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

Microbiology. 2000 Nov;146 ( Pt 11):2803-2814. doi: 10.1099/00221287-146-11-2803.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Carbohydrate Dehydrogenases / genetics
  • Carbohydrate Dehydrogenases / metabolism
  • Carbohydrate Epimerases / genetics
  • Carbohydrate Epimerases / metabolism
  • Chromosomes, Bacterial / genetics
  • Cloning, Molecular
  • DNA Primers / genetics
  • Genes, Bacterial*
  • Hydro-Lyases / genetics
  • Hydro-Lyases / metabolism
  • Mutation
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism
  • O Antigens / biosynthesis*
  • Oligosaccharides / biosynthesis*
  • Operon
  • Plasmids / genetics
  • Pseudomonas aeruginosa / classification
  • Pseudomonas aeruginosa / genetics*
  • Pseudomonas aeruginosa / metabolism*
  • Rhamnose / metabolism
  • Serotyping

Substances

  • DNA Primers
  • O Antigens
  • Oligosaccharides
  • Carbohydrate Dehydrogenases
  • dTDP-4-dehydrorhamnose reductase
  • Nucleotidyltransferases
  • glucose-1-phosphate thymidylyltransferase
  • Hydro-Lyases
  • dTDPglucose 4,6-dehydratase
  • Carbohydrate Epimerases
  • dTDP-4-ketorhamnose 3,5-epimerase
  • Rhamnose