Electrochemical Detection of Circadian Redox Rhythm inCyanobacterial Cells via Extracellular Electron TransferKoichi Nishio1, Tunanunkul Pornpitra1, Seiichiro Izawa1, Taeko Nishiwaki-ohkawa2, Souichiro Kato3,4,Kazuhito Hashimoto1 and Shuji Nakanishi1,*1Department of Applied Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656 Japan2Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan3Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904 Japan4Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo,Hokkaido, 062-8517 Japan

*Corresponding author: E-mail, nakanishi@light.t.u-tokyo.ac.jp; Fax, +81-3-5841-8751.(Received December 22, 2014; Accepted April 28, 2015)

Recent research on cellular circadian rhythms suggeststhat the coupling of transcription–translation feedbackloops and intracellular redox oscillations is essential forrobust circadian timekeeping. For clarification of themolecular mechanism underlying the circadian rhythm,methods that allow for the dynamic and simultaneous de-tection of transcription/translation and redox oscillationsin living cells are needed. Herein, we report that thecyanobacterial circadian redox rhythm can be electrochem-ically detected based on extracellular electron transfer(EET), a process in which intracellular electrons areexchanged with an extracellular electrode. As the EET-based method is non-destructive, concurrent detectionwith transcription/translation rhythm using bioluminescentreporter strains becomes possible. An EET pathway thatelectrochemically connected the intracellular region ofcyanobacterial cells with an extracellular electrode wasconstructed via a newly synthesized electron mediatorwith cell membrane permeability. In the presence of themediator, the open circuit potential of the culturemedium exhibited temperature-compensated rhythm withapproximately 24 h periodicity. Importantly, such circadianrhythm of the open circuit potential was not observed inthe absence of the electron mediator, indicating that theEET process conveys the dynamic information regardingthe intracellular redox state to the extracellular electrode.These findings represent the first direct demonstrationof the intracellular circadian redox rhythm of cyanobacterialcells.

Keywords: Circadian clock � Cyanobacteria �

Electrochemistry � Extracellular electron transfer � Redoxrhythm.

Abbreviations: EET, extracellular electron transfer; EM,mid-point potential; Chl, Chlorophyll; MPC, 2-methacryloy-loxyethyl phosphorylcholine; OCP, open circuit potential;PMF, poly(2-methacryloyloxyethyl phosphorylcholine-co-vinylferrocene); PQ, plastoquinone; SHE, standard hydro-gen electrode; TTFL, transcription–translation feedback loop.

Introduction

Circadian rhythms are responsible for the temporal organiza-tion of various biological processes, and are conserved in or-ganisms as diverse as cyanobacteria, fungi, algae, plants andmammals (Dunlap 1999, Dunlap et al. 2003). Circadian oscilla-tions have been modeled as a transcription–translation feed-back loop (TTFL) that controls the downstream metabolicrhythms accompanying the periodic changes in the intracellu-lar redox state at the transcriptional level. However, recent re-search has suggested that the metabolic (i.e. redox) rhythmsfeed back to the transcriptional control process, thereby con-structing robust circadian timekeeping systems (Rutter et al.2001, Wijnen and Young 2006, Eckel-Mahan and Sassone-Corsi2009, Aguilar-Armnal and Sassone-Corsi 2013). Thus, there hasbeen renewed interest in understanding the complementaryrelationship between the TTFL and the redox state in control-ling circadian rhythms (Asher and Schibler 2011, O’Neil andReddy 2011, O’Neil et al. 2011, Bass 2012, Edgar et al. 2012,Rey and Reddy 2013).

The cyanobacterium Synechococcus elongatus PCC7942 hasbeen used as a model species for investigating circadianrhythms (Ishiura et al. 1998, Iwasaki and Dunlap 2000,Nakajima et al. 2005), as various molecular biological methodol-ogies have been established in this species owing to its smallgenome size. Cyanobacterial circadian rhythm has beendescribed as a TTFL-based process involving three essentialkai genes that are specific to this group of microorganisms(Ishiura et al. 1998, Xu et al. 2000, Johnson et al. 2008). It wasalso demonstrated that a rhythm with a periodicity similar tothat of cyanobacterial circadian rhythm can be observed in atest tube containing only ATP and the three key Kai proteinsKaiA, KaiB and KaiC (Nakajima et al. 2005). Emerging evidencesuggests that TTFL is not the sole mechanism underlying cir-cadian periodicity. For example, a post-translational regulationcircuit was shown to be sufficient to generate oscillations ingrowing cyanobacteria (Teng et al. 2013). In addition, a closerelationship between the intracellular redox state and circadianrhythm has been also shown in cyanobacterial cells (Ivleva et al.2005, Kim et al. 2012).

Plant Cell Physiol. 0(0): 1–6 doi:10.1093/pcp/pcv066, Advance Access publication on 14 May 2015, available online at www.pcp.oxfordjournals.org! The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: journals.permissions@oup.com

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Herein, we report that the redox rhythm of S. elongatus cellscan be electrochemically detected by adding a membrane-per-meable electron mediator with an appropriate redox potentialto the culture medium, thereby enabling real-time detection ofcircadian redox rhythm without the genetic manipulation ofcells.

Results

Strategy for the electrochemical detection

For the direct detection of the redox rhythm in cyanobacterialcells, we first synthesized an electron mediator molecule withcell membrane permeability and low cytotoxicity. We assumedthat the redox balance between the oxidized and reducedforms of the electron mediator (Rox/red) within cells would os-cillate in synchronization with the cellular circadian redoxrhythms (step 1, Fig. 1a). Oscillations in the intracellular Rox/

red would lead to periodic changes in the Rox/red of the culturemedium as the electron mediator molecules cross the cellmembrane (step 2, Fig. 1a). Oscillation in the extracellularRox/red would affect, in turn, the redox potential of the culturemedium. As the redox potential of the medium can be easilymeasured electrochemically using an electrode located in theculture medium, the circadian redox rhythm would be detect-able as periodic changes in the open circuit potential (OCP) ofthe system (step 3, Fig.1a).

Fig. 1b shows the schematic of the experimental set-up. Asingle-chamber electrochemical system equipped with a tin-doped indium oxide (ITO) electrode (3 cm2) located at thebottom of the reactor as the working electrode was used forthe measurements. Synechococcus elongatus cells were injected

into the system, which contained culture medium supple-mented with electron mediator molecules, and were allowedto settle gradually on the ITO surface.

Synthesis of a new electron mediator

One candidate intracellular redox-active species of S. elongatusis a plastoquinone (PQ) located in the thylakoid membrane.The redox state of PQ is reported to regulate the cyanobacterialcircadian clock gene cluster, which is composed of kaiABC (Kimet al. 2012). In previous studies, we showed that the redox stateof PQ could be controlled by constructing an extracellular elec-tron transfer (EET) pathway to an extracellular electrode via theelectron mediator poly(2-methacryloyloxyethyl phosphoryl-choline-co-vinylferrocene) [(Poly(MPC-co-VF); PMF, Fig. 2a](Lu et al. 2014, Nishio et al. 2013), which is composed of bio-compatible MPC (Ishihara et al. 1998) and redox-active vinyl-ferrocene (VF). However, as the redox potential of PMF isrelatively high [0.5 V vs. a standard hydrogen electrode(SHE)], many intracellular redox species may have participatedin the EET process, thereby interfering with the electrochemicalsensing of the redox state of PQ.

To detect OCP rhythm, we therefore synthesized a newmediator, Me8–PMF (Fig. 2b), which contained eight methylgroups in the ferrocene ring (the synthetic scheme is shown inSupplementary Fig. S1). Me8–PMF was composed of 80 mol%MPC and 20 mol% octamethylferrocene, and had a weight-average mol. wt of 4.2 kDa. Synthesized Me8–PMF was solublein water, indicating that the hydrophilic MPC unit was suffi-cient to solubilize the hydrophobic octamethylferrocene unit.We then performed cyclic voltammetry to investigate the redoxpotential of Me8–PMF and PMF (Fig. 2c). The cyclic voltam-mograms showed a pair of positive and negative peaks corres-ponding to the oxidation of Fe2+ (to Fe3+) and reduction ofFe3+ (to Fe2+). The redox potential of a mediator can be esti-mated from the mid-point potential (EM) between the oxida-tion and reduction peak potentials. As can be seen in the solidcurve in Fig. 2c, the EM of Me8–PMF was located at 0.18 V vs.SHE, which was approximately 0.3 V more negative than that ofPMF (Fig. 2c, dashed curve; Nishio et al. 2013). The lower redoxpotential of Me8–PMF would allow this compound to receiveelectrons from important compounds involved in electrontransport, such as NAD (–0.3 V) and PQ (0.1 V), but not fromcomponents downstream of PQ, such as cytochrome (0.36 V)and plastocyanin (0.3 V). Thus, Me8–PMF was predicted tobe more sensitive to the redox state of PQ in S. elongatusthan PMF.

Extracellular electron transfer via Me8–PMF

We next examined whether Me8–PMF electrochemically inter-acts with intracellular PQ by measuring the fluorescence ofChlorophyll in PSII (Fig. 3). The fluorescence intensity of Chlis higher when PQ is in the reduced state as compared with theoxidized state, and thus can be used as a qualitative indicator ofthe redox state of PQ (Campbell et al. 1998, Lu et al. 2014).Based on previously developed methods, a modulated fluorom-eter was used to monitor the Chl fluorescence from S. elongatuscells. For this technique, cells were periodically irradiated with

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Fig. 1 Schematic for (a) explaining the detection principle of circadianredox rhythm and (b) the experimental set-up.

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620 nm light to excite the Chl of PSII, and only fluorescence thatexhibited periodic excitation was extracted using a lock-in amp-lifier. The redox state of PQ was estimated from the ratio ofinstantaneous fluorescence (F) to maximum fluorescence (Fm

0)

using the equation Y = (Fm0 – F)/Fm

0 (Campbell et al. 1998).Using this approach, PSII fluorescence from S. elongatus cellsappeared to change synchronously with the potential of theextracellular electrode, which alternated between +0.4 V (>EM)

(a) PMF

(b) Me8-PMF

(c)

PMF

Me8-PMF

Fig. 2 (a) and (b) Molecular structures of PMF and Me8–PMF, respectively. (c) Cyclic voltammograms of Me8–PMF (solid curve) and PMF(dashed curve). The scan rate was 10 mV s–1.

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and 0 V (<EM) (Fig. 3). Notably, in the absence of Me8–PMF,the intensity of PSII fluorescence did not change with thechange in electrode potential (Lu et al. 2014). In addition, theY value rapidly responded to changes in the applied potential.Taken together, these results suggested that Me8–PMF directlyor indirectly affected the redox state of PQ, as predicted.

Electrochemical detection of the circadianredox rhythm

Based on the above results, it was confirmed that an electro-chemical connection was established between intracellular PQand the extracellular electrode via the Me8–PMF mediator. Wetherefore attempted to detect OCP and TTFL rhythm in thepresence of Me8–PMF using an engineered strain of S. elongatusin which a bioluminescent reporter was introduced down-stream of the kaiBC and psbB gene promoter (see theSupplementary data). Cells subjected to three cycles oflight/dark with 24 h periodicity were transferred to the electro-chemical chamber, and the OCP was then measured. A repre-sentative time course of the OCP is shown in Fig. 4a (solidcurve), in which oscillations with 24 h periodicity were clearlyobserved. Notably, the amplitude of oscillations decreased withtime, a response we attributed to the reduced activity of the cellin the closed experimental system. The Q10 value of the OCPoscillation, which represents the factor by which the oscillationfrequency increases for every 10�C rise in temperature, wasestimated to be 1.1 (Fig. 5). Thus, it was concluded that theOCP oscillations exhibited a temperature compensationproperty.

Discussion

The OCP of the culture medium containing S. elongatus cellsand the electron mediator Me8–PMF clearly exhibited 24 hrhythm. When the S. elongatus cells were pre-cultured underanti-phase light/dark cycles, the observed OCP oscillation wasalso anti-phasic. Thus, the oscillatory phase of the OCP ap-peared to be determined by the phase of the light/dark cycles

used to culture cells prior to the measurements. In addition, aperiodic OCP rhythm was not observed when PMF (withoutmethyl groups) was used (Fig. 4b, solid curve), even though aclear bioluminescence rhythm was detected (dashed curve).This result indicates that the redox potential of the electronmediator is an important factor influencing the detection of thecircadian redox rhythm. Furthermore, the observed period ofthe bioluminescent rhythm is consistent with that of the

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Fig. 4 Representative time courses of OCP and bioluminescence inthe presence of (a) Me8–PMF and (b) PMF. Prior to the OCP meas-urements, the cells were subjected to light/dark cycles with 24 h peri-odicity to promote the synchronous oscillation of cells. The averagelevel of each trace was normalized to a value of 1.

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23 25 27 29 31 33 35Temperature / °C

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Fig. 5 Periods of the circadian OCP (diamonds) and bioluminescence(squares) oscillations in cultures of S. elongatus at differenttemperatures.

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Fig. 3 Representative time course of PSII fluorescence (Y)under + 0.4 V/0 V cycles (bar: black, 0.4 V; white, 0 V). The samplewas continuously illuminated during the potential cycling.

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concurrently measured OCP rhythm (Fig. 5), clearly indicatingthat the presence of Me8–PMF did not affect the period of thecircadian rhythm. Recently, Kim et al. (2012) reported that theaddition of oxidized quinones to the culture medium reset thecyanobacterial circadian clock. Taken together, these findingssuggest that the presence of Me8–PMF influenced the phase ofthe circadian rhythm. Detailed investigations of the effects ofMe8–PMF on the oscillation phase and TTFL control of circadianredox rhythm are currently under progress in our laboratory.

The results of the present study demonstrate that the cir-cadian redox rhythm of cyanobacterial cells can be detected asperiodic changes of the OCP, which is formed from the estab-lishment of an EET pathway between PQ and an extracellularelectrode via an electron mediator. Notably, this method ofmeasuring circadian redox rhythm does not require the geneticmanipulation of cells and can be performed with standard elec-trochemical equipment. As cellular redox rhythms are thoughtto be a common phenomenon among a wide range of organ-isms, the EET-based technique developed here for the directdetection of redox rhythms is expected to be generally applic-able to various types of cells, particularly those for which gen-etic engineering methods are not available. We anticipate thatthis new method can even be applied for human cells and maycontribute to the development of chronotherapy.

Materials and Methods

Synthesis of Me8–PMF

Me8–PMF was synthesized by a free-radical polymerization process with a,

a0-azobisisobutyronitrile as the initiator. The synthetic scheme

(Supplementary Fig. S1) and detailed protocol are described in the

Supplementary data. The proportion of MPC and octamethylferrocene in

the synthesized polymer was determined from UV-Vis absorption spectra.

The molecular weight was measured by gel permeation chromatography in a

mixture of methanol and water [70 : 30 (v/v)] containing 10 mM lithium brom-

ide. Poly(ethylene oxide) was used as a standard for calibration of the system.

Cell culture

Synechococcus elongatus PCC7942 was routinely pre-cultured in BG11 medium

in diurnal light/dark (12 h/12 h) cycles at 2,000 lux with shaking at 30�C.

Synechococcus elongatus PCC7942 carrying both PkaiBC-luxAB in neutral site

I and PpsbAI-luxCDE in neutral site II was used for bioluminescence measure-

ments, as described previously (Lu et al. 2014).

Electrochemical measurement

A single-chamber, three-electrode system was used for all electrochemical

measurements. In the system, glass coated with ITO (3 cm2) was placed at

the bottom of the reactor as the working electrode, and Ag/AgCl (saturated

KCl) and platinum wire were used as the reference and counter electrodes,

respectively. To perform electrochemical measurements, 4 ml of BG11 supple-

mented with mediator molecules (1 g l–1 Me8–PMF) was added into the reactor

chamber, which was then purged with N2/CO2 [98 : 2 (v/v)] gas for 5 min at

50 ml min–1. Synechococcus elongatus cells were then injected into the system

to give an optical density at 730 nm of 2.0. The light intensity within the reactor

was adjusted to 600 lux.

Detection of Chl fluorescence

Fluorescence from Chl in PSII was detected using a fiber-type fluorometer

(DUAL-PAM/F; Waltz) that was set at the bottom of the electrochemical

system. The system was irradiated with measuring light (620 nm) and actinic

light (460 nm) through the ITO electrode. Living S. elongatus cells in the elec-

trochemical system were periodically irradiated with 620 nm light to excite PSII

Chl, and the generated fluorescence was measured. Notably, only fluorescence

that followed periodic excitation was extracted using a lock-in amplifier.

The ratio of instantaneous to maximum fluorescence, Y = (Fm0 – F)/Fm

0 ,

which reflects the photochemical efficiency of open PSII centers under a

given light acclimation status, was also calculated.

In situ observations of bioluminescence and opencircuit potential

Bioluminescence from an engineered strain of S. elongatus expressing a bio-

luminescent reporter was measured using a Gene Light 55 GL-100 A lumin-

ometer (Microtec), which is a hybrid device consisting of light-emitting diode

lamps (white and red) and electrochemical equipment. The lamps were used to

irradiate the cultivation system, but were turned off for 1 min during the hourly

measurement of bioluminescence. After exposure to darkness for 1 min to

allow the Chl fluorescence to decay below the limit of detection, biolumines-

cence was automatically measured every hour by a photomultiplier through the

ITO electrode at the bottom of the reactor. The OCP was simultaneously

measured with bioluminescence using a galvanostat (BioLogic, VMP3). The

period of the OCP rhythm was determined by measuring the time interval

between the neighboring local maximum values of the OCP vs. time curves.

Supplementary data

Supplementary data are available at PCP online.

Funding

This work was supported by a Grant-in-Aid for SpeciallyPromoted Research [24000010]; the Japan Society for thePromotion of Science (JSPS) [Grant-in-Aid for JSPS Fellows23�9692].

Acknowledgments

The authors thank Lin Xiaojie, Dr. Tomohiro Konno and Dr.Kazuhiko Ishihara of the Department of Materials Engineering,The University of Tokyo for provision of MPC and help withgel-permeation chromatography.

Disclosures

The authors have no conflicts of interest to declare.

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