Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters
- 15 September 2008
- journal article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 190 (18), 6060-6069
- https://doi.org/10.1128/jb.00579-08
Abstract
Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy- d -mannuronic acid ( d -ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD, and WbpI has been proposed for the production of this sugar in P. aeruginosa , based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB to wbpI were present, but a putative dehydrogenase gene was missing; therefore, the biosynthetic mechanism for UDP- d -ManNAc3NAcA was unclear. Nonpolar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans . Thus, the genes that encode the putative oxidase, transaminase, N -acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa . Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO 1629 and wbpO 3150 , were shown to complement a wbpA knockout of P. aeruginosa . Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO 1629 and WbpO 3150 , and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP- d -GlcNAc, UDP- d -GalNAc, to a lesser extent, and UDP- d -Glc, to a much lesser extent. Together, these results suggest that B. pertussis produces UDP- d -ManNAc3NAcA through the same pathway proposed for P. aeruginosa , despite differences in the genomic context of the genes involved.Keywords
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