Measurement of Activity of Reactive Oxygen Species (ROS), Antioxidant Enzymes, and Cell Membrane Permeability

To mechanistically understand how herbicide exposure could affect the rate of conjugation, we measured changes in cell membrane permeability, antioxidant enzyme, and ROS activity in the absence and presence of herbicides for all recipient and donor strains. Bacterial ROS were detected with a DCFDA/H2DCFDA-cellular ROS detection assay kit (ab113851, Abcam, UK) using multimode microplate reader (Varioskan LUX2,Thermo Fisher Scientific, USA). Bacterial antioxidant enzymes, including catalase (CAT) and superoxide dismutase (SOD) activity were assayed with commercial kits manufactured by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and a microplate reader (Infinite^® 200 PRO, Tecan, Swiss). The hydrolysis rate of the o-nitrophenyl-β-D-galactopyranoside (ONPG) by cell was measured to assess cell permeability in terms of absorbance values at 420 nm as described in a previous study (Jin et al. 2020). Transmission electron microscope (TEM) was performed to characterize whether the cell morphology or membranes were changed under exposure of herbicide indicative of cell damage. The details for all these methods are described in Supplementary materials (Supplementary Texts 5–7). Each experiment was performed at least in triplicate.