Clone And Differential Expression Of Metal Resistance-related Genes In Acidithiobacillus Ferrooxidans

Many genes are annotated as metal transporter or binding proteins in the genome of Acidithiobacillus ferrooxidans（A. ferrooxidans） ATCC23270. In the work six metal-related genes （afe₀654, afe₀671, afe₀674, afe₁143, afe₁144and afe₂126） were the target genes. The relative expression of metal-related genes were studied when A. ferrooxidans were exposed to metal ions, which established the theoretical foundation for studying the molecular mechanism that A. ferrooxidans tolerated high concentration of metal ions.The results of clone showed the target genes existed in A.ferrooxid-ians DC. The sequencing and blasting results showed the sequences of the target genes are the same as those of A. ferrooxidans ATCC23270. Bio informatics analysis indicated that the protein encoded by afe₀654was predicted to be heavy metal binding protein; the proteins encoded by afe₀671and afe₁144were predicted to be tweleve transmembrane proteins, belonging to CzcA family; the protein encoded by afe₀674was a putative heavy metal efflux system protein; the protein encoded by afe₁143was a heavy metal efflux pump and the protein encoeded by afe₂126had eleven predicted transmembrane domains and were predicted to be localized to plasma membrane, belonging to bivalent cation transporter, NRAMP family. CzcA protein was the component of CzcCBA complex, which forms a continuous channel from the cytoplasm to the outside of the cell compsed by CzcA （a large inner membrane protein）, CzcC （a smaller outer membrane protein） and CzcB （a periplasmic coupling protein）. In addition, CzcCBA functioned as an ion/proton exchanger to efflux Cd2+, Zn2+and Co2+is a member of the metal-resistance family within the larger superfamily of chemiosmotic pumps are called RND （for Resistance, Nodulation and Division）.The tolerance levels of A. ferrooxidans DC to Mn2+, Zn2+and Cd2+were determined, which are0.52,0.42and0.16mol/L for iron-grown cells and0.38,0.18and0.08mol/L for sulfur-grown cells, respectively. The toxicity of same metal ion to sulfur-grown cells is greater than ferrous iron-grown cells and the toxicity of Cd2+is greater than that of Zn2+which is greater than that of Mn2+to A. ferrooxidans DC. The real-time quantitative PCR （RT-qPCR） was employed to study the differential expression of target genes when A. ferrooxidans strain DC was exposed to Mn2+, Zn2+and Cd2+. The results showed the relative expressions of afe0671, afe₁143and afe₁144were up-regulated in the presence of Mn2+, Zn2+and Cd2+. In the presence of Mn2+and Cd2+afe₀654and afe₀674were up-regulated while in the presence of Zn2+they were down-regulated in iron-grown cells and up-regulated in sulfur-grown cells. The afe₂126gene was up-regulated in A. ferrooxi-dans DC exposed to Mn2+and Zn2+and down-regulated exposed to Cd2+. According to the RT-qPCR results we predicted that the proteins encoded by afe₀671, afe₁143, afe₁144and afe₂126may relate with the transport of Mn2+, Zn2+and Cd2+. The proteins encoded by afe₀654and afe₀674may relate with the transport of Mn2+and Cd2+and how they transport Zn2+needed further study.