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% CO2 necessity for growth (CO2-dependence), a trait of Brucella ovis and most Brucella abortus biovars 1–4 strains. Although molecular tests are replacing phenotypic methods, CO2-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 CO2-dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO2 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 CO2-dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO2-dependent B. abortus and B. ovis, and that a modified CAII sequence explains the CO2-independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO2-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 CO2-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 materialThe online version of this article (10.1186/s13567-018-0583-1) contains supplementary material, which is available to authorized users.

A carbonic anhydrase pseudogene sensitizes selectBrucellalineages to low CO2tension

“…We report a forward genetic selection strategy to identify B. ovis mutants that grow spontaneously under standard atmospheric conditions, and have defined the genetic changes in these strains by whole genome sequencing. In agreement with recent results from Perez-Etayo and colleagues (27), we found that B. ovis mutants selected to grow in a standard atmosphere carry a frameshift mutation at the 3' end of a b-carbonic anhydrase gene, bcaABOV. Carbonic anhydrases catalyze the reversible hydration of CO2 to bicarbonate, an essential metabolic building block (28), and plasmidbased expression of the frameshifted bcaABOV allele or of functional Escherichia coli carbonic anhydrases, can or cynT, are sufficient to enable growth of B. ovis in a standard atmosphere.…”

“…Replacing bcaABOV with bcaABAB2308 enabled growth of B. ovis in 0.04% CO2 (Fig. 4C), consistent with Perez-Etayo et al (27). Replacing bcaABOV with the frameshifted bcaABAB pseudogene did not enable B. ovis growth in a standard atmosphere (Fig.…”

“…Similar bcaABOV mutations in B. ovis strains that grow without CO2 supplementation have been recently described by Perez-Etayo et al (27), though they have used the name carbonic anhydrase II (CAII) for the gene locus and product. For the purposes of this manuscript, we have chosen to use the bacterial genetic naming guidelines of Demerec et al (29), and thus refer to this gene as bcaABOV.…”

“…Our group and others (27) have shown that bcaA is a specific genetic determinant of growth under low pCO2. We have further shown that B. ovis and most B. abortus strains harbor non-functional bcaA pseudogenes containing distinct single-base insertion mutations that result in a frameshifted gene product.…”

“…Strains that failed to grow are indicated with "no growth". frameshifted mutant B. ovis BcaA1BOV protein is identical to B. suis BcaABSU1330 (locus tag: BR_RS08400; also known as Bs1330CAII (27) or bsCAII (34)) with the exception of one residue (Fig. S2A).…”