| The leaching environment is a extreme environment for the existence of acid and high concentration heavy metals. Leaching efficiency of the leaching bacteria depend on its oxidation ability and resistance mechanisms. Acidithiobacillus caldus is an obligated autotrophic sulfur-oxidizing bacterium. It obtains energy and reducing power by oxidizing inorganic reduced sulfur compounds, and fixes CO2 from atmosphere through Calvin cycle as the carbon source. It plays important roles in biohydrometallurgy, coal desulfurization and the treatment of waste water. It major participant in consortia of microorganisms used for the bioleaching of low-grade sulfide ores, so the activation mechanism of oxidation of elemental sulfur is an important issue to be addressed. Some studies show that activation of elemental sulfur oxidation is dependent on glutathione, while glutathione reductase is the key enzyme in the glutathione metabolism, it is essential for reduction of glutathione disulfide (GSSG) to the reduced form(GSH), necessary for protection of the cells against oxidative stress as an antioxidant and detoxification of xenobiotics. Therefore, analysis the biological function of glutathione reductase is very important and meaningful. In this study,we have done such works:Firstly, Alignment of GRs shows that the GR from A. caldus also contains the highly conserved segments and in which the highly conserved NADPH binding site sequence one arginine residue was replaced by an asparagine residue. The glutathione reductase (GR) from Acidithiobacillus caldus MTH-04 was cloned and expressed in E. coli BL21. The in vitro enzymatic activity assay of the expressed product showed that the recombinant protein has the GR enzymatic properties and its optimal activity at 30℃ and pH 7.0, combine the result of phylogenetic tree suggests that GR is located in the cytoplasm. The enzymatic activity of glutathione reductase(GR) was strongly inhibited by Cu2+, Zn2+, Cd2+, Ag+, Fe2+, Co2+, unaffected by Ca2+, Mg2+, Sn2+, Mn2+.Secondly, according to the remarkless gene knockout technique on A. caldus, we acquired the mutant strain lack of glutathione reductase gene, then we constructed a recombinant plasmids pJRD215-gr and conjugated to mutant. In addition, we constructed engineering bacteria containing tac promoter and plasmids with overexpressed gr for the further research.Thirdly, we measured and compared the growth differences between the A. caldus MTH-04 wild type, the mutants strain and overexpression strain on different sulfur compounds media including sulfur and tetrathionate as the sole sulfur source. The results show that there are no significant difference between this strains, but when presence of Cu2+, Zn2+, Na2SO4. with the increase of concentration, the growth delayed and the growth of gr mutant is lower than the wild, but the growth of the gr complemental strain was similar to the wild.Lastly, we inoculated A. caldus, the gr mutant and the overexpression strain to the middle exponential growth phase in Starkey-S0 medium, and the cells were harvested to extract the total RNA and measured by Real-time PCR. The result shows that:(1) when gr lacked, other enzymes are still able to generate GSSG and GSH, assert redox homeostasis in the organism, that is the reason why there are no significant different between this strains. (2) when presence of Cu2+ or Zn2+, this heavy metals will irreversible binding of the cation thiol groups of glutathione reductase, inhibited the strain growth, caused the oxidative damage, then the gene of thioredoxin antioxidant system will upregulated, the gene encode heavy metal transferase also upregulated. |
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