AUTHOR=Shi Yuping , Wu Wei , Yang Yinghui , Liu Xiao , Lin Jianqiang , Liu Xiangmei , Lin Jianqun , Pang Xin 

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

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1250330

DOI=10.3389/fmicb.2023.1250330

ISSN=1664-302X

ABSTRACT=<p><italic>Acidithiobacillus caldus</italic> 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 <italic>A. caldus</italic>. In this research, the methods of bioinformatics, gene expression, GR activity assays were used to detect and characterize the glutathione reductase gene from the <italic>A. caldus</italic> MTH-04 strain. Then, <italic>A. caldus gr</italic> knockout mutant and <italic>gr</italic> 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 <italic>gr</italic> gene F0726_RS04210 was detected in the genome of <italic>A. caldus</italic> MTH-04. The purified recombinant protein of F0726_RS04210 showed remarkable GR activity at optimal pH 7.0 and 30°C using <italic>in vitro</italic> assay. The evolutionary relationship of GR from <italic>A. caldus</italic> MTH-04 was close to that from <italic>Escherichia coli</italic> K12. Gene knockout or overexpression of <italic>gr</italic> in <italic>A. caldus</italic> did not affect the growth rate on S<sup>0</sup> medium, suggesting that GR did not play a key role in the activation of sulfur. Deletion of <italic>gr</italic> resulted in increased sensitivity to heavy metals (Cu<sup>2+</sup> and Zn<sup>2+</sup>) in <italic>A. caldus</italic>, and the <italic>gr</italic> 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 <italic>A. caldus</italic>.</p>