The O-antigen is a part of the outer membrane of Gram-negative bacteria and is related to bacterial virulence. It is one of the most variable cell constituents, and its structural diversity is almost entirely due to genetic variation of the O-antigen gene cluster. In this study, the O-antigen structure of Escherichia coli O62 was elucidated by chemical analysis and nuclear magnetic resonance spectroscopy, but showing not consistent with the O-antigen gene cluster between conserved genes galF and gnd reported earlier. The complete genome of E. coli O62 was then sequenced and analyzed, and another O-antigen gene cluster was found and characterized that correlated perfectly with the established O-antigen structure. A deletion and complementation experiment confirmed the functionality of the novel gene cluster and demonstrated that the O62-antigen is synthesized by the ABC transporter-dependent system. To our knowledge, this is the first report that the O-antigen gene cluster is positioned at a novel locus in E. coli. Comparative analysis indicated that E. coli O62 likely originated from E. coli O68 via an IS event resulting in the repression of the O68-antigen synthesis, followed by the acquisition of a novel O-antigen gene cluster from Enterobacter aerogenes.

characterization, O-antigen, Escherichia coli, bacterial polysaccharide structure, O-antigen gene cluster, ABC transporter-dependent pathway

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,5,2	aDGlcp	99.1	72.4	74.2	70.8	73.6	61.9
3,5	bDGalf	106.8	87.7	75.6	81.5	76.1	62.8
3,2	aDGlcp	99.4	72.4	74.2	70.8	73.6	61.9
3	bDGalf	106.4	87.7	76.2	82.0	77.1	62.6
	bDGalf	109.0	81.1	83.3	82.9	71.7	64.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,5,2	aDGlcp	5.09	3.57	3.71	3.45	3.76	3.77-3.88
3,5	bDGalf	5.32	4.21	4.24	4.16	4.00	3.81-3.81
3,2	aDGlcp	5.08	3.57	3.71	3.45	3.76	3.77-3.88
3	bDGalf	5.29	4.21	4.23	4.05	4.00	3.81-3.81
	bDGalf	5.24	4.33	4.21	4.14	3.91	3.69-3.69


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,5,2	aDGlcp	99.1/5.09	72.4/3.57	74.2/3.71	70.8/3.45	73.6/3.76	61.9/3.77-3.88
3,5	bDGalf	106.8/5.32	87.7/4.21	75.6/4.24	81.5/4.16	76.1/4.00	62.8/3.81-3.81
3,2	aDGlcp	99.4/5.08	72.4/3.57	74.2/3.71	70.8/3.45	73.6/3.76	61.9/3.77-3.88
3	bDGalf	106.4/5.29	87.7/4.21	76.2/4.23	82.0/4.05	77.1/4.00	62.6/3.81-3.81
	bDGalf	109.0/5.24	81.1/4.33	83.3/4.21	82.9/4.14	71.7/3.91	64.2/3.69-3.69

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

810.705 g/mol (C₃₀H₅₀R₂O₂₅, R = next and previous repeats, not counted in mol weight)