The CO2-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases

Abstract

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 2 necessity for growth (CO 2 -dependence), a trait of Brucella ovis and most Brucella abortus biovars 1–4 strains. Although molecular tests are replacing phenotypic methods, CO 2 -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 2 -dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO 2 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 2 -dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO 2 -dependent B. abortus and B. ovis , and that a modified CAII sequence explains the CO 2 -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 2 -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 2 -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.