PROJECT SUMMARY The ability to respond to stimuli is often considered to be a key characteristic of life. Cells can detect new conditions, transduce that information into a usable form, and execute an appropriate response. One common signal transduction strategy is to represent information by the specific and transient placement of phosphoryl groups on proteins. Errors in signal transduction can lead to diseases (e.g. cancer, diabetes), and drugs have been developed to block aberrant signaling processes. Understanding the mechanisms, regulation, and impact of protein phosphorylation is thus of fundamental interest, as well as of practical significance to human health. Microorganisms are the dominant form of life on Earth by many measures, including genetic diversity, raw numbers, environmental distribution, and evolutionary experience. Thus, it is logical to seek basic signal transduction principles in microbes. Our long-term goal is comprehensive understanding of signal transduction by two-component regulatory systems, which occur in microorganisms from all three phylogenetic domains, as well as plants. In a basic two-component system, a sensor kinase (SK) detects stimuli and autophosphorylates using ATP. A response regulator (RR) then catalyzes phosphotransfer from the SK (or from small molecules), which turns on the response. RR dephosphorylation, either self-catalyzed or stimulated by another protein, ends the response. Inclusion of histidine-containing phosphotransferase (Hpt) proteins results in more complex multi-step phosphorelays by adding an Hpt and a second RR onto the basic SK to RR scheme. The kinetics and directionality of phosphoryl group reactions are important to synchronize responses with stimuli. Genome sequencing presents a challenge (a rapidly widening gap between the number of known proteins and what can be studied experimentally) and an opportunity (diverse and extensive sequence data). To learn how to reveal properties of hundreds of thousands of two-component proteins from sequence data alone, our innovative research strategy focuses on amino acid sequence differences (rather than similarities) between the conserved domains of SKs, Hpts, or RRs. Our well-established and productive experimental approach integrates biochemistry, bioinformatics, biophysics, molecular biology, and structural biology. In this project, we will identify factors that affect the kinetics of self-catalyzed RR phosphorylation and dephosphorylation (AIM 1), SK-stimulated dephosphorylation of RRs (AIM 2), and phosphotransfer reactions between Hpts and RRs (AIM 3). We will also characterize the molecular mechanisms underlying each of these reactions. Antibiotic resistance of bacterial and fungal pathogens is a major and increasing threat to human health. RRs are central to most phosphotransfer reactions of two-component systems. Our study of binding of small molecules to RRs may influence design of therapeutic agents to disable critical two-component systems of microbial pathogens. The results of our project could also be used to predict or manipulate the signaling kinetics of two-component systems. Fundamental principles of signal transduction may emerge as well.