The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii.

The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNA-binding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana.

Rhythmic adenosine triphosphate release from the cyanobacterial circadian clock protein KaiC revealed by real-time monitoring of bioluminescence using firefly luciferase.

The cyanobacterial circadian clock is composed of three clock proteins, KaiA, KaiB and KaiC. This KaiABC clock system can be reconstituted in vitro in the presence of adenosine triphosphate (ATP) and Mg2+ , and shows circadian rhythms in the phosphorylation level and ATPase activity of KaiC. Previously, we found that ATP regulates a complex formation between KaiB and KaiC, and KaiC releases ATP from KaiC itself (PLoS One, 8, 2013, e80200). In this study, we examined whether the ATP release from KaiC shows any rhythms in vitro. We monitored the release of ATP from wild-type and ATPase motif mutants of KaiC as a bioluminescence in real time using a firefly luciferase assay in vitro and obtained the following results: (a) ATP release from KaiC oscillated even without KaiA and KaiB although period of the oscillation was not 24 hr; (b) ATP was mainly released from the N-terminal domain of KaiC; and (c) the ATP release was enhanced and suppressed by KaiB and KaiA, respectively. These results suggest that KaiC can generate basal oscillation as a core clock without KaiA and KaiB, whereas these two proteins contribute to adjusting and stabilizing the oscillation.

Circadian Clock in Arabidopsis thaliana Determines Flower Opening Time Early in the Morning and Dominantly Closes Early in the Afternoon.

Many plant species exhibit diurnal flower opening and closing, which is an adaptation influenced by the lifestyle of pollinators and herbivores. However, it remains unclear how these temporal floral movements are modulated. To clarify the role of the circadian clock in flower movement, we examined temporal floral movements in Arabidopsis thaliana. Wild-type (accessions; Col-0, Ler-0 and Ws-4) flowers opened between 0.7 and 1.4 h in a 16-h light period and closed between 7.5 and 8.3 h in a diurnal light period. In the arrhythmic mutants pcl1-1 and prr975, the former flowers closed slowly and imperfectly and the latter ones never closed. Under continuous light conditions, new flowers emerged and opened within a 23-26 h window in the wild-type, but the flowers in pcl1-1 and prr975 developed straight petals, whose curvatures were extremely small. Anti-phasic circadian gene expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYLE (LHY) and TIMING OF CAB EXPRESSION 1 (TOC1) occurred in wild-type flowers, but non-rhythmic expression was observed in pcl1-1 and prr975 mutants. Focusing on excised petals, bioluminescence monitoring revealed rhythmic promoter activities of genes expressed (CCA1, LHY and PHYTOCLOCK 1/LUX ARRHYTHMO, PCL1/LUX) in the morning and evening. These results suggest that the clock induces flower opening redundantly with unknown light-sensing pathways. By contrast, flower closing is completely dependent on clock control. These findings will lead to further exploration of the molecular mechanisms and evolutionary diversity of timing in flower opening and closing.

Lumi-Map, a Real-Time Luciferase Bioluminescence Screen of Mutants Combined with MutMap, Reveals Arabidopsis Genes Involved in PAMP-Triggered Immunity.

Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single-nucleotide polymorphisms (SNPs) for studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole-genome sequencing-based method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing the LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). Although five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas.

The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas.

Atomic force microscopy analysis of SasA-KaiC complex formation involved in information transfer from the KaiABC clock machinery to the output pathway in cyanobacteria.

The cyanobacterial clock oscillator is composed of three clock proteins: KaiA, KaiB and KaiC. SasA, a KaiC-binding EnvZ-like orthodox histidine kinase involved in the main clock output pathway, exists mainly as a trimer (SasA3mer ) and occasionally as a hexamer (SasA6mer ) in vitro. Previously, the molecular mass of the SasA-KaiCDD complex, where KaiCDD is a mutant KaiC with two Asp substitutions at the two phosphorylation sites, has been estimated by gel-filtration chromatography to be larger than 670 kDa. This value disagrees with the theoretical estimation of 480 kDa for a SasA3mer -KaiC hexamer (KaiC6mer ) complex with a 1:1 molecular ratio. To clarify the structure of the SasA-KaiC complex, we analyzed KaiCDD with 0.1 mmol/L ATP and 5 mmol/L MgCl2 (Mg-ATP), SasA and a mixture containing SasA and KaiCDD6mer with Mg-ATP by atomic force microscopy (AFM). KaiCDD images were classified into two types with height distribution corresponding to KaiCDD monomer (KaiCDD1mer ) and KaiCDD6mer , respectively. SasA images were classified into two types with height corresponding to SasA3mer and SasA6mer , respectively. The AFM images of the SasA-KaiCDD mixture indicated not only KaiCDD1mer , KaiCDD6mer , SasA3mer and SasA6mer , but also wider area "islands," suggesting the presence of a polymerized form of the SasA-KaiCDD complex.

Circadian oscillations of KaiA-KaiC and KaiB-KaiC complex formations in an in vitro reconstituted KaiABC clock oscillator.

The circadian clock is an endogenous biological mechanism that generates autonomous daily cycles in physiological activities. The phosphorylation levels of KaiC oscillated with a period of 24 h in an ATP-dependent clock oscillator reconstituted in vitro from KaiA, KaiB and KaiC. We examined the complex formations of KaiA and KaiB with KaiC in the KaiABC clock oscillator by fluorescence correlation spectrometry (FCS) analysis. The formation of KaiB-containing protein complex(es) oscillated in a circadian manner, with a single peak at 12 h and single trough at 24 h in the circadian cycle, whereas that of KaiA-containing protein complex(es) oscillated with two peaks at 12 and 24 h. FCS and surface plasmon resonance analyses showed that the binding affinity of KaiA for a mutant KaiC with Ala substitutions at the two phosphorylation sites considered to mimic the nonphosphorylated form of KaiC (np-KaiC) was higher than that for a mutant KaiC with Asp substitutions at the two phosphorylation sites considered to mimic the completely phosphorylated form of KaiC (cp-KaiC). The results from the study suggest that a KaiA-KaiB-cp-KaiC ternary complex and a KaiA-np-KaiC complex were formed at 12 and 24 h, respectively.

Importance of the monomer-dimer-tetramer interconversion of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro KaiABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N-terminal deletion, KaiB10-108 . KaiB10-108 was a monomer at 4 °C but a dimer at 35 °C. KaiB10-108 was able to drive normal clock oscillation in an in vitro reconstituted KaiABC clock oscillator at 25 °C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41 °C. Wild-type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1 m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells.

Phase-resetting mechanism of the circadian clock in Chlamydomonas reinhardtii.

Although the circadian clock is a self-sustaining oscillator having a periodicity of nearly 1 d, its period length is not necessarily 24 h. Therefore, daily adjustment of the clock (i.e., resetting) is an essential mechanism for the circadian clock to adapt to daily environmental changes. One of the major cues for this resetting mechanism is light. In the unicellular green alga Chlamydomonas reinhardtii, the circadian clock is reset by blue/green and red light. However, the underlying molecular mechanisms remain largely unknown. In this study, using clock protein-luciferase fusion reporters, we found that the level of RHYTHM OF CHLOROPLAST 15 (ROC15), a clock component in C. reinhardtii, decreased rapidly after light exposure in a circadian-phase-independent manner. Blue, green, and red light were able to induce this process, with red light being the most effective among them. Expression analyses and inhibitor experiments suggested that this process was regulated mainly by a proteasome-dependent protein degradation pathway. In addition, we found that the other clock gene, ROC114, encoding an F-box protein, was involved in this process. Furthermore, we demonstrated that a roc15 mutant showed defects in the phase-resetting of the circadian clock by light. Taken together, these data strongly suggest that the light-induced degradation of ROC15 protein is one of the triggers for resetting the circadian clock in C. reinhardtii. Our data provide not only a basis for understanding the molecular mechanisms of light-induced phase-resetting in C. reinhardtii, but also insights into the phase-resetting mechanisms of circadian clocks in plants.

Comparative analysis of kdp and ktr mutants reveals distinct roles of the potassium transporters in the model cyanobacterium Synechocystis sp. strain PCC 6803.

Photoautotrophic bacteria have developed mechanisms to maintain K(+) homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K(+) uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K(+) (Kdp). The contributions of each of these K(+) transport systems to cellular K(+) homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K(+) uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K(+) uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K(+) depletion. Correspondingly, Kdp-mediated K(+) uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K(+) required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K(+) concentration under conditions of limited K(+) in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K(+) availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K(+) levels under fluctuating environmental conditions.

Site-directed spin labeling-electron spin resonance mapping of the residues of cyanobacterial clock protein KaiA that are affected by KaiA-KaiC interaction.

The cyanobacterial clock proteins KaiA, KaiB and KaiC interact with each other to generate circadian oscillations. We have identified the residues of the KaiA homodimer affected through association with hexameric KaiC (KaiC6mer) using a spin-label-tagged KaiA C-terminal domain protein (KaiAc) and performing electron spin resonance (ESR) analysis. Cys substitution and/or the attachment of a spin label to residues located at the bottom area of the KaiAc concave surface, a KaiC-binding groove, hindered the association of KaiAc with KaiC6mer, suggesting that the groove likely mediates the interaction with KaiC6mer. The residues affected by KaiC6mer association were concentrated in the three areas: the concave surface, a lobe-like structure (a mobile lobe near the concave surface) and a region adjacent to both the concave surface and the mobile lobe. The distance between the two E254, D255, L258 and R252 residues located on the mobile lobe decreased after KaiC association, suggesting that the two mobile lobes approach each other during the interaction. Analyzing the molecular dynamics of KaiAc showed that these structural changes suggested by ESR analysis were possible. Furthermore, the analyses identified three asymmetries in KaiAc dynamic structures, which gave us a possible explanation of an asymmetric association of KaiAc with KaiC6mer.

High-throughput phenotyping of chlamydomonas swimming mutants based on nanoscale video analysis.

Studies on biflagellated algae Chlamydomonas reinhardtii mutants have resulted in significant contributions to our understanding of the functions of cilia/flagella components. However, visual inspection conducted under a microscope to screen and classify Chlamydomonas swimming requires considerable time, effort, and experience. In addition, it is likely that identification of mutants by this screening is biased toward individual cells with severe swimming defects, and mutants that swim slightly more slowly than wild-type cells may be missed by these screening methods. To systematically screen Chlamydomonas swimming mutants, we have here developed the cell-locating-with-nanoscale-accuracy (CLONA) method to identify the cell position to within 10-nm precision through the analysis of high-speed video images. Instead of analyzing the shape of the flagella, which is not always visible in images, we determine the position of Chlamydomonas cell bodies by determining the cross-correlation between a reference image and the image of the cell. From these positions, various parameters related to swimming, such as velocity and beat frequency, can be accurately estimated for each beat cycle. In the examination of wild-type and seven dynein arm mutants of Chlamydomonas, we found characteristic clustering on scatter plots of beat frequency versus swimming velocity. Using the CLONA method, we have screened 38 Chlamydomonas strains and detected believed-novel motility-deficient mutants that would be missed by visual screening. This CLONA method can automate the screening for mutants of Chlamydomonas and contribute to the elucidation of the functions of motility-associated proteins.

The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB(1-94)), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB(1-94) interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB(1-94) also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB(1-94) showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB(1-94).

Phase-dependent generation and transmission of time information by the KaiABC circadian clock oscillator through SasA-KaiC interaction in cyanobacteria.

Circadian clocks allow organisms to predict environmental changes of the day/night cycle. In the cyanobacterial circadian clock machinery, the phosphorylation level and ATPase activity of the clock protein KaiC oscillate with a period of approximately 24 h. The time information is transmitted from KaiC to the histidine kinase SasA through the SasA autophosphorylation-enhancing activity of KaiC, ultimately resulting in genome-wide transcription cycles. Here, we showed that SasA derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 has the domain structure of an orthodox histidine kinase and that its C-terminal domain, which contains a phosphorylation site at His160, is responsible for the autophosphorylation activity and the temperature- and phosphorylation state-dependent trimerization / hexamerization activity of SasA. SasA and KaiC associate through their N-terminal domains with an affinity that depends on their phosphorylation states. Furthermore, the SasA autophosphorylation-enhancing activity of KaiC requires the C-terminal ATPase catalytic site and depends on its phosphorylation state. We show that the phosphotransfer activity of SasA is essential for the generation of normal circadian gene expression in cyanobacterial cells. Numerical simulations suggest that circadian time information (free phosphorylated SasA) is released mainly by unphosphorylated KaiC during the late subjective night.

Aquaporin AqpZ is involved in cell volume regulation and sensitivity to osmotic stress in Synechocystis sp. strain PCC 6803.

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ΔaqpZ strain than the wild type. The ΔaqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ΔaqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity.

Plasma membrane aquaporin AqpZ protein is essential for glucose metabolism during photomixotrophic growth of Synechocystis sp. PCC 6803.

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity at pH values ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism.

N-terminal acetyltransferase 3 gene is essential for robust circadian rhythm of bioluminescence reporter in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a model species of algae for studies on the circadian clock. Previously, we isolated a series of mutants showing defects in the circadian rhythm of a luciferase reporter introduced into the chloroplast genome, and identified the genes responsible for the defective circadian rhythm. However, we were unable to identify the gene responsible for the defective circadian rhythm of the rhythm of chloroplast 97 (roc97) mutant because of a large genomic deletion. Here, we identified the gene responsible for the roc97 mutation through a genetic complementation study. This gene encodes a protein that is homologous to the subunit of N-terminal acetyltransferase (NAT) which catalyzes N-terminal acetylation of proteins. Our results provide the first example of involvement of the protein N-terminal acetyltransferase in the circadian rhythm.

Direct interaction between KaiA and KaiB revealed by a site-directed spin labeling electron spin resonance analysis.

In cyanobacteria, three clock proteins, KaiA, KaiB and KaiC, play essential roles in generating circadian oscillations. The interactions of these proteins change during the circadian cycle. Here, we demonstrated direct interaction between KaiA and KaiB using electron spin resonance spectroscopy. We prepared cystein (Cys)-substituted mutants of Thermosynechococcus elongatus KaiB, labeled specifically their Cys residues with spin labels and measured the ESR spectra of the labeled KaiB. We found that KaiB labeled at the 64th residue showed spectral changes in the presence of KaiA, but not in the presence of KaiC or bovine serum albumin as a negative control. KaiB labeled at the 101st residue showed no such spectral changes even in the presence of KaiA. The results suggest that KaiB interacts with KaiA in the vicinity of the 64th residue of KaiB. Further analysis demonstrated that the C-terminal clock-oscillator domain of KaiA is responsible for this interaction.

Functionally important structural elements of the cyanobacterial clock-related protein Pex.

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.

ATPase activity and its temperature compensation of the cyanobacterial clock protein KaiC.

KaiA, KaiB and KaiC constitute the circadian clock machinery in cyanobacteria. KaiC is a homohexamer; its subunit contains duplicated halves, each with a set of ATPase motifs. Here, using highly purified KaiC preparations of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 produced in Escherichia coli, we found that the N- and C-terminal domains of KaiC had extremely weak ATPase activity. ATPase activity showed temperature compensation in wild-type KaiC, but not in KaiC(S431A/T432A), a mutant that lacks two phosphorylation sites. We concluded that KaiC phosphorylation is involved in the ATPase temperature-compensation mechanism-which is probably critical to the stability of the circadian clock in cyanobacteria-and we hypothesized the following temperature-compensation mechanism: (i) The C-terminal phosphorylation sites of a KaiC hexamer subunit are phosphorylated by the C-terminal domain of an adjacent KaiC subunit; (ii) the phosphorylation suppresses the ATPase activity of the C-terminal domain; and (iii) the phosphorylated KaiC spontaneously dephosphorylates, resulting in the recover of ATPase activity.