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Journal of Bacteriology, September 2008, p. 6060-6069, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00579-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Erin L. Westman,1 Andrew Preston,2 Robert A. Field,3 and Joseph S. Lam1*

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada,1 School of Clinical Veterinary Science, University of Bristol, Langford, Bristol, United Kingdom,2 Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich, United Kingdom3

Received 25 April 2008/ Accepted 2 July 2008

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, wbpO1629 and wbpO3150, were shown to complement a wbpA knockout of P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO1629 and WbpO3150, 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.

* Corresponding author. Mailing address: University of Guelph, Department of Molecular and Cellular Biology, 50 Stone Road E., Science Complex Room 4244, Guelph, Ontario, Canada N1G 2W1. Phone: (519) 824-4120, ext. 53823. Fax: (519) 837-1802. E-mail: jlam{at}uoguelph.ca

{triangledown} Published ahead of print on 11 July 2008.

Journal of Bacteriology, September 2008, p. 6060-6069, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00579-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • King, J. D., Kocincova, D., Westman, E. L., Lam, J. S. (2009). Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa. Innate Immunity 15: 261-312 [Abstract]  
  • Westman, E. L., McNally, D. J., Charchoglyan, A., Brewer, D., Field, R. A., Lam, J. S. (2009). Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic Acid in Pseudomonas aeruginosa. J. Biol. Chem. 284: 11854-11862 [Abstract] [Full Text]  


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