Journal of Bacteriology, August 2004, p . 4972-4977, Vol . 186, No . 15

Circadian Rhythms in the Thermophilic Cyanobacterium Thermosynechococcus elongatus: Compensation of Period Length over a Wide Temperature Range

Kiyoshi Onai,^1,2, Megumi Morishita,^1, Shino Itoh,^1,3 Kazuhisa Okamoto,^1,4 and Masahiro Ishiura^1,2,3,4*

Center for Gene Research,¹ Bio-Oriented Technology Research Advancement Institution,² Division of Biological Science, Graduate School of Science, Nagoya University, Furo, Chikusa, Nagoya 464-8602,³ Aichi Science and Technology Foundation, Naka, Nagoya 460-0002, Japan⁴

Received 2 February 2004/ Accepted 5 May 2004

 
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°C) . Theses results indicate that T . elongatus has the circadian clock that is widely temperature compensated .

 
Circadian rhythms are endogenous daily fluctuations in physiological activities that have the following three characteristics: they are sustained under constant conditions with a period length of about 24 h; the phase of the rhythm is reset by environmental cues such as light-dark (LD) cycles and low-high temperature cycles; and the period of the rhythm is almost constant at physiological temperatures (5, 26) . Thus far, circadian rhythms have been observed only in mesophilic organisms; we have found no reports of circadian rhythms in thermophilic organisms .

The clock gene cluster kaiABC generates circadian rhythms in the mesophilic cyanobacterium Synechococcus sp . strain PCC 7942 (hereafter called Synechococcus) (8) . KaiA and KaiC proteins regulate the transcription of the kaiBC operon positively and negatively, respectively, and this feedback regulation is important for generating circadian oscillations (8) . Kai proteins interact with each other in all possible combinations (9), and KaiA enhances the phosphorylation of KaiC (10, 28, 29) . These mesophilic clock proteins are unstable, however, and their structure and function, their mutual interactions, and their reactions with their substrates in the circadian feedback processes remain unknown .

The thermophilic cyanobacteria Thermosynechococcus elongatus (30) and T . vulcanus (13), which were isolated from a Japanese hot spring, grow optimally at ca . 57°C, and their proteins are highly stable . The proteins can be easily purified, characterized for biochemical and biophysical properties, and crystallized . The photosynthetic reaction center protein complexes of photosystems I and II have been X-ray analyzed in T . elongatus (11, 31) and T . vulcanus (12) . Recently, we cloned the gene cluster kaiABC from both species (7; T . Uzumaki and M . Ishiura, unpublished data), and both showed high homology with the corresponding Synechococcus gene (8) . In T . elongatus, we determined the three-dimensional structure of KaiC by single-particle analysis of the purified protein by using electron microscopy (7), as well as the X-ray crystal structure of the C-terminal clock-oscillator domain of KaiA (28) .

We recently established efficient procedures for gene transfer and manipulation in T . elongatus and T . vulcanus (20) . We report here the development of a real-time bioluminescence monitoring system in T . elongatus, and we demonstrate circadian rhythms in a bioluminescent transgenic strain of T . elongatus in a wide range of ambient temperatures (30 to 60°C) .

 
Bacterial strains, culture conditions, and manipulation of DNA. The wild-type cells of T . elongatus BP-1 (30) were grown at 55°C under constant light from white fluorescent lamps at 50 µmol m^–2 s^–1 (hereafter called LL conditions) in BG-11 liquid medium (hereafter called liquid BG-11 [6]) and on BG-11 agar medium (hereafter called solid BG-11) that contained 1.5% Bacto Agar (Nippon BD, Tokyo, Japan) . To permit selection and maintenance of kanamycin-resistant (Km^r) transformants, liquid BG-11 and solid BG-11 were supplemented with kanamycin at final concentrations of 100 and 75 µg/ml, respectively . Escherichia coli strain DH5 (Takara, Kusatsu, Japan) was maintained at 37°C with or without 50 µg of ampicillin/ml in Luria-Bertani broth (LB) liquid medium and on LB solid medium containing 1.5% agar in LB medium (23) .

We performed DNA manipulations and sequencing by standard techniques (2, 23) and confirmed the nucleotide sequences of plasmid constructs and the subclones of PCR products by sequencing . We prepared plasmid DNAs by standard methods (23) .

Construction of a reporter plasmid and a reporter strain of T . elongatus. We amplified a 0.46-kb promoter region of the psbA1 gene (p_psbA1) of T . elongatus (17; K . Onai and M . Ishiura, unpublished results) by PCR with the genomic DNA of wild-type cells as a template and the primer set psbA1F1 (5'-TTCAGCACCCCAGAGATTTTCGGCAACGGC-3') and psbA1R1 M (5'-GGTCATATGCGTGATAAGTCCAAATATATT-3'; the NdeI site is underlined, and the CAT sequence complementary to the ATG initiation codon of the psbA1 gene is in italics) and subcloned it into pT7Blue-T (Novagen, Madison, Wis.), giving pT7BT/p_psbA1 . We amplified a 2.2-kb fragment carrying a luxAB gene set derived from Xenorhabdus luminescens strain ATCC 29999 (Xl luxAB; GenBank/EMBL/DDBJ accession no. M57416) by PCR with plasmid Xlux/pT7-3 (27) as a template and the primer set Xlux-F1 M (5'-CCATATGAAATTTGGAAACTTTTTGC-3'; the NdeI site is underlined, and the ATG initiation codon of the luxA gene is in italics) and Xlux-R1 M (5'-TTCTAGAAAGCTGCTGCTTTGTTGGC-3'; the XbaI site is underlined) and subcloned it into pT7Blue-T, yielding pT7BT/Xl luxAB . We excised the Xl luxAB gene segment from pT7BT/Xl luxAB as a 2.2-kb NdeI-XbaI fragment and ligated it with a larger NdeI-XbaI fragment of pT7BT/p_psbA1, producing pT7BT/p_psbA1::Xl luxAB . We excised a p_psbA1::Xl luxAB gene fusion segment from pT7BT/p_psbA1::Xl luxAB as a 2.7-kb BamHI-XbaI fragment, blunted it at the ends of the fragment by a filling-in reaction with Klenow fragment (Takara), and ligated it into the StuI site of the targeting plasmid vector pTS2_Te/km_Te (20) in the opposite direction to a Km^r gene cassette (P_cpcC::km_Te [20]), yielding pTS2_Te/PSBA1 (Fig. 1) . pTS2_Te/km_Te enabled us to transfer exogenous DNA fragments to a specific targeting site (TS2_Te) in the T . elongatus genome by a double crossover via homologous recombination with the P_cpcC::km_Te gene as a selective marker (20) .

 
We transformed the wild-type cells of T . elongatus with pTS2_Te/PSBA1 (a reporter vector) and pTS2_Te/km_Te (a control vector) by natural transformation and selected the resulting Km^r transformants as colonies as described previously (20) . Using the genomic DNA as a template and the primer set eTS2-F4 (5'-GCAATGCCCACAACTTCCCCCTCG-3') and eTS2-R3 (5'-TGGTGGGCTTAGTGATTGAGCGTA-3') (Fig . 1 [20]), we confirmed by PCR that the constructs had been inserted into the TS2_Te in the T . elongatus genome as expected . We assigned the name A073 to one of the Km^r transformants obtained by transformation with the control vector pTS2_Te/km_Te and used it as a control strain . We assigned the names A197 to A205 to nine genetically independent Km^r transformants obtained by transformation with the reporter vector pTS2_Te/PSBA1 . The growth and pigmentation of the Km^r transformants seemed normal .

Assay of bioluminescence in T . elongatus. At temperatures below 41°C, we monitored the bioluminescence rhythms from a single colony grown on solid BG-11 . At temperatures 55 and 60°C, we monitored the rhythms from liquid cultures because we could not detect bioluminescence from colonies grown at over 43°C, probably because X . luminescence luciferase was unstable at such high temperatures .

We assayed the bioluminescence from cells grown on solid BG-11 as follows . Cells were grown to colonies on solid BG-11 at 52°C for 3 days under LL conditions . Then 6-mm agar plugs carrying a single colony were punched out with a glass tube, and each plug was transferred to a well in a 96-well microplate (CulturePlate-96; Perkin-Elmer Life Sciences Japan, Tokyo, Japan) . Next, 20-µl aliquots of n-decanal (Sigma Japan, Tokyo) dissolved in salad oil (1% [vol/vol]; Nissin Foods, Tokyo, Japan) were added to the empty wells adjacent to the filled wells, and the plates were sealed with a plate seal (Perkin-Elmer Life Sciences Japan) so that a constant vapor pressure would be maintained . To synchronize the circadian clock of the cells, we placed the plates in the dark for 12 h at 30, 35, or 41°C (dark conditions) . After that, we returned the plates to LL conditions at their respective temperatures . The bioluminescence from each well carrying a colony was measured continuously by an automated bioluminescence-monitoring apparatus with photomultiplier tubes as photon detectors (K . Okamoto, K . Onai, T . Furusawa, and M . Ishiura, unpublished data) . The apparatus is composed of three units: (i) a platform integrated with a conveying system on which 10 sample plates are set under uniform light conditions and rotated through all plate positions once in each measuring cycle; (ii) a unit that measures the bioluminescence from each well of the plate; and (iii) an analyzing computer that collects and analyzes bioluminescence data automatically . Because we separated the temperature-sensitive controlling unit from the other units, the apparatus can measure bioluminescence at up to 50°C . The measurements were made for 3 s in the dark after the cells were exposed to 180 s of darkness to permit the decay of chlorophyll fluorescence, and then the plates were returned to LL conditions . This measuring process was repeated automatically every 1 to 2 h . The 183 s of darkness did not affect circadian rhythm (data not shown) .

We assayed the bioluminescence from cells grown in liquid medium as follows . We grew the cells to a stationary phase (2 x 10⁹ cells/ml) in liquid BG-11 at 55 or 60°C under LL conditions with shaking at 180 rpm, incubated them in the dark for 12 h to synchronize the circadian clock under the same conditions as those in LL conditions, except for illumination, and then returned them to LL conditions . Every 3 h, we removed 500-µl from each LL culture, transferred it into a 1.5-ml microcentrifuge tube, and removed two 150-µl aliquots . One aliquot was used to optically determine cell density (1 optical density at 730 nm unit corresponded to a cell density of 10⁹ cells/ml in our experimental conditions) . The other was used to measure bioluminescence, as follows . The culture was mixed with 10 µl of 1% n-decanal and transferred into wells on plates, and the plates were sealed as described above and incubated for 1 h at 30°C under LL conditions to allow de novo synthesis of active luciferase . The bioluminescence was then measured for 3 s at 30°C in the dark by the monitoring apparatus described above . The 1-h incubation at 30°C did not affect circadian rhythms (data not shown) .

We analyzed bioluminescence data by the visual-inspection-peak (VIP) method with linear regression (22) or the cosinor method (19) by using the RAP program (K . Okamoto, K . Onai, and M . Ishiura, unpublished data) . The RAP program does all of the following in real time: (i) displays the bioluminescence time course on a monitor, (ii) records time series data, (iii) analyzes high-throughput bioluminescence data, (iv) displays the analyzed results on a monitor, (v) performs statistical analysis of the analyzed results, and (vi) prints out the time course and the analyzed results during monitoring . The VIP method recognizes rhythm peaks defined as the middle time point between the last time point where a value increases continuously for more than three time points and the first time point at which it decreases continuously for more than three time points . The period length and peak phase of rhythms are calculated from the intervals between the adjacent two peaks of rhythms by the linear regression method . The cosinor method (19) is based on Fourier analysis .

Northern blot analysis. Cells of the psbA1-reporting strains of T . elongatus were grown on solid BG-11 in 90-mm dishes at 52°C for 3 days under LL conditions, placed in the dark for 12 h at 52°C to synchronize the circadian clock, and then returned to LL conditions at 52°C . At 3-h intervals, we harvested cells under LL conditions by washing each dish with 3 ml of liquid BG-11 and centrifuging the wash at 3,500 x g for 2 min at 4°C . The cells were resuspended in 100 µl of 10 mM EDTA (pH 8.0) and immediately frozen in liquid nitrogen . Total RNA was extracted from the frozen cells as previously described (15) . RNA was separated on 1.2% agarose gels containing formaldehyde by agarose gel electrophoresis, transferred to positive-charged nylon membranes (Hybond N+; Amersham Biosciences, Tokyo), and hybridized with [-³²P]dCTP-labeled psbA1-specific probe (nucleotides 1,112 to 1,592 under accession no. D14325) by standard techniques (2, 23) . Signals from hybridized bands were detected and quantified with a Bio-Image Analyzer (BAS2000; Fuji Photo Film Co., Ltd., Tokyo, Japan) . We normalized quantified signals against densities of ethidium bromide-stained rRNAs and analyzed the densitometric data by the cosinor method (19) by using the RAP program .

 
Bioluminescence rhythms observed in a psbA1 reporting strain of T . elongatus. In a previous study, we monitored gene expression continuously as bioluminescence in the mesophilic cyanobacteria Synechococcus (14) and Synechocystis sp . strain PCC 6803 (hereafter called Synechocystis) (1) . We used the luxAB gene set derived from Vibrio harveyi as a reporter gene at temperatures of 30°C . Because T . elongatus grows better at higher temperatures, we used as a reporter a gene set derived from X . luminescence (Xl luxAB) that encodes a thermostable luciferase . The Xl luxAB gene set encodes two different luciferase subunits—LuxA and LuxB—and Xl luciferase is more thermostable than the V . harveyi luciferase (27) . We obtained nine independent transformants of a psbA1-reporting strain (A197 to A205) that carried the p_psbA1::Xl luxAB gene fusion inserted into the targeting site TS2_Te in the genome and examined their bioluminescence under LL conditions at 41°C after an exposure to 12 h of darkness to synchronize the circadian clock .

Under LL conditions, the bioluminescence from each transformant oscillated rhythmically with a period length of 24.8 ± 0.3 h, and the phase of the rhythms was 23.2 ± 0.6 h after exposure to LL conditions (Table 1 and Fig . 2) . The rhythms were sustained for at least 10 days . The control transformant, A073, which did not have a bioluminescence-reporting gene, did not show any significant bioluminescence .

 
Resetting of bioluminescence rhythms by LD cycles. We examined whether the bioluminescence rhythms observed in the psbA1-reporting strain could be reset by daily light-dark (LD) cycles . We grew A205 transformant cells as colonies on solid BG-11, exposed samples to two 12-h light, 12-h dark (i.e., LD) cycles that were 12-h out of phase, and then transferred the cells to LL conditions at 41°C in order to monitor their bioluminescence rhythms . The two samples showed bioluminescence rhythms with opposite phases and, in both cases, bioluminescent peaks occurred 23.8 h after the onset of LL conditions and again 24.8 h later (Fig . 3) . These results indicate that the rhythms were reset, and their phases determined, by the LD cycle .

 
Temperature compensation of bioluminescence rhythms. We examined the effects of temperature on period length by using the A205 psbA1-reporting strain . The period lengths ± the standard deviation of the rhythms observed under LL conditions at 30, 35, 41, 55, and 60°C were 27.4 ± 1.0 h (n = 14), 24.7 ± 0.3 h (n = 6), 24.8 ± 0.7 h (n = 32), 24.6 ± 0.3 h (n = 7), and 22.3 ± 1.2 h (n = 5), respectively (Fig . 4) . The temperature coefficient (Q₁₀) for rhythm frequency (1/period length) was estimated to be 1.08 in this temperature range; especially, the period was almost constant (Q₁₀ = 1.00) in a temperature range between 35 and 55°C . These results indicate that the period length of the rhythms was compensated against extreme changes in ambient temperature .

 
Northern blot analysis of psbA1 mRNA. We demonstrated above that bioluminescence from the psbA1-reporting strain was controlled by the circadian clock . To determine whether the bioluminescence rhythms of the psbA1-reporting strain reflected rhythmic changes of promoter activity, we examined changes in the level of psbA1 mRNA in the cells of the A205 psbA1-reporting strain grown at 52°C under LL conditions (Fig . 5) . We found that the levels oscillated rhythmically and the rhythms were similar in period length and phase to the bioluminescence rhythms . We obtained essentially the same results from the cells of a nontransgenic wild-type strain of T . elongatus (data not shown) . Thus, the circadian rhythmic changes in bioluminescence level demonstrated here probably reflected the clock-controlled activities of the psbA1 promoter, although we did not examine the degradation of psbA1 mRNA .

 
The T . elongatus circadian clock period has wide temperature-compensation. The bioluminescence rhythms observed in the psbA1-reporting strain apparently reflected promoter activity (Fig . 5) and satisfied the three criteria for circadian rhythms: they had a period length of about 24 h that was persistent under constant conditions (Table 1 and Fig . 2), they were reset by LD cycles (Fig . 3), and the period showed temperature compensation (Fig . 4) . We therefore concluded that T . elongatus has a circadian clock and that the clock controls the activity of the psbA1 promoter .

In general, the rate of chemical, biochemical, and physiological processes varies with temperature, and their Q₁₀ values are usually between 2 and 3 . In contrast, the period length of circadian rhythms do not vary much with temperature, and the Q₁₀ is usually 0.9 to 1.3 (5, 21, 25) . The temperature compensation of the period length observed in mesophilic species is restricted to a range of <20°C (5); examples include eclosion rhythms of Drosophila sp . (4)., expansion and contraction rhythms of Cavernularia obesa colonies (16), leaf movement rhythms of Phaseolus multiflorus (3), and CAB2::LUC reporter expression rhythms of Arabidopsis thaliana (24) . The period length of bioluminescence rhythms observed in the mesophilic cyanobacteria Synechococcus (14) and Synechocystis (1) has a Q₁₀ of 1.1 within a range of only 10°C . The period we observed in T . elongatus, on the other hand, had a Q₁₀ of 1.08 within a range of more than 30°C and a Q₁₀ of 1.00 from 35 to 55°C, demonstrating perfect compensation within that wide range (Fig . 4) .

T . elongatus is a free-living, slowly mobile, unicellular organism that shows optimum growth at ca . 55°C (29; K . Onai, M . Morishita, and M . Ishiura, unpublished) . Its ability to compensate for wide temperature changes may support its survival in hot springs as well as in streams where 55°C temperatures are restricted to small areas . T . elongatus cells are viable at 30°C for at least 7 days . They are also viable at <30°C but cannot proliferate, and they can even withstand freezing (Onai et al., unpublished) .

We identified a gene cluster in T . elongatus homologous to the clock gene cluster kaiABC in Synechococcus (7; Uzumaki and Ishiura, unpublished), and we found that each of the kai genes exists as a single copy in the T . elongatus genome (18; Uzumaki and Ishiura, unpublished) . Presumably, the kaiABC gene cluster in T . elongatus has circadian clock functions . The large difference in the temperature range of temperature compensation between the Synechococcus and T . elongatus circadian clocks, however, suggests the existence of an additional system in T . elongatus that stabilizes the oscillations of the clock against large changes in ambient temperature .

The real-time monitoring system for bioluminescence rhythms in T . elongatus that we describe here provides great advantages for analyzing the circadian clock in the cyanobacterium . We can measure and analyze circadian rhythms within a wide ambient temperature range . The clock proteins and clock-related proteins prepared from T . elongatus are stable and provide excellent material for structure and function studies . Working with T . elongatus, we recently succeeded in determining the three-dimensional structure of KaiC (7) and the X-ray crystal structure of the C-terminal clock oscillator domain of KaiA (28) . Thus, T . elongatus is one of the best model organisms for molecular dissection of the circadian clock at the atomic level, especially the mechanisms for temperature compensation of period length .

 
We thank Edward Meighen (McGill University) for the gift of plasmid Xlux/pT7-3 carrying the Xl luxAB gene set . We also thank Hideaki Nakashima (Okayama University) and Shigeru Itoh (Nagoya University) for critical reading of the manuscript and Miriam Bloom (SciWrite Biomedical Writing and Editing Services) for professional editing .

This study was supported by grants to M.I . from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT); The Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN); Research for the Future Novel Gene Function Involved in Higher-Order Regulation of Nutrition-Storage in Plants (Japan Society for the Promotion of Science); Ground-based Research for Space Utilization (Japan Space Forum); The National Project on Protein Structural and Function Analyses (MEXT); and The Promoting Cooperative Research Project (Aichi Science and Technology Foundation) . The Division of Biological Science, Graduate School of Science, Nagoya University, is supported by a 21st COE grant from MEXT .

 
* Corresponding author . Mailing address: Center for Gene Research, Nagoya University, Furo, Chikusa, Nagoya 464-8602, Japan . Phone: 81-52-789-4527 . Fax: 81-52-789-4526 . E-mail: ishiura@gene.nagoya-u.ac.jp.

K.O . and M.M . contributed equally to this study .

1. Aoki, S., T . Kondo, and M . Ishiura. 1995 . Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp . strain PCC 6803 . J . Bacteriol . 177:5606-5611.
2. Ausubel, F . M., R . Brent, R . E . Kingston, D . D . Moore, J . G . Seidman, J . A . Smith, and K . Struhl. 1987 . Current protocols in molecular biology . Greene Publishing Associates/Wiley Interscience, New York, N.Y.
3. Bünning, E. 1931 . Untersuchungen über die autonomen tagesperiodischen Bewungen der Primärblätter von Phaseolus multiflorus. Jahrb . Wiss . Bot . 75:439-480.
4. Bünning, E. 1935 . Zur Kenntnis der endonomen Tagesrhythmik bei Insekten and bei Pflanzen . Ber . Deutsch. Bot . Gesellsch . 53:594-623.
5. Bünning, E. 1973 . The physiological clock: circadian rhythms and biological chronometry, 3rd ed . Springer-Verlag, New York, N.Y.
6. Castenholz, R . W. 1988 . Culturing methods for cyanobacteria . Methods Enzymol . 167:68-93.
7. Hayashi, F., H . Suzuki, R . Iwase, T . Uzumaki, A . Miyake, J.-R . Shen, K . Imada, Y . Furukawa, K . Yonekura, K . Namba, and M . Ishiura. 2003 . ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC . Genes Cells 8:287-296 .
8. Ishiura, M., S . Kutsuna, S . Aoki, H . Iwasaki, C . R . Andersson, A . Tanabe, S . S . Golden, C . H . Johnson, and T . Kondo. 1998 . Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria . Science 281:1519-1523 .
9. Iwasaki, H., Y . Taniguchi, M . Ishiura, and T . Kondo. 1999 . Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in cyanobacteria . EMBO J . 18:1137-1145 .
10. Iwasaki, H., T . Nishiwaki, Y . Kitayama, M . Nakajima, and T . Kondo. 2002 . KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria . Proc . Natl . Acad . Sci . USA 99:15788-15793 .
11. Jordan, P., P . Fromme, H . T . Witt, O . Klukas, W . Saenger, and N . Krauss. 2001 . Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution . Nature 411:909-917.
12. Kamiya, N., and J.-R . Shen. 2003 . Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-Å resolution . Proc . Natl . Acad . Sci . USA 100:98-103 .
13. Koike, H., and Y . Inoue. 1983 . Preparation of oxygen-evolving photosystem II particles from a thermophilic blue-green alga, p . 257-263 . In Y . Inoue, A . R . Crofts, N . Govindjee, N . Murata, G . Renger, and K . Satoh (ed.), The oxygen evolving system of photosynthesis . Academic Press, Inc., Tokyo, Japan.
14. Kondo, T., C . A . Strayer, R . D . Kulkarni, W . Taylor, M . Ishiura, S . S . Golden, and C . H . Johnson. 1993 . Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria . Proc . Natl . Acad . Sci . USA 90:5672-5676.
15. Mohamed, A., and C . Jansson. 1989 . Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803 . Plant Mol . Biol . 13:693-700.
16. Mori, S. 1960 . Influence of environmental and physiological factors on the daily rhythmic activity of a sea pen . Cold Spring Harbor Symp . Quant . Biol . 25:333-344.
17. Motoki, A., T . Shimazu, M . Hirano, and S . Katoh. 1995 . The two psbA genes from the thermophilic cyanobacterium Synechococcus elongatus. Plant Physiol . 108:1305-1306.
18. Nakamura, Y., T . Kaneko, S . Sato, M . Ikeuchi, H . Katoh, S . Sasamoto, A . Watanabe, M . Iriguchi, K . Kawashima, T . Kimura, Y . Kishida, C . Kiyokawa, M . Kohara, M . Matsumoto, A . Matsuno, N . Nakazaki, S . Shimpo, M . Sugimoto, C . Takeuchi, M . Yamada, and S . Tabata. 2002 . Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 . DNA Res . 9:123-130.
19. Nelson, W., Y . L . Tong, J . K . Lee, and F . Halberg. 1979 . Methods for cosinor-rhythmometry . Chronobiologia 6:305-323.
20. Onai, K., M . Morishita, T . Kaneko, S . Tabata, and M . Ishiura. 2004 . Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: a simple and efficient method for gene transfer . Mol . Genet . Genomics 271:50-59.
21. Pittendrigh, C . S. 1954 . On temperature independence in the clock system controlling emergence time in Drosophila. Proc . Natl . Acad . Sci . USA 40:1018-1029.
22. Pittendrigh, C . S., and S . Daan. 1976 . A functional analysis of circadian pacemakers in nocturnal rodents . I . The stability and liability of spontaneous frequency . J . Comp . Physiol . 106:223-252.
23. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory Press, Plainview, N.Y.
24. Somers, D . E., A . A . R . Webb, M . Pearson, and S . A . Kay. 1998 . The short-period mutant, toc1-1, alters circadian clock regulation of multiple outputs throughout development in Arabidopsis thaliana. Development 125:485-494 .
25. Sweeney, B . M., and J . W . Hastings. 1960 . Effects of temperature upon diurnal rhythms . Cold Spring Harbor Symp . Quant . Biol . 25:87-104.
26. Sweeney, B . M. 1987 . Rhythmic phenomena in plants, 2nd ed . Academic Press, Inc., San Diego, Calif.
27. Szittner, R., and E . Meighen. 1990 . Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium . J . Biol . Chem . 265:16581-16587 .
28. Uzumaki, T., M . Fujita, T . Nakatsu, F . Hayashi, H . Shibata, N . Itoh, H . Kato, and M . Ishiura. Crystal structure of the C-terminal clock-oscillator domain of the cyanobacterial KaiA protein . Nat . Struct . Mol . Biol., in press.
29. Williams, S . B., I . Vakonakis, S . S . Golden, and A . C . LiWang. 2002 . Structure and function from the circadian clock protein KaiA of Synechococcus elongatus: a potential clock input mechanism . Proc . Natl . Acad . Sci . USA 99:15357-15362 .
30. Yamaoka, T., K . Satoh, and S . Katoh. 1978 . Photosynthetic activities of a thermophilic blue-green alga . Plant Cell Physiol . 19:943-954.
31. Zouni, A., H . T . Witt, J . Kern, P . Fromme, N . Krauss, W . Saenger, and P . Orth. 2001 . Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution . Nature 409:739-743.

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