Fluorescence monitoring of cyanobacterial circadian clock components in vitro elucidates phase relationships, improves period/amplitude effect measurements, and uncovers mechanisms for robustness

The rising and setting of the sun, and the environmental changes in light and temperature that are associated with this rhythm, has shaped nearly all life on Earth. Biological clocks that adapt organism’s metabolism, physiology, and behavior to best take advantage of this circadian cycle have heavily influenced the evolution of plants, animals, and bacteria. Despite the importance of biological clocks, no clock mechanism is well understood at the molecular level. A model system that is uniquely suited for detailed mechanistic studies has been established in cyanobacteria, as it’s oscillator is composed of only three core proteins, KaiA, KaiB, and KaiC, and three output proteins SasA, CikA, and RpaA, and can be reconstituted in vitro. Here, real-time fluorescence spectroscopy has been utilized to assign phase relationships, measure period and amplitude effects from modulating core and output protein concentrations, and uncovered a paradigm breaking dose-dependent compensation for low KaiB concentrations by SasA