Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2015. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2015-12-22T16:56:46Z No. of bitstreams: 1 2015_HelenaRaíraMagaldiRibeiro.pdf: 3739850 bytes, checksum: c9003205f1c12d8c0454cb4be35594c9 (MD5) / Approved for entry into archive by Patrícia Nunes da Silva(patricia@bce.unb.br) on 2016-01-20T12:40:44Z (GMT) No. of bitstreams: 1 2015_HelenaRaíraMagaldiRibeiro.pdf: 3739850 bytes, checksum: c9003205f1c12d8c0454cb4be35594c9 (MD5) / Made available in DSpace on 2016-01-20T12:40:44Z (GMT). No. of bitstreams: 1 2015_HelenaRaíraMagaldiRibeiro.pdf: 3739850 bytes, checksum: c9003205f1c12d8c0454cb4be35594c9 (MD5) / Membros do domínio Archaea encontram-se amplamente distribuídos na natureza, sendo frequentemente detectados em sedimentos, solos e ambientes aquáticos. Por outro lado, estudos recentes evidenciam a necessidade do cultivo laboratorial desses organismos, especialmente daqueles de ambientes não extremos, já que muitos aspectos de sua biologia permanecem ainda desconhecidos. Por esta razão, neste trabalho aprimoramos metodologias visando a obtenção de culturas de archaeas a partir de amostras de aquário de água doce residencial, utilizando a água deste aquário para a confecção dos meios de cultura. Visando favorecer o crescimento de archaeas oxidantes de amônia (AOAs), os meios de cultura foram adicionados de cloreto de amônio e suplementados com diversos antibióticos e antifúngicos, a fim de torná-los altamente seletivos para archaeas. Onze tipos coloniais foram selecionados e caracterizados quanto à morfologia celular por diferentes técnicas de microscopia, revelando a presença de células cocóides diminutas, com diâmetro médio de 1 µM, algumas vezes apresentando projeções celulares provavelmente envolvidas na adesão. Para as análises de filogenia molecular, DNA destas colônias foram utilizados em ensaios de PCR específicos para os genes de rRNA 16S dos Domínios Archaea e Bacteria e também o gene amoA, específico de AOAs, seguidos de sequenciamento de DNA. Os resultados revelaram que todas as colônias correspondiam a co-cultivos de archaeas pertencentes ao Filo Thaumarchaeota e bactérias dos gêneros Pandorea ou Cupriavidus. Foi também verificado o potencial nitrificante de algumas amostras, uma vez que foram detectadas sequências do gene amoA em alguns dos tipos coloniais selecionados. Paralelamente, foi construída uma biblioteca de genes de rRNA16S de Archaea, a partir do DNA total extraído de uma amostra de água e elementos do aquário. As análises filogenéticas desta biblioteca sugerem que o aquário consiste em um ambiente pouco diverso em termos da comunidade de Archaea presente. ____________________________________________________________________________________ ABSTRACT / Members of Archaea are found on a vast range of environments, including soils, sediments, and aquatic habitats. Recent studies stress the need of cultivation of mesophilic Archaea, since several physiological and biochemical aspects of these organisms remain unknown due to cultivation-independent techniques intrinsic limitations. In this work, we refined methodologies in order to obtain archaeal cultures from a freshwater aquarium, using the water from the aquarium to prepare the culture media. In order to favor the growth of ammonia oxidizing archaea (AOAs), the media were supplemented with ammonium chloride, and a number of antibiotics and antifungals. Eleven colonies were selected and characterized microscopically, revealing the presence of small coccoid cells, with an average diameter of 1 µM, sometimes presenting cell appendages probably involved in cell adhesion. Molecular phylogeny analyses were performed by PCR experiments specifically directed to the 16S rRNA genes of Archaea and Bacteria, as well as to the amoA gene, found only on AOAs, followed by automated DNA sequencing. The results revealed that all colonies consisted of co-cultures of archaeal cells belonging to phylum Thaumarchaeote, with bacteria of the genera Pandoraea or Cupriavidus. Some colonies were also positive for the presence of amoA gene, suggesting a nitrification potential of these samples. A genomic 16S rRNA library was also constructed from a sample consisting of water and other elements of the aquarium. The phylogenetic analyses of this library suggest that probably, the aquarium corresponds to an environment with a small diversity, in terms of the archaeal community.

Methanogenesis is the biological production of methane and is utilized by methanogenic archaea (methanogens) to generate energy. This process is responsible for 70% of total atmospheric methane, a potent greenhouse gas and an important energy source (natural gas). In the future, reversing methanogenesis in an engineered methanogenic strain could be realized to efficiently convert natural gas into liquid fuels. Methyl coenzyme M reductase (Mcr) catalyzes the final reaction of methanogenesis in methanogens and the first reaction in the anaerobic oxidation of methane (AOM) carried out by the anaerobic methanotrophs (ANME). Cofactor F₄₃₀, a unique nickel-containing tetrapyrrole, serves as the prosthetic group and catalytic component of Mcr. Recently, multiple F₄₃₀ variants have been discovered in several methanogenic species, including Methanococcus maripaludis, Methanosarcina acetivorans, and Methanocaldococcus jannaschii. A novel variant reported here has an exact mass of 1008.3478, a similar absorption spectrum as unmodified F₄₃₀, and associates with purified Mcr from M. acetivorans. Based on the exact mass, this molecule is likely modified with a mercaptopropamide moiety. In some conditions, this modified F₄₃₀ comprises 30-50% of the total F₄₃₀ pool. We also report upon our work to identify the sulfur insertion enzyme required to produce methylthio-F₄₃₀ that functions with Mcr in ANME-1. We hypothesized that the insertion of the methylthio moiety is likely catalyzed by a methylthiotransferase (MTTase) homolog present in ANME. However, purified ANME MTTase does not appear to catalyze this reaction, and instead catalyzes the methylthiolation of N6-threonylcarbamoyladenosine (t6A) in tRNA. / Master of Science in Life Sciences / Methanogens are a unique but diverse group of microorganisms that produce methane to generate their energetic needs. The byproduct of their metabolism is methane gas, most of which escapes into the atmosphere. Methanogens produce 70% of Earth's atmospheric methane, which is a gas that has contributed to 20% of global warming since the start of the industrial era. However, methane, which makes up the majority of natural gas, is also an important source of energy, and natural gas generates 40% of the United States' electricity. An issue with natural gas is, as a gas, it readily leaks out in the extraction and transport process. A solution to this is to convert the gas into liquids, which do not display these negatives. It is possible, through a better understanding of how methanogens work, we could produce a methanogen strain that can efficiently convert methane into liquid fuels. The last methane-generating step in methanogenic metabolism uses a protein known as methyl-coenzyme M reductase (Mcr). To do this, Mcr uses a small molecule known as cofactor F₄₃₀. Recently, variants of the standard F₄₃₀ structure have been described, in both methanogens as well as another microbial group known as the anaerobic methanotrophs (ANME). ANME generate their energy through reversing methanogenic metabolism. The work here involves studying why and how methanogens and ANME make F₄₃₀ variants. The hope is this work will reveal either different functionalities of cofactor F₄₃₀ not previously known, or that they influence Mcr catalysis, potentially in the reverse (methane degradation) direction.

Orientador: Telma Teresinha Berchielli / Coorientador: Juliana Duarte Messana / Banca: Eduardo Henrique Bevitori Kling de Moraes / Banca: Otavio Rodrigues Machado Neto / Banca: Paulo Henrique Moura Dian / Banca: Yury Tatiana Granja Salcedo / Resumo: Exp. 1 e 2: Foi avaliado o efeito de dois planos nutricionais (PN) sobre o consumo, digestibilidade, desempenho e emissão de metano (CH4), parâmetros fermentativos, eficiência de síntese microbiana e bactérias ruminais de três grupos genéticos (GG), na fase de recria, em pastagem. Cento e sessenta e dois tourinhos Nelore, cruzados ½Senepol e cruzados ½Angus, foram utilizados em um delineamento em blocos casualizados, arranjo fatorial 2 × 3, com idade média de 10 ± 2 meses e peso corporal inicial de 262 ± 31 kg, no experimento de desempenho. Doze tourinhos, sendo quatro de cada GG (Nelore, Senepol e cruzados ½Angus), canulados no rúmen, com idade média de 16 ± 2 meses e peso corporal inicial de 411 ± 34 kg, no experimento de metabolismo. Ambos foram alocados em doze piquetes de Urochloa brizantha (A. Rich.) Stapf. cv. Xaraés, submetidos a dois PN: suplementação com mineral (SM) 30 g / 100 kg peso corporal (PC) ou concentrado (SC) 300 g / 100 kg de PC. No experimento de desempenho o PN com SC proporcionou maior consumo de nutrientes e digestibilidade de PB e EE e maior AOL e EG (P < 0,05). Cruzados ½Senepol apresentaram maior digestibilidade de MS, MO e PB que Nelore e ½Angus (P < 0,05). Porém, Nelore e ½Senepol SC apresentaram maior GMD, GMDc e GPH que os ½Angus (P < 0,05), e Nelore apresentaram maior AOL que cruzados ½Angus e ½Senepol (P < 0,05) e maior EG que cruzados ½Senepol (P < 0,05). Cruzados ½Senepol e Nelore que receberam SC apresentaram menor emissão de CH4 por kg GM... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Exp. 1 and 2: The objective of this study was evaluated effect of nutritional plans on the performance, intake, digestibility, enteric methane emissions, ruminal fermentation, microbial efficiency synthesis and rumen microbial bacteria of young bulls of different genetic group during the growth phase on pasture. One hundred and sixty-two Nellore young bulls, crossbred ½Senepol and crossbred ½Angus, were used in a randomized complete block design, 2 × 3 factorial arrangement, average age of 10 ± 2 months and initial body weight of 262 ± 31 kg, in the experiment performance. Twelve bulls, four of each GG (Nellore, Senepol and crossbred ½Angus), cannulas in the rumen, with a mean age of 16 ± 2 months and initial body weight of 411 ± 34 kg, in the metabolism experimente. Both animals were allocated in twelve paddocks of Urochloa brizantha (A. Rich.) Stapf. cv. Xaraés, and were submitted to two plans nutrition: supplement with mineral (SM) salt 30 g / 100 kg body weight (PC) or concentrate (CS) 300 g / 100 kg of BW. In the performance experiment the PN with CS provided higher nutrient consumption and digestibility of CP and EE and higher LM area and FD (P <0.05). Crossbred ½Senepol showed higher digestibility of DM, OM and CP than Nellore and ½Angus (P < 0.05). However, Nellore and ½Senepol SC presented higher ADG, ADGc and GWH than ½Angus (P < 0.05), and Nellore had higher LM area than crossbred ½Angus and ½Senepol (P < 0.05) and higher FD than crossbred ½Senepol (P < 0.05). Howe... (Complete abstract click electronic access below) / Doutor

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For many years, it has been understood that protein phosphorylation-dephosphorylation constitutes one of the most ubiquitous mechanisms for controlling the functional properties of proteins. Although originally believed to be a eukaryotic phenomenon, protein phosphorylation is now known to occur in all three domains of life <i>Eukarya, Bacteria,</i> and Archaea</i>. Very little is known, however, concerning the origins and evolution of protein phosphorylation-dephosphorylation. Knowledge of the structure and properties of the protein kinases resident in the members of the <i>Archaea</i> represents a key piece of this puzzle. The extreme acidothermophilic archaeon, <i>Sulfolobus solfataricus</i>, exhibits a membrane-associated protein kinase activity. Solubilization of the kinase activity requires the presence of detergent such as Triton X-100 or octyl glucoside, indicating its activity reside in an integral membrane protein. This protein kinase utilizes purine nucleotides as phosphoryl donors <i>in vitro</i> with a requirement for a divalent metal ion cofactor, favoring Mn⁺². A preference for NTPs over NDPs and for adenyl nucleotides over the analogous guanyl nucleotides was observed. The enzyme appears to be a glycoprotein that displays catalytic activity on SDS-PAGE corresponding to a molecular mass of ≈67 kDa, as well as an apparent molecular mass of –125 kDa on a gel filtration column. Challenged with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, reduced carboxyamidomethylated and maleylated lysozyme (RCM lysozyme), histone H4 proved, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels <i>in vitro</i>. All of the aforementioned substrates were phosphorylated on threonine, while histone H4 was phosphorylated on serine as well. When the phosphoacceptor threonine in the MLC peptide was substituted with serine an appreciable decrease in phosphorylation was noted. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to several known "eukaryotic" protein kinase inhibitors. Primary sequence motifs based on known conserved subdomains of eukaryotic protein kinases were used to search the genome of <i>S. solfataricus</i> for eukaryotic-like protein kinase sequences. Six hypothetical proteins were identified from <i>S. solfataricus</i> whose primary sequence exhibited noticeable similarities to eukaryotic protein kinases. The hypothetical protein encoded by <i>ORF sso0197</i> contained 7 putative subdomains, <i>ORFs sso0433, sso2291, sso2387</i>, and <i>sso3207</i> contained 8 putative subdomains, and <i>ORF sso3182</i>, contained 9 putative subdomains of the 12 characteristically conserved subdomains found within eukaryotic protein kinases. <i>ORF sso2387</i> was cloned and expressed in <i>Escherichia coli</i>. The expressed protein, SsPK2, was solubilized from inclusion bodies using 5 M urea. SsPK2 was able to phosphorylate casein, BSA, RCM lysozyme, and mixed histones <i>in vitro</i>. Phosphoamino acid analysis of casein, BSA, and mixed histones revealed that they were all phosphorylated on serine. SsPK2 underwent autophosphorylation on serine at elevated temperature using both purine nucleotide triphosphates as phosphoryl donors in vitro, but exhibited a noticeable preference for ATP. Autophosphorylate of SsPK2 also occurred at elevated temperature using a variety of divalent metals cofactors in order of Mn⁺² > Mg⁺² >> Ca²⁺ ≈ Zn⁺². Polycations such as polyLys stimulated the phosphorylation of exogenous substrates while polyanions such as poly(Glu:Tyr) were shown to inhibit the phosphorylation of exogenous substrates. Of the "eukaryotic" protein kinases inhibitors tested, only tamoxifen had any noticeable effect of the catalytic activity of SsPK2 towards itself and exogenous substrates. A truncated form of SsPK2 containing the perceived catalytic domain also exhibited protein kinase activity towards itself and exogenous substrates. The observed protein kinase activity for SsPK2trunk was similar to that observed for SsPK2. Proteins from the membrane fraction of <i>S. solfataricus</i> subject to phosphorylation <i>in vitro</i> on serine or threonine residues were identified using MALDI-MS / peptide fingerprinting techniques. Nine phosphoproteins were assigned a tentative identification using the ProFound protein search engine from Rockefeller University. The identity of two of nine phosphoproteins, a translational endoplasmic reticulum ATPase and an ≈ 42 kDa hypothetical protein, were determined with a relatively high degree of confidence. Collectively the results suggested MALDI-MS peptide mapping coupled with [³²P] labeling <i>in vivo</i> will have a tremendous potential for mapping out a major portion of the phosphoproteome of <i>S. solfataricus</i>. / Ph. D.

The archaeal RNA polymerase (RNAP) is similar to the eukaryotic RNAP-II in terms of subunit composition and overall protein structure. Despite its similarity, a new archaeal-specific Rpo13 subunit has been identified. Rpo13 occupies a position in the enzyme which, in RNAP-II, is filled by the eukaryotic-specific Rpb5 jaw domain. It has therefore been proposed to contact DNA, where the positively charged C-terminal tail might mediate protein-DNA interactions. Furthermore, analysis of archaeal genomes has identified a homologue of the eukaryotic RNAP-III-specific RPC34 subunit. RPC34 may associate with the single archaeal RNAP, modulating the specificity of the archaeal RNAP and re-directing it to a subset of genes such as non-coding genes, in analogy to the RNAP-III/RPC34 eukaryotic system involved in the transcription of 5S rRNA, tRNAs and other small RNAs. More importantly, the association of RPC34 with the single archaeal RNAP would define an archaeal enzyme which acts as a precursor of the eukaryotic RNAP-III. Electrophoretic mobility shift assay (EMSA) analysis of purified Rpo13 protein by recombinant means subsequently incubated with a double-stranded (ds)DNA sequence reveals the formation of protein-DNA complexes, where Rpo13’s binding to DNA is non-sequence specific but discriminatory to dsDNA, as no binding is observed in the presence of single-stranded (ss)DNA. Also, it is found that the ma jor determinant of DNA binding is the Rpo13’s positively charged C-terminal tail, since DNA binding is abolished with a Rpo13 mutant deficient in this tail. Furthermore, neither ma jor groove nor minor groove interacting compounds have a major impact on Rpo13’s binding to DNA, suggesting that Rpo13 may associate with the negatively charged DNA phosphate backbone. Moreover, in vitro transcription assays indicate that a transcription product is observed upon RNAP incubation with a bubble DNA oligo shown to make Rpo13 contacts in the RNAP-DNA crystal structure. In addition, while a GST-pulldown experiment suggests the existence of an interaction between the archaeal RNAP and RCP34 in vitro, co-immunoprecipitation assays argue against the existence of such interaction from an in vivo point of view. Finally, a chromatin immunoprecipitation (ChIP)-sequencing approach to analyse Rpo13’s genomic distribution versus the one of the bulk RNAP was undertaken. While the gel filtration elution profile analysis of Rpo13 in the S. acidocaldarius cell extract versus the one of recombinant Rpo13 suggests that there is a free Rpo13 pool in the cell extract, indicating that Rpo13 may be acting as a transiently-associated RNAP subunit displaying a factor-like function, the ChIP-sequencing approach reveals that Rpo13 is a bona fide RNAP subunit since it co-localises from a genomic point of view with the bulk RNAP.

L’isomère de l’uridine (U), la pseudouridine (?), est le nucléoside le plus fréquemment rencontré dans les ARN. La réaction de pseudouridylation dans les ARN est catalysée par des ARN:?-synthases qui, soit fonctionnent comme des enzymes protéiques, soit sont portées chez les eucaryotes et les archaea par des particules ribonucléoprotéiques (RNP). Chaque RNP est composée d’un RNA guide dit à boîtes H/ACA (sno/sca/sRNA chez les eucaryotes et sRNA chez les archaea) et d’un ensemble invariable de protéines : l’enzyme ARN:?-synthase Dyskérine/aCBF5 et les 3 protéines NOP10/aNOP10, GAR1/aGAR1 et NHP2/L7Ae. L’ARN guide assure la reconnaissance de l’ARN cible à modifier par appariement de bases. Les sRNA H/ACA identifiés chez l’archae Pyrococcus abyssi ont servi de modèles pour des études de structure-fonction basées sur i) la mesure de l’activité de sRNP H/ACA reconstituées avec les composants protéiques et nucléiques purifiés, ii) l’analyse de la structure 2D des ARN au sein des RNP par des sondes chimiques et enzymatiques et par dichroïsme circulaire, iii) les diverses structures 3D disponibles. Les déterminants moléculaires présents sur les ARN guides ont été précisés, ainsi que le rôle fonctionnel des différentes protéines, de leurs domaines structuraux et de résidus conservés. Nous montrons que l’association entre aNOP10 et L7Ae est cruciale pour l’activité des RNP. Nous avons identifié que aCBF5 pouvait catalyser la formation des résidus ?55 dans les ARNt et ?2603 dans l’ARN 23S sans l’intervention de sRNA. Nous avons étudié le rôle de résidus conservés de aNOP10 ainsi que du site actif et de la boucle ß7/ß10 de aCBF5 dans les activités guidée et non guidée de aCBF5. / Pseudouridine (?), uridine’s isomer, is the most abundant modified nucleoside found in structured RNAs. The reaction of pseudouridylation is either catalyzed by a limited number of standalone RNA:?-synthases, or in eukaryotes and archaea by multiple ribonucleoprotein particles (the so-called, box H/ACA sno/sca/sRNPs). Each RNP is composed of a specific box H/ACA RNA used as a guide to define the U residue for modification, and a common set of four proteins– the RNA:?-synthase Dyskerin/aCBF5, and the auxiliary proteins NOP10/aNOP10, GAR1/aGAR1, NHP2/L7Ae (respectively in human and archaea). Initially, 7 guide RNAs were identified in the archaeon P. abyssi, which were used as models for structure-function analyses. Activities of RNPs reconstituted with purified protein and RNA components were measured; RNA 2D structure within RNPs was investigated by chemical and enzymatic probing assays as well as by circular dichroism. By taking into consideration the various 3D structures recently resolved, we were able to pinpoint the RNA molecular determinants and to clarify the role played by each protein, their domains, and some of their conserved residues. Hence, interaction between aNOP10 and L7Ae was found to be crucial for RNP activity. Moreover, we show that the enzyme aCBF5 catalyzes pseudouridylation at the position 55 in tRNAs and the position 2603 in 23S rRNA without the use of any guide sRNA. The role of residues conserved in aNOP10, and in the active site and the ß7/ß10 loop of aCBF5 for the RNA and non-RNA guided activities were analyzed.

N-glycosylation is a metabolic process found in all three domains of life. It is the attachment of a polysaccharide glycan to asparagine (Asn) residues within the amino acid motif, Asn-Xaa-Ser/Thr. In the archaeon, Methanococcus maripaludis, a tetrasaccharide glycan was isolated from purified flagella and its structure determined by mass spectrometry analysis. The linking sugar to the protein is surprisingly, N-acetylgalactosamine (β-GalNAc), with the next proximal sugar a derivative of N-acetylglucosamine (β-GlcNAc), being named β-GlcNAc3Ac, and the third sugar a derivative of N-acetylmannosamine (β-ManNAc), with an attached threonine residue on the C6 carbon (β-ManNAc3NAm). The terminal sugar is an unusual diglycoside of aldulose ((5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose). Previous genetic analyses identified the glycosyltransferases (GTs) responsible for the transfer of the second and third sugars of the glycan, as well as the oligosaccharyltransferase (OST) which attaches the glycan to protein. Left unidentified were the first and fourth GTs, the flippase as well as any genes involved in glycan sugar biosynthesis and modification. In this work, genes suspected to be involved in the biosynthesis of N-linked sugars, as well as those that might encode the missing GTs and flippase were targeted for in-frame deletion. Mutants with a deleted annotated GT gene (MMP1088) had a small decrease in flagellin molecular weight as determined by immunoblotting. Mass spectrometry (MS) analysis confirmed that the N-linked glycan was missing the terminal sugar as well as the threonine found on the third sugar of wildtype cells. Mutants with a deleted gene annotated to be involved in acetamidino synthesis (a functional group that is present on the third sugar), also had a decrease in flagellin molecular weight. MS analysis determined that the N-linked glycan was missing the acetamidino group on the third sugar as well as its attached threonine, along with the terminal sugar. Both mutants were able to assemble functional flagella but had impaired motility compared to wildtype cells in mini-swarm agar. Deletions were also constructed in four other GT genes considered candidates in assembly of the linking sugar. However, none of these mutants had the expected decrease in flagellin molecular weight. With the work done in this study, the glycosyl transferase that attaches the last sugar of the M. maripaludis N-linked assembly pathway has been identified as well as a gene involved in the biosynthesis and modification of the glycan sugars. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-07-07 15:45:19.052