Attenuation of the posttranslational oscillator via transcription–translation feedback enhances circadian-phase shifts in Synechococcus

Abstract

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...

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

“…2B. Whereas the phase of the KaiC phosphorylation cycle was less affected by the light-to-dark transition, the KaiC phosphorylation rhythm was attenuated with lowered amplitude during extended darkness when the cells were transferred to the dark at subjective dawn compared with that at subjective dusk (9). The attenuation of the KaiC phosphorylation cycle is likely to arise from the lower KaiC/KaiA ratio during the dark, because the TTFL-based KaiC accumulation cycle exhibits a trough at subjective dawn to early subjective day (9).…”

“…Whereas the phase of the KaiC phosphorylation cycle was less affected by the light-to-dark transition, the KaiC phosphorylation rhythm was attenuated with lowered amplitude during extended darkness when the cells were transferred to the dark at subjective dawn compared with that at subjective dusk (9). The attenuation of the KaiC phosphorylation cycle is likely to arise from the lower KaiC/KaiA ratio during the dark, because the TTFL-based KaiC accumulation cycle exhibits a trough at subjective dawn to early subjective day (9). Based on these observations, we have proposed that (i) TTFL modulates the KaiC/KaiA ratio periodically in LL, (ii) it affects the state (robustness) of the KaiC phosphorylation cycle (or PTO) in the subsequent dark, and (iii) the following light exposure triggers phase shifts, depending on the state of the PTO in the dark (9).…”

“…Nocturnal timekeeping in Synechococcus was originally suggested by Xu et al (8), who reported that the circadian phase in the light was not affected after acclimation to a dark pulse ranging from 3 to 36 h, starting from hour 12 in the light. However, we have since demonstrated that after cells were acclimated to dark stimulation from hour 24 or 30 in the light and then recovered to LL, the circadian phase changed dramatically, such that the phase was set to that of light onset (9). In the present study, we applied this type of "wedge" experiments to the kaiC EE mutant strain and demonstrated that the kaiBC promoter rhythm in the mutant was set mainly to the dark offset (light onset).…”

Structure, function, and mechanism of the core circadian clock in cyanobacteria

“…In vivo, this PTO also functions within a TTFL framework; i.e. circadian variation of clock protein expression resulting in a more robust rhythm (4,(12)(13)(14). Together with three other effector proteins, the KaiABC oscillator comprises a self-contained minimal system that is capable of integrating timekeeping, entrainment and output signaling processes into the cyanobacterial cell.…”

Evolution of the circadian clock system in cyanobacteria: a genomic perspective

“…Циркадная система очень важна для обеспечения адаптационной и экологической пластичности цианобактерий, поскольку она контролирует основные физиоло-гические процессы, такие как фотосинтез, фиксация азота и клеточное деление (Johnson, Golden, 1999). Молекулярный механизм циркадных ритмов цианобактерий был тщательно изучен в последние два десятилетия (Hosokawa et al, 2013;Espinosa et al, 2015), однако о том, как этот механизм развивался и превратился в высокоэффективную и надежную циркадную систему, известно пока сравнительно немного.…”