Salt Tolerance Mechanism And Functional Characterization Of Transcriptional Regulators In Synechocystis Sp. PCC6803

Changing concentration of salt in environments has a significant impact on growth of photosynthetic cyanobacteria.In addition,large-scale cultivation of cyanobacteria in seawater directly for industrial application will save precious fresh water.However,how to improve salt tolerance of cyanobacteria is still challenging.To explore regulatory mechanisms may be involved for long-term salt stress responses,in our previous work,sll1734 encoding CO2 uptake-related protein(Cup A)and three genes encoding hypothetical proteins(i.e.,ssr3402,slr1339,and ssr1853)were found induced significantly after a 3-day salt stress,and the corresponding gene knockout mutants were found salt sensitive.To further decipher the mechanisms that these genes may be involved,in this study,we performed a comparative metabolomic analysis of the wild-type Synechocystis and the four salt-sensitive mutants using a gas chromatography-mass spectrometry(GC-MS)approach.A metabolomic data set that consisted of 60 chemically classified metabolites was then subjected to a weighted correlation network analysis(WGCNA)to identify the metabolic modules and hub metabolites specifically related to each of the salt-stressed mutants.The results showed that two,one,zero,and two metabolic modules were identified specifically associated with the knockout events of sll1734,ssr3402,slr1339,and ssr1853,respectively.The mutant-associated modules included metabolites such as lysine and palmitic acid,suggesting that amino acid and fatty acid metabolisms are among the key protection mechanisms against long-term salt stresses in Synechocystis.The metabolomic results were further confirmed by quantitative reverse-transcription PCR analysis,which showed the upregulation of lysine and fatty acid synthesis-related genes.The study provided new insights on metabolic networks involved in long-term salt stress response in Synechocystis.In the model cyanobacterium Synechocystis sp.PCC 6803,metabolic activities are regulated by transcriptional regulators(TRs).However,the functions of TRs are poorly understood.To address the issue,we constructed knockout mutants for 32 putative TR-encoding genes of Synechocystis,and comparatively analyzed their phenotypes and metabolic profiles using LC-MS-based metabolomics under autotrophic growth condition.The results showed that only four mutants of TR gene(i.e.,sll1872,slr0741,slr0395 and slr1871)showed differential growth patterns in BG11 medium when compared with the wild type;however,in spite of no growth difference observed for the remaining TR mutants,metabolomic profiling showed that they were visibly different at the metabolite level,suggesting significant functional diversity of TRs in Synechocystis.In addition,an integrative metabolomics and phylogenetic analyses of all TR mutants led to the identification of five pairs of TR genes that each pair shared close relationship in both phylogenetic and metabolomics clustering trees,suggesting possible conserved functions of these TRs during evolution.Moreover,more than a dozen pairs of TR genes with different origin and evolution were found with similar metabolomic profiles,suggesting a possible functional convergence of the TRs during genome evolution.Finally,attempt was also made to predict possible regulatory targets of TRs using protein-protein interaction network analysis.This study provided new insights into the regulatory functions and evolution of TR genes in Synechocystis.