Aerobic Gram negative bacteria such as Pseudomonas putida were reported to possess class A ATCases and to have a M.W. of 360 kD. The nucleotide sequence of the P. putida pyrBC was determined to answer this question once and for all. The expected regulatory gene was not found. It is shown that the P. putida pyrB gene is overlapped by pyrC by 4 bp. The P.putida pyrB is 1005 bp (335 aa) in length and the pyrC is 1275 bp (425 aa) long. Both of these genes complement E. coli mutants with their respective genotypes. Another finding borne out from the sequence is an effector binding site at the N-terminus of pyrB of P. putIda. The binding site shows that effectors compete with carbamoylphosphate for the active site. In this dissertation, it is shown that the ATCase of P.putida is a trimer of M.W. of 109 kD (3 x 36.4 kD) and that the gene encoding pyrB is overlapped by the pyrC gene which encodes DHOase. It is also shown that the pyrBC encoded enzymes copurify as a dodecameric complex with a M.W. of 484 kD.

Aspartate transcarbamoylase (ATCase) of Pseudomonas putida consists of two different polypeptides, PyrB and PyrC' (Schurr et al, 1995). The role of the PyrC' and the assembly of PyrB and PyrC' have been studied. The ATCase made in vitro of P.putida PyrB with P.putida PyrC', and of E.coli PyrB with P.putida PyrC ' were generated under two different conditions, denaturation and renaturation, and untreated. It was found that PyrC' plays a role in the enzymatic regulation by ATP, CTP and UTP. In addition to playing a role in substrate binding, the PyrB polypeptide is also involved in effector binding (Kumar et al., manuscript in preparation). The most energetically preferred form of the P.putida WT is a dodecamer with a molecular mass of 480 kDa. The ratio between the PyrB and the PyrC' is 1:1. In studies of nucleotide binding, it was discovered that the P.putida PyrB was phosphorylated by a protein kinase in the cell extract. In the presence of 20 mM EDTA, this phosphorylation was inhibited and the inhibition could be overcome by the addition of divalent cations such as Zn2+ and Mg2+. This result suggested that the phosphorylation reaction required divalent cations. In the CAD complex of eukaryotes, phosphorylations of the CPSase and the linker region between ATCase and DHOase did not occur in the presence of UTP and it was hypothesized (Carrey, 1993) that UTP and phosphorylation(s) regulated the conformational change in the enzyme complex. Therefore, the same idea was approached with P.putida ATCase, where it was found that 1.0 mM UTP inhibited the phosphorylation of PyrB by more than 50%. These results suggested that the regulation of the conformational change of the P.putida ATCase might be similar to that of CAD. Furthermore, peptide mapping for phosphorylation sites was performed on P.putida ATCase WT, WT --11 amino acids and WT --34 amino acids from the N-terminus of the PyrB polypeptide. The results showed that the phosphorylation sites were located on the fragment that contained amino acid number-35 to amino acid number-112 from the N-terminus of the PyrB polypeptide.

Tobacco-soil bacteria have evolved not only to tolerate high concentrations of nicotine, but to degrade it as a primary growth source. The genomes of several of these species have been sequenced, allowing for the identification of unique bacterial degradation pathways. In the Gram-negative bacteria, Pseudomonas putida, the nicotine-degrading gene cluster has been described; the encoded enzymes catabolize nicotine via the pyrrolidine pathway, ultimately forming malate and fumarate. In previous studies, the flavoenzyme, nicotine oxidoreductase (NicA2), has been identified as the first committed step of nicotine catabolism in this organism. Preliminary kinetic analysis reported that NicA2 has high specificity for S-nicotine, but a slow catalytic rate. Taking advantage of its unique evolutionary adaptation, we aim to refine the inherent catalytic function and structural features of NicA2 towards the development of a biotherapeutic for nicotine addiction, nicotine poisoning and tools for nicotine biosensor development. Our goal is to identify the factors contributing to the mechanistic and substrate-binding properties of NicA2 to improve its biotherapeutic potential. This work presents the first crystal structure of NicA2, resolved to 2.2 Å resolution, establishing it as a member of the flavin-dependent amine oxidase family with a conserved amine oxidase fold. Structural analysis identified a unique composition of the canonical aromatic cage (W427 and N462), which flanks the flavin isoalloxazine ring. Additionally, the X-ray crystallographic structure of the NicA2/S-nicotine complex was refined to 2.6 Å resolution, revealing a hydrophobic active site in support of a hydride-transfer mechanism. Analysis of enzyme activity with a series of substrate analogs and kinetic analysis of active-site residues reveal the determinants of substrate binding affording the remarkable specificity of this enzyme. Using site-directed mutagenesis of aromatic cage residues, along with analysis of the kinetics of the reductive and oxidative steps, we demonstrate that the rate-limiting reaction step is in the oxidative half-reaction. Structural analysis of an active-site variant revealed a secondary binding site consistent with kinetic analysis demonstrating substrate inhibition. Together, our findings provide kinetic and structural evidence for the catalytic mechanism of NicA2, expanding the possibilities for the generation of catalytically-efficient variants and supporting its role as a promising therapeutic strategy. / 2021-01-30T00:00:00Z

m-Bromotrifluorotoluene, 1, was subjected to microbial oxidation by Pp 39/D. The products from the reaction were isolated and identified by various spectral techniques. The absolute stereochemistry of the major metabolite has been determined as cis-ααα-trifluoromethyl 2R, 3S-dihydroxy-5-bromo-4,6-cyclohexadiene 2. The absolute stereochemistry was discovered by comparison of the products from the convergent synthetic pathway of 2 and the known diol metabolite from α,α,α-trifluorotoluene, 4. / Master of Science

In the presence of a solid-liquid or liquid-air interface, bacteria can choose between a planktonic and a sessile lifestyle. Depending on environmental conditions, cells swimming in close proximity to the interface can irreversibly attach to the surface and grow into three-dimensional aggregates where the majority of cells is sessile and embedded in an extracellular polymer matrix (biofilm). We used microfluidic tools and time lapse microscopy to perform experiments with the polarly flagellated soil bacterium Pseudomonas putida (P. putida), a bacterial species that is able to form biofilms. We analyzed individual trajectories of swimming cells, both in the bulk fluid and in close proximity to a glass-liquid interface. Additionally, surface related growth during the early phase of biofilm formation was investigated. In the bulk fluid, P.putida shows a typical bacterial swimming pattern of alternating periods of persistent displacement along a line (runs) and fast reorientation events (turns) and cells swim with an average speed around 24 micrometer per second. We found that the distribution of turning angles is bimodal with a dominating peak around 180 degrees. In approximately six out of ten turning events, the cell reverses its swimming direction. In addition, our analysis revealed that upon a reversal, the cell systematically changes its swimming speed by a factor of two on average. Based on the experimentally observed values of mean runtime and rotational diffusion, we presented a model to describe the spreading of a population of cells by a run-reverse random walker with alternating speeds. We successfully recover the mean square displacement and, by an extended version of the model, also the negative dip in the directional autocorrelation function as observed in the experiments. The analytical solution of the model demonstrates that alternating speeds enhance a cells ability to explore its environment as compared to a bacterium moving at a constant intermediate speed. As compared to the bulk fluid, for cells swimming near a solid boundary we observed an increase in swimming speed at distances below d= 5 micrometer and an increase in average angular velocity at distances below d= 4 micrometer. While the average speed was maximal with an increase around 15% at a distance of d= 3 micrometer, the angular velocity was highest in closest proximity to the boundary at d=1 micrometer with an increase around 90% as compared to the bulk fluid. To investigate the swimming behavior in a confinement between two solid boundaries, we developed an experimental setup to acquire three-dimensional trajectories using a piezo driven objective mount coupled to a high speed camera. Results on speed and angular velocity were consistent with motility statistics in the presence of a single boundary. Additionally, an analysis of the probability density revealed that a majority of cells accumulated near the upper and lower boundaries of the microchannel. The increase in angular velocity is consistent with previous studies, where bacteria near a solid boundary were shown to swim on circular trajectories, an effect which can be attributed to a wall induced torque. The increase in speed at a distance of several times the size of the cell body, however, cannot be explained by existing theories which either consider the drag increase on cell body and flagellum near a boundary (resistive force theory) or model the swimming microorganism by a multipole expansion to account for the flow field interaction between cell and boundary. An accumulation of swimming bacteria near solid boundaries has been observed in similar experiments. Our results confirm that collisions with the surface play an important role and hydrodynamic interactions alone cannot explain the steady-state accumulation of cells near the channel walls. Furthermore, we monitored the number growth of cells in the microchannel under medium rich conditions. We observed that, after a lag time, initially isolated cells at the surface started to grow by division into colonies of increasing size, while coexisting with a comparable smaller number of swimming cells. After 5:50 hours, we observed a sudden jump in the number of swimming cells, which was accompanied by a breakup of bigger clusters on the surface. After approximately 30 minutes where planktonic cells dominated in the microchannel, individual swimming cells reattached to the surface. We interpret this process as an emigration and recolonization event. A number of complementary experiments were performed to investigate the influence of collective effects or a depletion of the growth medium on the transition. Similar to earlier observations on another bacterium from the same family we found that the release of cells to the swimming phase is most likely the result of an individual adaption process, where syntheses of proteins for flagellar motility are upregulated after a number of division cycles at the surface. / Bakterien sind einzellige Mikroorganismen, die sich in flüssigem Medium mit Hilfe von rotierenden Flagellen, länglichen Fasern aus Proteinen, schwimmend fortbewegen. In Gegenwart einer Grenzfläche und unter günstigen Umweltbedingungen siedeln sich Bakterien an der Oberfläche an und gehen in eine sesshafte Wachstumsphase über. Die Wachstumsphase an der Oberfläche ist gekennzeichnet durch das Absondern von klebrigen, nährstoffreichen extrazellulären Substanzen, welche die Verbindung der Bakterien untereinander und mit der Oberfläche verstärken. Die entstehenden Aggregate aus extrazellulärer Matrix und Bakterien werden als Biofilm bezeichnet. In der vorliegenden Arbeit untersuchten wir ein Bodenbakterium, Pseudomonas putida (P. putida), welches in wässriger Umgebung an festen Oberflächen Biofilme ausbildet. Wir benutzten photolithographisch hergestellte Mikrokanäle und Hochgeschwindigkeits-Videomikroskopie um die Bewegung schwimmender Zellen in verschiedenen Abständen zu einer Glasoberfläche aufzunehmen. Zusätzlich wurden Daten über das parallel stattfindende Wachstum der sesshaften Zellen an der Oberfläche aufgezeichnet. Die Analyse von Trajektorien frei schwimmender Zellen zeigte, dass sich Liniensegmente, entlang derer sich die Zellen in eine konstante Richtung bewegen, mit scharfen Kehrtwendungen mit einem Winkel von 180 Grad abwechseln. Dabei änderte sich die Schwimmgeschwindigket von einem zum nächsten Segment im Mittel um einen Faktor von 2. Unsere experimentellen Daten waren die Grundlage für ein mathematisches Modell zur Beschreibung der Zellbewegung mit alternierender Geschwindigkeit. Die analytische Lösung des Modells zeigt elegant, dass eine Population von Bakterien, welche zwischen zwei Geschwindigkeiten wechseln, signifikant schneller expandiert als eine Referenzpopulation mit Bakterien konstanter Schwimmgeschwindkeit. Im Vergleich zu frei schwimmenden Bakterien beobachteten wir in der Nähe der Oberfläche eine um 15% erhöhte Schwimmgeschwindigkeit der Zellen und eine um 90 % erhöhte Winkel-geschwindigkeit. Außerdem wurde eine signifikant höhere Zelldichte in der Nähe der Grenzfläche gemessen. Während sich der Anstieg in der Winkelgeschwindigkeit durch ein Drehmoment erklären lässt, welches in Oberflächennähe auf den rotierenden Zellkörper und die rotierenden Flagellen wirkt, kann die Beschleunigung und Akkumulation der Zellen bei dem beobachteten Abstand nicht durch existierende Theorien erklärt werden. Unsere Ergebnisse lassen vermuten, dass neben hydrodynamischen Effekten auch Kollisionen mit der Oberfläche eine wichtige Rolle spielen und sich die Rotationsgeschwindigkeit der Flagellenmotoren in der Nähe einer festen Oberfläche grundsätzlich verändert. Unsere Experimente zum Zellwachstum an Oberflächen zeigten, dass sich etwa sechs Stunden nach Beginn des Experiments größere Kolonien an der Kanaloberfläche auflösen und Zellen für ca. 30 Minuten zurück in die schwimmende Phase wechseln. Ergebnisse von mehreren Vergleichsexperimenten deuten darauf hin, dass dieser Übergang nach einer festen Anzahl von Zellteilungen an der Oberfläche erfolgt und nicht durch den Verbrauch des Wachstumsmediums bedingt wird.

Made available in DSpace on 2014-12-17T15:01:58Z (GMT). No. of bitstreams: 1 MarileideMA.pdf: 625410 bytes, checksum: 4571e21e5bf4561ee450806713d749af (MD5) Previous issue date: 2005-03-11 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In the last decade, biological purification of gaseous waste has become an important alternative to many conventional methods of exhaust air treatment. More recently, biofiltration has proved to be an effective and inexpensive method for the treatment of air contaminated with volatile organic compounds (VOCs). A biofilter consists in a reactor packed with a porous solid bed material, where the microorganisms are fixed. During the biofiltration process, polluted air is transported through the biofilter medium where the contaminant is degraded. Within the biofilm, the pollutants in the waste gases are energy and carbon sources for microbial metabolism and are transformed into CO2, water and biomass. The bed material should be characterized by satisfactory mechanical and physical properties as structure, void fraction, specific area and flow resistance. The aim of this research was the biofilter construction and study of the biological degradation of ethanol and toluene, as well as the modeling of the process. Luffa cylindrica is a brazilian fiber that was used as the filtering material of the present work. The parameters and conditions studied were: composition of nutrients solution; effect of microflorae strains, namely Pseudomanas putida and Rhodococcus rhodochrous; waste gas composition; air flow rate; and inlet load of VOCs. The biofilter operated in diffusion regime and the best results for remotion capacity were obtained when a microorganisms consortion of Pseudomanas putida and Rhodococcus rhodochrous,were used, with a gas flow rate of 1 m3.h-1 and molar ratio nitrogene/phosphore N/P=2 in the nutrients solution. The maximum remotion capacity for ethanol was around 90 g.m-3.h-1 and 50 g.m-3.h-1 to toluene. It was proved that toluene has inhibitory effect on the ethanol remotion When the two VOCs were present in the same waste gas, there was a decrease of 40% in ethanol remotion capacity. Luffa cylindrica does not present considerable pressure drop. Ottengraf and van Lith models were used to represent the results obtained for ethanol and toluene, respectively. The application of the transient model indicated a satisfactory approximation between the experimental results obtained for ethanol and toluene vapors biofiltration and the ones predicted it / Na ultima d?cada, a purifica??o biol?gica de rejeitos gasosos tem sido uma importante alternativa em detrimento dos m?todos convencionais de tratamento. Mais recentemente, a biofiltra??o tem se mostrado um m?todo efetivo e de baixo custo para tratamento de ar contaminado com compostos org?nicos vol?teis (COVs). O biofiltro ? um reator que possui um leito filtrante constitu?do por um material poroso, onde est?o fixados os microrganismos. Durante o processo de biofiltra??o, o ar polu?do ? transportado ao longo do leito onde o contaminante ? degradado. No biofilme, os poluentes presentes no efluente gasoso ser?o a fonte de carbono e energia para os microrganismos e ap?s serem metabolizados ser?o transformados em CO2, ?gua e biomassa. O leito filtrante deve ser caracterizado por propriedades f?sicas e mec?nicas como estrutura, fra??o de vazios, ?rea especifica e resist?ncia ao fluxo gasoso. O principal objetivo deste trabalho de pesquisa foi a constru??o de um biofiltro e o estudo da degrada??o do etanol e tolueno, bem como a modelagem do processo. Luffa cylindrica ? uma fibra bastante encontrada no nordeste do Brasil e foi utilizada no presente trabalho como leito filtrante. Os par?metros e condi??es estudos foram: composi??o da solu??o nutriente; efeito da esp?cie de microrganismos, denominadas Pseudomanas putida e Rhodococcus rhodochrous; composi??o do efluente gasoso; taxa de fluxo gasoso e carga de entrada do COV. O biofiltro operou em regime difusional, sendo os melhores resultados para a capacidade de remo??o foram obtidos quando utilizado o cons?rcio de microrganismos de Pseudomanas putida e de Rhodococcus rhodochrous, com uma vaz?o volum?trica de ar de 1 m3.h-1 e raz?o molar nitrog?nio/fosforo N/P=2 na solu??o nutriente. A m?xima capacidade de elimina??o foi de 90 g.m-3.h-1 para o etanol e 50 g.m-3.h-1 para o tolueno. Constatou-se que o tolueno tem efeito inibit?rio sobre a degrada??o do etanol, quando os dois VOCs est?o presentes no mesmo efluente, houve uma queda de 40% na remo??o do etanol. A Luffa cylindrica n?o apresentou grandes valores de perda de carga. Os modelos de Ottengraf e van Lith representaram bem os resultados obtidos para o etanol e tolueno, respectivamente. A aplica??o do modelo transiente evidenciou uma aproxima??o satisfat?ria entre os resultados experimentais obtidos para a biofiltra??o de vapores de etanol e tolueno e aqueles previstos pelo referido modelo

O uso intensivo de produtos organoclorados contendo estruturas aromáticas em sua composição tem crescido de forma rápida nos últimos anos em face de sua ampla utilização em vários setores da indústria moderna. A decomposição de tais compostos é lenta, dada sua grande estabilidade química, tornando-os poluentes recorrentes do meio-ambiente. Diferentes estratégias estão disponíveis para tentar solucionar este problema. Os chamados processos de bioremediação estão entre elas e vem ganhando espaço dentre as possíveis escolhas em face de sua maior eficiência e por estarem baseados no uso de moléculas como enzimas, que não afetam o ambiente, para realizar a tarefa de degradação de substâncias tóxicas. Neste contexto se coloca a enzima clorocatecol 1,2-dioxigenase de Pseudomonas. putida, alvo de estudos deste trabalho. Nosso grupo tem trabalhado no entendimento do mecanismo de ação da enzima clorocatecol 1,2-dioxigenase de Pseudomonas. putida há alguns anos já que acreditamos que a utilização de forma mais eficiente e efetiva possível da enzima clorocatecol 1,2-dioxigenase de Pseudomonas. putida passa necessariamente pelo conhecimento acerca de maneiras de controle da atividade enzimática. Com isso, os resultados obtidos consistiram na otimização dos processos de expressão e purificação que permitiram a obtenção da proteína pura e com bom rendimento, após mudança no vetor de expressão. Foram feitos ensaios de atividade enzimática com diferentes substratos e desenvolvimento de um protocolo de caracterização cinética, para assim avaliar a existência de mecanismos de inibição/modulação da reação pelo substrato e/ou produto. Análise da estrutura secundária por meio de dicroísmo circular e dicroísmo circular com radiação síncrotron para avaliar a integridade estrutural frente da nova construção. Também foram realizados testes para separação dos ligantes enzima clorocatecol 1,2-dioxigenase de Pseudomonas putida para análise em espectrômetro de massas afim de se identificar as moléculas anfipáticas que podem estar presentes em seu sítio hidrofóbico. Por fim, ensaios de interação proteína-lipídio utilizando calorimetria diferencial de varredura, ressonância paramagnética eletrônica e dicroísmo circular indicam uma provável interação com modelos de membrana, principalmente, quando na presença de PIP2 (fosfatidilinositol-4,5-bisfosfato). / The use of chlorinated compounds bearing aromatic structures in their chemical composition has quickly grown in the last few years due to their general presence in processes of modern industry. The degradation of such compounds is slow, due to their high chemical stability, thus making them frequent polutants of the environment. Different strategies to tackle this problem are available. The so-called bioremediation methods are among those strategies and have been gaining many applications because of their higher efficiency and due to the use of enzymes, which do not affect the environment, to perform the degradation task. Chlorocatechol 1,2-dioxygenase from Pseudomonas putida is one of those enzymes and is the object of study of this project. Our group has been working on understanding the mechanism of action of Chlorocatechol 1,2-dioxygenase from Pseudomonas putida since we believe that a more efficient use of Chlorocatechol 1,2-dioxygenase from Pseudomonas putida necessarily involves knowledge about ways of controlling the enzymatic activity. Thus, our results consisted in the optimization of the expression and purification protocols that allow the production of pure protein in high yields after changing its expression vector. Assays of enzyme activity with different substrates and development of a protocol for kinetic characterization was also done to assess the existence of mechanisms of inhibition/modulation of the reaction by the substrate and/or product. Analysis of the secondary structure by circular dichroism and synchrotron radiation circular dichroism was also performed to assess the integrity of the new construction. Tests were also carried out to separate the amphipatic ligands present in the Chlorocatechol 1,2-dioxygenase from Pseudomonas putida structure for analysis in a mass spectrometer in order to identify which kind of amphipathic molecule may be present in enzyme hydrophobic site. Finally, lipid-protein interactions were investigated by means of differential scanning calorimetry, electron paramagnetic resonance and circular dichroism. The results indicated a potential interaction with model membranes, especially in the presence of PIP2 (phosphatidylinositol 4,5-bisphosphate).

O uso intensivo de produtos organoclorados contendo estruturas aromáticas em sua composição tem crescido de forma rápida nos últimos anos em face de sua ampla utilização em vários setores da indústria moderna. A decomposição de tais compostos é lenta, dada sua grande estabilidade química, tornando-os poluentes recorrentes do meio-ambiente. Diferentes estratégias estão disponíveis para tentar solucionar este problema. Os chamados processos de bioremediação estão entre elas e vem ganhando espaço dentre as possíveis escolhas em face de sua maior eficiência e por estarem baseados no uso de moléculas como enzimas, que não afetam o ambiente, para realizar a tarefa de degradação de substâncias tóxicas. Neste contexto se coloca a enzima clorocatecol 1,2-dioxigenase de Pseudomonas. putida, alvo de estudos deste trabalho. Nosso grupo tem trabalhado no entendimento do mecanismo de ação da enzima clorocatecol 1,2-dioxigenase de Pseudomonas. putida há alguns anos já que acreditamos que a utilização de forma mais eficiente e efetiva possível da enzima clorocatecol 1,2-dioxigenase de Pseudomonas. putida passa necessariamente pelo conhecimento acerca de maneiras de controle da atividade enzimática. Com isso, os resultados obtidos consistiram na otimização dos processos de expressão e purificação que permitiram a obtenção da proteína pura e com bom rendimento, após mudança no vetor de expressão. Foram feitos ensaios de atividade enzimática com diferentes substratos e desenvolvimento de um protocolo de caracterização cinética, para assim avaliar a existência de mecanismos de inibição/modulação da reação pelo substrato e/ou produto. Análise da estrutura secundária por meio de dicroísmo circular e dicroísmo circular com radiação síncrotron para avaliar a integridade estrutural frente da nova construção. Também foram realizados testes para separação dos ligantes enzima clorocatecol 1,2-dioxigenase de Pseudomonas putida para análise em espectrômetro de massas afim de se identificar as moléculas anfipáticas que podem estar presentes em seu sítio hidrofóbico. Por fim, ensaios de interação proteína-lipídio utilizando calorimetria diferencial de varredura, ressonância paramagnética eletrônica e dicroísmo circular indicam uma provável interação com modelos de membrana, principalmente, quando na presença de PIP2 (fosfatidilinositol-4,5-bisfosfato). / The use of chlorinated compounds bearing aromatic structures in their chemical composition has quickly grown in the last few years due to their general presence in processes of modern industry. The degradation of such compounds is slow, due to their high chemical stability, thus making them frequent polutants of the environment. Different strategies to tackle this problem are available. The so-called bioremediation methods are among those strategies and have been gaining many applications because of their higher efficiency and due to the use of enzymes, which do not affect the environment, to perform the degradation task. Chlorocatechol 1,2-dioxygenase from Pseudomonas putida is one of those enzymes and is the object of study of this project. Our group has been working on understanding the mechanism of action of Chlorocatechol 1,2-dioxygenase from Pseudomonas putida since we believe that a more efficient use of Chlorocatechol 1,2-dioxygenase from Pseudomonas putida necessarily involves knowledge about ways of controlling the enzymatic activity. Thus, our results consisted in the optimization of the expression and purification protocols that allow the production of pure protein in high yields after changing its expression vector. Assays of enzyme activity with different substrates and development of a protocol for kinetic characterization was also done to assess the existence of mechanisms of inhibition/modulation of the reaction by the substrate and/or product. Analysis of the secondary structure by circular dichroism and synchrotron radiation circular dichroism was also performed to assess the integrity of the new construction. Tests were also carried out to separate the amphipatic ligands present in the Chlorocatechol 1,2-dioxygenase from Pseudomonas putida structure for analysis in a mass spectrometer in order to identify which kind of amphipathic molecule may be present in enzyme hydrophobic site. Finally, lipid-protein interactions were investigated by means of differential scanning calorimetry, electron paramagnetic resonance and circular dichroism. The results indicated a potential interaction with model membranes, especially in the presence of PIP2 (phosphatidylinositol 4,5-bisphosphate).

Made available in DSpace on 2015-03-26T13:37:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 546542 bytes, checksum: c9dbde18124d512955d3a032831ab07f (MD5) Previous issue date: 2008-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / In most experiments dealing with biological control of plant diseases, plant growth-promoting rhizobacteria (PGPR) are delivered by seed microbiolization while bacterial phylloplane residents are delivered by spraying. In this work aimed to test several procedures for delivering the biocontrol agent Pseudomonas putida to tomato plants, as well as to study populational tendencies of the biocontrol agent in plant surfaces and to make attempts to establish correlations among populational tendencies and efficiency of biological control. The bacterium Pseudomonas putida (isolate UFV- 0073), autochthonous in tomato phylloplane, was previously selected for the biocontrol of tomato aerial part diseases. Four deliver modes were tested, as follow: 1- seed microbiolization (OD540 = 0,3) followed by planting; 2 - spraying tomato phylloplane with bacterial live propagules (OD540 = 0,3); 3 microbiolization and spraying seedlings with the antagonist (treatments 1 and 2) and 4 - spraying tomato phylloplane with the supernate obtained from bacterial culture (OD540 = 1.0). Results indicated that for the pathosystem tomato - Xanthomonas campestris pv. vesicatoria the most efficient deliver mode was to spray tomato phylloplane with propagule suspension of the antagonist while for its counterpart tomato- Pseudomonas syringae pv. tomato pathosystem, the best deliver modes were to spray the phylloplane with culture supernates and seed microbiolization along with phylloplane spray with a propagule suspension. In order to study populational tendencies for the biocontrol agent UFV-0073 in tomato phylloplane, a semi-selective culture medium was developed and tested based on the constitutive multiple resistance to antibiotics exhibited by the aforementioned isolate of P. putida. Under greenhouse conditions, measurement of population tendencies as a function of time indicated a population decrease in the sense that at day 0, the population was estimated as 8,92 x 107 CFU/g of leaf tissue and in day 5 this population went down to 4,13 x 105 CFU/g of leaf tissue, what means a population 215 times smaller that the original one. On the other hand, under field conditions, the populational tendency did decrease but in a slower rate and presented a tendency to equilibrium, with up and down population peaks in terms of recovered bacteria. So, at 0 day , the population was estimated as being 4,62 x 106 CFU/g leaf fresh weight and nine days after deliver it was 9,91 x 105 UFC/g, what means five times smaller than the population at 0 day. Last but not least, it was established a correlation between populational tendencies of the P. putida in the phylloplane and biocontrol of tomato bacterial speck (P. syringae pv. tomato), when results indicated that the biocontrol was effective up to the fifth day after deliver. This work provides a better understanding about the best deliver mode of P. putida while biocontrol agent for tomato diseases in the aerial parts as well as about time intervals for delivering the biocontrol agent under investigation. / Na maioria dos experimentos sobre controle biológico de fitopatógenos já descritos, Rizobactérias Promotoras de Crescimento em Plantas (PGPR s) são dispensadas por microbiolização de sementes e bactérias residentes do filoplano são dispensadas por pulverização da parte aérea da cultura. Procurou-se verificar, se outras formas de dispensa do agente de biocontrole são igualmente eficientes aos métodos clássicos de dispensa. Objetivou-se testar várias formas de dispensa do agente de biocontrole Pseudomonas putida no filoplano do tomateiro, estudar sua dinâmica populacional no filoplano e correlacionar a dinâmica populacional com o biocontrole de doenças da parte aérea da cultura. O isolado Pseudomonas putida (UFV-0073), autóctone do filoplano de tomateiro, foi selecionado para o controle biológico de doenças da parte aérea da cultura. Foram testadas quatro formas de dispensa do agente de biocontrole em plantas de tomate: 1- sementes microbiolizadas DO540nm= 0,3 e logo em seguida semeadas; 2-atomização do filoplano do tomateiro com a cultura bacteriana DO540nm= 0,3; 3- a aplicação dos tratamentos 1 e 2 conjuntamente e 4- atomização do filoplano com o sobrenadante DO540nm= 1,0. Para o patossistema tomateiro e Xanthomonas campestris pv. vesicatoria, a melhor forma de dispensa foi a pulverização do filoplano com propágulos do agente de biocontrole. No patossistema Pseudomonas syringae pv. tomato e plantas de tomate, as melhores formas de dispensa do agente de controle biológico foram a pulverização do filoplano com o sobrenadante da cultura e a microbiolização de sementes associada à pulverização do filoplano com propágulos do isolado. Para o estudo da dinâmica populacional do agente de biocontrole UFV-0073, no filoplano foi desenvolvido e testado um meio semi-seletivo tirando partido da resistência múltipla constitutiva a antibióticos. Em condições de casa-de-vegetação, houve uma tendência de queda na população do antagonista. No dia 0 (zero), a população do antagonista foi de 8,92 x 107 UFC/g de tecido foliar e no quinto dia após a dispensa sua população foi de 4,13 x 105, ou seja, 215 vezes menor do que a população inicial. Em condições de campo, a queda populacional do agente de biocontrole foi mais branda, revelando uma tendência ao equilíbrio, com alternância entre aumento e queda da quantidade de bactérias recuperadas. No dia zero, a população foi de 4,62 x 106 UFC/g de tecido foliar, nove dias após a dispensa a população passou a 9,91 x 105 UFC/g, cerca de 5 vezes menor do que a população inicial. Por fim, foi correlacionada a dinâmica populacional com o biocontrole de P. syringae pv. tomato, quando os resultados indicaram que o biocontrole foi efetivo até o quinto dia após a dispensa do agente de controle biológico no filoplano do tomateiro. Estudos dessa natureza são necessários para se estabelecer qual a melhor forma de dispensa e qual o intervalo entre as aplicações com o agente de biocontrole, quando realizadas pulverizações visando ao controle biológico de doenças. São necessários mais estudos com o agente de biocontrole, testando o controle contra outros patógenos e em condições de casa-devegetação e no campo.

Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans ce travail, nous nous sommes intéressés au développement de différents biocapteurs se basant sur l'immobilisation d'enzymes ou de bactéries sur des microélectrodes en vue d’une détection électrochimique. Nous avons montré les potentialités d’application de deux biocapteurs conductimétriques à base de protéinase K ou de protéinase K et de pronase à la détection des modifications de conformation de la myoglobine et de l’albumine de sérum bovin au cours de leur relargage à partir de microsphères de poly (ε-caprolactone). Nous avons également mis au point un biocapteur conductimétrique à base de catalase et d’alcool oxydase pour une détection rapide et sensible des alcools ainsi que deux biocapteurs à catalase pour la détection impédimétrique et conductimétrique du cyanure et l’étude des interactions catalase-cyanure. Nous avons enfin élaboré des biocapteurs bactériens à base de Pseudomonas putida F1 pour la détection du trichloroéthylène dans les eaux souterraines. Pour cela, une voie originale d’immobilisation des cellules, basée sur la fonctionnalisation du transducteur à l’aide de couches autoassemblées et d’anticorps, ainsi que l’utilisation de nanotubes de carbone, a été explorée / The development of biosensors is an expanding research area. Indeed, biosensors are rapid, selective and cost-effective analytical tools which find applications in various fields (environment, health, food,…). They are constituted of a sensitive biological element (antibody, enzyme, microorganism, DNA…), which can selectively recognize one analyte or a group of analytes, associated to an electrochemical, optical or thermal transducer. In this work, we developed different biosensors based on enzymes or bacteria immobilised onto microelectrodes in view of electrochemical detection. First, we demonstrated the potentialities of two conductometric biosensors based on proteinase K or proteinase K and pronase for the detection of myoglobin and bovine serum albumine conformation changes during their release from poly (ε-caprolactone) microspheres. Then, we elaborated a bi-enzymatic conductometric biosensor with catalase and alcohol oxidase as sensing elements, for a rapid and sensitive detection of alcohols. Catalase impedimetric and conductometric biosensors were also developed for cyanide detection and used for the study of catalase-cyanide interactions. Finally, we prepared Pseudomonas putida F1 whole cell biosensors for the determination of trichloroethylene in groundwaters. For that, an original route, including the functionalisation of the transducer with a self-assembled-monolayer and antibodies, and the use of single-wall carbon nanotubes, was investigated for cell immobilisation