Study On The Mechanism Of Horizontal Transfer Of Multidrug-resistant Plasmid RP4 Affecting The Metabolism Of Ammonia-oxidizing Bacteria

Bacterial antibiotic resistance caused by widespread use of antibiotics is a serious threat to human health.The direct cause of Antibiotic resistance of bacteria is acquiring Antibiotic resistant genes（ARGs）.As a new environmental pollutant,ARGs in water environment mainly come from domestic sewage,industrial wastewater and medical wastewater.Wastewater carrying ARGs will eventually converge into municipal sewage treatment systems,making it an important point source of antibiotic-resistant bacteria and ARGs contamination in the environment.The ammoxidation process mediated by ammoxidation bacteria（NH₄⁺-N→NH₂OH→NO₂^--N）is not only the first step but also the ring-limiting step in the process of biological nitrogen removal.The normal and stable ammoxidation reaction is the key to ensure the successful completion of biological nitrogen removal reaction.Horizontal transfer of ARGs is an important source of bacterial genetic diversity.However,bringing genetic diversity may also cause genetic conflicts in the host,and even interfere with the original metabolic process.However,the law of influence of ARGs conjugation transfer on ammonia nitrogen removal efficiency of ammoxidation bacteria is still unclear,and the related biological characteristics and molecular mechanism of action are still unclear.Therefore,a comprehensive analysis of the mechanism of influence of drug resistance gene conjugation transfer on ammoxidation metabolism can not only further explain the ecological risk of ARGs transmission.At the same time,it can improve and enrich the theory of biological nitrogen removal.In this study,the conjugate transfer plasmid RP4,which encode multiple drug resistance genes,and Nitrosomonas europaea（N.europaea）,an important model for the study of biological wastewater nitrogen removal process and greenhouse gas emissions,were selected as the research objects.The influence of RP4 plasmid on bacterial growth and metabolism after its entry into N.europaea through conjugation transfer was elucidated,and the mechanism of the influence of RP4 plasmid on N.europaea ammonia oxidation metabolism was analyzed from the aspects of functional proteins of ammonia oxidation,electron transport,energy production,quorum sensing and expression of related genes.The main research results are as follows:Firstly,we established a conjugative transfer model between donor（Escherichia coli HB101（RP4））and recipient（N.europaea ATCC25978）mediated by the RP4plasmid.The results showed that the RP4 plasmid in E.coli successfully entered N.europaea and became N.europaea（RP4）through natural conjugation transfer.The conjugation transfer frequency reaches 4.25×10^-4at 2 h,indicating that N.europaea has certain potential to receive ARGs.By studying the growth of N.europaea and N.europaea（RP4）under the same conditions,the results showed that carrying RP4plasmid had no significant effect on the growth of N.europaea.Second,by measuring NH₄⁺-N utilization and NO₂^--N production of the two strains of bacteria under different culture duration and ammonia nitrogen concentration,the results showed that carrying RP4 plasmid severely inhibited the ammonia oxidation capacity of N.europaea,and the inhibition degree was more than 40%.And NO₂^--N was hardly produced in N.europaea（RP4）.Using NH₂OH as nitrogen source,it was found that there was no significant difference in the utilization rate of NH₂OH by N.europaea,but no NO₂^--N was generated by N.europaea（RP4）.The content of N_XO in the culture system was detected by GC-MS,and it was found that the metabolized end products of N.europaea（RP4）were mainly greenhouse gases NO and N₂O.Thirdly,the results of studies on the permeability of cell membrane,the content of cellular porin and the expression of related regulatory genes of N.europaea（RP4）showed that the RP4 plasmid did not affect the permeability of cell membrane and the transport capacity of substrate.However,the expression of ccm A,rh50 and NE1029genes encoding transporters was inhibited to varying degrees.N.europaea studies on the activities of key functional enzymes in ammonia oxidation metabolism（ammonia monooxygenase and hydroxylamine oxidase）,enzyme activities related to electron transport and energy production,and regulatory gene expression showed that RP4plasmid almost completely inhibited the NO₂^--N formation process catalyzed by hydroxylamine oxidase,resulting in reduced electron release from NH₂OH oxidation.Therefore,the decrease of electrons received by ammonia monooxygenase directly reduces the utilization of ammonia nitrogen,which is specifically manifested in the decreased activity of AMO and HAO and the decreased activity of electron transport system.Fourthly,we also found that the expression of cyt L encoding cytochrome P460,which regulates the production of N₂O,was stimulated,which was consistent with the endogenous production of more N₂O by N.europaea（RP4）.The activity of Rubisco,a key rate-limiting enzyme of Calvin cycle,and the expression of cbb R and rbc L didn’t changed,indicating that RP4 plasmid did not affect the Calvin cycle,which was consistent with the conclusion that RP4 plasmid did not affect the growth of N.europaea.In addition,N.europaea（RP4）over-activated NADH-QH₂reductase,ATP synthase and citrate synthase in the electron transport system,promoting the accumulation of ATP and NADH.By detecting the contents of quorum sensing signal molecules,biofilms and extracellular polymers during the metabolism of N.europaea,it was found that RP4 plasmid promoted the secretion of signal molecules C₄-HSL and C₈-HSL in N.europaea,which intensified the accumulation of extracellular polymers and the generation of biofilms.This study preliminarily confirmed that RP4 plasmid can enter N.europaea through conjugation transfer and seriously inhibit the process of ammonia oxidation metabolism,and discussed the mechanism of RP4 plasmid’s influence on N.europaea ammonia oxidation metabolism.This study will improve the understanding and insight of the effects of ARGs pollution on environmental microorganisms,further reveal the role of ARGs in the biological treatment of sewage,and further explore the unknown field of ammonia oxidation metabolism process to enrich and improve the biological nitrogen removal mechanism.