Genetic characterization of a Tn5-disrupted glycosyltransferase gene homolog in Brucella abortus and its effect on lipopolysaccharide composition andvirulence

We constructed a rough mutant of Brucella abortus 2308 by transposon (Tn5) mutagenesis. Neither whole cells nor extracted lipopolysaccharide (LPS) ham this mutant, designated RA1, reacted with a Brucella O-side-chain-specific monoclonal antibody (MAb), Bru-38, indicating the absence of O-side-chain synthesis. Compositional analyses of LPS from strain RA1 showed reduced lev els of quinovosamine and mannose relative to the levels in the parental, wi ld-type strain, 2308. We isolated DNA flanking the Tn5 insertion in strain RA1 by cloning a 25-kb XbaI genomic fragment into pGEM-3Z to create plasmid pJM6. Allelic exchange of genomic DNA in B. abortus 2308 mediated by elect roporation of pJM6 produced kanamycin-resistant: clones that were not react ive with MAb Bru-38. Southern blot analysis of genomic DNA from these rough clones revealed Tn5 in a 25-kb XbaI genomic fragment. A homology search wi th the deduced amino acid sequence of the open reading frame disrupted by T n5 revealed limited homology with various glycosyltransferases. This B. abo rtus gene has been named wboA. Transformation of strain RA1 with a broad-ho st-range plasmid bearing the wild-type B. abortus wboA gene resulted in the restoration of O-side-chain synthesis and the smooth phenotype. B. abortus RA1 was attenuated for survival in mice. However, strain RA1 persisted in mice spleens for a longer time than the B. abortus vaccine strain RB51, but as expected, neither strain induced antibodies specific for the O side cha in.