Bacteria inhabit many environments, from hot dry desert regions to high pressure niches on the ocean floors, from high salt areas to cold latitudes, from inside plant root nodules to inside human cells. Studying the kinds of habitats different bacteria encounter and how they react to the challenges these environments offer is important to understand bacteria in their natural state, what kind of responses characterize them and how they can function when confronted with adversity. Here I present my research on Brucella ovis, a facultative intracellular pathogen that has evolved to withstand stressful and harmful situations, from nutrient limitation, to oxidative stress, to drastic drops in pH. One ambient factor that perturbs B. ovis homeostasis is the atmospheric level of carbon dioxide, as B. ovis cannot be cultured in laboratory conditions without CO2 supplementation. I examined the genetic underpinnings of the CO2 dependence of B. ovis growth, identifying mutations in a carbonic anhydrase gene (bcaA) as responsible for this particular metabolic requirement. B. ovis harbors a unique, non-functional pseudogene allele of bcaA (bcaAbov), and I found that some B. abortus lineages also harbor a bcaA pseudogene, thus rendering them dependent on CO2 supplementation for growth. My data explain why B. ovis and select strains of B. abortus require elevated CO2 levels for growth, which was first noted over a century ago. Transcription of one third of the genes in wild-type Brucella ovis change when cells are shifted from high to low CO2 conditions; gene expression is unchanged upon CO2 shift in a strain in which the pseudogene is restored to a functional bcaA. Thus, wild-type B. ovis has increased sensitivity to environmental levels of CO2 because of a carbonic anhydrase pseudogene. This sensitivity could help B. ovis better detect when it is inside and outside the host. I also present work in which I analyze Brucella ovis in the context of stationary phase, as a proxy to better understand the environment and related response that this pathogen encounters within the host. I discovered that cysE -which encodes for a serine O-acetyltransferase, involved in the first step of de novo cysteine biosynthesis- is required for B. ovis fitness in stationary phase. Deletion of cysE increases sensitivity to hydrogen peroxide and attenuates Brucella ovis in tissue culture infection models. Thus, sulfur and cysteine metabolism is important for resistance to the hostile environment within the intracellular niche and presents an intriguing target for drug development against Brucella infections.




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