Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria.

To allow continuous monitoring of the circadian clock in cyanobacteria, we previously created a reporter strain (AMC149) of Synechococcus sp. strain PCC 7942 in which the promoter of the psbAI gene was fused to Vibrio harveyi luciferase structural genes (luxAB) and integrated into the chromosome. Northern (RNA) hybridization and immunoblot analyses were performed to examine changes in abundance of the luxAB mRNA, the native psbAI mRNA, and the luciferase protein to determine whether bioluminescence is an accurate reporter of psbAI promoter activity in AMC149. Under constant light conditions, the mRNA abundances of both luxAB and psbAI oscillated with a period of approximately 24 h for at least 2 days. The expression of these two genes following the same pattern: both mRNAs peaked in the subjective morning, and their troughs occurred near the end of the subjective night. The amount of luciferase protein also oscillated with a period of approximately 24 h, and the protein rhythm is in phase with the bioluminescence rhythm. The rhythm of the luciferase mRNA phase-leads the rhythms of luciferase protein and in vivo bioluminescence by several hours. Comparable results were obtained with a short-period mutant of AMC149. Together, these results indicate that the bioluminescence rhythm in AMC149 is due primarily to circadian oscillation of psbAI promoter activity in this cyanobacterium.     

Characterization of plant circadian rhythms by employing Arabidopsis cultured cells with bioluminescence reporters

Recent intensive studies have begun to shed light on the molecular mechanisms underlying the plant circadian clock in Arabidopsis thaliana. During the course of these previous studies, the most powerful technique, elegantly adopted, was a real-time bioluminescence monitoring system of circadian rhythms in intact plants carrying a luciferase (LUC) fusion transgene. We previously demonstrated that Arabidopsis cultured cells also retain an ability to generate circadian rhythms, at least partly. To further improve the cultured cell system for studies on circadian rhythms, here we adopted a bioluminescence monitoring system by establishing the cell lines carrying appropriate reporter genes, namely, CCA1::LUC and APRR1::LUC, with which CCA1 (CIRCADIAN CLOCK-ASSOCIATED1) and APRR1 (or TOC1) (ARABIDOPSIS PSEUDO-RESPONSE REGULATORS1 or TIMING OF CAB EXPRESSION1) are believed to be the components of the central oscillator. We report the results that consistently supported the view that the established cell lines, equipped with such bioluminescence reporters, might provide us with an advantageous means to characterize the plant circadian clock.     

Circadian rhythms in the thermophilic cyanobacterium Thermosynechococcus elongatus: compensation of period length over a wide temperature range

Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies. We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium T. elongatus BP-1 that uses a bacterial luciferase gene set (Xl luxAB) derived from Xenorhabdus luminescens as a bioluminescence reporter gene. A promoter region of the psbA1 gene of T. elongatus was fused to the Xl luxAB gene set and inserted into a specific targeting site in the genome of T. elongatus. The bioluminescence from the cells of the psbA1-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes. The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature. The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60 degrees C). Theses results indicate that T. elongatus has the circadian clock that is widely temperature compensated.     

Circadian rhythm of the cyanobacterium Synechocystis sp. strain PCC 6803 in the dark.

The cyanobacterium Synechocystis sp. strain PCC 6803 exhibited circadian rhythms in complete darkness. To monitor a circadian rhythm of the Synechocystis cells in darkness, we introduced a PdnaK1::luxAB gene fusion (S. Aoki, T. Kondo, and M. Ishiura, J. Bacteriol. 177:5606-5611, 1995), which was composed of a promoter region of the Synechocystis dnaK1 gene and a promoterless bacterial luciferase luxAB gene set, as a reporter into the chromosome of a dark-adapted Synechocystis strain. The resulting dnaK1-reporting strain showed bioluminescence rhythms with a period of 25 h (on agar medium supplemented with 5 mM glucose) for at least 7 days in darkness. The rhythms were reset by 12-h-light-12-h-dark cycles, and the period of the rhythms was temperature compensated for between 24 and 31 degrees C. These results indicate that light is not necessary for the oscillation of the circadian clock in Synechocystis.     

Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.     

Improvement of the bioluminescence reporter system for real-time monitoring of circadian rhythms in the cyanobacterium Synechocystis sp. strain PCC 6803

Circadian rhythm is a self-sustaining oscillation whose period length coincides with the 24-hour day-night cycle. A powerful tool for circadian clock research is the real-time automated bioluminescence monitoring system in which a promoter region of a clock-controlled gene is fused to a luciferase reporter gene and rhythmic regulation of the promoter activity is monitored as bioluminescence. In the present study, we greatly improved the bioluminescence reporter system in the cyanobacterium Synechocystis sp. strain PCC 6803. We fused an 805-bp promoter region of the dnaK gene seamlessly to the luxA coding sequence and integrated the P(dnaK)::luxAB fusion gene into a specific intergenic region of the Synechocystis genome (targeting site 1). The resulting new reporter strain, PdnaK::luxAB(-), showed 12 times the bioluminescence intensity of the standard reporter strain, CFC2. Furthermore, we generated strain PdnaK::luxAB(+), in which the P(dnaK)::luxAB fusion gene and the selection-marker spectinomycin resistance gene are transcribed in opposite directions. The PdnaK::luxAB(+) strain showed 19 times the bioluminescence intensity of strain CFC2. The procedures used to increase the bioluminescence intensity are especially useful for bioluminescence monitoring of genes with low promoter activity. In addition, these reporter constructs facilitate bioluminescence monitoring of any gene because the promoter fragments they contain can easily be replaced by digestion with unique restriction enzymes. They would therefore contribute to a genome-wide analysis of gene expression in Synechocystis.     

A compact multi-channel apparatus for automated real-time monitoring of bioluminescence

We have developed a multi-channel apparatus for automated monitoring of bioluminescence in real time. We designed this apparatus to be compact (230 mm wide, 600 mm deep, and 227.5 mm high) so that it can be operated in a relatively small commercially-available incubator. The apparatus can process 20 samples at maximum in a single run, providing enough processibility in small-scale experiments. We verified the reliability and sensitivity of the apparatus by observing circadian bioluminescence rhythms over one week from a bioluminescent reporter strain (E9) of the cyanobacterium Synechococcus sp. strain PCC 7942 [Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S., Johnson, C.H., Kondo, T., Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria, Science, 281 (1998) 1519-1523]. Our apparatus allows flexible experimental designs and will be effectively used for the studies of gene expression in various purposes.     

Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803.

The expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity. A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome. Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light. The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light. The period of the rhythm was temperature compensated between 25 and 35 degrees C. Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms. Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp. strain PCC 6803.     

A portable integrated automatic apparatus for the real-time monitoring of bioluminescence in plants

Real-time monitoring of gene expression by a bioluminescence reporter gene is a powerful method for large-scale, detailed analysis of gene expression in living cells and large-scale screening of mutants. We have developed a portable, compact, integrated automatic bioluminescence-monitoring apparatus that can continuously monitor 960 individual plant seedlings or micro-organism colonies under uniform light conditions at temperatures up to 50 °C. The apparatus gave reproducible and reliable results for both bioluminescence photon counts and period length of bioluminescence rhythms of Arabidopsis reporter strain. Using the apparatus, we measured bioluminescence rhythms in the thermophilic cyanobacterium Thermosynechococcus at temperature up to 43 °C. We also monitored the expression of the flowering regulator gene CONSTANS in Arabidopsis as bioluminescence in high time resolution under different photoperiodic conditions. The high-throughput bioluminescence-monitoring apparatus developed here is a powerful tool for real-time monitoring of gene expression and gene function.     

A promoter-trap vector for clock-controlled genes in the cyanobacterium Synechocystis sp. PCC 6803

We constructed a promoter-trap vector pPT6803-1 to isolate circadian clock-controlled promoters in the cyanobacterium Synechocystis sp. strain PCC 6803. The vector contains a promoterless luciferase gene set (luxAB) from Vibrio harveyi that is targeted to a specific site of the Synechocystis genome as a reporter for gene expression. A library was constructed in pPT6803-1 by introducing the genomic DNA fragments upstream of luxAB to transform Synechocystis cells. Of approximately 10,000 Synechocystis transformants, at least 55 (#1-55) showed circadian rhythms of bioluminescence under continuous illumination. Clones #19, #22, and #26 exhibited obviously different waveforms of bioluminescence from each other. Deletion analysis and primer extension experiments mapped the promoters for the clpP, slr1634, and rbpP genes that are responsible for bioluminescence from #19, #22, and #26, respectively.     

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides

Circadian rhythms are known to exist in all groups of eukaryotic organisms as well as oxygenic photosynthetic bacteria, cyanobacteria. However, little information is available regarding the existence of rhythmic behaviors in prokaryotes other than cyanobacteria. Here we report biological rhythms of gene expression in a purple bacterium Rhodobacter sphaeroides by using a luciferase reporter gene system. Self-bioluminescent strains of Rb. sphaeroides were constructed, which produced a bacterial luciferase and its substrate, a long chain fatty aldehyde, to sustain the luminescence reaction. After being subjected to a temperature or light entrainment regime, the reporter strains with the luciferase genes driven by an upstream endogenous promoter expressed self-sustained rhythmicity in the constant free-running period. The rhythms were controlled by oxygen and exhibited a circadian period of 20.5 h under aerobic conditions and an ultradian period of 10.6-12.7 h under anaerobic conditions. The data suggest a novel endogenous oscillation mechanism in purple photosynthetic bacteria. Elucidation of the clock-like behavior in purple bacteria has implications in understanding the origin and evolution of circadian rhythms.     

A compact multi-channel apparatus for automated real-time monitoring of bioluminescence

We have developed a multi-channel apparatus for automated monitoring of bioluminescence in real time. We designed this apparatus to be compact (230 mm wide, 600 mm deep, and 227.5 mm high) so that it can be operated in a relatively small commercially-available incubator. The apparatus can process 20 samples at maximum in a single run, providing enough processibility in small-scale experiments. We verified the reliability and sensitivity of the apparatus by observing circadian bioluminescence rhythms over one week from a bioluminescent reporter strain (E9) of the cyanobacterium Synechococcus sp. strain PCC 7942 [Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C.R., Tanabe, A., Golden, S.S., Johnson, C.H., Kondo, T., Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria, Science, 281 (1998) 1519–1523]. Our apparatus allows flexible experimental designs and will be effectively used for the studies of gene expression in various purposes.     

Characterization of transcriptional oscillation of an Arabidopsis homolog of PnC401 related to photoperiodic induction of flowering in Pharbitis nil

AtC401 is an Arabidopsis homolog of PnC401 that is related to photoperiodic induction of flowering in Pharbitis nil. These genes show free-running rhythms. To study the free-running rhythm of AtC401, we fused a firefly luciferase reporter to the AtC401 promoter and transformed it into Arabidopsis plants. The observed bioluminescence oscillated under continuous light and continuous dark only with sucrose supplementation. The free-running period of bioluminescence was temperature-compensated between 22 degrees C and 30 degrees C. Light-pulse experiments under continuous darkness produced a phase-response curve typical of circadian rhythms. We conclude that rhythmic expression of AtC401 is controlled by a circadian oscillator.     

Global orchestration of gene expression by the biological clock of cyanobacteria

Prokaryotic cyanobacteria express robust circadian (daily) rhythms under the control of a central clock. Recent studies shed light on the mechanisms governing circadian rhythms in cyanobacteria and highlight key differences between prokaryotic and eukaryotic clocks.     

Conserved expression profiles of circadian clock-related genes in two Lemna species showing long-day and short-day photoperiodic flowering responses

The Lemna genus is a group of monocotyledonous plants with tiny, floating bodies. Lemna gibba G3 and L. paucicostata 6746 were once intensively analyzed for physiological timing systems of photoperiodic flowering and circadian rhythms since they showed obligatory and sensitive photoperiodic responses of a long-day and a short-day plant, respectively. We attempted to approach the divergence of biological timing systems at the molecular level using these plants. We first employed molecular techniques to study their circadian clock systems. We developed a convenient bioluminescent reporter system to monitor the circadian rhythms of Lemna plants. As in Arabidopsis, the Arabidopsis CCA1 promoter produced circadian expression in Lemna plants, though the phases and the sustainability of bioluminescence rhythms were somewhat diverged between them. Lemna homologs of the Arabidopsis clock-related genes LHY/CCA1, GI, ELF3 and PRRs were then isolated as candidates for clock-related genes in these plants. These genes showed rhythmic expression profiles that were basically similar to those of Arabidopsis under light-dark conditions. Results from co-transfection assays using the bioluminescence reporter and overexpression effectors suggested that the LHY and GI homologs of Lemna can function in the circadian clock system like the counterparts of Arabidopsis. All these results suggested that the frame of the circadian clock appeared to be conserved not only between the two Lemna plants but also between monocotyledons and dicotyledons. However, divergence of gene numbers and expression profiles for LHY/CCA1 homologs were found between Lemna, rice and Arabidopsis, suggesting that some modification of clock-related components occurred through their evolution.     

Functional conservation of clock-related genes in flowering plants: overexpression and RNA interference analyses of the circadian rhythm in the monocotyledon Lemna gibba

Circadian rhythms are found in organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCIATED1, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1, GIGANTEA (GI), and EARLY FLOWERING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYH1, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis.     

The circadian clock of cyanobacteria

A circadian clock, with physiological characteristics similar to those of eukaryotes, functions in the photosynthetic prokaryote, cyanobacteria. The molecular mechanism of this clock has been efficiently dissected using a luciferase reporter gene that reports the status of the clock. A circadian clock gene cluster, kaiABC, has been cloned via rhythm mutants of cyanobacterium, Synechococcus, and many clock mutations mapped to the three kai genes. Although kai genes do not share any homology with clock genes so far identified in eukaryotes, analysis of their expression suggests that a negative feedback control of kaiC expression by KaiC generates the circadian oscillation and that KaiA functions as a positive factor to sustain this oscillation. BioEssays 22:10-15, 2000.