Department of Health Laboratory Technology, School of Public Health and Management, Chongqing Medical University, Medical 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, China.
Department of Health Laboratory Technology, School of Public Health and Management, Chongqing Medical University, Medical 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, China; Innovation Center for Social Risk Governance in Health, Chongqing Medical University, Chongqing 400016, China.
Ecotoxicol Environ Saf. 2018 Aug 15;157:417-423. doi: 10.1016/j.ecoenv.2018.03.079. Epub 2018 Apr 11.
To find an efficient chromium (VI) resistance system, with a highly efficient, economical, safe, and environmentally friendly chromium-removing strain, ChrA, ChrB, and ChrAB fragments of the chromium (VI) resistance gene in Serratia sp. S2 were cloned, and their prokaryotic expression vectors were constructed and transformed into E. coli BL21. The anti-chromium (VI) capacity and characteristics of engineered bacteria, role of ChrA and ChrB genes in the anti-chromium (VI) processes, and the mechanism of chromium metabolism, were explored.
The PCR technique was used to amplify ChrA, ChrB, and ChrAB genes from the Serratia sp. S2 genome. ChrA, ChrB, and ChrAB genes were connected to the prokaryotic expression vector pET-28a and transferred into E. coli BL21 for prokaryotic expression. Cr-absorption and Cr-efflux ability of the engineered strains were determined. The effects of respiratory inhibitors and oxygenated anions on Cr-efflux of ChrA and ChrB engineered strains were explored.
ChrA, ChrB, and ChrAB engineered strains were constructed successfully; there was no significant difference between the control strain and the ChrB engineered strain for Cr-metabolism (P > 0.05). Cr-absorption and Cr-efflux of ChrA and ChrAB engineered strains were significantly stronger than the control strain (P < 0.05). Oxyanions (sulfate and molybdate) and inhibitors (valinomycin and CN) could significantly inhibit the Cr-efflux capacities of ChrA and ChrAB engineered strains (P < 0.05), while NADPH could significantly promote such capacities (P < 0.05).
The Cr-transporter, encoded by ChrA gene, confer the ability to pump out intracellular Cr on ChrA and ChrAB engineered strains. The ChrB gene plays a positive regulatory role in ChrA gene regulation. The Cr-metabolism ability of the ChrAB engineered strain is stronger than the ChrA engineered strain. ChrA and ChrAB genes in the Cr-resistance system may involve a variety of mechanisms, such as sulfate ion channel and respiratory chain electron transfer.
寻找一种高效、经济、安全、环保的铬去除菌株，克隆鞘氨醇单胞菌 S2 中铬（VI）抗性基因的 ChrA、ChrB 和 ChrAB 片段，并构建其原核表达载体，转化大肠杆菌 BL21。探讨工程菌的抗铬（VI）能力及特性、ChrA 和 ChrB 基因在抗铬（VI）过程中的作用、铬代谢机制。
采用 PCR 技术从鞘氨醇单胞菌 S2 基因组中扩增 ChrA、ChrB 和 ChrAB 基因，将 ChrA、ChrB 和 ChrAB 基因连接到原核表达载体 pET-28a 上，转入大肠杆菌 BL21 进行原核表达，测定工程菌的 Cr 吸收和 Cr 流出能力，探讨呼吸抑制剂和含氧阴离子对 ChrA 和 ChrB 工程菌 Cr 流出的影响。
成功构建了 ChrA、ChrB 和 ChrAB 工程菌；对照菌株与 ChrB 工程菌 Cr 代谢无显著差异（P>0.05）。ChrA 和 ChrAB 工程菌的 Cr 吸收和 Cr 流出能力明显强于对照菌株（P<0.05）。含氧阴离子（硫酸盐和钼酸盐）和抑制剂（缬氨霉素和 CN）可显著抑制 ChrA 和 ChrAB 工程菌的 Cr 流出能力（P<0.05），而 NADPH 可显著促进这种能力（P<0.05）。
ChrA 基因编码的 Cr 转运蛋白赋予 ChrA 和 ChrAB 工程菌将细胞内 Cr 泵出的能力。ChrB 基因对 ChrA 基因的调控起正向调控作用。ChrAB 工程菌的 Cr 代谢能力强于 ChrA 工程菌。Cr 抗性系统中的 ChrA 和 ChrAB 基因可能涉及多种机制，如硫酸盐离子通道和呼吸链电子传递。
