The circadian clock orchestrates metabolism and cell division and imposes important consequences to health and diseases, such as obesity, diabetes, and cancer. It is well established that phosphorylation-dependent circadian rhythms are the result of circadian clock protein interactions, which regulate many intercellular processes according to time of day. The phosphorylation-dependent circadian rhythm undergoes a succession of phases: Phosphorylation Phase → Transition Phase → Dephosphorylation Phase. Each phase induces the next phase. However, the mechanism of each phase and how the phosphorylation and dephosphorylation phases are prevented from interfering with each other remain elusive. In this research, we used a newly developed isotopic labeling strategy in combination with a new type of nuclear magnetic resornance (NMR) experiment to obtain the structural and dynamic information of the cyanobacterial KaiABC oscillator system. This system is uniquely suited for the mechanistic studies: mixing KaiA, KaiB KaiC, and ATP generates a self-sustained ~24 h rhythm of KaiC phosphorylation in vitro. Our data strongly suggest that the dynamic states of KaiC underpin the timing mechanism of cyanobacterial oscillator.

The cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator comprised of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. A mutant of a clock gene in the input pathway, cikA, has a cell division defect, and the circadian clock inhibits the cell cycle for a short period of time during each cycle. However, the interaction between the circadian cycle and the cell cycle and the molecular mechanisms underlying it have been poorly understood. In addition, the subcellular localization of clock proteins and possible localization dynamics, which are critical in the timing circuit of eukaryotic clock systems and might also shed light on the interaction between circadian cycle and cell cycle, have remained largely unknown. A combination of genetics, cell biology, and microscopy techniques has been employed to investigate both questions. This work showed that the cell division defect of a cikA mutant is a function of the circadian clock. High ATPase activity of KaiC coincides with the inhibition of cytokinesis by the circadian clock. CikA likely represses KaiC's ATPase activity through an unknown protein, which in cikA's absence stimulates both the ATPase and autokinase activities independently of KaiA or KaiB. SasA-RpaA acts as an output in the control of cell division, and the localization of FtsZ is the target, although it still remains to be seen how RpaA, directly or indirectly, inhibits FtsZ localization. The project also showed that clock proteins are localized to the cell poles. KaiC is targeted to the cell pole in a phosphorylation-dependent manner. KaiB and CikA are also found at the poles independently of KaiC. KaiA likely only localizes to the cell pole during the dephosphorylation phase, which is dependent on both KaiB and KaiC, specifically on the phosphorylation of KaiC at S431. Overall, significant progress was made in both areas and this project sheds light on how the circadian oscillator operates in cyanobacterial cells and interacts with another fundamental cellular function.