labA: a novel gene required for negative feedback regulation of the cyanobacterial circadian clock protein KaiC

Abstract

In the cyanobacterium Synechococcus elongatus PCC 7942, circadian timing is transmitted from the KaiABC-based central oscillator to the transcription factor RpaA via the KaiC-interacting histidine kinase SasA to activate transcription, thereby generating rhythmic circadian gene expression. However, KaiC can also repress circadian gene expression, including its own. The mechanism and significance of this negative feedback regulation have been unclear. Here, we report a novel gene, labA (low-amplitude and bright), that is required for negative feedback regulation of KaiC. Disruption of labA abolished transcriptional repression caused by overexpression of KaiC and elevated the trough levels of circadian gene expression, resulting in a low-amplitude phenotype. In contrast, overexpression of labA significantly lowered circadian gene expression. Furthermore, genetic analysis indicated that labA and sasA function in parallel pathways to regulate kaiBC expression, whereas rpaA functions downstream from labA for kaiBC expression. These results suggest that temporal information from the KaiABC-based oscillator diverges into a LabA-dependent negative pathway and a SasA-dependent positive pathway, and then converges onto RpaA to generate robust circadian gene expression. It is likely that quantitative information of KaiC is transmitted to RpaA through LabA, whereas SasA mediates the state of the KaiABC-based oscillator.[Keywords: Circadian clock; cyanobacteria; KaiC; labA; RpaA; SasA] Supplemental material is available at http://www.genesdev.org. The circadian clock is an endogenous timing system that controls various biological activities with a period of ∼24 h. Most organisms use self-sustained oscillation to coordinate with and adapt to daily environmental changes. KaiA, KaiB, and KaiC have been identified as essential components for circadian oscillation in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus). KaiC, an autokinase and autophosphatase, is the central component of the cyanobacterial circadian clock and interacts with KaiA and KaiB Iwasaki et al. 1999;Nishiwaki et al. 2000;Taniguchi et al. 2001). KaiA enhances the autokinase activity of KaiC (Iwasaki et al. 2002;Williams et al. 2002;Uzumaki et al. 2004) and/or inhibits its autophosphatase activity (Kitayama et al. 2003;Xu et al. 2003), while KaiB attenuates the activity of KaiA (Williams et al. 2002;Kitayama et al. 2003;Xu et al. 2003). KaiC phosphorylation oscillates with a period of ∼24 h when the three recombinant Kai proteins are incubated in vitro in the presence of ATP . This oscillation period is refractory to changes in temperature, an important feature of circadian rhythms ). Thus, Kai-based chemical oscillation is thought to be the basic circadian timing loop in Synechococcus Tomita et al. 2005).KaiC also interacts with a sensory histidine kinase, SasA . Autophosphorylation of SasA is enhanced in response to KaiC binding (Smith and Williams 2006;Takai et al. 2006), and this phosphate group is then transferred to the putati...

Cyanobacterial daily life with Kai-based circadian and diurnal genome-wide transcriptional control in Synechococcus elongatus

“…3A and S1). Although the kaiA (1,15), pex (19), labA (20), and ldpA (21) genes did not fulfill our 2-filtration criteria for cycling genes, they each showed low-amplitude rhythms peaking at subjective dusk and dawn as illustrated in Fig. 3A.…”

Robust circadian clocks from coupled protein-modification and transcription–translation cycles

“…RpaA activates kaiBC expression. Deletion of rpaA, which encodes a putative response regulator, reduces kaiBC expression (40)(41)(42). Because neither promoters nor transcription or chromosomecompaction factors have been identified that interact with RpaA (42), we make the phenomenological assumption that RpaA directly activates kaiBC expression (40).…”

“…RpaA is activated by KaiC when KaiC is in the active state. RpaA is activated via phosphorylation by the histidine kinase SasA, whose activity is in turn stimulated by KaiC (38,42); inactivation of SasA reduces kaiBC expression (40)(41)(42). Moreover, RpaA phosphorylation occurs 4-8 h before the peak of KaiC phosphorylation (42).…”

A Cyanobacterial Circadian Clock Based on the Kai Oscillator

“…We recently reported that the novel gene labA is required for the negative feedback regulation by KaiC. Disruption of labA abolished the transcriptional repression caused by overexpression of KaiC and elevated the trough levels of circadian gene expression, resulting in a low-amplitude phenotype (Taniguchi et al 2007). In contrast, overexpression of labA significantly lowered the levels of circadian gene expression.…”