MOLECULAR-CLONING AND CHARACTERIZATION OF CGS, THE BRUCELLA-ABORTUS CYCLIC BETA(1-2) GLUCAN SYNTHETASE GENE - GENETIC COMPLEMENTATION OF RHIZOBIUM-MELILOTI NDVB AND AGROBACTERIUM-TUMEFACIENS CHVB MUTANTS

The animal pathogen Brucella abortus contains a gene, cgs, that comple mented a Rhizobium meliloti nodule development (ndvB) mutant and an Ag robacterium tumefaciens chromosomal virulence (chvB) mutant. The compl emented strains recovered the synthesis of cyclic beta(1-2) glucan, mo tility, virulence in A. tumefaciens, and nitrogen fixation in R. melil oti; all traits were strictly associated with the presence of an activ e cyclic beta(1-2) glucan synthetase protein in the membranes. Nucleot ide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino a cids (316.2 kDa) and with 51% identity to R, meliloti NdvB. Four regio ns of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2300 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB, Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly co nserved in Rhizobium, are not necessary for cyclic beta(1-2) glucan sy nthesis. Confirmation of the identity of this protein as B, abortus cy clic beta(1-2) glucan synthetase was done by the construction of a R. abortus Tn3-HoHo1 insertion mutant that does not form cyclic beta(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result su ggests that cyclic beta(1-2) glucan may be a virulence factor in Bruce lla infection.