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

Abstract

KaiA, KaiB, and KaiC clock proteins from cyanobacteria and ATP are sufficient to reconstitute the KaiC phosphorylation rhythm in vitro, whereas almost all gene promoters are under the control of the circadian clock. The mechanism by which the KaiC phosphorylation cycle drives global transcription rhythms is unknown. Here, we report that RpaA, a potential DNA-binding protein that acts as a cognate response regulator of the KaiC-interacting kinase SasA, mediates between KaiC phosphorylation and global transcription rhythms. Circadian transcription was severely attenuated in sasA (Synechococcus adaptive sensor A)- and rpaA (regulator of phycobilisome-associated)-mutant cells, and the phosphotransfer activity from SasA to RpaA changed dramatically depending on the circadian state of a coexisting Kai protein complex in vitro. We propose a model in which the SasA–RpaA two-component system mediates time signals from the enzymatic oscillator to drive genome-wide transcription rhythms in cyanobacteria. Moreover, our results indicate the presence of secondary output pathways from the clock to transcription control, suggesting that multiple pathways ensure a genome-wide circadian system.

Circadian rhythms are endogenous biological timing processes that are observed ubiquitously in organisms from cyanobacteria to green plants and mammals (1). Cyanobacteria are the simplest organisms known to exhibit such rhythms (2). In the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter referred to as Synechococcus), essentially all gene promoters are under the control of the circadian clock, as demonstrated by promoter-trap experiments (3, 4). Generation of such transcription rhythms requires three clock genes: kaiA, kaiB, and kaiC (4, 5). Recently, we demonstrated that the circadian rhythm of KaiC phosphorylation persists even in the absence of transcription/translation processes (6). Moreover, the circadian oscillation of KaiC phosphorylation was reconstituted in vitro by incubating three recombinant Kai proteins with ATP (7). The period of oscillation in vitro was stable despite temperature change (temperature compensation), and the circadian periods observed in vivo in KaiC mutant strains were consistent with those measured in vitro (7). Thus, a transcription/translation feedback loop is not essential for the circadian timing mechanism, but the biochemical network among Kai proteins must drive the core oscillation.

How does the Kai-based chemical oscillator drive circadian rhythms in genome-wide transcription in cyanobacteria? Kai proteins do not show any similarity to eukaryotic circadian clock proteins or to any known DNA-binding motifs. One possibility could be KaiC-mediated activation of gene expression by means of a two-component regulatory system, including a KaiC-binding histidine kinase (HK), SasA (8). In sasA (Synechococcus adaptive sensor A)-inactivated strains, kaiBC expression is lowered dramatically, and circadian transcription rhythms are attenuated severely. Although SasA is not necessary to drive basic oscillation, it may function in a connection between the KaiC phosphorylation cycle and transcription. To examine this possibility, the identification and characterization of the as yet unknown cognate response regulator(s) (RRs) of SasA is essential.

Here, we report a presumed DNA-binding RR, RpaA, as a SasA partner that is involved in the Kai-based circadian system. RpaA is required for genome-wide transcription rhythms. Moreover, our biochemical analysis shows that autophosphorylation of SasA and phosphotransfer from SasA to RpaA are altered, depending on some circadian states of the coexisting Kai protein complex. Our results strongly suggest that a circadian timing signal is primarily mediated by the SasA–RpaA two-component regulatory system acting from the posttranslational oscillator to the transcription machinery.

Results

Identification of RpaA as a Potential SasA Partner.

In the complete genome sequence of S. elongatus PCC 6301, which is highly homologous to that of S. elongatus PCC 7942, we identified 24 RR genes, including hybrid sensory kinase genes (Table 2, which is published as supporting information on the PNAS web site) and 13 HK genes. To search for a cognate RR of SasA, we performed genetic deletion of each RR gene individually in a S. elongatus PCC 7942 strain harboring a kaiBC bioluminescence reporter (P_kaiBC::luxAB) by substituting the gene of interest with a kanamycin-resistant gene or by insertion of a spectinomycin-resistant gene. We succeeded in inactivating 22 of 24 RR genes, whereas null mutant strains lacking syc2234_d (ycf29) or syc0104_c (ycf27, rpaB) were not obtained, suggesting that the two latter genes were essential under our experimental conditions (Table 2). Then temporal kaiBC gene expression patterns were monitored in the 22 RR mutants under continuous light (LL) conditions after 12-h dark treatment to synchronize the clock. We found that disruption of the syc1409_d gene dramatically affected the P_kaiBC::luxAB bioluminescence profile (Fig. 1A). The gene syc1409_d encodes an OmpR (outer membrane protein regulator) family RR harboring presumed DNA-binding motifs and is highly homologous to the Synechocystis rpaA (regulator of phycobilisome-associated) gene (9) (Fig. 5, which is published as supporting information on the PNAS web site). Although RpaA is somehow responsible for energy transfer from phycobilisome to photosystem I in Synechocystis sp. PCC 6803 (9), its underlying mechanism and the cognate sensory kinase are unknown.

Fig. 1.

Genome-wide transcription rhythms are attenuated in the sasA-null and rpaA-null mutants. (A) Arrhythmia in the absence of sasA or rpaA at standard LL conditions. WT, sasA-disrupted (ΔsasA, green trace) and rpaA-disrupted (ΔrpaA, red trace) cells that carry kaiBC-, psbAI-, purF-, sigC-, and sigD6-reporter cassettes were grown for 3–4 d on solid medium in LL at 50 μE·m⁻²·s⁻¹ to give 30–60 colonies (0.2-mm diameter). After a 12-h dark treatment, cells were transferred to LL at 50 μE·m⁻²·s⁻¹. The bioluminescence was measured by a photomultiplier tube. For clarity, bioluminescence profiles of the reporter strains lacking sasA or rpaA are shown on magnified scales (ΔsasA, pale blue; ΔrpaA, orange; magnified scale is shown on the right). (B) Residual rhythmicity from sasA- or rpaA-null mutants under dim LL conditions at 15 μE·m⁻²·s⁻¹.

As shown in Fig. 1A, genetic deletion of the Synechococcus rpaA gene lowered kaiBC promoter activity dramatically and nullified rhythmicity under LL conditions with standard light intensity [50 microeinsteins (μE)·m⁻²·s⁻¹], reminiscent of the sasA-null phenotype (8). Examining a larger set of clock-controlled promoters revealed that inactivation of either of the sasA or rpaA genes similarly nullified rhythmic expression of the kaiA, psbAI, purF, sasA, cikA, rpaA, srrB, rpoD6, and sigC genes (Fig. 1A; see also Fig. 6, which is published as supporting information on the PNAS web site).

To confirm that the lowered bioluminescence indeed accompanied reduced kaiBC mRNA levels, we performed northern (RNA) blot analyses under standard LL conditions. As shown in Fig. 2A, circadian fluctuation in kaiBC mRNA level was not evident in either mutant strain, whereas a robust circadian change peaking at subjective dusk was observed in the WT strain. The levels of kaiBC mRNAs were lowered in the mutants to ≈10% of that in WT cells at peak, correlating well with the bioluminescence profiles shown in Fig. 1A.

Fig. 2.

Expression of kaiBC mRNA and KaiBC proteins in the rpaA and sasA mutants. (A) Northern blot analysis. The kaiBC mRNA levels were examined in WT, sasA-disrupted (ΔsasA), and rpaA-disrupted (ΔrpaA) strains at 4-h intervals during hours 0–48 in LL (80 μE·m⁻²·s⁻¹). Densitometric data of the blot also are shown. Multiple bands are caused by mRNA degradation, and separations are caused by the presence of rRNA. (B) Western blot analysis. Accumulated levels of KaiA, KaiB, KaiC, and SasA were examined. Cells were collected every 4 h from hour 0 to hour 24 in LL. Proteins (0.5 μg) were prepared from cells of WT (○); sasA-deficient (▴); and rpaA-null strains (■); subjected to SDS/PAGE on 10% gels; and then analyzed by immunoblotting using each anti-Kai antiserum or anti-SasA antiserum.

In WT cells, the KaiB and KaiC levels oscillate under LL conditions in a circadian manner, whereas KaiA and SasA proteins accumulate constitutively (8, 10). Consistent with our bioluminescence and Northern blot analyses, circadian rhythms in the KaiB and KaiC levels were abolished with lowered expression levels in the rpaA-null mutant and in the sasA-null mutant (8), whereas the KaiA and SasA accumulation profiles were unaffected (Fig. 2B).

Circadian rhythm in the KaiC phosphorylation state is observed in WT cells by Western blot analysis (ref. 8 and Fig. 2B). Interestingly, accumulated KaiC protein was phosphorylated constitutively without evident circadian modulation in both sasA and rpaA mutant strains (Fig. 2B). Because KaiA is known to enhance the accumulation of phosphorylated KaiC (11–13), the constitutively elevated KaiA/KaiC ratio may account for the enhanced KaiC phosphorylation levels in both mutant strains. These results also suggest that both the SasA protein and the RpaA protein are necessary for accumulating the levels of KaiB and KaiC that are sufficient for driving a robust KaiC phosphorylation rhythm.

Phosphorelay from KaiC to the SasA–RpaA Two-Component System.

How do the SasA and RpaA proteins modulate kaiBC gene expression? In general, sensory kinases function with autophosphorylation and phosphotransfer activities at the beginning of a phosphorelay system that terminates by modifying function of the cognate RR. The closely similar circadian phenotypes and clock protein behaviors in sasA-null and rpaA-null mutants strongly suggest that SasA and RpaA are bona fide cognate partners in a two-component regulatory system. To validate this possibility, we examined phosphotransfer between SasA and RpaA in vitro. Initially, purified recombinant SasA protein was incubated with or without recombinant RpaA protein in the presence of [γ-³²P]ATP at 25°C for 60 min and then subjected to SDS/PAGE and autoradiography. SasA was radiolabeled in the presence of ATP because of its autophosphorylation activity (Fig. 3A Top), as shown in ref. 14. When the in vitro phosphorylation experiment was performed in the presence of RpaA, SasA's autophosphorylation signal was attenuated (Fig. 3A Middle). However, no incorporation of radioactive phosphate into RpaA could be detected, presumably because of rapid dephosphorylation of RpaA by SasA, as demonstrated for a typical Escherichia coli two-component system, EnvZ and OmpR (15). Interestingly, Smith and Williams (16) recently observed that the addition of KaiC accelerates in vitro SasA autophosphorylation activity. Thus, we tested whether addition of a recombinant KaiC protein could modify potential phosphotransfer activity from SasA to RpaA. KaiC activated the in vitro autophosphorylation activity of SasA dramatically and enhanced phosphotransfer from SasA to RpaA (Fig. 3A). We also tested whether aspartate phosphorylation of RpaA by means of SasA might be modified depending on the circadian phosphorylation/complex state of KaiC. To address this question, we reconstituted a KaiC phosphorylation cycle in vitro by incubating KaiA, KaiB, and KaiC recombinant proteins with ATP (7), and we then tested the effect of the reaction mixture on the SasA-to-RpaA phosphotransfer activity (see Materials and Methods). Initially, we confirmed that the phosphorylation state of KaiC was cyclically alternated on SDS/PAGE gels in the presence of KaiA, KaiB, and ATP (see autoradiogram in Fig. 3B). These reaction mixtures were collected every 4 h, immediately frozen, and stored. Then we performed in vitro SasA–RpaA phosphorylation/phosphotransfer experiments in the presence of each Kai reaction mixture (Fig. 3B). We found that both SasA autophosphorylation and SasA-to-RpaA phosphotransfer activities were modulated depending on the phase of the Kai reaction cycle in vitro. Both activities were maximal when the KaiC phosphorylation became active during the in vitro circadian cycle (Fig. 3B). These results strongly suggest that the Kai protein complex regulates the activity of RpaA protein cyclically in cells according to their complex/phosphorylation state and thereby activates (or represses) expression of RpaA's target genes in a circadian manner.

Fig. 3.

In vitro reconstitution of SasA–RpaA phosphorelay activated by phosphorylated KaiC. (A) Autophosphorylation of SasA and SasA–RpaA phosphorelay in the presence or absence of KaiC. (Top) SasA protein was incubated in the absence of RpaA, in the presence of RpaA, or in the presence of RpaA and KaiC in a buffer containing [γ-³²P]ATP. Each reaction was stopped at the indicated times and then analyzed by SDS/PAGE followed by autoradiography. (Middle and Bottom) Densitometric analyses of [³²P]SasA signals in the absence (▴) and presence (●) of RpaA or in the presence of RpaA plus KaiC (■) and of [³²P]RpaA signals in the presence of SasA only (×) and SasA plus KaiC (♦) are shown. (B) KaiC-mediated phosphotransfer signaling between SasA and RpaA in vitro. KaiC was incubated with KaiA and KaiB, collected at the indicated times, and stored. The time-fractionated samples were then incubated with SasA and RpaA in the presence of [γ-³²P]ATP at 25°C for 30 min. SDS/PAGE and autoradiography were performed. Below the autoradiogram, the densitometric analyses are shown as follows. (Top) Relative ratio of phosphorylated KaiC to total KaiC. (Middle) Relative amounts of [³²P]SasA. (Bottom) Relative amounts of [³²P]RpaA. Two independent experiments were performed for each combination; the black lines show plots of average values.

Residual Transcription Rhythms in the Absence of SasA or RpaA.

In sasA-null mutant strains, a residual unstable kaiBC bioluminescence rhythm with a period length shortened by ≈3 h is observed under LL with lower light intensity [15 μE·m⁻²·s⁻¹ (8)]. This light-intensity-dependent attenuation of the bioluminescence rhythm in the sasA-null strain was also observed for many clock-controlled genes (Table 1 and Figs. 1B and 6B). In the WT strain, purF::luxAB bioluminescence rhythm peaks at subjective dawn, in contrast to dusk-peaking genes such as kaiBC. Interestingly, such phase-angle difference among clock-controlled genes was abolished in sasA-null mutant cells (Figs. 1B and 6B). Thus, SasA is involved in phase control in this cyanobacterial circadian system, but the mechanism that drives different phasic expression profiles remains obscure. By contrast, almost all gene promoter activities remain arrhythmic in the rpaA-null mutants (Table 1 and Figs. 1B and 6B). Most HKs have both kinase and phosphoryl-protein phosphatase activities (17). Thus, the activity of its cognate RR can remain residually even in the absence of the phosphatase activity of HK, possibly accounting for the more severe phenotype in the rpaA-null mutants than in the sasA-null mutants. Nevertheless, the residual rhythmicity in the sasA-null mutants demonstrates the presence of some cryptic output pathways that are sustained without SasA function.

Table 1.

Bioluminescence rhythms of PkaiBC/PpsbAI-reporter strains

Genotype	Reporter cassette	Light condition, μE·m−2·s−1	Number rhythmic	Average amplitude, mean ± SD	Average period, mean ± SD
WT	PkaiBC::luxAB	50	19/19 (100)	11.9 ± 3.4	24.8 ± 0.4
	PpsbAI::luxAB	50	19/19 (100)	9.1 ± 3.9	25.6 ± 0.6
	PkaiBC::luxAB	15	8/8 (100)	5.5 ± 1.0	25.6 ± 0.3
	PpsbAI::luxAB	15	8/8 (100)	2.3 ± 0.7	25.3 ± 0.7
ΔsasA	PkaiBC::luxAB	50	0/91 (0)	n.r.	n.r.
	PpsbAI::luxAB	50	6/32 (18)	1.2 ± 0.1	22.8 ± 0.5
	PkaiBC::luxAB	15	32/32 (100)	1.7 ± 0.2	23.0 ± 0.3
	PpsbAI::luxAB	15	24/24 (100)	1.3 ± 0.1	22.7 ± 0.7
ΔrpaA	PkaiBC::luxAB	50	0/35 (0)	n.r.	n.r.
	PpsbAI::luxAB	50	0/32 (0)	n.r.	n.r.
	PkaiBC::luxAB	15	3/24 (12)	1.2 ± 0.1	22.6 ± 0.5
	PpsbAI::luxAB	15	5/24 (20)	1.3 ± 0.2	23.0 ± 0.7

Values under “Number rhythmic” indicate the number of rhythmic culture plates from the total that had an analytically extracted amplitude >1.1 (percentages are given in parentheses). Amplitude was defined as an average peak-to-trough bioluminescence signal ratio during the second and third cycles from each culture plate. “Average amplitude” is the average amplitude extracted from all of the culture plates. “Average period” is the average period extracted from all of the culture plates showing a circadian period in a population. n.r., no rhythmicity.

Next, we examined KaiB, KaiC, and SasA accumulation and KaiC phosphorylation profiles in the sasA and rpaA mutants under lower-light LL conditions. As shown in Fig. 6C, KaiB and KaiC protein levels in both mutants remained lower than in the WT strain. Again, the phosphorylated form of KaiC was dominant over the nonphosphorylated form. Therefore, the residual rhythmicity observed only under LL in both mutants cannot be caused simply by the attenuated accumulation of KaiB and KaiC proteins (see Discussion).

Growth of sasA- or rpaA-Disrupted Cells Under Light/Dark (LD) Cycles.

Previously, we observed that inactivation of sasA did not affect the growth rate of Synechococcus under various LL conditions, whereas the sasA-disrupted strain grows much slower than the WT or a kaiABC-deficient strain under LD cycles [periodic alteration of 12-h light and 12-h darkness (8)]. These results suggested an additional role for SasA in adapting the cell's metabolism specifically to natural light and dark transitions. We examined whether RpaA is also involved in this type of SasA-mediated physiology by comparing the growth phenotypes of the WT, sasA-deficient, and rpaA-deficient strains under LL and LD conditions. As shown in Fig. 7, which is published as supporting information on the PNAS web site, both sasA-deficient and rpaA-deficient strains grow as well as the WT strain under LL conditions, whereas they grow much slower than the WT cells under LD cycles. These results further support the hypothesis that SasA and RpaA are bona fide two-component partners, participating in both circadian output regulation and metabolic control.

Discussion

From a Posttranslational Oscillator to Transcription Rhythms.

Our results strongly support the hypothesis that the SasA–RpaA two-component regulatory system is the primary clock output that is necessary for coordinating genome-wide circadian gene expression with proper phase relationship and period length and for maintaining the robust oscillation of KaiC phosphorylation (Fig. 4).

Fig. 4.

Schematic representation of the SasA–RpaA functions in the cyanobacterial circadian system. See Discussion for details.

There are LxxxExxxL, LR, and TxxGxGY DNA-binding motifs that are characteristic of OmpR-type proteins in the RpaA aminoacyl sequence (ref. 9 and Fig. 5). E. coli OmpR protein regulates only a subset of genes responding to osmotic stimuli (15). Therefore, it is plausible that Kai-mediated activation of the presumed DNA-binding protein RpaA accelerates transcription of its target gene(s), although biochemical analysis of its DNA-binding activity has not yet been reported. Considering the genome-wide transcription rhythms regulated by the SasA–RpaA system (Fig. 1), one possible class of RpaA target genes may include master transcription regulators such as sigma factor genes and/or transcription factors for driving transcriptional cascades. In this context, any dramatic reduction in the promoters of sigC and rpoD6 genes in the rpaA-null mutant (Fig. 1) would severely affect a number of downstream genes. Another class of potential targets would be factors that regulate chromosome compaction (18). In any case, the search for the RpaA-binding promoter will provide a further clue to understanding the cyanobacterial circadian system. It should be noted that RpaA did not exhibit significant affinity to the kaiBC promoter by the gel-shift analysis in our experimental conditions (data not shown).

Our biochemical analysis demonstrated that the magnitude of SasA–RpaA phosphorelay changed depending on the phase of the coexisting Kai protein cycle (Fig. 3B). Interestingly, the time course profiles demonstrated that ³²P incorporation into SasA and RpaA was maximal in the presence of the Kai mixture before the KaiC phosphorylation peak was reached. Therefore, it is unlikely that KaiC, with a minimal or maximal phosphorylation state in itself, is most effective at accelerating the phosphorelay from SasA to RpaA. Instead, some biochemical reaction states that enhance the KaiC phosphorylation process would be more important for activating the SasA–RpaA system. In this respect, under LL conditions, KaiC phosphorylation peaks at hour 16 in LL in Synechococcus, whereas transcription rhythms of most genes peak at LL hour 10–12, at which time the SasA–RpaA activities presumably become maximal.

Presence of Secondary Output Pathway(s).

Because the circadian KaiC phosphorylation cycle can be reconstituted with three Kai proteins and ATP in vitro in the absence of SasA and RpaA (7), nullification of KaiC protein cycles in the sasA and rpaA mutants seems primarily caused by dramatic reductions in the accumulated levels of KaiB and KaiC. Nevertheless, residual unstable kaiBC promoter rhythmicity was observed, if not always, in both sasA and rpaA mutants exclusively under dim LL conditions (Table 1). Because the KaiC phosphorylation rhythm was obscure in these mutants under such low-light conditions (Fig. 6C), very weak posttranslational cycles in KaiC phosphorylation and/or Kai–protein interactions may be sufficient to drive transcription cycles. Note that in the recently isolated kaiC^pr1 mutant, the amplitude of KaiC phosphorylation rhythm becomes very low, whereas that of the P_kaiBC::luxAB bioluminescence rhythm remains less affected (19). Although we previously suggested that the KaiC phosphorylation cycle was the core process for generating a basic oscillation (6, 7), these results may implicate that an additional, as yet unknown process also contributes to transcription rhythms. Alternatively, it is possible that only a small set of cells produced sufficient amounts of KaiB and KaiC proteins stochastically to gain both phosphorylation and bioluminescence rhythms, whereas bulk biochemical analysis might fail to detect such a single-cell level phosphorylation rhythm.

As documented above, sasA-null mutants produce unstable, short-period transcription rhythms, whereas rpaA-null mutants are more arrhythmic under dim LL conditions (Table 1 and Figs. 1B and 6B). This phenotypic difference between sasA and rpaA mutants may be explained by the more severe phenotype of RR mutants than that of cognate HK mutants (see above). Another HK may sense time signals from KaiC to mediate phosphorelay to RpaA in the absence of SasA. Furthermore, very unstable, low-amplitude rhythms with low penetrance in the rpaA-null mutants (Table 1) suggest that there may be another minor clock output pathway functioning without the SasA–RpaA function (Fig. 4).

SasA and RpaA as Adaptive Systems for LD Cycles.

Growth is severely suppressed in a sasA-disrupted strain (8) as well as in a rpaA-disrupted strain only under LD cycles (Fig. 7). These results suggest that sasA is important for normal growth under natural diurnal conditions. In a different unicellular cyanobacterial strain, Synechocystis sp. PCC 6803, rpaA was originally identified by mutant screening for abnormal energy transfer from phycobilisomes to photosystems (9): Disruption of rpaA and rpaB affected energy transfer from phycobilisome to photosystem I and photosystem II, respectively. Although both OmpR-type RRs were originally thought to belong to the Ycf27 family of RR members, the same researchers showed that RpaA could not be the member responsible by reexamining its sequence in detail (20). As documented above, we failed to obtain mutant cells in which rpaB was disrupted in Synechococcus. Instead, we generated an inducible dominant-negative rpaB mutant and found that this mutant was also defective in growth under LD cycles but that its circadian cycling remained essentially unaffected (H.I., unpublished data). Therefore, the effect of sasA and rpaA disruption on circadian function alone cannot explain the adaptation to diurnal growth and vice versa.

The Synechocystis rpaA gene, also known as rre31, is involved in the induction of a subset of genes by hyperosmotic and salt stresses (21). Microarray analysis suggested that a candidate partner of Rre31 in the stress response would be Hik33 (21). Therefore, we tried to generate a Synechococcus strain that was deficient in the hik33 homolog but could not obtain null mutant cells, suggesting its essential role in our standard LL and LD conditions (H.I. and R.K., unpublished data). The effects of signal cross-talk among His-to-Asp phosphorelay factors on circadian systems and other cellular functions would be interesting topics to be further addressed.

Materials and Methods

Bacterial Strains, Media, and Culture.

S. elongatus PCC 7942 and its derivatives used in this study are listed in Tables 2 and 3, which are published as supporting information on the PNAS web site. These strains were grown in a modified BG-11 liquid medium [BG-11M (8)] for RNA and protein preparations or on BG-11 solid media (1.5% agar) for bioluminescence assays at 30°C under LL by using white fluorescent lamps (50 μmol·m⁻²·s⁻¹ or 15 μmol·m⁻²·s⁻¹).

Construction of Bioluminescence Reporter Plasmids.

Targeting plasmids for various bioluminescence reporters were constructed by using the MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen, Carlsbad, CA). Briefly, the upstream region of the gene of interest (listed in Table 3) was fused to a promoterless Vibrio harveyi luxAB operon with a spectinomycin-resistant gene (Ω). This promoter–reporter cassette was cloned into the XhoI site of the neutral site I (22) segment from the Synechococcus genome.

Genetic Deletion of RR Genes.

Genetic disruption of three hybrid kinase genes that contained the RR domain with the HK motifs (syc0532_d, syc0681_c, and syc2278_d) was performed by insertion of the Ω cassette. Briefly, each of the coding segments were cloned into the pGEM-T vector (Promega, Madison, WI). Then the Ω cassette was inserted into the RR genes on each vector by using the GPS-M Mutagenesis System (NEB, Beverly, MA) (Table 2). Each of the other 21 RR genes (a list is shown in Table 2) was deleted by substitution with a kanamycin-resistant gene (km^r) as described in ref. 23. Each of the resulting inactivation cassettes and vectors was introduced into a kaiBC::luxAB reporter strain, NUC42 (24).

Bioluminescence Monitoring.

Bioluminescence assays and analysis were performed as described in ref. 8.

Northern Blot Analysis.

Total RNA (5 μg) from Synechococcus cells was prepared by using the hot phenol method, followed by Northern blotting analysis using a digoxigenin-labeled kaiBC probe as described in ref. 8.

Western Blot Analysis.

Western blotting analysis was performed as described in ref. 6.

Bacterial Expression and Purification of the KaiA, KaiB, KaiC, SasA, and RpaA Proteins.

Recombinant KaiA, KaiB, and KaiC proteins were produced in E. coli and purified as described in ref. 7. For the expression and purification of SasA and RpaA, we cloned the S. elongatus sasA and rpaA genes into the BamHI–SmaI and EcoRI sites of the pGEX-6P-1 vector (Amersham Pharmacia Biosciences, Buckinghamshire, U.K.), respectively, and then introduced them to E. coli strain BL21. Cells expressing either the SasA or RpaA protein were collected and disrupted in an extraction buffer (300 mM NaCl/20 mM Tris·HCl/1 mM EDTA, pH 8.0) by sonication. After addition of benzonase nuclease (Merck, Darmstadt, Germany) and 1% Triton X-100, the homogenate was centrifuged at 38,000 × g. The supernatant was applied to a glutathione Sepharose 4B column (Amersham Pharmacia Biosciences), washed with five column volumes of the buffer, and then applied with PreScission Protease (Amersham Pharmacia Biosciences) to remove the GST tag. SasA and RpaA were eluted with one column volume of extraction buffer, and the eluent was diluted and applied to a Resource Q column (Amersham Pharmacia Biosciences). After washing with the buffer (60 mM NaCl/20 mM Tris·HCl/0.5 mM EDTA, pH 8.0), proteins were eluted with a 60–450 mM NaCl gradient. SasA was further purified by Superose 6 gel filtration chromatography with buffer (150 mM NaCl/20 mM Tris·HCl/0.5 mM EDTA, pH 8.0). Protein concentration was determined by the Bradford method with BSA as a standard. Purity of SasA and RpaA was ≥95% as determined by SDS/PAGE.

Phosphotransfer Assays.

For the experiments shown in Fig. 3A, phosphotransfer assays were performed as described in ref. 15 with slight modifications. In the presence or absence of 1.7 μM KaiC and RpaA proteins, SasA (1.7 μM) was incubated in 10 μl of TEDG buffer [20 mM Tricine-NaOH/0.5 mM EDTA/0.5 mM DTT/10% glycerol (vol/vol), pH 8.0] containing 0.05 mM [γ-³²P]ATP (10,000 cpm/pmol), 5 mM MgCl₂, and 150 mM KCl. After incubation at 25°C, the reaction was stopped by addition of 5 μl of an SDS buffer. After heating at 95°C for 5 min, samples were subjected to SDS/PAGE by using 10% gels. The radioactive levels of phosphorylated proteins were analyzed by using a BAS2000 Image Analyzer (Fuji, Tokyo, Japan). For the experiments shown in Fig. 3B, KaiC (17 μM) was incubated with KaiA (6 μM) and KaiB (17 μM) in a reaction buffer (20 mM Tris·HCl/150 mM KCl/0.5 mM EDTA/5 mM MgCl₂/1 mM ATP, pH 8.0) at 30°C. Aliquots (1 μl) of the reaction mixture were collected at the indicated times and stored at −80°C. The time-sampled Kai reaction mixture was then incubated with SasA (1.7 μM) and RpaA (1.7 μM) in 10 μl of reaction buffer [20 mM Tricine-NaOH/0.5 mM EDTA/0.5 mM DTT/10% glycerol (vol/vol)/0.1 mM [γ-³²P]ATP (10,000 cpm/pmol)/5 mM MgCl₂/150 mM KCl, pH 8.0] at 25°C for 30 min. SDS/PAGE and autoradiography were performed as described above.

Abbreviations

LL

continuous light

LD

light/dark

μE

microeinstein

HK

histidine kinase

RR

response regulator