Light-Driven Changes in Energy Metabolism Directly Entrain the Cyanobacterial Circadian Oscillator Michael J. Rust, et al. Science 331, 220 (2011); DOI: 10.1126/science.1197243 This copy is for your personal, non-commercial use only. If you wish to distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here. Permission to republish or repurpose articles or portions of articles can be obtained by following the guidelines here. The following resources related to this article are available online at www.sciencemag.org (this infomation is current as of January 13, 2011 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http://www.sciencemag.org/content/331/6014/220.full.html Supporting Online Material can be found at: http://www.sciencemag.org/content/suppl/2011/01/12/331.6014.220.DC1.html This article cites 27 articles, 17 of which can be accessed free: on January 13, 2011 http://www.sciencemag.org/content/331/6014/220.full.html#ref-list-1 This article appears in the following subject collections: Physiology http://www.sciencemag.org/cgi/collection/physiology www.sciencemag.org Downloaded from Science (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. Copyright 2011 by the American Association for the Advancement of Science; all rights reserved. The title Science is a registered trademark of AAAS. REPORTS ation of KaiC (4, 5). Because ADP cannot serve Light-Driven Changes in Energy as the phosphoryl donor in the kinase reaction, physiological changes in the relative amounts of Metabolism Directly Entrain the ATP and ADP caused by changes in light have the potential to modulate the activity of enzymes that consume ATP, and might directly affect the Cyanobacterial Circadian Oscillator function of the circadian clock (21). To test this, we developed an experimental system to mimic 1 2 ’ 1† Michael J. Rust, * Susan S. Golden, Erin K. O Shea in vitro the changes in nucleotide ratio we had observed in living cyanobacteria. We initiated Circadian clocks are self-sustained biological oscillators that can be entrained by environmental cues. oscillator reactions with purified KaiA, KaiB, Although this phenomenon has been studied in many organisms, the molecular mechanisms of entrainment remain unclear. Three cyanobacterial proteins and adenosine triphosphate (ATP) are sufficient to generate oscillations in phosphorylation in vitro. We show that changes in illumination that A induce a phase shift in cultured cyanobacteria also cause changes in the ratio of ATP to adenosine diphosphate (ADP). When these nucleotide changes are simulated in the in vitro oscillator, they cause 4 phase shifts similar to those observed in vivo. Physiological concentrations of ADP inhibit kinase activity in the oscillator, and a mathematical model constrained by data shows that this effect is 3 sufficient to quantitatively explain entrainment of the cyanobacterial circadian clock. 2 ircadian clocks, based on self-sustained tensity tends to have opposite effects on the 1 biological oscillators with a ~24-hour pe- circadian clocks of nocturnal versus diurnal or- bioluminescence (arb) riod, allow organisms to anticipate regular ganisms (9). 0 C 24 36 48 60 72 variations in the environment caused by Earth’s We reasoned that the link to metabolism might time (hours) rotation (1). For a circadian system to be most be especially important in photoautotrophic cya- useful to the organism, it must function robustly nobacteria, as these microorganisms are entirely B 6 in the face of fluctuations in the environment reliant on photosynthesis to extract energy from on January 13, 2011 but also accept appropriate input signals to stay the environment (10). Darkness elicits profound 5 entrained with the true diurnal cycle. Therefore, changes in the physiology of S. elongatus,beyond a fundamental problem is to elucidate the mo- the direct effects on photosynthesis. When cultures 4 lecular mechanisms that allow faithful transduc- are removed from the light, global transcription 3 tion of timing information into the oscillator. and translation rates drop and large changes in the 2 The cyanobacterium Synechococcus elongatus superhelicity of endogenous DNA are observed PCC 7942 has a core circadian oscillator that (11, 12). 1 bioluminescence (arb) can be reconstituted in vitro from three purified Cyanobacteria growing in the light primarily 0 24 36 48 60 72 proteins: KaiA, KaiB, and KaiC (2). KaiC is an synthesize adenosine triphosphate (ATP) by photo- www.sciencemag.org time (hours) enzyme that catalyzes the phosphorylation and phosphorylating adenosine diphosphate (ADP) at dephosphorylation of two of its own residues the thylakoid membrane (13),andchangesincul- C in an ordered pattern (3). KaiA and KaiB work ture illumination have been reported to affect the 100 together to modulate the activity of KaiC in a relative abundance of adenine nucleotides (14–16). phosphorylation-dependent manner, resulting in We therefore tested whether incubating synchron- 80 stable oscillations in the amount of phosphorylated ized cultures in the dark under conditions sufficient KaiC (4–6). We sought to identify light-dependent to cause a phase shift in the circadian clock would 60 clock input pathways in living cyanobacteria and result in changes in the concentrations of ATP and Downloaded from to study their mechanism in the reconstituted in ADP. We monitored changes induced by a dark 40 vitro oscillator. pulse in the circadian rhythm of an engineered % ATP / (ATP + ADP) Causal links between metabolic activity and strain of S. elongatus with a bioluminescent reporter 0 5 10 15 circadian clocks have previously been described. of clock-driven transcription (17). In agreement time (hours relative to dark) The circadian clock in the rat liver can be entrained with previous studies, an 8-hour dark pulse applied by feeding (7), and adenosine monophosphate– during the subjective day (Fig. 1A) induced a phase Fig. 1. Sustained drop in the ATP/ADP ratio and activated protein kinase has been implicated in shift in the circadian rhythm, and the clock was a phase shift in the circadian clock in response to clock function in liver tissue (8). A classical result refractory to a dark pulse applied during the subjec- a pulse of darkness. (A) Bioluminescence rhythms kaiBC luxAB connecting clocks and metabolism is Aschoff’s tive night (Fig. 1B) (18–20). These phase shifts in of a reporter under circadian control (P :: ) in synchronized cultures either grown in constant Rule—the observation that varying the light in- the transcriptional reporter were mirrored by shifts light (black symbols) or subjected to an 8-hour in the rhythm of KaiC phosphorylation, as mea- pulse of darkness (shaded region) at t =29hours sured by immunoblotting (fig. S1). Irrespective of 1 (blue symbols). Bars show the range of duplicate Howard Hughes Medical Institute, Center for Systems Bi- when the dark pulse was applied, these cultures ology, Department of Molecular and Cellular Biology, and measurements from the same experiment. Time is Department of Chemistry and Chemical Biology, Harvard experienced large changes in their adenine measured from the release of cultures into con- 2 University, Cambridge, MA 02138, USA. Center for Chro- nucleotide pool (Fig. 1C). After 2 to 3 hours in stant light; arb, arbitrary units. (B)Sameas(A) nobiology, Division of Biological Sciences, University of the dark, the ATP/(ADP + ATP) ratio had fallen except that the dark pulse was applied at t =40 California, San Diego, La Jolla, CA 92093, USA. to nearly 50% and remained low for the duration hours. (C) Relative levels of ATP and ADP extracted *Present address: Department of Molecular Genetics and Cell of the dark pulse; when illumination was re- from cultures before, during (shaded region), and Biology and Institute for Genomics and Systems Biology, Uni- versity of Chicago, Chicago, IL 60637, USA. stored, this ratio returned to ~85% within 1 hour. after a dark pulse in the conditions shown in (A) †To whom correspondence should be addressed. E-mail: The mechanism of the KaiABC protein os- (blue symbols) and in (B) (red symbols). Bars in- [email protected] cillator relies on ordered multisite phosphoryl- dicate the range from duplicate cultures. 220 14 JANUARY 2011 VOL 331 SCIENCE www.sciencemag.org REPORTS Fig. 2. Phase shifts in the in vitro KaiABC A 60 oscillator caused by changing the ATP/ADP ratio. C (A) Phase shift induced by adding an amount of 50 60 ADP equal to the amount of ATP in the reaction buffer ~6 hours before peak phosphorylation 50 (blue symbols) relative to a control (black 40 symbols). The shaded region indicates the time % P-KaiC after addition of ADP but before the addition of 30 40 pyruvate kinase to convert ADP to ATP. (B) Same % P-KaiC as (A) except that ADP was added ~6 hours after 20 30 peak phosphorylation (red symbols). (C) Phase 12 24 36 48 60 shifts induced by a pulse of ADP in the in vitro time (hours) oscillator reaction (lower panel) shown adjacent B 10 to the rhythm of KaiC phosphorylation in the 60 control (upper panel) to indicate where in the 5 cycle the pulses were applied. Differences in 50 phase were measured by fitting the data to a 0 sinusoidal function. (D) Effect of addition of 40 mixtures of nucleotides to lower the ATP/ADP -5 ratio (colored symbols) to various levels relative % P-KaiC 30 to a control that received only ATP (black symbols). phase advance (hours) -10 Total adenine nucleotide concentration was the same 24 36 48 across all reactions. The shaded region indicates the 20 time before addition of pyruvate kinase. (E)Quan- 12 24 36 48 60 pulse time (hours) time (hours) tification of phase shifts from the experiment shown in (D).