Functional Characterization Of Slr0643in The Cyanobacterium Synechocystis Sp. PCC6803

Regulated intra-membrane proteolysis (RIP) is a conserved mechanism that regulatessignal transduction across membrane by recruiting membrane-bound proteases to cleave thetransmembrane domains of substrate. As the first identified protease that conducting RIP,metalloprotease Site-2-Protease (S2P) has received extensive studies during the past decadeand more and more S2P like proteases have been identified and studied in different organismsthough some of their substrates and related Site-1-Proteases (S1Ps) remain elusive. Here therecently researches on the S2P cascades and the variation as well as conservation werediscussed. Other than the diverse S1Ps, the conserved catalytic motif and prevalence of aminoacids of low helical propensity in the substrates TM segment suggest a conserved catalyticconformation and mechanism in S2P homologs, thus shed light on the future researches on S2Pcascades.So far limited research has been reported about S2P homologs in photosynthetic organism.Based on the understanding of S2P cascades, I analyzed the distribution of S2P homologs indifferent strains of cyanobacteria. Every cyanobacterial species contains genes encodingsite-2-protease (S2P) homolog, which prokaryotic homologs play essential roles in regulatingstress response through intramembrane proteolysis of membrane-bound anti-sigma factors.Thus, I chose the model cyanobacteria Synechocystis sp. PCC6803to study Slr0643, one of theS2P homologs in Synechocystis. Here, gene of Slr0643, one of four S2P homologs inSynechocystis sp. PCC6803, was insertionally disrupted to explore its physiological role. Onlypartially segregated disrupted mutant was obtained, indicating its indispensability for growth.The partially disrupted mutant could not grow at pH6.5and slr0643gene expression wastransiently induced after pH transfer from7.5to6.5, which demonstrated the pivotal role offully functional Slr0643in acid acclimation. The physiological data suggested that the growthof the disruptive mutant was retarded under pH6.5maybe caused by the impairedphotosynthesis system Ⅱ. DNA microarray and quantitative RT-PCR analyses figured outgenes involved in early acid acclimation and revealed differentially expressed genes due toslr0643disruption. Most interestingly, the acid inducibility of SigH operon(sll0856-sll0857-sll0858) was significantly diminished in slr0643mutant thus rendered it the potential target of Slr0643. This observation, together with analysis of differential expression inother sigma factors, implies that Slr0643play an important role in regulating acid acclimationmost probably through its regulation on SigH operon. Meanwhile, in order to study the proteaseproperty of Slr0643in vivo and in vitro, I generated the antiserum against Slr0643andexpressed this multiple transmembranes protease in E.coli successfully. Then I found thatGST-slr0643show metalloprotease characters to cleave β-casein when a proper buffer systemwas used. I believe all these works presented here will facilitate the further characterization onregulation and function of Slr0643dramatically.