The soil-borne, rhizosphere-competent, filamentous fungus Trichoderma virens is a well-known biocontrol agent able to control pathogenic fungi through the production of antibiotics, the induction of systemic resistance in host plants, or by directly parasitizing the competing fungus. Competition for iron is another means by which Trichoderma can hinder competing microorganisms, and siderophores are a means by which microorganisms obtain iron. In silico analysis of the T. virens genome suggested that two genes putatively encoding extracellular siderophore-producing nonribosomal peptide synthetases (NRPSs) were present. In this study, a disruption was created in one of the genes, TvNPS6, to create a mutant unable to produce the NRPS TvNps6 (DeltaTvnps6). Previously, a mutant (DeltaTvsidD) had been generated with a disruption in the second gene (TvSIDD) encoding an NRPS thought to be involved in siderophore biosynthesis. A double mutant (DeltaDeltaTvsidDTvnps6) was generated by transformation of a DeltaTvsidD strain with a vector targeting disruption of TvNPS6. This resulted in transformants disrupted within both the putative siderophore-producing NRPSs. Thus, three mutants were available for analysis of the role of these genes in the ecology of T. virens. Transformants were confirmed by PCR and Southern blotting analysis. Phenotypic characterization of the mutants included both HPLC analysis of siderophore production, growth on agar and in liquid media, conidiation, germination in the presence of hydrogen peroxide, biocontrol against Pythium ultimum, in vitro confrontation against Rhizoctonia solani and growth with iron chelators to determine the contribution of reductive iron assimilation (RIA) compared to that of siderophores. The HPLC analysis demonstrated that T. virens Gv 29-8 (wild-type) produced a single siderophore peak when grown in an iron-depleted medium. This peak was not present in the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 mutants but was apparent with the DeltaTvsidD mutants. From the HPLC analysis, T. virens evidently produces a coprogen-type siderophore. Few differences were observed in the other phenotypic tests, though hydrogen peroxide showed some small inhibitory effects towards the DeltaTvnps6 mutants. The addition of chelators, which inhibit RIA, exerted some negative effects on all strains growing under iron-limited media, particularly the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 strains. This study demonstrated that although T. virens has two genes putatively encoding siderophore producing NRPSs, only the TvNPS6 gene was required for extracellular siderophore production. The greater sensitivity of the mutants towards the iron chelators suggests that unlike other other fungi studied, Trichoderma virens utilizes RIA, rather than siderophore production, as the primary means by which the fungus obtains iron in an iron-limited environment.

As agrarian society developed, the most fertile soils able to sustain the nutritional requirements needed for high crop yield were assigned to farming, while the more penurious soils were left to uphold the forest ecosystems. Some temperate forests are developed on acidic soils considered to be nutrient poor, as much of the inorganic nutrients are entrapped in poorly weatherable soil minerals and not easily accessed by plant roots. In an undisturbed ecosystem, the largest contribution of available nutrients comes from the recycling of organically bound nutrients via the decomposition of dead plant material. If biomass is removed, for instance with a more intensified exploitation of the forest ecosystems including whole tree harvesting, this source of nutrients is consequently decreased. The importance of soil mineral weathering as a source of nutrients, and especially that promoted by soil biota, is thereby emphasized. This thesis addresses biotic parameters associated with mineral weathering. Different aspects of soil solution sampling strategies and analysis of different organic ligands as well as biomarkers for the estimation of fungal biomass were investigated. These chemical parameters were also evaluated as indicators of microbial activity in relation to mineral nutrient availability in soil. With the assumption that the current nutrient status of a soil will affect the microbial interest of certain minerals as sources of inorganic nutrients, a mineral amendment trial was performed in a Swedish boreal forest soil. Overall, the amended soil presented good nutrient status, but with a possible shortage of iron. Due to this, it was hypothesized that the amended mineral with the highest iron content i.e. biotite would cause an elevation of microbial activity in its vicinity when compared to the bulk soil. The level of microbial activity in the vicinity of the amended minerals was evaluated via quantification of organic acids and siderophores, as well as estimation of fungal biomass and enzymatic activity. The highest microbial activity was measured for the O horizon of the investigated podzol, although nothing indicated an elevated association with the amended minerals. In the E horizon, however, elevation in microbial activity was observed in the vicinity of the biotite mineral when compared with bulk soil, although only a few of the investigated parameters differed significantly when evaluated separately.   To enable this study, a highly sensitive analytical method employing liquid chromatography and mass spectrometry was developed to quantify a number of hydroxamate siderophores. On-line pre-concentration enabled detection of these organic ligands in the pico-molar range – a necessity when analyzing natural samples. Furthermore, an analytical method was developed for the estimation of fungal biomass via quantification of chitin-derived glucosamine, which also employed liquid chromatography and tandem mass spectrometry. Unlike currently available methods, the one presented in this thesis did not involve analyte derivatization, which resulted in high sample throughput while simultaneously avoiding complications involved with the additional derivatization procedure. The distribution of a group of organic ligands known as aromatic low molecular mass organic acids was also studied in a boreal forest podzol soil. Different sampling and samples preparation techniques, namely tension-lysimeters, soil centrifugation and liquid-soil extraction, were compared when analyzing soil solution components. Significant differences in analyte amount and species type were found between these sampling techniques. Some of the differences could be accounted for by variation in soil composition at different depths of the investigated podzol, but others could be attributed to structural differences within the studied analyte group. This clearly illustrated the intricacy of sampling and analysis when working with a sample matrix as complex and diverse as soil. As previously, liquid chromatography and mass spectrometry was used to quantify the analytes of interest. A highly sensitive analytical method was developed that was able to detect eleven aromatic low molecular mass organic acids in the nano-molar range. High selectivity was ensured by applying multiple reaction monitoring enabled by collision induced fragmentation of the analytes. / FORE

La phase aqueuse de l’atmosphère et plus précisément les gouttelettes de nuage est un des milieux les plus concentrés et réactifs de l’atmosphère au sein duquel les composés présents peuvent subir de nombreuses transformations, principalement par voie photochimique. De plus, elle a la propriété d’être oxydante due à la présence d’espèces radicalaires telles qu’OH ou HO2 et de composés tels que le peroxyde d’hydrogène et le fer.La présence avérée de microorganismes métaboliquement actifs dans l’atmosphère a soulevé de nombreuses questions et plus récemment sur leur rôle dans les processus atmosphériques. Ces organismes pourraient modifier la composition des nuages en utilisant comme substrat les composés carbonés représentant une part importante des composés présents dans les nuages. De plus, ils sont suspectés de jouer un rôle dans la capacité oxydante des nuages en impactant des composés clés de la réactivité chimique tels que le fer ou le peroxyde d’hydrogène. L’objectif de ces travaux de thèse était de se focaliser sur les interactions des microorganismes avec deux espèces oxydantes de la phase aqueuse des nuages, le fer et le peroxyde d’hydrogène.Tout d’abord, un intérêt particulier a été porté au cycle du fer et à sa complexation dans les nuages, de nature encore très incertaine à ce jour. Dans l’idée d’apporter des premiers éléments de réponse quant à cette complexation, un large screening réalisé sur des microorganismes des nuages a été effectué afin d’évaluer leur capacité à produire des sidérophores. Les résultats obtenus suggèrent l’éventuelle présence de sidérophores dans les eaux de nuage comme molécules chélatantes du fer(III) ce qui pourrait impacter la chimie du fer dans la phase aqueuse des nuages.Il a ensuite été question de s’intéresser au peroxyde d’hydrogène. Dans une première approche, les paramètres et mécanismes responsables de la transformation biotique et abiotique de H2O2 dans les eaux de nuage ont été étudiés, ainsi que ses effets sur le métabolisme énergétique des microorganismes. Dans une deuxième approche, les modifications du métabolisme microbien face à H2O2 ont été approfondies à travers une approche métabolomique. Les résultats ont ainsi suggéré que le peroxyde d’hydrogène module fortement le métabolisme énergétique des microorganismes des nuages. Les microorganismes sont capables de gérer une condition de stress oxydant mais qu’en même temps ce stress induit une réorganisation de leur métabolisme. Il a également été montré que diverses voies métaboliques telles que le métabolisme des sucres, acides carboxyliques, lipides, acides aminés, peptide et glutathion sont impactées.Intégrer ces données biologiques dans des modèles de chimie atmosphérique pour améliorer la quantification de cette modulation sur la chimie atmosphérique apparait comme une des perspectives les plus importantes à envisager. Pour cela, des constantes cinétiques de biodégradation de quatre composés majeurs des nuages ont été déterminées. Les sorties du modèle nous permettront de mieux évaluer l’impact du métabolisme microbien sur la chimie des nuages. / The aqueous phase of the atmosphere and, more precisely, cloud droplets is one of the most reactive environments of the atmosphere within which the compounds present can be transformed especially by photochemical reactions. In addition, it contains many radical species such as HO, HO2, hydrogen peroxide or iron which explains its oxidizing power.The presence of metabolically active microorganisms in the atmosphere raised many questions and, currently, on their role in atmospheric processes. These organisms could modify the composition of clouds using carbon compounds as substrate that represented an important part of compounds present in clouds. They are also suspected to play a role in the oxidative capacity of clouds by impacting key compounds of chemical reactivity such as iron or hydrogen peroxide.The objective of this work was to focus on the interactions between cloud microorganisms and two oxidant species of clouds aqueous phase, iron and hydrogen peroxide.First, the cycling of iron and its complexation still very uncertain was studied. In order to provide responses we achieved a screening to evaluate the capacity of cloud microorganisms to produce siderophores. The results obtained suggest the possible presence of siderophores in cloud water as chelating molecules of iron (III) which could have a strong impact on iron chemistry in cloud aqueous phase.Then, we focused on hydrogen peroxide. The parameters and mechanisms responsible for the biotic and abiotic transformation of H2O2 in cloud water were studied, as well as its effects on energetic metabolism of microorganisms. The modifications of the microbial metabolism in the presence of H2O2 were pursued using metabolomics. The results suggest that H2O2 strongly modulate the energetic metabolism of cloud microorganisms. They are able to handle oxidative stress conditions but at the same time this stress induces a reorganization of their metabolism. Various metabolic pathways such as sugar, carboxylic acids, lipids, amino acids, peptide and glutathione metabolism are impacted.One of the important perspectives to consider is the integration of these biological data into atmospheric chemistry models in order to improve the quantification of this modulation on atmospheric chemistry. For this, biodegradation rate constants of four major compounds present in clouds were determined. The output will allow us to assess better the impact of microbial metabolism on clouds chemistry.

A Terra Preta Antropogênica (TPA) ocorre somente em sítios arqueológicos na região amazônica. Estes solos de origem antrópica foram enriquecidos em nutrientes, provavelmente pelo manejo de restos orgânicos e do fogo pelas populações pré-colombianas. Em TPA, a presença de material orgânico estável e a grande atividade biológica indicam que este tipo de solo pode ser um local de alta diversidade microbiana, constituindo numa fonte de germoplasma microbiano. Contudo, atualmente não se tem conhecimento da biologia e, sobretudo da estrutura e diversidade das comunidades microbianas deste solo. O conhecimento sobre a diversidade microbiana trará compreensão das funções exercidas pelas comunidades microbianas no solo e o conhecimento das suas interações com outros componentes da biodiversidade, além de benefícios econômicos e estratégicos, como a descoberta de microrganismos potencialmente exploráveis nos processos biotecnológicos. Metabólitos secundários, como antibióticos e toxinas microbianas, podem ser considerados como produtos naturais de importância ecológica. Muitos metabólitos secundários produzidos por fungos e bactérias desenvolvem atividades multifuncionais através da via não-ribossomal, pelas enzimas peptídeo sintetase e policetídeo sintase, que são enzimas multidomínio e podem estar envolvidas na produção de sideróforos e antibióticos. Este estudo teve como objetivo a busca por peptídeos não-ribossômicos produzidos por bactérias isoladas de TPA e suas caracterizações bioquímica e molecular. Através de cultivo, 150 isolados foram selecionados de TPA para análise taxonômica por amplificação e seqüenciamento do gene 16S rRNA, após análise de restrição por enzima de restrição para se conhecer o polimorfismo genético dos isolados. De acordo com o seqüenciamento pôde-se agrupar esses isolados em 17 grupos. Os resultados indicaram a presença dos gêneros Pseudomonas, Bacillus, Arthrobacter, Janthinobacterium, Staphylococcus e Massili. Metabólitos extracelulares foram extraídos dos cultivos bacterianos, usando acetato de etila e clorofórmio, seguidos pela concentração dos extratos, para determinação da capacidade antimicrobiana e a produção de sideróforo foi avaliada usando-se cromoazurol S (CAS). Os resultados mostraram que treze dos 17 isolados apresentaram genes NRPS ou PKS, ou para ambos os genes, associados à produção de peptídeos não-ribossômicos. De todos os isolados estudados, 97% apresentaram produção de sideróforos e algumas espécies dos gêneros Bacillus, Pseudomonas, Arthrobacter e Janthinobacterium mostraram positivas em inibir o crescimento de cinco bactérias testes. A produção de sideróforos do tipo hidroxamato foi positiva para 8 isolados e 7 isolados para o tipo catecol, sendo que 2 isolados não apresentaram reação. Os resultados obtidos em espectrometria de massas Q-TOF indicaram a presença do antibiótico fenazina para o isolado Pseudomonas putida BCM 20, considerado de importância no controle biológico de diversas bactérias patogênicas. / The Anthropogenic Dark Earth (ADE) occurs mainly in archeological sites in the Amazon region. These soils from anthropic origin were enriched with nutrients, probably by the management of organic residues and fire produced by pre-Colombian populations. In ADE, the presence of stable organic matter and high biological activity indicate that this type of soil can be a site of a highly microbial diversity, appointing as a source of microbial germplasm. However, at the present time there is little knowledge about its biology, and especially the structure and diversity of the microbial communities from these soils. The knowledge about microbial diversity will promote a better understanding of the function promoted by the soil microbial community and the knowledge of its interaction with components from biodiversity, likewise the economical and strategic benefits, as for example the discovery of potential microorganisms for exploring biotechnological processes. Secondary metabolites, like antibiotics and microbial toxins, may be considered as natural products of ecological importance. Many of the secondary metabolites produced by fungi and bacteria develop multifunctional activities via non-ribosomal for peptide enzymes and polypeptide synthesis, that are multi-domain enzymes and can be involved in the production of siderophore and antibiotics. This study had the objective of searching for non-ribosomal peptides produced by bacteria isolated from ADE and its biochemical and molecular characterization. Through cultivation in selective media, 150 strains were isolated from ADE for taxonomic analysis by amplification and sequencing of the 16S rRNA gene, after the restriction analysis by restriction enzyme for knowing the genetic strains polymorphism. From the sequencing, it was possible to select these strains in 17 groups, with predominant genera - Pseudomonas, Bacillus, Arthrobacter, Janthinobacterium, Staphylococcus and Massili. Extracellular metabolites were extracted from the cultivated bacteria, using ethylacetate and chloroform, followed by detection of antimicrobial metabolites from bacterial supernatants. Siderophore production was evaluated using ChromoBlue S (CAS). The results from the strains representing the 17 groups showed that at least thirteen strains presented NRPS or PKS gene, or for both genes, associated to the production of non-ribosomal peptides. From all the studied strains, it was found that 97% presented the production of siderophore and that several species from the genera Bacillus, Pseudomonas, Arthrobacter and Janthinobacterium proved to be positive on bacterial growth inhibition. The production of hydroxamate-type siderophore was positive to eight strains and seven strains for the cathecol type, and two strains did not present any reaction. The results obtained from the Q-TOF mass spectrometry confirmed the production of 6-hydroxyphenazine-1-carboxylic acid from the isolated identified as Pseudomonas putida BCM 20. This compound belongs to a fully studied group of antibiotics for biological control against several pathogenic bacteria.

Au cours de notre travail de thèse, nous avons isolé la seconde espèce du genre Gemmata, Gemmata massiliana à partir de l'eau d'un réseau hospitalier. Nous avons étudié sa distribution dans deux réseaux hospitaliers à Marseille après avoir mis au point une PCR temps réel spécifique. Les échantillons d'eau filtrée recueillis dans l'unité de soins intensifs et des échantillons d'eau non-filtrée recueillis dans des fauteuils dentaires, des réservoirs et des points d'utilisation ont été testés. Au total, 2,2% des échantillons d'eau filtrée ont été positifs contre 11,3% des points d'eau non-filtrée, dont 14,1% des fauteuils dentaires, 5,9% des points d'utilisation et 8% des échantillons de réservoirs. Les patients hospitalisés peuvent être exposés à G. massiliana par l'intermédiaire de l'eau de l'hôpital. Le rôle des Gemmata comme pathogènes opportunistes méritait donc d'être exploré. Nous avons détecter de l'ADN de Planctomycetes dans 2 échantillons testés de sang de patients leucémiques, mais toutes les tentatives d'isolement ont échoué. Nous avons travaillé à améliorer les conditions de culture des Gemmata. Notre analyse du génome de G. obscuriglobus et de G. massiliana a indiqué l'absence de certains composants essentiels dans la voie du métabolisme du fer, les sidérophores et l'enzyme ferriréductase. La culture des Gemmata en présence du surnageant filtré d'Escherichia coli contenant les sidérophores et l'enzyme ferriréductase, améliorait significativement la croissance des Gemmata par rapport à la culture sur une gélose standard. L'amélioration des techniques de culture nous permettra par la suite de mieux aborder l'étude de pathogénicité. / During our thesis work, we isolated a second Gemmata species; Gemmata massiliana from a hospital water network in France. We studied its distribution in two hospitals water network in Marseille after developing a real-time PCR. Filtered water collected at the intensive care unit and non-filtered water collected from dental chairs, tanks and usage points were tested. In total, 2.2% filtered water samples tested positive versus 11.3% non-filtered points, including 14.1% dental chairs, 5.9% usage points and 8% tank specimens. We concluded that hospitalized patients may be exposed to G. massiliana through hospital water, especially the non-filtered water. The role of Gemmata as opportunistic pathogen deserved to be explored. Additionally, using 16S rRNA gene-based specific Planctomycetes primers, enabled us to detect DNA Planctomycetes in 2 of 100 blood samples tested of leukemic patients. In one of the positive specimens DNA of a Gemmata-related bacterium was detected, unfortunately all isolation attempts proved futile. Therefore, we worked at improving the Gemmata species culture conditions to optimize their isolation from clinical samples. Genome analysis indicated that Gemmata organisms do not encode some essential iron pathway components, siderophores and ferric reductase. On this basis, we have shown that culture of G. obscuriglobus and G. massiliana in the presence of Escherichia coli filtered supernatant containing siderophores and extracellular ferric reductase, significantly improved the two species growth compared to their culture on a standard agar. The improvement of Gemmata species culture techniques will allow us to better address the pathogenicity study.

In analytical chemistry the main goal is normally to determine the identity and/or concentration of one or more species in a sample. The samples analyzed are often natural samples, containing numerous different species in a complex matrix and the choice of technique for multi-elemental detection is in general inductively coupled plasma spectrometry. The chemical forms of an element can affect many of its characteristics e.g. toxicity, which makes speciation analysis important. Therefore, determination of the identity and quantity of an element is still important, but for many applications measurements of total element concentration provides insufficient information. To be able to perform speciation analysis, separation, identification and/or characterization of the various forms of elements in the sample has to be accomplished. Speciation analysis has been employed in a wide range of disciplines, including for example environmental science, biology and clinical chemistry. This thesis describes work to improve and understand the elemental speciation analysis with liquid chromatography coupled to plasma spectrometry and also highlights the importance and potential of the synergy between atomic spectrometry and molecular mass spectrometry. The combination of the matrix tolerant, robust and very sensitive plasma spectrometry used together with molecular mass spectrometry, which provides structural information and the possibility to identify unknown species, is demonstrated to be a very powerful tool for speciation analysis. In this thesis methods are developed for on-line sample clean-up and pre-concentration coupled to liquid chromatography and plasma spectrometry, which makes handling of small sample volumes easier and also decreases the risk of contamination. The problems associated with organic modifiers in plasma spectrometry are also addressed. Applications of speciation analysis are exemplified by analysis of aluminium-chelated siderophores in field-soil solutions and organic phosphorous species in aquatic sediments. The possibility to analyze un-dissolved samples as slurries with minimal sample preparation is also discussed.

Arsenic (As) is a toxic metalloid and the remediation of soils and waters from this contaminant as well as the prevention of future contamination are still pending tasks in Chile. There are bacteria able to live in environments polluted with arsenic, as they have tolerance mechanisms for this metalloid, or even can use it for energy metabolism. The potential tolerance mechanisms include the production of siderophores, metabolites with chelating activity that can decrease the toxicity of metals and metalloids. Although a correlation between siderophore production and metalloid tolerance has been described, the structure of arsenic-binding siderophores and their implications in tolerance have not been elucidated yet. In this work, it is proposed that bacteria isolated from contaminated environments produce arsenic-binding siderophores. The main aims of this work are to study the production of the siderophores by arsenic-tolerant bacteria, to characterize these compounds and to determine their relation with tolerance to arsenic. Fourteen arsenic-tolerant bacteria were isolated from contaminated water, From these, four strains belonging to the species Rhodococcus erythropolis, Arthrobacter oxydans and Kocuria rosea were selected, in addition to the previously isolated Rhodococcus erythropolis S43, for a more detailed study. The isolates were used to produce siderophore extracts, which were then evaluated for their iron- and arsenic-binding activity. To detect the latter, a new method (As-mCAS) was set up, based on the Chrome Azurol S (CAS) test, an assay to detect iron-chelating activity of siderophores. After testing the extracts, R. erythropolis S43 was selected as the strain with the best arsenic-binding activity. For the subsequent chemical characterization, siderophores were produced under control conditions (iron-free M9 medium) and under stress conditions with arsenic (iron-free M9 medium with sodium arsenite). HPLC analysis of the extracts for both culture conditions showed the presence of a single compound with both an iron-chelating and an arsenic-binding activity. Analyses by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) for both culture conditions suggested the main presence of the siderophore heterobactin B. In addition, the genome of strain S43 was sequenced. A cluster of ars-genes was predicted, probably responsible for the arsenic-tolerance of the strain. In addition, a complete gene cluster for heterobactin production was found. However, no significant difference was obtained in the expression of these determinants in the presence or absence of arsenic, suggesting that the production of this siderophore in strain S43 is not responsible for the tolerance to the metalloid.