The Functional Study Of Slr0895 Transcriptional Regulator In Glucose Metabolism And Reactive Oxidative Stress Acclimation In Synechocystis Sp.PCC 6803

Given its extraordinary ability to use sunlight and carbon dioxide to grow,cyanobacteria are now considering as emerging green host to produce value-added products.The bottleneck of the effort to eploit this host is to face the adverse natural condition such as high light during its growth.Like other organism,cyanobacteria also respond to the adverse condition by generating reactive oxygen species(ROS).The effect of these ROS on the glucose metabolic pathway could prevent to optimize product production because most of the pathways are fed by intermediates of the glucose metabolic pathway.Thus understanding the mechanism of genetic mechinaries that sense adverse condition and mediate interaction between glucose metabolic pathway and other pathways in cyanobacteria is pivotal to establish it as suitable host.Similarly,cynaobacteria is also simple and ideal platform to understand interaction of glucose metabolism and other cellular activities such as phosynthesis,chlorophyll metabolism that takes place in plant under adverse physiological condition.To understand the cellular mechanism in such adverse conditions,I incubated culture of cyanobacterium Synechocystis sp.PCC 6803(here after Synechocystis only)in prolonged dark(72 h)mediated heterotrophic condition,[GLC(+)DARK] and then shifted into mixotrophic condition in high light,named [GLC(+)DARK-LIGHT].The results showed that the GLC(+)DARK-LIGHT condition could generate oxidative stress and prevent Synechocystis wild type(WT)cells from growth.In contrast,a deletion mutant of slr0895(D95),which encodes a Tet R family HTH type transcriptional regulator,was found growth well with different glucose uptake,glycogen and pigment contents from the WT under the GLC(+)DARK-LIGHT condition.The complementation study by expressing slr0895 in the D95 mutant(C95 strain)restored growth characteristics and phenotypes of WT under the GLC(+)DARK-LIGHT condition.In addition,D95 mutant was found with a decreased ROS content in compared with WT and C95 grown under the GLC(+)DARK-LIGHT condition,and further q RT-PCR analysis revealed that both sod B and slr0896 expression increased in D95 in GLC(+)DARK-LIGHT condition.With a hypothesis that Slr0895 may be involved in ROS response regulation,I fist applied a Chip-PCR analysis to determine possible target of Slr0895,the results showed that slr0896 gene was negatively controlled by Slr0895.Deletion of both slr0895 and slr0896 showed increased ROS and restored phenotype of WT,further demonstrating slr0896 plays key roles in ROS mitigation in D95.Furthermore,increased expression of positive regulator of glucose metabolism,sig E,and some other genes in the sig E regulon was also found in D95 mutant under the GLC(+)DARK-LIGHT condition.Finally,metabolomics analysis using LC-MS and GC-MS showed active ROS response and mitigation,as well as shifted glucose metabolism from glycolytic to the oxidative pentose phosphate(OPP)pathway under the GLC(+)DARK-LIGHT condition.Together,the results suggested that Slr0895 was involved in perceiving signals from ROS under physiological condition,and then co-ordinate gene expression of stress removal and glucose metabolism.This is the first ever study in cyanobacteria,in best of my knowledge,that transcriptional factor could sense ROS,generated in the presence of glucose under adverse condition,and co-ordinate glucose metabolism.These findings could be important to understand ROS signaling and glucose metabolism in plant under similar consition as well as to generate strain cyanobacteria with improved ROS tolerant.