A atrazina, um herbicida triazínico amplamente utilizado no controle de ervas daninhas, é um potencial contaminante ambiental, e possui como principal via de degradação uma via biológica. A linhagem Pseudomonas sp. ADP é o micro-organismo de referência no processo de degradação da atrazina em ambientes contaminados, pois contém em seu genoma os genes atzA, atzB, atzC, atzD, atzE e atzF, os quais codificam as enzimas responsáveis pelo processo de degradação. Bactérias gram-positivas também possuem capacidade para degradar a atrazina, mas iniciam a via de degradação através do gene trzN, análogo ao atzA. O objetivo do presente trabalho foi obter e analisar isolados bacterianos e consórcios formados por duas ou mais bactérias com capacidade para degradação completa do herbicida atrazina, como também analisar os efeitos da atrazina na comunidade bacteriana do solo. Duas bactérias gram-negativas, A01 e A02, foram isoladas de amostras de solo e foram identificadas como pertencentes aos gêneros Achromobacter e Pseudomonas, respectivamente, através do sequenciamento do gene 16S rRNA. Ambos os micro-organismos apresentaram potencial para biodegradação da atrazina em meio sólido, mas somente o isolado Pseudomonas sp. apresentou todos os genes atzA, atzB, atzC, atzD, atzE e atzF, que codificam as enzimas da via completa de degradação da atrazina. O isolado Achromobacter sp. apresentou somente os genes atzA, atzB e atzC, que representam a via inicial de degradação da atrazina até a formação de ácido cianúrico como metabólito. Um ensaio utilizando o método Southern Blot foi realizado para verificar se os genes atz detectados nos isolados do estudo são plasmidiais, sendo que apenas o isolado Pseudomonas sp. apresentou plasmídeo. A expressão dos genes atzA, atzB, atzC e atzD foi avaliada pelo método Northern Blot, contudo apenas o isolado Pseudomonas sp. apresentou expressão diferencial após tratamento com atrazina. Análises em HPLC/DAD e LC-MS/MS demonstraram que o isolado Pseudomonas sp. apresenta um perfil de degradação da atrazina semelhante ao perfil do micro-organismo padrão Pseudomonas sp. ADP, sendo apto a degradar 99% de atrazina in vitro em 24 horas. Já o isolado Achromobacter sp. apresentou um perfil de degradação lento, com início do processo após 24 horas. Os três metabólitos iniciais formados pela degradação da molécula de atrazina foram detectados em amostras contendo tanto o isolado Pseudomonas sp. quanto o isolado Achromobacter sp. O consórcio bacteriano composto pelos dois isolados deste estudo não apresentou eficiência de degradação superior às culturas puras. Por fim, um experimento de campo foi realizado com o objetivo de analisar os efeitos da atrazina na comunidade bacteriana do solo. O herbicida atrazina foi aplicado ao solo e amostras foram coletadas para análise através das técnicas de qPCR e Sequenciamento de Nova Geração. Foi possível observar que a abundância dos genes responsáveis pelo início da via de degradação da atrazina se altera ao longo do tempo, sendo que o aumento mais expressivo foi observado no gene trzN, comumente encontrado em bactérias gram-positivas com capacidade para degradar a atrazina. O sequenciamento do gene 16S rRNA indicou que a aplicação de atrazina ao solo não provocou mudanças significativas na comunidade bacteriana. As amostras apresentaram alta diversidade antes e após o tratamento com atrazina e a análise ii da abundância relativa mostrou pequenas diferenças na abundância de famílias após quatro e oito semanas de aplicação da atrazina ao solo. Assim, é possível sugerir que a aplicação do herbicida atrazina ao solo nas doses recomendadas não provoca danos significativos na estrutura da comunidade bacteriana do solo. / Atrazine, a triazine herbicide widely used to control broadleaf weeds, is a potential contaminant of soils, groundwater, rivers, lakes and oceans. Its main route of degradation is a biological pathway. Pseudomonas sp. ADP is a standard bacterium in the process of atrazine mineralization in contaminated environments, because it possesses a plasmid that contains the genes atzA, atzB, atzC, atzD, atzE and atzF, which encode the enzymes responsible for the atrazine degradation process. Gram-positive bacteria also have the ability to degrade atrazine, but the degradation starts through the trzN gene, wich is analogue to atzA. The aim of the present work was to obtain and analyze a bacterial isolate or a consortium formed by two or more bacteria capable of completely degrade the herbicide atrazine and to analyze the effects of atrazine on the soil bacterial community. Two gram-negative microorganisms, A01 and A02, were isolated from soil samples and were identified as Achromobacter sp. and Pseudomonas sp., respectively, through the sequencing of the 16S rRNA gene. Both microorganisms showed potential to degrade atrazine on solid medium, but only the isolate Pseudomonas sp. presented the genes atzA, atzB, atzC, atzD, atzE and atzF that are essential for the biodegradation process. Achromobacter sp. presented only the atzA, atzB and atzC genes, which represent the initial pathway of atrazine degradation that leads to the formation of cyanuric acid. An assay using Southern Blot was performed to verify if the atz genes detected in the isolates were located on plasmid, however only Pseudomonas sp. showed a plasmid. The atz gene expression was evaluated through Northern Blot methodology, but only Pseudomonas sp. showed differential expression after atrazine induction. Analyzes in HPLC/DAD and LC-MS/MS demonstrated that the isolate Pseudomonas sp. presents an atrazine degradation profile similar to the profile of Pseudomonas sp. ADP and is capable of degrading 99% of atrazine in vitro. The strain Achromobacter sp. presented a slow degradation profile and started the degradation process after 24 hours of incubation. The three initial metabolites formed after atrazine degradation were detected in samples containing both Pseudomonas sp. and Achromobacter sp. The bacterial consortium composed of the two isolates of this study did not show higher degradation efficiency than pure cultures. Lastly, a field experiment was performed in order to study the effects of atrazine on the soil bacterial community. The herbicide atrazine was applied to the soil and samples were collected to be analysed using qPCR and Next Generation Sequencing. It was possible to observe that the abundance of the atz genes that initiate the degradation process is changed over time. A significant increase was observed on trzN, which is commonly found in gram-positive bacteria that is capable of degrading atrazine. 16S rRNA gene sequencing indicated that atrazine application to soil do not cause significant changes in the bacterial community. Soil samples presented high diversity before and after atrazine treatment and the relative abundance analysis showed slight differences in families abundance after four and eight weeks of atrazine application to soil. Therefore, the results suggest that the use of atrazine in recommended doses does not cause significant damage to the structure of the soil bacterial community.

L’atrazine, un des herbicides les plus utilisés pour contrôler le développement des plantes adventices dans les cultures, a conduit à la contamination de l’environnement. L’exposition chronique à cet herbicide a conduit à l’émergence de populations microbiennes du sol capables de dégrader l’atrazine et de l’utiliser comme une source d’azote pour leur croissance. Ces populations microbiennes sont responsables de la biodégradation accélérée (BDA) de l’atrazine, un service écosystémique contribuant à diminuer la persistance de cet herbicidedans l’environnement. L’objectif de ce travail était d’étudier les mécanismes génétiques et physiologiques responsables du fonctionnement et de l’amélioration de ce service écosystémique. Nous avons appliqué une démarche expérimentale allant des gènes codant la dégradation à des communautés microbiennes afin d’identifier les processus adaptatifs impliqués dans l’évolution de la fonction de BDA de l’atrazine.Le premier volet a consisté à évaluer l’importance de mutations accumulées dans le gène atzA dans la transformation de l’atrazine en hydroxyatrazine catalysée par AtzA. Le séquençage de gènes atzA de différents isolats bactériens dégradant l’atrazine (Pseudomonas sp. ADP WT, Pseudomonas sp. ADP Ps et différents Chelatobacter heintzii) a montré que la séquence du gène atzA était très conservée. Toutefois quatre mutations non silencieuses ont pu être identifiées (1 chez Pseudomonas sp. ADP MSE et 3 chez Chelatobacterheintzii). La modélisation de la structure de la protéine AtzA a permis de montrer que trois des mutations étaient situées dans des régions importantes (site actif, poche de liaison avec l’atrazine et liaison avec le métalFe2+. [...] Le second volet a consisté à étudier la plasticité de la voie de biodégradation de l’atrazine dans deux conditions opposées : (i) la première visait à évaluer la persistance de la capacité de dégradation en absence de pression de sélection et (ii) la seconde visait à évaluer l’évolution de la capacité de dégradation en présence d’une pression de sélection élevée. Pour conduire ces études, des manipulations d’évolution expérimentale sur Pseudomonas sp. ADP ont été menées. (i) L’exposition à l’acide cyanurique, intermédiaire métabolique de l’atrazine, a conduit à la sélection d’une population nouvellement évoluée capable de croître plus rapidement dans un milieu de culture ne contenant que l’acide cyanurique comme source d’azote. Cette population est caractérisée par une délétion d’une région de 47 kb du plasmide ADP1 contenant les gènes atzABC. Les analyses conduites ont permis de conclure que le gain de compétitivité de la population évoluée résidait dans la perte du fardeau génétique représenté par la région de 47 kb, la capacité de dégradation de l’acide cyanurique restant inchangée. (ii) L’exposition à l’atrazine a conduit à la sélection d’une populationnouvellement évoluée caractérisée par l’insertion du plasmide ADP1 en quasi-totalité sur le chromosome bactérien. [...] Le troisième volet a consisté à développer un outil permettant d’évaluer, à l’échelle d’une communauté microbienne synthétique, l’évolution du potentiel génétique dégradant. Pour ce faire quatre souches dégradantes dont une, Arthrobacter sp. TES6, isolée au cours de cette étude, ont été choisies. [...] Ces travaux montrent que la fonction de biodégradation accélérée de l’atrazine est très versatile et qu’elle est en constante évolution. Il met en évidence que le principal facteur pilotant cette évolution est le niveau d’exposition des populations dégradantes au pesticide. / Atrazine, one of the most used herbicide to control the development of weeds in crop, has led to the contamination of the environment. Repeated exposure to this herbicide resulted in the emergence of microbial populations able to degrade atrazine and to use it as a nitrogen source for its growth. These microbial populations are responsible for accelerated biodegradation of atrazine (BDA), a key ecosystemic service diminishing the persistence of this herbicide in the environment. The aim of this PhD work was to study genetic and physiological mechanisms responsible for functioning and improving of this ecosystemic service. We applied an experimental approach starting from genes to communities degrading atrazine in order to identify processes of adaptation involved in the evolution of accelerated biodegradation function.The first part of the PhD aimed at evaluating the importance of accumulation of single mutations in the atzA gene for the activity of AtzA transforming atrazine to hydroxyatrazine. Sequencing or atzA genes amplified from different atrazine-degrading isolates (Pseudomonas sp. ADP WT, Pseudomonas sp. ADP Ps and differents Chelatobacter heintzii) showed that atzA sequence was conserved. However, four non synonymous mutations were identified (1 for Pseudomonas sp. ADP Ps and 3 for Chelatobacter heintzii). Modeling of AtzA structure showed that three mutations were located in important regions (active site, interaction with atrazine and with the metal Fe2+). [...] The second part aimed at studying the plasticity of the atrazine-degrading pathway in two opposed conditions: (i) one aiming at evaluating the persistence of degrading capability in absence of selection pressure and (ii) a second one aiming at evaluating the evolution of degrading capability under high selection pressure exerted by atrazine. With these aims, experimental evolutions were carried out with Pseudomonas sp. ADP. (i) We showed that cyanuric acid exposure led to the selection of a newly-evolved population characterized by increased growing ability on culture medium containing this substance as nitrogen source. This population is characterized by the deletion of a 47 kb region containing atzABC genes from ADP1. We showed that increased fitness of newly-evolved population was due to the selective loss of the genetic burden represented by the 47 kb region, the cyanuric acid degrading ability remaining unchanged. (ii) Atrazine exposure led to the selection of population characterized by the insertion of ADP1 plasmid in the bacterial chromosome. [...] The third part aimed at developing a tool allowing monitoring the evolution of atrazine-degrading genetic potential at the scale of a synthetic microbial community. To do so four degrading strains among which, one was isolated in this study, were chosen. [...] Altogether, these results showed that the atrazine accelerated biodegradation function is highly versatile and under constant evolution. Furthermore, they highlight that the exposure to atrazine is the key parameter driving the evolution of degrading population