Supporting Information for Biotechnology Letters
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Biodegradation of thifensulfuron-methyl by Ochrobactrum sp. ZWS16 in liquid
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medium and soil
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Weisong Zhao, Li Xu, Dongzhi Li, Xuefeng Li, Chengju Wang, Mingqi Zheng,
Canping Pan, Lihong Qiu
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College of Science, China Agricultural University, Beijing 100193, China;
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Corresponding author:
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Lihong Qiu, College of Science, China Agricultural University, Beijing 100193,
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China.
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E-mail: lihongqiuyang@126.com
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Telephone number: 86-10-62733924
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Fax number: 86-10-62734294;
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Corresponding author. Tel.:+86 10 62733924; fax: +86 10 62734294
E-mail address:lihongqiuyang@126.com
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Supplementary Results
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Supplementary Figure Legends
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Supplementary Fig. 1 Morphology observation of strain ZWS16. (a) the colonial morphology
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of strain ZWS16 on LB agar plate, (b) morphological characteristics of strain ZWS16 by
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scanning electron microscope (SEM 20, 000×).
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Supplementary Fig. 2 Phylogenetic analysis of isolated strain and related species by the
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neighbor-joining method. Numbers in parentheses represent the GenBank accession numbers of
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the sequences. Bootstrap values obtained with 1000 resamplings were indicated as percentages at
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the nodes. The scale bars represent 0.0005 substitutions per site.
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Supplementary Fig. 3 Degradation of thifensulfuron-methyl under different inoculum amounts
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of strain ZWS16 in liquid medium. Data expressed as mean ± standard deviation.
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Supplementary Fig. 4 Base peak chromatograms of different samples. (a) medium blank, (b)
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medium+ZWS16, (c) medium+ZWS16+thifensulfuron-methyl. A, B, C, D and E are indication
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of metabolites.
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Supplementary Fig. 5 Mass spectrometry of thifensulfuron-methyl and its metabolites in
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positive or negative ion mode.
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Supplementary Materials and Methods
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Chemicals and Media
Thifensulfuron-methyl
(98.2%
purity),
ethametsulfuron-methyl
(96%
purity),
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pyrazosulfuron-ethyl (97% purity), bensulfuron methyl (96% purity), triasulfuron (95% purity),
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metsulfuron-methyl (96% purity) and tribenuron-methyl (75% purity) were provided from Pesticide
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Toxicology Laboratory of China Agricultural University. Nicosulfuron (97.8% purity) was obtained
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from Zibo Nab Agrochemicals Limited, Shandong, China. Methanol and acetonitrile were
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chromatographic grade and purchased from Mreda Technology Inc. (USA). Other chemicals used
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were analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China).
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Minimal salt medium (MSM) contained 1.5 g K2HPO4/l, 0.5 g KH2PO4/l, 1 g NaCl/l, 0.2 g
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MgSO4•7H2O /l, 0.025 g FeSO4/l, 0.02 g CaCl2/l and 1.0 g NH4NO3/l, pH 7. Lysogeny broth medium
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(LB) contained 10 g peptone/l, 5 g yeast extract/l and 5 g NaCl/l, pH 7. Solid medium plates were
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prepared by adding 1.5% (w/v) agar into the above liquid medium. All media were sterilized by
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autoclaving at 121°C for 30 min. MSM was used to isolate thifensulfuron-methyl-degrading strain.
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LB was used to conduct degradation experiments.
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Enrichment and isolation of bacteria
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Soil sample was collected from sulfonylurea herbicides-contaminated soil in Tangshan, Hebei,
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China, where has been exposed to sulfonylurea herbicides for more than 10 years. Approximately 5 g
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of soil sample was placed to an 250 ml flask containing 100 ml of MSM with 10 mg
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thifensulfuron-methyl/l and incubated at 30℃ on a rotary shaker at 180 rpm in the dark. After 7 days,
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5 ml of enrichment culture was then transferred into fresh MSM containing 10 mg
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thifensulfuron-methyl/l and incubated at conditions mentioned above. After seven transfers, HPLC
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was used to determine the concentration of thifensulfuron-methyl. The final enrichment culture was
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serially diluted with sterile distilled water and spreaded on MSM agar plates containing 200 mg
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thifensulfuron-methyl/l. Different bacterial colonies were picked and further purified by streaking
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plate method, and tested for their thifensulfuron-methyl-degrading ability. Strain ZWS16, with
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thifensulfuron-methyl-degrading ability, was selected for further studies.
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Morphological, 16S rRNA gene and phylogenetic analysis
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Strain ZWS16 was identified on the basis of its morphological and its 16S rRNA gene sequence.
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The cell morphology was investigated by scanning electron microscope (SEM, S-3400N, Hitachi,
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Japan). The genomic DNA of strain ZWS16 was extracted by TIANamp Bacteria DNA Kit (Tiangen
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Biotech Co., Ltd. Beijing, China), according to the manufacturer’s instructions and used as PCR
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template. The 16S rRNA gene was amplified in a Mastercycler gradient (Eppendorf, Germany) with
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the
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(5′-GGTTACCTTGTTACGACTT-3′). Conditions for PCR were: 5 min of denaturation at 95℃,
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followed by 35 cycles of 94℃ for 1 min, 54℃ for 1min, and 72℃ for 1min, plus an additional 10
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min cycle at 72℃. The amplified 16S rRNA products were electrophoresed in a 1.0% agarose gel,
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collected and purified using a gel extraction kit (TIANgel Midi purification Kit, Tiangen Biotech Co.,
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Ltd. Beijing, China). The PCR products were ligated into the vector pGEM-T (Promega Co., Madison,
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USA), and then transformed into Escherichia coli DH5α cells (Tiangen Biotech Co., Ltd. Beijing,
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China). The cloned insert was sequenced (Beijing Sunbiotech Co., Ltd. Beijing, China). The
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determined sequence was deposited to GenBank database and compared with different 16S rRNA
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gene sequences in GenBank by BLAST program. Multiple alignments of sequences were carried out
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using Clustal W, and phylogenetic tree was analyzed by MEGA 5.10 software. The distance was
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calculated using the maximum composite likelihood model. An unrooted tree was built by the
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neighbor-joining method.
universal
primers:
27F
(5′-AGAGTTTGATCCTGGCTCAG-3′)
and
1492R
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Degradation of other sulfonylurea herbicides by strain ZWS16
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In order to determine its ability to degrade other sulfonylurea herbicides, strain ZWS16 was
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inoculated into medium supplemented with 10 mg herbicide/l (nicosulfuron, triasulfuron,
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pyrazosulfuron-ethyl,
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tribenuron-methyl). The herbicides were extracted and determined as mentioned above. The control
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uninoculated with strain ZWS16 was carried out under the same conditions.
bensulfuron-methyl,
ethametsulfuron-methyl,
metsulfuron-methyl,
and
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Identification of thifensulfuron-methyl and its metabolites
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The mass spectrometer was equipped with an Electronic Spray Ion (ESI) source, and operated in
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the positive and negative mode. The ESI-MS interface was operated under the conditions as follow:
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gas temperature of 350℃, dry gas flow of 5 L/min, the nebulizer nitrogen gas pressure was 15 psi,
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and the capillary voltage was set to 3500V. Full scans were obtained by scanning from m/z 100 to 500.
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A reverse-phase Welch materials XB-C18 column (4 mm×250 mm, 5μm) was used. The sample
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volume injected was maintained at 10 μl. The flow rate was 0.5 ml/min. The mobile phase, containing
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acetonitrile (A) and 0.1% v/v acetic acid in water (B) elutes according to the following gradient
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conditions: time=0-1 min, 30% v/v A; time= 1-8 min, 80% v/v A; time=8-15 min, 30% v/v A. The
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ultraviolet detection wavelength was 241 nm.
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Biodegradation of thifensulfuron-methyl in soil
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The soil sample was collected at depth of 5-20 cm beneath the surface in a farm that had never
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been treated with sulfonylurea herbicides at Shangzhuang Experimental Station of China Agricultural
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University (Beijing, China). Soil was air-dried, passed through a 1.43 mm sieve before use. The soil
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sample was sterilized by autoclaving at 121℃ for 30 min in three times (ref. 1).
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Thifensulfuron-methyl in acetonitrile was mixed into 500 g sterilized soil until a final
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concentration of 10 mg/kg, after acetonitrile was volatilized (overnight), strain ZWS16 was
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inoculated into the soil and homogenized by vigorous stirring, and incubated at 30℃ in the dark
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during the experiment (T2); A same volume of sterile deionized water was added into the same
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amount of contaminated soil and was used as control (T1). The moisture content of the soils was 20%
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(w/w) and adjusted by periodically added sterile deionized water during the experiment. Three
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replicates were prepared for each treatment.
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The concentration of thifensulfuron-methyl in soils was determined at different intervals by
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methods as followed: approximately 10 g of soil samples was extracted with 10 ml acetonitrile
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(include 0.1% acetic acid, v/v) and 5 ml sterile distilled water, and fully homogenized and let stand
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for 1 min, then 4 g anhydrous magnesium sulfate and 1.5 g sodium chloride was added, and placed
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into ice-water bath for 1-2 min. The mixture was shaken for 1 h at 250 rpm on a rotary shaker at room
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temperature and then centrifuged for 5 min at 4000 g. The supernatant of organic solvent was
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transferred into vials through 0.22 μm membrane filter for HPLC analysis.
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Statistical analysis
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The statistical significance of data were evaluated by the ANOVA test (P = 0.05). Experimental
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results were processed using SigmaPlot 12. Custom software (LC/MSD Trap 5.2) was used for
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analysis of the mass spectrometry data. Biodegradability tests were carried out independently in
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triplicate and average values were calculated. The first-order rate constant for the degradation data
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was obtained using equation Eq. (1) as follows:
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ln ct  kt  ln c0
(1)
The half-life was determined by equation Eq. (2) as follows:
T0.5  0.6931/ k
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(2)
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where c0 is the thifensulfuron-methyl concentration at zero time, ct is the thifensulfuron-methyl
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concentration at time t, k is the rate constant and T 0.5 is the half-life.
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Supplementary Results and discussion
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Strain isolation and identification
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Sulfonylurea herbicides have been widely used in the control of broadleaf weeds in cereal crops
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for many years in the city of Tangshan, Hebei, China. It was probable that bacteria has adapted to
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sulfonylurea herbicides-contaminated environment. A strain designated ZWS16, which could degrade
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more than 90% of 50.0 mg thifensulfuron-methyl/l within 10 days under neutral condition, was
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isolated and selected for further study. Colonies grown on LB agar plate were opaque, dry and white
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(Supplementary Fig. 1a). The cells of strain ZWS16 were rod-like, with size of 0.9-1.1 × 0.4-0.5 μm
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(Supplementary Fig. 1b).
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A 1490 bp fragment of 16S rRNA was obtained from strain ZWS16, which was sequenced and
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deposited in GenBank database with accession number KM051418. In order to identify the phylogeny
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of ZWS16, bacteria from different species close to ZWS16 were chosen to construct the phylogenetic
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tree based on 16S rRNA gene sequences by the neighbor-joining method (Supplementary Fig. 2). The
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phylogenetic analysis revealed that strain ZWS16 clustered closely with Ochrobactrum sp.
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(AB840687.1). On the basis of the above characters, strain ZWS16 was identified as Ochrobactrum
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sp. The genus Ochrobactrum sp. is known that could degrade chlorothalonil (ref. 2), PAHs (ref. 3)
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and glyphosate (ref. 4). To our knowledge, this is the first reported on the biodegradation of
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thifensulfuron-methyl by Ochrobactrum sp.
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Reference list
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1.
Luo S, Tao CJ, Piao XY, Shi J, Jiang H, Liu XL (2008) HPLC determination of the degradation
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of flumorph in soil. Chinese J Anal Chem 36:7-11
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2. Liang B, Li R, Jiang D, Sun JQ, Qiu JG, Zhao YF, Li SP, Jiang JD (2010) Hydrolytic dechlorination
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of chlorothalonil by Ochrobactrum sp. CTN-11 isolated from a chlorothalonil-contaminated soil.
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Curr Microbiol 61:226-233
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3. Arulazhagan P, Vasudevan N (2011) Biodegradation of polycyclic aromatic hydrocarbons by a
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halotolerant bacterial strain Ochrobactrum sp. VA1. Mar Pollut Bull 62:388-394
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4. Sviridov AV, Shushkova TV, Zelenkova NF, Vinokurova NG, Morgunov IG, Ermakova IT,
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Leontievsky AA (2012) Distribution of glyphosate and methylphosphonate catabolism systems
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in soil bacteria Ochrobactrum anthropi and Achromobacter sp. Appl Microbiol Biot 93:787-796
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a
b
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Supplementary Fig. 1
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Ochrobactrum sp. KD2009-45 (FN645728.1)
Ochrobactrum sp. DGG-1-3 (JX966415.1)
ZWS16 (KM051418)
98 Ochrobactrum sp. strain: 182J (AB840687.1)
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Bacillus sp. B3 (EU281629.1)
Bacillus sp. D2 (EU281633.1)
Gluconacetobacter sp. SS-1 (GU247753.1)
Alpha proteobacterium NBM4 (HQ703920.1)
0.0005
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Supplementary Fig. 2
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Supplementary Fig. 3
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Supplementary Fig. 4
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Thifensulfuron-methyl
O
S
N
N
O
OH
N
NH
N
O
S
O
O
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Thifensulfuron-methyl
S
O
N
N
O
OH
N
NH
N
O
S
O
O
177
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Compound A
O
S
O
OH
N
NH2
S
O
O
179
Compound B
S
O
N
N
O
OH
N
NH
N
OH
S
O
O
180
Compound C
S
HO
N
N
O
OH
N
NH
N
O
S
O
O
181
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Compound C
S
HO
N
N
O
OH
N
NH
N
O
S
O
O
182
S
Compound D
HO
OH
N
S
O
NH
O
NH
OH
MW=279
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Compound E
S
N
N
OH
N
NH
O
N
S
MW=329
O
O
184
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Supplementary Fig. 5
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