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.circadian clock ͉ cyanobacteria ͉ genome-wide expression ͉ KaiC ͉ light:dark

Background:The circadian output pathway in cyanobacteria is mediated by a two-component system consisting of SasA/RpaA. Results: An additional response regulator, RpaB, directly binds to clock-regulated promoters during the night. Conclusion: RpaB is also a key regulator of the circadian output pathway; RpaA and RpaB function cooperatively. Significance: Clarification of output pathway details is crucial for understanding the circadian clock.

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 ...

Circadian rhythms are endogenous biological timing processes that are ubiquitous in organisms ranging from cyanobacteria to humans. In the photoautotrophic unicellular cyanobacterium Synechococcus elongatus PCC 7942, under continuous light (LL) conditions, the transcription-translation feedback loop (TTFL) of KaiC generates a rhythmic change in the accumulation of KaiC relative to KaiA clock proteins (KaiC/KaiA ratio), which peak and trough at subjective dawn and dusk, respectively. However, the role of TTFL in the cyanobacterial circadian system remains unclear because it is not an essential requirement for the basic oscillation driven by the Kai-based posttranslational oscillator (PTO) and the transcriptional output mechanisms. Here, we show that TTFL is important for the circadian photic resetting property in Synechococcus. The robustness of PTO, which is exemplified by the amplitude of the KaiC phosphorylation cycle, changed depending on the KaiC/KaiA ratio, which was cyclic under LL. After cells were transferred from LL to the dark, the clock protein levels remained constant in the dark. When cells were transferred from LL to continuous dark at subjective dawn, the KaiC phosphorylation cycle was attenuated with a lower KaiC/KaiA ratio, a higher KaiC phosphorylation level, and a lower amplitude than that in cells transferred at subjective dusk. We also found that the greater the degree to which PTO was attenuated in continuous dark, the greater the phase shifts upon the subsequent light exposure. Based on these results, we propose that TTFL enhances resetting of the Kai-based PTO in Synechococcus.M ost organisms exhibit circadian oscillations in their physiological activities with periods of ∼24 h. The unicellular cyanobacterium Synechococcus elongatus PCC 7942 is an obligate photoautotroph and the simplest model organism in circadian biology. In Synechococcus, essentially all promoter activities exhibit circadian rhythms that are regulated under continuous light (LL) conditions by the three clock genes kaiA, kaiB, and kaiC (1-3). In eukaryotic model organisms, the core process that generates and maintains self-sustaining circadian oscillations is reported to be driven by transcription-translation feedback loops (TTFLs), whereby rhythmic expression of clock gene products regulates the expression of associated genes in ∼24-h cycles (4). In Synechococcus, however, the importance of the presence of TTFL is unclear because this feedback loop is not necessary for basic oscillation and output mechanisms. Under dark conditions, kaiA and kaiBC gene expression levels are rapidly down-regulated to zero whereas the KaiC phosphorylation cycle persists in the dark, even in the presence of excess transcription-translation inhibitors (5). Therefore, TTFL in the kai genes is not essential for basic oscillation. The reconstitution of the temperaturecompensated KaiC phosphorylation rhythm in vitro by incubating recombinant KaiA, KaiB, and KaiC with ATP (6) supports this conclusion. Previously, we showed that the posttranslati...

bCyanobacteria are unique organisms with remarkably stable circadian oscillations. These are controlled by a network architecture that comprises two regulatory factors: posttranslational oscillation (PTO) and a transcription/translation feedback loop (TTFL). The clock proteins KaiA, KaiB, and KaiC are essential for the circadian rhythm of the unicellular species Synechococcus elongatus PCC 7942. Temperature-compensated autonomous cycling of KaiC phosphorylation has been proposed as the primary oscillator mechanism that maintains the circadian clock, even in the dark, and it controls genome-wide gene expression rhythms under continuous-light conditions (LL). However, the kaiC EE mutation (where "EE" represents the amino acid changes Ser431Glu and Thr432Glu), where phosphorylation cycling does not occur in vivo, has a damped but clear kaiBC expression rhythm with a long period. This suggests that there must be coupling between the robust PTO and the "slave" unstable TTFL. Here, we found that the kaiC EE mutant strain in LL was hypersensitive to the dark acclimation required for phase shifting. Twenty-three percent of the genes in the kaiC EE mutant strain exhibited genome-wide transcriptional rhythms with a period of 48 h in LL. The circadian phase distribution was also conserved significantly in most of the wild-type and kaiC EE mutant strain cycling genes, which suggests that the output mechanism was not damaged severely even in the absence of KaiC phosphorylation cycles. These results strongly suggest that the KaiC phosphorylation cycle is not essential for generating the genome-wide rhythm under light conditions, whereas it is important for appropriate circadian timing in the light and dark.