1 Department of Biology, The University of St Thomas, St Paul, MN 55105, USA
2 Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843-3258, USA
Correspondence
Susan S. Golden
sgolden{at}tamu.edu
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ABSTRACT |
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Present address: Department of Biology, The University of Utah, Salt Lake City, UT 84112, USA.
These authors contributed equally to this work.
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INTRODUCTION |
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In S. elongatus, the circadian pacemaker consists of the products of at least three genes, kaiA, kaiB and kaiC. The kai locus is expressed from two promoters one upstream of kaiA (PkaiA, monocistronic message) and one upstream of kaiB (PkaiBC, dicistronic kaiBC message) which drive transcription in the same circadian phase in wild-type cells, with peak expression at the time that corresponds to dusk when cells are kept in continuous light (subjective dusk) (Ishiura et al., 1998). Central to models for the mechanism of eukaryotic circadian clocks are delayed transcriptionaltranslational feedback loops that regulate the timing of clock-gene expression. These feedback loops involve positive-effector proteins that stimulate core clock-gene expression. The core clock components then negatively regulate their own expression, to generate an oscillation of gene expression and clock-protein production that results in a 24 h regulatory mechanism (Dunlap, 1999
; Dunlap et al., 2004
; Harmer et al., 2001
; Young & Kay, 2001
). Some aspects of the regulation of clock genes in S. elongatus are reminiscent of autoregulatory feedback models. Previous work has shown that KaiA is required for expression from PkaiBC, and that overexpression of kaiA enhances expression from PkaiBC, suggesting a role in positive activation (Ishiura et al., 1998
). KaiC is required for normal levels of expression from its own promoter; however, overexpression of kaiC blocks expression from PkaiBC, suggesting a role in negative autoregulation (Ishiura et al., 1998
). Hence, these data were interpreted to be consistent with eukaryotic circadian-timing models. Recent data have shown the mechanism to be more complex (Iwasaki et al., 2002
; Tomita et al., 2005
; Xu et al., 2003
).
There has been increasing evidence that the feedback loops central to eukaryotic circadian-clock models are not necessary for rhythms to persist in the cyanobacterial clock. Expression of kaiC from an Escherichia coli consensus promoter (Ptrc, inducible by IPTG) was shown to complement a kaiC null strain, which demonstrates that cis elements in the kaiBC promoter sequence are not specifically required to maintain rhythmicity in the cells (Xu et al., 2003). Some mutants of S. elongatus affect the phase relationship between PkaiA and PkaiBC expression, but do not disrupt the self-sustained rhythmicity of the circadian clock itself. For example, the cpmA (circadian phase modifier) mutation results in expression of PkaiA 10 h out of phase from PkaiBC (Katayama et al., 1999
). Similarly, null mutations in any of the known group 2 sigma factors, which are important for promoter recognition and subsequent transcription in S. elongatus, also show differential effects on circadian expression (Nair et al., 2002
; Tsinoremas et al., 1996
). In particular, sigC inactivation lengthens the period of expression from PkaiA, but not from PkaiBC, by 2 h; thus, the peak expression from PkaiA with respect to PkaiBC falls out of phase by up to 8 h (Nair et al., 2002
). This finding suggests that the relative timing of transcriptional activity, at least from the kaiA promoter, is not important for generating circadian rhythms.
In this study we tested directly to determine whether the coordinated timing of transcription from the kai promoters is important for circadian timekeeping in S. elongatus. The native transcriptional regulation of kai genes was bypassed to force expression in an unusual phase. Expressing the kaiA gene or kaiBC dicistron from the purF promoter (Min et al., 2004), such that expression was delayed by 12 h with respect to wild-type expression timing, forced a peak at subjective dawn (class 2) rather than the normal subjective dusk (class 1). This work shows that individual kai loci, expressed independently from a class 2 promoter, function normally in the core circadian timing mechanism, and support downstream circadian rhythms in S. elongatus. Therefore, coincident transcriptional patterns of expression of the kai genes are not necessary for precise and persistent circadian rhythms in the cyanobacterium.
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METHODS |
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DNA manipulations and sequencing.
Plasmid DNA for sequencing and subcloning was prepared with a QIAprep miniprep kit (QIAGEN). Sequencing was performed with BigDye terminator mix (Applied Biosystems) according to the manufacturer's instructions. PCR primers were generated, and sequencing reactions were run, at the Gene Technology Laboratory (Institute of Developmental and Molecular Biology, Texas A&M University). Restriction endonuclease digestions were performed according to manufacturer's instructions (New England Biolabs). DNA fragments for subcloning were purified from agarose gel slices by the CONCERT rapid gel extraction system (Invitrogen).
Construction of kai mutant strains.
An in-frame deletion of each kai gene was generated by removing codons for 254, 79 and 409 amino acids of the kaiA, kaiB and kaiC products, respectively (see Fig. 1a for further details of the deletions). Each in-frame deletion construct was cloned into pRL278, a sacB hit-and-run negative selection vector (Andersson et al., 2000
), used to transform AMC541 (Golden et al., 1987
), and crossed into the chromosome via homologous recombination to replace each wild-type kai gene with the deleted allele (Clerico et al., 2005
; Golden, 1988
). Chromosomal DNA from each deletion strain was prepared (Golden et al., 1987
), and regions surrounding each deletion were amplified by PCR. Amplification products were desalted using the CONCERT rapid PCR purification system (Invitrogen), and sequenced to verify the in-frame deletion of each kai gene.
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Construction of a neutral site I Gateway vector.
A Gateway technology-compatible (Invitrogen) NS1 vector, pAM3110, was designed by amplifying the recombination cassette from pDONR221 (Invitrogen), which includes the sequences for attP1, the ccdB gene, a Cm-resistance gene, and attP2. The primers anneal 25 bp upstream of the M13 forward sequence and 15 bp downstream of the M13 reverse sequence, and incorporate MluI and XhoI restriction sites, respectively. The PCR product was cloned into MluI/XhoI-digested pAM2314 to create pAM3110. Exogenous DNA to be recombined at NS1 of the S. elongatus chromosome can be amplified with primers containing the attB1 and attB2 sequences, and cloned into pAM3110 using a BP Clonase reaction. The recombinant construct is inserted adjacent to an Sp/Sm-resistance -cassette; both the selectable marker and the recombinant construct are flanked by S. elongatus NS1 DNA.
Construction of ectopic kai alleles.
The kaiA gene and its native promoter region (PkaiA : : kaiA) (Ishiura et al., 1998) were amplified using PCR, and cloned into the NS1 cloning vector pAM1303 digested with BamHI and NotI. The kaiA segment in this plasmid (pAM2246) extends from 312 bp upstream of the kaiA GTG start codon to 80 bp downstream of the kaiA TGA stop codon, and was generated using one primer that incorporates a BglII restriction site and one primer that incorporates a NotI restriction site. PpurF : : kaiA was constructed as follows: an 88 bp PpurF promoter fragment (Min et al., 2004
) was amplified by PCR using primers to incorporate an upstream restriction site (BamHI) and a downstream restriction site (MluI). PpurF was cloned into pUCBM20 as a BamHIMluI fragment, creating pAM2380. A promoterless kaiA gene was amplified using PCR with primers flanking the kaiA gene from 84 bp upstream of the kaiA GTG start codon and from 80 bp downstream of the kaiA TGA stop codon, incorporating MluI and NotI restriction sites, respectively, and subsequently cloned into the MluI/NotI sites of pAM2380 to create PpurF : : kaiA (pAM2383). This construct was cloned as an EcoRINotI fragment into the NS1 cloning vector pAM1303 to create pAM2482.
The kaiBC native promoter (Ishiura et al., 1998) was amplified along with either the kaiB gene alone (PkaiBC : : kaiB) or kaiBC (PkaiBC : : kaiBC). In both cases the upstream primer annealed 494 bp upstream of the kaiB ATG start, and incorporated a BamHI site. The downstream primer had an end point 30 bp downstream of the kaiB TAA stop codon for PkaiBC : : kaiB, and 146 bp downstream of the kaiC TAG stop codon for PkaiBC : : kaiBC; both incorporated a NotI restriction site. Insertion of each fragment into BamHI- and NotI-digested pAM1303 generated pAM2245 (PkaiBC : : kaiB) and pAM3109 (PkaiBC : : kaiBC). The PkaiBC : : kaiC allele was generated as for PkaiBC : : kaiBC, except that pAM1979 (the kaiB deletion construct) served as the template, and the downstream primer had an end point 46 bp downstream of the kaiC TAG stop codon.
The PpurF : : kaiBC construct used the same class 2 promoter segment as described for the generation of PpurF : : kaiA. A promoterless kaiBC operon was amplified by PCR using primers to flank kaiBC from 68 bp upstream of the kaiB ATG start codon to 146 bp downstream of the kaiC TAG stop codon. Amplification incorporated an upstream (MluI) and downstream (NotI) restriction site. The resultant PCR product was cloned downstream of the purF promoter in pAM2380 to create pAM3135. The entire PpurF : : kaiBC construct was amplified from pAM3135 using one primer that contains an attB1 sequence and one primer that contains an attB2 sequence. The purified PCR product was then used in a BP Clonase recombination reaction with pAM3110 to create pAM3139.
Measurement of in vivo bioluminescence.
Automated measurement of bioluminescence from the various S. elongatus luc reporter strains was performed by adding 10 µl 100 mM firefly luciferin (Biosynth) to each inoculated agar pad in a 96-well plate. The cultures were allowed to grow for 24 h in LL. Each black plate was then placed on the Packard TopCount 96-well microplate scintillation and bioluminescence counter, exposed to two light/dark cycles for synchronization, and placed in LL (Andersson et al., 2000; Canales et al., 2005
). Because light intensity has a minor effect on circadian period, and because absolute expression levels (but not circadian parameters) vary with cell number (Ditty et al., 2003
; Katayama et al., 2003
), all mutant and control analyses reported here were calculated from samples carefully paired on the monitoring device for equivalent illumination, from samples grown in parallel under identical conditions.
Data analysis.
All data acquired by the TopCount were graphed and analysed by the Import and Analysis (I&A) Excel interface (S. A. Kay Laboratory, The Scripps Research Institute, La Jolla, CA, USA; Plautz et al., 1997). The circadian period and standard deviations of all reporter fusions were calculated from at least five cycles of data obtained in LL by FFT-NLLS, a Fast Fourier statistical package associated with I&A (Plautz et al., 1997
). Each strain was measured in at least three independent experiments. The circadian periods of the total number of individual wells of each strain from all experiments were used to calculate the mean and SD values displayed in Tables 2 and 3
. Figs 1(c), 2(b), 3(b), 4(a) and 4(b)
show representative traces of each strain. The circadian phase of each trace is determined by the time of peak bioluminescence expression with respect to the time the cells were placed in LL after the synchronizing LD cycles. Reporters termed class 1 peak at subjective dusk, with their first peak at 12 h after their entry into LL, and peaking again approximately every 24 h. Class 2 reporters peak at subjective dawn; after an initial acute increase, peak expression occurs 24 h after entering LL.
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Equal amounts of total protein (1020 µg) from each whole-cell extract sample were separated by SDS-PAGE (12·5 %) for immunoblotting. Protein was transferred to 0·2 µm Protran nitrocellulose membranes (Schleicher & Schuell) by capillary transfer for KaiA and KaiB detection, as previously described (Michel et al., 2001), or to 0·45 µm Protran nitrocellulose membranes (Schleicher & Schuell) by semi-dry blot transfer, according to the manufacturer's instructions (Bio-Rad), for KaiC. Polyclonal rabbit antiserum to KaiA and KaiB was used at dilutions of 1 : 2000 and 1 : 1000, respectively, and was detected using peroxidase-conjugated goat anti-rabbit IgG (Calbiochem). Polyclonal chicken antiserum to KaiC (Aves Labs) was used at a dilution of 1 : 2000, and detected with peroxidase-conjugated goat anti-chicken IgY (Aves Labs). Detection of Kai-antibody binding was visualized with the SuperSignal West Pico chemiluminescent substrate detection system (Pierce), according to the manufacturer's instructions. Under the stated conditions, the phosphorylated and unphosphorylated KaiC proteins are not resolved. Therefore, KaiC immunoblots detect the total level of KaiC present in the cell.
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RESULTS AND DISCUSSION |
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Previous experiments have shown that kaiA expression has a positive effect on PkaiBC, because in the absence of KaiA the amount of expression from a PkaiBC reporter decreases (Ishiura et al., 1998). Therefore, it was expected that an in-frame deletion of kaiA would cause a decrease in the amount of KaiB and KaiC proteins expressed in this mutant background. This expected phenotype was reported by the PkaiBC : : luc promoter fusion in the
kaiA strain (AMC702); levels of bioluminescence from the reporter are depressed to below background levels (Fig. 1c
). However, noticeably elevated levels of KaiB and KaiC protein were detected in the
kaiA strain (Fig. 1b
). The data are consistent with the hypothesis that a negative element was removed from the PkaiBC promoter region in the process of deleting the kaiA gene, as the stop codon of the kaiA gene is only 90 bp upstream of the start codon for kaiB (Ishiura et al., 1998
). Others have observed evidence for the presence of a negative regulatory element in this region (Kutsuna et al., 2005
). The elevated level of KaiB and KaiC protein production was not detected at the transcriptional level by the PkaiBC : : luc promoter fusion, because the reporter includes this putative negative element.
An insertional mutant of kaiA (kaiA, AMC1161) was generated to inactivate the gene without affecting potential regulatory elements of kaiBC (Fig. 1a
). As in the
kaiA mutant background, rhythms of bioluminescence from the PkaiBC : : luc reporter are arrhythmic in the
kaiA strain (Fig. 1c
). Conversely, protein levels of KaiB and KaiC are decreased relative to wild-type in
kaiA (Fig. 1b
). Therefore, this kaiA insertion mutant strain was used for further complementation studies.
KaiC has been shown to act as a negative regulator of the kaiBC promoter; when KaiC is overexpressed, expression from a PkaiBC reporter decreases significantly (Ishiura et al., 1998). However, regulation of the kaiBC promoter is more complicated in that KaiC is also required for normal levels of kaiBC expression. This necessity is demonstrated in the slightly lower levels of KaiB protein in the
kaiC mutant background (Fig. 1b
).
Complementation of kaiA with class 2 PpurF : : kaiA restores wild-type circadian rhythms
Because mutants of S. elongatus have been identified that uncouple the transcriptional phasing of the two circadian clock kai operons (Katayama et al., 1999; Nair et al., 2002
), the effect of directly changing the transcriptional phasing of kaiA (to 12 h out of phase with that of kaiBC) was examined. Expression of an ectopic copy of kaiA from either its native promoter or the heterologous purF promoter in an otherwise wild-type strain shortened the circadian period by about an hour (Table 3
). Each kaiA construct was also tested for its ability to complement the
kaiA strain (AMC1161). Expression of either PkaiA : : kaiA or PpurF : : kaiA in the
kaiA strain (AMC1233 and AMC1234, respectively) restored circadian rhythmicity with periodicities very near that of the PkaiBC : : luc reporter in a wild-type background (Table 3
). Also, the phase of the rhythm was not altered when kaiA was produced 12 h out of phase from its native timing (Fig. 2b
). Levels of Kai proteins in these strains are comparable to those in the wild-type (Fig. 2a
). Therefore, the peak of the oscillation in transcription of kaiA does not have a substantial effect on the properties of the cyanobacterial circadian rhythm.
PpurF : : kaiBC can restore wild-type rhythms to a kaiBC strain
Throughout the circadian cycle, the overall level of KaiA protein remains constant, or fluctuates with low amplitude, over a 24 h time scale (Xu et al., 2000). Therefore, altering the expression of the kaiA gene may not result in a change in the amount of KaiA protein over time, and thus would not alter the phasing of the clock itself. The KaiB and KaiC proteins, however, accumulate in a circadian fashion with peak protein levels lagging approximately 4 h after the peaks in mRNA levels, and these oscillations, along with changes in phosphorylation levels, have been proposed to be required for rhythmicity (Ishiura et al., 1998
; Tomita et al., 2005
; Xu et al., 2003
). Therefore, the necessity for peak transcriptional activity of kaiB and kaiC to occur at subjective dusk was examined.
An ectopic copy of the entire kaiBC dicistron, expressed from either its native promoter or the purF promoter, was tested for its ability to complement a strain that lacks both kaiB and kaiC. In a wild-type background a second copy of kaiBC from either promoter had little effect on the circadian rhythm of the cells (AMC1276 and AMC1277, Table 2). In the
kaiBC strain expression of PkaiBC : : kaiBC from NS1 (AMC1282) drives PkaiBC : : luc expression in class 1 phase, peaking at subjective dusk (Fig. 3b
) with a period very close to that of wild-type (Table 3
). Moreover, expressing kaiBC from the heterologous purF promoter in the
kaiBC strain (AMC1283), such that transcription would peak 12 h out of phase from its wild-type pattern and from expression of kaiA, did not alter the period of luciferase expression from a PkaiBC : : luc reporter (Table 2
). In addition, the phase of peak bioluminescence from PkaiBC : : luc was not altered, despite the fact that these core clock genes are being transcriptionally activated 12 h out of phase from their normal time (Fig. 3b
). Levels of Kai proteins in these complemented strains are similar to those seen in wild-type (Fig. 3a
).
Pervasive effects of PpurF : : kaiBC expression on both class 1 and class 2 reporters
To determine the effect of mistimed kaiBC expression on promoters whose activities are not central to the clock, well-characterized class 1 (PpsbAI : : luc) and class 2 (PpurF : : luc) reporters were measured for rhythms in bioluminescence in the PpurF : : kaiBC-complemented strain. Consistent with the results for the PkaiBC : : luc reporter, both PpsbAI : : luc (STC101) and PpurF : : luc (STC100) reporters displayed rhythms with wild-type period and phasing (Table 2, Fig. 4
). In LL, the peak of expression from the purF reporter is 12 h out of phase from PpsbAI : : luc. However, in the LD cycles prior to release into LL, PpurF : : luc, and all known class 2 reporters (S. Canales & S. Golden, unpublished results), peak only 4 h before class 1 rhythms, as has been previously described (Liu et al., 1996
). The class 2 rhythms exhibited by PpurF : : luc in the PpurF : : kaiBC-complemented strain demonstrate that the purF promoter is still recognized as class 2, and is driving kaiB and kaiC in that phase from NS1. A PkaiA : : luc reporter also maintained its wild-type circadian properties in this complemented background (data not shown).
Conclusions
We have demonstrated that the promoter elements of the kai locus are not specifically needed to support circadian control of gene expression; in fact, the synchrony of expression of kai loci is not needed to maintain rhythms. Either kaiA or kaiBC can be expressed from the class 2 purF promoter, 12 h out of phase from the other kai locus, and preserve the persistence and precision of the circadian mechanism. However, cis elements do exist that regulate the level of expression of these genes: overexpression of kaiC causes a decline in the activity from PkaiBC (Ishiura, 1998), and in the absence of kaiC, bioluminescence levels do not reach the high peak levels seen in wild-type (Fig. 1c
). The bioluminescence rhythm from a PkaiBC reporter has a high amplitude as compared to other reporters, such as PpsbAI (compare Fig. 1c
and Fig. 4a
), and declines to near background levels at its trough. The negative regulatory element may assist in the robust, high-amplitude expression from PkaiBC, because removal of a portion of the promoter region results in increased levels of KaiB and KaiC proteins in the
kaiA strain. When this element is missing, as in the
kaiA strain, the trough levels may be increased such that there is a higher basal level of kaiBC expression, which would account for the observed increase in protein level.
The amounts of each Kai protein affect the period length of the rhythm. Expression of a second copy of kaiA in a wild-type background shortened the circadian rhythm by about an hour (Table 3). Previous experiments have demonstrated that KaiA increases the rate at which KaiC is autophosphorylated by about 2·5-fold in vitro (Williams et al., 2002
). Having two copies of kaiA, as seen in AMC1099 and AMC1101, may lead to an increase in the phosphorylation state of KaiC protein, and increase the rate of progression through the circadian cycle, which may account for the shortened period in these strains. The regulation is more complicated, though, as expression of an ectopic copy of kaiB in a wild-type strain (AMC1271) also shortens the period of circadian rhythmicity in LL (Table 2
). KaiB abrogates the stimulatory effect of KaiA on the autophosphorylation of KaiC; a second copy of kaiB would not be expected to cause the same effect on the period length as the ectopic copy of kaiA. The presence of an additional copy of kaiC in the wild-type strain (AMC1273) causes a slight lengthening of the period of the rhythm (Table 2
). The stability of KaiC and its complexes has been shown to be a determining factor of the period length in the cell (Kageyama et al., 2002
; Xu et al., 2003
). The extra copy of kaiC and the resulting increased level of KaiC protein present in the cell may allow more periodosome scaffolds to form, assembly and disassembly of which could lengthen the cycle.
These data and those from other laboratories provide increasing evidence that the mechanisms for maintaining the clock in cyanobacteria are not dependent on the negative and positive limbs of a transcriptionaltranslational feedback loop. In addition to the ability to sustain circadian rhythms when kai genes are expressed from heterologous promoters (Xu et al., 2003; this work), S. elongatus continues to tell time in the dark, when de novo expression of the kai genes, and thus protein synthesis, is not detectable (Tomita et al., 2005
; Xu et al., 2003
). Under these conditions, which would disrupt a transcriptionaltranslational feedback loop, circadian rhythms in the phosphorylation state of KaiC continue (Tomita et al., 2005
). Moreover, the Kai proteins can establish a temperature-compensated circadian rhythm of KaiC phosphorylation in vitro, directly demonstrating that there is a post-translational clock mechanism in the cyanobacterium (Nakajima et al., 2005
). Thus, an understanding of the post-translational dynamics of core clock proteins will be needed to elucidate the stabilizing and time-keeping properties of the Kai-based clock.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 March 2005;
revised 5 May 2005;
accepted 9 May 2005.
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