The role of the Synechocystis sp. PCC 6803 homolog of the circadian clock output regulator RpaA in day–night transitions

Abstract

Cyanobacteria exhibit rhythmic gene expression with a period length of 24 hours to adapt to daily environmental changes. In the model organism Synechococcuselongatus PCC 7942, the central oscillator consists of the three proteins KaiA, KaiB and KaiC and utilizes the histidine kinase SasA and its response regulator RpaA as output-signaling pathway. Synechocystis sp. PCC 6803 contains in addition to the canonical kaiAB1C1 gene cluster two further homologs of the kaiB and kaiC genes. Here, we demonstrate that the SasA-RpaA system interacts with the KaiAB1C1 core oscillator only. Interaction with KaiC2 and KaiC3 proteins was not detected, suggesting different signal transduction components for the clock homologs. Inactivation of rpaA in Synechocystis sp. PCC 6803 leads to reduced viability of the mutant in light-dark cycles, especially under mixotrophic growth conditions. Chemoheterotrophic growth of the ∆rpaA strain in the dark was abolished completely. Transcriptomic data revealed that RpaA is mainly involved in the regulation of genes related to CO - acclimation in the light and to carbon metabolism in the dark. Further, our results indicate a link between the circadian clock and phototaxis.

Synechocystis KaiC3 Displays Temperature- and KaiB-Dependent ATPase Activity and Is Important for Growth in Darkness

“…Characterization of the ATPase activity of KaiC3 was of special interest, because ATP hydrolysis defines the period length and temperature compensation of the Kai oscillator (4). We further demonstrate that the ΔkaiC3 mutant strain has a growth defect under chemoheterotrophic growth conditions, which is similar to but less pronounced than those of ΔkaiAB1C1 and ΔrpaA strains (22,39). Our data support the idea of a function of KaiC3 and KaiB3 in fine-tuning the central oscillator composed of KaiA 6803 , KaiB1, and KaiC1 in Synechocystis.…”

“…As in vitro analyses suggested an interaction of KaiC3 with KaiB1 and KaiC1, we aimed at elucidating a putative role of KaiC3 and its cross talk with the KaiC1-based system in the cell. Clock factors are reported to be essential for cell viability in light-dark cycles (18,22,24). For assessing growth of mutant strains lacking kai genes, we used a spot plating assay (56).…”

“…As KaiC3 homologs are also present in nonphotosynthetic bacteria (27), we were interested in the growth of the ΔkaiC3 strain in the dark. The Synechocystis wild-type strain used here, in contrast to those used in previous studies (48), is able to grow in complete darkness when supplemented with glucose (22). We therefore analyzed the viability of ΔkaiC3 cells via spot assays in constant light and in complete darkness on agar plates containing 0.2% glucose (Fig.…”

“…Conversely, it is also possible that KaiC3 function is controlled by the KaiAB1C1 clock system. Köbler et al (22) demonstrated that solely KaiC1, but not KaiC3, interacts with the main output histidine kinase SasA in the Synechocystis timing system. Thus, in Synechocystis, only the main oscillator feeds timing information into the SasA-RpaA output system to control the expression of many genes involved in dark growth (22).…”

“…In Synechocystis, deletion of the kaiAB1C1 gene cluster causes reduced growth in light-dark rhythms, even when grown as a single culture, demonstrating a more pronounced phenotype than S. elongatus, where only a competitive growth disadvantage was observed (39). Deletion of rpaA also reduced growth in light-dark cycles, which further implies metabolic orchestration by the Synechocystis timer (22). The reported number of oscillating genes in Synechocystis varies largely among different studies, which is likely due to differences in growth conditions and strain variations (40)(41)(42)(43).…”