Two polysaccharides were isolated from Escherichia coli O12, the major being identified as the O12-antigen and the minor as the K5-antigen. The polysaccharides were studied by sugar analysis, Smith degradation, and one- and two-dimensional (1)H and (13)C NMR spectroscopy. As a result, the following structure of the O12-polysaccharide was elucidated, which, to our knowledge, has not been hitherto found in bacterial carbohydrates: →2)-β-d-Glcp-(1→6)-α-d-GlcpNAc-(1→3)-α-l-FucpNAc-(1→3)-β-d-GlcpNAc-(1→. The →4)-β-d-GlcpA-(1→4)-α-d-GlcpNAc-(1→ structure established for the K5-polysaccharide (heparosan) is previously known. Functions of genes in the O-antigen biosynthesis gene cluster of E. coli O12 were assigned by comparison with sequences in the available databases and found to be consistent with the O12-polysaccharide structure.

Escherichia coli, glycosyltransferase, O-antigen gene cluster, O-Polysaccharide structure

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
1,3,2	Ac	175.4-175.7	23.4-23.5
1,3	aLFucpN	99.0	50.7	69.0	72.4	68.1	16.6
1,2	Ac	175.4-175.7	23.4-23.5
1	bDGlcpN	102.2	56.9	79.8	69.8	77.1	62.1
	Subst	103.8	81.0	61.5	70.2	81.0	63.8


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
1,3,2	Ac	-	1.98-2.04
1,3	aLFucpN	5.01	4.11	3.90	3.77	4.42	1.19
1,2	Ac	-	1.98-2.04
1	bDGlcpN	4.64	3.85	3.68	3.53	3.47	3.78-3.94
	Subst	4.93	3.91	3.70-3.82	3.66-3.92	3.86	3.60-3.68


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
1,3,2	Ac		23.4-23.5/1.98-2.04
1,3	aLFucpN	99.0/5.01	50.7/4.11	69.0/3.90	72.4/3.77	68.1/4.42	16.6/1.19
1,2	Ac		23.4-23.5/1.98-2.04
1	bDGlcpN	102.2/4.64	56.9/3.85	79.8/3.68	69.8/3.53	77.1/3.47	62.1/3.78-3.94
	Subst	103.8/4.93	81.0/3.91	61.5/3.70-3.82	70.2/3.66-3.92	81.0/3.86	63.8/3.60-3.68

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@@H]1[C@H](O[C@H]2[C@H](O)[C@@H](CO)O[C@@H](OCC(CO)C3OCC(CO)O3)[C@@H]2NC(C)=O)O[C@@H](C)[C@@H](O)[C@H]1O

568.573 g/mol (C₂₃H₄₀N₂O₁₄)