ABSTRACT: The rotation of the Earth results in predictable environmental changes. To address this daily rhythm, organisms from all kingdoms of life have evolved diverse timing mechanisms. The three proteins KaiA, KaiB, and KaiC constitute the central timing mechanism that drives circadian oscillations in cyanobacteria. In addition to this standard protein oscillator, Synechocystis sp. PCC 6803, one of the main model organisms for cyanobacterial research, harbors several, diverged clock homologs. The nonstandard KaiB3-KaiC3 system has been suggested to impact the metabolic switch in response to darkness. Here, we demonstrate the direct interaction of KaiC3 with Sll0485, a potential new chimeric KaiA homolog that we named KaiA3. At the N-terminus, KaiA3 is similar to the NarL-type response regulator receiver domain. However, its similarity to canonical NarL transcription factors drastically decreases in the C-terminal domain, which resembles the circadian clock protein, KaiA. In line with this, we detected the formation of a high molecular weight complex of KaiA3 together with KaiC3 and KaiB3, and a KaiA3-mediated stimulation of KaiC3 phosphorylation in vitro. Phosphorylation of KaiC3 was rhythmic over 48 h in vitro in the presence of KaiA3 and KaiB3 as well as in light-dark entrained cells released to free-running conditions. Deletion of the kaiA3 gene leads to KaiC3 dephosphorylation, and results in growth defects during mixotrophic growth and in darkness. Further, our analyses identified KaiA3 in other bacterial species, representing the first potential KaiA homolog outside the cyanobacterial phylum. In summary, we suggest that KaiA3 is a novel, nonstandard KaiA homolog, thereby extending the KaiB3-KaiC3 system in Cyanobacteria and potentially other prokaryotes. We suggest that KaiA3B3C3, together with the canonical KaiAB1C1 system, mediates the auto-/heterotrophic switch in the facultative heterotroph, Synechocystis.
Philipp Spaet 