The cyanobacterial circadian oscillator can be reconstituted in vitro by mixing three clock proteins, KaiA, KaiB and KaiC, with ATP. KaiC is the only protein with circadian rhythmic activities. In the present study, we tracked the complex formation of the three Kai proteins over time using blue native (BN) polyacrylamide gel electrophoresis (PAGE), in which proteins are charged with the anionic dye Coomassie brilliant blue (CBB). KaiC was separated as three bands: the KaiABC complex, KaiC hexamer and KaiC monomer. However, no KaiC monomer was observed using gel filtration chromatography and CBB-free native PAGE. These data indicate two conformational states of KaiC hexamer and show that the ground-state KaiC (gs-KaiC) is stable and competent-state KaiC (cs-KaiC) is labile and degraded into monomers by the binding of CBB. Repeated conversions from gs-KaiC to cs-KaiC were observed over 24 h using an in vitro reconstitution system. Phosphorylation of KaiC promoted the conversion from gs-KaiC to cs-KaiC. KaiA sustained the gs-KaiC state, and KaiB bound only cs-KaiC. An E77Q/E78Q-KaiC variant that lacked N-terminal ATPase activity remained in the gs-KaiC state. Taken together, ATP hydrolysis induces the formation of cs-KaiC and promotes the binding of KaiB, which is a trigger for circadian oscillations.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins: KaiA, KaiB, and KaiC. Through interactions among the three Kai proteins, the phosphorylation states of KaiC generate circadian oscillations in vitro in the presence of ATP. Here, we characterized the complex formation between KaiB and KaiC using a phospho-mimicking mutant of KaiC, which had an aspartate substitution at the Ser431 phosphorylation site and exhibited optimal binding to KaiB. Mass-spectrometric titration data showed that the proteins formed a complex exclusively in a 6:6 stoichiometry, indicating that KaiB bound to the KaiC hexamer with strong positive cooperativity. The inverse contrast-matching technique of small-angle neutron scattering enabled selective observation of KaiB in complex with the KaiC mutant with partial deuteration. It revealed a disk-shaped arrangement of the KaiB subunits on the outer surface of the KaiC C1 ring, which also serves as the interaction site for SasA, a histidine kinase that operates as a clock-output protein in the regulation of circadian transcription. These data suggest that cooperatively binding KaiB competes with SasA with respect to interaction with KaiC, thereby promoting the synergistic release of this clock-output protein from the circadian oscillator complex.

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...

Although organisms are exposed to various pressure and temperature conditions, information remains limited on how pressure affects biological rhythms. This study investigated how hydrostatic pressure affects the circadian clock (KaiA, KaiB, and KaiC) of cyanobacteria. While the circadian rhythm is inherently robust to temperature change, KaiC phosphorylation cycles that were accelerated from 22 h at 1 bar to 14 h at 200 bars caused the circadian-period length to decline. This decline was caused by the pressure-induced enhancement of KaiC ATPase activity and allosteric effects. Because ATPase activity was elevated in the CI and CII domains of KaiC, while ATP hydrolysis had negative activation volumes (Δ V ≠ ), both domains played key roles in determining the period length of the KaiC phosphorylation cycle. The thermodynamic contraction of the structure of the active site during the transition state might have positioned catalytic residues and lytic water molecules favourably to facilitate ATP hydrolysis. Internal cavities might represent sources of compaction and structural rearrangement in the active site. Overall, the data indicate that pressure differences could alter the circadian rhythms of diverse organisms with evolved thermotolerance, as long as enzymatic reactions defining period length have a specific activation volume.

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.