Frankia est une actinobactérie symbiotique de 8 familles de plantes actinorhiziennes. Elle est connue par sa capacité à fixer l'azote moléculaire. La taxonomie et la phylogénie du genre Frankia reste incomplète et à explorer. Les objectifs de cette thèse sont d'apporter des connaissances supplémentaires sur la position phylogénétique et l'évolution des différents groupes d'infectivité du genre Frankia. Dans un premier temps, une phylogénie moléculaire basée sur les gènes glnII, nifH, gyrB et des ITS 16S-23S de l'ADNr a été réalisée. Le résultat de cette étude souligne la présence de quatre groupes de Frankia : (i) le groupe 1 associe les souches infectives des Betulaceae, Myricaceae et Casuarinaceae ; (ii) le groupe 2 des microsymbiotes obligatoires associés aux Coriariaceae, Datiscaceae, Rosaceae et Ceanothus (Rhamnaceae); (iii) le groupe 3 de souches d'Elaeagnaceae, Rhamnaceae, Myricaceae et Gymnostoma (Casuarinaceae) et (iv) le groupe 4, à position ancestrale, renferme les souches atypiques non fixatrices d'azote et/ou non infectives. Le groupe 3 aurait émergé à partir du groupe 4, alors que les groupes 1 et 2 sont les groupes qui ont émergé plus récemment. Dans cette thèse, nous avons montré que la concaténation des séquences des trois gènes (glnII, nifH et gyrB) semble être un outil puissant pour une meilleure étude évolutive afin de contourner l'influence du phénomène de transfert horizontal des gènes sur la phylogénie du genre Frankia. Par ailleurs, nous avons remarqué que la faible variabilité génétique est associée à la régression de taille des génomes de Frankia et coïncide avec des transitions de mode de vie symbiotique et une répartition géographique restreinte (le cas des Frankia–Casuarina et des Frankia non cultivables du groupe 2). Dans un second temps, nous avons focalisé nos recherches sur le modèle Frankia-Coriaria. Nous avons défini quatre groupes de Frankia endosymbiotes et deux groupes pour les Coriaria en se basant sur les séquences de trois marqueurs, glnA (glutamine synthétase), dnaA (amorceur de réplication des chromosomes) et l'IGS nifD-K (l'espace intergénique entre les gènes nifD et nifK codant pour les sous-unités alpha et beta de la protéine molybdène-fer) pour les Frankia microsymbiotes et deux régions d'ADN, matK (maturase chloroplastique) et ITS1- 2 (ARNr 18S - ITS1 - ARNr 5.8S - ITS2 –ARNr 28S) pour la plante hôte. L'analyse phylogénétique de deux partenaires symbiotiques, Frankia et son hôte respectif, montre l'absence de cospéciation. Ce résultat est cohérent avec celui de dernier chapitre dont nous avons montré, pour la première fois, l'occurrence de Frankia compatibles avec Coriaria dans un sol tunisien, dépourvu de la plante hôte depuis plus de deux siècles. Ce résultat est un bon argument de l'indépendance de Frankia microsymbiote de la plante hôte Coriaria et met en question la non cultivabilité des Frankia du groupe 2 / Frankia is an actinobacterium best known for its ability to fix molecular nitrogen and infect the roots of 8 actinorhizal plant families. The Taxonomy and the phylogeny of the Frankia genus remain incomplete and have to be more explored. The objective of this thesis is to provide additional knowledge on the phylogeny and evolution of different Frankia groupes. Firstly, the molecular phylogeny based on the analysis of glnII, gyrB, nifH genes, and 16S–23S rRNA internally transcribed spacer (ITS) sequences was carried out. The result of this study emphasized the presence of four Frankia clusters: (i) cluster 1 for Frankia associated with Betulaceae, Myricaceae and Casuarinaceae (ii) cluster 2 contains Frankia microsymbionts associated with Coriariaceae, Datiscaceae, Rosaceae and Ceanothus (Rhamnaceae), (iii) cluster 3 for Frankia of Elaeagnaceae, Rhamnaceae, Myricaceae and Gymnostoma (Casuarinaceae) and (iv) cluster 4 including atypical Frankia strains that are non-infective and/or non-nitrogen-fixing was positioned at a deeper branche followed by groupes 3. While clusters 1 and 2 appeared to have diverged more recently. The present study demonstrates the utility of phylogenetic analyses based upon concatenated gyrB, nifH and glnII sequences to resolve previously unresolved or poorly resolved nodes and will help describing species among the genus Frankia. The variation of the average pairwise distance within and between the clusters allows us to suggest a gradual erosion of Frankia diversity concomitantly with a shift from saprophytic non infective/non-effective to facultative and symbiotic lifestyle. Then, we focused on the cluster 2 of non-culturable Frankia in general and special focus on Frankia associated with Coriaria. The absence of cospeciation between the uncultured Frankia microsymbionts and the disjunct actinorhizal Coriaria species has been shown. These results were obtained following analyze of three bacterial genes; glnA (glutamine synthetase), dnaA (chromosome replication initiator) and the nifD-K IGS (intergenic spacer between genes coding respectively for nitrogenase molybdenum-iron alpha and beta subunits) and two DNA region of the host plants; matK (chloroplast-encoded maturase K) and the intergenic transcribed spacers (nuclear-encoded 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA).This result is consistent with the last chapter in which we showed, for the first time, the occurrence of compatible Frankia with Coriaria in a Tunisian soil, devoid of the host plant for more than two centuries. This represents a first argument for the independence of Frankia nodulating Coriaria to their host plants

Haloferax mediterranei es un microorganismo perteneciente al Dominio Archaea que fue aislado por primera vez en las Salinas de Santa Pola, Alicante. Esta arquea halófila es capaz de crecer con glucosa como única fuente de carbono y con nitrato como única fuente de nitrógeno. En el interior celular, el nitrato se convierte en amonio mediante la nitrato y nitrito reductasas asimilativas. En función de su disponibilidad intracelular el amonio puede asimilarse mediante dos vías: a altas concentraciones de amonio, este se asimila mediante la vía de la glutamato deshidrogenasa (GDH), mientras que a bajas concentraciones de amonio, este se asimila por el ciclo glutamina sintetasa (GS)-glutamato sintasa (GOGAT). La GS (EC 6.3.1.2) es una enzima clave tanto en la asimilación de amonio como en la biosíntesis de glutamina y de otros aminoácidos. Está presente en todos los Dominios de la vida, considerándose como un buen reloj molecular de la evolución ya que su secuencia muestra homología en todos los organismos. La familia de la GS está dividida en tres clases (GSI, GSII y GSIII) dependiendo de su secuencia, el gen que la codifique y del organismo al que corresponda. El genoma de Hfx. mediterranei contiene tres marcos de lectura abiertos que muestran homología con la GS y están codificados por los genes glnA-1, glnA-2 y glnA-3. Los genes glnA-2 y glnA-3 se encuentran localizados muy próximos en el genoma, únicamente separados por 1,6 kb, y a su vez dichos genes están separados del gen glnA-1. La proteína codificada por el gen glnA-1 tiene una identidad del 51,9% y del 49,1% con las proteínas codificadas por los genes glnA-2 y glnA-3, respectivamente. Las proteínas GlnA-2 y GlnA-3 muestran una identidad entre sí de un 60,9%. La proteína GlnA-1 mantiene parcial o completamente conservadas las tres secuencias consenso características de las GS, mientras que las proteínas GlnA-2 y GlnA-3 únicamente mantienen parcialmente conservada una de ellas (putative ATP_binding región signature PS00181). Además, en base a la presencia de los dominios conservados se determinó que las tres proteínas GlnA de Hfx mediterranei presentan el dominio catalítico PF00120 (Gln-synt_C) utilizado para la identificación de las GS tipo l. Analizando los aminoácidos presentes en cada una de las tres proteínas GlnA, se identificó que, de los 18 residuos aminoacídicos característicos de las GSI conservados universalmente, la secuencia de la proteína GlnA-1 presenta todos ellos, además del residuo de adenilación. Sin embargo, 8 de los residuos clave para la biosíntesis de glutamina se encuentran sustituidos por otros en las proteínas GlnA-2 y GlnA-3, careciendo además ambas proteínas del residuo de adenilación. El análisis del transcriptoma en función de la fuente de nitrógeno de la cepa mutante de deleción del gen glnA-1 (HM26-ΔglnA-1) reveló que los genes glnA-2 y glnA-3 no pueden sustituir la función del gen glnA-1. Además, estos últimos genes presentaron un perfil de expresión diferente al de glnA-1 en la cepa parental de Hfx. mediterranei HM26. En condiciones limitantes de nitrógeno, la deleción del gen glnA-1 afectó al nivel de expresión de genes involucrados en diferentes procesos metabólicos perteneciendo en su mayoría al metabolismo del nitrógeno, al metabolismo de las vesículas de gas y los relacionados con los sistemas CRISPR, sistemas de transporte y con reguladores transcripcionales. El estudio de expresión a nivel transcripcional en función de la fuente de nitrógeno de los genes glnA mediante RT-PCR reveló que los genes glnA-1 y glnA-2 se expresan en todas las condiciones analizadas (medio complejo, medio definido con amonio o nitrato 40 mM y en medio definido carente de nitrógeno) mientras que el gen glnA-3 no se expresa en estas condiciones. El estudio de expresión a nivel traduccional en función de la fuente de nitrógeno de las proteínas GlnA mediante Western blotting confirmó que la proteína GlnA1 se expresa en todas las condiciones analizadas (en medio complejo, y en los medios definidos con amonio, nitrato o glutamina 40 mM y en medio definido carente de fuente de nitrógeno) durante todas las etapas de crecimiento analizadas. Del mismo modo, este análisis de expresión reveló que la proteína GlnA-2 se expresa en todas las condiciones analizadas excepto al emplear medio complejo, pudiendo estar implicado algún mecanismo de regulación postranscripcional en la expresión de la proteína GlnA-2 en esta condición. Asimismo, la proteína GlnA-3 no mostró expresión en ninguna de estas condiciones analizadas ni en presencia de otras fuentes de nitrógeno (glutamato) o de carbono (citrato) ensayadas. La proteína recombinante GlnA-1, obtenida mediante expresión heteróloga, mostró una mayor actividad GS que las proteínas GlnA-2 y GlnA-3. Sin embargo, las proteínas recombinantes GlnA-2 y GlnA-3 mostraron valores elevados de actividad y-glutamil putrescina sintetasa mientras que GlnA-1 solo tiene cierta actividad residual cuando se emplea putrescina como sustrato. Los niveles de actividad y-glutamil putrescina sintetasa de las proteínas GlnA-2 y GlnA-3 son unas 10 veces superiores a los valores de actividad GS. En los extractos de Hfx. mediterranei R4 crecidos en presencia de nitrato 40 mM como fuente de nitrógeno, en los cuales se expresan tanto la proteína GlnA-1 como GlnA-2, se observó una actividad GS mayor a la detectada en los extractos obtenidos a partir medio complejo. Por otra parte, Hfx. mediterranei R4 fue capaz de crecer con putrescina como única fuente de nitrógeno a diferentes concentraciones (20 - 250 mM), aunque las densidades ópticas alcanzadas fueron menores que las observadas al emplear otras fuentes de nitrógeno. En extractos proteicos obtenidos a partir de cultivos de Hfx. mediterranei empleando nitrato 40 mM o putrescina a diferentes concentraciones la actividad y-glutamil putrescina sintetasa fue mucho mayor a la detectada al emplear medio complejo como fuente de nitrógeno, condición donde la expresión de la proteína GlnA-2 no fue detectada. Para completar el análisis de las proteínas GlnA-2 y GlnA-3 se generaron mutantes de deleción de los genes glnA-2 y glnA-3 a partir de la cepa parental de Hfx. mediterranei HM26 (ΔpyrE2) mediante la técnica pop-in/pop-out. Los mutantes obtenidos del gen glnA-2 resultaron ser mutantes homocigotos mientras que los mutantes del gen glnA-3 resultaron ser heterocigotos. La cepa mutante HM26-ΔglnA-2 se caracterizó fisiológicamente en diferentes medios de cultivos: medio complejo y medio definido con amonio, nitrato o glutamina como fuentes de nitrógeno. El análisis estadístico de los parámetros de crecimiento reveló que existían diferencias de crecimiento significativas entre la cepa parental HM26 y la cepa mutante HM26-ΔglnA-2 al emplear altas concentraciones de amonio y/o nitrato. El análisis de expresión a nivel traduccional de la cepa mutante HM26-ΔglnA-2 confirmó que la deleción del gen glnA-2 no afectó a la expresión de la proteína GlnA-1 ni a la ausencia de expresión de GlnA-3. Sin embargo, la deleción del gen glnA-2 afectó de forma significativa a la actividad y-glutamil putrescina sintetasa al emplear nitrato 40 mM como fuente de nitrógeno, confirmando la función del gen glnA-2 como una y-glutamil putrescina sintetasa más que una glutamina sintetasa.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.

The goal of this project was to develop novel bacterial biomarkers for use in an industrial context. These biomarkers would be used to determine aluminium industry activity impact on a local ecosystem. Sediment bacterial communities of the Saguenay River are subjected to industrial effluent produced by industry in Jonquière, QC. In-situ responses of these communities to effluent exposure were measured and evaluated as potential biomarker candidates for exposure to past and present effluent discharge. Bacterial community structure and composition between control and affected sites were investigated. Differences observed between the communities were used as indicators of a response to industrial activity through exposure to effluent by-products. Diversity indices were not significantly different between sites with increased effluent exposure. However, differences were observed with the inclusion of algae and cyanobacteria. UniFrac analyses indicated that a control (NNB) and an affected site (Site 2) were more similar to one another with regard to community structure than either was to a medially affected site (Site 5) (Figure 2.4). We did not observe a signature of the microbial community structure that could be predicted with effluent exposure. Microbial community function in relation to bacterial mercury resistance (HgR) was also evaluated as a specific response to the mercury component present in sediments. Novel PCR primers and amplification conditions were developed to amplify merP, merT and merA genes belonging to the mer-operon which confers HgR (Table 5.6). To our knowledge, the roles of merP and merT have not been explored as possible tools to confirm the presence of the operon. HgR gene abundance in sediment microbial communities was significantly correlated (p < 0.05) to total mercury levels (Figure 3.4) but gene expression was not measurable. We could not solely attribute the release of Hg0 from sediments in bioreactor experiments to a biogenic origin. However, there was a 1000 fold difference in measured Hg0 release between control and affected sites suggesting that processes of natural remediation may be taking place at contaminated sites (Figure 3.7). Abundance measurements of HgR related genes represent a strong response target to the mercury immobilized in sediments. Biomarkers built on this response can be used by industry to measure long term effects of industrially derived mercury on local ecosystems. The abundance of mer-operon genes in affected sites indicates the presence of a thriving bacterial community harbouring HgR potential. These communities have the capacity to naturally remediate the sites they occupy. This remediation could be further investigated. Additional studies will be required to develop biomarkers that are more responsive to contemporary industrial activity such as those based on the integrative oxidative stress response.