The CO-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases.

Brucella bacteria cause brucellosis, a major zoonosis whose control requires efficient diagnosis and vaccines. Identification of classical Brucella spp. has traditionally relied on phenotypic characterization, including surface antigens and 5-10% CO necessity for growth (CO-dependence), a trait of Brucella ovis and most Brucella abortus biovars 1-4 strains. Although molecular tests are replacing phenotypic methods, CO-dependence remains of interest as it conditions isolation and propagation and reflects Brucella metabolism, an area of active research. Here, we investigated the connection of CO-dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO to the bicarbonate used by anaplerotic and biosynthetic carboxylases. Based on the previous demonstration that B. suis carries two functional CAs (CAI and CAII), we analyzed the CA sequences of CO-dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO-dependent B. abortus and B. ovis, and that a modified CAII sequence explains the CO-independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO-independent growth of B. suis in rich media but not of B. abortus in rich media or B. suis in minimal media. Finally, we also found that insertion of a heterologous active CAII into B. ovis reverted the CO-dependence but did not alter its virulence in the mouse model. These results allow a better understanding of central aspects of Brucella metabolism and, in the case of B. ovis, provide tools for large-scale production of diagnostic antigens and vaccines.