Only a few hyperthermophilic archaea are found to have a complete nonoxidative stage of pentose phosphate pathway (NOPPP), which indicates that most archaeal hyperthermophiles are partially missing pentose metabolizing enzymes. However, very limited research has been done in this interesting field. Although few hyperthermophilic enzymes in PPP have been studied in detail, the enzymatic activities reported previously were underestimated because assays were performed at temperatures much lower than 80°C. The commercially available auxiliary enzymes used in those assays were not thermostable, which limited assay temperature as well. The substrates used in those assays are extremely expensive, which is another factor limiting the study in this area. In this project, an inexpensive and accurate assay system was developed to overcome these limitations. Genes encoding several auxiliary enzymes for PPP enzyme assays were cloned from selected hyperthermophiles and overexpressed in E.coli Rossetta2 TM (DE3). These enzymes were glyceraldehydes-3-phosphate dehydrogenase from Thermotoga maritima (TmGAPDH), triosephosphate isomerase from Pyrococcus furiosus (PfTIM), glycerophosphate dehydrogenase from Pyrococcus furiosus (PfG3PDH) and Aeropyrum pernixK1 (ApG1PDH), transketolase from T. maritima (TmTK), xylose isomerase from T. maritima (TmXI) and Thermotoga neapolitana (TpXI), and xylulose kinase from T. maritima (TmXuK). Their activities were determined under anaerobic conditions at 80°C in both cell free extracts and for purified enzymes. The assay system contained the following parts: A) TmGAPDH, TmXI or TpXI, TmXuK (TmTK), PfTIM, and PfG3PDH or ApG1PDH as auxiliary enzymes for TK (XuK) assay; B) TmGAPDH, PfTIM, and PfG3PDH or ApG1PDH for transaldolase (TAL) assay. D-xylose, instead of the traditional assay substrate xylulose-5-phosphate (xylulose), was used as the substrate in this assay system for the determination of TK (XuK) activity. The activities of XuK, TK, and TAL were also determined in several hyperthermophilic bacteria and archaea. All enzymes served as paradigms to prove the feasibility of using the new assay system for other hyperthermophilic PPP enzymes. The species of hyperthermophiles used in this study were T. maritima, P. furiosus, Thermococcus guaymasensis, T. neapolitana, Thermotoga hypogea and T. petrophila. Two different methods were tested for the TAL assay (part B), with either TmGAPDH or PfTIM plus TmG3PDH as the auxiliary enzymes. Similar characteristics were obtained using both methods. The existence of TAL in all selected hyperthermophiles might indicate that the existence of the PPP is functioning among them. The XuK assays in selected hyperthermophiles were successfully conducted using the new assay system (part A). T. petrophila showed the highest XuK activity (0.3 U/mg), indicating the feasibility of the assay system’s application in the determination of hyperthermophilic PPP enzymes at high temperatures (80°C). TmTK activity may also be determined using the new assay system if the auxiliary enzyme XuK activity would be higher. Therefore, the new assay system developed in this project demonstrates dual functions for both XuK and TK assays in hyperthermophiles.

Only a few hyperthermophilic archaea are found to have a complete nonoxidative stage of pentose phosphate pathway (NOPPP), which indicates that most archaeal hyperthermophiles are partially missing pentose metabolizing enzymes. However, very limited research has been done in this interesting field. Although few hyperthermophilic enzymes in PPP have been studied in detail, the enzymatic activities reported previously were underestimated because assays were performed at temperatures much lower than 80°C. The commercially available auxiliary enzymes used in those assays were not thermostable, which limited assay temperature as well. The substrates used in those assays are extremely expensive, which is another factor limiting the study in this area. In this project, an inexpensive and accurate assay system was developed to overcome these limitations. Genes encoding several auxiliary enzymes for PPP enzyme assays were cloned from selected hyperthermophiles and overexpressed in E.coli Rossetta2 TM (DE3). These enzymes were glyceraldehydes-3-phosphate dehydrogenase from Thermotoga maritima (TmGAPDH), triosephosphate isomerase from Pyrococcus furiosus (PfTIM), glycerophosphate dehydrogenase from Pyrococcus furiosus (PfG3PDH) and Aeropyrum pernixK1 (ApG1PDH), transketolase from T. maritima (TmTK), xylose isomerase from T. maritima (TmXI) and Thermotoga neapolitana (TpXI), and xylulose kinase from T. maritima (TmXuK). Their activities were determined under anaerobic conditions at 80°C in both cell free extracts and for purified enzymes. The assay system contained the following parts: A) TmGAPDH, TmXI or TpXI, TmXuK (TmTK), PfTIM, and PfG3PDH or ApG1PDH as auxiliary enzymes for TK (XuK) assay; B) TmGAPDH, PfTIM, and PfG3PDH or ApG1PDH for transaldolase (TAL) assay. D-xylose, instead of the traditional assay substrate xylulose-5-phosphate (xylulose), was used as the substrate in this assay system for the determination of TK (XuK) activity. The activities of XuK, TK, and TAL were also determined in several hyperthermophilic bacteria and archaea. All enzymes served as paradigms to prove the feasibility of using the new assay system for other hyperthermophilic PPP enzymes. The species of hyperthermophiles used in this study were T. maritima, P. furiosus, Thermococcus guaymasensis, T. neapolitana, Thermotoga hypogea and T. petrophila. Two different methods were tested for the TAL assay (part B), with either TmGAPDH or PfTIM plus TmG3PDH as the auxiliary enzymes. Similar characteristics were obtained using both methods. The existence of TAL in all selected hyperthermophiles might indicate that the existence of the PPP is functioning among them. The XuK assays in selected hyperthermophiles were successfully conducted using the new assay system (part A). T. petrophila showed the highest XuK activity (0.3 U/mg), indicating the feasibility of the assay system’s application in the determination of hyperthermophilic PPP enzymes at high temperatures (80°C). TmTK activity may also be determined using the new assay system if the auxiliary enzyme XuK activity would be higher. Therefore, the new assay system developed in this project demonstrates dual functions for both XuK and TK assays in hyperthermophiles.

It was estimated that more than one hundred open reading frames in Pyrococcus furiosus and Thermotoga maritima could encode flavoproteins based on the results of motif search and comparison of genomic annotation to the experimentally characterized flavoproteins. However, only a few flavoproteins have been characterized from those anaerobic hyperthermophiles. It was found T. maritima and Thermotoga hypogea were able to grow in the presence of micromolar level of oxygen. As part of an oxygen removal system, the presence of NADH oxidase was detected in both microorganisms. In T. hypogea, NADH oxidase activity was constant regardless of the presence of oxygen, while in T. maritima it was increased in the presence of oxygen. The purified T. hypogea NADH oxidase was a flavin adenine dinucleotide (FAD)-containing homodimer with subunit molecular mass of 50 kDa. In addition to NADH oxidase activity, it also demonstrated activity of dihydrolipoamide dehydrogenase (DLDH), which is probably involved in glycine decarboxylation. The purified NADH oxidase from T. maritima was a heterodimeric protein of two subunits with molecular weight of 54 and 46 kDa, which were identified to be encoded by TM1432 and TM1433, respectively. Each subunit bore one FAD and the large subunit had one bacterioferritin-associated ferredoxin (BFD)-like [2Fe-2S]-center. Although the T. maritima NADH oxidase had very unusual oxygen sensitivity, the oxygen inactivated enzyme could be fully recovered by incubating with reducing reagents anaerobically. The NADH oxidases from both T. hypogea and T. maritima catalyzed the reduction of oxygen only to hydrogen peroxide. NADH-dependent peroxidase activities were detected in both T. maritima and T. hypogea, suggesting the presence of a multi-component oxygen detoxification system in Thermotoga species. In addition to its NADH oxidase activity, the enzyme from T. maritima exhibited FAD-linked glycerol-3-phosphate dehydrogenase (FAD-GPDH) activity. Along with the glycerol kinase, the FAD-GPDH took part in glycerol utilization in T. maritima. Ferredoxin NAD+ oxidoreductase (FNOR) activity was detected in T. maritima using an NADH:benzyl viologen oxidoreductase (BVOR) assay. The purified enzyme was a homodimeric FAD-containing protein with subunit molecular mass of 37 kDa. The purified enzyme was very active in catalyzing the reduction of BV and methyl viologen (MV) using either NADH or NADPH as electron donor and could indeed catalyze the reduction of NAD+ with the reduced ferredoxin from T. maritima. The purified enzyme was further identified to be encoded by TM0869 and annotated as thioredoxin reductase (TrxR). T. maritima TrxR could not use commercially available thioredoxin (Trx) from Spirulina, but the Trx purified from T. maritima. T. maritima Trx was identified to be encoded by TM0868 and annotated as glutaredoxin (Grx)-like protein, which showed both thioredoxin (Trx) and Grx activity. The purified T. maritima TrxR could catalyze the Trx-dependent reduction of both insulin and DTNB using NAD(P)H as electron donor. The identified Trx-TrxR system in T. maritima is the first one characterized in hyperthermophilic bacteria. T. hypogea has great potential in microbial hydrogen production. The key enzyme involved in this process, hydrogenase, has not been studied yet. The growth-dependent hydrogenase activity was detected in T. hypogea, from which a homotetrameric hydrogenase was purified. The purified T. hypogea hydrogenase did not contain any flavin prosthetic group as speculated, but [Fe-S]-centers. The hydrogenase could catalyze both BV and MV-dependent hydrogen oxidation and MV-dependent hydrogen evolution. Neither NAD(P)H nor NAD(P) could be used as electron carrier for this enzyme. T. hypogea hydrogenase could utilize ferredoxin as electron carrier for both production and oxidation of hydrogen, which suggests that the purified hydrogenase plays an important role in hydrogen metabolism of T. hypogea. It was concluded that flavoproteins can be involved at least in several very important cellular processes such as detoxification of oxygen, utilization of glycerol, redox regulation, and hydrogen metabolism in hyperthermophiles.

Only a few hyperthermophilic archaea are found to have a complete nonoxidative stage of pentose phosphate pathway (NOPPP), which indicates that most archaeal hyperthermophiles are partially missing pentose metabolizing enzymes. However, very limited research has been done in this interesting field. Although few hyperthermophilic enzymes in PPP have been studied in detail, the enzymatic activities reported previously were underestimated because assays were performed at temperatures much lower than 80??C. The commercially available auxiliary enzymes used in those assays were not thermostable, which limited assay temperature as well. The substrates used in those assays are extremely expensive, which is another factor limiting the study in this area. In this project, an inexpensive and accurate assay system was developed to overcome these limitations. Genes encoding several auxiliary enzymes for PPP enzyme assays were cloned from selected hyperthermophiles and overexpressed in E.coli Rossetta2 TM (DE3). These enzymes were glyceraldehydes-3-phosphate dehydrogenase from Thermotoga maritima (TmGAPDH), triosephosphate isomerase from Pyrococcus furiosus (PfTIM), glycerophosphate dehydrogenase from Pyrococcus furiosus (PfG3PDH) and Aeropyrum pernixK1 (ApG1PDH), transketolase from T. maritima (TmTK), xylose isomerase from T. maritima (TmXI) and Thermotoga neapolitana (TpXI), and xylulose kinase from T. maritima (TmXuK). Their activities were determined under anaerobic conditions at 80?? iv C in both cell free extracts and for purified enzymes. The assay system contained the following parts: A) TmGAPDH, TmXI or TpXI, TmXuK (TmTK), PfTIM, and PfG3PDH or ApG1PDH as auxiliary enzymes for TK (XuK) assay; B) TmGAPDH, PfTIM, and PfG3PDH or ApG1PDH for transaldolase (TAL) assay. D-xylose, instead of the traditional assay substrate xylulose-5-phosphate (xylulose), was used as the substrate in this assay system for the determination of TK (XuK) activity. The activities of XuK, TK, and TAL were also determined in several hyperthermophilic bacteria and archaea. All enzymes served as paradigms to prove the feasibility of using the new assay system for other hyperthermophilic PPP enzymes. The species of hyperthermophiles used in this study were T. maritima, P. furiosus, Thermococcus guaymasensis, T. neapolitana, Thermotoga hypogea and T. petrophila. Two different methods were tested for the TAL assay (part B), with either TmGAPDH or PfTIM plus TmG3PDH as the auxiliary enzymes. Similar characteristics were obtained using both methods. The existence of TAL in all selected hyperthermophiles might indicate that the existence of the PPP is functioning among them. The XuK assays in selected hyperthermophiles were successfully conducted using the new assay system (part A). T. petrophila showed the highest XuK activity (0.3 U/mg), indicating the feasibility of the assay system???s application in the determination of hyperthermophilic PPP enzymes at high temperatures (80??C). TmTK activity may also be determined using the new assay system if the auxiliary enzyme XuK activity would be higher. Therefore, the new assay system developed in this project demonstrates dual functions for both XuK and TK assays in hyperthermophiles.

Hyperthermophiles grow optimally at 80 ??C and above, and many of them have the ability to utilize various carbohydrates as carbon source and produce ethanol as an end product. Alcohol dehydrogenase (ADH) is a key enzyme responsible for alcohol production, catalyzing interconversions between alcohols and corresponding ketones or aldehydes. ADHs from hyperthermophiles are of great interests due to their thermostability, high activity and enantioselectivity. The gene encoding ADH from hyperthermophilic archaeon Thermococcus guaymasensis was cloned, sequenced and over-expressed. DNA fragments of the genes encoding the ADHs were amplified directly from the corresponding genomic DNA by combining the use of conventional and inverse PCRs. The entire gene was detected to be 1092 bp and the deduced amino acid sequence had a total of 364 amino acids with a calculated molecular mass of 39463 Dalton. The enzyme belonged to the family of zinc-containing ADHs with catalytic zinc only. It was verified that the enzyme had binding motifs of catalytic zinc only (GHEX2GX5GX2V, residues 62-76) and coenzyme NADP (GXGX2G, residues 183-188). The tertiary structural modeling showed two typical domains, one catalytic domain close to amino-terminal (N-terminal) end and one coenzyme-binding domain close to carboxy-terminal (C-terminal) end. Since its codon usage pattern seemed to be different from that of Escherichia coli, the enzyme was over-expressed in the E. coli codon plus strain using pET-30a vector. The recombinant enzyme was detected to be soluble and active (1073 U/mg), which was virtually the same to the native enzyme (1049 U/mg). The recombinant ADH possessed almost identical properties with the native enzyme. The optimal pHs for ethanol oxidation and acetaldehyde reduction were 10.5 and 7.5 respectively, while the activity for alcohol oxidation was much higher than that of aldehyde reduction. The enzyme activity was inhibited in the presence of 100 ??M Zn2+ in the assay mixture and it has a half-life of 6 hours after exposure to air. Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 ??C. The gene encoding an alcohol dehydrogenase from T. hypogea was cloned, sequenced and over-expressed. The gene sequence (1164 bp) was obtained successfully by sequencing all the DNA fragments amplified from PCR. The deduced amino acid sequence was found to have high degrees of identity (~72%) to iron-containing ADHs from Thermotoga species and harbored typical iron and NADP-binding motifs, Asp195His199His268His282 and Gly39Gly40Gly41Ser42, respectively. The structural modeling showed that N-terminal domain of ThADH contained ??/??-dinucleotide-binding motif and its C-terminal domain was ??-helix-rich region including iron-binding motif. The gene encoding T. hypogea ADH was functionally expressed in E. coli using the vector pET-30a. The recombinant protein was expressed optimally in E. coli grown in the presence of 1 mM ferrous and induced by 0.4-0.6 mM IPTG. The recombinant enzyme was found to be soluble, active and thermostable, and had a subunit size of 43 kDa revealed by SDS-PAGE analyses. The native ADH from T. hypogea was purified to homogeneity for comparative analysis using a three-step liquid chromatography while the recombinant ADH over-expressed in E. coli was isolated by a simpler procedure including one-hour heat treatment. The activity of the purified recombinant enzyme was 69 U/mg and presented almost identical properties with the native enzyme. The optimal pHs for ethanol oxidation and acetaldehyde reduction were 11.0 and 8.0 respectively, while activity for alcohol oxidation were higher than that of aldehyde reduction. The enzyme was oxygen sensitive and it had a half-life (t1/2) of 20 minutes after exposed to air. The enzyme remained 50% activity after incubation at 70 ??C for 2 hours. Successful high-level expression of T. hypogea ADH in E. coli will significantly facilitate further study on the catalytic mechanism of iron-containing ADHs. In summary, both zinc- and iron-containing ADHs from two hyperthermophiles were successfully cloned, sequenced and overexpressed in mesophilic host E. coli, and such a high-level expression of ADH genes provides possibilities for three dimensional structural analysis by X-ray crystallography and enzyme modification by mutagenesis, which will help further explore mechanisms of catalysis and protein thermostability of iron and zinc-containing ADHs and their potential applications in biotechnology.

Extreme thermophiles and hyperthermophiles are microorganisms capable of growing optimally at 65-79??C and 80??C plus, respectively. Many of the enzymes isolated from them are thermostable, which makes them a potential resource for research and industrial applications. An increasing number of hyper/thermophiles is shown to be able to produce ethanol as an end-metabolite. Despite characterization of many alcohol dehydrogenases (ADHs) with a potential role in the production of ethanol, to date there has been no significant progress in identifying the enzymes responsible for the production of acetaldehyde, which is an intermediate in production of ethanol from pyruvate.<br> Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles is a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus.<br> The bifunctional and TPP-containing POR/PDC enzyme was isolated and characterized from the ethanol-producing hyperthermophilic archaeon Thermococcus guaymasensis (Topt=88??C), as well as the bacteria Thermotoga hypogea (Topt=70??C) and Thermotoga maritima (Topt=80??C). The T. guaymasensis enzyme was purified anaerobically to homogeneity as judged by SDS-PAGE analysis. POR and PDC activities were co-eluted from each of the chromatographic columns, and the ratio of POR to PDC activities remained constant throughout the purification steps. All of the enzyme activities were CoA- and TPP-dependent and highly sensitive toward exposure to air. The apparent kinetic parameters were determined for the main substrates, including pyruvate and CoA for each activity. Since the genome sequence of T. guaymasensis and T. hypogea were not available, sequences of the genes encoding POR were determined via primer walking and inverse PCR.<br> A novel enzyme capable of catalyzing the production of acetaldehyde from pyruvate in hyperthermophiles was also characterized. The enzyme contained TPP and flavin and was expressed as recombinant histidine-tagged protein in the mesophilic host Escherichia coli. The new enzyme was a bifunctional enzyme catalyzing another reaction as the major reaction besides catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde.<br> Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g. Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles used in this study.<br> In conclusion, no commonly-known PDCs was found in hyperthermophiles, but two types of acetaldehyde-producing enzymes were present in various bacterial and archaeal hyperthermophiles. Although the deduced amino acid sequences from different hyperthermophiles are quite similar, the levels of POR and PDC activities appeared to vary significantly between the archaeal and bacterial enzymes, which most likely reflects the different physiological implications of each activity.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13792号 / 工博第2896号 / 新制||工||1427(附属図書館) / 26008 / UT51-2008-C708 / 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

This dissertation research aims to show that there are deterministic microbial distribution patterns based on quantifiable environmental thresholds by determining and rationalizing the relative abundances of hyperthermophilic methanogens, autotrophic iron(III) oxide reducers, and heterotrophic sulfur reducers within deep-sea hydrothermal vents. Organisms of these metabolisms are predicted to be relatively more abundant in different regions depending on environmental conditions such as reduction potential, organic carbon, and hydrogen availability. The relative abundances of these metabolic groups within samples from the Endeavour Segment and Axial Volcano in the northeastern Pacific Ocean were determined. Iron(III) oxide reducers were detected in nearly all samples while methanogens were generally not present or present in concentrations lower than those of the iron(III) reducers. To determine growth constraints and the effect of hydrogen concentration on hyperthermophilic methanogen growth kinetics, Methanocaldococcus jannaschii and two new Methanocaldococcus field isolates were grown at varying hydrogen concentrations. The hydrogen-dependent growth kinetics for all three strains were statistically indistinguishable, exhibiting longer doubling times and lower maximum cell concentrations with decreasing hydrogen concentrations until growth ceased below 17-23 μM. This minimum hydrogen concentration for hyperthermophilic methanogenesis was correlated with field microbiology and fluid geochemistry data from the Endeavour Segment and Axial Volcano. Anomalously high methane concentrations and thermophilic methanogens were only observed in fluid samples where hydrogen concentrations were above this predicted threshold. Aside from anomalous sites, methanogens are predicted to be hydrogen limited, and may rely on hydrogen produced by heterotrophs as suggested by in situ sampling and co-culture experiments. Models and kinetic experiments suggest that iron(III) oxide reducers are not hydrogen limited under the same conditions. A Methanocaldococcus strain that we isolated from Axial Volcano and used in our hydrogen threshold experiments was bioenergetically modeled over its range of growth temperatures, pH, NaCl concentrations, and NH4Cl concentrations. Its methane production rates and growth energies were largely constant but increased at superoptimal temperatures and when nitrogen was limiting. The results of this research demonstrate that the rates of and constraints on metabolic processes can be used to predict the distribution and biogeochemical impact of hyperthermophiles in deep-sea hydrothermal vent systems.

La sécrétion de vésicules membranaires (VMs) constitue un processus physiologique important qui a particulièrement été étudié chez les Bactéries et les Eucaryotes. La récente découverte de la production de VMs chez les Archées souligne cependant que ce phénomène est universel et suggère que le dernier ancêtre commun aux trois domaines, LUCA (Last Universal Common Ancestor), produisait certainement des VMs. Les VMs des Archées n’ayant pour le moment été étudiées que chez certaines Crénarchées (ex : G/ Sulfolobus), nous avons entrepris de caractériser les VMs produites par un groupe d’Euryarchées hyperthermophiles anaérobies, les Thermococcales. Dans la première partie de cette étude, nous avons examiné le mécanisme de production ainsi que la composition en lipides et en protéines des VMs de trois espèces de Thermococcales: Thermococcus kodakaraensis, Thermococcus gammatolerans et Thermocococus sp. 5-4. Nous avons observé que les VMs sont sécrétées par un processus de bourgeonnement à partir de l’enveloppe cellulaire similaire à la formation des ectosomes par les cellules eucaryotes. De plus, les VMs sont fréquemment libérées en groupes, formant de grosses protubérances ou des filaments ressemblant aux nanopodes récemment décrits chez les Bactéries. Des différences de structure et de composition protéique sont observées entre les VMs des trois souches étudiées. Cependant, les VMs et les membranes cellulaires d’une même souche ont des compositions protéique et lipidique très proches, confirmant que les VMs sont produites à partir des membranes des cellules. Les VMs et les membranes cellulaires des trois souches comportent notamment un récepteur de peptides de la famille OppA (Oligopeptide-binding protein A) et des homologues de cette protéine ont été identifiés dans les VMs de certaines souches de Sulfolobus.Les VMs sécrétées par les Thermococcales sont associées à de l’ADN et cette association les protègent contre la thermodégradation. Nous montrons dans notre étude que les cellules de T. kodakaraensis transformées avec le plasmide navette plC70 relâchent des VMs comportant ce plasmide. De façon intéressante, ces VMs peuvent être utilisées pour transférer pLC70 à des cellules dénuées de plasmides, suggérant que les VMs pourraient être impliquées dans le transfert d’ADN entre cellules à haute température.Dans la seconde partie de cette étude, nous nous sommes particulièrement intéressés à la souche Thermococcus nautilus, une Thermococcale produisant des VMs associées de manière sélective à deux plasmides contenus dans la cellule. L’un d’eux correspond notamment à un génome viral défectueux de la lignée d’adenovirus PRD1. Ceci indique que les VMs peuvent être un moyen de transport pour des génomes viraux et suggère que la production de VMs par des cellules ancestrales pourraient avoir joué un rôle dans l’apparition des virus.En plus d’être impliquées dans le transport de plasmides/virus, les VMs produites par T. nautilus exercent un effet toxique sur certaines souches de Thermococcales, probablement dû au convoyage de toxines. Même si ces « thermococcines » nécessitent d’être caractérisées, il s’agit de la première mise en évidence d’une activité toxique liée aux VMs chez les Thermococcales. / Secretion of membrane vesicles (MVs) is an important physiological process that has been extensively studied in Bacteria and Eukarya. The recent discovery that Archaea produce MVs shows that this process is universal and suggests that the Last Universal Common Ancestor, LUCA, certainly produced MVs. As these archaeal MVs have been only studied in some Crenarchaeota (ex: G/ Sulfolobus), we started characterizing MVs produced by Thermococcales, a group of hyperthermophilic anaerobic Euryarchaeota.In the first part of this study we examined the mechanism of production as well as the protein and lipid composition of MVs produced by three strains of Thermococcales: Thermococcus kodakaraensis, Thermococcus gammatolerans and Thermocococus sp. 5-4. We observed that MVs are released by a budding process from the cell envelope that is similar to ectosome formation in eukaryotic cells. Moreover, clusters of MVs often form filamentous structures and protuberances on cell surfaces, resembling recently described bacterial nanopods. Differences in structure are observable between MVs of the three species, as well as in their protein composition. However, MVs and cell membranes from the same species have a quite similar protein and lipid composition, confirming that MVs are produced from cell membranes. A major protein present in cell membranes and MVs from the three strains is the oligopeptide-binding proteins (OppA), which has homologues in MVs from Sulfolobus species. Thermococcales MVs harbor DNA and protect this DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harboring this plasmid. Interestingly, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature. In the second part of this study, we were specially interested in the strain Thermococcus nautilus, a Thermococcale that produces MVs selectively enriched in two plasmids from the cell. Notably, one of them corresponds to the genome of a defective virus from PRD1-adenovirus lineage. This indicates that MVs can be used as vehicles for the transport of viral genomes and suggests that production of MVs by ancestral cells could have played a role in the origin of viruses.In addition to be involved in transport of plasmids/viruses, MVs from T. nautilus display a toxic effect on some strains of Thermococcales, maybe due to the delivery of toxins. Even if these “thermococcins” remain to be characterized, this is the first time that a toxic activity associated with MVs has been shown in Thermococcales.

Les archées sont principalement connues pour leur capacité à croître et survivre dans des conditions extrêmes de température, pression, pH, etc. qui sont hostiles à l’homme. Néanmoins, il est désormais clair que les archées sont aussi présentes de manière ubiquitaire dans divers environnements. L’étude détaillée des différents aspects de la biologie de ces microorganismes a amené à des découvertes pour le moins inattendues comme celle de la virosphère associée aux archées qui est unique. En effet, plusieurs virus infectant les archées ont été isolés et présentent une incroyable diversité tant au niveau morphologique que génomique et ne ressemblent aucunement aux virus connus de bactéries ou d’eucaryotes. Récemment, l’analyse en détails du cycle viral a mis à jour de nouveaux mécanismes d’interactions avec la cellule hôte. Au cours de mes travaux de thèse, nous nous sommes intéressés aux systèmes virus-hôtes présents dans les milieux hyperthermiques et acidophiles en sélectionnant les virus fusiforme et filamenteux SSV1 et SIRV2 en tant que modèles d’étude. Tout d’abord, nous avons défini une nouvelle classification des virus fusiformes en basée sur l’analyse comparative des protéines structurales et des génomes viraux. L’ensemble des virus considérés forme un réseau global malgré le fait qu’ils ont été isolés dans des environnements distincts ; qu’ils infectent des hôtes qui sont distant phylogénétiquement parlant et que certains de leurs virions présentent une certaine pléomorphicité. Ensuite, la caractérisation en détails de l’architecture des virions fusiformes de SSV1 a révélé qu’ils étaient enveloppés, composés de protéines de capside glycosylées et contenaient le complexe nucléoprotéique. Finalement, nous nous sommes concentrés sur la manière dont les virus d’archées interagissent avec la cellule hôte. Alors que les virions de SIRV2 semblent utiliser une stratégie pour l’entrée qui est similaire aux bactériophages dits flagellotrophiques ; on observe que les virions de SSV1 emploient un mécanisme de sortie qui rappelle le bourgeonnement des virus eucaryotes enveloppés. L’ensemble de ces recherches participent à une meilleure compréhension de la biologie des archées ainsi que de leurs virus et permettent de définir des cibles intéressantes pour de futures études. / Although, archaea were initially regarded as exotic microorganisms capable of growing in conditions which are hostile to humans, it became clear that they are ubiquitous and abundant in various environments. Detailed studies focusing on different aspects of archaeal biology have led to many unexpected discoveries, including the unique virosphere associated with archaea. Indeed, highly diverse viruses characterized by uncommon virion shapes and mysterious genomic contents have been isolated that typically do not resemble viruses of either bacteria or eukaryotes. Recent analysis of the sequential events of the viral cycle resulted in major breakthroughs in the field. In the framework of my PhD studies, I have focused on two model hyperthermo-acidophilic virus-host systems, the spindle-shaped SSV1 and rod-shaped SIRV2, both infecting organisms of the genus Sulfolobus. Initially, we defined structure-based lineages for all known spindle-shaped viruses isolated from highly divergent hosts and residing in very different environments. Then, we provided insights into the architecture of spindle-shaped viruses by showing that SSV1 virions are composed of glycosylated structural proteins and contain a lipid envelope. Finally, we focused on virus-host interplay. Whereas SIRV2 virions appear to use a similar entry strategy as flagellotrophic bacteriophages, SSV1 virions employ an exit mechanism reminiscent of the budding of eukaryotic enveloped viruses. Collectively, these studies shed light on the biology of archaeal viruses and help to define interesting targets that should be the focus of intensive research in the next future.