Studies On The Copper Resistant Mechanism Based On Cop Family In Acidithiobacillus Ferrcoxidans

China's copper mineral resources have the characteristics of low taste,complexity and difficult to handle.Bio-metallurgy has great advantages in processing this kind of low-grade ores,and can also effectively leach metals from waste ores and slag.Compared with traditional metallurgical technology,it has characteristics of low cost,less energy consumption and less environmental pollution.In order to improve the effective utilization of mineral resources in China,it is necessary to construct efficient bioleaching strains and carry out the research and application of bio-metallurgical technology.During the process of biological leaching,the metallurgical microorganisms will be poisoned by the continuous dissolution and accumulation of copper ions,which leads to the decrease of biomining efficiency.Therefore,the construction of stable genetic engineering bacteria with high resistance to copper is an effective way to improve the efficiency of bio-metallurgy.Acidithiobacillus ferrooxidans?A.ferrooxidans?is a kind of dominant species in bioleaching.It can tolerate 800 mM of copper ions,which is the most resistant strains among all leaching bacteria and can be used to study the mechanism of copper resistance.However,only a copper resistance model was proposed in this bacteria,whic was estabolised based on bioinformatics,proteomics and transcriptome studies and the function identifications of some genes in E.coli deficent strains.Therefore,further study on copper resistance-related genes in the model can not only enrich the copper resistance mechanism of this bacterium,but also provide theoretical basis and experimental data for the construction of efficient copper-resistant strains.In the copper resistance model of A.ferrooxidans,the Cop system,Cus system.polyP system and some copper ion transporters are the key factors that enable the bacterium to tolerate high copper ions.Among them,Cop system is composed of three copper iron tranport related P-ATPases and their genes are AFE 2779?copAl?,AFE₂439?copA2?,and AFE₂021?copB?,respectively.It has been reported that the heterologous expression of copAl and copB in E.coli deficient strains could improve the copper resistance of the defective strains.However,the roles of CopA1 and CopB in the copper resistance are still uncertain in A.ferrooxidans.According the transcriptome data of A.ferrooxidans conducted in our lab previously,it was also found that copAl and copB were up-regulated under the stimulation of copper.Therefore,the two genes were selected to study further to explore the roles of CopAl and CopB in the copper resistance in A.ferrooxidans.Gene knockout is an effective method for study of gene function.In order to study the role of these two genes in copper resistance of A.ferrooxidans,the markerless knockout method was used to construct knockout mutants of copA1 and copB genes.First,primers were designed according to the sequences of the two genes in the genome of A.ferrooxidans ATCC 23270 at NCBI,and the up-and down-stream homologous arms of the gene were amplified by PCR.After digestion with corresponding restriction enzymes,the two homologous arms were ligated to pUC19 for sequencing and then subcloned to plasmid pK19,respectively.The corresponding recombinant plasmids pK19-copA1 and pK19-copB were then transfered to A.ferrooxidans ATCC 23270 by conjugation.Under the selection of kanamycin,the single-crossovers were obtained.Plasmid pMSD1-I-SceI was transfer to the obtained single-crossovers,respectively,to induce the occurance of second homologous recombination.Finally,A.ferrooxidans AcopAl and A.ferrooxidans AcopB were succesfully screened and obtained.The growth curves of A.ferrooxidans ATCC 23270 wild-type strain and its two gene knockout mutants when grown on sulfur powder as energy source,and the ferrous oxidation curves when grown on ferrous iron as energy source were detected,respectively.The results showed that the knockout of copAl and copB gene alone could only decrease the copper resistant property of the mutants to a certain extent,but did not make the mutants lose their copper resistance property completely.Then,the RT-qPCR method was used to analyze the differences of expression of the copper resistance related genes.The results showed that more genes were differently expressed when grown on ferrous than sulfur powder as the energy source.In both knockout mutants,copC,copZ and ompA were up-regulated and omp49 was down-regulated.The ppK1?ppK2 and cusA were up-regulated only in A.ferrooxidans?copA1.The above results indicated that the copper resistance system in A.ferrooxidans was controled by multiple anti-copper systems at the same time,and the damage to one system might be compensated by strengthening other anti-copper systemsIn summary,based on the construction of A.ferrooxidans ?copA1 and ?copB,the properties of the knockout mutants on copper resistance and the differential expression of copper resistance related genes were detected.The results indicated that the Cop system,Cus system and polyP system,as well as the outer membrane proteins encoded by omp40?tonB and ompA,play a certain role in the copper resistance mechanism of A.ferrooxidans.The damage of Cop system can be remedied by strengtherning other copper resistance systems.Since all genes of Cop systerm were not knocked out at the same time,further work need to be done to evaluate the role of Cop system in the copper resistance in A.ferrooxidans.Anyway,A ferrooxidans ? cop ?1 and ? copB constructed in this paper provided direct experimental materials for studying the roles of the two genes in copper resistance in A.ferrooxidans.