7,8-Linoleate diol synthase (7,8-LDS) of the take-all pathogen of wheat, Gaeumannomyces graminis, converts linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8-HPODE) by 8-dioxygenase activity (8-DOX), and further isomerizes the hydroperoxide to 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by hydroperoxide isomerase activity. Sequence alignment showed homology to prostaglandin H synthase (PGHS), and both enzymes share structural and catalytic properties. The 8-DOX of 7,8-LDS was successfully expressed in Pichia pastoris and in insect cells (Sf21). Site-directed mutagenesis confirmed His379 as the proximal heme ligand and Tyr376 as a residue, which forms a tyrosyl radical and initiates catalysis. Furthermore, mutagenesis suggested His203 could be the proposed distal histidine, and Tyr329 of catalytic relevance for substrate positioning at the active site. Aspergilli are ubiquitous environmental fungi. Some species, in particular Aspergillus fumigatus, are responsible for invasive aspergillosis, which is a life-threatening disease for immunocompromised patients. A. fumigatus and A. nidulans metabolized linoleic acid to 8R-HPODE, 10R-hydroperoxyoctadecadienoic acid (10R-HPODE), 5S,8R-dihydroxyoctadecadienoic acid, and 8R,11S-dihydroxyoctadecadienoic acid. When the genomes of certain Aspergilli strains were published, several species showed at least three homologous genes (ppoA, ppoB, ppoC- psi producing oxygenases) to 7,8-LDS and PGHS. Gene deletion identified PpoA as an enzyme with 8-DOX and 5,8-hydroperoxide isomerase activities, designated 5,8-LDS in homology to 7,8-LDS. In the same way, PpoC was identified as a 10-dioxygenase (10-DOX), which converts linoleic acid to 10R-HPODE. 10-DOX differs from LDS, since it dioxygenates linoleic acid at C-10, after hydrogen abstraction at C-8 and double bond migration. 10-DOX was cloned and expressed in insect cells. Leu384 and Val388 were found to be critical for dioxygenation at C-10. Mutation to the homologous residues of 5,8- and 7,8-LDS (Leu384Val, Val388Leu) increased oxygen insertion at C-8. LDS and 10-DOX are fusion proteins with a dioxygenase and a hydroperoxide isomerase (cytochrome P450) domain with a cysteine heme ligand. The P450 domain of 10-DOX lacked the crucial cysteine heme ligand and was without hydroperoxide isomerase activity. LDSs and 10-DOX are newly characterized heme containing fungal dioxygenases, with homology to PGHS of vertebrates. Their metabolites regulate reproduction, development, and act as signal molecules with the host after pathogen attack.

O acúmulo de poluentes orgânicos persistentes é um dos principais problemas de contaminação do meio ambiente em todo o mundo. As atividades industriais e os avanços tecnológicos na maioria dos setores de produção contribuem com a crescente liberação de compostos poluentes, altamente tóxicos, biocumulativos e resistentes à degradação física, química, fotolítica e biológica. Esses poluentes são o resultado do uso indevido de pesticidas de um modo geral, da subprodução não planejada de sintéticos tóxicos como dioxinas policloradas e de uma lista enorme de atividades que promovem a combustão incompleta da matéria orgânica e despejam no ambiente uma vasta quantidade de hidrocarbonetos aromáticos persistentes. A biorremediação é uma estratégia biotecnológica que vem lançar luz sobre as técnicas de revitalização de sítios contaminados. É baseada na utilização de microorganismos, ou suas enzimas, para reduzir ou eliminar perigos ambientais contaminantes em substâncias inertes, CO2 e água. As oxigenases são uma classe de enzimas amplamente estudadas por suas propriedades catalíticas únicas, sendo a clorocatecol 1,2- dioxigenase de Pseudomonas putida um interessante alvo biotecnológico para a biorremediação devido a sua ampla especificidade a substratos aromáticos, aromáticos halogenados e dialogenados altamente poluentes, biocumulativos e carcinogênicos. Nesse trabalho, o protocolo de expressão heteróloga em E. coli foi implementado em nosso laboratório e a purificação realizada em coluna de afinidade através do uso de resina de quitina. Os cristais de cor marrom avermelhado da enzima clorocatecol 1,2-dioxigenase Pseudomonas putida foram obtidos na presença de polietilenogligol e acetato de magnésio durante o período de 10 dias, utilizando-se a técnica de difusão de vapor em gota sentada. A estrutura cristalográfica da enzima Pp 1,2-CCD foi determinada através da utilização da técnica MR-SAD, utilizando átomos de ferro, cofator da proteína, como agentes espalhadores e as coordenadas da enzima 3-clorocatecol 1,2-dioxigenase de Rhodococcus opacus como modelo de busca. O modelo final, contendo 3 moléculas na unidade assimétrica foi refinado a 3.4 Å de resolução. A enzima se enovela na forma dimérica (Fe3+)2, e apresenta de um modo geral, dois domínios catalíticos compostos de fitas-beta e uma região de loops e um domínio de ligação composto por hélices formando um túnel hidrofóbico. Como a primeira clorocatecol de bactérias gram-negativa a ser descrita para essa classe de enzimas, será possível investigar as diferenças conformacionais que levam aos mecanismos de catálise, amplo a diversos substratos, e avaliar características de seu funcionamento e ativação, como uma importante ferramenta para validação do papel desta proteína dentro do processo de biorremediação. / Accumulation of persistent organic pollutants is one of the most important environmental problems worldwide. Industrial activities and technological advances contribute to the spread of pollutants, highly toxic, bioaccumulative, and resistant to physical, chemical, photolytic and biological degradation. The persistent organic pollutants are consequence of the inappropriate use of pesticides, the production of toxic synthetic compounds such as polychlorinated biphenyls and a large list of activities promote incomplete combustion of which organic matter and release a large amount of persistent aromatic hydrocarbons to the environment. Bioremediation is a biotechnogical strategy that has shed light on revitalization techniques of contaminated sites. It is based on the application of microorganisms, or their enzymes, to eliminate or reduce environmental contaminants into inert substances, CO2 and water. The oxygenases are a class of largely studied enzymes due to their catalytic properties, being chlorocatechol 1,2-dioxygenase from Pseudomonas putida an interesting biotechnological target for bioremediation due to its specificity to a broad spectrum of highly polluted aromatic substrates. In the present work, heterologous expression protocol has been implemented in our laboratory and purification was performed by using affinity column in chitin resin. Brown-reddish crystals of chlorocatechol 1,2-dioxygenase from Pseudomonas putida were obtained in presence of polyethylene glycol (PEG) and magnesium acetate after 10 days, by utilizing vapor diffusion techniques in sitting drops. The crystallographic structure of Pp 1,2-CCD has been solved by MR-SAD technique, using iron atoms, the enzyme cofactor, as scattering centers and the coordinates of 3- chlorocatechol 1,2-dioxygenase of Rhodococcus opacus as the search model. The final model, containing three molecules in the asymmetric unit, has been refined up to 3.4 Å resolution. The enzyme folds in the dimeric form (Fe3+)2, and shows two catalytic domains composed of -strands and a loop region, and a helical domain that pack together forming a hydrophobic channel. As being the first member of the chlorocatechol dioxygenase family of enzymes from a gram-negative bacteria to be solved, it will be possible to explore the crystallographic structure of chlorocatechol 1,2- dioxygenase from Pseudomonas putida in order to investigate the conformational differences that explain its mechanism of action, on a broad spectrum of substrates, and evaluate the functional features, as an important tool to validate the role of this enzyme in the bioremediation process.

O intenso uso de compostos orgânicos em conjunto com o grande avanço industrial culminou em um enorme acúmulo de poluentes orgânicos no meio ambiente. Dentre estes poluentes têm-se destacado a presença de hidrocarbonetos aromáticos altamente tóxicos e resistentes à degradação física, química, fotolítica e biológica. Desta maneira, uma nova forma de combater a presença deste tipo de composto no meio ambiente têm sido estudada: o uso de microorganismos, naturais ou geneticamente modificados, capazes de transformá-los em substâncias inertes, como CO2 e água. Tal metodologia é denominada biorremediação. Dentres estes microorganismos destacam-se bactérias dos gêneros Pseudomonas, Aeromonas, Beijerinckia, dentre outros, que têm sido estudadas para esta finalidade. A enzima clorocatecol 1,2-dioxigenase (Pp 1,2-CCD) é uma das proteínas expressas por bactérias do gênero Pseudomonas putida, sendo responsável pela clivagem de hidrocarbonetos aromáticos através da incorporação de ambos os átomos de uma molécula de oxigênio à estrutura do anel aromático, sendo a proteína escolhida para desenvolvermos o presente trabalho. Mais especificamente, nos interessa estudar como o mecanismo de ação da referida enzima é controlado por moléculas extrínsecas, como fosfolipídios. Tal interesse pela interação entre a enzima e fosfolipídios surgiu recentemente quando da obtenção da primeira estrutura cristalográfica de uma enzima da família da CCD (dioxigenases intradióis). Nesta estrutura foi observado um sítio de ligação por monômero para fosfolipídios, o que fez com que várias questões relativas à influência desses sobre a atividade da enzima fossem levantadas. Nosso objetivo foi fazer uso das técnicas de Dicroísmo Circular (CD), Calorimetria e Ressonância Paramagnética Eletrônica (RPE) para estudar alterações conformacionais da enzima e de sua cinética induzidas por moléculas de fosfolipídio, e assim, obter informações que correlacionem as mudanças estruturais com o mecanismo de atividade enzimática da enzima. Os resultados obtidos através do uso daquelas técnicas em conjunto com protocolos que possibilitam a delipidação da enzima mostraram que a presença do fosfolipídios na estrutura da enzima tem influência sobre a atividade enzimática. Quando retiramos o fosfolipídio/ácido graxo, pudemos visualizar uma pequena mudança na estrutura secundária da enzima, um aumento da entalpia de reação, bem como um aumento na velocidade de reação, enquanto que a afinidade da enzima pelo substrato diminuiu. Pudemos também observar uma maior estabilidade térmica da enzima quando na ausência do fosfolipídio/ácido graxo e não foi observado interação da Pp 1,2-CCD com modelos micelares constituídos por lisofosfolipídios. Um breve estudo realizado sobre o papel da força iônica na atividade e na estabilidade térmica da proteína mostrou que na ausência de NaCl, em pH 8, a enzima se mostrou mais ativa, com uma afinidade pelo substrato maior e neste ambiente com baixa força iônica foi observado uma pequena interação da enzima com modelos micelares carregados negativamente. Assim, pudemos concluir que as moléculas anfipáticas, retiradas com os processos de delipidação, apesar de modificarem muito pouco a estrutura secundária da enzima, ainda assim instauram modificações na sua função de catálise do substrato catecol. Esta informação juntamente com os dados sobre inibição do processo reacional ocasionada pelo produto da reação formam um novo conjunto de dados que pode ser utilizado para se alcançar o objetivo mais geral de se controlar a atividade biológica da Pp 1,2-CCD. / The intensive use of organic compounds in conjunction with the industrial advances led to a huge accumulation of organic pollutants in the environment. Among these pollutants it has been noticed the presence of aromatic hydrocarbons that are highly toxic and resistant to physical, chemical and biological degradation. Thus, a new way to deal with the presence of this compounds in the environment has been studied: the use of microorganisms, natural or genetically modified, that can turn them into inert substances such as CO2 and water. This methodology is called bioremediation. Among those microorganisms, bacteria from the gender Pseudomonas, Aeromonas, Beijerinckia, among others, have been studied for this purpose. The enzyme chlorocatechol 1,2-dioxygenase (Pp 1,2-CCD) is one of the proteins expressed by Pseudomonas putida bacteria, being responsible for the cleavage of aromatic hydrocarbons through the incorporation of both atoms of a molecule of oxygen into the aromatic ring structure, being the protein chosen for investigation in this work. More specifically, we are interested in studying how the mechanism of action of this enzyme is controlled by extrinsic molecules such as phospholipids. The interest in the interaction between the enzyme and phospholipids arose recently when the first crystal structure of an enzyme of the intradiol dioxygenase family was reported. In this structure it was observed a binding site for a phospholipid per monomer, which raised many issues concerning its influence on the activity of the enzyme. Our goal was to use the techniques of Circular Dichroism (CD), calorimetry and Electron Magnetic Resonance (EMR) to study enzyme conformational changes and kinetics alterations induced by phospholipid molecules, thus gathering information on the structure-function correlation. The results obtained through those experimental techniques in conjunction with the use of protocols for protein delipidation showed that the presence of phospholipids/fatty acids in the structure of the enzyme play a role in enzyme activity. Upon removal of the phospholipid/fatty acids, we observed small changes in the secondary structure of the enzyme, an increase of the enthalpy of reactions as well as an increase in the reaction rate, whereas the affinity of the enzyme for the substrate decreased. We also observed a higher thermal stability of the Pp 1,2-CCD in the absence of the phospholipids/fatty acids, but no interaction was observed between the Pp 1,2-CCD and lysophospholipid micelles. A brief study of the function of ionic strength on the activity and thermal stability of the protein showed that in the absence of NaCl, at pH 8, the enzyme is more active, showing a greater affinity for the substrate and a low interaction was observed between Pp 1,2-CCD and negatively charged micelles. This information along with the data on the inhibition capacity of the reaction product are a new set of data that can be used to achieve the more general goal of controlling Pp 1,2-CCD biological activity.

Triptofano (TRP), um amino ácido essencial, é metabolizado por duas vias principais, a via das quinureninas e a via serotonérgica. Em ambas as vias há a possibilidade de formação de compostos ativos no sistema imune que se caracterizam pelas ações imunossupressoras e indutoras de tolerância. Na via serotonérgica há a formação de serotonina (5-HT) e em alguns tecidos de melatonina (MEL). Este composto pode ainda ser oxidado por ação de peroxidases aos seus produtos de abertura de anel indólico o AFMK (N1-acetil-n2-formil-5-metoxiquinuramina) e AMK (N1-acetil-5-metoxiquinuramina). Já na via das quinureninas, o TRP é diretamente metabolizado à N-formilquinurenina (NFK) e este é rapidamente deformilado a quinurenina (QUIN). Neste projeto avaliamos qual o efeito do choque endotóxico induzido por injeção endovenosa de LPS (1 mg/kg) sobre a biodisponibilidade de TRP e formação de seu metabólito QUIN. Este estudo foi realizado em condições controle e na vigência de sobrecarga de TRP (administração subcutânea de 0,8 mg/kg). Utilizamos ratos machos Wistar com 30 dias separados em quatro grupos: GI (controle), GII (LPS), GIII (TRP) e GIV (TRP+LPS). TRP (0,8 mg/Kg) foi injetado por via subcutânea nos tempos 0 e 2 horas. Quando injetado, LPS (1 mg/kg) foi administrado por via intravenosa no tempo 2 horas. Após 1 hora da última administração, sangue e cérebro foram coletados. O cérebro foi seccionado em três regiões: cerebelo, córtex e mesencéfalo, os quais foram processados para obtenção de homogenatos. Tanto os homogenatos quanto o soro foram tratados com acetona para extração de TRP e seus metabólitos. A análise destes compostos foi realizada por cromatografia líquida de alta eficiência (HPLC). A administração de TRP elevou significativamente a sua concentração no soro e no SNC. Quando da administração de LPS no grupo que já havia recebido sobrecarga de TRP (GIV) houve uma marcada elevação de TRP e de QUIN séricos e das regiões do SNC, especialmente na região do córtex. Concluímos que na vigência de choque endotóxico há um aumento da biodisponibilidade de TRP, tanto no soro como no SNC e que há um aumento da metabolização deste pela rota das quinureninas, possivelmente via IDO. Estes resultados contribuem para a compreensão da toxicidade de TRP, especialmente relevante no caso em que haja um choque endotóxico concomitante e evidencia o córtex como uma região mais susceptível para os efeitos tóxicos do TRP. / Tryptophan (TRP) is an essential amino acid, metabolized by two main paths; the kynurenine and the serotonergic pathways. In both, there is the possibility of generation of biologic active compounds, especially on the immune system leading to immunosuppression and tolerance. In the serotonergic path there is the formation of serotonine (5-HT) and in some tissues of melatonine (MEL). The latter can be oxidized by the action of peroxidases to its indole ring opening product AFMK (N1-acetil-n2-formil-5-methoxikynuramine) and AMK (N1-acethyl-5-methoxykynuramine). In the kynurenine path, TRP is metabolized to N-formylkynurenine (NFK) that is deformilated to kynurenine (KYN). In this study we evaluated the effect of a endotoxic skock induced by an intravenous injection of LPS (1 mg/kg) on the bioavailability of TRP and formation of KYN. This study was carried out in control conditions and on TRP overload (subcutaneous administration of 0,8 mg/Kg). One month old male Wistar rats were divide in four groups: GI(control), GII(LPS), GIII(TRP) and GIV (TRP+LPS). TRP (0,8 mg/kg) was subcutaneously injected at zero and 2h times. When injected, LPS (1mg/kg) was intravenously administered at 2 h. After one hour from the last administration, blood and brain were collected. Brain is separated in cerebellum, midbrain and cortex and was lysed for the preparation of homogenates. Both, serum and homogenates were extracted in acetone; TRP and KYN were analyzed by HPLC. TRP overload caused a significant increase in its concentration in serum and brain. When LPS was administered in conjunction with TRP overload (GIV) there was a remarkable increase in TRP and KYN in serum and brain, especially in cortex. Our conclusion is that in the bioavailability of TRP, in serum and in brain, and its metabolization to kynurenine is increased by inflammation. IDO is probably involved in this condition. Our results contribute to the knowledge of TRP toxicity, particularly with a concomitant inflammation and demonstrate the cortex as a region of more susceptibility to TRP toxicity.

A indoleamina 2,3-dioxigenase (IDO) é uma enzima, produzida pelas células trofoblásticas e, por sua capacidade de catabolizar o triptofano, inibe a proliferação das células T maternas, participando desta forma como importante mecanismo da tolerância materno-fetal. Contudo, pouco se sabe se a ação da IDO é influenciada por outras substâncias presentes no micro-ambiente uterino e, por esta razão, formulou-se a hipótese de que esta enzima poderia ter sua ação alterada nesse contexto e, desta forma, avaliou-se o comportamento da expressão da IDO frente à adição de alguns desses componentes presentes no útero gestante. Neste trabalho, células uterinas, de placentas e de embriões de ratas e camundongos fêmeas prenhes e não prenhes foram mantidas em cultivo e a elas adicionadas estradiol, progesterona, interferon , triptofano e 1-metil-D- triptofano, avaliando-se a expressão da IDO pela técnica de citometria de fluxo nos períodos de 4, 24 e 48 horas. Os resultados demonstraram que as diferenças mais significativas na expressão da IDO entre as ratas prenhes e não prenhes, foram observadas após a adição de progesterona (19,24%), interferon (11.22%) e triptofano (23,53%), nas ratas prenhes. Já nos camundongos fêmeas prenhes e não prenhes, as maiores expressões de IDO foram observadas no primeiro grupo pela adição de progesterona (11,33%), estradiol (9,98%) e interferon (21,78%). Considerando esses resultados, podemos concluir que a expressão da IDO pelas células uterinas, placentárias e embrionárias em cultivo sofre influência dos fatores testados, o que permite novas hipóteses para melhor compreensão da participação dessa enzima na tolerância materno-fetal, particularmente em relação a sua interação com hormônios e citocinas presentes no útero gestante. Além disso, poderá colaborar na elaboração de possíveis tratamentos de enfermidades, relacionadas à manutenção e sucesso da gestação onde haja envolvimento do sistema imunológico materno e dos mecanismos de tolerância. / Indoleamine 2,3-dioxygenase (IDO) is an enzyme produced by trophoblast cells and due to its ability to catabolize tryptophan, inhibits the proliferation of maternal T cells, thus playing an important role as one of the mechanisms of maternal-fetal tolerance. However, little is known whether the action of IDO is influenced by substances present in the pregnant uterine microenvironment. This study evaluated the behavior of the IDO expression in cultured placental and embryonic cells from mice and rats in face of the addition of estradiol, progesterone, interferon, tryptophan and 1-methyl-D-tryptophan, by flow cytometry, at 4, 24 and 48 hours periods. The results showed that high expressions of IDO were observed in pregnant rats cells after the addition of progesterone (19.24%), interferon (11.22%) and tryptophan (23.53%). In mice, high expressions of IDO were observed in the pregnant group cells by the addition of progesterone (11.33%), estradiol (9.98%) and interferon- (21.78%). Considering these results, we may conclude that the expression of IDO by cultured placental and embryonic cells from mice and Wistar rats is indeed influenced by factors present in pregnant uterus, which provides additional information to better understand IDO role in the maternal-fetal tolerance, particularly on its interactions with reproductive hormones and cytokines. Additionally, it may contribute to the establishment of possible treatments for pregnancy-losses related to the maternal immune system response and mechanisms of tolerance.

L’indoléamine-2,3-dioxygénase (IDO) est une enzyme du catabolisme du tryptophane suspectée de jouer un double rôle lors des infections en contribuant aux défenses innées de l’hôte et en régulant la réponse immunitaire. IDO est exprimée au cours de l’infection par le virus de l’hépatite C (VHC). Cependant, les mécanismes moléculaires conduisant à l’expression de IDO lors de l’hépatite C et l’impact de IDO sur la réplication virale et sur la réponse immunitaire ne sont pas connus. Dans ce travail de thèse, nous avons montré que le VHC stimule l’expression de IDO dans les hépatocytes.L’expression de IDO était transitoire et coïncidait avec l’expression des interférons (IFNs) de types I et III et avec la transcription de gènes stimulés par les IFNs. L’expression de IDO était également augmentée dans les hépatocytes exposés à la présence de lymphocytes T CD4+ activés et producteurs d’IFN-γ. L’expression hépatique de IDO diminuait la réplication virale, ce qui suggère que IDO limite la diffusion du VHC dans le foie au cours de l’hépatite C. Grâce à des expériences de silencing, nous avons montré que IDO contribue à l’effet antiviral de l’IFN-α sur le VHC. L’expression de IDO était régulée par l’activation des facteurs de transcription IRF-1 et STAT-1 dans les hépatocytes infectés par le VHC. En plus de son effet antiviral sur le VHC, l’expression hépatique de IDO inhibait significativement la prolifération des lymphocytes T CD4+ activés, suggérant un rôle immunorégulateur de IDO au cours de l’hépatite C. Nos données suggèrent donc que IDO joue un double jeu lors de l’hépatite C, en limitant la réplication virale et en régulant la réponse immunitaire adaptative de l’hôte. Notre travail ouvre la voie à des expérimentations in vivo et à des études cliniques visant à préciser la place des inhibiteurs pharmacologiques de IDO dans l’arsenal thérapeutique contre le VHC. / Indoleamine-2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that plays a dual role during infectious diseases by contributing to the innate defenses against pathogens and by regulating the immune response. IDO is expressed in patients with hepatitis C virus (HCV) infection. However, the molecular mechanism of IDO induction in HCV infection and its role in the antiviral immune response remain unknown. Using primary human hepatocytes, we have shown that HCV infection stimulates IDO expression. IDO gene induction was transient and coincided with the expression of type I and type III interferons (IFNs) and IFN-stimulated genes (ISGs) in HCV-infected hepatocytes. IDO expression was also stimulated when the hepatocytes were incubated with IFN-γ-secreting CD4+ T cells. Expression of IDO prior to HCV infection significantly impaired HCV replication in hepatocytes, suggesting that IDO limits the spread of HCV in the liver. By using siRNA-mediated IDO knockdown experiments, we have shown that IDO contributes to the IFN-α-antiviral effect on HCV replication. IDO expression was regulated by IRF-1 and STAT-1 in HCV-infected hepatocytes. Hepatic IDO expression also had a significant inhibitory effect on CD4+ T cell proliferation, suggesting an immunoregulatory role of IDO during HCV infection. Our data suggest that hepatic IDO plays a dual role during HCV infection by retarding viral replication and also regulating host immune responses. This work paves the way for in vivo experiments and clinical studies aiming to determine the relevance of pharmacological inhibition of IDO during HCV infection.

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.

Polycyclic aromatic hydrocarbons, produced by anthropological and natural activities, are hazardous through formation of oxidative radicals and DNA adducts. Growth of Mycobacterium sp. strain KMS, isolated from a contaminated soil, on the model hydrocarbon pyrene induced specific proteins. My work extends the study of isolate KMS to the gene level to understand the pathways and regulation of pyrene utilization. Genes encoding pyrene-induced proteins were clustered on a 72 kb section on the KMS chromosome but some also were duplicated on plasmids. Skewed GC content and presence of integrase and transposase genes suggested horizontal transfer of pyrene-degrading gene islands that also were found with high conservation in five other pyrene-degrading Mycobacterium isolates. Transcript analysis found both plasmid and chromosomal genes were induced by pyrene. These processes may enhance the survival of KMS in hydrocarbon-contaminated soils when other carbon sources are limited. KMS also grew on benzoate, confirming the functionality of an operon containing genes distinct from those in other benzoate-degrading bacteria. Growth on benzoate but not on pyrene induced a gene, benA, encoding a benzoate dioxygenase α-subunit, but not the pyrene-induced nidA encoding a pyrene dioxygenase α-subunit; the differential induction correlated with differences in promoter sequences. Diauxic growth occurred when pyrene cultures were amended with benzoate or acetate, succinate, or fructose, and paralleled delayed expression of nidA. Single phase growth and normal expression of benA was observed for benzoate single and mixed cultures. The nidA promoters had potential cAMP-CRP binding sites, suggesting that cAMP could be involved in carbon repression of pyrene metabolism. Growth on benzoate and pyrene requires gluconeogenesis. Intermediary metabolism in isolate KMS involves expression from genes encoding a novel malate:quinone oxidoreductase and glyoxylate shunt enzymes. Generation of C3 structures involves transcription of genes encoding malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Carbon source modified the transcription patterns for these genes. My findings are the first to show duplication of pyrene-degrading genes on the chromosome and plasmids in Mycobacterium isolates and expression from a unique benzoate-degrading operon. I clarified the routes for intermediary metabolism leading to gluconeogenesis and established a potential role for cAMP-mediated catabolite repression of pyrene utilization.

No description available.

No description available.