| Acetamiprid is a high-efficient,low-toxicity and widely-used chloronicotinyl insecticide,one member of the neonicotinoid insecticides.At present,the neonicotinoid insecticide is the top one used insecticide in the world,and acetamiprid is one of the important kind of neonicotinoid insecticides,which are widely used in rice,vegetables,cotton,fruit trees and tea leaves to protect them from aphids,thrips,plant hoppers and some lepidoptera pests.Due to the widespread use and high water solubility,it is easy to enter the surface and underground water via soil.In addition,the acute toxicity of acetamiprid on bees and its residues on tea leaves and cotton have been reported.Microbial degradation is the key way for acetamiprid to be eliminated in environments.In recent years,studies regarding to the degradation of acetamiprid by microorganisms have been reported.However,most of them focused on degrading-strain isolation and metabolic pathways,only one enzyme--nitrile hydratase involved in acetamiprid degradation is reported.Consequently,it is both theoretically and practically significant to study the metabolic pathway of acetamiprid by microorganisms and the key gene involved in acetamiprid degradation,as well as the ecological process during the bioremediation of acetamiprid-contaminated sites.In this research,a high efficient acetamiprid-degrading strain that can use acetamiprid as the sole carbon source,Pigmentiphaga sp.D-2,was isolated from a wastewater-treatment pool in an acetamiprid-manufacturing factory.The strain D-2 could degrade 99%of 50 mg·L-1 acetamiprid within 72 h.The optimum growth and degradation temperature for the strain were 37? and the optimum growth and degradation pH values were 7.The acetamiprid metabolic pathway by strain D-2 was analyzed by HPLC-ESI-MS and MS-MS,leading to discover two metabolic pathways:?,breakage of the C-N bond of the acetamiprid to generate IM1-4;?,dechlorination and demethylation of acetamiprid to generate '-cyano-N-methyl-N-(pyridin-3-ylmethyl)imidoformamide,which is reported for the first time.Protein purification combining with genomic sequencing was used to clone the key gene involved in the degradation of acetamiprid.Target protein(Ama)was obtained by a 3-step purification procedure:ammonium sulfate precipitation,Q-Sepharose FF chromatography and Superdex-200 gel filtration.Analysis of peptide fingerprint and the genomic sequence of strain D-2 identified the target gene was orf05630,located in Scaffold26.The ORF analysis found that ama contains alpha(amal)and beta(ama2)subunits.The corresponding protein was searched against the GenBank database using the BLASTP program.The results revealed that Ama alpha subunit shares 35%identity with N,N-dimethylformamidase alpha subunit from Paracoccus aminophilus,Ama beta subunit shares 56%identity with N,N-dimethylformamidase beta subunit from Paracoccus aminophilus.The results revealed that ama shares 56%identity with N,N-dimethylformamidase beta subunit from Paracoccus aminophilus.Primers were designed to amplify the acetamiprid amidase(ama)which contains alpha and beta subunits.The alpha subunit gene is 372 bp in length,encoding a 123-amino acid protein with a calculated molecular mass of 13.5 kDa,the beta subunit gene is 2294 bp in length,encoding a 764-amino acid protein with a calculated molecular mass of 84.2 kDa.Ama was expressed in Escherichia coli BL21(DE3)and purified using Ni-NTA affinity chromatography column.The purified Ama showed the activity of breaking the C-N bond of the acetamiprid to generate IM1-4.The optimum pH value for Ama activity was 7.5;the optimum temperature for Ama activity was 37?55?.The enzyme can be activated by Al3+,Fe3+,Fe2+ and Li+(0.1 mM,respectively),partial inhibited by Cu2+(0.1 mM),and strongly inhibited by Hg2+(0.1 mM).The degradation of acetamiprid by strain D-2 in different soil and at different concentrations of acetamiprid was studied.It was discovered that when the concentration of acetamiprid is lower(50 mg·kg-1 dry soil),30-40%of the acetamiprid was naturally degraded.The inoculation of strain D-2(108 cfu·g-1 dry soil)into the acetamiprid-contaminated soils could increase the degrading efficiency,and the degradation rate reached more than 90%.It was found that,strain D-2 worked more efficently in acidic soil.The MiSeq high-throughput sequencing was used to study the ecological process during the bioremediation of acetamiprid at different concentrations in different soils by strain D-2.The Alpha diversity of three kinds of soil indicated that the application of acetamiprid could reduce the abundance and diversity of the soil microbial community in acidic soil and neutral soil;the inoculation of strain D-2 could increase the abundance and diversity of the soil microbial community,indicating the strain D-2 could remove the acetamiprid toxicity and increase the microbial diversity of soil.While in alkaline soil,neither the application of acetamiprid nor the inoculation of strain D-2 could change the microbial communities.The Beta diversity of three kinds of soils indicated that the application of acetamiprid only changed the microbial community structure in alkaline soil,but the inoculation of strain D-2 could change the microbial community structure in both acidic and alkaline soils.Based on the analysis of the bacteria phylum in three soils,it could be concluded that in acidic soil,the percentage of Proteobacteria increased greatly when high concentration of acetamiprid and strain D-2 were applied;in neutral soil,the application of high concentration of acetamiprid or the inoculation of strain D-2 could increase the percentage of Actinobacteria;in alkaline soil,the percentage of Proteobacteria increased when high concentration of acetamiprid was applied.According to the analysis of the reported genus showing acetamiprid degradation ability,the application of acetamiprid could increase the percentage of Pseudoxanthomonas in the three kinds of soils;the percentage of Pseudoxanthomonas,Pseudomonas and Stenotrophomonas increased when acetamiprid and strain D-2 were applied.The results indicated that the acetamiprid degrading bacteria will increase under the selection pressure of acetamiprid in soil.The inoculation of strain D-2 played a critical role in the degradation of acetamiprid in soil. |
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