Isolation and characterization of Bradyrhizobium sp. SR1 degrading two β-triketone herbicides
Sana Romdhane 1,2,3 • Marion Devers-Lamrani3 • Fabrice Martin-Laurent3 •
Christophe Calvayrac2 • Emilie Rocaboy-Faquet1 • David Riboul 4,5 •
Jean-François Cooper2 • Lise Barthelmebs 1
Received: 27 February 2015 / Accepted: 13 April 2015
Ⓒ Springer-Verlag Berlin Heidelberg 2015
Abstract In this study, a bacterial strain able to use sulcotrione, a β-triketone herbicide, as sole source of carbon and energy was isolated from soil samples previously treated with this herbicide. Phylogenetic study based on16S rRNA gene sequence showed that the isolate has 100 % of similarity with several Bradyrhizobium and was accordingly designated as Bradyrhizobium sp. SR1. Plasmid profiling revealed the presence of a large plasmid (>50 kb) in SR1 not cured under nonselective conditions. Its transfer to Escherichia coli by electroporation failed to induce β-triketone degrading capac- ity, suggesting that degrading genes possibly located on this plasmid cannot be expressed in E. coli or that they are not plasmid borne. The evaluation of the SR1 ability to degrade various synthetic (mesotrione and tembotrione) and natural (leptospermone) triketones showed that this strain was also able to degrade mesotrione. Although SR1 was able to entirely
Responsible editor: Robert Duran
Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-4544-1) contains supplementary material, which is available to authorized users.
* Lise Barthelmebs [email protected]
1 Biocapteurs Analyses Environnement (BAE), University of Perpignan Via Domitia, 66860 Perpignan, France
2 Laboratoire de Chimie des Biomolécules et de l’Environnement– CRIOBE–USR 3278 CNRS EPHE, University of Perpignan Via Domitia, 66860 Perpignan, France
3 INRA, UMR 1347 Agroécologie, Pole Ecoldur, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France
4 INPT, ENSIACET, Université de Toulouse, 31432 Toulouse, France
5 Laboratoire de Génie Chimique (LGC UMR 5503), CNRS, 4 allée Emile Monso, BP 84234, 31432 Toulouse, France
dissipate both herbicides, degradation rate of sulcotrione was ten times higher than that of mesotrione, showing a greater affinity of degrading-enzyme system to sulcotrione. Degradation pathway of sulcotrione involved the formation of 2-chloro-4-mesylbenzoic acid (CMBA), previously identified in sulcotrione degradation, and of a new metabolite identified as hydroxy-sulcotrione. Mesotrione degradation pathway leads to the accumulation of 4-methylsulfonyl-2-nitrobenzoic acid (MNBA) and 2-amino-4 methylsulfonylbenzoic acid (AMBA), two well-known metabolites of this herbicide. Along with the dissipation of β-triketones, one could observe the decrease in 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition, indicating that toxicity was due to parent molecules, and not to the formed metabolites. This is the first report of the isolation of bacterial strain able to transform two β-triketones.
Keywords β-Triketone . Sulcotrione . Mesotrione . Biodegradation . Bradyrhizobium sp. SR1
The widespread use of herbicides over the last decades to con- trol weeds and to ensure quality and high crop yields led to the contamination of the environment. Among popular plant pro- tection products, β-triketones, which replaced atrazine banned in several European countries (Chaabane et al. 2008; Calvayrac et al. 2012), are used as selective pre- and postemergence her- bicides for a wide range of broad-leaved and grass weeds in maize (Mitchell et al. 2001). Sulcotrione (2-[2-chloro- 4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione) and mesotrione (2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3- cyclohexanedione) (Fig. 1) released in 1993 and 2003, respec- tively, are chemically derived from leptospermone, a natural
Fig. 1 Molecular structure of β- triketones herbicides
phytotoxin of the Californian bottlebrush plant, Callistemon spp. (Dayan et al. 2007). The target site for triketone herbicides was identified as the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD), which is found in a wide range of eu- karyotic and prokaryotic organisms being involved in the ca- tabolism of tyrosine. In plants, this enzyme catalyzes the con- version of p-hydroxyphenyl pyruvate (HPP) to homogentisate, which is the biosynthesis precursor of plastoquinone and α- tocopherol (Schulz et al. 1993; Lee et al. 1997; Meazza et al. 2002; Rocaboy et al. 2014). HPPD inhibition causes disruption of carotenoids biosynthesis and results in foliage bleaching of treated plants. All triketones are HPPD competitive inhibitors that form a tight complex with the enzyme by a mechanism of coordination to the active-site iron atom. Maize is known to exhibit natural tolerance to triketones using a nontarget mech- anism based on rapid metabolic detoxification, involving a ring hydroxylation by the cytochrome P450 monooxygenase (P450), associated to a less sensitive form of the maize HPPD to triketones (Mitchell et al. 2001; Siehl et al. 2014).
Several works have detailed the environmental profile of triketone herbicides. Half-life (DT50) in field varies between 16 and 122 days for sulcotrione and 6 and 34 days for mesotrione, depending on pH and organic matter content of soil (Rouchaud et al. 1998; Dyson et al. 2002; Chaabane et al. 2005; Crouzet et al. 2010). Although these herbicides are con- sidered to have a favorable environmental and toxicological profiles (Shaner 2004), these molecules are of concern due to their potential toxicity for any living organisms harboring HPPD enzyme (Schulz et al. 1993; Wu et al. 2011; Joly et al. 2013). The toxicity of sulcotrione and mesotrione has been demonstrated on model organisms, such as Tetrahymena pyriformis and Vibrio fischeri (Microtox® bioassay). These two herbicides showed a toxicity level equal to or higher than atrazine (Bonnet et al. 2008; Joly et al. 2013). Recently, higher cytotoxicity and genotoxicity of sulcotrione mixture after
photodegradation were shown in Allium cepa test as compared to the parent molecule (Goujon et al. 2014).
Microbial degradation is considered as the major dissipa- tion process limiting the accumulation of pesticides in the environment (Anderson 1984; Walker and Welch 1991; Topp 2003; Arbeli and Fuentes 2007). For a range of pesti- cides, their repeated application was shown to lead to the selection of microbial populations able to use them as sources of nutrients and energy for their growth. This phenomenon is relying on the acquisition of degradation capacities through genetic mechanisms such as horizontal transfer, mutation, and genetic rearrangements (van der Meer et al. 1992). To date, only few papers reported the isolation and the characterization of bacterial strains able to degrade the two synthetic β- triketones. To our best knowledge, only one bacterial strain identified as Pseudomonas sp. 1OP has been found to be able to degrade sulcotrione (Calvayrac et al. 2012, 2014). Two Bacillus sp. strains isolated from agricultural soil and cloud water (Durand et al. 2006; Batisson et al. 2009) have been shown to completely and rapidly degrade mesotrione. A new mesotrione-degrading bacteria isolated from aquatic environ- ment was identified as Pantoea ananatis (Pileggi et al. 2012). Contrary to the two Bacillus sp. strains, mesotrione was not a carbon source to P. ananatis, and glucose was re- quired to start growth and degradation, suggesting a cometabolic process. Recently, Escherichia coli DH5-α was shown to possess a mechanism of adaptation to mesotrione involving glutathione-S-transferase (GST) en- zyme (Olchanheski et al. 2014). Similarly to P450, GST enzymes have been commonly characterized in plants as nontarget based mechanisms for herbicide resistance (Ma et al. 2013). Then, E. coli DH5-α mesotrione degradation could be considered as an oxidative and nonspecific stress response. Until now, nothing is known about catabolic genes involved in sulcotrione and mesotrione degradation.
In this study, we report the isolation of a sulcotrione- degrading bacterial strain from an agricultural soil. Taxonomic and genetic characterization of the bacterial isolate was performed by 16S rRNA sequencing, plasmid profiling, and cloning. The capacity of the strain to degrade aerobically a range of synthetic (mesotrione, tembotrione) and natural (leptospermone) β-triketones was estimated. Metabolic path- ways of sulcotrione and mesotrione were also investigated. The toxicity of these two herbicides and their related metabo- lites was estimated using HPPD inhibition test.
Materials and methods
Soil sampling and characteristics
Soil was collected from the surface layer (0–10 cm) of an experimental field located at the University of Perpignan, France. Soil samples were sieved (2 mm) and stored at 4 °C until use. The composition and characteristics of the soil was
13.9 % clay, 60.5 % silt, 25.6 % sand, 20 % soil humidity,
1.7 % organic matter, 0.9 % organic carbon, 0.98 g.kg−1 total nitrogen, 15.5 meq 100 g−1 cation exchange capacity (CEC), 214 % Ca2+/CEC and pH in water 8.1.
Chemicals and media
Analytical standards of sulcotrione (98.8 % purity) and 1,3- cyclohexanedione (CHD) (97.0 % purity) were purchased from Sigma-Aldrich (France). 2-Chloro-4-mesylbenzoic acid (CMBA) (95.0 % purity) was purchased from Acros Organics (France). Standards of mesotrione and tembotrione were pur- chased from Dr. Ehrenstorfer GmbH (Germany) as well as mesotrione metabolites 4-methylsulfonyl-2-nitrobenzoic acid (MNBA) and 2-amino-4-methylsulfonylbenzoic acid (AMBA). Leptospermone was kindly provided by F. E. Dayan ( USDA, University of Mississippi, USA). Acetonitrile, methanol, and dichloromethane (HPLC quality) were purchased from Sigma-Aldrich and hydrochloric acid (38.0 %) from Prolabo (France).
β-Triketones solutions were prepared in water at initial concentration of 1 g L−1 (pH 9). They were then sterilized using a 0.2-μm filter, and finally added to mineral salts medi-
um (MS) (Rousseaux et al. 2001) composed by: K2HPO4 (1.6 g L−1), KH2PO4 (0.4 g L−1), NaCl (0.1 g L−1), MgSO4 7H2O (0.2 g L−1), CaCl2 (0.02 g L−1), FeSO4, 7H2O
(5 mg L−1), H3BO3 (2 mg L−1), MnSO4, H2O (1.8 mg L−1), ZnSO4 (0.2 mg L−1), CuSO4 (0.1 mg L−1), Na2MoO4
(0.25 mg L−1), biotin (0.04 mg L−1), and thiamine (0.1 mg L−1). Each β-triketone herbicide or each metabolite
was used as sole carbon source in liquid MS medium. Yeast
extract mannitol medium (YM) containing (per liter) 2.5 g of yeast extract, 0.5 g of K2HPO4, 0.1 g of NaCl, 0.2 g of
MgSO4⋅7H2O, 0.1 g of CaCO3, and 10 g of mannitol (Vincent 1970) and TY medium composed by (per liter) 5 g
tryptone, 3 g yeast extract, and 66 mg CaCl2 were used as a rich medium. Luria–Bertani medium (LB) [tryptone (10 g L−1), yeast extract (5 g L−1), and NaCl (5 g L−1)] was
used for E. coli plating.
Enrichment, isolation, and screening of microbial strains
Enrichment of sulcotrione-degrading bacteria was performed by adding 10 g of soil in an Erlenmeyer flask containing 90 mL of sterilized MS medium supplemented with
sulcotrione at a final concentration of 35 mg L−1
(0.106 mM). The culture was incubated at 28 °C with contin-
uous shaking at 150 rpm. After five enrichments, a serial di- lution of the obtained bacterial consortium was done in MS liquid in order to reduce the culture microbial diversity. The
10−3–10−8 dilutions were plated on both MS-sulcotrione and
TY solid medium. Plates were incubated at 28 °C. Degrading
capacity of dilution cultures was monitored. When about 50 % of the initial sulcotrione concentration was degraded, a new dilution series was started until three to four different growing colonies were obtained on TY plates. Each isolated colony was inoculated in liquid MS-sulcotrione to assess their degrading capacity. Regularly, 1-mL aliquot was sampled to monitor sulcotrione disappearance in the culture medium. Noninoculated cultures were used as control. A pure strain degrading the sulcotrione was isolated, and named SR1.
One-milliliter aliquots of liquid culture were analyzed by high-performance liquid chromatography (HPLC) Jasco ap- paratus equipped with a Phenomenex Luna C18 column (150×3.0 mm; 5 μm) and a diode array detector Jasco 875- UV set. The mobile phase was a mixture of water (AW) and
acetonitrile (ACN) acidified by 0.1 % formic acid, delivered at a flow rate of 0.5 mL min−1. A gradient method was used in order to improve molecules and metabolites separation: 0–
4 min 70 % AW–30 % A-ACN, from 4 to 7 min 40 % AW– 60 % A-ACN maintained for 15 min, then back to 70 % AW– 30 % A-ACN.
Metabolites identification was accomplished using liquid chromatography–mass spectrometry (LC-MS) methodology. Primarily, Thermo Fisher Scientific LC-MS device, Accela HPLC coupled to a LCQ Fleet equipped with an electrospray ionization source and a 3D ion-trap analyzer was used. A full scan range in both positive and negative ion mode from m/z 60 to 300 in both positive was carried out, using the following gradient system: 90 %/10 % (AW/A-ACN) from 0 to 5 min, then 20 %/80 % (AW/A-ACN) maintained to 16 min. Confirmation of the nature of sulcotrione byproducts was car- ried out using an LC–high-resolution mass spectrometry
device: LC-HRMS Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher). Ions were generated using an electrospray ion source (ESI), and analysis was done in both positive and negative modes. The chromatographic system was equipped with a Luna PFP 2 (150×2.00 mm; 3 μm, Phenomenex). 0.1 % formic acid in Milli-Q water, and 0.1 % formic acid in ACN were used as mobile phase. The elution gradient were 3 % A-ACN (5 min), 3–95 % A- ACN(20 min), 95 % A-ACN (5 min), 95-3 % A-ACN
(1 min), and 3 % A-ACN (5 min). Mass spectra analysis was effectuated using Xcalibur (version 2.0, Thermo Fisher) and MetAlign (version 041011, Arjen Lommen). For LC-MS analysis, aliquots were extracted from the bacterial culture medium as described by Calvayrac et al. (2012).
Purification was performed using a semi-preparative de- vice (WATERS 1525 binary HPLC pump, Waters 248 DAD) equipped with a column ODS Uptisphere 5 μm. Mobile phase was a mixture of water and acetonitrile acidified by 0.1 % formic acid, delivered as follows: 20 % A-ACN (5 min), 95 % A-ACN until 22 min, then 20 % A-ACN until 35 min. The structure of sulcotrione metabolite was confirmed using 1H NMR (400 MHz, CDCl3).
Characterization of a sulcotrione-degrading isolate
Bacterial genomic DNA of SR1 strain was extracted by stan- dard alkaline lysis method from one pure colony and used as template for PCR extraction reactions. The amplification re- action was carried out in 50 μl reaction volume containing
1.25 U of GoTaq DNA polymerase (Promega, USA) using 50 ng of DNA as template. 16S rRNA gene was amplified using universal primers 27f (5′-AGA GTT TGA TCH TGG CTC AG-3′) and 1492r (5′-TAC GGH TAC CTT GTT ACG ACT T-3′) (Gürtler and Stanisich 1996). PCR reactions were carried out in a thermocycler (Gradient Cycler, MJ Research, USA) under the following conditions: 5 min at 94 °C, follow- ed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, and a 72 °C cycle for 15 min. The concentration of 16S rRNA amplicon was quantified using a spectrophotome- ter (Biophotometer, Eppendorf, Germany), and DNA quality was checked on 1 % agarose gel in 1× TBE buffer. The purity of the isolated bacteria was checked by 16S rRNA restriction fragment length polymorphism (RFLP). Restriction profiles were obtained by PCR products treatment using restriction enzymes AluI and RsaI (Fermentas, France). Separation of digestion products was effectuated on high-resolution 3 % agarose gel (MP Q-BIOgene, USA). DNA Molecular Weight Marker VIII (Roche Applied Science, France) was used to estimate the restriction fragments size.
16S rRNA amplicon was sequenced using the Sanger tech- nology (Beckman Coulter Genomics). Sequence was deposit- ed in GenBank under accession number KP683073. SR1 16S rRNA sequence was compared to closest sequences retrieved
from GenBank database using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/ BLAST/) of the National Center for Biotechnology Information (NCBI). For phylogenetic analysis, multiple alignments were done with ClustalX program (Thompson et al. 1997), and neighbor-joining tree was calculated with the NJPlot software (Perrière and Gouy 1996).
Degradation of β-triketones herbicides by the isolated strain SR1
Capacity of SR1 to degrade β-triketone herbicides was esti- mated in resting cell experiments. SR1 was grown in YM incubated at 28 °C under agitation at 150 rpm. Bacterial cul- ture was centrifuged at 4500g for 10 min at 4 °C. The pellet was then washed twice with MS medium and resuspended in 100 mL of MS containing herbicides at a concentration of
35 mg L−1 and an OD620 of 0.5 nm. Aliquots were regularly collected and frozen at −20 °C until analysis. Resting cell experiments were realized in triplicate. Noninoculated MS
liquid controls supplemented either in β-triketone herbicide or in degradation product were maintained under the same conditions. As a negative control, similar experiments were carried out with four other Bradyrhizobium strains (B. japonicum USDA 110, Bradyrhizobium sp. LL13, Bradyrhizobium USDA 127, and Bradyrhizobium sp. G49) never exposed to sulcotrione.
SR1 strain plasmid curing assays
In order to establish a relationship between the degradation capacity and the presence of the plasmid, a curing experiment was carried out. The isolated strain was diluted (1/100 and 1/1000) and grown in YM medium without pesticide until the end of exponential growth phase. An aliquot was taken, and plasmid profile was done using Eckhardt method as pre- viously described (Devers et al. 2007). Ten transfers (about 80 generations) were accomplished. Degradation capacity of the isolate was assessed after each transfer using resting cells ex- periment to monitor the evolution of sulcotrione-degrading ability.
SR1 strain plasmid isolation and transfer
SR1 strain was grown in YM liquid medium at 28 °C for 3 days with shaking at 150 rpm. Fifty milliliters of the grown culture was used to extract its plasmid using the Qiagen Plasmid midi kit (Qiagen, France). The plasmid was Tn5- labeled using the EZ-Tn5™
E. coli TransforMax™ EC100D™ pir+ cells according to the manufacturer’s recommendations (Epicentre, USA). Transformants harboring SR1 plasmid was cultivated
overnight in LB medium containing Kanamycine (50 mg L−1). SR1 Tn5-labeled plasmid was also introduced in electrocompetent cells of Bradyrhizobium sp. G49 by elec-
troporation. Resting cell experiments were then carried out at 37 and 28 °C, respectively, for E. coli and Bradyrhizobium sp. G49 as previously described to assess sulcotrione degradation.
HPPD activity monitoring using colorimetric assay
Aliquots, regularly collected from SR1 resting cells, incubated with 35 mg L−1 of sulcotrione or mesotrione were analyzed with the colorimetric free cells bioassay developed by
Rocaboy et al. (2014). Aliquots were firstly centrifuged (13, 500 rpm for 15 min) to remove SR1 cells from the superna- tant. Each well of the 96-well microplate was filled with 12.5 or 25 μL of supernatant, and mixed with E. coli HPPD-
induced cells and 1.2 mg mL−1 tyrosine. Triketone-positive controls were performed using 35 mg L−1 sulcotrione or mesotrione solution instead of resting cells supernatants. MS
was added for controls. All tests were performed in triplicate. After 12 h of incubation, optical density of each well was measured at 405 nm.
Isolation and characterization of sulcotrione-degrading bacteria
Enrichment culture was conducted using the soil of Perpignan that was previously exposed to sulcotrione (Chaabane et al. 2008) and shown to have a microflora able to degrade this compound with a half-life time of 8 days (Calvayrac et al. 2012). After five enrichment cycles, a mixed bacterial culture able to degrade sulcotrione was obtained (data not shown). After serial dilution of enrichment cultures and plating on both nutrient agar TY and MS agar containing sulcotrione as the sole carbon source, four pure isolates were obtained. The eval- uation of their sulcotrione-degrading capacities revealed that only one isolate named SR1 was capable of efficiently de- grade sulcotrione. The purity of SR1 strain was verified using RFLP fingerprinting of PCR-amplified 16S rDNA in both rich and MS medium (data not shown). This isolate was a Gram- negative, aerobic, motile, and rod-shaped bacterium.
SR1 16S rRNA sequence (1353 bp) showed 100 % of similarity with several Bradyrhizobium strains including
B. elkanii strain SEMIA 6399, B. japonicum strain SEMIA 6154 and Bradyrhizobium sp. CMVU02. None of these iso- lates are known to degrade pollutants such as pesticides. Besides, resting cell experiments carried out with four Bradyrhizobium strains never previously exposed to sulcotrione showed that they were unable to degrade this her- bicide (data not shown). Phylogenetic distances with other
known triketones-degrading bacteria are presented in Fig. 2. Based on phylogenetic analysis, this isolate was named Bradyrhizobium sp. SR1. One could observe that the two known sulcotrione degraders belonged to the phylum of Proteobacteria in the α- and γ-Proteobacteria classes (Bradyrhizobium sp. SR1 and Pseudomonas sp. 1OP, respec- tively), the two mesotrione degraders belonged to the γ- Proteobacteria (E. coli DH5-α and P. ananatis) as well, and the two others belonged to the Firmicutes phylum (Bacillus sp. MES11 and Bacillus sp. 3B6).
Degradation of sulcotrione by Bradyrhizobium sp. SR1
Degradation kinetics of sulcotrione was established in resting cell experiments in MS medium supplemented with 35 mg L−1 of sulcotrione. One could observe that all the
sulcotrione was completely degraded within 48 h in SR1 cul-
ture, while the concentration of sulcotrione remained stable in the control indicating the chemical stability of this molecule under our conditions (Fig. 5a, Supplementary data Fig. 1-A). Sulcotrione dissipation could be described by a first-order
kinetic (C=120e−0.075t, R2=0.913, n=3) after a lag phase es- timated to about 4 h. Half-life time was estimated to about
13 h (Supplementary data Fig. 1-A). During the degradation of sulcotrione by Bradyrhizobium sp. SR1, we searched for the two-known sulcotrione metabolites: CHD and CMBA. Under our experimental conditions, we did not detect CHD. However as previously reported for Pseudomonas sp. 1OP (Calvayrac et al. 2012), CMBA was identified using mass spectral in negative mode (ESI m/z 234) as the major metab- olite. The apparition of this by-product is concomitant with the dissipation of sulcotrione with a molar recovery close to 90 % all during the time course of the herbicide degradation (Fig. 3a). Concomitantly with the appearance of CMBA (after 4 h of incubation), we observed the formation of another me- tabolite having a retention time different from those of CMBA and sulcotrione ones. This metabolite cannot be detected any- more when the sulcotrione was fully dissipated (Supplementary data Fig. 1-A). The LC-MS analysis corre- sponding to this peak (ESI m/z 343) allowed us to propose hydroxy-sulcotrione as an intermediate metabolite of sulcotrione that finally gives CMBA (Fig. 4). In order to con- firm this hypothesis, the metabolite was purified by semipreparative chromatography from a large amount of SR1 resting cells prepared in MS-sulcotrione medium. Analysis of the metabolite in high-resolution LC-MS revealed the presence of a hydroxyl group on α-carbon of the carbonyl functional group. Additional proton NMR analysis of the pu- rified metabolite clearly confirmed the absence of the hydrox- yl group on the aromatic ring. Further resting cell experiments showed that SR1 was not able to degrade CMBA and CHD.
Resting cell experiments were also performed with higher concentrations of sulcotrione (100, 200, and 400 mg L−1).
Fig. 2 Neighbor-joining phylogenetic analysis based on the multiple alignment of 16S rRNA sequences of Bradyrhizobium sp. SR1 with sulcotrione degrading strains (marked in bold), mesotrione degrading strains (underlined), and nondegrading strains. Nonavailable 16S rRNA sequences were replaced by their closest relative. Bootstrap values higher than 900 iterations out of 1000 are marked as black circles. Accession numbers of used strains are Bradyrhizobium sp. SR1 (KP683073), Bradyrhizobium elkanii strain SEMIA 6405 (FJ025109.1), Bradyrhizobium japonicum strain USDA 110 (AF363150.1), Sinorhizobium meliloti strain SEMIA 116 (FJ025128.1), Rhizobium etli
CFN 42 (REU28916), Sphingomonas wittichii RW1 16S (NR_ 074268.1), Burkholderia pseudomallei (NR_074340.1), Ralstonia pickettii DSM 6297T (LN681565.1), Xanthomonas oryzae strain LMG 5047 (NR_026319.1), Escherichia coli DH5-α (KC768803.1), Pantoea ananatis strain CSA37 (KM091726.1), Pantoea ananatis LMG 20103 (NR_103927.1), Pseudomonas aeruginosa strain DSM 50071 (NR_ 026078.1), Pseudomonas sp. 1OP (JF303892.1), Bacillus sp. MES11(EU864320.1), Bacillus macroides (AF157696), Staphylococcus aureus strain ATCC (NR_115606.1), and Lactobacillus salivarius strain ATCC 11741(AF089108.2)
After 48 h of incubation, 66 and 48 mg L−1 of sulcotrione were degraded for an initial concentration of 100 and 200 mg L−1, respectively, corresponding to a degradation rate of 66 and 24 %. For these two concentrations, sulcotrione
biodegradation was completed after 5 days of incubation. However, at the highest concentration (400 mg L−1), SR1 strain was not able to degrade anymore sulcotrione.
Degradation of other triketone herbicides by Bradyrhizobium sp. SR1
The ability of SR1 strain to degrade other synthetic (mesotrione and tembotrione) or natural triketones (leptospermone) was investigated in resting cell experiments showing that SR1 was
only able to degrade mesotrione. However, the time required to get the full disappearance of 35 mg L−1 mesotrione (i.e., 20 days) was ten times longer than that required to dissipate
the same quantity of sulcotrione (i.e., 2 days) under the same conditions. Kinetics of first order was also observed (C = 102e−0.255t, R2=0.993, n=3) with a half-life time estimated to
be about 3 days (Supplementary data Fig. 1-B).
Chromatographic analysis clearly showed that mesotrione biotransformation resulted in the generation of MNBA and AMBA, the two known mesotrione by-products (Durand et al. 2006). MNBA (ESI m/z 244) and AMBA (ESI m/z 214) appeared after about 24 h of incubation and then was
accumulated in the medium up to 20 and 6 mg L−1, respec- tively, by the end of the experiment. The cumulative profiles
of metabolites during mesotrione transformation confirmed a quantitative transformation of mesotrione into MNBA and AMBA (Fig. 3b). Resting cell experiments showed that none of these two metabolites could be degraded by SR1.
Microbial toxicity of sulcotrione and mesotrione metabolites
In order to estimate the microbial toxicity of sulcotrione and mesotrione metabolites formed by Bradyrhizobium sp. SR1, HPPD inhibition capacity was assessed from the supernatants of resting cells using a colorimetric bioassay. The bioassay principle is that HPPD inhibitors decreased the ability of a recombinant E. coli clone to produce a melanine-like pigment resulting from tyrosine catabolism through HPP and
Fig. 3 Degradation kinetics of a sulcotrione and b mesotrione by Bradyrhizobium sp. SR1 in resting cell experiments. Cumulative formation of metabolites during sulcotrione and mesotrione degradation is represented. Standard deviations are indicated (n=3)
homogentisic acid. As expected, at T0, similar HPPD inhibi- tion capacities were observed in SR1 culture and in the non- inoculated sulcotrione control (Fig. 5a). For 4 h, the HPPD inhibition capacity of the SR1 supernatant aliquots from sulcotrione resting cells was stable averaging 18±1.30 %. Thereafter, the HPPD inhibition decreased with time being negligible after 48 h of incubation. A significant linear regres- sion (R2=0.92) between the decrease in sulcotrione and HPPD inhibition was observed (Fig. 5b). Similarly, no HPPD inhibi- tion was observed with SR1 supernatant aliquots from mesotrione resting cells after 20 days of incubation when no more mesotrione remained (data not shown).
Genetic localization of sulcotrione degradation in Bradyrhizobium sp. SR1
Attempts were done to identify the genetic localization of sulcotrione degradation in SR1. Keeping in mind that pesti- cide degradation by soil bacteria is often supported by large catabolic plasmids, this hypothesis was explored. Thus, a plas- mid profile of the strain SR1 was performed and revealed the presence of a unique plasmid. Its size was estimated to be superior to 50 kb in comparison with a plasmid of known size (Fig. 6). Curing experiments were performed by cultivating SR1 isolate on a rich medium in the absence of selection
pressure exerted by sulcotrione. After about 80 generations, the strain did neither lose its plasmid nor its capacity to de- grade sulcotrione in resting cell experiments. In addition, the plasmid pSR1, labeled with Tn5, was successfully transferred to E. coli by electroporation (Fig. 6). However, transformed
E. coli were not able to degrade sulcotrione in resting cell experiment. Furthermore, the insertion of the labeled plasmid into a nondegrading B. japonicum G49 was also assessed, but this transformation was unsuccessful.
In this study, we report the isolation of SR1, a sulcotrione- degrading strain isolated from the soil of Perpignan, which was regularly exposed to this herbicide and known to host a microflora able to transform sulcotrione to CHD and CMBA (Chaabane et al. 2008). SR1 was able to use sulcotrione as the sole source of carbon and energy for its growth. 16S rRNA phylogeny analysis indicated that SR1 strain belongs to the α- Proteobacteria class, Bradyrhizobium genus and was named Bradyrhizobium sp. SR1. Interestingly, although this genus is mainly known for N-fixing symbiosis developed with legumi- nous plants, several studies have reported the involvement of various Bradyrhizobium strains in pesticide degradation
Fig. 4 Proposed scheme for the degradation of sulcotrione by Bradyrhizobium sp. SR1
Fig. 5 a HPPD inhibitor capacity of SR1 resting cells incubated with 35 mg L−1 of sulcotrione and collected at different time (T0 to T96h) using colorimetric free cell bioassay. Control: negative control including
SR1 resting cells incubated without sulcotrione. Sulco: noninoculated
sulcotrione-positive control at a concentration of 35 mg L−1. Standard deviations are indicated (n=3). b Linear regression between sulcotrione
dissipation and HPPD inhibition
including herbicides such as 2,4-D (Kamagata et al. 1997; Huong et al. 2007), pentoxazone (Satsuma et al. 2000), sima- zine (Ozawa et al. 2004), and insecticide like methoxychlor (Satsuma et al. 2013).
Our work has demonstrated that SR1 was not only able to degrade sulcotrione but also mesotrione another herbicide be- longing to the β-triketone family. To our best knowledge, this is the first report describing the isolation and the characteriza- tion of a bacterial strain able to degrade these two herbicides from triketone class. Up to now, only one bacterial isolate,
Fig. 6 Plasmid profiles from (1 and 4) Sphingomonas sp. SH., used as molecular weight marker, (2) Bradyrhizobium sp. SR1, and (3) E. coli pSR1::EZTn5. The size of the plasmid is indicated in kb
Pseudomonas sp. 1OP, also isolated from the soil of Perpignan, was shown to degrade sulcotrione and to use it as sole carbon and energy sources under aerobic conditions (Calvayrac et al. 2012). One could observe that, as previously shown for Pseudomonas sp. 1OP by these authors, sulcotrione degradation with SR1 was leading to equimolar formation of CMBA metabolite. However, during the time course of sulcotrione degradation, we observed the transient accumula- tion of hydroxy-sulcotrione. This compound was previously detected as a metabolite resulting from abiotic degradation (i.e., photocatalysis treatment) of sulcotrione (Jovic et al. 2013). This observation led us to propose that SR1 degrada- tion pathway is organized in two-step process: sulcotrione being first transformed to hydroxy-sulcotrione through oxida- tion, which is then transformed to CMBA and consecutively accumulated as a dead-end metabolite (Fig. 4). This is in ac- cordance with Chaabane et al. (2008) reporting the accumu- lation of CMBA in the soil of Perpignan as a result of micro- bial transformation of sulcotrione. However, CHD the other degradation product of sulcotrione observed in the soil of Perpignan (Chaabane et al. 2008) was not detected in the culture media of strain SR1 neither by HPLC/UV nor by HPLC/mass spectrometry. Similar observation was done for Pseudomonas sp. 1OP (Calvayrac et al. 2012). As previously suggested, this discrepancy might be caused by abiotic trans- formation of CHD or to β-diketone oxidation catalyzed by enzymatic C–C bound breaking (Calvayrac et al. 2012). CMBA and CHD degrading tests have been carried out in resting cell experiments showing that SR1 was unable to de- grade these two metabolites. SR1 was not able to degrade
CHD probably because degradation pathway of this metabo- lite is inducible.
To date, several mesotrione-degrading bacteria have been isolated from different environments (i.e., water, soil, and clouds), but only a limited number of strains, all belonging to Bacillus genus, used this herbicide as a carbon source (Durand et al. 2006; Batisson et al. 2009; Pileggi et al. 2012). For Bacillus sp. 3B6, the major pathway of mesotrione degradation was initiated by a reduction of the nitro group of mesotrione resulting in the formation of several transforma- tion products ( M2, M 3, and M 4 as described in Supplementary data Fig. 2) leading to the formation of AMBA (Durand et al. 2010). A minor pathway leading almost directly to AMBA by an oxidative cleavage of mesotrione followed by a reduction of the nitro group was also observed. Although in the literature MNBA and AMBA have been de- scribed as the two main metabolites of mesotrione (Alferness and Wiebe 2002), MNBA was barely detected in Bacillus sp. 3B6 either because it was formed in an amount below its detection limit or because it was transformed since its forma- tion in the medium (Durand et al. 2010). Similar mesotrione- degrading pathway was observed in Bacillus sp. MES11 (Batisson et al. 2009). According to our observations, the mesotrione degradation pathway of Bradyrhizobium sp. SR1 differed from that of Bacilli degraders, since (i) the three me- tabolites (M2, M3, and M4) resulting from the reduction of the nitro group of mesotrione were not detected during mesotrione degradation and (ii) both MNBA and AMBAwere detected in the culture media. Additionally, resting cell exper- iments showed, that contrarily to Bacilli mesotrione- degrading strains, SR1 did not transform MNBA to AMBA. To explain this discrepancy, one could hypothesize, on the one hand, that SR1 strain harbors two distinct mesotrione- degradation pathways resulting in the formation of both MNBA and AMBA. On the other hand, mesotrione could be transformed into MNBA following the minor mesotrione- degradation pathway previously described in Bacilli strains, which is then transformed to AMBA (Supplementary data Fig. 2). Further studies are necessary to determine the pathway of degradation of mesotrione in strain SR1.
Although SR1 was able to degrade both sulcotrione and mesotrione, sulcotrione rate of degradation was ten times higher than that of mesotrione. This reveals that the selection driven by sulcotrione exposure led to the selection of SR1 harboring degrading enzymes highly specific for this herbi- cide. Among the other triketones tested, SR1 was only able to degrade mesotrione, which is having a chemical structure di- verging from sulcotrione only by one substituent, –NO2 in- stead of –Cl in position 2 of the aromatic group. Concerning tembotrione, the presence of 2,2,2-trifluoroethoxymethyl group in position 3 on the aromatic ring may contribute to the steric hindrance of the benzoyl 3-substituent on the enzy- matic binding (Calvayrac et al. 2014) compromising its
interaction with the catalytic site of SR1-degrading enzymes. On the contrary, the absence of the aromatic ring in the leptospermone may dramatically reduce its affinity for SR1 catabolic enzymes. A few number of pesticide-degrading strains are known to metabolize molecules belonging to the same family depending on the affinity of the degrading- enzyme to the pesticide (Karns et al. 1986; Strong et al. 2002; Karpouzas et al. 2005). As an example, biochemical studies carried out with Mycobacterium brisbanense strain JK1, a diuron-degrading bacterial isolate, showed that PuhA displayed the highest affinity for diuron, while this enzyme had lower affinities for other phenylureas such as chlorotoluron, isoproturon, linuron, and monolinuron (Khurana et al. 2009). It was suggested that the substrate spec- ificity of PuhA was predominantly determined by steric fac- tors, which could be the case for the enzymatic triketone deg- radation system of SR1.
The SR1 strain was not able to degrade sulcotrione at high concentrations. This might be explained by a possible dose- depending toxic effect of sulcotrione most likely resulting from the inhibition of a potential HPPD enzyme implicated in tyrosine metabolism of SR1. The presence of an hppd gene in B. japonicum USDA110 phylogenetically related to SR1 (Fig. 2) supports this hypothesis. Furthermore, the toxicity of triketone molecules and their related metabolites was estimat- ed by monitoring HPPD inhibition. As expected, the obtained results confirmed that sulcotrione and mesotrione toxicity due to HPPD inhibition was mainly linked to parent molecule and revealed that the main metabolites displayed weak HPPD in- hibitory capacities. These last observations are in contradic- tion with a previous study showing that CMBA metabolite displayed a higher toxicity than sulcotrione using Microtox assays with V. fischeri (Bonnet et al. 2008). Therefore, one could conclude that SR1-degrading activity is, on the one hand, beneficial to soil living organisms having sulcotrione- sensitive HPPD enzymes by removing the exposure to sulcotrione but, on the other hand, deleterious to soil organ- isms sensitive to sulcotrione metabolites, such as CMBA, which is accumulated during sulcotrione transformation. This ambivalent contribution of pesticide degraders was al- ready reported for a range of pesticides that are transformed to toxic intermediates (i.e., phenylurea transformation to ani- line derivatives, for review, see Hussain et al. 2015). For en- vironmental safety, the ideal situation would be to have a synergetic cooperation between different microbial popula- tions able to completely mineralize sulcotrione in soil.
Genes coding for degrading enzymes involved in different pesticide metabolisms, such as 2,4-D, isoproturon, and atra- zine, are well known to form catabolic islands on plasmids spread all over the degrading community by horizontal gene transfer (Assinder and Williams 1990; Udikovic-Kolic et al. 2012; Gu et al. 2013). Recently, Calvayrac et al. (2014) showed that genetic system responsible for sulcotrione
degradation by Pseudomonas sp. 1OP was also plasmid borne. Several attempts were done in this work to determine if the degrading genes were localized on the large plasmid harbored by SR1. We showed that SR1 plasmid was not elim- inated by curing experiment carried out on rich medium and that sulcotrione-degrading ability was maintained without pesticide selection pressure. The stability of this plasmid might be explained by the presence of essential genes for SR1 survival and growth. This is in agreement with previous studies showing that plasmids cannot be cured in rhizobia mainly because of the presence of genes essential for free- living growth (Finan et al. 1986). Although pSR1::Tn5 was successfully transferred to E. coli, the transconjugant was not able to degrade sulcotrione. Based on this result, one could hypothesize that either catabolic genes are not plasmid borne or, for some reasons, genes located on SR1 plasmid are not expressed in the transconjugant.
We report the isolation and characterization of Bradyrhizobium sp. SR1 from an agricultural soil, which is able to biotransform two triketone herbicides. The dissipation of sulcotrione and mesotrione led to the accumulation of different known by-prod- ucts, CMBA and MNBA/AMBA. Interestingly, hydroxy- sulcotrione was identified as a new metabolite, and a metabolic pathway was proposed. Further studies are required to identify and describe genes coding for enzymes involved in the trans- formation of these two triketones.
Acknowledgments This work was supported by the French BAgence National de la Recherche^ under TRICETOX project, number ANR-13- CESA-0002. Leptospermone was kindly provided by Franck E. Dayan
(Natural Products Utilization Research Unit, USDA). The authors would like to thank Nathalie Bontemps (LCBE-CRIOBE-USR 3278 CNRS EPHE, UPVD) for sulcotrione metabolite analysis. The authors declare that they have no conflict of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Compliance with ethical standards All authors agreed to be listed and have approved of the manuscript, its content, and its submission to Environmental Science and Pollution Research. It has not been submitted or published elsewhere, whether partly or fully. All authors are in agree- ment with the ethical rules of ESPR.
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