Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be →2)-β-D-Galf-(1→6)-α-D-Glcp-(1→3)-α-L-Rhap-(1→3)-α-D-GlcpNAc-(1→, with an O-acetyl group on C-2 of the rhamnose and a side chain α-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.

structure, O-antigen, rfb, gene cluster, Escherichia coli K12

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3,3,6	bDGalf	107.2	87.5	76.8	83.4	71.4	64.2
3,3	aDGlcp	98.1	72.1	74.1	70.2	71.4	66.7
3,2	Ac	173.5	22.7
3	aLRhap	99.7	69.7	74.5	71.7	69.9	17.7
2	Ac
6	aDGlcp	99.3	72.7	74.3	70.8	73.1	61.8
	aDGlcpN	96.5	54.0	80.8	69.6	72.6	67.4


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3,3,6	bDGalf	5.09	4.18	4.29	4.05	3.91	3.71
3,3	aDGlcp	4.97	3.56	3.72	3.56	4.02	3.95
3,2	Ac	-	2.14
3	aLRhap	4.93	5.17	3.96	3.62	4.13	1.30
2	Ac
6	aDGlcp	5.02	3.59	3.78	3.45	3.78	3.90
	aDGlcpN	4.99	4.20	3.78	3.72	4.02	3.84


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3,3,6	bDGalf	107.2/5.09	87.5/4.18	76.8/4.29	83.4/4.05	71.4/3.91	64.2/3.71
3,3	aDGlcp	98.1/4.97	72.1/3.56	74.1/3.72	70.2/3.56	71.4/4.02	66.7/3.95
3,2	Ac		22.7/2.14
3	aLRhap	99.7/4.93	69.7/5.17	74.5/3.96	71.7/3.62	69.9/4.13	17.7/1.30
2	Ac
6	aDGlcp	99.3/5.02	72.7/3.59	74.3/3.78	70.8/3.45	73.1/3.78	61.8/3.90
	aDGlcpN	96.5/4.99	54.0/4.20	80.8/3.78	69.6/3.72	72.6/4.02	67.4/3.84

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](OC[C@H]2O[C@H](O[C@@H]3[C@@H](O)[C@H](C)O[C@@H](O[C@@H]4[C@@H](NC(C)=O)[C@@H](*)O[C@H](CO[C@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@H]4O)[C@@H]3OC(C)=O)[C@H](O)[C@@H](O)[C@@H]2O)O[C@@H]([C@H](O)CO)[C@@H]1O

877.796 g/mol (C₃₄H₅₅R₂NO₂₅, R = next and previous repeats, not counted in mol weight)