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
4,4,3	aDGalp	96.6	69.5	70.6	70.4	72.0	62.0
4,4	bLRhap	101.8	68.4	78.6	71.6	73.4	18.0
4,2	Ac	175.0-175.7	23.0-23.6
4	bDGlcpN	103.2	57.1	74.8	78.2	75.7	61.9
5	Ac	175.0-175.7	23.0-23.6
7	Ac	175.0-175.7	23.0-23.6
	aXLegp	174.3	97.0	40.6	77.7	52.0	71.1	54.3	67.5	20.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7	H8	H9
4,4,3	aDGalp	5.12	3.85	3.95	4.01	4.18	3.70-3.72
4,4	bLRhap	4.86	4.27	3.69	3.46	3.43	1.33
4,2	Ac	-	1.95-2.01
4	bDGlcpN	4.57	3.62	3.67	3.64	3.49	3.81-3.93
5	Ac	-	1.95-2.01
7	Ac	-	1.95-2.01
	aXLegp	-	-	1.90-2.46	4.00	3.81	4.27	3.84	3.84	1.13


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7	C8/H8	C9/H9
4,4,3	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
4,4	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
4,2	Ac		23.0-23.6/1.95-2.01
4	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
5	Ac		23.0-23.6/1.95-2.01
7	Ac		23.0-23.6/1.95-2.01
	aXLegp			40.6/1.90-2.46	77.7/4.00	52.0/3.81	71.1/4.27	54.3/3.84	67.5/3.84	20.4/1.13

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

845.802 g/mol (C₃₃H₅₅N₃O₂₂)