A Carbonic Anhydrase Pseudogene Sensitizes Select Brucella Lineages to Low CO₂ Tension

Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO₂ supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO₂ are determined by mutations in the carbonic anhydrase gene, bcaA Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO₂ tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that "rescue" a functional bcaA reading frame, which enables growth under low CO₂ and enhances the growth rate under high CO₂ Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO₂ tension, while frame-rescued B. ovis mutants are insensitive to CO₂ shifts. B. ovis initiates a gene expression program upon CO₂ downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO₂ tension relative to that of other Brucella lineages. CO₂-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.IMPORTANCEBrucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO₂ tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO₂ limitation.