Study Of Copper Resistance Related Genes In Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans,as an important microorganism in bioleaching industry,is also one of the most studied organisms in the bioleaching microbial community.Although A.ferrooxidans can survive in a higher concentration of copper ion compared with most other bioleaching microorganisms,the underlying genetic mechanism is still largely unknown.In this paper,the t tolerance on five heavy metal ions Cu²⁺,Zn²⁺, Cd²⁺,Pb²⁺,and Ag⁺ of the A.ferrooxidans strain DC and 26^# was studied, the results showed that the tolerance on Cu²⁺ and Ag⁺ is quite different between the strain 26^# and DC,the tolerance on Cu²⁺ of the strain 26^# is 5.5 times more than the strain DC while the tolerance on Ag⁺ is 2 times.The whole genome of A.ferrooxidans has been sequenced by Integrated Genomics Inc,and from the annotated information,there are four genes involved in copper homeostasis.In this research,the relationship between the gene Afe1073(the Locus is AFE₁073) and copper homeostasis was investigated.The copper resistance gene Afe1073 of strain DC and strain 26^# were successfully cloned and sequence analysis indicated that there are two amino acids that is A(248) and S(754) of strain 26^# which are different from the standard strain ATCC23270.Real-time PCR was used to detect the different expression of Afe1073 in different concentration of copper ions between two strains DC and 26^#.The results showed that Afe1073 was intensively responsive to copper,the expression was enhanced while the Cu²⁺ concentration increased.Moreover,the expression of Afe1073 increase up to 4610 times in strain DC and 518 times in strain 26^# at the highest concentration the bacteria could tolerate.The bioinformatics analysis showed that the sequence of protein expressed by Afe1073 had all conserved domains of P1b1-type ATPase which was a kind of heavy metal pump.Furthermore,to be unexpected the molecular modeling revealed that the amino acids,which were supported to participate in determination which kind of heavy metal the protein pumped were responsible for the gate of copper ion channel in the transmembrane area of the protein,and the mutation amino acid S(754) in strain 26^# was located in the area.The study about the effects of Ag⁺ on the Afe1073 expression found that the highest concentration that the bacteria could tolerate increased when Ag⁺ existed.The result of Real-time PCR suggest that the gene Afe1073 is also very sensitive to Ag⁺ and its expression amounts increased hundreds or even thousands fold.All of these results confirmed that Afe1073 plays important role in copper homeostasis.The results lead us to a further step for understanding the copper homeostasis mechanism in this extremophilic microorganism,and provide a theoretical foundation for us to upgrade the bioleaching technique through improving copper resistance of A. ferrooxidans.