Isolation And Identification Of Pendimethalin-degrading Bacterium,Metabolic Pathway Study And Clonging Of The Gene Responsible For Pendimethalin Nitroreduction

Pendimethalin[N-?1-ethylpropyl?-2,6-dinitro-3,4-xylidine],a selective pre-emergence dinitroaniline herbicide,is widely used to control a variety of annual gramineous grasses and certain annual broadleaf weeds.It is the third largest herbicide and the largest selective herbicide used in the world.Pendimethalin is very stable and persist in the environment,and shows high toxicity to aquatic invertebrates and terrestrial invertebrate.Furthermore,pendimethalin is a probable carcinogenesis.Therefore,great concern and interest have been raised regarding the environmental behavior,degradation mechanism and ecological security of pendimethalin.Normally,pendimethalin is faded from environment abiotically and biotically,including volatilization,photo-degradation and biodegradation.Microbial metabolism is the most important factor in the degradation of pendimethalin in the environment.And up to now,some pendimethalin-degrading bacteria and fungi strains have been reported,and several intermediate metabolites have been identified.However,the complete metabolic pathway of pendimethalin degradation by organisms is still not clear,and the biochemical and genetic foundation of microbial degradation of pendimethalin has not been elucidated.Furthermore,herbicide degradation or detoxification gene can be used to construct herbicide-resistant transgenic crops.However,patented and desirable herbicide degradation or detoxification gene is very lacking in China.Herbicide-tolerant transgenic engineering is an effective way to control weeds and reduce agriculture costs.Glyphosate-resistant transgenic crops,which was introduced with a glyphosate-resistance gene?CP4 epsps?,have been widely planted.However,the continued use of glyphosate-resistant crops has resulted in the worldwide occurrence of glyphosate-resistant weeds.Pendimethalin can effectively kill glyphosate-resistant weeds.Therefore,pendimethalin-degrading gene have potential application in the genetic engineering of herbicide-resistant plants.In this study,a pendimethalin-degrading strain Y3,which was identified as Bacillussubtilis,was isolated from activated sludge by enrichment technology.The metabolic intermediates of pendimethalin was identified by HPLC?High Performance Liquid Chromatography?and MS/MS?tandem mass spectrometry?,and two new intermediates was found.A nitroreductase gene pnr responsible for reduction of aromatic ring C-6-nitro group of pendimethalin was cloned from strain Y3.pnr was expressed in Escherichia coli BL21?DE3?,and PNR was purified and characterized.In conclusion,the present study provides scientific basis for deep research of degradation mechanism of pendimethalin,high-efficient degrading strain for bioremediation of pendimethalin residue-contaminated soil and water,and patented and desirable degradation gene for herbicide-resistant engineering.The main results were showed as following:1 Isolation and identification of pendimethalin-degradating bacterium.A bacterium strain Y3,capable of efficiently degrading pendimethalin,was isolated from activated sludge treating pendimethalin-manufacturing wastewater by enrichment culture method.Strain Y3 could ulitize pendimethalin as the sole carbon source,and degrade about 82%of 100 mg/L pendimethalin within 36 h of incubation,and 100%pemdimethalin within 60 h of incubation.Strain Y3 was Gram-positive,spore-forming bacterium.Colonies grown on LB agar plate were white,dry,fold and the border was irregular.The phylogenetic analysis of 16S rRNA gene sequences indicated that strain Y3 belonged to the genus Bacillus and formed a subclade with Bacillus subtilis subsp.inaquosorum KCTC 13429T?99.9%similarity?,Bacillus subtilis subsp.subtilis NCIB 3610T?99.4%similarity?and Bacillus subtilis subsp.spizizenii NRRL B-23049T?99.1%similarity?.DNA-DNA hybridization experiments showed that the DNA-DNA relatedness value between strain Y3 and its closest phylogenetic neighbours Bacillus subtilis KCTC 13429T were 75.3%,which was above the threshold of 70%that was suggested for species delineation.Thus,on the basis of phenotypic and phylogenetic properties,strain Y3 was identified as Bacillus subtilis.The optimal temperature and pH for the degradation of pendimethalin in strain Y3 were 30 ? and pH 7.5,respectively.Strain Y3 could also degrade three other major members of dinitroaniline herbicides,including butralin,oryzalin and trifluralin,and diphenylether herbicides lactofen and benzofluorfen.The degradation efficiency towards pendimethalin were similar to those of butralin and oryzalin,but was significant higher than that of trifluralin.2 Identification of the metabolic intermediates of pendimethalin and study of its metabolic pathway.Three metabolic products with retention time being 8.92 min,4.85 min and 4.5 min,respectively,were identified by HPLC during the degradation of pendimethalin by strain Y3.The[M+H]+ of the three products were m/z 252.17,m/z 266.15 and m/z 250.12,respectively.Based on the MS/MS spectrum data,the three products were identified as 6-aminopendimethalin,5-amino-2-methyl-3-nitroso-4-?pentan-3-ylamino?benzoic acid and 8-amino-2-ethyl-5-?hydroxymethyl?-1,2-dihydroquinoxaline-6-carboxylic acid.A partial metabolic pathway of pendimethalin in strain Y3 was proposed.Firstly,one of the nitro-group of pendimethalin was reduced to amino group through nitroreduction to generate 6-aminopendimethalin,which was subsequently transformed to 5-amino-2-methyl-3-nitroso-4-?pentan-3-ylamino?benzoic acid by nitroreduction at the nitro-group connected to C-2 and carboxylation of arylmethyl group at C-4.The latter was further transformed to 8-amino-2-ethyl-5-?hydroxymethyl?-1,2-dihydroquinoxaline-6-carboxylic acid.3 Cloning and characterization of the gene responsible for pendimethalin nitroreduction in strain Y3.In order to clone the corresponding gene,the pendimethalin nitroreductase?PNR?was purified from the cell extract of strain Y3 by ammonium sulfate precipitation,DEAE-sepharose chromatography,hydrophobic interaction chromatography and sephadex G-75 gel filtration.The PNR activity of the crude cell extract of strain Y3 was 0.38 U/mg protein only.After four purification steps,the specific activity of the purified protein was 18.5 U/mg protein,with a purification fold of 48.7.The purified protein was subjected to MALDI-TOF mass spectrometry analysis,the sequences of acquired peptide fragments were used to compared with the deduced proteins from the draft genome of strain Y3,an ORF?orf03879,630 bp in length?annotated as a nitroreductase was figured out.The ORF was expressed in Escherichia coli BL21?DE3?,and the recombinant protein was purified to homogeneity by Ni²⁺-NTA resin,it was a functional homodimer with subunit molecular mass of about 23 kDa and showed the nitroreductase activity against pendimethalin,indicating that the ORF coding for PNR.UHPLC-MS/MS and 1H NMR analysis indicated that the product was 6-aminopendimethalin.All the above results suggested that orf03879 coding for PNR in strain Y3.The phylogenetic analysis revealed that PNR was a member of type I nitroreductases subfamily B1.PNR showed 100%identity to an uncharacterized NAD?P?H nitroreductase YdgI from B.subtilis subsp.strain 168.Among the characterized nitroreductases,PNR showed the highest identity?only 35%?to nitrobenzoate nitroreductase PnbA of Lactobacillus plantarum WCFS1;it also shared 31%,29%and 28%amino acid sequence identities with the quinone reductase DrgA of Synechocystis sp.,the NAD?P?H-flavin oxidoreductase Frase I of Vibrio fischeri,and the NAD?P?H-dependent oxidoreductase PnrB of Pseudomonas putida,respectively.Quantitative real-time PCR analysis suggested that the pnr gene was constitutive expression in strain Y3.4 Characteristics of the recombinant PNRThe highest activity of PNR were observed at pH 7.0 and 35 ?.PNR was easily lost its activity when pH above 8.8 or below 5.0,or temperature above 50 ?.PNR activity was enhanced by?1.0 mM?Mg²⁺,Ca²⁺,Fe²⁺,and Fe3+,and inhibited by?1.0 mM?Cd²⁺,Hg²⁺,Cu²⁺,Ag+,and Zn²⁺.Besides,?1.0 mM?Li+,K+,Co²⁺,Mn²⁺,and Ni²⁺ had no obvious effects on PNR activity.100.0 mM EDTA also had no obvious effect on PNR activity,while 100.0 mM SDS was supposed to be an inhibitor.100.0 mM DTT could strongly enhance PNR activity.PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides,including butralin,oryzalin,and trifluralin.However,the number of reduced nitro groups was two instead of one,which differed from the nitroreduction of pendimethalin by PNR and which may be due to the symmetry in the chemical structures of the two nitro groups.The Vmax/Km of PNR towards pendimethalin,trifluralin,oryzalin and butralin were 33.63,33.34,33.31 and 31.10,respectively.