Rhythms In Cyanobacteria Research Articles

A topological mechanism for robust and efficient global oscillations in biological networks

Long and stable timescales are often observed in complex biochemical networks, such as in emergent oscillations. How these robust dynamics persist remains unclear, given the many stochastic reactions and shorter time scales demonstrated by underlying components. We propose a topological model that produces long oscillations around the network boundary, reducing the system dynamics to a lower-dimensional current in a robust manner. Using this to model KaiC, which regulates the circadian rhythm in cyanobacteria, we compare the coherence of oscillations to that in other KaiC models. Our topological model localizes currents on the system edge, with an efficient regime of simultaneously increased precision and decreased cost. Further, we introduce a new predictor of coherence from the analysis of spectral gaps, and show that our model saturates a global thermodynamic bound. Our work presents a new mechanism and parsimonious description for robust emergent oscillations in complex biological networks.

A Logic NAND Gate for Controlling Gene Expression in a Circadian Rhythm in Cyanobacteria.

To enable circadian control of gene expression in cyanobacteria, we constructed a genetic logic gate (NAND) using orthogonal promoters via modular CRISPR interference. The NAND gates were tested in Synechococcus elongatus PCC 7942 using a fluorescent reporter. The NAND gate dynamics were characterized based on the affinity of the dCas9 complex to the output element. Upon connecting tight gene repressions with the circadian promoter (the purF gene; peak expression at dawn), inversed peak expressions were obtained as an output of the NAND gate although the retroactivities were shown in the ON and OFF states. A dark-responsive genetic element of the NAND gate was also expanded to an AND gate in S. elongatus PCC 7942. These cyanobacterial NAND and AND gates could facilitate the control of gene expressions in dynamic metabolic engineering technologies, thereby enabling the cyanobacteria to serve as biosolar cell factories.

Sketching Biological Phenomena and Mechanisms

In many fields of biology, both the phenomena to be explained and the mechanisms proposed to explain them are commonly presented in diagrams. Our interest is in how scientists construct such diagrams. Researchers begin with evidence, typically developed experimentally and presented in data graphs. To arrive at a robust diagram of the phenomenon or the mechanism, they must integrate a variety of data to construct a single, coherent representation. This process often begins as the researchers create a first sketch, and it continues over an extended period as they revise the sketch until they arrive at a diagram they find acceptable. We illustrate this process by examining the sketches developed in the course of two research projects directed at understanding the generation of circadian rhythms in cyanobacteria. One identified a new aspect of the phenomenon itself, whereas the other aimed to develop a new mechanistic account. In both cases, the research resulted in a paper in which the conclusion was presented in a diagram that the authors deemed adequate to convey it. These diagrams violate some of the normative "cognitive design principles" advanced by cognitive scientists as constraints on successful visual communication. We suggest that scientists' sketching is instead governed by norms of success that are broadly explanatory: conveying the phenomenon or mechanism.

Circadian Rhythms in Cyanobacteria.

Life on earth is subject to daily and predictable fluctuations in light intensity, temperature, and humidity created by rotation of the earth. Circadian rhythms, generated by a circadian clock, control temporal programs of cellular physiology to facilitate adaptation to daily environmental changes. Circadian rhythms are nearly ubiquitous and are found in both prokaryotic and eukaryotic organisms. Here we introduce the molecular mechanism of the circadian clock in the model cyanobacterium Synechococcus elongatus PCC 7942. We review the current understanding of the cyanobacterial clock, emphasizing recent work that has generated a more comprehensive understanding of how the circadian oscillator becomes synchronized with the external environment and how information from the oscillator is transmitted to generate rhythms of biological activity. These results have changed how we think about the clock, shifting away from a linear model to one in which the clock is viewed as an interactive network of multifunctional components that are integrated into the context of the cell in order to pace and reset the oscillator. We conclude with a discussion of how this basic timekeeping mechanism differs in other cyanobacterial species and how information gleaned from work in cyanobacteria can be translated to understanding rhythmic phenomena in other prokaryotic systems.

High-throughput and quantitative approaches for measuring circadian rhythms in cyanobacteria using bioluminescence.

The temporal measurement of a bioluminescent reporter has proven to be one of the most powerful tools for characterizing circadian rhythms in the cyanobacterium Synechococcus elongatus. Primarily, two approaches have been used to automate this process: (1) detection of cell culture bioluminescence in 96-well plates by a photomultiplier tube-based plate-cycling luminometer (TopCount Microplate Scintillation and Luminescence Counter, Perkin Elmer) and (2) detection of individual colony bioluminescence by iteratively rotating a Petri dish under a cooled CCD camera using a computer-controlled turntable. Each approach has distinct advantages. The TopCount provides a more quantitative measurement of bioluminescence, enabling the direct comparison of clock output levels among strains. The computer-controlled turntable approach has a shorter set-up time and greater throughput, making it a more powerful phenotypic screening tool. While the latter approach is extremely useful, only a few labs have been able to build such an apparatus because of technical hurdles involved in coordinating and controlling both the camera and the turntable, and in processing the resulting images. This protocol provides instructions on how to construct, use, and process data from a computer-controlled turntable to measure the temporal changes in bioluminescence of individual cyanobacterial colonies. Furthermore, we describe how to prepare samples for use with the TopCount to minimize experimental noise and generate meaningful quantitative measurements of clock output levels for advanced analysis.

Intersubunit communications within KaiC hexamers contribute the robust rhythmicity of the cyanobacterial circadian clock.

Circadian rhythms, endogenous oscillations with periods of ~24 h, are found in many organisms, and they enhance fitness in alternating day/night environments. In cyanobacteria, three clock proteins, KaiA, KaiB, and KaiC, control the timekeeping mechanism. KaiC, the central component of the system, is a hexameric ATPase that also has autokinase and autophosphatase activities. It has been assumed that KaiC’s hexameric structure was critical for regulation of the circadian clock; however, the underlying molecular mechanism of such regulation has remained unclear. Recently, we elucidated the regulation of KaiC’s activities by its phosphorylation state, in the context of its hexameric structure. We found that local interactions at subunit interfaces regulate KaiC’s activities by coupling the nucleotide-binding states. We also discovered the mechanism of regulation by intersubunit communication in KaiC hexamers. Our observations suggest that intersubunit communication precisely synchronizes KaiC subunits to avoid dephasing, and contributes to the robustness of circadian rhythms in cyanobacteria [Kitayama, Y. et al. Nat. Commun. 4:2897 doi: 10.1038/ncomms3897 (2013)].

1P279 時計タンパク質による概日リズムの同調機構(25. 非平衡・生体リズム,ポスター,第52回日本生物物理学会年会(2014年度))

1P279 時計タンパク質による概日リズムの同調機構(25. 非平衡・生体リズム,ポスター,第52回日本生物物理学会年会(2014年度))

KaiC intersubunit communication facilitates robustness of circadian rhythms in cyanobacteria

The cyanobacterial circadian clock is the only model clock to have been reconstituted in vitro. KaiC, the central clock component, is a homohexameric ATPase with autokinase and autophosphatase activities. Changes in phosphorylation state have been proposed to switch KaiC’s activity between autokinase and autophosphatase. Here we analyse the molecular mechanism underlying the regulation of KaiC’s activity, in the context of its hexameric structure. We reconstitute KaiC hexamers containing different variant protomers, and measure their autophosphatase and autokinase activities. We identify two types of regulatory mechanisms with distinct functions. First, local interactions between adjacent phosphorylation sites regulate KaiC’s activities, coupling the ATPase and nucleotide-binding states at subunit interfaces of the CII domain. Second, the phosphorylation states of the protomers affect the overall activity of KaiC hexamers via intersubunit communication. Our findings indicate that intra-hexameric interactions play an important role in sustaining robust circadian rhythmicity.

Hierarchy of models: From qualitative to quantitative analysis of circadian rhythms in cyanobacteria

A hierarchy of models, ranging from high to lower levels of abstraction, is proposed to construct "minimal" but predictive and explanatory models of biological systems. Three hierarchical levels will be considered: Boolean networks, piecewise affine differential (PWA) equations, and a class of continuous, ordinary, differential equations' models derived from the PWA model. This hierarchy provides different levels of approximation of the biological system and, crucially, allows the use of theoretical tools to more exactly analyze and understand the mechanisms of the system. The Kai ABC oscillator, which is at the core of the cyanobacterial circadian rhythm, is analyzed as a case study, showing how several fundamental properties-order of oscillations, synchronization when mixing oscillating samples, structural robustness, and entrainment by external cues-can be obtained from basic mechanisms.

Elucidating gene signatures that control the circadian rhythm in cyanobacteria using bioinformatics methods

Background The circadian rhythm, or biological “clock,” allows the organism to anticipate and prepare for the changes in the physical environment. Studies have found that the internal clock consists of an array of genes and the protein products they encode, which regulate various physiological processes throughout the body. Cyanothece sp. ATCC 51142 is an organism that has both photosynthetic (producing oxygen) and nitrogen fixing ability. It has developed a temporal regulation in which N2 fixation and photosynthesis occur at different times throughout a diurnal cycle with very high levels of CO2 fixation during the light and high levels of N2 fixation in the dark. The mechanisms underlying the circadian rhythm and the signature genes elucidating this mechanism are addressed in this research.

Expression, purification, crystallization and preliminary X-ray analysis of the KaiC-like protein PH0187 from the hyperthermophilic archaeonPyrococcus horikoshiiOT3

KaiC is the central protein in the circadian rhythm in cyanobacteria. The 28 kDa KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of PH0187 were obtained using a reservoir solution consisting of 1.0 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic pH 5.3 (the final pH value of the reservoir solution was 4.8) and diffracted X-rays to 2.75 Å resolution. The crystal of PH0187 belonged to space group P6(3)22, with unit-cell parameters a=b=239.1, c=106.5 Å. The crystal contained four PH0187 molecules in the asymmetric unit.

Dual KaiC-based oscillations constitute the circadian system of cyanobacteria

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB, and KaiC proteins are essential for the generation of circadian rhythms. Both in vivo and in vitro, phosphorylation of KaiC is regulated positively by KaiA and negatively by KaiB and shows circadian rhythmicity. The autonomous circadian cycling of KaiC phosphorylation is thought to be the basic pacemaker of the circadian clock and to control genome-wide gene expression in cyanobacteria. In this study, we found that temperature-compensated circadian oscillations of gene expression persisted even when KaiC was arrested in the phosphorylated state due to kaiA overexpression. Moreover, two phosphorylation mutants showed transcriptional oscillation with a long period. In kaiA-overexpressing and phosphorylation-deficient strains, KaiC oscillated and transient overexpression of phosphorylation-deficient kaiC reset the phase of the rhythm. These results suggest that transcription- and translation-based oscillations in KaiC abundance are also important for circadian rhythm generation in cyanobacteria. Furthermore, at low temperature, cyanobacteria can show circadian rhythms only when both the KaiC phosphorylation cycle and the transcription and translation cycle are intact. Our findings indicate that multiple coupled oscillatory systems based on the biochemical properties of KaiC are important to maintain robust and precise circadian rhythms in cyanobacteria.

The complete sequence and functional analysis of pANL, the large plasmid of the unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942

Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin–antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.

1P-299 多重分子修飾による記憶、ヒステリシス、振動とそのシアノバクテリアの概日リズムへの応用(数理生物学,第46回日本生物物理学会年会)

1P-299 多重分子修飾による記憶、ヒステリシス、振動とそのシアノバクテリアの概日リズムへの応用(数理生物学,第46回日本生物物理学会年会)

A sequential program of dual phosphorylation of KaiC as a basis for circadian rhythm in cyanobacteria

The circadian phosphorylation cycle of the cyanobacterial clock protein KaiC has been reconstituted in vitro. The phosphorylation profiles of two phosphorylation sites in KaiC, serine 431 (S431) and threonine 432 (T432), revealed that the phosphorylation cycle contained four steps: (i) T432 phosphorylation; (ii) S431 phosphorylation to generate the double-phosphorylated form of KaiC; (iii) T432 dephosphorylation; and (iv) S431 dephosphorylation. We then examined the effects of mutations introduced at one KaiC phosphorylation site on the intact phosphorylation site. We found that the product of each step in the phosphorylation cycle regulated the reaction in the next step, and that double phosphorylation converted KaiC from an autokinase to an autophosphatase, whereas complete dephosphorylation had the opposite effect. These mechanisms serve as the basis for cyanobacterial circadian rhythm generation. We also found that associations among KaiA, KaiB, and KaiC result from S431 phosphorylation, and these interactions would maintain the amplitude of the rhythm.

Monomer-shuffling and allosteric transition in KaiC circadian oscillation.

Circadian rhythms in living organisms have long been attributed solely to a transcription-translation loop comprising a negative or positive feedback. The rhythms in cyanobacteria are known to be modulated by kaiC, kaiA and kaiB genes. It was recently shown, however, that their product proteins KaiC, KaiA and KaiB are sufficient to reconstitute the circadian rhythm in the phosphorylation level of KaiC in vitro. It has since been unclear why such an oscillatory behavior can occur in the absence of the apparent transcription-translation feedback. In the meantime, it has been reported that the monomer exchange between KaiC hexamers occurs in a phosphorylation-dependent manner, which suggests that the monomer shuffling is also involved in the circadian rhythm (H. Kageyama et al., Mol. Cell, 23, 161 (2006)). To further clarify the role of the monomer shuffling, we have performed a computational modeling of interactions among Kai proteins assuming the allosteric transition of KaiC hexamer as well as the monomer shuffling. The results show that the existence of both monomer shuffling and allosteric transition can synchronize the phosphorylation level of the KaiC hexamers, and stabilizes its oscillation.

A Mathematical Model for the Kai-Protein–Based Chemical Oscillator and Clock Gene Expression Rhythms in Cyanobacteria

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.

Integrating the Circadian Oscillator into the Life of the Cyanobacterial Cell

In two decades, the study of circadian rhythms in cyanobacteria has gone from observations of phenomena in intractable species to the development of a model organism for mechanistic study, atomic-resolution structures of components, and reconstitution of a circadian biochemical oscillation in vitro. With sophisticated biochemical, biophysical, genetic, and genomic tools in place, the circadian clock of the unicellular cyanobacterium Synechococcus elongatus is poised to be the first for which a systems-level understanding can be achieved.

A Model for the Circadian Rhythm of Cyanobacteria that Maintains Oscillation without Gene Expression

An intriguing property of the cyanobacterial circadian clock is that endogenous rhythm persists when protein abundances are kept constant either in the presence of translation and transcription inhibitors or in the constant dark condition. Here we propose a regulatory mechanism of KaiC phosphorylation for the generation of circadian oscillations in cyanobacteria. In the model, clock proteins KaiA and KaiB are assumed to have multiple states, regulating the KaiC phosphorylation process. The model can explain 1), the sustained oscillation of gene expression and protein abundance when the expression of the kaiBC gene is regulated by KaiC protein, and 2), the sustained oscillation of phosphorylated KaiC when transcription and translation processes are inhibited and total protein abundance is fixed. Results of this work suggest that KaiA and KaiB strengthen the nonlinearity of KaiC phosphorylation, thereby promoting the circadian rhythm in cyanobacteria.

A KaiC-associating SasA–RpaA two-component regulatory system as a major circadian timing mediator in cyanobacteria

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.