Genetic organisation of the lipopolysaccharide O-antigen biosynthesis region of Brucella melitensis 16M (wbk)

Brucella spp. are Gram-negative, facultative intracellular bacteria that ca use a zoonotic world-wide disease. As in other Gram-negative bacteria, its S-LPS (smooth lipopolysaccharide) is a major determinant of virulence. The Brucella melitensis 16M LPS O-antigen is a homopolymer of 4-formamido-4,6, dideoxymannose. In this study, the previously cloned 14-kb wbk gene cluster was sequenced, and seven open reading frames (ORFs) as well as four insert ion sequences were identified. Six of the seven ORFs are homologous to LPS biosynthesis genes from other organisms. The gmd, per and wbkC gene product s are predicted to be involved in 4-formamido-4,6,dideoxymannose synthesis. By deletion experiments, we demonstrated that the putative formyltransfera se WbkC is absolutely required for the O-side-chain production. The wbkA ge ne product is similar to several mannosyltransferases and is probably invol ved in the polymerisation of the B. melitensis O-side-chain. We also identi fied two genes (wzm and wzt) encoding proteins with high similarity to seve ral two-component ABC (ATP-binding cassette) transporters. Their implicatio n in O-antigen translocation across the inner membrane was confirmed by gen e replacement. Finally, no function has been assigned to the wbkB gene eith er by homology search or functionally, because deletion of wbkB did not int erfere with the O-antigen structure. The seven ORFs have a low G + C conten t, indicating that they might have been acquired by lateral transfer from a progenitor with more A + T rich DNA. (C) 2000 Editions scientifiques et me dicales Elsevier SAS.