Global repression by KaiC as a master process of prokaryotic circadian system

Yoichi Nakahira*, Mitsunori Katayama†, Hiroshi Miyashita‡, Shinsuke Kutsuna§, Hideo Iwasaki, Tokitaka Oyama, and Takao Kondo¶

Division of Biological Science, Graduate School of Science, Nagoya University, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Furo-cho, Chikusa, Nagoya 464-8602, Japan

Communicated by J. Woodland Hastings, Harvard University, Cambridge, MA, November 18, 2003 (received for review August 24, 2003) A kaiABC clock was previously identified from cya- site of pTS2KPtrc::kaiC (pTS2KPtrc::kaiCB). NUC0203 was nobacterium Synechococcus elongatus PCC 7942, and the feedback transformed with pTS2KPtrc::kaiCB to generate cells harboring regulation of kai was proposed as the core mechanism an IPTG-inducible kaiCB cassette in the neutral site II site generating circadian oscillation. In this study, we confirmed that (NUC0204: PkaiBC::luxAB͞kaiBCϪϩPtrc::kaiCB). NUC0205 the Kai-based oscillator is the dominant circadian oscillator func- (complemented kaiABC), NUC0206 (kaiBC-inactivated), and tioning in . We probed the nature of this regulation NUC0207 (kaiBC-inactivated and Ptrc-driven kaiCB), all carry- and found that excess KaiC represses not only kaiBC but also the ing the Ptrc::luxAB reporter in NSI, were constructed by the rhythmic components of all genes in the . This result same methods used to create the PkaiBC::luxAB reporter strains. strongly suggests that the KaiC protein primarily coordinates The Ptrc-driven, kaiA strain (NUC0209: kaiAϪϩPtrc::kaiA) was genomewide gene expression, including its own expression. We generated by the transformation of a kaiA-inactivated strain (5) also found that a promoter derived from E. coli is feedback with pTS2KPtrc::GTGkaiA (S.K., unpublished data). controlled by KaiC and restores the complete in kaiBC-inactivated arrhythmic mutants, provided it can express kaiB Promoter Trap. For promoter-trap experiments, Ϸ0.6-kb SauII- and kaiC genes at an appropriate level. Unlike eukaryotic models, IAI-digested Synechococcus genomic DNA fragments were specific regulation of the kaiBC promoter is not essential for cloned into pNS2CmLuxTer4⍀PL-I, which contained a chlor- cyanobacterial circadian oscillations. amphenicol-resistant (Cmr) gene and promoter-less luxAB genes in a neutral site II target sequence (H.M., unpublished data). ϫ 5 n various model organisms, the core mechanisms controlling The resulting 5.0 10 independent clones were used as a Icircadian clocks that mark time in cells have been traced to promoter-trap library. Wild-type Synechococcus and several –translation feedback regulation of specific ‘‘clock clock mutants, in which the PpsbAI::luxAB reporter had been genes’’ (1). In eukaryotic models, cis-acting elements and trans- replaced with a kanamycin resistance gene, were transformed acting factors controlling clock genes are thought to be clock- with library DNA. For promoter trap in kaiC-overexpresser cells Ϸ specific components. We previously identified a kaiABC clock (Fig. 1), 0.5- to 2.2-kb SauIIIAI-digested Synechococcus genomic ϩ gene cluster from the cyanobacterium Synechococcus elongatus DNA fragments were cloned into pAM717DdluxAB( ), a de- r PCC 7942 and proposed the feedback regulation of kai genes as rivative of pAM1852 containing promoter-less luxAB and Cm the core mechanism governing circadian oscillations. In this genes in an NSI targeting sequence (M.K., unpublished data). ϫ 5 model, kaiBC expression is regulated negatively by KaiC and The resulting 2.7 10 independent clones were used as the positively by KaiA (2). However, the mechanism governing second promoter-trap library. A kaiC-inducible strain, kaiBC gene expression is not yet well understood. Previously, we NUC0201, was generated by transformation of Synechococcus reported that the expression of every gene displays circadian with pTS2Ptrc::kaiC (2). NUC0201 was then transformed with the second promoter-trap library. Bioluminescent colonies were transcription in this prokaryote (3), but the molecular mecha- PLANT BIOLOGY nisms responsible for this phenomenon also remain unclear. In screened by using a cooled charge-coupled device camera system this study, we probed the nature of these regulations with (6). Among the 8,000 Cm-resistant colonies, 638 bright bio- following findings. Overexpression of kaiC repressed not only luminescent colonies were subjected to bioluminescence rhythm kaiBC but the majority of genes in the genome. When expressed assays. under the control of a synthetic promoter (Ptrc) derived from Escherichia coli, kaiB and kaiC could restore the complete Assay of Bioluminescence. Bioluminescence assays were per- circadian rhythm in kaiBC-inactivated arrhythmic mutants. formed with the cooled charge-coupled device camera system These results strongly suggest that the KaiC protein primarily and a photomultiplier-based quantitative system (6). coordinates genomewide gene expression, including its own Western and Northern Analyses. Proteins and mRNA were ex- expression, through a circadian feedback loop. tracted from cell grown in continuous or batch culture systems Materials and Methods (see Fig. 3), then analyzed as described (5, 7). Bacterial Strains. A kaiC-inducible Synechococcus strain, used for

promoter trap analysis, was generated by transformation of Abbreviations: IPTG, isopropyl-␤-D-thiogalactopyranoside; LL, continuous light. NUC42 (4) with pTS2KPtrc::kaiC (2). NUC43 was a kaiABC- *Present address: Faculty of Human and Environment, Kyoto Prefectural University, Hangi- deleted strain carrying a PkaiBC::luxAB reporter construct (4). cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan. NUC0202 is a wild-type reporter strain generated by restoring †Present address: Department of Life Sciences (Biology), University of Tokyo, Komaba 3-8-1, the kai gene cluster missing from NUC43 with pCkaiABC (2). Meguro-ku, Tokyo 153-8902, Japan. The kaiBC-null mutant Synechococcus strain NUC0203 ‡Present address: Pharmaceutical Research Center Kyoto, Bayer Yakuhin, Ltd., Kunimidai, Ϫ (PkaiBC::luxAB͞kaiBC ) was generated by disruption of the Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan. kaiC gene in a kaiB-inactivated strain (ref. 2 and S.K., unpub- §Present address: Faculty of Science, Yokohama City University, Seto 22-2, Kanazawa-ku, lished data). A DNA fragment containing a Shine–Dalgarno Yokohama 237-0063, Japan. sequence and the ORF of kaiB was amplified by PCR using ¶To whom correspondence should be addressed. E-mail: [email protected]. pTSKPtrc::kaiB as the template, then introduced into the SalI © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307411100 PNAS ͉ January 20, 2004 ͉ vol. 101 ͉ no. 3 ͉ 881–885 Downloaded by guest on September 27, 2021 Fig. 1. KaiC represses circadian rhythms of promoter activities. Approximately 640 clones carrying random promoter::luxAB reporters were isolated by promoter-trapping from IPTG-inducible kaiC cells. The circadian bioluminescence rhythms of these clones and the PkaiBC::luxAB reporter strain were analyzed on solid media under continuous light (LL) conditions of 46 ␮mol⅐mϪ2⅐sϪ1 at 30°C in the absence or presence of 1 mM IPTG (administered at the time point indicated by arrows). The bioluminescence profiles of nine representative clones are shown here. Maximum bioluminescence intensity during the experiment, normalized to the sample colony numbers, is shown for each rhythm. Rhythmic and nonrhythmic components are shown with yellow and blue backgrounds, respectively. Abscissa, hours in LL.

Results and Discussion thiogalactopyranoside (IPTG) abolished the normal rhythms of The kai Oscillator as the Dominant Oscillator. As every promoter all clones, while also severely lowering their bioluminescence. assayed exhibited period lengths identical to that of the kai Quantitative analyses of bioluminescence for representative oscillator, we expected that the kai oscillator would dominate all promoters (Fig. 1) disclosed that Synechococcus promoters can gene expression rhythms. However, it remains possible that be classified into high- and low-amplitude promoter groups. Five alternate oscillators coincide or synchronize their periods with to 10 percent of the promoters (e.g., PkaiBC,PsigC, and PchlB) that of the kai oscillator. A population of promoters exhibits an were classified as high-amplitude promoters, characterized by altered period length in a sigma factor mutant background sharp bioluminescence peaks with complete extinction at trough (sigCϪ) (8), suggesting the possibility that another oscillator can phases. The remaining 90% of promoters were low-amplitude promoters exhibiting broader peaks; in this subset, a significant regulate these promoters if linkage of SigC to the kai oscillator level of bioluminescence was retained even during trough is disconnected. To address this issue, we applied a promoter phases. PkaiA,PclpC, and PpsbAI belong to this group. The trap to various kai mutants (Fig. 5 A and B, which is published activity of low-amplitude promoters exhibited both rhythmic and as supporting information on the PNAS web site) and the ⌬ nonrhythmic components, whereas high-amplitude promoters kaiABC (Fig. 5C) strain. Irrespective of the gene mutated, the possessed only the rhythmic component. Excess KaiC completely bioluminescence profiles of each of the several hundred pro- eliminated the rhythmic components of both groups, but did not moters examined displayed identical period lengths (Fig. 5B) and affect the nonrhythmic ones (Fig. 1). Thus, excess KaiC pre- arrhythmia in all arrhythmic mutants (Fig. 5 A and C). These sumably suppresses gene expression regulated by the circadian results indicate that the kai oscillator is indeed the dominant clock without affecting expression leading to nonrhythmic ac- oscillator orchestrating gene expression in the whole genome of tivity. Interestingly, Synechococcus grew well regardless of kaiC Synechococcus. overexpression. The lack of growth impairment implies that the expression of the group of high-amplitude genes and the rhyth- Global Gene Repression by KaiC. How does the kai oscillator mic component of the low-amplitude gene group are not essen- influence all cellular promoters? To examine whether repression tial for survival. It is likely that nonrhythmic expressions of by KaiC is specific to the kaiBC promoter, we applied the low-amplitude genes unaffected by KaiC are sufficient for promoter trap to a Synechococcus strain carrying a kaiC over- Synechococcus housekeeping metabolism. expression construct (Ptrc::kaiC). In the absence of inducer, all of the 640 bioluminescence transformants tested exhibited cir- Circadian Oscillation by E. coli Promoter. Repression of high- cadian rhythms of various bioluminescence intensities, phases, amplitude promoters by KaiC raised the possibility that the waveforms, and amplitudes, as reported (3). As expected from cis-element(s) of the kaiBC promoter may be dispensable for our clock model (2), kaiC overexpression by isopropyl-␤-D- rhythm generation. To test this possibility, we inactivated kaiBC,

882 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307411100 Nakahira et al. Downloaded by guest on September 27, 2021 Fig. 2. Quantitative assay of IPTG dose-dependent restoration of the circadian Ϫ rhythm in a kaiBC strain. A kaiABC gene cluster was introduced back into a Fig. 3. Expression profiles of KaiC protein in kaiBCϪϩPtrc::kaiCB cells. (A) kaiABC-deleted strain (⌬kaiABCϩkaiABC, top trace). Ptrc::kaiCB was introduced Circadian rhythm of KaiC accumulation kaiBCϪϩPtrc::kaiCB cells. Cells carrying a Ϫ into a kaiBC-deleted strain (kaiBC ϩPtrc::kaiCB, lower traces). PkaiBC::luxAB (A) PkaiBC::luxAB reporter were grown in a continuous liquid culture system in the and Ptrc::luxAB (B) reporter cells are shown. Assays for bioluminescence rhythms presence of 10 ␮M IPTG under LL conditions after a 12-h dark treatment, as and presentation of data are the same as described in Fig. 1. Note that the scale described (2). (Upper) Bioluminescence from the PkaiBC reporter was measured for the bioluminescence profile differs among panels. In the lower 12 panels, the every 30 min. Cells were harvested every 4 h, from hour 48 to hour 96 in LL. (Lower) concentrations of IPTG indicated in the figure were administered in the solid Western blotting analysis was performed by using an anti-KaiC antiserum. P-KaiC medium 48–60 h before experimental LL. and KaiC bands are phosphorylated KaiC and nonphosphorylated KaiC, respec- tively (5). (B) ⌬kaiABC, ⌬kaiABCϩkaiABC, kaiBCϪ, and kaiBCϪϩPtrc::kaiCB cells were grown in liquid BG-11 medium in LL of 30 ␮mol⅐mϪ2⅐sϪ1 at 30°C. IPTG (10 or then introduced kaiC and kaiB back into the strain under the 100 ␮M) was administered to kaiBCϪϩPtrc::kaiCB cells as indicated. Total protein control of the E. coli trc promoter (kaiBCϪϩPtrc::kaiCB). In this (5 ␮g), prepared from cells collected at hour 12 in LL after 12 h of darkness, was strain, kaiC and kaiB are inducible by IPTG in a dose-dependent analyzed by Western blotting. manner. We monitored bioluminescence from a PkaiBC::luxAB

reporter after treatment with variable IPTG concentrations PLANT BIOLOGY to that required for rhythm restoration of the PkaiBC::luxAB (Fig. 2A). In the absence of IPTG, bioluminescence from the reporter, the circadian rhythm was again restored. Surprisingly, PkaiBC::luxAB reporter was stronger than that of control cells, ␮ presumably due to the absence of KaiC. Bioluminescence was at 9 M IPTG, the waveform representing the Ptrc::luxAB reduced to the background levels by 100 ␮M IPTG treatment. rhythm changed from a low-amplitude type to a high-amplitude Although bioluminescence profiles were completely arrhythmic one, similar to the PkaiBC::luxAB reporter. in either the absence or presence of 100 ␮M IPTG, intermediate IPTG concentrations (from 6 to 15 ␮M) could restore circadian Feedback Regulation on the trc Promoter. We next examined rhythms. The presence of 9 ␮M IPTG produced a restored Ptrc-driven accumulation of KaiC protein. In the presence of 10 ␮ Ϫϩ rhythm very similar to that observed in control cells M IPTG, kaiBC Ptrc::kaiCB cells exhibited circadian (⌬kaiABCϩkaiABC). rhythms of KaiC expression peaking at circadian time 16–20 We monitored PkaiBC activity under conditions where Ptrc (Fig. 3A), similar to the pattern seen in wild-type cells (5, 9). We also confirmed that KaiB and KaiC proteins accumulated in the drives kaiCB transcription rather than PkaiBC (Fig. 2A). To Ϫ monitor expression of kaiC and kaiB in Ptrc::kaiCB strain, we kaiBC ϩPtrc::kaiCB cells in an IPTG-inducible, dose- replaced the PkaiBC::luxAB reporter with a Ptrc::luxAB reporter dependent manner. KaiC levels in cells with the rhythm restored (Fig. 2B). As seen for all promoters, Ptrc::luxAB showed a (by 10 ␮M IPTG) were similar to those in wild-type cells (Fig. circadian bioluminescence rhythm in the control cells (Fig. 2B, 3B). The correlation of KaiC levels in the native and artificial top trace). The broad peaks and relatively high expression levels oscillators suggests that appropriate KaiC levels coincide with evident during trough phase for Ptrc::luxAB indicated that this the proper generation of circadian oscillation. rhythm belonged to the low-amplitude group. As Ptrc is a strong IPTG-induced KaiC accumulation (Fig. 3B) indicated that promoter, kaiBCϪϩPtrc::kaiCB cells exhibited significant biolu- kaiCB was expressed as expected. Indeed, bioluminescence of minescence even in the absence of IPTG induction. Elevation of the Ptrc::luxAB reporter increased in an IPTG dose-dependent IPTG to 50 ␮M lowered the average level of bioluminescence to manner in the absence of the Ptrc::kaiCB construct (unpublished background levels, as observed for PkaiBC::luxAB (Fig. 2A). In data). However, trc promoter activity assayed by biolumines- the presence of 6–18 ␮M IPTG concentrations, a similar range cence responded in an inverse manner in the presence of the

Nakahira et al. PNAS ͉ January 20, 2004 ͉ vol. 101 ͉ no. 3 ͉ 883 Downloaded by guest on September 27, 2021 Ptrc::kaiCB construct (Fig. 2B). As depicted by the scales of each diagram in Fig. 2B, bioluminescence of Ptrc::kaiCB was lowered as IPTG concentration was elevated. The apparent activity in the absence of the inducer (probably due to a leaky trc promoter) does not support KaiC accumulation. On the other hand, Ptrc-driven bioluminescence was greatly diminished by 50 ␮M IPTG treatment, whereas KaiC level (Fig. 3) increased in an IPTG dose-dependent manner. This apparent contradiction can be reconciled by assuming negative feedback regulation of Ptrc by KaiC. Because we assayed the Ptrc activities from 48–60 h after an administration of IPTG, the initial response of Ptrc to IPTG was not monitored in our experiments. Instead, Fig. 2B should depict the activities attained by feedback regulation, if KaiC from the Ptrc::kaiCB construct affects the Ptrc activity. Under such tight feedback regulation, Ptrc activities that drive kaiCB would be lowered by the accumulated KaiC to a level that balances the positive (induction by IPTG) and negative (repression by KaiC) pres- sures. If KaiC could efficiently repress the promoter, then the Fig. 4. The circadian system of cyanobacteria with a genomewide oscillator stronger the IPTG induces the promoter, the stronger the ␮ by Kai proteins. The circadian system of cyanobacteria proposed from the promoter activity is repressed. In the presence of 50 M IPTG, current study is illustrated schematically. Ptrc would be permitted to drive the kaiCB gene only in sufficient amounts that compensate the degradation of kaiCB mRNA or KaiC. The response of Ptrc to IPTG, as shown in Fig. 2B, rather tioning in cyanobacteria. KaiC represses not only kaiBC but also implies that Ptrc functions as a key component of the feedback the rhythmic components of all genes in the genome. Surpris- loop. ingly, an E. coli-derived promoter can close the Kai-based Complementation of circadian oscillations through the intro- feedback loop to function in the same manner as the endogenous duction of per and tim clock genes under the control of consti- circadian oscillator. These results imply that specific regulation tutive promoters has been reported in clock-null mutants of of the kaiBC promoter is not essential for circadian oscillation, Drosophila (10, 11). The generation of oscillations was thought provided that a kaiBC promoter supports sufficient RNA poly- to result from the interactions between synthesized clock pro- merase activity. Promoter-specific regulation, however, may be teins. Recently, Xu et al. (12) also reported complementation of important for low-amplitude genes that respond to noncircadian 24-h oscillations in cyanobacteria by the Ptrc promoter fused to factors or genes that display circadian rhythms of diverse phase kaiBC. With quantitative analyses of Ptrc, we demonstrated that relationship (ref. 3 and this study). We propose a revised model Ptrc, which is normally feed-forward regulated as a downstream of circadian system regulation in cyanobacteria (Fig. 4). All of promoter of the clock, switched to be feedback-controlled by the genes in the genome can be divided into high- and low- promoting expression of the kaiCB genes. Moreover, it should be amplitude gene groups. In addition to kai promoters, all pro- noted that the feedback loop generates a circadian oscillation at moters undergo transcriptional repression by KaiC (or a KaiC- an appropriate level of IPTG induction (9 ␮M). Thus, any associated complex). KaiC likely binds to DNA structures to promoter, even promoters derived from E. coli, may function as regulate transcription by changing the condensation and͞or a key regulator of circadian feedback loops, provided that it can supercoiling status of Synechococcus chromosomes. Because express kaiB and kaiC genes at an appropriate level. We also KaiC belongs to the superfamily of bacterial DNA , determined that the kaiA promoter was replaceable by the Ptrc including RecA and the DnaB DNA helicase (15), we predict an promoter. The circadian rhythm of kaiA-inactivated cells was interaction between KaiC and DNA. Mori et al. (16) recently complemented by the introduction of the Ptrc::kaiA construct reported that KaiC binds to DNA in vitro. In addition, DNA (Fig. 6, which is published as supporting information on the supercoiling in Chlamydomonas chloroplast changes with a PNAS web site). Thus, two of the kai cluster promoters can be diurnal rhythm in cells growing in alternating 12-h dark-12-h replaced with the Ptrc promoter. light periods (17), and histone acetylation has been implicated in circadian gene regulation in mammals (18). Circadian Characteristics of Ptrc Driven Rhythm. An important Recent cDNA microarray studies in Drosophila (19, 20), criterion of circadian rhythms is that the period length is not mouse (21, 22), Neurospora (23), Arabidopsis (24), and Lingulo- greatly altered by temperature changes (13); Synechococcus dinium (formerly Gonyaulax) (25) revealed that considerable satisfies this criterion (14). The period lengths of Ϫϩ numbers of genes (2–7% of total genes) are controlled by the kaiBC Ptrc::kaiCB cell rhythms were not altered at tempera- in eukaryotes. A recent enhancer trap experi- tures between 25–35°C (Fig. 7, which is published as supporting ment also revealed that one-third of Arabidopsis genes are information on the PNAS web site). The Q10 values for Ϫϩ ␮ ␮ clock-regulated (26). Thus, circadian regulation is far more kaiBC Ptrc::kaiCB cells were 0.95 and 0.97 for 6 M and 9 M extensive than previously expected. In cyanobacteria, our results IPTG, respectively, similar to the values observed for wild-type imply that the circadian oscillator and the core metabolism are cells (Ϸ1.0). Moreover, a 5-h pulse in darkness, resetting the highly interdependent, that is, the oscillator regulates or influ- phase of the Synechococcus rhythm, also shifted the phase of the ences whole cellular metabolism and the circadian oscillator is Ptrc-driven rhythms (data not shown). These results indicate that based on an integration of cellular metabolism. kaiBCϪϩPtrc::kaiCB cells generate circadian rhythms physio- logically equivalent to those of the wild-type. Thus, the proper- We thank Dr. M. Ishiura (Nagoya University) for construction of the ties of circadian rhythm are not ascribed to the kai gene promoter trap library used in Fig. 5. This research was supported in part promoters but to the Kai proteins themselves. by Japanese Ministry of Education, Culture, Sports, Science, and Tech- nology Grants-in-Aid 11233203, 15GS0308, and COE 14COEA01 (to Genome-Based Clock Model. In this study, we revealed that the T.K.), and Japanese Society for Promotion of Science Grants-in-Aid Kai-based oscillator is the dominant circadian oscillator func- 13680778 and 15370074 (to H.I.) and 200203019 (to Y.N.).

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