As cianobactérias são microrganismos fotossintetizantes oxigênicos com ampla plasticidade metabólica e estrutural, que apresentam potencial biotecnológico para exploração na biossorção de metais pesados e biodegradação de poluentes orgânicos. Devido as suas fortes interações com cátions e ao contínuo suprimento de biomassa barata,as cianobactérias podem ser candidatas promissoras à biossorventes para remoção de metais e radionuclídeos. Dessa maneira, numa tentativa de encontrar uma cianobactéria com esse perfil para remover 226Ra de uma mina de urânio desativada da Unidade de Tratamento de Minérios (UTM) pertencente às Indústrias Nucleares do Brasil (INB), Caldas, MG, doze linhagens de cianobactérias foram isoladas desse ambiente. Essas linhagens foram morfologicamente caracterizadas como Aphanothece sp. CENA75, Rhabdoderma sp. CENA114, Synechococcus cf. lividus CENA79, Aphanocapsa cf. holsatica CENA80, Geitlerinema acutissimum CENA85, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81, Leptolyngbya cf. tenerrima CENA76, Leptolyngbya sp. CENA83, Phormidium formosum CENA86, Phormidium violaceum CENA82 e Nostoc sp. CENA87. A análise molecular dos isolados, baseada em seqüências quase completas do gene RNAr 16S (1325 pb), estava de acordo com a análise morfológica, com exceção das linhagens Rhabdoderma sp. CENA114 e Phormidium violaceum CENA82. As seqüências de RNAr 16S dessas duas linhagens mostraram valores baixos de identidades (<92%) com seqüências do GenBank, o que pode representar novas espécies de cianobactérias. Altas percentagens de identidades (>96%) das seqüências do gene de RNAr 16S foram encontradas entre as linhagens restantes e as do GenBank. A árvore filogenética construída usando o método ?Neighbour Joining? mostrou que as linhagens unicelulares das ordens Chroococcales e as filamentosas da Oscillatoriales eram polifiléticas, conforme já relatado. A distribuição e abundância da população de cianobactérias nos efluentes da UTMINB foram investigadas pelo método da contagem de células viáveis (número mais provável, NMP). O NMP mostrou uma população de cianobactérias variando de 4.0 x 100 to ?2.4 x 108 cells?mL-1. Os locais Cava da Mina, com pH médio de 3,88, e o sistema de tratamento da usina, com pH 8,0, mostraram os mais baixos e mais altos valores de NMP, respectivamente. Para identificar os isolados de cianobactérias prejudiciais, um teste imunológico (ELISA) foi realizado para detectar microcistinas, uma hepatotoxina que causa envenenamento em humanos. A produção de microcistinas foi detectada em três isolados, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81 e Leptolyngbya cf tenerrima CENA76. Esse resultado é inédito, pois não há relatos dos gêneros Pseudanabaena e Leptolyngbya como produtores de microcistinas. / Cyanobacteria are oxygenic photosynthetic microorganisms with wide metabolic and structural plasticity, which have biotechnological potential for exploration in metals biosorption and organic pollutants biodegradation. Due to its strong interactions with cations and a reliable supply of cheap biomass, cyanobacteria may be a promising biosorbent candidate for removing metals and radionuclides. In this way, in an attempt to find a cyanobacteria with this profile to remove 226Ra from a deactivated uranium mine effluents of the Ores Treatment Unit (UTM) belonging to the Nuclear Industries of Brazil (INB), Caldas, MG, twelve cyanobacterial strains were isolated from this environment. These strains were characterized morphologically as Aphanothece sp. CENA75, Rhabdoderma sp. CENA114, Synechococcus cf. lividus CENA79, Aphanocapsa cf. holsatica CENA80, Geitlerinema acutissimum CENA85, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81, Leptolyngbya cf. tenerrima CENA76, Leptolyngbya sp. CENA83, Phormidium formosum CENA86, Phormidium violaceum CENA82 and Nostoc sp. CENA87. The molecular analysis of the isolates, based on the sequences of nearly complete 16S rRNA gene (1325 bp), was in agreement with the morphological analysis, with exception of Rhabdoderma sp. CENA114 and Phormidium violaceum CENA82 strains. The 16S rRNA sequences of these two strains showed low identities scores (<92%) with sequences from GenBank, which may represent novel cyanobacterial species. High percentages of identities (>96%) of 16S rRNA gene sequences were found between the remaining strains and of the GenBank. The phylogenetic tree of 16S rRNA sequences constructed using Neighbour-Joining method showed that unicellular strains of the orders Chroococcales and filamentous Oscillatoriales were polyphyletic, as reported earlier. The distribution and abundance of cyanobacterial population in the effluents of UTMINB were investigated by viable cells counting (most probable number, MPN) method. The MPN showed a cyanobacterial population range from 4.0 x 100 to ?2.4 x 108 cells?mL-1. The locations of the Pit Mine with pH 3.88 and the Plant System Treatment with pH 8.0 showed the lowest and highest MPN values, respectively. To identify harmful cyanobacterial isolates, an immunological test (ELISA) was carried out to detect microcystins, a hepatotoxin which cause human poisoning. Microcystins production was found in three isolates, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81 and Leptolyngbya cf. tenerrima CENA76. This is a novel result since there is no report for both genera, Pseudanabaena and Leptolyngbya, as microcystin producers. Based on the obtained results, the Aphanothece CENA75 strain found in all UTM-INB effluents sampled, including in the Pit Mine location, which has an acidic pH (average of 3.88) and high level of uranium (5.68 mg?L-1) and radium, was selected for the 226Ra biosorption assays. The experiments performed in pH 3.5 and 5.0 showed that dried biomass of Aphanothece CENA75 behaves as a weakly acid resin. The ratio (final concentration/initial concentration) of 226Ra adsorption after 135 min in pH 3.5 and 5.0 was 0.86 and 0.82, respectively. These results showed that the dried biomass of Aphanothece CENA75 adsorbed low amount of 226Ra in both studied pH values. However, the increase of the radionuclide retention in pH 5.0 suggests that more adsorption may occur in pH above of this value.

A microbiota intestinal humana desempenha papel essencial no organismo saudável, pois sintetiza vitaminas, influencia no desenvolvimento e maturação do sistema imune da mucosa intestinal, além de exercer importante função protetora, competindo por nutrientes e receptores com bactérias patogênicas. A colonização desta microbiota se inicia na criança recém-nascida e alcança estabilidade em torno do segundo ano de vida, com consequência para a saúde da criança e do adulto. As diferenças na composição da microbiota estão relacionadas a diferentes níveis de contaminação ambiental e de diferentes fatores endógenos. O objetivo do nosso estudo foi analisar a microbiota fecal de crianças com idade entre 2 dias a 1 ano de idade, que vivem em baixas condições socioeconômicas em São Paulo, Brasil. Foram coletadas amostras de fezes de crianças saudáveis, nos seguintes pontos pós-nascimento: 2º e 7º dias, 1 mês, 3 meses, 6 meses e 1 ano de vida. O DNA bacteriano foi extraído diretamente a partir das amostras de fezes e as bibliotecas 16S rRNA foram construídas utilizando 2 iniciadores bactéria-específicos. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em bibliotecas de gene 16S rRNA. Os principais grupos filogenéticos identificados foram Escherichia, Clostridium, Streptococcus e Bacteroides, do 1º ao 30º dia de vida. A partir do 3º mês, Streptococcus e bactérias não cultiváveis, além do gênero Escherichia, ganharam relevância na microbiota. Estes dados, em conjunto com as informações nutricionais, intercorrências clínicas e ambientais, sugerem a influência da contaminação ambiental e interpessoal no aumento da complexidade na composição da microbiota fecal. Essa abordagem molecular permitiu a análise da microbiota fecal do grupo selecionado, encontrando perfil bacteriano diferente do que é descrito nos países desenvolvidos. / The human intestinal microbiota plays essential role in healthy body since it synthesizes vitamins, influences on the development and maturation of the immune system of the intestinal mucosa. Furthermore, it also plays an important protective function competing for nutrients and receptors with pathogenic bacteria. The colonization of this microbiota starts in the newborn child and achieves stability around the second year of life, with consequence for the health of children and adults. The differences in the microbiota composition are related to different levels of environmental contamination and different endogenous factors. The aim of our study was to analyze the fecal microbiota of children ranging from 2º days to 1º year old living in low socioeconomic status in São Paulo, Brazil. We collected fecal samples of healthy children at the following points after birth: 2º e 7º days, 1 month, 3 months, 6 months and one year of life. Bacterial DNA was extracted directly from stool samples, and the 16S rRNA libraries were made using 2 bacterium-specific primers. The clones were randomly selected, and partially sequenced and analyzed based on 16S rRNA libraries. The main phylogenetic groups identified were Escherichia, Clostridium, Streptococcus, Bacteroides ranging from the 1º to 30º days of life. From the third month Streptococcus and uncultured bacteria, and, besides, Escherichia gender gained relevance in the microbiota. These data together with nutritional information, environmental and clinical intercurrents suggest the influence of interpersonal and environmental contamination in the increase of complexity in fecal microbiota composition. This molecular approach allowed the fecal microbiota analysis. This bacterial profile is different from described in developed countries.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Coal is a fossil fuel extracted from underground by mining processes with the greatest availability in the world. In the state of Rio Grande do Sul, Brazil, the main source is the Candiota Mine with reserves of a billion tonnes likely to be mined in the open. This activity results in built environments with a high degree of disorganization, acid pH, heavy metal toxicity and susceptible to erosion. Exposing this weathering and sulphide minerals facilitates the formation of acid mine drainage, changing and affecting the quality of water, soil and air. Aiming to restore and preserve the desirable environmental conditions it is necessary to recover those mining areas. An alternative is the introduction of nitrogen-fixing bacteria that benefit the growth of leguminous plants. In this perspective, this study aimed to investigate the biodiversity of nitrogen fixing bacteria in soils built after coal mining. Contruidos soil samples were collected and after, in solution, added on substrate with plants-baits siratro and vetch to perform the isolation of nitrogen-fixing bacteria (BNF) through nodes. After the isolates were subjected to the pH (3.0, 4.0, 5.0, 7.0 and 9.0) testing and heavy metals (Cd, Cu, Pb, Ni and Zn in 3 different dosages ). Later isolates efficient at pH test were subjected to molecular characterization and verification of Noda and nifH genes. The data obtained showed a large number of isolates tolerant to different pH values and different metals with different concentrations. Molecular characterization showed 4 phyla α-β-γ-Proteobacteria and Firmicutes, whichever phylum α-proteobacteria, mainly Bradyrhizobium sp. Due greater adaptation in acidic environments, the predominant characteristic of degraded areas after coal mining. / O carvão é o combustível fóssil extraído do subsolo por processos de mineração com a maior disponibilidade no mundo. No estado do Rio Grande do Sul, Brasil, a principal fonte é a Mina de Candiota, com reservas de um bilhão de toneladas passíveis de serem mineradas a céu aberto. Essa atividade resulta em ambientes construídos com elevado grau de desestruturação, pH ácido, toxicidade de metais pesados e susceptível a processos erosivos. A exposição deste às condições atmosféricas e a minerais sulfetados possibilita a formação de drenagem ácida de mina, alterando e comprometendo a qualidade da água, do solo e do ar. Visando reestabelecer e preservar as condições ambientais desejáveis torna-se necessário a recuperação dessas áreas de mineração. Uma alternativa é a introdução de bactérias fixadoras de nitrogênio que beneficiam o crescimento de plantas leguminosas. Nesta perspectiva, este trabalho teve por objetivo investigar a biodiversidade de bactérias fixadoras de nitrogênio em solos construídos após a mineração de carvão. As amostras de solos contruídos foram coletadas e após, em solução, adicionadas em substrato com plantas-iscas de siratro e de ervilhaca para realizar o isolamento das bactérias fixadoras de nitrogênio (FBN) através dos nódulos. Após, os isolados foram submetidos ao teste de pH (3,0; 4,0; 5,0; 7,0 e 9,0) e de metais pesados (Cd, Cu, Pb, Ni e Zn, em 3 dosagens diferentes). Posteriormente os isolados mais eficientes no teste de pH foram submetidos a caracterização molecular e verificação dos genes nodA e nifH. Os dados obtidos apresentaram um amplo número de isolados tolerantes a diversos valores de pH e diferentes metais com variadas concentrações. A caracterização molecular demonstrou 4 filos α-β-γ-Proteobacteria e Firmicutes, prevalecendo o filo α-proteobacteria, principalmente por Bradyrhizobium sp., devido maior adaptação em ambientes ácidos, característica predominante das áreas degradadas após a mineração de carvão.

The giant barrel sponge, Xestospongia muta is an iconic and essential species of the coral reefs in South Florida. The sponge has primary roles providing ecosystem services and creating unique habitats for diverse microbial communities. On April 27, 2012 an outbreak of Sponge Orange Band Disease (SOB) was detected off the coast of South Florida. The disease begins with sponge bleaching, followed by mesohyl or “mesohyl” necrosis and often total mesohyl disintegration. Sampling from two diseased populations at Boynton Beach and Fort Lauderdale, FL took place on May 11th and May 29th, 2012. Each of the nine diseased sponges from Boynton Beach and the five diseased sponges from Fort Lauderdale had three separate mesophyl samples collected to examine the effects of disease progression on the microbial community. These included healthy mesohyl from a diseased sponge (HoD), the boundary layer which captured the advancing line of diseased mesohyl (BL) and diseased mesohyl from a diseased sponge (D). Mesohyl from three sponges with no visible signs of SOB disease were also collected from each sampling location to use for healthy controls (HC). Sequencing of the V4 region of the 16S rRNA gene was performed on all of these samples via the “454” pyrosequencing on a Titanium GS FLX platform. The microbial communities associated with the diseased samples revealed a microbiome shift that followed the progression of Sponge Orange Band Disease (SOB) and was dominated by Bacteroidetes, Protebacteria and Chloroflexi. No singular or group of microbes were solely found within the infected mesohyl of Xestospongia muta from both sampling site populations; therefore there is no unequivocal candidate as a definite microbial causative SOB agent. But there were bacteria associated with disease progression that included Armatimonadetes, Caldithrix, Chlorobi, Fibrobacteres, Fusobacteria, GN02, KSB3, OP1, OP2, OP8, Planctomycetes, SR1, TM6, Tenericutes, Verrucomicrobia, WPS-2 and ZB3. Verrucomicrobia and Plantomycetes increased significantly within the D and the BL populations, which was consistent within all the diseased sponges. This study provides a deep sequencing profile of microbial communities within Xestospongia muta affected with SOB Disease and provides a new insight into the sponge healthy microbiome.

Marine micro-organisms are important components of various biogeochemical cycles, complex food webs and ecological niches. Metagenomic sequencing can provide rapid profile of metabolic activities within the sponge and resident microbes. However, the study of metatranscriptomes from sponges using high throughput sequencing technology has only recently begun. Through this study we isolated, characterized and compared metatranscriptome profiles of Axinella corrugata host and sponge-specific microbial communities using 454 pyrosequencing technology. Four cDNA libraries (two eukaryotic and two prokaryotic) were generated from Axinella corrugata sponge samples collected in December 2009 and May 2010, and were characterized to a) reveal which metabolic genes were actively expressed and b) reveal possible interactions between the sponge and its microbial symbionts. The techniques used for isolation of mRNA and cDNA normalization also helped in optimization of whole-transcriptome amplification. More than 130,000 ESTs were generated for the two seasonal sponge samples and the metagenomic data sets were analyzed using bioinformatics tool, MG-RAST. Several stress-related transcripts were found which can increase our understanding of sensitivity of the sponge to changes in physical parameters in nature. The involvement of the sponge and its microbial consortia is depicted through actively expressed nitrogen and sulfur metabolism genes. Novel genes involved in several functional pathways may be discovered upon further studying hypothetical genes found across all four metagenomic data sets. Metatranscriptomic data sheds light on the functional role of microbes within the sponges and the extent of their involvement in sponge metabolism. 16S rRNA analysis was also carried out using genomic DNA of the same samples, to better elucidate the bacterial taxa abundance in the sponge. This study provides a profile of active mRNA trancripts in Axinella corrugata which include eukaryotic as well as prokaryotic sequences. The data analysis of this research provides new information at the cross-disciplinary interface between molecular biology and computational science.

Doctor of Philosophy / Division of Biology / Brett K. Sandercock / Gastrointestinal microbiota play a vital role in maintaining organismal health, through facilitating nutrient uptake, detoxification and interactions with the immune system. Shorebirds vary widely in life-history characteristics, such as habitat, migration and breeding system, but the dynamics of their gut microbial communities are unknown. In my dissertation, I investigated composition and dynamics of gut microbiota in migratory shorebirds from embryos to 10 day old chicks, and determined environment and host-related factors affecting gut microbial communities of adults. First, I tested whether precocial chicks from three species of arctic-breeding shorebirds acquire gut microbiota before or after hatching using next-generation sequencing. In addition, I documented the dynamics of gut microbial establishment. I showed that gut microbiota were absent in shorebird embryos before hatching, but that stable gut communities established within the first three days after hatching. In addition, gut microbiota of young shorebird chicks were more similar to the environmental microbiome than later in life, suggesting that the environment is a likely source for microbial recruitment. After reaching adulthood, shorebirds migrate long distances, potentially exposing them to a wide range of microorganisms. Host phylogeny and environmental factors have both been identified as drivers of gut microbiota composition in birds in previous studies. The second part of my project aimed to compare the relative importance of host and environmental factors that underlie variation in gut microbiota composition in eight species of migratory shorebirds sampled across the North American Arctic. I found that sampling site was the main driver of variation in gut microbiota of Arctic-breeding shorebirds, and that site-related variation in gut microbiota of shorebirds was a result of differences in core bacterial taxa that occurred in more than half of the analyzed samples. A relatively large influence of local environment on gut microbiota composition of chicks and adults lead to the question: how does site affect pathogen prevalence in shorebirds? Migratory behavior has been hypothesized to have evolved as a response to variation in climatic conditions and food availability, to avoid predation, and to reduce risk of exposure to pathogens. The migratory escape hypothesis predicts avoidance of high disease prevalence areas through migration, and has been proposed as one of the main reasons that many bird species migrate to the Arctic for breeding. To test the migratory escape hypothesis in shorebirds, I screened for prevalence of seven known avian pathogens in shorebirds at different stages of migration. I did not detect the majority of pathogens we tested for, with the exception of Campylobacter jejuni and C. coli. Prevalence of C. jejuni in shorebirds was linked to sampling sites but not shorebird species. My dissertation is the first comprehensive study to broadly characterize the gut microbiota in shorebirds. Overall, local environment emerged as an important factor in shaping microbiota composition in Arctic-breeding shorebirds throughout my dissertation research. The role of local environment in shaping gut microbiota invites future investigations of the interactions among shorebirds and the microorganisms present in their environment, as well as the functions gut microbiota perform within their shorebird hosts.

Translation is a crucial factor in determining the rate of protein biosynthesis; for this reason, bacterial species typically evolve features to improve translation efficiency. Biosynthesis is a finely tuned cellular process aimed at providing the cell with an appropriate amount of proteins and RNAs to fulfill all of its metabolic functions. A key bacterial feature for faster recognition of the start codon on mRNA is the binding between the anti-Shine-Dalgarno (aSD) sequence on prokaryotic ribosomes at the 3’ end of the small subunit (SSU) 16S rRNA and Shine-Dalgarno (SD) sequence, a purine-rich sequence located upstream of the start codon in the mRNA. This binding helps to facilitate positioning of initiation codon at the ribosomal P site. This pairing, as well as factors such as the location of aSD binding relative to the start codon and the sequence of the aSD motif can heavily influence translation efficiency. The objective of this thesis is to understand the SD-aSD interactions and how changes in aSD sequences can affect SD sequences in addition to the underlying impact these changes have on the translational efficiency of prokaryotes. In chapter two, we hypothesized that differences in the prevalence of SD motifs between B. subtilis and E. coli arise as a result of changes in the free 3' end of 16S rRNA which may have led B. subtilis and E. coli to evolve differently. E. coli is expected to be more amenable to the acquisition of SD motifs that do not perfectly correspond with its free 3’ 16S rRNA end than B. subtilis. Further, we proposed that the evolutionary divergence of these upstream sequences may be exacerbated in B. subtilis by the absence of a functional S1 protein. Based on the differences between E. coli and B. subtilis, we were able to identify SD motifs that can only perfectly base pair in one of the two species and are expected to work well in one species, but not the other. Furthermore, we determine the frequency and proportion of these specific SD motifs that are expected to be preferentially present in one of the two species. Our motif detection is in keeping with the expectation that the predicted five categories of SD that are associated with B. subtilis and are expected to be less efficient in E. coli exhibit greater usage in the former than latter. Similarly, the predicted category of SD motifs associated with the E. coli 16S rRNA 3’ end is used more frequently in E. coli.Across prokaryote genomes, translation initiation efficiency varies due to codon usage differences whereas among genes, translation initiation varies because different genes vary in SD strength and location. In chapter 3 we hypothesized that there is differential translation initiation between 16 archaeal and 26 bacterial genomes. Translation initiation was found to be more efficient in Gram-positive than in Gram-negative bacteria and also more efficient in Euryarchaeota than in Crenarchaeota. We assessed the efficiency of translation initiation by measuring: i) the SD sequence’s strength and position and ii) the stability of the secondary structure flanking the start codon, which both affect accessibility of the start codon

Hajj, the annual Islamic pilgrimage to Makkah, Saudi Arabia, is a unique mass gathering event that brings more than 2 million individuals from around the world. Several public health considerations, such as the spread of infectious diseases, must be taken into account with this large temporary influx of people. Gastrointestinal diseases, such as diarrhea, are common at Hajj, yet little is known about the etiology. The human gut microbiome, collection of organisms residing within the intestinal tract, has been under intense study recently, since next generation DNA sequencing technologies allow for extensive surveying of genetic material found in complex biological samples, such as those containing many different organisms. Thus, using 16S rRNA and metagenomic shotgun sequencing, we have characterized the gut microbiome of over 612 pilgrims with and without diarrhea. Several metadata factors, such as hospitalization and different comorbidities, were found to have significant effects on the overall gut microbiome composition. Metagenomic shotgun sequencing efforts revealed the presence of antimicrobial resistance genes originating from disparate regions from around the world. This study provides a snapshot of information concerning the health status of the gut microbiome of Hajj pilgrims and provides more context to the investigation of how to best prepare for mass gathering events.

The intestinal microflora is an extensive ecosystem of microorganisms that consists of symbiotic and pathogenic species. The microflora is responsible for many important functions in the human body. An unquestionable function is that it affects the health state of the host. The higher the biodiversity, the greater the benefit for the host. However, it is necessary to point out that this should not include a high diversity of pathogenic bacterial species. There are many "beneficial" species, especially from the Bifidobacterium and Lactobacillus families. In recent decades, the popularity of supplementing these "beneficial" species with various supplementary diets (e.g. probiotics) has been growing a lot. The presented diploma thesis deals with pilot studies of liquid commercial probiotic preparations from American companies Ascended Health (not available on the Czech market) and their effects on the human microbiome. The study involved 9 volunteers who provided 70 fecal samples before, during, and after use of the studied products. Two methods were used to determine the biodiversity of intestinal bacterial species. Both are based on the identification by bacterial DNA that encodes gene 16S rRNA. The first method uses PCR-DGGE technique and then identification by Sanger sequencing. The second method...

The aim of this work is proposal of new method for bacteria classification based on sequences of marker genes. For this purpose was chosen 10 marker genes. Resulting MultiGene classifier processes data set by dividing it in several groups and choosing gene for each group which can distinguish this group with best results. This work describes implementation of MultiGene classifier and its results in comparison with other bacteria classifiers and with classification based entirely on gene 16S rRNA.