Genetic basis for lipopolysaccharide O-antigen biosynthesis in bordetellae - PubMed (original) (raw)

Genetic basis for lipopolysaccharide O-antigen biosynthesis in bordetellae

A Preston et al. Infect Immun. 1999 Aug.

Abstract

Bordetella bronchiseptica and Bordetella parapertussis express a surface polysaccharide, attached to a lipopolysaccharide, which has been called O antigen. This structure is absent from Bordetella pertussis. We report the identification of a large genetic locus in B. bronchiseptica and B. parapertussis that is required for O-antigen biosynthesis. The locus is replaced by an insertion sequence in B. pertussis, explaining the lack of O-antigen biosynthesis in this species. The DNA sequence of the B. bronchiseptica locus has been determined and the presence of 21 open reading frames has been revealed. We have ascribed putative functions to many of these open reading frames based on database searches. Mutations in the locus in B. bronchiseptica and B. parapertussis prevent O-antigen biosynthesis and provide tools for the study of the role of O antigen in infections caused by these bacteria.

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Figures

FIG. 1

FIG. 1

Arrangement of genes in the insert in pBgl-br and the different frames of the coding sequences. wbmA to wbmU are novel, while the remaining genes have been previously described (1), including wlb (LPS band A biosynthesis locus), waaC (heptosyltransferase), waaA (2-keto-3-deoxyoctulosonic acid transferase), and baf (Bvg accessory factor). The DNA to the left of wlbL is present in B. bronchiseptica. The region to the left of wlbL extending to wbmH is also present in B. parapertussis. DNA sequence information beyond this region is not available although whole-genome DNA sequencing of B. parapertussis is under way. In B. pertussis, the wbm locus is replaced by an IS (arrow). The positions of the _Bst_EII restriction sites used for the construction of deletion mutants are indicated by asterisks. wbm contains at least three different transcriptional units, suggested by the direction of the genes. The coding sequences of wbmA and wbmB, wbmH to wbmF, wbmL and wbmK, wbmO to wbmM, wbmP and wbmQ, and wbmR to wbmU may comprise translationally linked units; the coding sequences of adjacent genes within these units overlap. Position numbers are in thousands.

FIG. 2

FIG. 2

(A) SDS-PAGE–silver stain analysis of LPS from B. pertussis Tohama I (lane 1), B. bronchiseptica wild type (lane 2) and mutant (lane 3), and B. parapertussis wild type (lane 4) and mutant (lane 5). The markers indicate the migratory positions of the O antigen of B. bronchiseptica (a, lane 2) and B. parapertussis (b, lane 4), band A of B. pertussis (c, lane 1) and B. bronchiseptica (c, lanes 2 and 3), band B of B. pertussis (d, lane 1) and B. bronchiseptica (d, lanes 2 and 3), the novel structure expressed by the B. parapertussis mutant (d, lane 5), and the truncated band B of B. parapertussis (e, lanes 4 and 5). Wild-type B. bronchiseptica and B. parapertussis both expressed O antigen whereas the mutants did not. (B) Western blot analysis of a replica of the gel shown in panel A with monoclonal antibody BL-2, which recognizes an epitope in band A. This analysis demonstrates that the B. bronchiseptica O-antigen mutant was not affected in band A expression and that the novel structure expressed by the B. parapertussis O antigen mutant was not a full band A structure.

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