An In Vitro Approach to Elucidating Clock-Modulating Metabolites

Amidst the discussions regarding how the information about light or darkness is transduced to the cyanobacterial circadian clock, it was found that it turned out to be affected by metabolites that reflect photosynthetic activity. Reconstituting the bacterial timekeeper in a test tube containing only KaiA, KaiB, KaiC, ATP, and magnesium enabled a detailed functional analysis of the circadian clock. Here we added CikA, which competes with KaiA for the common binding site on KaiB. Quinone is a redox-sensing metabolite used as an electron shuttle in both photosynthesis and cellular respiration, and its oxidized form as a proxy for darkness becomes acutely abundant at the onset of sunset. Applying a dark pulse to cyanobacteria forces their gene expression rhythm to be shifted in a phase-dependent manner. In our attempt to mimic this process in vitro, oxidized quinone inactivated KaiA and CikA and generated a phase advance and delay respectively, in agreement with the in vivo data. Additionally, magnesium showed a role in inhibiting the KaiC phosphorylation in vitro. A possible history of clock evolution can be suggested from this finding since magnesium could directly modulate the KaiC-only pacemaker, a supposed timekeeping relic of the past that gave rise to today’s intricate KaiABC clock.