Cold temperatures lead to nullification of circadian rhythms in many organisms. Two typical scenarios explain the disappearance of rhythmicity: the first is oscillation death, which is the transition from self-sustained oscillation to damped oscillation that occurs at a critical temperature. The second scenario is oscillation arrest, in which oscillation terminates at a certain phase. In the field of nonlinear dynamics, these mechanisms are called the Hopf bifurcation and the saddle-node on an invariant circle bifurcation, respectively. Although these mechanisms lead to distinct dynamical properties near the critical temperature, it is unclear to which scenario the circadian clock belongs. Here we reduced the temperature to dampen the reconstituted circadian rhythm of phosphorylation of the recombinant cyanobacterial clock protein KaiC. The data led us to conclude that Hopf bifurcation occurred at similar to 19 degrees C. Below this critical temperature, the self-sustained rhythms of KaiC phosphorylation transformed to damped oscillations, which are predicted by the Hopf bifurcation theory. Moreover, we detected resonant oscillations below the critical temperature when temperature was periodically varied, which was reproduced by numerical simulations. Our findings suggest that the transition to a damped oscillation through Hopf bifurcation contributes to maintaining the circadian rhythm of cyanobacteria through resonance at cold temperatures.

Background: Most organisms, especially photoautotrophs, alter their behaviours in response to day-night alternations adaptively because of their great reliance on light. Upon light-to-dark transition, dramatic and universal decreases in transcription level of the majority of the genes in the genome of the unicellular cyanobacterium, Synechococcus elongatus PCC 7942 are observed. Because Synechococcus is an obligate photoautotroph, it has been generally assumed that repression of the transcription in the dark (dark repression) would be caused by a nocturnal decrease in photosynthetic activities through the reduced availability of energy (e.g. adenosine triphosphate (ATP)) needed for mRNA synthesis.
Results: However, against this general assumption, we obtained evidence that the rapid and dynamic dark repression is an active process. Although the addition of photosynthesis inhibitors to cells exposed to light mimicked transcription profiles in the dark, it did not significantly affect the cellular level of ATP. By contrast, when ATP levels were decreased by the inhibition of both photosynthesis and respiration, the transcriptional repression was attenuated through inhibition of RNA degradation. This observation indicates that Synechococcus actively downregulates genome-wide transcription in the dark. Even though the level of total mRNA dramatically decreased in the dark, Synechococcus cells were still viable, and they do not need de novo transcription for their survival in the dark for at least 48 hours.
Conclusions: Dark repression appears to enable cells to enter into nocturnal dormancy as a feed-forward process, which would be advantageous for their survival under periodic nocturnal conditions.

The filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest models of a multicellular system showing cellular differentiation. In nitrogen-deprived culture, undifferentiated vegetative cells differentiate into heterocysts at similar to 10-cell intervals along the cellular filament. As undifferentiated cells divide, the number of cells between heterocysts (segment length) increases, and a new heterocyst appears in the intermediate region.
To understand how the heterocyst pattern is formed and maintained, we constructed a one-dimensional cellular automaton (CA) model of the heterocyst pattern formation. The dynamics of vegetative cells is modeled by a stochastic transition process including cell division, differentiation and increase of cell age (maturation). Cell division and differentiation depend on the time elapsed after the last cell division, the "cell age". The model dynamics was mathematically analyzed by a two-step Markov approximation. In the first step, we determined steady state of cell age distribution among vegetative cell population. In the second step, we determined steady state distribution of segment length among segment population. The analytical solution was consistent with the results of numerical simulations. We then compared the analytical solution with the experimental data, and quantitatively estimated the immeasurable intercellular kinetics. We found that differentiation is initially independent of cellular maturation, but becomes dependent on maturation as the pattern formation evolves. Our mathematical model and analysis enabled us to quantify the internal cellular dynamics at various stages of the heterocyst pattern formation. (C) 2015 Elsevier Ltd. All rights reserved.

Cyanobacteria are unique organisms with remarkably stable circadian oscillations. These are controlled by a network architecture that comprises two regulatory factors: posttranslational oscillation (PTO) and a transcription/translation feedback loop (TTFL). The clock proteins KaiA, KaiB, and KaiC are essential for the circadian rhythm of the unicellular species Synechococcus elongatus PCC 7942. Temperature-compensated autonomous cycling of KaiC phosphorylation has been proposed as the primary oscillator mechanism that maintains the circadian clock, even in the dark, and it controls genome-wide gene expression rhythms under continuous-light conditions (LL). However, the kaiC(EE) mutation (where "EE" represents the amino acid changes Ser431Glu and Thr432Glu), where phosphorylation cycling does not occur in vivo, has a damped but clear kaiBC expression rhythm with a long period. This suggests that there must be coupling between the robust PTO and the "slave" unstable TTFL. Here, we found that the kaiC(EE) mutant strain in LL was hypersensitive to the dark acclimation required for phase shifting. Twenty-three percent of the genes in the kaiC(EE) mutant strain exhibited genome-wide transcriptional rhythms with a period of 48 h in LL. The circadian phase distribution was also conserved significantly in most of the wild-type and kaiC(EE) mutant strain cycling genes, which suggests that the output mechanism was not damaged severely even in the absence of KaiC phosphorylation cycles. These results strongly suggest that the KaiC phosphorylation cycle is not essential for generating the genome-wide rhythm under light conditions, whereas it is important for appropriate circadian timing in the light and dark.

This is a hybrid installation that creates an interface between people and cyanobacteria. The project integrates the photosynthetic activity of these microorganisms, the dynamics of environmental light and the bioelectrical activity of the participants. Using cell culture and computer vision, this work renders the photosynthetic activity of cyanobacteria in the form of an organic structure. It also produces dynamic geometries of solar energy by analysing environmental data. Simultaneously, this work transforms the activity of the nervous system of each participant into 'light' to stimulate the cells. As a result, visitors and cyanobacteria influence each other giving subsistence to a dynamic feedback system.

Circadian rhythms are endogenous biological timing processes that are ubiquitous in organisms ranging from cyanobacteria to humans. In the photoautotrophic unicellular cyanobacterium Synechococcus elongatus PCC 7942, under continuous light (LL) conditions, the transcription-translation feedback loop (TTFL) of KaiC generates a rhythmic change in the accumulation of KaiC relative to KaiA clock proteins (KaiC/KaiA ratio), which peak and trough at subjective dawn and dusk, respectively. However, the role of TTFL in the cyanobacterial circadian system remains unclear because it is not an essential requirement for the basic oscillation driven by the Kai-based posttranslational oscillator (PTO) and the transcriptional output mechanisms. Here, we show that TTFL is important for the circadian photic resetting property in Synechococcus. The robustness of PTO, which is exemplified by the amplitude of the KaiC phosphorylation cycle, changed depending on the KaiC/KaiA ratio, which was cyclic under LL. After cells were transferred from LL to the dark, the clock protein levels remained constant in the dark. When cells were transferred from LL to continuous dark at subjective dawn, the KaiC phosphorylation cycle was attenuated with a lower KaiC/KaiA ratio, a higher KaiC phosphorylation level, and a lower amplitude than that in cells transferred at subjective dusk. We also found that the greater the degree to which PTO was attenuated in continuous dark, the greater the phase shifts upon the subsequent light exposure. Based on these results, we propose that TTFL enhances resetting of the Kai-based PTO in Synechococcus.

Background: The cyanobacterial circadian program exerts genome-wide control of gene expression. KaiC undergoes rhythms of phosphorylation that are regulated by interactions with KaiA and KaiB. The phosphorylation status of KaiC is thought to mediate global transcription via output factors SasA, CikA, LabA, RpaA, and RpaB. Overexpression of kaiC has been reported to globally repress gene expression.
Results: Here, we show that the positive circadian component KaiA upregulates "subjective dusk" genes and that its overexpression deactivates rhythmic gene expression without significantly affecting growth rates in constant light. We analyze the global patterns of expression that are regulated by KaiA versus KaiC and find in contrast to the previous report of KaiC repression that there is a "yin-yang" regulation of gene expression whereby kaiA overexpression activates "dusk genes" and represses "dawn genes," whereas kaiC overexpression complementarily activates dawn genes and represses dusk genes. Moreover, continuous induction of kaiA latched KaiABC-regulated gene expression to provide constitutively increased transcript levels of diverse endogenous and heterologous genes that are expressed in the predominant subjective dusk phase. In addition to analyzing KaiA regulation of endogenous gene expression, we apply these insights to the expression of heterologous proteins whose products are of potential value, namely human proinsulin, foreign luciferase, and exogenous hydrogenase.
Conclusions: Both KaiC and KaiA complementarily contribute to the regulation of circadian gene expression via yin-yang switching. Circadian patterns can be reprogrammed by overexpression of kaiA or kaiC to constitutively enhance gene expression, and this reprogramming can improve 24/7 production of heterologous proteins that are useful as pharmaceuticals or biofuels.

The filamentous, heterocystous cyanobacterium Anabaena sp. strain PCC 7120 is one of the simplest multicellular organisms that show both morphological pattern formation with cell differentiation (heterocyst formation) and circadian rhythms. Therefore, it potentially provides an excellent model in which to analyze the relationship between circadian functions and multicellularity. However, detailed cyanobacterial circadian regulation has been intensively analyzed only in the unicellular species Synechococcus elongatus. In contrast to the highest-amplitude cycle in Synechococcus, we found that none of the kai genes in Anabaena showed high-amplitude expression rhythms. Nevertheless, similar to 80 clock-controlled genes were identified. We constructed luciferase reporter strains to monitor the expression of some high-amplitude genes. The bioluminescence rhythms satisfied the three criteria for circadian oscillations and were nullified by genetic disruption of the kai gene cluster. In heterocysts, in which photosystem II is turned off, the metabolic and redox states are different from those in vegetative cells, although these conditions are thought to be important for circadian entrainment and timekeeping processes. Here, we demonstrate that circadian regulation is active in heterocysts, as shown by the finding that heterocyst-specific genes, such as all1427 and hesAB, are expressed in a robust circadian fashion exclusively without combined nitrogen.

One of the key characteristics of all circadian rhythms is that the free-running period remains stable under a relatively broad range of ambient temperatures, referred to as "temperature compensation" of the period. Outside of the range of temperature compensation, circadian clocks stop running and are arrested at a certain phase. Based on bifurcation theory, Hopf bifurcation and saddle-node bifurcation are plausible scenarios of circadian arrhythmia at low temperature. We focused on a biochemical circadian oscillation, KaiC phosphorylation rhythms, which can be reconstituted in a test tube by mixing the three protines, KaiA, KaiB, and KaiC in the presence of ATP. The KaiC phosphorylation rhythm in vitro is the simplest circadian oscillation to observe directly and precisely dynamics of circadian oscillator. We found that the phenomena of nullification of KaiC phosphorylation rhythm by low temperature was explained by theory of Hopf bifurcation.

The objective of our research was to predict cell fates of a multicellular system, accompanied by cellular differentiation. To fulfill this objective, we sought to distinguish the differentiated and undifferentiated cells of filamentous cyanobacteria (Anabaena sp. PCC 7120) using Raman imaging. This technique indicated Raman bands of the cellular system, in which several bands were assigned to vibrations of beta-carotene and scytonemin. We applied principal component analysis (PCA) to the Raman spectra to determine the PC1 and PC2 loading plots and their scores. The data points obtained for heterocyst tended to converge along the bottom of the scatterplot whereas those for vegetative cells were more widely distributed in the PC plane. This indicates that the chemical compositions of a heterocyst were relatively stable. As vegetative cells are capable of proliferation or differentiation, they may transit and exist in several states including the pseudo-differentiated state. The results suggest that the chemical compositions of a vegetative cell fluctuated according to its cellular condition. In conclusion, the results of Raman imaging indicate that the diverse states of vegetative cells are localized in a specific state through differentiation.

The circadian clock of cyanobacteria is composed of KaiA, KaiB, and KaiC proteins, and the SasA-RpaA two-component system has been implicated in the regulation of one of the output pathways of the clock. In this study, we show that another response regulator that is essential for viability, the RpaA paralog, RpaB, plays a central role in the transcriptional oscillation of clock-regulated genes. In vivo and in vitro analyses revealed that RpaB and not RpaA could specifically bind to the kaiBC promoter, possibly repressing transcription during subjective night. This suggested that binding may be terminated by RpaA to activate gene transcription during subjective day. Moreover, we found that rpoD6 and sigF2, which encode group-2 and group-3 sigma factors forRNApolymerase, respectively, were also targets of the RpaAB system, suggesting that a specific group of sigma factors can propagate genome-wide transcriptional oscillation. Our findings thus reveal a novel mechanism for a circadian output pathway that is mediated by two paralogous response regulators.

Circadian rhythms are a fundamental property of most organisms, from cyanobacteria to humans. In the unicellular obligately photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are controlled by the KaiABC-based clock under continuous light conditions. When Synechococcus cells are transferred from the light to continuous dark (DD) conditions, the expression of most genes, including the clock genes kaiA and kaiBC, is rapidly down-regulated, whereas the KaiC phosphorylation cycle persists. Therefore, we speculated that the posttranslational oscillator might not drive the transcriptional circadian output without de novo expression of the kai genes. Here we show that the cyanobacterial clock regulates the transcriptional output even in the dark. The expression of a subset of genes in the genomes of cells grown in the dark was dramatically affected by kaiABC nullification, and the magnitude of dark induction was dependent on the time at which the cells were transferred from the light to the dark. Moreover, under DD conditions, the expression of some dark-induced gene transcripts exhibited temperature-compensated damped oscillations, which were nullified in kaiABC-null strains and were affected by a kaiC period mutation. These results indicate that the Kai protein-based posttranslational oscillator can drive the circadian transcriptional output even without the de novo expression of the clock genes.

In the cyanobacterium Synechococcus elongatus PCC 7942, the central oscillator of the circadian system consists of three genes (kaiA, kaiB, kaiC) and their protein products. In the presence of ATP, the interactions among these proteins drive a temperature-compensated circadian rhythm of KaiC phosphorylation in vitro. The temporal information from this protein-based chemical oscillator regulates expression from essentially all gene promoters in vivo. Some insights have been reported that describe key factors involved in the transduction of temporal information from the central oscillator via circadian output pathways. Moreover, recent studies have demonstrated that the rhythm in gene transcription is sustained, even in the absence of rhythmic KaiC phosphorylation, suggesting that several interconnected output pathways are integrated into the robust circadian oscillatory system in cyanobacteria.

In the unicellular cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild-type strains, > 30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was severely attenuated in kaiABC-null strains. Although it has been proposed that KaiC globally represses gene expression, our analysis revealed that dawn-expressed genes were up-regulated by kaiC-overexpression so that the clock was arrested at subjective dawn. Transfer of cells to DD conditions from LL immediately suppressed expression of most of the genes, while the clock kept even time in the absence of transcriptional feedback. Thus, the Synechococcus genome seems to be primarily regulated by light/dark cycles and is dramatically modified by the protein-based circadian oscillator.

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation.

The use of synthetic biology to design artificial gene circuits is an important approach for understanding the principles underlying the complicated dynamic behaviours of biomolecular networks, such as genetic switching and biological rhythms. The synthetic approach is also useful in systems biology in that it can be used to create artificial bypasses for processes related to cellular phenomena of interest for their easier analysis. To validate the role of transcription feedback in the cyanobacterial circadian system, we propose an experimental design for a 'semi-synthetic' approach that involves transplantation of the kaiABC genes into Escherichia coli and the construction of chimeric transcriptional outputs. The design principle and preliminary results are discussed. Copyright © 2008 Inderscience Enterprises Ltd.

In the cyanobacterium, Synechococcus elongatus, most promoters are regulated by a circadian clock under continuous light (LL) conditions. Nevertheless, the basic circadian oscillation is primarily generated by alternating KaiC phosphorylation/dephosphorylation reactions at the posttranslational level. Indeed, the KaiC phosphorylation cycle was recently reconstituted in vitro by incubating KaiA, KaiB, and KaiC proteins with ATP. However, the molecular dynamics of this chemical oscillation and the mechanism that drives the circadian transcription/translation rhythms remain unknown. In this report, the KaiC phosphorylation cycle and the gene regulatory network in the cyanobacterial circadian system have been modeled. The model reproduces the robust KaiC phosphorylation cycle in the absence of de novo gene expression as is observed in vitro, as well as its coupling to transcriptional/translational feedback in LL conditions in vivo. Moreover, the model is consistent with most previous experiments, including various combinations of genetic knockout or overexpression of kai genes. It also predicts that multiple KaiC phosphorylation states and dynamic Kai protein interactions may be required for the cyanobacterial circadian system.

Among the σ70 family bacterial σ factors, group 2 σ factors have similar promoter recognition specificity to group 1 (principal) σ factors and express and function under specific environmental and physiological conditions. In general, the cyanobacterial genome encodes more than four group 2 σ factors, and the unicellular Synechococcus elongatus PCC 7942 (Synechococcus) has five group 2 σ factors (RpoD2-6). In this study, we analyzed expression of group 2 σ factors of Synechococcus at both mRNA and protein levels, and we showed that the rpoD3 expression was activated only by high light (1,500 μmol photons m -2 s-1) among the various stress conditions examined. After high light shift, rpoD3 mRNA accumulated transiently within the first 5 min and diminished subsequently, whereas RpoD3 protein increased gradually during the first several hours. We also found that the rpoD3 deletion mutant rapidly lost viability under the same conditions. Analysis of the rpoD3 promoter structure revealed the presence of an HLR1 (high light-responsive element 1) sequence, which was suggested to be responsible for the high light-induced transcription under the control of the NblS (histidine kinase)-RpaB (response regulator) two-component system (Kappell, A. D., and van Waasbergen, L. G. (2007) Arch. Microbiol. 187, 337-342), at +6 to +23 with respect to the transcriptional start site. Here we demonstrated that recombinant RpaB protein specifically bound to HLR1 of the rpoD3 and hliA genes in vitro, and overexpression of a truncated RpaB variant harboring only the phosphoreceiver domain derepressed the transcription in vivo. Thus, we have concluded that phosphorylated RpaB are repressing the rpoD3 and hliA transcription under normal growth conditions, and the RpaB dephosphorylation induced by high light stress results in transcriptional derepression. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

The use of synthetic biology to design artificial gene circuits is an important approach for understanding the principles underlying the complicated dynamic behaviors of biomolecular networks, such as genetic switching and biological rhythms. The synthetic approach is also useful in systems biology in that it can be used to create artificial bypasses for processes related to cellular phenomena of interest for their easier analysis. To validate the role of transcription feedback in the cyanobacterial circadian system, we propose an experimental design for such a "semi-synthetic" approach that involves transplantation of the kaiABC genes into Escherichia coli and the construction of chimeric transcriptional outputs. The design principle and some preliminary results have been reported.

KaiA, KaiB, and KaiC clock proteins from cyanobacteria and ATP are sufficient to reconstitute the KaiC phosphorylation rhythm in vitro, whereas almost all gene promoters are under the control of the circadian clock. The mechanism by which the KaiC phosphorylation cycle drives global transcription rhythms is unknown. Here, we report that RpaA, a potential DNA-binding protein that acts as a cognate response regulator of the KaiC-interacting kinase SasA, mediates between KaiC phosphorylation and global transcription rhythms. Circadian transcription was severely attenuated in sasA (Synechococcus adaptive sensor A)- and rpaA (regulator of phycobilisome-associated)-mutant cells, and the phosphotransfer activity from SasA to RpaA changed dramatically depending on the circadian state of a coexisting Kai protein complex in vitro. We propose a model in which the SasA-RpaA two-component system mediates time signals from the enzymatic oscillator to drive genome-wide transcription rhythms in cyanobacteria. Moreover, our results indicate the presence of secondary output pathways from the clock to transcription control, suggesting that multiple pathways ensure a genome-wide circadian system.

KaiA, KaiB, and KaiC are essential proteins of the circadian clock in the cyanobacterium Synechococcus elongatus PCC 7942. The phosphorylation cycle of KaiC that occurs in vitro after mixing the three proteins and ATP is thought to be the master oscillation governing the circadian system. We analyzed the temporal profile of complexes formed between the three Kai proteins. In the phosphorylation phase, KaiA actively and repeatedly associated with KaiC to promote KaiC phosphorylation. High levels of phosphorylation of KaiC induced the association of the KaiC hexamer with KaiB and inactivate KaiA to begin the dephosphorylation phase, which is closely linked to shuffling of the monomeric KaiC subunits among the hexamer. By reducing KaiC phosphorylation, KaiB dissociated from KaiC, reactivating KaiA. We also confirmed that a similar model can be applied in cyanobacterial cells. The molecular model proposed here provides mechanisms for circadian timing systems.

Kai proteins globally regulate circadian gene expression of cyanobacteria. The KaiC phosphorylation cycle, which persists even without transcription or translation, is assumed to be a basic timing process of the circadian clock. We have reconstituted the self-sustainable oscillation of KaiC phosphorylation in vitro by incubating KaiC with KaiA, KaiB, and adenosine triphosphate. The period of the in vitro oscillation was stable despite temperature change (temperature compensation), and the circadian periods observed in vivo in KaiC mutant strains were consistent with those measured in vitro. The enigma of the circadian clock can now be studied in vitro by examining the interactions between three Kai proteins.

An autoregulatory transcription-translation feedback loop is thought to be essential in generating circadian rhythms in any model organism. In the cyanobacterium Synechococcus elongatus, the essential clock protein KaiC is proposed to form this type of transcriptional negative feedback. Nevertheless, we demonstrate here temperature-compensated, robust circadian cycling of KaiC phosphorylation even without kaiBC messenger RNA accumulation under continuous dark conditions. This rhythm persisted in the presence of a transcription or translation inhibitor. Moreover, kinetic profiles in the ratio of KaiC autophosphorylation-dephosphorylation were also temperature compensated in vitro. Thus, the cyanobacterial clock can keep time independent of de novo transcription. and translation processes.

In the cyanobacterium Synechococcus elongatus PCC 7942, KaiA, KaiB, and KaiC are essential proteins for the generation of a circadian rhythm. KaiC is proposed as a negative regulator of the circadian expression of all genes in the genome, and its phosphorylation is regulated positively by KaiA and negatively by KaiB and shows a circadian rhythm in vivo. To study the functions of KaiC phosphorylation in the circadian clock system, we identified two autophosphorylation sites, Ser-431 and Thr-432, by using mass spectrometry (MS). We generated Synechococcus mutants in which these residues were substituted for alanine by using site-directed mutagenesis. Phosphorylation of KaiC was reduced in the single mutants and was completely abolished in the double mutant, indicating that KaiC is also phosphorylated at these sites in vivo. These mutants lost circadian rhythm, indicating that phosphorylation at each of the two sites is essential for the control of the circadian oscillation. Although the nonphosphorylatable mutant KaiC was able to form a hexamer in vitro, it failed to form a clock protein complex with KaiA, KaiB, and SasA in the Synechococcus cells. When nonphosphorylatable KaiC was overexpressed, the kaiBC promoter activity was only transiently repressed. These results suggest that KaiC phosphorylation regulates its transcriptional repression activity by controlling its binding affinity for other clock proteins.

A kaiABC clock gene cluster was previously identified from cyanobacterium Synechococcus elongatus PCC 7942, and the feedback regulation of kai genes was proposed as the core mechanism generating circadian oscillation. In this study, we confirmed that the Kai-based oscillator is the dominant circadian oscillator functioning in cyanobacteria. We probed the nature of this regulation and found that excess KaiC represses not only kaiBC but also the rhythmic components of all genes in the genome. This result strongly suggests that the KaiC protein primarily coordinates genomewide gene expression, including its own expression. We also found that a promoter derived from E. coli is feedback controlled by KaiC and restores the complete circadian rhythm in kaiBC-inactivated arrhythmic mutants, provided it can express kaiB and kaiC genes at an appropriate level. Unlike eukaryotic models, specific regulation of the kaiBC promoter is not essential for cyanobacterial circadian oscillations.

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA-KaiC complex during the late subjective night. KaiB-KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA-enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein-protein interactions and post-translational modification of Kai proteins in the cyanobacterial clock system.

Physical interactions among clock-related proteins KaiA, KaiB, KaiC, and SasA are proposed to be important for circadian function in the cyanobacterium Synechococcus elongatus PCC 7942. Here we show that the Kai proteins and SasA form heteromultimeric protein complexes dynamically in a circadian fashion. KaiC forms protein complexes of similar to350 and 400-600 kDa during the subjective day and night, respectively, and serves as a core of the circadian protein complexes. This change in the size of the KaiC-containing complex is accompanied by nighttime-specific interaction of KaiA and KaiB with KaiC. In various arrhythmic mutants that lack each functional Kai protein or SasA circadian rhythms in formation of the clock protein complex are abolished, and the size of the protein complexes is dramatically affected. Thus, circadian-regulated formation of the clock protein complexes is probably a critical process in the generation of circadian rhythm in cyanobacteria.

Cyanobacterial clock proteins KaiA and KaiC are proposed as positive and negative regulators in the autoregulatory circadian kaiBC expression, respectively. Here, we show that activation of kaiBC expression by kaiA requires KaiC, suggesting a positive feedback control in the cyanobacterial clockwork. We found that robust circadian phosphorylation of KaiC. KaiA was essential for in vivo KaiC phosphorylation and activated in vitro KaiC autophosphorylation. These effects of KaiA were attenuated by the kaiA2 long period mutation. Both the long period phenotype and the abnormal KaiC phosphorylation in this mutant were suppressed by a previously undocumented kaiC mutation. We propose that KaiA-stimulated circadian KaiC phosphorylation is important for circadian timing.

Both regulated expression of the clock genes kaiA, kaiB, and kaiC and interactions among the Kai proteins are proposed to be important for circadian function in the cyanobacterium Synechococcus sp, strain PCC 7942. We have identified the histidine kinase SasA as a KaiC-interacting protein. SasA contains a KaiB-like sensory domain, which appears sufficient for interaction with KaiC. Disruption of the sasA gene lowered kaiBC expression and dramatically reduced amplitude of the kai expression rhythms while shortening the period. Accordingly, sasA disruption attenuated circadian expression patterns of all tested genes, some of which became arrhythmic. Continuous sasA overexpression eliminated circadian rhythms, whereas temporal overexpression changed the phase of kaiBC expression rhythm. Thus, SasA is a close associate of the cyanobacterial clock that is necessary to sustain robust circadian rhythms.

A negative feedback control of kaiC expression by KaiC protein has been proposed to generate a basic oscillation of the circadian clock in the cyanobacterium Synechococcus sp, PCC 7942, KaiC has two P loops or Walker's motif As, that are potential ATP-/GTP-binding motifs and DXXG motifs conserved in various GTP-binding proteins. Herein, we demonstrate that in vitro KaiC binds ATP and, with lower affinity, GTP, Point mutation by site-directed mutagenesis of P loop 1 completely nullified the circadian rhythm of kaiBC expression and markedly reduced ATP-binding activity. Moreover, KaiC can be autophosphorylated in vitro. These results suggest that the nucleotide-binding activity of KaiC plays important roles in the generation of circadian oscillation in cyanobacteria.

The kai gene cluster, which is composed of three genes, kaiA, kaiB and kaiC, is essential for the generation of circadian rhythms in the unicellular cyanobacterium Synechococcus sp, strain PCC 7942. Here we demonstrate the direct association of KaiA, KaiB and KaiC in yeast cells using the two-hybrid system, in vitro and in cyanobacterial cells. KaiC enhanced KaiA-KaiB interaction iii vitro and in yeast cells, suggesting that the three Kai proteins were able to form a heteromultimeric complex. We also found that a long period mutation kaiA1 dramatically enhanced KaiA-KaiB interaction in vitro. Thus, direct protein-protein association among the Kai proteins may be a critical process in the generation of circadian rhythms in cyanobacteria.

Cyanobacteria are the simplest organisms known to have a circadian clock. A circadian crock gene cluster kaiABC was cloned from the cyanobacterium Synechococcus. Nineteen clock mutations were mapped to the three kai genes. Promoter activities upstream of the kaiA and kaiB genes showed circadian rhythms of expression, and both kaiA and kaiBC messenger RNAs displayed circadian cycling. inactivation of any single kai gene abolished these rhythms and reduced kaiBC-promoter activity. Continuous kaiC overexpression repressed the kaiBC promoter, whereas kaiA overexpression enhanced it. Temporal kaiC overexpression reset the phase of the rhythms. Thus, a negative feedback control of kaiC expression by KaiC generates a circadian oscillation in cyanobacteria, and KaiA sustains the oscillation by enhancing kaiC expression.

Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short cireadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions-circadian period regulation, phase resetting, and cell division-attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.

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Cyanobacteria are the simplest organisms known to exhibit circadian rhythms and have provided experimental model systems for the dissection of basic properties of circadian organization at the molecular, physiological, and ecological levels. This review focuses on the molecular and genetic mechanisms of circadian rhythm generation in cyanobacteria. Recent analyses have revealed the existence of multiple feedback processes in the prokaryotic circadian system and have led to a novel molecular oscillator model. Here, the authors summarize current understanding of, and open questions about, the cyanobacterial oscillator.

A circadian rhythm in the accumulation of the core clock protein KaiC has been proposed to be important for proper circadian timing in the cyanobacterium Synechococcus elongatus PCC 7942 under continuous light conditions. Cycling in the abundance of the KaiC protein is delayed to the rhythm of its mRNA by similar to8 h, consistent with the proposed function of KaiC as a negative feedback regulator of kaiBC transcription. Here, we present temporal profiles of the synthesis and degradation of KaiC protein that determine the rhythm of its accumulation. The rate of KaiC synthesis shows a robust circadian oscillation, which is delayed to the mRNA rhythm slightly and advances the rhythm of KaiC accumulation by similar to6 h. The stability of KaiC protein also shows circadian fluctuations, such that KaiC degradation is suppressed during the mid-subjective night. These results suggest that transcriptional, translational, and posttranslational processes are important for the proper circadian changes in KaiC accumulation. Moreover, the turnovers of the phosphorylated and non-phosphorylated forms of KaiC show robust circadian rhythms with an anti-phase relationship to each other. Interestingly, when translation was inhibited, KaiC degradation and phosphorylation proceeded within at least 4 h in a circadian phase-dependent manner. Thus, the circadian timing seems flexible even when any perturbation in protein synthesis occurs.

Circadian rhythms, endogenous biological oscillations with a period of about a day, have been observed in innumerable physiological processes in most of organisms from cyanobacteria to higher plants and mammals. These rhythms are thought to be adaptive to daily environmental changes on the earth. These rhythms are daily fluctuations in cellular, physiological, and behavioral parameters, which are observed ubiquitously in many organisms. The circadian system is composed schematically of a central oscillator and inputs to and outputs from the clock. The cyanobacterium is the simplest organism known to harbor circadian clocks, and now becomes one of most successful model organisms for comprehensive understanding the oscillatory system at the molecular level. Molecular genetic studies on circadian clocks in the cyanobacterium Synechococcus identified two histidine kinases, SasA, and CikA. SasA interacts with a well-established clock protein KaiC and is necessary to sustain robust circadian oscillation, whereas CikA functions in a photic input pathway to the clock. Thus, multiple His-to-Asp signaling pathways are likely to play important roles in the Synechococcus circadian system. © 2003 Elsevier Inc. All rights reserved.

kaiABC, a gene cluster, encodes KaiA, KaiB and KaiC proteins that are essential to circadian rhythms in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. Kai proteins can interact with each other in all possible combinations. This study identified two KaiA-binding domains (C-KABD1 and C-KABD2) in KaiC at corresponding regions of its duplicated structure. Clerk mutations on the two domains and kaiA altered the strength of C-KABD-KaiA interactions assayed by the yeast two-hybrid system. Thus, interaction between KaiA and KaiC through C-KABD1 and C-KABD2 is likely important for circadian timing in the cyanobacterium. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Circadian rhythms have been observed in innumerable physiological processes in most of organisms. Recent molecular and genetic studies on circadian clocks in many organisms have identified and characterized several molecular regulatory factors that contribute to generation of such rhythms. The cyanobacterium is the simplest organism known to harbor circadian clocks, and it has become one of most successful model organisms for circadian biology In this review, we will briefly summarize physiological observations and consideration of circadian rhythms in cyanobacteria, molecular genetics of the clock using Synechococcus, and current knowledge of the input and output pathways that support the cellular circadian system. Finally, we will document some current problems in the studies on the cyanobacterial circadian clock.

Interactions of wild-type and Tyr83 mutant (Ys3F, Y83S, Y83L, and Y83H) plastocyanins (PCs) with lysine peptides as models for the PC interacting site of cytochrome f have been studied by absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies and electrochemical measurements. The spectral and electrochemical properties of PCs corresponded well with each other; species having a longer wavelength maximum for the S(Cys) pi --> Cu 3d(x2)-(y2) charge transfer (CT) band observed around 600 nm and a stronger intensity for the 460-nm absorption band exhibited stronger intensities for the positive Met --> Cu 3d(x2-y2) and negative His pi(1) --> Cu 3d(x2-y2) circular dichroism (CD) bands at about 420 and 470 nm, respectively, a lower average v(Cu-S) frequency, a smaller \ A(parallel to)\ EPR parameter, and a higher redox potential, properties all related to a weaker Cu-S(Cys) bond and a more tetrahedral planar geometry for the Cu site. Similarly, on oligolysine binding to wild-type and several Tyr83 mutant PCs, a longer absorption maximum for the 600-nm CT band, a stronger intensity for the 460-nm absorption band, stronger 420-nm positive and 470-nm negative CD bands, and a lower average v(Cu-S) frequency were observed, suggesting that PC assumes a slight more tetrahedral geometry on binding of oligolysine. Since changes were observed for both wild-type and Tyr83 mutant PCs, the structural change due to binding of oligolysine to PC may not be transmitted through the path of Tyr83-Cys84-copper by a cation-pi interaction which is proposed for electron transfer.

Common regulatory patterns have emerged among the feedback loops lying within circadian systems. Significant progress in dissecting the mechanism of clock resetting by temperature and the role of the WC proteins in the Neurospora light response has accompanied documentation of the importance of nuclear localization and phosphorylation-induced turnover of FRQ to this circadian cycle. The long-awaited molecular description of a transcription/translation loop in the Synechococcus circadian system represents a quantal step forward, followed by the identification of additional important proteins and interactions, finally, the adaptive significance of rhythms in Synechococcus and by extension in all clocks nicely ties up an extraordinary year.

The zfx gene encoding a zinc-containing ferredoxin from Thermoplasma acidophilum strain HO-62 was cloned and sequenced. It is located upstream of two genes encoding an archaeal homolog of nascent polypeptide-associated complex alpha subunit and a tRNA nucleotidyltransferase. This gene organization is not conserved in several euryarchaeoteal genomes, The multiple sequence alignments of the zfx gene product suggest significant sequence similarity of the ferredoxin core fold to that of a low potential 8Fe-containing dicluster ferredoxin without a zinc center. The tightly bound zinc site of zinc-containing ferredoxins from two phylogenetically distantly related Archaea, T. acidophilum HO-62 and Sulfolobus sp. strain 7, was further investigated by x-ray absorption spectroscopy. The zinc K-edge x-ray absorption spectra of both archaeal ferredoxins are strikingly similar, demonstrating that the same zinc site is found in T. acidophilum ferredoxin as in Sulfolobus sp, ferredoxin, which suggests the structural conservation of isolated zinc binding sites among archaeal zinc-containing ferredoxins, The sequence and spectroscopic data provide the common structural features of the archaeal zinc-containing ferredoxin family.

To reveal the regulatory mechanism of G(2)-arrest of cell division in embryos at the diapause stage of the silkworm, Bombyx mori, the profiles for mRNA levels of a Bombyx homologue of Cdc2 and a novel Bombyx Cdc2-related kinase (Bcdrk) were examined during ovarian development, early embryogenesis and diapause stage, using a reverse transcription/polymerase chain reaction system. Although levels of mRNAs for Cdc2 and Bcdrk were higher in paired ovaries during the early pupal stage, the levels decreased at the middle stage. Both mRNAs became abundant in oocytes toward the maturation. From oviposition up to the stage just before cellular blastoderm, high levels of both mRNAs were maintained, and thereafter the levels declined. In diapausing eggs kept at 25 degrees C, mRNA levels remained lower, whilst in diapause eggs exposed to 5 degrees C from 2 days after oviposition, in order to break diapause, relatively higher levels of mRNAs were found. In eggs treated with HCl to avert the entry into diapause, mRNA levels per egg for Cdc2 and Bcdrk increased 2 days after treatment, although these increases were not observed in terms of mRNA levels per actin mRNA. These results were discussed in relation to the activities of nuclear/cellular division in ovaries and eggs. In addition, Bcdrk was suggested to play a role similar to that in Bombyx Cdc2 kinase, because the changing profile for levels of Bcdrk mRNA resembled that for cdc2 mRNA.

In diapausing eggs of the silkworm, Bombyx mori, embryonic cells are arrested at G2 phase. The ability to undertake cell division is resumed in the course of dispause termination caused by such a treatment as acclimation to 5°C. As an initial trial to investigate the relationship between diapause end embryonic cell cycling, we have cloned and sequenced two Bombyx cDNAs encoding two distinct cdc2-related Ser/Thr protein kinases. One (Bm cdc2) encoded a 37.0 kDa protein which had all of the domains characteristic of other Cdc2 kinase. The other (Bcdrk) encoded a 45.1 kDa protein that was most similar to Drosophila and human cdc2-related protein kinases (Dcdrkprotein and PISSRLE kinase). Northern blot analysis was carried out to examine levels of Bm cdc2 end Bcdrk mRNA during embryogenesis of non-diapause eggs. The result demonstrated that the mRNA level of Bm cdc2 appeared to correspond to the activity of nuclear/cellular division in non-diapause eggs, and that the developmental profile in the level of Bcdrk mRNA was somewhat different from that of Bm cdc2 mRNA.

単細胞性シアノバクテリア，概日時計研究の最も単純なモデル生物である。期間中に以下の結果を得た
①Kai蛋白質リン酸化振動のin vitro再構成系では，低温でリズムを停止する過程で自由継続リズムから減衰振動に移行し，周期の安定性を保証するHopf分岐に従うことを明らかにした。②従来必須の時計遺伝子とされてきたkaiA遺伝子の機能欠失株が転写レベルの減衰振動を示すことを見いだした。③特殊な実験プロトコールを導入することでシアノバクテリアがUV耐性リズムを示すこと，糖代謝と密接な関連があることを示した。

好熱菌Thermus thermophilusの「細菌型mitoNEET（TthNEET）」は、II型糖尿病改善薬ピオグリタゾン標的候補として発見された哺乳類mitoNEET鉄硫黄蛋白質のホモログであり、クラスター近傍残基も保存されている。先の研究でTthNEET遺伝子完全破壊株につき、「グルコース感受性」が増強され増殖阻害されることを見出した。本研究では、変異導入株の構造解析、表現型解析等を行い、このグルコース感受性生育阻害が、mitoNEET型クラスターによるレドックス制御とは直接相関しないことを見出した。またプロテオーム解析等から、TthNEET欠損と相関変動する泳動スポットを検出した。

独自に池から分離した複数のシアノバクテリアが，寒天培地上で著しく複雑かつ興味深いコロニー・パターンを形成することを見出した。本研究では，とりわけGeitlerinemaとPseudanabaenaの二種類について，定量的な顕微鏡観測や，環境条件に対するコロニーパターンの変化を調べ，それぞれ定性的なコロニーパターン形成モデルを提案し，その一部はシミュレーションを併用することで検証した。また，それぞれの株についてコロニーパターンが変化する変異株を分離し，ゲノム配列の解読により，変異の同定を試みた。

MitoNEETは、II型糖尿病改善薬のミトコンドリア標的膜酵素として同定され、創薬ターゲットとしても期待されているが、生理機能は不明である。本研究では、遺伝子操作系およびオミックス解析系が整っている高度好熱菌とシアノバクテリアを主材料として、（１）細菌型および真核細胞ミトコンドリアのmitoNEETホモログの構造生物学解析、（２）好熱菌とシアノバクテリアのmitoNEETホモログ欠損株等の機能生理解析を行った。微生物ホモログとして初めて構造機能生理相関に関する新知見を得たほか、真核生物mitoNEETにつき薬剤結合型構造の強度データ収集にも成功し、当該分野における新たな展望が開けた。

個人レベルでゲノムデータを解読できる、ポスト・ゲノム時代のバイオメディア・アートに関する調査研究を行なった。バイオメディア・アートのポータルサイト「Bioart.jp」を立ち上げた。バイオアートの父と呼ばれるアーティストのジョー・デイビスを日本に招聘し、ワークショップ等を開催した。最終年度には3つの展覧会を開催し、本とカタログを出版した。

1.生命情報科学とデザインアートに関する研究については、2012年4月にオーストラリアのWester nAustralia大学のSymbioticA(バイオ・アート・センター)とコラボレーションした。その目的は、日本のバイオアートを紹介し、アートと生物の融合、可能性を模索することであり、その結果はオーストラリアで発表した。
2.生命科学技術を援用するバイオメディアアートの動向に関する調査研究については、生物のバイオマテリアル(生きている細胞)とリボソーム及びタンパク質等の人工細胞(soft interfaces)の研究を行い、バイオアートの可能性を追求した。研究結果については、学会発表で論文発表を行い、そのうち2本の論文は学会誌掲載。
3.日本におけるバイオアートの展開に関する調査研究については、芸術と生物学の接点がよりよく理解される為に外国と国内で学会発表を積極的で行った。その目的は、社会に対してバイオアートの言語、技術、概念を説明し、また海外に対して日本におけるバイオアートの現状と可能性を提示するものであった0
4.生命情報アートの可能性の模索に関しては、2012年度にベルギー、オーストラリア、カナダ、ロシア、2013年度に日本、イタリアで、アートとバイオテクノロジーの分野でのプレゼンテーションをした。

kai遺伝子欠損株においてもkloと名付けた少数の遺伝子群では、不安定な転写振動リズムが観察される。これらの発現に影響を与える複数の遺伝子座を同定した。野生株に置いてはklo遺伝子群は顕著な転写リズムを示さない。いっぽう、今回見出した複数の遺伝子座の多重変異体では、顕著にklo遺伝子群の発現が活性化され、また転写振動も回復していた。この変異体では、ゲノムワイドなkai依存性の転写振動の多くにも少なからぬ影響が見られた。

単細胞性シアノバクテリアSynechococcus PCC 7942を用い、時計遺伝子の新規転写翻訳を伴わない転写リズムの存在を、あらゆる生物に先駆けて初めて明らかにした。また、kai遺伝子などの高振幅遺伝子の発現の新たな制御因子として、二成分制御系のRpaB蛋白質を同定し、生化学的な解析を行い、論文発表した。さらに、多細胞性シアノバクテリアAnabaena PCC 7120を用い、ゲノムワイドな発現プロファイリング、発光レポーターの開発などを通じ、その概日特性や分化細胞特異的な概日転写制御を初めての明らかにし、論文発表した。

目的:
単細胞性シアノバクテリアは概日リズムを示す最も単純な生物群である。申請者は連続明条件下におけるゲノムワイドな転写リズムを示すが,夜間には時計遺伝子を含むほとんどのmRNAが直ちに分解されるダイナミックなゲノム発現動態を明らかにした。このことから,シアノバクテリアの概日時計は暗期中で転写を制御しないと考えられたが,最近一部の暗誘導遺伝子群の暗誘導に時刻依存性,時計遺伝子依存性が見られた。このことは,従来真核生物にのみ想定されていた概日時計を介する光周的な遺伝子発現が,原核生物においても存在することを初めて示している。さらに,暗期中でも減衰振動発現を示す遺伝子を発見し,時計遺伝子の転写が停止する夜間においても概日時計が転写を調節できることが明らかになった。本研究では,これらの解析を踏まえ,シアノバクテリアの明暗応答と概日応答の統合的制御の分子機構を解明することを目的とした。
このため,とくに時計遺伝子kaiABCの転写翻訳が停止する暗期中で,digA遺伝子など少数の遺伝子群が概日転写振動(ただし減衰振動)を示すことに着目し,この発現パターンがシグマ因子に依存することを明らかにした(投稿中)。また,レポーターの作成も試みたが,暗期特有のマスキングがかかっているためか,あるいはレポーター蛋白質が発現しにくいためか暗誘導の転写レポーター作成には至っておらず,今後の課題として残った。いっぽう,その過程で時計遺伝子の概日発現の一細胞観測に成功し,コロニー内での位相の差(ばらつき)を確認した。

シアノバクテリアの概日リズム,とくに転写翻訳に依存しない翻訳後振動子によるゲノムワイドな転写制御に関する解析を行った。連続明ではゲノム上の約1/3-1/2の遺伝子群に顕著な転写レベルの蓄積リズムが観られ,その位相分布,kai 依存性などを解析した。また,連続明では殆どの遺伝子の転写が劇的に低下し,プロテオームとトランスクリプトームの相関性に著しい乖離が生じること,ごく一部の遺伝子発現は暗期に活性化され,その多くはkai依存性がみられることなどを明らかにした。これらは,あらゆる生物種において,新規の概日遺伝子発現を欠く条件での初めての概日時計依存的転写調節の観察例である。

本研究は,多細胞性シアノバクテリアAnabaena sp.PCC7120の窒素欠乏下における細胞分化(ヘテロシスト分化)・パターン形成のダイナミクスを明らかにするための観測操作系の確立を目指すものであった。その前提として,まず顕微鏡下で培養しつつ,分化誘導を行い,その様子(明視野像,光合成活性,遺伝子発現パターン)をタイムラプス観測するための同時多点同時モニタリング系をほぼ確立し,ヘテロシスト分化に関わる細胞系譜解析を行った。その結果,窒素欠乏処理直後の光合成活性,細胞分化の中枢遺伝子であるhetR遺伝子の初期のばらつきと,最初にヘテロシストを生み出すことになる細胞の位置には相関はなく,窒素欠乏処理直後の細胞のばらつきが後々直接の影響を及ぼしているわけではないことが示唆された。実際,hetR発現パターンの推移を詳しく観察することで,hetR発現強度には一過的な増減が多く観察されること,細胞分裂が揺らぎの一端を担っていることなどが確認された。
さらに一細胞レベルで分化誘導実験を行うことで,パターン形成ダイナミクスの摂動実験を行うことが本研究計画の主要なテーマであった。そこで,MEMS(micro-electro mechanical system)技術を用い,複雑な流路を備えたPDMS-ガラスを素材とするマイクロデバイスを何パターンか作製した。とくに空気バルブを多用し,流路の切り替えを実現したPDMS-ガラスデバイスを作製した。かなり複雑な構成にしてしまったために,デバイスに脆弱な点もあり改善の余地が残ったが,従来にない微小空間流路培養系が確立し,デバイス内での通常の細胞分化を確認することが出来た。今後は,今回作製したデバイスを用い,分化誘導をきたす2-oxoglutarateを局所的に投与する実験を開始する予定である。

真核生物型の概日振動遺伝子ネットワークの再構成に関しては、昨年に引き続き、再構成された短周期振動に影響を与えるパラメータセットの同定を目指した。さらに、新たな周期解析方法を導入して統計的な解析も行った。培地を高栄養なものに変え、フィードバックループを始動する際にpositive因子の発現誘導を短時間にすると、12時間を越えるような長周期振動が観察された。また、positive因子とnegative因子の挙動を波長の異なるルシフェラーゼを用いて同時モニターできる系を開発し、両者の振動がほぼ同位相であることを見出した。これは理論的な予想とは反する結果であった。この他、ハイスループットな実験系の確立を目指し、96ウェルマイクロプレートリーダーの導入を行い、観察系の細胞密度が低い場合に明瞭な振動が出やすいことを見出した。細胞毎に振動の周期・位相・振幅が異なるため集団レベルで同調した観察結果が得られにくかったのだと推測している。
原核生物型の概日振動分子ネットワークの再構成に関しては、昨年度に引き続き、大腸菌へのkai遺伝子群(シアノバクテリアの時計遺伝子群)の移植実験を行った。今回はkaiAとkaiBCを独立に制御することで、さまざまな発現比での動態を追った。その結果、培養条件(細胞増殖速度)依存的にリン酸化上昇・脱リン酸化促進ダイナミクスが得られており、条件検討によってはリン酸化振動の再構成は可能であると思われるが、実現には至らなかった。一方、時刻情報を遺伝子発現に変換するSasA-RpaA系の大腸菌に移植してもKaiCによるリン酸化ジレーの変化は観察できなかった。リン酸化の一過的な変化などについては興味深い観察結果も得られたので論文にまとめた(Int.J.Bioinf.Res. Appl., 印刷中)。

シアノバクテリアの時計蛋白質KaiA, KaiB, KaiCを適当な濃度比でin vitroで混合することで,24時間周期のKaiCリン酸化の概日リズムを再溝成することができる。しかし,実際の細胞は,細胞分裂・増殖に伴う細胞内外のノイズ外乱のために,in vitroのようなstaticな場ではない。シアノバクテリアでは,増殖条件でのみ,kai遺伝子を含む大多数の遺伝子の転写翻訳リズムが観察されるが,kai遺伝子の転写振動は,コアの蛋白質振動ループの安定化に寄与している可能性が考えられる。この検証には転写翻訳ループ(すなわち時計遺伝子自体の転写リズム)のある条件と無い条件を再構成して,そのリン酸化振動の安定性解析をすればよい。そこで,概日リズムを示さない細胞として大腸菌に対し,kaiAもしくはkaiBCを,trcプロモーターもしくはbadプロモーターに連結した形質転換体を複数ヴァージョン作製し,KaiC蛋白質の量的変動・リン酸化の経時変化を検討した。その結果,さまざまな条件下でリン酸化プロファイルの変動に相違が見られた(Tozakiら,論文印刷中)。ただし,当面の目標は,大腸菌内でリン酸化振動を再構成することであるが,いまだ成功していない。理由としては,Kai蛋白質群の大腸菌内での発現量が,振動可能な範囲を逸脱していること,大腸菌内の細胞環境がシアノバクテリアのそれとは異なる可能性などが考えられる。
さらに,マイクロアレイを用いた概日発現および明暗応答プロファイルの解析を続け,連続明条件下における発現位相が,明暗サイクル下でのそれと多くの場合乖離すること,比較的少数にとどまる暗誘導遺伝子群が,実際に蛋白質レベルでも誘導を受けること,例外的ではあったが,連続明条件下,高振幅で周期的に発現するnon-coding RNAが存在することなどを明らかにした。

概日時計は時計遺伝子の発現のフィードバック制御によるとされており、概日振動の周期長や安定性を説明するためには細胞の遺伝子発現の包括的解析が必要で、それを基にシステム的解析が必要と考えられた。当初の計画ではDNAチップと生物発光レポーターで遺伝子発現の包括的解析を可能として、シアノバクテリア細胞の概日システムの分子機構の全体像を解明することを目的とした。シアノバクテリアの概日時計による遺伝子発現制御の包括的解析を2つの方法(SynechococcusのGeneChipの開発、生物発光による遺伝子発現解析)で可能とし、振動サイクルでの時間経過について解析を行なった。その結果、当初の期待と予測をほぼ裏付ける成果を得ることができた。この成果は、新たなタンパク質による時計モデルに基づいて、概日システムを解析する時には、その基礎となろう。
一方、全く予想していなかった転写の停止した暗期中でのKaiCリン酸化サイクルを見出し、さらに3つのタンパク質とATPのみでこれを再構成した。これは極めてインパクトの大きな発見で、概日時計の基本モデルに大きな修正を迫るものである。そこで、期間の後半はin vitroの再構成系を利用したKaiCのリン酸化サイクルの生化学解析を中心として行い、1)KaiA,KaiBの相互作用2)KaiCのリン酸化プログラムの解明、3)周期を規定するKaiCのATPase活性の発見、4)KaiCリン酸化リズムの自己同調機構などの極めて新規な成果を得た。いずれもタンパク質自体が高度な機能をもつことを示したものであるが、特に概日時計の特徴である24時間の周期長とその安定性がKaiCのATPase反応に基づくことやKaiC6量体間でその時間情報が交換されることなど、大きなインパクトをもつ結果が得られ、概日時計のデザイン原理を理解する可能性をもつものである。

私たちは,シアノバクテリアでは概日リズム発生の主要因が転写翻訳フィードバックではないことを最近明らかにした(Tomtia et al.2005)。さらに,in vitroではKaiA, KaiB, KaiCの三者でKaiCのリン酸化振動に十分であることを示した(Nakajima et al.2005)。本研究では,このリン酸化振動を大腸菌に移植できるかどうかを試み,さらに人工的な入出力系を付与出来るかどうかを試すことが主要目的である。
いまのところ,誘導性プロモーターの下流にkai遺伝子群を組み込み,大腸菌株に導入したが,まだリン酸化振動の再構成には成功していない。しかしながら,出力系のデザインのため,KaiC結合ヒスチジンキナーゼSasAのパートナーとなる応答因子の探索を行い,有力な候補SyelRR16を得た。sasA同様,syelRR16欠失株においても転写リズムは著しく減衰した。興味深いことに,SasAの自己リン酸化ならびにSyelRR16へのリン酸基転移反応は,KaiCのリン酸化リズムの程度に応じて顕著に変化することが明らかになった。SyelRR16は典型的なOmpR型DNA結合蛋白質であることから,KaiABC蛋白質による翻訳後修飾レベルの基本振動が,SasA->SyelRR16のリン酸化リレーと介して転写調節に変換されることが強く示唆された。このシステムを用いれば,Kai振動をSasA->SyelRR16を介して下流遺伝子発現に変換し,自由に振動系を調節出来る可能性がある。

シアノバクテリアの時計蛋白質KaiA,KaiB,KaiCの組替え蛋白質をATPと試験管内で混合してインキュベートすることでKaiCのリン酸化の概日振動がin vitroで発生することを示した(Science,2005)。このことは,生物リズムの新しい展開を約束するだけでなく,新たな蛋白質機能を発見したという意味で蛋白質科学,新たな長周期振動発生機構の発見という意味で動的物理化学(非線形科学),数理生物学に画期的な材料を提供し,さらにその反応が極めて遅く,ATP消費も極端に少ないことから,ある種の新規機能性材料開発にも示唆を与えうる可能性がある。理論モデルについては,複数の理論系研究グループと共同研究を行い,実験系と理論系の研究者の相互乗り入れを図った。これらの研究成果は,現在投稿中である(Miyoshi et al.,submitted to BMC Bioinformatics ; Kurosawa et al. submitted to Biophysical J)。
真核生物型のキメラ蛋白質を用いた,人工振動ネットワークの形成(担当・松本)については,キメラ蛋白質の分解速度,プロモーター活性の強弱,培養条件などを振り,いわゆる「inhibitor-activator系」を用いたモデルを取り入れることによって,いくつかの周期発現振動を開発することに成功した。現在,早稲田大学から,博士研究員が松本研究室に出向し,ショウジョウバエ培養細胞の取り扱いや基本的な技術を習得しつつある。岩崎グループでは,現在-細胞レベルの発光・蛍光長期モニタリングシステムを構築しつつあり,より精度の高い遺伝子発現の挙動や揺らぎの観測・制御を目指す予定である。

比較的長大で,しかも非常に安定な周期性を示す概日(サーカディアン)リズムは,従来,多くの遺伝子・蛋白質からなる複雑なネットワークの結果として生じると考えられてきた。
概日リズムの研究分野の特色は,その領域形成の過程から極めて学際的で,たとえば現在盛んに主張される「実験と理論の共存」が,比較的古くから積極的かつ継続的に図られてきた分野と言える。そのため,生理学・生態学・分子生物学などによる実験研究の蓄積と,振動発生における理論的研究が同時に進められてきた。現在,生命科学の最前線で概日リズムが比較的露出度が高いように見えるのは,システム生物学など「生命科学における実験と理論の融合」が新たに注目されてくる中で,その代表的なモデルケースと位置づけられるからである。
さて,すでに1965年にGoodwinは転写・翻訳プロセスのネガティヴ・フォードバック制御により転写振動を起こす可能性に言及している。しかし,実際に実験を伴って提案されたのは,1990年にHardin,Rosbashらがハエの生物時計遺伝子の発現リズム機構が最初である。以降,カビ,植物,哺乳類などでも,基本的に生物時計の発振機構は転写・翻訳フィードバックに集約されるとの報告が相次ぎ,時間生物学のドグマとして定着した。
しかし,私たちはシアノバクテリアの時計蛋白質KaiCの概日リン酸化リズムが,特定の条件下(連続暗条件下や転写・翻訳阻害剤投与下)では転写翻訳フィードバックを必要としないことを最近明らかにした(1)。さらに,三つの時計蛋白質KaiA,KaiB,KaiCの組み替え蛋白質をATPと特定の濃度でインキュベートするだけで,温度補償性を伴うKaiCのリン酸化振動を,試験管内で再薄成できることも示した(世界初の概日振動の試験管内再構成)(2)。これらの発見は,従来の概日リズムの分子機構に関し,いくつかの根本的な見直しを迫り,また新たな視座を提供するものである。

本研究は、二成分制御系を介した転写プロファイルの解析、無機物質同化系を制御する転写因子の同定と機能解析、概日時計遺伝子を介したゲノムワイドのグローバルな転写制御の分子機構の解析を行い、ラン藻(シアノバクテリア)に特有の転写制御ネットワークを明らかにすることを目的とする。17年度までに得られた主な成果は以下の通りである。
(1)絶対光独立栄養型ラン藻シネココッカスSynechococcus PCC 6301株の全ゲノム配列(2,696,255bp)を決定した。この中に、2つのrRNA遺伝子オペロン(rrnAとrrnB)、45のtRNA遺伝子、4つの低分子RNA遺伝子、およびを2525個のタンパク質遺伝子をアノテートした。二成分制御系遺伝子を37個同定した。全タンパク質遺伝子の10%の遺伝子は他の生物種のゲノムに存在しないことを明らかにした。さらに、シネココッカスSynechococcus 6301株のゲノムの全遺伝子および遺伝子間領域をカバーするオリゴDNAチップを作製した。
(2)他のラン藻種にはなく、Synechococcus 7942株/6301株にのみ存在する遺伝子群の中に、潜在的な硝酸輸送活性をもつ新規トランスポータの遺伝子(syc1147)とその活性を制御する二成分シグナル伝達系の遺伝子群(syc2277-syc2278)を同定した。
(3)時計関係の研究での最大の研究成果として,連続暗期中で転写・翻訳活性が極端に低下し,kai遺伝子の転写翻訳が完全に停止してもKaiC蛋白質のリン酸化振動が完全に持続することを発見した。さらに、KaiABC蛋白質の試験管内リン酸化振動発生を世界で初めて明らかにした。これらの研究は,シアノバクテリアに限らず哺乳類,植物を含む生物時計の分子機構の常識を一変させ,大きな反響を呼んだ。

シアノバクテリアの概日振動は時計蛋白質KaiCによるkaiBCプロモーターのフィードバック制御を骨格として発生していると考えられている(Ishiura et al. 1998)。この様式は真核生物の時計モデルと共通であるが、これまでのところ、真核生物の様に時計遺伝子の上流領域に対するKaiCの特異的な制御は確認されていない。そこで、シアノバクテリアの概日システムの特徴を裏付けている機構を探るべく、解析を行ない、以下の結果を得た。1)KaiCの過剰発現はkaiBCプロモーターのみならずすべてのプロモーターのリズム成分だけを選択的に抑制する。2)kaiBC欠損株において、大腸菌由来のプロモーターによりkaiCBを発現させた場合でも、発現レベルを調整すると、ほぼ完全な概日振動を回復することが出来た。従って、シアノバクテリアではKaiCは遺伝子発現の包括的制御を行っており、細胞の遺伝子発現を包括的に制御するとともに、概日振動の発生を実現していると考えられる。これを裏付けるためシアノバクテリアのKaiC蛋白質の細胞内の時計蛋白質の分布を調べ、KaiC蛋白質は膜画分と核様体に多く存在することを明らかにし,さらにKaiCのDNA結合能についても詳細な生化学データが蓄積しつつある。

本研究では,シアノバグテリアの概日リズムにおけるリン酸化制御の解明を目的とした。シアノバクテリアの時計蛋白質は,KaiA,KaiB,KaiCが知られるが,その機能はまだ不明である。今回,KaiCのリン酸化制御を中心に多くの生化学的・分子遺伝学的成果を得ることが出来た。
まず,KaiCがセリンスレオニン残基で自己リン酸化し,細胞質中で顕著なリン酸化リズムを示すことを報告した(岩崎ら,PNAS)。さらに,そのリン酸化はKaiAにより促進され(岩崎ら,PNAS),KaiBにより抑制される(北山ら,EMBOJ.)ことが明らかとなった。さらにこの知見と対応して,KaiA-KaiB-KaiCは他の時計関連因子SasAとともに主観的夜に巨大な複合体を形成していること(景山ら,JBC),さらにKaiC蛋白質中の二つのKaiA結合領域を同定してその重要性を検証する(谷口ら,FEMS Lett.)など,Kai蛋白質間相互作用とリン酸化制御が繋がったことは,シアノバクテリアの時計蛋白質の性質の理解に飛躍的な発展をもたらしたと言えよう。
のみならず,分子遺伝学的な解析から,KaiCが従来提唱されていたネガティヴフィードバック作用に加え,ポジティヴフィードバック制御にも関与していることが示唆され(岩崎ら,PNAS),さらに複雑で巧妙な概日時計の仕掛けが垣間見えてきた。

A. 多細胞性シアノバクテリアの時間パターン（概日システム）の解析：　細胞分化パターニングを示す糸状性シアノバクテリアAnabaena における概日システムの解析を行った。マイクロアレイ解析を複数回行い、単細胞性シアノバクテリアとは，時計遺伝子の発現パターンや概日発現遺伝子群の発現位相分布に大きな違いがみられることを明らかにした。さらに、そこから二つの高振幅概日発現遺伝子群を抽出し、バクテリアルルシフェラーゼ遺伝子を用いた生物発光レポーター株を作製し、リアルタイムモニタリングを行うことで詳細な概日発現リズムの状態を解析することができ、連続明条件下における周期の温度補償性や暗パルスに対する光位相応答を確認した。B. シアノバクテリアの細胞分化パターニングの解析：　Anabaena の細胞分化の中枢遺伝子hetR, patS は，確証されたわけではないが，転写因子および，その活性を阻害するオリゴペプチドをコードする。自己活性化ループと自己抑制がカップリングし，抑制因子が拡散性というTuring モデルが当てはまりそうだが，妥当性は検討されていない。そこで，顕微鏡下での連続培養観測系を立ち上げ，バクテリアでは世界初となる細胞分化系譜を構築し，位置情報の決定が初期値依存的ではなく，細胞間相互作用を介して動的に決定されることを明らかにした（PLoSONE,2009）。この成果を踏まえ，マイクロデバイス工学を援用した微小流路を用い，特定の分化制御・パターン制御遺伝子候補を局所的に発現させたり，分化誘導に重要な影響を与える代謝産物などを添加できる系を構築することを目標とし、複数のデザインのデバイスを用いて解析を行った。その結果、細胞フィラメントの流路への導入には成功するが、デバイス内での成長阻害が見られており、まだ詳細な解析には至っていない。今後引き続き検討する必要がある。C. シアノバクテリアのコロニー・パターン形成の解析：　申請者が西早稲田キャンパスの池から単離した、複雑なコロニーパターンを形成する2種（Pseuanabaena, Geitlerinema）を対象とし、これらのパターンがどのようなプロセスと原理で生成するのかを解析した。まず、さまざまな環境条件下での運動パターンの定量的な観測を行い，モルフォロジーダイアグラムの構築を行った。その結果、Geitlerinemaは比較的安定な集団軌道を自律的に形成し、特徴的な渦状コロニーを形成するのに対し、Pseudanabaenaはバンドル状、円盤状、彗星状の三つの形態を動的に遷移しながら複雑かつ流動的に多様なコロニーパターンを形成することを見いだし、それらの動的パターンの定量的な解析を進めた。D. シアノバクテリアを用いたバイオメディア・アートの試み：　シアノバクテリアのパターン形成・運動過程を長期間撮影することで得られる映像に加え，細胞を用いた絵画・彫刻表現や，電気回路との接続による新たな形式のメディア芸術の展開を図った。この際，科学者・芸術家の双方にとって新しいこと（サイエンスとアートが未分化であり，双方を相補的に展開できること），ときとしてファインアートにおける造形行為を相対化する側面を持つこと，科学と芸術の境界面を鋭くえぐり出すものであること，新たな規範的な造形美の可能性を追求すること，をコンセプトとし、さまざまな作品を発表することが出来た。2010 年度はオーストリア・リンツにおいて個展およびライトアートビエンナーレへの招待展示、オランダ・ハーグにおけるオランダ国際ビエンナーレにおける招待展示を行った。2011年度はTokyo Designers Weekに多摩美術大学、慶應義塾大学SFCとの共同でバイオアートに関する展示、また岡本太郎生誕100周年記念展（岡本太郎美術館）での大規模な作品の展示を行い、多くの注目を集めた。metaPhorestと呼ばれる、生命論に興味を持つ芸術家たちがアーティスト・イン・レジデンス的に研究室に集うプラットフォームを本格化されることで、国内外でも有数の生命美学の拠点の一つを形成しつつあると考えている。岩崎は、さらに2010年に米国NSF+英国ESPRCの主宰で実現した、合成生物学に関わる芸術・デザインの国際プロジェクトSynthetic Aestheticsのメンバー（約400名の応募中12名）に選ばれ、生命美学に関するオーストラリアのアーティストとの共同プロジェクトを開始した。

多細胞性シアノバクテリアAnabaena sp. PCC 7120は、窒素欠乏条件下でヘテロシストと呼ばれる細胞を、およそ10細胞に一つの割合で分化する。ヘテロシストは窒素固定に特化した細胞である。光合成によって生じる酸素に対して感受性があり、不可逆的に機能損傷を受ける窒素固定酵素が発現し、光合成系機能が抑制される。このヘテロシスト分化は、多細胞体制に見られる最も単純な秩序だった分化パターン（形態形成）の優れた研究モデルであり、分子遺伝学的解析から分化促進因子HetR、分化抑制因子PatSなど、多くの関連制御因子群が同定されてきた。　私たちは、これらの発生分化パターンの一細胞レベルでの動態を観察するため、蛍光顕微鏡と高感度の冷却CCDカメラを用いて詳細なタイムラプス解析を行い、バクテリアの細胞系譜解析を実現した。また、hetR遺伝子発現動態をリアルタイムで観察し、細胞系譜と合わせて詳細に解析した。その結果、細胞分化パターニングは、pre-deterministicに決っているのではなく、細胞間相互作用を伴って動的に運命決定されていくこと、細胞分裂を阻害しても、等間隔の細胞分化パターンを維持できることなどを見いだした。これは、細胞分裂が分化に決定的な役割を果たすとの先行研究でのモデルを覆す重要な知見である。　また、私たちは単細胞性シアノバクテリアを用いた概日リズムの研究で、さまざまな解析を行ってきた。その経験を踏まえ、Anabaenaにおける概日リズムの解析に着手し、概日リズムの発現に必須なkai遺伝子の相同遺伝子群の欠損株の作成や、ゲノムワイドな概日発現リズムを探索するためのマイクロアレイ解析を行った。マイクロアレイ解析についても今後さらに解析が必要であるが、単細胞性シアノバクテリアで得られた結果とは異なり、kai遺伝子の発現リズムがそれほど明確ではないこと、にもかかわらず一部の遺伝子の発現は高振幅リズムを示すことが分かってきた。また、kai遺伝子破壊株では、一部の概日発現遺伝子の発現リズムの異常が観察されたが、再現性を確認することが必要である。

多細胞性シアノバクテリアのモデル種としてAnabaena PCC 7120の細胞分化の中枢遺伝子の一つhetR，patSは，それぞれ転写因子およびその転写調節因子をコードしている。HetRは自身の転写の促進因子であり，patSの転写も促進する。いっぽう，PatSは拡散性のオリゴペプチドで，HetRの働きを抑制し，ネガティブフィードバックを生み出す。したがって，自己活性化ループと自己抑制ループのカップリングしたネットワークを含む，さらに抑制因子が拡散性であるようなネットワークであり，そのままTuring patternによる固定波形成ネットワーク・モデルに妥当している。当初は，この遺伝子ネットワークを異種生物である大腸菌に組み込み，大腸菌をテストチューブとして遺伝子機能やそのダイナミクスを解析することを目標とした。 そこで，まず大腸菌にhetRオペロンとpatSを遺伝子導入し，転写レベルの発現活性化・抑制を再現出来るかをチェックした。この際，レポーターとしてバクテリアルシフェラーゼを用い，遺伝子発現の経時変化のリアルタイムモニターを行ったが，発光を確認できず，hetRオペレーターは大腸菌では働かないことが明らかになった。 そこで，現実のシアノバクテリアにおけるhetRの遺伝子発現のリアルタイムモニタリング系の構築のほうが重要であると判断し，蛍光顕微鏡観察条件下で連続的にバクテリアを長期培養・遺伝子発現モニタリングできる系の開発に成功した。これにより，バクテリアの発生に関する細胞系譜を初めて取得することに成功した。　これらの成果は，バクテリアを用いた形態形成の研究を飛躍的に向上させるものとして，国際的にも大きなインパクトを与えつつある。