Lipopolysaccharides on the cell surface of Gram-negative bacteria are an important factor in pathogenicity, and the O-specific polysaccharide chain (O-polysaccharide, O-antigen) defines the immunospecificity of different bacterial strains. Cronobacter turicensis is an emerging foodborne pathogen which causes severe invasive infections in neonates. In this study, a new O serotype, C. turicensis O2, was established, the structure and genetics of the O-antigen were investigated, and a serotype-specific gene was identified. Sugar and methylation analyses, and nuclear magnetic resonance spectroscopy indicated that the O-polysaccharide contains D-galactose (D-Gal), N-acetyl-D-glucosamine (D-GlcNAc), L-rhamnose (L-Rha) and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (di-N-acetyllegionaminic acid, Leg5Ac7Ac). The structure of the tetrasaccharide repeat of the O-polysaccharide was established as: [Formula: see text]. The O-antigen gene cluster of C. turicensis O2 was sequenced and compared with related gene clusters from available databases. Putative genes for the synthesis of L-Rha and Leg5Ac7Ac, and genes encoding sugar transferases and O-antigen processing genes (wzx and wzy) were found. The tentatively assigned functions of the O-antigen genes were in agreement with the structure of the O-polysaccharide. In addition, primers based on the wzy gene were shown to be specific for C. turicensis O2 in a screen against 145 strains.

legionaminic acid, O-antigen gene cluster, O-Polysaccharide structure, Cronobacter turicensis, specific gene

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7	C8	C9
4,3,3,5	Ac	175.0-175.7	23.0-23.6
4,3,3,7	Ac	175.0-175.7	23.0-23.6
4,3,3	aXLegp	174.3	101.0	41.3	79.0	51.4	72.9	55.0	68.7	19.5
4,3	aDGalp	96.6	69.5	70.6	70.4	72.0	62.0
4	bLRhap	101.8	68.4	78.6	71.6	73.4	18.0
2	Ac	175.0-175.7	23.0-23.6
	bDGlcpN	103.2	57.1	74.8	78.2	75.7	61.9


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
4,3,3,5	Ac	-	1.95-2.01
4,3,3,7	Ac	-	1.95-2.01
4,3,3	aXLegp	-	-	1.80-3.01	3.66	3.80	3.88	3.86	3.99	1.18
4,3	aDGalp	5.12	3.85	3.95	4.01	4.18	3.70-3.72
4	bLRhap	4.86	4.27	3.69	3.46	3.43	1.33
2	Ac	-	1.95-2.01
	bDGlcpN	4.57	3.62	3.67	3.64	3.49	3.81-3.93


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
4,3,3,5	Ac		23.0-23.6/1.95-2.01
4,3,3,7	Ac		23.0-23.6/1.95-2.01
4,3,3	aXLegp			41.3/1.80-3.01	79.0/3.66	51.4/3.80	72.9/3.88	55.0/3.86	68.7/3.99	19.5/1.18
4,3	aDGalp	96.6/5.12	69.5/3.85	70.6/3.95	70.4/4.01	72.0/4.18	62.0/3.70-3.72
4	bLRhap	101.8/4.86	68.4/4.27	78.6/3.69	71.6/3.46	73.4/3.43	18.0/1.33
2	Ac		23.0-23.6/1.95-2.01
	bDGlcpN	103.2/4.57	57.1/3.62	74.8/3.67	78.2/3.64	75.7/3.49	61.9/3.81-3.93

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1C[C@](O[C@H]2[C@@H](O)[C@@H](CO)O[C@H](O[C@@H]3[C@@H](O)[C@H](C)O[C@H](O[C@@H]4[C@@H](CO)O[C@@H](*)[C@H](NC(C)=O)[C@H]4O)[C@@H]3O)[C@@H]2O)(C(=O)O)O[C@@H]([C@H](NC(C)=O)[C@@H](C)O)[C@@H]1NC(C)=O

827.787 g/mol (C₃₃H₅₃R₂N₃O₂₁, R = next and previous repeats, not counted in mol weight)