Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-27T14:23:05Z No. of bitstreams: 1 texto completo.pdf: 166311 bytes, checksum: 83322a34768e7a94f65989a1722e7533 (MD5) / Made available in DSpace on 2017-04-27T14:23:05Z (GMT). No. of bitstreams: 1 texto completo.pdf: 166311 bytes, checksum: 83322a34768e7a94f65989a1722e7533 (MD5) Previous issue date: 2004-08-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Três procariotas obtidos de filoplano de tomateiro (Bacillus cereus, Pseudomonas putida, Novosphingobium capsulatum), previamente selecionados como bons antagonistas de patógenos da cultura foram formulados em cinco formas distintas e avaliados quanto a sua efetividade como agentes de biocontrole. Quatro das cinco formulações, consistiam de produtos comerciais (Agromil-Raiz, Agromil-S, Ecolife 40 and ISR 2000) os quais propágulos dos antagonistas foram incorporados. A quinta formulação foi proposta no presente trabalho e consiste de uma solução de nutrientes com estratos de parede de células de Saccharomyces cerevisiae e goma xantana. Os efeitos deletérios das formulações sobre o crescimento dos antagonistas bem como a sobrevivência foram investigados. Os resultados indicam que Agromil-S e ISR 2000 tem efeito inibitório sobre o crescimento dos antagonistas os outros produtos não interferem significantemente na multiplicação dos antagonistas. A vida de prateleira dos antagonistas na formulação proposta foi estudada e nenhuma foi eficiente em aumentar a sobrevivência quando comparadas com o controle. O biocontrole experimental de doenças do tomateiro induzidas por três patógenos Corynespora cassiicola, Pseudomonas syringae pv. tomato, e oidio respectivamente, em casa de vegetação foi tentado para todos aos antagonistas e para todas as formulações, o controle foi alcançado embora em alguns casos não se encontrou diferença significativa entre o tratamento e o controle. Mais esforços e pesquisas devem ser realizados afim de melhorar a proteção de células a serem dispensadas em plantas. / To improve each antagonist efficiency as control agent of tomato leaf diseases, a study was carried out on five distinct formulae consisting of additives and one out of three autochthonous, isolated prokaryotic residents (Bacillus cereus, Pseudomonas putida, Novosphingobium capsulatum) of the tomato phylloplane and already selected as good antagonists to tomato pathogens. Four of these five formulations consisted of commercial products (Agromil-Raiz, Agromil-S, Ecolife 40 and ISR 2000) to which each antagonist living propagules were incorporated. The fifth of these combinations consisted of a nutrient solution amended with a Saccharomyces cerevisiae cell wall extract along with xanthan gum. Therefore, under consideration were the effects of each formula on each antagonist protective action and survival. Results of in vitro studies indicated that Agromil-S and ISR 2000 inhibited the antagonists' growth while the others didn't interfere in a significant way with the antagonists’ multiplication. In shelf live experiments, none of the studied formulation was efficient enough to increase significantly the antagonist survival when compared to controls. Under greenhouse conditions, every antagonist, in all formulas, controlled the tomato leaf diseases caused by Corynespora cassiicola, Pseudomonas syringae pv. tomato, or Oidium sp., although differences between some treatments and controls were not significant. We feel that with this we have only begun our efforts to find ways to improve protection of the antagonist’s living cells being delivered to tomato plants, and which will render them eventually well protected against leaf pathogens. . / Dissertação importada do Alexandria

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.

The aim of this work is study of utilization of PHA producing bacteria in bioremediation technologies. For this study were used bacteria Pseudomonas putida KT2440 and two isolates from soil contaminated by petroleum - Pseudomonas gessardii (D2) a Pseudomonas fulva (D3). The experimental part describes especially study of feather biodegradation using selected microbial strains. All the tested bacterial strains were capable of feather degradation and utilization as the sole carbon source. During biodegradation experiment, we monitored weight loss of feather, protease and keratinase activity, concentration of bacterial biomass and PHA content as well as pH. The highest biodegradation ability and keratinase activity was observed in Pseudomonas putida. None of tested bacteria accumulated detectable amount of PHA during growth on waste feather, nevertheless, bacterial biomass grown during feather degradation can be used as an inoculum for PHA production on waste frying oil and octanoic acid. Using this experimental setup, high PHA content (54% of cell dry weight) was achiaved in Pseudomonas putida. Another part of the thesis deals with biodegradation of petroleum oil. The highest capability of growth on this carbon source were determined in Pseudomonas fulva.

With a growing global population, food security and waste management strategies are needed (FAO, 2002b); insects have been promoted for these goals. Black soldier fly larvae (BSFL) are endorsed for their ability in decomposition and use as feed (Bava et al., 2019; Swett Walker, 2018). Studies show the life cycle of BSFL is impacted by bacterial supplementation (Franks et al., 2021; Kooienga et al., 2020). We seek to determine the effects of supplementing BSFL diet with Pseudomonas putida 3P. We conducted two forms of a 10-day bench-top study observing larval mass, frass conversion, C:N content, and ammonia production with supplementation of Pseudomonas putida 3P in single and double-doses. Supplementation with P. putida results in roughly 1.5X larger larval wet and dry mass, variable concentrations of ammonia, and approximately 1.15X smaller C:N ratios in frass. This suggests interruption of larval rearing may affect volatile ammonia concentrations and inhibit P. putida behavior.

Im Rahmen der vorliegenden Arbeit wurden die Wechselwirkungen im System Bakterium –Tensid – Schadstoff mittels kalorimetrischer Untersuchungen (ITC, DSC) sowie mit XPS-Analysen und durch Zeta-Potential-Messungen an Bakterienoberflächen charakterisiert. Für die Untersuchungen wurden zwei Gram-positive Rhodococcus-Stämme und ein Gram-negativer Pseudomonas putida-Stamm verwendet. Als Biotenside wurden das Rhamnolipid JBR 425 und der von Rhodococcus erythropolis B7g produzierte Trehalosetetraester (THL-4) ausgewählt. Das synthetische Tensid SDS diente als Referenzsubstanz. Aus den kalorimetrischen Experimenten konnte eine starke Wechselwirkung zwischen den Tensiden und den aktiven Bakterienkulturen abgeleitet werden. THL-4 führte beim Wachstum der Rhodococcen auf n-Hexadecan zur Verkürzung der lag-Phase. SDS wies hingegen eine toxische Wirkung für die Bakterienstämme auf. Thermodynamische Betrachtungen ergaben, dass Wechselwirkungen des SDS mit den Bakterienzellen gegenüber der Mizellbildung bevorzugt werden.

Im Rahmen der vorliegenden Arbeit wurden die Wechselwirkungen im System Bakterium –Tensid – Schadstoff mittels kalorimetrischer Untersuchungen (ITC, DSC) sowie mit XPS-Analysen und durch Zeta-Potential-Messungen an Bakterienoberflächen charakterisiert. Für die Untersuchungen wurden zwei Gram-positive Rhodococcus-Stämme und ein Gram-negativer Pseudomonas putida-Stamm verwendet. Als Biotenside wurden das Rhamnolipid JBR 425 und der von Rhodococcus erythropolis B7g produzierte Trehalosetetraester (THL-4) ausgewählt. Das synthetische Tensid SDS diente als Referenzsubstanz. Aus den kalorimetrischen Experimenten konnte eine starke Wechselwirkung zwischen den Tensiden und den aktiven Bakterienkulturen abgeleitet werden. THL-4 führte beim Wachstum der Rhodococcen auf n-Hexadecan zur Verkürzung der lag-Phase. SDS wies hingegen eine toxische Wirkung für die Bakterienstämme auf. Thermodynamische Betrachtungen ergaben, dass Wechselwirkungen des SDS mit den Bakterienzellen gegenüber der Mizellbildung bevorzugt werden.

An exceptionally important stress response of Pseudomonas putida strains to toxic chemicals is the induction of efflux pumps that extrude solvents, as well as other toxicants, into the surrounding medium. However, the bacterial tolerance mechanisms are still not fully understood, thus in this thesis metabolomic approaches were used to detect and identify metabolites involved in P. putida DOT-T1E tolerance to abiotic stresses, in particular focussing on the role of efflux pumps. To elucidate any metabolome alterations several strains of P. putida, including the wild type DOT-T1E, and the efflux pump knockouts DOT-T1E-PS28 and DOT-T1E-18, were challenged with different levels of propranolol. Fourier-transform infrared (FT-IR) spectroscopy, which provided a rapid, high-throughput metabolic fingerprint of P. putida strains, was used to investigate any phenotypic changes resulting from exposure to propranolol. FT-IR data illustrated phenotypic changes associated with the presence of propranolol within the cell that could be assigned to the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling on the same samples was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during growth of bacteria following this toxic perturbation with propranolol. GC-MS revealed significant changes in ornithine levels which can be directly linked to bacterial tolerance mechanisms, and alterations in the levels of several other metabolites which were also modified in response to propranolol exposure. Moreover, the effect of the organic solvent toluene was also investigated using the same approach. Examination of FT-IR data indicated that protein and fatty acids were the most affected components of P. putida strains due to the presence of toluene within the cell. Moreover, application of GC-MS allowed for the identification and quantification of several metabolites which were differentially produced or consumed in the presence of toluene. To investigate the role of efflux pumps in P. putida DOT-T1E, several analytical techniques were employed including Raman spectroscopy, gas and liquid chromatography to identify and quantify the level of propranolol or toluene in P. putida cells. These analyses showed that propranolol and toluene accumulated in the mutant P. putida DOT-T1E-18 (lacking the TtgABC pump) at higher levels in comparison with the levels found in the wild-type DOT-T1E and the mutant DOT-T1E-PS28 (lacking the TtgGHI pump), indicating the key role of efflux pumps in solvent tolerance. Furthermore, the effect of Mg2+ and Ca2+ on the stabilisation of the toluene tolerance of P. putida DOT-T1E strains was examined in order to elucidate whether divalent cations interact with efflux pumps or other resistant mechanisms to improve solvent tolerance. FT-IR analysis suggested that the influence of divalent cations on the stabilisation of the toluene tolerance could be due to the contribution of metal ions towards other tolerance mechanisms such as lipopolysaccharide (LPS) instead of enhancing the activity of efflux pumps. In conclusion, this thesis presents evidence that phenotypic fingerprinting and metabolic profiling approaches in combination with chemometric methods can generate valuable information on phenotypic responses occurring within microbial cultures subjected to abiotic stress.

No description available.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2010-2011 / Le but de cette étude était d'en apprendre davantage sur les relations antagonistes entre Helminthosporium solani et les souches E30 et 94-19 de Pseudomonas putida. Les travaux présentés dans ce mémoire montrent que la souche 94-19 produit des composés antifongiques quel que soit le milieu de culture utilisé alors que la souche E30 produit des composés antifongiques seulement en présence de cellules fongiques vivantes, en particulier en présence de cellules vivantes de H. solani. La présence des gènes de voies de biosynthèse de la pyrrolnitrine, de la pyolutéorine et du 2,4-diacétylphloroglucinol chez la souche 94-19 suggère l'implication de ces molécules dans l'activité antagoniste de cette bactérie envers H. solani. Aucun de ces gènes n'a été retrouvé chez la souche E30. Les travaux réalisés suggèrent plutôt qu'un composé thermostable de faible poids moléculaire serait impliqué dans l'activité antagoniste de la souche E30 envers H. solani.

No description available.