Muitos fatores, como variações sazonais, tipos de tecido vegetal, cultivares e espécies de hospedeiro, tipo de solo, interação com microrganismos benéficos ou patógenos entre outros, afetam a estrutura e a composição da comunidade bacteriana das plantas. A introdução de plantas geneticamente modificadas (PGM) foi somada ao conjunto desses fatores, podendo acarretar efeitos diretos e indiretos sobre a comunidade bacteriana. Cana-de-açúcar é uma cultura de grande importância no Brasil; a área de cultivo está em expansão devido aos incentivos para a produção de álcool. Uma bactéria potencialmente importante para a cultura de cana-de-açúcar é a Methylobacterium, um importante endófito de diversas culturas de interesse econômico e que, em cana-de-açúcar, pode melhorar a germinação de sementes, promover um aumento do peso da planta e da área foliar, além do número de internódios. Dessa forma, o trabalho teve como objetivos: i) avaliar os efeitos da canade- açúcar transgênica resistente a insetos e a herbicida sobre a comunidade bacteriana associada; ii) avaliar se o suposto efeito causado pela cana-de-açúcar transgênica resistente a herbicida deriva diretamente do transgene ou dos tratos culturais diferenciados aos quais a planta transgênica é submetida; iii) avaliar a interação entre Methylobacterium spp. e cana-de-açúcar. Analisando a densidade bacteriana das plantas de cana-de-açúcar convencionais e transgênicas não foi possível constatar diferenças relacionadas à introdução dessas plantas. Analisando somente quanto ao manejo de ambos os experimentos, foi possível observar que diferenças em trato cultural ou manejo de plantas, decorrentes ou não da transgenia, podem influenciar a comunidade bacteriana. Por meio de ARDRA, foi possível observar distribuição diferenciada dos ribotipos com a introdução das PGMs. No experimento realizado somente com plantas transgênicas para resistência a Imazapyr, foi possível notar que, em 17 meses, a presença da planta transgênica e a aplicação do herbicida Imazapyr podem ter resultado na redução da densidade bacteriana associada à rizosfera de cana-de-açúcar. Por meio de DGGE, foi visto que o estado fisiológico da planta foi a maior fonte de variação. Novamente por meio de ARDRA, foram observados ribotipos cuja presença foi afetada pelo cultivo da planta transgênica. Esses ribotipos diferentemente distribuídos poderiam resultar em alterações na atividade bacteriana dessas plantas, uma vez que esses ribotipos podem representar grupos funcionais importantes. A colonização de Methylobacterium spp. em cana-de-açúcar foi analisada por meio de microscopia eletrônica de varredura, reisolamento e microscopia óptica de fluorescência. Foi visto que as linhagens utilizadas colonizam cana-de-açúcar, sendo que os pontos de maior colonização são os flanges cuticulares e as regiões pilosas da raiz. Outros estudos são necessários para o melhor aproveitamento dessa bactéria na cultura de cana-de-açúcar. / Many factors, such as seasonal variations, kinds of vegetal tissue, cultivares and host species, kind of soil, interaction with beneficial or pathogen microorganisms, among others, affect the structure and composition of plants bacterial community. The introduction of genetically modified plants (GMP) was added to the set of these factors, making it possible to cause direct and indirect effects on the bacterial community. Sugarcane is a very important crop in Brazil; the cultivation area is expanding due to incentives to alcohol production. A potentially important bacterium for sugar cane cultivation is the Methylobacterium, which is an important endophyte for several cultures of economic interest and which can improve seed germination in sugar cane, promote an increase of plant weight and foliar area, and also the internodes. Thus, the work had as objectives: i) to assess the effects of transgenic sugarcane resistant to insects and herbicide on the associated bacterial community; ii) to assess if the presumed effect caused by transgenic sugarcane resistant to herbicides derives directly from the transgene or differentiated cultural handlings to which the genetically modified plant is undertaken; iii) to assess the interaction between Methylobacterium ssp. and sugarcane. Analyzing the bacterial density of conventional and transgenic sugarcane plants it was not possible to see differences related to the introduction of these plants. Analyzing only in relation to the handling of both experiments, it was possible to see that differences in cultural wielding or handling of plants derived or not from transgenia can influence the bacterial community. By means of ARDRA, it was possible to see a differentiated distribution of ribotypes with the introduction of GMPs. In the experiment made only with transgenic plants for Imazapyr resistance, it was possible to see that in 17 months, the presence of the transgenic plant and the application of Imazapyr herbicide can bring result regarding the bacterial density reduction associated to sugarcane rizosphere. By means of DGGE, it was seen that the physiological status of the plant was the greatest variation source. Again, by means of ARDRA, ribotypes whose presence was affected by transgenic plant cultivation were observed. If distributed differently, these ribotypes can represent important functional groups. The Methylobacterium ssp. Colonization in sugarcane was analyzed and by means of scanning electronic microscopy, re-isolation, and fluorescence optical microscopy. It was observed that the utilized lineages colonize sugarcane being cuticle flange and perilous regions of root the highest colonization points. Other studies are needed to a better good use of this bacterium in sugarcane culture.

L'efficacité des antibiotiques actuellement utilisés dans le monde entier est en baisse à un rythme inquiétant. La majorité des produits actifs naturels sont isolés des ascomycètes ou des Actinobactéries. Parmi les 10 000 antibiotiques connus, plus de la moitié sont produits par des bactéries du genre Streptomyces. Il est donc intéressant de rechercher de nouvelles molécules actives dans des niches encore sous explorés, tels que les symbioses microbiennes mutualistes. Ainsi, les lichens sont des organismes complexes abritant des communautés bactériennes à la surface et, plus rarement, à l'intérieur de leurs thalles et constituent un modèle d’étude pour la découverte de nouvelles molécules d’intérêts. Une optimisation des conditions d'extraction des lichens a été développée. Le profilage chimique par LC / MS de neuf lichens (2 à algues vertes : Roccella fuciformis et R. phycopsis et de 7 cyanolichens: Lichina confinis, L. pygmaea, Leptogium lichenoides, Synalissa symphorea, Collema auriforme, C. cristatum et C. fuscovirens) ont été effectués et comparés avec des approches de «molecular network". Cela a permis de souligner la similitude chimique entre tous les cyanolichens d’une part et les espèces lichéniques à algues vertes d’autre part. Une étude chimique plus approfondie de R. fuciformis et R. phycopsis a été par la suite effectuée et dix composés différents ont été isolés et identifiés. Neuf d'entre eux ont été isolés et identifiés par RMN et des voies de fragmentation ont été proposés pour cinq d'entre eux. Une étude de localisation in situ de leurs métabolites majeurs respectifs (érythrine et acide roccellique pour R. phycopsis et érythrine, acide léprarique et acetylportentol pour R. fuciformis) a été réalisée et a démontré leur emplacement spécifique au sein des thalles lichéniques. Les communautés bactériennes cultivables associées à trois lichens de la côte bretonne (France) (Roccella fuciformis, Lichina confinis, L. pygmaea) et un lichen terrestre récolté en Autriche (Collema auriforme) ont été étudiées afin de trouver de nouveaux métabolites d'intérêts. L'abondance et la diversité des communautés bactériennes associées à ces lichens a été montré: 247 souches ont été isolées et identifiées par l’étude du gène de l'ARNr 16S. Ainis, plus de 30% de toutes les souches expriment des gènes permettant la production potentielle des composés bioactifs et 12% appartiennent probablement à de nouvelles espèces bactériennes. Les métabolites secondaires de deux bactéries cultivables associées ont été étudiés (MOLA1488, Streptomyces sp. et MOLA1416, Hoeflea sp.) et certains métabolites spécialisés actifs ont été isolés (des dicétopipérazines, des alcaloïdes, des dérivés phénoxazine par exemple ...) présentant des propriétés biologiques intéressantes. Enfin, pour mettre en évidence les interactions possibles entre les lichens et leurs bactéries associées, une approche de culture (extraits lichéniques et bactéries associées) a été réalisée à partir de 4 souches bactériennes les plus abondantes associées à Roccella fuciformis pour (1) évaluer l'impact de ces métabolites sur la croissance de ces quatre souches et également, (2) à évaluer la capacité de bioconversion de l'acide leprarique et de l’érythrine. Ces bactéries ont montré la capacité de bio-converser l’érythrine en acide orsellinique, mais aucun des quatre métabolites testés n’a affecté leur croissance. / Efficiency of currently used antibiotics is worldwide decreasing at a worrying rate, while we are faced with new and emerging pathogens. The majority of active natural products are isolated from the Ascomycetes or from the Actinobacteria. Among the 10000 known antibiotics, more than half are produced by bacteria of one single genus, Streptomyces. It is therefore most interesting to search for novel active molecules in yet under explored niches, such as mutualistic microbial symbioses. Lichens are complex organisms harboring bacterial communities on the surface and, more rarely, inside their thalli and present a model to discover new biomolecules. Optimization of extraction conditions of lichens has been developed. Chemical profiling by LC / MS of nine lichens (2 green algae Roccella fuciformis and R. phycopsis and 7 cyanolichens: Lichina confinis, L. pygmaea, Leptogium lichenoides, Synalissa symphorea, Collema auriforme, C. cristatum and C. fuscovirens) were made and compared with a "Molecular network" approach. This has allowed to identify the chemical similarity between all cyanolichens and between two lichen species containing green algae. On the other hand, further chemical study on R. fuciformis and R. phycopsis was conducted and ten different compounds were isolated. Nine of them have been isolated and identified by NMR and mass fragmentation pathways have been highlighted for five of them. In situ localization of their major respective metabolites (erythrin and roccellic acid for R. phycopsis and erythrin, lepraric acid and acetylportentol for R. fuciformis) was performed and showed a specific location in the lichen thallus. We focused also on the cultivable bacterial communities associated to three lichens from Brittany coast (France) (Roccella fuciformis, Lichina confinis, L. pygmaea) and one inland lichen from Austria (Collema auriforme) to find new secondary metabolites of interest. The abundance and the diversity of the bacterial communities associated to these lichens were showed: 247 strains were isolated and identified by 16S rRNA gene analysis. More than 30% of all strains express potential bioactive compounds and 12% represent probably new species. The secondary metabolites patterns of their cultivable associated bacteria were studied (MOLA1488, Streptomyces sp. and MOLA1416, Hoeflea sp.) and some active secondary metabolites were isolated (e.g. dicetopiperazines, pyrrole alkaloïds, phenoxazine derivatives …) showing biological properties. Finally, to highlight potential interactions between lichens and their associated bacteria, an approach of culture (lichen extracts and bacteria) was performed from 4 most abundant bacterial strains associated with Roccella fuciformis to (1) assess the impact of major metabolites (compounds 4) of this lichen on the growth of these four strains by a an optimized method of viability using MTT; and also to evaluate (2) the ability to bioconversion of these four strains of lepraric acid and erythrin. These bacteria have shown the ability to metabolize erythrin in orsellinic acid, but none of the four tested metabolites has affected their growth.

"Où peut-on trouver des microbes, et comment survivent-ils dans ces lieux ?" sont des questions essentielles afin de comprendre la vie sur Terre. Les populations bactériennes du sol sont connues pour jouer un rôle important dans les cycles biogéochimiques, l'entretien des sols, les effets climatiques et l'agriculture.Dans ce travail, j'ai utilisé la technique de pyroséquençage, via le produit d’une PCR d’ADNr 16S amplifiée extraite d’ADN totale, afin de révéler les populations bactériennes présentes dans quatre environnements inhabituels et oligotrophes différents:A. Les écosystèmes saumâtres sont largement distribués sur Terre et sont représentés par des systèmes aquifères salés et des sols salins. Nous avons examiné la composition bactérienne des sédiments des estuaires, sols saumâtres et des échantillons de sol sablonneux de la région de Camargue, échantillonnés pendant deux années consécutives. Les membres appartenant au phylum Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Acidobacteria et Actinobactéries ont été trouvés principalement dans les sols et sédiments. Nous avons constaté que les membres de ces groupes bactériens étaient associés principalement à des bactéries halophiles, sulfatoréductrices (SRB), nitratoréductrices et coliformes, dont leurs proportions ont probablement été affectées par la salinité et leurs localisations géographiques.B. Les bactéries associées à la rhizosphère des plantes sont connues pour jouer un rôle essentiel dans les cycles biogéochimiques, la nutrition des plantes et la lutte biologique contre les maladies végétales. Nous avons examiné les populations bactériennes de la rhizosphère du riz (Oryza sativa) en fin de croissance dans la région de la Camargue en 2013 et 2014. Les populations bactériennes les plus abondantes se sont révélées être des membres appartenant au phylum Proteobacteria, Acidobacteria, Chloroflexi et Gemmatimonadetes. Les genres bactériens auxquels appartiennent ces différents phylums sont connus pour participer dans des processus biogéochimiques du sol, tel que la nitrification, la dénitrification, l'oxydation, ainsi que comme agents de control biologique. Les proportions bactériennes trouvées varient considérablement en fonction de leur localisation géographique et selon l’année d’échantillonnage.C. Nous avons examiné les sols de surface de "Padza de Dapani" situés sur l'île de Mayotte au large de la côte est de l'Afrique, car cette région n’est pas un vrai désert, mais y ressemble due à l’érosion du sol. Les sols de Mayotte sont acides, oligotrophes et minéralisées, et leur population bactérienne principale appartient aux phylums des Actinobactéries, Proteobacteria et Acidobacteria. Un fait intéressant, les membres des genres Acinetobacter, Arthrobacter, Burkholderia et Bacillus sont prédominants dans nos échantillons, comme observé dans des déserts (asiatiques) chauds et jouant probablement un rôle dans la minéralisation des sols, expliquant la désertification.D. Les régions arides de la Terre constituent > de 30% de la surface continentale et les sols oligotrophes sont soumis à des facteurs environnementaux difficiles tels que la faible pluviométrie moyenne annuelle, l'exposition aux UV et les grandes fluctuations de température. Nous avons examiné les populations bactériennes présentes dans la rhizosphère des plantes pionnières et les sols de surface du désert de Jizan d'Arabie Saoudite. Les phylums bactériens les plus abondants appartiennent aux groupes des Bacteroidetes, Proteobacteria et Firmicutes qui diffèrent entre la rhizosphère des plantes étudiées par rapport à la surface du sol, à l'exception de la plante "Panicum Turgidum" qui contient des proportions élevées (70%) des membres appartenant au genre Flavobacterium. / “What microbes are where, and how do they live there” is now an essential question to understand life on Earth, even when comparing seemingly similar ecosystems in different locations. Soil bacterial populations are known to play important roles in biogeochemical cycles, soil maintenance, climatic effects and agriculture. I used pyrosequencing of PCR amplified 16S rDNA from total extracted DNA in order to reveal the bacterial populations living in four different unusual and oligotrophic environments: A. Saline areas are widely distributed on Earth’s and are represented by both saline lakes and saline soils. We examined the bacterial composition of estuary sediments, brackish and sandy soil samples from the Camargue region (Rhône delta in southern France) sampled in two consecutive years. Members belonging to the Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Acidobacteria and Actinobacteria phyla were found principally in saline sediment and soil samples. We found that members from these phyla were associated principally to halophilic bacteria, sulphate reducing bacteria (SRB), nitrate reducing bacteria and coliforms, and that their varying proportions were likely affected by salinity and geographical location. B. Bacterial populations associated with the rhizosphere of plants are known to play essential roles in biogeochemical cycles, plant nutrition and disease biocontrol. We examined the bacterial populations of the rhizosphere of rice (Oryza sativa) growing in the Camargue region in 2013 and 2014. The most abundant bacterial populations were found to be members belonging to the Proteobacteria, Acidobacteria, Chloroflexi and Gemmatimonadetes phyla. The genera members belong these phyla were found to participate in soil biogeochemical processes such as nitrification, denitrification, oxidation, as well as act as biocontrol agents. The bacterial populations were found to significantly vary by geographical location as well by year of collection. C. We examined the surface soils from “Padza de Dapani” on the island of Mayotte off the east coast of Africa, as this region is not a true (hot) desert, but resembles one due to extensive soil erosion. In the acidic, oligotrophic and mineralized soil samples from Mayotte, members of the Actinobacteria, Proteobacteria and Acidobacteria phyla dominated the bacterial populations. Interestingly, members of the genera Acinetobacter, Arthrobacter, Burkholderia and Bacillus were found to be predominant in our samples, as is also observed in hot (Asian) deserts and may play roles in soil mineral weathering, thus helping to understand desertification processes. D. Earth’s arid regions comprise >30% of the continental surface and the oligotrophic soils are subjected to harsh environmental factors such as low average annual rainfall, high UV exposure and large temperature fluctuations. We examined the bacterial populations present in the rhizosphere of pioneer plants and surface soils in the Jizan desert of Saudi Arabia. The most abundant bacterial phyla belonged to the Bacteroidetes, Proteobacteria and Firmicutes phyla that were different between the rhizosphere of plant versus these from surface sand, with the exception of the plant “Panicum Turgidum”, which contain in its rhizosphere high proportions (70%) of members belonging to the Flavobacterium genus.

Deep-sea anoxic brine pools are one of the most extreme ecosystems on Earth, which are characterized by drastic changes in salinity, temperature, and oxygen concentration. The interface between the brine and overlaying seawater represents a boundary of oxic-anoxic layer and a steep gradient of redox potential that would initiate favorable conditions for divergent metabolic activities, mainly methanogenesis and sulfate reduction. This study aimed to investigate the diversity of Bacteria, particularly sulfate-reducing communities, and their ecological roles in the interfaces of five geochemically distinct brine pools in the Red Sea. Performing a comprehensive study would enable us to understand the significant role of the microbial groups in local geochemical cycles. Therefore, we combined culture-dependent approach and molecular methods, such as 454 pyrosequencing of 16S rRNA gene, phylogenetic analysis of functional marker gene encoding for the alpha subunits of dissimilatory sulfite reductase (dsrA), and single-cell genomic analysis to address these issues. Community analysis based on 16S rRNA gene sequences demonstrated high bacterial diversity and domination of Bacteria over Archaea in most locations. In the hot and multilayered Atlantis II Deep, the bacterial communities were stratified and hardly overlapped. Meanwhile in the colder brine pools, sulfatereducing Deltaproteobacteria were the most prominent bacterial groups inhabiting the interfaces. Corresponding to the bacterial community profile, the analysis of dsrA gene sequences revealed collectively high diversity of sulfate-reducing communities. Desulfatiglans-like dsrA was the prevalent group and conserved across the Red Sea brine pools. In addition to the molecular studies, more than thirty bacterial strains were successfully isolated and remarkably were found to be cytotoxic against the cancer cell lines. However, none of them were sulfate reducers. Thus, a single-cell genomic analysis was used to study the metabolism of uncultured phyla without having them in culture. We analysed ten single-cell amplified genomes (SAGs) of the uncultivated euryarchaeal Marine Benthic Group E (MBGE), which contain a key enzyme for sulfate reduction. The results showed the possibility of MBGE to grow autotrophically only with carbon dioxide and hydrogen. In the absence of adenosine 5’-phosphosulfate reductase, we hypothesized that MBGE perform sulfite reduction rather than sulfate reduction to conserve energy.

Organism succession during ecosystem development has been well studied for aboveground plant communities while the associated pattern of change in microbial communities remains largely unknown. A study was conducted along developmental sand-dune chronosequences bordering Lake Michigan at Wilderness State Park and Altamaha river valley of southeast Georgia with the hypothesis that soil bacterial communities will follow a pattern of change that is associated with soil, plant, and ecosystem development. This study site included 5 replicate sites along 14 dunes ranging in age from 105 to 500,000 years since deposition. The microbial composition and diversity in the soil was studied using bacterial tag-encoded FLX amplicon pyrosequencing of the 16S rRNA gene. As hypothesized, Bray-Curtis ordination indicated that bacterial community assembly changed along the developmental gradient at both sites. However, there was no seasonal effect at Michigan sites despite likely differences in plant carbon inputs. At the Michigan site, soil Ca, Mg levels and pH showed a significant log-linear correlation with soil development (r = 0.83, 0.84 and 0.81, respectively). Bacterial diversity represented by Simpson’s reciprocal index (Simpson’s 1/D) showed a steady decline from the youngest to the oldest dunes with the largest decline (212 to 58) during the initial stages of soil development (105 to 450 years). The change in plant species abundance was higher in the youngest sites than the older sites. This change was significantly correlated with the change in microbial community distribution (p < 0.0001; r = 0.56). Similarly, at Georgia sites, soil development showed significant log-linear correlation with soil base cations (Ca and Mg) (r = 0.93and 0.95). However, diversity indices and PLFA failed to show any particular change in trend across the developing chronosequences. When the results from both sites were used to study bacterial spatial patterns, local geochemical features were found to be a dominant factor in driving bacterial community structure, while geographic distance as a single factor could contribute to some community variation at a scale (50 – 1700 km). The results suggest that soil nutrients and plant community could be a strong driving force in shaping microbial community assembly across a developing soil ecosystem.

A biogeografia estuda a distribuição dos organismos em relação ao espaço e ao tempo, favorecendo a compreensão dos mecanismos que geram e mantém a diversidade, especiação, extinção e dispersão das espécies. Dentre as florestas tropicais, a Mata Atlântica constitui um mosaico vegetal de grande diversidade, onde a filosfera representa um dos habitats mais comuns para os microrganismos. O objetivo do presente trabalho foi avaliar a estrutura e diversidade da comunidade bacteriana da filosfera de Maytenus robusta no Parque Estadual Carlos Botelho, Parque Estadual Ilha do Cardoso e Estação Ecológica de Assis, no estado de São Paulo, Brasil. As folhas foram coletadas em duas épocas do ano, seca e chuvosa. A estrutura da comunidade bacteriana foi avaliada através de PCR-DGGE da região V3 do gene rRNA 16S e a diversidade por sequenciamento da região V1-V3 do mesmo gene. A similaridade entre a estrutura de comunidades de Bacteria foi determinada com base na presença ou ausência das bandas detectadas no gel após PCR-DGGE. O agrupamento hierárquico gerado com o coeficiente de Jaccard e o método UPGMA mostrou a existência de comunidades bacterianas distintas na filosfera de M. robusta nas áreas amostradas. A existência de padrões biogeográficos foi determinada através de análises de regressão, usando os dados de similaridade da estrutura das comunidades bacterianas e os de distância geográfica entre as árvores amostradas. A correlação negativa observada nas avaliações fornece evidências para suportar a hipótese de que a similaridade entre as comunidades bacterianas da filosfera de plantas da mesma espécie diminui com o aumento da distância entre as árvores, dentro de um mesmo bioma. A avaliação espaço temporal da composição da comunidade bacteriana, realizada pela análise NMDS, demonstrou que houve efeito espacial mas no temporal na estrutura das comunidades bacterianas da filosfera de M. robusta. A afiliação taxonômica de 1.470 sequências de clones do gene rRNA 16S de Bacteria, obtidas da filosfera de M. Robusta, nas diferentes áreas e épocas, e a comparação múltipla das bibliotecas, mostraram que as comunidades bacterianas na filosfera foram distintas umas das outras, sendo os filos Proteobacteria e Acidobacteria os mais frequentes. Somente 1% das Unidades Taxonômicas Operacionais foram comuns entre os indivíduos avaliados. Com base nos resultados obtidos, pode-se inferir que, em cada bioma, plantas da mesma espécie possuem comunidades bacterianas únicas, sugerindo a existência de endemismo, altos níveis de especiação e baixa dispersão das comunidades bacterianas nas áreas avaliadas. / Biogeography studies the distribution of organisms in relation to space and time, favoring the understanding of the mechanisms that generate and keep the diversity, speciation, extinction and dispersion of species. Among the tropical forests, the Atlantic Forest constitutes a highly diverse vegetation mosaic, in which the phyllosphere represents one of the most common habitats for microorganisms. The goal of this work was to evaluate the structure and diversity of the bacterial community from the phyllosphere of Maytenus robusta in the Carlos Botelho State Park, Ilha do Cardoso State Park and Assis Ecologic Station, São Paulo state, Brazil. The leaves were collected in two different seasons of the year, dry season and rainy season. The structure of the bacterial community was evaluated through PCR-DGGE of the 16S rRNA gene V3 region, and the diversity by sequencing of the V1-V3 region of the same gene. The similarities between the structures of the bacterial community were determined based on the presence or absence of bands detected in the gels after PCR-DGGE. The hierarchical clustering generated using the Jaccard coefficient and the UPGMA method showed the existence of distinct bacterial communities in the M. robusta phyllosphere of the sampled areas. The existence of biogeographic patterns was determined through regression analyses, using the community structure similarity data geographic distance among the sampled trees. The negative correlation observed in most of the cases provides evidence to support the hypothesis that the similarity between the bacterial communities from phyllosphere of plants of the same species decreases as the distance among trees increased, within the same biome. The spacial-temporal evaluation of the structure of the bacterial communities, performed by the NMDS analyses, showed the occurrence of spacial but not temporal effects on the structure of the bacterial communities of M. robusta phyllosphere. The taxonomic affiliation of 1,470 bacterial 16S rRNA gene clones obtained from the M. robusta phyllosphere, in different areas and seasons, as well as the multiple comparisons of libraries showed that the bacterial communities in the phyllosphere were distinct from each other, and that Proteobacteria and Acidobacteria phyla were the most frequent. Only 1% of the bacterial Operational Taxonomic Units were common among the individuals evaluated. Based on the results obtained it is possible to conclude that, in each biome, plants of same species have unique bacterial communities, suggesting the existence of endemism, high levels of speciation and low dispersal of bacterial communities in the evaluated areas.

Terra Preta Antropogênica (TPA) é um dos tipos de solos mais \"férteis\" do mundo. A TPA recebe esta denominação por ser encontrada em sítios arqueológicos onde viveram grupos pré-históricos. São pequenas faixas de solos que apresentam altas concentrações de nutrientes, matéria orgânica e encontram-se distribuídas aleatoriamente pela região Amazônica. A verdadeira origem destes solos ainda não está bem esclarecida. Devido à falta de informações sobre sua diversidade bacteriana, este trabalho estudou a diversidade bacteriana em amostras de solos TPA coletadas em duas regiões: Lagoa Balbina - sítio Terra preta (Amazônia Central- Amazonas) e Floresta Nacional de Caxiuanã - sítio arqueológico Mina I (Amazônia Oriental - Pará), através de técnicas moleculares independentes de cultivo. O DNA genômico total das amostras de solo foi extraído e usado como molde em uma reação de PCR utilizando oligonucleotídeos específicos do gene 16S rRNA para o Domínio Bactéria. O produto de PCR amplificado foi clonado no vetor pGEM-T e 980 clones foram selecionados e comparados com o banco de dados de 16S rRNA do RDP II e GenBank (NCBI-EUA). Os resultados apresentaram predominância com microrganismos não-conhecidos representando 41,6 % das seqüências de solo TPA- Balbina, 68,3 % das seqüências de ADJ-Balbina, 84,8% das seqüências de solo TPAMina e 47,7 % das seqüências de ADJ-Mina. O filo mais predominante nas amostras TPABalbina foi Firmicutes, representando 37,1% do total de seqüências analisadas. Os filos em destaque foram Proteobacteria (9,6%), seguidos de Verrucomicrobia (5,6%), Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) e Nitrospira (0,5%). Por outro lado, em ADJ-Balbina destacaram-se os filos Proteobacteria 15,1%, Acidobacteria (12,5%), Firmicutes (2,3%), Nitrospira (1,1%) e Verrucomicrobia (0,8%). Em TPA-Mina, os filos apresentados foram Proteobacteria (6,5%), Acidobacteria (4,7%), Firmicutes (1,4%), Nitrospira (1,1%), Planctomycetes (1,1%) e Verrucomicrobia (0,4%). Contudo, na biblioteca ADJ-Mina verificou a presença dos filos Acidobacteria (27,2%), Proteobacteria (14,2%), Firmicutes (3,8%), Verrucomicrobia (3,8%), Nitrospira (1,3%), Planctomycetes (1,3%), Actinobacteria (0,4%) e Gemmatimonadetes (0,4%). O pH do solo pode ser um dos atributos do solo que pode ter influência direta na diversidade bacteriana dos solos estudados, assim como pode ter efeito uma floresta natural sobre as populações microbianas em seu solo, fato observado na adjacência do solo Terra Preta em Caxiuanã - PA. A estimativa da riqueza de UTOs pelo Bootstrap corroborou diretamente os valores de diversidade obtidos pelos índices de Simpson e Shannon. De um modo geral, uma maior probabilidade de ocorrência de UTOs únicas empregadas pelo estimador Jackknife se correlacionou com uma maior percentagem de baixas frequências de filotipos nas quatro bibliotecas. Os métodos não-paramétricos ACE e Chao1 para a estimativa da riqueza de UTOs também corroboraram com os valores obtidos com o estimador Jackknife. / Anthropogenic Dark Earth (ADE) is one of the most fertile soils in the world. ADE soils have received this nomination due to the pre-historical origin of these archaeological sites, established by pre-colombian populations. ADEs are small areas of soil which present high nutrient and organic matter contents and are randomly distributed throughout the Amazonian region. The true origin of these soils is not known yet. Due to the lack of information concerning the bacterial diversity, this work studied the bacterial diversity in ADE soils collected from two regions: Lagoa Balbina - site Terra Preta (Central Amazonia- Amazonas state) and National Forest of Caxiuanã - archaeological site Mina I (Oriental Amazonia - Pará state), using culture-independent molecular techniques. The total genomic DNAs extracted from the soil samples were used as templates in the PCR reactions using the universal primers for the 16S rRNA bacterial gene. The PCR-products were cloned into the pGEM-T vector and 980 clones were selected and searched using the GenBank (NCBI-USA) and the RDP II program. Data analyses indicated predominance of unknown microorganisms, representing 41.6 % among the sequences from ADE-Balbina, 68.3 % from Adjacent-Balbina, 84.8% from ADE-Mina and 47.7 % from Adjacent-Mina. The predominant phylum in ADEBalbina was Firmicutes, representing 37.1% of the total sequences from that site, followed by Proteobacteria (9.6%), Verrucomicrobia (5.6%) Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) and Nitrospira (0.5%). On the other hand, in the adjacent soil ADJ-Balbina, the predominant phylla were Proteobacteria (15.1%), Acidobacteria (12.5%), Firmicutes (2.3%), Nitrospira (1.1%) and Verrucomicrobia (0.8%). In the Oriental Amazon, the prevalent phylla from the ADE-Mina soil were Proteobacteria (6.5%), Acidobacteria (4.7%), Firmicutes (1.4%), Nitrospira (1.1%), Planctomycetes (1.1%) and Verrucomicrobia (0.4%). In the ADJ-Mina verificou a presença dos filos Acidobacteria 27.2%, Proteobacteria 14.2%, Firmicutes 3.8%, Verrucomicrobia 3.8%, Nitrospira 1.3%, Planctomycetes 1.3%, Actinobacteria 0.4% e Gemmatimonadetes 0.4%. The soil pH may be of the soil attributes which may have directly influenced the bacterial diversity in those soils, as well as the above-ground vegetation from the natural forest in Caxiuanã-Pará. The estimates of the Operational Taxonomic Units (OTUs) richness using Bootstrap directly corroborated the diversity values obtained from the Simpson and Shannon indexes. Unique UTOs using Jackknife estimator were correlated with a higher percentage of the low frequencies of phylla in all the four clone libraries. The nonparametric ACE and Chao1 methods to estimate the OTUs richness also corroborated the Jackknife values.

Terra Preta Antropogênica (TPA) é um dos tipos de solos mais \"férteis\" do mundo. A TPA recebe esta denominação por ser encontrada em sítios arqueológicos onde viveram grupos pré-históricos. São pequenas faixas de solos que apresentam altas concentrações de nutrientes, matéria orgânica e encontram-se distribuídas aleatoriamente pela região Amazônica. A verdadeira origem destes solos ainda não está bem esclarecida. Devido à falta de informações sobre sua diversidade bacteriana, este trabalho estudou a diversidade bacteriana em amostras de solos TPA coletadas em duas regiões: Lagoa Balbina - sítio Terra preta (Amazônia Central- Amazonas) e Floresta Nacional de Caxiuanã - sítio arqueológico Mina I (Amazônia Oriental - Pará), através de técnicas moleculares independentes de cultivo. O DNA genômico total das amostras de solo foi extraído e usado como molde em uma reação de PCR utilizando oligonucleotídeos específicos do gene 16S rRNA para o Domínio Bactéria. O produto de PCR amplificado foi clonado no vetor pGEM-T e 980 clones foram selecionados e comparados com o banco de dados de 16S rRNA do RDP II e GenBank (NCBI-EUA). Os resultados apresentaram predominância com microrganismos não-conhecidos representando 41,6 % das seqüências de solo TPA- Balbina, 68,3 % das seqüências de ADJ-Balbina, 84,8% das seqüências de solo TPAMina e 47,7 % das seqüências de ADJ-Mina. O filo mais predominante nas amostras TPABalbina foi Firmicutes, representando 37,1% do total de seqüências analisadas. Os filos em destaque foram Proteobacteria (9,6%), seguidos de Verrucomicrobia (5,6%), Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) e Nitrospira (0,5%). Por outro lado, em ADJ-Balbina destacaram-se os filos Proteobacteria 15,1%, Acidobacteria (12,5%), Firmicutes (2,3%), Nitrospira (1,1%) e Verrucomicrobia (0,8%). Em TPA-Mina, os filos apresentados foram Proteobacteria (6,5%), Acidobacteria (4,7%), Firmicutes (1,4%), Nitrospira (1,1%), Planctomycetes (1,1%) e Verrucomicrobia (0,4%). Contudo, na biblioteca ADJ-Mina verificou a presença dos filos Acidobacteria (27,2%), Proteobacteria (14,2%), Firmicutes (3,8%), Verrucomicrobia (3,8%), Nitrospira (1,3%), Planctomycetes (1,3%), Actinobacteria (0,4%) e Gemmatimonadetes (0,4%). O pH do solo pode ser um dos atributos do solo que pode ter influência direta na diversidade bacteriana dos solos estudados, assim como pode ter efeito uma floresta natural sobre as populações microbianas em seu solo, fato observado na adjacência do solo Terra Preta em Caxiuanã - PA. A estimativa da riqueza de UTOs pelo Bootstrap corroborou diretamente os valores de diversidade obtidos pelos índices de Simpson e Shannon. De um modo geral, uma maior probabilidade de ocorrência de UTOs únicas empregadas pelo estimador Jackknife se correlacionou com uma maior percentagem de baixas frequências de filotipos nas quatro bibliotecas. Os métodos não-paramétricos ACE e Chao1 para a estimativa da riqueza de UTOs também corroboraram com os valores obtidos com o estimador Jackknife. / Anthropogenic Dark Earth (ADE) is one of the most fertile soils in the world. ADE soils have received this nomination due to the pre-historical origin of these archaeological sites, established by pre-colombian populations. ADEs are small areas of soil which present high nutrient and organic matter contents and are randomly distributed throughout the Amazonian region. The true origin of these soils is not known yet. Due to the lack of information concerning the bacterial diversity, this work studied the bacterial diversity in ADE soils collected from two regions: Lagoa Balbina - site Terra Preta (Central Amazonia- Amazonas state) and National Forest of Caxiuanã - archaeological site Mina I (Oriental Amazonia - Pará state), using culture-independent molecular techniques. The total genomic DNAs extracted from the soil samples were used as templates in the PCR reactions using the universal primers for the 16S rRNA bacterial gene. The PCR-products were cloned into the pGEM-T vector and 980 clones were selected and searched using the GenBank (NCBI-USA) and the RDP II program. Data analyses indicated predominance of unknown microorganisms, representing 41.6 % among the sequences from ADE-Balbina, 68.3 % from Adjacent-Balbina, 84.8% from ADE-Mina and 47.7 % from Adjacent-Mina. The predominant phylum in ADEBalbina was Firmicutes, representing 37.1% of the total sequences from that site, followed by Proteobacteria (9.6%), Verrucomicrobia (5.6%) Acidobacteria (2,5%), Gemmatimonadetes (2,5%), Actinobacteria (0,5%) and Nitrospira (0.5%). On the other hand, in the adjacent soil ADJ-Balbina, the predominant phylla were Proteobacteria (15.1%), Acidobacteria (12.5%), Firmicutes (2.3%), Nitrospira (1.1%) and Verrucomicrobia (0.8%). In the Oriental Amazon, the prevalent phylla from the ADE-Mina soil were Proteobacteria (6.5%), Acidobacteria (4.7%), Firmicutes (1.4%), Nitrospira (1.1%), Planctomycetes (1.1%) and Verrucomicrobia (0.4%). In the ADJ-Mina verificou a presença dos filos Acidobacteria 27.2%, Proteobacteria 14.2%, Firmicutes 3.8%, Verrucomicrobia 3.8%, Nitrospira 1.3%, Planctomycetes 1.3%, Actinobacteria 0.4% e Gemmatimonadetes 0.4%. The soil pH may be of the soil attributes which may have directly influenced the bacterial diversity in those soils, as well as the above-ground vegetation from the natural forest in Caxiuanã-Pará. The estimates of the Operational Taxonomic Units (OTUs) richness using Bootstrap directly corroborated the diversity values obtained from the Simpson and Shannon indexes. Unique UTOs using Jackknife estimator were correlated with a higher percentage of the low frequencies of phylla in all the four clone libraries. The nonparametric ACE and Chao1 methods to estimate the OTUs richness also corroborated the Jackknife values.

Orientador: Valéria Maia Merzel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T14:04:11Z (GMT). No. of bitstreams: 1 Silva_CynthiaCanedoda_D.pdf: 9736339 bytes, checksum: 506af9b64177978ae4c3c5c0588e1b28 (MD5) Previous issue date: 2010 / Resumo: Os efluentes de refinarias contêm vários compostos tóxicos, que podem provocar sérios danos aos seres vivos se não tratados previamente. Os fenóis são poluentes encontrados em efluentes de diversas indústrias, incluindo as refinarias de petróleo, e são conhecidos como um dos compostos mais tóxicos encontrados no meio ambiente. Portanto, há necessidade constante de buscar formas de remover ou reduzir a presença destas substâncias nos efluentes. Este trabalho teve como objetivos analisar e explorar a diversidade microbiana presente em lodo de sistemas de tratamento de efluentes de refinarias petróleo, através da construção de bibliotecas de genes RNAr 16S e bibliotecas metagenômicas em vetores do tipo fosmídeo. Análises filogenéticas das bibliotecas de genes RNAr 16S e metagenômicas mostraram uma comunidade bacteriana bastante diversa, com predominância do Filo Proteobacteria, seguido de Actinobacteria, Planctomycetes, Verrucomicrobia, Cloroflexi e Bacteroidetes. Dentre os gêneros mais abundantes, se destacaram Thauera, Comamonas, Diaphorobacter e Thiobacillus. Espécies pertencentes a estes gêneros realizam o processo de desnitrificação e espécies de Thauera e Comamonas estão relacionadas com a degradação de compostos aromáticos. A grande diversidade bacteriana refletiu em uma ampla diversidade metabólica, sendo obtido um grande número de resultados para genes relacionados com processos biológicos importantes para o tratamento de efluentes, como o metabolismo de nitrogênio, enxofre, fósforo, além de genes relacionados com o catabolismo de compostos aromáticos, tais como benzoato, bifenil e fenol. Adicionalmente, foram observadas possíveis novas sequências para os genes que codificam enzimas-chaves responsáveis pela meta-clivagem de fenol e outros aromáticos, como fenol hidroxilase e catecol 2,3-dioxigenase. No presente estudo foram também realizadas triagens funcionais dos 13.200 clones para enzimas hidrolíticas, com detecção de dois hits para esterase e um para lipase. Este trabalho mostrou que a bioprospecção genômica microbiana é uma estratégia inovadora na área de tratamento de efluentes, que pode contribuir para o conhecimento da microbiota responsável pela degradação dos poluentes. / Abstract: Refinery wastewater has several toxic compounds that may cause serious damage to life. Phenolic compounds are found in several industrial effluents, including petroleum refinery, and represent a significant environmental toxicity hazard. Therefore, there is a constant need to search for new technologies able to remove and reduce the presence of these substances in wastewater. This study aimed to analyze and explore the microbial diversity present in sludge from refinery wastewater treatment systems through the construction of 16S rRNA genes libraries and metagenomic libraries in fosmid vectors. Sequencing and phylogenetic analyses of libraries showed a highly diverse bacterial community, with predominance of the Proteobacteria phylum, followed by Actinobacteria, Planctomycetes, Verrucomicrobia, Cloroflexi and Bacteroidetes. The most abundant genera were Thauera, Comamonas, Diaphorobacter and Thiobacillus. Species belonging to these genera are facultative anaerobic and may be involved with the denitrification process, and Thauera and Comamonas species are related with degradation of aromatic compounds. This bacterial diversity reflected in a broad metabolic diversity. Analysis of the metagenomic data derived from pyrosequencing revealed a lot of hits related with biological processes of great relevance for wastewater treatment, such as genes for nitrogen, sulfur and phosphorus metabolism, in addition to genes related to the catabolism of aromatic compounds, such as benzoate, biphenyl and phenol. Additionally, sequences representing possible new genes encoding for key-enzymes responsible for the meta-cleavage of phenol and other aromatic compounds, such as phenol hydroxylase and cathecol 2,3-dioxygenase, were found in the fosmid libraries. Finally, high-throughput functional screening of 13,200 clones for hydrolytic enzymes yielded two positive hits for lipase and one for esterase. The data gathered together in this work showed that microbial genomic prospection is an innovative strategy for the wastewater treatment system that may undoubtedly contribute to the knowledge of microorganisms responsible by pollutant degradation. / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

A biogeografia estuda a distribuição dos organismos em relação ao espaço e ao tempo, favorecendo a compreensão dos mecanismos que geram e mantém a diversidade, especiação, extinção e dispersão das espécies. Dentre as florestas tropicais, a Mata Atlântica constitui um mosaico vegetal de grande diversidade, onde a filosfera representa um dos habitats mais comuns para os microrganismos. O objetivo do presente trabalho foi avaliar a estrutura e diversidade da comunidade bacteriana da filosfera de Maytenus robusta no Parque Estadual Carlos Botelho, Parque Estadual Ilha do Cardoso e Estação Ecológica de Assis, no estado de São Paulo, Brasil. As folhas foram coletadas em duas épocas do ano, seca e chuvosa. A estrutura da comunidade bacteriana foi avaliada através de PCR-DGGE da região V3 do gene rRNA 16S e a diversidade por sequenciamento da região V1-V3 do mesmo gene. A similaridade entre a estrutura de comunidades de Bacteria foi determinada com base na presença ou ausência das bandas detectadas no gel após PCR-DGGE. O agrupamento hierárquico gerado com o coeficiente de Jaccard e o método UPGMA mostrou a existência de comunidades bacterianas distintas na filosfera de M. robusta nas áreas amostradas. A existência de padrões biogeográficos foi determinada através de análises de regressão, usando os dados de similaridade da estrutura das comunidades bacterianas e os de distância geográfica entre as árvores amostradas. A correlação negativa observada nas avaliações fornece evidências para suportar a hipótese de que a similaridade entre as comunidades bacterianas da filosfera de plantas da mesma espécie diminui com o aumento da distância entre as árvores, dentro de um mesmo bioma. A avaliação espaço temporal da composição da comunidade bacteriana, realizada pela análise NMDS, demonstrou que houve efeito espacial mas no temporal na estrutura das comunidades bacterianas da filosfera de M. robusta. A afiliação taxonômica de 1.470 sequências de clones do gene rRNA 16S de Bacteria, obtidas da filosfera de M. Robusta, nas diferentes áreas e épocas, e a comparação múltipla das bibliotecas, mostraram que as comunidades bacterianas na filosfera foram distintas umas das outras, sendo os filos Proteobacteria e Acidobacteria os mais frequentes. Somente 1% das Unidades Taxonômicas Operacionais foram comuns entre os indivíduos avaliados. Com base nos resultados obtidos, pode-se inferir que, em cada bioma, plantas da mesma espécie possuem comunidades bacterianas únicas, sugerindo a existência de endemismo, altos níveis de especiação e baixa dispersão das comunidades bacterianas nas áreas avaliadas. / Biogeography studies the distribution of organisms in relation to space and time, favoring the understanding of the mechanisms that generate and keep the diversity, speciation, extinction and dispersion of species. Among the tropical forests, the Atlantic Forest constitutes a highly diverse vegetation mosaic, in which the phyllosphere represents one of the most common habitats for microorganisms. The goal of this work was to evaluate the structure and diversity of the bacterial community from the phyllosphere of Maytenus robusta in the Carlos Botelho State Park, Ilha do Cardoso State Park and Assis Ecologic Station, São Paulo state, Brazil. The leaves were collected in two different seasons of the year, dry season and rainy season. The structure of the bacterial community was evaluated through PCR-DGGE of the 16S rRNA gene V3 region, and the diversity by sequencing of the V1-V3 region of the same gene. The similarities between the structures of the bacterial community were determined based on the presence or absence of bands detected in the gels after PCR-DGGE. The hierarchical clustering generated using the Jaccard coefficient and the UPGMA method showed the existence of distinct bacterial communities in the M. robusta phyllosphere of the sampled areas. The existence of biogeographic patterns was determined through regression analyses, using the community structure similarity data geographic distance among the sampled trees. The negative correlation observed in most of the cases provides evidence to support the hypothesis that the similarity between the bacterial communities from phyllosphere of plants of the same species decreases as the distance among trees increased, within the same biome. The spacial-temporal evaluation of the structure of the bacterial communities, performed by the NMDS analyses, showed the occurrence of spacial but not temporal effects on the structure of the bacterial communities of M. robusta phyllosphere. The taxonomic affiliation of 1,470 bacterial 16S rRNA gene clones obtained from the M. robusta phyllosphere, in different areas and seasons, as well as the multiple comparisons of libraries showed that the bacterial communities in the phyllosphere were distinct from each other, and that Proteobacteria and Acidobacteria phyla were the most frequent. Only 1% of the bacterial Operational Taxonomic Units were common among the individuals evaluated. Based on the results obtained it is possible to conclude that, in each biome, plants of same species have unique bacterial communities, suggesting the existence of endemism, high levels of speciation and low dispersal of bacterial communities in the evaluated areas.