Membrane topology mapping of the O-antigen flippase (Wzx), polymerase (Wzy), and ligase (WaaL) from Pseudomonas aeruginosa PAO1 reveals novel domain architectures

Abstract

Biosynthesis of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows the Wzy-dependent pathway, requiring the integral inner membrane proteins Wzx (O-antigen [O-Ag] flippase), Wzy (O-Ag polymerase), and WaaL (O-Ag ligase). For an important first step in deciphering the mechanisms of LPS assembly, we set out to map the membrane topology of these proteins. Random and targeted 3'wzx, wzy, and waaL truncations were fused to a phoA-lacZalpha dual reporter capable of displaying both alkaline phosphatase and beta-galactosidase activity. The results from truncation fusion expression and the corresponding differential enzyme activity ratios allowed for the assignment of specific regions of the proteins to cytoplasmic, transmembrane (TM), or periplasmic loci. Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. Our data revealed unique TM domain properties in these proteins, particularly for Wzx, indicating the potential for a charged pore. Novel periplasmic and cytoplasmic loop domains were also uncovered, with the latter in Wzy and WaaL revealing tracts consistent with potential Walker A/B motifs.

FIG 1

Topological map of Wzx from P. aeruginosa PAO1 based on phoA-lacZα fusion analysis (GenBank accession no. 15598349). Colored residues represent the amino acid positions of each truncation used. Residue colors denote the subcellular localization of a given truncation: blue, periplasm; purple, TM; and red, cytoplasm. Truncation letter colors denote the method of truncation generation: white, random; green, interval scanning; black, targeted (periplasm and cytoplasm); orange, targeted (TM). All TMS are labeled (X1 to X12). The AP/BG enzyme normalized activity ratios (NARs) for representative residues (see Table S1 in the supplemental material) are displayed in rectangles. Amino acid identity is displayed above/below each NAR for quantified residues.

FIG 2

Topological map of Wzy from P. aeruginosa PAO1 based on analysis of 105 phoA-lacZα fusions (GenBank accession no. 15598350). Colored residues represent the amino acid positions of each truncation used. Residue colors denote the subcellular localization of a given truncation: blue, periplasm; purple, TM; red, cytoplasm. Truncation letter colors denote the method of truncation generation: white, random; black, targeted (periplasm and cytoplasm); orange, targeted (TM). All TMS are labeled (Y1 to Y14). The AP/BG enzyme NARs for representative residues (see Table S2 in the supplemental material) are displayed in rectangles. Amino acid identity is displayed above/below each NAR for quantified residues.

FIG 3

Topological map of WaaL from P. aeruginosa PAO1 based on analysis of 66 phoA-lacZα fusions (GenBank accession no. 15600192). Colored residues represent the amino acid positions of each truncation used. Residue colors denote the subcellular localization of a given truncation: blue, periplasm; purple, TM; red, cytoplasm. Truncation letter colors denote the method of truncation generation: white, random; black, targeted (cytoplasm); orange, targeted (TM). All TMS are labeled (L1 to L12). The AP/BG enzyme NARs for representative residues (see Table S3 in the supplemental material) are displayed in rectangles. Amino acid identity is displayed above/below each NAR for quantified residues.

FIG 4

Fluorescence micrographs of P. aeruginosa PAO1 expressing C-terminal GFP fusions of Wzx, Wzy, and WaaL from respective pHERD26T clones. Images were captured at ×400 as described in Materials and Methods. FM, fluorescence micrograph; DIC, differential interference contrast. White bar = 15 µm.