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.

21Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation 22 strategies and differences in the presence of circadian clock proteins. In Synechococcus elongatus 23 PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity 24 of KaiC correlates with the period of the clock and mediates temperature compensation. 25 Synechocystis sp. PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins 26 were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore 27 characterized the enzymatic activity of KaiC3 as a representative of non-standard KaiC homologs 28 in vitro. KaiC3 displayed ATPase activity, which were lower compared to the Synechococcus 29 elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature-dependent. Hence, KaiC3 is 30 missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid 31 analysis showed that KaiC3 interacts with KaiB3, KaiC1 and KaiB1. Further, KaiB3 and KaiB1 32reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in 33 constant darkness is completely abolished after deletion of ∆kaiAB1C1 and reduced in the 34 absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a 35 crosstalk between the KaiC1 and KaiC3 based systems. 36 Importance: The circadian clock influences the cyanobacterial metabolism and deeper 37 understanding of its regulation will be important for metabolic optimizations in context of 38 industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock 39 systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is 40 required. Synechocystis PCC 6803 represents a major cyanobacterial model organism and harbors 41 phylogenetically diverged homologs of the clock proteins, which are present in various other non-42 3 cyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple 43 Synechocystis Kai proteins and characterize enzymatic activities of the non-standard clock 44 homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness 45 suggesting its involvement in the regulation of non-photosynthetic metabolic pathways. 46 Introduction: 47 Cyanobacteria have evolved the circadian clock system to sense, anticipate and respond to 48 predictable environmental changes based on the rotation of Earth and the resulting day-night 49 cycle. Circadian rhythms are defined by three criteria: (i) oscillations with a period of about 24 50 hours without external stimuli, (ii) synchronization of the oscillator with the environment and (iii) 51 compensation of the usual temperature dependence of biochemical reactions, so that the period 52 of the endogenous oscillation does not depend on temperature in a physiological range (2). The 53 cyanobacterial circadian clock system has been studied in much detail in the unicellular model 54 cyanoba...

Cyanobacteria form a heterogeneous bacterial group with diverse lifestyles, acclimation strategies, and differences in the presence of circadian clock proteins. In Synechococcus elongatus PCC 7942, a unique posttranslational KaiABC oscillator drives circadian rhythms. ATPase activity of KaiC correlates with the period of the clock and mediates temperature compensation. Synechocystis sp. strain PCC 6803 expresses additional Kai proteins, of which KaiB3 and KaiC3 proteins were suggested to fine-tune the standard KaiAB1C1 oscillator. In the present study, we therefore characterized the enzymatic activity of KaiC3 as a representative of nonstandard KaiC homologs in vitro. KaiC3 displayed ATPase activity lower than that of the Synechococcus elongatus PCC 7942 KaiC protein. ATP hydrolysis was temperature dependent. Hence, KaiC3 is missing a defining feature of the model cyanobacterial circadian oscillator. Yeast two-hybrid analysis showed that KaiC3 interacts with KaiB3, KaiC1, and KaiB1. Further, KaiB3 and KaiB1 reduced in vitro ATP hydrolysis by KaiC3. Spot assays showed that chemoheterotrophic growth in constant darkness is completely abolished after deletion of ΔkaiAB1C1 and reduced in the absence of kaiC3. We therefore suggest a role for adaptation to darkness for KaiC3 as well as a cross talk between the KaiC1- and KaiC3-based systems. IMPORTANCE The circadian clock influences the cyanobacterial metabolism, and deeper understanding of its regulation will be important for metabolic optimizations in the context of industrial applications. Due to the heterogeneity of cyanobacteria, characterization of clock systems in organisms apart from the circadian model Synechococcus elongatus PCC 7942 is required. Synechocystis sp. strain PCC 6803 represents a major cyanobacterial model organism and harbors phylogenetically diverged homologs of the clock proteins, which are present in various other noncyanobacterial prokaryotes. By our in vitro studies we unravel the interplay of the multiple Synechocystis Kai proteins and characterize enzymatic activities of the nonstandard clock homolog KaiC3. We show that the deletion of kaiC3 affects growth in constant darkness, suggesting its involvement in the regulation of nonphotosynthetic metabolic pathways.

The putative circadian clock system of the facultative heterotrophic cyanobacterial strain Synechocystis sp. PCC 6803 comprises the following three Kai-based systems: a KaiABC-based potential oscillator that is linked to the SasA-RpaA two-component output pathway and two additional KaiBC systems without a cognate KaiA component. Mutants lacking the genes encoding the KaiAB1C1 components or the response regulator RpaA show reduced growth in light/dark cycles and do not show heterotrophic growth in the dark. In the present study, the effect of these mutations on central metabolism was analyzed by targeted and non-targeted metabolite profiling. The strongest metabolic changes were observed in the dark in ΔrpaA and, to a lesser extent, in the ΔkaiAB1C1 mutant. These observations included the overaccumulation of 2-phosphoglycolate, which correlated with the overaccumulation of the RbcL subunit in the mutants, and taken together, these data suggest enhanced RubisCO activity in the dark. The imbalanced carbon metabolism in the ΔrpaA mutant extended to the pyruvate family of amino acids, which showed increased accumulation in the dark. Hence, the deletion of the response regulator rpaA had a more pronounced effect on metabolism than the deletion of the kai genes. The larger impact of the rpaA mutation is in agreement with previous transcriptomic analyses and likely relates to a KaiAB1C1-independent function as a transcription factor. Collectively, our data demonstrate an important role of homologs of clock proteins in Synechocystis for balanced carbon and nitrogen metabolism during light-to-dark transitions.

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