Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. Current detection and treatment strategies for invasive aspergillosis, as well as other invasive fungal infections, are poor. Iron has been shown to be essential for Aspergillus fumigatus virulence. Furthermore, mechanisms in the iron regulatory network are believed to be potential drug targets since iron management in fungi is vastly different from that in mammals and other eukaryotes. Therefore a better understanding of iron homeostasis in Aspergillus fumigatus could help improve drug therapies for invasive aspergillosis. In this research a discrete model of iron uptake, storage and utilization in Aspergillus fumigatus with particular focus on siderophore-mediated iron acquisition is constructed. The model predicts oscillations in gene expression as the fungus adapts to a switch from an iron depleted to an iron replete environment. The model is validated via in vitro experiments. / Master of Science

O ferro é essencial para a sobrevivência das bactérias, estando envolvido em vários processos metabólicos. Apesar de sua baixa solubilidade, as bactérias dispõem de sistemas eficientes e específicos para captação, utilização e armazenamento desse metal, ao mesmo tempo controlando adequadamente a concentração de ferro livre no meio, para evitar seus potenciais efeitos tóxicos. Xylella fastidiosa é uma bactéria gram-negativa que coloniza o xilema de uma diversidade de plantas cultivadas e silvestres em várias partes do mundo, sendo o agente causador de uma série de doenças em plantas economicamente importantes como citros, videiras e café. Alguns mecanismos de virulência de X. fastidiosa já foram elencados, entre esses a formação de biofilme, que promoveria a oclusão do xilema e consequente estresse hídrico para a planta. Postula-se que a captação de ferro por X. fastidiosa, causando redução da concentração desse metal no xilema das plantas infectadas, seja uma das estratégias utilizadas pela bactéria em seu processo de infecção. Nosso objetivo neste trabalho foi de investigar o processo de captação de ferro por X. fastidiosa e avaliar em maior detalhe o efeito da concentração de ferro no crescimento e na expressão de fatores de virulência dessa bactéria, expandindo os estudos anteriormente realizados em nosso grupo. Para tal propusemos: (i) avaliar a capacidade de X. fastidiosa produzir sideróforos, pequenas moléculas quelantes de ferro; (ii) investigar, em condições de cultivo com diferentes concentrações de ferro, o perfil de expressão de alguns genes de X. fastidiosa que possam ser integrantes do stimulon do ferro; (iii) analisar o efeito da concentração de ferro no crescimento e formação de biofilme por X. fastidiosa. Observamos que X. fastidiosa parece produzir um composto quelante de ferro que pode ser um sideróforo e elencamos alguns genes que poderiam estar envolvidos na síntese desse composto. Confirmamos que ferro é necessário para o crescimento de X. fastidiosa, sendo que em alta concentração de ferro foi observada maior deposição de biofilme pela cultura bacteriana. Interessantemente também foi observado que as células crescidas em meio com pouco ferro secretaram mais exopolissacarídeos totais na fração do biofilme, o que estabelece uma relação entre as concentrações de ferro no meio com a expressão de um fator de virulência essencial para o desenvolvimento da infecção por X. fastidiosa. / Iron is an essential nutrient for bacteria, being involved in many metabolic processes. Despite its low solubility, bacteria present efficient and specific systems for uptake, utilization and storage of iron, meanwhile controlling metal concentration adequately to prevent potential toxic effects of free iron. Xylella fastidiosa is a Gram-negative bacterium which colonizes the xylem of a wide range of crops and wild species of plants worldwide. This bacterium is the agent of several diseases affecting economically important crops, such as citrus, grapevine and coffee. Among the proposed X. fastidiosa virulence mechanisms is the formation of biofilm which may occlude xylem vessels causing hydric stress. It is postulated that iron uptake might cause reduction of its concentration within the xylem of infected plants therefore constituting a virulence strategy during bacterial infection process. Our aims in this work were to investigate the process of iron uptake in X. fastidiosa and to evaluate the effect of iron concentration in the growth and morphology of this bacterium, expanding previous studies. For this we set to: (i) investigate the capacity of X. fastidiosa in producing siderophores, small biomolecules utilized for iron quelation; (ii) evaluate, under culture conditions at different iron concentrations, the expression profile of candidate genes that might be part of the iron stimulon; (iii) analyse the effect of iron concentration in growth and biofilm formation of X. fastidiosa. We observed that X. fastidiosa seems to produce a compound with iron quelating ability, such as a siderophore, and we point to a group of genes that might be involved in the synthesis of this compound. We confirmed that iron is necessary for growth of X. fastidiosa. At high concentrations of iron we observed higher biofilm production. Interestingly, we also observed that when grown in low iron concentration, X. fastidiosa secretes more exopolysaccharides (EPS) associated with the biofilm, suggesting a link between iron concentration and expression of an essential virulence factor to the infection process of X. fastidiosa.

Thesis (S.M.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), 2008. / Includes bibliographical references (p. 54-59). / (cont) An increased understanding of heterotrophic bacterial strategies for acquiring nutrients and trace elements is critical for elucidating their impact on biogeochemical cycling in the ocean. It is estimated that iron is a limiting nutrient for phytoplankton growth in over 30% of the open ocean, but still little is known about bacterial strategies for iron acquisition. Siderophore (Fe ligand) production by bacteria may play a major role in influencing the bioavailability of iron in the ocean. Despite the importance of siderophores in the environment, only limited information from a select group of bacteria is available. On a cruise through the Costa Rica Dome (CRD) upwelling region in July 2005, a library of 867 isolates from five depth profiles inside and outside of the dome was obtained and screened for siderophore production using the Chrome Azurol-S (CAS) assay. Phylogenetic affiliation of 134 isolates was determined by sequencing the 16s rDNA gene, and determined that gamma proteobacteria such as Alteromonas, Pseudoalteromonas, Halomonas, and Marinobacter dominated the collection, while alpha-proteobacteria such as Roseobacter were also represented. The isolates obtained from stations in the CRD showed greater siderophore-producing capabilities between 55m and 100m while strains isolated from outside the CRD had shallower peak (-8-35m) production. Functional group determination showed that hydroxamate production dominated from 50-150m, while hydroxamate and catechol production is roughly equal in shallower waters. By characterizing the siderophores produced by these isolates and determining the genetic make-up of the population, these findings further our understanding of how heterotrophic microbes affect biogeochemical processes and the competitive nature of nutrient acquisition. / by Whitney B. Krey. / S.M.

Le développement de l'industrie et de l'agriculture a entraîné l'accumulation d'éléments métalliques dans les sols. Dans ces écosystèmes, la fraction vivante intervient de façon importante dans la modification de la rétention des contaminants. Parmi les techniques de décontamination, la phytoremédiation apparaît comme un outil de choix. Si un des inconvénients majeurs de cette technique réside dans des durées de traitement pouvant atteindre plusieurs années, l'ajout de micro-organismes couplé aux plantes améliorerait les prélèvements et réduirait les temps de décontamination. Cette étude s'est intéressée aux possibilités de développer des procédés de phytoremédiation associés à des bactéries productrices de sidérophores, molécules ayant une forte affinité pour le fer mais également pour d'autres métaux. Dans ce contexte, un système de complexité croissante, jusqu'au modèle complet comprenant les constituants du sol, les bactéries, les métaux contaminants et les plantes, a été étudié avec pour objectif la compréhension des processus intervenant dans ces différentes interactions. Ces travaux ont conduit, dans un premier temps, à la compréhension, notamment par une approche de biologie moléculaire, des interactions intervenant entre des minéraux du sol, des argiles et des oxy-hydroxydes de fer, et une bactérie productrice de sidérophores, Pseudomonas aeruginosa. Les résultats ont mis en évidence le rôle majeur de l'un de ces sidérophores, la pyoverdine, ainsi que du biofilm, dans la dissolution des minéraux par cette bactérie. Une étude en fluorimétrie et dosages des métaux a, quant à elle, montré que la pyoverdine était capable de complexer des métaux contaminants adsorbés sur les argiles ou présents dans la structure cristalline des oxy-hydroxydes de fer. Enfin, ces travaux ont abouti à la mise en place du système d'étude dans sa globalité. Si la pyoverdine ou les bactéries productrices de sidérophores n'ont pas amélioré le prélèvement par les plantes du cadmium et du nickel, des résultats prometteurs ont cependant été obtenus pour le cuivre. De plus, les bactéries productrices de sidérophores s'avèrent être des cibles intéressantes en phytostabilisation.

O ferro é essencial para a sobrevivência das bactérias, estando envolvido em vários processos metabólicos. Apesar de sua baixa solubilidade, as bactérias dispõem de sistemas eficientes e específicos para captação, utilização e armazenamento desse metal, ao mesmo tempo controlando adequadamente a concentração de ferro livre no meio, para evitar seus potenciais efeitos tóxicos. Xylella fastidiosa é uma bactéria gram-negativa que coloniza o xilema de uma diversidade de plantas cultivadas e silvestres em várias partes do mundo, sendo o agente causador de uma série de doenças em plantas economicamente importantes como citros, videiras e café. Alguns mecanismos de virulência de X. fastidiosa já foram elencados, entre esses a formação de biofilme, que promoveria a oclusão do xilema e consequente estresse hídrico para a planta. Postula-se que a captação de ferro por X. fastidiosa, causando redução da concentração desse metal no xilema das plantas infectadas, seja uma das estratégias utilizadas pela bactéria em seu processo de infecção. Nosso objetivo neste trabalho foi de investigar o processo de captação de ferro por X. fastidiosa e avaliar em maior detalhe o efeito da concentração de ferro no crescimento e na expressão de fatores de virulência dessa bactéria, expandindo os estudos anteriormente realizados em nosso grupo. Para tal propusemos: (i) avaliar a capacidade de X. fastidiosa produzir sideróforos, pequenas moléculas quelantes de ferro; (ii) investigar, em condições de cultivo com diferentes concentrações de ferro, o perfil de expressão de alguns genes de X. fastidiosa que possam ser integrantes do stimulon do ferro; (iii) analisar o efeito da concentração de ferro no crescimento e formação de biofilme por X. fastidiosa. Observamos que X. fastidiosa parece produzir um composto quelante de ferro que pode ser um sideróforo e elencamos alguns genes que poderiam estar envolvidos na síntese desse composto. Confirmamos que ferro é necessário para o crescimento de X. fastidiosa, sendo que em alta concentração de ferro foi observada maior deposição de biofilme pela cultura bacteriana. Interessantemente também foi observado que as células crescidas em meio com pouco ferro secretaram mais exopolissacarídeos totais na fração do biofilme, o que estabelece uma relação entre as concentrações de ferro no meio com a expressão de um fator de virulência essencial para o desenvolvimento da infecção por X. fastidiosa. / Iron is an essential nutrient for bacteria, being involved in many metabolic processes. Despite its low solubility, bacteria present efficient and specific systems for uptake, utilization and storage of iron, meanwhile controlling metal concentration adequately to prevent potential toxic effects of free iron. Xylella fastidiosa is a Gram-negative bacterium which colonizes the xylem of a wide range of crops and wild species of plants worldwide. This bacterium is the agent of several diseases affecting economically important crops, such as citrus, grapevine and coffee. Among the proposed X. fastidiosa virulence mechanisms is the formation of biofilm which may occlude xylem vessels causing hydric stress. It is postulated that iron uptake might cause reduction of its concentration within the xylem of infected plants therefore constituting a virulence strategy during bacterial infection process. Our aims in this work were to investigate the process of iron uptake in X. fastidiosa and to evaluate the effect of iron concentration in the growth and morphology of this bacterium, expanding previous studies. For this we set to: (i) investigate the capacity of X. fastidiosa in producing siderophores, small biomolecules utilized for iron quelation; (ii) evaluate, under culture conditions at different iron concentrations, the expression profile of candidate genes that might be part of the iron stimulon; (iii) analyse the effect of iron concentration in growth and biofilm formation of X. fastidiosa. We observed that X. fastidiosa seems to produce a compound with iron quelating ability, such as a siderophore, and we point to a group of genes that might be involved in the synthesis of this compound. We confirmed that iron is necessary for growth of X. fastidiosa. At high concentrations of iron we observed higher biofilm production. Interestingly, we also observed that when grown in low iron concentration, X. fastidiosa secretes more exopolysaccharides (EPS) associated with the biofilm, suggesting a link between iron concentration and expression of an essential virulence factor to the infection process of X. fastidiosa.

Pseudomonas aeruginosa est une bactérie à Gram-négatif, pathogène et opportuniste, responsable de nombreuses et sévères infections chez l’homme. Ce microorganisme comme la plupart des organismes vivants a besoin de fer pour sa croissance ainsi que d’autres métaux biologiques comme le zinc, le cuivre, le nickel, le manganèse, le cobalt, le molybdène, le vanadium et d’autres. Afin d’acquérir le fer P. aeruginosa produit deux sidérophores majeurs, la pyoverdine (PVD), souvent considérée comme sidérophore principal, et la pyochéline (PCH). Lors de cette thèse nous avons pu démontrer les enzymes de biosynthèse de ces sidérophores adoptent une organisation spécifique aux pôles des bactéries. De plus, l’étude de la composition en métaux de P. aeruginosa dans différentes conditions de cultures a pu démontrer que la bactérie adapte sa concentration intracellulaire en métaux selon la composition du milieu extracellulaire. / Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It is responsible for a wide range of human diseases. Iron is an essential element for this organism, like for nearly all other organisms. To a lower extent this organisms needs other metals such as zinc, copper,nickel, manganese and other for its survival. To acquire iron, P. aeruginosa secrets two major siderophores, pyoverdine (PVD) and pyochelin (PCH). During the thesis we have shown that the enzymes involved in the biosynthesis of theses siderophores adopt a specific localization at the bacterial cell poles. Furthermore, the study of the metal composition of P. aeruginosa in different growth conditions has shown that this bacterium is able to adaptits internal metal concentration to the extracellular metal availability.

Pseudomonas is a diverse genus of Gram-negative bacteria that includes pathogens of plants, insects, and humans as well as environmental strains with no known pathogenicity. Pseudomonas fluorescens itself encompasses a heterologous group of bacteria that are prevalent in soil and on foliar and root surfaces of plants. Some strains of P. fluorescens suppress plant diseases and the genomic sequences of many biological control strains are now available. I used a combination of bioinformatic and phylogenetic analyses along with mutagenesis and biological assays to identify and compare the TonB-dependent outer-membrane proteins (TBDPs) of ten plant-associated strains of P. fluorescens and related species. TBDPs are common in Gram-negative bacteria, functioning in the uptake of ferric-siderophore complexes and other substrates into the cell. I identified 14 to 45 TBDRs in each strain of P. fluorescens or P. chlororaphis. Collectively, the ten strains have 317 TBDPs, which were grouped into 84 types based upon sequence similarity and phylogeny. As many as 13 TBDPs are unique to a single strain and some show evidence of horizontal gene transfer. Putative functions in the uptake of diverse groups of microbial siderophores, sulfur-esters, and other substrates were assigned to 28 of these TBDP types based on similarity to characterized orthologs from other Pseudomonas species. Redundancy of TBDP function was evident in certain strains of P. fluorescens, especially Pf-5, which has three TBDPs for ferrichrome/ferrioxamine uptake, two for ferric-citrate uptake and three for heme uptake. Five TBDP types are present in all ten strains, and putative functions in heme, ferrichrome, cobalamin, and copper/zinc uptake were assigned to four of the conserved TBDPs. The fluorescent pseudomonads are characterized by the production of pyoverdine siderophores, which are responsible for the diffusible UV fluorescence of these bacteria. Each of the ten plant-associated strains of P. fluorescens or P. chlororaphis has three to six TBDPs with putative roles in ferric-pyoverdine uptake (Fpv). To confirm the roles of the six Fpv outer membrane proteins in P. fluorescens Pf-5, I introduced deletions into each of the six fpv genes in this strain and evaluated the mutants and the parental strain for heterologous pyoverdine uptake. I identified at least one ferric-pyoverdine that was taken up by each of the six Fpv outer-membrane proteins of Pf-5. By comparing the ferric-pyoverdine uptake assay results to a phylogenetic analysis of the Fpv outer-membrane proteins, I observed that phylogenetically-related Fpv outer-membrane proteins take up structurally-related pyoverdines. I then expanded the phylogenetic analysis to include nine other strains within the P. fluorescens group, and identified five additional types of Fpv outer-membrane proteins. Using the characterized Fpv outer-membrane proteins of Pf-5 as a reference, pyoverdine substrates were predicted for many of the Fpv outer-membrane proteins in the nine other strains. Redundancy of Fpv function was evident in Pf-5, as some pyoverdines were recognized by more than one Fpv. It is apparent that heterologous pyoverdine recognition is a conserved feature, giving these ten strains flexibility in acquiring iron from the environment. Overall, the TBDPs of the P. fluorescens group are a functionally diverse set of structurally-related proteins present in high numbers in many strains. While putative functions have been assigned to a subset of the proteins, the functions of most TBDPs remain unknown, providing targets for further investigations into nutrient uptake by P. fluorescens spp.. The work presented here provides a template for future studies using a combination of bioinformatic, phylogenetic, and molecular genetic approaches to predict and analyze the function of these TBDPs. / Graduation date: 2012

A pouca solubilidade do ferro impõe desafios para sua captação por bactérias e outros organismos. Uma solução eficaz para este problema é a utilização de sideróforos próprios ou exógenos para solubilizar o ferro do ambiente ou de proteínas do hospedeiro e transportá-lo para o interior da célula bacteriana. Neste trabalho, identificamos vias de produção e captação de ferro por sideróforos e definimos o papel destas moléculas na virulência da bactéria Chromobacterium violaceum, um abundante componente da microbiota de solo e água que ocasionalmente causa graves infecções em humanos. Por meio de análises in silico, vários genes relacionados com a síntese e captação de sideróforos foram encontrados no genoma de C. violaceum ATCC 12472 em dois clusters de síntese de metabólitos secundários. Obtenção de linhagens mutantes de vários destes genes e caracterização destas linhagens por ensaios de CAS, curvas de crescimento em carência de ferro e ensaios de estimulação de crescimento revelaram que C. violaceum produz sideróforos endógenos. Essa produção mostrou-se dependente do percursor comum 2,3-DHBA produzido pelas enzimas codificadas pelos genes entCEBA (CV_1485-84-83-82) e de duas enzimas sintetases de peptídeo não ribossomais (NRPSs CV_1486 e CV_2233), as quais provavelmente montam dois sideróforos distintos do tipo catecolato. Cada sideróforo foi captado por um receptor dependente de TonB (RDTB) específico, com o sideróforo produzido via NRPS CV_1486 sendo captado pelo RDTB CV_1491, e o sideróforo produzido via NRPS CV_2233 sendo captado pelo RDTB CV_2230, uma vez que mutantes sem esses RDTBs acumularam sideróforos no meio externo no ensaio de CAS. Além de seus sideróforos endógenos, C. violaceum foi capaz de utilizar xenosideróforos do tipo catecolato de outras bactérias via o RDTB CV_1491. Ensaios de infecção em camundongos revelaram que tanto a síntese quanto a captação de seus sideróforos endógenos são importantes para a virulência de C. violaceum, pois as linhagens mutantes que não produzem sideróforos (?CV_1485-84- 83-82, ?CV_1485-84-83-82/1486::pNPT e ?CV_1486/2233::pNPT) ou são incapazes8 de captá-los (?CV_2230/1491) tiveram sua virulência diminuída em relação a linhagem selvagem. Os dados mostrando que o mutante que não capta ambos sideróforos de C. violaceum teve atenuação mais acentuada da virulência e induziu menor produção de NET em ensaios com neutrófilos in vitro sugerem que o acúmulo de sideróforos na infecção pode ser benéfico para o hospedeiro. Por fim, demonstramos a possibilidade de gerar mutantes de transposon em C. violaceum e ao realizarmos varredura de uma coleção destes mutantes identificamos ao menos um potencial novo fator de transcrição envolvido na regulação da síntese de sideróforo nesta bactéria. Portanto, os dados obtidos neste trabalho revelaram que C. violaceum utiliza-se de diferentes sideróforos endógenos para captação de ferro e que estas moléculas são importantes para seu estabelecimento no hospedeiro. / The low solubility of iron imposes challenges for its uptake by bacteria and other organisms. An effective solution to this problem is the use of own or exogenous siderophores to solubilize the iron from environmental or host sources and transport it into the bacterial cell. In this work, we identified pathways for production and uptake of siderophores, and we defined the role of these molecules in virulence of the bacterium Chromobacterium violaceum, an abundant component of the microbiota of soil and water, which occasionally causes serious infections in humans. By performing an in silico analysis, we found several genes related with synthesis and uptake of siderophores in the genome of C. violaceum ATCC 12472 within two secondary metabolite biosynthesis gene clusters. Obtaining mutant strains from several of these genes and characterizing these strains by CAS assays, growth curves under iron deficiency and growth stimulation assays revealed that C. violaceum produces endogenous siderophores. This production was shown to be dependent on the common precursor 2,3-DHBA produced by the enzymes encoded by the genes entCEBA (CV_1485-84-83-82) and on two non-ribosomal peptide synthetase enzymes (NRPSs CV_1486 and CV_2233), which probably build two distinct catecholate siderophores. Each siderophore was picked up by a specific TonB-dependent receptor (RDTB), with the siderophore produced via NRPS CV_1486 being picked up by RDTB CV_1491, and the siderophore produced via NRPS CV_2233 being picked up by RDTB CV_2230, since mutants without those RDTBs accumulated siderophores in the external environment in the CAS assays. In addition to its endogenous siderophores, C. violaceum was able to use catecholate-type xenosiderophores from other bacteria via the RDTB CV_1491. Infection assays in mice revealed that both the synthesis and the uptake of its endogenous siderophores are important for the virulence of C. violaceum, since mutant strains that do not produce siderophores (?CV_1485-84-83- 82, ?CV_1485-84-83- 82/1486 :: pNPT and ?CV_1486/2233 :: pNPT) or are unable to uptake them (?CV_2230/1491) had their virulence decreased relative to the wild type strain. The data showing that the mutant strain unable to uptake both siderophores of10 C. violaceum had more pronounced attenuation of virulence and induced lower NET production in in vitro neutrophil assays suggest that the accumulation of siderophores in the infection may be beneficial to the host. Finally, we demonstrated the possibility of generating transposon mutants in C. violaceum, and by screening a collection of these mutants we identified at least one potential novel transcription factor involved in the regulation of siderophore synthesis in this bacterium. Therefore, the data obtained in this work revealed that C. violaceum uses different endogenous siderophores for iron uptake and that these molecules are important for its establishment in the host.

A pouca solubilidade do ferro impõe desafios para sua captação por bactérias e outros organismos. Uma solução eficaz para este problema é a utilização de sideróforos próprios ou exógenos para solubilizar o ferro do ambiente ou de proteínas do hospedeiro e transportá-lo para o interior da célula bacteriana. Neste trabalho, identificamos vias de produção e captação de ferro por sideróforos e definimos o papel destas moléculas na virulência da bactéria Chromobacterium violaceum, um abundante componente da microbiota de solo e água que ocasionalmente causa graves infecções em humanos. Por meio de análises in silico, vários genes relacionados com a síntese e captação de sideróforos foram encontrados no genoma de C. violaceum ATCC 12472 em dois clusters de síntese de metabólitos secundários. Obtenção de linhagens mutantes de vários destes genes e caracterização destas linhagens por ensaios de CAS, curvas de crescimento em carência de ferro e ensaios de estimulação de crescimento revelaram que C. violaceum produz sideróforos endógenos. Essa produção mostrou-se dependente do percursor comum 2,3-DHBA produzido pelas enzimas codificadas pelos genes entCEBA (CV_1485-84-83-82) e de duas enzimas sintetases de peptídeo não ribossomais (NRPSs CV_1486 e CV_2233), as quais provavelmente montam dois sideróforos distintos do tipo catecolato. Cada sideróforo foi captado por um receptor dependente de TonB (RDTB) específico, com o sideróforo produzido via NRPS CV_1486 sendo captado pelo RDTB CV_1491, e o sideróforo produzido via NRPS CV_2233 sendo captado pelo RDTB CV_2230, uma vez que mutantes sem esses RDTBs acumularam sideróforos no meio externo no ensaio de CAS. Além de seus sideróforos endógenos, C. violaceum foi capaz de utilizar xenosideróforos do tipo catecolato de outras bactérias via o RDTB CV_1491. Ensaios de infecção em camundongos revelaram que tanto a síntese quanto a captação de seus sideróforos endógenos são importantes para a virulência de C. violaceum, pois as linhagens mutantes que não produzem sideróforos (?CV_1485-84- 83-82, ?CV_1485-84-83-82/1486::pNPT e ?CV_1486/2233::pNPT) ou são incapazes8 de captá-los (?CV_2230/1491) tiveram sua virulência diminuída em relação a linhagem selvagem. Os dados mostrando que o mutante que não capta ambos sideróforos de C. violaceum teve atenuação mais acentuada da virulência e induziu menor produção de NET em ensaios com neutrófilos in vitro sugerem que o acúmulo de sideróforos na infecção pode ser benéfico para o hospedeiro. Por fim, demonstramos a possibilidade de gerar mutantes de transposon em C. violaceum e ao realizarmos varredura de uma coleção destes mutantes identificamos ao menos um potencial novo fator de transcrição envolvido na regulação da síntese de sideróforo nesta bactéria. Portanto, os dados obtidos neste trabalho revelaram que C. violaceum utiliza-se de diferentes sideróforos endógenos para captação de ferro e que estas moléculas são importantes para seu estabelecimento no hospedeiro. / The low solubility of iron imposes challenges for its uptake by bacteria and other organisms. An effective solution to this problem is the use of own or exogenous siderophores to solubilize the iron from environmental or host sources and transport it into the bacterial cell. In this work, we identified pathways for production and uptake of siderophores, and we defined the role of these molecules in virulence of the bacterium Chromobacterium violaceum, an abundant component of the microbiota of soil and water, which occasionally causes serious infections in humans. By performing an in silico analysis, we found several genes related with synthesis and uptake of siderophores in the genome of C. violaceum ATCC 12472 within two secondary metabolite biosynthesis gene clusters. Obtaining mutant strains from several of these genes and characterizing these strains by CAS assays, growth curves under iron deficiency and growth stimulation assays revealed that C. violaceum produces endogenous siderophores. This production was shown to be dependent on the common precursor 2,3-DHBA produced by the enzymes encoded by the genes entCEBA (CV_1485-84-83-82) and on two non-ribosomal peptide synthetase enzymes (NRPSs CV_1486 and CV_2233), which probably build two distinct catecholate siderophores. Each siderophore was picked up by a specific TonB-dependent receptor (RDTB), with the siderophore produced via NRPS CV_1486 being picked up by RDTB CV_1491, and the siderophore produced via NRPS CV_2233 being picked up by RDTB CV_2230, since mutants without those RDTBs accumulated siderophores in the external environment in the CAS assays. In addition to its endogenous siderophores, C. violaceum was able to use catecholate-type xenosiderophores from other bacteria via the RDTB CV_1491. Infection assays in mice revealed that both the synthesis and the uptake of its endogenous siderophores are important for the virulence of C. violaceum, since mutant strains that do not produce siderophores (?CV_1485-84-83- 82, ?CV_1485-84-83- 82/1486 :: pNPT and ?CV_1486/2233 :: pNPT) or are unable to uptake them (?CV_2230/1491) had their virulence decreased relative to the wild type strain. The data showing that the mutant strain unable to uptake both siderophores of10 C. violaceum had more pronounced attenuation of virulence and induced lower NET production in in vitro neutrophil assays suggest that the accumulation of siderophores in the infection may be beneficial to the host. Finally, we demonstrated the possibility of generating transposon mutants in C. violaceum, and by screening a collection of these mutants we identified at least one potential novel transcription factor involved in the regulation of siderophore synthesis in this bacterium. Therefore, the data obtained in this work revealed that C. violaceum uses different endogenous siderophores for iron uptake and that these molecules are important for its establishment in the host.

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.