O objetivo deste estudo foi avaliar a remoção do surfactante não iônico álcool etoxilado de cadeia não ramificada (AE) em reator anaeróbio de leito fluidificado preenchido com areia como material suporte, em escala de bancada (1,2 L) com TDH de 18 horas, recirculação e fluxo contínuo. O reator foi inoculado com lodo proveniente de reator UASB utilizado no tratamento de dejetos de suinocultura e alimentado com substrato sintético acrescido de surfactante não iônico GENAPOL® C-100 (Sigma-Aldrich®) como fonte de (AE). Análises de monitoramento da concentração do surfactante não iônico AE e matéria orgânica, bem como, dos parâmetros físico-químicos foram realizadas para observar e quantificar a estabilidade do reator, na remoção e degradação do surfactante. A operação do reator foi dividida em cinco etapas: inoculação (535±121 mg/L de DQO), adaptação da biomassa (600±70 mg/L de DQO), Fase I (4,7 mg/L de AE e 623±65 mg/L de DQO), Fase II (22,5 mg/L de AE e 735±87 mg/L de DQO), Fase III (51,4 mg/L de AE e 697±68 mg/L de DQO), Fase IV (107,4 mg/L de AE e 845±87 mg/L de DQO) e Fase V (97,9 mg/L de AE e 882±126 mg/L de DQO). Aplicação das técnicas de PCR/DGGE e pirosequenciamento da região do rRNA 16S foi realizada para constatar a diversidade microbiana nas fases operacionais IV e V. A eficiência média de remoção de matéria orgânica e AE foi de 88% e 99%, respectivamente, durante a operação do reator. A similaridade das populações dos Domínios Archaea e Bacteria foi de 74% e 59%, respectivamente, para as amostras da Fase IV (com sacarose) e Fase V (sem sacarose). A sacarose não alterou o comportamento físico-químico do reator de leito fluidificado, mas este co-substrato influenciou, tanto, na produção de ácidos orgânicos voláteis, quanto, na diversidade dos microrganismos envolvidos na degradação do AE. Por meio da análise de pirosequenciamento das amostras das Fases IV e V do material suporte e separador de fases do reator foram identificados 83 gêneros dos quais 18 foram relacionados com a degradação de surfactante não iônico, bem como, seus subprodutos. Obteve-se maior abundância relativa para os seguintes gêneros: Sporomusa, Geobacter, Desulfobulbus, Synergistes, Sedimentibacter, Holophaga, Serpens e Azonexus. Observou-se elevada diversidade filogenética e similaridade com sequências de bactérias relacionadas com a degradação de surfactante não iônico AE. / The aim of this study was to evaluate the removal of nonionic alcohol ethoxylate non-branched (AE) in anaerobic fluidized bed reactor filled with sand as support material, on a bench scale (1.2 L) with 18 hours of TDH, recirculation and continuous flow. The reactor was inoculated with sludge from a UASB reactor used in the treatment of swine manure and fed with synthetic substrate plus nonionic GENAPOL® C-100 (Sigma-Aldrich®) as a source of AE. Monitoring analysis of the nonionic surfactant AE concentration and organic matter, as well as the physicochemical parameters were performed to observe and quantify the reactor stability, in the removal and degradation of the surfactant. The reactor operation was divided into five phases: inoculation (535±121 mg/L of COD), biomass adaptation (600±70 mg/L of COD), Phase I (4,7 mg/L of AE and 623±65 mg/L of COD), Phase II (22,5 mg/L of AE and 735±87 mg/L of COD), Phase III (51,4 mg/L of AE and 697±68 mg/L of COD), Phase IV (107,4 mg/L of AE and 845±87 mg/L of COD) and Phase V (97,9 mg/L of AE and 882±126 mg/L of COD). Application of the techniques PCR/DGGE and pyrosequencing of the 16S rRNA region were performed to observe the microbial diversity in the operational phases IV and V. The average removal efficiency of organic matter and AE was 88% and 99%, respectively, during the reactor operation. The populations similarity of the Archaea and Bacteria Domains were 74% and 59%, respectively, for samples from Phase IV (with sucrose) and Phase V (without sucrose). Sucrose did not alter the physical-chemical behavior of the fluidized bed reactor, but this co-substrate influenced both in the volatile fatty acids production, as in the diversity of microorganisms involved in the AE degradation. Through the pyrosequencing analysis of samples from Phases IV and V of the reactor (support material and phase separator), 83 genera were identified of which 18 were related to the nonionic surfactant degradation, as well as its byproducts. Highest relative abundance values were obtained for the following genera: Sporomusa, Geobacter, Desulfobulbus, Synergistes, Sedimentibacter, Holophaga, Serpens and Azonexus. A high phylogenetic diversity and similarity to sequences of bacteria related to the degradation of nonionic AE surfactant were observed.

O objetivo deste estudo foi avaliar a remoção do surfactante não iônico álcool etoxilado de cadeia não ramificada (AE) em reator anaeróbio de leito fluidificado preenchido com areia como material suporte, em escala de bancada (1,2 L) com TDH de 18 horas, recirculação e fluxo contínuo. O reator foi inoculado com lodo proveniente de reator UASB utilizado no tratamento de dejetos de suinocultura e alimentado com substrato sintético acrescido de surfactante não iônico GENAPOL® C-100 (Sigma-Aldrich®) como fonte de (AE). Análises de monitoramento da concentração do surfactante não iônico AE e matéria orgânica, bem como, dos parâmetros físico-químicos foram realizadas para observar e quantificar a estabilidade do reator, na remoção e degradação do surfactante. A operação do reator foi dividida em cinco etapas: inoculação (535±121 mg/L de DQO), adaptação da biomassa (600±70 mg/L de DQO), Fase I (4,7 mg/L de AE e 623±65 mg/L de DQO), Fase II (22,5 mg/L de AE e 735±87 mg/L de DQO), Fase III (51,4 mg/L de AE e 697±68 mg/L de DQO), Fase IV (107,4 mg/L de AE e 845±87 mg/L de DQO) e Fase V (97,9 mg/L de AE e 882±126 mg/L de DQO). Aplicação das técnicas de PCR/DGGE e pirosequenciamento da região do rRNA 16S foi realizada para constatar a diversidade microbiana nas fases operacionais IV e V. A eficiência média de remoção de matéria orgânica e AE foi de 88% e 99%, respectivamente, durante a operação do reator. A similaridade das populações dos Domínios Archaea e Bacteria foi de 74% e 59%, respectivamente, para as amostras da Fase IV (com sacarose) e Fase V (sem sacarose). A sacarose não alterou o comportamento físico-químico do reator de leito fluidificado, mas este co-substrato influenciou, tanto, na produção de ácidos orgânicos voláteis, quanto, na diversidade dos microrganismos envolvidos na degradação do AE. Por meio da análise de pirosequenciamento das amostras das Fases IV e V do material suporte e separador de fases do reator foram identificados 83 gêneros dos quais 18 foram relacionados com a degradação de surfactante não iônico, bem como, seus subprodutos. Obteve-se maior abundância relativa para os seguintes gêneros: Sporomusa, Geobacter, Desulfobulbus, Synergistes, Sedimentibacter, Holophaga, Serpens e Azonexus. Observou-se elevada diversidade filogenética e similaridade com sequências de bactérias relacionadas com a degradação de surfactante não iônico AE. / The aim of this study was to evaluate the removal of nonionic alcohol ethoxylate non-branched (AE) in anaerobic fluidized bed reactor filled with sand as support material, on a bench scale (1.2 L) with 18 hours of TDH, recirculation and continuous flow. The reactor was inoculated with sludge from a UASB reactor used in the treatment of swine manure and fed with synthetic substrate plus nonionic GENAPOL® C-100 (Sigma-Aldrich®) as a source of AE. Monitoring analysis of the nonionic surfactant AE concentration and organic matter, as well as the physicochemical parameters were performed to observe and quantify the reactor stability, in the removal and degradation of the surfactant. The reactor operation was divided into five phases: inoculation (535±121 mg/L of COD), biomass adaptation (600±70 mg/L of COD), Phase I (4,7 mg/L of AE and 623±65 mg/L of COD), Phase II (22,5 mg/L of AE and 735±87 mg/L of COD), Phase III (51,4 mg/L of AE and 697±68 mg/L of COD), Phase IV (107,4 mg/L of AE and 845±87 mg/L of COD) and Phase V (97,9 mg/L of AE and 882±126 mg/L of COD). Application of the techniques PCR/DGGE and pyrosequencing of the 16S rRNA region were performed to observe the microbial diversity in the operational phases IV and V. The average removal efficiency of organic matter and AE was 88% and 99%, respectively, during the reactor operation. The populations similarity of the Archaea and Bacteria Domains were 74% and 59%, respectively, for samples from Phase IV (with sucrose) and Phase V (without sucrose). Sucrose did not alter the physical-chemical behavior of the fluidized bed reactor, but this co-substrate influenced both in the volatile fatty acids production, as in the diversity of microorganisms involved in the AE degradation. Through the pyrosequencing analysis of samples from Phases IV and V of the reactor (support material and phase separator), 83 genera were identified of which 18 were related to the nonionic surfactant degradation, as well as its byproducts. Highest relative abundance values were obtained for the following genera: Sporomusa, Geobacter, Desulfobulbus, Synergistes, Sedimentibacter, Holophaga, Serpens and Azonexus. A high phylogenetic diversity and similarity to sequences of bacteria related to the degradation of nonionic AE surfactant were observed.

Thiomicrospira crunogena, a deep-sea hydrothermal vent chemolithoautotroph, uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately autotrophic Gammaproteobacterium would use a ‘wishbone’ version of the citric acid cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the fully oxidative version to minimize carbon loss as carbon dioxide. However, upon examination of its complete genome sequence, it became apparent that this organism did not fulfill this expectation. Instead of a wishbone pathway, T. crunogena appears to run a fully oxidative CAC. The cycle is ‘locked’ in the oxidative direction by replacement of the reversible enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both oxidoreductases was confirmed via assays on T. crunogena cell extracts. To determine whether this peculiar CAC was novel, complete genome sequences of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated Microbial Genome database. Genes catalyzing steps in the CAC were collected from each organism and vetted for paralogs that had adopted an alternative, ‘non-CAC’ function through genome context and cluster analysis. Alignments were made with the remaining sequences and were verified by comparing them to curated alignments at Pfam database and examination of active site residues. Phylogenetic trees were constructed from these alignments, and instances of horizontal gene transfer were determined by comparison to a 16S tree. These analyses verified that the CAC in T. crunogena is indeed unique, as it does not resemble any of the canonical cycles of the six classes of proteobacteria. Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes encoded by genes that are likely to have been acquired via horizontal gene transfer. The gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with those present in the Cyanobacteria, while those encoding oxoglutarate: acceptor oxidoreductase cluster among the Firmicutes, and malate: quinone oxidoreductase clusters with the Epsilonproteobacteria.

Bacteria swim towards improving conditions by controlling flagellar activity via signals (CheY) sent from chemosensory protein clusters, which respond to changing stimuli. The best studied chemotactic bacterium, E. coli, has one transmembrane chemosensory protein cluster controlling flagellar behaviour. R. sphaeroides has two clusters, one transmembrane and one cytoplasmic. The roles of the two clusters in regulating swimming and chemosensory behaviour are explored here. Newly-developed software was used to measure the effect of deleting or mutating each chemotaxis protein on unstimulated swimming and on the chemosensory response to dynamic change. New behaviours were identified by using much larger sample sizes than previous studies. R. sphaeroides chemotaxis mutants were classified as (i) stoppy unresponsive; (ii) smooth unresponsive or (iii) stoppy inhibited compared to wildtype swimming and chemosensory behaviour. The data showed that the ability to stop during free-swimming is not necessarily connected to the ability to respond to a chemotaxis challenge. The data suggested a new model of connectivity between the two chemosensory pathways. CheY₃ and CheY₄ are phosphorylated by the transmembrane polar cluster in response to external chemoeffector concentrations. CheY₆-P produced by the cytoplasmic cluster is a requirement for chemotaxis, whether or not the polar cluster is able to produce CheY₆-P. CheY₆-P stops the motor, whereas CheY_3,4-P allow smooth swimming. When chemoeffector levels fall, the signals through CheY_3,4 fall, allowing CheY₆-P to bind and stop the motor. As the polar cluster adapts to the fall by the action of the adaptation proteins CheB₁ and CheR₂, the concentration of CheY_3,4-P increases again, to compete with CheY₆-P and allow periods of smooth swimming. Under aerobic conditions, the cytoplasmic cluster controls the basal stopping frequency and does not appear to respond to external chemoeffector changes. The role of the adaptation proteins in resetting the signalling state in R. sphaeroides is unclear, particularly the roles of the proteins associated with the cytoplasmic cluster, CheB₂ and CheR₃. Tandem mass spectrometry was used to identify glutamate and glutamine (EQ) sites on the cytoplasmic R. sphaeroides chemoreceptor TlpT that are deamidated and methylated by the R. sphaeroides adaptation homologues. In E. coli, adaptation sites are usually EQ/EQ pairs. However the sites reported in TlpT vary at the first residue in the pair. Mutation of the putative EQ adaptation sites caused changes in adaptation, suggesting that CheY₆-P levels are controlled and reset by CheB₂ and CheR₃ controlling the adaptation state of TlpT.

Over the past century, dust emissions have increased in frequency and intensity due to anthropogenic influences and extended droughts. Dust transports microbes, nutrients, heavy metals and other materials that may then change the biogeochemistry of the receiving environments. The purpose of this study was to find whether unique bacterial communities may provide distinct fingerprints of dust sources in the Western USA. We collaborated with the National Wind Erosion Research Network (NWERN) to identify bacterial core communities (core) of dust from ten NWERN sites, and compared communities to location, soil, and regional characteristics. In order of importance, precipitation levels (F = 43, P = 0.0001, Df = 2, r2 = 0.25), location (F = 16, P = 0.0001, Df = 5, r2 = 0.23), soil texture (F = 14, P = 0.0001, Df = 3, r2 =0.12), seasonality (F = 11, P = 0.0001, Df = 2, r2 = 0.064), and elevation (F = 5.7, P = 0.0002, r2 = 0.033) determined bacterial community composition. Bacterial core communities were defined as taxa present in at least 50% of samples at each site and offered predictable patterns of dust communities in terms of abundant (> 1% relative abundance) and rare (< 1% relative abundance) signatures. We found distinct bacterial core communities that reflected dust source systems, for example, sites contaminated with heavy metals contained Romboutsia, Turicibacter, Clostridium sensu stricto 1, Geodermatophilus, and Microvirga. Sites with association to plants and biocrusts contained Methylobacterium-Methylorubrum, Bradyrhizobium, Paenibacillus thermoaerophilus, Cohnella, and bacterial families Solirubrobacteraceae, Sphingobacteraceae, and Myxococcaceae. The presence of Sphingomonas, Stenotrophomonas, Rhodococcus, and Phenylobacterium were found in hydrocarbon contaminated soils. High stress (UV radiation and desiccation) sites contained Deinococcus, Blastococcus, and Modestobacter. We found that seasonal changes affected microbial community composition in five NWERN sites (CPER, HAFB, Jornada, Red Hills, and Twin Valley) (p < 0.05), while no seasonal effects on bacterial distribution were observed at Moab. Our results identify that the use of core microbiomes may offer a fingerprinting method to identify dust source regions.

Bacterial genomes provide records of the molecular processes associated with emergence and evolution of different bacterial lifestyles. This thesis is based on whole-genome comparisons within the genus Bartonella, an excellent model system for studies of host- and vector-specificity and infection outcome in animal-associated bacteria. The louse-borne human specialist and trench fever agent Bartonella quintana was contrasted to the flea-borne generalist relatives Bartonella henselae and Bartonella grahamii, which cause asymptomatic infection in cat and mouse respectively. While B. henselae is commonly isolated from humans, and causes cat scratch disease, there is only one reported case of B. grahamii human infection. The gene complements of the three species are nested like Russian dolls with the smaller genome (B. quintana) being entirely contained in the medium sized (B. henselae), which in turned is contained in the largest (B. grahamii). Size differences reflect differences in the horizontally and vertically acquired gene content, and in the number of genus- and species- specific genes, owing to differential impact of bacteriophages and plasmids, and to different degrees of genome decay. These processes can be attributed to the three distinct lifestyles. Comparisons with other alpha-proteobacteria suggest that the Bartonella genus as a whole evolved from plant-associated species, and that horizontal transfer, in particular of genes involved in interaction with the host, played a key role in the transition to animal intracellular lifestyle. The long-term genome decay associated with this lifestyle is most advanced in the host-restricted B. quintana. The broad host-range species B. grahamii has the largest genome and the largest proportion of auxiliary DNA of the three, probably because it has access to a larger gene pool. In encodes all the known pathogenicity determinants found in the genomes of B. henselae and B. quintana, suggesting that these genes primarily evolved to facilitate colonization in the reservoir host.

Gene Transfer Agents (GTAs) are phage-like pthesiss that are produced by many alpha proteobacteria in late stationary growth phase and are capable of transferring chromosomal genes (termed "constitutive transduction"). Examination of alpha proteobacterial genomic sequences indicated widespread occurrence of GTA-like elements. The goal of this study was to investigate gene transfer potential of GTAs of marine alpha proteobacteria in culture as well as in natural marine environments. Another goal was to determine the potential of bacterial symbionts from zooxanthellae and coral to genetically transfer beneficial properties between symbionts. Ruegeria mobilis (ID 45A6) was isolated from cultures of the coral endosymbiotic dinoflagellate, Symbiodinium spp. A goal of the research was to determine if GTAs from this isolate have the capability of transferring genes to environmental recipients and have an impact on settlement of coral larvae. Little is known about coral settlement cues, yet there may be contributions from the extensive symbiotic relationship of coral reef-associated bacteria. Several gene transfer experiments in different environments were performed using transformed isolates of Ruegeria mobilis containing a transposon marker gene. Experiments were also performed using GTAs from the Ruegeria mobilis isolate to observe any impact GTAs have on coral larval settlement, using larvae from the brooding coral, Porites astreoides, and from the reef building coral, Montastraea faveolata. Gene transfer frequencies from statistically significant gene transfer experiments resulted in an average of 2.92 × 10-1 (transfer recipients to total viable population). Coral settlement experiments resulted in a statistically significant increase in larval settlement with the addition of GTAs for 80% of the executed experiments. The entire study has demonstrated that GTA-mediated gene exchange is much higher than any other mode of horizontal gene transfer and it has been established that these genes can be exchanged between bacterial taxa. GTAs can also have an impact on coral larval settlement mechanisms that are not yet completely understood. GTA-mediated beneficial gene exchange may be an important driver in adaptation to an evolving planet.

Made available in DSpace on 2015-04-17T14:55:19Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 1356123 bytes, checksum: 7115ed47a5956fbd95bdffd700b48424 (MD5) Previous issue date: 2012-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Trichogramma species are identified by the morphology of the male genitalia, making the identification difficult due to the small size of the individual (0.25 mm) and/or the presence of cryptic species. Therefore molecular biology constitutes a good alternative for the identification of Trichogramma species and is also used in the identification of Proteobacteria of the genus Wolbachia, known to induce parthenogenesis in Trichogramma species. The purpose of this thesis was to use molecular methods to identify Trichogramma species, verify the diversity of Trichogramma pretiosum populations in 11 cities in Brazil and its association with bacteria of the genus Wolbachia. The samples of Trichogramma pretiosum species and Trichogramma populations were submitted to extraction of genomic DNA for PCR using the forward primer ITS-2- 5 TGTGAACTGCAGGACACATG-3 and the reverse primer IT2-5 GTCTTGCCTGCTCTGCTCTGAG.-3`. The DNA products obtained from Trichogramma species and populations of T. pretiosum were purified and sequenced. To detect the presence of bacteria of the Wolbachia genus in T. pretiosum populations, the amplifications were done using wsp specific primers, with a final reaction volume of 25 μl. The size of PCR products was determined using a molecular weight marker 100 bp using a negative control without DNA, the remaining components and a positive control containing Wolbachia. It can be verified that it was possible to extract DNA from all the Trichogramma species with quantities and qualities sufficient for electrophoretic profiles with genomic DNA concentrations ranging from 15.3 to 50ng/μl. Using PCR, the fragments of the ITS2 region of ribosomal DNA were amplified and one can identify the species of Trichogramma with the sequencing by using the similarity search of the BLAST program in the GenBank database (NCBI - National Center for Biotechnology Information) and dendrogram analysis performed according to the genetic distance matrix, from which three groups were formed, the first by T. exigum, the second by T. pretiosum and the third by T. galloi. With the results of the query by similarity using the BLAST program in the GenBank database a maximum identification average of 92,2% was obtained regarding the species of Trichogramma pretiosum, confirming the correct sequencing. The size of the sequences ranged from 355 to 503 bp. The average G + C content (guanine + cytosine) was 53,2%. After aligning the 11 sequences of T. pretiosum, 391 sites were found conserved, reflecting 73% of the total of 536 sites found, confirming the similarity between samples, as well as confidence in the sequences obtained. The average found for this distance matrix was 0.30. According to the dendrogram obtained from the genetic distance matrix using the neighbor-joining method one can verify the presence of four groups, the first formed by the TPAES, TPPARS, TPRMT TPCVMT populations and the second by the TPSPMT population, the third group by the TPPPE, TPMVCE, TPPPB, TPPPMT, TPJSP populations, and the fourth by the TPPLMT population, being the most genetically distant population. The geographical distance did not affect the genetic similarity or dissimilarity between parasitoids having no significant difference between geographic distance and genetic similarity of the T. pretiosum samples in the locations collected. Of the T. pretiosum populations analyzed, the presence of viii endobacterias occurred in the population from the state of Espírito Santo, the first local record of the presence of this α proteobacteria for the species. / As espécies de Trichogramma são identificadas por meio da morfologia da genitália do macho, sendo dificultada pelo tamanho reduzido do indivíduo (0,25 mm) e/ou a presença de espécies crípticas. Por esta razão, a biologia molecular se constituí em uma boa alternativa para a identificação de espécies de Trichogramma, sendo também utilizada na identificação de proteobactéria do gênero Wolbachia, conhecida por induzir partenogênese em espécies de Trichogramma. O objetivo desta tese foi utilizar métodos moleculares para identificação de espécies de Trichogramma, verificar a diversidade de populações de Trichogramma pretiosum em 11 municípios no Brasil e sua associação com a bactéria do gênero Wolbachia. As amostras de espécies do gênero Trichogramma e de populações de Trichogramma pretiosum foram submetidas à extração de DNA genômico para a realização de PCR utilizando o primer direto ITS-2- 5 TGTGAACTGCAGGACACATG-3 e o reverso IT2-5 GTCTTGCCTGCTCTGCTCTGAG.-3`. Os produtos de DNA obtidos das espécies de Trichogramma e das populações de T. pretiosum foram purificados e submetidos ao seqüenciamento. Para detectar a presença de bactérias do gênero Wolbachia em populações de T. pretiosum, as amplificações foram realizadas com primers específicos wsp, com um volume final da reação de 25 μl. O tamanho dos produtos de PCR foi determinado utilizando um marcador de peso molecular de 100 pb, sendo utilizado um controle negativo sem DNA e o restante dos componentes e um controle positivo com a presença de Wolbachia. Pode-se verificar que foi possível extrair DNA de todas as espécies de Trichogramma estudadas com quantidades e qualidades suficientes para obter perfis eletroforéticos, com concentrações do DNA genômico variando entre 15,3 e 50 ng/μl. Por meio da técnica de PCR, os fragmentos da região ITS2 do DNA ribossomal foram amplificados, sendo possível identificar as espécies de Trichogramma pelo sequenciamento, sendo realizada a busca por similaridade utilizando o programa BLAST, no banco de dados do GenBank (NCBI National Center for Biotechnology Information) e feita a análise de dendrograma de acordo com a matriz de distância genética, onde foi obtido três grupos, o primeiro formado pela espécie T. exigum, o segundo pela espécie T. pretiosum e o terceiro pela espécie T. galloi. Com o resultado da busca por similaridade utilizando o programa BLAST, no banco de dados do GenBank, obteve-se uma média de 92,2% de máxima identificação referente à espécie de Trichogramma pretiosum, confirmando o correto sequenciamento. O tamanho das sequências variaram de 355 a 503 pb. A média do conteúdo G + C (Guanina + Citosina) foi de 53,2%. Após o alinhamento das 11 sequências de T. pretiosum, foram encontrados 391 sítios conservados, que refletem 73 % do total de 536 sítios encontrados, confirmando a semelhança entre as amostras, bem como a confiança nas sequências obtidas. A média encontrada para essa matriz de distância foi de 0,30. De acordo com o dendrograma obtido a partir da matriz de distância genética pelo método do vizinho próximo (Neighbor-Joining), pode-se verificar a presença de quatro grupos: o primeiro, formado pelas populações TPAES, TPPARS, TPRMT e TPCVMT; o segundo, pela população de TPSPMT; o terceiro grupo, pelas populações de TPPPE, TPMVCE, TPPPB, TPPPMT, TPJSP e o quarto pela população de TPPLMT, sendo a população mais distante geneticamente. A distância geográfica não afetou a similaridade ouvi dissimilaridade genética entre os parasitóides, não existindo, diferenças significativas entre distância geográfica e a similaridade genética das amostras de T. pretiosum nas localidades coletadas. Das populações de T.pretiosum analisadas, a presença da endobactérias ocorreu na população proveniente do Estado do Espírito Santo, sendo o primeiro registro local da presença dessa α proteobactéria para a espécie.

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Neves.pdf: 1385774 bytes, checksum: ffa92314f32d6b5261145ca973e972c7 (MD5) Previous issue date: 2013-03-11 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The microorganisms of the community aquatic system of the Solimões and Negro rivers, as well as the diversity of these ecosystems have not been thoroughly investigated so far. The aim of this study was to characterize the bacterial microbiota of the Negro River in relation to seasonality, both in its quantitative aspect (forming units count colonies - CFUs) and qualitative (taxonomic identification via DNA sequencing RNA encoding small ribosomal subunit SSU -rRNA). Negro River water samples were collected at five different points on 4 and 5 depths dates from different times of the year, and all samples CFUs were counted. Total DNA was extracted from the biomass contained in the water of the Negro in full periods (N2) and receding (RN3) and analyzed spectrophotometrically and by agarose gel electrophoresis 0.8%. They were amplified variable regions V3 and V4 SSU-rRNA PCR (Reaction Polymerase Chain DNA) and the amplicons were sequenced by pyrosequencing using equipment 454 (Roche®). Files (reading DNA) of RN2 and Rio Negro RN3 libraries were submitted to the Mothur program suite for quality control, alignment of sequences, calculation of genetic distances and definition of Operational Taxonomic Units - OTUs, plus an estimate of the richness and diversity of species. The CFU count was possible to compare the number of culturable bacteria in the different collection points and due to seasonality. By molecular analysis we found that the phylum Proteobacteria proved dominant in these two collections of the river (full and low tide), the dominant genera of this phylum in full were Limnohabitans, methylomonas, and other expressive filo was Acidobacteria the GP3 race. In the ebb period abundant genera were Polynucleobacter, Acinetobacter and Curvibacter within the phylum Proteobacteria. Rarefaction curves demonstrated that RN3 library is more diverse in number of species than the RN2 library and confirmed by Shannon and InvSimpson index. It can be seen from the ACE 1 indexes and Cha RN3 the library is richer in species compared to RN2. / A comunidade de microrganismos do sistema aquático dos rios Solimões e Negro, bem como a diversidade destes ecossistemas não foram exaustivamente estudadas até o momento. O objetivo deste trabalho foi caracterizar a microbiota bacteriana do rio Negro em relação a sazonalidade, tanto em seu aspecto quantitativo (contagem de Unidades Formadoras de Colônias - UFCs) e no qualitativo (identificação taxonômica via sequenciamento do DNA codificador do RNA de pequena subunidade ribossomal SSU-rRNA). Amostras de água do rio Negro foram coletadas em cinco diferentes pontos, em 4 profundidades e em 5 datas de diferentes épocas do ano, sendo que de todas amostras as UFCs foram contadas. O DNA total foi extraído da biomassa contida na água do rio Negro em períodos de cheia (RN2) e vazante (RN3) e analisado espectrofotometricamente e por eletroforese em gel de agarose 0,8%. Foram amplificadas as regiões variáveis V3 e V4 do SSU-rRNA por PCR (Reação em Cadeia da DNA Polimerase) e os amplicons foram sequenciados por pirosequenciamento utilizando o equipamento 454 (Roche®). Os arquivos brutos das bibliotecas RN2 e RN3 do Rio Negro foram submetidas à suíte do programa Mothur para o controle de qualidade, alinhamento das sequências, cálculo das distâncias genéticas e definição das Unidades Taxonômicas Operacionais – OTUs, além de, estimativa da riqueza e diversidade das espécies. Pela contagem de UFCs foi possível comparar o número de bactérias cultiváveis nos diferentes pontos de coleta e em função da sazonalidade. Pela análise molecular verificou-se que o filo Proteobacteria mostrou-se dominante nestas duas coletas do rio (cheia e vazante), os gêneros dominantes deste filo na cheia foram Limnohabitans, Methylomonas, e outro filo expressivo foi Acidobacteria do gênero Gp3. No período de vazante os gêneros abundantes foram Polynucleobacter, Acinetobacter e Curvibacter, dentro do filo Proteobacteria. As curvas de rarefação demonstraram que a biblioteca RN3 é mais diversa em número de espécies do que a biblioteca RN2 e confirmado pelo índice de Shannon e InvSimpson. Pode-se observar a partir dos índices de ACE e Cha 1 que a biblioteca RN3 é mais rica em espécies comparada com a RN2.

A diversidade bacteriana pode ser estudada, combinando técnicas convencionais e técnicas que empreguem tecnologias modernas para sua melhor compreensão. O objetivo do trabalho foi analisar a diversidade de bactérias cultiváveis e não cultiváveis em amostras de água do mar coletadas no Canal de São Sebastião no período de agosto/2005 a março/2007. As bactérias marinhas foram quantificadas em Agar marinho e identificadas por seqüenciamento do gene 16S rDNA. A concentração dos grupos a-, b-, g- e s-proteobacteria foi verificada através da técnica de FISH. A comunidade total foi analisada através da construção de três bibliotecas mensais (novembro/2006, fevereiro/2006, fevereiro/2007). O seqüenciamento identificou 87% das bactérias marinhas como Vibrio sp. A técnica de FISH detectou maior concentração de b-proteobacteria (10,2%), em relação ao número de células totais (DAPI) que variou de 7,0x106 a 2,3x107 céls/mL. As bibliotecas de clones foram compostas pelos filos Proteobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Fusobacteria, Verrucomicrobia e Chloroflexi. / Microbial diversity can be studied by a combination of techniques of both conventional and modern approaches for better understanding. The aim of this study was analyze marine bacteria culturable and nonculturable diversity from seawater samples collected at São Sebastião Channel during August 2005 to March 2007. Marine bacteria were quantified using Marine Agar and identified by 16S rRNA sequencing. Concentration of a-, b-, g- e s-proteobacteria group was verified through three clones library monthly (November 2006, February 2006, February 2007). The sequencing identified 87% of marine bacteria such as Vibrio sp. The FISH technique to detect higher concentration of b-proteobacteria (10.2%), compared to number total cells (DAPI) which range from 7.0 x 106 to 2.3 x 107 cells/mL. Clones library were composed of the phylum Proteobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Fusobacteria, Verrucomicrobia e Chloroflexi.