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Involvement of the rml locus in core oligosaccharide and O polysaccharide assembly in Pseudomonas aeruginosa

Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1¹
Canadian Bacterial Diseases Network, Networks of Centers of Excellence, Heritage Medical Research Building, Hospital Drive, Calgary, Alberta, Canada T2N 4N1²
Center for Infection and Biomaterials Research, NU13-143, Toronto General Hospital, Toronto, Ontario, Canada M5G 2C4³
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A OR6⁴

Author for correspondence: Joseph S. Lam. Tel: +1 519 824 4120 ext. 3823. Fax: +1 519 837 1802. e-mail: jlam{at}uoguelph.ca

L-Rhamnose (L-Rha) is a component of the lipopolysaccharide (LPS) core, several O antigen polysaccharides, and the cell surface surfactant rhamnolipid of Pseudomonas aeruginosa. In this study, four contiguous genes (rmlBDAC) responsible for the synthesis of dTDP-L-Rha in P. aeruginosa have been cloned and characterized. Non-polar chromosomal rmlC mutants were generated in P. aeruginosa strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extracted from the mutants was analysed by SDS-PAGE and Western immunoblotting. rmlC mutants of both serotype O5 and serotype O6 synthesized a truncated core region which was unable to act as an attachment point for either A-band or B-band O antigen. A rmd rmlC PAO1 double mutant (deficient in biosynthesis of both D-Rha and L-Rha) was constructed to facilitate structural analysis of the mutant core region. This strain has an incomplete core oligosaccharide region and does not produce A-band O antigen. These results provide the genetic and structural evidence that L-Rha is the receptor on the P. aeruginosa LPS core for the attachment of O polysaccharides. This is the first report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of P. aeruginosa LPS, and provides further insight into the mechanisms of LPS biosynthesis and assembly in this bacterium.

Keywords: L-rhamnose, Pseudomonas aeruginosa, lipopolysaccharide, rml, rmd

Abbreviations: Gm, gentamicin; Rha, rhamnose

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