Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica

The Bordetella pertussis wlb locus (wlb(pe), formerly bpl) is required for the biosynthesis of a trisaccharide that, when attached to the B. pertussis lipopolysaccharide (LPS) core (band B), generates band A LPS. The equivalent loci in Bordetella bronchiseptica (wlb(br)) and Bordetella parapertussis (wlb(pa)) were identified and cloned. The wlb(br) and wlb(pa) loci differ from wlb(pe) in that they lack the insertion sequence that defines the right-hand terminus of wlb(pe). Deletion of 12 kb of DNA containing the whole wlb locus (Delta wlb) by allelic exchange in each of the three bordetellae had no effect on band B biosynthesis, whereas band A biosynthesis was prevented in B. pertussis and B. bronchiseptica. In B. bronchiseptica and B. parapertussis, Delta wlb mutants also lacked O-antigen. Reintroduction of the wlb(pe) or wlb(br) loci on a shuttle vector into the three Delta wlb mutants restored the wildtype LPS phenotype in the B. pertussis and B. bronchiseptica mutants. In the case of a. parapertussis, which normally does not synthesize an apparent band A structure, introduction of the wlb(pe) or wlb(br) loci now enabled the generation of band A. This suggests that the attachment point for band A trisaccharide on the LPS core is present in B. parapertussis, and further suggests that the wild-type wlb(pa) locus is not fully functional. Introduction of the wlb(pa) locus into the Delta wlb(pe), Delta wlb(br) and Delta wlb(pa) mutants had interesting consequences. The B. bronchiseptica and B. parapertussis recipients were now able to biosynthesize O-antigen, but no band A was generated. In the B. pertussis recipient, a truncated band A was expressed consistent with a mutation in the wlbH gene, but on Western blotting the expression of a small amount of full-length band A was also seen. Evidence that the wlbH(pa) protein is not fully functional was provided by the failure of the wlb(pa) locus to fully complement a B. pertussis wlbH (Delta wlbH(pe)) mutant. This was supported by DNA sequence data showing that a single amino acid, conserved between homologous proteins from a range of bacteria, is altered in the B. parapertussis WlbH protein.