| Abstract:During bioleaching process, the high concentration of heavy metal ions in solution will have a significant toxicity on the growth of microorganisms. Therefore, it is very important to determine the molecular mechanisms that allow these bacteria to resist extremely high concentrations of heavy metals in their environment.Here we determined the tolerance levels of seventeen A. ferrooxidans strains response to copper, zinc respectively. Additionally eight metal resistance related genes (afe-0022, afe-1143, afe-0326, afe-0329, afe-0602, afe-0603, afe-0604and afe-1788) were amplified to identify whether those genes existed in the genome of seventeen A. ferrooxidans strains. Real-time quantitative PCR (RT-qPCR) was used to analyze the transcriptional expression levels of the metal resistance related genes when six A. ferrooxidans strains (DX5, DY15, DY26, YTW, GD-0, GD-B) with different metal-tolerance exposed to0.15M Cu2+,0.2M Zn2+respectively.The effects of0.15M Cu2+,0.2M Zn2+on ferrous oxidation activity and cells growth of seventeen A. ferroxidans strains showed that these strains could divide into three groups. The high copper-resistance strains had7(strains DY26, DY30, DY32, DX5, GF, GD-A, GD-9). The moderate-resistance strains had5(strains DY15, DY18, BY-01, GD-B, BN), and the copper sensitive strains included5strains (strains JYC-3, YTW, GD-0, DC, ATCC23270). There10strains (DY32, GF, BY-01, JYC-3, YTW, GD-A, GD-B, GD-9, BN, ATCC23270) had high zinc tolerance levels. The resistance levels of4strains (DX5, DY26, DC, GD-0) were moderate, while3strains (DY15, DY18, DY30) were sensitive to zinc ion. The results of classification showed that there are4strains (DY32, GF, GD-A, GD-9) have both copper and zinc high resistance levels. The other13strains had different tolerance levels response to copper and zinc.PCR amplification experiment showed that among eight metal resistance related genes there are seven target genes present in the genome of seventeen A. ferrooxidans strains. But gene afe-0604presented in the genome of8strains (DC, YTW, GD-0, GD-A, GD-B, GD-9, BN and ATCC23270). The other9strains (DX5, DY15, DY18, DY26, DY30, DY32, GF, BY-01and JYC-3) did not show the amplified band of gene afe-0604.Bioinformatics analysis showed that gene afe-0022code cation channel protein. The protein encoded by gene afe-0326was cation efflux system protein. The copper-translocating p-type ATPase was encoded by gene afe-0329. Gene afe-1143coded MFP subunit of heavy metal efflux transporter. Genes afe-0602, afe-0603and afe-0604were the component of cation ABC transporter. Gene afe-0602coded periplasmic cation-binding protein. The ATP-binding protein encoded by gene afe-0603. Gene afe-0604coded permease protein with seven predicted transmembrane helix. The membrane-associated zinc metalloprotease with five predicted transmembrane helix was encoded by gene afe-1788.The results of RT-qPCR showed that, the expressions of four target genes (afe-0022, afe-0326, afe-0329and afe-1143) were up-regulated when four A. ferrooxidans strains (DX5, DY15, DY26, GD-B) exposed to0.15M Cu2+. Under0.15M Cu2+, the expressions of two target genes(afe-0326and afe-1143) were up-regulated in two copper-sensitive strains (YTW, GD-0), while the expressions of two target genes(afe-0022and afe-0329) had no significantly change. It was worth mentioning that the relative expression of gene afe-1143in two copper-sensitive strains (YTW, GD-0) higher than the other four copper-resistant strains. What is more, the relative expressions of two target genes(afe-0326and afe-0022) in six A. ferrooxidans strains response to0.15M Cu2+showed the same trend with the resistance levels. The expression levels of four target genes(afe-0602, afe-0603, afe-0604, afe-1788) in six strains response to0.2M Zn2+had a small increase, but didn’t show a clear correlation between the zinc-tolerance levels of six strains.According to the results of RT-qPCR and bioinformatics analysis, the proteins encoded by afe-0022, afe-0326, afe-0329, and afe-1143related with Cu transport of A. ferrooxidans strains. The proteins encoded by genes afe-0602, afe-0603, afe-0604, and afe-1788how to transport Zn2+needed further study. |
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