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
4,4	bLRhap	101.45	71.52	73.57	73.01	73.01	17.57
4,2	Ac
4	bDGlcpN	102.77	56.92	74.48	78.30	75.22	62.22
	aDGalpA	101.64	69.70	70.59	80.31	72.54	175.76


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,4	bLRhap	4.83	4.06	3.58	3.36	3.36	1.32
4,2	Ac
4	bDGlcpN	4.64	3.70	3.70	3.61	3.42	3.74-3.92
	aDGalpA	5.21	3.75	4.04	4.38	4.37	-


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,4	bLRhap	101.45/4.83	71.52/4.06	73.57/3.58	73.01/3.36	73.01/3.36	17.57/1.32
4,2	Ac
4	bDGlcpN	102.77/4.64	56.92/3.70	74.48/3.70	78.30/3.61	75.22/3.42	62.22/3.74-3.92
	aDGalpA	101.64/5.21	69.70/3.75	70.59/4.04	80.31/4.38	72.54/4.37	

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@H]2[C@@H](C(=O)O)O[C@H](O)[C@H](O)[C@H]2O)O[C@H](CO)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)[C@H]2O)[C@@H]1O

543.475 g/mol (C₂₀H₃₃NO₁₆)