The Research On The Chromosomal Toxin-antitoxin Genes Ssr1114/slr0664 And Ssl2920/ssl2921 Of Synechocystis Sp. PCC6803

Toxin-antitoxin gene systems(TA systems), widely distributed on the chromosome of free-living bacteria, are assumed to be involved in or mediate cell growth inhibition or cell death as a prokaryotic cell stress factor. However, only a few chromosomal TA systems in the chromosome of E.coli have been demonstrated by experimental test, such as relBE and mazEF systems on E.coli chromosome can mediate growth suppression and cell death induced by environmental stresses. Because the sequence of TA systems in bacteria has a low homology, not many research has been done, the biological function of chromosome TA systems is still under debate. Therefore, the study on the TA system in different bacteria has an important siganificance to interpret its physiological function and reveal the molecular mechanism of bacteria adaptation to environmental stresses. In this thesis, the characteristics of two pairs of toxin-antitoxin genes ssr1114/slr0664 and ssl2920/ssl2921 in the chromosome of Synechocystis sp.PCC6803 are studied. It includes:1) Cloning the sequences of genes slr0664 or ssl2921, and ssr1114/slr0664 or ssl2920/ssl2921 to the downstream of the promoter PpetE which were induced by copper ion in the Synechocystis sp.PCC6803. The recombination plasmids induced by copper were constructed and transfer into the ssr1114/slr0664, ssl2920/ssl2921 deletion mutants, then the expression regulation mutants were constructed. After that, the cells growth characteristic of these expression regulation mutants were analyzed after copper ion induced.2) The ssr1114/slr0664 and ssl2920/ssl2921 were co-expressed after induced in E.coli and purified by using affinity capture technique. The co-expressed products Ssr1114 and Slr0664, Ss12920 and Ss12921 were identified by using spectrometric mass analysis, thereby proved the cross-interaction between Ssr1114 and Slr0664, Ss12920 and Ss12921.3) With the help of the plasmid which has two promoters induced by IPTG and arabinose respectively in E.coli, we cloned the protease gene and the toxic-antitoxic genes under the control of promoter P_Iac and P_BAD, respectively, the protease Lons or ClpP2s/ClpXs and TA protein Ssr1114/Slr0664 or Ss12920/Ss12921 were expressed after heterogeniously induced. The protease whether which degrades the antitoxic proteins were identified by analyze the cell growth and Western blot. This study has the following conclusions:1) The products of slr 0664 and ssl2921 induced by copper ion showed toxic effect on the cells of PCC6803. However, when ssr1114/slr0664 and ssl2920/ssl2921 were co-expressed induced by copper, the toxicity can be inhibited. According to the results, slr0664 and ssl2921 are toxic genes, and the corresponding upstream genes ssr1114 and ssl2920 are antitoxic genes.2) The interaction between Ssr1114 and Slr0664, Ssl2920 and Ssl2921 are proved by co-express and co-purified recombinant protein Ssr1114 and Slr0664, Ssl2920 and Ssl2921. It suggested that the antitoxic protein Ssr1114 and Ssl2920 interact with Slr0664 and Ssl2921 by form complex, thereby inhabited the toxicityof the toxic protein to the cells.3) It was observed clearly that the antitoxic protein Ssr1114 was degraded by both proteases Lons and ClpP2s/ClpXs from Synechocystis sp.PCC6803, butthe antitoxic protein Ssl2920 was not sensitive to these proteases. It is guessed that antitoxic protein Ssl2920 can be degraded by other proteases in the Synechocystis sp.PCC6803.