Fig. 7: Phosphorylation of KaiC3 in Synechocystis (PCC-M) wild-type, kaiA3 mutant (ΔkaiA3), and kaiA3 overexpression (kaiA3-OE) strains grown under photoautotrophic conditions.

Samples were collected every 6 h from cells grown in a 12 h LD cycle, followed by LL. Whole-cell extracts were separated using Phos-tag SDS-PAGE and immunodecorated with a KaiC3-specific antiserum. Representative blots are shown in (a). We detected 4-5 bands which partially overlapped or were slightly shifted compared to the bands detected in the ∆kaiC3 strain (12 h time point was loaded). The two indicated prominent bands were absent in the ∆kaiC3 strain. b Whole cell extracts of Synechocystis wild-type (WT), kaiA3 mutant (ΔkaiA3), and the overexpression (kaiA3-OE) strain grown for 6 or 24 h in a 12 h LD cycle were loaded together with in vitro phosphorylated recombinant KaiC3 (KaiA3-KaiC3/6 h), which was generated by 6 h incubation with 4.2 µM KaiA3 at 30 °C. c KaiC3 was dephosphorylated (KaiC3/λ-PP) using Lambda phosphatase and analyzed alongside with in vitro phosphorylated KaiC3 (KaiA3-KaiC3/6 h). Based on the band pattern of recombinant KaiC3, we assigned the indicated bands to fully phosphorylated (PP-KaiC3) and non-phosphorylated KaiC3 (NP-KaiC3). The control reactions in (b) and (c) were performed at least twice on different gels. d The ratio of PP-KaiC3 to NP-KaiC3 measured in wild-type and ΔkaiA3 mutant is plotted as average (line) with dots indicating biological replicates (n = 4). Data derived from two biological replicates of kaiA3-OE are presented as individual curves. The white and dark gray boxes represent the light and dark periods, respectively, and the light gray box represents the subjective night. Source data are provided as a Source Data file.