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Regulation of Hydroxylation and Nitroreduction Pathways during Metabolism of the Neonicotinoid Insecticide Imidacloprid by Pseudomonas putida Tian-Qi Lu,† Shi-Yun Mao,† Shi-Lei Sun,† Wen-Long Yang,† Feng Ge,*,‡ and Yi-Jun Dai*,† †

Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing 210023, People’s Republic of China ‡ Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing 210042, People’s Republic of China ABSTRACT: Imidacloprid (IMI) is mainly metabolized via nitroreduction and hydroxylation pathways, which produce different metabolites that are toxic to mammals and insects. However, regulation of IMI metabolic flux between nitroreduction and hydroxylation pathways is still unclear. In this study, Pseudomonas putida was found to metabolize IMI to 5-hydroxy and nitroso IMI and was therefore used for investigating the regulation of IMI metabolic flux. The cell growth time, cosubstrate, dissolved oxygen concentration, and pH showed significant effect on IMI degradation and nitroso and 5-hydroxy IMI formation. Gene cloning and overexpression in Escherichia coli proved that P. putida KT2440 aldehyde oxidase mediated IMI nitroreduction to nitroso IMI, while cytochrome P450 monooxygenase (CYP) failed to improve IMI hydroxylation. Moreover, E. coli cells without CYP could hydroxylate IMI, demonstrating the role of a non-CYP enzyme in IMI hydroxylation. Thus, the present study helps to further understand the environmental fate of IMI and its underlying mechanism. KEYWORDS: aldehyde oxidase, hydroxylation, imidacloprid, nitroreduction, Pseudomonas putida

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INTRODUCTION Imidacloprid (IMI, Figure 1), the first commercial neonicotinoid insecticide, is currently one of the most widely used insecticides in the world, with applications in more than 100 countries for more than 140 agricultural crops.1,2 However, IMI exhibits high persistence in soil with a half-life of up to 229 days, and 80%−98% of the IMI residues enter into the soil during crop treatment, thereby resulting in soil and water contaminations.3−6 It has been reported that IMI adversely affects the integrity of the ecosystems and is particularly toxic to bees and other beneficial insects.7−11 Because of its harmful effects on bees, IMI had been subjected to a partial 2-year ban from December 2013 in the European Union.6 Nevertheless, IMI is still widely used in the rice- and tea-planting regions of Asia, where its extensive application has caused many problems, such as pest resistance and persistence in crops, vegetables, and tea.6,12−14 IMI metabolism has been studied in soils, plants, mammals, and microorganisms, and has been found to comprise two major pathways, namely, an oxidation pathway that produces 5hydroxy and an olefin IMI and nitroreduction pathway that produces nitroso (NNO), guanidine (NNH), and urea (O) IMI metabolites (Figure 1).1 Metabolite toxicity studies have revealed that the insecticidal activity of the olefin IMI product formed by the hydroxylation of IMI is 10-fold higher than that of IMI against whitefly and aphids.15,16 In contrast, while the guanidine and urea IMI products formed via the nitroreduction pathway do not possess any insecticidal properties, the guanidine IMI has been reported to exhibit higher levels of mammalian toxicity than IMI.17,18 Thus, IMI metabolism through the nitroreduction pathway could lead to © XXXX American Chemical Society

increased environmental toxicity and greater risk to the ecosystem. Therefore, it is very crucial to study the regulation of the IMI metabolic flux between the nitroreduction and hydroxylation pathways. In a previous study on IMI metabolism in soil, Sarkar et al.19 examined the persistence of IMI in three different types of soil collected from West Bengal, India, and reported that IMI had half-lives of 28.7−47.8 days, and that olefin and urea metabolites were detected in all the three soil samples. Similarly, Singh and Singh20 demonstrated that IMI residues in treated soil from the Agricultural Research Station, Jaipur, India, had an average half-life of 40.96 days. In our previous study, we had reported that IMI had a half-life of 61.3 days in unsterilized soil from Nanjing, China, and detected the metabolites olefin and guanidine IMI. In contrast, almost no IMI degradation was detected in sterilized soil.21 Moreover, Cycoń et al.22 observed that IMI had a half-life of 88.8 days in unsterilized soil from Poland, while nonbiotic degradation of IMI was noted in both control and sterilized soil subjected to pesticide treatments. These results proved that microbial metabolism represents the major pathway for the degradation of IMI in soil. Studies on the interactions between IMI and soil microorganisms have revealed that the presence of IMI in soil may induce changes in the structure, genetic diversity, and catabolic enzyme activities of soil microbial communities and promote evolution of bacteria capable of degrading IMI among Received: March 25, 2016 Revised: May 25, 2016 Accepted: May 27, 2016

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DOI: 10.1021/acs.jafc.6b01376 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Various pathways for the microbial metabolism of IMI. The superscripts indicate cited references.

the inhabiting microflora.20,23−25 So far, seven bacteria, namely, Bacillus alkalinitrilicus, Klebsiella pneumoniae BCH1, Leifsonia sp. PC-21, Mycobacterium sp. MK6, Pseudomonas sp. 1G, Pseudomonas sp. RPT 52, Pseudoxanthomonas indica CGMCC 6648, and Stenotrophomonas maltophilia CGMCC 1.1788, have been reported to metabolize IMI via nitroreduction and/or hydroxylation pathway (Figure 1).14,15,26−31 However, regulation of the IMI metabolic flux between the nitroreduction and hydroxylation pathways has not been investigated. In the present study, we initially screened the microorganisms capable of transforming IMI via the hydroxylation and nitroreduction pathways and studied the key factors involved in regulating the metabolic flux of IMI through these two pathways. Furthermore, as mammalian aldehyde oxidase (AOX) and cytochrome P450 monooxygenase (CYP) have been proven to be the IMI nitroreductase and hydroxylase, respectively,1 we cloned and overexpressed the homogeneous aox and cyp from the target bacterium in Escherichia coli Rosetta (DE3) cells and examined IMI metabolism in the constructed E. coli strains. The present study on microbial metabolism of IMI will help in understanding the molecular mechanism of microbial degradation of IMI. The behavior of IMI in soil depends on the physicochemical parameters of the soil, such as organic matter, insecticide residue, pH, temperature, and time.5,23,24 As microbial degradation is the main mechanism of IMI degradation in soil,21,24 the persistence of IMI bioefficacy against horsebean aphid Aphis craccivora following its application in sterilized and nonsterilized soil was compared to evaluate the effects of soil microbial activity on IMI persistence in soil. Interestingly, the aphid mortality following IMI application in unsterilized soil at 14 days was found to be 10% higher than that in sterilized soil, indicating that soil microorganisms may have an impact on the insecticidal activity of IMI after soil application.21 Subsequently, a strategy to pretransform IMI was developed in which IMI was pretransformed in the resting cells transformation broth of Stenotrophomonas maltophilia CGMCC 1.1788 with succinate as a cosubstrate for 8 days. After the incubation period, the

solution, containing untransformed IMI and the metabolites 5hydroxy and olefin IMI, was diluted and applied to soils, and the insecticidal activity against A. craccivora was determined to be 2.2-fold higher than that of the control without pretransformed IMI application.32 Therefore, pretreatment of IMI through the hydroxylation pathway may reduce IMI application dosage and consequently decrease the persistence of IMI residues in the environment.32 In the present study, we examined the regulation of the IMI metabolic flux between nitroreduction and hydroxylation pathways to help in the development of a management strategy to increase the flux of hydroxylation pathway and reduce the flux of nitroreduction pathway to decrease the amount of IMI used in agricultural applications as well as alleviate its ecotoxicity, thus lowering its environmental impact.

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MATERIALS AND METHODS

Chemicals. IMI was kindly provided by Prof. Jue-Ping Ni of Jiangsu Pesticide Research Institute, Nanjing, China (>98% purity). 5Hydroxy and olefin IMI were synthesized according to the methods described in our previous study.15 The mass data of 5-hydroxy IMI (purity of 98%) at m/z were as follows: 114.0, 167.0, 224.0, 250.0, 271.0 (parent ion), 307.0. The 13C nuclear magnetic resonance (NMR) data of 5-hydroxy IMI were as follows (δ): 41.8, 50.6, 80.3, 124.5, 132.9, 139.7, 149.6, 149.7, 158.9. The 1H NMR data of 5hydroxy IMI were as follows (δ): 3.37 (dd, 1H, J = 12.0 Hz, J = 2.4 Hz), 3.84 (dd, 1H, J = 12.0 Hz, J = 7.6 Hz), 4.42 (s, 1H, J = 16.1 Hz), 4.58 (d, J = 16.1 Hz), 5.25 (ddd, 1H J = 7.5 Hz, J = 7.5 Hz, J = 2.5 Hz), 6.82 (d, 1H, J = 7.5 Hz), 7.51 (d, 1H, J = 8.2 Hz), 7.82 (dd, 1H, J = 8.2 Hz, J = 2.4 Hz), 8.38 (d, 1H, J = 2.4 Hz), 9.03 (b, 1H). Nitroso IMI was synthesized according to the methods reported by Kanne et al.33 The mass data of nitroso IMI (purity of 97%) at m/z were as follows: 162.0, 197.0, 239.0 (parent ion), 296.0, 396.0. The 13C NMR data of nitroso IMI were as follows (δ): 41.5, 44.6, 46.6, 124.5, 132.0, 139.9, 149.8, 150.0, 170.0. The 1H NMR data of nitroso IMI were as follows (δ): 3.68 (s, 4H), 4.57(s, 2H), 7.53 (d, 1H, J = 8.0 Hz), 7.72 (dd, 1H, J = 8.0 Hz, J = 2.1 Hz), 8.36 (d, 1H, J = 2.1 Hz), 9.03 (b, 1H). Acetonitrile for high performance liquid chromatography (HPLC) analysis was purchased from TEDIA Co. Ltd. (Fairfield, OH, U.S.A.). All of the other reagents used were of analytical grade B


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Journal of Agricultural and Food Chemistry and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Strains and media. Pseudomonas putida KT2400 was kindly provided by Prof. Jian He of Nanjing Agricultural University, China. For cell culture, Luria−Bertani (LB) medium (pH 7.2) containing 10.0 g of peptone, 5.0 g of yeast extract, and 10.0 g of NaCl in 1.0 L of deionized water was used. The mineral salt medium (MSM) for enrichment of IMI-degrading microorganisms contained 0.45 g of IMI, 10.0 g of glucose, 2.13 g of Na2HPO4, 1.36 g of KH2PO4, 0.50 g of MgSO4·7H2O, and 10 mL of metal ion solution in 1.0 L of deionized water (pH 7.5). The metal ion solution contained 0.30 g of H3BO3, 0.10 g of KI, 0.40 g of CaCl2·2H2O, 0.04 g of CuSO4·5H2O, 0.20 g of FeSO4·7H2O, 0.40 g of MnSO4·7H2O, 0.20 g of NaMoO4·2H2O, and 10.0 mL of concentrated HCl in 1.0 L of deionized water. Isolation and Identification of the IMI-Transforming Bacterium. Soil samples were collected from Nanchang in Jiangxi province, Wuxi, Suzhou and Nanjing in Jiangsu province. A total of 2.0 g of soil sample were added into a 100 mL flask containing 18 mL of sterilized MSM broth and glass beads. The flask was shaken in a rotary shaker at a speed of 200 rpm for 2 h, and then 5 mL of the mixture were inoculated into a 500 mL flask containing 200 mL of MSM broth. The culture was incubated at 30 °C on a rotary shaker at 220 rpm and sampled every week. The IMI residue in the samples were monitored by HPLC analysis and the culture broth, which is able to degrade IMI, was spread onto an MSM agar plate and incubated for 1 week. Subsequently, single colonies were streak-plated onto LB agar plates and incubated at 30 °C. Meanwhile, the bacteria preserved in our laboratory, which were previously isolated from soil as per the abovementioned procedure, were examined for their ability to transform IMI via hydroxylation and nitroreduction pathways, through resting cells transformation of IMI. Metabolites were detected by HPLC.28 The bacterial isolates were identified by 16S rRNA gene sequence analysis. The 16S rRNA gene was amplified by colony PCR. The PCR system comprised appropriate bacterial colony, 2.5 μL of Taq buffer (10×), 1 μL of MgCl2 (25 mmol/L), 1 μL of each primer K1 (5′AACTGAAGAGTTTGATCCTGGCTC-3′) and K2 (5′-TACGGTTACCTTGTTACGACTT-3′), 2.5 μL of dNTP (2.5 mmol/L each), 0.5 μL of rTaq DNA polymerase (2 U/μL, Takara, Dalian, China), and 16.5 μL of sterilized distilled water. The total volume of the reaction mixture was 25 μL. The mixture broth was primarily denatured for 5 min at 95 °C. The cycling conditions were as follows (for both the PCRs): denaturation for 1 min at 95 °C, annealing for 1 min at 59 °C, and extension for 2 min at 72 °C. A final 10 min extension step at 72 °C was performed after 25 cycles. The PCR products were checked using agarose gel electrophoresis. The PCR product was ligated to plasmid pMD18-T by using the pMD18-T vector cloning kit (Takara, Dalian, China, code No: 6011) and transformed into Escherichia coli DH5α cells. The purification of the target plasmid and sequencing of 16S rRNA gene were conducted by Springen Biotech Co., Ltd. (Nanjing, China). The obtained DNA sequence was compared by alignment with those in the GenBank database using the nucleotide blast tool. A neighbor-joining phylogenetic tree based on the 16S rRNA gene sequence was constructed by using the MEGA 6.0 software (www.megasoftware.net). IMI Biotransformation. The bacterial isolates and the preserved bacteria were streaked onto LB agar plates and incubated at 30 °C until single colonies appeared. Subsequently, a single colony was inoculated into a 20 mL test tube containing 3 mL of LB broth and incubated in a rotary shaker at 220 rpm for 24 h at 30 °C. Then, 1 mL of the broth was poured into a 500 mL flask containing 100 mL of LB broth and the flask was incubated under the above-mentioned culture conditions. For the transformation of IMI in resting cells, the cells that were precultivated for 6 h were harvested by centrifugation at 6000g for 10 min and washed with 0.2 mol/L sodium phosphate buffer (pH 8.0). The cell pellet was resuspended in the same buffer containing 450 mg/L IMI and 10 mmol/L glucose. The cell density at 600 nm was adjusted to 5.0, and 2 mL of the broth were added into a 50 mL centrifuge tube and incubated in rotary shaker at 220 rpm for 96 h at 30 °C. The above-mentioned preparation of IMI transformation is considered to be the standard resting cell transformation. The

transformation broth without IMI or bacteria was used as substrate IMI control and bacterium control, respectively.15,34 To test the effect of precultivated cell growth period on the resting cell transformation of IMI, the incubation time was adjusted to 2, 6, and 12 h, which represented the cells at early, middle, and late stages of the logarithmic phase, respectively. To examine the effects of different cosubstrates on the transformation of IMI in resting cells, glucose was replaced with other carbohydrates and organic acids at a concentration of 10 mmol/L. The transformation broth without a cosubstrate was used as a control. To evaluate the effect of dissolved oxygen concentration in the transformation broth on IMI transformation, the volume of the transformation broth in 50 mL centrifuge tube was varied from 2 to 10 mL. To evaluate the effect of pH on the transformation of IMI, the pH of the phosphate buffer used for cell suspension was adjusted from 5 to 9. The other conditions were the same as those employed in the standard resting cells transformation. After transformation for 96 h, the samples for HPLC analysis were centrifuged at 10 000g for 10 min to remove the cell pellets, and the supernatant was collected and filtered through a membrane (pore size, 0.22 μm) and diluted five times with HPLC mobile phase. Cloning and Overexpression of aox and cyp from P. putida KT2400 in E. coli Rosetta cells. The genomic DNA was extracted using a bacterial genomic DNA extraction kit ver 2.0 (Takara). The primer pairs P1 (5′-GAATTCATGAGCGCGATCTCTCCC AATG3′) and P2 (5′-CTCGAGTCATACCTGACTGTTGTCTTTCAG-3′) (underlined bases indicate the restriction enzyme sites of EcoRI and XhoI, respectively) were used for cloning the full length of aox of P. putida KT2440 (GenBank accession number: NC_002947.3, located at site from 3743190 to 3746928).35,36 The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., China. The PCR system comprised 0.5 μL of genomic DNA template of P. putida KT2440, 0.3 μL of each primer P1 and P2, 2 μL of dNTP (2.5 mmol/L each), 5 μL of PrimerSTAR buffer (Mg2+ plus, 5 × ), 16.6 μL of sterilized distilled water, and 0.3 μL of PrimerSTAR DNA polymerase (2.5 U/μL, Takara). The total volume of the reaction mixture was 25 μL. After an initial denaturation for 5 min at 95 °C, the cycling conditions were as follows (for both the PCRs): denaturation for 1 min at 95 °C, annealing for 1 min at 60 °C, and extension for 4 min at 72 °C. A final 10 min extension step at 72 °C was performed after 30 cycles. The PCR products were checked using agarose gel electrophoresis and then purified by agarose gel DNA purification kit ver. 2.0 (Takara). An adenine residue was added to the 3′-terminus of the purified PCR products using the reaction mixture comprising 37 μL of the PCR product, 5 μL of PCR buffer (10×), 4 μL of dATP (2.5 mmol/L), 3 μL of MgCl2 (25 mmol/L), and 1 μL of rTaq DNA polymerase (5 U/ μL, Takara). The products with added adenine residue were further purified and then ligated into the pMD19-T vector (Takara) using the TA PCR cloning kit (Takara, cat. no. D101A), and the positive plasmid was sequenced. The plasmid pMD19-T containing the correct sequence of aox was purified by using a MiniBEST plasmid purification kit ver. 4.0 (Takara). To 20 μL of the solution containing the target plasmid, 4 μL of buffer (10 × ), 0.8 μL of EcoRI (Takara, D1040A), 0.8 μL of XhoI (Takara, D1094A), and 14.4 μL of autoclaved deionized water were added. The reaction mixture was incubated at 37 °C for 6 h, and aox (3.7 kb) was separated by 1% agarose gel electrophoresis and recovered using agarose gel DNA purification kit ver. 2.0 (Takara). The above-mentioned procedure was also employed for the linearization and purification of plasmid pET28a (5.3 kb). Subsequently, 5.0 μL of the aox solution (50 ng/μL) and the linearized pET28a (16 ng/μL) were mixed with 1 μL of T4 DNA ligase buffer (10×) and 0.5 μL of T4 DNA ligase (Takara) and incubated at 16 °C for 20 h. The transformation of aox into E. coli Rosetta competent cells was performed according to the method of Ge et al.20 For the construction of the cyp (PP_1955, located at site from 2211866 to 2213101) recombinant pET28a, ClonExpress II one step cloning kit (Vazyme Biotech, Nanjing, China) was used. The primer pairs P3 (5′-ACAGCAAATGGGTCGCGGATCCGAATTCATGGC


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Figure 2. HPLC and LC−MS analyses of the transformation of IMI by P. putida Z-4. AAATACTTGATCGTCCTCAAG-3′) and P4 (5′-ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTACCAGATTACTTGCAAAGAGAGG-3′) (underlined bases indicate the restriction enzyme site of EcoRI and XhoI, respectively), which have a pET28a homologous base located before the restriction enzyme site, were used. The PCR system comprised 0.5 μL of P. putida KT2440 genomic DNA, 0.4 μL of each primer P3 and P4, 2 μL of dNTP (2.5 mmol/L each), 5 μL of PrimerSTAR buffer (Mg2+ plus, 5×), 15 μL of sterilized distilled water, and 0.2 μL of PrimerSTAR DNA polymerase (2.5 U/μL, Takara). The total volume of the reaction mixture was 25 μL. After an initial denaturation for 5 min at 95 °C, the cycling conditions were as follows (for both the PCRs): denaturation for 1 min at 95 °C, annealing for 50 s at 55 °C, and extension for 1.5 min at 72 °C. A final 10 min extension step at 72 °C was performed after 30 cycles. The linearized pET28a digested by EcoRI and XhoI and the PCR product PP_1955 were recombined in a system comprising 4 μL of CE buffer (5×), 5 μL of pET28a (28.37 ng/μL), 1 μL of PP_1955 (51 ng/μL), 2 μL of Exnase II (Vazyme Biotech), and 8 μL of autoclaved deionized water. This recombination system (20 μL) was incubated at 37 °C for 30 min and then in an ice-bath for 5 min, and the product was transformed into the E. coli Rosetta competent cells and screened in LB agar plate with kanamycin and chloromycetin. The recombinant pET28a was verified by DNA sequencing and subsequently induced to overexpress CYP. Overexpression of the recombinant aox and cyp was accomplished according to the method of Ge et al.37 To analyze enzyme solubility, the cell pellets were resuspended in 0.2 culture volumes of 0.2 mol/L sodium phosphate buffer and disrupted by sonication (10 s) for 3 min at 4 °C. After sonication, the total protein sample was collected from the cell suspension, and the soluble protein sample was collected from the supernatant by centrifugation at 12 000g for 20 min. SDS-PAGE was employed for checking the overexpression of AOX and CYP, and a standard molecular weight protein mixture was used as the reference. The gels were stained with Coomassie brilliant blue for protein detection. HPLC and Liquid Chromatography−Mass Spectrometry Analyses. An Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, U.S.A.) equipped with an Agilent HC-C18 column (4.6 × 250 mm) was used for the quantitative analysis of IMI and its metabolites. The column was eluted at a flow rate of 1 mL/min under isocratic conditions using a mobile phase consisting of water, acetonitrile, and 0.01% acetic acid (water/ acetonitrile =75:25, v/v). The signal was monitored at a wavelength of 269 nm using an Agilent G1314A UV detector. The injection volume of the sample was 20 μL. Under these HPLC conditions, the hydroxylation metabolites, 5-hydroxy IMI, olefin IMI, and IMI

appeared at the retention times of 6.0, 6.9, and 9.6 min, respectively, while the nitroreduction metabolites, nitroso, guanidine, and urea IMI appeared at the retention times of 5.6, 4.1, and 6.2 min, respectively. The limit of detection of IMI, 5-hydroxy IMI, and nitroso IMI were 2.4, 16.5, and 14.0 ng/mL respectively. Liquid chromatography−mass spectrometry (LC−MS) was conducted using an Agilent 1290 infinity LC with a G1315B diode-array detector and an Agilent 6460 Triple Quadrupole LC−MS system (Agilent Technologies) equipped with an electrospray ionization source, operated in the positive and negative ionization modes. The column, mobile phase, and monitored wavelength were similar to those described for HPLC, except the flow rate for the elution of the column, which was set to 0.6 mL/min. Under these conditions, the standard nitroso IMI, 5-hydroxy IMI, and IMI had retention times of 9.1, 11.1, and 15.8 min, respectively.

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RESULTS Screening of Bacteria That Could Transform IMI via Hydroxylation and Nitroreduction Pathways. The MSM broth inoculated with soil collected from Suzhou city showed about 20% of IMI degradation after incubation for 4 weeks, and the other soil extracts hardly degrade IMI. Among about 300 colonies isolated from the MSM broth, a strain named Z-5 showed an IMI degradation rate of 14%. Furthermore, HPLC analysis indicated that the IMI transformation by strain Z-5 produced only one product at a retention time of 6.9 min, which is similar to the retention time of standard 5-hydroxy IMI. Among the 80 preserved strains, except strain Z-9 (Pseudoxanthomas indica CGMCC 6648) with the highest IMI-hydroxylating capacity reported,28 a strain named Z-4 exhibited IMI degradation rate of 10% in resting cells transformation of IMI in 96 h. The IMI transformation by Z4 produced two metabolites (P1 and P2) that had the retention times of 5.6 and 6.9 min, respectively (Figure 2C), and the two peaks did not appear in the bacterium control (Figure 2A) and substrate IMI control (Figure 2B). LC−MS analysis of the metabolites P1 and P2 produced by Z-4 revealed the retention times of 9.2 and 11.1 min, respectively (Figure 2D). Furthermore, metabolite P1 (Figure 2E) exhibited peaks with m/z values of 238, 320, 296, 196, and 129 for [M−H]+, [M+2CH3CN−H]+, an unknown adduct, [M−Cl−NO−H+Na]+, and [M−Cl−C3H5N4O−H+K]+, respectively. Metabolite P2 (Figure 2F) presented peaks with m/ D


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Journal of Agricultural and Food Chemistry z values of 270, 352, 223, and 166 for [M−H]+, [M +2CH3CN−H]+, an unknown fragment ion, and [M− C3H5N4O3+CH3CN−H], respectively. The IMI substrate (Figure 2G) showed peaks with m/z values of 254 and 312 for [M−H]+ and [M+NaCl−H]+, respectively. The mass data recorded for metabolites P1 and P2 were noted to be consistent with those recorded for the standard nitroso and 5-hydroxy IMI metabolites. Therefore, it was concluded that strain Z-4 transformed IMI to the corresponding nitroso and 5-hydroxy IMI metabolites via the nitroreduction and hydroxylation pathways, respectively. Taxonomic Identification of Strain Z-4 by 16S rRNA Gene Sequence Analysis. Nucleotide BLAST analysis of the 16S rRNA gene sequences indicated that the strain Z-4 shared 100% sequence identity with P. putida KT2440 and was clustered with P. putida KT2440 in the phylogenetic tree (Figure 3). P. putida KT2440 is a typical strain of the genus Pseudomonas and its genomic DNA has been sequenced.36

Table 1. IMI Reduction and Formation of 5-Hydroxy and Nitroso IMI Metabolites at Different Phases of the Cell Culturea content (mg/L) cell growth time (h)

reduced IMI

5-hydroxy

nitroso

2 6 12

85.9 ± 14.8a 14.7 ± 1.8b 6.3 ± 1.5c

20.4 ± 7.0a 4.0 ± 1.1b 1.1 ± 0.2c

0.7 ± 0.3a 4.8 ± 0.5b 3.7 ± 1.1c

a

The bacteria were precultivated in LB broth for the indicated time and the cells were subsequently collected, washed, suspended in phosphate buffer containing IMI with a final OD600 of 5. These transformation systems were incubated on a rotary shaker at 220 rpm at 30 °C for 96 h. The data represent the mean values of triplicates. The mean values (±SD) within a column followed by different superscripts are significantly different at p ≤ 0.05 according to Duncan’s test.

We further examined the effect of cell growth period on IMI transformation using two other bacterial strains, S. maltophilia CGMCC 1.1788 and P. indica CGMCC 6648, which have been proven to be capable of hydroxylating IMI.15,28 Interestingly, 5hydoxy IMI formation by S. maltophilia CGMCC 1.1788 did not vary with different cell growth periods, whereas that by P. indica CGMCC 6648 increased from 67.3 to 264.2 mg/L when the cell growth period was extended from 4 h (early stage of logarithmic phase) to 18 h (late stage of logarithmic phase). Furthermore, P. indica CGMCC 6648 did not produce nitroso IMI under any of the cell growth periods tested. In contrast, while the resting cells of S. maltophilia CGMCC 1.1788 precultivated for 4 h did not produce nitroso IMI, those precultivated for 18 h produced 1.1 mg/L nitroso IMI in the presence of 100 mmol/L glucose. As shown in Table 2, P. putida Z-4 transformed IMI via a cometabolism mechanism, as evidenced by the enhanced

Figure 3. Neighbor-joining phylogenetic tree of P. putida Z-4, other members of the genus Pseudomonas, and representatives of some other taxa based on 16S rRNA gene sequence comparisons Bootstrap percentages from 1000 replicates are shown at nodes. The sequence of S. maltophilia was used as the outgroup. Bar, 2% sequence divergence.

Table 2. Effect of Co-substrate on IMI Reduction as Well as Hydroxylation and Nitroreduction of IMI by P. putida Z-4a content (mg/L) co-substrate glucose fructose sucrose lactose maltose succinate citrate malate pyruvate control

Effect of Cell Growth Phase, Cosubstrate, Dissolved Oxygen Concentration, and pH on the IMI Metabolic Pathway of P. putida Z-4. As strain Z-4 hardly degraded IMI under the growing culture transformation conditions, a resting cells transformation experiment was employed for further investigations. The cells grown for 2, 6, and 12 h presented OD600 values of 0.22, 1.99, and 4.75, indicating that the cells were at the early, middle, and late stage of the logarithmic phase, respectively. Subsequently, these cells were prepared for the resting cells IMI transformation experiment with the OD600 value adjusted to 5 and transformed for 96 h. As shown in Table 1, the resting cells precultivated for 2 h presented the highest activity for IMI degradation, reducing 85.9 mg/L IMI at an IMI degradation rate of 20% and producing 20.4 mg/L 5hydroxy IMI and 0.7 mg/L nitroso IMI. However, 5-hydroxy IMI formation dramatically decreased to 1.1 mg/L when the precultivation period was extended to 12 h, whereas the production of nitroso IMI peaked, reaching a concentration of 4.8 mg/L, at 6 h of cell growth. The final byproduct 6chloronicotinic acid, which peaked at 6.3 min, could not be detected.

reduced IMI 48.5 57.2 54.4 57.2 68.6 39.0 41.7 55.3 35.1 5.2

± ± ± ± ± ± ± ± ± ±

9.6bc 5.0c 10.9c 4.1c 13.7d 4.0b 7.6b 6.4c 7.7b 2.0a

5-hydroxy 2.8 5.9 5.5 10.4 16.1 0.2 0.4 ND ND 0.4

± ± ± ± ± ± ±

0.3b 1.1c 1.1c 1.4d 3.7e 0.1a 0.1a

± 0.1a

nitroso 3.9 5.7 2.6 0.7 0.5 2.4 8.5 3.2 2.8 0.4

± ± ± ± ± ± ± ± ± ±

0.3c 0.5d 0.5b 0.2a 0.2a 0.8b 2.7e 0.5bc 0.5b 0.0a

a

The P. putida Z-4 cells were grown for 6 h. The OD600 value of the resting cells for IMI transformation was adjusted to 5. The amount of cosubstrate added to each experiment was 10 mmol/L. The data represent the mean values of triplicates. The mean values (±SD) within a column followed by different superscripts are significantly different at p ≤ 0.05 according to Duncan’s test.

degradation of IMI following the addition of a carbohydrate or organic acid as the cosubstrate. The addition of a cosubstrate increased IMI degradation from 6.7- to 13.2-fold, when compared with that noted in the corresponding control without a cosubstrate. The examined carbohydrate cosubstrates increased 5-hydroxy IMI formation, whereas the organic acid E


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Phylogenetic analysis of the 16S rRNA gene sequence indicated that P. putida Z-4 had 100% similarity with the typical strain P. putida KT2440 (Figure 3). As P. putida KT2440 could transform IMI to produce two metabolites, 5-hydroxy and nitroso IMI, the genomic DNA determining P. putida KT2440 was used for cloning the genes coding the IMI-metabolizing enzymes. Human and fruit fly cytochrome P450 3A4 and 6G1 have been reported to be responsible for IMI hydroxylation, while rabbit liver AOX has been identified with IMI nitroreductase with the function of transforming IMI to nitroso IMI and aminoguanidine (NNH2).12,39 A cyp gene and an aox gene cluster were detected in the genomic DNA of P. putida KT2440. The cyp gene (PP_1955, GenBank accession number: NC_002947) had a length of 1236 bp and its translating protein PP_1955 showed 14.68% and 14.1% amino acid identity with cytochrome P450 3A4 and 6G1, respectively. The PP_1955 was noted to have the highest identity of 23% with cytochrome P450 21A2 (GenBank accession number: M26856) by using P450 BLAST server (http://blast.uthsc.edu/ ). The aox had a length of 3739 bp, and the corresponding three subunit coding genes were 528-, 1002-, and 2217-bp long, respectively. Thus, the KT2440 AOX comprised three independent subunits involving a small subunit (175 amino acids, accession number: NP_745451), molybdopterin-binding subunit (333 amino acids, accession number: NP_745452), and large subunit (738 amino acids, accession number: NP_745453) (Figure 4). Unlike the KT2440 AOX, the rabbit AOX (NP_001075459) exhibited only one peptide chain with a length of 1334 amino acids. This peptide chain was composed of five conserved domains, including a 2Fe-2S binding domain, a FAD binding domain in molybdopterin dehydrogenase, a CO dehydrogenase flavoprotein C-terminal domain, aldehyde oxidase and xanthine dehydrogenase, an a/b hammerhead domain, and a molybdopterin-binding domain of aldehyde dehydrogenase (Figure 4). The cyp and aox were cloned into plasmid pET28a and overexpressed in the E. coli Rosetta cells. SDS-PAGE indicated that the small (I), molybdopterin-binding (II), and large (III) subunits of AOX were obviously overexpressed in the E. coli Rosetta cells (Figure 5, Lane 3) with a calculated molecular weight of 18.6, 35.6, and 79.5 kDa, respectively. The subunits II and III could be observed in the partially soluble fraction (Lane 4), whereas the subunit I could not be detected. The control E. coli cells without aox in pET28a did not produce the three proteins (Lane 1). As shown in Figure 6A, HPLC analysis indicated that the E. coli cells overexpressing AOX transformed IMI to 5-hydroxy and nitroso IMI, whereas the control E. coli cells could only transform IMI to 5-hydroxy IMI (Figure 6B). Heterologous expression of AOX proved that the AOX of P. putida KT2400 is IMI nitroreductase with the ability to mediate IMI nitroreduction to produce nitroso IMI. In the presence of citrate as the cosubstrate, the E. coli cells overexpressing AOX produced 3.4 ± 0.73 mg/L nitroso IMI in 96 h. SDS-PAGE of the total protein and soluble protein samples revealed the presence of CYP in the total proteins (Lane 5), but not in the soluble protein fraction (Lane 6), indicating that CYP was overexpressed with a major inclusion body. A decrease in the temperature and concentration of the inducer, isopropyl β-D-thiogalactoside, did not improve the solubility of CYP. HPLC analysis indicated that the E. coli cells overexpressing CYP showed a decrease in 5-hydroxy IMI formation, when compared with the control E. coli cells without CYP,

cosubstrates, including malate and pyruvate, did not enhance 5hydroxy IMI formation. On the one hand, maltose was the best cosubstrate for the formation of 5-hydroxy IMI metabolite (16.1 mg/L), but could not improve nitroso IMI formation. Citrate was found to be the best cosubstrate for the formation of nitroso IMI metabolite (8.5 mg/L), but could not enhance 5hydroxy IMI formation. The variation in the volumes of the transformation broth had no effect on the reduction of IMI by P. putida Z-4 (Table 3; p > Table 3. Effect of the Transformation Broth Volume on the Transformation of IMI by P. putida Z-4 Resting Cellsa content (mg/L) volume (mL)

reduced IMI

5-hydroxy

nitroso

2 5 10

18.7 ± 4.2a 17.9 ± 3.2a 17.6 ± 4.1a

3.5 ± 0.6a 1.9 ± 0.5b 0.9 ± 0.1c

9.1 ± 0.6a 10.5 ± 0.6b 11.8 ± 1.1c

a The P. putida Z-4 cells precultivated for 6 h were used for the preparation of resting cells for the transformation of IMI. The data represent the mean values of triplicates. The mean values (±SD) within a column followed by different superscripts are significantly different at p ≤ 0.05 according to Duncan’s test.

0.05). However, when the volume of the transformation broth was increased from 2 to 10 mL, the production of 5-hydroxy and nitroso IMI was significantly decreased from 3.5 to 0.9 mg/ L and slightly increased from 9.1 to 11.8 mg/L, respectively. These results indicated that the concentration of dissolved oxygen had opposite effects on IMI hydroxylation and nitroreduction. IMI is stable at a pH range from 4 to 9,38 and the IMI substrate control did not exhibit IMI degradation. Therefore, the variation in IMI degradation and metabolite formations by P. putida Z-4 at different pH values could have resulted from the effect of pH on the activities of bacterial enzymes involved in hydroxylation and nitroreduction. As shown in Table 4, 65.2 Table 4. Effect of the Initial pH on IMI Transformation by P. putida Z-4 Resting Cellsa content (mg/L) pH 5 6 7 8 9

reduced IMI

5-hydroxy

± ± ± ± ±

ND ND 5.2 ± 0.3a 4.2 ± 0.6b 1.1 ± 0.7c

28.8 31.3 65.2 31.0 34.3

5.9a 5.8a 6.7b 5.4a 3.2a

nitroso 0.15 1.5 3.2 2.2 1.4

± ± ± ± ±

0.1a 0.1b 0.4d 0.3c 0.1b

a

A 0.2 mol/L Na2HPO4/NaH2PO4 solution was used as the phosphate buffer. The P. putida Z-4 cells precultivated for 6 h were used for the preparation of resting cells for the transformation of IMI. A transformation period of 96 h was employed. The data represent the mean values of triplicates. The mean values (±SD) within a column followed by different superscripts are significantly different at p ≤ 0.05 according to Duncan’s test.

mg/L IMI was degraded at pH 7, while only 31.3 and 31.0 mg/ L IMI was degraded at pH 6 and 8, respectively. Moreover, at pH 5 and 6, P. putida Z-4 did not produce 5-hydroxy IMI. Similarly, acidic pH also inhibited 5-hdroxy IMI formation by S. maltophilia CGMCC 1.1788 (data not shown). Cloning and Functional Overexpression of aox and cyp from P. putida KT2440 in E. coli Rosetta Cells. F


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Figure 4. Structure of KT2440 aox and the conserved domains of the AOX subunits of KT2440 AOX and rabbit AOX.

Figure 5. SDS-PAGE analysis of AOX and CYP of P. putida KT2440 overexpressed in E. coli Rosetta cells Lanes 1, 3, and 5 represent the total proteins of E. coli Rosetta (DE3) cells that overexpressed the plasmid pET28a (control), pET28a-aox, and pET28a-cyp, respectively. Lanes 2, 4, and 6 indicate the soluble protein fraction of E. coli Rosetta (DE3) cells that overexpressed the plasmid pET28a, pET28a-aox, and pET28a-cyp, respectively. Lane M shows the standard protein markers. I, II, and III represent the three subunits of AOX. CYP indicates P450 monooxygenase PP_1955. Figure 6. HPLC analysis of IMI transformation by the E. coli Rosetta cells overexpressing AOX (A) and control (B).

which may have resulted from KT2440 CYP lacking the ability to hydroxylate IMI as well as the overexpressed CYP competing with the endogenic IMI hydroxylase of E. coli cells. Interestingly, the E. coli cells without CYP40 could hydroxylate IMI (Figure 6B), suggesting the involvement of a non-CYP enzyme in IMI hydroxylation in E. coli.

repeated dosages of IMI confirmed that midges, ostracods, and mayflies disappeared, and their populations did not recover when the IMI residues in water were >1 ppb.35 Similarly, Van Dijk et al.11 reported that IMI concentrations as low as 0.01 ppb led to a significant reduction in the number of macroinvertebrates in surface waters. In soil and water environments, hydroxylation and nitroreduction are the two major pathways of IMI metabolism, and microbial degradation is predominant.19,21,38 In the present study, we found that IMI degradation and its metabolic flux via hydroxylation and

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DISCUSSION IMIthe most widely used insecticide in the world, with 22 000 tons produced annually13is a nicotinic acetylcholine receptor agonist, which is toxic even at low concentrations.18 Experiments in aquatic model ecosystems treated with single or G


Article

Journal of Agricultural and Food Chemistry nitroreduction pathways by P. putida Z-4 were regulated by the bacterial growth phase, cosubstrate, dissolved oxygen concentration, and pH. Furthermore, the other IMI-degrading bacteria, S. maltophilia CGMCC 1.1788 and P. indica CGMCC 6648, showed different aspects of IMI metabolism under these culture conditions. For example, P. putida Z-4 metabolized IMI to nitroso IMI at a pH range of 5−9 (Table 4), while S. maltophilia CGMCC 1.1788 only produced nitroso IMI under alkaline pH and P. indica CGMCC 6648 could not transform IMI to nitroso IMI (data not shown). Therefore, it is reasonable to speculate that IMI fate in soil is significantly dependent on bacterial diversity, bacterial growth stage, and environmental conditions (e.g., pH, amount of dissolved oxygen, etc.). Co-metabolism is frequently observed during the degradation of xenobiotics by microorganisms, and the presence of a cosubstrate such as carbohydrate or organic acid, which acts as an energy source for the regeneration of cofactor NAD(P)H,41 can enhance the xenobiotics degradation rate.29 We previously found that NAD(P)H enhanced IMI hydroxylation and thus increased IMI degradation.34 Furthermore, Dick et al.35 proved that NADPH slightly increases the rate of nitroso IMI formation in mammalian metabolism of IMI. Accordingly, in the present study, as shown in Table 2, the cosubstrates enhanced 5-hydroxy and/or nitroso IMI formation, which might also be related to cofactor NAD(P)H regeneration. Moreover, as shown in Table 1, the duration of precultivation of cells had significant effects on IMI degradation and 5hydroxy and nitroso IMI formation. We speculate that the different cell growth periods may have different NAD(P)H regeneration efficacies, leading to different IMI metabolic flux between nitroreduction and hydroxylation pathways. Cometabolic degradation of IMI has been observed in other bacterial strains such as Leifsonia sp. PC-21, Pseudomonas 1G, S. maltophilia CGMCC 1.1788, and P. inidca CGMCC 6648,26,28,29,41 and some researchers suggest that the complete set of genes responsible for IMI degradation may not be found within a single bacterium since the commercial use of IMI.22,29 Therefore, IMI-degrading bacteria are comparatively difficult to screen and the isolates usually show low IMI degradation rate. Hydroxylation and nitroreduction of IMI by P. putida Z-4 were noted to be inversely associated with the dissolved oxygen concentration in the resting cells transformation broth. The formation of nitroso IMI slightly increased with the increase in the volume of the transformation broth, whereas 5-hydroxy IMI formation was significantly decreased. The oxygen atom in the hydroxyl group of 5-hydroxy IMI is derived from atmospheric oxygen dissolved in the transformation broth, which is increased by reducing the volume of the transformation broth.15 Oxygen-sensitive nitroreduction of IMI to nitroso, guanidine, and urea IMI metabolites has been reported in Pseudomonas sp. 1G by Pandey et al.29 under microaerophilic conditions. As Pseudomonas sp. 1G and P. putida Z-4 belong to the same genus, it is highly likely that both employ similar mechanisms for IMI nitroreduction by AOX to nitroso IMI metabolite in an oxygen-sensitive manner.38,39 These findings are in accordance with the previous report that IMI nitroreduction pathway is favored under anaerobic conditions in soil and water systems.38 The guanidine IMI produced via nitroreduction pathway is more toxic to mammalian species and is more water-soluble than IMI, and therefore may pose an increased risk to the environment.18

It has been reported that the rabbit AOX could convert IMI to an aminoguanidine metabolite in a two-step reduction via the nitroso intermediate.35 However, aminoguanidine metabolite could not be detected in the present study, similar to that noted in a previous study on IMI degradation by Pseudomonas sp. 1G.29 Gene cloning and overexpression of aox in E. coli proved that the AOX from P. putida KT2440 is responsible for nitroreduction of IMI to nitroso IMI (Figure 6A). Unlike the rabbit aox containing only one gene, KT2440 aox was noted to be composed of three subunit genes with a full length of 3.7 kb. Therefore, it may be difficult to accurately overexpress KT2440 aox in E. coli (Figure 5), resulting in low nitroreduction activity of the AOX-overexpressing E. coli cells to convert IMI to nitroso IMI. Another reason for the low IMI nitroreduction activity of AOX in P. putida KT2440, P. putida Z-4, and AOXoverexpressing E. coli cells may be the inhibition of AOX activity by the nitroso IMI metabolite, which has been reported by Dick et al.35 in rabbit liver cytosol AOX transformation of IMI. Mammalian CYPs are known to be involved in the hydroxylation of IMI.42 Although P. putida KT2440 can transform IMI to 5-hydroxy IMI, PP_1955 may be excluded from IMI hydroxylase. Genomic analysis of P. indica CGMCC 6648 and S. maltophilia CGMCC 1.1788 (ATCC 13637), the two IMI-hydroxylating bacteria with higher hydroxylation ability than P. putida KT2440, indicated the absence of CYPcoding gene in their genomic DNA. Similarly, the E. coli Rosetta cells without CYP40 could also hydroxylate IMI (Figure 6B). Hence, it can be concluded that a non-CYP enzyme may be responsible for IMI hydroxylation. Among the 13 monooxygenase-coding genes in E. coli,40 we have knocked out 10 nonlethal monooxygenase genes and overexpressed three monooxygenase genes; however, we could not obtain a positive mutated strain. The IMI hydroxylation ability of P. indica CGMCC 6648, which has only eight monooxygenasecoding genes in its genomic DNA, is more than 10 folds higher than that of P. putida KT2440.28 These eight monooxygenasecoding genes have been cloned into plasmid pET28a and overexpressed in E. coli Rosetta cells to further identify the IMI hydroxylase. In conclusion, the present study found that P. putida degraded the neonicotinoid insecticide, IMI, and that the IMI metabolic flux between nitroreduction and hydroxylation was regulated by pH, oxygen, cosubstrate, and cell growth duration. In addition, KT2440 AOX was proven to catalyze IMI nitroreduction to nitroso IMI. These findings could help to further understand the fate of IMI in the environment and formulate novel strategies for microbial remediation of IMI contamination in soil environments.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86 25 85287298; fax: +86 25 85287298; e-mail: [email protected] (F.G.). *Tel: +86 25 85891731; fax: +86 25 85891067; e-mail: [email protected] (Y.-J.D.). Funding

This research was financed by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the National Science Foundation of China (31570104) and the Academic Natural Science Foundation of Jiangsu Province (14KJA180004). H


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Journal of Agricultural and Food Chemistry Notes

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The authors declare no competing financial interest.

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