Study On The Substrates Of S2P Protease In Synechocystis Sp. PCC6803

It is a conserved mechanism in bacteria that controlling the freedom of extracytoplasmicfounction Sigma factors (ECF factors) in response to extracytoplasmic stress. On normalcondition, ECF factor was restrained by anti-factor, a transmembrane protein which wasusually co-transcribed with sigma factor. Extracytoplasmic stress will cause metalloproteasesite-2protease (S2P) cleavage follow after site-1protease cleaves transmembraneanti-sigma factor to release sequestered sigma factor in response to extracytoplasmic stress.As the first identified protease that conducting Regulated Intramembrane Proteolysis (RIP),S2P has received extensive studies during the past decade and more and more S2Phomologues have been identified in different organisms. There are four S2P homologs inSynechocystis sp.PCC6803:Sll0862, Slr0643, Sll0528, Slr1821, but their founction remainselusive. Phenotype analysis of Slr0643disrupted mutant revealed that the mutant stopgrowing when it was subjected to acid environment, indicating slr0643is essential in acidacclimation. DNA microarray and quantitative RT-PCR analysis of mutant and wild-typestrains at pH7.5versus pH6.5identified genes involved in early acid acclimation andrevealed genes expressed different due to Slr0643disruption. Most interestingly, analysis ofmicroarray date revealed the close relationship between Slr0643disruption and expressionof SigH operon. Thus it is suggested that Slr0643/Sll0857/SigH might act through anS2P/Anti-Sigma factor/Sigma factor mechanism to play a role in acid acclimation.In this study, using RT-PCR we found sll0857-sll0858is co-transcript with sigH factor(sll0856). Results of yeast two-hybrid experiment indicate that there are close interactionbetween Sll0856and Sll0857. In vivo, we constructed the mutants0856-0857-tag/WT(△P/WT) and0856-0857-tag/△0643-Kmr(△P/0643) which Sll0857–Nwas fused with HA tag, Sll0857-C was fused with6His tag and Sll0856-C was fused withC-myc tag. And in vitro, we expressed and purified the putative substrates of the site-2proteases of Synechocystis sp. PCC6803in order to identify the corresponding relationshipbetween S2Ps and substrates in vitro. These works will lay the foundation for the further research of the relationship among Slr0643/Sll0857/SigH.