Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Objetivou-se com esse trabalho selecionar bactérias residentes de filoplano de maracujazeiro como possível agente de biocontrole da mancha-bacteriana que tem como agente causal a bactéria Xanthomonas axonopodis pv. passiflorae (Xap) e estudar os mecanismos de ação envolvidos no controle biológico. A primeira etapa de seleção baseou-se em testes in vivo com 224 isolados obtidos a partir de folhas sadias de maracujazeiro amarelo coletadas em pomares comerciais, sendo 102 oriundos do estado de Roraima, 72 do Pará e 50 de São Paulo. Estes foram testados em casa-de-vegetação no controle das bactérias fitopatogénicas originadas dos estados de Roraima (Xap-RR), São Paulo (Xap-SP) e Pará (Xap-PA). Após esta etapa, foram selecionados os residentes de filoplano RR-46; RR-29; RR-133; RR-98 e RR-14, oriundo de Roraima e SP-11; SP-18; SP-22; SP-48 e SP-28, provenientes de São Paulo devido apresentarem baixos níveis de severidade da doença 2,3; 2,8; 2,9; 3,5; 3,6; 3,2; 3,25; 3,55; 4,25 e 6,25%, respectivamente. Não houve nenhum antagonista detectado como promissor proveniente do estado do Pará. Foram selecionados os isolados RR-14; RR-29; RR-46; RR-98; RR-133; SP-11; SP-18; SP-22; SP-28 e SP-48, para a segunda etapa de seleção. Esta realizada, in vitro onde os isolados foram submetidos aos ensaios para verificação da utilização de fontes únicas de carbono para verificação de sobreposição de nicho, antibiose por difusão em meio de cultura, produção de sideróforos e influencia na atividade da enzima peroxidase na planta. Os resultados demonstraram que os isolados RR-98 e RR-113 foram capazes de competir por nicho somente contra Xap-RR, através da sobreposição de nicho. Na antibiose, por difusão em meio de cultura o isolado RR-29, foi capaz de inibir três isolados de Xap, provenientes de RR, SP, PA e o isolado SP-28 inibiu apenas as duas últimas. A produção de sideróforos foi observada somente pelos isolados RR-29 e SP-28. Nenhum antagonista foi capaz de influenciar no aumento da atividade de peroxidases nas plantas de maracujazeiro o que indica que não são capazes de induzirem resistência. A sobreposição de nicho, competição por ferro e/ou antibiose são fatores que explicam a capacidade de controle da mancha-bacteriana, mediadas pelos isolados RR-98 e RR-133, RR-29 e SP-28. / The mass selection in vivo is a step of great importance that there be no targeting of biological control mechanisms involved, but only about the efficacy of the biocontrol agent. The objective of this work was to select residents phylloplane bacteria passion fruit as possible biocontrol agent of bacterial spot which is the causal agent Xanthomonas axonopodis pv. passiflorae. 224 isolates were obtained from healthy leaves of yellow passion fruit collected from commercial orchards, and 102 from the state of Roraima, 72 from Pará and 50 from São Paulo. The isolation was done using a half of definitive leave soaked in sterile saline (0.85% NaCl) with 0,3% Tween 80 and maintained in a shaker for 20 minutes, followed by serial dilutions and seeding in Petri dishes containing culture medium 523. Four steps to select for isolates of Roraima, three for isolates from São Paulo and three isolates from Pará were conducted in greenhouse so there would be a confirmation of the antagonistic efficacy of these isolates. Disease severity ranged between 1,1% and 15,3% for isolates from Roraima, 1,15% to 27,5% for isolates from São Paulo and 1,5% to 36,4% for isolates Pará were selected residents phylloplane RR-43, RR-29, RR-133, RR-98 and RR-14, derived from Roraima and SP-11, SP-18, SP-22, SP-48 and SP-28, coming from São Paulo because they presented low levels of disease severity 2,3; 2,8; 2,9; 3,5; 3,6; 3,2; 3,25; 3,55; 4,25 and 6.25%, respectively. There was no antagonist detected from Pará. Were selected isolates RR-43, RR-29, RR-133, RR-98, RR-14 SP-11, SP-18, SP-22, SP-48 and SP-28, for in vitro assays.

Les circuits et réseaux d’eau subissent épisodiquement des problèmes de contamination par des microorganismes tels que Legionella pneumophila, conduisant à la dégradation de la qualité microbiologique de l’eau circulante. De nouveaux moyens de traitements doivent être mis en place de façon notamment à limiter l’usage de biocides chimiques. Ce travail de thèse s’inscrit dans cette problématique et a pour objectif d’identifier de nouveaux composés actifs contre L. pneumophila en tenant compte de son comportement et de ses interactions au sein de son microenvironnement. De manière à obtenir des composés produits par des souches bactériennes appartenant à la même niche écologique que L. pneumophila, une campagne de prélèvement d’eau a été réalisée. Un total de 273 isolats environnementaux ont été isolés et testés contre L. pneumophila. Les résultats obtenus indiquent que 178 de ces isolats (65%) présentent une activité anti-Legionella. Quatre souches ont ensuite été sélectionnées (Aeromonas bestiarum SW257, Rahnella aquatilis SW265, Flavobacterium spp. PW52 et Pseudomonas spp PW329) et les composés actifs produits ont été caractérisés. A. bestiarum SW257 produit un peptide anti-Legionella. Flavobacterium sp. PW52 produit un mélange de composés anti-Legionella ayant des propriétés tensioactives, les flavolipides. Pseudomonas sp. PW329 produit des composés organiques volatiles, identifiés par SPME-GC-MS, actifs contre L. pneumophila. Enfin, R. aquatilis SW265 produit un sidérophore à activité anti-Legionella. / Water is essential to sustain life and water sources used for human consumption must be biologically safe, to avoid any risk for health. Indeed, the most common and widespread health risk associated with drinking water are infectious diseases caused by pathogenic microorganisms such as Legionella pneumophila. However, more efforts are needed to control disinfection by-products and minimize people exposure to potentially hazardous chemicals while maintaining adequate disinfection to ensure good water quality. Thus, this work aimed to find natural antibacterial compounds to control L. pneumophila growth using bacteria from freshwater environments. Environmental aquatic bacteria were sampled from five freshwater sources to get a large culturable bacterial collection. A total of 273 bacterial isolates were recovered and screened for their ability to produce anti-Legionella compounds. Among those, 178 (65%) were shown to be active against L. pneumophila. Four strains (Aeromonas bestiarum SW257, Rahnella aquatilis SW265, Flavobacterium spp. PW52, and Pseudomonas spp PW329) were next selected for the characterization of their active compounds. A. bestiarum SW257 produces an anti-Legionella peptide, and Flavobacterium spp PW52 produces a mixture of anti-Legionella compounds with surface active properties, named flavolipids. Finally Pseudomonas sp. PW329 delivers many volatile organic compounds, and R. aquatilis SW26 produces a anti-Legionella siderophore.

L’apport de fer est essentiel pour la plupart des organismes vivants, incluant la majorité des bactéries pathogènes. Cependant, le fer libre est toxique : il est lié à des protéines de stockage et de transport (e.g. ferritine, hémoprotéines…) et voit son homéostasie finement régulée. Afin d’extraire le fer de ces protéines, les bactéries utilisent divers systèmes tels que des protéines de surface ou encore des sidérophores. Bacillus cereus est une bactérie Gram-positive sporulante, pathogène opportuniste chez l’homme, 2ème cause en France de toxi-infection alimentaire collective. Chez B. cereus, la protéine de surface IlsA et le sidérophore bacillibactine (BB) sont impliqués dans l’acquisition du fer de la ferritine exogène et elles sont importantes pour l’infection de l’insecte modèle Galleria mellonella. Mes travaux présentaient deux parties : tout d’abord, l’étude de l’import du complexe BB-Fe3+ dans la cellule par FeuA, protéine de liaison de ce complexe à la surface de la bactérie, souligne le rôle central du couple BB-FeuA. La délétion des gènes codants pour ces deux molécules limite l’acquisition par B. cereus du fer de la ferritine, de l’hème, de l’hémoglobine et du fer inorganique in vitro. En revanche, elle présente un phénotype de virulence in vivo comparable à la souche de référence dans le cas d’injection intra-hémocœlique de larves de G. mellonella. Ce résultat surprenant suggère un probable rétrocontrôle sur l’expression de facteurs de virulence lorsque B. cereus ne produit ni BB ni FeuA, et se trouve par conséquent fortement carencé en fer. Le second volet de mes travaux s’intéresse à l’expression des gènes liés à l’homéostasie du fer in vivo, au cours de l’infection de l’intestin de larves de G. mellonella axéniques. Nous avons choisi une approche de type microgénomique, en prélevant les échantillons par microdissection laser, sur de façon à prélever de petits échantillons dans une zone définie, puis en analysant l’expression de quelques gènes ciblés par RT-qPCR et ddPCR à 3h et 16h post ingestion. Nos résultats montrent que : i) la colonisation intestinale de G. mellonella est impactée lorsque B. cereus est dépourvu du couple BB-FeuA ; ii) ilsA est exprimé lors de l’infection intestinale ; iii) les gènes ciblés impliqués dans l’homéostasie du fer sont activés dès le début de l’infection, suggérant un rôle dans l’adaptation et la pathogénicité ; iv) une faible modulation de l’expression est observée entre les deux temps. Ces travaux ouvrent de nouvelles connaissances fondamentales sur l’homéostasie du fer et des perspectives quant à l’utilisation de nouvelles techniques pour l’étude in situ des interactions hôte-pathogène. / Iron acquisition is essential for most living organisms, including many pathogenic bacteria. However, free iron is toxic: it is bound into storage or transport proteins (e.g. ferritin, hemoproteins…) and iron homeostasis is tightly regulated. To scavenge iron from these sources, bacteria possess several systems to acquire the bound iron, by surface proteins or siderophores. Bacillus cereus is a sporeforming Gram-positive bacterium, opportunistic human pathogen, 2nd cause of food-borne disease in France. It has been demonstrated that the B. cereus surface protein IlsA and the siderophore bacillibactin (BB) are involved in iron acquisition from ferritin and that these two molecules are important for infection of the insect model G. mellonella. My thesis project focused on two parts: first the study of the BB-Fe3+ complex import into the cell by the siderophore binding protein FeuA highlights the central role of both BB and FeuA. The deletion of the genes encoding for these two molecules limits iron acquisition by B. cereus from ferritin, heme, hemoglobin and inorganic iron in vitro. On the other hand, the virulence phenotype during intra-haemocelic infection of G. mellonella is similar to the Wild-type strain. These results suggest a possible feedback on the expression of virulence factor genes when B. cereus is unable to synthetize both BB and FeuA, and therefore are under high stress. The second part of my work focused on the expression of genes involved in iron homeostasis in vivo, during gut infection of germ-free larvae of G. mellonella. We chose to perform a microgenomic approach, using laser-capture microdissection to get small samples in targeted areas, and then analysing the expression of chosen genes by RT-qPCR and ddPCR at two time points post ingestion The results show that : i) the colonisation of G. mellonella gut is impacted when B. cereus is deprived of both BB and FeuA ; ii) ilsA is expressed during gut infection ; iii) iron homeostasis is involved in adaptation and pathogenicity from the early step of infection of the insect gut ; iv) only weak gene expression modulation occured between the two timepoints This work gives new fundamental knowledge about B. cereus iron homeostasis, and highlights the use of new techniques regarding the in situ study of host-pathogen interactions.

No description available.

Diazotrophic microbes reside in soybean nodules; however, other non-nitrogen fixing bacteria are a part of the interior nodule microbiome. Results from a previous greenhouse study show that a novel species of Pseudomonas associates with soybean nodules as a plant-growth promoting rhizobacteria (PGPR). This study observes the soybean growth promoting potential of Pseudomonas spp. in a field setting. Additionally, this study observed differences in soybean growth promotion based on amending the plant with isolated strains or a mixed culture of the species' strains. Two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were either amended with isolated strains of the novel Pseudomonas spp. (referred to as PAMW1 and BUMW2 in this study), a mix of the two strains, or an uninoculated control. The study recorded measurements to observe growth, yield, and nitrogen fixation differences. The study uses two-way factorial ANOVAs and non-parametric, multivariate analyses to determine differences in growth promotion among samples. Soybean amended with PAMW1 has greater shoot mass, biomass, and height than other treatments. Through nonmetric multidimensional scaling (NMS), samples amended with a mixed culture or PAMW1 may be different regarding growth promotion relative to the non-amended samples. Univariate results support the hypothesis that the novel Pseudomonas spp. benefit soybean in a field setting. However, it is inconclusive whether a mixed culture amendment of multiple strains alters the overall growth promotion of soybean compared to samples amended with isolated strains. / Master of Science / Soil hosts a relatively abundant and diverse community of microorganisms. Moreover, the area of soil that interacts closely with plant roots and their associated exudates, called the rhizosphere, has a significantly greater microbial abundance than surrounding bulk soil. Interactions between microbes and the plant often promote plant growth because of secondary metabolites produced by these beneficial microbes. One particular bacterial species, belonging to the Pseudomonas genus, was discovered and extracted from the soybean nodule interior. Nitrogen-fixing bacteria predominantly reside in the soybean nodule, yet this microorganism cannot fix nitrogen. Although trace amounts of non-nitrogen-fixing bacteria reside in the soybean nodule, this novel species has a relatively high abundance. This study determines the benefits of this species in the soybean nodule. Following positive results in a greenhouse study, this field experiment observes variance in soybean growth and productivity based on their received bacterial amendment. For this study, two soybean cultivars were either amended with an isolated strain of this species, a mix of the two strains, or left uninoculated to serve as a control. Numerous recorded measurements serve as indices of soybean growth and productivity. The results suggest that this novel Pseudomonas species benefits the plant by significantly improving biomass. With further research, this species can potentially serve as an environmentally sensitive and sustainable alternative to fertilizers through its ability to promote soybean growth.

Liquid chromatography (LC) coupled to mass spectrometry (MS) offers highly selective and sensitive analysis of a wide variety of compounds. However, the use of hyphenated experimental set-ups implies that many parameters may have an effect on the studied response. Therefore, in order to determine optimized experimental conditions it is of vital importance to incorporate systematic procedures during method development. In this thesis, a generic stepwise optimization strategy is proposed that aims at high chromatographic quality, as well as high mass spectrometric response. The procedure comprises (i) screening experiments to identify the most important parameters, (ii) LC studies to ensure sufficient chromatographic separation, (iii) extended infusion experiments in order to maximize precursor signal(s), and in the case of tandem MS (iv) extended infusion experiments to determine optimal conditions for collision induced dissociation and when applicable also ion trap settings. Experimental design and response surface methodology is used throughout the procedure. Further, the general applicability of LC-MS is demonstrated in this thesis. Specifically, a novel quantitative column-switched LC-MS method for ferrichrome, ferrichrysin and ferricrocin determination is presented. Using the method it was shown how the siderophore content varies with depth in podzolic soil profiles in the north and south of Sweden. The parallel approach using LC coupled to both inductively coupled plasma (ICP) mass spectrometry, and electrospray ionization (ESI) tandem MS is also evaluated as a tool to identify unknown siderophores in a sample. Additionally, different trypsin digestion schemes used for LC-ESI-MS peptide mapping were compared. By multivariate data analysis, it was clearly shown that the procedures tested induce differences that are detectable using LC-ESI-MS. Finally, the glutathione S-transferase catalyzed bioactivation of the prodrug azathioprine was verified using LC-MS.

Different aspects of interaction of metals other than iron with metallophore mixtures were elucidated. Metal chelation besides iron and greater than with pure desferrioxamine B is documented by enhanced performance of soil mineral dissolution following application of diverse metallophore mixtures. New insights from genome to product of metallophores from Gordonia rubripertincta CWB2 under different cultivation conditions and under metal stress, particularly induced by rare earth elements are given. This evaluation led to increase by a factor of 13. Structure analyses showed that the strain produces citrate and different desferrioxamines. A robust RP-HPLC-based method quantified metal complexation. Further metal immobilization methods for metallophores for metal extraction from solutions with low concentrations were established. Moreover, catechol and hydroxamate metallophores from Arthrobacter oxydans ATW2 and Kocuria rosea ATW4 demonstrate the enhancement of Ge and rare earth element bioavailability, plant growth promotion and phytoextraction potential.:CONTENTS Abstract ..........................................................................................................1 CHAPTER 1 Introduction ......................................................................................5 CHAPTER 2 Mobilization of trace elements in soil extracts by bacterial siderophores in dependence of the pH-value..................................31 CHAPTER 3 Cultivation dependent formation of siderophores by Gordonia rubripertincta CWB2........................................................................41 CHAPTER 4 Data on metal-chelating, -immobilization and biosorption properties by Gordonia rubripertincta CWB2 in dependency on rare earth adaptation.......................................................................................67 CHAPTER 5 Analysis of desferrioxamine-like siderophores and their capability to selectively bind metals and metalloids: development of a robust analytical RP-HPLC method. ..........................................................97 CHAPTER 6 Secondary metabolites released by the rhizosphere bacteria Arthrobacter oxydans and Kocuria rosea enhance plant availability and soil–plant transfer of germanium (Ge) and rare earth elements (REEs) .........................................................................................120 CHAPTER 7 Conclusion remarks and further perspectives ...............................154 References .......................................................................................................162 Acknowledgement .........................................................................................185 Curriculum Vitae ..........................................................................................187 List of publications ..........................................................................................189 List of oral and poster presentations ..........................................................190 List of proceedings ..........................................................................................191 / En este trabajo se han dilucidado diferentes aspectos de la interacción de los metales con las mezclas de metalóforos desde distintos ángulos. La quelación de metales por debajo del hierro se pone de manifiesto en la mejora del rendimiento de la disolución de los minerales del suelo debido a la aplicación de diversas mezclas de metanóforos en comparación con la deferoxamina B pura. Se ofrecen nuevos conocimientos desde el genoma hasta el producto de los metanóforos de Gordonia rubripertincta CWB2 en diferentes condiciones de cultivo y bajo estrés por metales, especialmente inducido por elementos de tierras raras. Esta evaluación condujo a una sobreproducción de un factor de 13. El análisis estructural muestra que la cepa produce citrato y diferentes desferrioxaminas. Un método robusto basado en RP-HPLC cuantificó la complejación de metales. Se establecieron otros métodos de inmovilización de metales para la extracción de metales a partir de soluciones poco concentradas. Además, los metalóforos de catecol e hidroxamato de Arthrobacter oxydans ATW2 y Kocuria rosea ATW4 demuestran la mejora de la biodisponibilidad de GE y REE, la promoción del crecimiento vegetal y el potencial de fitoextracción.:CONTENTS Abstract ..........................................................................................................1 CHAPTER 1 Introduction ......................................................................................5 CHAPTER 2 Mobilization of trace elements in soil extracts by bacterial siderophores in dependence of the pH-value..................................31 CHAPTER 3 Cultivation dependent formation of siderophores by Gordonia rubripertincta CWB2........................................................................41 CHAPTER 4 Data on metal-chelating, -immobilization and biosorption properties by Gordonia rubripertincta CWB2 in dependency on rare earth adaptation.......................................................................................67 CHAPTER 5 Analysis of desferrioxamine-like siderophores and their capability to selectively bind metals and metalloids: development of a robust analytical RP-HPLC method. ..........................................................97 CHAPTER 6 Secondary metabolites released by the rhizosphere bacteria Arthrobacter oxydans and Kocuria rosea enhance plant availability and soil–plant transfer of germanium (Ge) and rare earth elements (REEs) .........................................................................................120 CHAPTER 7 Conclusion remarks and further perspectives ...............................154 References .......................................................................................................162 Acknowledgement .........................................................................................185 Curriculum Vitae ..........................................................................................187 List of publications ..........................................................................................189 List of oral and poster presentations ..........................................................190 List of proceedings ..........................................................................................191 / Es wurden verschiedene Aspekte der Wechselwirkung von anderen Metallen als Eisen mit Metallophormischungen untersucht. Die Chelatbildung mit anderen Metallen als Eisen, die größer ist als bei reinem Desferrioxamin B, wird durch eine verbesserte Leistung bei der Auflösung von Bodenmineralien nach Anwendung verschiedener Metallophormischungen dokumentiert. Es werden neue Erkenntnisse vom Genom bis zum Produkt von Metallophoren aus Gordonia rubripertincta CWB2 unter verschiedenen Anbaubedingungen und unter Metallstress, insbesondere induziert durch Seltene Erden, gegeben. Diese Auswertung führte zu einer Steigerung um den Faktor 13. Strukturanalysen zeigten, dass der Stamm Citrat und verschiedene Desferrioxamine produziert. Eine robuste RP-HPLC-basierte Methode quantifizierte die Metallkomplexierung. Weitere Methoden zur Metallimmobilisierung von Metallophoren für die Metallextraktion aus Lösungen mit niedrigen Konzentrationen wurden etabliert. Darüber hinaus zeigen Catechol- und Hydroxamat-Metallophore aus Arthrobacter oxydans ATW2 und Kocuria rosea ATW4 die Verbesserung der Bioverfügbarkeit von Ge und Seltenen Erden, die Förderung des Pflanzenwachstums und das Phytoextraktionspotenzial.:CONTENTS Abstract ..........................................................................................................1 CHAPTER 1 Introduction ......................................................................................5 CHAPTER 2 Mobilization of trace elements in soil extracts by bacterial siderophores in dependence of the pH-value..................................31 CHAPTER 3 Cultivation dependent formation of siderophores by Gordonia rubripertincta CWB2........................................................................41 CHAPTER 4 Data on metal-chelating, -immobilization and biosorption properties by Gordonia rubripertincta CWB2 in dependency on rare earth adaptation.......................................................................................67 CHAPTER 5 Analysis of desferrioxamine-like siderophores and their capability to selectively bind metals and metalloids: development of a robust analytical RP-HPLC method. ..........................................................97 CHAPTER 6 Secondary metabolites released by the rhizosphere bacteria Arthrobacter oxydans and Kocuria rosea enhance plant availability and soil–plant transfer of germanium (Ge) and rare earth elements (REEs) .........................................................................................120 CHAPTER 7 Conclusion remarks and further perspectives ...............................154 References .......................................................................................................162 Acknowledgement .........................................................................................185 Curriculum Vitae ..........................................................................................187 List of publications ..........................................................................................189 List of oral and poster presentations ..........................................................190 List of proceedings ..........................................................................................191