Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

Mol Microbiol. 1998 Jun;28(6):1103-19. doi: 10.1046/j.1365-2958.1998.00871.x.

Abstract

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of alpha1-->2, alpha1-->3, alpha1-->3 linked D-rhamnose (Rha). The A-band polysaccharide is assembled by the alpha-D-rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two alpha1-->3 linked Rha residues and one alpha1-->2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3' end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3' end of the cluster and are predicted to encode a coenzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D-galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Amino Acid Sequence
  • Bacterial Proteins*
  • Base Sequence
  • Blotting, Southern
  • Blotting, Western
  • Carbohydrate Sequence
  • Cloning, Molecular
  • DNA Helicases*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Escherichia coli Proteins
  • Genes, Bacterial
  • Genetic Complementation Test
  • Glycosyltransferases / chemistry
  • Glycosyltransferases / genetics*
  • Glycosyltransferases / metabolism*
  • Lipopolysaccharides / biosynthesis*
  • Molecular Sequence Data
  • Multigene Family
  • Mutation
  • O Antigens / chemistry
  • Plasmids / genetics
  • Pseudomonas aeruginosa / classification
  • Pseudomonas aeruginosa / enzymology*
  • Pseudomonas aeruginosa / genetics*
  • Restriction Mapping
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Serotyping

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Lipopolysaccharides
  • O Antigens
  • Glycosyltransferases
  • WbpL protein, Pseudomonas
  • Adenosine Triphosphatases
  • UvrD protein, E coli
  • DNA Helicases

Associated data

  • GENBANK/AF010181
  • GENBANK/AF010182
  • GENBANK/AF010183
  • GENBANK/AF010184
  • GENBANK/AF010185
  • GENBANK/U50396