Extradiol dioxigenases são enzimas que catalisam a clivagem oxidativa de ligações C-C entre grupos hidroxila fenólicos adjacentes utilizando catecóis como substratos. Esta classe de enzimas é bem caracterizada em bactérias, onde catalisam a degradação de compostos aromáticos. Na maioria das plantas Caryophyllales, como a beterraba, primavera e a maravilha, L-3,4-diidroxifenilalanina (L-DOPA) extradiol dioxigenases (DODAs) catalisam a clivagem oxidativa de L-DOPA na posição 4,5 gerando o ácido betalâmico, aldeído precursor das betalaínas, uma classe de pigmentos naturais que substitui as antocianinas na pigmentação dessas espécies. Alguns fungos basidiomicetos também produzem betalaínas, como o agário-das-moscas (Amanita muscaria). Nesse organismo, DODA é capaz de catalisar uma clivagem adicional na posição 2,3 da L-DOPA, formando muscaflavina, um isômero do ácido betalâmico que dá origem a uma outra classe de pigmentos naturais: as higroaurinas. Desde a caracterização do gene dodA, o qual codifica para a DODA de A. muscaria (AMAMU), não existem relatos na literatura que explorem a promiscuidade catalítica desta enzima, sua relação com outras linhagens de DODAs e a síntese quimioenzimática de betalaínas a partir desta enzima. Dessa forma, buscamos contextualizar as relações filogenéticas e funcionais entre AMAMU e diferentes linhagens de DODAs, bem como estabelecer um método que viabilize a clonagem, expressão heteróloga e caracterização funcional destaenzima. As análises filogenéticas revelaram que AMAMU possui uma evolução convergente com DODAs de plantas e bactérias e que, apesar de AMAMU ser funcionalmente homóloga à DODA da bactéria Escherichia coli, esta última apresenta homologia com DODAs de plantas. Logo, não há uma relação direta entre a sequência primária de DODAs e sua função. Nós também demonstramos que não há uma relação entre a expressão de transcritos de BvDODA1, e de seu parálogo BvDODA2, e a diferença de pigmentação entre variedades de beterrabas amarelas e vermelhas. A clonagem da sequência codificadora (CDS) publicada para o gene dodA de A. muscaria resultou na retenção do primeiro íntron, o que impedia a sua expressão. Então, uma nova CDS de 558 nucleotídeos foi proposta para este gene, a qual inclui um códon de início da tradução que se mantém na fase de leitura e codifica para uma proteína de 185 resíduos, 43 a menos que AMAMU. A expressão desta CDS resultou na proteína recombinante AmDODA, capaz de catalisar a síntese de ácido betalâmico e muscaflavina a partir de L-DOPA e D-DOPA. AmDODA possui um tamanho aproximado de 22 kDa, com um pH ótimo de atividade de 8,5 e uma constante de Michaelis (KM) de 3,7 ± 0,9 mmol L-1 e de velocidade máxima (Vmax) de 3,3 ± 0,4 µ mol min-1 mg-1. Sua utilização foi demonstrada na síntese quimioenzimática de betalaínas-modelo com potencial aplicação como sondas para microscopia confocal de fluorescência de dois fótons. Neste contexto, esta Tese explora os aspectos moleculares, bioquímicos e biológicos da DODA do fungo A. muscaria e traz importantes contribuições acerca da pigmentação por betalaínas na natureza. / Extradiol dioxigenases are enzymes that catalyze the oxidative cleavage of C-C bonds between adjacent phenolic hydroxyl groups using catechols as substrates. This class of enzymes is well characterized in bacteria, where they catalyze the degradation of aromatic compounds. In most plants of the Order Caryophyllales, such as beet, paperflower and four o\'clock flower, L-3,4-dihydroxyphenylalanine (L-DOPA) extradiol dioxygenases (DODAs) catalyze the oxidative 4,5-cleavage of L-DOPA generating the betalamic acid, an aldehyde precursor of the betalains, a class of natural pigments that replaces anthocyanins in the pigmentation of these species. Some basidiomycete fungi also produce betalains, such as the fly agaric (Amanita muscaria). In this organism, DODA is able to catalyze an additional 2,3-cleavage of L-DOPA, yielding muscaflavine, an isomer of betalamic acid that gives rise to another class of natural pigments: the hygroaurins. Since the characterization of the dodA gene, which encodes the A. muscaria DODA (AMAMU), there are no reports in the literature that explore the catalytic promiscuity of this enzyme, its relation to other DODAs and the chemoenzymatic synthesis of betalains from this enzyme. Thus, we seek to contextualize the phylogenetic and functional relationships between AMAMU and different DODA lineages, as well as to establish a method that enable the cloning, heterologous expression and functional characterization of this enzyme. Phylogenetic analysis revealed that AMAMU has a convergent evolutionwith plant and bacterial DODAs and that although AMAMU is functionally homologous to the DODA of the Escherichia coli bacteria, this latter is homologous to the plant DODAs. Therefore, there is no direct relationship between the primary sequence of DODAs and their function. We have also shown that there is no relationship between the expression of BvDODA1 transcripts, and its BvDODA2 paralogue, and the pigment difference between yellow and red beet varieties. Cloning of the published coding sequence (CDS) for the dodA gene of A. muscaria resulted in the retention of the first intron, which prevented its expression. Then, a new CDS of 558 nucleotides was proposed for this gene, which includes a translation start codon that remains in the open reading frame and encodes for a protein 185 residues long, 43 less than AMAMU. Expression of this CDS resulted in the recombinant AmDODA protein, able to catalyze the synthesis of betalamic acid and muscaflavine from L-DOPA and D-DOPA. AmDODA has an approximate size of 22 kDa, with an optimum activity pH of 8.5 and a Michaelis constant (KM) of 3.7 ± 0.9 mmol L-1 and a maximum velocity (Vmax) of 3.3 ± 0.4 µmol min-1 mg-1. Its use was demonstrated in the chemoenzymatic synthesis of betalains-model with potential application as probes for confocal microscopy of two-photon fluorescence. In this context, this thesis explores the molecular, biochemical and biological aspects of the DODA of the fungus A. muscaria and brings important contributions about the pigmentation by betalains in nature.

Os inibidores de BRAF (iBRAFs) e de MEK (iMEK), inauguraram uma nova classe de medicamentos, a terapia direcionada, no combate ao melanoma metastático. Entretanto, os pacientes adquirem resistência ao tratamento em poucos meses. Além disso, a imunoterapia vem ganhando espaço no tratamento do câncer, incluindo o melanoma, porém, com alguns aspectos inexplorados. Dentro deste tema, a enzima IDO vem despertando um grande interesse pela participação nos mecanismos de imunotolerância, imunoescape e progressão tumoral. A IDO é responsável pelo consumo e depleção do triptofano, produzindo a quinurenina. Ela está presente em diversos tipos celulares, incluindo células do sistema imune e células tumorais. Este trabalho objetivou avaliar a expressão de IDO durante a progressão da doença - desde do nevo até o melanoma metastático e também avaliar a regulação de IDO induzido por IFN-γ após tratamento com iBRAF em linhagens parentais e resistentes ao iBRAF, buscando-se os mecanismos moleculares. Por fim, objetivou-se entender os efeitos do 1-metil-triptofano (1-MT), um inibidor de IDO, tanto na sua capacidade de inibir a atividade de IDO quanto na sua influência na capacidade clonogênica. O estudo de bioinformática sobre o repositório público GSE12391 mostrou que o nível de expressão gênica de IDO foi superior nos estágios mais avançado da doença. Além disso, todas amostras de melanoma primário de pacientes apresentaram a imunomarcação de IDO, enquanto que nenhuma amostra de nevo apresentou tal marcação. Adicionalmente, a ocorrência de IDO se deu nos infiltrados linfoides, em células mononucleares do sistema imune. Duas análises de bioinformática de expressão gênica demonstraram que a IDO estava expressa positivamente na fase de resistência ao iBRAF. Ademais, os resultados de expressão proteica mostraram que a inibição de via MAPK (tanto por iBRAF quanto por iMEK) conseguiu modular a expressão de IDO, sendo que a maioria das linhagens apresentou uma diminuição de IDO. A atividade de IDO, medida através da produção de quinurenina, por HPLC se mostrou em consonância com os resultados de expressão proteica, exceto pela linhagem WM164 que não apresentou atividade enzimática, embora a proteína estivesse presente. Por fim, o 1-MT conseguiu inibir de maneira eficiente a enzima IDO, bloqueando a produção de quinurenina. Além de que, o 1-MT reduziu a capacidade clonogênica de maneira dose-dependente. Portanto, conclui-se que a expressão de IDO é crescente conforme a progressão do melanoma, que a inibição da via MAPK regulou a expressão de IDO e que o 1-MT reduz a capacidade clonogênica, além da sua função primária de inibir IDO / BRAF and MEK inhibitors (BRAFi and MEKi) has launched a new class of medication, the target therapy, to combat metastatic melanoma. Nevertheless, patients acquired resistance to the treatment in few months. Additionally, immunotherapy has been gaining space in cancer treatment, including melanoma, but some aspects need to be explored. Inside this theme, IDO enzyme has called the attention due to its participation in the mechanisms of immune tolerance, scape and tumor progression. IDO is responsible for tryptophan consume e depletion, producing kynurenine. It is present in different cells, including cells from immune system and tumor cells. This work purposed evaluate IDO expression during disease progression - since nevus until metastatic melanoma and also, evaluate IFN-γ-induced IDO regulation after BRAFi treatment in parental and resistant melanoma cell lines, seeking the molecular mechanisms. Lastly, it was evaluated the effects of 1-methyltryptopahn (1-MT), an IDO inhibitor, by its ability to inhibit IDO and also by its influency on the clonogenic capability. Bioinformatic study performed on GSE12391 showed that gene expression level of IDO was superior in the most advanced stages of the disease. Additionally, all sample of patient\'s primary melanoma presented IDO immunostaining, whereas, no nevus samples presented such staining. Besides, IDO occurrence was in the lymphoid infiltrates, in mononuclear cells from immune system. Two bioinformatic analysis of gene expression demonstrated that IDO was differentially overexpressed during BRAFi resistance stage. Moreover, protein expression results presented that MAPK pathway inhibition (both by BRAFi and by MEKy) was able to modulate IDO expression, and most of the cell lines presented an IDO downregulation. IDO activity, measured through kynurenine production, by HPLC was consonant with protein expression results, except by WM164 cell line, which did not present enzymatic activity, albeit the protein was present. By the end, 1-MT could inhibit efficiently IDO enzyme, blocking kynurenine production. Furthermore, 1-MT reduced clonogenic capability in a dosedependent manner. Therefore, it was concluded that IDO expression increases along with melanoma progression, MAPK pathway inhibition regulated IDO expression and 1-MT reduced clonogenic capability, besides its primary function of IDO inhibitor.

A indoleamina 2,3-dioxigenase (IDO) desempenha um papel importante na tolerância materno-fetal devido á sua ação de catabolizar o triptofano e, consequentemente, impedir a proliferação de linfócitos T, que necessitam desse aminoácido para se manter. Hormônios da reprodução também participam do processo de sobrevivência do feto alogênico, como a progesterona que bloqueia o estímulo mitogênico da proliferação de células T, modula a produção de anticorpos, favorece a produção de IL-10, etc. e o estradiol, que pode modular o perfil imune Th1 ou Th2 na gestação, dependendo de sua concentração. Contudo, a existência ou não de correlação da ação da IDO com esses hormônios ou vice-versa ainda encontra-se pouco evidenciada. Desta forma este trabalho verificou a influência da expressão da IDO e às ações de hormônios da reprodução e citocinas no ciclo celular de células oriundas de fetos e placenta de gestação a termo de fêmeas de ratas e camundongos em cultivo. Este estudo foi realizado em complementação a um trabalho anterior, no qual foi realizada uma avaliação da expressão da IDO por citometria de fluxo em células uterinas, de placentas e de embriões de ratas e camundongos fêmeas prenhes e não prenhes que foram mantidas em cultivo e suplementadas com estradiol, progesterona, interferon γ, triptofano e 1-metil-DL- triptofano. A avaliação das fases do ciclo celular foi realizada pela citometria de fluxo. De acordo com os resultados, em relação ao efeito dos tratamentos no comportamento das células no ciclo celular, podemos observar que, em ratas prenhes e não prenhes, ao adicionar estradiol, houve maior predominância das células em fase G1 nos períodos de 4 e 24 horas, bem como no grupo de camundongos fêmeas prenhes. Algumas células uterinas de ratas não prenhes tratadas com estradiol, assim como as tratadas com progesterona e interferon γ progrediram no ciclo para fase de síntese nos tempos de 24 e 48 horas. Com a adição de triptofano podemos notar nos grupos de ratas não prenhes, camundongos fêmeas prenhes e não prenhes, um aumento da quantidade de células na fase G1 nos três períodos analisados. No grupo de ratas prenhes foi observado uma predominância celular em fase G1 nos tempos de 4 e 24 horas, sendo que em 48 horas, as células sofreram fragmentação de DNA. As células que foram suplementadas com 1- metil DL - triptofano + triptofano mantiveram o mesmo comportamento no ciclo celular , se mantendo em fase G1 nos tempos de 4, 24 e 48 horas grupos prenhes e não prenhes de ratas e nos tempos de 4 e 24 horas nos grupos de camundongos fêmeas prenhes e não prenhes, estando com seu DNA fragmentado e em fase de síntese, respectivamente, no tempo de 48 horas. A suplementação dos diversos fatores aos cultivos celulares permitiu observar alterações na dinâmica do ciclo celular, representada principalmente por um aumento de células em fase G1 nos grupos de células placentárias e embrionárias que receberam progesterona, estradiol, interferon γ e triptofano e apresentaram um significativo aumento da expressão de IDO, e que permite inferir que provavelmente a síntese de RNAm esteja correlacionada à produção da IDO. / The indoleamine 2,3-dioxygenase (IDO) plays an important role in maternal-fetal tolerance due to its capacity to catabolize tryptophan and thereby preventing the proliferation of T lymphocytes, necessary for their maintenance. Reproductive hormones are also involved in the process of survival of semi-allogeneic fetus, as progesterone that blocks mitogenic stimulation of T cell proliferation, modulates antibodies production, promotes production of IL-10, etc. and estradiol, that can modulate the Th1 or Th2 immune profile during pregnancy, depending on its concentration. However, there is very few evidences regarding a possible correlation between IDO expression and these hormones; thus this study examined the influence of the expression of IDO and the actions of reproductive hormones and cytokines in the cell cycle of cells derived from fetuses and placenta at term gestation of female rats and mice in culture. This study was conducted as a complement to an earlier work, in which an evaluation was made of the IDO expression by flow cytometry in uterine cells, placentas and embryos of pregnant rats and mice and non-pregnant females that were maintained in culture and supplemented with estradiol, progesterone, interferon γ, tryptophan and 1-methyl-DLtryptophan. The cell cycle evaluation was conducted by flow cytometry. According to the results, regarding the effect of treatments on the behavior of the cells in the cell cycle, it was observed that in pregnant and non-pregnant rats after the addition of estradiol, there is a greater predominance of cells in G1 phase at periods of 4 and 24 hours, that also was noted in the group of pregnant female mice. Uterine cells from non-pregnant female rats treated with estradiol and progesterone as well as treated with interferon γ progressed to the phase of synthesis on the cycle, at 24 and 48 hours. With the addition of tryptophanin the groups of non-pregnant rats, pregnant and non-pregnant mice , an increased amount of cells in the G1 phase were observed in the three periods analyzed. In the group of pregnant rats a predominance of cells in the G1 phase was observed in periods of 4 and 24 hours, and 48 hours, where the cells undergone DNA fragmentation. In pregnant and non-pregnant rats the cells supplemented with 1 - methyl - DL - tryptophan + tryptophan remained at G1 phase in periods of 4, 24 and 48 hours and 4 and 24 hours for cells from pregnant and non-pregnant mice , that show ragmented DNA followed by synthesis at 48 hours period. Supplementation of the various factors to cell cultures allowed to observe dynamic changes in the cell cycle, represented primarily by an increase of cells in G1 phase in groups of embryonic and placental cells that received progesterone, estradiol, interferon γ and tryptophan and showed a significant increase in the expression of IDO, that allows us to infer that the mRNA synthesis is probably correlated to the production of IDO.

Pseudomonas aeruginosa (Pa) est un pathogène opportuniste responsable d’infections pulmonaires aigues graves chez les malades prédisposés. Devant l’émergence croissante de la résistance aux antibiotiques, le développement de thérapeutiques alternatives adjuvantes est indispensable et nécessite la compréhension des interactions hôte-pathogènes au cours de l’infection. La voie métabolique de dégradation du tryptophane appelée voie des kynurénines produit chez l’hôte des métabolites aux propriétés immunomodulatrices connues. Récemment, l’existence de cette voie a été mise en évidence chez Pa, bien que la nature et la quantité de métabolites produits ne soient pas parfaitement connus. La production bactérienne de kynurénines pourrait interférer avec la mise en place de la réponse immunitaire de l’hôte et sa régulation au cours des différentes phases de l’infection, altérant la balance immunitaire pulmonaire au profit du pathogène. A ce titre, la voie des kynurénines de Pa constituerait une cible thérapeutique potentielle. L’objectif de ce travail de thèse est d’étudier l’implication de la voie des kynurénines de Pa dans la virulence bactérienne et la réponse immune de l’hôte dans un modèle murin d’agression respiratoire aiguë. Pour cela, les souris sont infectées avec des souches sauvages de Pa, avec des souches mutantes ΔkynA, non productrices de kynurénines, et des souches ΔkynU, surproductrices de kynurénines. Les interactions potentielles avec la voie des kynurénines de l’hôte sont explorées en inhibant la première enzyme de la voie métabolique, l’indoleamine-2,3-dioxygenase (IDO). Enfin, le rôle du récepteur arylhydrocarbone (AhR), récepteur connu des kynurénines et impliqué dans l’immunité pulmonaire, est exploré en comparant la réponse à l’infection de souris AhR KO à celle des souris sauvages. Dans ce travail, nous décrivons tout d’abord la production des différents métabolites de la voie des kynurénines de Pa in vitro et in vivo dans le modèle d’infection respiratoire aigue, en décrivant pour la première fois la production d’acide kynurénique et de 3-hydroxy-kynurénine pour cette bactérie. Ensuite, nous montrons que les kynurénines bactériennes interfèrent avec la réponse immune de l’hôte, en majorant le recrutement cellulaire alvéolaire, tout en atténuant le niveau d’inflammation et l’activation des cellules présentatrices d’antigènes. Enfin, nous rapportons que l’IDO et l’AhR sont impliqués dans cette immunomodulation, faisant des kynurénines bactériennes des agents du dialogue hôte-pathogène au cours de l’infection respiratoire aigue. A la lumière de ces résultats, la voie des kynurénines pourrait constituer une cible thérapeutique d’intérêt dans les infections respiratoires à P. aeruginosa. / Pseudomonas aeruginosa (Pa) is a Gram-negative bacteria frequently involved in healthcare-associated pneumonia and considered as a « problem-pathogen ». To face the announced post-antibiotic era due to increasing resistance and lack of new antibiotics, new treatment strategies have to be developed. During pneumonia, lung injury results from both bacterial-mediated virulence and host response. Modulation of an overreacting host response could be an alternative therapeutic target in Pa-induced lung infection. Kynurenines are small molecules resulting from tryptophan degradation with reported immunomodulatory properties. Pa is known to produce kynurenine, but the functional enzymes, types and amounts of secreted metabolites are poorly known. Interestingly, many host cells also possess the kynurenine pathway, whose metabolites are known to control immune system homeostasis. The following experiments aim to determine whether bacterial metabolites can interfere with the host’s immune response, leading to a possible immunomodulatory interplay between bacteria and host kynurenine pathways, impacting on the pathophysiology of P. aeruginosa infection. To that goal, we use a murin model of acute lung injury. Mice were infected with WT strain of Pa, compared to mutant strains unable to produce kynurenine (ΔkynA), and mutant strains overproducing them (ΔkynU). Moreover, we studied the interactions between bacterial and host kynurenine pathways by inhibiting the first enzyme of the host pathway called indoleamine-2,3-dioxygenase (IDO). Finally, we assessed the role of the arylhydrocarbon receptor (AhR), a known receptor to kynurenine involved in lung immunity, using AhR KO mice. First, we assess types and levels of metabolites produced by Pa in an in vitro model, and the relevance of this production in vivo. We show for the first time that Pa is able to secrete kynurenine at clinically relevant levels, and other metabolites such as kynurenic acid and 3 OHkynurenine, what was unknown to date. Second, we show that bacterial metabolites were able to modulate the host innate immune response, by increasing alveolar recruitment of neutrophils, associated with decreased inflammatory cytokines levels and impairment of antigen-presenting cells activation. Finally, we report that IDO and AhR are involved in this kynurenine-mediated immunomodulation. These data suggest that pulmonary infection with a bacteria highly expressing the kynurenine pathway enzymes could lead to an imbalance in the immune response to infection, thus constituting a potential therapeutic target to improve Pa-induced pneumonia outcome.

Polychlorinated biphenyls (PCBs) are carcinogenic, persistent, and bioaccumulative contaminants that pose risks to human and environmental health. PCB biodegradation by indigenous microbial communities could be a cost-effective and an environmental-friendly bioremediation strategy for in situ PCB removal. A comprehensive understanding of the microbial PCB degradation at the contaminated site is required for the acceptance and optimization of using microbial PCB degradation as the site clean-up strategy. This thesis describes investigations of the aerobic and anaerobic microbial degradation of PCBs under both field and laboratory conditions. The microbial PCB degradation potential in sediments from Indiana Harbor and Ship Canal (IHSC), a site that was historically contaminated by PCBs, was explored by analyzing the PCB congener distributions and microbial communities in two core sediment samples. PCB congener analysis suggested the possibility of in situ dechlorination in deep sediments. Molecular analysis of biomarker genes revealed the potential of both aerobic and anaerobic PCB degradation in sediments. Microbial communities were characterized by the combination use of terminal restriction fragment length polymorphism (T-RFLP), clone library, and pyrosequencing. These methods elucidated the dominant role of Proteobacteria, especially Acidovorax and Acinetobacter in sediments. To improve the microbial PCB degradation, phytoremediation with switchgrass (Panicum vigratum) was employed under laboratory conditions. Congener analysis showed that both phytoextraction and microbial PCB degradation contributed to the enhanced PCB removal in the presence of switchgrass. Bioaugmentation with Burkholderia xenovorans LB400 was performed to further promote aerobic PCB degradation. The presence of LB400 was associated with improved degradation of PCB 52, but not PCB77 or PCB 153. Increased abundance of the biphenyl dioxygenase gene, which is indicative of aerobic PCB degradation, and its transcript were observed after bioaugmentation, suggesting active aerobic PCB degradation. To promote the anaerobic PCB degradation, redox cycling (alternating flooding and non-flooding) was performed. Redox cycling was found to improve the removal of PCB 153 in unplanted soils and to increase the dechlorinating Chloroflexi population. Characterization of the microbial community by T-RFLP and clone library revealed that Proteobacteria and Acidobacteria were dominant. Species that contain dechlorination potential were identified, including Geobacter and Clostridium, suggesting that their possible role in PCB dechlorination. The research described in this thesis provides scientific knowledge and evidence for the feasibility of employing bioremediation including natural attenuation, phytoremediation, and bioaugmentation to clean up PCB contamination. Such information will be critical in selecting and optimizing remediation strategies for PCB contaminated sites.

2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl compounds and nitrite, with oxygen as the electron acceptor. Although nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in chemical industry for a quick and effective way of synthesizing common reagents. Consequently, the biochemical and biophysical analysis of 2-nitropropane dioxyganase has a potential for bioremediation purposes. In this study, recombinant enzyme is purified to high levels, allowing for detailed characterization. The biochemical analysis of 2-nitropropane dioxygenase presented in this study has established that enzyme utilizes alkyl nitronates as substrates by forming an anionic flavosemiquinone in catalysis. The enzyme is inhibited by halide ions, does not contain iron and has a positive charge located close to the N(1)-C(2)=O locus of the isoalloxazine moiety of the FMN cofactor.

In dieser Arbeit wurden PCR-Primer für den Nachweis von Genen des bakteriellen Chloraromaten-Abbaus entwickelt. Als Zielgene wurden hierfür die Gene der Chlorcatechol-1,2-Dioxygenasen und Chlormuconat-Cycloisomerasen des modifizierten ortho-Weges ausgesucht. Die entwickelten Primer wurden an verschiedenen Chloraromaten abbauenden Bakterien getestet. Es gelang dabei erstmals, Fragmente von Chlormuconat-Cycloisomerase-Genen aus alpha-Proteobakterien zu erhalten. Mit den neu entwickelten Primern zum Nachweis der Chlorcatechol-1,2-Dioxygenase-Gene wurden aus den Stämmen Burkholderia sp. 3CB-1 und Rhodococcus opacus 1CP auch Fragmente amplifiziert, die nur relativ geringe Ähnlichkeiten zu bereits bekannten Genen aufwiesen. Um die Sequenzdatenbasis für das Primerdesign zu erweitern, wurden außerdem Chlorcatechol-Gencluster aus zwei Vertretern der alpha-Proteobakterien kloniert und sequenziert. Aus dem Stamm Sphingomonas sp. TFD44 konnten dabei zwei verschiedene Gencluster charakterisiert werden, von denen nur eines einen kompletten Satz der Chlorcatechol-Gene enthielt. Die beiden Gencluster aus dem anderen Stamm, Sphingomonas sp. EML146, wiesen Homologien zu diesem Gencluster auf. Die Konstruktion einer Knockout-Mutante und Proteinanreicherungen ergaben Hinweise auf weitere Chlorcatechol-Abbaugene in Sphingomonas sp. TFD44.

The main aim of this work was the study of bacteria capable to degrade the pyridine monocarboxylic acids. Achromobacter sp. strain JS18 capable to utilize 3-hydroxypyridine- 2-carboxylic acid was selected by screening of microorganisms hydroxylating the pyridine ring at unusual positions or transforming pyridine derivatives . The strain 5HP consuming 5- hydroxypyridine-2-carboxylic acid as a sole carbon and energy source was isolated from soil. The 16S rRNA-based phylogenetic analysis showed that the isolate belongs to Pusillimonas genus. It was found that picolinic, nicotinic and dipicolinic acids were metabolized via three distinct inducible pathways in Achromobacer sp. JS18. The appropriate biodegradation routes of these acids as well as 3-hydroxypyridine-2-carboxylic acid were was proposed. Nicotinic acid, 5-hydroxypicolinic acid and 3-hydroxypyridine induced three distinct metabolic pathways in Pusillimonas sp. 5HP cells. All pathways had the same intermediate – 2,5-dihydroxypyridine. For the first time 5-hydroxypicolinate 2-monooxygenase, which catalyzed oxidative decarboxylation of 5-hydroxypicolinic acid, was discovered, partially purified and characterized. The analysis of Sinorhizobium sp. L1 cells showed that 3-hydroxypyridine and nicotinic acid were degraded via different metabolic pathways. The Sinorhizobium sp. L1 cells converted 3-hydroxymethylpyridine to nicotinic acid. 3-hydroxypyridine and nicotinic acid induced biosynthesis of distinct isoforms of 2... [to full text] / Šio darbo metu buvo tiriamos bakterijos, galinčios skaidyti piridino monokarboksirūgštis. Netradicinėse vietose hidroksilinančių ir/ar hidroksilintų pikolino ir nikotino rūgščių skaidyme dalyvaujančių mikroorganizmų atranka leido identifikuoti Achromobacter sp. JS18 bakteriją, skaidančią 3-hidroksipiridino-2-karboksirūgštį, ir iš dirvožemio išskirti 5HP kamieną, galintį panaudoti 5-hidroksipiridino-2-karboksirūgštį vieninteliu anglies ir energijos šaltiniu. 16S rRNR geno analizės duomenimis 5HP kamienas priklauso Pusillimonas genčiai. Tai pirmoji žinoma bakterija, galinti skaidyti 5- hidroksipiridino-2-karboksirūgštį. Nustatyta, kad Achromobacter sp. JS18 bakterijose yra indukuojami trys skirtingi piridino monokarboksirūgščių skaidymo keliai. Pasiūlyti pikolino, nikotino, dipikolino rūgščių ir 3-hidroksipiridino-2-karboksirūgšties, skaidymo keliai. Pusillimonas sp. 5HP ląstelėse pirmą kartą aptikta ir dalinai išgryninta 5- hidroksipikolinato 2-monooksigenazė, katalizuojanti 5-hidroksipiridino-2-karboksirūgšties oksidacinį dekarboksilinimą, susidarant 2,5-dihidroksipiridinui. 5HP bakterijose yra indukuojami trys (3-hidroksipiridino, 5-hidroksipikolino ir nikotino rūgščių) skaidymo keliai, susidarant bendram metabolitui – 2,5-dihidroksipiridinui. Tiriant Sinorhizobium sp. L1 ląstel÷s, buvo nustatyta, kad 3-hidroksipiridino ir nikotino rūgšties skaidymas vyksta skirtingais keliais. 3-Hidroksimetilpiridinas L1 ląstelėse yra oksiduojamas iki nikotino rūgties ir skaidymas vyksta... [toliau žr. visą tekstą]

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