Mastitis represents one of the most significant health problems of dairy herds. The two major causative agents of this disease are Escherichia coli and Staphylococcus aureus. Of the first, its lipopolysaccharide (LPS) is thought to play a prominent role during infection. Here, we report the O-antigen (OPS, O-specific polysaccharide) structure of the LPS from bovine mastitis isolate E. coli 1303. The structure was determined utilizing chemical analyzes, mass spectrometry as well as 1D and 2D NMR spectroscopy methods. The O-repeating unit was characterized as -[→4)-β-D-Quip3NAc-(1→3)-α-L-Fucp2OAc-(1→4)-β-D-Galp-(1→3)-α-D-GalpNAc-(1→]- in which the O-acetyl substitution was non-stoichiometric. The nucleotide sequence of the O-antigen gene cluster of E. coli strain 1303 was also determined. In large parts, this determinant located between the gnd and galF genes comprises a presently unknown DNA sequence with 13 putative open reading frames. The O-antigen of E. coli 1303 was shown to substitute O-7 of the terminal L,D-heptose of the K-12 core oligosaccharide. Interestingly, the non-OPS-substituted core oligosaccharide represented a truncated version of the K-12 outer core, namely terminal L,D-heptose and glucose were missing, however, it possessed a third Kdo residue in the inner core. On the base of structural and genetic data we show that the mastitis isolate E. coli strain 1303 represents a new subtype of serotype O5 and possesses the K-12 core type which is rather uncommon among human and bovine isolates.

Lipopolysaccharide, structure, O-antigen, Escherichia coli, NMR spectroscopy, gene cluster, Staphylococcus aureus, mastitis

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,4,3,3	Ac
3,4,3	bDQuip3N	105.06	73.86	57.89	77.47	73.38	19.25
3,4	aLFucp	96.89	68.38	80.02	72.96	67.94	16.36
3	bDGalp	106.13	70.31	66.33	78.93	75.91	62.36
2	Ac
	aDGalpN	98.42	49.07	78.88	69.91	72.41	62.36


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,4,3,3	Ac	-	1.95
3,4,3	bDQuip3N	4.74	3.31	4.07	3.51	3.66	1.36
3,4	aLFucp	5.06	3.97	4.11	4.02	4.33	1.18
3	bDGalp	4.47	3.62	4.11	3.65	3.63	3.74
2	Ac	-	2.02
	aDGalpN	5.25	4.36	3.91	4.29	4.11	3.74


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,4,3,3	Ac	NMR TSV error 2: unequal length of 13C and 1H datasets
3,4,3	bDQuip3N	105.06/4.74	73.86/3.31	57.89/4.07	77.47/3.51	73.38/3.66	19.25/1.36
3,4	aLFucp	96.89/5.06	68.38/3.97	80.02/4.11	72.96/4.02	67.94/4.33	16.36/1.18
3	bDGalp	106.13/4.47	70.31/3.62	66.33/4.11	78.93/3.65	75.91/3.63	62.36/3.74
2	Ac	NMR TSV error 2: unequal length of 13C and 1H datasets
	aDGalpN	98.42/5.25	49.07/4.36	78.88/3.91	69.91/4.29	72.41/4.11	62.36/3.74

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

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

698.672 g/mol (C₂₈H₄₆R₂N₂O₁₈, R = next and previous repeats, not counted in mol weight)