| In this study,we have characterized a novel ArsR/Smt B family regulator,AcsR（Acidithiobacillus caldus copper-sensitive repressor）.The characterization of the acsR operon,transcriptional analysis,determination of AcsR properties,copper binding and competitive experiments,site-directed mutation,and electrophoretic mobility shift assays have provided us with molecular insights into copper resistance in A.caldus and broadened our understanding of the ArsR/Smt B family member that senses copper.The main study contents were listed as follows:（1）A.caldus ACAty＿RS15375 gene was identified by upregulated-transcriptional level under copper stress.Through bioinformatics analysis,ACAty＿RS15375 gene was determined to be a member of the ArsR/Smt B family.When compared to another ArsR/Smt B family Zn（II）-sensitive protein,Smt BSe,the amino acids in response to Zn（II）were replaced with other residues,indicating that it may not bind Zn.It was determined that the potential promoter of the acsR gene was PIII,with fluorescence intensities much higher than that of the control and the other potential promoters PI and PII.By constructing the p JN19R-promoter-acsR-EGFP plasmid,it was found that the fluorescence value was significantly inhibited in the absence of copper ions,while the inhibition was not significantly alleviated in the presence of copper ions.（2）By constructing recombinant plasmid p ET28a-AcsR,the soluble heterologous of the recombinant AcsR protein was expressed in Escherichia coli BL21.After desalting and purification,the recombinant protein was a dimer in Tris-HCl solution.The circular dichroic spectrum data analysis showed that the secondary structure of apo-AcsR comprised anα-helix（36.5%）,an extended strand（17.3%）,aβ-turn（18.6%）,and a random coil（27.6%）.Under anaerobic conditions,copper ion titration experiments showed that the apo-AcsR monomer binds two Cu（Ⅰ）molecules in vitro（4CuⅠper dimer）.In an effective bicinchoninic acid（BCA）competition system,the dissociation constant KD was 2.55±0.32×10-9 M,indicating that apo-AcsR competes weakly for Cu（I）.AcsR C10A,C14A,C62A,and C64A mutants were constructed by site-directed mutagenesis.Size exclusion chromatography profiles showed that the original assembly of all mutants remained intact,indicating that the mutation of a single cysteine residue would not alter the dimeric structure state.Adding～2 mol equivalents of Cu SO4（relative to mutants AcsR C10A,C14A,and C62A）resulted in saturation.However,saturation was reached after adding～1 mol equiv.of Cu（I）to the AcsR C64A mutant,suggesting that Cys64 is an essential amino acid that can binding Cu（I）.The KD values were 3.24±0.15,4.17±0.38,3.93±0.27,and 6.05±0.47×10-9 M for AcsR C10A,C14A,C62A,and 64A,respectively.（3）It was determined that AcsR would bind to the P2245 and P2270 DNA fragments.The results revealed that as the concentration of AcsR increased,the amount of DNA-protein complex became higher.We put a concentration gradient of Cu SO4 to see if Cu controls how well this DNA-protein complex binds.The results showed that apo-AcsR couldn’t bind to DNA when there was more Cu SO4.These results showed that there was a form of negative regulation between apo-AcsR and the DNA repeats. |
