Institute of Biology III, Faculty of Biology, University of Freiburg, 79104, Freiburg, Germany.
Institute for Synthetic Microbiology, Biology Department, Heinrich Heine University Düsseldorf, 40225, Düsseldorf, Germany.
Nat Commun. 2024 Sep 3;15(1):7674. doi: 10.1038/s41467-024-51914-5.
The circadian clock of cyanobacteria, which predicts daily environmental changes, typically includes a standard oscillator consisting of proteins KaiA, KaiB, and KaiC. However, several cyanobacteria have diverse Kai protein homologs of unclear function. In particular, Synechocystis sp. PCC 6803 harbours, in addition to a canonical kaiABC gene cluster (named kaiAB1C1), two further kaiB and kaiC homologs (kaiB2, kaiB3, kaiC2, kaiC3). Here, we identify a chimeric KaiA homolog, named KaiA3, encoded by a gene located upstream of kaiB3. At the N-terminus, KaiA3 is similar to response-regulator receiver domains, whereas its C-terminal domain resembles that of KaiA. Homology analysis shows that a KaiA3-KaiB3-KaiC3 system exists in several cyanobacteria and other bacteria. Using the Synechocystis sp. PCC 6803 homologs, we observe circadian oscillations in KaiC3 phosphorylation in vitro in the presence of KaiA3 and KaiB3. Mutations of kaiA3 affect KaiC3 phosphorylation, leading to growth defects under both mixotrophic and chemoheterotrophic conditions. KaiC1 and KaiC3 exhibit phase-locked free-running phosphorylation rhythms. Deletion of either system (∆kaiAB1C1 or ∆kaiA3B3C3) alters the period of the cellular backscattering rhythm. Furthermore, both oscillators are required to maintain high-amplitude, self-sustained backscatter oscillations with a period of approximately 24 h, indicating their interconnected nature.
蓝藻的生物钟可以预测日常环境变化，通常包括一个由 KaiA、KaiB 和 KaiC 蛋白组成的标准振荡器。然而，一些蓝藻具有功能不明确的不同 Kai 蛋白同源物。特别是，集胞藻 PCC 6803 除了具有一个典型的 kaiABC 基因簇（命名为 kaiAB1C1）外，还拥有另外两个 kaiB 和 kaiC 同源物（kaiB2、kaiB3、kaiC2、kaiC3）。在这里，我们鉴定了一个嵌合 KaiA 同源物，命名为 KaiA3，由位于 kaiB3 上游的基因编码。在 N 端，KaiA3 与响应调节受体结构域相似，而其 C 端结构域与 KaiA 相似。同源性分析表明，KaiA3-KaiB3-KaiC3 系统存在于几种蓝藻和其他细菌中。使用集胞藻 PCC 6803 同源物，我们观察到在存在 KaiA3 和 KaiB3 的情况下，KaiC3 磷酸化在体外呈现出昼夜节律振荡。kaiA3 的突变会影响 KaiC3 的磷酸化，导致在混养和化养条件下的生长缺陷。KaiC1 和 KaiC3 表现出相位锁定的自由运行磷酸化节律。缺失任何一个系统（∆kaiAB1C1 或 ∆kaiA3B3C3）都会改变细胞反向散射节律的周期。此外，两个振荡器都需要维持具有约 24 小时周期的高振幅、自维持反向散射振荡，表明它们的相互关联性质。
