O-antigen (O-polysaccharide), a part of the outer membrane of Gram-negative bacteria, is one of the most variable cell constituents and is related to bacterial virulence. O-antigen diversity is almost entirely due to genetic variations in O-antigen gene clusters. In this study, the O-polysaccharide structures of Salmonella O55 and Escherichia coli O103 were elucidated by chemical analysis and nuclear magnetic resonance spectroscopy. It was found that the O-polysaccharides have similar pentasaccharide O-units, which differ only in one sugar (glucose versus N-acetylglucosamine) and in the N-acyl group (acetyl versus 3-hydroxybutanoyl) on 3-amino-3,6-dideoxy-d-galactose (d-Fuc3N). The Salmonella O55 antigen gene cluster was sequenced and compared with the E. coli O103 antigen gene cluster reported previously. The two gene clusters were found to share high-level similarity (DNA identity ranges from 53% to 76%), except for two putative acyl transferase genes (fdtC in Salmonella O55 and fdhC in E. coli O103) which show no similarity. Replacement of the fdtC gene in Salmonella O55 with the fdhC gene from E. coli O103 resulted in production of a modified O-antigen, which contains a 3-hydroxybutanoyl derivative of Fuc3N in place of 3-acetamido-3,6-dideoxygalactose. This finding strongly suggests that fdhC is a 3-hydroxybutanoyltransferase gene. The sequence similarity level suggested that the O-antigen gene clusters of Salmonella O55 and E. coli O103 originate from a common ancestor, and this evolutionary relationship is discussed.

Lipopolysaccharide, O-antigen, O-antigen gene cluster, Escherichia coli O103, Salmonella O55

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,6,2	bDGlcp	101.1	82.3	77.2	71.1	76.8	62.2
3,4,6,3	lR3HOBut	174.3	45.8	65.8	23.2
3,4,6	bDFucp3N	103.5	74.2	55.6	71.3	72.6	16.2
3,4,2	Ac	175.0	22.8
3,4	aDGlcpN	99.2	54.9	71.3	70.1	72.1	68.8
3,2	Ac	175.3	22.9
3	aDGalpN	98.0	50.7	68.0	77.7	72.6	60.9
2	Ac	174.9	23.4
	bDGlcpN	102.2	55.5	79.4	71.7	76.6	61.8


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,6,2	bDGlcp	4.71	3.46	3.49	3.40	3.38	3.75-3.92
3,4,6,3	lR3HOBut	-	2.50	4.25	1.28
3,4,6	bDFucp3N	4.55	3.90	4.19	3.65	3.85	1.25
3,4,2	Ac	-	2.09
3,4	aDGlcpN	4.95	3.96	3.87	3.86	4.21	3.93-4.03
3,2	Ac	-	2.07
3	aDGalpN	5.49	4.27	3.91	4.06	3.92	3.71-3.75
2	Ac	-	2.05
	bDGlcpN	4.82	3.76	3.75	3.69	3.40	3.78-3.93


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,6,2	bDGlcp	101.1/4.71	82.3/3.46	77.2/3.49	71.1/3.40	76.8/3.38	62.2/3.75-3.92
3,4,6,3	lR3HOBut		45.8/2.50	65.8/4.25	23.2/1.28
3,4,6	bDFucp3N	103.5/4.55	74.2/3.90	55.6/4.19	71.3/3.65	72.6/3.85	16.2/1.25
3,4,2	Ac		22.8/2.09
3,4	aDGlcpN	99.2/4.95	54.9/3.96	71.3/3.87	70.1/3.86	72.1/4.21	68.8/3.93-4.03
3,2	Ac		22.9/2.07
3	aDGalpN	98.0/5.49	50.7/4.27	68.0/3.91	77.7/4.06	72.6/3.92	60.9/3.71-3.75
2	Ac		23.4/2.05
	bDGlcpN	102.2/4.82	55.5/3.76	79.4/3.75	71.7/3.69	76.6/3.40	61.8/3.78-3.93

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

1002.971 g/mol (C₄₀H₆₆R₂N₄O₂₅, R = next and previous repeats, not counted in mol weight)