See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/223473143 Isolation and characterization of a metsulfuron-methyl degrading bacterium Methylopila sp. S113 Article in International Biodeterioration & Biodegradation · December 2007 DOI: 10.1016/j.ibiod.2007.02.005 CITATIONS READS 37 201 6 authors, including: Xing Huang Jian He Nanjing Agricultural University 236 PUBLICATIONS 3,700 CITATIONS 61 PUBLICATIONS 776 CITATIONS SEE PROFILE SEE PROFILE Ji-Quan Sun Inner Mongolia University 57 PUBLICATIONS 572 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Xenobiotic compound degradation View project Bacterial resource of alkaline-saline soil View project All content following this page was uploaded by Xing Huang on 04 March 2020. The user has requested enhancement of the downloaded file. ARTICLE IN PRESS 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 Lià 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 lÀ1 initially added metsulfuron-methyl was depleted after 72 h when a culture was inoculated with 104 cells lÀ1 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 lÀ1 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 lÀ1) 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 lÀ1 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 lÀ1 bensulfuronmethyl in represent the major sulfonylurea herbicides removal M9 medium. The objective of this study was to isolate new bacteria 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 À1 À1 (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 10À6 cells) was inoculated into 100 ml MSM methyl (98.0% purity), thifensulfuron-methyl (96.0% purity), bensulfur- medium with the selected herbicide (50 mg lÀ1) 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 Á 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 À1 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 lÀ1) as the sole carbon source and incubated at 30 1C on a rotary (15,000 lb inÀ2). 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 lÀ1 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 À70 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 lÀ1 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 lÀ1 of the tested herbicide. The reaction medium was incubated at methyl was spread onto minimal salts agar plate containing 50 mg lÀ1 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.