PROJECT SUMMARY Signal transduction and protein phosphorylation are vital for life. The ability to respond to stimuli is considered a key characteristic of life. Cells detect new conditions, transduce relevant information into a usable form, and execute appropriate responses. Errors in signal transduction can cause reduced fitness or disease. A common strategy is to represent information by the specific and transient placement of phosphoryl groups on proteins. Many pathogens use phosphorylation to regulate virulence in response to host parameters. Thus, understanding the mechanisms, regulation, and impact of phosphoryl group transfer among microbial signaling proteins is of fundamental interest, and of practical significance to infectious diseases of humans. Kinetics of two-component regulatory system (TCS) phosphotransfer reactions are crucial to biological function. TCSs are widely used for signal transduction by microorganisms and plants (but not humans) to control virulence, physiology, development, behavior, etc. Sensor kinases detect stimuli and internalize input on intracellular dimerization and histidine phosphorylation (DHp) domains using phosphoryl groups. Receiver domains catalyze transfer of phosphoryl groups from sensor kinases to themselves and from themselves to water to modulate response regulator output. Receiver domains can also reversibly exchange phosphoryl groups with histidine-containing phosphotransfer (Hpt) domains to create more complex phosphorelays. The kinetics of phosphotransfer reactions are crucial to synchronize responses with stimuli and can differ by many orders of magnitude between TCSs that control biological functions operating on different timescales. Innovative uses of protein sequence data to design experiments that provide insight into all TCSs. We deliberately confront a key challenge arising from the genomic era - the large and increasing gap between the number of identified TCSs and the number that can reasonably be studied experimentally. Our long-term goal to comprehensively understand the molecular mechanisms of signal transduction in TCSs demands a fundamentally different and less granular approach than studying one TCS at a time. We focus on aspects of DHp, Hpt, and receiver domains that vary between TCSs, using bioinformatics to guide our experiments to unravel the factors governing TCS specialization. We will determine mechanisms and factors that modulate phosphotransfer reactions between DHp, Hpt, and receiver domains (AIM 1); DHp phosphatase activity (AIM 2); and receiver domain autocatalytic reactions, which form the basis of TCS signaling (AIM 3). High impact on TCSs and beyond. TCS proteins are among the most abundant in nature. Completion of our Aims will help predict the properties of uncharacterized TCSs from amino acid sequence features, facilitate systems and synthetic biology applications of TCSs, and potentially aid design of antibiotics that target TCSs. Robust predictive capability ...