The archaeal flagellum is a rotating prokaryotic motility apparatus used for swimming motility and adhesion; however, it is more closely related to the bacterial type IV pilus system than its bacterial namesake. Methanococcus maripaludis is a highly flagellated, obligately anaerobic methanogen and is used as the archaeal model system during this study. The identified structural genes of the archaeal flagella are transcribed by a single fla operon; however, the interactions between the majority of the Fla proteins has yet to be elucidated. In this work, several techniques were attempted to determine the protein-protein interactions between Fla proteins, including membrane fractionation experiments and in vitro dimerization assays. Evidence from these experiments suggests that two proteins, FlaC and FlaE, have the ability to self-associate. The M. maripaludis flagella system is also used as a model for the study of the N-linked glycosylation pathway in the domain, due to the presence of a tetrasaccharide N-linked to flagellin monomers. Previous work has identified several of the processes involved in the assembly of this glycan, including glycosyltransferases, the oligosaccharide transferase and several of the key components involved in the biosynthesis of the sugar residue precursors. However, many of the enzymes responsible for biochemical modifications to the sugar residues remain to be determined. The operon structure of the genes between mmp1080 and mmp1095 was experimentally confirmed using RT-PCR, and each of the operons contains at least one gene involved in the biosynthesis of the N-linked glycan. In-frame deletions of genes in this region were characterized for effects on the N-linked glycan. Evidence suggests that Mmp1082 and Mmp1083 are acting in conjunction with Mmp1081 in the addition of an acetamidino functional group to the third sugar residue. Mmp1085 was determined to be a methyltransferase responsiblefor the methylation of the terminal sugar residue. Additionally, Mmp1087 and Mmp1094 were identified as potentially having an effect on the glycan. Though this work, the breadth of knowledge in regards to both the archaeal flagella and the N-linked glycosylation process in the domain has been increased. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-01-28 11:50:05.542

Orientador: Jackson Antônio Marcondes de Souza / Coorientador: Daniel Guariz Pinheiro / Coorientador: Luciano Takeshi Kishi / Resumo: A torta de filtro apresenta bagaço residual oriundo do processo de extração do caldo de cana-de-açúcar e quando armazenada por longos períodos, se torna um habitat ideal para o desenvolvimento de comunidades microbianas que atuam na desconstrução da lignocelulose. Nossas análises de dados de sequenciamento de DNA metagenômico sugerem que a torta de filtro armazenada por 40 dias possui uma microbiota com características funcionais e ecológicas exclusivas em relação a outros ambientes com elevada disposição de material lignocelulósico. Assim como em ambientes de compostagem, os filos mais abundantes são Actinobacteria, Proteobacteria, Firmicutes e Bacteroidetes. Dentre os principais genes que estes micro-organismos possuem, estão Glicosiltransferases, Carboidrato Esterases e Glicosil Hidrolases, que atuando em conjunto, são passíveis de desconstruírem a lignocelulose e participarem na liberação de açúcares menores, ácidos orgânicos e outros nutrientes. Neste trabalho, identificamos novas enzimas da família AA10 que oxidam a celulose cristalina, demostrando o potencial deste ambiente em possibilitar a adaptação de micro-organismos que expressam enzimas capazes de desestruturar a celulose altamente condensada, possibilitando a liberação de moléculas de glicose. A comunidade microbiana pode acessar nutrientes como Fósforo e Nitrogênio através da despolimerização da biomassa vegetal ou decomposição da microbiota morta. No ciclo biogeoquímico do nitrogênio, a evaporação de amônia é ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The filter cake presents residual bagasse from the process of extracting the sugarcane juice and when stored for long periods, it becomes an ideal habitat for the development of microbial communities that act in the deconstruction of lignocellulose. Our analyzes of metagenomic DNA sequencing data suggest that the filter cake stored for 40 days has a microbiota with unique functional and ecological characteristics compared to other environments with high lignocellulosic material. Thus in composting environments, the most abundant phyla are Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Glycosyltransferases, Carbohydrate Estersases and Glycoside Hydrolases, which act together, are capable of deconstructing lignocellulose and participate in the release of smaller sugars, organic acids and other nutrients. In this work, we identify new enzymes of the AA10 family that oxidize crystalline cellulose, demonstrating the potential of this environment to enable the adaptation of microorganisms that express enzymes capable of destabilizing highly condensed cellulose, allowing the release of glucose molecules. The microbial community can access nutrients such as Phosphorus and Nitrogen through the depolymerization of the plant biomass or decomposition of the dead microbiota. In the biogeochemical cycle of nitrogen, the evaporation of ammonia is reduced by the assimilation of this substance by the microbial community, and ammonia is produced by ammonification of nitrate and... (Complete abstract click electronic access below) / Doutor

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13793号 / 工博第2897号 / 新制||工||1428(附属図書館) / 26009 / UT51-2008-C709 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 濵地 格 / 学位規則第4条第1項該当

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2018. / Submitted by Fabiana Santos (fabianacamargo@bce.unb.br) on 2018-09-03T20:38:24Z No. of bitstreams: 1 2018_ThiagoRodriguesdeOliveira.pdf: 3205780 bytes, checksum: 3bcc450ab74953bdffd4e6c59d584dc0 (MD5) / Approved for entry into archive by Fabiana Santos (fabianacamargo@bce.unb.br) on 2018-09-11T16:56:29Z (GMT) No. of bitstreams: 1 2018_ThiagoRodriguesdeOliveira.pdf: 3205780 bytes, checksum: 3bcc450ab74953bdffd4e6c59d584dc0 (MD5) / Made available in DSpace on 2018-09-11T16:56:29Z (GMT). No. of bitstreams: 1 2018_ThiagoRodriguesdeOliveira.pdf: 3205780 bytes, checksum: 3bcc450ab74953bdffd4e6c59d584dc0 (MD5) Previous issue date: 2018-09-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). / A presença de uma camada proteica de superfície (camada S) é comum em organismos procarióticos. As proteínas que compõem essa camada apresentam a propriedade de se auto-organizar em arranjos simétricos estáveis, que as confere um alto potencial biotecnológico. Apesar de presente em quase todas as archaeas, existem poucos relatos do uso de proteínas de camada S desses organismos em estudos aplicados. Além disso, os estudos descrevendo a estrutura de proteínas desse tipo são escassos. Assim, esse trabalho tem o objetivo de caracterizar estruturalmente as proteínas de camada S da archaea halófila Haloferax volcanii, utilizá-las para a construção de envelopes celulares biomiméticos para o estudo de membranas e a expressão dessa proteína em E. coli para construção de fusões gênicas de interesse. As proteínas foram obtidas a partir da cultura de células de H. volcanii e caracterizadas por ensaios de espalhamento de luz dinâmico, dicroísmo circular e espectroscopia de fluorescência. Microscopia eletrônica de transmissão foi utilizada para observar a formação do arranjo cristalino das proteínas purificadas, bem como de envelopes celulares de H. volcanii. Foram produzidas monocamadas lipídicas por Langmuir-Blodgett utilizando os lipídios DPPE, DPPC e DPhPE. A estrutura secundária da proteína é afetada pelo pH, de maneira que uma maior quantidade de folhas beta foi detectada em valores mais altos. Esse mesmo fator influencia também a sua estrutura terciária, de maneira que em valores mais alcalinos as regiões próximas aos resíduos de triptofano da proteína interagem mais com o meio. No entanto, ensaios de espalhamento de luz dinâmico indicaram formação de oligômeros em todos os pHs testados, havendo picos monodispersos, indicando a capacidade de cristalização da proteína em todas as condições. No entanto, imagens de microscopia revelam que os arranjos formados nessas condições não estão de acordo com o normalmente observado na superfície celular desse organismo, algo que foi somente possível em condições de alta salinidade. A concentração de íons de sódio, magnésio e cálcio no meio afeta a estrutura secundária e terciária da proteína, influenciando assim a sua propriedade de auto-arranjo. Nos ensaios de Langmuir-Blodgett, foi observada a cristalização das proteínas sobre as diferentes superfícies lipídicas, sugerindo assim um possível potencial no uso dessas proteínas para produção de envelopes celulares artificiais. Foi também possível a expressão de proteínas de camada S de H. volcanii em E. coli. / The presence of a protein surface layer, known as the S-layer, is commonly found in prokaryotic cell envelopes. This layer is composed of proteins that self-assemble into a paracrystalline surface structure, a property that has been extensively used in biotechnological research. Despite its detection in almost all archaea described to date, there are very few reports in the literature using these proteins for applied purposes. Furthermore, studies describing structural aspects of these proteins are scarce. Thus, the objective of the present study was to investigate the structural properties of the Haloferax volcanii S-layer protein, use them for production of biomimetic S-layer supported lipid platforms and heterologous expression of S-layer fusion proteins in E. coli. The S-layer proteins were purified directly from H. volcanii cells and their structural properties were evaluated through dynamic light scattering, circular dichroism and fluorescence spectroscopy. Transmission electron microscopy was used for analyses of the protein’s self-assembly properties as well as H. volcanii cell envelope preparations. Lipid monolayers using DPPE, DPPC and DPhPE were produced through Langmuir-Blodgett. The protein’s secondary structure is affected by pH values in the medium, with higher beta sheet detection in more alkaline values. This factor also affects the protein’s tertiary structure, with higher tryptophan exposure to the environment in higher pH. Dynamic light scattering essays revealed oligomer formation in all pH values evaluated, indicating that the protein’s self-assembly process occurs in these conditions. However, micrograph images showed that these oligomers do not resemble the lattice found on the H. volcanii cell surface, and correct lattice formation was only achieved in higher salt concentrations. The concentrations of sodium, magnesium and calcium ions in the environment affect the protein’s secondary and tertiary structures, which influences the protein’s functional properties. On the Langmuir-Blodgett essays, an increase of surface pressure was detected on all lipid monolayers tested, indicating a potential use of the H. volcanii S-layer proteins in artificial lipid platform production. It was also possible to isolate the protein’s encoding gene and heterologous protein expression was successful in E. coli cells.

Made available in DSpace on 2014-06-12T18:02:57Z (GMT). No. of bitstreams: 2 arquivo3678_1.pdf: 1377857 bytes, checksum: 7d15f520b2b07e3bad5591816c1345df (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A água, depois de utilizada para o abastecimento público e nos processos produtivos, retorna com sua composição alterada, constituindo o esgoto. Os esgotos quando lançados aos corpos d água sem tratamento adequado podem causar graves problemas de saúde pública e ambientais como a eutrofização. Reatores biológicos anaeróbios tipo UASB (Reator Anaeróbio de Fluxo Ascendente e Manta de Lodo) são uma alternativa interessante para o tratamento dos esgotos domésticos, combinando baixos custos de implantação e simplicidade operacional. No entanto, o sucesso do processo depende do tipo, adaptação e atividade da população microbiana presente em seu interior. Diante disto, este trabalho vem contribuir para o melhor entendimento do consorcio microbiano em reator tipo UASB para tratamento de esgoto, realizando a identificação dos microrganismos pertencentes aos domínios Bactéria e Archaea por meio da análise das seqüências dos genes rRNA 16S. Neste sentido, foram coletadas amostras de lodo dos cinco níveis do reator UASB localizado na ETE Mangueira, Recife/PE, para posterior extração de DNA. Em seguida foi realizada a clonagem e sequenciamento dos produtos de PCR provenientes dos genes rRNA 16S heterogêneos. A comparação das seqüências com os bancos de dados de rRNA 16S, NCBI e RDP, indicaram a ocorrência de representantes dos filos Actinobacteria (42%), Proteobacteria (13%), Chloroflexi (9%), Firmicutes (6%) e Bacteroidetes (4%) para o domínio Bacteria, e representantes das ordens Methanomicrobiales (43%), Methanobacteriales (17%) e Methanosarcinales (12%) para Archaea, distribuídos distintamente ao longo dos cinco níveis do reator. Do total das seqüências, 26% das de Bacteria e 17% das de Archaea não apresentaram similaridade com seqüências já conhecidas, levando a crer que possivelmente sejam pertencentes a microrganismos ainda não descritos

Master of Science / Department of Geology / Matthew Kirk / Biodegradation of crude oil to methane has long been known to exist in shallow petroleum reservoirs. It is only in the past decade, however, in which the concept of in-reservoir petroleum biodegradation has changed from a model emphasizing aerobic crude-oil degradation (with oxygen delivered down from meteoric waters) to a more recent model in which crude-oil degradation is driven by anaerobic processes (methanogenic microorganisms). In this study, we examine controls on microbial conversion of crude oil and coal into methane in middle-Pennsylvanian strata in the Cherokee Basin, Kansas, USA and how access to oil or coal influence microbial communities. Specifically, we considered the following hypotheses: 1) microorganisms in the basin are capable of generating methane by degrading crude oil or coal and 2) potential controls on the rate of methane formation include microbial diversity, formation water chemistry, nutrient abundance, and carbon dioxide abundance. To test these hypotheses, we used three sets of laboratory experiments constructed of materials from the Cherokee basin, Kansas. One set tested environmental controls on methane generation from oil, another from coal, and a third was a control experiment that utilized methanogenic substrates rather than oil or coal. In the experiments with oil and coal, environmental factors tested ammonium/phosphate availability, feedlot wastewater injection, and carbon dioxide abundance. Our experiments also tested the influence of salinity, by including materials from a well producing water with relatively low salinity and a well producing water with relatively high salinity. The cultures were allowed to incubate from approximately 75 to 170 days, during which headspace of oil and coal bioreactors were sampled periodically and analyzed for methane concentrations. Post incubation analyses included microbial DNA sequencing. We determined that a higher concertation of methanogens existed in the lower salinity well, which has higher potential for practical stimulatory injection. Of methane produced, the only significant (Mann Whitney) treatment had access to oil in lower salinity formation water. Access to coal resulted in no significant results. Microbial diversity, in the form of methanogenic archaea abundance, formation water chemistry (salinity), and wastewater nutrient often correlated with increased, yet insignificant, rates of methane production, while carbon dioxide abundance showed no benefit. Of methanogenic substrates consumed, we determined that most Cherokee basin methanogens preferred methanol over hydrogen and acetate.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23230号 / 工博第4874号 / 新制||工||1761(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19034号 / 農博第2112号 / 新制||農||1031(附属図書館) / 学位論文||H27||N4916(農学部図書室) / 31985 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 准教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20712号 / 工博第4409号 / 新制||工||1685(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 浜地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21276号 / 工博第4504号 / 新制||工||1700(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 跡見 晴幸, 教授 森 泰生, 教授 梅田 眞郷 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM