Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in Pseudomonas aeruginosa PAO1 via modulation of cell surface attachment Theresa Lindhout, 1 Peter C. Y. Lau, 1 Dyanne Brewer 1,2 and Joseph S. Lam 1 Correspondence Joseph S. Lam jlam@uoguelph.ca 1 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada 2 Mass Spectrometry Facility, University of Guelph, Guelph, ON N1G 2W1, Canada Received 1 May 2009 Revised 11 June 2009 Accepted 3 July 2009 In many Gram-negative bacterial species, rough strains producing truncated lipopolysaccharide (LPS) generally exhibit defects in motility compared with smooth strains. However, the role that LPS plays in bacterial motility is not well understood. The goal of this study was to examine the relationship between LPS defects and motility of Pseudomonas aeruginosa. P. aeruginosa wild- type strain PAO1 and three isogenic mutants with defects in the rmlC, migA and wapR genes and producing truncated core oligosaccharide were investigated in terms of motility, attachment to glass and flagella expression. Compared with the wild-type, the three mutants showed significant retardation in both swarming motility on 0.5 % soft-agar plates and swimming motility on 0.3 % soft-agar plates. Moreover, attachment to abiotic surfaces was observed to be stronger in these mutants. The assembly of flagella appeared to be intact in these strains and the ability of individual cells to swim was unaffected. Flagellin proteins prepared from mutants rmlC and rmd, defective in the production of TDP-L-rhamnose and GDP-D-rhamnose, respectively, were compared and a change in molecular mass was observed only in the rmlC mutant. These data indicated that L-rhamnose, and not its enantiomer, D-rhamnose, is incorporated into the flagellin glycan of P. aeruginosa PAO1. The nucleotide-activated sugar precursor TDP-L-rhamnose is therefore shared between LPS biosynthesis and flagellin glycosylation in P. aeruginosa PAO1. Our results suggest that although biochemical precursors are shared by LPS and flagellin glycan biosynthesis, LPS truncations probably alter flagella-mediated motility in P. aeruginosa by modulating cell-surface attachment but not flagella synthesis. INTRODUCTION Pseudomonas aeruginosa is a motile, Gram-negative bacterium capable of adapting to survive in a wide range of environments. In addition, it is an important oppor- tunistic pathogen that can colonize human tissues, often causing severe to fatal infections in individuals with cystic fibrosis, compromised immune systems and burn wounds. LPS is an integral component of the outer leaflet of the outer membrane of this organism, where it functions in maintaining cell envelope stability, forming a permeability barrier, and participating in pathogenesis. Each LPS molecule is divided into three regions: lipid A, core oligosaccharide (OS) and O polysaccharide (O antigen). The core OS of P. aeruginosa LPS can be further divided into two regions, inner and outer core OS (Sadovskaya et al., 2000). In P. aeruginosa PAO1 the outer core OS is composed of three D-glucose (D-Glc) residues, one L- rhamnose (L-Rha), and one N-(L-alanyl)-D-galactosamine (Fig. 1). Four enzymes encoded by genes in the rmlBDAC operon catalyse the conversion of glucose 1-phosphate to dTDP-L- Rha, which is the precursor for the L-Rha present in the outer core OS (Rahim et al., 2000). L-Rha is transferred to the core OS by one of two putative rhamnosyltransferases, MigA and WapR, which attach L-Rha to D-Glc with distinct linkages. The result is two glycoforms of the core OS that exist simultaneously on the cell surface (Poon et al., 2008). MigA is associated with the transfer of L-Rha to an a-D-Glc residue on the core OS through an a-1,6 linkage Abbreviations: OS, oligosaccharide; TEM, transmission electron micro- socopy; UPLC, ultraperformance liquid chromatography. Four supplementary movie files, showing swimming of wild type and migA, wapR and rmlC mutant strains, are available with the online version of this paper. Microbiology (2009), 155, 3449–3460 DOI 10.1099/mic.0.030510-0 030510 G 2009 SGM Printed in Great Britain 3449