KaiA-stimulated KaiC phosphorylation in circadian timing loops in cyanobacteria

Abstract

Cyanobacterial clock proteins KaiA and KaiC are proposed as positive and negative regulators in the autoregulatory circadian kaiBC expression, respectively. Here, we show that activation of kaiBC expression by kaiA requires KaiC, suggesting a positive feedback control in the cyanobacterial clockwork. We found that robust circadian phosphorylation of KaiC. KaiA was essential for in vivo KaiC phosphorylation and activated in vitro KaiC autophosphorylation. These effects of KaiA were attenuated by the kaiA2 long period mutation. Both the long period phenotype and the abnormal KaiC phosphorylation in this mutant were suppressed by a previously undocumented kaiC mutation. We propose that KaiA-stimulated circadian KaiC phosphorylation is important for circadian timing.

Fig 1.

(A) kaiBC promoter (P_kaiBC) activity was monitored with a luciferase reporter gene set fused to P_kaiBC in the wild-type (WT; black), kaiA⁻ (blue), kaiB-inactivated (kaiB⁻; yellow), kaiC⁻ (red), and kaiABC-cluster-depleted (kaiABC⁻; green) strains. The bioluminescence profiles of four independent clones for each mutant were measured in LL after two cycles of 12 h light:12 h dark (LD) alternations with a photomultiplier tube as described (3). Representative bioluminescence profiles normalized to that of the wild type are shown. (B) The effect of overexpression of the kaiA gene on P_kaiBC activity in the wild-type and kaiC-disrupted strains. Bioluminescence from P_kaiBC reporter strains carrying a P_trc:kaiA construct (for kaiA overexpression) was monitored. Cells were grown on agar plates as described above and treated with 500 μM isopropyl β-d-thiogalactopyranoside or water after 50 h in LL (arrows). (C) The effect of overexpression of the kaiC gene on P_kaiBC activity in wild-type and kaiA-disrupted strains.

Fig 2.

Suppression of kaiA2 long period phenotype by the kaiC15 mutation. kaiBC expression profiles in the wild-type, kaiABC⁻, kaiA2, kaiC2;kaiC15, and kaiC15 strains are shown. Measurement of the bioluminescence and representation of data are the same as described for Fig. 1.

Fig 3.

KaiA-stimulated KaiC autokinase activity. (A) Autophosphorylation of the KaiC protein with [γ-³²P]ATP in the presence or absence of KaiA, KaiB, and BSA. Autokinase assays were performed at 30°C for 60 min and then analyzed by SDS/PAGE and autoradiography. (B) Determination of phosphorylated aminoacyl species for KaiC autophosphorylation by the 2D thin-layer electrophoresis assay. Crosses indicate the sample origin positions. (C) Separation of phosphorylated and nonphosphorylated forms of KaiC by SDS/PAGE on 10% gels (acrylamide:N,N′-methylenebisacrylamide = 29.8:0.2). KaiC incubated with [γ-³²P]ATP was subjected to SDS/PAGE followed by CBB staining (Left) and autoradiography (Right). (D) The effect of KaiA on the mobility of the KaiC protein on 7.5% SDS-polyacrylamide gels. (E) Effects of KaiA2 mutant proteins on KaiC autokinase activity. The graph shows the time course profile of KaiC autophosphorylation with or without the wild-type or kaiA2-mutation-introduced KaiA proteins (KaiA and KaiA2, respectively).

Fig 4.

(A) KaiC protein expression profile examined by Western blotting. Cells were cultured in a continuous culture system in BG-11 liquid medium to maintain an OD₇₃₀ of 0.18 under LL (50 μE⋅m⁻²⋅s⁻¹, in which E indicates an einstein, 1 mol of photons). After two LD cycles, cells were returned to LL conditions and then collected every 4 h. Total proteins were extracted by sonication of cell pellets in SDS-sample buffer and then subjected to SDS/PAGE with 10% gels (acrylamide:N,N′-methylenebisacrylamide = 29.8:0.2), followed by Western blotting assay (4.0 μg protein per lane). (B) λPPase assay. Soluble protein extracts were prepared from Synechococcus culture collected at hours 4 and 16 under LL (LL 4 and LL 16), immunoprecipitated with anti-KaiC IgG, digested with 100 or 1,000 units of λPPase in the presence or absence of λPPase inhibitors (NaF and Na₃VO₄), and then subjected to Western blotting with anti-KaiC antisera. (C) Expression of clock proteins in wild-type, kaiA-null, and kaiC-null mutant strains by Western blotting. Note that no circadian fluctuation was observed in both kai-gene mutants in terms of the state of KaiC phosphorylation throughout the circadian cycle (data not shown). We show here Western blots using cells collected at LL 16, when the level of KaiC phosphorylation is maximal in the wild-type strain. (D) KaiC phosphorylation profile in kaiA⁻, kaiA-overexpressing (ox-kaiA), and kaiC-overexpressing (ox-kaiC) cells (Western blotting). Cells were cultured in BG-11 liquid medium (100 μM isopropyl-β-d-thiogalactopyranoside) and collected at LL 16. Total proteins (8 μg) were extracted from the collected cells and subjected to 10% gels followed by Western blotting assay. (E) Circadian KaiC phosphorylation profiles in the wild-type, kaiA2, kaiC15, and kaiA2;kaiC15 strains. Total proteins (8 μg) were analyzed by Western blotting as described above.

Fig 5.

A model for KaiA–KaiC functions in the Synechococcus clock. KaiA protein activates KaiC autokinase activity. The state of KaiC phosphorylation would regulate kaiBC expression. KaiC protein (complex) might affect its own promoter indirectly through controlling a clock output mechanism, such as regulation of the state of chromosome condensation (2), or by regulating SasA kinase activity (6). Phosphorylation also possibly affects the stoichiometry of clock protein complex(es) and/or the turnover of the KaiC protein (complex), which will affect the equilibrium state between nonphosphorylated and phosphorylated forms of KaiC. Because KaiC autophosphorylates at multiple residues, multiple phosphorylated forms of KaiC may be able to play different biochemical roles.