O-antigen is part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, and contributes the major antigenic variability to the cell surface. Screening for the Escherichia coli O-serogroup is the conventional method for identifying E. coli clones. In this study, we investigated the structural characteristics of the E. coli O99 O-antigen and the organization of the genes involved in its synthesis. On the basis of sugar and methylation analysis and nuclear magnetic resonance spectroscopy data, we established the structure of the branched hexasaccharide repeat unit of the O-polysaccharide. This unit consists of four d-rhamnose (d-Rha) moieties in the backbone and two d-glucose (d-Glc) moieties in the side chain, as shown below: [carbohydrate structure: see text]. The O-antigen gene cluster of E. coli O99, which was located between galF and gnd, was found to contain putative genes for the synthesis of d-Rha, genes encoding sugar transferases, and ATP-binding cassette (ABC) transporter genes (wzm and wzt). Our findings indicate that in E. coli O99, the synthesis and translocation of the O-antigen occurs by an ABC transporter-dependent process.

O-antigen, O-antigen gene cluster, Escherichia coli O99

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,2,2,2	aDGlcp	101.1	73.1	74.3	70.8	73.7	61.9
3,2,2	aDRhap	102.2	78.5	78.6	73.3	71.1	?
3,2	aDRhap	102.1	79.6	71.3	73.7	70.8	?
3	aDRhap	102.3	79.0	71.5	73.6	70.7	?
2	aDGlcp	102.3	73.1	74.2	70.7	73.7	61.7
	aDRhap	102.3	79.5	78.2	73.4	70.9	?


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,2,2,2	aDGlcp	5.22	3.54	3.75	3.42	3.68	3.74-3.78
3,2,2	aDRhap	5.16	4.20	3.95	3.74	3.77	1.28
3,2	aDRhap	5.11	4.08	3.89	3.48	3.70	1.27
3	aDRhap	5.19	4.07	3.89	3.50	3.82	1.31
2	aDGlcp	5.15	3.54	3.74	3.45	3.66	3.76-3.78
	aDRhap	5.25	4.13	3.97	3.74	3.88	1.33


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,2,2,2	aDGlcp	101.1/5.22	73.1/3.54	74.3/3.75	70.8/3.42	73.7/3.68	61.9/3.74-3.78
3,2,2	aDRhap	102.2/5.16	78.5/4.20	78.6/3.95	73.3/3.74	71.1/3.77	?/1.28
3,2	aDRhap	102.1/5.11	79.6/4.08	71.3/3.89	73.7/3.48	70.8/3.70	?/1.27
3	aDRhap	102.3/5.19	79.0/4.07	71.5/3.89	73.6/3.50	70.7/3.82	?/1.31
2	aDGlcp	102.3/5.15	73.1/3.54	74.2/3.74	70.7/3.45	73.7/3.66	61.7/3.76-3.78
	aDRhap	102.3/5.25	79.5/4.13	78.2/3.97	73.4/3.74	70.9/3.88	?/1.33

The spectrum also has 4 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

908.85 g/mol (C₃₆H₆₀R₂O₂₆, R = next and previous repeats, not counted in mol weight)