Low temperature nullifies the circadian clock in cyanobacteria through Hopf bifurcation.

Cold temperatures lead to nullification of circadian rhythms in many organisms. Two typical scenarios explain the disappearance of rhythmicity: the first is oscillation death, which is the transition from self-sustained oscillation to damped oscillation that occurs at a critical temperature. The second scenario is oscillation arrest, in which oscillation terminates at a certain phase.

Tracking and visualizing the circadian ticking of the cyanobacterial clock protein KaiC in solution.

The circadian clock in cyanobacteria persists even without the transcription/translation feedbacks proposed for eukaryotic systems. The period of the cyanobacterial clock is tuned to the circadian range by the ATPase activity of a clock protein known as KaiC. Here, we provide structural evidence on how KaiC ticks away 24 h while coupling the ATPase activity in its N-terminal ring to the phosphorylation state in its C-terminal ring.

Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus.

In the unicellular cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild-type strains, >30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk.

Nonparametric entrainment of the in vitro circadian phosphorylation rhythm of cyanobacterial KaiC by temperature cycle.

The three cyanobacterial Kai proteins and ATP are capable of generating an autonomous rhythm of KaiC phosphorylation in a test tube. As the period is approximately 24 hours and is stable in a wide temperature range, this rhythm is thought to function as the basic oscillator of the cyanobacterial circadian system. We have examined the rhythm under various temperature cycles and found that it was stably entrained by a temperature cycle of 20-28 hours.

Regulation of circadian clock gene expression by phosphorylation states of KaiC in cyanobacteria.

Three clock proteins--KaiA, KaiB, and KaiC--have been identified as essential components of the circadian oscillator in cyanobacteria, and Kai-based chemical oscillation is thought to be the basic circadian timing mechanism in Synechococcus elongatus PCC 7942. Transcription and translation of kaiBC in cyanobacterial cells was quantitatively studied to elucidate how these processes are coupled to the chemical oscillator using a strain in which circadian oscillation is under the control of IPTG (isopropyl-beta-D-thiogalactopyranoside). The kinetics of repression of kaiBC promoter triggered by IPTG allowed estimation of transient response at 10 h.

ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria.

Self-sustainable oscillation of KaiC phosphorylation has been reconstituted in vitro, demonstrating that this cycle is the basic time generator of the circadian clock of cyanobacteria. Here we show that the ATPase activity of KaiC satisfies the characteristics of the circadian oscillation, the period length, and the temperature compensation. KaiC possesses extremely weak but stable ATPase activity (15 molecules of ATP per day), and the addition of KaiA and KaiB makes the activity oscillate with a circadian period in vitro.

Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro.

Kai proteins globally regulate circadian gene expression of cyanobacteria. The KaiC phosphorylation cycle, which persists even without transcription or translation, is assumed to be a basic timing process of the circadian clock. We have reconstituted the self-sustainable oscillation of KaiC phosphorylation in vitro by incubating KaiC with KaiA, KaiB, and adenosine triphosphate.