Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(β1-3)]GlcNAc(β1-6)[Glc(β1-3)][ManNAc(α1-4)]GalNA c(α1-4)ManNAc(β1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm.

cell wall polysaccharide, Biofilm, Bacillus cereus

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
6,4,2	aDGalp	100.81	69.93	70.72	70.02	70.46	68.19
6,4	xR2HOGlt	180.00	80.72	32.67	30.05	178.70
6,2	Ac	?	23.52
6	aDFucpN4N	98.36	51.49	68.29	54.80	66.90	17.05
2	Ac	?	23.38
	bDGlcpN	102.52	56.91	73.39	70.92	75.63	66.61


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
6,4,2	aDGalp	4.987	3.813	3.948	3.918	4.131	3.633-3.903
6,4	xR2HOGlt	-	3.959	2.523	2.066	-
6,2	Ac	-	2.077
6	aDFucpN4N	4.915	4.104	4.102	4.323	4.295	1.132
2	Ac	-	2.045
	bDGlcpN	4.599	3.697	3.583	3.595	3.632	3.746-3.942


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
6,4,2	aDGalp	100.81/4.987	69.93/3.813	70.72/3.948	70.02/3.918	70.46/4.131	68.19/3.633-3.903
6,4	xR2HOGlt		80.72/3.959	32.67/2.523	30.05/2.066	
6,2	Ac		23.52/2.077
6	aDFucpN4N	98.36/4.915	51.49/4.104	68.29/4.102	54.80/4.323	66.90/4.295	17.05/1.132
2	Ac		23.38/2.045
	bDGlcpN	102.52/4.599	56.91/3.697	73.39/3.583	70.92/3.595	75.63/3.632	66.61/3.746-3.942

The spectrum also has 2 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

681.645 g/mol (C₂₇H₄₃R₂N₃O₁₇, R = next and previous repeats, not counted in mol weight)