A novel amperometric biosensor for ß-triketone herbicides based on hydroxyphenylpyruvate dioxygenase inhibition: A case study for sulcotrione.

An amperometric biosensor was designed for the determination of sulcotrione, a ß-triketone herbicide, based on inhibition of hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme allowing the oxidation of hydroxyphenylpyruvate (HPP) in homogentisic acid (HGA). HPPD was produced by cloning the hppd gene from Arabidopsis thaliana in E. coli, followed by overexpression and purification by nickel-histidine affinity. The electrochemical detection of HPPD activity was based on the electrochemical oxidation of HGA at +0.1 V vs. Ag/AgCl, using a poly(3,4-ethylenedioxythiophene) polystyrene sulfonate-modified screen-printed electrode. Assays were performed at 25°C in 0.1 M phosphate buffer pH 8 containing 0.1M KCl. The purified HPPD was shown to display a maximum velocity of 0.51 µM(HGA) min(-1), and an apparent K(M) of 22.6 µM for HPP. HPPD inhibition assays in presence of sulcotrione confirmed a competitive inhibition of HPPD, the calculated inhibition constant K(I) was 1.11.10(-8) M. The dynamic range for sulcotrione extended from 5.10(-10) M to 5.10(-6) M and the limit of detection (LOD), estimated as the concentration inducing 20% of inhibition, was 1.4.10(-10) M.

Isolation and characterization of Bradyrhizobium sp. SR1 degrading two ß-triketone herbicides.

In this study, a bacterial strain able to use sulcotrione, a ß-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 ß-triketone degrading capacity, 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 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 ß-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 ß-triketones.

Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of ß-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 µM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.