Deletion of the BCSP31 gene of Brucella abortus by replacement

The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain.