(1. 南京工业大学 生物与制药工程学院，南京 211816；
2. 中南大学 资源加工与生物工程学院生物冶金教育部重点实验室，长沙 410083)
摘 要: 为了查明Cu2+胁迫影响Acidithiobacillus ferrooxidans(At. ferrooxidans)亚铁氧化代谢机制，本研究设计了由低到高Cu2+浓度(0~25 g/L)胁迫环境，利用邻菲啰啉分光光度法、实时荧光定量PCR和循环伏安法等分析手段分别探究了不同Cu2+浓度胁迫下At. ferrooxidans亚铁氧化率、亚铁氧化电子传递链rus operon相关基因表达及胞外电子传递的响应情况。结果表明：Cu2+胁迫浓度由0 g/L增加至25 g/L时，At. ferrooxidans菌亚铁氧化活性显著降低(25 g/L Cu2+，对应下24 h亚铁氧化抑制率约为50%)，rus operon相关基因表达显著下调(尤其是细胞内膜蛋白编码基因Cyc1和细胞色素c氧化酶编码基因CoxBACD分别下调12.4倍和4~6倍)，Fe2+氧化胞外传递电子能力也降低(循环伏安曲线还原峰值29.0 μA(对照组，0 g/L Cu2+)和26.5 μA(25 g/L Cu2+))。综上所述，高浓度Cu2+胁迫引起At. ferrooxidans菌亚铁氧化代谢Rus operon相关基因表达显著下调，造成胞外电子传递速率降低，最终导致At. ferrooxidans菌亚铁氧化率降低。
关键字: Acidithiobacillus ferrooxidans；重金属胁迫；亚铁氧化代谢；胞外电子传递
（1. Faculty of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing 211816, China;
2. Key Laboratory of Biometallurgy, Ministry of Education, School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China）
Abstract:The effects of Cu2+ stress (0-25 g/L) on the ferrous oxidation efficiency, expression of Rus operon genes and extracellular electron transfer of Acidithiobacillus ferrooxidans(At. ferrooxidans) were investigated by using phenanthroline spectrophotometry, real-time quantitative PCR and cyclic voltammetry. The obtained results indicate that the increased concentrations of Cu2+ stress from 0 g/L to 25 g/L inhibit the ferrous oxidation activity of At. ferrooxidans. Under the stress of 25 g/L Cu2+, the 24 h ferrous oxidation inhibition efficiency is about 50% in comparison to the control (0 g/L Cu2+). The high concentration of Cu2+ stress also decreases the transcriptional levels of Rus operon genes. Especially, Cyc1 and CoxBACD are decreased by 12.4 fold and 4-6 fold, respectively. Meanwhile, the decreased rate of extracelluler electron transfer in At. ferrooxidans under the stress of 25 g/L Cu2+ (26.5 μA) is found in comparison to that in the control (0 g/L Cu2+, 29.0 μA). In conclusion, the high concentration of Cu2+ stress results in the down-regulated expression of Rus operon genes related to Fe2+ oxidation, afterwards inhibiting the electron transport from Fe2+ oxidation, and finally leading to a decrease in the rate of ferrous oxidation.
Key words: Acidithiobacillus ferrooxidans; metal stress; ferrous oxidation metabolism; extracellular electron transfer
