Construction of a Brucella abortus RecA mutant and its survival in mice

Abstract

To determine if RecA plays a role in the virulence of Brucella abortus, a B. abortus RecA mutant was constructed and its survival was examined in mice. The recA gene was cloned from a B. abortus genomic DNA library by complementation of an Escherichia coli recA mutant in the presence of methyl methanesulfonate (MMS). The nucleotide sequence of recA was determined and the deduced protein sequence possesses extensive conservation with other RecA proteins of Gram-negative bacteria. A deletion plasmid was constructed in a suicide vector by deleting a segment of recA and inserting a kanamycin resistance gene. The deletion plasmid was introduced into B. abortus strain 2308, a virulent strain, by electroporation. Replacement of recA with the kanamycin resistance fragment was confirmed by Southern blot analysis. The RecA mutant was more sensitive than the parental strain to killing by MMS. When administered intraperitoneally to BALB/c mice, numbers of bacteria per spleen were consistently lower in animals infected with the RecA mutant than with the parental strain. However, both the RecA mutant and parental strain persisted in mice through 100 days post-infection. These results indicate that RecA is not crucial for persistence of B. abortus in mice.