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

The main objective of this study was to improve our understanding of biological filtration (biofilm type) treatment for manganese (Mn) removal in drinking water. Biological filtration treatment involves biofilms of Mn(II)-oxidizing microorganisms attached to solid filter material that remove and immobilize dissolved Mn(II) in raw water by conversion to black MnO2(s) precipitates. Mn-biological filtration is an emerging green technology that can serve as an alternative to conventional physicochemical treatments but its full potential is hindered by various factors. These include lack of understanding the: (1) optimal removal conditions for Mn, (2) mechanisms for Mn releases of the accumulated Mn in the biofilter, and (3) effects of recalcitrant natural organic matter (NOM) on biofiltration. Confounding these issues is the unknown identity of the diverse microbial communities which occupy the biofilms attached to the filter media. To investigate these issues, biological Mn removal was studied in laboratory bench scale reactors using a new Mn(II)-oxidizing bacterium isolate, Pseudomonas Putida EC112. The main research hypothesis formulated that the transition metal catalyst, MnO2(s), can increase the bioavailable carbon and energy from recalcitrant NOM (e.g., humic acids (HA)) to biological filters. Mn and HA can be found in most natural waters, including groundwaters, lakes and streams. To test the hypothesis, the potential for strain EC112 growth and Mn(II) oxidation utilizing the organic substrate products from the oxidation reaction between HA and MnO2(s) was assessed. Biological Mn(II)-oxidation kinetics were investigated in batch (suspended cell) and continuous flow (biofilm) bioreactors at optimal pH and temperature conditions for strain EC112. Batch kinetics was successfully characterized with the Monod model. Continuous flow steady-state kinetics was modeled with a single, zero-order kinetic parameter. Enhanced Mn(II) removal capacity was observed for strain EC112 in batch and continuous flow reactors in the presence of HA and MnO2. The effect of MnO2(s) on HA biodegradability was studied and optimal conditions for biodegradation were identified. Biofilter Mn(II) releases were observed during the continuous flow bioreactor experiments. Release conditions were identified and releases modeled using pseudo first-order kinetics. Changes in HA structure induced by MnO2(s) oxidation were studied with Fourier transform infrared (FT-IR) and proton nuclear magnetic spectroscopy (1H-NMR).

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to promoter affinity for σ54-holoenzyme, ii) that σ54 is under significant competition with other σ-factors in the cell, and iii) that mutants of σ70, and the beta- and beta prime-subunits of RNA polymerase that can bypass the need for ppGpp and DksA in vivo have defects that would favour the formation of σ54-RNA holoenzyme over that with σ70, and that mimic the effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. A purely passive model for ppGpp/DksA regulation of σ54-dependent transcription that functions through their potent negative effects on transcription from powerful σ70-stringent promoters is presented.

TOL plasmids of Pseudomonas putida encode enzymes for the degradation of toluene and related aromatics. These genes are organized into two operons regulated by the Xy1R and Xy1S transcriptional activators. Previous analysis of the TOL pDK1 catechol-2,3-dioxygenase gene (xy1E) and a comparison of this gene to xy1E from the related TOL plasmid pWW0, revealed the existance of a substantial level of sequence homology (82%).

Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.

"Bacteria during the course of their life undergo a lot of developments on their surface. The changes that occur inside a cell result in the production of a variety of biopolymers on the cell surface. These polysaccharides have been found to play a major role in deciding the adhesive or repulsive nature of a bacterial cell. Based on the application the adhesive nature of a cell sometimes needs to be manipulated such that bacteria are required to have higher adhesions for bioremediation applications and in the case of bioreactors bacteria must not stick to walls to avoid fouling. In order to control adhesions of a cell to a variety of substrates, knowledge of the polysaccharides present on its surface is needed. Therefore the goal of the present study is to detect the sugars present on the surface of Pseudomonas putida KT2442 using Atomic force microscopy and to relate properties of the polysaccharides to bacterial adhesion. Previous experiments suggested that cellulose and other sugars were produced by Pseudomonas putida KT2442. Thus the cells were grown to late exponential phase and treated with cellulase to degrade any cellulose, if present, on the surface of the cells. Control experiments were done on untreated cells and cells that were not treated with cellulase but were centrifuged, since centrifugation is a part of the cellulase treatment and may also affect the bacterial surface. An appropriate (Steric) fitting model for the atomic force microscope (AFM) approach curves was applied to calculate the height and density of the polymer brush layer present on the cell surface. There was a decrease in the density of the polymer brush and increase in the height of the brush upon treatment with cellulase. Centrifugation alone did not affect the approach curves. From looking at the retraction curves it verified the results got from the approach curves and indicated stretching out of the polymer brush to greater distances after the treatment with cellulase. Another batch of cells was treated with dextranase to check for the presence of dextran on the cell surface. Dextranase treated cells behaved identical to the control cells, suggesting that dextran is not one of the polysaccharides present on the bacterial surface. No change was observed in retraction curves data for dextranase treated and untreated cells."

Microorganisms are capable of producing advanced biofuels that can be used as ‘drop-in’ alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms for improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. First, using genomic DNA from Marinobacter aquaeolei, we constructed a transgenic library that we expressed in Escherichia coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance. Additionally, we used genomic DNA from Pseudomonas putida KT2440, which has innate solvent-tolerance properties, to create transgenic libraries in an E. coli host. We exposed cells containing the library to pinene, selecting for genes that improved tolerance. Importantly, we found that expressing the sigma factor RpoD from P. putida greatly expanded the diversity of tolerance genes recovered. With low expression of rpoDP. putida, we isolated a single pinene tolerance gene; with increased expression of the sigma factor our selection experiments returned multiple distinct tolerance mechanisms, including some that have been previously documented and also new mechanisms. Interestingly, high levels of rpoDP. putida induction resulted in decreased diversity. We found that the tolerance levels provided by some genes are highly sensitive to the level of induction of rpoDP. putida, while others provide tolerance across a wide range of rpoDP. putida levels. This method for unlocking diversity in tolerance screening using heterologous sigma factor expression was applicable to both plasmid and fosmid-based transgenic libraries. These results suggest that by controlling the expression of appropriate heterologous sigma factors, we can greatly increase the searchable genomic space within transgenic libraries. This dissertation describes a method of effectively screening genomic DNA from multiple organisms for genes to mitigate biofuel stress and shows how tolerance genes can improve bacterial growth in the presence of toxic biofuel compounds. These identified genes can be targeted in future studies as candidates for use in biofuel production strains to increase biofuel yields.