The type R3 core oligosaccharide predominates in the lipopolysaccharides from enterohemorrhagic E. coli isolates including O157:H7. The R3 core biosynthesis (waa) genetic locus contains two genes, waaD and waaJ, that are predicted to encode glycosyltransferases involved in completion of the outer core. Through determination of the structures of the lipopolysaccharide core in precise mutants and biochemical analyses of enzyme activities, WaaJ was shown to be a UDP-glucose:(galactosyl) LPS a-1,2-glucosyltransferase and WaaD a UDP-glucose:(glucosyl) LPS a-1,2-glucosyltransferase. The residue added by WaaJ was identified as the ligation site for O polysaccharide and this was confirmed by determination of the structure of the linkage region in serotype O157 lipopolysaccharide. The initial O157 repeat unit begins with a N-acetylgalactosamine residue in a -anomeric configuration, whereas the biological repeat unit for O157 contains a-linked N-acetylgalactosamine residues. With the characterization of WaaJ and WaaD, the activities of all of the enzymes encoded by the R3 waa locus are either known or predicted from homology data with a high level of confidence. However, when core oligosaccharide structure is considered, the origin of an additional a-1,3-linked N-acetylglucosamine residue in the outer core is unknown. The gene responsible for a non-stoichiometric a-1,7-linked N-acetylglucosamine substituent in the heptose (inner core) region was identified on the large virulence plasmids of E. coli O157 and Shigella flexneri serotype 2a. This is the first plasmid-encoded core oligosaccharide biosynthesis enzyme reported in E. coli.
p.31242, Fig. 4, structure 1, table II
13C NMR, 1H NMR, EI-MS, methylation, NMR-2D, DNA sequencing, SDS-PAGE, MS/MS, composition analysis
oligosaccharide 1 isolated from N,O-deacylated E. coli CWG653 (serotype O157 wzy mutant) LPS by HPAEC
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6
0,6,6,4 aXKdop
0,6,6,5,3,3,3,2,2 aDGlcp
0,6,6,5,3,3,3,2,4,3,4,3 aDRhap4N 103.6 70.1 68.5 55.2 67.8 18.1
0,6,6,5,3,3,3,2,4,3,4 aLFucp 100.6 68.5 79.1 73.1 68.2 16.4
0,6,6,5,3,3,3,2,4,3 bDGlcp 105.2 74.8 75.5 76.4 77.6 60.9
0,6,6,5,3,3,3,2,4 bDGalpN 102.1 53.4 83.0 68.4 76.6 ?
0,6,6,5,3,3,3,2 aDGlcp 92.7 76.1 71.4 78.0 71.9 61.6
0,6,6,5,3,3,3,3 aDGlcpN
0,6,6,5,3,3,3 aDGalp
0,6,6,5,3,3 aDGlcp
0,6,6,5,3,4 P
0,6,6,5,3,7 aXLDmanHepp
0,6,6,5,3 aXLDmanHepp
0,6,6,5,4 P
0,6,6,5 aXLDmanHepp
0,6,6 aXKdop
0,6,4 P
0,6 bDGlcpN
0 aDGlcpN
P
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6
0,6,6,4 aXKdop
0,6,6,5,3,3,3,2,2 aDGlcp
0,6,6,5,3,3,3,2,4,3,4,3 aDRhap4N 5.09 4.07 4.02 3.13 4.06 1.35
0,6,6,5,3,3,3,2,4,3,4 aLFucp 4.98 3.91 3.91 3.89 4.42 1.18
0,6,6,5,3,3,3,2,4,3 bDGlcp 4.64 3.41 3.63 3.57 3.60 3.87-3.94
0,6,6,5,3,3,3,2,4 bDGalpN 4.62 3.25 3.86 4.23 3.75 ?
0,6,6,5,3,3,3,2 aDGlcp 5.51 3.79 4.06 3.87 3.94 3.84-3.95
0,6,6,5,3,3,3,3 aDGlcpN
0,6,6,5,3,3,3 aDGalp
0,6,6,5,3,3 aDGlcp
0,6,6,5,3,4 P
0,6,6,5,3,7 aXLDmanHepp
0,6,6,5,3 aXLDmanHepp
0,6,6,5,4 P
0,6,6,5 aXLDmanHepp
0,6,6 aXKdop
0,6,4 P
0,6 bDGlcpN
0 aDGlcpN
P
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6
0,6,6,4 aXKdop
0,6,6,5,3,3,3,2,2 aDGlcp
0,6,6,5,3,3,3,2,4,3,4,3 aDRhap4N 103.6/5.09 70.1/4.07 68.5/4.02 55.2/3.13 67.8/4.06 18.1/1.35
0,6,6,5,3,3,3,2,4,3,4 aLFucp 100.6/4.98 68.5/3.91 79.1/3.91 73.1/3.89 68.2/4.42 16.4/1.18
0,6,6,5,3,3,3,2,4,3 bDGlcp 105.2/4.64 74.8/3.41 75.5/3.63 76.4/3.57 77.6/3.60 60.9/3.87-3.94
0,6,6,5,3,3,3,2,4 bDGalpN 102.1/4.62 53.4/3.25 83.0/3.86 68.4/4.23 76.6/3.75 ?/?
0,6,6,5,3,3,3,2 aDGlcp 92.7/5.51 76.1/3.79 71.4/4.06 78.0/3.87 71.9/3.94 61.6/3.84-3.95
0,6,6,5,3,3,3,3 aDGlcpN
0,6,6,5,3,3,3 aDGalp
0,6,6,5,3,3 aDGlcp
0,6,6,5,3,4 P
0,6,6,5,3,7 aXLDmanHepp
0,6,6,5,3 aXLDmanHepp
0,6,6,5,4 P
0,6,6,5 aXLDmanHepp
0,6,6 aXKdop
0,6,4 P
0,6 bDGlcpN
0 aDGlcpN
P
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