PROJECT SUMMARY Enabling High-Throughput Analysis and Single-Cell Imaging of Bacterial Signals Our research aims to understand how bacteria perceive chemical signals to regulate different behaviors. We have invented different types of biosensors to rapidly measure key signaling molecules in bacteria, including one for cyclic di-GMP. This signal controls whether bacteria attach to surfaces, form sticky biofilms, and secrete toxins. One of our major goals is to identify nutrients, other chemicals, and environmental inputs that change cyclic di-GMP levels in different bacteria. We recently demonstrated a successful approach that combines structure-based bioinformatics analysis and experimental screening. However, the discovery of primary inputs remains challenging because each bacterium harbors many cyclic di-GMP signaling enzymes, the signal is transiently produced, highly charged, and low in abundance, and the screening method remains a key bottleneck. Thus, this proposal will develop next-generation fluorescent biosensors to enhance high-throughput, quantitative screening of enzyme activity directly in cells (Aim 2). These biosensors then will be applied to discover primary inputs for a widespread small molecule binding domain associated with cyclic di-GMP and other signaling enzymes (Aim 3). In addition, towards understanding environmental factors that regulate cyclic di-GMP, this proposal will develop a new type of biosensor to perform in situ imaging of cyclic di-GMP in biofilms (Aim 3). In the long term, this project aims to inform personalized diets to treat inflammatory bowel diseases and promote gut health. For this renewal of the project, the original scope also has been expanded to study the permeability of small molecules into bacterial cells. The permeability process includes passive permeation, active uptake, and active efflux mechanisms, and is critical to bacterial growth, signaling, and antibiotic resistance. This proposal will develop a high-throughput assay that enables real-time monitoring of small molecule permeability in cells (Aim 1). The assay will be applied to understand both the molecular structures and genetic factors that affect accumulation of fluorescent dyes and of clinical antibiotics inside cells. In the long term, this new aim will improve chemical biology tools that use these dyes and antibiotics treatments.