The structure of the O-antigen polysaccharide (PS) of Escherichia coli O115 has been investigated using a combination of component analysis together with 1D and 2D NMR spectroscopy experiments. The repeating unit of the O-antigen was elucidated using the O-deacetylated PS and has the following branched pentasaccharide structure: →3)[β-L-Rhap-(1→4)]-β-D-GlcpNAc-(1→4)-α-D-GalpA-(1→3)-α-D-Manp-(1→3)-β-D-GlcpNAc-(1→. Cross-peaks of low intensity, corresponding to a β-L-Rhap-(1→4)-β-D-GlcpNAc-(1→ structural element, were present in the NMR spectra and attributed to the terminal part of the polysaccharide; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Analysis of the NMR spectra of the native polysaccharide revealed O-acetyl groups distributed over different positions of the l-Rhap residue (~0.70 per repeating unit) as well as at O-2 and O-3 of the d-GalpA residue (~0.03 and ~0.25 per repeating unit, respectively), which is in agreement with the presence of two acetyltransferases previously identified in the O-antigen gene cluster (Wang et al. 2010). In addition, the four glycosyltransferases initially identified in the O-antigen gene cluster of E. coli O115 were analyzed using BLAST, and the function of two of them predicted based on similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as Escherichia coli O58 and O152.

Lipopolysaccharide, NMR, structure, O-acetylation, O-antigen gene cluster, Escherichia coli O115

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,4,2	Ac	175.24	23.51
3,3,4,4	bLRhap	101.93	71.32	73.52	73.01	73.01	17.57
3,3,4	bDGlcpN	102.82	56.47	77.61	76.76	74.95	62.22
3,3	aDGalpA	101.64	69.70	70.59	80.36	72.54	175.76
3	aDManp	102.56	70.88	80.29	66.18	74.06	61.51
2	Ac	175.01	23.39
	bDGlcpN	100.56	56.05	81.60	71.83	76.63	61.91


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,4,2	Ac	-	2.08
3,3,4,4	bLRhap	4.82	4.18	3.62	3.37	3.37	1.32
3,3,4	bDGlcpN	4.58	3.80	4.08	3.63	3.42	3.73-3.89
3,3	aDGalpA	5.20	3.71	4.03	4.34	4.37	-
3	aDManp	5.11	4.12	3.86	3.85	3.63	3.81
2	Ac	-	2.01
	bDGlcpN	4.68	3.60	3.77	3.53	3.39	3.74-3.94


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,4,2	Ac		23.51/2.08
3,3,4,4	bLRhap	101.93/4.82	71.32/4.18	73.52/3.62	73.01/3.37	73.01/3.37	17.57/1.32
3,3,4	bDGlcpN	102.82/4.58	56.47/3.80	77.61/4.08	76.76/3.63	74.95/3.42	62.22/3.73-3.89
3,3	aDGalpA	101.64/5.20	69.70/3.71	70.59/4.03	80.36/4.34	72.54/4.37	
3	aDManp	102.56/5.11	70.88/4.12	80.29/3.86	66.18/3.85	74.06/3.63	61.51/3.81
2	Ac		23.39/2.01
	bDGlcpN	100.56/4.68	56.05/3.60	81.60/3.77	71.83/3.53	76.63/3.39	61.91/3.74-3.94

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

890.795 g/mol (C₃₄H₅₄R₂N₂O₂₅, R = next and previous repeats, not counted in mol weight)