Evaluation for genetic robustness in the machinery of period determination of the cyanobacterial circadian clock

细长聚球藻PCC7942是一种用于生物钟分析的蓝藻模型。生物发光报告系统可用于有效监测该菌株蓝藻菌落的昼夜节律。通过遗传分析发现，蓝藻的生物钟基本上只由三个时钟基因组成：KaiA、Kaib和KaiC。在这些基因座中，特别是在kaiC中，发现了各种改变周期长度从16小时到60小时以上的突变。最近，人们发现在试管中将它们的纯化蛋白与三磷酸腺苷混合后，KaiC的周期磷酸化/去磷酸化具有很强的昼夜周期。也就是说，蓝藻的生物钟是一种化学振荡器，不需要转录/翻译等生物过程/网络。我们试图在实验室里对这个简单系统的遗传稳健性进行实验评估，以对抗三个KAI基因座的突变。利用一组在这些基因座上随机引入氨基酸替换的约300个单一突变，我们开始尝试评估这些突变在野生型背景下对周期长度表型的影响。我们发现，与Kaib和KaiC基因相比，KaiA基因突变对昼夜节律的影响要小得多。这表明这三个时钟基因之间的遗传稳健性是不同的。此外，还发现超过90%的KaiB和KaiC突变在节律中表现出表型。因此，与其他遗传位点相比，这些基因座的遗传稳健性明显较低。利用体外振荡系统分析了其稳健性的分子基础。结果表明，这三个时钟分量可以获得较强的周期性，这一结论与仅在这些基因座上的突变具有表型的事实是一致的。

Synechococcus elongatus PCC 7942 is a model cyanobacterium for analysis of circadian clock. A bioluminescent reporter system can be used for effective monitoring of circadian rhythms of cyanobacterial colonies of this strain. Through a genetic analysis, it was found that the circadian clock of cyanobacteria basically comprises only three clock genes : kaiA, kaiB, and kaiC. In these loci, especially in kaiC, various mutations that altered period lengths ranging from 16 to over 60 hours were identified. Recently, it was found that mixing purified proteins of them and ATP in a test tube brought about a robust cyclic phosphorylation/dephosphorylation of KaiC with a circadian period. Namely, the circadian clock of cyanobacteria is a chemical oscillator that does not require biological processes/networks such as transcription/translation. We have tried to experimentally evaluate the genetic robustness of this simple system against mutations in the three kai loci in the laboratory. Using a set of〜300 single mutations with randomly introduced amino acid substitutions in these loci, we began trying evaluation of effects of those mutations in wildtype background on the phenotype of period lengths. We found that mutations in kaiA had much smaller effects on the circadian rhythm than those in kaiB and kaiC. This suggested that the genetic robustness among the three clock genes is diverged. Further, it was found more than 90% of mutations in kaiB and kaiC showed phenotypes in the rhythm. Thus, it was shown that the genetic robustness of these loci are remarkably low compared to other genetic loci. The molecular basis for the robustness was also analyzed using the in vitro oscillation system. It was shown that the robust periodicity can be attained by the three clock components and this conclusion is consistent with the fact that mutations only in these loci show phenotypes on it.