Characterization、Function And Regulation Mechanism Of Copper-sensitive Operon Repressor CsoR In Acidithiobacillus Caldus

Acidithiobacillus caldus is a typical strain for leaching copper ore.Although this strain has high copper resistance,the large amount of copper ions accumulated during the leaching process will still greatly affect the leaching efficiency of the strain.The copper-sensitive operon repressor（CsoR）can regulate many detoxification-related genes to resist extreme copper stress.The in-depth study of this protein has important theoretical value and applications for the construction of high-efficiency copper-resistant strains and the improvement of the leaching rate of A.caldus value.In this study,CsoR derived from A.caldus was used as the object,and a series of studies on the properties,functions,and possible regulatory mechanisms of CsoR^Ac were carried out using molecular biology,biochemistry,and EMSA techniques.（1）This study successfully identified CsoR derived from A.caldus.CsoR^Ac has highly conserved Cu（Ⅰ）-ligands（Cys33,His58,and Cys62）,nearly all-helical structure,and amino acid sequence that constitutes the"core"region.（2）PⅢis the promoter of cso R,but the binding activity of PⅢand CsoR^Ac in vivo is weak.The PⅢpromoter activity is the highest in E.coli DH5α,which is 1.5 times that of the negative control.In addition,the PⅡpromoter and CsoR^Ac showed a certain in vivo binding activity.When induced by Cu（Ⅱ）,Co（Ⅱ）,Cd（Ⅱ）,and Zn（Ⅱ）,the fluorescence intensity of EGFP from E.coli DH5α（p JN19-ProⅡ-EGFP）was increased by about 3,5.7,2.6,and 1.8times compared with the self-control group,respectively.However,the results of EMSA showed that the PⅡpromoter and CsoR^Ac had no in vitro binding activity.（3）CsoR^Ac recombinant protein can achieve soluble intracellular expression in E.coli BL21（DE3）.The molecular weight of recombinant CsoR^Ac is about 13.5 k Da,the structure is mainlyαhelix,and it is a tetramer in native conditions.Each mol of CsoR^Ac monomer can bind 0.8 mol of Cu（Ⅰ）,and CsoR^Ac is still in the state of tetramer after being combined with Cu（Ⅰ）.The affinities of CsoR^Ac and Cu（Ⅰ）in the BCS/BCA system are 2.26×10^-18 M and 0.53×10^-15 M,respectively.The site-directed mutations of key amino acids Cys33,His58,and Cys62 basically did not change the secondary structure and assembly state of CsoR^Ac,nor did it have much impact on the Cu（Ⅰ）binding ability of the protein.The affinity of CsoR^Ac and Cu（Ⅰ）remained the same order of magnitude as apo-CsoR^Ac.（4）CsoR^Ac binds to the target DNA in the form of a tetramer,but has a low affinity.Only by means of a highly sensitive fluorescent labeling method can the blocking band be observed.In addition,CsoR^Ac can combine with the P08430 fragment to generate two retarded bands.The ratio of DNA to protein in the complex is 1:4 and 1:8,respectively.