Franck Clémence Thiour-Mauprivez

Emmanuel Dayan

Hugo Terol

Marion Devers

Christophe Calvayrac

Fabrice Martin-Laurent

Lise Barthelmebs
email: barthelm@univ-perp.fr

Thiour-Mauprivez Clémence

Dayan Franck Emmanuel

Terol Hugo

Devers- Lamrani Marion

Calvayrac Christophe

Martin-Laurent Fabrice

BARTHELMEBS Lise

Maria Esperanza

Martinez Romero

R Etli Cfn42

Fergual O'gara

P Fluorescens F113

Sylvie Mazurier

P Fluorescens Ctr1015

Assessing the effects of β-triketone herbicides on HPPD from environmental bacteria using a combination of in silico and microbiological approaches

Keywords: pyomelanin, 4-hydroxyphenylpyruvate dioxygenase, soil bacteria, β-triketone herbicides, tembotrione, molecular docking

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Herbicides account for 60% of the volume of all plant protection products applied worldwide [START_REF] Dayan | Current Status and Future Prospects in Herbicide Discovery[END_REF], and for 30% in the European Union Member States (EU Eurostats). Following atrazine ban in Europe in 2003, β-triketones herbicides have been commonly used as pre-and post-emergent selective compounds and are among the top five most used herbicides on maize crops. β-triketones herbicides are synthetized based on the structure of leptospermone, a natural phytotoxin produced by the Californian bottlebrush plant, Callistemon citrinus [START_REF] Beaudegnies | Herbicidal 4-hydroxyphenylpyruvate dioxygenase inhibitors-A review of the triketone chemistry story from a Syngenta perspective[END_REF]. Sulcotrione (2-(2-chloro-4-methylsulfonylbenzoyl)cyclohexane-1,3dione) was the first β-triketone herbicide commercialized [START_REF] Schulz | SC-0051, a 2-benzoyl-cyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase[END_REF], followed by mesotrione (2-[4-(methylsulfonyl)-2-nitrobenzoyl]cyclohexane-1,3-dione) and, more recently, by tembotrione (2-[2chloro-4-methylsulfonyl-3-(2,2,2-trifluoroethoxymethyl)benzoyl]cyclohexane-1,3-dione) [START_REF] Mitchell | Mesotrione: a new selective herbicide for use in maize[END_REF] (Table 1).

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1. 13.11.27) is the biochemical target of βtriketones herbicides. This enzyme is a non-heme iron enzyme involved in the second reaction of the tyrosine catabolism pathway by converting 4-hydroxyphenylpyruvate (HPP) in homogentisate (HGA) via oxidative decarboxylation, aromatic ring hydroxylation and carboxymethyl group rearrangement [START_REF] Moran | 4-Hydroxyphenylpyruvate dioxygenase[END_REF].

HGA is a key precursor of α-tocopherol and plastoquinone, that acts as an essential cofactor for phytoene desaturase, allowing plants to produce carotenoids required to protect them from light and free radicals [START_REF] Norris | Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation[END_REF].

HPPD is ubiquitous in almost all living organisms [START_REF] Santucci | 4-Hydroxyphenylpyruvate Dioxygenase and Its Inhibition in Plants and Animals: Small Molecules as Herbicides and Agents for the Treatment of Human Inherited Diseases[END_REF]. In non-photosynthetic organisms, including humans, HPPD is also involved in tyrosine degradation, leading to HGA synthesis, the latter is involved in the synthesis pathway of fumarate, one of the intermediates of the Krebs cycle, and acetoacetate, involved in lipid biosynthesis [START_REF] Dixon | Enzymes of tyrosine catabolism in Arabidopsis thaliana[END_REF].

In some microorganisms, HPPD activity is linked to pyomelanin production, a pigment accumulating following oxidation and polymerization of HGA. This brown pigment is easily observable on culture media supplemented with L-tyrosine, and its production has been described in various microbial species such as fungi and yeasts [START_REF] Schmaler-Ripcke | Production of pyomelanin, a second type of melanin, via the tyrosine degradation pathway in Aspergillus fumigatus[END_REF][START_REF] Vasanthakumar | Pyomelanin production in Penicillium chrysogenum is stimulated by L-tyrosine[END_REF][START_REF] Almeida-Paes | L-tyrosine induces the production of a pyomelanin-like pigment by the parasitic yeast-form of Histoplasma capsulatum[END_REF][START_REF] Perez-Cuesta | Melanin and pyomelanin in Aspergillus fumigatus: from its genetics to host interaction[END_REF] and in numerous bacterial species such as Legionella pneumophila, Shewanella oneidensis, Streptomyces avermitilis, Burkholderia cenocepacia, Bacillus thuringiensis, Alteromonas stellipolaris, Rubrivivax benzoatilyticus, Klebsiella pneumoniae, Alacaligenes faecalis, Enterobacter sp. and Vibrio angillarum [START_REF] Steinert | The Lly protein is essential for phydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila[END_REF][START_REF] Brownlee | Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide[END_REF][START_REF] Keith | Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate[END_REF][START_REF] Turick | The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1[END_REF][START_REF] Zhu | Isolation and identification of a gene encoding 4hydroxyphenylpyruvate dioxygenase from the red-brown pigment-producing bacterium Alteromonas stellipolaris LMG 21856[END_REF][START_REF] Singh | Isolation of pyomelanin from bacteria and evidences showing its synthesis by 4-hydroxyphenylpyruvate dioxygenase enzyme encoded by hppD gene[END_REF][START_REF] Batallones | Disruption of hmgA by DNA Duplication is Responsible for Hyperpigmentation in a Vibrio anguillarum Strain[END_REF][START_REF] Tan | A single point mutation in hmgA leads to melanin accumulation in Bacillus thuringiensis BMB181[END_REF][START_REF] Mekala | Pyomelanin production: Insights into the incomplete aerobic l-phenylalanine catabolism of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2[END_REF]. Some of these studies report either a protective role of pyomelanin against environmental stresses, such as reactive oxygen species for the soil-borne plant pathogen Ralstonia solanacearum [START_REF] Ahmad | Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum[END_REF], light for L. pneumophila [START_REF] Steinert | The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis[END_REF], ultraviolet irradiation for the marine bacteria Vibrio splendidus [START_REF] Liang | Dual functions of a 4-hydroxyphenylpyruvate dioxygenase for Vibrio splendidus survival and infection[END_REF], or a modification in virulence for pathogenic bacteria [START_REF] Batallones | Disruption of hmgA by DNA Duplication is Responsible for Hyperpigmentation in a Vibrio anguillarum Strain[END_REF],

highlighting the importance of deciphering HPPD function in microbes.

Nonetheless, microbial HPPD structure and β-triketone herbicide binding mode are only described in two microorganisms : Pseudomonas fluorescens F113 (also called Pseudomonas kilonensis F113 [START_REF] Vacheron | Differential Contribution of Plant-Beneficial Functions from Pseudomonas kilonensis F113 to Root System Architecture Alterations in Arabidopsis thaliana and Zea mays[END_REF]) and Streptomyces avermitilis [START_REF] Brownlee | Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide[END_REF][START_REF] Serre | Crystal structure of Pseudomonas fluorescens 4hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway[END_REF]. Studies on binding modes of -triketones were mainly performed on HPPD plant models such as Arabidopsis thaliana [START_REF] Moran | 4-Hydroxyphenylpyruvate dioxygenase[END_REF][START_REF] Meazza | The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase[END_REF][START_REF] Dayan | Beta-triketone inhibitors of plant phydroxyphenylpyruvate dioxygenase: modeling and comparative molecular field analysis of their interactions[END_REF]. Most inhibition studies are performed on recombinant plants or bacterial HPPD cloned and expressed in Escherichia coli strains [START_REF] Rocaboy-Faquet | Novel bacterial bioassay for a highthroughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors[END_REF][START_REF] Neuckermans | A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors[END_REF][START_REF] Liu | Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19[END_REF]. Only a few studies take an interest in deciphering the potential inhibition of soil microbial HPPD by βtriketone herbicides under agronomical conditions [START_REF] Thiour-Mauprivez | Assessing the effects of β-triketone herbicides on the soil bacterial and hppd communities: a lab-to-field experiment[END_REF]. Also, the effect of β-triketone herbicides on microbial pigmentation production has been tested only with few pure strains and always using high herbicide concentration (from 12 to 200 the recommended field dose (RfD)) [START_REF] Turick | The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1[END_REF][START_REF] Mekala | Pyomelanin production: Insights into the incomplete aerobic l-phenylalanine catabolism of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2[END_REF][START_REF] Almeida-Paes | Biosynthesis and Functions of a Melanoid Pigment Produced by Species of the Sporothrix Complex in the Presence of l-Tyrosine[END_REF].

As the use of β-triketone herbicides on crops continues to expand, it is important to assess their effect on "non-target organisms" [34], and especially on the HPPD activity of soil microbiota. We could wonder if the protective role of pyomelanin production against environmental stresses observed in some bacterial species, could be modified by the application of β-triketone herbicides on crops. Such an effect could affect the balance of soil microbiota and alter, for example, microbial populations playing a key role in plant growth, such as Plant Growth Promoting Rhizobacteria (PGPR), and consequently could have an impact on soil ecosystem services.

The aim of this study is to decipher the possible impacts of three triketone molecules on the HPPD enzymatic activities of different bacterial strains belonging to various genera. Six environmental bacterial strains were selected and tested for their pyomelanin production. Strains were exposed to 1 or 10 RfD of sulcotrione. Three strains, selected according to their different sensitivities to sulcotrione, were then exposed, in a whole-cell assay, to increasing doses of three β-triketone herbicides, namely sulcotrione, mesotrione and tembotrione. As the calculated EC50 (half-maximum Effective Concentration) varied depending the strain and the molecule considered, in silico molecular docking was performed to document the binding mode of β-triketones with bacterial HPPD and to explain, at a molecular level, the differences observed in the whole-cell assay when working at a population level.

Material and methods

Bacterial strains and culture conditions B. cereus ATCC14579, P. fluorescens F113, P. fluorescens CTR1015 and P. fluorescens NEM419 were cultured in a Lysogenic Broth (LB: 10 g/l tryptone; 5 g/l yeast extract; 10 g/l NaCl). S. oneidensis MR-1 was grown in a Tryptic Soy Broth (TSB: 17 g/l tryptone; 3 g/l soy; 5 g/l NaCl; 2.5 g/l K2HPO4; 2.5 g/l glucose) and R. etli CFN42 in Tryptone Yeast extract media supplemented with CaCl2 (TY+CaCl2: 5 g/l tryptone; 3 g/l yeast extract; 0.9 g/l CaCl22H2O; pH=6.8).

Pyomelanin production

In order to test their ability to produce pyomelanin, each of the six bacterial strains were grown in their appropriate culture medium supplemented with 0.9 g/l of tyrosine at 24 °C with constant shaking at 120 rpm. Samples of each culture were collected at T0, 48 h, 72 h, and a last point between 6 to 10 days depending of the strain, and centrifugated at 5,000 rpm for 10 min. The absorbance spectra of cell-free supernatant were recorded in a UV-Vis spectrophotometer in the range of 250 to 600 nm, as soluble pyomelanin can be quantified at different wavelengths, ranging from 400 to 430 nm [START_REF] Singh | Isolation of pyomelanin from bacteria and evidences showing its synthesis by 4-hydroxyphenylpyruvate dioxygenase enzyme encoded by hppD gene[END_REF][START_REF] Mekala | Pyomelanin production: Insights into the incomplete aerobic l-phenylalanine catabolism of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2[END_REF][START_REF] Ahmad | Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum[END_REF][START_REF] Rocaboy-Faquet | Novel bacterial bioassay for a highthroughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors[END_REF][START_REF] Turick | Melanin Production and Use as a Soluble Electron Shuttle for Fe(III) Oxide Reduction and as a Terminal Electron Acceptor by Shewanella algae BrY[END_REF].

The E. coli BL21 strain, known as a non-producing pyomelanin strain, was added to this experiment as negative control, and the E. coli BL21 HPPD, modified to produce pyomelanin, as the positive control [START_REF] Rocaboy-Faquet | Novel bacterial bioassay for a highthroughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors[END_REF].

Bacterial resting cells preparation

Resting cells were prepared for each of the six strains in Phosphate Buffer (PB) in order to work at constant biomass, in a unique solution, whatever the strain. Each bacterial strain was grown overnight at 28°C and 110 rpm in its appropriate culture medium. Overnight cultures were diluted and incubated in a new fresh appropriate medium during three to eight-hour according to its growth capacity, to reach the exponential phase of growth. Then, cells were harvested by centrifugation for 5 min at 5,000 rpm, washed twice in 20 mM PB, and then resuspended in a volume of PB calculated to have an OD600 of 3.

In this way, biomass was similar in all the resting cells.

Colorimetric method for HPPD activity monitoring using whole-cell assay in a 96-well multiplate format To assess the HPPD activity of the six selected strains, a colorimetric test in a 96-well multiplate format was developed. 96-well-microplates were all filled with 50 µL of resting cells, and completed with 125 µL of L-Tyrosine 1.8 g/l (pH 9), and with 75 µL of herbicide solutions diluted in PB for the assays (A) or with 75 µL of PB for the positive control (PC). For the negative control (NC), only 200 µL of PB were added to the resting cells. Herbicide concentrations ranged from 0.05 to 15 times of the RfD for each molecule tested; corresponding to: 0.23 to 67.5 µM for sulcotrione; 0.22 to 66 µM for mesotrione and 0.17 to 52.5 µM for tembotrione. All herbicides solutions were prepared in PB. All controls and assays were performed in triplicate. Microplates were covered with a Breathseal sealer (Greiner Bio-One TM , Germany) and incubated at 28°C and 110 rpm. After 48 hours of incubation, OD430, and OD600

were measured using an Epoch 2 Microplate Spectrophotometer (Greiner Bio-One TM , Germany).

Pyomelanin detection was measured at OD430 rather than OD400 because of a better signal observed at this wavelenght due to the resting cell mode done in PB. PB solely had the same absorbance as empty wells and was not included to our controls. In the second experiment, microplates were first centrifuged at 6000 rpm for 6 minutes and OD430 of the supernatant was measured.

For each triplicate, normalized OD430 (OD430N) was calculated according to:

OD430N = OD430A (or OD430PC) -OD430NC
where OD430A is the mean OD430 measured at various concentrations of herbicide, OD430PC is the mean OD430 measured at 0 µM of herbicide (i.e. PC) and OD430NC is the mean OD430 measured for the NC.

The enzymatic activity percentage, Act. %, was calculated according to the equation:

Act. %=( OD430A OD430PC
) × 100

Dose-response curves

For each herbicide tested, EC50 was calculated using the "drc" package in R [START_REF] Ritz | Dose-Response Analysis Using R[END_REF]. A three-parameter log-logistic function with lower limit 0 was applied to our datasets, corresponding to this equation:

𝑓(𝑥) = 0 + 𝑑 -0 1 + exp (𝑏(log(𝑥) -log(𝑒))
where b, d and e are estimated by the model with a p<0.05.

In silico analysis

Protein sequences were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with WP_000810922.1 corresponding to the accession number of B. cereus ATCC14579 HPPD sequence and WP_011072057.1, WP_011425038.1 and WP_014347821.1 corresponding to the HPPD sequences of S. oneidensis MR-1, R. etli CFN42 and P. fluorescens F113 respectfully. The protein sequences of both P. fluorescens CTR1015 and P. fluorescens NEM419 are not available yet. Protein sequences of L. pneumophila (WP_011216180.1), P. aeruginosa (PWU35210.1), A. salmonicida (WP_059112190.1), V. parahaemolyticus (WP_057619320.1) and V. anguillarum (QEH62709.1) were also added to the analysis. Sequences were aligned using ClustalW [START_REF] Thompson | Multiple sequence alignment using ClustalW and ClustalX[END_REF].

Homology modelling

Homology models of each of the HPPDs were obtained using the multiple alignment function of Modeller version 9.22 [START_REF] Webb | Comparative Protein Structure Modeling Using MODELLER[END_REF][START_REF] Webb | Protein Structure Modeling with MODELLER[END_REF], including the crystal structures of HPPD from Streptomyces avermitilis and from Arabidopsis thaliana (1t47 and 5yy6, respectively) [START_REF] Brownlee | Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide[END_REF][START_REF] Yang | Structural basis for herbicidal inhibitor selectivity revealed by comparison of crystal structures of plant and mammalian 4-hydroxyphenylpyruvate dioxygenases[END_REF]. The crystal structure of A. thaliana was included because it incorporates the conformational change known to occur upon binding of the inhibitors. Preliminary models with the lowest DOPE scores were selected for further refinement using GROMACS (version 2018.3) [START_REF] Hess | GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation[END_REF][START_REF] Abraham | GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers[END_REF]. The structures were relaxed to correct for steric clashes or inappropriate geometries prior to molecular dynamics simulation. The final topology of the homology models were evaluated using MolProbity [START_REF] Davis | MolProbity: all-atom contacts and structure validation for proteins and nucleic acids[END_REF][START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF].

Docking of herbicides

The structures of mesotrione and tembotrione were downloaded as sdf files from PubChem (pubchem.ncbi.nlm.nih.gov), edited with Spartan18 (Wavefunction Inc., Irvine CA 92612) to include delocalized -1 charges over two of the three keto groups, and saved as saved as pdb files. The structures were converted to Autodock compatible pdbqt format using AutoDockTools (version 1.5.6). These herbicides were docked to the catalytic domain of HPPD using Autodock4 [START_REF] Morris | AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility[END_REF][START_REF] Forli | Computational protein-ligand docking and virtual drug screening with the AutoDock suite[END_REF], using the iron metal dication (with a +0.6 partial charge) as an anchor [START_REF] Chen | MolProbity: all-atom structure validation for macromolecular crystallography[END_REF] and the following conserved residues (T164, S202, P215, N217, Q240, L295, F312, F321, L341, F333, N337, F338). The binding energy of the herbicides on the bacterial HPPD models were obtained from the dock.dlg file. Proteins and ligand interactions were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) [START_REF] Delano Wl Pymol | DeLano Sceintifics[END_REF].

Results and discussion

Pyomelanin production in the six bacterial strains

In order to study the potential effect of β-triketones on microbial HPPD activity, six environmental bacterial strains were selected: Pseudomonas fluorescens F113, as the HPPD study model [START_REF] Serre | Crystal structure of Pseudomonas fluorescens 4hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway[END_REF], two P.

fluorescens strains CTR1015 and NEM419 as PGPR bacteria models isolated from soil [START_REF] Robin | Diversity of root-associated fluorescent pseudomonads as affected by ferritin overexpression in tobacco[END_REF][START_REF] Delorme | Pseudomonas lini sp. nov., a novel species from bulk and rhizospheric soils[END_REF],

Rhizobium etli strain CFN42 as a symbiotic bacteria model [START_REF] Piñero | Tyrosinase from Rhizobium etli is involved in nodulation efficiency and symbiosis-associated stress resistance[END_REF], the well-studied pyomelanin producer Shewanella oneidensis MR-1 [START_REF] Ahmad | Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum[END_REF] and Bacillus cereus ATCC14579 the only Gram-positive bacterium of this study [START_REF] Tian | Complementary screening, identification and application of a novel class II 5-enopyruvylshikimate-3-phosphate synthase from Bacillus cereus[END_REF].

A first experiment was performed to validate that these six bacterial strains produce pyomelanin. As previously described, soluble pyomelanin can be quantified measuring absorbance at different wavelengths, ranging from 400 to 430 nm [START_REF] Turick | The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1[END_REF][START_REF] Singh | Isolation of pyomelanin from bacteria and evidences showing its synthesis by 4-hydroxyphenylpyruvate dioxygenase enzyme encoded by hppD gene[END_REF][START_REF] Mekala | Pyomelanin production: Insights into the incomplete aerobic l-phenylalanine catabolism of a photosynthetic bacterium, Rubrivivax benzoatilyticus JA2[END_REF][START_REF] Ahmad | Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum[END_REF][START_REF] Rocaboy-Faquet | Novel bacterial bioassay for a highthroughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors[END_REF]. Spectrophotometric analysis of cell-free supernatants of the six strains showed a significant increase of OD400 between the different sampling time, similar to what observed for P. fluorescens F113 (Supplementary Information 1a). Final OD400 were reported for the six tested strains, and for the two controls, validating the pyomelanin production for the studied bacterial strains (Supplementary Information 1b).

Development of a multiplate colorimetric whole-cell assay for HPPD activity analysis

To assess the HPPD activity of the six selected strains, a colorimetric test in a 96-well multiplate format was developed. Resting cells were prepared for each of the six strains in PB and added to the assay in order to work at constant biomass, in a unique solution whatever the strain. Then, OD430, characterizing the melanin-like pigment, and thus, reflecting the HPPD activity [START_REF] Singh | Isolation of pyomelanin from bacteria and evidences showing its synthesis by 4-hydroxyphenylpyruvate dioxygenase enzyme encoded by hppD gene[END_REF][START_REF] Rocaboy-Faquet | Novel bacterial bioassay for a highthroughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors[END_REF] was, for each strain, directly measured in each well containing bacterial resting cells, PB and tyrosine. P. fluorescens F113 HPPD expressed the highest production of pyomelanin pigment with an OD430N of 1.43 ± 0.11, followed by P. fluorescens CTR1015 and NEM419 with OD430N of 0.86 ± 0.01 and 0.74 ± 0.02 respectively, and then S. oneidensis MR-1, B. cereus ATCC14579 and R. etli CFN42 with OD430N of 0.70 ± 0.12; 0.66 ± 0.07 and 0.55 ± 0.03 respectively (Supplementary Information 2). These results were similar to those obtained in the first experiment, validating the miniaturization of the assay, and the pigmentation measurement in PB.

Studing the effect of β-triketones on HPPD activity using the colorimetric whole-cell assay

The resting cells of the six bacterial strains of our study were exposed or not to 1RfD or 10RfD of sulcotrione. The HPPD enzymatic activity percentage (Act. %) was calculated for each strain and is depicted in Figure 1. In B. cereus ATCC14579, S. oneidensis MR-1 and P. fluorescens CTR1015, HPPD activity clearly decreased with increasing doses of sulcotrione, with a decrease of, at least, 31 % at 1RfD, and of, at least, 74 % at 10RfD. In R. etli CFN42, HPPD activity decreased of 15% at 1RfD and 12% at 10RfD. In Pseudomonas NEM 419 enzymatic activity remained unchanged at 1RfD but decreased of 50% at 10RfD. On the contrary, HPPD activity of P. fluorescens F113 slightly decreased in response to sulcotrione exposure but not significantly compared to the control.

There is indeed a strain-dependant response to sulcotrione, even within strains belonging to the same genus. To characterize more accurately the inhibition of bacterial HPPD activity in response to sulcotrione, two bacterial strains were exposed to increasing doses of this herbicide, more finely between 0 and 10RfD. P. fluorescens F113, in which the HPPD is well described and which is known to have an ecological role in rhizosphere, was considered in these experiments [START_REF] Vacheron | Differential Contribution of Plant-Beneficial Functions from Pseudomonas kilonensis F113 to Root System Architecture Alterations in Arabidopsis thaliana and Zea mays[END_REF][START_REF] Serre | Crystal structure of Pseudomonas fluorescens 4hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway[END_REF]. As P. fluorescens F113 HPPD activity does not seem to be affected by sulcotrione, we also exposed S. oneidensis MR-1 to this herbicide, a strain in which the HPPD activity was well-documented [START_REF] Turick | The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1[END_REF]. The obtained results confirmed our previous observations: P. fluorescens F113 OD430N appeared constant even with 10RfD of sulcotrione (Figure 2.A) whereas the one of S. oneidensis MR-1 was drastically decreasing proportionally to the RfD of sulcotrione, losing around 50% of HPPD activity from 2RfD of sulcotrione (Figure 2.B). This first assay informed us about the existence of tolerant HPPD activities, such as the one of P. fluorescens F113, and sensitive one, such as the one of S. oneidensis MR-1. This phenomenon has already been reported for bacterial strains carrying acetohydroxyacid synthase (AHAS), the target enzyme of the sulfonylurea herbicides (SN). Enterobacteriaceae, carrying AHAS isoform 1 are tolerant to SN, whereas other bacteria carrying isoform 2 or 3 are sensitive to those herbicides [START_REF] Thiour-Mauprivez | Assessing the effects of β-triketone herbicides on the soil bacterial and hppd communities: a lab-to-field experiment[END_REF][START_REF] Petric | Nicosulfuron application in agricultural soils drives the selection towards NS-tolerant microorganisms harboring various levels of sensitivity to nicosulfuron[END_REF]. Concerning P. fluorescens F113, one may hypothesize either such a difference between several bacterial HPPD isoforms, or a possible detoxification phenomenon supporting resistance to β-triketones specific to this bacterial strain. In this latter case, detoxification could be achieved by the transformation of the active compound. Although P. putida 1OP is known to degrade sulcotrione and used it as a source of carbon [START_REF] Calvayrac | Isolation and characterisation of a bacterial strain degrading the herbicide sulcotrione from an agricultural soil[END_REF], the transformation of β-triketones by P. fluorescens F113 has not yet been described. Detoxification can also be achieved by efflux pumps that are pumping herbicides out of the cell. As an example, a putative major facilitator superfamily (MFS) transporter encoded by the yhhS gene conferred resistance in an E. coli strain to glyphosate [START_REF] Staub | Bacterial glyphosate resistance conferred by overexpression of an E. coli membrane efflux transporter[END_REF]. Efflux pumps are commonly described in Gram-negative and especially in P. aeruginosa where they impart resistance to antibiotics [START_REF] Li | The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria[END_REF][START_REF] Rampioni | Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence[END_REF]. Other studies have tested the HPPD activity of P. aeruginosa strains in presence of NTBC, a β-triketone used in human medicine, and inhibitory effects are observed at high doses of the inhibitor. For the DKN343 and 1111 isolates, NTBC (up to 900 µM) showed no inhibitory effects on HPPD activity and in the PAO1 mutant, P. aeruginosa hmga::tn, 300 µM of NTBC are required to inhibit the enzyme activity [START_REF] Ketelboeter | Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress[END_REF]. The same authors showed that the expression of the MexAB-OprM efflux pump is required for resistance to NTBC in DKN343 [START_REF] Ketelboeter | Characterization of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexanedione resistance in pyomelanogenic Pseudomonas aeruginosa DKN343[END_REF]. This efflux pump is known to pump out a broad range of antibiotics, detergents or organic solvents [START_REF] Chen | Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa[END_REF]. We can then hypothesize that a similar efflux pump can allow β-triketone such as sulcotrione, to be pumped out by P. fluorescens F113.

Anyhow, further studies reporting a possible expression of genes encoding efflux pumps in parallel with an exposition to sulcotrione could be needed to reinforce this hypothesis.

Designing dose-response curves on three bacterial models

In order to refine our results, we decided to expose to other -triketone herbicides three of the six bacterial strains: a Gram-negative harbouring a sensitive HPPD activity (S. oneidensis MR-1), a tolerant one (P. fluorescens F113), and the only Gram-positive (B. cereus ATCC14579). Using the colorimetric whole-cell assay, resting cells of S. oneidensis MR-1, B. cereus ATCC14579 and P. fluorescens F113 were exposed to increasing doses of sulcotrione, mesotrione and tembotrione. The pre-screening at high doses of herbicides allowed us to select specific inhibitory concentrations ranging from 0to 10RfD for sulcotrione (Figure 3.A), 0to 0.5RfD for mesotrione (Figure 3.B) and 0to 0.07RfD for tembotrione (Figure 3.C). As previously showed with sulcotrione, the HPPD activity of P. fluorescens F113 is not inhibited by any of the three β-triketones tested (Figure 3.A, 3.B and 3.C) even at high doses, up to 15RfD (data not shown). At these concentrations, a clear dose-dependent response was observed in B. cereus ATCC14579 and S. oneidensis MR-1 which allowed us to calculate EC50 for the different herbicides. Tembotrione appears to have the strongest inhibiting effect on the HPPD activity of both bacteria. Indeed, EC50 are not even calculable due to a too rapid decrease of HPPD activity. However, we can assume that, for both B. cereus ATCC14579 and S. oneidensis MR-1, EC50 is below 0.02RfD of tembotrione, corresponding to 0.07 µM, and that tembotrione has a slightly higher effect on the HPPD activity of S. oneidensis MR-1. Similarly, mesotrione has a lower effect on the HPPD activity of B. cereus ATCC14579 with an EC50 of 2.2 ± 0.3 µM than on the one of S. oneidensis MR-1 for which the EC50 cannot be calculated because a too rapid decrease of its HPPD activity but can be easily graphically estimated below 0.2RfD of mesotrione, being 0.88 µM. Sulcotrione appears to be the active compound with the lowest effect whatever the strain considered with an EC50 of 8.5 ± 3.3 µM in B. cereus ATCC14579 and 8.8 ± 2.8 µM in S. oneidensis MR-1. This experiment confirmed our previous results with a strain-dependant response but also highlights a molecule-dependant response. Indeed, β-triketones were ranked as follows from the highest to the lowest inhibitory effect: tembotrione, mesotrione and sulcotrione. Tembotrione appears then to have the lowest EC50 in both B. cereus ATCC14579 and S. oneidensis MR-1. This molecule was shown to be more toxic on the earthworm Dendrobaena veneta than glyphosate and nicosulfuron [START_REF] Hackenberger | Acute and subchronic effects of three herbicides on biomarkers and reproduction in earthworm Dendrobaena veneta[END_REF]. Even though the cited study did not compare tembotrione to other β-triketones, it provides insights in its potential toxicity. When cloned in E. coli, the topramezone-resistant HPPD of the bacterium Sphingobium sp. TPM-19 exhibits a higher resistance to mesotrione (EC50 of 4.2 µM) compared to tembotrione (EC50 of 2.5 µM) [START_REF] Liu | Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19[END_REF] suggesting that, as observed in our study, tembotrione is more active than mesotrione on HPPD activity even for resistant HPPDs. This higher activity might be due to the fluorinated tail (-O-CH2-CF3) which increases its lipophilicity and favors the passage of hydrophilic and lipophilic barriers [START_REF] Dumas | Fate and ecotoxicological impact of new generation herbicides from the triketone family: An overview to assess the environmental risks[END_REF]. However, tembotrione physico-chemical properties such as solubility or pKa do not explain its little or no effect on the HPPD enzymatic activity of P. fluorescens F113. One can notice that the MexAB-OprM efflux pump previously cited is not yet described neither in B. cereus ATCC14579 nor in S. oneidensis MR-1, suggesting that these bacteria cannot extrude tembotrione once it has entered the cell. Regarding a possible biodegradation mechanism of tembotrione, the trifluoromethyl group added to this compound could make difficult its biodegradation. Moreover, it is known that, in soils, half-life time (DT50) of sulcotrione, mesotrione and tembotrione are comprised between 3.2 and 144 days depending on the soil tested [START_REF] Dumas | Fate and ecotoxicological impact of new generation herbicides from the triketone family: An overview to assess the environmental risks[END_REF]. A possible abiotic or biotic degradation in our culture conditions is, then, quite unlikely.

Taken together, these hypotheses can explain the fact that tembotrione harbors a higher inhibitory effect on the HPPD activities of certain bacteria than sulcotrione does.

Beyond the cellular level, one can imagine that such strain-and molecule-dependent responses may also be due to molecular changes in the studied HPPD. As it is the case in plants where maize harbors a resistant HPPD [START_REF] Fritze | The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase[END_REF], bacteria could also harbor HPPD with different sensitivities to β-triketones.

In silico HPPD sequences analysis

We then conducted in silico analysis in order to identify possible differences in the HPPD sequences of the tested strains and, following that, molecular docking was led to identify different binding responses.

HPPD protein sequences of the three bacteria previously studied were aligned (Supplementary Information 3), together with the HPPD of R. etli CFN42, which was available online and was part of our first experiment, and five others HPPD sequences from different bacteria strains, whose pyomelanin production has been described [START_REF] Steinert | The Lly protein is essential for phydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila[END_REF][START_REF] Batallones | Disruption of hmgA by DNA Duplication is Responsible for Hyperpigmentation in a Vibrio anguillarum Strain[END_REF][START_REF] Pavan | Glycerol inhibition of melanin biosynthesis in the environmental Aeromonas salmonicida 34melT[END_REF][START_REF] Bolognese | Bacterial melanin production by heterologous expression of 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas aeruginosa[END_REF]. Even though these nine sequences have relatively low similarity, ranging from 35.26 to 87.39%, they retain some highly conserved regions. Firstly, the catalytic domain, located in the C-terminus involves a triad consisting of one glutamate (E) and two histidine (H) residues. This triad is highly conserved in bacteria, plant and mammalian forms of HPPD, because these three residues coordinate the binding of a metal dication (either Fe or Co) involved in the catalytic function of HPPD [START_REF] Serre | Crystal structure of Pseudomonas fluorescens 4hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway[END_REF][START_REF] Fritze | The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase[END_REF] (Figure 4A), thus playing an essential role in enzyme function.

Always in the C-terminal region, the motifs H-I/V/L/L-A and F-F/I/V/L-E, common across HPPD from different bacterium phyla are identified. They are typical from the active site of the enzyme and are conserved among 2506 bacterial protein sequences [START_REF] Thiour-Mauprivez | Design of a degenerate primer pair to target a bacterial functional community: The hppd bacterial gene coding for the enzyme targeted by herbicides, a study case[END_REF]. Additionally, the binding domain of the substrate HPP is a highly conserved area formed by mostly hydrophobic residues (T164, S202, P215, N217, Q240, L295, F312, F321, F333, N337, F338, L341 based on sequence of P. fluorescens HPPD) (Figure 4C and Supplementary Information 3 and 4). These 12 amino acids present almost 100% of identity with just one exception for the HPPD sequence of B. cereus in the position 164, where a Valine replace Tyrosine. This binding domain plays an important role in positioning HPP in the proper position for the region specific hydroxylation reaction catalyzed by HPPD [START_REF] Moran | 4-Hydroxyphenylpyruvate dioxygenase and hydroxymandelate synthase: Exemplars of the α-keto acid dependent oxygenases[END_REF]. β-triketone herbicides are known to interact at this interphase by forming a stable complex between two of their keto groups towards a bidentate association with the active-site metal ion by coordinate as well as this hydrophobic domain [START_REF] Brownlee | Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide[END_REF]. Amino acid sequence comparison of the bacterial HPPD revealed a highly conserved active site and could not explain the differences of inhibition previously observed between the different strains of our study when working on the whole-cell assay. Similar observations were done when HPPD sequence of maize, whose HPPD is naturally tolerant to β-triketone, was compared with other HPPD sequences from β-triketone sensible plants as A. thaliana [START_REF] Fritze | The crystal structures of Zea mays and Arabidopsis 4-hydroxyphenylpyruvate dioxygenase[END_REF].

To go further, molecular docking was then carried out with the HPPD from bacterial strains either sensitive or tolerant to β-triketone to explore the complex formed between bacterial enzyme and inhibitors.

Docking of sucotrione, mesotrione and tembotrione to microbial HPPD

The capacity of the three β-triketone compounds to fit within the substrate binding pocket of the microbial HPPD of S. oneidensis MR-1, B. cereus ATCC14579 and P. fluorescens F113 was estimated by the binding energy. This parameter was determined for each docking experiments as the average of the energy of the correct orientations between the herbicide and the ligand based on homology model of P. fluorescens F113. For example, the docking results obtained with mesotrione coordinated on the metal dication on homology model of P. fluorescens F113 generated 25 different poses. On these 25 poses generated by the docking algorithm, 18 were in an orientation most similar to that observed with inhibitors co-crystalized with HPPD, 3 were in the less favourable pose and 4 in the least desirable orientation (Supplementary Information 5). Regardless of the poses, all docking resulted in proper interaction with the iron metal dication (Supplementary Information 6).

As we can observed, molecular docking revealed that sulcotrione, mesotrione and tembotrione are able to bind with HPPD of B. cereus ATCC14579, S. oneidensis MR-1 and P. fluorescens F113. Whatever the strain, docking results suggest that tembotrione and sulcotrione binds more tightly to the bacterial HPPDs tested than mesotrione. Regarding the number of docking achieving the preferred binding pose (similar to green pose in Supplementary Information 6), both sulcotrione and mesotrione docked correctly and more frequently than tembotrione. These latter observations do not correspond to the one obtained with whole-cell assay experiments where tembotrione had a higher inhibitory potential than sulcotrione and mesotrione whatever the strains. Interestingly, Moran et al., suggest that HPPD inhibitor selectivity is based more on bioavailability of the molecule than on direct affinity for HPPD from a specific organism [START_REF] Moran | 4-Hydroxyphenylpyruvate dioxygenase[END_REF]. This observation could explain the apparent discrepancy observed in the results between docking and whole-cell experiments.

Our results also showed that the binding force of the three tested β-triketones with the HPPD of P. fluorescens F113 is lower than the one of the two others strains, suggesting that these three compounds are less able to fit the substrate binding pocket of the HPPD of P. fluorescens F113 than that of the two others microbial HPPD. It is noteworthy that these docking predictions agree with the measurement done with our colorimetric whole-cell assay.

In fact, P. fluorescens F113, was tolerant to the tested β-triketones despite the fact that docking predicted that these active compounds can interact with the active site of its HPPD, even if this HPPD presents the lowest binding force of interaction. Therefore, this poor interaction with the HPPD enzyme of P. fluorescens F113 cannot only explained the β-triketone tolerance of this strain. Thus, in silico analysis is useful to understand and describe molecular interaction between the active compound and the target. However, it is not enough to fully predict the tolerance or the sensitivity of the bacterial HPPD to βtriketones because there are a range of other abiotic and biotic processes involved in the response bacterial cells to β-triketones. A new screening of bacterial strains having either tolerant or sensitive HPPD activity have to be done to gain more information and validate the docking results obtained with the three HPPD sequences of this study. Furthermore, additional experiments analysing the inhibitor binding kinetics on purified HPPDs from these microorganisms would yield important information regarding their affinity. Our docking provides insight on the Kon values of the herbicides, but it is well known that HPPD inhibitors bind very tightly to the target site [START_REF] Garcia | Inhibition of p-Hydroxyphenylpyruvate Dioxygenase by the Diketonitrile of Isoxaflutole: A Case of Half-Site Reactivity[END_REF]. Consequently, measuring the Koff values might be even more insightful. Indeed, compounds with high Koff values (binding for a very short time) may not be very active. On the other hand, compounds with low Koff values (tight binding or irreversible inhibition) would potentially be more active [START_REF] Siehl | Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette[END_REF]. However, this is beyond the scope of this paper.

Conclusion

At a molecular level, molecular docking reveals that tembotrione and sulcotrione appear to have a stronger binding force to the bacterial HPPD enzymes than mesotrione. However, in silico study shows that tembotrione binds less frequently than the two other molecules. At a cellular level, whole-cell assays show that inhibitory potential is also molecule dependent but with tembotrione on the top step of the podium. Such differences allow us to think that in silico and in vivo approaches complement each other.

HPPD enzymatic activity inhibition also appears strain-dependent. At a molecular level, P. fluorescens F113 shows a lower binding force to herbicides than B. cereus ATCC14579 and S. oneidensis MR-1, confirming the observations made at the cellular level. By combining approaches at both cellular and molecular levels, this is the first study that assesses the effect of agronomical doses of three β-triketone active compounds on the HPPD enzymatic activities of six environmental bacterial strains.

At the community level, a recent lab-to-field study demonstrates that, neither sulcotrione nor its formulated product, Decano ® , has an effect on the abundance and the diversity of both total and hppd bacterial communities [START_REF] Thiour-Mauprivez | Assessing the effects of β-triketone herbicides on the soil bacterial and hppd communities: a lab-to-field experiment[END_REF]. As we measure that tembotrione has a lower EC50 than sulcotrione in some bacteria, conducting a lab-to-field experiment with tembotrione and one of its formulated products could be interesting. Actually, working at a lower biological level (i.e. whole-cell assay coupled with molecular docking) should be of interest, prior to conducting microcosm or field experiments, which are more difficult to set up.

Whole-cell assay developed in this study could be applied to other herbicides and pesticides that also target an enzyme harbored by non-target organisms and, more precisely, by microorganisms, like EPSPSynthase, targeted by Glyphosate or ALSynthase targeted by sulfonylurea herbicides [START_REF] Thiour-Mauprivez | Effects of herbicide on nontarget microorganisms: Towards a new class of biomarkers?[END_REF], as there is a need to develop new tools for pesticide risk assessment on soil microorganisms [START_REF] Martin-Laurent | ECOFUN-MICROBIODIV: an FP7 European project for developing and evaluating innovative tools for assessing the impact of pesticides on soil functional microbial diversity--towards new pesticide registration regulation?[END_REF][START_REF] Karpouzas | LOVE TO HATE' pesticides: felicity or curse for the soil microbial community? An FP7 IAPP Marie Curie project aiming to establish tools for[END_REF].

Figure 2 .

 2 Figure 2. OD430N in P. fluorescens F113 (A) and S. oneidensis MR-1 (B) in regard to increasing RfD of sulcotrione. Each value is obtained by subtracting the mean OD430 obtained at various concentrations of sulcotrione (i.e. OD430A) from the mean OD430 obtained in negative controls (i.e. OD430NC) and is depicted on this figure as "OD430N". Error bars are calculated from the standard deviation for each series.

Figure_2

  Figure_2

Figure 3 .

 3 Figure 3. HPPD relative activity (Act. %) in B. cereus ATCC14579, S. oneidensis MR-1 and P. fluorescens F113 exposed to sulcotrione (A), mesotrione (B) and tembotrione (C). Act. % represents the percentage obtained by dividing the mean OD430 obtained at various concentrations of sulcotrione, mesotrione or tembotrione (i.e. OD430A) from the mean OD430 obtained in the positive control (i.e. OD430PC). The grey dotted line corresponds to 50% of activity. Error bars are calculated from the standard deviation for each series. Significant differences between means are indicated by different letters (Kruskal-Wallis tests, p<0.05).

Figure 3

 3 Figure 3 revised

Figure 4 .

 4 Figure 4. A) Residues involved in stabilizing the metal dication (Fe 2+ ) within the HPPD catalytic domain of P. fluorescens in pink (1cxj), A. thaliana in green (6isd) and H. sapiens in blue (3isq). B) Alignment of the 4 bacterial models derived from the HPPD sequences of P. fluorescens F113, B. cereus ATCC14579, S. oneidensis MR-1 and R. etli CFN42. C) Amino acid residues lining the substrate binding pocket of HPPD are highly conserved of the 4 bacterial homology models.Numbering is based on sequence of P. fluorescens HPPD.

Figure_4

  Figure_4

Table 1 .

 1 β-triketone molecules used as herbicides. CMBA: 2-chloro-4-methyl sulfonyl benzoic acid; CHD: 1,3-cyclohexanedione MNBA: 4-methylsulfonyl-2-nitro benzoic acid; AMBA: 2-amino-4methylsulfonylbenzoic acid TCMBA: 2-(2-chloro-4-mesyl-3-(2,2,2-trifluoroethoxy) methyl)