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.