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This protocol could be used to analyze Kai-protein complexes in Clear-native PAGE perform Western Blot transfers of large native protein complexes with a semi dry blotting system perform the immunodetection of his-tagged proteins with a single monoclonal anti-his antibody conjugated to horseradish peroxidase (HRP) that catalyses the chemical reaction of the substrate ECL generating a chemiluminescence signal

This protocol can be used to - express his-tagged proteins in E. coli cells by using a T7-polymerase based expression system - Lyse cells with sonification - purify target protein by Ni-affinity chromatography with nickel magnetic beads

Synechococcus elongatus PCC 7942 is the model organism for circadian research in Cyanobacteria, and many breakthroughs in our understanding of the clock came through an early focus on a single model organism. However, Cyanobacteria are known for their diversity, and blanket generalization from a single species may be inaccurate. Bioinformatic sequence and data analyses combined with in-depth biochemical, structural, and physiological characterization revealed a range of timing systems and gave insights into shared and distinct features of the clock in different cyanobacteria. Those clock systems range from a largely reduced system in Prochlorococcus, which lacks the core factor KaiA along with central input and output factors, to an extended system in Synechocystis sp. PCC 6803, which harbors two additional Kai systems with rather unknown functions. In between, there are more cyanobacterial strains for which circadian rhythmicity was shown. Moreover, homologs of cyanobacterial clock proteins are present in some Bacteria and Archaea, opening questions about the evolution of circadian rhythmicity in prokaryotes. In this chapter, we will highlight common aspects and key differences of timing systems that are based on Kai proteins.

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., 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.

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 CO2 - acclimation in the light and to carbon metabolism in the dark. Further, our results indicate a link between the circadian clock and phototaxis.

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. 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 non-standard KaiC homologs in vitro. KaiC3 displayed ATPase activity, which were lower compared to 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 crosstalk between the KaiC1 and KaiC3 based systems.

This protocol can be used for: (i) heterologous expression of GST-tagged proteins from pGEX-6P1 based expression vectors in E. coli. (ii) purification of recombinant proteins via affinity chromatography using glutathione-agarose or glutathione-sepharose (GST tagged protein can be eluted with glutathione. Alternatively, the tag can be cleaved off by prescission protease) (iii) further purification of the eluted protein via anion exchange chromatography This protocol was modified from Wiegard A, Dörrich AK, Deinzer HT, Beck C, Wilde A, Holtzendorff J, Axmann IM: Biochemical analysis of three putative KaiC clock proteins from Synechocystis sp. PCC 6803 suggests their functional divergence. Microbiology 2013, 159, 948-958 Snijder J, Schuller JM, Wiegard A, Lössel, P, Schmelling NM, Axmann IM, Plitzko JM, Förster F, Heck AJR: Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state. Science 2017, 355(6330):1181-1184

This protocol describes how to detect synthesis of [α32P]ATP from [α32P]ATP by recombinant KaiC proteins. Radioactive nucleotides are separated via thin layer chromatography using TLC PEI Cellulose F plates as stationary phase and LiCl as soluble phase. The principle of this method is based on Egli et al. (Egli M, Mori T, Pattanayek R, Xu Y, Qin X, Johnson CH.2012. Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism. Biochemistry 51:1547-58.)

100x BG11 (500 ml) 74.79 g NaNO3 3.75 g MgSO4 × 7H2O 1.8 g CaCl2 × 2H2O 0.30 g Citric Acid 0.28 ml 0,5 M Na2EDTA (pH 8) Add Aqua bidest (ddH2O) to a final volume of 500 ml. Aliquot (5x100 ml) and autoclave. Store at 4°C. Trace-Metal-Mix (500 ml) 1.43 g H3BO3 900 mg MnCl2 × 4H2O 110 mg ZnSO4 × 7H2O 195 mg Na2MoO4 × 2H2O 39.5 mg CuSO4 × 5H2O 24.7 mg Co(NO3)2 × 6H2O Put some ddH2O into the bottle; dissovle the salts seperately and fill up to 500 ml. Sterile filter and aliquot the solution (10 x 50 ml). Store at 4°C. 2x BG11 (1 L) 20 ml 100x BG11 2 ml K2HPO4 × 3H2O (30 mg/ml) 2 ml Na2CO3 (20 mg/ml) 2 ml Trace-Metal-Mix Add 900 ml deionized water, afterwards add the solutions, fill up to 1 L with deionized water and autoclave. Store at RT. Before use add 2 ml of Fe-Ammonium-Citrate (6 mg/ml). 1xBG11 (1 L) 10 ml 100xBG11 10 ml TES pH 8 1 ml K2HPO4 (30 mg/ml) 1 ml Na2CO3 (20 mg/ml) 1 ml Trace-Metal-Mix Add 900 ml deionized water, afterwards add the solutions, fill up to 1 L with deionized water and autoclave. Store at RT. Before use add 1 ml of Fe-Ammonium-Citrate (6 mg/ml).

This protocol can be used to investigate protein-protein interaction via yeast two-hybrid experiments. It describes the yeast-two hybrid method relying on the activity of the histidine-synthetase. If the proteins interact, the yeast cells are able to grow on selective medium without histidine.

This protocol can be used to investigate protein-protein interaction via yeast two-hybrid experiments. It describes the yeast-two hybrid method relying on a color change using β-galactosidase activity.

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

This protocol can be used to: (i) express Strep-tagged proteins in E.coli (ii) lyse cells (iii) purify Strep-tagged proteins via gravity flow affinity chromatography using eitherStrep-tactin XT superflow or Strep-Tactin resin

This protocol can be used to analyse ATPase activity of KaiC proteins. Produced ADP is separated chromatographically using HPLC and monitored via its absorption at 260 nm.

This protocol can be used to further purifiy (Kai) proteins via size exclusion chromatographyusing eitherHiPrep 16/60 Sephacryl S-300 HR column (120 ml column volume), HiPrep 16/60 Sephacryl S-200 HR (120 ml column volume) orSuperdex 200 Increase 10/300 GL (24 ml column volume)