除草剂对质粒介导ARGs接合转移的影响及机制  

Effects and mechanisms of herbicides on horizontal transfer of antibiotic resistance genes through plasmid-mediated conjugation

作　　者：孙凤飞 王秀 毛雨梦 刘珂 刘娟 SUN Feng-fei;WANG Xiu;MAO Yu-meng;LIU Ke;LIU Juan(Institute of Organic Contaminant Control and Soil Remediation,College of Resources and Environmental Sciences,Nanjing Agricultural University,Nanjing 210095,China;Institute of Animal Husbandry and Poultry Science,Nanjing 210017,China)

机构地区：[1]南京农业大学资源与环境科学学院,土壤有机污染控制与修复研究所,江苏南京210095 [2]南京市畜牧家禽科学研究所,江苏南京210017

出　　处：《中国环境科学》2024年第11期6426-6434,共9页China Environmental Science

基　　金：国家自然科学基金资助面上项目(42377018);江苏省自然科学基金资助面上项目(BK20221519)。

摘　　要：本研究以同时携带四环素、氨苄青霉素和卡那霉素3种抗生素抗性基因(ARGs)的可移动质粒RP4为供体质粒(保存于供体菌E.coli DH5α中),以E.coli HB101为受体菌,以丁草胺、百草敌和草铵膦作为除草剂代表,研究了不同浓度除草剂对可移动质粒介导的ARGs接合转移的影响,并从细菌细胞膜通透性、胞内ROS含量以及与接合转移相关调控基因的转录水平等角度,阐明了其作用机制.结果表明,100~800mg/L的丁草胺以及40~160mg/L的百草敌都可促进质粒RP4的接合转移.供试浓度范围内,丁草胺皆可刺激细菌胞内产生ROS,百草敌则使ROS水平下降.丁草胺和百草敌处理下,受体菌表面皆出现明显的皱缩,且随着浓度升高,出现孔洞甚至断裂现象.400~800mg/L的丁草胺和40~80mg/L的百草敌皆可通过提高细胞膜通透性,下调kor A基因的转录,同时上调trb Bp、trf Ap和tra A基因的转录,进而促进RP4的接合转移.供试浓度下,草铵膦对RP4的接合影响甚微,对细菌细胞膜通透性、胞内ROS水平以及接合相关调控基因转录水平的影响也不大.研究结果提示需要关注农田环境中除草剂的使用对ARGs传播的风险,并建议通过控制除草剂的种类及施用浓度来控制ARGs在农田生态系统中的接合转移.The spread of antibiotic resistance genes(ARGs)in farmland ecosystem is directly related to human health,and has attracted much attention in recent years.Herbicides are common chemical reagents widely used in farmland ecosystem,and until now,little information has been known about its effects and mechanisms on horizontal gene transfer(HGT)of ARGs between bacteria.In this study,with Escherichia coli DH5α(carrying RP4 plasmid with TetR,AmpR,KanR)as the donor and E.coli HB101as the recipient,a series of diparental conjugation experiments were conducted to investigate the effects of butachlor,paracetamol and glufosinate on HGT of ARGs via plasmid-mediated conjugation.Furthermore,the mechanisms involved were also clarified from cell membrane permeability,intracellular ROS content,and the transcription levels of genes related to conjugation.The results showed that 100∼800mg/L of butachlor and 40∼160mg/L of paracetamol could promote the conjugation transfer of plasmid RP4.Under test concentrations,butachlor stimulated the intracellular production of ROS,while paraquat reduced the ROS level.Under the treatment of butachlor or paracetamol,the surface of the recipient bacterium appeared obvious shrinkage,and with the increase of concentration,the pore and even break appeared.Butachlor(400∼800mg/L)and paraquat(40∼80mg/L)could both increase cell membrane permeability,inhibit the korA transcription and promote the transcription of trbBp、trfAp and traA,thus promoting the conjugation transfer of RP4.Under test concentrations,there was only a little effect of glufosinate on RP4 conjugation,as well as cell membrane permeability,intracellular ROS content and transcription of conjugation-related regulatory genes.This study indicated that widespread use of herbicides in agricultural production could affect the HGT of ARGs via plasmid-mediated conjugation,and the application of reasonable types and concentrations of herbicides could reduce the ARG transmission in both soil environments and plants.

关 键 词：除草剂 可移动质粒RP4 抗生素抗性基因 水平迁移 二亲接合 

分 类 号：X53[环境科学与工程—环境工程]