Project Summary Circadian rhythms are daily oscillations in behavior with a nearly 24-hour period that are generated by an internal biological clock. Across many organisms, health and fitness are impaired when the circadian clock does not appropriately synchronize with the daily cycles in the external environment. It is thus critical to understand how clocks respond to challenging fluctuating environments with intermittent or irregular inputs that are typical of modern life. This problem is conceptually challenging because circadian clocks are complex systems. In general, there is a core oscillator consisting of biochemical circuitry that generates a rhythmic daily signal, and the timing of this rhythm can be adjusted by input signals that communicate information about the environment to the oscillator. However, this oscillator is also embedded in the rest of cellular physiology, and so its response to a changing environment is likely contingent on the status of metabolism and other signaling pathways. We are using the bacterial model organism Synechococcus elongatus to crack the problem of clock-environment interaction because this organism has the remarkable feature that the core oscillator can be reconstituted in vitro using purified proteins. We will thus use a reductionistic approach to build up to an integrated mathematical model of clock function in the intact cell when subject to environmental fluctuations. In Aim 1, we will study the purified test tube oscillator, collecting a large data set of kinetic measurements on the core clock proteins at various temperatures, metabolite concentrations, and protein stoichiometries. Using advanced statistical approaches, we will then constrain a model of elementary reactions to uncover how temperature compensation, metabolic sensing, and entrainment function in the core oscillator. In Aim 2, we will study how the clock shifts in response to environmental fluctuations in the living cell. Here we will use a novel assay to isolate the history-dependence of clock sensitivity that is absent from the core oscillator. We will then use a genetic analysis to find the key pathways used to modulate clock sensitivity in vivo. These data will then be incorporated into a expanded mathematical model that describes the function of the clock in vivo when environmental conditions fluctuate. In Aim 3, we will develop a deep mutagenic scanning approach, to find the clock phenotype and competitive growth defects of 10,000s of point mutations in the clock genes simultaneously. This will not only allow us to discover critical interaction sites on the clock proteins, but also to obtain a comprehensive list of period mutants and mutants that disrupt temperature compensation. Because this assay is based on the transcriptional feedback loops ubiquitous in circadian clocks, it can be generally applied to other clock systems as well.