A chimeric KaiA-like regulator extends the nonstandard KaiB3-KaiC3 clock system in bacteria

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.