A carbonic anhydrase pseudogene sensitizes selectBrucellalineages to low CO2tension

Abstract

AbstractBrucellaare 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 increased CO2tension to cultivate particular species, which confounded early efforts to isolateB. abortusfrom diseased cattle. Differences in the capacity ofBrucellaspecies to assimilate CO2are determined by mutations in the carbonic anhydrase gene,bcaA. Ancestral single nucleotide insertions inbcaAhave resulted in frameshifted pseudogenes inB. abortusandB. ovislineages, which underlie their inability to grow under the low CO2tension of a standard atmosphere. Incubation of wild-typeB. ovisin air selects for mutations that “rescue” a functionalbcaAreading frame, which enables growth under low CO2and enhances growth rate in high CO2. Consistent with this result, we show that heterologous expression of functionalE. colicarbonic anhydrases enablesB. ovisgrowth in air. Growth of wild-typeB. ovisis acutely sensitive to a reduction in CO2tension, while frame-rescuedB. ovismutants are completely insensitive to CO2shifts. Wild-typeB. ovisinitiates a gene expression program upon CO2downshift that resembles the stringent response and results in activation of itsvirBtype IV secretion system. Our study provides evidence that loss-of-function insertion mutations inbcaAsensitize the response ofB. ovisandB. abortusto reduced CO2tension relative to otherBrucellalineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.ImportanceBrucellaspp. are highly related, but exhibit differences in animal host preference that must be determined by genome sequence differences.B. ovisand the majority ofB. abortusstrains require increased CO2tension to be cultivatedin vitro, and harbor conserved insertional mutations in the carbonic anhydrase,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 inbcaAthat restore its consensus reading frame and rescue its function. Loss ofbcaAfunction has evolved independently in theB. ovisandB. abortuslineages, and results in a dramatically increased sensitivity to CO2limitation.