Polymorphism at the dnaK locus of Brucella species and identification of a Brucella melitensis species-specific marker

The dnaK gene and surrounding sequences from reference strains of the six Brucella species were amplified by the polymerase chain reaction (PCR) with primers chosen according to the published sequence of the B. ovis dnaK gene and studied for polymorphism with nine restriction endonucleases. The restriction patterns were identical for ail species with all restriction endonucleases tested except for B. melitensis strain 16M that showed a different pattern with EcoRV, consistent with the presence of a single site instead of two for the other Brucella species. The absence of the second EcoRV site for B. melitensis 16M was confirmed by DNA sequence analysis. The second EcoRV site in other Brucella species was located in a 12-bp segment, which was missing from the published dnak sequence of B. ovis, between the stop codon of the dnaK gene and its putative transcription terminator sequence. The difference between B. ovis strain 63/290 and B. melitensis 16M was due to an additional base-pair in B. melitensis 16M. Subsequently, 71 other field, vaccinal and reference strains of the six Brucella species and their different biovars were studied for restriction fragment length polymorphism (RFLP) of the dnaK locus with EcoRV. The presence of a unique EcoRV site was specific to B. melitensis strains. Southern blot analysis of whole genomic DIVA digested with EcoRV and with the dnaK gene used as probe also detected a distinct pattern for B. melitensis. These results indicate that both PCR-RFLP and Southern blot analysis of the dnaK locus can be used to distinguish B. melitensis strains from the other Brucella species and may be useful for typing and diagnostic purposes as well.