Diverse Sensory Mechanisms in Bacterial PAS-Containing One-Component Systems

All living organisms must constantly sense and respond to environmental changes to survive. Bacteria possess a vast repertoire of molecular signaling systems that enable rapid adaptation to fluctuations in temperature, light, redox state, and nutrient availability. Among these, one-component systems (OCSs)—proteins that couple stimulus detection and response within a single protein—represent a compact and versatile mode of regulation. Within this class, Per-ARNT-Sim (PAS) domain-containing OCSs are especially widespread and diverse, yet many remain uncharacterized with respect to their activating ligands and signaling mechanisms. Understanding how PAS domains detect stimuli and transmit conformational signals to their respective output domains not only provides fundamental insight into bacterial physiology but also offers opportunities to engineer tunable molecular switches for biotechnology. In this dissertation, I combined bioinformatics, structural biology, and biophysical methods to identify and characterize new OCS transcription factors and enzymes with distinct sensory modalities. From a genome-wide search, I discovered three novel systems: 1) FG219, a heme-binding transcription factor, which undergoes large-scale conformational transitions upon changes in redox state. Structural analyses using NMR, HDX–MS, and X-ray crystallography revealed redox sensitive structural rearrangements and led to proof-of-concept experiments for development ofin vivo biosensor for oxidative stress. 2) BO253, an OCS protein which harbors an unusually large ligand-binding pocket, where crystallographic detection of a PEG molecule within the cavity provided clues to the chemical nature of potential physiological ligands. 3) CU228, a constitutively dimeric OCS, which served as a model for developing a pipeline to identify small-molecule stabilizers of orphan receptors, integrating both protein- and ligand-based detection strategies. In a separate, collaborative project, I studied TdcA, a temperature-sensitive PAS containing OCS diguanylate cyclase from a clinical isolate of a pathogenic Pseudomonas aeruginosa, which mediates temperature sensitive biofilm formation, related to the temperature increase of host invasion. Through temperature-resolved HDX–MS and infrared spectroscopy, I identified thermolabile regions responsible for temperature sensing, which were functionally validated in vivo by collaborators. Together, these studies expand our understanding of the diversity and conservation of the structure and mechanism of PAS-mediated signaling and demonstrate how insights from basic bacterial biology can guide synthetic and biomedical applications. By elucidating new modes of ligand and redox sensing, establishing generalizable screening frameworks, and identifying potential antibiotic targets, this work advances both the fundamental principles and practical utility of one-component signal transduction systems.