Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation

Abstract

In Escherichia coli, phosphoryl substituents in the lipopolysaccharide core region are essential for outer membrane stability. Mutation of the core glucosyltransferase encoded by waaG (formerly rfaG) resulted in lipopolysaccharide truncated immediately after the inner core heptose residues, which serve as the sites for phosphorylation. Surprisingly, mutation of waaG also destabilized the outer membrane. Structural analyses of waaG mutant lipopolysaccharide showed that the cause for this phenotype was a decrease in core phosphorylation, an unexpected side effect of the waaG mutation.

FIG. 1

Structure of the LPS from E. coli strains with an R1-type core. Core residues are designated by sugar abbreviation and number to facilitate identification. Abbreviations are as follows: GlcN, d-glucosamine; Hep, l-glycero-d-manno-heptose; P, phosphate; EtNP, 2-aminoethyl phosphate; Glc, d-glucose; and Gal, d-galactose. All sugars are in the pyranose configuration, and the linkages are α unless otherwise indicated. The assignment of function to genes encoding core glycosyltransferases and phosphotransferases has been reported previously (2, 3, 7, 19). Of particular note, the waaG gene product (indicated by an asterisk) is the glucosyltransferase for GlcI.

FIG. 2

Deduced structure of the LPS from CWG303 (waaG). The two major oligosaccharides observed after complete deacylation of CWG303 LPS differ only in the presence or absence of a phosphate (P) residue on Hep residue E (R = OH in OS-1; R = P in OS-2). The major oligosaccharides after mild acid hydrolysis of CWG303 LPS again differ only by phosphorylation on Hep residue E (R = OH in OS-3; R = P or 2-aminoethyl diphosphate in OS-4). The P substitution on Hep residue E (HepI) occurs in about 40% of LPS molecules, with 30% of these P residues being further modified by PEtN.

FIG. 3

SDS-PAGE gel (12.5% polyacrylamide) of Sarkosyl-insoluble outer membrane fractions. Lane 1, F470 (parent); lane 2, CWG303 (waaG); lane 3, CWG296 (waaP). Molecular mass markers (in kilodaltons) are indicated to the left of the gel, and major proteins are labeled on the right.