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Reconstruction of the ancient cyanobacterial proto-circadian clock system KaiABC

ABSTRACT: Earlier in its history, the Earth used to spin faster than it does today. How ancient organisms adapted to the short day/night cycles during that time remains unclear. In this study we reconstruct and analyse the ancient circadian clock system KaiABC (anKaiABC) of cyanobacteria that existed ~0.95 billion years ago, when the daily light/dark cycle was approximately 18 hour-long. Compared to their contemporary counterparts, anKaiABC proteins had different structures and interactions. The kinase, phosphatase, and adenosine triphosphatase (ATPase) activities of anKaiC were lower, while the anKaiA and anKaiB proteins were less effective at regulating the KaiC/anKaiC phosphorylation status. We provide evidence indicating that the anKaiABC system does not endogenously oscillate, but it can be entrained by an 18 hour-long light/dark cycle. A Synechococcus strain expressing ankaiABC genes exhibits better adaptation to 9-h light/9-h dark cycles (LD9:9) that mimic the ancient 18-h day/night cycles, whereas the kaiABC-expressing strain preferentially adapts to the LD12:12 contemporary conditions. These findings suggest that, despite its lack of self-sustaining circadian oscillation, the proto-circadian system may have mediated adaptation of ancient cyanobacteria to the 18 hour-long light/dark cycles present 0.95 billion years ago.

SUBMITTER: Silin Li

PROVIDER: S-SCDT-10_1038-S44318-025-00425-0 | biostudies-other |

REPOSITORIES: biostudies-other

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Ancient cyanobacterial proto-circadian clock adapted to short day-night cycles ~ 0.95 billion years ago

Project description: Not available

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Frequency doubling in the cyanobacterial circadian clock.

Project description:Organisms use circadian clocks to generate 24-h rhythms in gene expression. However, the clock can interact with other pathways to generate shorter period oscillations. It remains unclear how these different frequencies are generated. Here, we examine this problem by studying the coupling of the clock to the alternative sigma factor sigC in the cyanobacterium Synechococcus elongatus Using single-cell microscopy, we find that psbAI, a key photosynthesis gene regulated by both sigC and the clock, is activated with two peaks of gene expression every circadian cycle under constant low light. This two-peak oscillation is dependent on sigC, without which psbAI rhythms revert to one oscillatory peak per day. We also observe two circadian peaks of elongation rate, which are dependent on sigC, suggesting a role for the frequency doubling in modulating growth. We propose that the two-peak rhythm in psbAI expression is generated by an incoherent feedforward loop between the clock, sigC and psbAI Modelling and experiments suggest that this could be a general network motif to allow frequency doubling of outputs.

| S-EPMC5199125 | biostudies-literature

ATP hydrolysis by KaiC promotes its KaiA binding in the cyanobacterial circadian clock system.

Project description:The cyanobacterial clock is controlled via the interplay among KaiA, KaiB, and KaiC, which generate a periodic oscillation of KaiC phosphorylation in the presence of ATP. KaiC forms a homohexamer harboring 12 ATP-binding sites and exerts ATPase activities associated with its autophosphorylation and dephosphorylation. The KaiC nucleotide state is a determining factor of the KaiB-KaiC interaction; however, its relationship with the KaiA-KaiC interaction has not yet been elucidated. With the attempt to address this, our native mass spectrometric analyses indicated that ATP hydrolysis in the KaiC hexamer promotes its interaction with KaiA. Furthermore, our nuclear magnetic resonance spectral data revealed that ATP hydrolysis is coupled with conformational changes in the flexible C-terminal segments of KaiC, which carry KaiA-binding sites. From these data, we conclude that ATP hydrolysis in KaiC is coupled with the exposure of its C-terminal KaiA-binding sites, resulting in its high affinity for KaiA. These findings provide mechanistic insights into the ATP-mediated circadian periodicity.

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Dynamic localization of the cyanobacterial circadian clock proteins.

Project description:BackgroundThe cyanobacterial circadian clock system has been extensively studied, and the structures, interactions, and biochemical activities of the central oscillator proteins (KaiA, KaiB, and KaiC) have been well elucidated. Despite this rich repository of information, little is known about the distribution of these proteins within the cell.ResultsHere we report that KaiA and KaiC localize as discrete foci near a single pole of cells in a clock-dependent fashion, with enhanced polar localization observed at night. KaiA localization is dependent on KaiC; consistent with this notion, KaiA and KaiC colocalize with each other, as well as with CikA, a key input and output factor previously reported to display unipolar localization. The molecular mechanism that localizes KaiC to the poles is conserved in Escherichia coli, another Gram-negative rod-shaped bacterium, suggesting that KaiC localization is not dependent on other clock- or cyanobacterial-specific factors. Moreover, expression of CikA mutant variants that distribute diffusely results in the striking delocalization of KaiC.ConclusionsThis work shows that the cyanobacterial circadian system undergoes a circadian orchestration of subcellular organization. We propose that the observed spatiotemporal localization pattern represents a novel layer of regulation that contributes to the robustness of the clock by facilitating protein complex formation and synchronizing the clock with environmental stimuli.

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Quinone sensing by the circadian input kinase of the cyanobacterial circadian clock.

Project description:Circadian rhythms are endogenous cellular programs that time metabolic and behavioral events to occur at optimal times in the daily cycle. Light and dark cycles synchronize the endogenous clock with the external environment through a process called entrainment. Previously, we identified the bacteriophytochrome-like circadian input kinase CikA as a key factor for entraining the clock in the cyanobacterium Synechococcus elongatus PCC 7942. Here, we present evidence that CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms, varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC during resetting of circadian phase by a dark pulse. The abundance of CikA varies inversely with light intensity, and its stability decreases in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The pseudo-receiver domain of CikA is crucial for sensitivity to DBMIB, and it binds the quinone directly, a demonstration of a previously unrecognized ligand-binding role for the receiver fold. Our results suggest that resetting the clock in S. elongatus is metabolism-dependent and that it is accomplished through the interaction of the circadian oscillator with CikA.

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Damped Oscillating Phosphoryl Transfer Reaction in the Cyanobacterial Circadian Clock.

Project description:Most organisms have circadian clocks to ensure the metabolic cycle to resonate with the rhythmic environmental changes without "damping," or losing robustness. Cyanobacteria is the oldest and simplest form of life that is known to harbor this biological intricacy. Its KaiABC-based central oscillator proteins can be reconstituted inside a test tube, and the post-translational modification cycle occurs with 24 h periodicity. KaiC's two major phosphorylation sites, Ser-431 and Thr-432, become phosphorylated and dephosphorylated by interacting with KaiA and KaiB, respectively. Here, we mutate Thr-432 into Ser to find the oscillatory phosphoryl transfer reaction damps. Previously, this mutant KaiC was reported to be arrhythmic in vivo. However, we found that the mutant KaiC gradually loses the ability to run in an autonomous manner and stays constitutively phosphorylated after 3 cycles in vitro.

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Protocols for in vitro reconstitution of the cyanobacterial circadian clock.

Project description:Circadian clocks are intracellular systems that orchestrate metabolic processes in anticipation of sunrise and sunset by providing an internal representation of local time. Because the ~24-h metabolic rhythms they produce are important to health across diverse life forms there is growing interest in their mechanisms. However, mechanistic studies are challenging in vivo due to the complex, that is, poorly defined, milieu of live cells. Recently, we reconstituted the intact circadian clock of cyanobacteria in vitro. It oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of individual clock proteins and promoter DNA simultaneously under defined conditions without user intervention. We found that reproducibility of the reactions required strict adherence to the quality of each recombinant clock protein purified from Escherichia coli. Here, we provide protocols for preparing in vitro clock samples so that other labs can ask questions about how changing environments, like temperature, metabolites, and protein levels are reflected in the core oscillator and propagated to regulation of transcription, providing deeper mechanistic insights into clock biology.

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The cyanobacterial circadian clock follows midday in vivo and in vitro.

Project description:Circadian rhythms are biological oscillations that schedule daily changes in physiology. Outside the laboratory, circadian clocks do not generally free-run but are driven by daily cues whose timing varies with the seasons. The principles that determine how circadian clocks align to these external cycles are not well understood. Here, we report experimental platforms for driving the cyanobacterial circadian clock both in vivo and in vitro. We find that the phase of the circadian rhythm follows a simple scaling law in light-dark cycles, tracking midday across conditions with variable day length. The core biochemical oscillator comprised of the Kai proteins behaves similarly when driven by metabolic pulses in vitro, indicating that such dynamics are intrinsic to these proteins. We develop a general mathematical framework based on instantaneous transformation of the clock cycle by external cues, which successfully predicts clock behavior under many cycling environments.

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Bayesian modeling reveals metabolite-dependent ultrasensitivity in the cyanobacterial circadian clock.

Project description:Mathematical models can enable a predictive understanding of mechanism in cell biology by quantitatively describing complex networks of interactions, but such models are often poorly constrained by available data. Owing to its relative biochemical simplicity, the core circadian oscillator in Synechococcus elongatus has become a prototypical system for studying how collective dynamics emerge from molecular interactions. The oscillator consists of only three proteins, KaiA, KaiB, and KaiC, and near-24-h cycles of KaiC phosphorylation can be reconstituted in vitro. Here, we formulate a molecularly detailed but mechanistically naive model of the KaiA-KaiC subsystem and fit it directly to experimental data within a Bayesian parameter estimation framework. Analysis of the fits consistently reveals an ultrasensitive response for KaiC phosphorylation as a function of KaiA concentration, which we confirm experimentally. This ultrasensitivity primarily results from the differential affinity of KaiA for competing nucleotide-bound states of KaiC. We argue that the ultrasensitive stimulus-response relation likely plays an important role in metabolic compensation by suppressing premature phosphorylation at nighttime.

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Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

Project description:KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix-coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation.

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