Gene knockout of glutathione reductase results in increased sensitivity to heavy metals in Acidithiobacillus caldus

Acidithiobacillus caldus plays an important role in bioleaching of low-grade metal ore. It can promote the release of heavy metals in mining-associated habitats and survive in high concentrations of heavy metals. Functions of glutathione reductase (GR) in cell defense against reactive oxygen species caused by heavy metals have been elucidated in some eukaryotic cells and bacteria; however, no information is available in A. caldus. In this research, the methods of bioinformatics, gene expression, GR activity assays were used to detect and characterize the glutathione reductase gene from the A. caldus MTH-04 strain. Then, A. caldus gr knockout mutant and gr overexpression strain were constructed, and the heavy metal tolerant properties and transcriptional levels of ROS related genes of them were compared to study the function of GR. The results showed that, a putative gr gene F0726_RS04210 was detected in the genome of A. caldus MTH-04. The purified recombinant protein of F0726_RS04210 showed remarkable GR activity at optimal pH 7.0 and 30°C using in vitro assay. The evolutionary relationship of GR from A. caldus MTH-04 was close to that from Escherichia coli K12. Gene knockout or overexpression of gr in A. caldus did not affect the growth rate on S⁰ medium, suggesting that GR did not play a key role in the activation of sulfur. Deletion of gr resulted in increased sensitivity to heavy metals (Cu²⁺ and Zn²⁺) in A. caldus, and the gr overexpression strain showed enhanced tolerance to heavy metals. Furthermore, transcription analysis also revealed strong correlations between GR and the antioxidant pathway. The above results suggest that GR can play an important role in heavy metal tolerance in A. caldus.