ChrA基因在大肠杆菌中的表达及其抗铬特性

摘要

目的 研究铬(Ⅵ)转运蛋白编码基因ChrA工程菌的抗铬(Ⅵ)能力及铬(Ⅵ)抗性机制.方法 以沙雷氏菌S2基因组作为模板,PCR扩增ChrA基因,连接表达载体pET-28a(+),转化至E.coli BL21表达.测定ChrA工程菌的Cr(Ⅵ)抗性、摄取能力和外排Cr(Ⅵ)能力,探索铬(Ⅵ)负载时间、含氧阴离子(硫酸盐、钼酸盐、钒酸盐、钨酸盐)和呼吸抑制剂(缬氨霉素、寡霉素、CN-、NADH)对其抗Cr(Ⅵ)能力的影响,分析ChrA蛋白转运Cr(Ⅵ)的机制路径.结果 成功构建pET-28a(+)-ChrA工程菌,并表达出膜蛋白ChrA;ChrA工程菌的Cr(Ⅵ)吸收能力低于对照株(P<0.05),Cr(Ⅵ)外排能力高于对照株(P<0.05);工程菌在50 mg/L Cr2O72-中负载30 min,新鲜溶液中释放10 min后,菌体中的Cr(Ⅵ)外排量达到20%,但随着铬(Ⅵ)负载时间延长,外排量逐渐减少;工程菌外排能力受含氧阴离子硫酸盐和钼酸盐显著抑制(P<0.05),推测ChrA蛋白是通过硫酸盐通道运输Cr(Ⅵ),而钨酸盐和钒酸盐对其Cr(Ⅵ)外排无明显抑制或促进作用;在抑制剂类别中,K+载体缬氨霉素、CN-显著性抑制其Cr(Ⅵ)外排,NADH促进其Cr(Ⅵ)外排(P<0.05),而寡霉素无明显抑制或促进作用,表明ChrA蛋白外排铬(Ⅵ)是利用质子动力通过细胞膜化学渗透作用把铬离子泵出细胞外.结论 ChrA蛋白具有将Cr(Ⅵ)从胞浆转运至胞外的作用,ChrA的表达提高了大肠杆菌的铬(Ⅵ)抗性能力.%Objective To construct a genetically engineered Escherichia coli expressing chromate (Cr) ion transporter ChrA and test its Cr resistance capacity. Methods ChrA gene was cloned by PCR from the DNA template of Serratia sp. S2 and linked with the prokaryotic vector pET-28a(+).The recombinant vector was transformed into E.coli BL21(DE3)cells for expression of ChrA protein. Cr (VI) risistance and Cr (VI) uptake and efflux of the engineered bacteria were tested, and the effects of Cr loading time, oxyanions (ulfate, molybdate, vanadate, tungstate), and respiratory inhibitors (valinomycin, CN-, oligomycin, and NADH) on Cr (VI) efflux were examined to analyze the pathway of Cr (VI) transport by ChrA protein. Results The engineered E.coil strain was successfully constructed.Experiments using cell suspensions showed a lowered Cr2O72-uptake but an increased efflux capacity of ChrA-engineered bacteria compared with the control strain(P<0.05).The engineered E.coil cells in exponential growth incubated for 30 min in the presence of 50 mg/L Cr2O72-showed a total displacement of Cr(VI)of 20% after resuspension in PBS at 10 min,but chromate efflux decreased subsequently as the incubation time extended.Oxyanions sulfate and molybdate significantly inhibited chromate efflux in the engineered bacteria (P<0.05), whereas tungstate and vanadate did not obviously affect chromate efflux; chromate efflux was significantly inhibited by K+ionophore valinomycin and CN-,enhanced by NADH(P<0.05),but not affected by oligomycin,suggesting the role of chromate transporter ChrA as a chemiosmotic pump that extrudes chromate using the proton-motive force.Conclusion ChrA can efficiently transport chromate ions from the cytoplasm to enhance chromate resistance of the genetically engineered E.coli.