(1. 南京工業(yè)大學(xué) 生物與制藥工程學(xué)院，南京 211816；
2. 中南大學(xué) 資源加工與生物工程學(xué)院生物冶金教育部重點(diǎn)實(shí)驗(yàn)室，長沙 410083)
摘 要: 為了查明Cu2+脅迫影響Acidithiobacillus ferrooxidans(At. ferrooxidans)亞鐵氧化代謝機(jī)制，本研究設(shè)計(jì)了由低到高Cu2+濃度(0~25 g/L)脅迫環(huán)境，利用鄰菲啰啉分光光度法、實(shí)時(shí)熒光定量PCR和循環(huán)伏安法等分析手段分別探究了不同Cu2+濃度脅迫下At. ferrooxidans亞鐵氧化率、亞鐵氧化電子傳遞鏈rus operon相關(guān)基因表達(dá)及胞外電子傳遞的響應(yīng)情況。結(jié)果表明：Cu2+脅迫濃度由0 g/L增加至25 g/L時(shí)，At. ferrooxidans菌亞鐵氧化活性顯著降低(25 g/L Cu2+，對應(yīng)下24 h亞鐵氧化抑制率約為50%)，rus operon相關(guān)基因表達(dá)顯著下調(diào)(尤其是細(xì)胞內(nèi)膜蛋白編碼基因Cyc1和細(xì)胞色素c氧化酶編碼基因CoxBACD分別下調(diào)12.4倍和4~6倍)，F(xiàn)e2+氧化胞外傳遞電子能力也降低(循環(huán)伏安曲線還原峰值29.0 μA(對照組，0 g/L Cu2+)和26.5 μA(25 g/L Cu2+))。綜上所述，高濃度Cu2+脅迫引起At. ferrooxidans菌亞鐵氧化代謝Rus operon相關(guān)基因表達(dá)顯著下調(diào)，造成胞外電子傳遞速率降低，最終導(dǎo)致At. ferrooxidans菌亞鐵氧化率降低。
關(guān)鍵字: Acidithiobacillus ferrooxidans；重金屬脅迫；亞鐵氧化代謝；胞外電子傳遞
（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
