L'étude des virus d'archaea, la manière dont ils sont capables d'infecter leurs hôtes et éventuellement de réaliser le transfert de certains gènes est d'intérêt pour mieux comprendre les mécanismes moléculaires qui ont permis le brassage de l'information génétique dans le phylum des archaea. Notre modèle d'étude est le fusellovirus SSV1 qui infecte certaines souches du genre Sulfolobus, dont Sulfolobus shibatae et Sulfolobus solfataricus. Le cycle viral de SSV1 est actuellement très peu connu, mais comprend l'intégration du génome viral dans celui de l'hôte à un site spécifique et la production de particules virales, sans lyse cellulaire, lors d'irradiation UV des cultures infectées. Nous avons initié l'étude de ce virus en analysant les propriétés biochimiques de son intégrase. Nous avons ainsi montré que l'intégrase virale était un membre à part entière de la famille des tyrosine recombinases, une classe de recombinases spécifiques de site que l'on trouve chez les procaryotes eubactériens mais également chez les eucaryotes. L'étude biochimique de l'intégrase de SSV1 nous a permis de mettre en évidence le caractère hybride du mécanisme de recombinaison spécifique de site chez les archaea. En effet, si l'organisation des sites de recombinaison est similaire à celle de systèmes phagiques eubactériens, celle du site actif de la recombinase est de type eucaryote, puisqu'il est assemblé à l'interface de deux protomères fournissant chacun des résidus intervenant dans la catalyse. Nous continuerons l'étude de l'organisation spatiale de ce système mosaïque en cristallisant l'intégrase sur un site synthétique. Une autre originalité du système archaéen est que le gène de l'intégrase est disrupté lors de l'intégration du génome viral dans celui de son hôte. Cette particularité, conservée chez tous les fusellovirus séquencés à ce jour, ouvre de nombreuses hypothèses quant au rôle de la recombinaison spécifique de site dans leur cycle réplicatif. La compréhension du mode de réplication, du maintien et de la mobilisation de ces virus lors de signaux environnementaux tels que l'irradiation UV est essentielle non seulement pour évaluer leur rôle dans la plasticité des génomes d'archaea, mais également pour leur utilisation future comme outils génétique chez leurs hôtes naturels, les Sulfolobales.<br />Nous exploiterons les résultats obtenus in vitro pour évaluer le rôle de l'intégration dans le maintien du virus sous forme stable, en replaçant des mutations inactivant soit l'intégrase soit le site viral de recombinaison dans le génome de SSV1. Le devenir de ces virus recombinants réintroduits dans Sulfolobus solfataricus sera évalué et nous permettra de déterminer si l'intégration du génome viral dans celui de son hôte est essentiel au maintien et à l'amplification du virus. Une analyse biochimique réciproque consistera à déterminer si une forme tronquée de l'intégrase (correspondant au produit de la disruption intégrative) est fonctionnelle pour le processus d'excision. La directionalité des évènements d'intégration et d'excision peut reposer soit sur la forme active de la recombinase (tronquée ou non) soit, et de manière non exclusive, par l'intervention de protéines accessoires fournies soit par l'hôte soit par le virus. L'identification de ces partenaires éventuels sera réalisée en utilisant des approches biochimiques classiques (co-immunoprécipitation, affinité, séquence peptidique) dans différentes conditions de croissance induisant ou non la production virale. Les résultats seront confrontés aux informations obtenues par les analyses transcriptomiques des effets des radiations réalisées sur Sulfolobus mais également Thermococcus ou Pyrococcus. L'analyse du pool de gènes induits lors d'une irradiation devrait contribuer à l'identification des facteurs de l'hôte intervenant dans la production virale en réponse au stress.<br />Outre le rôle de l'intégration dans le cycle viral, nous évaluerons dans une approche plus globale le rôle des différentes protéines codées par le virus. En effet, sur les 34 protéines potentiellement produites par SSV1 seules 4 ont une fonction identifiée. Par ailleurs, l'analyse comparative des différents génomes de fusellovirus montre que seules 18 ORFs (dont l'intégrase) sont communes à tous ces virus, suggérant que les protéines correspondantes assurent les fonctions minimales essentielles au développement viral. Chacune de ces ORFs sera délétée par LI-PCR. Cette stratégie devrait nous permettre de nous affranchir d'effets secondaires transcriptionnels liés à l'organisation polycistronique du génome viral. L'effet de l'inactivation de chaque ORF sera évalué en prenant en compte différentes étapes du développement viral (stabilité du génome dans la cellule hôte, production de particules virales, infectivité...). Nous espérons ainsi définir la fonction de ces protéines qui n'ont pour l'heure aucun homologue dans le vivant.

<p>The desert ant Cataglyphis fortis lives in the Sahara desert where it is exposed to extreme temperatures up to 70° C. In other words, the organism is considered as a thermophile. Until now the genome remains unknown but the fact that C. fortis provides heat stable proteins makes it very interesting in the field of protein studies and maybe even therapeutical research later on. This thesis focuses on trying to find genes that are expressed in C. fortis. Different genes were chosen and capable primers designed. After fishing for the enzyme GAPDH a fragment was found and sequenced. The sequence showed 31% homology on amino acid level with protein disulfide isomerase (PDI) in Apis mellifera (honey bee) and Drosophila melanogaster (fruitfly). The received sequence can be used to design new primers that match exactly. Gene fishing can also be continued by using the other primers that were designed during this project.</p>

The desert ant Cataglyphis fortis lives in the Sahara desert where it is exposed to extreme temperatures up to 70° C. In other words, the organism is considered as a thermophile. Until now the genome remains unknown but the fact that C. fortis provides heat stable proteins makes it very interesting in the field of protein studies and maybe even therapeutical research later on. This thesis focuses on trying to find genes that are expressed in C. fortis. Different genes were chosen and capable primers designed. After fishing for the enzyme GAPDH a fragment was found and sequenced. The sequence showed 31% homology on amino acid level with protein disulfide isomerase (PDI) in Apis mellifera (honey bee) and Drosophila melanogaster (fruitfly). The received sequence can be used to design new primers that match exactly. Gene fishing can also be continued by using the other primers that were designed during this project.

If small isoprenoids, the starting component of carotenoids, can be efficiently excreted from thermophilic cyanobacteria, they could help satisfy the demand for sustainably produced hydrocarbons. This is the driving force behind wanting to understand the response and regulation of isoprenoid pathways to environmental stimuli in the thermophilic cyanobacterium, Thermosynechococcus elongatus, BP-1. The portion of the isoprenoid pathway studied here is the carotenoid pathway since these products are critical to adaptation and they encompass the largest pool of isoprenoid compounds in cyanobacteria. Although synthetic biology in cyanboacteria has improved in recent years, there are many undiscovered metabolic complexities that make large-scale commercial production challenging. To address this need, I quantify and report for the first time metabolic shifts within the carotenoid pathway of BP-1 due to combined effects of temperature, pH and blue light. I show that metabolism shifts from the dicyclic into the monocyclic carotenoid pathway in response to pH, and that decreasing temperature drives flux into the end products of both pathways. Also, I report that the productivity of an uncommon carotenoid, 2-hydroxymyxol 2’-fucoside (HMF), approached 500 μg/L-day in cultures grown at 45 °C, high light intensity, and pH 8. In order to further elucidate these responses, I analyzed 42 RNAseq samples taken over time of BP-1 induced by cold and heat stress and compared these results to metabolomics data. I showed that crtR and crtG, two central carotenogenesis genes, are transcriptionally controlled and used weighted gene co-expression network analysis (WGCNA) to determine eight separate co-expressed modules of biological significance. Among the co-regulated heat response and cold response genes there were three and five non-coding RNA, respectively, providing targets for future investigation. Using subtractive genomics and transcriptional data I narrowed the potential missing steps of the myxol pathway in cyanobacteria to seven unknown BP-1 genes, two of which were confirmed not to be involved in the missing step(s). Finally, by generating a ΔcrtG mutant and testing it under different environmental parameters, I showed that HMF does not protect against high pH or low temperature (despite up-regulation at these conditions), and that CrtG has a higher affinity for monocyclic than dicyclic carotenoids. / text

Techn. Hochsch., Diss., 2002--Aachen.

Biocatalysis is a field rapidly expanding to meet a demand for green and sustainable chemical processes. As the use of enzymes for synthetic chemistry becomes more common, the construction of multistep enzyme reactions is likely to become more prominent providing excellent cost and productivity benefits. However, the design and optimisation of multistep reactions can be challenging. An enzyme toolbox of well-characterised enzyme parts is critical for the design of novel multistep reactions. Furthermore, while whole-cell biocatalysis offers an excellent platform for multistep reactions, we are limited to the use of mesophilic host organisms such as Escherichia coli. The development of a thermophilic host organism would offer a powerful tool allowing whole-cell biocatalysis at elevated temperatures. This study aimed to investigate the construction of a multistep enzyme reaction from well-characterised enzyme parts, consisting of an esterase, a carboxylic acid reductase and an alcohol dehydrogenase. A novel thermostable esterase Af-Est2 was characterised both biochemically and structurally. The enzyme shows exceptional stability making it attractive for industrial biocatalysis, and features what is likely a structural or regulatory CoA molecule tightly bound near the active site. Five carboxylic acid reductases (CARs) taken from across the known CAR family were thoroughly characterised. Kinetic analysis of these enzymes with various substrates shows they have a broad but similar substrate specificity and that electron rich acids are favoured. The characterisation of these CARs seeks to provide specifications for their use as a biocatalyst. The use of isolated enzymes was investigated as an alternative to whole-cell biocatalysis for the multistep reaction. Additional enzymes for the regeneration of cofactors and removal of by-products were included, resulting in a seven enzyme reaction. Using characterised enzyme parts, a mechanistic mathematical model was constructed to aid in the understanding and optimisation of the reaction, demonstrating the power of this approach. Thermus thermophilus was identified as a promising candidate for use as a thermophilic host organism for whole-cell biocatalysis. Synthetic biology parts including a BioBricks vector, custom ribosome binding sites and characterised promoters were developed for this purpose. The expression of enzymes to complete the multistep enzyme reaction in T. thermophilus was successful, but native T. thermophilus enzymes prevented the biotransformation from being completed. In summary, this work makes a number of contributions to the enzyme toolbox of well-characterised enzymes, and investigates their combination into a multistep enzyme reaction both in vitro and in vivo using a novel thermophilic host organism.

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.

This thesis describes the characterization of SsoPox, a lactonase with promiscuous phosphotriesterase activity from the hyperthermophilic archaeon, Sulfolobus solfataricus, and the potential of the phosphotriesterase from Brevundimonas diminuta (PTEBd) to function as an organophosphate sensor. Arg-223 and Tyr-99 of SsoPox are not essential for lactonase activity, however substitution of a phenylalanine in place of Tyr-97 abolished lactonase activity while reducing paraoxonase activity by 20-fold. Substrate specificity of SsoPox can be modulated through the partial blockage of the hydrophobic binding tunnel adjacent to the active site. The specificity constant for N-(3-oxo-decanoyl)-L-homoserine lactone decreased 37-fold when a phenylalanine was introduced in place of Leu-226. PTEBd was expressed and purified from Pseudomonas putida and, like SsoPox, can be immobilized to Disruptor paper. The immobilized enzyme can be used to detect five organophosphates at concentrations as low as 50 μM. Incubation of PTEBd-immobilized sensors at different temperatures proved that the enzyme is stable for at least 40 days at 23.5 degrees Celsius without any detectable change in activity.

Many double-stranded DNA viruses use a packaging motor during maturation to recognize and transport genetic material into the capsid. In terminase motors, the TerS complex recognizes DNA, while the TerL motor packages the DNA into the capsid shell. Although there are several models for DNA recognition and translocation, how the motor components assemble and power DNA translocation is unknown. Using the thermophilic P74-26 bacteriophage model system, we discover that TerL uses a trans-activated ATP hydrolysis mechanism. Additionally, we identify critical residues for TerL ATP hydrolysis and DNA binding. With a combination of x-ray crystallography, SAXS, and molecular docking, we build a structural model for TerL pentamer assembly. Apo and ATP analog-bound TerL ATPase domain crystal structures show ligand-dependent conformational changes, which we propose power DNA translocation. Together, we assimilate these findings to build models for both motor assembly and DNA translocation. Additionally, with the P76-26 system, we identify the TerS protein as gp83. I find that P74-26 TerS is a nonameric ring that stimulates TerL ATPase activity while inhibiting TerL nuclease activity. Using cryoEM, I solve 3.8 Å and 4.8 Å resolution symmetric and asymmetric reconstructions of the TerS ring. I observe in P74-26 TerS, the conserved C-terminal beta-barrel is absent, and instead the region is flexible or unstructured. Furthermore, the helix-turn-helix motifs of P74-26 TerS are positioned differently than those of known TerS structures, suggesting P74-26 uses an alternative mechanism to recognize DNA.

No description available.