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

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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.