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Global Gene Repression by Kaic As a Master Process of Prokaryotic Circadian System

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Global Gene Repression by Kaic As a Master Process of Prokaryotic Circadian System Global gene 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 gene cluster 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 genes 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 cyanobacteria. 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 genome. 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kaiA-inactivated strain (5) also found that a promoter derived from E. coli is feedback with pTS2KPtrc::GTGkaiA (S.K., unpublished data).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 circadian rhythm 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 transcription–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
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