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Isolation and characterization of a metsulfuron-methyl degrading bacterium sp. S113

Article in International Biodeterioration & Biodegradation · December 2007 DOI: 10.1016/j.ibiod.2007.02.005

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International Biodeterioration & Biodegradation 60 (2007) 152–158 www.elsevier.com/locate/ibiod

Isolation and characterization of a metsulfuron-methyl degrading bacterium Methylopila sp. S113

Xing Huang, Jian He, Jiquan Sun, Jijie Pan, Xiaofei Sun, Shunpeng LiÃ

Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, PR China

Received 14 September 2006; received in revised form 29 January 2007; accepted 12 February 2007 Available online 12 April 2007

Abstract

The bacterium S113, capable of degrading metsulfuron-methyl, was isolated from metsulfuron-methyl-treated soil. The isolate was identified as Methylopila sp. according to its phenotypic features and 16S rDNA phylogenetic analysis. This strain could utilize metsulfuron-methyl as the sole carbon or nitrogen source. More than 97% of the 50 mg l1 initially added metsulfuron-methyl was depleted after 72 h when a culture was inoculated with 104 cells l1 of strain S113. This strain could also degrade bensulfuron-methyl, thifensulfuron-methyl and ethametsulfuron-methyl. Cell-free extract of S113 was able to metabolize metsulfuron-methyl and other sulfonylurea herbicides. The metsulfuron-methyl degrading enzyme(s) was(ere) constitutively expressed and was(ere) not induced by metsulfuron-methyl. Inoculation of strain S113 into soil was found to promote the removal of metsulfuron-methyl in soil. r 2007 Elsevier Ltd. All rights reserved.

Keywords: Sulfonylurea herbicides; Metsulfuron-methyl; Biodegradation; Methylopila sp.

1. Introduction processes in soil. The fact that sulfonylureas removal was faster and more effective in non-sterile soil compared with Sulfonylurea herbicides belong to a class of chemicals sterile soil suggested the involvement of bacterial degrading used for weed control. They are used on a wide range of activities in soil (Walker et al., 1989; Ismail and Lee, 1995; crops such as rice, wheat, barley, soybean, cotton, potato Li et al., 1999). Few reports on microbial degradation and corn (Brown, 1990). The target enzyme of sulfonylurea of sulfonylurea herbicides have been published so far. herbicides is acetolactate synthase (ALS), which catalyzes Zanardini et al. (2002) isolated Pseudomonas fluorescens the first common reaction in the biosynthesis of the strain B2 capable of co-metabolically degrading approxi- branched amino acids valine, leucine and isoleucine (Blair mately 21% of the initially added 100 mg l1 metsulfuron- and Martin, 1988; Brown, 1990). Sulfonylurea herbicides methyl within 2 weeks. Boschin et al. (2003) reported that were introduced into China in 1989, and soon became 33% of metsulfuron-methyl (100 mg l1) was degraded by a among the most frequently used herbicides because of their strain of Aspergillus niger within 28 days incubation under high herbicidal activity at low application rates. Some of laboratory conditions. Yu et al. (2005) obtained a fungal the sulfonylurea herbicides, such as metsulfuron-methyl, isolate, MD, capable of utilizing metsulfuron-methyl as the chlorsulfuron and ethametsulfuron-methyl, persist for a sole carbon and energy source. In this case, 79% of the long time in soil, and the residues in soil can significantly added metsulfuron-methyl at concentration of 10 mg l1 damage rotation crops (Moyer et al., 1990; Kotoula et al., in mineral salts medium was degraded within 7 days. 1993; Flaburiari and Kristen, 1996; Nicholls and Evans, Brevibacterium sp. BH isolated by Zhu et al. (2005) can 1998). Chemical hydrolysis and microbial metabolism remove 80% of an initial 200 mg l1 bensulfuronmethyl in represent the major sulfonylurea herbicides removal M9 medium. The objective of this study was to isolate new ÃCorresponding author. Tel./fax: 86 25 84396314. that can potentially degrade metsulfuron-methyl (methyl E-mail address: [email protected] (S. Li). 2-[[(4-methoxy-6-methyl-1,3,5-triazine-2-yl) aminocarbonyl]

0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2007.02.005 ARTICLE IN PRESS X. Huang et al. / International Biodeterioration & Biodegradation 60 (2007) 152–158 153 aminosulfonyl] benzoate). We described here the isolation www.ncbi.nlm.nih.gov/BLAST/). The neighbor-joining (NJ) (Saitou and and characteristics of Methylopila sp. S113, a new isolate Nei, 1987) method (implemented in MEGA2.0, Kumar et al., 2001) was used for phylogenetic analysis with the model of Kimura-2-Parameter. capable of utilizing metsulfuron-methyl as the sole carbon The robustness of the tree topology was assessed by bootstrap analysis, or nitrogen source. Experiments were also conducted to with 1000 resembling replicates. evaluate the potential of this strain to remove metsulfuron- methyl in soil. 2.4. Removal of metsulfuron-methyl and other sulfonylurea herbicide by S113 in mineral salts medium 2. Materials and methods Metsulfuron-methyl, thifensulfuron-methyl, bensulfuron-methyl, etha- metsulfuron-methyl, chlorsulfuron, and pyrazosulfuron-ethyl were se- 2.1. Chemicals and soil lected as substrates. Strain S113 grown in TY liquid medium 1 1 (bactotryptone 5 g l , yeast extract 3 g l , CaCl2 0.6 mM, pH7.2) was Soil sample was collected from the surface layer (0–10 cm) from an centrifuged, washed, and suspended in MSM medium. After the optical agricultural field located in the city of Yangzhou, Jiangsu, China. The soil density at 600 nm (OD600) had been adjusted to 1.0, 1 ml bacterial had been exposed to sulfonylurea herbicides during 10 years. Metsulfuron- inoculum (corresponding to 106 cells) was inoculated into 100 ml MSM methyl (98.0% purity), thifensulfuron-methyl (96.0% purity), bensulfur- medium with the selected herbicide (50 mg l1) as the sole carbon source. on-methyl (95.0% purity), ethametsulfuron-methyl (97.0% purity), All cultures were incubated at 30 1C and 150 rpm on a rotary shaker. chlorsulfuron (99.0% purity), and pyrazosulfuron-ethyl (97.0% purity) Samples were collected from the cultures at an interval of 12 h and the were purchased from Changzhou Agrochemical factory, Changzhou, concentration of the selected herbicide was determined by HPLC Jiangsu Province, China. Methanol was chromatographic pure grade. following the protocol described below. Each treatment was performed Other chemicals used were analytical grade. in three replicates, and the control experiment without microorganism was carried out under the same conditions. 2.2. Enrichment and isolation 2.5. Removal of metsulfuron-methyl and other sulfonylurea The mineral salt medium (MSM) had the following composition (per herbicide by cell-free extracts liter): NaCl, 1.0 g; NH4NO3, 1.0 g; K2HPO4, 1.5 g; KH2PO4, 0.5 g; MgSO4 7H2O, 0.1 g; FeSO4, 0.025 g; trace element solution 10 ml (Ferrari Cells in TY liquid medium grown to the stationary phase were 1 et al.,1994); pH7.0. NH4NO3 was removed and glucose (1.0 g l ) was harvested by centrifugation (12,000g, 10 min) at 4 1C, washed twice with supplemented when metsulfuron-methyl was used as the sole nitrogen 10 mM sodium phosphate buffer (pH 7.2), and resuspended in the same source. About 1.0 g of the soil sample was added to an Erlenmeyer flask buffer at a concentration equivalent to an OD600 of 5.0. This cell (250 ml) containing 100 ml MSM with the addition of metsulfuron-methyl suspension was passed three times through a chilled French pressure cell (50 mg l1) as the sole carbon source and incubated at 30 1C on a rotary (15,000 lb in2). Cell debris and unbroken cells were removed by shaker at 150 rpm for about 7 days. About 5 ml of enrichment culture was centrifugation (30,000g, 45 min) at 4 1C. The supernatant was passed then subcultured five times into fresh MSM containing 50 mg l1 through a cellulose acetate filter with a pore size of 0.2 mm and metsulfuron-methyl every 7 days. Metsulfuron-methyl removal was immediately stored at 70 1C. measured by HPLC in the culture from the fifth transfer. The enrichment The assay to quantify removal of metsulfuron and other herbicides by capable of degrading metsulfuron-methyl was serially diluted in MSM, cell-free extract was performed in 0.2 M sodium phosphate buffer. Each and transferred to fresh MSM containing 50 mg l1 metsulfuron-methyl. reaction vial comprised 20 ml of the cell-free extract prepared as described The loss of metsulfuron-methyl was again measured over time. The above in 5 ml of 0.2 M sodium phosphate buffer (pH 7.2) containing highest dilution that still exhibited degradation capability for metsulfuron- 50 mg l1 of the tested herbicide. The reaction medium was incubated at methyl was spread onto minimal salts agar plate containing 50 mg l1 30 1C. At regular intervals, the reaction was stopped by the addition of metsulfuron-methyl. After incubation at 30 1C for 3 days, the colonies acetonitrile. The concentration of the herbicide remaining in the reaction were selected to verify their degrading capabilities. One strain, designated vial was measured by HPLC following the protocol described below. S113, was selected for further investigation. Control samples containing boiled extract were treated and analyzed in the same way. Protein concentrations were determined using the Bradford 2.3. Identification of the strain assay with bovine serum albumin (Sigma, Beijing, China) as the protein standard. All experiments were carried out in triplicate. In each case, 1 U of enzyme activity was defined as the amount of enzyme removing 1 nmol The strain S113 was identified with reference to Bergey’s Manual of of each tested herbicide in 1 h at 30 1C. Determinative Bacteriology (Holt et al., 1994). Partial 16S rDNA sequence was amplified by PCR using the following primers: 2.6. Enzyme induction 50-AGAGTTTGATCCTGGCTCAG-30 as forward and 50-TACGGT- TACCTTGTTACGACTT-30 as the reverse (Lane, 1991). Taq DNA polymerase was purchased from TaKaRa biotechnology (Dalian, China) The capacity of metsulfuron-methyl to induce the metsulfuron-methyl Co., Ltd. PCR reactions were carried out with a PTC 200 gradient cycler degrading enzymes was examined following to the protocol described (MJ Research, Waltham, MA, 02451-2173) under the following condi- previously by Mulbry (1994). Cells grown to the stationary phase in TY tions: 3 min at 95 1C; 30 cycles of 1 min at 94 1C, 1 min at 52 1C and 1 min medium were harvested by centrifugation (12,000g, 10 min), washed twice at 72 1C; plus an additional 10 min cycle at 72 1C. PCR fragments were with 10 mM phosphate buffer (pH 7.2), and suspended in minimal medium 1 ligated into the linear vector pMD18-T (TaKaRa Biotechnology, Dalian containing 50 mg l metsulfuron-methyl or in the same medium contain- Co., Ltd., China) after purification by agarose gel electrophoresis, and ing no metsulfuron-methyl, and incubated at 30 1C for 24 h. The cells were then transformed into competent Escherichia coli DH5a cells. The collected by centrifugation, pressed, and assayed for hydrolytic activity as recombinant plasmid in positive clones was extracted and used as the described above. template for direct sequencing of 16S rDNA fragment by using an automatic sequencer (Applied Biosystems, model 3730). The partial 16S 2.7. Removal of metsulfuron-methyl in soil rDNA sequence has been submitted to the GenBank database with the accession number DQ916067. This sequence was compared to known Soil samples were collected from the top 0–10 cm from the Agricultural sequences found in the GenBank database using Blast (http:// farm, Weigang Campus, Nanjing Agricultural University, Nanjing, China. ARTICLE IN PRESS 154 X. Huang et al. / International Biodeterioration & Biodegradation 60 (2007) 152–158

1 The soil has never been treated with the metsulfuron-methyl. Soil samples initially added 50 mg l metsulfuron-methyl after 72 h of were dried at room temperature, and then sieved to 5 mm and stored at incubation. 4 1C. Soil samples were sterilized as described by Zhang et al. (2006). Sub- samples (100 g) of fresh soil and sterile soil were weighed, and the solution On TY agar plates, this strain forms colonies of of metsulfuron-methyl was added to obtain a final concentration of approximately 2 mm in diameter after 3 days incubation 10 mg kg1 soil, and mixed well. One set of fresh soil and of sterile soil at 30 1C. The colonies are circular, convex, lustrous, and were inoculated with S113 (106 cells g1). Another set of uninoculated soil white. Strain S113 is a Gram-negative, colorless rod was kept as a control. The inoculum was thoroughly mixed into the soils (0.5–0.7 mm wide and 1.1–1.3 mm long). Methyl red and under sterile conditions, and the moisture was adjusted to 65% (w/w of 7 dry weight of soil). Each soil microcosm was incubated at 30 1C in the Voges Proskauer assays are negative. Strain S113 is dark. At intervals of 0, 3, 5, 10, 15 and 25 days, 10 g of the soil samples oxidase and urease positive, and weakly catalase positive. were collected and the concentration of metsulfuron-methyl was detected. It metabolizes sugars to produce acids and it produces H2S Experiments were conducted in triplicate. during growth in nutrient broth. It does not produce indole or NH3 production. The strain is sensitive to gentamycin, 2.8. Quantification of sulfonylurea herbicides by HPLC streptomycin, and tetracycline. A 1.5 kb 16S rDNA fragment of S113 was deposited For metsulfuron-methyl extraction from liquid culture, a 1 ml sample in the GenBank database under accession number removed from 100 ml liquid culture was extracted with 4 ml dichlor- DQ916067. Multiple alignments revealed that the 16S omethane. The extract was dried over anhydrous Na2SO4 and then evaporated at room temperature. The residues were dissolved in 200 mlof rDNA sequence of S113 was closely related to that of methanol. An aliquot of the solution (20 ml) was injected into a HPLC Methylopila capsulata (AF004844, 96% of similarity) and system for detection. Methylosulfonomonas methylovora strain M2 (U62893, For metsulfuron-methyl extraction from soil, 10 g of soil sample was 94% of similarity). To identify the phylogeny of strain extracted with 50 ml of PBS-acetonitrile (80:20, V:V), the mixture was S113, strains from different genera were chosen to shaken for one hour at 200 rpm on a rotary shaker and then centrifuged. The supernatant was decanted into a glass bottle, and the organic solvent construct the phylogenetic tree based on 16S rDNA was removed by rotary evaporation in a water bath at 35 1C and a pressure sequences (Fig. 1). The phylogenetic analysis revealed that of 40 mbar (Bossi et al., 1999). The aqueous extract was extracted using the strain S113 clustered closely with M. capsulata the procedure described above. (AF004844). According to its phenotypic features, physio- Metsulfuron-methyl concentration in the extracts was determined by logical and biochemical characteristics and 16S rDNA reverse-phase high-performance liquid chromatography (HPLC, 600 Controller, Rheodyne 7725i Manual injector and 2487 Dual k Absorbance phylogenetic analysis, strain S113 can be identified as Detector; Waters Co., Milford, MA). Detection of metsulfuron-methyl belonging to the Methylopila sp. The bacterium is was recorded at 254 nm. The separation column for the HPLC (internal classified as belonging to the phylum , the diameter, 4.6 mm; length, 25 cm) was filled with Kromasil 100-5C18. The class , the order Rhizobiales and the mobile phase was methanol:water (70:30, V:V), and the flow rate was family . This genus has been described by 1.0 ml min1. The concentrations of the other sulfonylurea herbicides were determined using HPLC conditions identical to those described for Doronina et al. (1998). metsulfuron-methyl. Each analysis was repeated three times. 3.2. Removal of metsulfuron-methyl and other sulfonylurea 2.9. Recovery assay herbicides by S113

Triplicate analyses were conducted with liquid culture at levels of 0.1, The growth of strain S113 on liquid MSM supplemented 1.0, 10.0 and 50 mg l1 sulfonylurea herbicides, and with soil at levels of with metsulfuron-methyl and its ability to remove metsul- 0.1, 1.0 and 10.0 mg kg1 metsulfuron-methyl. Extraction and analysis furon-methyl are shown in Fig. 2. After incubation for were performed as described above. The recovery of sulfonylurea 1 1 72 h, more than 97% of the 50 mg l metsulfuron-methyl herbicides from liquid culture at levels of 0.1, 1.0, 10.0 and 50 mg l initially added to the medium was removed by strain S113 were determined to be 88.677.9%, 91.374.7%, 97.172.4%, 98.571.8%. The recovery of metsulfuron-methyl from soil at levels of 0.1, 1.0 and when metsulfuron-methyl was the sole carbon source. 10.0 mg kg1 were determined to be 85.677.1%, 89.175.7%, 95.474.1%. However, when metsulfuron-methyl was used as the sole These data indicate that the extraction procedure was efficient in nitrogen source it took 102 h of incubation to reach the extracting metsulfuron-methyl residues from liquid culture and soil. same level of removal (97% of 50 mg l1metsulfuron- methyl). No significant change in metsulfuron-methyl 3. Results concentration was observed in cultures that were not inoculated. Metsulfuron-methyl depletion was associated 3.1. Strain isolation and identification to a concomitant increase of OD600 from about 0.01 to 0.3 in both cultures with or without an added nitrogen source. A total of 22 bacterial isolates from the enrichment grew This indicates that metsulfuron-methyl serves as carbon on MSM plates supplemented with metsulfuron-methyl as and nitrogen sources for S113 growth. the sole carbon source. Four of these isolates were found to When thifensulfuron-methyl, bensulfuron-methyl, and possess the ability to remove metsulfuron-methyl in liquid ethametsulfuron-methyl were used as the sole source of culture. Strain S113 was selected for further study due to its carbon, they were rapidly removed within 72 h. Thifensulfur- relatively high potential to remove metsulfuron-methyl. on-methyl was removed more rapidly than bensulfuron- This strain was able to degrade more than 97% of the methyl and ethametsulfuron-methyl. Ethametsulfuron-methyl ARTICLE IN PRESS X. Huang et al. / International Biodeterioration & Biodegradation 60 (2007) 152–158 155

37 sporium AJ458478 AJ458495 96 Methylosinus sporium AJ458489

76 Methylosinus sporium AJ458488 Methylosinus sporium Y18946 Methylosinus sporium AJ458468 83 99 90 Methylosinus sporium AJ458486 Methylocystis aldrichii DQ364433 Methylocystis parvus AJ458508 99 96 Methylocystis parvus Y18945 99 67 Methylocystis parvus AJ458476

98 Methylosinus trichosporium AJ458496 Methylosinus trichosporium AJ458482 93 99 99 Methylosinus trichosporium AJ458485 Methylocapsa acidiphila AJ278726 Methylobacterium or ganophilum AB175639

99 Agrobacterium rhizogenes AY945955 Rhizobium tropici D11344

89 Methylopila capsulata AF004844 S113

99 Methylosulfonomonas methylovora U62893 Methylopila helvetica AF227126 16 99 Albobacter methylovorans AF273213

0.01

Fig. 1. Phylogenetic analysis of the isolate S113 and related species by the neighbor-joining approach. Bootstrap values (%) are indicated at the nodes. The scale bars represent 0.01 substitutions per site.

60 0.4 was removed more slowly than bensulfuron-methyl. Under the same conditions, chlorsulfuron and pyrazosulfuron-ethyl 50 were not removed by strain S113 (Fig. 3). 0.3 -1 40 3.3. Removal of metsulfuron-methyl by cell-free extracts 30 0.2 600 OD The capacity of cell extracts to remove metsulfuron- 20 methyl was evaluated using the assay described in Section Metsulfuron-methyl concentration mg l 0.1 2. When 20 ml of cell extract prepared as described in 10 Section 2 was added to 5 ml of reaction buffer containing 1 0 0 50 mg l metsulfuron-methyl the substrate was almost

0 completely depleted after 24 h incubation at 30 1C. Under 12 24 36 48 60 72 84 96 102 Time(d) the same conditions, no substrate depletion was recorded when boiled cell-free extracts were added to the reaction removal of metsulfuron-methyl as sole nitrogen source buffer. This shows that metsulfuron-methyl was trans- removal of metsulfuron-methyl as sole carbon source formed by soluble enzymes from cell-free extracts of strain growth on metsulfuron-methyl as sole nitrogen source S113. The enzymatic activity responsible for metsulfuron- growth on metsulfuron-methyl as sole carbon source methy removal was stable. About 40% of the original 1 Fig. 2. Removal of metsulfuron-methyl by strain S133 and concomitant activity remained when the extract was incubated at 30 C growth in minimal medium containing the herbicide as sole carbon or for 192 h. The crude extract could be stored at 70 1C for nitrogen source. Error bars represent the standard error of three replicates. several months with no loss of activity. ARTICLE IN PRESS 156 X. Huang et al. / International Biodeterioration & Biodegradation 60 (2007) 152–158

60 Table 1 Sulfonylurea herbicide degrading activitya of the cell-free extracts of strain S113 50

) 1

-1 Sulfonylurea herbicide Concentration Activity (U mg of (nM) protein)b 40 Metsulfuron-methyl 13.1 0.5470.03 Thifensulfuron-methyl 12.9 43.0072.1 30 Bensulfuron-methyl 12.2 2.5470.34 Ethametsulfuron-methyl 12.2 0.9270.07 Chlorsufuron 13.9 0 20 Pyrazosulfuron-ethyl 12.1 0

a Herbicides concentration(mgl Mean of triplicates7standard error (SE). 10 bOne unit of enzyme activity is the amount of enzyme that removes 1 nmol of each of the tested herbicide per hour at 30 1C. 0 020406080 Time(h) -1 12

ethametsulfuron-methyl bensulfuron-methyl 10 thifensulfuron-methyl pyrazosulfuron-ethyl 8 chlorsufuron metsulfuron-methyl 6 Fig. 3. Removal of metsulfuron-methyl, thifensulfuron-methyl, bensul- furon-methyl, thifensulfuron-methyl, pyrazosulfuron-ethyl, chlorsufuron 4 by S113 in minimal medium. Error bars represent the standard error of three replicates. 2

Metsulfuron-methyl concentration mg l 0 0 10203040 The capacity of metsulfuron-methyl to induce the degrading enzyme(s) was tested by comparing the activity Time(d) of cell-free extracts from cells grown in TY medium or in Sterilized soil minimal medium containing metsulfuron-methyl as sole growth substrate. There was no difference in enzyme Sterilized soil+S113 activities between the cell-free extracts prepared from cells Non-sterilized soil grown in TY or in minimal medium. Therefore, the Non-sterilized soil+S113 metsulfuron-methyl degrading enzymes are constitutively expressed in strain S113. Fig. 4. Removal of metsulfuron-methyl in inoculated and uninoculated Table 1 shows the removal activity of the cell-free soil microcosms. Error bars represent the standard error of three extract with different herbicides. Thifensulfuron-methyl, replicates. bensulfuron-methyl, and ethametsulfuron-methyl could be removed by cell-free extract, while chlorsulfuron and pyrazosulfuron-ethyl could not. contribution of the indigenous micro flora to metsulfuron- methyl removal. 3.4. Removal of metsulfuron-methyl in soil 4. Discussion Only 11.9% of the initially added 10 mg l1 metsulfuron- methyl was removed in uninoculated fresh soil after 30 d of In the present study, strain S113 was isolated from incubation. In contrast, 76.9% of the added substrate was contaminated soil, and was identified as Methylopila sp. removed after 30 d for fresh soil inoculated with strain S113 This genus was first described by Doronina et al. (1998), (Fig. 4). Therefore, metsulfuron-methyl removal improved and only a few strains have been reported in the literature. significantly in soils inoculated with strain S113. The So far, Methylopila sp. TES which is able to degrade removal in fresh uninoculated soil was superior to that in isoproturon (Talaat et al., 2004), is the only other isolate of the uninoculated sterile soil microcosms, suggesting that this genus known to degrade an herbicide, but strain S113 indigenous microbial flora in the soil may play a role in is the only one able to metabolize metsulfuron-methyl. metsulfuron-methyl degradation. Similarly, metsulfuron- Up to now, only a few microorganisms able to methyl removal was slightly better in fresh soil inoculated metabolize metsulfuron-methyl have been isolated from with strain S113 than in inoculated sterile soil suggesting a soils exposed to herbicide. P. fluorescens B2 and a strain of ARTICLE IN PRESS X. Huang et al. / International Biodeterioration & Biodegradation 60 (2007) 152–158 157 the fungal species A. niger were found to degrade Blair, A.M., Martin, T.D., 1988. A review of the activity, fate and metsulfuron-methyl co-metabolically (Zanardini et al., mode of action of sulfonylurea herbicides. Pesticide Science 22, 2002; Boschin et al., 2003). The only strain reported to 195–219. Boschin, G., Agostina, A.D., Arnoldi, A., Marotta, E., Zanardini, E., utilize metsulfuron-methyl as the sole carbon source is the Negri, M., Valle, A., Sorlini, C., 2003. Biodegradation of fungal strain MD (Yu et al., 2005). This strain removes chlorsulfuron and metsulfuron-methyl by Aspergillus niger in labora- 1 79% of metsulfuron-methyl at concentration of 10 mg l tory conditions. Journal of Environment Science Health B 38, in 7days. Strain S113, described in this paper, can utilize 737–746. metsulfuron-methyl as the sole carbon or nitrogen source, Bossi, R., Vejrup, K., Jacobsen, C.S., 1999. Determination of sulfonylurea degradation products in soil by liquid chromatography- and more than 97% of metsulfuron-methyl at a concentra- 1 ultraviolet detection followed by confirmatory liquid chromatogra- tion of 50 mg l was removed within 72 h of incubation. phy-tandem mass spectrometry. Journal of Chromatography A 855, S113 can also degrade bensulfuron-methyl, thifensulfuron- 575–582. methyl and ethametsulfuron-methyl. Our investigation did Brown, H.M., 1990. Mode of action, crop selectivity, and soil relations of not allow us to determine whether these compounds are the sulfonylurea herbicides. Pesticide Science 29, 263–281. completely mineralized or only partially transformed by Doronina, N.V., Trotsenko, Y.A., Krausova, V.I., Boulygina, E.S., Tourova, T.P., 1998. Methylopila capsulata gen. nov., sp. nov., a strain S113. However, although our data did not allow novel non-pigmented aerobic facultatively methylotrophic determining the metabolic pathway used by the bacteria to bacterium. International Journal of Systematic Bacteriology 48, metabolize metsulfuron-methyl (methyl 2-[[(4-methoxy- 1313–1321. 6-methyl-1,3,5-triazine-2-yl)aminocarbonyl]aminosulfonyl]- Ferrari, A., Brusa, T., Rutili, A., Canzi, E., Biavati, B., 1994. Isolation benzoate), data indicate that strain S113 is at least able to and characterization of Methanobrevibacter oralis sp. nov. Current Microbiology 29, 7–12. cleave the triazine portion of the molecule since the Flaburiari, A., Kristen, U., 1996. The influence of chlorsulfuron and substrate can supply both the carbon and the nitrogen metsulfuron methyl on root growth and on the ultrastructure of root required for growth. tips of germinating maize seeds. Plant Soil 180, 19–28. Zanardini et al. (2002) proposed a pathway for the Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1994. degradation of metsulfuron by P. fluorescens B2 that led to Bergey’s Manual of Determinative Bacteriology, ninth ed. Williams and Wilkins, Baltimore. the cleavage of the sulfonylurea bridge. This strain was Ismail, B.S., Lee, H.J., 1995. Persistence of metsulfuron-methyl in two capable of degrading metsulfuron-methyl and chlorsulfur- soils. Journal of Environment Science and Health, Part B 30, on. Unlike, P. fluorescens B2 strain S113 is unable to 485–497. metabolize chlorsulfuron, which might suggest that Methy- Karpouzas, D.G., Walker, A., 2000. Factors influencing the ability of lopila sp. S113 possesses a metsulfuron-methyl metabolic Pseudomonas putida epI to degrade ethoprophos in soil. Soil Biology and Biochemistry 32, 1753–1762. pathway that differs from that of P. fluorescens B2 or that Kearney, P.C., Karns, J.S., Muldoon, M.T., Ruth, J.M., 1986. Couma- the substrate specificities of the pathway enzymes differ phos disposal by combined microbial and UV-ozonation reactions. between these strains. More investigations will be required Journal of Agricultural Food Chemistry 34, 702–706. to elucidate the pathway used by strain S113 to metabolize Kotoula, S.E., Eleftherohorinos, I.G., Gagianas, A.A., Sficas, A.G., 1993. metsulfuron. Phytotoxicity and persistence of chlorsulfuron, metsulfuron-methyl, triasulfuron and tribenuron-methyl in three soils. Weed Research 33, Bioremediation is a cost-effective method to degrade 355–367. toxic compounds into innocuous products. Successful Kumar, B., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: removal of pesticides from soil by implanted bacteria Molecular Evolutionary Genetics Analysis Software. 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