Clone And Overexpression Of The Copper And Cadmium-resistance Related Genes In Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans?A.ferrooxidans?,as an important microorganism in bioleaching industry,is also the only studied Acidithiobacillus organisms both with the oxidation abilities of Fe²⁺ and sulfur.Although A.ferrooxidans can survive in a higher concentration of heavy metal ions compared with most other germs,the underlying genetic mechanism is still unknown.It is important to intense research of heavy metal resistant mechanism of the bacteria that can exist in extreme environments.Currently,there are few reports focused on the genetics research for A.ferrooxidans metabolic mechanisms of copper and cadmium.Many A.ferrooxidans metal resistance related genes researches mostly focus on the bioinformatics prediction,but lack the support with corresponding experimental data.Therefore,three metal resistance related genes were selected from A.ferrooxidans genome annotation to build overexpression strain,and provide the experimental data to explore the specific function of genes.The primary subjects of which were as follows:1.A strain of Acidithiobacillus ferrooxidans named L1 was isolated from the acid mine drainage from a coal mine in Agan Town,Lanzhou City,Gansu Province.It was classified as Gram-negative bacteria,the shape of which was short rod according to Gram staining and scanning electron microscopy.The stain with high sulfur-oxidation ability could also use Fe²⁺ as energy resource.As far as the bacterial growth period was concerned,the lag phase lasted for a relative long time of 43 h and then the stationary phase was monitored to last about 14 h.After being cultured for 70 h,the bacterial activity was noticed to decrease gradually.On the other hand,the inhibition effects of copper and cadmium on the growth of A.ferrooxidans L1 strain was Cu²⁺<Cd²⁺ under the same condition,and the resistance against Cu²⁺ and Cd²⁺ were up to 0.04 mol/L and 0.08 mol/L,respectively.2.The physicochemical properties,structural feature,functional domains,phylogenesis as well as the metabolic pathway of the gene-encoded amino acid sequences were analyzed by bioinformatics methods.Proteins AFE-1948,AFE-1862,AFE-RS11495 which were expressed correspondingly by genes afe-1948,afe-1862,afe-RS11495 were distributed in extracellular or cell membrane and more likely to participate in the metal ions adsorption or transport mechanism.3.In order to explore the function characteristics of AFE-1948,AFE-1862,AFE-RS11495 from the A.ferrooxidans L1,the genes of afe-1948,afe-1862,afe-RS11495 were cloned by PCR amplification technique from A.ferrooxidans L1 isolated by our laboratory.The genes were transformed into E.coli BL21?DE3?,and the optimal expressing conditions were defined according to sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?.4.In order to further examine the metal resistance of these three genes,overexpression analysis was used to determine the change of metal resistances after transferred into the receptor bacteria.The result showed that: When this bacterium was exposed to Cu²⁺ or Cd²⁺,comparing with the control group,the cell biomass decreased,at the same time the content of intracellular target protein was increased.The result indicated that resistance of the receptor E.Coli BL21 significantly weakened,and may result in intracellular heavy metals content increasing.