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.
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.
Department of Microbiology and Canadian Bacterial Diseases Network, University of Guelph, ON, Canada.
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Rocchetta
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Burrows
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Pacan
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Lam
D000251:Adenosine Triphosphatases / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D019081:O Antigens / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012703:Serotyping
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D019081:O Antigens; D016695:Glycosyltransferases; C114818:WbpL protein, Pseudomonas; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases