Interaction Between The Components Of Two-component Systems As Well As The Components Of Toxin-antitoxin Systems And Other Protein In The Cyanobacterium Synechocystis Sp.PCC 6803

Both two-component system (TCS) and toxin-antitoxin system (TAS) are important regulatory elements mediating bacterial adaptation to specific stresses, widely existing in varieties of bacteria. A typical TCS consists of a histidine kinase (HK) and a response regulator (RR). When the HK senses specific environmental stress, the conserved histidine residue of the HK are phosphorylated, and then the phosphate group is transferred to the aspartate residue of the downstream RR. The activated RR regulates the expression of the target gene (s) and the product(s) make bacterial cells adapt to specific stress through regulating metabolism processes. A typical type II TAS is made up of a stable toxin and an unstable antitoxin. The toxin regulate growth rate by targeting to important cellular process and facilitate bacteria to adapt to various stresses. The activity of toxins is regulated by antitoxins at the transcriptional and protein level s. There are a large number of TCS and TAS in cyanobacteria which have unique ability to adapt to various environmental stresse. There are 44 HK genes,42 RR genes and at least 34 TCS genes on the chromosome of the model cyanobacteria Synechocystis PCC6803. In addition to several well-studied TCS, most of them remain to be elucidated for their composition and physiologic roles. We previously found that some TAS show atypical regulatory patterns of toxin activity, and guess that there may be other proteins invoived in the regulation of TA activities.Protein-protein interaction plays important roles in cellular signal transduction, regulation of protein activity and assemble of macro-molecule complex. To explore the composition of TCS and regulation of T A S activity, we analyze the interaction between TCS components as well as TAS components and other proteins using yeast two-hybrid system technique.The main contents are as follows:(1) Construction of expressing plasmids for yeast two-hybrid system analysis. PCR amplifications were performed for the genes enconding thel3 pairs of TCS,13 TAS and 15 non-TAS proteins, respecitively. The amplified products were cloned into the expression plasmids of yeast two-hybrid system, constructing corresponding yeast-expression recombinant plasmids。(2) Analyses of the interaction between TCS components, as well as TAS components and other proteins by yeast two-hybrid system technique. The constructed plasmids were co-transferred into yeast cells, obtaining the recombinant yeast strains. According to the phenotypic characteristics of recombinant strains in different selective media, we anslyzed the interaction between TCS, and TAS toxin or anti-toxin and other cellular proteins.(3) Construction of TCS component deletion mutant and analysis of growth phenotype. According to the results of the yeast two-hybrid system analyses, we selected a pair of TCS components which interact with each other to construct deletion mutants by gene knockout technique and analyzed the growth phenotype of the deletion mutants under the tested stress conditions.The following conclusions are obtained according to the research above:(1) We found the interaction between HiklO and Rre3, Hik13 and Rre18, Hik38 and Hik28,Hik16 and Rre17,Hik5 and Rre42,Hik7 and Rre5,8 TAS components and 10 non-TCS proteins,as well as TAS components of VapBC10 and VapBC12.(2) The Rre3-deletion and Hik10-deletion mutants showed normally grew under high salt conditions, but their growths were significantly inhibited under Cd stress compared with the wild-type strain. The results suggest that Hik10 and Rre3 may constitute a functional TCS mediating Synechocystis to adapt to Cd stress.