Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus

Abstract

In the unicellular cyanobacterium Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock under continuous light (LL) conditions. Here, we used high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild-type, kaiABC-null, and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild-type strains, >30% of transcripts oscillated significantly in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was severely attenuated in kaiABC-null strains. Although it has been proposed that KaiC globally represses gene expression, our analysis revealed that dawn-expressed genes were up-regulated by kaiC-overexpression so that the clock was arrested at subjective dawn. Transfer of cells to DD conditions from LL immediately suppressed expression of most of the genes, while the clock kept even time in the absence of transcriptional feedback. Thus, the Synechococcus genome seems to be primarily regulated by light/dark cycles and is dramatically modified by the protein-based circadian oscillator.

Fig. 1.

Analysis of genome-wide circadian transcription profiles under LL conditions. (A and B) Variations in the oscillatory indexes; amplitude (ordinate) and a cosine-fitting correlation score (correlation P value; abscissa) from each transcript under LL conditions in wild-type (WT; A) and in kaiABC-null (ΔkaiABC; B) strains. Definition of each score is described in SI Methods. Briefly, a higher amplitude indicates the standard deviation (SD) normalized to the mean value representing larger fluctuations, and a lower P value means better correlation to a periodic (sinusoidal) waveform with a 24 h period. Eight hundred “cycling genes” were identified with an amplitude of >0.1 and a P value of <0.1. For more stringent filtration, the lower amplitude and higher P value from 2 independent experiments were used to ensure reproducibility. The numbers in the panel indicate scores of 12 rhythmic or arrhythmic genes: point 1, kaiA; point 2, kaiB; point 3, kaiC; point 4, sasA; point 5, rpaA; point 6, cikA; point 7, rpoD5/sigC; point 8, rpoD6; point 9, purF; point 10, pilH/rre7; point 11, ctaC; and point 12, opcA. (C and D) Phase distributions of the peak expression times of 800 circadianly expressed genes. The numbers of ORFs (C) or the amplitudes from 2 independent experiments (D) are plotted on the ordinate.

Fig. 2.

Clock-controlled expression profiles in wild-type and kaiABC-null strains under LL conditions. (A and B) Expression profiles of 800 cycling genes sorted by peak time in wild-type strains. The colors in descending order from red to black to green represent normalized data. For diagram A, the average and SD over 2 cycles are 0.0 and 1.0, respectively, whereas for diagram B the data were normalized to the mean value. (C) Expression profiles of the clock-controlled genes in kaiABC-null mutant strains. The sorting order of the genes is the same as for A and B. The data were normalized to the mean value of expression levels in wild-type strains. One of 2 independent experiments in each strain is shown (see Fig. S2 for results from the other series of experiments).

Fig. 3.

Temporal expression profiles of representative clock-related genes and sigma factor genes. Temporal profiles of clock-related genes and sigma factor genes in LL. Expression profiles of genes in wild-type (black) and kaiABC-null (red) strains from 2 independent experiments are shown. The number on the ordinate indicates relative expression level. (A) mRNA profiles of clock genes, kaiA, kaiB, kaiC, and clock-controlled genes, cikA, ldpA, sasA, rpaA, pex, labA and cpmA examined by microarray analysis. (B) Temporal profiles of the principal (Group 1) sigma factor gene, rpoD1/sigA, and high-amplitude Group 2 sigma factor genes, rpoD6, rpoD5/sigC and sigF2. For nomenclature of sigma factors, see ref. . rpoA encodes the alpha subunit of RNA polymerase.

Fig. 4.

Overinduction of KaiC arrests the clock at subjective dawn. (A) Ptrc::kaiC cells were cultured in the presence (red) or absence (blue) of 100 μM IPTG from hour 25 (CT 1) for 8 h in LL after 12 h darkness and subjected to microarray analysis. Solid lines show the average mRNA level of the 2 independent experiments shown in dots. Dashed lines show the circadian expression profiles of corresponding genes in wild-type strains. Results for representative subjective dusk genes (kaiC, sigC, cikA), low-amplitude/arrhythmic genes (rpaA, sasA, ldpA) and subjective dawn genes (pilH/rre7, syc1137_d/Synpcc7942_0377, purF) are shown. (B) The expression level of each clock-controlled gene in Ptrc::kaiC cells in the presence of IPTG for 8 h was compared with that in the absence of IPTG at hour 33 in LL. The ordinate indicates the peak time of each of the 800 cycling genes as shown in Fig. 1D. Two hundred sixty-nine cycling genes that show significantly different expression between WT and Ptrc::kaiC cells are plotted on a logarithmic scale as black filled circles (Student's t test; P < 0.05) and the other genes are plotted as gray open circles.

Fig. 5.

Suppression of genome-wide transcription under DD conditions. (A) Organization of Synechococcus expression profiles in DD from ZT 12 sorted by induction levels in the dark in the absence or presence of rifampicin. The data were normalized to the value at ZT 12. (B) Double plot of one LD cycle reconstructed by connecting data from hours 4–12 in LL and hours 0–12 in DD. The data were normalized to the average level over one LD cycle. (C) Total mRNA accumulation levels estimated from the sum of mRNA hybridization signals normalized to genomic DNA signals (see SI Methods) under LL (white circles) and DD in the absence (black) or presence (red) of rifampicin. Maximal total signals under LL were normalized to 1,000. (D) Population of nocturnally accumulating or diurnally accumulating transcripts in either subjective dawn (sDawn) or subjective dusk (sDusk) genes or low-amplitude/arrhythmic genes. Transcripts were defined as light-accumulating (yellow) or dark-accumulating (black) if the sum of the expression levels at ZT 4, 8 and 12 was larger or smaller than that at ZT 16, 20 and 24. Because the effect of LD transition is generally higher than the moderate changes in low-amplitude genes under LL, data from 97 higher-amplitude genes were used to identify 87 genes peaking at subjective dusk and 10 peaking at subjective dawn. Data for the 2418 remaining low-amplitude/arrhythmic (LA/AR) genes out of 2515 total genes are also shown.