/5.22	54.1/4.09	76.3/4.02	77.5/4.03	71.9/3.94	68.1/4.07-4.12
4,2	Ac
4	bDManpN	99.7/4.91	55.0/4.51	73.8/4.10	75.3/3.74	76.3/3.51	62.2/3.77-3.84
2	Ac
3	aDGalp	98.8/5.64	70.0/3.82	70.6/3.74	72.2/4.00	72.2/3.84	61.9/3.76
	bDGlcpN	101.9/4.67	55.3/3.92	76.6/3.92	78.1/4.10	76.3/3.54	61.6/3.77-3.84

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OC[C@H]1O[C@H](O[C@@H]2[C@@H](CO)O[C@@H](O[C@@H]3[C@@H](CO)O[C@@H](*)[C@H](NC(C)=O)[C@H]3O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@@H](NC(C)=O)[C@H]2O)[C@H](NC(C)=O)[C@@H](O[C@H]2O[C@H](CO)[C@H](O)[C@H](O)[C@H]2O)[C@@H]1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O

1096.005 g/mol (C₄₂H₆₉R₂N₃O₃₀, R = next and previous repeats, not counted in mol weight)