Curtin Water Quality Research Centre, Department of Chemistry, Curtin University, GPO Box U1987, Perth, 6845, Australia.
School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, 61005, Republic of Korea.
Water Res. 2020 Sep 1;182:115921. doi: 10.1016/j.watres.2020.115921. Epub 2020 May 16.
This study investigated the degradation and deactivation of an extracellular ampicillin resistance gene (amp) encoded in plasmid pUC19 during exposure to UV, OH (generated by UV/HO), and combined exposure to UV and OH (and/or SO) using UV/HO and UV/SO. The degradation rates of amp measured by quantitative polymerase chain reaction increased with increasing target amplicon length (192-851 bps). The rate constants for the degradation of pUC19 (2686 bps) were calculated as 0.26 cm/mJ for UV and 1.5 × 10 Ms for OH, based on the degradation rates of amp amplicons and assuming an equal sensitivity of DNA damage across the entire plasmid. DNA repair-proficient Escherichia coli (E. coli) AB1157 strain (wild-type) and its repair-deficient mutants including AB1886 (uvrA), AB2463 (recA), AB2480 (uvrA, recA), and DH5α (recA, endA) were applied as recipient cells in gene transformation assays. Results suggested that the elimination efficiency of transforming activity during UV and OH exposure was dependent on the type of DNA repair genes in recipient E. coli strains. Losses of transforming activity were slower than the degradation of pUC19 by a factor of up to ∼5 (for E. coli DH5α), highlighting the importance of DNA repair in recipient cells. The degradation rates of amp amplicons were much larger (by a factor of ∼4) in UV/HO and UV/SO than UV direct photolysis, indicating the significant contribution of OH and SO to the gene degradation. Not only UV and SO, but also OH contributed to the degradation of amp during UV/SO, which was attributed to the conversion of SO to OH and a 10-fold larger reactivity of OH towards amp as compared to SO. However, the enhanced gene degradation by radicals did not lead to a faster elimination of gene transforming activity during UV/HO and UV/SO, suggesting that UV- and radical-induced DNA damage were not additive in their contributions to losses of gene transforming activity. Wastewater effluent organic matter (EfOM) accelerated the degradation of amp during UV irradiation by means of reactive species production through indirect photolysis reactions, whereas EfOM mainly acted as a radical scavenger during UV/HO and UV/SO treatments.
本研究考察了在暴露于 UV、OH（由 UV/HO 产生）以及 UV/HO 和 UV/SO 联合暴露下，质粒 pUC19 中编码的胞外氨苄青霉素抗性基因（amp）的降解和失活情况。通过定量聚合酶链反应测量的 amp 降解率随目标扩增子长度的增加（192-851 bps）而增加。基于 amp 扩增子的降解率，并假设整个质粒的 DNA 损伤敏感性相等，计算出 pUC19（2686 bps）的降解速率常数分别为 0.26 cm/mJ（UV）和 1.5×10 Ms（OH）。DNA 修复功能正常的大肠杆菌（E. coli）AB1157 菌株（野生型）及其修复缺陷突变体，包括 AB1886（uvrA）、AB2463（recA）、AB2480（uvrA、recA）和 DH5α（recA、endA），被用作基因转化测定中的受体细胞。结果表明，在 UV 和 OH 暴露期间，转化活性的消除效率取决于受体大肠杆菌菌株中 DNA 修复基因的类型。转化活性的丧失速度比 pUC19 的降解速度慢，因子高达 5（对于大肠杆菌 DH5α），这突出了受体细胞中 DNA 修复的重要性。与 UV 直接光解相比，amp 扩增子在 UV/HO 和 UV/SO 中的降解速度要大得多（因子约为 4），这表明 OH 和 SO 对基因降解有重要贡献。不仅 UV 和 SO，而且 OH 在 UV/SO 期间也有助于 amp 的降解，这归因于 SO 向 OH 的转化以及 OH 对 amp 的反应性比 SO 大 10 倍。然而，自由基引起的基因降解并没有导致在 UV/HO 和 UV/SO 期间更快地消除基因转化活性，这表明在基因转化活性的损失中，UV 和自由基诱导的 DNA 损伤并不是相加的。废水有机物（EfOM）通过间接光解反应产生的活性物种加速了 UV 照射下 amp 的降解，而 EfOM 在 UV/HO 和 UV/SO 处理期间主要作为自由基清除剂。
