Effect Of Mancozeb On The Conjugative Transfer Of The Antibiotic Resistance Plasmid And Its Regulatory Mechanism

The problem of combined pollution of pesticide residues and antibiotic resistance genes（ARGs）in greenhouse agricultural environment is becoming more and more serious,which poses a potential threat to the ecological environment and human health.Previous studies have reported that non-antibiotic environmental pollutants,such as disinfectants,nanomaterials,heavy metals and so on,could enhance the abundance of ARGs in environment by promoting horizontal gene transfer.However,the dissemination and regulation mechanism of ARGs under the stress of pesticides are still unclear.In this study,the typical pesticide mancozeb was used as the target compound to construct suitable and stable intra-genera and inter-genera conjugative transfer systems of multidrug-resistant plasmid RP4.We studied the conjugative transfer frequency of plasmid RP4 by the traditional plate culture method with selective labeling,the reactive oxygen species（ROS）,cell membrane permeability,and m RNA expression levels of related regulatory genes in bacterial cells by transmission electron microscopy（TEM）,flow cytometry,and reverse transcription PCR,and the transcription expression levels of donor and recipient bacteria and key regulatory genes in the inter-genera conjugative transfer system by transcriptome sequencing（RNA-seq）technique under mancozeb stress.These results will provide a scientific basis for revealing the dissemination mechanism of ARGs in the pesticide-polluted environment,and also provide a new understanding of the comprehensive assessment of the ecological risk of mancozeb pollution.The main results are summarized as follows:（1）E.coli MG1655 carrying plasmid RP4 was used as the donor,and E.coli HB101or P.putida KT2440 were used as the intra-and inter-genera recipients,respectively.The conjugative transfer frequency of plasmid RP4 increased first and then decreased with the increasing exposure concentration of mancozeb（0～10 mg/L）.The maximum conjugative frequency of plasmid RP4(intra-genera:2.40×10^-4;inter-genera:9.31×10^-5)was observed under 1 mg/L mancozeb exposure,which was 5.55-fold and 8.97-fold higher than that in the controls,respectively.The conjugative transfer frequency was decreased by the high concentration of mancozeb（10 mg/L）,which is largely due to the lower bacteria survival for both donors and recipients.In addition,mancozeb mainly exists as a parent rather than its metabolites or heavy metals Mn and Zn ions during the conjugative transfer process（0-8 h）,indicating that mancozeb parent played a major role in promoting the conjugative transfer of plasmid RP4.（2）The intracellular ROS levels of E.coli MG1655,E.coli HB101,and P.putida KT2440 under mancozeb stress were 1.41～2.12,1.09～1.77,and 1.34～11.92 times higher than those in the controls,respectively.After adding ROS scavenger thiourea,the frequency of intra-and inter-genera conjugative transfer significantly decreased when compared to the mancozeb treatment（p<0.05）,indicating that ROS generation is one of the important mechanisms of promoting the conjugative transfer of plasmid RP4 under the stress of mancozeb.TEM results showed that the cell membrane of donor and recipient bacteria was damaged to a certain extent when exposed to 1 mg/L mancozeb.In addition,flow cytometry results showed that the cell membrane permeability of E.coli MG1655,E.coli HB101,and P.putida KT2440 increased with the increasing exposure concentration of mancozeb（0～10 mg/L）,which was 1.03～4.28,1.34～3.37,and 2.76～6.44times higher than that of the controls,respectively.RT-qPCR results showed that mancozeb could promote the conjugative transfer of plasmid RP4 through the following ways:1)The expression levels of negative regulatory factors korA,korB,and trbA were decreased,and those of the conjugative transfer system genes trb Bp,trf Ap,tra F,and tra J were increased;2)The expression level of negative regulatory gene kilA was increased to reduce the vertical transfer of plasmid RP4;3)The expression levels of outer membrane encoding genes ompA,ompC,ompF in the donor and recipient bacteria were increased to improve cell membrane permeability;and 4)The expression levels of oxidative stress genes（rpos,soxS,soxR,and oxyR）and SOS response genes（lexA and umuD）were up-regulated,resulting in the accumulation of ROS in the donor and recipient cells.（3）The RNA-seq results showed that the transcription levels of conjugative transfer-related genes tra-and trb-in the plasmid RP4 were up-regulated when exposed to 1 mg/L mancozeb,and those of some important functional genes related to the cell membrane,ROS production,SOS response,and pilin synthesis were also up-regulated in the donor and recipient strains,which jointly promoted the increase of conjugative transfer frequency of plasmid RP4.Differential expression gene（DEGs）analysis showed that there were 92 DEGs in the donor E.coli MG1655 and 253 DEGs in the recipient P.putida KT2440 under the stress of mancozeb;GO enrichment analysis showed that the DEGs in the donor were mainly related to the ion binding function,and the DEGs in the recipient was mainly related to the intracellular steady-state expression function;KEGG pathway enrichment analysis showed that the DEGs in the donor were mainly enriched in the pathways of glyoxylate and dicarboxylate metabolism and ABC transporters,and the DEGs in the recipient were mainly enriched in the pathways of cell membrane-related peptidoglycan biosynthesis.