Characterization Of Site-2-Protease Sll0528 And Sigma Factor SigH In The Stress Response Of Cyanobacterium Synechocystis Sp. PCC6803

It is a widespread and conserved transmembrane signal transduction mechanismin eukaryotes and prokaryotes that S2P(site-2-protease) regulates sigma factor inresponse to environmental stress. S2 P homologs shared conserved catalytic motif indifferent cyanobacterium, but the function is unkown. Hence, here this thesis mainlystudied the roles of S2 P Sll0528 and sigma factor Sig H in different stress acclimation,and the relationship between S2 P Slr0643 and Sig H under acid stress inSynechocystis sp. PCC6803.Transcription data indicated that Sll0528 expressed significantly in many stresscondition, however the exactly mechanism is still unknown. Firstly, I studied theproteolytic activity of Sll0528 and confirmed it is a metalloprotease Site-2-Protease(S2P). Then, gene of Sll0528 was totally replaced by chloramphenicol to explore itsphysiological role. Knock-out of Sll0528 gene in wild type Synechocystis sp. PCC6803 increased their sensitivity to salt, cold and hyperosmotic stress, suggested it maybe crucial to their acclimation; while the mutant is unsensitive to acid, highlight andglucose stress. The Δsll0528 mutant was revealed by retarded growth, reducedpigments, lost water with a smaller cell and disrupted photosystems in 0.9M Na Clwhich suggested the photosynthesis is inhibited. The physiological data in somehowexplains why Δsll0528 mutant grew slowly under salt stress. When addition with1 m M trehalose under the salt stress, photosystems in wild-type can totally recovered,and 75% recovery in Δsll0528 mutant. It indicated that Sll0528 may be involved inthe synthesis of compatible solute glucosylglycerol(GG) in response to salt stress.Quantitative RT-PCR analysis figured out that the expression of genes Sig G and Sig Hwas induced in Δsll0528 mutant under salt stress which suggests the downstream geneof Sll0528 in regulation of slat acclimation is Sig G and Sig H, while Sig F may be insomehow remedy the deficiency of Sll0528.Totally knock-out of Sig H mutant increased their sensitivity to cold stress(17℃), as revealed by retarded growth, reduced pigments, no more unsaturated fattyacid produced and disrupted photosystems. Quantitative RT-PCR analyses upon wildtype figured out the expression of Sig H was induced under cold for about 0.5h. Thephenotype of Δsig H mutant and Δsll0528 mutant was similar under cold stressindicated that Sll0528 may be regulate the Sig H in response to cold stress.Formerly study on Slr0643 mutant suggested that Slr0643 may cleave proteinSll0857 to release Sig H in response to acid stress, but the definite function remainselusive. Based on the research upon Slr0643, I demonstrated the proteolyticrelationship between Slr0643 and Sll0857 through co-expression in vitro, andanalyzed the phenotype similarity of Δslr0643 and Δsig H mutant under acid stress.The results indicated that Slr0643 protease can cleave the Sll0857 protein specifically,and the HExx H structure made sense. Δsig H mutant grew slowly under acid stress,which is different with the lethal in Δslr0643 mutant. These results indicated thatSig H may be not the single downstream sigma factor, another sigma factor maybeinvolved in response to acid stress.The results in this thesis not only lay the foundation for further investigation ofS2 P homologs involved in regulating signal transduction across membrane inSynechocystis sp. PCC6803, but also provide hints for the functional analysis of otherS2 Ps in photosynthetic organisms.