A Carbonic Anhydrase Pseudogene Sensitizes Select Lineages to Low CO Tension

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 CO supplementation to cultivate particular species, which confounded early efforts to isolate from diseased cattle. Differences in the capacity of species to assimilate CO are determined by mutations in the carbonic anhydrase gene, Ancestral single-nucleotide insertions in have resulted in frameshifted pseudogenes in and lineages, which underlie their inability to grow under the low CO tension of a standard atmosphere. Incubation of wild-type in air selects for mutations that "rescue" a functional reading frame, which enables growth under low CO and enhances the growth rate under high CO Accordingly, we show that heterologous expression of functional carbonic anhydrases enables growth in air. Growth of is acutely sensitive to a reduction in CO tension, while frame-rescued mutants are insensitive to CO shifts. initiates a gene expression program upon CO 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 sensitize the response of and to reduced CO tension relative to that of other lineages. CO-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts. spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. and the majority of strains require high CO tension to be cultivated and harbor conserved insertional mutations in the carbonic anhydrase gene, , 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 that restore its consensus reading frame and rescue its function. Loss of function has evolved independently in the and lineages and results in a dramatically increased sensitivity to CO limitation.