Screening The Interaction Proteins Of Clock Proteins In Microcystis Aeruginosa

It is the endogenous circadian clock of the cyanobacteria,plants and animals that control many cellular processes and confer an adaptive advantage for these organisms on a competitive environment.Cyanobacteria are the simplest organisms known to exhibit circadian rhythms.The proteins KaiA,KaiB and KaiC encoded by kai gene cluster are essential for circadian clock regulation.The KaiC phosphorylation cycle regulated by the interactions among Kai proteins generates the timing mechanism of circadian rhythms.The protein-protein association among the Kai proteins and other proteins may be a critical process in the generation of circadian rhythms.To learn more about how cyanobacteria tell time,the proteins interacted with each Kai in Microcustis aeruginosa PCC7806 were respectively identified by coimmunoprecipitation or pull-down.O ne protein interacted with KaiA,four proteins interacted with KaiB and two proteins interacted with KaiC were picked out respectively.The protein interaction with KaiA was Alpha/beta superfamily hydrolase.Four proteins interacted with KaiB were dissimilatory sulfite reductase alpha subunit,unnamed protein product(named B6),hypothetical protein Amuc₁273 and 3-dehydroquinate synthase. Two proteins interacted with KaiC were Putative UDP-2,3- diacylglucosa- mine hydrolase and unkown protein(named C4).It was indicated that B6 was universal stress protein A(USPA) and C4 belonged to DUF820 superfamily which may have nuclease activity by bioinformatics.The interactions of KaiB with USPA and KaiC with C4 in vitro were both further confirmed by pull-down.Only the interaction of KaiB with USPA in vivo was detected by BacterioMatch Two-Hybrid System while the interaction of KaiC with C4 wasn't detected.C4 and C196 have been predicted to belong to the PD-(D/E)xK superfamily of nucleases.Therefore,their DNA cleavage activities were analyzed.Both C4 and C196 can lead to DNA degradation in suitable condition.C4 is most active in L and K buffer while optimum buffers of C196 were K and M buffer.The "optimum" temperature for C4 and C196 enzymes was 61℃and higher temperatures above 64℃lead to a sharp decrease in reaction rates.