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

L-Rhamnose (L-Rha) is a component of the lipopolysaccharide (LPS) core, sev eral O antigen polysaccharides, and the cell surface surfactant rhamnolipid of Pseudomonas aeruginosa. In this study, four contiguous genes (rmIBDAC) responsible for the synthesis of dTDP-L-Rha in P. aeruginosa have been clon ed and characterized. Non-polar chromosomal rmIC mutants were generated in P. aeruginosa strains PAO1 (serotype O5) and PAK (serotype O6) and LPS extr acted from the mutants was analysed by SDS-PACE and Western immunoblotting. rmIC mutants of both serotype O5 and serotype O6 synthesized a truncated c ore region which was unable to act as an attachment point for either A-band or B-band O antigen. A rmd rmIC PAO1 double mutant (deficient in biosynthe sis of both D-Rha and L-Rha) was constructed to facilitate structural analy sis of the mutant core region. This strain has an incomplete core oligosacc haride 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. ae ruginosa LPS core for the attachment of O polysaccharides. This is the firs t report of a genetically defined mutation that affects the synthesis of a single sugar in the core oligosaccharide region of P. aeruginosa LPS, and p rovides further insight into the mechanisms of LPS biosynthesis and assembl y in this bacterium.