Characterization Of The Function And Mechanism Of Synechocystis Sp.PCC 6803 Site-2-protease Sll0528 In Oxidative Stress Acclimation

Most environmental stresses that cyanobacteria face will cause elevated levels of reactive oxygen species(ROS)within cells.Therefore,ROS is considered to be a universal trigger of stress responses in cyanobacteria.As a photosynthetic autotroph with short culture period and straightforward genetic manipulation,cyanobacteria Synechocystis sp.PCC6803 is usually regarded as a model organism for studying energy metabolism,stress signal transduction and energy sources biotechnology.There are four Site-2-Proteases in Synechocystis sp.PCC6803:Slr0643,Sll0862,Sll0528 and Slr1821,and they response to various environmental stresses.Previous studies have found that Sll0528 involved in salt,ammonium,sorbitol,heat and other stress responses,but its function and mechanism in oxidative stress acclimation have not been reported yet.Here,the phenotypic characteristics of wild type strain WT,sll0528overexpression strain OE0528 and its knockout strain?sll0528 were investigated under methylene blue,H₂O₂ and menadione.The enzyme and non-enzyme mechanisms against oxidative stress were tracked.The expression and regulation of genes related to oxidative stress were analyzed,and the function and mechanism of sll0528 in oxidative stress acclimation were explored by using widely targeted metabolomics as well.The research conclusions obtained are as follows:(1)Sll0528 is involved in the response to oxidative stress.The knockout of gene sll0528caused higher ROS accumulation in strains under Na Cl,NH₄Cl,and sorbiol stresses,indicating that Sll0528 might participate in a variety of abiotic stress responses by regulating intracellular ROS levels.The growth status of WT,?sll0528 and OE0528 under menadione and H₂O₂showed that OE0528 had the highest survival rate under menadione and H₂O₂stresses,while the growth of?sll0528 was seriously inhibited,revealing that Sll0528 responded to H₂O₂stress and superoxide anion O₂^-stress caused by menadione.While,the survival rate of OE0528,WT and?sll0528 under methylene blue decreased consistently with the increase of its concentrations,suggesting that Sll0528 might be not essential for the response to singlet oxygen¹O₂ stress caused by methylene blue.Besides,further investigations found that OE0528 and WT could quickly eliminate intracellular H₂O₂,but the photosynthetic pigment of WT was damaged more seriously than OE0528,suggesting that Sll0528 may participate in H₂O₂stress by clearing H₂O₂,promoting photosynthetic pigment synthesis and photosynthetic reparation.(2)The function and mechanism of Sll0528 in menadione acclimation.The overexpression of gene sll0528 enhanced the tolerance of OE0528 to menadione.Specifically,the intracellular ROS of OE0528 was stable and maintained at a low level under menadione,different from the2-3 times increased in WT and?sll0528.The activity of SOD enzyme in OE0528 recovered more quickly than in WT and?sll0528 after it was inhibited by menadione.The carotenoids in OE0528 was more accumulated than that in WT and?sll0528,and there was almost no reduction under menadione in OE0528.The genes sll0528,sodB(encoding superoxide dismutase)and katG(encoding hydroperoxidase)in OE0528 were rapidly up-regulated under menadione and expressed superlatively about 6,2 and 13 times higher than WT respectively,which were detected by RT-qPCR.In addition,the positive role of Sll0528 in menadione acclimation was confirmed by constructing a complementary strain?sll0528/pJA2-sll0528 of?sll0528 and verifying its phenotype.(3)The functions of Sll0528 in response to oxidative stress based on metabolomics.The widely targeted metabolomics analysis of OE0528 and WT treated with menadione for 15min revealed that hydrangenol,S-adenosylmethionine and glutathione were significantly upregulated in OE0528.Based on the literature data and the expression of drgA(encoding NAD(P)H:quinone oxidoreductase)determined by RT-qPCR,hydrangenol was suggested to induce the expression of NAD(P)H:quinone oxidoreductase,which is necessary to convert menadione to nontoxic hydroquinone through two-electron reduction pathway.The up-rugulated S-adenosylmethionine was speculated to synthesize more glutathione in OE0528,since S-adenosylmethionine is a precursor of glutathione synthesis.In addition,58 metabolites were significantly down-regulated in OE0528,including D-maltose,nicotinamide adenine dinucleotide,4-aminobutyric acid,L-aspartic acid and so on.The enrichment analysis of the pathways with differentially accumulated metabolites revealed that OE0528 down-regulated the nicotinate and nicotinamide metabolism pathway and cofactor synthesis pathway,because marker metabolites such as 6-hydroxynicotinate and nicotinamide adenine dinucleotide located in these two pathways were significantly down regulated,which might directe to synthesize more hydrangenol to relieve oxidative stress.OE0528 weakened amino acid metabolism pathway and purine,pyrimidine metabolism pathway through down-regulating L-ornithine,4-aminobutyric acid,adenine,cytidine and other marker metabolites,which might convert to synthesize more glutathione.OE0528 weakened pathways like glycolysis through down-regulating sucrose,D-glucose-6-phosphate and fructose-1,6-bisphosphate and other sugars,which could alleviate the endogenous ROS pressures.The results of this study provided a theoretical basis for further exploring the mechanism of S2P gene family members to participate in stress response,and lay a foundation for the construction of genetically engineered algae strains chassis that can tolerate to various environmental stresses in the future.