KaiA调控蓝藻生物钟的分子机制研究进展

蓝藻生物钟系统主要包括输入途径、核心振荡器和输出途径3部分,核心振荡器主要由时钟蛋白KaiA、KaiB、KaiC构成。3种蛋白之间的相互作用产生节律信号及调控输入、输出信号进而维持生物振荡的精确与稳定。文中围绕蓝藻生物钟核心振荡器及核心振荡器组成蛋白的结构、功能与相互作用特点,结合本实验室近期取得的研究成果,针对时钟蛋白KaiA调节KaiC的酶活性、介导核心振荡器的时相重置、与CikA竞争KaiB的结合位点等方面近年来的研究进展进行了综述。     

Stoichiometric interactions between cyanobacterial clock proteins KaiA and KaiC

We determined the stoichiometry of KaiA-KaiC interactions. Using immunoblotting and two-dimensional Native- and SDS-PAGE (2DNS-PAGE) analysis, we demonstrated that the reaction products of KaiA-KaiC interactions in the presence of ATP consisted of only phosphorylated KaiC whereas in the presence of the unhydrolyzable analogue 5'-adenylylimidodiphosphate (AMPPNP) they consisted of KaiA and KaiC. In the presence of ATP, the KE (molar ratio of KaiA dimer to KaiC hexamer giving half saturation in the enhancement of KaiC phosphorylation) was 0.25, and IAsys affinity biosensor analysis demonstrated that 1 molecule of KaiA dimer interacted with 1 molecule of KaiC hexamer. In the presence of AMPPNP, the ratio of KaiA dimer to KaiC hexamer in KaiA-KaiC complexes was determined to be 2 by 2DNS-PAGE, Native-PAGE/Scatchard plot, and IAsys analyses. These results suggest that 2 molecules of KaiA dimer can interact with 1 molecule of KaiC hexamer, and that interactions of at least 1 molecule of KaiA dimer with 1 molecule of KaiC hexamer are enough to enhance the phosphorylation of KaiC by KaiA at an almost saturated level.     

Genome-wide identification of major protein families of cyanobacteria and genomic insight into the circadian rhythm

Cyanobacteria are among the most abundant organisms present on earth and are considered to be one of the oldest known clades. Cyanobacteria are oxygenic photosynthetic bacteria and are well known as promising renewable sources of energy; therefore, it is important to understand aspects of their genomes in detail across species. Advances in sequencing technology and the availability of several cyanobacterial genomes have provided an excellent opportunity to understand the diversity and evolution of the cyanobacterial genome. Here, we compared the genomes of 62 different phototrophic cyanobacteria. Evaluation of genetic diversity of all the cyanobacteria species studied revealed that evolution from their common ancestors was polyphyletic. In addition, the genomes were very diverse and varied among species, and significant genomic diversity was observed at the species and strain level. Overall, we identified 56 different protein families of cyanobacteria species/strains and found that they varied significantly among strains of a species. The circadian clock proteins KaiA, KaiB and KaiC (KaiABC complex proteins) of cyanobacteria were found to be present and consistent in the majority of cyanobacterial species while absent in a few others. Evolutionary analysis of the KaiABC protein complex showed that the KaiA protein has a high frequency of polymorphism, and multiple alleles were found to be present at high frequency. These results demonstrated that evolution of phosphorylation events occurred via KaiA in the KaiABC complex. Furthermore, multiple sequence alignment showed that KaiA, KaiB and KaiC proteins are highly conserved in nature. Our results provide direct information regarding the presence of different protein or protein families in cyanobacteria. The information presented here will serve as a starting point to explore the genetic diversity of cyanobacteria with the potential to play important roles in biotechnological applications.