Three major output pathways from the KaiABC-based oscillator cooperate to generate robust circadian kaiBC expression in cyanobacteria.

Circadian kaiBC expression in the cyanobacterium Synechococcus elongatus PCC 7942 is generated by temporal information transmission from the KaiABC-based circadian oscillator to RpaA, a putative transcriptional factor, via the SasA-dependent positive pathway and the LabA-dependent negative pathway which is responsible for feedback regulation of KaiC. However, the labA/sasA double mutant has a circadian kaiBC expression rhythm, suggesting that there is an additional circadian output pathway. Here we describe a third circadian output pathway, which is CikA-dependent. The cikA mutation attenuates KaiC overexpression-induced kaiBC repression and exacerbates the low-amplitude phenotype of the labA mutant, suggesting that cikA acts as a negative regulator of kaiBC expression independent of the LabA-dependent pathway. In the labA/sasA/cikA triple mutant, kaiBC promoter activity becomes almost arrhythmic, despite preservation of the circadian KaiC phosphorylation rhythm, suggesting that CikA largely accounts for the residual kaiBC expression rhythm observed in the labA/sasA double mutant. These results also strongly suggest that transcriptional regulation in the labA/sasA/cikA triple mutant is insulated from the circadian signals of the KaiABC-based oscillator. Based on these observations, we propose a model in which temporal information from the KaiABC-based circadian oscillator is transmitted to gene expression through three separate output pathways.