Studies On The Copper Resistant Mechanism Of Cop And Cus Systems In Acidithiobacillus Ferrooxidans And Preliminary Study On Copper Biosensor

Compared to the traditional metallurgical methods,bioleaching technology has characteristics of low cost and is environmental friendly.It has a bright future in processing low-grade ores,which is of great significance to China's sustainable development.But the biological leaching is accompanied by the continuous dissolution and accumulation of copper ions,which has a toxic effect on the leaching microorganisms and leads to the decrease of biomining efficiency.Acidithiobacillus ferrooxidans,a dominant species in bioleaching,is also reported to be the most copper-tolerate microorganism.So,it is an excellent material for studying the copper resistance mechanism of bioleaching bacteria.However,little is known on the copper resistance mechanism of A.ferrooxidans.There is only a copper resistance model obtained by bioinformatics and omics analysis and constructed with reference to the copper resistance mechanism of E.coli.Moreover,the anti-copper components in the model lack the experimental verification in A.ferrooxidans itself.Therefore,more work needs to be done to study the copper resistance genes of A.ferrooxidans,which is helpful to improve the copper resistance mechanism of this bacteria,and also to provide theoretical basis and experimental data for constructing high-efficient copper-resistant strains.According to the copper resistance mechanism model of A.ferrooxidans,three systems are known to work together to maintain the copper ion balance in cells.They are the Cop,Cus and polyP systems.In the Cop system,CopA1,CopA2 and CopB are P-ATPase,and their roles had been previously verified in our laboratory.CopD is a membrane protein located on the inner membrane.The copD is up-regulated by copper ion stimulation,but its function is not yet clear.To research the function of the gene,it was knocked out by the markerless knockout method established in our laboratory.First,the two homology arms of the gene were amplified and ligated to pUC19 plasmid for sequencing,subsequently subcloned to a suicide plasmid to construct the knockout plasmid pK19TG-copD.Then,the knockout plasmid was transferred to A.ferrooxidans ATCC 23270 by conjugation,and the single crossovers could be obtained by using the selection of kanamycin.The restriction enzyme I-Sce I encoded by the I-Sce I gene on plasmid pMSD1-PTScel was used to induce the second homologous recombination in the single crossovers,and the mutant A.ferrooxidans ?copD was successfully obtained.Compared with A.ferrooxidans ATCC 23270 wild type,the growth properties of A.ferrooxidans ?copD were characterized when grown on sulfur powder and ferrous iron added with different concentrations of copper ion,respectively.The results indicated that the ?copD knockout mutant grew better than the wild type when supplemented with low concentration of copper ion.The growth was inhibited only when the concentration of copper ion increased.Combined with the analysis of the changes of gene expression,the results indicated that CopD may be related to the absorption of copper ions in cells.Overexpression is also an effective method to study gene function.The previous qRT-PCR results and transcriptome data indicate that the Cus system is likely to play an important role in the copper tolerance of A.ferrooxidans.So,the cusCBA operon was selected to overexpress on plasmid pJRD215 under control of Ptac in A.ferrooxidans,and the gene overexpression strain A.ferrooxidans(pJRD215-PTcusCBA)was constructed.Compared with A.ferrooxidans(pJRD215),the growth curves of A.ferrooxidans(pJRD215-PTcusCBA)were measured when grown on sulfur powder and ferrous iron supplemented with different concentrations of copper ion,respectively.The differential expression of copper resistance related genes under copper induction was analyzed.The results showed that the overexpression of cusCBA can significantly improve the copper tolerance of A.ferrooxidans.Therefore,CusCBA are proved to play an important role in the copper resistance of A.ferrooxidans.Five copper resistance related genes with low response to other heavy metal ions were screened by qRT-PCR and their promoters were studied further.By using gusA gene as a reporter,the activities of the promoters were characterized by detecting the GusA activities in E.coli DH5?.The changes on the activity were also detected in response to the stimulation of copper ion.The promoter of the cus operon was successfully screened and demonstrated very high activity and highest response to copper stimulation.It showed potential applicable for future study to construct high-efficent copper resistance strains and copper sensor in A.ferrooxidans.In summary,the function of CopD and CusCBA in the copper resistance in A.ferrooxidans was characterized by constructing A.ferrooxidans ?copD mutant and cusCBA gene overexpression strain of A.ferrooxidans(pJRD215-PTcusCBA),respectively.CopD was speculated to play roles in absorbing copper ions,while CusCBA were proved to play an important role in the copper resistance of A.ferrooxidans.In addition,a promoter from A.ferrooxidans with high response to copper ion stimulation was screened by using the gusA reporter gene in E.coli,and it could be used for subsequent study in A.ferrooxidans.