Pseudomonas Putida Metabolizes The Nicotinic Insecticide Imidacloprid

A series of ecological problems caused by the abuse of neonicotinoid pesticide imidacloprid(IMI)are attracting people's close attention.IMI is mainly metabolized in different ways through the nitroreduction and hydroxylation pathways.Metabolite toxicity studies revealed that the insecticidal activity of the olefin IMI product formed by the hydroxylation of IMI is 20-fold greater than that of IMI.In contrast,the guanidine IMI product formed via the nitroreduction pathway exhibited much higher levels mammalian toxicity than IMI.With this in mind,research directed towards developing a deeper understanding of the processes involved in the microbial regulation of the IMI metabolic flux between the nitroreduction and hydroxylation pathways could therefore assist in the formulation of an integrated plan for the effective management of IMI and decrease the environmental toxicity.The microbial activity towards the degradation of IMI plays an important role in determining the fate of IMI found in environment.In this paper,bacterial strains which can transform the neonicotinoid pesticide IMI through the nitroreduction and hydroxylation pathways were screened and identified and its IMI degradation mechanism was studied.A new imidacloprid transfromation bacterium named as Z-4 separated from our strain preservation library was identified with Pseudomonas putida by 16S rRNA gene analysis.Nucleotide BLAST analysis of the 16S rRNA sequences indicated that the strain Z-4 shared a 100%sequence identity with P.putida KT2440.HPLC analysis showed that P.putida Z-4 converted IMI to two new products peaked with retention time of 5.6 and 6.8 min respectively.Liquid chromatography-mass spectrometry(LC-MS)analysis indicated that the two metabolites were nitroso IMI and 5-hydroxy IMI.The above results showed that P.putida Z-4 transforned IMI(=N-NO2)via a nitroreduction pathway to produce nitroso(=N-NO)IMI and a hydroxylation pathway to produce 5-hydroxy IMI.Compared with Stenotrophomonas maltophilia CGMCC 1.1788 and Pseudoxanthomonas indica CGMCC 6648 which have been reported before,IMI degrading efficiency by P.putida Z-4 is lower.Therefore,IMI degradation conditions of P.putida Z-4 have been optimized,besides that,S.maltophilia CGMCC 1.1788,P.indica CGMCC 6648 and P.putida Z-4 have been investigated their IMI degradation characteristics.Maltose was found to be the best co-substrate for the formation of the 5-hydroxy IMI metabolite(16.1 mg/L),leading to a 40.1-fold increase in the amount of the 5-hydroxy IMI metabolite compared with the corresponding control reaction containing no co-substrate.while citrate was found to be the best co-substrate for the formation of the nitroso IMI metabolite(8.5 mg/L),leading to a 21.2-fold increase in the amount of the nitroso IMI metabolite.It is noteworthy that the hydroxylation of IMI by S.maltophilia CGMCC 1.1788 was found to be insensitive to the length of time allowed for the pre-culture of the cells.In contrast,increasing the pre-culture times for the P.indica CGMCC 6648 and P.putida Z-4 cells led to an increase and decrease in the hydroxylation of IMI,respectively.The amounts of the 5-hydroxy and olefin IMI metabolites produced following the pre-culture of P.indica CGMCC 6648 with IMI for 18 h were 3-fold higher than those obtained following a pre-culture time of 4 h.The amounts of the 5-hydroxy metabolites produced following the pre-culture of P.putida Z-4 with IMI for 2 h were 12-fold higher than those obtained following a pre-culture time of 12 h.The formation of the 5-hdroxy IMI metabolite by P.putida Z-4 or S.maltophilia CGMCC 1.1788 could there be increased by increasing the amount of dissolved oxygen in the medium.The formation of the nitroso IMI metabolite by P.putida Z-4 in the current study increased slightly as the amount of dissolved oxygen decreased.S.maltophilia CGMCC 1.1788 did not produce olefin IMI and nitroso IMI under acidic or neutral conditions and P.Z-4 did not produce 5-hydroxy IMI under acidic conditions.Mammalian cytochrome P450 enzymes and aldehyde oxidase(AOX)are the IMI hydroxylase and nitroreductase respectively.In this paper,we found that the typical strain P.putida KT2440 could also metabolize IMI to produce the corresponding 5-hydroxy and nitroso IMI metabolites.One CYPs 450 gene,fifteen monooxygenase genes and one aox was found in the genomic DNA of P.putida KT2440,aox was composed of 3739 nucleotides encoding a multimeric protein composited by small subunit,molybdopterin-binding subunit and large subunit.We constructed an expression plamid pET28a-aox which was consists of pET28a and aox from PCR.SDS-PAGE analysis indicates that the three subunits of AOX all could be over-expressd in E.coli Rosetta,but they exist in the form of inclusion body.HPLC analysis showed E.coli Rosetta with successful over-expressed aox could transform IMI to nitroso IMI,and the control group without over-expressing AOX cannot produce nitroso IMI.Our study shows that AOX from P.putida has the activity of IMI nitroreduction.The enzyme activity is low because of inclusion body existing.It's enzyme activity hasn't been improved by altering IPTG concentration and temperature of expression and so on.In this study we also tried to knockout aox in P.putida KT2440 by the suicide carrier pEX18Gm via twice homologous recombination,but we can't get double exchange strain through homologous recombination using 300 bp and 500 bp homologous arms because aox reaches a length of 3.7 kb.