Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide.
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.
Abeyrathne
Priyanka D
PD
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Daniels
Craig
C
Poon
Karen K H
KK
Matewish
Mauricia J
MJ
Lam
Joseph S
JS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019729:Carbon-Oxygen Ligases / Q000235:genetics / Q000502:physiology*; D002473:Cell Wall / Q000502:physiology; D003001:Cloning, Molecular; D017353:Gene Deletion; D005816:Genetic Complementation Test; D009154:Mutation; D019081:O Antigens / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure
D001426:Bacterial Proteins; D019081:O Antigens; C095976:O antigen, Pseudomonas; D019729:Carbon-Oxygen Ligases