This project will define the mechanisms within a 24-h (circadian) biological clock that synchronize the endogenous circadian oscillator with natural day/night cycles by integrating genetic, biochemical, biophysical, and cell biological approaches in a cyanobacterial model system. The 2.7 Mb genome of the model organism Synechococcus elongatus is fully sequenced, a global functional genomics project is underway, sophisticated genetic tools are available, 3-dimensional structures are known for the core clock components, and the basic oscillation can be recapitulated in vitro. These resources offer exceptional potential for a comprehensive mechanistic understanding of biological timekeeping. Our findings predict a model in which input pathways reset the cyanobacterial clock in response to environmental cues by modulating the phosphorylation state of the circadian oscillator protein KaiC, which is stimulated by the C- terminal domain of the oscillator protein KaiA. The KaiC phosphorylation state oscillates during the circadian cycle, and is essential for formation of higher order complexes that assemble and disassemble once per circadian cycle. In the prior funding period we identified the following: a key integrator of environmental signals (CikA), the domain of KaiA on which this information impinges, 3-dimensional structures of key domains, other interacting components in the signal transduction pathway, and evidence of cellular redox state sensing by clock components. The proposed project will test our entrainment model by defining the steps from environmental sensing to interaction with the oscillator that enable synchronization of the clock with the external daily cycle. The Specific Aims are to: (1) identify the molecular events downstream of the CikA histidine protein kinase;(2) define the biochemical functions of known and candidate input pathway members to define the molecular signals that flow through the clock;and (3) define the physical interactions of CikA with partners in the cell, including intracellular localization of a functional clock complex. Lay summary: The project will provide new insights into how an organism's circadian clock becomes synchronized with the environment and will show how circadian clocks can be adjusted by external stimuli, which is of relevance for the design of therapeutic interventions. The research will reveal the functions of novel domains of regulatory proteins that also operate in pathogenic bacteria.