Regensburg, Universiẗat, Diss., 2003.

Cette thèse porte sur l'écophysiologie et la physiologie d'une bactérie hyperthermophile et microaérophile, Aquifex aeolicus, cultivée dans différentes conditions d'oxygénation. Au cours de ce travail, trois systèmes expérimentaux (jarres, microcosmes et fermenteur) ont été testés : (1) le nouveau système de jarres qui été mis au point est muni de microplaques de 24 puits couplé à un robot. Il permet d'étudier un grand nombre de facteurs trophiques ou de formulations de milieux de cultures tout en conservant une atmosphère de composition constante, (2) pour l'étude de facteurs trophiques gazeux, l'utilisation des microcosmes a été montrée peu adaptée, amenant même dans certains cas, à des interprétations erronées, et, 3) le fermenteur reste le meilleur outil pour étudier l'influence de la concentration en O2 dissous (pO2) sur le métabolisme d'A. aeolicus. A partir des cinétiques de croissance obtenues en fermenteur, il a été établi que la densité de biomasse est constante et que la vitesse de croissance est maximale pour une pO2 comprise, respectivement, entre 0.006 et 6 mg/L et, entre 1 et 2 mg/L. Pour des pO2 supérieures à 2 mg/L, il a été montré que l'oxygène a un effet toxique sur la croissance d'A. aeolicus. Pour des conditions optimales d'oxygénation (pO2=1.5 mg/L) et lorsque l'H2 (100 mL/min) limite la croissance, le catabolisme énergétique est alors dévié vers la consommation du thiosulfate. En effet, pour les débits d'H2 de 450 et 100 mL/min, d'une part 97 et 79 % de l'O2 sont respectivement réduits par l'hydrogène et d'autre part 3 et 21 % de l'O2 sont respectivement réduits par le thiosulfate. / This manuscript addresses the physiology and ecophysiology of the microaerophilic hyperthermophilic bacterium Aquifex aeolicus, grown under different oxygen-supply conditions. Three experimental systems, jar, microcosm and fermentor were tested in those experiments: (1) a newly-engineered jar system containing 24-well microplates coupled to an automated controller. This system allows to study a broad spectrum of trophic factors or culture media formulations while maintaining a constant atmospheric composition; (2) microcosm systems were, here, proved ill-adapted to studying gas-phase trophic factors, and in some cases even to leading to false interpretations; 3) the fermentor system remains the best tool to studying the influence of dissolved O2 concentration (pO2) on A. aeolicus metabolism. Based on in-fermentor growth kinetic curves, we established that biomass density was maximum and constant at a pO2 in the range 0.006 to 6 mg/L and growth rate was maximum at a pO2 of about 2 mg/L. At a pO2 over 2 mg/L, oxygen level had a toxic effect on A. aeolicus growth. Under optimal oxygen supply (pO2 = 1.5 mg/L) and when H2 (100 mL/min) is the growth-limiting factor, energy catabolism is diverted towards thiosulfate consumption: at H2 flow-rates of 450 and 100 mL/min, 97% and 79% of O2 is reduced by hydrogen while 3% and 21% of O2 is reduced by thiosulfate, respectively. Under over-oxygenation conditions (pO2 = 10.5 and 12 mg/L), growth was correlated to high thiosulfate consumption whereas the expression of genes encoding hydrogenases was significantly downregulated and hydrogenase activity was null.

Lateral gene transfer (LGT) is an important factor contributing to the evolution of prokaryotic genomes. The Aquificae are a hyperthermophilic bacterial group whose genes show affiliations to many other lineages, including the hyperthermophilic Thermotogae, the Proteobacteria, and the Archaea. Here I outline these scenarios and consider the fit of the available data, including two recently sequenced genomes from members of the Aquificae, to different sets of predictions. Evidence from phylogenetic profiles and trees suggests that the ?-Proteobacteria have the strongest affinities with the three Aquificae analyzed. However, this phylogenetic signal is by no means the dominant one, with the Archaea, many lineages of thermophilic bacteria, and members of genus Clostridium and class ?-Proteobacteria also showing strong connections to the Aquificae. The phylogenetic affiliations of different functional subsystems showed strong biases: as observed previously, most but not all genes implicated in the core translational apparatus tended to group Aquificae with Thermotogae, while a wide range of metabolic systems strongly supported the Aquificae - ?-Proteobacteria link. Given the breadth of support for this latter relationship, a scenario of ?-proteobacterial ancestry coupled with frequent exchange among thermophilic lineages is a plausible explanation for the emergence of the Aquificae.

Le soufre est utilisé à des fins bioénergétiques par des micro-organismes tels que la bactérie hyperthermophile Aquifex aeolicus qui nécessite pour sa croissance de l'oxygène, de l'hydrogène et un composé soufré indispensable. Une soufre réductase réduisant des chaînes de soufre, une Sulfure Quinone Oxydoréductase (SQR) oxydant l'H2S et une Soufre Oxygénase Réductase (SOR) oxydant et réduisant simultanément des chaînes de soufre ont été caractérisées chez cette bactérie. L'organisation de certaines de ces enzymes dans des supercomplexes membranaires a également été démontrée.Nous avons montré qu'Aq_477, précédemment caractérisée comme une soufre transférase de la famille des rhodanèses, est capable (i) de « charger » des chaînes de soufre ; (ii) d'interagir avec deux partenaires (la soufre réductase et la SOR) ; (iii) de leur présenter ce substrat. Ceci conduit à une optimisation du métabolisme. Nous avons ainsi démontré l'implication directe d'Aq_477, rebaptisée SbdP pour Sulfur -binding -donating Protein, dans le métabolisme énergétique du soufre de la bactérie. Une analyse poussée du génome nous a permis de construire un nouveau modèle suggérant notamment un recyclage des composés soufrés entre différents systèmes enzymatiques. La recherche de l'existence d'un niveau d'organisation des complexes respiratoires supérieur aux supercomplexes chez Aquifex aeolicus nous a conduits à développer de nouvelles méthodes d'étude permettant de proposer plusieurs pistes de recherche. Enfin, nous avons montré l'existence d'un nanocompartiment protéique constitué de l'encapsuline Aq_1760, dans lequel vient s'ancrer la ferritine atypique à domaines en tandem Aq_331. / Sulfur can be used in energy metabolism by microorganisms as electron donor and acceptor. The hyperthermophilic bacterium Aquifex aeolicus, which need oxygen, hydrogen and an essential sulfur compound for its growth presents sulfur reduction and oxidation pathways linked to the energy synthesis. A sulfur reductase (reduction of sulfur chains), a Sulfide Quinone Oxidoreductase (SQR, oxidation of H2S) and a Sulfur Oxygenase Reductase (SOR, simultaneous oxidation and reduction of sulfur chains) have been characterized in this bacterium. It has also been shown that some of these enzymes are organized in membrane-bound supercomplexes.We have demonstrated that Aq_477, previously characterized as a sulfurtransferase belonging to the rhodanese superfamily, can load long sulfur chains and acts as a sulfur donor for its partners (sulfur reductase and SOR) which use these sulfur chains as substrate, thus optimizing the metabolism. These results show that Aq_477, renamed SbdP for Sulfur -binding -donating Protein, is involved in the sulfur energy metabolism of Aquifex aeolicus. The identification in the genome of some new proteins potentially involved in this metabolism permitted us to propose a new model which suggests a recycling of sulfur compounds between different enzymatic systems. We also looked for an organization level of respiratory complexes higher than supercomplexes, which led us to develop new study methods and propose several research trails. Finally, we have shown the existence of protein nanocompartment constituted by the encapsulin Aq_1760, in which the atypical tandem-domain ferritin Aq_331 is anchored.

<p>Members of the thioredoxin superfamily of proteins catalyze disulfide bond reduction and oxidation using the active site C-X-X-C sequence. In hyperthermophilic organisms, cysteine side chains were expected in low abundance since they were not believed to endure the high temperatures under which they grow. Recently it has been found that disulfide bonds in hyperthermophiles are more frequent, the higher the growth temperature of the organism. This is perhaps used as an adaptation to high temperature in order to stabilize proteins under harsh conditions. A protein with sequence and structural similarities to mesophilic members of the thioredoxin superfamily, called protein disulfide oxidoreductases (PDO), has been found in the genomes of recently sequenced hyperthermophilic genomes. In this study PDOs from the hyperthermophiles Aquifex aeolicus (AaPDO) and Pyrococcus furiosus (PfPDO) have been investigated. The molecular weight is about 26 kDa and their structures are comprised of two homologous thioredoxin folds, referred to as the N-unit and the C-unit, each containing a C-X-X-C motif. The sequence identity between the two units and the two proteins is low, but they are still structurally very similar. The function of these proteins in vivo is unknown. As a first step in characterizing the activity of these proteins, the redox characteristics of these domains will be investigated. During this project, the genes for AaPDO and PfPDO have been cloned into overexpression vectors, expressed in E. coli and purified to homogeneity. To allow for individual study of the activities of two units, mutated proteins were prepared in which the cysteine residues of the N-unit (AaPDOnm and PfPDOnm) and of the C-unit (AaPDOcm and PfPDOcm) and purified. Circular dichroism spectra recorded of the wild type and mutants indicate that all purified proteins are folded and that the N- and C-unit active site mutants are structurally similar to the corresponding wild type proteins.</p>

Members of the thioredoxin superfamily of proteins catalyze disulfide bond reduction and oxidation using the active site C-X-X-C sequence. In hyperthermophilic organisms, cysteine side chains were expected in low abundance since they were not believed to endure the high temperatures under which they grow. Recently it has been found that disulfide bonds in hyperthermophiles are more frequent, the higher the growth temperature of the organism. This is perhaps used as an adaptation to high temperature in order to stabilize proteins under harsh conditions. A protein with sequence and structural similarities to mesophilic members of the thioredoxin superfamily, called protein disulfide oxidoreductases (PDO), has been found in the genomes of recently sequenced hyperthermophilic genomes. In this study PDOs from the hyperthermophiles Aquifex aeolicus (AaPDO) and Pyrococcus furiosus (PfPDO) have been investigated. The molecular weight is about 26 kDa and their structures are comprised of two homologous thioredoxin folds, referred to as the N-unit and the C-unit, each containing a C-X-X-C motif. The sequence identity between the two units and the two proteins is low, but they are still structurally very similar. The function of these proteins in vivo is unknown. As a first step in characterizing the activity of these proteins, the redox characteristics of these domains will be investigated. During this project, the genes for AaPDO and PfPDO have been cloned into overexpression vectors, expressed in E. coli and purified to homogeneity. To allow for individual study of the activities of two units, mutated proteins were prepared in which the cysteine residues of the N-unit (AaPDOnm and PfPDOnm) and of the C-unit (AaPDOcm and PfPDOcm) and purified. Circular dichroism spectra recorded of the wild type and mutants indicate that all purified proteins are folded and that the N- and C-unit active site mutants are structurally similar to the corresponding wild type proteins.

Ribonuclease III is a highly-conserved bacterial enzyme that cleaves double-stranded (ds) RNA structures, and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, those crystals involved complexes containing either cleaved RNA, or a mutant RNase III that is catalytically inactive. In addition, neither the biochemical properties of A. aeolicus (Aa)-RNase III, nor the reactivity epitopes of its cognate substrates are known. The goal of this project is to use Aa-RNase III, for which there is atomic-level structural information, to determine how RNase III recognizes its substrates and selects the target site. I first purified recombinant Aa-RNase III and defined the conditions that support its optimal in vitro catalytic activity. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of 70-85°C, a pH optimum of 8.0, and with either Mg2+ or Mn2+ supports efficient catalysis. Cognate substrates for Aa-RNase III were identified and their reactivity epitopes were characterized, including the specific bp sequence elements that determine processing reactivity and selectivity. Small RNA hairpins, based on the double-stranded structures associated with the Aquifex 16S and 23S rRNA precursors, are cleaved in vitro at sites that are consistent with production of the immediate precursors to the mature rRNAs. Third, the role of the dsRBD in scissile bond selection was examined by a mutational analysis of the conserved interactions of RNA binding motif 1 (RBM1) with the substrate proximal box (pb). The individual contributions towards substrate recognition were determined for conserved amino acid side chains in the RBM1. It also was shown that the dsRBD plays key dual roles in both binding energy and selectivity, through RBM1 responsiveness to proximal box bp sequence. The dsRBD is specifically responsive to an antideterminant (AD) bp in pb position 2. The relative structural rigidity of both dsRNA and dsRBD rationalizes the strong effect of an inhibitory bp at pb position 2: disruption of one RBM1 side chain interaction can effectively disrupt the other RBM1 side chain interactions. Finally, a cis-acting model was developed for subunit involvement in substrate recognition by RNase III. Structurally asymmetric mutant heterodimers of Escherichia coli (Ec)-RNase III were constructed, and asymmetric substrates were employed to reveal how RNase III can bind and deliver hairpin substrates to the active site cleft in a pathway that requires specific binding configurations of both enzyme and substrate. / Chemistry

Thermal stability of theG37 tRNA methyltransferase proteins from Thermotoga maritima and Aquifex aeolicus have been compared using Differential Scanning Calorimetry. It was shown that the Thermotoga protein is remarkably stable and is denatured at temperatures in excess of 100 degrees Centigrade. The Aquifex aeolicus protein was less stable, denaturing broadly at temperatures between 55oC and 100oC. In contrast, the mesophilic E. coli protein was completely denatured at 55oC. Enzymatic activity of the proteins was measured at various temperatures. Both the Thermotoga and Aquifex enzymes are active at ambient temperatures, and display a significant decrease in activity when the temperature is raised above 50oC. This may relate to subtle changes in protein structure causing an effect on the tRNA based assay. Both enzymes contain inter subunit disulfide bonds which might contribute to thermal stability. Assays of the enzymes in the presence of high concentrations of Dithiothreitol (DTT) did not significantly reduce activity at higher temperatures, but did stimulate activity at lower temperatures. Site directed mutagenesis of non -conserved protein sequences within Thermotoga maritima were initiated in order to determine what structures might confer heat stability on the protein. Alanine mutagenesis of lysine residues 103,104 led to reduced catalytic activity, but did increased activity at higher temperatures. Aspartate is the most common residue at the relative position 166 in the variable loop of most TrmD genes. It has been shown that in E. coli this is essential for catalytic activity and possibly the residue which carries out N1 deprotonation on residue G37 in tRNA. In Thermotoga glutamate is present at this position. Alanine mutagenesis of this residue did not eliminate activity suggesting another nearby residue may function in this capacity in the Thermotoga TrmD protein.