A Carbonic Anhydrase Pseudogene Sensitizes Select Brucella Lineages to Low CO ₂ Tension

Abstract

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. IMPORTANCE Brucella 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.

Polymorphisms in Brucella Carbonic Anhydrase II Mediate CO2 Dependence and Fitness in vivo

“…Brucella species also carry a second CA, CA I, but the polymorphisms found at both the DNA and protein levels do not allow us to infer CO 2 dependence. This result is in agreement with that reported by Pérez-Etayo et al (2018) and Varesio et al (2019) and further extends the range of strains tested.…”

“…Moreover, although an active CA I alone is enough to support CO 2 -independent growth of B. suis in rich media, it is not able to do it in minimal media or to support CO 2 -independent growth of B. abortus at all. A similar result was also obtained by Varesio et al (2019) that identified BcaA BOV (CA II) as the enzyme responsible for the growth of B. ovis in a standard, unsupplemented atmosphere (0.04% CO 2 ), in this case, by whole-genome sequencing (WGS) of CO 2 -independent mutants. Interestingly, they also reported that a CO 2 downshift B. ovis initiates a gene expression program that resembles the stringent response and results in transcriptional activation of its type IV secretion system.…”

Pseudogene-Derived lncRNAs and Their miRNA Sponging Mechanism in Human Cancer

“…However, compared with other members of lncRNAs, transcribed pseudogenes-derived lncRNAs have not been paid attention previously. Recent studies have demonstrated that transcribed pseudogenederived lncRNAs play important roles in multiple biological processes, such as cell proliferation, cell cycle, cell migration and cell death (Lai et al, 2019;Oliveira-Mateos et al, 2019;Varesio et al, 2019).…”

Brucella oviscysteine biosynthesis contributes to peroxide stress survival and fitness in the intracellular niche

“…A B. ovis RB Tn-himar library was built and mapped as described (16). Briefly, E. coli APA752 (a WM3064 donor strain carrying a pKMW3 mariner transposon library) was conjugated into B. ovis bcaA1 (17) under atmospheric CO2 conditions. Kan resistant colonies were collected, grown to OD600 = 0.6 and frozen in 1 ml aliquots.…”

“…The host environment inhabited by B. ovis is quite restricted: it is sexually transmitted and has a specific tropism for the male genital tract in rams (13)(14)(15). We previously constructed a randomly barcoded (RB) library of B. ovis Tn-himar mutants (16,17), and in this present study we set out to develop this barcoded mutant library as a tool to identify B. ovis genes with fitness defects in stationary phase culture. We measured the relative fitness of RB Tn-himar mutants as a function of growth phase in a complex medium and discovered that disruption of the cysteine biosynthesis gene, cysE, resulted in the largest stationary phase fitness defect in our experiment.…”