Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

Abstract

Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.

FIGURE 1.

Ye O:3 LPS outer core. A, genetic organization of the OC gene cluster (nucleotide sequence GenBank^TM accession number Z47767). The genes are drawn to scale, and locations of relevant restriction sites are indicated. B, structure of the OC hexasaccharide. The different enzymes involved in OC biosynthesis are indicated, i.e. the enzymes for the UDP-Sugp and UDP-GalpNAc biosynthesis are given in parentheses above the respective residues and the six GTases beside the catalyzed glycosidic linkages. The Glcp(I), Glcp(II), GalpNAc(I), and GalpNAc(II) residues are indicated. The minimal structures functioning as phage and enterocoliticin receptor and the mAb 2B5 epitope are also marked.

FIGURE 2.

Silver-stained DOC-PAGE and immunoblotting analysis of LPSs of OC mutants together with phage and enterocoliticin sensitivities of OC mutants. Upper panel, silver staining; middle panel, immunoblotting with mAb 2B5; bottom panel, phage and enterocoliticin sensitivities. Lanes are as follows: Wt, YeO3-R1; Δ+c, YeO3-c-OC-R/pRV16NP; Δ, YeO3-c-OC-R; O, YeO3-wbcO1-R; Q, YeO3-c-wbcQ1-R; N, YeO3-c-wbcN-R1; M, YeO3-c-OC-R/pRV16NPwbcM1; L, YeO3-c-OC-R/pRV16NPwbcL1; K, YeO3-c-OC-R/pRV16NPwbcK2.

FIGURE 3.

Three-dimensional model of WbcK. a, overall fold; b, closer view of the active site structure. The N-terminal GT-A domain is colored green and the conserved helix of the C-terminal domain with lighter green. The most unreliable area of the fold is colored gray. Glycerol (GOL) is bound into the acceptor sugar site in the SpsA x-ray structure and was modeled to the WbcK model. The residues involved in UDP, metal ion, and glycerol binding are shown as sticks. The carbon atoms of residues involved in UDP and metal ion binding are colored cyan, and those of residues in the acceptor binding site are colored green.