Estimation du potentiel génétique hydroxylant d'un sol par PCR quantitative : un nouvel outil d'évaluation et de prédiction de la pollution d'un sol par des micropolluants organiques

Diplôme : Dr. d'UniversitéDuring the last two centuries, industrialisation has led to increasing contamination of the environment by xenobiotics, notably aromatic compounds (i.e. PAHs, pesticides). These widespread pollutants are a major threat affecting soil quality and human health. In this context, numerous policies have been drawn up notably in Europe, to monitor the threats of pollution to ecosystems and to detect and implement measures to counterbalance the damage. Nevertheless, the European Commission and scientific community are still confronted with a lack of appropriate tools to monitor biodiversity in relation to environmental processes affecting global warming and soil protection. This study proposes tools to assess the biodiversity of functional bacterial communities involved in the process of natural and xenobiotic aromatics biodegradation in soil environments. The major route for the degradation of aromatic compounds is the ß-ketoadipate pathway. We developed two molecular markers i.e. two primer sets targeting two key genes (pcaH and catA) of the ß-ketoadipate pathway, encoding enzymes responsible for the ring cleavage of catechol and protocatechuate. The PCAH molecular marker targeted a sequence from the ß-subunit of 3,4-PCD encoded by the pcaH gene. PCAH was shown to amplify a wide diversity of pcaH sequences in the soil environment, thereby highlighting its interest in characterising the pca bacterial community responsible for the degradation of protocatechuate. This tool was applied to assess the structure, diversity and density of the pca bacterial community. When tested in experimental fields, it revealed changes in the structure, diversity and density of the pca bacterial community in response to organic amendments. This work showed that PCAH could constitute a tool of prime interest for assessing the impact produced by extrinsic factors such as agricultural practices on biodiversity of the pca community. The CATA molecular marker targeted a fragment of the 1,2-CTD encoding gene (catA). The wide diversity of catA sequences was revealed. At this stage in the development of pca and cat bioindicators, we can only provide tools to describe the structure, diversity and density of these bacterial communities. The use of PCR-RFLP fingerprinting and q-PCR assays is recommended to assess the impact of environmental stresses on these functional bacterial communities.Les deux derniers siècles ont été marqués par une industrialisation responsable d’une contamination accrue de l’environnement par des xénobiotiques, notamment les composés aromatiques (HAPs et pesticides). Ces composes sont très communs et présentent un risque important aussi bien pour la qualité des sols que la santé humaine. Ainsi, de nombreuses mesures politiques ont été établies, en Europe, pour la surveillance des menaces liées à la pollution des écosystèmes et la mise en place de mesure pour contrebalancer les effets notoires de telles contaminations. Néanmoins, la commission Européenne et la communauté scientifique demeurent confrontées au manque d’outil de contrôle de la biodiversité en lien avec les processus environnementaux affectant le changement climatique et la protection des sols. De ce fait, cette étude propose l’évaluation de la biodiversité fonctionnelle des communautés bactériennes impliquées dans les processus de biodégradation des polluants organiques dans le sol. La voie principale, connue pour la biodegradation de ces composés, est la voie des the ß-ketoadipate. Nous avons ainsi développés deux marqueurs moléculaires (deux couples d’amorces PCR) ciblant deux gènes clés (pcaH et catA) de la voie des ß-ketoadipate, codant pour les enzymes responsables de la fission des cycles aromatiques. Le marqueur moléculaire PCAH a pour cible la sous unité ß de la protocatéchuate 3,4-dioxygénase codée par le gène pcaH. Ce couple d’amorce permet d’amplifier des fragments de gènes provenant d’une grande diversité de bactéries issues du sol. Ceci montre l’intérêt de pouvoir caractériser la communauté bactérienne pca responsable de la biodégradation du protocatéchuate. Cet outil a été développé pour décrire ma structure, la diversité et la densité de la communauté bactérienne pca, montrant ainsi, sur une étude de cas, l’impact de l’amendement sur ces trois composantes de la biodiversité. Ce travail a montré que PCAH représente un outil pertinent pour étudier et estimer l’impact provoqué par des perturbations extrinsèques telles que les pratiques agricoles sur la biodiversité de cette communauté bactérienne. D’autre part, le marqueur moléculaire CATA a pour cible un fragment du gène codant la catéchol 1,2-dioxygénase (catA) et permet d’amplifier une diversité importante de séquences. A ce stade de l’étude, le développment de pca et cat comme bioindicateur se limite à la description de la la structure, diversité et densité de ces communautés

Estimation of the density of the protocatechuate-degrading bacterial community in soil by real-time PCR

International audienceThe β-ketoadipate pathway is the major route for degradation of aromatic compounds by various soil microorganisms. Protocatechuate 3,4-dioxygenase, a key enzyme of this pathway and which is encoded by pcaGH genes, catalyses the ring cleavage of protocatechuate. Microorganisms harbouring pcaGH genes are widely distributed in the environment but little is known about their relative abundance within the total microflora. Hence, this paper reports the development of a real-time PCR assay to quantify the bacterial pcaH sequence that encodes the β sub-unit of the protocatechuate 3,4-dioxygenase. This real-time PCR assay was linear over seven orders of magnitude with a calculated efficiency of 99% and sensitive up to 102 copies of the pcaH sequence per assay. Real-time PCR analyses performed on six soils with different physico-chemical properties, revealed pcaH densities ranging from 103 to 104 copies of pcaH ng−1 of soil DNA, which corresponded to approximately 0.2–10.9% of the total bacterial community. The sequencing of real-time PCR amplicons yielded 48 deduced amino acid sequences that exhibited 44–100% identity to known bacterial PcaH sequences, thereby confirming the accuracy of this real-time PCR assay

Detection and organization of atrazine-degrading genetic potential of seventeen bacterial isolates belonging to divergent taxa indicate a recent common origin of their catabolic functions

International audienceA collection of 17 atrazine-degrading bacteria isolated from soils was studied to determine the composition of the atrazine-degrading genetic potential (i.e. trzN, trzD and atz) and the presence of IS1071. The characterization of seven new atrazine-degrading bacteria revealed for the first time the trzN-atzBC gene composition in Gram-negative bacteria such as Sinorhizobium sp. or Polaromonas sp. Three main atrazine-degrading gene combinations (i) trzN–atzBC, (ii) atzABC–trzD and (iii) atzABCDEF were observed. The atz and trz genes were often located on plasmids, suggesting that plasmid conjugation could play an important role in their dispersion. In addition, the observation of these genes (i) on the chromosome, (ii) on the same DNA fragment but on different plasmids and (iii) on DNA fragments also hybridizing with IS1071 suggests that transposition may also contribute to disperse the atrazine-degrading genes

Structure, diversity and density of the protocatechuate-degrading bacterial community - Impact of long-term organic amendments

International audienc

Isolation and characterization of an isoproturon mineralizing Sphingomonas sp. strain SH from a French agricultural soil

International audienceThe phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized in an agricultural soil in France that had been periodically exposed to IPU. Enrichment cultures from samples of this soil isolated a bacterial strain able to mineralize IPU. 16S rRNA sequence analysis showed that this strain belonged to the phylogeny of the genus Sphingomonas (96% similarity with Sphingomonas sp. JEM-14, AB219361) and was designated Sphingomonas sp. strain SH. From this strain, a partial sequence of a 1,2-dioxygenase (catA) gene coding for an enzyme degrading catechol putatively formed during IPU mineralization was amplified. Phylogenetic analysis revealed that the catA sequence was related to Sphingomonas spp. and showed a lack of congruence between the catA and 16S rRNA based phylogenies, implying horizontal gene transfer of the catA gene cluster between soil microbiota. The IPU degrading ability of strain SH was strongly influenced by pH with maximum degradation taking place at pH 7.5. SH was only able to mineralize IPU and its known metabolites including 4-isopropylaniline and it could not degrade other structurally related phenylurea herbicides such as diuron, linuron, monolinuron and chlorotoluron or their aniline derivatives. These observations suggest that the catabolic abilities of the strain SH are highly specific to the metabolism of IPU

Use of PCAH as a molecular marker for estimating the diversity of the protocatechuate-degrading bacterial community in soil environment

International audienceMicroorganisms degrading phenolic compounds play an important role in soil carbon cycling. The pcaH gene encoding a key ring-cleaving enzyme of the β -ketoadipate pathway was selected as a functional marker. Using a degenerate primer pair, pcaH fragments were cloned from two soils. The RFLP screening of 150 pcaH clones yielded 68 RFLP families. Comparison of 86 deduced amino acid sequences displayed 70 % identity to known PcaH sequences. Phylogenetic analysis results in two major groups mainly related to PcaH sequences from Actinobacteria and Proteobacteria phyla. This gene constitutes a suitable molecular marker to study the diversity of this functional group