Abstract
Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterised the enzymatic activity of KaiC3 as a representative of non-standard KaiC homologs in vitro. KaiC3 displayed ATP synthase and ATPase activities, which were lower compared to the Synechococcus 7942 KaiC protein. ATP hydrolysis showed no temperature compensation. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1 and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a crosstalk between the KaiC1 and KaiC3 based systems.
Importance The circadian clock is known to influence the cyanobacterial metabolism globally and a deeper understanding of its regulation and fine-tuning will be important for metabolic optimizations in context of industrial applications. Due to the heterogeneity of cyanobacteria, the characterization of clock systems in organisms apart from the standard circadian model Synechococcus elongatus sp. PCC 7942 is required. Synechocystis sp. PCC 6803 is of special interest, because it represents a major cyanobacterial model organism and harbors diverged homologs of the clock proteins, which are present in various other non-cyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize the enzymatic activities of the non-standard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness suggesting its involvement in the regulation of non-photosynthetic metabolic pathways.
Introduction
Cyanobacteria have evolved the circadian clock system to sense, anticipate and respond to predictable environmental changes based on the rotation of Earth and the resulting day-night cycle. Circadian rhythms are defined by three criteria: (i) oscillations with a period of about 24 hours without external stimuli, (ii) synchronization of the oscillator with the environment and (iii) compensation of the usual temperature dependence of biochemical reactions, so that the period of the endogenous oscillation does not depend on temperature in a physiological range (1). The cyanobacterial circadian clock system has been studied in much detail in the unicellular model cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). Its core oscillator is composed of three proteins, which are unique to prokaryotes: KaiA, KaiB, and KaiC (from now on KaiA7942, KaiB7942 and KaiC7942; please note that some of the hereafter cited information were gained studying proteins from Thermosynechococcus elongatus BP-1 though) (2). The level of KaiC7942 phosphorylation and KaiC7942’s ATPase activity represent the key features of the biochemical oscillator. KaiA7942 stimulates autophosphorylation and ATPase activity of KaiC7942, whereas KaiB7942 binding to the complex inhibits KaiA7942 action, stimulates autodephosphorylation activity and reduces ATPase activity of KaiC7942 (3–5). As a consequence of dynamic interactions with KaiA7942 and KaiB7942, KaiC7942 rhythmically phosphorylates and dephosphorylates with a 24-hour period (2).
KaiC7942 consists of two replicated domains (CI and CII) which assemble into a hexamer forming an N-terminal CI ring and a C-terminal CII ring (6–8). Phosphorylation takes place in the CII ring (9), whereas ATP hydrolysis occurs in both rings (10). In the CII ring, ATP hydrolysis is part of the dephosphorylation mechanism (11, 12). ATP hydrolysis in the CI ring correlates with the period of the clock and temperature compensation and is further required for a conformational change of KaiC7942, which allows binding of KaiB7942 (4, 13, 14). The levels of phosphorylation and ATP hydrolysis of the KaiC7942 serve as the read-out for regulatory proteins, which orchestrate the circadian output (15, 16). For a recent review on the functioning of the KaiABC7942 system, see Swan et al. (17).
Cyanobacteria represent one of the most diverse prokaryotic phyla(18) and little is known about timekeeping mechanisms in other cyanobacteria than Synechococcus 7942. A core diurnal genome has been described (19), but temporal coordination varies and, based on genomic analyses, large variations in the cyanobacterial clock systems can be expected (20–25).
The cyanobacterial model strain Synechocystis sp. PCC 6803 (from now on Synechocystis 6803) contains a standard kai operon, encoding homologs of the kaiA, kaiB and kaiC genes (in the following designated kaiA6803, kaiB16803 and kaiC16803) as well as two additional kaiB and kaiC genes (26). The kaiC26803 and kaiB26803 genes form an operon, whereas kaiC36803 and kaiB36803 are orphan genes. Bioinformatic studies revealed that all cyanobacteria, which do contain at least one kaiB gene, always encode orthologs of KaiB1 and KaiC1. Additional KaiB3 and KaiC3 homologs are present in about one third of cyanobacterial genera, with a slightly higher occurrence of KaiC3. KaiC3 is also present in bacteria and archaea, showing a higher distribution than KaiC1 and KaiC2 homologs (19). The reported number of oscillating genes in Synechocystis 6803 varies largely between different studies, which is likely due to differences in growth conditions (27–30). These results imply that Synechocystis 6803 might be able to fine-tune its circadian rhythm in response to environmental conditions. In line with this, Aoki and Onai (21) suggested that KaiC36803 and KaiB36803 modulate the amplitude and period of the KaiAB1C16803 oscillator, whereas, to our knowledge, no function for kaiC2B26803 has been described so far.
The three KaiC proteins from Synechocystis 6803 show high conservation of the kinase motif in the CII domain and all three proteins were shown to exhibit autophosphorylation activity (19, 23). KaiA6803 stimulates the autophosphorylation of KaiC16803, but does not affect the phosphorylation of the two other KaiC homologs. Based on sequence analysis and experimental validation of kinase activity, Schmelling et al. (19) concluded that autokinase and autophosphatase activities are highly conserved features of all cyanobacterial KaiC homologs. Likewise, ATPase motifs in the N-terminal CI domain of KaiC are highly conserved in all cyanobacterial KaiC homologs (19). However, ATP hydrolysis was only characterised for several KaiC1 homologs and one non-cyanobacterial KaiC2 protein (10, 31–33).
In a natural day and night cycle, Synechococcus 7942 orchestrates its metabolism in a precise temporal schedule, with the metabolism being adjusted by the clock but also feeding input information to the clock (34–36). Knockout of the Synechococcus 7942 kai genes leads to a growth disadvantage. When grown in competition with the wild type under light-dark conditions, the clock deficient mutant cells are eliminated from the culture (37). In Synechocystis 6803 deletion of the kaiAB1C1 gene cluster causes reduced growth in light-dark rhythms, even when grown as single culture (38). A comparably strong impact on growth in a light-dark cycle was observed in both strains after deletion of the regulator of phycobilisome association A (RpaA) (34, 39), the master transcription factor of circadian gene expression. In addition, studies on single nucleotide polymorphisms identified the rpaA gene as one of three genes responsible for the faster growth of Synechococcus elongatus UTEX 2973 compared to Synechococcus 7942(40). Using a transposon library, David et al. (41) showed that KaiA, despite being non-essential for growth in light-dark cycles, strongly contributes to the fitness of Synechococcus 7942 under these conditions. Decreased fitness most likely occurs due to reduced phosphorylation of RpaA in the kaiA knockout strain (41). Overall, these data demonstrate the value of the cyanobacterial clock system for metabolic orchestration under natural conditions, with the clock system considered especially important for the transition from light to darkness (42).
Here, we aim at a detailed biochemical characterization of KaiC36803 and the role of Synechocystis 6803 KaiB6803 homologs in modulation of KaiC36803 function. Further, we demonstrate that the ΔkaiC3 mutant strain has a growth defect under chemoheterotrophic growth conditions, which is similar, but less pronounced compared to ΔkaiAB1C1 and ΔrpaA strains (38, 39). Our data support the idea of a function of KaiC36803 and KaiB36803 in fine-tuning the central oscillator composed of KaiA6803, KaiB16803 and KaiC16803 in Synechocystis 6803.
Results
Recombinant kaiC36803 displays ATP synthase activity
Previous bioinformatic analysis predicted that kinase, dephosphorylation and ATPase activities are conserved in KaiC36803 (19). So far, only the kinase activity of KaiC36803 was experimentally confirmed which differed from the activity of KaiC7942 by lacking stimulation by KaiA7942 and KaiA6803 (23). Therefore, we aimed at the characterization of further predicted activities of KaiC36803.
The first step in KaiC7942 dephosphorylation is regarded as a reversal of phosphorylation: KaiC7942 transfers its bound phosphoryl group to ADP and thereby synthesizes ATP (11, 12). To investigate reversibility of intrinsic phosphorylation, we tested whether KaiC36803 can synthesize [α-32P]ATP from [α-32P]ADP (Fig. 1). As control, we used phosphorylated and dephosphorylated KaiC7942 (Fig. S1.). Immediately after adding [α-32P]ADP, all KaiC proteins started to synthesize [α-32P]ATP. Phosphorylated as well as non-phosphorylated KaiC7942 both showed higher initial ATP synthesis than KaiC36803 (Fig. 1). After incubating phosphorylated and dephosphorylated KaiC7942 for 2 hours at 30°C with [α-32P]ADP, a relative [α-32P]ATP level of 26-28 % was detected. KaiC36803 was also capable of intrinsic ATP synthesis, but compared to KaiC7942 less than one third of relative [α-32P]ATP was formed. This can be explained by two hypotheses: (i) KaiC36803 showed lower phosphotransferase activity or (ii) higher consumption of the produced ATP. The latter would require that KaiC36803 exhibits higher ATPase activity than KaiC7942. Therefore, we next examined ATP hydrolysis activity of KaiC36803.
kaiC36803 displays ATPase activity
ATP hydrolysis can be measured by quantifying ADP production in the presence of KaiC over time (4). Conservation of WalkerA and WalkerB motifs in the CI domain of KaiC36803 proteins suggested their capability to hydrolyze ATP (19). We confirmed the predicted ATPase activity in vitro using Strep-KaiC36803, which produced 8.5 ± 1.0 ADP molecules per monomer and day (mean ± s.d., Fig. 2). This activity was about 45 % lower than the one measured for KaiC7942 (19.1 ± 3.3 ADP per day (4)). Hence, the above-observed lower level of net ATP production by KaiC36803 was not based on a higher ATP consumption, but due to lower dephosphorylation activity per se. In KaiC7942, the ATPase activity changes after substitution of the phosphorylation sites (4). We therefore generated variants of Strep-KaiC36803, in which residues S423 and T424 are either replaced with aspartate and glutamate (Strep-KaiC36803-DE) or replaced with two alanine residues (Strep-KaiC36803-AA). Strep-KaiC36803-AA, showed more than two fold increased ATPase activity, whereas ATP hydrolysis by Strep-KaiC36803-DE was only slightly different from the wild-type protein (Fig. 2) and not reduced as reported for KaiC7942 (4).
kaiC36803 interacts with kaiB36803 and components of the standard KaiAB1C16803 oscillator
In vitro and in silico studies suggested that KaiA6803, KaiB16803 and KaiC16803 form the standard clock system of Synechocystis 6803 (21, 23). Since Aoki and Onai (21) suggested that KaiC36803 might modulate the main oscillator function, we performed protein-protein interaction studies in order to reveal a possible crosstalk between the multiple Kai proteins. First, interaction was determined by yeast-two hybrid analysis using KaiC16803, KaiC36803, KaiB16803 and KaiB36803 fused to AD and BD domains, respectively. The color change of the colonies indicates β-galactosidase activity and is an estimate for the interaction of the respective two proteins. As expected, these experiments showed self-interaction of KaiC36803 (Fig. 3A), since KaiC homologs are known to form hexamers (6, 8). In addition, an interaction of KaiC36803 with KaiB36803 was detected (Fig. 3A) which is in line with bioinformatic analysis showing frequent co-occurrence of kaiC3 and kaiB3 genes in genomes (19). We could not detect an interaction between KaiC36803 and KaiA6803 by yeast-two hybrid analysis (Fig. 3B), but detected a heteromeric interaction between KaiC16803 and KaiC36803 using different protein fusion variants (Fig. 3C). These results confirm previous in vivo data, which showed co-purification of KaiA6803 with KaiC16803 but not with KaiC36803, and a weak interaction between the two KaiC homologs KaiC16803 and KaiC36803 (23). Besides KaiC16803, also its respective KaiB protein, KaiB16803, showed an interaction with KaiC36803 in our analysis (Fig. 3C), corroborating the hypothesis that there is a cross talk between the putative KaiC36803-B36803 system and the core oscillator KaiAB1C16803. Such a possible cross talk via the KaiB proteins was further supported by our in vitro pull-down assays, in which KaiC36803 interacted with KaiB36803 (Fig. S3B,D) and further with KaiB16803 (Fig. S3B,D). Also KaiC16803 interacted with both, KaiB16803 and KaiB36803 homologs (Fig. S3,A,C). One must take into account that based on the in vitro pull-down assays using Synechocystis 6803 whole cell extracts we cannot exclude indirect interactions. As we have shown that KaiC16803 and KaiC36803 interact with each other, both proteins could bind as a hetero-hexamer to the GST-tagged KaiB6803 proteins and therefore co-elute from the affinity matrix.
ATPase activity of kaiC36803 is reduced in the presence of kaiB16803 and KaiB36803
The interaction of KaiC36803 with KaiB16803 and KaiB36803 (Fig. 3 and Fig. S3) suggested a regulation of KaiC36803 activity by these two KaiB proteins. We therefore measured ATP hydrolysis by Strep-KaiC36803 in the presence of KaiB16803 and KaiB36803 proteins, respectively (Fig. 4A). After size exclusion chromatography KaiB16803 was mainly eluted as tetramer (44 and 72 kDa, respectively, depending on the column), whereas KaiB36803 was eluted as monomer (13/23 kDa) and tetramer (41/70 kDa) (Fig. S4). Therefore, the monomeric and tetrameric KaiB36803 fractions were tested separately. In all measurements, the ATPase activity was linear and showed no oscillations. ATP hydrolysis was reduced by 55 % after the addition of the KaiB36803 monomer, but not affected by the KaiB36803 tetramer. The KaiB16803 tetramer also inhibited the ATPase activity of Strep-KaiC36803, though to a lesser extend (35 % reduction compared to Strep-KaiC36803 alone, Fig. 4). Because ATPase activity of KaiC7942 is influenced by KaiA7942 (4), we also investigated the effect of KaiA6803 on KaiC36803, but in line with the lack of interaction in our yeast two-hybrid analysis data (Fig. 3B), ATPase activity of Strep-KaiC36803 was not clearly affected by KaiA6803 (Fig. 4A).
ATPase activity of kaiC36803 is temperature dependent
True circadian clocks are characterized by temperature compensated oscillations, which ensure robust time measurements under temperature fluctuations. In the Synechococcus 7942 KaiABC clock, overall temperature compensation is derived from KaiC7942’s ATPase activity, which is stable between 25 and 35 °C (Q10=1.2, (4)). We therefore asked whether N-Strep-KaiC36803, as a representative of non-standard KaiC homologs, shows temperature compensation as well. Measurements at 25, 30 and 35 °C, however, revealed a temperature-dependent ADP production by KaiC36803 (Q10=2.4, Fig. 4B; activation energy = 66.5 kJ mol−1, Fig. S5A). Hence, KaiC36803 is lacking a characteristic feature of circadian oscillators. In accordance, dephosphorylation of Strep-KaiC36803 was higher at 25 °C than at 30 and 35 °C (Fig. S5B)
The non-standard kaiC36803 protein supports growth of Synechocystis 6803 cells in the dark
As in vitro analyses suggested an interaction of KaiC36803 with KaiB16803 and KaiC16803, we aimed at elucidating the role of KaiC36803 and its crosstalk with the putative main oscillator in the cell. Clock factors are reported to be essential for cell viability in light-dark cycles (34, 39, 41). Accordingly, deletion of the kaiAB1C1 cluster of Synechocystis 6803 resulted in growth defects in light-dark cycles (38). Lower viability of the ΔkaiAB1C1 strain was more pronounced under photomixotrophic (0.2 % glucose) compared to photoautotrophic conditions, whereas deletion of kaiC3 had no effect on cell viability in light-dark cycles (38). As KaiC3 homologs are also present in non-photosynthetic bacteria (19), we were interested in the growth of the ΔkaiC3 strain in the dark. The Synechocystis 6803 WT strain used here, in contrast to previous studies (43), is able to grow in complete darkness when supplemented with glucose (39). We therefore analyzed the viability of ΔkaiC3 cells via spot assays (44) in constant light and in complete darkness on agar plates containing 0.2 % glucose (Fig. 5). Under photomixotrophic growth conditions with continuous illumination, wild type and ΔkaiC3 showed similar viability, whereas the viability of the ΔkaiAB1C1 strain was reduced. Further, in complete darkness, wild type and ΔkaiC3 displayed detectable growth, while growth of ΔkaiAB1C1 was abolished completely. Growth inhibition of the ΔkaiAB1C1 mutant under mixotrophic conditions in the light and in the dark highlights the importance of the KaiAB1C16803 oscillator for the switch between these two different metabolic modes of Synechocystis 6803. Comparing wild-type and ΔkaiC3 strains, the latter showed reduced growth in complete darkness. Thus, KaiC36803 seems to be linked to dark adaption of Synechocystis 6803 cells, yet not as essential as the core oscillator KaiAB1C16803.
Discussion
Characterization of enzymatic activities of KaiC36803
Previous sequence analysis suggested that the three enzymatic activities of KaiC7942, which are autophosphorylation, autodephosphorylation and ATPase activity, are conserved in all cyanobacterial KaiC proteins (19). This hypothesis is supported by experiments demonstrating autophosphorylation of all KaiC6803 proteins (23), different cyanobacterial KaiC1 and KaiC3 homologs (19, 31, 33), and even non-cyanobacterial KaiC homologs from Rhodopseudomonas palustris (32), Legionella pneumophila (45) as well as two thermophilic Archaea (19). In the current paper, we provide experimental evidence that dephosphorylation and ATPase activities are conserved in KaiC36803 as well. However, the level of the ATPase activity seems to vary between KaiC proteins. While ATPase activity of the KaiC from Prochlorococcus marinus MED4 was reported to have the same activity as KaiC7942 (31), we show here that the ATPase activity of KaiC36803 is lower. In contrast, ATP hydrolysis activity of the KaiC2 homolog from Legionella pneumophila and KaiC1 homolog from Gloeocapsa sp. PCC 7428 were reported to be elevated when compared to the standard KaiC7942 protein (32, 33).
ATPase activity of KaiC36803 further differed from that of KaiC7942, by lacking temperature compensation (Fig. 4). Therefore, KaiC36803 cannot be the core component of a true circadian oscillator. This is further supported by differences in the ATP hydrolysis rate between the phosphomimetic variants of KaiC7942 and KaiC36803. ATPase activity of the true clock protein KaiC7942 depends on its phosphorylation status. In the KaiC7942-AA variant, which mimics the dephosphorylated state of KaiC7942, the ATPase activity is higher than average, whereas, in KaiC7942-DE, mimicking the phosphorylation state, it is diminished (4). In our analysis, ATP hydrolysis by Strep-KaiC36803-AA was similarly increased in comparison to Strep-KaiC36803. However, Strep-KaiC36803-DE did not show reduced activity as was shown for KaiC7942-DE, implying that the phosphorylation state of KaiC36803 does not influence KaiC36803’s ATPase activity.
However, the ATPase activity of Strep-KaiC36803 was affected decisively by the addition of KaiB36803 and KaiB16803. Both KaiB homologs led to decreased ATPase activity of Strep-KaiC36803, with KaiB36803 having a stronger impact (Fig. 4A). KaiB7942 exists as a monomer or a tetramer in solution (46). As a tetramer, KaiB7942 subunits adopt a unique fold, which was also observed in crystals of KaiB16803 (47). Notably, KaiB7942 binds as a monomer to KaiC7942, in which it adopts a different, thioredoxin-like fold (48–50). The formation of KaiB7942 tetramers is concentration-dependent and thereby ensures that the effective monomer concentration is stable over a broad range of total KaiB7942 concentrations (46). This points at a putative mechanism how KaiC36803 might fine-tune the standard oscillator: Binding of KaiB16803 to KaiC36803 will reduce the free KaiB16803 concentration in the cell and thereby influence the KaiAB1C16803 oscillator. In contrast, only the monomeric form of KaiB36803 had a regulatory effect on ATP hydrolysis of KaiC36803 suggesting that KaiB36803 bound as a monomer to KaiC36803, but no transition from tetramer to monomer occurred. This finding is surprising, because residues K57, G88 and D90, which are important for fold switching of KaiB7942 (50), are conserved in KaiB36803. However, we show only in vitro data here. In the cell, protein concentrations as well as spatial and temporal separation of the different proteins might have an influence on these interactions. In addition, we do not take possible heteromeric interactions among the KaiB proteins into account.
Function of KaiC36803 in an extended network
Our interaction studies imply a crosstalk between KaiC16803 and KaiC36803 (Fig. 6). KaiC1 is believed to form a standard oscillator together with KaiA6803 and KaiB16803 (21, 23), which is supported by the interaction with KaiB16803 observed in this paper (Fig. 3C, Fig. S3). We further hypothesize that KaiC36803 acts in a separate non-circadian regulatory system together with KaiB36803. In accordance with bioinformatic analysis, which showed a significant co-occurrence of KaiB3 and KaiC3 in Cyanobacteria (19), KaiB36803 had a stronger effect on the ATPase activity of KaiC36803 than KaiB16803 (Fig. 4A). Since KaiC36803 is able to form hetero-oligomers with KaiC16803 and was shown to interact with KaiB16803 as well, interference between the KaiC36803-KaiB36803 system and the standard oscillator is very likely. However, we exclude that KaiA6803 is involved in the putative crosstalk by four reasons: (i) KaiA6803 did neither stimulate ATP hydrolysis nor kinase activity of KaiC36803 (this paper and Wiegard et al. (23)); (ii) We were not able to show an interaction of KaiC36803 and KaiA6803 using different approaches (this paper and Wiegard et al. (23)); (iii) KaiA interacting residues are not conserved in cyanobacterial KaiC3 homologs, and (iv) KaiC3 homologs are present in organisms which do not harbor KaiA (19).
The growth defect of the mutant strains, ΔkaiAB1C1 and ΔkaiC3, in complete darkness suggests that the here proposed putative KaiC3-KaiB3 system and the KaiAB1C16803 oscillator may target similar cellular functions. The metabolic network of cyanobacteria is described as temporally partitioned with extensive effects of day-night transitions, involving shifts in ATP and reductant levels and alterations of the carbon flux (41). In Synechococcus 7942, environmental signals can be fed into the main clock output system via the transcriptional regulator RpaB (51). In contrast to Synechococcus 7942, Synechocystis 6803 is able to grow in complete darkness, which adds another layer of complexity to day-night transitions and demand for further regulatory elements. The here observed impaired mixotrophic growth of the kaiC36803 mutant in darkness supports the idea of KaiC36803 and KaiB36803 functioning as such additional elements to adjust the state of the main Synechocystis 6803 oscillator. Conversely, it is also possible that KaiC36803 function is controlled by the KaiAB1C16803 clock system. Köbler et al. (39) demonstrated that solely KaiC16803, but not KaiC36803, interacts with the main output histidine kinase SasA in the Synechocystis 6803 timing system. Thus, in Synechocystis 6803, only the main oscillator feeds timing information into the SasA-RpaA output system to control the expression of many genes involved in dark growth (39). The output pathway for KaiC36803 is unknown so far and it might be possible that the only function of KaiC36803 is to modulate the function of the main oscillator in response to a yet unknown input factor.
Material and Methods
Cloning, expression and purification of recombinant Kai proteins
In this paper, we name the Synechocystis kai genes according to the definition introduced by Schmelling et al. (19) (Please note that naming of KaiB3/KaiB2 and KaiC3/KaiC2 is not consistent in the literature). Genes encoding KaiB16803 (ORF slr0757) and KaiB36803 (ORF sll0486) were amplified from genomic Synechocystis 6803 wild type DNA using specific primers (Table S1) and Phusion Polymerase (New England Biolabs). After restriction digest with BamHI and NotI, amplified fragments were inserted into pGEX-6P1 (GE Healthcare) and the resulting plasmids were used for heterologous expression. For production of recombinant KaiC7942 as well as KaiA6803, KaiB16803, KaiB36803 and KaiC36803, we used pGEX-based plasmids described in Wiegard et al. (23) (see also Table S2 for a list of all plasmids used in this study). A detailed protocol of expression and purification can be found on protocols.io (https://dx.doi.org/10.17504/protocols.io.48ggztw). Briefly, proteins were expressed as GST-fusion proteins in E. coli BL21 [DE3] or NEB Express (New England Biolabs) and lysed in 50 mM Tris/HCl (pH8), 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT (+ 5 mM MgCl2, 1 mM ATP for KaiC proteins). Purification was performed via batch affinity chromatography using glutathione agarose 4B (Macherey and Nagel) or glutathione sepharose 4B (GE Healthcare) in the same buffer. Finally, the GST-tag was cleaved off using PreScission protease (GE Healthcare) in 50 mM Tris/HCl (pH8), 150 mM NaCl, 1 mM EDTA, 1 mM DTT (+ 5 mM MgCl2, 1 mM ATP for KaiC proteins). If homogeneity of the proteins was not sufficient, they were further purified via anion exchange chromatography using a MonoQ or ResourceQ column (GE Healthcare Life Sciences) using 50 mM Tris/HCl (pH8), 1 mM EDTA, 1 mM DTT (+ 5 mM MgCl2, 1 mM ATP for KaiC proteins) and a 0-1M NaCl gradient.
To produce Strep-KaiC36803 variants with amino acid substitutions, the kaiC36803 gene in the pGEX-kaiC36803 vector was modified by site directed mutagenesis using the Quick-Change Site-Directed Mutagenesis Kit (Stratagene) or Q5 Site-Directed Mutagenesis Kit (New England Biolabs). Base triplets encoding S423 and T424 were changed to code for alanine or for aspartate and glutamate resulting in kaiC36803-AA and kaiC36803-DE genes, respectively. To generate kaiC36803-catE1-catE2, two subsequent site-directed mutagenesis reactions were performed to exchange nucleotides encoding E67 and E68 as well as nucleotides encoding E310 and E311 all with bases encoding glutamine (all primers used for mutagenesis are listed in Table S1). Afterwards, kaiC36803 WT and modified kaiC36803 genes were amplified with KOD-Plus-Neo polymerase (Toyobo) using pASK-kaiC3 primers (Table S1). Amplicons were digested with SacII and HindIII and ligated into the respective restriction sites of pASK-IBA5plus (IBA Life sciences). For purification of recombinant Strep-KaiC7942, the pASK-IBA-5plus based vector described in Oyama et al. (14) was used. Strep-KaiC36803 proteins were expressed in E. coli Rosetta gamiB (DE3) or Rosetta gami2 (DE3) cells (Novagen). Expression of Strep-KaiC7942 was carried out in E. coli DH5α. Cells were cultured in LB medium containing 100 μg ml−1 ampicillin with vigorous agitation at 37 °C. At OD600nm 0.33-0.68 protein expression was induced with 200 ng ml−1 anhydrotetracycline and the strains were further incubated as following: Strep-KaiC7942: 7h at 37°C, Strep-KaiC36803-WT: 5h at 35°C or 3.5h at 37°C, Strep-KaiC36803-AA and Strep-KaiC36803-catE1-catE2-: 18°C overnight, Strep-KaiC36803-DE: 25°C overnight. Cells were harvested and lysed by sonication in ice-cold buffer W [20mM Tris/HCl (pH8), 150 mM NaCl, 5 mM MgCl2, 1 mM ATP (+ 2 mM DTT for Strep-KaiC36803 proteins)] including protease inhibitors (protease inhibitor cocktail, Roche or Nacalai). Soluble proteins were loaded on self-prepared columns packed with Strep-Tactin XT superflow or Strep-Tactin Sepharose (IBA lifesciences) and purified under gravity flow. After washing with buffer W, Strep-KaiC proteins were eluted with ice cold buffer W + 50 mM D(+)biotin (for Strep-Tactin XT Superflow) or ice cold buffer W + 2.5 mM desthiobiotin (for Strep tactin Superflow). See https://dx.doi.org/10.17504/protocols.io.meac3ae for a detailed protocol.
All proteins used for ATPase activity measurements were further purified via size exclusion chromatography. Strep-KaiC36803 and Strep-KaiC7942 proteins were applied on a Sephacryl S300 HR HiPrep 16/60 Sephacryl column (GE Healthcare) and separated in 20 or 50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 2 mM DTT, 1 mM ATP and 5 mM MgCl2. For separation of KaiB16803 and KaiB36803, a Sephacryl S200 HR HiPrep 16/60 column (GE Healthcare) or Superdex 200 Increase 10/30 GL column (GE Healthcare) and 20 or 50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 2 mM DTT as running buffer were used. KaiA6803 was purified on a Superdex 200 Increase 10/30 GL column (GE Healthcare) in 20 mM Tris/HCl (pH 8.0), 150 mM NaCl, 2 mM DTT. See https://dx.doi.org/10.17504/protocols.io.mdtc26n for further details.
ATPase activity
For ATPase measurements, KaiC proteins fused to an N-terminal Strep-tag were used and all Kai proteins were purified via size exclusion chromatography (see above). 3.45 μM of Strep-KaiC36803 variants were incubated in ATPase buffer (20 mM Tris/HCl (pH8), 150 mM NaCl, 1 mM ATP, 5 mM MgCl2) at 25 °C, 30 °C or 35 °C for 24 hours. To analyze the influence of KaiA6803 and KaiB6803 proteins on KaiC36803 ATPase activity, we mixed 0.2 mg ml−1 KaiC36803 with 0.04 mg ml−1 KaiA6803 or 0.04 mg ml−1 KaiB6803 and incubated the mixtures for 24 hours at 30 °C. Monomeric and oligomeric KaiB36803 were analyzed separately. To monitor ADP production, every 3, 4 or 6 hours 2 μl of the reaction mixture were applied on a Shim-Pack-VP-ODS column (Shimadzu) and separated using 100 mM phosphoric acid, 150 mM triethylamine, 1 % acetonitrile as running buffer. ADP production per monomer KaiC and 24 hours was calculated using a calibration curve. A detailed protocol can be found on protocols.io (https://dx.doi.org/10.17504/protocols.io.mebc3an). The Q10 value was calculated from ATPase measurements at 25 °C and 35 °C, using the formula
ATP synthase activity
To investigate dephosphorylation via ATP synthesis, we used KaiC proteins which were expressed as GST-fusion proteins and subsequently cleaved off their GST-tag. 3 μM KaiC in ATP synthesis buffer (20 mM Tris/HCl(pH8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 0.5 mM ATP) was mixed with 0.8 μCi ml−1 [α32P]ADP and stored at −20 °C or incubated for 2 hours at 30 °C. As a control, the same experiment was performed in the presence of 0.5 mM ADP. After 20-fold dilution, nucleotides in a 0.5 μl reaction mixture were separated via thin layer chromatography using TLC PEI Cellulose F plates (Merck Millipore) and 1 M LiCl as solvent. [α-32P]ADP and [γ-32P]ATP were separated in parallel to identify the signals corresponding to ADP and ATP, respectively. Dried plates were subjected to autoradiography and signals were analysed using a Personal Molecular Imager FX system (Bio-Rad) and ImageLab software (Bio-Rad). For each reaction mixture, the relative intensity of [α-32P]ATP was calculated as percentage of all signals in the corresponding lane. Because [α-32P]ATP was already synthesized during mixing of the samples, the relative ATP intensity measured in the −20 °C sample containing 0.5 mM ADP was subtracted for normalization. The principle of this method is based on Egli et al. (12). A detailed protocol is available on protocols.io (https://dx.doi.org/10.17504/protocols.io.48qgzvw).
Yeast two-hybrid assays
For yeast two-hybrid assays, vectors containing the GAL4 activation domain (AD) and the GAL4 DNA-binding domain (BD) were used. Genes of interest were amplified from Synechocystis 6803 wild type genomic DNA with the Phusion Polymerase (NEB) according to manufacturer’s guidelines. Indicated restriction sites were introduced via the oligonucleotides listed in Table S1. Vectors and PCR fragments were cut with the respective restriction enzymes (Thermo Fisher Scientific) and the gene of interest was ligated into the vector, leading to a fusion protein with an AD- or BD-tag either at the N- or C-terminus. All constructed plasmids are listed in Table S2. Transformation of yeast cells was performed according to manufacturer's guidelines using the Frozen‑EZ Yeast Transformation II Kit (Zymo Research) and cells were selected on complete supplement mixture (CSM) lacking leucine and tryptophan (-Leu -Trp) dropout medium (MP Biochemicals) at 30 °C for 3–4 days. Y190 (Clontech) cells were used for measuring β‑galactosidase activity. Formed colonies were spotted on a second plate (CSM ‑Leu ‑Trp) and incubated for 2 days. Afterwards a colony-lift filter assay was performed as described by Breeden et al. (52). A detailed protocol can be found on protocols.io (https://dx.doi.org/10.17504/protocols.io.v7ve9n6).
Bacterial strains and growth conditions
Wild type Synechocystis 6803 (PCC-M, re-sequenced, (53)) and the kaiC3 deletion mutant (38) were cultured photoautotrophically in BG11 medium (54) supplemented with 10 mM TES buffer (pH 8) under constant illumination with 50 μmol photons m−2s−1 of white light (Philips TLD Super 80/840) at 30 °C. Cells were grown either in Erlenmeyer flasks with constant shaking (140 rpm) or on plates (0.75 % Bacto-Agar, Difco) supplemented with 0.3 % thiosulfate. Detailed recipes can be found on protocols.io (https://dx.doi.org/10.17504/protocols.io.wj5fcq6).
Spot assays
Experiments were performed as previously described (44). Strains were propagated mixotrophically on BG11 agar plates with the addition of 0.2 % (w/v) glucose. Dilution series of cell cultures started with OD750nm 0.2 and OD750nm 0.4, followed by incubation of the plates for 6 or 28 days under constant light conditions and in complete darkness, respectively.
Author contributions
I.M. Axmann, A. Wilde, A. Wiegard, C. Köbler and K. Terauchi conceived the project. A. Wiegard, C. Köbler A.K. Dörrich and K. Oyama designed, performed and analyzed experiments. I.M Axmann, A. Wilde and K. Terauchi supervised the study. All authors interpreted and discussed the data. A. Wiegard, C. Köbler and A. Wilde wrote the manuscript. I.M. Axmann, C. Azai, K. Terauchi, A.K. Dörrich, and K. Oyama commented essentially on the manuscript. All authors approved the manuscript.
This manuscript contains supplementary information
Supplementary methods
Table S1. Oligonucleotides used in this study.
Table S2. Plasmids used in this study.
Figure S1. Initial phosphorylation level of KaiC proteins.
Figure S2. Original scans of the KaiC3 yeast two-hybrid interactions.
Figure S3. Interaction of KaiB6803 and KaiC6803 proteins in pull down analysis.
Figure S4. Purification of KaiB1 and KaiB3 proteins used for ATPase analysis
Figure S5. ATPase activity and dephosphorylation of KaiC36803 are temperature dependent
Acknowledgement
We thank Junko Moriwaki, Nancy Sauer, Jennifer Andres, Lukas Pohlig, Thomas Volkmer and Megumi Fujimoto for technical assistance. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2048/1 – Project ID: 390686111 to A. Wiegard and I.M. Axmann. The work was financially supported by Heine Research Academies - Travel Grants, and EMBO ASTF 145 to A. Wiegard, by grants AX 84/1-3 and EXC 1028 from the German Research Foundation to I.M. Axmann, by JSPS Grants-in-Aid 16H00784, 17K19247 and 19K05833 to K. Terauchi, and by grant WI2014/5-3 from the German Research Foundation to A. Wilde.