El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Based on the review of different research studies, we could assess that, due to their unique biological features, microbes, specifically bacteria, could be used to repair damaged soils with heavy metal and toxic compound contents. Furthermore, these microorganisms are metabolically capable to oxidize cyanide and its by-products to generate less-toxic compounds at the end of the process. This research proposal seeks to improve conventional mine closure designs, thus counteracting their negative short-term, medium-term, and long-term after-effects to the environment. The proposed technique as a solution, therefore, is microbial remediation, using pseudomonas fluorescens bacteria to oxidize this compound to non-toxic components. It will ensure operational continuity for the deposits and, in turn, the sustainability of the entire mining industry.

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Industrial waste pollution is a problem of considerable magnitude and of great importance to modern industry. One critical aspect of waste pollution is reduction of dissolved oxygen in natural waters resulting from the oxygen uptake of bacteria while digesting organic wastes. Fundamental information concerning the effect of various physical and chemical factors on the rate of oxygen uptake by bacteria was sought by investigating the rate of oxygen uptake by Pseudomonas fluorescens, a common water organism, as a function of the concentration of substrate and the temperature of environment. The substrates were D-glucose, D-xylose, L-arabinose and acetate, and the temperatures investigated ranged from 15 to 37 ºC. Oxygen uptakes were determined manometrically using the direct Warburg method in conjunction with resting cell techniques. In all tests 3.0 milligrams or Pseudomonas fluorescens (stated as dry bacterial protoplasm) were suspended in 2.5 milliliters of 0.05 molar phosphate buffer of pH 6.8. At 25 °C, for all substrates with the exception of acetate, the rate of oxygen uptake is dependent on concentration in the lower concentration ranges, and follows the Michaelis-Menten equation. Saturation concentrations stated as millimoles per test were 0.0500 for glucose and 0.600 for xylose. Saturation had not been reached in the case or arabinose at the highest concentration tested of 0.8000 millimoles per test. The rate or oxygen uptake increased with acetate concentration up to 0.1000 millimoles per test, and at concentrations above this value a decrease in rate of uptake was observed. A special equation was developed to cover the latter case. When substrate concentrations were held constant and temperature of the environment was varied from 15.0 to 37.0 °C the rate of oxygen uptake increased with temperature in all cases. For glucose the rate of change of the rate of oxygen uptake was 15.8 ± 1.6 microliters per hour per degree centigrade between 15.0 and 20.0 °C, 2.4 ± 0.2 microliters per hour per degree centrigrade between 20.0 and 25.0 °C, and 11.7 ± 1.2 microliters per hour per degree centigrade between 25.0 and 37.0 °C. When glucose concentration was held constant at 0.1000 millimole. For xylose the rate of change of the rate of oxygen uptake was 18.0 ± 1.8 microliters per hour per degree centigrade between 15.0 and 18.0 ºC, 3.9 ± 0.4 microliters per hour per degree centigrade between 18.0 and 35.0 °C, and 16.5 ± 1.6 microliters per hour per degree centigrade between 35.0 and 37.0 ºC when xylose concentration was held constant at 0.400 millimole. For arabinose the rate of change of the rate of oxygen uptake was 10.4 ± 1.0 microliters per hour per degree centigrade between 15.0 and 17.5 °C, 0.4 ± 0.0 microliters per hour per degree centigrade between 17.5 and 30.0 ºC, and 2.4 ± 0.2 microliters per hour per degree centigrade between 30.0 and 37.0 ºC when arabinose concentration was held constant at 0.400 millimole. For acetate the rate of change of the rate of oxygen uptake was 19.7 ± 2.0 microliters per hour per degree centigrade between 15.0 and 18.0 ºC, 2.8 ± 0.3 microliters per hour per degree centigrade between 18.0 and 28.0 °C, and 12.5 ± 1.5 microliters per hour per degree centigrade between 28.0 and 37.0 °C when the acetate concentration was held constant at 0.0500 millimole. / Ph. D.

One of the challenges in biology is placing a function on the myriad of gene sequences having become available from rapid advances in genome sequencing. One such example is a gene cluster (Nit1C) found in bacteria that is tied to the unusual ability of certain bacteria to grow when supplied cyanide as the sole nitrogen source. The term cyanotrophs has been applied to such bacteria, for which a genetic linkage between cyanotrophy and Nit1C was demonstrated for 10 separate bacteria. In addition to growth, cyanide induced the expression of Nit1C genes in all organisms tested, and in one case, deletion of one of the Nit1C genes (nitC) caused a loss of growth. Of the ten bacteria able to grow cyanotrophically, all gave evidence of harboring Nit1C on their genome except for two (Pseudomonas fluorescens Pf11764 and P. monteilii BCN3), which were sequenced and the presence of Nit1C was also confirmed. A broader search of bacteria identified 270 separate strains with the cluster, all limited to bacteria spanning the phyla Firmicutes, Actinobacteria, Proteobacteria and Cyanobacteria. Remarkably, many examples of a single representative of a given taxon contained Nit1C, most poignantly displayed by Pf11764 and PmBCN3; the interpretation being the cluster was likely acquired by horizontal gene transfer in response to cyanide as an environmental cue. Consistent with its absence in Archaea is the time line for the emergence of cyanide producing organisms (cyanogens) on earth dating back only 400-500 million years.

Les polluants atmosphériques de type oxydes d’azote (NOx), principalement constitués du NO, NO2 et leurs dérivés, représentent une énorme menace d’un point de vue environnemental et sanitaire. Leurs propriétés chimiques sont largement exploitées à l’échelle du vivant pour leur rôle dans divers processus de signalisation (systèmes nerveux et cardiovasculaire) ou l’élimination de pathogènes (système immunitaire). Néanmoins, des dérégulations dans la production cellulaire ou l’apport exogène de ces composés est à l’origine de nombreuses pathologies humaines (e.g. pulmonaires), généralement attribuées à la pollution. Toutefois, un grand nombre de microorganismes aéroportés sont continuellement exposés à ces composés délétères, intimement connectés aux espèces réactives de l’oxygène (ROS). Ainsi l’hypothèse de l’ensemble de ce travail a porté sur l’impact du NO2, NOx majoritairement retrouvés dans l’atmosphère, sur une souche aéroportée de P. fluorescens, espèce désormais associée aux voies aériennes et potentiellement pathogène. A l’issue d’une exposition à 45 ppm de NO2, la survie de P. fluorescens MFAF76a est significativement impactée suggérant un effet bactériostatique, conforté par l’impact observé sur le métabolisme énergétique. De plus, le NO2 induit un stress d’enveloppe via la perte d’un glycérophospholipide (UGP) et le remaniement de divers composants membranaires (LPS, peptidoglycane, acides gras). La pompe à efflux MexEF-OprN semblent participer à la stabilisation de la membrane et pourraient être également impliquée dans l’efflux des oxydes d’azotes, mécanismes confortés par l’étude d’un mutant MFAF76a-oprN. La porine majoritaire OprF semble également contribuer à la stabilisation de la membrane externe, néanmoins son implication reste à confirmer. De plus, une interconnexion entre ROS et NOx dans la signalisation (OxyR, IscR), et les mécanismes de détoxification, a été observée. La flavohémoprotéine Hmp semble être un élément crucial dans la détoxification des NOx chez P. fluorescens comme l’illustre un mutant MFAF76a-hmp. Les similitudes importantes entre les effets connus du NO et ceux observés lors d’une exposition au NO2 suggèrent une conversion non enzymatique du NO2, une fois pénétré dans la cellule, en NO. Désormais, une étude plus approfondie est nécessaire afin de décrypter (i) les mécanismes impliqués dans la régulation de la pompe à efflux RND MexEF-OprN et de la flavohémoprotéine Hmp, (ii) d’autres acteurs intervenant dans la réponse au stress d’enveloppe et la détoxification ainsi que (iii) le devenir de NO2 dans la cellule. / Nitrogen oxides (NOx) atmospheric pollutants, mainly constituted of NO, NO2 and derived compounds, are a big threat to the environment and health. Their chemical properties are largely exploited at the cellular scale for their role in diverse physiological processes such as signalisation (nervous and cardiovascular systems) or in pathogens eradication (immunity system).However, dysregulation in production pathways or exogenous input of these compounds lead to several pathologies (e.g. respiratory diseases), usually attributed to atmospheric pollution. However, a wide range of airborne microorganisms are constantly exposed to these deleterious compounds, intimately connected to reactive oxygen species (ROS). Thus, the hypothesis of this work deals with the impact of NO2, the main atmospheric NOx, on an airborne P. fluorescens, a strain usually neglected but yet associated with human airways, and potentially pathogenic. Following an exposure to 45 ppm of NO2, the survival of P. fluorescens MFAF76a is severely impaired, suggesting a bacteriostatic effect, as comforted by NO2 impact on energetic metabolism. Moreover, an exposure to NO2 induces an envelope stress through the loss of an Unknown Glycerophospholipid (UGP) and the reorganisation of membrane constituents (LPS, peptidoglycan, fatty acids). The efflux pump MexEF-OprN is involved in membrane stabilization and could also efflux NOx, as highlighted by a MFAF76a-oprN mutant. The major porin OprF could also contribute in external membrane stabilisation, however its implication is still under investigation. Moreover, ROS and NOx are interconnected as illustrated by their shared signalisation (OxyR, IscR) and detoxification pathways. The flavohemoprotein Hmp is a crucial element in the detoxification of NOx in P. fluorescens as illustrated in an MFAF76a-hmp mutant. The similarities between the known effects of NO and those observed in the case of an exposure to NO2, suggest a non-enzymatic conversion of NO2, following cell penetration, into NO. Henceforth, deeper studies are required to decode (i) the mechanisms involved in the regulation of the RND efflux pump MexEF-OprN and the flavohemoprotein Hmp, (ii) other relevant actor implicated in the envelope stress response and in detoxification pathways as well as (iii) the fate of NO2 within the cell.

Le système de sécrétion de type VI (SST6) est un complexe multi-protéique permettant l’export d’effecteurs. Ce mécanisme est impliqué à la fois dans la virulence envers les cellules eucaryotes, dans l’activité antibactérienne mais également dans l’acquisition d’ions présents dans ’environnement. Ainsi, le SST6 joue un rôle important dans l’adaptation et la compétition, éléments essentiels dans la colonisation et la persistance au sein d’une niche écologique. Actuellement, très peu d’études portent sur l’importance du SST6 chez des souches environnementales, contrairement aux nombreuses études portant sur des pathogènes tels que Pseudomonas aeruginosa, Burkholderia thailandensis, Vibrio cholerae ou Escherichia coli. Mon sujet de recherche avait pour objectif de caractériser le ou les rôles du SST6 de la souche environnementale Pseudomonas fluorescens MFE01. Ces travaux ont permis d’appréhender certaines fonctions du SST6 de cette souche. Le génome de MFE01 ne comporte qu’un seul cluster de gènes du SST6 où sont regroupés les gènes codant pour la machinerie du SST6 (le « core-component ») à l’exception des gènes hcp. Les protéines Hcp sont des éléments structuraux du SST6 dont elles forment le tube interne qui permet le transfert des effecteurs. Différents gènes hcp sont disséminés sur le chromosome et parmi ces « hcp » orphelins, hcp2 et hcp3 codent respectivement pour les protéines Hcp2 et Hcp3. Ces deux Hcp sécrétées par le SST6, sont associées à l’activité antibactérienne de MFE01 sur différentes souches pathogènes et environnementales, tels que P. aeruginosa, P. fluorescens MFN1032 et Pectobacterium atrosepticum. La protéine Hcp1, codée par le gène orphelin hcp1, est impliquée dans l’inhibition de mobilité de souche compétitrice. Hcp1 permettrait la sécrétion d’au moins deux toxines qui perturberaient l’assemblage du flagelle. Chez MFE01Δhcp1 et MFE01ΔtssC (TssC est un élément de la gaine contractile du SST6), ces toxines seraient accumulées dans le cytoplasme, inhibant ainsi ’assemblage de leur propre flagelle. La surproduction du régulateur FliA, qui contrôle notamment l’assemblage du filament flagellaire, restaure la mobilité chez ces deux mutants. En parallèle, le SST6 de la souche MFE01 est essentiel à la formation et la maturation de biofilm mais également à la compétition bactérienne en biofilm mixte. Ce système interviendrait dans la communication bactérienne indispensable au comportement social, requis lors de l’élaboration des biofilms. / Type VI secretion system (T6SS) is a multiproteic apparatus that secreted proteinaceous effectors. T6SS participate in a variety of functions, whose eukaryote virulence, antibacterial activity or metal ion uptake. These capacities conferring an advantage in adaptation and competition, crucial to colonization or persistence within ecological niche. As well, only a few studies have focused on the T6SS functions of environmental strains, contrary to numerous studies dealing with pathogens as Pseudomonas aeruginosa, Burkholderia thailandensis, Vibrio cholerae or Escherichia coli. The purpose of my research project was to characterize the T6SS function(s) of the environmental strain Pseudomonas fluorescens MFE01. This work had led to understand the various functions of T6SS of MFE01 strain. This strain has a single T6SS cluster where all the core component proteins were gathered, except hcp genes. Three orphan hcp genes where found and are scattered in genome. Hcp proteins form the inner tube allowing effectors secretion. Both Hcp2 and Hcp3 proteins were involved in antibacterial activity on pathogens or environmental strains like P. aeruginosa, P. fluorescens or Pectobacterium atrosepticum. Characterization of Hcp1 proteins role constituted a major focus of this project. Hcp1 proteins participate to motility inhibition of competitive strains through T6SS. Hcp1 may be associated with secretion of at least two toxins perturbing the flagellar filament assembly. In MFE01Δhcp1 and MFE01ΔtssC mutants (Tss is a contractile sheath constituent), these toxins may be accumulated into cytoplasm and perturb assembly of their own flagella. Interestingly, overproduction of FliA flagellar regulator, which controls assembly of flagellar filament, restores motility of both mutants. Simultaneously, T6SS of MFE01 strain contributes to maturation and biofilm formation but also in bacterial competition within mixed biofilm. T6SS may be a mean of bacterial communication and thus coordinate a social behavior, primordial for biofilm formation.

Les Streptomyces sont des bactéries communes des écosystèmes forestiers tempérés. Elles sont présentes au sein de différentes niches écologiques telles que la rhizosphère des plantes, la mycorhizosphère ou encore la minéralosphère. Dans ces niches, elles interagissent avec de nombreux autres genres bactériens et différents champignons symbiotiques, pathogènes et saprophytes. Nous avons entrepris une étude sur l'interaction entre deux souches bactériennes modèles du sol Streptomyces ambofaciens ATCC23877 et Pseudomonas fluorescens BBc6R8. Des expériences de cocultures sur différents milieux gélosés ont révélé que chaque souche bactérienne affecte la production de métabolites secondaires chez l'autre partenaire. Ainsi, P. fluorescens inhibe la capacité de S. ambofaciens à produire la kinamycine, un « effet ping-pong » a été mis en évidence. D'autre part, sur un milieu déficient en fer, P. fluorescens est capable d'utiliser les desferrioxamines et la coelicheline produites par S. ambofaciens en tant que xénosiderophores et ne produit alors plus ses propres sidérophores, la pyoverdine et l’énantio-pyochéline. Enfin, au cours des études d’interactions, P. fluorescens BBc6R8 s’est révélée capable d’inhiber la production du pigment bleu diffusible γ-actinorhodine chez Streptomyces coelicolor A3(2) M145 en acidifiant le milieu par la production d’acide gluconique. L’ensemble de ces résultats nous permet de proposer différentes hypothèses sur l’importance écologique de ces trois interactions en les replaçant au sein du contexte de l’écosystème sol / Streptomyces are common bacteria in temperate forest ecosystems, which inhabit various ecological niches such as plant rhizosphere and mycorrhizosphere, as well as mineralosphere. In these niches, they interact with many other bacterial genera and different symbiotic, pathogenic and saprotrophic fungi. We have initiated a study on interaction between two soil model bacteria strains Streptomyces ambofaciens ATCC23877 and Pseudomonas fluorescens BBc6R8. Cocultures experiments on different agar media have revealed that each bacteria affects the production of secondary metabolites in the other partner. Thus, P. fluorescens inhibits the ability of S. ambofaciens to produce kanamycin, a "ping-pong effect" was highlighted. On the other hand, on iron deficient medium, P. fluorescens is able to use the desferrioxamines and coelichelin produced by S. ambofaciens as xenosiderophores and does no longer produce its own siderophores, pyoverdin and enantio-pyochelin. In another way, during pairwise co-culture experiments, Pseudomonas fluorescens BBc6R8 was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas strains and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. Altogether these results prompt us to propose some hypotheses on the ecological significance of these three interactions by replacing them in the soil ecosystem context