The prokaryotic Synechoccus elongatus circadian clock

Cyanobacteria are photoautotrophic prokaryotes that exhibit an exceptional variety of forms, faculties, and functions. They are found in nearly every habitat accessible to sunlight. In addition to their common photosynthetic characteristics, they represent the simplest organisms known to exhibit circadian rhythms. Physiological studies regarding the temporal separation of nitrogen fixation and oxygenic photosynthesis were among the first to suggest that cyanobacteria have endogenous timing mechanisms (Golden et al., 1997). Indeed, the cyanobacterial clock regulates many physiological processes, such as photosynthesis, nitrogenase activity, amino acid uptake, cell division or carbohydrate synthesis (for review, see (Golden, 2007). Figure 3 shows examples for the visualization of S.elongatus endogenous

Figure 3. Gene expression reflected by bioluminescence from a Ф(kaiB-luc+) reporter (left) and chromosome compaction rhythms (right) in S.elongatus (red, autofluorescence from S. elongatus;

green, DAPI-stained DNA). Cells are sampled at the indicated times during the light/dark cycle (upper) and the second free-running cycle (lower). These Figures are issued from (Smith and Williams, 2006).

rhythmicity.

The generation of a strain with an insertion of the luciferase coding sequence downstream the promoter of the circadian psbA1 gene, which encode a key component of the photosynthetic apparatus, allowed isolating a variety of clock mutants with bioluminescence as a readout. The genes corresponding to the mutants were shown to be comprised in a gene cluster composed of three genes, kaiA, kaiB and kaiC. Transcription of these three genes is rhythmic. Overexpression of kaiA increases the expression kaiBC, which share a single promoter and are co-transcribed. In contrast, overexpression of kaiC suppresses the expression of kaiBC (Ishiura et al., 1998). KaiA acts thus as a positive element that enhances KaiBC expression. KaiC, in turn, represses its own transcription as well as that of kaiB (Figure 4). KaiA protein levels do not oscillate, whereas KaiB and KaiC protein levels cycle.

This transcription/translation-based model had to be reconsidered when Tomita and co-workers observed that KaiC is phosphorylated in a circadian manner, even in absence of transcription and translation (Tomita et al., 2005). In the same perspective, an in vitro experiment assembling recombinant KaiA, KaiB and KaiC in the presence of ATP, revealed robust oscillations in KaiC phosphorylation with a period close to 24 hours for a few days

(Nakajima et al., 2005). KaiC possesses auto-kinase and auto-phosphatase activities that are regulated by KaiA and KaiB. KaiA dimers transiently bind KaiC hexamer units, and this promotes KaiC phosphorylation. Once phosphorylated, KaiC associates with KaiB, which inactivates KaiA and induces the auto-phosphatase activity of KaiC (Figure 5). These findings place KaiC phosphorylation at the heart of the S.elongatus clock. However, a recent study by the same group revealed that KaiA-overexpressing cyanobacteria, which have constitutively phosphorylated KaiC, show circadian gene expression (Kitayama et al., 2008). In fact, the KaiC phosphorylation cycle and the transcription/translation cycle appear to compensate each other, depending on the conditions, in order to maintain robust and precise circadian rhythm.

KaiC also interacts with a histidine protein kinase SasA (Synechococcus adaptive sensor A).

In turn, SasA mediates the activation of RpaA (regulator of phycobilisome-associated A), a DNA-binding protein that is believed to be necessary for coordinating genome-wide circadian gene expression with proper phase relationships and period lengths and for maintaining the robust oscillation of KaiC phosphorylation (Figure 5) (Takai et al., 2006).

Another study suggests that a cyclic change in genome topology (see Figure 3) could explain the universal rhythmicity of promoter activity in S. elongatus and the residual rhythmicity observed in sasA and rpaA mutants (Smith and Williams, 2006). Thus, the clock could include output pathways from the oscillator to gene expression using transcriptional modulation via SasA and RpaA and through whole scale remodeling of the chromosome.

Cyanobacterial circadian rhythms can be synchronized by light, nutrients and Figure 4. KaiC negatively regulates its own (kaiBC) expression to generate a molecular feedback loop. In contrast, KaiA activates kaiBC expression as a positive element to make the loop oscillate. This model implies circadian fluctuation in the levels of the Kai proteins.

This Figure is issued from (Harmer et al., 2001)

Figure 5. The oscillation in KaiC phosphorylation results from the opposing actions of KaiA, which stimulates KaiC autokinase activity, and KaiB, which abolishes the positive effect of KaiA. Temporal information is transduced from the Kai oscillator to SasA through the stimulation of SasA autophosphorylation by KaiC. SasR (RpaA) is activated by SasA via transfer of a phosphoryl group.

RpaA is necessary for overt rhythmicity of gene expression. Additionally, the Kai oscillator regulates the compaction rhythm of the cyanobacterial nucleoid (double-stranded loop), which would control accessibility of transcriptional machinery to promoter regions. An input to the oscillator is provided by CikA, which influences the state of KaiC phorphorylation in response to light variations. This Figure is issued from (Ditty et al., 2003)

period) have been identified as factors needed to transmit indirectly light cues to the circadian clock. CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC (Ivleva et al., 2006). LdpA protein is also sensitive to the redox state of the cell, and this has been suggested to affect not only CikA, but also KaiA (Ivleva et al., 2005). This suggest that in S.elongatus, the clock receives at least some information about light not directly from a photoreceptor, but indirectly from a signal that reflects the redox state of the cell, which in photosynthetic organisms greatly depends on light intensity.

Important evidence emanating from the studies on the cyanobacterial clock is the

observation that circadian rhythm can be detected also in cells that are dividing faster than circadian frequency. This implies that DNA replication and cell division do not interfere with circadian oscillations, and that the oscillators pass time to daughter cells (Kondo et al., 1997).