Structural and functional basis of bacterial transcriptional regulation

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Project Summary / Abstract Antibiotic resistance poses an increasingly prevalent public health challenge, but advances in the development of new drugs has not progressed commensurately. A significant impediment lies in that many fundamental biochemical processes in bacteria remain poorly understood. The primary goal of the proposed research is to illuminate how bacteria respond and adapt to changes within their environments from a structural and mechanistic standpoint. Using the genetically tractable Gram positive organism Bacillus subtilis as our model system, we will focus our efforts on elucidating the molecular basis for copper-dependent transcriptional regulation and uncovering the drivers of enzyme specificity during the environmental stress response. Copper is required for bacterial survival, but excess levels of this transition metal can be toxic. Therefore, copper levels must be carefully controlled. The mechanisms by which copper export occurs have been extensively studied, but relatively little is known about copper import. We hypothesize that Cu uptake is regulated by Cu-dependent transcriptional repressors and the proteins under their control. Here, we will focus on a suite of proteins implicated in these processes, studying them at a detailed structural, molecular, and cellular level to better understand how they regulate copper uptake. In parallel, we will investigate what causes a family of very closely related kinases to specifically regulate distinct functions. Many bacterial species, including Bacillus subtilis and a number of pathogenic strains, encode one or more kinases that contain a domain termed the Bergerat fold. Despite their structural commonalities, enzymes with this domain exhibit strong preferences for their physiological binding partners. However, the molecular basis for how such specificity is conferred has yet to be investigated. We hypothesize that variations within the Bergerat fold influence substrate specificity and kinetics and can be targeted by small molecule inhibitors. By integrating tools from structural biology, microbiology, biochemistry, and computational studies, we will be poised to reveal new paradigms in transcriptional control. The completion of the proposed studies will catalyze our future investigations into elucidating analogous pathways in pathogenic strains, uncovering novel members of these families in bacterial phylogeny, and designing small molecule inhibitors.