Si-Min Zhou1, Lan-Lan Dong, Yuan He, Hong Xiao. 1. Research Center for Medicine and Social Development, Innovation Center for Social Risk Governance in Health, School of Public Health and Management, Chongqing Medical University, Chongqing 400016, China. E-mail: 934293515@qq.com.
Abstract
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.
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.