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

Abstract

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.

Damped circadian oscillation in the absence of KaiA in Synechococcus

“…Under our experimental conditions, dark pulses of 2 or 4 h gave rise to slight phase shifts in the WT strain, whereas dark pulses of 6 h advanced the phase during the subjective day (hour 8 in LL, Fig. 2a), as reported previously 26 . Conversely, the phases of bioluminescence rhythms in kaiA − strains were much more dramatically shifted against dark pulses, especially those of 4 or 6 h (Fig.…”

“…In this study, to verify whether any specific phosphorylation state of KaiC is required for the damped oscillation, we introduced mutations into kaiC to constitutively mimic either the hypophosphorylated or hyperphosphorylated form of KaiC in kaiA − strains. When kaiA is intact, substitution of KaiC phosphorylation sites (S431 and T432) with phosphomimic glutamate residues (kaiC EE ) does not abolish transcriptional rhythms, but it sustains rhythms with a lengthened period of 48 h 26,31 .…”

“…It is interesting that the kaiA-less oscillation and the oscillation in the kaiC EE mutant share some similarity in their circadian characteristics: they are less temperature-compensated, subservient to the intact KaiA-including timing system, hypersensitive to photic entraining signals, and more dependent on TTFL 26,50 . They support (i) unnecessity of KaiC phosphorylation for driving imperfect oscillations, and (ii) contribution of KaiC phosphorylation cycle in the intact Kai system for robust circadian timing with KaiA.…”

Circadian Oscillators: Around the Transcription–Translation Feedback Loop and on to Output

“…Most studies agree that the PTO (now reconstituted in another cell type [88]) is the necessary-and-sufficient core oscillator. However, even absent the KaiABC-based PTO, the TTFL is capable of driving temperature-compensated rhythmic gene expression (albeit with a long 48 hr period length) of a substantial portion of the genome [89], suggesting that while the PTO is required for robustness and proper periodicity it may cooperate with the TTFL for output. Two recent analyses have tied the slow rate of ATP hydrolysis in the PTO to rates of structural changes in the Kai proteins, via a slow peptide isomerization in KaiC [90] and a slow and rare shift between folded states of KaiB that affects its ability both to capture KaiA (and thereby promote KaiC dephosphorylation) and to bind to and activate CikA, thereby influencing output [32].…”

Giving Time Purpose: The Synechococcus elongatus Clock in a Broader Network Context

“…This view is in contrast to the dominant output model that the oscillator induces transcriptional activation rather than repression. Intriguingly, cells that express a KaiC variant unable to undergo phosphorylation cycling, poised at the stage where we now realize KaiB and SasA compete for KaiC binding, still exhibited transcriptional rhythms of gene expression, albeit with a 48-h rather than 24-h period (87). Nakahira et al showed that there is a global down-regulation of gene expression when kaiC is over-expressed (58), supporting this paradigm shift.…”