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
3,3,4,4,2	%Ac
3,3,4,4,3	%Ac
3,3,4,4,4	%Ac
3,3,4,4	bLRhap	99.85	73.53	72.37	73.39	73.34	17.48
3,3,4	bDGlcpN
3,3,2	3%Ac
3,3,3	25%Ac
3,3	aDGalpA	101.44	67.82	73.36	78.06	72.63	175.39
3	aDManp
2	Ac
	bDGlcpN


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,4,2	Ac
3,3,4,4,2	%Ac
3,3,4,4,3	%Ac
3,3,4,4,4	%Ac
3,3,4,4	bLRhap	5.08	5.49	3.82	3.35	3.47	1.35
3,3,4	bDGlcpN
3,3,2	3%Ac
3,3,3	25%Ac
3,3	aDGalpA	5.28	3.92	5.26	4.44	4.45	-
3	aDManp
2	Ac
	bDGlcpN


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,4,2	Ac
3,3,4,4,2	%Ac
3,3,4,4,3	%Ac
3,3,4,4,4	%Ac
3,3,4,4	bLRhap	99.85/5.08	73.53/5.49	72.37/3.82	73.39/3.35	73.34/3.47	17.48/1.35
3,3,4	bDGlcpN
3,3,2	3%Ac
3,3,3	25%Ac
3,3	aDGalpA	101.44/5.28	67.82/3.92	73.36/5.26	78.06/4.44	72.63/4.45	
3	aDManp
2	Ac
	bDGlcpN

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

1100.98 g/mol (C₄₄H₆₄R₂N₂O₃₀, R = next and previous repeats, not counted in mol weight)