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Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON. / Doctor of Philosophy / Metabolic engineering is the use of genetic engineering to modify microorganisms in order to produce high yields of valuable commodity chemicals. The goal of this research is to develop new methods to improve genetic modification and selection of microbial cells. The specific objectives were to: (i) develop new tools for DNA assembly and manipulation, (ii) utilize small synthetic RNA to control protein expression level, (iii) use Raman spectroscopy to study phenotypic responses to environmental changes and (iv) enrich for microorganisms that detoxify dangerous toxins. First, a new technique for DNA assembly, named λ-PCR (lambda-PCR), was developed. This method allows the easy manipulation of plasmid DNA with high-efficiency and low-cost compared to traditional techniques. Second, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system in order to modulate (downregulate or overexpress) fluorescent protein expression. Next, Raman spectroscopy was used to assess phenotypic response of cyanobacterial cells to different environmental modifications (light settings, salts, sugar, etc…). Finally, genomic library was used to discover and characterize enzymes capable of degrading a mycotoxin.

As lagoas salino-alcalinas (salinas) da sub-região Nhecolândia do Pantanal, Mato Grosso do Sul, combinam valores de pH elevados com a presença de altas concentrações de sal, assemelhando-se aos lagos de soda da África Oriental. O entendimento atual dos mecanismos físicos, químicos e biológicos nestes ambientes extremos do Brasil é limitado. Embora os micro-organismos estejam envolvidos nos processos biogeoquímicos em ecossistemas aquáticos, investigações sobre os grupos bacterianos que contribuem para a diversidade e funções específicas nessas salinas inexistem. Assim, a presente dissertação centrou-se na avaliação da comunidade bacteriana de duas salinas (Salina Verde e Salina Preta), localizadas na sub-região da Nhecolândia. Especificamente, investigou-se a diversidade e a estrutura das comunidades bacterianas, os perfis metabólicos das lagoas e genes funcionais que codificam enzimas relacionadas a transformação do nitrogênio, mercúrio, selênio e arsênio. As amostras de água foram coletadas durante a estação seca (setembro de 2012) na Salina Verde (pH 9,5, E.C. 2575 mS cm-1), caracterizada pela presença constante de floração de cianobactérias e na Salina Preta (pH 8,9, E.C. 1500 mS cm-1), sem registro de ocorrência de floração. As amostragens foram realizadas em triplicatas em duas profundidades (superfície e fundo) e duas vezes no dia (10:00 h e 15:00 h) devido à ocorrência natural de saturação de oxigênio observada na Salina Verde. O DNA total de cada amostra ambiental foi extraído e a diversidade bacteriana e funcionalidade foram acessadas por pirosequenciamento do gene de 16S RNAr e sequenciamento metagenômico. A análise de PCR quantitativa do gene de 16S RNAr foi realizada de forma a quantificar a comunidade bacteriana. A abundância bacteriana foi maior na Salina Verde do que na Salina Preta (1010 e 109 cópias mL-1, respectivamente). As sequências parciais do gene de 16S RNAr obtidas no pirosequenciamento mostraram a dominância de táxons do gênero Anabaenopsis sp. na floração da Salina Verde, englobando até 92% do total de sequências. A comunidade bacteriana da Salina Preta apresentou os maiores índices de diversidade e riqueza, sendo dominantes os filos Proteobacteria, Bacteroidetes, Acidobacteria e Verrucomicrobia. Apenas a Salina Preta mostrou diferenças na comunidade bacteriana de acordo com as profundidades amostradas. Na superfície desta lagoa, os filos Actinobacteria e Verrucomicrobia predominaram, enquanto no fundo, prevaleceram os filos Proteobacteria e Chlamydiae. A temperatura foi detectada como o fator abiótico que influenciou a heterogeneidade espacial da Salina Preta. Por sua vez, a alcalinidade e o pH foram os fatores que impulsionaram as diferenças e variações das comunidades bacterianas em ambas as lagoas. Genes bacterianos envolvidos nos ciclos biogeoquímicos do nitrogênio, mercúrio e arsênio foram encontrados nas salinas Verde e Preta, sugerindo uma elevada redundância funcional nas transformações desses elementos. Não foram encontrados genes microbianos envolvidos no ciclo do selênio. Os dados gerados revelaram uma comunidade microbiana taxonômica e funcionalmente complexa que habita as salinas. Os resultados deste estudo fornecem uma avaliação aprofundada baseada em abordagens independentes de cultivo, sendo este um passo importante na compreensão da dinâmica funcional desses ambientes no Pantanal brasileiro. / The saline-alkaline lakes (salinas) of the Nhecolândia sub-region of the Pantanal, Mato Grosso do Sul state, combine high pH values with the presence of high salt concentrations, resembling the soda lakes of East Africa. The current understanding of physical, chemical and biological mechanisms in these extreme environments is limited. Although microorganisms are involved in biogeochemical processes in aquatic ecosystem, researches on the bacterial groups that contribute to diversity and specific functions in these salinas are scarce. This dissertation therefore focused on the evaluation of bacterial community of two salinas (Salina Verde and Salina Preta) located in the Nhecolândia subregion. Specifically, it was investigated the diversity and structure of bacterial communities, the metabolic profile of the lakes and functional genes that encode the nitrogen, mercury and arsenic-transforming enzymes. Water samples were collected during the dry season (September 2012) from Salina Verde (pH 9.5, E.C. 2575 mS cm-1), characterized by constant presence of cyanobacterial bloom, and from Salina Preta (pH 8.9, E.C. 1500 mS cm-1), with no report of bloom occurrence. Triplicate samplings were carried out in two depths (surface and bottom) and twice a day (10 AM and 3 PM) due to naturally occurrence of oxygen saturation, observed at Salina Verde. Total DNA of each environmental sample was extracted and bacterial diversity and functionality were accessed by 16S rRNA gene pyrosequencing and metagenomic sequencing. Analysis of quantitative PCR of the 16S rRNA gene was performed in order to quantify the bacterial community. Bacterial abundance was higher in the Salina Verde than in the Salina Preta (1010 and 109 copies mL-1, respectively). The partial sequences of the 16S rRNA gene obtained in the pyrosequencing revealed the genus Anabaenopsis sp. as the dominant taxa in the Salina Verde bloom, encompassing up to 92% of the total bacteria. Bacterial community of the Salina Preta showed the highest diversity and richness index, with dominant phyla Proteobacteria, Bacteroidetes, Acidobacteria and Verrucomicrobia. Only the Salina Preta showed differences in bacterial community in accordance with the depths sampled. On the surface of this lake, the phyla Actinobacteria and Verrucomicrobia predominated, while in the bottom, Proteobacteria and Chlamydiae prevailed. The temperature was detected as the abiotic factor influencing the spatial heterogeneity at Salina Preta. On the other hand, alkalinity and pH were the factors driving the differences and variation of bacterial community in both lakes. Bacterial genes involved in the biogeochemical cycles of nitrogen, mercury and arsenic were found in Salina Verde and Salina Preta, suggesting a high metabolic redundancy in the transformation these elements. No microbial genes involved in selenium cycle were found. The data showed a taxonomic and functional complex microbial community inhabiting salinas. The results of this study provide a detailed assessment based on culture-independent approaches, which is a stepping stone to understand the functional dynamics of these environments in the Brazilian Pantanal.

Dans le contexte de la bioéconomie, la découverte et la caractérisation des enzymes capables de dégrader la paroi végétale est particulièrement intéressante pour l’utilisation de la biomasse lignocellulosique dans l’industrie. A cet égard, la métagénomique fonctionnelle est un outil puissantpour découvrir de nouvelles enzymes à partir d’écosystèmes microbiens variés, comme l’illustrent les travaux sur le tube digestif du termite Pseudacanthotermes militaris. Cette étude a fourni une mine d’informations et identifié un hypothétique locus d’utilisation du xylane (XUS), codant pour cinq glycosides hydrolases (GH) et une carbohydrate esterase (CE) de Bacteroidales.Le XUS du métagénome de Pseudacanthotermes militaris contient une xylanase de la famille GH10 qui possède une organisation modulaire complexe dans laquelle la séquence du domaine GH10 est interrompue par une insertion de deux carbohydrate binding modules (CBM). Des travaux préliminaires ont montré que cette enzyme modulaire, désignée Pm25, est active sur xylane. Par conséquent, un des objectifs de cette étude a été la caractérisation détaillée des propriétés biochimiques et catalytiques de Pm25. Le rôle des CBM a également été examiné en quantifiant les interactions protéines-sucres et permettant ainsi une meilleure compréhension du rôle spécifique de ces modules, les résultats obtenus permettent de cerner l’impact de la modularité de Pm25 sur ses propriétés fonctionnelles.Dans une deuxième partie de l’étude, nous avons entrepris d’étudier la fonction de Pm25 dans le contexte du cluster XUS. Pour ce faire, nous avons étudié les enzymes adjacentes à Pm25 sur le locus,une autre GH10, une GH11, une GH115 et une GH43. La comparaison des paramètres cinétiques et une étude détaillée des produits d’hydrolyse ont été analysés par spectrométrie de masse et ont révélé que la GH10 et la GH11 étaient les enzymes clefs de la dépolymérisation en étant 20 fois plus efficaces que Pm25. En parallèle, nous avons développé un protocole pour l’utilisation de la micro-thermophorèse (MST) pour quantifier les interactions CBM-sucres, une approche intéressante qui nécessite peut d’échantillon et de ligand contrairement à d’autres méthodes biophysiques. Dans l’ensemble, cette étude a révélé le rôle important de Pm25 et ses homologues dans les locus d’utilisation des xylanes chez les Bacteroidetes et a permis d’identifié le sens de cette architecture particulière. / In the context of bioeconomy, the discovery and study of plant-cell wall degrading enzymes is particularly relevant for the use of lignocellulosic biomass for industrial purposes. In this respect, functional metagenomics has proven to be a powerful tool to discover new enzymes from a variety of microbial ecosystems, as exemplified by work performed on the gut of the termite Pseudacanthotermes militaris. This study provided a wealth of information and identified an interesting hypothetical xylan utilization system, encoding five glycoside hydrolases (GH) and one carbohydrate esterase (CE) annotated from bacteroidales. The Pseudacanthotermes militaris-derived putative XUS cluster contains a GH10 xylanase that displays a quite complex modular arrangement wherein the GH10 catalytic module contains two insertional carbohydrate binding modules (CBM). During the preliminary work, this modular enzyme, designated Pm25, was shown to be active on xylan, thus in the present research we set out to more thoroughly characterize its biochemical and catalytic properties.The role of the CBM was also investigated, quantifying protein-carbohydrate interactions and thus providing better insight into the specific role of the modules. Taken together, the results obtained provide insight into how Pm25 modularity translates into functional properties. In second part of our study, we set out investigate the function of Pm25 in the context of the XUS cluster. To achieve this we studied a xylan utilization system, which is constituted by another GH10, GH11, GH115 and GH43. The comparison of kinetic parameters and a detailed end product analysis by mass spectrometry showed that GH10 and GH11 outweigh over 20 fold Pm25 catalytic efficiency. In parallel, we developed the use of MicroScale Thermophoresis (MST) to quantify CBM-carbohydrates interactions, an interesting approach requiring smaller concentration of proteinsand ligands compared to other biophysical methods. Overall this study highlighted the important role of Pm25 homologs in the xylan utilization system in Bacteroidetes, and pinpointed the meaning of its unusual architecture.

Les bactéries et champignons des sols sont impliqués dans différentes fonctions des écosystèmes terrestres. Ils sont notamment investis dans la formation des sols, la stabilité des agrégats et les successions végétales. La Nouvelle- Calédonie est un archipel subtropical, classé comme hotspot de biodiversité et dont un tiers de la surface est recouvert par les substrats ultramafiques. Ces milieux sont caractérisés par de faibles concentrations en nutriments (N, K, P) et de fortes concentrations en métaux lourds (Ni, Co, Cr, Mn). Les écosystèmes présents sur ces substrats sont originaux et diversifiés. Ils sont aussi fortement menacés par l’activité minière. Cependant, pour pouvoir correctement restaurer ces milieux et relancer les dynamiques végétales, il est important de connaître les communautés microbiennes associées à ces écosystèmes ainsi que les facteurs les structurant. Ce travail de recherche a permis d’améliorer nos connaissances sur les communautés microbiennes issues de différents écosystèmes des sols ultramafiques néo-calédoniens, ainsi que sur les interactions existantes entre ces microorganismes et les facteurs biotiques et abiotiques. Les résultats obtenus ont montré que chaque formation végétale et chaque site possèdent une communauté microbienne qui lui est propre, d’où l’intérêt de conserver et protéger les écosystèmes calédoniens. De plus, ces travaux ont aussi montré la capacité des communautés bactériennes et fongiques de servir de bio-indicateurs, et plus particulièrement les communautés fongiques qui sont plus sensibles aux perturbations et variations de la couverture végétale. Il a aussi été démontré qu’en maquis ou forêts monospécifiques, les communautésectomycorhiziennes possèdent des fonctions similaires dans la production d’enzymes de dégradation de la matière organique. Ces travaux ont permis une meilleure connaissance des communautés microbiennes associées auxformations végétales des substrats ultramalfiques ainsi que des facteurs les structurant. Cela devrait améliorer la mise en place des futurs chantiers de restauration de ces écosystèmes. / Soil bacteria and fungi play different functions in terrestrial ecosystems. They are implicated in soil formations, aggregate stability, and plant succession. New Caledonia is a subtropical archipelago, classified as a biodiverse hotspot and a third of its surface is covered by ultramafic soils. These soils are characterised by low concentrations of nutrients (N, K, P) and high concentrations of heavy metals (Ni, Co, Cr, Mn). Ecosystems present in these soils are origina and diversified but strongly threatened by mining activity. It is a necessity to restore these ecosystems after ore exploitation. However, to correctly restorethese environments and relaunch plant dynamics, it is important to identify the microbial communities associated with these ecosystems as well as the structuring factors.This research enabled us to improve our knowledge of microbial communities from different ecosystems on New Caledonian ultramafic substrates, as well as the interactions which exist between these microorganisms and biotic and abiotic factors. Results obtained showed that each plant formation and each site possessed its own microbial community,hence the interest in conserving and protecting New Caledonian ecosystems. Moreover, these works also showed the capacity of bacterial and fungal communities to be used as bioindicators, and more particularly fungal communities which are more sensitive to disturbance and plant cover variations. It has also been demonstrated that in monospecific maquis and rainforests, ectomycorrhizal communities have similar functions in the production of degradative enzymes of organic matter. This research improved understanding of microbial communities associated with plant formations on ultramafic substrates as well as structuring factors. This should improve the implementation of future restoration projectson these ecosystems.

Submitted by Liliane Ferreira (ljuvencia30@gmail.com) on 2018-04-02T14:29:46Z No. of bitstreams: 2 Dissertação - Marcus Vinícius Forzani Araújo - 2017.pdf: 1549598 bytes, checksum: 0807ed298bd63e9574018c8c399c3ca5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-02T14:41:18Z (GMT) No. of bitstreams: 2 Dissertação - Marcus Vinícius Forzani Araújo - 2017.pdf: 1549598 bytes, checksum: 0807ed298bd63e9574018c8c399c3ca5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-02T14:41:18Z (GMT). No. of bitstreams: 2 Dissertação - Marcus Vinícius Forzani Araújo - 2017.pdf: 1549598 bytes, checksum: 0807ed298bd63e9574018c8c399c3ca5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-10-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Characterized as extremely important, the soil is a complex environment and it shelters a great diversity of microorganisms. However, little is known about the diversity and ecology of the soil microbiota. Thus, the first part of this dissertation reviews the methodological evolution used to characterize the diversity and abundance of microorganisms found in soil. The second part consists of the application of two methodologies reviewed in the previous chapter, serial dilution and solid medium plating, to estimate free-living nitrogen fixing microorganisms, and fumigation-extraction to estimate soil microbial biomass (BMS). The last part employs the most modern microbial soil characterization technique, the metagenomics of 16S rRNA. Hence, our initial hypothesis was that sugarcane fields’ soils would have better soil microbiological indicators than grasslands’ soils. The results confirmed that the hypothesis was partially correct, and it was possible to find about 140% more free-living diazotrophic colony-forming units (CFUs) and a 17% richer alpha diversity in sugarcane fields’ soils than in grasslands’ soils. The beta diversity between sugarcane plantations and pastures presented clear differences. However, sugarcane fields’ soils obtained about 25% less BMS than grasslands’ soils. In relation to the bacterial phyla, the grasslands have more Actinobacteria, Chloroflexi and Planctomycetes and sugarcane fields have a greater number of TM7 and bacteria that were not identified, being Proteobacteria and Acidobacteria the dominating phyla in both types of soil. Although the results of nitrogen fixers and microbial biomass appear to be conflicting, it is an indication that the diazotrophic community undergoes with a diverse biotic and abiotic influences than the total community of soil microorganisms, and thus respond differently. / Caracterizado como de extrema importância, o solo é um ambiente complexo e que abriga uma grande diversidade de micro-organismos. Entretanto ainda pouco se sabe sobre a diversidade e ecologia da microbiota do solo. Deste modo, a primeira parte desta dissertação revisa a evolução metodológica empregada para caracterizar a diversidade e abundância dos micro-organismos encontrados no solo. A segunda parte consiste na aplicação de duas metodologias revisadas no capítulo anterior, a de diluição seriada e plaqueamento em meio sólido, para estimar micro-organismos fixadores de nitrogênio de vida-livre, e a fumigação-extração, para estimar a biomassa microbiana do solo (BMS). E a última parte emprega a técnica mais moderna de caracterização das comunidades microbianas de solo, a técnica de metagenômica de 16S rRNA. À vista disso, a nossa hipótese inicial era que solos de canavial teriam indicadores microbiológicos de solo melhores do que solos de pastagem. Os resultados comprovaram que a hipótese estava parcialmente correta, sendo possível encontrar cerca de 140% a mais de Unidades Formadoras de Colônias (UFCs) de diazotróficos de vida-livre e uma diversidade alfa 17% mais rica em solos de canaviais do que em solos de pastagens. A diversidade beta entre canaviais e pastagens apresentou diferenças nítidas. Entretanto, os solos de canaviais obtiveram cerca de 25% a menos de biomassa microbiana do solo do que solos de pastagens. Em relação aos filos bacterianos, os pastos possuem mais Actinobacteria, Chloroflexi e Planctomycetes e canaviais possuem maior número de TM7 e bactérias que não foram identificados, sendo Proteobacteria e Acidobacteria os filos dominantes nos dois tipos de solo. Apesar de parecerem conflitantes os resultados de fixadores de nitrogênio e biomassa microbiana, é um indicativo de que a comunidade de diazotróficos sofrem influências bióticas e abióticas diversas do que a comunidade total de micro-organismos do solo, e desta forma, respondem de forma diferente.

As lagoas salino-alcalinas (salinas) da sub-região Nhecolândia do Pantanal, Mato Grosso do Sul, combinam valores de pH elevados com a presença de altas concentrações de sal, assemelhando-se aos lagos de soda da África Oriental. O entendimento atual dos mecanismos físicos, químicos e biológicos nestes ambientes extremos do Brasil é limitado. Embora os micro-organismos estejam envolvidos nos processos biogeoquímicos em ecossistemas aquáticos, investigações sobre os grupos bacterianos que contribuem para a diversidade e funções específicas nessas salinas inexistem. Assim, a presente dissertação centrou-se na avaliação da comunidade bacteriana de duas salinas (Salina Verde e Salina Preta), localizadas na sub-região da Nhecolândia. Especificamente, investigou-se a diversidade e a estrutura das comunidades bacterianas, os perfis metabólicos das lagoas e genes funcionais que codificam enzimas relacionadas a transformação do nitrogênio, mercúrio, selênio e arsênio. As amostras de água foram coletadas durante a estação seca (setembro de 2012) na Salina Verde (pH 9,5, E.C. 2575 mS cm-1), caracterizada pela presença constante de floração de cianobactérias e na Salina Preta (pH 8,9, E.C. 1500 mS cm-1), sem registro de ocorrência de floração. As amostragens foram realizadas em triplicatas em duas profundidades (superfície e fundo) e duas vezes no dia (10:00 h e 15:00 h) devido à ocorrência natural de saturação de oxigênio observada na Salina Verde. O DNA total de cada amostra ambiental foi extraído e a diversidade bacteriana e funcionalidade foram acessadas por pirosequenciamento do gene de 16S RNAr e sequenciamento metagenômico. A análise de PCR quantitativa do gene de 16S RNAr foi realizada de forma a quantificar a comunidade bacteriana. A abundância bacteriana foi maior na Salina Verde do que na Salina Preta (1010 e 109 cópias mL-1, respectivamente). As sequências parciais do gene de 16S RNAr obtidas no pirosequenciamento mostraram a dominância de táxons do gênero Anabaenopsis sp. na floração da Salina Verde, englobando até 92% do total de sequências. A comunidade bacteriana da Salina Preta apresentou os maiores índices de diversidade e riqueza, sendo dominantes os filos Proteobacteria, Bacteroidetes, Acidobacteria e Verrucomicrobia. Apenas a Salina Preta mostrou diferenças na comunidade bacteriana de acordo com as profundidades amostradas. Na superfície desta lagoa, os filos Actinobacteria e Verrucomicrobia predominaram, enquanto no fundo, prevaleceram os filos Proteobacteria e Chlamydiae. A temperatura foi detectada como o fator abiótico que influenciou a heterogeneidade espacial da Salina Preta. Por sua vez, a alcalinidade e o pH foram os fatores que impulsionaram as diferenças e variações das comunidades bacterianas em ambas as lagoas. Genes bacterianos envolvidos nos ciclos biogeoquímicos do nitrogênio, mercúrio e arsênio foram encontrados nas salinas Verde e Preta, sugerindo uma elevada redundância funcional nas transformações desses elementos. Não foram encontrados genes microbianos envolvidos no ciclo do selênio. Os dados gerados revelaram uma comunidade microbiana taxonômica e funcionalmente complexa que habita as salinas. Os resultados deste estudo fornecem uma avaliação aprofundada baseada em abordagens independentes de cultivo, sendo este um passo importante na compreensão da dinâmica funcional desses ambientes no Pantanal brasileiro. / The saline-alkaline lakes (salinas) of the Nhecolândia sub-region of the Pantanal, Mato Grosso do Sul state, combine high pH values with the presence of high salt concentrations, resembling the soda lakes of East Africa. The current understanding of physical, chemical and biological mechanisms in these extreme environments is limited. Although microorganisms are involved in biogeochemical processes in aquatic ecosystem, researches on the bacterial groups that contribute to diversity and specific functions in these salinas are scarce. This dissertation therefore focused on the evaluation of bacterial community of two salinas (Salina Verde and Salina Preta) located in the Nhecolândia subregion. Specifically, it was investigated the diversity and structure of bacterial communities, the metabolic profile of the lakes and functional genes that encode the nitrogen, mercury and arsenic-transforming enzymes. Water samples were collected during the dry season (September 2012) from Salina Verde (pH 9.5, E.C. 2575 mS cm-1), characterized by constant presence of cyanobacterial bloom, and from Salina Preta (pH 8.9, E.C. 1500 mS cm-1), with no report of bloom occurrence. Triplicate samplings were carried out in two depths (surface and bottom) and twice a day (10 AM and 3 PM) due to naturally occurrence of oxygen saturation, observed at Salina Verde. Total DNA of each environmental sample was extracted and bacterial diversity and functionality were accessed by 16S rRNA gene pyrosequencing and metagenomic sequencing. Analysis of quantitative PCR of the 16S rRNA gene was performed in order to quantify the bacterial community. Bacterial abundance was higher in the Salina Verde than in the Salina Preta (1010 and 109 copies mL-1, respectively). The partial sequences of the 16S rRNA gene obtained in the pyrosequencing revealed the genus Anabaenopsis sp. as the dominant taxa in the Salina Verde bloom, encompassing up to 92% of the total bacteria. Bacterial community of the Salina Preta showed the highest diversity and richness index, with dominant phyla Proteobacteria, Bacteroidetes, Acidobacteria and Verrucomicrobia. Only the Salina Preta showed differences in bacterial community in accordance with the depths sampled. On the surface of this lake, the phyla Actinobacteria and Verrucomicrobia predominated, while in the bottom, Proteobacteria and Chlamydiae prevailed. The temperature was detected as the abiotic factor influencing the spatial heterogeneity at Salina Preta. On the other hand, alkalinity and pH were the factors driving the differences and variation of bacterial community in both lakes. Bacterial genes involved in the biogeochemical cycles of nitrogen, mercury and arsenic were found in Salina Verde and Salina Preta, suggesting a high metabolic redundancy in the transformation these elements. No microbial genes involved in selenium cycle were found. The data showed a taxonomic and functional complex microbial community inhabiting salinas. The results of this study provide a detailed assessment based on culture-independent approaches, which is a stepping stone to understand the functional dynamics of these environments in the Brazilian Pantanal.

A metagenômica trouxe novas perspectivas ao estudo de comunidades microbianas no ambiente, permitindo explorar tanto a diversidade taxonômica de microrganismos ainda não-cultivados, como o acesso direto a genes e vias metabólicas. Neste trabalho, foram construídas bibliotecas metagenômicas a partir de amostras de sedimentos de mangue da Baía de Guanabara (RJ), impactadas com hidrocarbonetos de petróleo e metais pesados. Proteobacteria (33,3%), bactérias afiliadas a redutoras-de-sulfato (29,7%) e Firmicutes (20%) representaram os grupos principais nas amostras ambientais, baseado em análises filogenéticas de rDNA 16S, ao passo que isolamentos seletivos utilizando diesel e naftaleno permitiram a recuperação preferencial de delta-Proteobacteria e actinomicetos. Bibliotecas metagenômicas dos sedimentos enriquecidos com óleo diesel, com insertos entre 25 e 35 Kb clonados em fosmídeos, foram triadas para detecção de genes catabólicos de monoxigenases (alkB1) e expressão de epóxido-hidrolases, esterases, lipases e monoxigenases em ensaios de alto desempenho (HTS, high throughput screening). Clones reativos a alkB1 foram detectados, porém não foram funcionais nas condições de HTS testadas. Nas bibliotecas de fosmídeos triadas, vários clones apresentaram atividade enzimática, sendo que dois apresentaram atividade de lipase-esterase com alta seletividade, elevada taxa de conversão de substratos e excesso enantiomérico (ee >99%). Os resultados de HTS comprovaram a eficiência do uso da clonagem direta de DNA ambiental na expressão de vias metabólicas de interesse com potencial de aplicação biotecnológica. / Metagenomics brought a new perspective to the study of microbial communities in the environment, enabling access to the taxonomic diversity of uncultured microorganisms, as well as direct access to genes and metabolic pathways. In the current study, metagenomic libraries were constructed from mangrove sediment samples of the Guanabara Bay (RJ, Brazil), impacted with oil hydrocarbons and heavy metals. Proteobacteria (33.3%), sulfate-reducing affiliated bacteria (29.7%) and Firmicutes (20%) represented the main groups in the environmental samples based upon 16S rDNA phylogenetic analysis, whereas selective isolation using diesel and naphtalene yielded delta-Proteobacteria and actinomycetes. Metagenomic libraries of diesel-enriched sediment samples, with 25 to 35 Kb fosmid inserts, were screened for detecting monooxigenase genes (alkB1) and expression of epoxide hydrolases, esterases, lipases and monooxigenases in high throughput screening (HTS) assays. Clones reactive to the alkB1 probe were detected, but were not functional under the HTS conditions used. Several functional clones were detected in the clone library, and two showed lipase-esterase activity with high rates of substrate conversion and enantiomeric ratio (ee >99%). The results obtained on HTS showed the efficiency of the direct cloning of environmental DNA for the expression of metabolic pathways with potential biotechnological application.

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