Fig. 1: Feasibility studies of in vitro oscillators for extant and ancestral Kai proteins.

a Phylogenetic tree of extant double-domain KaiC homologs. Cyanobacteria in freshwater, marine cyanobacteria, proteobacteria, bacteria, and euryarchaeota/crenarchaeota are colored green, blue, cyan, yellow, and red, respectively. Group III and IV KaiCs from freshwater cyanobacteria (gray entries) are originated from secondary copies of group I kaiC gene or lateral gene transfer. The three numbers preceding the name of each entry indicate the ID number, the total number of amino acids, and the sequence identity with KaiC^Se, respectively. A^*B^*C^*: sets of species-specific KaiA^*, KaiB^*, and KaiC^*. A^SeB^*C^*: sets of KaiA^Se, KaiB^*, and KaiC^*. A^SeB^SeC^*: sets of KaiA^Se, KaiB^Se, and KaiC^*. Rhythmic sets are indicated by filled circles, whereas arhythmic sets are labeled with crosses (see each result in Supplementary Fig. 3). Prep. C^* and Prep. A^*B^* indicate whether species-specific Kai proteins were adequately expressed and purified (open circles), prepared at a level that could be analyzed (triangles), or not prepared at all (crosses). Ex. A^*B^* indicates the classification of the existence of kaiA^* and kaiB^* genes; e.g., kaiA^* and kaiB^* coexist (ab), only kaiA^* exists (a–), only kaiB^* exists (–b), and neither exists (–). Symbols α–η represent evolutionarily important branching points for which the amino acid sequence of ancestral KaiC was restored (Fig. 2a and Supplementary Fig. 4a). In vitro rhythm assays of A^*B^*C^* (circles) or A^SeB^*C^* (diamonds) at 30 °C (blue) and 40 °C (red) as functions of incubation time (IT) for b Cyanothece sp. PCC7822 (KaiC^CP, ID: 007) in group I, c Oscillatoriales cyanobacterium JSC 12 (KaiC^Os, ID: 002) in group I, d Synechococcus sp. RCC307 (KaiC^SR, ID: 046) in group II, e Synechococcus sp. WH 7803 (KaiC^WH, ID: 056) in group II, f Rhodospirillum rubrum F11 (KaiC^Rr, ID: 104) in group III, g Cyanothece sp. ATCC 51142 (KaiC^Cy2, ID: 085) in group III, and h Flavobacterium johnsoniae UW101 (KaiC^Fj, ID: 212) in group IV. Shown are the representative examples of the fraction of phosphorylated KaiC (P-KaiC) from multiple experiments summarized in Supplementary Fig. 3. AR indicates that the period could not be determined (arrhythmic). In vitro rhythm assays for i A^γB^α/βC^α (circles) and A^SeB^SeC^α (diamonds), j A^γB^α/βC^β (circles) and A^SeB^SeC^β (diamonds), k A^γB^γC^γ (circles) and A^SeB^SeC^γ (diamonds), l A^ζB^γC^ζ (circles) and A^SeB^SeC^ζ (diamonds), m A^δB^δC^δ (circles) and A^SeB^SeC^δ (diamonds), n A^γB^εC^ε (circles) and A^SeB^SeC^ε (diamonds), and o A^γB^ηC^η (circles) and A^SeB^SeC^η (diamonds). Plots are shown as means ± s.d. of three replicates (see Table 1 for details on the number of replicates and their sources). Blue circles and gray diamonds correspond to the data taken at 30 °C, and red circles correspond to the data taken at 40 °C.