Wu Xueling, Hu Qi, Hou Dongmei, Miao Bo, Liu Xueduan
School of Minerals Processing & Bioengineering, Central South University, Changsha, China.
J Gen Appl Microbiol. 2010;56(6):491-8. doi: 10.2323/jgam.56.491.
Locus afe_0454 from Acidithiobacillus ferrooxidans (At.ferrooxidans) is annotated as related to copper resistance in The Institute for Genomic Research database. In our study, two At.ferrooxidans strains, 26(#) and DC, with different levels of copper ion resistance were isolated from acid mine drainages at two major copper mines in China, and their copper-resistance capacity was determined. The 26(#) strain had a copper-tolerance level of 0.22 mol/L, whereas the DC strain had a lower copper-tolerance level of 0.04 mol/L. The mutant 26(#) was generated from strain 26(#), and its copper-tolerance level was 0.25 mol/L. Using real-time quantitative reverse transcription polymerase chain reaction, differential expression of the afe_0454 gene during copper ion stress of these three strains was investigated. The results showed that the expression of afe_0454 was increased under copper ion stress, indicating that the afe_0454 gene is sensitive to copper levels. Furthermore, the afe_0454 gene expression ratio varied in the different copper-resistant strains. Gene expression was highest in the highest copper-resistant strain. The deduced amino acid sequence of the afe_0454 gene was 56.87% non-polar, indicating the AFE_0454 protein was hydrophobic. Searching with the AFE_0454 protein in The Institute for Genomic Research database showed that the structure of the copper resistance protein D (CopD), which transports copper ions outside of the cell, had the highest sequence identity (46%). Bioinformatics analysis showed that the AFE_0454 protein has eight transmembrane helixes and was predicted to be localized to the plasma membrane. These results strongly suggested that the AFE_0454 protein is likely a transmembrane protein and might be directly involved in copper ion resistance.
在基因组研究所数据库中，嗜酸氧化亚铁硫杆菌（At.ferrooxidans）的位点afe_0454被注释为与铜抗性相关。在我们的研究中，从中国两个主要铜矿的酸性矿井排水中分离出了两株对铜离子抗性水平不同的嗜酸氧化亚铁硫杆菌菌株，即26(#)和DC，并测定了它们的抗铜能力。26(#)菌株的耐铜水平为0.22 mol/L，而DC菌株的耐铜水平较低，为0.04 mol/L。突变体26(#)由26(#)菌株产生，其耐铜水平为0.25 mol/L。利用实时定量逆转录聚合酶链反应，研究了这三株菌株在铜离子胁迫下afe_0454基因的差异表达。结果表明，在铜离子胁迫下afe_0454的表达增加，表明afe_0454基因对铜水平敏感。此外，afe_0454基因的表达率在不同抗铜菌株中有所不同。在抗铜能力最强的菌株中基因表达最高。afe_0454基因推导的氨基酸序列中56.87%为非极性，表明AFE_0454蛋白具有疏水性。在基因组研究所数据库中用AFE_0454蛋白进行搜索发现，将铜离子转运到细胞外的抗铜蛋白D（CopD）的结构具有最高的序列同一性（46%）。生物信息学分析表明，AFE_0454蛋白有8个跨膜螺旋，预计定位于质膜。这些结果有力地表明，AFE_0454蛋白可能是一种跨膜蛋白，可能直接参与铜离子抗性。
