Identification And Analysis 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 germs, the underlying genetic mechanism is still unknown.Recently, the genome of A. ferrooxidans has been sequenced by Integrated Genomics Inc, and from the annotated information, there are four genes involved in copper homeostasis. The result of sequence alignment suggested that two of them, Afe0329 and Afe0663 (the Locus is AFE-0329 and AFE-0663 respectively), were high homologous (94.43% identity). In this article, we focus on these two high homologous genes, study the relationship between them and copper homeostasis, and identify the differences between them. To test these two genes relationship with copper homeostasis, Real-time PCR was carried out, it showed that genes Afe0329 and Afe0663 were intensive response to copper; with PCR-RFLP cloning approach, the differential gene expression between these high homologous genes in a genome was successfully identified for the first time, it revealed that compared with Afe0663, Afe0329 expressed more RNA in presence of copper ion medium, RFLP profile showed that 96% of lanes were products of Afe0329; analyzed by bioinformatics, the sequence of protein expressed by Afe0329 had all conserved domains of P1b3-type ATPase which was a kind of heavy metal pump, and to be unexpected, the Molecular modeling revealed that the amino acids, which were supported to participated 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; the P1b-type ATPase disrupted Escherichia coli could be partially rescued by complementation by plasmid carrying Afe0329. With Reverse transcriptase PCR, the transcripton of copper homeostasis relative gene Afe0329 in A. ferrooxidans standard strain ATCC23270 was investigated, the results showed that gene Afe0330 and Afe0331 were cotranscribed with Afe0329, they were in a single transcripton; the further analysis of genes in this transcripton was analyzed employed by Vector NTI, Blast, TMHMM Server, PSORTb software and so on, it showed protein AFE0330 which expressed by gene Afe0330 was a cytoplasmic protein, no significant homologous sequences of Afe0330 or Afe0331 had been obtained by Blast analysis, the function of Afe0330 or Afe0331 should be more studied; from the DNA of different strains, the transcripton of Afe0329 was amplified using special primer pairs to identify the universality of it in the genome of A. ferrooxidans strains, the results showed the transcripton of Afe0329 was universal in genome of A. ferrooxidans strains.All of these results confirm that compared with Afe0663, gene Afe0329 which cotranscriped with Afe0330 and Afe0331, have more important role in copper homeostasis. The results lead us to a further step for understanding the copper homeostasis mechanism in this extremophilic microorganism, and lay a theoretical foundation for us to upgrade the bioleaching technique through improving copper resistance of A. ferrooxidans.