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Journal of Bacteriology, May 2005, p. 3002-3012, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3002-3012.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Characterization of WaaL, a Ligase Associated with Linking O-Antigen Polysaccharide to the Core of Pseudomonas aeruginosa Lipopolysaccharide

Priyanka D. Abeyrathne, Craig Daniels,{dagger} Karen K. H. Poon, Mauricia J. Matewish, and Joseph S. Lam*

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received 17 November 2004/ Accepted 26 January 2005

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.

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Phone: (519) 824-4120, ext. 53823. Fax: (519) 837-1802. E-mail: jlam{at}uoguelph.ca.

{dagger} Present address: Centre for Infection and Biomaterials Research, Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada.

Journal of Bacteriology, May 2005, p. 3002-3012, Vol. 187, No. 9
0021-9193/05/$08.00+0     doi:10.1128/JB.187.9.3002-3012.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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