Division of Biological Science, Graduate School of Science, Nagoya University and CREST, Japan Science and Technology Corporation (JST), Furo-cho, Chikusa, Nagoya 464-8602, Japan1
First Department of Internal Medicine, Nagoya University, School of Medicine Tsurumai 65, Showa, Nagoya 466-8550, Japan2
Author for correspondence: Takao Kondo. Tel: +81 52 789 2498. Fax: +81 52 789 2963. e-mail: kondo{at}bio.nagoya-u.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: circadian clock, kai genes, kaiBC expression, amplitude of rhythm, bioluminescence rhythm
Abbreviations: LL, constant illumination; PMT, photomultiplier tube
These authors contributed equally to this work.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyanobacteria are the simplest organisms that display circadian rhythms (Golden et al., 1998 ; Kondo & Ishiura, 2000
; Iwasaki & Kondo, 2000
). We have previously introduced a bioluminescence reporter gene into the unicellular cyanobacterium Synechococcus elongatus PCC 7942 to monitor circadian gene expression (Kondo et al., 1993
), isolated various clock mutants (Kondo et al., 1994
), and both identified and cloned the clock gene cluster kaiABC, composed of kaiA, kaiB and kaiC (Ishiura et al., 1998
). While no similarity was found between Kai proteins and the clock proteins of other organisms, disruption of any single kai gene completely abolished circadian rhythm. Experiments in which kai genes are overexpressed suggest that KaiC represses its own (kaiBC) expression while KaiA enhances it. Thus, KaiC and KaiA have been proposed to be negative and positive elements, respectively, in the molecular feedback loop of kaiBC expression (Ishiura et al., 1998
).
Nineteen distinct mutants exhibiting a wide range of period lengths and arrhythmia mapped their mutations onto the kaiC gene (Ishiura et al., 1998 ). In light of this, it seems highly likely that understanding the biochemistry of the KaiC protein will be key in understanding circadian characteristics. Previous studies have shown that KaiA and KaiC interact with each other in the yeast two-hybrid system, in vitro, and in Synechococcus; closer examination revealed that the two half-units of KaiC (CI and CII, the first and second half domains, respectively) both interact with KaiA (Iwasaki et al., 1999
). The ATP-binding and autophosphorylation capacities of KaiC have also been shown to be important parts of the circadian timing mechanism (Nishiwaki et al., 2000
). Furthermore, two KaiA-binding domains (CKABD1 and CKABD2) in KaiC were found on the C-terminal regions of CI and CII, respectively, by using the yeast two-hybrid system and an in vitro interaction assay (Taniguchi et al., 2001
). More recently, SasA, a regulatory element consisting of a two-component sensory kinase, has been found to amplify the kai-based oscillations through its interaction with KaiC (Iwasaki et al., 2000
).
In previous work (Ishiura et al., 1998 ), only four KaiA mutants were identified after chemical mutagenesis. While the phenotypes of these mutants include arrhythmia, which indicates that KaiA is crucial in the establishment or maintenance of circadian rhythm, further mutational analysis is necessary to understand the function of KaiA. In this study, we performed PCR-based mutagenesis on the kaiA gene and screened for abnormal rhythmic phenotypes. Using targeting of gene integration, followed by high throughput screening for rhythmic phenotypes with bioluminescence, we obtained
400 mutants and analysed their phenotypes. Point mutations of representative kaiA mutants were also identified and mapped onto the KaiA protein. These mutations were concentrated in two distinct regions. We discuss the implications of our findings on the potential role of KaiA in generating circadian oscillations in cyanobacteria.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
DNA sequence of mutants.
We directly sequenced mutant kaiA genes after in vitro amplification of kaiA segments by PCR. Synechococcus cells were mixed in a tube with 0·1 mm zircon/silica beads and lysed by shaking with a Multi-Beads Shocker (Yasui Kikai). Genomic DNA was extracted by phenol extraction and purified by ethanol precipitation. Segments carrying kaiA were amplified by PCR using the following pair of primers: 818(3)up (5'- ACCGGCCACGTAGGGCTGTCC-3') (-409 to -389 of kaiA) and 715(1)low (described above). DNA sequence was then analysed with a DNA sequence kit and an ABI PRISM model 310 sequencer.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
KaiA mutations extend the period
As shown in period histograms (Fig. 2A and B
), the most striking feature of kaiA mutagenesis is an almost complete lack of short-period mutations. We found 301 long period mutants (period >27 h) and 92 arrhythmic mutants, whereas only one mutant showed a short-period phenotype and the oscillation rapidly damped in this mutant. Based on these statistical results, it can be concluded that mutations in kaiA generally extend the period, whereas periods can be both extended and shortened by mutations in kaiC (Ishiura et al., 1998
) and kaiB (our unpublished data). This tendency also holds true for the four kaiA mutants obtained by chemical (EMS) mutagenesis (Ishiura et al., 1998
). The one exceptional mutant (F224S) that showed a short period phenotype of 22 h (Fig. 3C
) is of particular interest; the biochemical properties of this mutant KaiA protein must be analysed to shed further light on the mechanism underlying the cyanobacterial oscillator.
|
|
Medium-amplitude mutants also sustained robust rhythmicity with period lengths of up to 40 h. As shown in Fig. 3B, the peak kaiBC expression was again little changed (-20% to +10%), whilst 1030% of peak kaiBC expression levels remained at trough phases. Thus, as with the high-amplitude group (Fig. 3A
), period extension up to 40% can be induced without a large change in kaiBC expression. These observations imply that the role of the KaiA protein is to control the period length of the circadian oscillation without activation of kaiBC expression. Direct examination of the biochemical activity of KaiA should be performed to address the molecular mechanism for this periodicity modulation. The phase of rhythm, once reset by 12 h darkness, was again not altered by these mutations (Fig. 3B
).
KaiA mutations lower kaiBC expression
The periods of lower amplitude mutants spanned an even wider range than those in the high-amplitude group, with period lengths as long as 50 h (Fig. 2B). Severe low-amplitude mutants (peak-to-trough<3) and arrhythmic mutants are shown in Fig. 3C
. Low-amplitude mutants in Fig. 3C
still sustain stable rhythms with 2450 h periods, whereas PkaiBC activity was considerably elevated at the trough phase and kaiBC expression at peak phase was lowered by 3050%. Exceptionally, kaiBC expression was elevated in the A245D mutant but this mutant failed to extend the period length. Mutants with very long periods are shown in Fig. 3D
. Note that kaiBC expression was significantly lowered in these very long-period mutants. Thus, the long periods of these mutations were induced not only as a direct effect of altered KaiA biochemistry, but also as an indirect effect of lowering kaiBC expression.
Arrhythmic mutants displayed various profiles. Some mutants showed normal peak and troughs during the first cycle, but were then damped through elevation of the trough level. Other mutants were arrhythmic from the beginning of LL. An important feature of these mutants is that PkaiBC activity was significantly lowered; in general, the level of PkaiBC reporter expression was lower than 10% of wild-type. As KaiA is known to enhance kaiBC expression (Ishiura et al., 1998 ), it is likely that the mutant KaiA proteins in this group could not sustain rhythmicity because they fail to activate kaiBC expression.
In this study, we have quantitatively examined the PkaiBC activity of KaiA mutants by normalizing bioluminescence data with colony size and number. From these assays, we found that most KaiA mutations lowered PkaiBC activity to varying degrees. As random mutation generally lowers the native function, these results would be consistent with a role for KaiA in enhancing PkaiBC activity (Ishiura et al., 1998 ). On the other hand, it appears that the level of kaiBC expression contributes little to period determination, but is important in maintaining robustness of the circadian rhythm.
Note also that the phase of the rhythm reset by 12 h darkness was changed in some low-amplitude mutants (Fig. 3C; D136V, D136Y and A245D). This suggests that KaiA is also involved in the phase-resetting mechanism of the cyanobacterial clock.
Clustering of mutations on the Kai map
We subsequently sequenced 45 mutants and found that 39 mutants have a single point mutation while five mutants overlapped with other mutants. Each point mutation was mapped to a residue in KaiA. As illustrated in Fig. 4, we were able to identify in KaiA an evident cluster of period mutations (residues 239245) and another weak one of period mutations (residues 113119). This suggests that cluster regions of KaiA are important in controlling the period length of the circadian oscillation, since most mutations that mapped to these regions have long period phenotypes (arrhythmia-causing mutations have not been found in these regions so far). The regions of KaiA that sustain rhythm by enhancing kaiBC expression are likely located elsewhere, because the mutations in arrhythmic mutants mapped to other parts of KaiA. These mutants (C53S, V76A, F178S, F224S, E274K) are also very interesting because these residues are essential for enhancing kaiBC expression. To make more detailed and reliable assessments of the different regions of KaiA, more mutations need to be identified.
Three very-long-period mutants (phenotype similar to those shown in Fig. 3D) carried double mutations in the kaiA sequence. Three other mutations that substantially affect KaiA all induced arrhythmicity. These included a missing start codon (GTG
GCG), a stop codon insertion (Q117) and a frame-shift at residue 143. The severe phenotype resulting from these mutations is also compatible with a crucial role for KaiA in sustaining the cyanobacterial circadian rhythm. Such a function could be interaction(s) of KaiA with other clock proteins (especially with KaiC, Iwasaki et al., 1999
; Taniguchi et al., 2001
), or alteration of phosphorylation of KaiC by KaiA (T. Nishiwaki, Y. Kitayama, H. Iwasaki & T. Kondo and H. Iwasaki, Y. Kitayama & T. Kondo, unpublished data).
Function of KaiA
By examining kaiBC expression of many KaiA mutants, we found that the KaiA protein could extend the period to some extent without lowering kaiBC expression. KaiA may interact with other clock proteins (especially with KaiC, Iwasaki et al., 1999 ; Taniguchi et al., 2001
), possibly altering the phosphorylation of KaiC (Nishiwaki et al., 2000
) or forming hetero-complexes of clock proteins (Iwasaki et al., 1999
). KaiA function is not yet well understood; analysis of the mutants identified in this research and, in particular, identification of regions in which they cluster, will lead to further elucidation of its biochemical mechanism of action. It should be noted that mutations can enhance or diminish protein activity (e.g. enzymic or binding activity), although it is assumed generally that most random mutations lower protein activity. In contrast to mutations in KaiB or KaiC, the vast majority of KaiA mutations extended the period and only rarely shortened it. The statistical observations made in this study lead us to conclude that the biochemical function of the KaiA protein is to move the clock operation forward.
On the other hand, in KaiA mutants with severe phenotypes, kaiBC expression was lowered by 4050% in very-long-period mutants and by 90% in arrhythmic KaiA mutants compared to wild-type. As our previous study (Ishiura et al., 1998 ) revealed that a function of KaiA was to activate kaiBC transcription and that kaiBC expression was severely lowered in a kaiA- strain, our observations for severe KaiA mutants are compatible with our previous study (Ishiura et al., 1998
). Clearly, arrhythmic mutants obtained in this study were very important for dissection of these actions. Interestingly, the site of arrhythmic or severe low-amplitude mutants was not included in the region in which moderate-period mutants clustered. These results also suggest the dual action of KaiA in the circadian time-keeping process in cyanobacteria.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bustos, S. A. & Golden, S. S. (1991). Expression of the psbDII gene in Synechococcus sp. strain PCC 7942 requires sequences downstream of the transcription start site. J Bacteriol 173, 7525-7533.[Medline]
Diaz, J. J., Rhoads, D. D. & Roufa, D. J. (1991). PCR-mediated chemical mutagenesis of cloned duplex DNAs. Biotechniques 11, 204206, 208, 210211.[Medline]
Dunlap, J. C. (1999). Molecular bases for circadian clocks. Cell 96, 271-290.[Medline]
Golden, S. S., Johnson, C. H. & Kondo, T. (1998). The cyanobacterial circadian system: a clock apart. Curr Opin Microbiol 1, 669-673.[Medline]
Ishiura, M., Kutsuna, S., Aoki, S., Iwasaki, H., Andersson, C. R., Tanabe, A., Golden, S. S., Johnson, C. H. & Kondo, T. (1998). Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria. Science 281, 1519-1523.
Iwasaki, H. & Kondo, T. (2000). The current state and problems of circadian clock studies in cyanobacteria. Plant Cell Physiol 41, 1013-1020.
Iwasaki, H., Taniguchi, Y., Ishiura, M. & Kondo, T. (1999). Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in cyanobacteria. EMBO J 18, 1137-1145.
Iwasaki, H., Williams, S. B., Kitayama, Y., Ishiura, M., Golden, S. S. & Kondo, T. (2000). A KaiC-interacting sensory histidine kinase, SasA, necessary to sustain robust circadian oscillation in cyanobacteria. Cell 101, 223-233.[Medline]
Kondo, T. & Ishiura, M. (2000). The circadian clock of cyanobacteria. Bioessays 22, 10-15.[Medline]
Kondo, T., Strayer, C. A., Kulkarni, R. D., Taylor, W., Ishiura, M., Golden, S. S. & Johnson, C. H. (1993). Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc Natl Acad Sci USA 90, 5672-5676.[Abstract]
Kondo, T., Tsinoremas, N. F., Golden, S. S., Johnson, C. H., Kutsuna, S. & Ishiura, M. (1994). Circadian clock mutants of cyanobacteria. Science 266, 1233-1236.[Medline]
Narusaka, Y., Narusaka, M., Satoh, K. & Kobayashi, H. (1999). In vitro random mutagenesis of the D1 protein of the photosystem II reaction center confers phototolerance on the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 274, 23270-23275.
Nishiwaki, T., Iwasaki, H., Ishiura, M. & Kondo, T. (2000). Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria. Proc Natl Acad Sci USA 97, 495-499.
Pittendrigh, C. S. (1993). Temporal organization: reflections of a Darwinian clock-watcher. Annu Rev Physiol 55, 17-54.
Prentki, P. & Krisch, H. M. (1984). In vitro insertional mutagenesis with a selective DNA fragment Gene 29, 303-313.[Medline]
Taniguchi, Y., Yamaguchi, A., Hijikata, A., Iwasaki, H., Kamagata, K., Ishiura, M., Go, M. & Kondo, T. (2001). Two KaiA-binding domains of cyanobacterial circadian clock protein KaiC. FEBS Lett 496, 86-90.[Medline]
Young, M. W. & Kay, S. A. (2001). Time zones: a comparative genetics of circadian clocks. Nat Rev Genet 9, 702-707.
Received 7 January 2002;
revised 15 May 2002;
accepted 17 May 2002.