In vitro evolution of an atrazine-degrading population under cyanuric acid selection pressure: Evidence for the selective loss of a 47kb region on the plasmid ADP1 containing the atzA, B and C genes

International audience; The adaptation of microorganisms to pesticide biodegradation relies on the recruitment of catabolic genes by horizontal gene transfer and homologous recombination mediated by insertion sequences (IS). This environment-friendly function is maintained in the degrading population but it has a cost which could diminish its fitness. The loss of genes in the course of evolution being a major mechanism of ecological specialization, we mimicked evolution in vitro by sub-culturing the atrazine-degrading Pseudomonas sp. ADP in a liquid medium containing cyanuric acid as the sole source of nitrogen. After 120 generations, a new population evolved, which replaced the original on…

Rapport final du projet Antibiotools - Des outils pour caractériser et suivre les antibiotiques et antibiorésistances dans les écosystèmes aquatique (PNREST Anses, 2017/3 ABR/22)

Transcriptomic and proteomic responses of Microbacterium sp. C448 exposed to sulfamethazine antibiotic

International audience; The Microbacterium sp. C448 was isolated from a soil regularly exposed to sulfamethazine(SMZ), for its ability to partly mineralise this antibiotic and other related sulfonamides.The aim of our study was to explore its metabolic adaptation towards exposure to SMZenvironmental (10 mg/L) and medicinal (250 mg/L) concentrations. Its transcriptomic andproteomic responses were analysed by focusing on the degradation regulon (sad genes) andresistance genes (folP and sul1).The transcriptomic and proteomic results were essentially congruent whatever theconcentrations tested. In culture conditions, exposure to the highest concentration of SMZ led tothe highest sad expression …

Evolution of genetic degradation potential of pesticide-degrading bacterial communities

International audience

Genomic approach to search for IPU degrading gene: catA as a possible gene target responsible for 4- isopropyl aniline degradation

Affiche, résumé étendu; Genomic approach to search for IPU degrading gene: catA as a possible gene target responsible for 4- isopropyl aniline degradation

Ecotoxicological impact of pesticides on natural microbial communities responsible for pesticide biodegradation in river sediments

International audience; The general use of pesticides in various crops allows ensuring the quality of the crop production but has environmental impact, pesticide residues being often found in water resources, notably river in the agrosystems. As a result of repeated exposure to pesticides microorganisms can adapt to their degradation being responsible for enhanced degradation, an environmental friendly process diminishing the persistence of pesticide residues in the environment. This process can occur in the soil but also in the sediment of the river regularly exposed to pesticide contamination. To our best knowledge the ecotoxicological impact of multiple pesticides contamination on the de…

Importance of genetic plasticity for microbial adaptation to pesticide biodegradation: in vitro evolution of Pseudomonas sp. ADP under atrazine or cyanuric acid selection pressure as case studies

International audience

Genetic plasticity of atrazine-degrader: evidence for the deletion of atzABC genes in pseudomonas sp. adp in response to cyanuric acid selection pressure

International audience; Several bacterial strains are known to metabolize s-triazines herbicides among which atrazine reported worldwidel to contaminate soil and water resources. The complete mineralization of atrazine to carbon dioxide and ammonium is catalyzed by 6 hydrolytic enzymes encoded by atzABCDEF genes located on the plasmid pADP1 in Pseudomonas ADP. The atzABC genes code for the upper pathway transforming atrazine to cyanuric acid, while the atzDEF genes code for the lower pathway transforming cyanuric acid to simple compounds. In order to study the genetic plasticity of the atrazine catabolic pathway, we performed an in vitro evolution study consisting in applying a continuous s…

Predicting pesticide biodegradation potential from microbial community composition: new tools for bioremediation

Bioaugmentation is receiving increasing attention as a green technology to treat contaminatedareas by inoculating specific biodegrading microorganisms. However, our understanding of therole of microbial community composition and structure in the expression of contaminantdegradation potential is yet to improve. It could help making wise choice for microorganisms –community or specific strain – to be inoculated in contaminated soils with consideration to theirindigeneous microbiota.Here we tried to predict the microbial degradation of two herbicides, glyphosate andisoproturon by means of penalized regression and machine learning methods routinely used ingenomic selection. To this end, we cond…

Regulation of bacterial and fungal MCPA degradation at the soil–litter interface

Abstract Much is known about mechanisms and regulation of phenoxy acid herbicide degradation at the organism level, whereas the effects of environmental factors on the performance of the phenoxy acid degrading communities in soils are much less clear. In a microcosm experiment we investigated the small-scale effect of litter addition on the functioning of the MCPA degrading communities. 14 C labelled MCPA was applied and the functional genes tfdA and tfdAα were quantified to characterise bacterial MCPA degradation. We identify the transport of litter compounds as an important process that probably regulates the activity of the MCPA degrading community at the soil–litter interface. Two possi…

DIAGSOL : Développement d’un outil microbien pour évaluer l’exposition des sols agricoles aux herbicides β-tricétones

International audience; En 2011, en France, plus de 2 millions d’hectares de champs de maïs ont été traités avec desherbicides β-tricétones. Qualifiés de « respectueux de l’environnement » en raison d’une efficacité àfaible dose, de récentes études ont démontré un effet transitoire des β-tricétones sur la diversitébactérienne des sols (Romdhane et al., 2016). L’utilisation récurrente des β-tricétones pourrait, àterme, perturber la diversité bactérienne et les fonctions écosystémiques des sols qu’elle soutient.Il est essentiel d’évaluer l’exposition des microorganismes des sols aux β-tricétones.L’enzyme 4-hydroxyphénylpyruvate dioxygénase (4-HPPD), cible de ces herbicides chez les plantesadv…

Molecular analysis of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHs

International audience; A PCR-based molecular tool was developed to estimate the diversity of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHS. A degenerate primer pair specific to catA sequences was designed by multiple alignment of known sequences coding a key intermediate of the β-ketoadiapate pathway degrading catechol, namely catechol 1,2-dioxygenase. The specificity of this primer pair was assessed in 21 pure strains by PCR and sequencing. Comparison of the 16S rDNA and catA phylogenies revealed an absence of congruence between these two genes. The primer set was able to amplify catA sequences in DNA extracts from an industrial soil highly …

Response of soil bacterial and hppd communities to tembotrione herbicide

Herbicides used in agriculture aim to prevent weed growth but are known to end up in contactwith soil microorganisms, thus defined as non-target organisms. Tembotrione, a recentlymarketed β-triketone herbicide, is known to inhibit the 4-HydroxyPhenylPyruvateDioxygenase(4-HPPD) in weeds. This enzyme is also found in numerous soil microorganisms, such as somePGPR and symbiotic bacteria, that play a key role in maintenance of ecosystem services.In this study, one of the major concerns is to assess whether tembotrione could have toxiceffects on soil microorganisms and could disturb soil microbial community dynamic andstructure. To investigate the possible impacts of this herbicide on these comm…

Antibiotrophie : une fonction émergente favorisant l’implantation des bactéries antibiorésistantes dans les sols

National audience; La fonction antibiotrophique, permettant l’utilisation d’antibiotiques comme source nutritive, arécemment été découverte chez des bactéries antibiorésistantes environnementales. Pourvérifier si leur avantage sélectif en environnements pollués s’en trouvait augmenté, nous avonsconduit une expérience en microcosmes dans laquelle Microbacterium sp. C448 (C448),présentant des capacités de résistance et de dégradation vis-à-vis des sulfamides, a été inoculéedans quatre sols supplémentés ou non par l’antibiotique sulfaméthazine (SMZ) et/ou du lisier.Après un mois d’incubation, C448 n’a été détectée que dans les sols traités avec la SMZ,indiquant une faible compétitivité de la s…

Predicting pesticide biodegradation potential from microbial community composition : new tools for bioremediation

Bioaugmentation is receiving increasing attention as a green technology to treat contaminated areas by inoculating specific biodegrading microorganisms. However, our understanding of the role of microbial community composition and structure in the expression of contaminant degradation potential is yet to improve. It could help making wise choice for microorganisms – community or specific strain – to be inoculated in contaminated soils with consideration to their indigeneous microbiota. Here we tried to predict the microbial degradation of two herbicides, glyphosate and isoproturon by means of penalized regression and machine learning methods routinely used in genomic selection. To this end,…

Nouveaux outils en bioremédiation : prédiction du potentiel de dégradation d’une communauté à partir de sa composition et création de communautés multidégradantes par coalescence

En bioremédiation, la bioaugmentation consiste en l’introduction de microorganismes dans un milieu contaminé afin d’augmenter et/ou de suppléer la population bactérienne en place, dans l’objectif de le dépolluer. Cette approche est cependant limitée par la difficulté de choisir des microorganismes efficients - communauté ou souche spécifique – à inoculer, en lien avec les conditions abiotiques et les microorganismes indigènes du milieu pollué.Dans ce travail nous avons cherché à prédire le potentiel microbien de dégradation de deux pesticides, le glyphosate et l’isoproturon, de communautés microbiennes telluriques en utilisant des méthodes statistiques issues de la prédiction génomique. Pou…

Antibiotrophy: Key Function for Antibiotic-Resistant Bacteria to Colonize Soils—Case of Sulfamethazine-Degrading Microbacterium sp. C448

Chronic and repeated exposure of environmental bacterial communities to anthropogenic antibiotics have recently driven some antibiotic-resistant bacteria to acquire catabolic functions, enabling them to use antibiotics as nutritive sources (antibiotrophy). Antibiotrophy might confer a selective advantage facilitating the implantation and dispersion of antibiotrophs in contaminated environments. A microcosm experiment was conducted to test this hypothesis in an agroecosystem context. The sulfonamide-degrading and resistant bacterium Microbacterium sp. C448 was inoculated in four different soil types with and without added sulfamethazine and/or swine manure. After 1 month of incubation, Micro…

Bacterial hppd: a biomarker of exposure of soils to beta-triketone herbicides?

National audience; β-triketone herbicides are among the most used herbicides in corn crop to control broadleaf weeds.These herbicides inhibit the 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) and lead to bleaching anddeath of weeds. This enzyme is not only found in plants but in all living organisms, includingmicroorganisms where it takes part to the tyrosine degradation pathway. Thus, microorganismsclassified as “non-target organisms” by current EU regulation for pesticide authorization, might beimpacted by β-triketones, with possible domino effect on microbial functions supporting soilecosystem services (Thiour-Mauprivez et al. 2019). Since microorganisms have been proposed by EFSAas key-d…

Characterization of an isoproturon mineralizing bacterial culture enriched from a French agricultural soil.

The phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized by a bacterial culture isolated from an agricultural soil regularly exposed to IPU. Molecular analysis of the bacterial culture by DNA fingerprinting, cloning and sequencing of the 16S rRNA genes revealed that it consisted of six different members among whom the dominant was related to Sphingomonas sp. Six bacterial strains belonging to genera Ancylobacter, Pseudomonas, Stenotrophomonas, Methylobacterium, Variovorax and Agrobacterium were isolated from the IPU-degrading culture. None of these were able to degrade IPU in pure culture and only the intact culture sustained th…

atzgene expressions during atrazine degradation in the soil drilosphere

One of the various ecosystemic services sustained by soil is pollutant degradation mediated by adapted soil bacteria. The pathways of atrazine biodegradation have been elucidated but in situ expression of the genes involved in atrazine degradation has yet to be demonstrated in soil. Expression of the atzA and atzD genes involved in atrazine dechlorination and s-triazine ring cleavage, respectively, was investigated during in situ degradation of atrazine in the soil drilosphere and bulked samples from two agricultural soils that differed in their ability to mineralize atrazine. Interestingly, expression of the atzA gene, although present in both soils, was not detected. Atrazine mineralizati…

Caractérisation de populations microbiennes tolérantes au chlordécone à partir de sols contaminés des Antilles

Le chlordécone (CLD), un insecticide organochloré utilisé en Martinique et en Guadeloupe pendant 20 ans afin de lutter contre le charançon du bananier, contamine des sols agricoles des Antilles posant un problème environnemental qui est devenu un problème majeur de santé publique. Il n’existe que très peu d’évidence rapportant la biodégradation de cet insecticide, classé depuis 2009 comme polluant organique persistant (POP) par la convention de Stockholm. L’objectif de ce travail est de rechercher des éléments indiquant la biotransformation du CLD dans les sols contaminés des Antilles. Des cultures par enrichissement ont été établies à partir d’échantillons de sols contaminés pour isoler de…

Exposure of benthic microbial communities to pharmaceuticals and resulting adaptation including tolerance, biodegradation and antibiotic resistance: advances and challenges

International audience; Since the early 1920’s, pharmaceuticals, including antibiotics, have been massively producedand consumed for the benefit of both human and animal health. Pharmaceuticals residues havethen reached the aquatic environment through diffuse and point (wastewater) sources. Amongthe pharmaceutical residues, the ubiquitous presence of antibiotics could exert a selectivepressure on microbial communities leading to the acquisition and dissemination of antibioticresistance in the environment.We present here the synthesis of recent research projects (e.g. PANDORE, Antibio-tools,Antibiotox, PharmaTox...) investigating the dissemination of pharmaceuticals, includingantibiotics, in…

Genomics based approach to identify the genes involved in ipu mineralization in sphingomonas sp.sh

Phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralised in aFrench agricultural soil previously exposed to IPU. A bacterial strain able to metabolise IPU was isolated from this soil adapted toIPU mineralization.

Biodegradation of synthetic β-triketone herbicide

Evidence of atrazine mineralization in a soil from the Nile Delta: Isolation of Arthrobacter sp. TES6, an atrazine-degrading strain

International audience; The s-triazine herbicide atrazine was rapidly mineralized (i.e., about 60% of C-14-ring-labelled atrazine released as (CO2)-C-14 within 21 days) by an agricultural soil from the Nile Delta (Egypt) that had been cropped with corn and periodically treated with this herbicide. Seven strains able to degrade atrazine were isolated by enrichment cultures of this soil. DNA fingerprint and phylogenetic studies based on 165 rRNA analysis showed that the seven strains were identical and belonged to the phylogeny of the genus Arthrobacter (99% similarity with Arthrobacter sp. AD38, EU710554). One strain, designated Arthrobacter sp. strain TES6, degraded atrazine and mineralized…

Fate, biodegradation and ecotoxicological impact of the bioherbicide leptospermone on soil bacterial community

International audience

Isolement de champignons d’un andosol de Guadeloupe contaminé à la chlordécone : caractérisation de leurs tolérances

Physiological and genetic characterization of an isoproturon mineralizing bacterial strain isolated from a French agricultural soil: Genomic approach to search for isoproturon degrading genes

Metabolic response of Microbacterium sp. C448 exposed to environmental and medicinal concentrations of sulfamethazine antibiotic

International audience

Impact écotoxicologique de pesticides sur les communautés microbiennes naturelles responsables de la biodégradation de pesticides dans les sédiments de rivière

National audience; L’utilisation quasi généralisée de pesticides dans de nombreuses cultures permet d’assurer une certaine qualité de la production végétale mais elle a des conséquences environnementales causant la contamination des eaux de surface, notamment les rivières circulant dans les agrosystèmes. Suite à l’exposition répétée aux pesticides, une fraction de la microflore est capable de s’adapter pour les dégrader conduisant à la mise en place de la biodégradation accélérée, un processus intéressant du point de vue environnemental parce qu’il diminue la persistance des pesticides dans l’environnement. La biodégradation accélérée peut avoir lieu dans les sols mais également dans les sé…

Mécanismes d'évolution de pseudomonas sp. adp sous pression de sélection exercée par l'atrazine

L’atrazine est un pesticide qui est largement utilisé dans le monde depuis une quarantaine d’années. Son application répétée aaboutit non seulement à la contamination des eaux mais également à l’apparition de bactéries telluriques capables d’utiliserce xénobiotique comme source d’azote pour leur croissance. La caractérisation des gènes atz responsables de la dégradationde cette molécule a révélé qu’ils étaient largement dispersés et très conservés au sein du monde bactérien traduisant ainsid’une évolution et d’une dispersion récente de ces gènes, probablement concomitantes avec l’application d’atrazine dansl’environnement

Processes involved in the adaptation of soil microbiota to enhance biodegradation of pesticides: atrazine-degrading community as a study case

International audience