Mild acid degradation of the lipopolysaccharide of Escherichia coli O96 afforded a mixture of two polysaccharides. The following structure of the pentasaccharide repeating unit of the major polymer was established by sugar analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy: [Formula: see text]. The O-antigen gene cluster of E. coli O96 between conserved galF and gnd genes was found to be consistent with this structure, and hence, the major polysaccharide represents the O96-antigen. The O96-antigen structure and gene cluster are similar to those of E. coli O170, and two proteins encoded in the gene clusters of both bacteria were putatively assigned a function of galactofuranosyltransferases. The minor polymer has the same structure as a peptidoglycan-related polysaccharide reported earlier in Providencia alcalifeciens O45 and several other O-serogoups of this species (Ovchinnikova OG, Liu B, Kocharova NA, Shashkov AS, Kondakova AN, Siwinska M, Feng L, Rozalski A, Wang L, Knirel YA. Biochemistry (Moscow) 2012;77:609-15) →4)-β-D-GlcpNAc-(1→4)-β-D-GlcpNAc3(Rlac-lAla)-(1→ where Rlac-lAla indicates (R)-1-[(S)-1-carboxyethylaminocarbonyl]ethyl.

O-antigen, Escherichia coli, O-polysaccharide, bacterial polysaccharide structure, O-antigen gene cluster, Peptidoglycan-related polysaccharide

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,5,4,4	bDGalf	109.1	82.5	78.1	84.6	72.1	64.2
3,5,4	aDGlcpA	99.2	81.1	71.6	79.5	72.4	175.1
3,5,2	Ac	175.5-176.1	23.3-23.4
3,5	bDGlcpN	102.2	57.1	75.4	77.6	75.4	62.0
3	bDGalf	109.4	82.4	77.2	82.7	79.0	62.5
2	Ac	175.5-176.1	23.3-23.4
	bDGlcpN	103.8	56.4	82.3	69.6	77.0	62.2


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,5,4,4	bDGalf	5.00	4.08	4.05	4.09	3.82	3.65-3.68
3,5,4	aDGlcpA	5.70	3.75	3.77	3.67	4.15	-
3,5,2	Ac	-	2.04-2.05
3,5	bDGlcpN	4.68	3.71	3.88	3.72	3.58	3.76-3.91
3	bDGalf	5.02	3.99	4.26	4.14	3.96	3.69-3.69
2	Ac	-	2.04-2.05
	bDGlcpN	4.74	3.83	3.63	3.49	3.47	3.76-3.93


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,5,4,4	bDGalf	109.1/5.00	82.5/4.08	78.1/4.05	84.6/4.09	72.1/3.82	64.2/3.65-3.68
3,5,4	aDGlcpA	99.2/5.70	81.1/3.75	71.6/3.77	79.5/3.67	72.4/4.15	
3,5,2	Ac		23.3-23.4/2.04-2.05
3,5	bDGlcpN	102.2/4.68	57.1/3.71	75.4/3.88	77.6/3.72	75.4/3.58	62.0/3.76-3.91
3	bDGalf	109.4/5.02	82.4/3.99	77.2/4.26	82.7/4.14	79.0/3.96	62.5/3.69-3.69
2	Ac		23.3-23.4/2.04-2.05
	bDGlcpN	103.8/4.74	56.4/3.83	82.3/3.63	69.6/3.49	77.0/3.47	62.2/3.76-3.93

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

906.794 g/mol (C₃₄H₅₄R₂N₂O₂₆, R = next and previous repeats, not counted in mol weight)