Oscillations in supercoiling drive circadian gene expression in cyanobacteria

Abstract

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA transcript abundance with 24-h periodicity under continuous light conditions. The mechanism underlying these oscillations remains elusive--neither cis nor trans-factors controlling circadian gene expression phase have been identified. Here, we show that the topological status of the chromosome is highly correlated with circadian gene expression state. We also demonstrate that DNA sequence characteristics of genes that appear monotonically activated and monotonically repressed by chromosomal relaxation during the circadian cycle are similar to those of supercoiling-responsive genes in Escherichia coli. Furthermore, perturbation of superhelical status within the physiological range elicits global changes in gene expression similar to those that occur during the normal circadian cycle.

Fig. 1.

Circadian gene expression and topology in S. elongatus. (A) Heat map of circadian gene expression (1748/2724 = 64% of total predicted ORFs) ordered by phase. Each gene is normalized between 0 and 1 such that yellow (1) and blue (0) indicate a peak and trough of expression, respectively. Circadian genes were identified on the chromosome and both endogenous plasmids. (B) Oscillations in the superhelicity of an endogenous plasmid (pANS) from same time-course as expression analysis. Each superhelical state corresponds to a unique state of gene expression suggesting that oscillations in supercoiling can drive oscillations in gene expression.

Fig. 2.

Sequence characteristics of genes suggest supercoiling mediated control. Genes that are monotonically relaxation activated (blue) and monotonically relaxation repressed (green) have increased and decreased AT content (in promoter and coding region), respectively, compared to all of the genes in the genome. Genes were aligned by translational start site and the mean AT content along −2,000–2,000 was calculated with a smoothing window of 100 nucleotides.

Fig. 3.

Manipulation of supercoiling via gyrase inhibition results in expected changes in gene expression. (A) Canonical subjective dawn (purF, red) and subjective dusk (kaiC, purple) genes oscillate with opposite phase in gene expression. Superimposed is the circadian oscillation in superhelicity of the endogenous pANS plasmid (black). Relaxed supercoiling states have lower relative mobility than negatively supercoiled states. KaiC expression is decreased during circadian relaxation while purF expression is increased. (B) Quantification of pANS plasmid supercoiling before and after addition of novobiocin at T = 64 h (blue) superimposed on supercoiling changes during the circadian cycle (black). Supercoiling was measured 5, 10, 15, 30, 60, 90, 150, 240, 330, and 480 min after novobiocin addition. Novobiocin addition immediately relaxes the pANS plasmid to a level similar to the most relaxed state during the circadian cycle. (C) Gyrase inhibition at T = 64 h in the circadian cycle results in an immediate change in gene expression due to drug-induced relaxation. Genes that have higher expression during relaxed circadian times immediately increase in gene expression (purF, red) whereas genes that have lower expression during relaxed circadian times immediately decrease in gene expression (kaiC, purple).

Fig. 4.

Genome-wide changes in expression due to gyrase inhibition induced relaxation. Scatter plot of the change in gene expression due to circadian relaxation versus drug-induced relaxation for circadian genes. The normal relaxation induced change in gene expression (x axis) is highly correlated with the rate of gene expression due to drug-induced relaxation (y axis) suggesting that supercoiling plays a primary role in dictating circadian gene expression. The linear best fit line is shown in red with Pearson correlation coefficient 0.85.