Intrinsic period stability of the cyanobacterial circadian oscillator across in vitro and in vivo conditions

Mar 18, 2026Proceedings of the National Academy of Sciences of the United States of America

Stable internal timing of the cyanobacteria’s daily clock inside cells and in lab tests

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Abstract

The period of over 20 KaiC period mutants ranged from 15 to 60 hours and was insensitive to environmental conditions.

Circadian rhythms can maintain an approximately 24-hour period with precision, regardless of external factors like temperature.
The reliability of in vivo circadian rhythms is influenced by both a protein-based oscillator and additional feedback mechanisms.
KaiC ATPase activity correlates more strongly with circadian frequency in controlled lab conditions than in living organisms.
The KaiC protein-based oscillator is suggested to encode a reliable circadian period independently of rhythm amplitude.
Intrinsic properties of the circadian clock may help maintain stability and precision in timing within living cells.

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A defining feature of circadian clocks that enables adaptation to Earth's rotation is their ability to sustain an approximately 24 h period with precision, regardless of environmental factors such as temperature. This remarkable reliability of circadian timing can be reconstituted in vitro using only three cyanobacterial clock proteins: KaiA, KaiB, and KaiC. In vivo circadian rhythms, however, are governed not only by this Kai protein-based oscillator but also by transcription-translation feedback loops and additional clock components. The contribution of the Kai protein-based circadian oscillator to the overall reliability of the in vivo circadian rhythm remains unclear. In this study, we compared over 20 KaiC period mutants with periods ranging from 15 to 60 h under in vitro and in vivo conditions. In both cases, the period was insensitive to environmental conditions, suggesting a compensatory mechanism independent of metabolic state or rhythm amplitude. The ATPase activity of KaiC, the pacemaker of the cyanobacterial circadian clock, exhibited a stronger correlation with in vitro circadian frequency than in vivo circadian frequency. These results indicate that the KaiC ATPase-driven protein-based circadian oscillator inherently encodes a reliable circadian period independent of rhythm amplitude or environmental conditions. This intrinsic property likely plays a critical role in preserving the precision and stability of circadian timing in vivo while being influenced by the intracellular environment.

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Intrinsic period stability of the cyanobacterial circadian oscillator across in vitro and in vivo conditions

Abstract

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