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.M ost organisms exhibit circadian oscillations in their physiological activities, with a period of ≈24 h, as part of their adaptation to external environmental changes. The unicellular cyanobacterium Synechococcus elongatus PCC 7942 is an obligate photoautotroph and the simplest model organism in circadian biology. In S. elongatus, most genes display circadian expression rhythms that are regulated by three clock genes, kaiA, kaiB, and kaiC, under continuous light (LL) conditions (1, 2). When the cells are transferred to continuous dark (DD) conditions after 12 h in the light, expression of the kaiA and kaiBC genes is rapidly downregulated to zero, whereas the KaiC phosphorylation cycle persists in the dark, even in the presence of excess transcription/translation inhibitors (3). Therefore, the basic oscillation is generated via a posttranslational process and does not need a translation/transcription feedback loop in the kai genes. The reconstitution of the temperature-compensated KaiC phosphorylation rhythm in vitro when KaiC is incubated with KaiA, KaiB, and ATP (4) supports this conclusion. Kitayama et al. (5) demonstrated rhythmic kaiBC expression and KaiC accumulation with a lengthened period of ≈60 h, even after two phosphorylation sites in KaiC (Ser-431 and Thr-432) were replaced with Glu. However, the unstable rhythm observed in the mutant was not robust under different culture conditions and different ambient temperatures (6). In eukaryotic model organisms, the core process that generates and maintains self-sustaining circadian oscillations is reported to be driven ...

Genome-Wide and Heterocyst-Specific Circadian Gene Expression in the Filamentous Cyanobacterium Anabaena sp. Strain PCC 7120

“…strain PCC 6803, a DNA microarray analysis identified ϳ50 significantly rhythmic genes, whereas rhythmic clock gene expression was limited to low-amplitude kaiA expression (29). Moreover, we recently found that in Synechococcus the expression rhythms of only a subset of genes are dampened in the dark, even when de novo kai gene expression is turned off (9).…”

“…This was originally thought to be the core of the circadian oscillation in Synechococcus (6). Although we have demonstrated that the kaiBC expression rhythm is not an essential requirement for circadian regulation (8,9), most previous molecular and physiological studies have successfully used a bioluminescent reporter (luciferase) to monitor the rhythm of the high-amplitude kaiBC promoter activity under LL (10,11). In Synechococcus, KaiA activates the autophosphorylation of KaiC, and this activation is antagonized by KaiB.…”

The circadian oscillator in Synechococcus elongatus controls metabolite partitioning during diurnal growth

“…However, the degree to which the circadian clock and light availability independently affect metabolic events is poorly understood. In S. elongatus, only two studies have investigated the behavior of mutants that lack a functional clock under an LD cycle (21,25). The available studies investigate these effects only over a lightto-dark transition, so currently there is an incomplete understanding of the circadian influence on cellular events over a full 24-h LD cycle.…”

Hypersensitive Photic Responses and Intact Genome-Wide Transcriptional Control without the KaiC Phosphorylation Cycle in the Synechococcus Circadian System

“…However, the circadian cycling of KaiC phosphorylation persisted when Synechococcus cells were maintained under continuous-dark conditions (DD) in the absence of de novo transcription and translation of the kai clock genes (3). Even during DD, the cyanobacterial clock regulates part of the transcriptional output, dependent on Kai protein-based posttranslational oscillation (PTO) (4). Moreover, the self-sustained oscillation of KaiC phosphorylation was reconstituted in vitro by incubation with Kai proteins and ATP (5).…”