Chromosomal and Plasmid-encoded Enzymes Are Required for Assembly of the R3-type Core Oligosaccharide in the Lipopolysaccharide of Escherichia coli O157:H7

Abstract

The type R3 core oligosaccharide predominates in the lipopolysaccharides from enterohemorrhagic Escherichia 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) lipopolysaccharide ␣-1,2-glucosyltransferase, and WaaD was shown to be a UDP-glucose:(glucosyl)lipopolysaccharide ␣-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 an N-acetylgalactosamine residue in a ␤-anomeric configuration, whereas the biological repeat unit for O157 contains ␣-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 ␣-1,3-linked N-acetylglucosamine residue in the outer core is unknown. The gene responsible for a nonstoichiometric ␣-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.

Phosphoethanolamine substitution in the lipid A of Escherichia coli O157 : H7 and its association with PmrC

“…However, YhjW (EptB) was recently reported to be responsible for the addition of PEtN to the second Kdo residue (Reynolds et al, 2005), and YijP (CptA) in S. Typhimurium was involved in the addition of PEtN to the phosphorylated heptose-I residue (Tamayo et al, 2005). E. coli O157 : H7 possesses a plasmid-borne shf locus consisting of shf-wabBecf3-msbB2 (Kim et al, 2004;Kaniuk et al, 2004). The Ecf3 of pO157 is highly homologous (83 % identity) to YijP, suggesting the same function as that of CptA of S. Typhimurium.…”

“…The Ecf3 of pO157 is highly homologous (83 % identity) to YijP, suggesting the same function as that of CptA of S. Typhimurium. In addition, WabB shows a UDP-Nacetylglucosamine : (heptose) LPS a-1,7-N-acetylglucosamine transferase that is responsible for a non-stoichiometric Nacetylglucosamine substitution in the heptose-III residue (Kaniuk et al, 2004). Therefore, the shf locus seems to be a module involved in O157 LPS lipid A-core modifications, and is also suggested to play a role in the survival and persistence of E. coli O157 : H7 in cattle and in the environment (Yoon et al, 2005).…”

“…E. coli O157 : H7 possesses a 92 kb plasmid pO157 that carries the shf locus (also known as ecf) consisting of shf-wabB-ecf3-msbB2 (Boerlin et al, 1998;Kim et al, 2004;Yoon et al, 2004;Kaniuk et al, 2004). The ecf3 gene of this shf locus encodes a hypothetical protein highly homologous (83 % identity) to YijP, which is one of the potential PmrC homologues.…”

The C-terminal Domain of the Escherichia coli WaaJ Glycosyltransferase Is Important for Catalytic Activity and Membrane Association

“…E. coli CWG350 is a waaJ::aacC1 (gentamycin r ) mutant derived from F653 (6). WaaJ derivatives were expressed in E. coli TOP10 (F Ϫ mcrA ⌬(mrr-hsdRMS-mcrBC) f80⌬lacZM15 ⌬lacX74 deoR recA1 araD139 ⌬(ara-leu)7697 galU galK rpsL (Str r ) endA1 nupG) and E. coli BL21 (DE3) (B F Ϫ hsdS B (r B Ϫm B Ϫ) gal dcm ompT (DE3)); both were purchased from Novagen.…”

“…These enzymes transfer hexose residues to a lipid A-core acceptors in reactions that, unlike O antigen biosynthesis, do not involve polyprenol-linked donors (1). WaaJ is required for the addition of an ␣-1,2-glucose to the outer core OS in R3 E. coli (6) and Salmonella enterica serovar Typhimurium (7), which becomes the point of attachment for O antigen ( Fig. 1) (8,9).…”