Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide

Abstract

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.

FIG. 1.

Comparison of Kyte and Doolittle hydropathy plots of WaaL proteins from various bacteria. The x axis corresponds to the amino acid residue, while the y axis corresponds to the relative hydropathy index. Each of the proteins contains 11 potential membrane-spanning domains, which are indicated above the x axis as numbered bars. (A) E. coli WaaL accession number AAC69648, (B) K. pneumoniae WaaL accession number AAD37765, (C) P. aeruginosa strain PA14 WaaL accession number ZP_00141473, (D) P. aeruginosa strain PAO1 WaaL (PA4999) accession number NP_253686.

FIG. 2.

pa4999 organization in PAO1 genome and strategy for PCR amplification and cloning of the putative waaL, pa4999. The gene was amplified as two parts using the PEP strategy. The first PCR product (using primers P1 and P3) was digested with SmaI and SalI restriction enzymes and cloned into the pEX18AP plasmid using the same restriction enzymes. The second part of pa4999 was amplified by using primers P4 and P5 that include the 3′ end of pa5000. Finally, this PCR product was used as a template to amplify the second half of pa4999 using P4 and P2 as primers. After that, PCR product was digested with SalI and PstI restriction endonucleases and ligated into the first part of pa4999 gene construct.

FIG. 3.

SDS-PAGE and Western immunoblotting analysis of LPS prepared from strain PAO1, mutant PAO1waaL, and complemented transconjugant. LPS from all strains were prepared by the HB method (A to D). Panel A is the silver-stained SDS-PAGE gel, and panels B to D are Western immunoblots with various monoclonal antibodies (MAb). LPS from the mutant PAO1waaL is deficient in the low-molecular-weight core-plus-one O-antigen band. Although panels B, C, and D showed the presence of LPS bands that reacted with MAb N1F10 (A-band specific), MAb MF15-4 (B-band specific), and MAb 18-19 (core-plus-one O-antigen), respectively, we suspect that these bands are undecaprenol lipid carrier-linked glycolipids that have not been ligated to core lipid A. These types of glycolipids are sensitive to phenol treatment, as described in our earlier studies (50). Panels E and F are Western immunoblotting analysis of LPS from strain PAO1, waaL mutant, and complemented transconjugant prepared using the hot-aqueous phenol method.

FIG. 4.

Determination of twitching motility using the sub-agar-surface translocation assay. The zones of motility cells are as follows: (A) P. aeruginisa PAO1, (B) mutant PAO1waaL, (C) PA14, and (D) mutant PA14waaL. Where the cells are nontwitching, only a small “needle-point” at the origin of inoculation can be visualized.

FIG. 5.

Examination of swimming motility of P. aeruginosa strains. Panels A and B represent the parent strains, waaL mutants, and complemented transconjugants of PAO1 and PA14, respectively. Cells were inoculated with a toothpick from an overnight LB agar plate onto a swim plate and photographed after 18 h incubation at 30°C.

FIG. 6.

Electron microscopic analysis of flagella and pili from (A) PAO1, (B) mutant PAO1waaL, (C) PA14, and (D) mutant PA14waaL. Open arrows indicate the flagella and closed arrows indicate the pili. Bars equal 653.59 nm for A, 861.11 nm for B, 1503.26 nm for C, and 1638.88 nm for D. Note that in the inset of Panel B, only a few PAO1waaL mutant bacteria within a group of cells were found to possess flagella. No pili could be discerned on the surfaces of any of the mutant bacteria examined.