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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.   Electronic supplementary material  The online version of this article (10.1186/s13567-018-0583-1) contains supplementary material, which is available to authorized users.

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