Matsukami Yusuke, Oyama Katsuaki, Azai Chihiro, Onoue Yasuhiro, Fujita Yuichi, Terauchi Kazuki
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Sci Rep. 2024 Dec 28;14(1):30949. doi: 10.1038/s41598-024-81991-x.
A circadian clock is reconstituted in vitro by incubating three proteins, KaiA, KaiB, and KaiC from the non-nitrogen-fixing cyanobacterium Synechococcus elongatus PCC 7942 in the presence of ATP. Leptolyngbya boryana is a filamentous cyanobacterium that grows diazotrophically under microoxic conditions. Among the aforementioned proteins, KaiC is the main clock oscillator belonging to the RecA ATPase superfamily. Genomic studies have revealed the presence of many genes encoding KaiC family ATPases in archaea and bacteria; however, very few have been analyzed in detail. For example, the L. boryana genome encodes two kaiC homologs designated as LbkaiC1 (LBWT_14830) and LbkaiC2 (LBWT_17950). LbKaiC1 is highly similar to KaiC from S. elongatus PCC 7942 compared with LbKaiC2. LbKaiC1 and LbKaiC2 were purified as Strep-tag fusion proteins. LbKaiC1 formed a hexamer and exhibited autophosphorylation, autodephosphorylation, and ATPase activities. Furthermore, it exhibited circadian phosphorylation rhythm in the presence of KaiA and KaiB from S. elongatus PCC 7942, indicating that LbKaiC1 is the central oscillator of the circadian clock in L. boryana. The temporal separation of nitrogen fixation from photosynthesis may be supported by the circadian rhythm generated by LbKaiC1 in L. boryana. LbKaiC2 had low ATPase activity, which depended on temperature, and its autophosphorylation activity was not detected like a circadian oscillator KaiC. Although the function of LbKaiC2 remains unknown, this work will provide comprehensive understanding of KaiC family ATPases.
通过在ATP存在的情况下孵育来自非固氮蓝藻细长聚球藻PCC 7942的三种蛋白质KaiA、KaiB和KaiC，可在体外重建生物钟。博氏鞘丝藻是一种丝状蓝藻，在微氧条件下进行固氮生长。在上述蛋白质中，KaiC是属于RecA ATP酶超家族的主要生物钟振荡器。基因组研究表明，古菌和细菌中存在许多编码KaiC家族ATP酶的基因；然而，很少有基因被详细分析。例如，博氏鞘丝藻基因组编码两个KaiC同源物，分别命名为LbkaiC1（LBWT_14830）和LbkaiC2（LBWT_17950）。与LbKaiC2相比，LbKaiC1与细长聚球藻PCC 7942的KaiC高度相似。LbKaiC1和LbKaiC2被纯化成为带有链霉亲和素标签的融合蛋白。LbKaiC1形成六聚体，并表现出自磷酸化、自去磷酸化和ATP酶活性。此外，在存在细长聚球藻PCC 7942的KaiA和KaiB的情况下，它表现出昼夜节律性磷酸化，这表明LbKaiC1是博氏鞘丝藻生物钟的核心振荡器。博氏鞘丝藻中LbKaiC1产生的昼夜节律可能支持了固氮与光合作用在时间上的分离。LbKaiC2的ATP酶活性较低，且依赖于温度，并且未检测到其作为生物钟振荡器KaiC的自磷酸化活性。尽管LbKaiC2的功能仍然未知，但这项工作将提供对KaiC家族ATP酶的全面理解。
