Synechocystis KaiC3 displays temperature and KaiB dependent ATPase activity and is important for growth in darkness

Abstract

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...

Two circadian oscillators in one cyanobacterium

“…Thus, we propose that Sll0485 is a novel KaiA-like homolog linked to the KaiB3-KaiC3 system. Deletion of sll0485 led to impaired viability during nonphotoautotrophic growth, in line with previous studies on the KaiB3-KaiC3 system (31). Hence, we introduced the name KaiA3 for Sll0485 and will use it from here onwards.…”

“…Recombinant KaiC3 was produced with an N-terminal Strep-tag (31). Strep-KaiC3 (monomer: 58 kDa) migrated as one band at ~ 400 kDa, in line with the size of the hexamer (348 kDa).…”

“…KaiA3-His6 (monomer: 35 kDa) migrated as one single band of approximately 100 kDa size, confirming the capability of KaiA3 to form homodimers. Recombinant KaiB3 (monomer: 12 kDa) was recently shown to form dimers and tetramers of 13 and 41 kDa size, respectively, after size exclusion chromatography (31). KaiB3 displayed three distinct bands in native gels.…”

“…Phenotypical analysis hinted at similar cellular functions of both timing systems. While deletion of kaiC3 had no effect on growth in light-dark cycles, the cells displayed growth defects under chemoheterotrophic conditions compared to that of the WT (29,31). The impairment was less severe in comparison with a ΔkaiAB1C1-deficient strain, which completely lost the ability to grow in the dark.…”

“…The impairment was less severe in comparison with a ΔkaiAB1C1-deficient strain, which completely lost the ability to grow in the dark. Furthermore, Wiegard et al verified KaiA-independent autophosphorylation, autodephosphorylation, and ATPase activity of Synechocystis KaiC3; enzymatic activities suggested to be conserved in all cyanobacterial KaiC proteins (22,31). However, compared to Synechococcus KaiC, KaiC3 ATPase activity was found to be diminished and lacked temperature compensation, an essential feature of true circadian oscillations (2,31).…”