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The rotation of the Earth results in predictable environmental changes that pose challenges for organisms and force them to adapt. To address this daily rhythm, organisms from all kingdoms of life have evolved diverse timing mechanisms. In the cyanobacterium Synechococcus elongatus PCC 7942, the three proteins KaiA, KaiB, and KaiC constitute the central timing mechanism that drives circadian oscillations. In addition to the standard oscillator, named KaiAB1C1, Synechocystis sp. PCC 6803 harbors several, diverged clock homologs. The nonstandard KaiB3-KaiC3 system was suggested to impact the metabolic switch in response to darkness. Here, we demonstrate the direct interaction of KaiC3 with Sll0485, which is a potential new chimeric KaiA homolog that we named KaiA3. The existence of a functional link between these proteins is further supported by the co-occurrence of genes encoding KaiA3 with the KaiB3–KaiC3-like gene products in 10 cyanobacterial and five other bacterial species. KaiA3 is annotated as a NarL-type response regulator due to its similarity to the response regulator receiver domains. However, its similarity to canonical NarL drastically decreases in the C-terminal domain, which resembles the circadian clock protein KaiA. In line with this, we detected the stimulation of KaiC3 phosphorylation by KaiA3 in vitro. Furthermore, we showed that deletion of the kaiA3 gene led to growth defects during mixotrophic growth conditions and, like a kaiC3-deficient mutant, viability was impaired during chemoheterotrophic growth in complete darkness. In summary, we suggest KaiA3 as a novel, nonstandard KaiA homolog within the cyanobacterial phylum, extending the KaiB3-KaiC3 system in Cyanobacteria and other prokaryotes.Significance StatementMany organisms that are subjected to day and night cycles are able to predict daily changes using a circadian clock system. Cyanobacteria use a relatively simple timekeeper, a protein-based oscillator consisting of KaiA, KaiB and KaiC. Several cyanobacteria and other prokaryotes harbor additional sets of oscillator components that, however, lack the KaiA protein. Our study identified a divergent KaiA homolog that is functionally and genetically linked to potential KaiB3-KaiC3 timekeepers. The data show that the nonstandard KaiA homolog affects the phosphorylation of KaiC3 from the cyanobacterium Synechocystis, suggesting that a similar function exists in other prokaryotes.

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 nontargeted 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.

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.