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No description available.

Three distinct families of PTPs, the conventional (cPTPs), low molecular weight (LMW PTPs), and Cdc25 PTPs, have converged upon a common catalytic mechanism and active site sequence, mainly, the phosphate-binding loop encompassing the PTP signature motif (H/V)<b>C</b>(X)₅<b>R</b>(S/T) and an essential Asp residue on a surface loop. There is little sequence similarity among the three families of phosphatases. All known LMW PTP remove phosphoryl groups esterified to the hydroxyl amino acid: tyrosine, whereas all members of the Cdc25 family are dual-specificity protein phosphatases that dephosphorylate all the hydroxyl amino acids: tyrosine, serine and threonine. The cPTP family primarily functions as tyrosine phosphatases, but it also includes dual-specific members. ORFs encoding potential cPTPs have been identified in five archaeal species: <i>Methanobacterium thermoautotrophicum</i>, <i>Methanococcus jannaschii</i>, <i>Thermococcus kodakaraensis</i>, <i>Pyrococcus horikoshii</i>, and <i>S. solfataricus</i>. Only one has been partially characterized, <i>Tk</i>-PTP from <i>T. kodakaraensis</i>. Hence, our current body of knowledge concerning the functional properties and physiological roles of these enzymes remains fragmented. The genome of <i>S. solfataricus</i> encodes a single conventional protein tyrosine phosphatase, SsoPTP. SsoPTP is the smallest known archaeal PTP (18.3 kDa) with a primary amino acid sequence that conforms to the cPTP protein tyrosine phosphatase paradigm, HCX₅R(S/T). Relatively little is known about its mode of action " whether it follows the conventional PTP mechanism or employs a different route for catalysis " or its physiological role. ORF <i>sso2453</i> from the genome of <i>Sulfolobus solfataricus</i>, encoding a protein tyrosine phosphatase, was cloned and its recombinant protein product, SsoPTP, was expressed in <i>E. coli</i> and purified by immobilized metal affinity chromatography. SsoPTP displayed the ability to dephosphorylate protein-bound phosphotyrosine as well as protein-bound phosphoserine/phosphothreonine. SsoPTP hydrolyzed both isomers of naphthyl phosphate, an indication of dual specificity. The four conserved residues within the presumed active site sequence: Asp⁶⁹, His⁹⁵, and Arg¹⁰², and the invariant Gln¹³⁹ residue were essential for catalysis, as it was predicted for the established members of the PTP family in both bacteria and eukaryotes. A substrate trapping protein variant, SsoPTP-C96S/D69A, was constructed to isolate possible SsoPTP substrates present in <i>S. solfataricus</i> cell lysates. Several potential substrates were isolated and identified by mass spectroscopy. / Ph. D.

No / Raman spectroscopy is a rapid nondestructive technique providing spectroscopic and structural information on both organic and inorganic molecular compounds. Extensive applications for the method in the characterization of pigments have been found. Due to the high sensitivity of Raman spectroscopy for the detection of chlorophylls, carotenoids, scytonemin, and a range of other pigments found in the microbial world, it is an excellent technique to monitor the presence of such pigments, both in pure cultures and in environmental samples. Miniaturized portable handheld instruments are available; these instruments can be used to detect pigments in microbiological samples of different types and origins under field conditions.

Au cours des vingt dernières années, les études sur les sédiments marins profonds ont révélé la présence et l'activité de communautés microbiennes inattendues. Il est maintenant formellement établi que la biosphère profonde héberge de nombreux représentants des domaines des Archaea et des Bacteria. Cependant,les micro-eucaryotes et plus particulièrement les champignons n’ont été que très peu étudiés dans ces écosystèmes singuliers. Dans ce contexte, des approches moléculaire et culturale ont été utilisées afin de caractériser la diversité des communautés fongiques des sédiments marins profonds en utilisant le bassin de Canterbury comme modèle d’étude. Les résultats principaux obtenus lors de ce travail de thèse sont les suivants : (i) L’approche moléculaire basée sur l’ADN a fourni la preuve directe que les communautés fongiques peuvent persister jusqu’à la profondeur record de 1740 mètres sous la surface du plancher océanique. (ii) Des approches complémentaires ciblant les ARNr et les ARNm ont permis de préciser leur activité métabolique et d’obtenir de premiers indices sur les fonctions de ces champignons à 350m sous la surface du plancher océanique, principalement liées à la croissance, à l’adaptation aux contraintes environnementales in situ et aux interactions entre communautés microbiennes. (iii) L’approche culturale a permis de constituer une collection de culture de 183 isolats fongiques avec des caractéristiques écophysiologiques témoignant leur capacité d’adaptation aux conditions in situ. (iv) Le potentiel biotechnologique des isolats obtenus a été estimé via la recherche de gènes impliqués dans la synthèse de métabolites secondaires et a permis de positionner cette collection d’organismes originaux comme une ressource d’intérêt biotechnologique potentiel. Ce travail qui témoigne de la persistance et de l’activité des communautés fongiques dans les sédiments marins profonds élargit notre vision de la diversité microbienne dans ces milieux et soulève des hypothèses sur le rôle écologique des champignons au sein de la biosphère profonde. / Over the past two decades, investigations on deep marine sediments have revealed the occurrenceand activity of unexpected microbial communities. Many representatives of Archaea and Bacteria were reportedbut micro-eukaryotes and especially fungal communities are still poorly studied in this ecosystem. In this underexplored context, molecular- and culture-based approaches were used to characterize the diversityof fungal communities in deep subsurface sediments using the Canterbury Basin as a model system. The main results of this work are: (i) The molecular DNA-based approach provided direct evidence that the fungal communities persist until the record depth of 1,740 meters below sea floor. (ii) Supplementary approaches targeting rRNA and mRNA revealed their metabolic activity and highlighted first hints into the fungal functions at350 meters below sea floor, mainly related to growth, adaptation to in situ environmental constraints andmicrobial interactions. (iii) The culture based approach allowed establishing a culture collection of 183 fungal isolates with ecophysiological characteristics indicating their ability to adapt to in situ conditions. (iv) This culture collection seems to represent a reservoir of secondary metabolites as many genes involved in secondary metabolites pathways were revealed. The fungal collection established may be considered as an untapped resource to explore for biotechnological applications. This work demonstrating the persistence and activity of fungal communities in deep subsurface sediments (i)broadens our view of microbial diversity in these environments and (ii) raises hypotheses about the ecologicalroles of fungi in the deep biosphere

Os elementos genéticos móveis (mobile genetic elements MGEs), são elementos extremamente importantes para plasticidade e evolução dos genomas. Uma das classes mais importantes de MGEs são as sequências de inserção (insertion sequences - IS). Estes elementos são encontrados em bactérias e archaea e apresentam grande diversidade de famílias e mecanismos de movimentação. Uma família interessante de IS é IS200/605, esta família encontra-se distribuída em bactérias, archaea e vírus e utiliza substratos de DNA de fita simples para seu processo de transposição. Neste trabalho, através da análise de dados públicos de transcritoma identificamos RNAs sobrepostos ao 3\' de genes tnpB de IS200/605 em archaea e bactérias, estes transcritos foram chamados de sense overlapping transcripts (sotRNAs). As extremidades 5\' e 3\' dos sotRNAs foram mapeadas através de dados de RNA-seq de pequenos RNAs (sRNA-seq) e validadas através da técnica de C-RACE. Análises de sequência e estrutura secundária demonstraram que estes RNAs apresentam um motivo conservado chamado de RE-like. Utilizando a sequência consenso deste motivo pudemos identificar RNAs intergênicos derivados de IS200/605 em H. salinarum NRC-1. Para caracterização funcional, construímos linhagens superexpressando os RNAs VNG_sot0042 e VNG_R0052, ambos contendo o motivo RE-like. Curvas de crescimento, utilizando as linhagens construídas demonstraram que a superexpressão destes RNAs aumenta o crescimento de H. salinarum demonstrando sua funcionalidade. Devido a presença do motivo RE-like, extremidades 5\' e 3\' determinadas e fenótipo visualizado em curva de crescimento padrão, o sotRNA VNG_sot0042 foi estudado mais a fundo. Realizamos experimentos de RNA-seq para avaliar o impacto desta superexpressão no transcritoma de H. salinarum NRC-1, assim como experimentos de SILAC para identificação de proteínas parceiras em larga escala. Nestes ensaios identificamos proteínas e genes associados ao processo de adesão, geração de células persistentes e resistência a metais pesados. Ensaios de adesão e sobrevivência a metais pesados demonstraram que a linhagem de superexpressão apresenta maior capacidade de aderir a vidro e maior sobrevivência em diversas condições de estresse. Deste modo, podemos sugerir que ncRNAs derivados de IS200/605 são importantes moléculas regulatórias em H. salinarum NRC-1 e nos ajudam a compreender a manutenção de IS200/605 e seus derivados nos genomas de procariotos. / Mobile genetic elements (MGEs) are extremely important for plasticity and evolution of genomes. Their impacts are diverse and could be related to antibiotic resistence or symbiosis. One of the most important class of MGEs are insertion sequences (ISs). These elements are found widespread throughout bacteria and archaea, presenting a great diversity of families. An interesting family is the IS200/605, this family is found widespread in bacteria, archaea and viruses and is divided in three subgroups according to it\'s genetic composition: IS200 with tnpA gene alone, IS605 with tnpA and tnpB and IS1341 with tnpB only. Another interesting aspect is the utilization of single-stranded DNA as a substrate during the transposition process. In this work, through the analysis of public available transcriptomic data we identified transcripts that overlaps the 3\' end of tnpB in IS200/605 in both bacteria and archaea. These transcripts were named sense overlapping transcripts (sotRNAs). Sequence and secondary structure analysis showed a conserved motif present in sotRNAs, the RE-like motif. Using the consensus sequence of this motif we identified novel intergenic ncRNAs containing this motif that are derived from IS200/605. For functional characterization we overexpressed a sotRNA (VNG_sot0042) and a intergenic (VNG_R0052), both containing the RE-like motif. Standard growth curves demonstrated that the overexpression of these ncRNAs improve H. salinarum growth showing that this RNA is functional. To further evaluate the impact of the overexpressions we prepared RNA-seq libraries of the strain overexpressing VNG_sot0042 and in parallel performed SILAC experiments to identify potential protein-RNA interaction partners. Differentially regulated genes and interacting proteins associated with adhesion and persistent cells generation were found. Adhesion and survival assays showed that the lineage overexpressing VNG_sot0042 has a better capability to adhere in glass surfaces and survive more in diverse stressful conditions.

DNA is constantly subjected to chemical and mechanical damage. The ability to repair the lesions sustained is essential for all life. Double stranded DNA (dsDNA) breaks are especially toxic as both antiparallel strands of DNA are severed. The most high fidelity mechanism available to repair this damage is homologous recombination, a mechanism that uses homology from the sister chromatid to replace any lost information. Key proteins involved in maintaining genomic stability this way are conserved in all domains of life. One such component is the Mre11/Rad50 complex that is involved in the initial recognition of damage and recruitment of subsequent repair factors. Understanding the function of this DNA repair complex and any associated proteins has implications for human cancers and aging. The proteins of thermophilic archaea present an excellent opportunity to study these systems in a robust, tractable and eukaryote-like system. Archaea are in many ways biochemically unique, for example they are the only domain capable of methanogenesis. However archaea share a high level of homology with eukaryotes in many essential cellular processes such as DNA replication, homologous recombination and protein degradation. In thermophilic archaea the mre11/rad50 genes are clustered in an operon with the herA/nurA genes that form a helicase/nuclease complex. This has lead to speculation that the four proteins work together during homologous recombination to produce the 3' overhangs required by RadA to identify homology. As part of this investigation I have performed extensive bioinformatic searches of a variety of archaeal/bacterial systems. These analyses have revealed operonic linkages to other known recombinational helicase/nucleases, such as AddAB and RecBCD. These genomic linkages are especially prevalent in thermophilic organisms suggesting their functional relevance is particularly acute in organisms exposed to a high amount of genomic stress. Comparison of the evolutionary trees, constructed for each protein, makes a single genomic linkage event the most likely scenario, but cannot definitively exclude other possibilities. Exhaustive attempts were made to demonstrate an interaction between Mre11/Rad50 and HerA/NurA. Despite analysis by nickel/cobalt pulldown, immunoprecipitation, analytical gel filtration, ITC and OCTET an interaction could not be confirmed or definitively dismissed. However in the process an interesting Rad50 tetrameric assembly was identified and attempts were made to crystalize it. Hexameric helicases and translocases are key to the replication and DNA packaging of all cellular life and multiple viruses. The hexameric translocase HerA is a robust model for investigating the common features of multimeric ATPases as it is extremely stable and experimentally tractable. Here it is revealed that HerA exists in a dynamic equilibrium fluctuating between hexameric and heptameric forms with rapidly interchanging subunits. This equilibrium can be shifted to heptamer by buffering conditions or towards the hexamer by the physical interaction with the partnering nuclease NurA, raising the possibility that these alternate states may play a role in translocase assembly or function. A novel C-terminal brace, (revealed by a collaborative crystallographic structure) is investigated; as well as stabilizing the assembly, this brace reaches over the ATPase active site of its neighbouring subunit. It is seemingly involved in the conversion of energy generated by ATP hydrolysis into physical movement in the central channel of the hexamer. The regulation of homologous recombination is extremely important to prevent aberrant activity, resulting in mutations and genome reorganization. In eukaryotic organisms, it is well established that post-translational modifications and protein turnover at the proteosome play important roles in this control. In particular, there is significant interest currently in the ubiquination-proteasome destruction pathway as a mechanism for extracting DNA repair components from chromatin at the termination of the DNA repair process. To date no Ubiquitin proteins have been identified in the Archaea, however related proteins URMs/SAMPs (Ubiquitin Related Modifier/Small Archaeal Modifier Protein) have previously been identified. URMs are thought to have evolved from a common antecedent to eukaryotic ubiquitin and likely represent an evolutionary 'missing link' in the adaption of sulphur transfer proteins for covalent modifications. There has been speculation that Urm1 may play a similar role to ubiquitin in the proteasome degradation pathway and we have recently provided evidence to corroborate this. Here the potential for modification of Mre11/Rad50/HerA/NurA by Urm1 was investigated. Indeed Rad50 shows evidence of clear urmylation both in vivo and in vitro. Western blotting and mass spec analysis confirmed the covalent attachment of Urm1 to Rad50. Furthermore I present preliminary evidence that this urmylation can lead to the destruction of Rad50 via a direct physical interaction with the proteasome. This is the first evidence of such a regulatory system for Rad50. Investigating the urmylation and destruction of Rad50 was closely linked to investigating the archaeal proteasome, a close homologue of the eukaryotic proteasome. To date the majority of archaeal core proteasomes examined were believed to consist of only two subunits; alpha and beta. The subunits are arranged into heptameric rings, which then form an alpha/beta/beta/alpha stack with a single channel running through the centre of all four rings. Here we reveal that in Sulfolobus species the inner catalytic chambers are made up of mixed beta rings composed of two subunits. The first plays a crucial structural role but appears catalytically inert, while the second conveys catalytic activity. Here we investigate an inactive complex, containing only the structural beta subunit, and an active complex, containing both beta subunits. First, electron microscopy was performed on both complexes revealing the expected four-layered toroidal stack. Both complexes were subsequently investigated crystallographically. A 3.8 Å structure was determined for the inactive complex. As well as being one of the few archaeal core proteasome structures, this is also an important first step towards structurally investigating the novel three-subunit proteasome. The discovery of active and inactive beta subunits in the archaea brings them even closer to eukaryotic proteasomal systems, making the archaea even more valuable as model systems.

As Terras Pretas de Índio (TPI), também conhecida por Amazonian Dark Earth, apresentam horizonte A antrópico, elevado pH, nutrientes importantes para o crescimento das plantas, elevado teor de carvão pirogênico e intensa atividade biológica quando comparadas aos seus solos de origem. A comunidade microbiana do solo é essencial para o funcionamento dos ecossistemas, sendo fundamentais em processos de decomposição da matéria orgânica, na disponibilização de nutrientes para as plantas e na ciclagem de nutrientes. Este estudo teve como objetivo acessar as estruturas e composição das comunidades de Bacteria e Archaea em fragmentos de carvão pirogênico (provenientes de TPI), TPI e solo adjacente (ADJ) utilizando as técnicas moleculares. Os solos foram coletados em quatro sítios arqueológicos (Balbina, Barro Branco, Costa do Açutuba e Hatahara), localizados na Amazônia Central. Em geral, as TPIs apresentaram elevados valores de pH, P e Ca corroborando com resultados anteriores em TPIs. As amostras de TPI dos sítios Balbina, Barro Branco e Hatahara apresentaram maior número de cópias do gene 16S rRNA de Bacteria quando comparadas com as amostras de solo ADJ. Os resultados obtidos pelas técnicas de fingerprinting (Terminal Restriction Fragment Length Polymorphism - T-RFLP e Denaturing Gradient Gel Electrophoresis - DGGE) utilizando o gene 16S rRNA de Bacteria e Archaea revelaram que as estruturas das comunidades bacterianas em fragmentos de carvão apresentaram diferenças significativas quando comparados com os solos ADJ. A partir da técnica de pirosequenciamento, os filos Proteobacteria, Actinobacteria e Crenarchaeota apresentaram predominância nos fragmentos de carvão. Observou-se também que em fragmentos de carvão, os microorganismos encontrados podem estar diretamente relacionados aos ciclos do N e C. Além disso, a presença desses micro-organismos em fragmentos de carvão pode favorecer a microbiota do solo e, consequentemente, sua qualidade. Neste sentido, o carvão pode também servir como elemento de recuperação em ambientes degradados, agindo como condicionador do solo sendo esta uma alternativa promissora no manejo de solos agrícolas. / Amazonian Dark Earth (ADE), also known as Terra Preta de Índio, present tropical A horizon, high pH, important nutrients for plant growth, high pyrogenic charcoal levels and intense biological activity when compared to its soil of origin. Soil microbial community is essential for ecosystem function, which is involved in fundamental processes such as the decomposition of organic matter, availability of nutrients to plants and the nutrient cycling. The objective of this study was to assess the structure and composition of bacterial and archaeal communities in fragments of pyrogenic charcoal (from ADE), ADE and adjacent soil (ADJ) using molecular techniques. The soils were collected in four archaeological sites (Balbina, Barro Branco, Costa do Açutuba and Hatahara), located in the central Amazon. In general, ADE presented high values of pH, P and Ca agreeing with previous results in ADE. ADE samples in Balbina, Barro Branco and Hatahara sites presented higher bacterial 16S rRNA gene copy number in comparison with ADJ soil samples. The results obtained with the fingerprinting techniques (Terminal Restriction Length Polymorphism, T-RFLP and Denaturing Gradient Gel Electrophoresis, DGGE) using the bacterial and archaeal 16S rRNA genes revealed that bacterial community structure in charcoal fragments differed significantly when compared to the ADJ soils. Using the pyrosequencing technique, the phyla Proteobacteria, Actinobacteria and Crenarchaeota were predominant in charcoal fragments. It was also observed that microorganisms from charcoal fragments may be directly related to the N and C cycles. Furthermore, the presence of these microorganisms in charcoal fragments may favor the soil microbiota and, consequently, its quality. In this context, pyrogenic charcoal can serve as an element to recover degraded areas acting as soil conditioner and a promising alternative to the management of agricultural soils.

L’étude du microbiote digestif a connu un regain d’intérêt au début des années 2000, avec l’avènement des techniques moléculaires. La culturomics a démontré sa complémentarité depuis 2010 en réduisant une partie des biais des méthodes moléculaires. Une revue sur les techniques d’étude du microbiote digestif et l’analyse du microbiote des sujets africains. Les études de métagénomique en Afrique ont révélé une augmentation de la biodiversité, en particulier des Spirochaetes et des Prevotella chez les africains par rapport aux occidentaux. Sur les 1162 bactéries isolées par culturomics, 476 n'étaient pas africaines, 445 étaient communes et 241 étaient d’origine africaine dont 68 nouvelles espèces. Pour ma participation au travail de culturomics, 102750 colonies testées par MALDI-TOF,ont permis d'identifier 377 espèces incluant 40 nouvelles espèces,17 nouveaux genres et 2 nouvelles familles.Ces nouvelles espèces ont été décrites par taxonogenomics ou new species announcement.Les archaea méthanogènes ont une prévalence de 97,4% pour M. smithii et associés à des pathologies comme l’abcès du cerveau,les parodontites etc. La culture est fastidieuse et nécessitait une source extérieure d’hydrogène. Sous enceinte anaérobie, nous avons cultivé avec succès M. smithii à partir d’un milieu de culture liquide inoculé d’échantillon de selle. L’isolement en culture pure a été un succès sur milieu gélosé en réalisant une coculture avec Bacteroides thetaiotaomicron. Nous avons aussi testé avec succès la coculture de M. smithii avec d’autres bactéries productrices d’hydrogène connues. Les tests de chromatographie en phase gazeuse montraient que ces souches produisaient de l’hydrogène. / The study of the digestive microbiota was a renewed interest in the early 2000s, with the advent of molecular techniques. The culturomics has demonstrated its complementarity since 2010 by reducing some of the biases of molecular methods. A review on the techniques of studying the digestive microbiota and the analysis of the microbiota of African subjects. Metagenomic studies in Africa have revealed an increase in biodiversity, especially Spirochaetes and Prevotella among Africans compared to Westerners. Of the 1162 bacteria isolated by culturomics, 476 were non-African, 445 were common, and 241 were of African origin, including 68 new species. For my participation in the work of culturomics, 102750 colonies tested by MALDI-TOF, identified 377 species including 40 new species, 17 new genera and 2 new families. These new species have been described by taxonogenomics or new species announcement.Methanogenic archaea have a prevalence of 97.4% for M. smithii and associated with pathologies such as brain abscess, periodontitis and so on. The cultivation is tedious and required an external source of hydrogen. Under anaerobic enclosure, we successfully cultivated M. smithii from a liquid culture medium inoculated with a stool sample. The isolation in pure culture was a success on agar medium by performing a coculture with Bacteroides thetaiotaomicron. We have also successfully tested the co-culture of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen.

Bulge-helix-bulge (BHB) elements guide the enzymatic splicing machinery that in Archaea excises introns from tRNAs, rRNAs from their primary precursor, and accounts for the assembly of piece-wise encoded tRNAs. This processing pathway renders the intronic sequences as circularized RNA species. Although archaeal transcriptomes harbor a large number of circular small RNAs, it remains unknown whether most or all of them are produced through BHB-dependent splicing. We therefore conduct a genome-wide survey of BHB elements of a phylogenetically diverse set of archaeal species and complement this approach by searching for BHB-like structures in the vicinity of circularized transcripts. We find that besides tRNA introns, the majority of box C/D snoRNAs is associated with BHB elements. Not all circularized sRNAs, however, can be explained by BHB elements, suggesting that there is at least one other mechanism of RNA circularization at work in Archaea. Pattern search methods were unable, however, to identify common sequence and/or secondary structure features that could be characteristic for such a mechanism.