Determination of glycosyltransferase specificities for the Escherichia coli O111 O antigen by a generic approach

Abstract

We describe a bacterial strain developed to facilitate the determination of glycosyltransferase (GT) specificities for O antigens of known structure and gene cluster sequence. For proof of principle for the approach, the strain was used to determine the specificity of the Escherichia coli O111 O-antigen GT genes.

FIG. 1.

The E. coli O111 O antigen and gene cluster. At the top is the structure (based on data from reference 24). At the bottom is a depiction of the gene cluster showing GDP-colitose pathway genes manB, manC, colA, and colB; processing genes wzx and wzy; and GT genes wbdH, wbdL, and wbdM (adapted from reference 27). Molecular sizes are indicated below.

FIG. 2.

Representation of the Wzy-dependent mechanism for LPS synthesis. The O unit is assembled on UndPP in the periplasm, and one such O unit is shown. Each O unit is then translocated to the periplasmic face by Wzx (step 1), then polymerized by Wzy (four events shown as steps 2 to 5), and then added to lipidA/core by WaaL. P, phosphate; O1 to O5, five steps information of O antigen; L, ligation step.

FIG. 3.

(A) Rhamnose induction of wecA and effect on O-antigen synthesis. Results of SDS-PAGE of LPS from strain P5814 carrying the O111 gene cluster (pPR2105). Lane 1, not induced by rhamnose; lane 2, rhamnose induced. (B) Results of SDS-PAGE of LPS from P5814 carrying pPR2102 and derivatives. Lanes 1 to 4, all with rhamnose induction, contain strain P5814 carrying, in lane 1, no plasmid; lane 2, pPR2102; lane 3, pPR2104; lane 4, pPR2103. Lane 5, strain P5814 carrying pPR2105 with no rhamnose induction. Arrows indicate direction of electrophoresis. Arrowheads indicate bands comprising LPS with a single complete or incomplete O unit or O-antigen polymer that are referred to in the text. Note that lane 5 is the same as lane 1 in Fig. 3A.