Robust Circadian Oscillations in Growing Cyanobacteria Require Transcriptional Feedback

Keeping in Synch Although it differs from mammalian clocks, the circadian clock of cyanobacteria is a valuable model for understanding how such clocks function. At the heart of the cyanobacterial clock is a posttranslational regulation (PTR) circuit in which the phosphorylation of the clock protein KaiC oscillates. This circuit is apparently sufficient for generating rhythms, but it is connected to a transcriptional-translational (TTR) feedback loop more similar to the one that functions in mammals. This TTR loop is, at least in some conditions, dispensable. To understand the role of the TTR circuit, Teng et al. (p. 737) engineered cyanobacteria so that the circadian behavior of individual cells in a population of growing cells could be monitored. Cells engineered to lack the TTR mechanism had rhythmic clocks but fell out of synch with the other cells in a population over time. The experimental results together with mathematical modeling indicate that the TTR mechanism is important to allow cells to robustly stay in rhythm with one another in the absence of synchronizing external cues. The cyanobacterial clock uses one circuit for rhythms and a second circuit for intercellular synchronous oscillations. The remarkably stable circadian oscillations of single cyanobacteria enable a population of growing cells to maintain synchrony for weeks. The cyanobacterial pacemaker is a posttranslational regulation (PTR) circuit that generates circadian oscillations in the phosphorylation state of the clock protein KaiC. Layered on top of the PTR is transcriptional-translational feedback regulation (TTR), common to all circadian systems, consisting of a negative feedback loop in which KaiC regulates its own production. We found that the PTR circuit is sufficient to generate oscillations in growing cyanobacteria. However, in the absence of TTR, individual oscillators were less stable and synchrony was not maintained in a population of cells. Experimentally constrained mathematical modeling reproduced sustained oscillations in the PTR circuit alone and demonstrated the importance of TTR for oscillator synchrony.

[1]  S. Kay,et al.  Molecular bases of circadian rhythms. , 2001, Annual review of cell and developmental biology.

[2]  Tetsuya Mori,et al.  Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC , 2003, The EMBO journal.

[3]  I. Mihalcescu,et al.  Cyanobacterial clock, a stable phase oscillator with negligible intercellular coupling , 2007, Proceedings of the National Academy of Sciences.

[4]  S. Kay,et al.  Time zones: a comparative genetics of circadian clocks , 2001, Nature Reviews Genetics.

[5]  E. O’Shea,et al.  The molecular clockwork of a protein‐based circadian oscillator , 2009, FEBS letters.

[6]  Stanly B. Williams,et al.  Circadian rhythms in gene transcription imparted by chromosome compaction in the cyanobacterium Synechococcus elongatus. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Alexander van Oudenaarden,et al.  Stochastic gene expression out-of-steady-state in the cyanobacterial circadian clock , 2007, Nature.

[8]  D. Virshup,et al.  Post-translational modifications regulate the ticking of the circadian clock , 2007, Nature Reviews Molecular Cell Biology.

[9]  Erin K O'Shea,et al.  A high resolution map of a cyanobacterial transcriptome , 2011, Genome Biology.

[10]  T. Kondo,et al.  Regulation of Circadian Clock Gene Expression by Phosphorylation States of KaiC in Cyanobacteria , 2007, Journal of bacteriology.

[11]  Jennifer J. Loros,et al.  Chronobiology: Biological Timekeeping , 2009 .

[12]  P. Cluzel,et al.  The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities. , 2012, Lab on a chip.

[13]  Alexander van Oudenaarden,et al.  References and Notes Supporting Online Material Circadian Gating of the Cell Cycle Revealed in Single Cyanobacterial Cells , 2022 .

[14]  P. R. ten Wolde,et al.  Robust circadian clocks from coupled protein-modification and transcription–translation cycles , 2010, Proceedings of the National Academy of Sciences.

[15]  T. Kondo,et al.  Cyanobacterial circadian pacemaker: Kai protein complex dynamics in the KaiC phosphorylation cycle in vitro. , 2006, Molecular cell.

[16]  Tetsuya Mori,et al.  Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Michael J Rust,et al.  References and Notes Supporting Online Material Materials and Methods Figs. S1 to S8 Tables S1 to S3 References Ordered Phosphorylation Governs Oscillation of a Three-protein Circadian Clock , 2022 .

[18]  M. Gillette,et al.  BIOLOGICAL TIMEKEEPING. , 2009, Sleep medicine clinics.

[19]  Vikram Vijayan,et al.  Oscillations in supercoiling drive circadian gene expression in cyanobacteria , 2009, Proceedings of the National Academy of Sciences.

[20]  T. Kondo,et al.  Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in Vitro , 2005, Science.

[21]  J. Ashby.  References and Notes , 1999 .

[22]  S. Golden,et al.  Detection of rhythmic bioluminescence from luciferase reporters in cyanobacteria. , 2007, Methods in molecular biology.

[23]  A. Goldbeter,et al.  Robustness of circadian rhythms with respect to molecular noise , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S. Golden,et al.  Expression of the psbDII gene in Synechococcus sp. strain PCC 7942 requires sequences downstream of the transcription start site , 1991, Journal of bacteriology.

[25]  Tetsuya Mori,et al.  Coupling of a Core Post-Translational Pacemaker to a Slave Transcription/Translation Feedback Loop in a Circadian System , 2010, PLoS biology.

[26]  Andrew J. Millar,et al.  Circadian rhythms persist without transcription in a eukaryote , 2010, Nature.

[27]  Eldon Emberly,et al.  Hourglass model for a protein-based circadian oscillator. , 2006, Physical review letters.

[28]  C. Johnson,et al.  Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. , 1998, Science.

[29]  E. O’Shea,et al.  Two antagonistic clock-regulated histidine kinases time the activation of circadian gene expression. , 2013, Molecular cell.

[30]  Takao Kondo,et al.  A KaiC-associating SasA–RpaA two-component regulatory system as a major circadian timing mediator in cyanobacteria , 2006, Proceedings of the National Academy of Sciences.

[31]  Jürgen Kurths,et al.  Detection of n:m Phase Locking from Noisy Data: Application to Magnetoencephalography , 1998 .

[32]  D. Gillespie.  Exact Stochastic Simulation of Coupled Chemical Reactions , 1977 .

[33]  Stanislas Leibler,et al.  Resilient circadian oscillator revealed in individual cyanobacteria , 2004, Nature.

[34]  S. Golden,et al.  Specialized techniques for site-directed mutagenesis in cyanobacteria. , 2007, Methods in molecular biology.

[35]  A. B. Reddy,et al.  Circadian Clocks in Human Red Blood Cells , 2010, Nature.

[36]  Takao Kondo,et al.  No Transcription-Translation Feedback in Circadian Rhythm of KaiC Phosphorylation , 2005, Science.

[37]  Takao Kondo,et al.  Dual KaiC-based oscillations constitute the circadian system of cyanobacteria. , 2008, Genes & development.

[38]  J. Dunlap.  Molecular Bases for Circadian Clocks , 1999, Cell.

[39]  Tetsuhiro S. Hatakeyama,et al.  Circadian transcriptional regulation by the posttranslational oscillator without de novo clock gene expression in Synechococcus , 2011, Proceedings of the National Academy of Sciences.

[40]  Takao Kondo,et al.  Global gene repression by KaiC as a master process of prokaryotic circadian system. , 2004, Proceedings of the National Academy of Sciences of the United States of America.