Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties

Abstract

WaaL is an inner membrane glycosyltransferase that catalyzes the transfer of O-antigen polysaccharide from its lipid-linked intermediate to a terminal sugar of the lipid A-core oligosaccharide, a conserved step in lipopolysaccharide biosynthesis. Ligation occurs at the periplasmic side of the bacterial cell membrane, suggesting the catalytic region of WaaL faces the periplasm. Establishing the membrane topology of the WaaL protein family will enable understanding its mechanism and exploit it as a potential antimicrobial target. Applying oxidative labeling of native methionine/cysteine residues, we previously validated a topological model for Escherichia coli WaaL, which differs substantially from the reported topology of the Pseudomonas aeruginosa WaaL, derived from the analysis of truncated protein reporter fusions. Here, we examined the topology of intact E. coli and P. aeruginosa WaaL proteins by labeling engineered cysteine residues with the membrane-impermeable sulfhydryl reagent polyethylene glycol maleimide (PEG-Mal). The accessibility of PEG-Mal to targeted engineered cysteine residues in both E. coli and P. aeruginosa WaaL proteins demonstrates that both ligases share similar membrane topology. Further, we also demonstrate that P. aeruginosa WaaL shares similar functional properties with E. coli WaaL and that E. coli WaaL may adopt a functional dimer conformation.

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol‐pal mutants

“…Cells lacking WzzE are known to lose modality in the ECA polymer, giving rise to a more random distribution of chain lengths (Barr et al, 1999). We validated this observation; there are relatively higher levels of shorter chain ECA, presumably including Lipid III ECA precursors, in the ∆wzzE It has previously been reported that the terminal GlcNAc moiety attached to the E. coli K-12 LPS core oligosaccharide is derived from und-PP-GlcNAc (i.e., Lipid I ECA ) via the action of WaaL (Feldman et al, 1999;Ruan et al, 2018). Since WaaL accepts different und-PP-linked substrates, it may be possible that corresponding modifications of LPS can occur in strains accumulating Lipid II/III ECA ; these modified LPS might contribute to restored OM barrier function in the tol-pal mutants.…”

“…However, since deletion of either wecA (does not restore OM barrier the GlcNAc moiety from und-PP-GlcNAc (Lipid I ECA ) can itself be transferred onto LPS in a WaaL-dependent manner (Feldman et al, 1999;Ruan et al, 2018). We reasoned it is likely that the incomplete sugar subunits on Lipid II/III ECA could also be ligated onto LPS, therefore, affecting the physical properties of the OM via LPS modification.…”

Identification of the lipopolysaccharide O‐antigen biosynthesis priming enzyme and the O‐antigen ligase in Myxococcus xanthus: critical role of LPS O‐antigen in motility and development

“…The amino acid sequences of WaaL ligases are not highly conserved, but they are all predicted integral IM proteins with eight or more TMH (Raetz and Whitfield, 2002). The WaaL ligases of Pseudomonas aeruginosa and E. coli (WaaL Pa and WaaL Ec respectively) contain a partially periplasmic Wzy_C domain, responsible for catalytic activity and 12 TMHs (Islam et al, 2010;Pan et al, 2012;Ruan et al, 2012;Ruan et al, 2018) ( Fig. 1E).…”

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function inEscherichia coli tol-palmutants

“…However, since deletion of either wecA (does not restore OM barrier function) or other wec genes (restore OM barrier function) would result in the loss of full length ECALPS, we ruled out the role of ECALPS in rescuing the OM permeability defect. It has been reported that the GlcNAc moiety from und-PP-GlcNAc (Lipid I ECA ) can itself be transferred onto LPS in a WaaL-dependent manner (Feldman et al, 1999;Ruan et al, 2018). We reasoned it is likely that the incomplete sugar subunits on Lipid II/III ECA could also be ligated onto LPS, therefore affecting the physical properties of the OM via LPS modification.…”

“…It has previously been reported that the terminal GlcNAc moiety attached to the E. coli K-12 LPS core oligosaccharide is derived from und-PP-GlcNAc (i.e. Lipid I ECA ) via the action of WaaL (Feldman et al, 1999;Ruan et al, 2018). Since WaaL accepts different und-PP-linked substrates, it may be possible that corresponding modifications of LPS can occur in strains accumulating Lipid II/III ECA ; these modified LPS might contribute to restored OM barrier function in the tol-pal mutants.…”