Recent research in microbial ecology has focused on how aquatic bacterial communities are assembled. Only a few of these studies follow a “Gleasonian” approach where the roles of single bacterial populations are in focus. In this thesis, novel molecular tools were used to describe the distribution and evolutionary relationships of microbes in productive aquatic environments. Many new phylogenetic groups of bacteria were identified, likely representing bacterial populations restricted to productive freshwaters. I also addressed the dynamics and functional role of individual bacterial populations in eutrophic lakes and brackish environments with a focus on either biogeochemically significant or potentially pathogenic representatives. Flavobacteria blooms were observed, on occasions characterized by high heterotrophic production. In addition to high temporal dynamics microbial community composition and function differed on the spatial scale, as exemplified by free-living and Cyanobacteria-associated habitats. At the community scale, microbial processes, such as biomass production and substrate uptake could be predicted from the presence and absence of individual bacterial populations. I also studied the niches of potentially pathogenic Vibrio populations in various coastal waters. Using a novel culture-independent method, a V. cholerae population was detected along the entire Swedish coastline. Results from an environmental survey and a laboratory mesocosm experiment reveal that phytoplankton-derived dissolved organic matter enhance the growth of V. cholerae and other Vibrio spp. and hence create a largely overlooked niche for these heterotrophic bacteria. This thesis and future work on the role of individual bacterial populations will facilitate predictions of biogeochemical cycles and the distribution of bacteria in the context of global climate change and local eutrophication.

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the 30S ribosomal subunit, has been traced to the arm/rmt family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosylmethionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA in the 30S ribosomal subunit. Neither 16S rRNA alone nor intact 70S ribosome is an efficient substrate for armA methyltransferase. To more fully characterize this family of enzymes, xiii we have investigated the substrate requirements of ArmA. We determined the Mg2+ dependence of ArmA activity and found that the enzyme could recognize both translationally active and translationally inactive forms of 30S subunits. To identify the site of interaction between ArmA and the 30S subunit, we used hydroxyl radical cleavage of 16S rRNA mediated by ferrous iron chelated to several sites on the ArmA molecule that were mutated to cysteine. This data suggests that significant conformational changes in 30S structure are involved in binding of ArmA. We hypothesized that a precursor intermediate in the biogenesis of the 30S subunit might be the optimal substrate for ArmA enzymes in vivo. To test this, we prepared 30S particles partially depleted of proteins by treatment with increasing concentrations of LiCl and assayed them for ArmA methylation. Even low concentrations of LiCl alter the 30S particles and greatly diminish their susceptibility to methylation. Additionally, a previously identified pre-30S particle isolated from an E. coli culture was assayed for its ability to support methylation by ArmA and found to be inferior to intact 30S particles as a methylation substrate. Thus, testing of immature particles prepared from in vitro and in vivo sources suggest that ArmA works very late in the 30S biogenesis pathway. Initiation factor 3 (IF3), a factor that only binds fully mature 30S particles, does not inhibit the ArmA methylation, while kasugamycin methyltransferase (KsgA) abolishes ArmA activity by sharing the same binding site with ArmA. From aforementioned experiments, we conclude that ArmA is most active toward 30S ribosomal subunits that are at or very near full maturation.

PURPOSE: The purpose of this study is to describe the subgingival bacterial biodiversity in untreated chronic periodontitis patients through the use of next generation 16S rRNA molecular analysis, and to determine similarities or differences between deep and shallow pockets within the same patients. METHODS: The analysis involved paired subgingival plaque samples from 24 subjects diagnosed with Generalized Moderate to Severe Chronic Periodontitis. One sample was selected from a single site having a probing depth >5 mm (i.e. Deep Site), and the other from a site with a probing depth <3mm (i.e. Shallow Site) within each subject. Bacterial DNA amplification of the V4-V6 region of the 16S rRNA was performed. The amplicons were sequenced via 454 Roche Genome Sequencer FLX System. The identified sequences were evaluated, and then compared to calculated false discovery rates. RESULTS: A total of 119 independent microbial genera were identified within the samples analyzed. Seven genera were identified to be statistically significant (p<0.05) in their association to deep or shallow sites following t-test and boot strap randomization: Actinomyces (p=0.004), Methylobacterium (p=0.028), Veillonella (p=0.028), and Rothia (p=0.038), and Streptococcus (p=0.033) in Shallow sites; while Mycoplasma (p=0.007) and Fusobacterium (p=0.016) were associated with deep sites. However, taking into account the calculated false discovery rates, it is suggested that none of the 119 microbial genera identified in this study were significantly associated with either deep nor shallow sites. CONCLUSION: The microbial genera identified within this study to be associated with deep and shallow sites follows the traditional pattern anticipated from the literature. However, the calculated false discovery rates suggest that these results may have occurred by chance and not due to a true difference.

The aim of presented work is to provide characteristics of the karyotypes of scorpions of the genus Euscorpius. Genus Euscorpius is a typical representative of scorpions in Europe. Its occurrence is wide throughout Europe. Until now, 18 species of this genus have been described. In this work six species were karyologically analyzed and one species was shown to possess only basic diploid number of chromosomes: E. carpathicus - 2n=90, E. concinnus - 2n=88, E. hadzii - 2n=68, E. sicanus - 2n=66, E. tergestinus - 2n=60, E. naupliensis - 2n=60, E. italicus - 2n=36. Description of the karyotypes revealed that all species studied exhibit achiasmatic meiosis; no presence of sex chromosomes was detected. The basic hypothesis of phylogenetic relationships and karyotype evolution of the genus Euscorpius was outlined. High interspecies variability in chromosome total count was found and by analysis of the 16S rRNA gene the taxonomic status of the species was confirmed. Hence, it seems that cytogenetic methods can contribute to the understanding of species diversity and differentiation of possible cryptic species within the genus Euscorpius.

A conversão de áreas de florestas da Amazônia em áreas agrícolas e pastoris desregula processos relacionados ao estoque de carbono, sendo considerada depois da queima de combustíveis fósseis a atividade que mais contribui com a emissão de gases do efeito estufa, dentre os quais se encontra o metano. A produção de metano é intermediada pelas arquéias metanogênicas, que atuam na decomposição anaeróbia da matéria orgânica. Portanto, para compreender as alterações do fluxo desse gás no ecossistema amazônico, é necessário que as comunidades microbianas envolvidas nesse processo sejam estudadas. Dessa forma, o presente estudo teve por objetivo monitorar e caracterizar comunidades de arquéias metanogênicas, por análises de enriquecimento destas comunidades, em amostras de solo provenientes de floresta primária, floresta secundária e pastagem da região amazônica. As amostras de solo foram colocadas em meio enriquecido com a adição de acetato, metanol ou H2:CO2, separadamente, para estimular os metabolismos aceticlástico, metilotrófico e hidrogenotrófico. O monitoramento desses cultivos foi realizado por análises de emissão de metano por cromatografia gasosa, quantificação do gene mcrA pela técnica de PCR quantitativo (qPCR) e caracterização da comunidade metanogênica por meio de microscopia e sequenciamento da região V4-V5 do gene 16S rRNA. Analisando a emissão de metano entre os três tipos de fontes de carbono para as três amostras de solo, os enriquecimentos com metanol apresentaram uma produção maior de metano em relação as amostras com o acetato e muito superior aos cultivos com atmosfera de H2:CO2. A maior média de produção de metano ocorreu nos enriquecimentos com metanol, indicando que a via metilotrófica embora considerada alternativa, pode ser importante na produção de metano no solo amazônico. Por meio da técnica de qPCR foi possível quantificar o gene mcrA das amostras de pastagens logo no tempo inicial da incubação, o que não foi possível para as amostras florestais. No tempo final, o número de cópias desse gene foi similar para os três perfis de solo. Foi possível observar pela caracterização fenotípica dos enriquecimentos agregados de células característicos do gênero Methanosarcina, gênero que foi identificado posteriormente pelo sequenciamento do gene 16S rRNA, além de células em formatos de bastonetes e cocos. Os resultados do sequenciamento permitiram identificar 7 grupos distintos de arqueias metanogênicas, afiliados aos filos Euryarchaeota e Bathyarchaeota. Nas amostras iniciais de pastagens foram identificadas sequências que se afiliaram a todos esses grupos, enquanto as amostras florestais apresentaram sequencias afiliadas apenas ao gênero Methanosarcina. A composição final da comunidade das amostras de pastagens foi similar a inicial, porém mais abundante. Os enriquecimentos de amostras de solo de floresta primária e secundária apresentaram uma composição distinta, devido ao enriquecimento de grupos que não foram identificados no início da incubação. Os resultados obtidos mostraram que embora as arqueias metanogênicas estejam em baixa abundância nos solos florestais, podem ser enriquecidos quando submetidos a condições favoráveis, atingindo produção de metano e alcançando composição similar as amostras de pastagens. / Amazonian forest conversion into agricultural and livestock areas disrupts processes related to carbon stock, being considered, after the fossil fuels burning, the activity contributes most to greenhouse gases emission, of which is methane. Methane production is mediated by methanogenic archaea, acting in organic matter anaerobic decomposition. Therefore, to understand the changes in the flow of this gas in the Amazonian ecosystem, it is necessary to study microbial communities involved in this process. This study aims to monitor and characterize methanogenic archaeal communities by population enrichment from soil samples collected in primary, secondary and pasture of the Amazon region. Soil samples were placed into an enriched medium and received separately acetate, methanol, and H2:CO2 to stimulate the three metabolism types: acetoclastic, methylotrophic and hydrogenotrophic. Monitoring was performed by methane emission analysis by gas chromatography, mcrA quantification by the quantitative PCR and the community characterization was performed by microscopy and sequencing of the V4-V5 region of the 16S rRNA gene. Analyzing the methane emission by the three types of carbon sources in the three soil samples, methanol enrichments presented a higher methane yield than the acetate samples and much larger than cultures with H2:CO2. These results indicate that methylotrophic pathway, although considered as an alternative, may be important in methane production in the Amazonian soil. Was possible to quantify the mcrA gene by qPCR from pasture samples at the initial incubation time, which was not possible for forest samples. In incubation final time, copies number of this gene was similar for the three soil profiles. The phenotypic characterization of enrichments revealed aggregated cells, characteristic of the genus Methanosarcina, later identified by 16S rRNA sequencing. The cells in rod-shaped and cocci formats were also observed. Was identify by sequencing 7 different methanogenic archaeas groups affiliated with Euryarchaeota and Bathyarchaeota phylum. In the initial pasture samples, were identified sequences affiliated with all these groups, while forest samples presented sequences affiliated with only a Methanosarcina genus. Pasture samples showed a final community composition similar to initial, however more abundant. Soil samples enrichment from primary and secondary forest presented a distinct composition due to groups enrichment that was not identified at incubation beginning. These results showed that although methanogenic archaeas are in low abundance in forest soils, they can be enriched when submitted to favorable conditions, archive methane production and reaching similar composition pasture samples.

One of the main focus of coral reef ecology has been to shed light on the importance of all microbial members of coral holobiont and how their interactions contribute to the coral’s resilience. However, knowledge is lacking about the composition of microbial communities inhabiting the surface mucus layer of corals including Fungia fungites, a species that lives under stressful conditions close to fish farms in Vietnam. I investigated the prokaryotic communities that are thriving in Fungia fungites surface mucus layer (SML) in the wild and how they were affected upon antibiotics and nitrogen stress using 16S rRNA gene-based techniques. Firstly, I observed a significant alteration in the composition of microbial communities due to antibiotics effect, with exposed communities featuring lower richness and α-diversity in contrast to the controls. Further, mucosal microbial communities were found to be mostly dominated by Proteobacteria (especially of the classes of Alphaproteobacteria and Gammaproteobacteria) and less by Bacteroidetes (Flavobacteriia). Results from this study suggest a developed antibiotic resistance of Alteromonadales and Campylobacterales indicated by their increased abundance upon antibiotics effect. Moving forward, future studies should focus on exploring also the contribution of non-prokaryotic microbial members of Fungia fungites holobiont and how antibiotic resistance can potentially influence coral’s health. The results support that Fungia fungites SML microbial communities are strongly affected by antibiotics exposure and call for future research to focus on the function of these microbial communities and how they can contribute to the coral’s resilience.

Orientador: Marcos Rogério André / Resumo: Micoplasmas hemotrópicos (hemoplasmas) são bactérias Gram-negativas que parasitam a superfície de eritrócitos de uma ampla variedade de mamíferos. Mycoplasma wenyonii e ‘Candidatus Mycoplasma haemobos’ são espécies de hemoplasmas relatadas em bovinos, podendo causar anemia hemolítica e redução na produção de carne e leite e, consequentemente, prejuízos econômicos. O presente estudo teve como objetivo investigar a ocorrência de hemoplasmas em bovinos de corte no Pantanal brasileiro, área endêmica para tripanossomíase bovina na América do Sul. Adicionalmente, objetivou-se caracterizar molecularmente os genótipos de hemoplasmas encontrados. Para tal, amostras de sangue e soro de 400 bovinos de corte foram colhidas em cinco propriedades do município de Corumbá, sub-região da Nhecolândia, estado de Mato Grosso do Sul, centro-oeste brasileiro. As amostras de sangue foram submetidas à extração de DNA e a ensaios de PCR convencional para hemoplasmas baseados no gene 16S rRNA. As sequências obtidas foram submetidas a inferências filogenéticas, análises de distância e de diversidade genotípica. O Ensaio Imunoenzimático Indireto (iELISA) mostrou a presença de anticorpos IgG anti-Trypanosoma vivax em 89,75% dos animais amostrados, confirmando a endemicidade para o referido agente na região sob estudo. Dentre as 400 amostras de sangue de bovinos testadas, 2,25% (9/400) mostraram-se positivas na cPCR para hemoplasmas. A análise filogenética das sequências obtidas confirmou a presença de DN... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hemotrophic mycoplasmas (haemoplasmas) are Gram-negative bacteria that parasitize the surface of erythrocytes of a wide variety of mammals. Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos' are hemoplasma species reported in cattle, which can cause hemolytic anemia and reduction in meat and milk production and, consequently, economic losses. The present study aimed to investigate the occurrence of haemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. In addition, the work aimed to characterize molecularly the genotypes of haemoplasmas found. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in the municipality of Corumbá, sub-region of Nhecolândia, in the state of Mato Grosso do Sul, central-western Brazil. Blood samples were subjected to DNA extraction and cPCR assays for haemoplasmas based on 16S rRNA gene. The sequences obtained were submitted to phylogenetic inferences, distance analysis and genotype diversity. The Indirect Immunoenzymatic Assay (iELISA) showed the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity for this agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive in cPCR for haemoplasmas. Phylogenetic analysis of the sequences obtained confirmed the presence of DNA 'C. M. haemobos' and M. wenyonii in 0.5% (2/400) and 1.75% (7/400) animals, r... (Complete abstract click electronic access below) / Mestre

Submitted by WALLYNSON EDUARDO SILVA ALMEIDA null (wallynson@yahoo.com.br) on 2017-02-24T16:09:51Z No. of bitstreams: 1 Dissertação_Wallynson_Eduardo_Silva_Almeida.pdf: 1951842 bytes, checksum: 7628e3c85a4699b4530e564dcd6cc11a (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-07T12:39:08Z (GMT) No. of bitstreams: 1 almeida_wes_me_jabo.pdf: 1951842 bytes, checksum: 7628e3c85a4699b4530e564dcd6cc11a (MD5) / Made available in DSpace on 2017-03-07T12:39:08Z (GMT). No. of bitstreams: 1 almeida_wes_me_jabo.pdf: 1951842 bytes, checksum: 7628e3c85a4699b4530e564dcd6cc11a (MD5) Previous issue date: 2017-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os microrganismos têm participação ativa nas transformações do fósforo (P) no solo, influenciando sua disponibilidade para as plantas e seu fluxo na natureza. As transformações resultam de decomposição e mineralização de compostos orgânicos, imobilização na microbiomassa e solubilização das formas inorgânicas dos minerais. A base desse estudo consistiu em avaliar 10 isolados bacterianos, visando caracterizá-los quanto: padrão de crescimento em meio Tryptone Yeast (TY) (padrão de crescimento em meio NBRIP (National Botanical Research Institute’s Phosphate); solubilização de fosfato de hidróxido de cálcio (Ca5OH(PO4)3); variação do pH nos diferentes meios; sequenciamento parcialmente do gene 16S rRNA, e influência do crescimento, em casa de vegetação, para as culturas da soja (Glycine max) e milho (Zea mays). Os melhores solubilizadores em 120 h de cultivo foram os isolados LGA- 11-V0522, 33,81 mg/ml, Chromotacterium Violaceum, LGA13-V20F, 32,65 mg/ml, Arthrobacter echigonensis, LGA1-V4-V20J, 32,19 mg/ml, Arthrobacter echigonensis, e LGA05-V0513, 30,96 mg/ml, Bacilus Cereus. Em casa de vegetação, os isolados que apresentaram melhores resultados foram, com maior desenvolvimento de parte aérea, LGA13-V20F, 22,67 cm, Arthrobacter echigonensis, LGA09-V20L, 22,0 cm, Bacillus thuringiensis, LGA07-V0508, 21,25 cm, Bacillus thuringiensis, e LGA12-V05D 19,25, Bacillus thuringiensis. / The microorganisms have an active participation in the transformations of the phosphorus (P) in the soil, influencing its availability to the plants and their flow in nature. The transformations result from decomposition and mineralization of organic compounds, immobilization in the microbiomass and solubilization of inorganic forms of minerals. The base of this study was to evaluate 10 bacterial isolates, aiming to characterize them as: growth pattern in Tryptone Yeast (TY) medium (growth pattern in NBRIP medium; solubilization of calcium hydroxide phosphate Ca5OH (PO4) 3), pH variation in the different media, partial sequencing of the 16S rRNA gene, and growth influence in the greenhouse for soybean (Glycine max) and maize (Zea mays) crops.The best solubilizers In 120 h of culture were the isolates LGA-11-V0522, 33.81 mg / ml, Chromotacterium Violaceum, LGA13-V20F, 32.65 mg / ml, Arthrobacter echigonensis, LGA1-V4-V20J, 32.19 mg / ml , Arthrobacter echigonensis, and LGA05- V0513, 30.96 mg / ml, Bacilus Cereus, and LGA13-V20F, 22.67 cm. In the greenhouse, LGA09-V20L, 22.0 cm, Bacillus thuringiensis, LGA07-V0508, 21.25 cm, Bacillus thuringiensis, and LGA12-V05D 19.25, Bacillus thuringiensis.

<p>Recent research in microbial ecology has focused on how aquatic bacterial communities are assembled. Only a few of these studies follow a “Gleasonian” approach where the roles of single bacterial populations are in focus. In this thesis, novel molecular tools were used to describe the distribution and evolutionary relationships of microbes in productive aquatic environments. Many new phylogenetic groups of bacteria were identified, likely representing bacterial populations restricted to productive freshwaters. I also addressed the dynamics and functional role of individual bacterial populations in eutrophic lakes and brackish environments with a focus on either biogeochemically significant or potentially pathogenic representatives. <i>Flavobacteria</i> blooms were observed, on occasions characterized by high heterotrophic production. In addition to high temporal dynamics microbial community composition and function differed on the spatial scale, as exemplified by free-living and <i>Cyanobacteria</i>-associated habitats. At the community scale, microbial processes, such as biomass production and substrate uptake could be predicted from the presence and absence of individual bacterial populations. I also studied the niches of potentially pathogenic <i>Vibrio </i>populations in various coastal waters. Using a novel culture-independent method, a <i>V. cholerae</i> population was detected along the entire Swedish coastline. Results from an environmental survey and a laboratory mesocosm experiment reveal that phytoplankton-derived dissolved organic matter enhance the growth of <i>V. cholerae</i> and other <i>Vibrio</i> spp. and hence create a largely overlooked niche for these heterotrophic bacteria. This thesis and future work on the role of individual bacterial populations will facilitate predictions of biogeochemical cycles and the distribution of bacteria in the context of global climate change and local eutrophication.</p>

<p>ABSTRACT</p><p>Nontypable Haemophilus influenzae is a common cause of otitis, sinusitis and conjunctivitis. It is the most common bacterial pathogen associated with chronic obstructive pulmonary disease (COPD). Studies have shown that nonpathogenic Haemophilus haemolyticus are often mistaken for Haemophilus influenzae due to an absent hemolytic reaction on blood agar. Distinguishing H. haemolyticus from H. influenzae is important to prevent unnecessary antibiotic use, and to understand the role of H. influenzae in clinical infections. In this study, PCR-primers for amplifying 16S rDNA sequences were used to set up a method for distinguishing H. haemolyticus from H. influenzae. The aim was to use the method for analyzing apparent H. influenzae strains, to investigate if some strains were in fact H. haemolyticus. However, because of problems with unspecific primerannealing,no conclusions could be drawn regarding misclassification of H. haemolyticus.</p><p>Moraxella catarrhalis is the second most common bacterial pathogen associated with COPD. It also causes otitis and sinusitis. An important virulence factor of M. catarrhalis is the outer membrane protein Moraxella catarrhalis IgD-binding protein (MID). One part of the protein; MID764-913 , has been shown to function as an adhesin, and this part has been fragmented to further investigate its adhesive properties. The aim of this second, independent study, was to express some of these proteinfragments by cloning in E. coli. The time spent on this project was too short, and no proteins could be expressed duing this period.</p>