Circadian transcriptional regulation by the posttranslational oscillator without de novo clock gene expression in Synechococcus

Abstract

Circadian rhythms are a fundamental property of most organisms, from cyanobacteria to humans. In the unicellular obligately photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are controlled by the KaiABC-based clock under continuous light conditions. When Synechococcus cells are transferred from the light to continuous dark (DD) conditions, the expression of most genes, including the clock genes kaiA and kaiBC, is rapidly down-regulated, whereas the KaiC phosphorylation cycle persists. Therefore, we speculated that the posttranslational oscillator might not drive the transcriptional circadian output without de novo expression of the kai genes. Here we show that the cyanobacterial clock regulates the transcriptional output even in the dark. The expression of a subset of genes in the genomes of cells grown in the dark was dramatically affected by kaiABC nullification, and the magnitude of dark induction was dependent on the time at which the cells were transferred from the light to the dark. Moreover, under DD conditions, the expression of some dark-induced gene transcripts exhibited temperature-compensated damped oscillations, which were nullified in kaiABC-null strains and were affected by a kaiC period mutation. These results indicate that the Kai protein-based posttranslational oscillator can drive the circadian transcriptional output even without the de novo expression of the clock genes.

Fig. 1.

kaiABC nullification alters dark-induced gene expression. (A) Northern hybridization analysis of the temporal expression profiles of the kaiBC operon and four representative dark-induced genes under 12 h/12 h LD conditions. Wild-type and kaiABC-null (ΔkaiABC) mutant cells were collected at the indicated times. Note that hspA and digA are differentially regulated by the kai genes. (B) Of the genes in the genome, 167 (7%) were identified as kai-dependent dark-induced genes and were categorized into four groups, types I–IV. The colors for the microarray data represent the expression levels, in descending order from red to black to green.

Fig. 2.

Circadian gating of dark-induced gene expression. (A) Day-length dependence of dark-induced profiles. Northern blotting analysis of three representative genes from each group of kai-dependent dark-induced genes (Fig. 1B) under the dark. Type I: pilT, chlB, and dpsA; type II: digA, gap1, and digC; type III: pilH/rre7, clpB, and digE; and type IV: hspA, psb28-2, and digD. When the cells were transferred to the dark after 12 h in the light, the expression profile of each gene was quite different in the WT (black line) and kaiABC-null mutant (red line) strains. When the WT cells were transferred to the dark after 24 h in the light, the expression profiles (green line) changed, becoming essentially similar to those in the mutant strain. (B) Dark-induced profiles of the digB and lrtA genes, which are categorized as kai-independent genes (kai-indep). Data are shown as in panel A. (C) Schematic representation of the experimental schedule. After the cells were entrained in two 12 h/12 h LD cycles, they were placed in the light for the indicated times, ranging from 6 h to 36 h in increments of 6 h, and were then transferred to the dark conditions. (D) Northern blotting analysis of the digA and hspA genes in the dark when they had been transferred after 6 h (L6D) to 36 h (L36D) under LL. The cells were collected at hours 0, 0.5 (30 min), 1, 2, 4, 8, and 12 in the dark. (E) Time of day-dependent induction levels of digA and hspA mRNAs after 4 h in the dark. The mean values and SDs of four independent experiments are shown. These data were initially normalized to the level at hour 0 in the light; the average level after 4 h in the dark under L12D conditions was then deemed to be 1. (F) Densitometric data for the time of day-dependent DigA and HspA protein accumulation in the dark, when the cells had been transferred to the dark after 12 h (L12D) or 24 h (L24D) in the light, analyzed by Western blotting.

Fig. 3.

Temperature-compensated, kaiC-dependent damped oscillations in digA and pilH/rre7 expression under DD. (A) Temporal expression profiles of the digA and hspA genes in the WT and kaiABC-null (ΔkaiABC) strains under DD after the cells were transferred from 12 h in the light (L12D). Asterisk shows the second peak of the damped digA mRNA cycle observed in the WT strain. (B) Temporal digA expression profile under DD at different temperatures (37 °C, 30 °C, and 25 °C). Note that the standard temperature in all other experiments described in the text was 30 °C. (C) Densitometric analysis of the temporal digA expression profile under DD at different temperatures in two independent experiments. (D and E) The kaiBC, digA, and pilH/rre7 mRNA expression profiles (D) and the KaiC phosphorylation cycles (E) in the rpoD5-null (ΔrpoD5) mutant strain and the kaiC^S157P;ΔrpoD5 double mutant strain. For the experiments shown in A and B, the short-period mutant strain was transferred to the dark at hour 10.5 (estimated subjective dusk = CT 12, considering its period length of 21 h) from the light, after two 12 h/12 h LD cycles, whereas the WT strain was transferred to the dark at hour 12 (CT 12).

Fig. 4.

Schematic representation of the differential coordination of the Kai protein-based posttranslational oscillator and the transcription/translation feedback process under light and dark conditions.