Copper (Cu) is required by marine microbes for essential biological processes, including photosynthesis and nitrogen fixation, but can be toxic above a certain threshold. Copper bioavailability in seawater is regulated by complexation with dissolved organic ligands of unknown source and structure. Culturing experiments have demonstrated the production of high-affinity Cu-binding ligands by marine algae in response to metal stress or limitation, suggesting they function either as metal ‘sponges’ to reduce copper toxicity or ‘carriers’ that promote uptake. The goal of my thesis research was to develop methods for the recovery and characterization of Cu ligands from seawater that could then be applied to natural samples to investigate sources and structures of recovered ligands. Using natural seawater spiked with model Cu ligands, I developed an immobilized Cu(II)-ion affinity chromatography (Cu(II)-IMAC) protocol which was shown to be effective in quantifying an operationally defined subset of natural Cu ligands. I then applied Cu(II)-IMAC to seawater collected along transects in the Canadian Arctic and NE Pacific Ocean to assess the abundance of this ligand pool across a diverse set of samples. Ligand distribution profiles and their covariance with other components of seawater (e.g. chlorophyll) were consistent with in situ biological production of some Cu-binding ligands. Model ligands spiked into seawater and recovered by Cu(II)-IMAC were also used to develop protocols for structural characterization of Cu ligands by solid-phase extraction (SPE) and tandem mass spectrometry (MS/MS). This research provides new tools for the isolation and characterization of copper ligands in natural samples, and new insights into the biogeochemical cycling and ecological significance of Cu in the ocean. / Graduate

Les ions métalliques (Fe, Zn, Cu, Mn, Ni, Co, Mo…) jouent un rôle vital dans un grand nombre de processus biologiques. Leur carence et leur excès entraînent des défauts de croissance voire la mort. Pour survivre et croître, certaines bactéries ont développé des stratégies pour acquérir les métaux dans leur environnement basées sur la synthèse de ligands complexant les métaux. Les plantes, elles, stockent les métaux et les transportent entre les tissus sous des formes chimiques particulières et un rôle grandissant pour les petits complexes métalliques semble se révéler. La spéciation des métaux gouvernerait donc leur biodisponibilité pour les consommateurs en ce qui concerne les plantes comestibles. Cette thèse a pour but le développement de méthodes analytiques pour l’identification et la quantification des formes chimiques des métaux chez les bactéries et les plantes.Les techniques de séparation chromatographique, comme la chromatographie d’exclusion stérique (SEC) et la chromatographie d’interaction hydrophile (HILIC), furent couplées à la spectrométrie de masse (MS) avec une ionisation préalable par plasma induit (ICP MS) ou par électrospray pour une détection élémentaire et moléculaire respectivement. La méthodologie analytique développée ici, qui est basée sur l’utilisation de l’HILIC avec détection double par ICP MS et électrospray MS, s’est avérée cruciale pour la découverte et la caractérisation de deux nouveaux métallophores, la staphylopine chez Staphylococcus aureus et la pseudopaline chez Pseudomonas aeruginosa. Ces métallophores sont synthétisés et exportés par les bactéries et servent pour l’acquisition des métaux de transition divalents dans les milieux très pauvres en métaux grâce à leur pouvoir complexant.Des techniques dédiées de préparation d’échantillons, incluant le fractionnement des extraits solubles à l’eau, furent mises en place pour l’analyse du fer dans le maïs. Les approches instrumentales développées, notamment après le fractionnement du fer l’échantillon, permirent l’identification par spectrométrie de masse de complexes du fer avec le citrate, le muginéate et le phytate dans les grains de maïs. Il existe aussi une différence significative dans les concentrations de certaines polyamines entre certaines variétés de graines contenant du fer faiblement biodisponible et celles contenant du fer fortement biodisponible. La plus grande abondance de complexe fer-muginéate dans les extraits gastro-intestinaux des variétés à fer fortement biodisponible semblerait indiqué un rôle important pour ce complexe dans la biodisponibilité du fer. Finalement, des ratios molaires plus faibles entre le phytate extrait et le fer qui lui est associé sembleraient aussi être un des facteurs lié à une plus grande biodisponibilité du fer. / Metal (Fe, Zn, Cu, Mn, Ni, Co, Mo…) ions play a vital role in a large number of biological processes. Both their deficiency and excess will lead to severe growth impairment or death. In order to survive and to grow, bacteria developed strategies to acquire metals from the environment based on the synthesis of metal complexing ligands. Plants store metals and transport them between tissues in particular chemical forms and a rising role for small metal complexes seems to be revealed. Therefore their speciation governs the bioavailability of the metals to consumers in the case of edible plants. This thesis aims at the development of analytical methods for the identification and quantification of metal chemical forms in bacteria and in plants.Chromatographic separation techniques, such as size exclusion chromatography (SEC) and hydrophilic interaction chromatography (HILIC) were coupled to mass spectrometry (MS) using ionization in an inductively coupled plasma (ICP MS) or electrospray for elemental and molecular detection, respectively. The developed analytical methodology based on HILIC with dual ICP MS and electrospray MS detection turned out to be crucial for the discovery and characterization of two novels metallophores, staphylopine in Staphylococcus aureus and pseudopaline in Pseudomonas aeruginosa. These metallophores are synthesized and exported by the bacteria and serve for the uptake of divalent transition metal in metal scarce conditions by means of chelation. Dedicated samples preparation techniques, including fractionation of water soluble extracts, were developed for iron analyses in maize. The developed instrumental approaches following the iron fractionation methods allowed the identification by mass spectrometry of citrate, mugineate and phytate complexes with iron in maize kernels. There was also a significant difference in some polyamine concentrations observed between some kernels with low and high bioavailable iron. The higher abundance of the iron-mugineate complex in the gastrointestinal extract in high bioavailable maize variety possibly indicates that iron-mugineate would have an important role in iron biodisponibility. Finally, lower molar ratios between extracted phytate and iron associated to it would seem to be also a key aspect for higher iron bioavailability.

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