Differential Gene Expression and Bioinformatics Analysis of Copper Resistance-Related Gene afe_1948 from Acidithiobacillus sp. L1

Abstract

Acidithiobacillus sp. L1, isolated from a coal mine in Agan Town, Lanzhou City, was introduced in this study. The gene afe_1948 relevant to copper resistance from the test bacterial strain was cloned and transformed into Escherichia coli BL21 (DE3) for heterologous prokaryotic expression, and analyzed by SDS-PAGE to evaluate its optimal expression conditions in E. coli. The expressions of afe_1948 in the presence of copper or cadmium were detected by qRT-PCR. The bioinformatics online analysis software was used to predict the physicochemical properties, structural features, functional domains, and possible metabolic pathways of the protein encoded by afe_1948. The result indicated that putative product AFE_1948 is a stable hydrophilic protein that locates on the cytoplasmic membrane or outside the cell. The gene afe_1948 is 1320 bp in length and encodes a total of 439 amino acids. In the experiment, the target gene was noticed to be very sensitive to copper and cadmium. Exposure to different Cu²⁺ concentration (0.05, 0.1, and 0.2 mol/L) resulted in the up-regulation of relative mRNA expression levels of afe_1948 in Acidithiobacillus sp. L1 by 2, 4, and 11-fold compared with the control, respectively. Similarly, the relative mRNA expression levels when exposed to Cd²⁺ at 0.02, 0.04, and 0.06 mol/L were increased by 2, 11, and 23-fold, respectively. To further understand the function of the gene, the variation of resistance to Cu²⁺ or Cd²⁺ after transferring into the recipient strain was examined by overexpression analysis. In the presence of 1.0 mmol/L Cu²⁺ or 0.2 mmol/L Cd²⁺, the highest relative expression levels regarding AFE_1948 were respectively determined to be more than 23% or close to 25% in recombinant E. coli cells. The assay demonstrated that the biomass of recombinant cells decreased significantly in the presence of Cu²⁺ or Cd²⁺ with different concentrations, but the content of intracellular AFE_1948 was enhanced, which may be contributed to a strong induction by the increase of the quantity of heavy metals entering into the cell and introduction of the resistance-related gene.

Keywords:

• Acidithiobacillus sp. L1
• afe_1948
• heavy metal resistance
• qRT-PCR
• bioinformatics analysis

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (No. 31460032) and The Youth Talent Support Program of Lanzhou University of Technology (No. 2018).