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Microbes preserve membrane functionality under fluctuating environmental conditions by modulating their membrane lipid composition. Although several studies have documented membrane adaptations in Archaea, the influence of most biotic and abiotic factors on archaeal lipid compositions remains underexplored. Here, we studied the influence of temperature, pH, salinity, the presence/absence of elemental sulfur, the carbon source, and the genetic background on the core lipid composition of the hyperthermophilic neutrophilic marine archaeon Pyrococcus furiosus. Every growth parameter tested affected the core lipid composition to some extent, the carbon source and the genetic background having the greatest influence. Surprisingly, P. furiosus appeared to only marginally rely on the two major responses implemented by Archaea, i.e., the regulation of the ratio of diether to tetraether lipids and that of the number of cyclopentane rings in tetraethers. Instead, this species increased the ratio of glycerol monoalkyl glycerol tetraethers (GMGT, aka. H-shaped tetraethers) to glycerol dialkyl glycerol tetrathers (GDGT) in response to decreasing temperature and pH and increasing salinity, thus providing for the first time evidence of adaptive functions for GMGT. Besides P. furiosus, numerous other species synthesize significant proportions of GMGT, which suggests that this unprecedented adaptive strategy might be common in Archaea.Significance statementWe describe here the membrane adaptive strategies the hyperthermophilic, neutrophilic, and marine model archaeon Pyrococcus furiosus implements in response to one of the largest sets of environmental stressors tested to date, including temperature, pH, salinity, presence/absence of elemental sulfur, carbon source, and genetic background. In contrast to the other archaea investigated so far, which response mainly involves the modulation of their diether/tetraether ratio and/or of their average number of cyclopentane rings, P. furiosus regulates its monoalkyl (so called H-shaped) to dialkyl tetraether ratio. Our study thus provides for the first time evidence of adaptive functions of archaeal monoalkyl tetraethers towards low temperature and pH and high salinity.

International audience; The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes a putative 3-methlyadenine DNA glycosylase II (Tg-AlkA). Herein, we report biochemical characterization and catalytic mechanism of Tg-AlkA. The recombinant Tg-AlkA can excise hypoxanthine (Hx) and 1-methlyadenine (1-meA) from dsDNA with varied efficiencies at high temperature. Notably, Tg-AlkA is a bi-functional glycosylase, which is sharply distinct from all the reported AlkAs. Biochemical data show that the optimal temperature and pH of Tg-AlkA for removing Hx from dsDNA are ca.70°C and ca.7.0-8.0, respectively. Furthermore, the Tg-AlkA activity is independent of a divalent metal ion, and Mg 2+ stimulates the Tg-AlkA activity whereas other divalent ions inhibit the enzyme activity with varied degrees. Mutational studies show that the Tg-AlkA W204A and D223A mutants abolish completely the excision activity, thereby suggesting that residues W204 and D223 are involved in catalysis. Surprisingly, the mutations of W204, D223, Y139 and W256 to alanine in Tg-AlkA lead to the increased affinity for binding DNA substrate with varied degrees, suggesting that these residues are flexible for conformational change of the enzyme. Therefore, Tg-AlkA is a novel AlkA that can remove Hx and 1-meA from dsDNA, thus providing insights into repair of deaminated and alkylated bases in DNA from hyperthermophilic Thermococcus.

Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature. Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bo nd of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave AP-containing ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. Phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs. KEY POINTS: We first reported a novel DNA glycosylase with bifunctionality. Tba UDG247 possesses an AP lyase activity.

We announce the draft genome sequence of Collimonas pratensis PMB3(1), isolated from the Scleroderma citrinum mycorrhizosphere. In addition to its mineral-weathering effectiveness and antifungal activity, this strain is characterized by genomic features that give it great potential as a biocontrol and plant growth-promoting agent in nutrient-poor soils.

International audience; The hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes two uracil DNA glycosylases (UDGs): Tba UDG247 and Tba UDG194. Herein, we characterized biochemically Tba UDG194. Compared with Tba UDG247, Tba UDG194 exhibits different biochemical characteristics. At >85 °C, >90 cleavage percentage was observed, suggesting that Tba UDG194 can remove uracil from DNA at physiological temperature of its host. Thus, the enzyme is the most thermophilic glycosylase among all the reported UDGs. Furthermore, the optimal pH of the enzyme activity was estimated to be 10, which is higher than that of Tba UDG247. Similar to Tba UDG247, Tba UDG194 activity is independent on a divalent metal ion. Mn2+, Zn2+ and Cu2+ display inhibitory effect on the enzyme activity at varied degreed whereas Mg2+ and Ca2+ have no detectable effect on the enzyme activity. In addition, Tba UDG194 is a salt-tolerant enzyme that retains compromised activity at 600 mM NaCl. Furthermore, Tba UDG 194 displays the following substrate preference: U ≈ U/G > U/T > U/A > U/C. The Arrhenius activation energy was estimated to be 20.1 ± 3.4 kcal/mol, theoretically representing the energy barrier for uracil removal from DNA by Tba UDG194. Overall, our observations suggest that Tba UDG194 might be involved in removal of uracil in DNA in Thermococcus cells.

International audience; Recent studies show that NucS endonucleases participate in mismatch repair in several archaea and bacteria. However, the function of archaeal NucS endonucleases has not been completely clarified. Here, we describe a NucS endonuclease from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans (Tga NucS) that can cleave uracil (U)-and hypoxanthine (I)-containing dsDNA at 80°C. Biochemical evidence shows that the cleavage sites of the enzyme are at the second phosphodiester on the 5′-site of U or I, and at the third phosphodiester on the 5′-site of the opposite base of U or I, creating a double strand break with a 4-nt 5′-overhang.The ends of the cleaved product of Tga NucS are ligatable, possessing 5′-phosphate and 3′-hydroxyl termini, which can be utilized by DNA repair proteins or enzymes. Tga NucS displays a preference for U/G-and I/T-containing dsDNA over other pairs with U or I, suggesting that the enzyme is responsible for repair of U and I in DNA that arise from deamination. Biochemical characterization of cleaving U-and I-containing DNA by Tga NucS was also investigated. The DNA-binding results show that the enzyme exhibits a higher affinity for normal, U-and I-containing dsDNA than for normal, U-and I-containing ssDNA. Therefore, we present an alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease in hyperthermophilic archaea.

International audience; (about 150 words) During the last decades, high pressure has been an important physical parameter not only to study biomolecules, but also for its biotechnological applications. High pressure affects organism's ability to survive by altering most of cell's macromolecules. These effects can be used, for example, to inactivate microorganisms, enhance enzymatic reactions or to modulate cell activities. Moreover, some organisms are capable to growth under high pressures thanks to their adaptation at all cellular levels. Such adaptation confers a wide range of potentially interesting macromolecules still to be discovered. In this chapter, we firstly present the different effects of pressure on cells and the diverse strategies used to cope against this harsh environment. Secondly, we explored the pressure biotechnological applications on pressure-sensitive and adapted-pressure organisms.

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65–70 °C and retains its DNA ligation activity even after heated at 100 °C for 2 h, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0–10.0, and its optimal pH is 6.0–9.0. Tba ligase activity is dependent on a divalent metal ion: Mn2+, Mg2+, or Ca2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Tba ligase is ATP-dependent and can also use UTP as a weak cofactor; however, the enzyme with high concentrations could function without an additional nucleotide cofactor. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylated, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. In addition, the mismatches at the first position 3′ to the nick suppress Tba ligase activity more than those at the first position 5′ to the nick. The enzyme also discriminates more effectively mismatches at 3′ to the nick than those at 5′ to the nick in a ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.

International audience; The hyperthermophilic piezophile Thermococcus barophilus displays a strong stress response characterized by the accumulation of the organic osmolyte mannosylglycerate during growth under sub-optimal pressure conditions (0.1 MPa). Taking advantage of this known effect, the impact of osmolytes in piezophiles in an otherwise identical cellular context was investigated, by comparing T. barophilus cells grown under low or optimal pressures (40 MPa). Using neutron scattering techniques, we studied the molecular dynamics of live cells of T. barophilus at different pressures and temperatures. We show that in presence of osmolytes, cells present a higher diffusion coefficient of hydration water and an increase of bulk water motions at high temperature. In absence of osmolytes, the T. barophilus cellular dynamics is more responsive to high temperature and high hydrostatic pressure. These results give therefore clear evidences for a protecting effect of osmolytes on proteins.

International audience; Archaea, the most extremophilic domain of life, contain ether and branched lipids which provide extraordinary bilayer properties. We determined the structural characteristics of diether archaeal-like phospholipids as functions of hydration and temperature by neutron diffraction. Hydration and temperature are both crucial parameters for the self-assembly and physicochemical properties of lipid bilayers. In this study, we detected non-lamellar phases of archaeal-like lipids at low hydration levels, and lamellar phases at levels of 90% relative humidity or more exclusively. Moreover, at 90% relative humidity, a phase transition between two lamellar phases was discernible. At full hydration, lamellar phases were present up to 70ᵒC and no phase transition was observed within the temperature range studied (from 25 °C to 70 °C). In addition, we determined the neutron scattering length density and the bilayer's structural parameters from different hydration and temperature conditions. At the highest levels of hydration, the system exhibited rearrangements on its corresponding hydrophobic region. Furthermore, the water uptake of the lipids examined was remarkably high. We discuss the effect of ether linkages and branched lipids on the exceptional characteristics of archaeal phospholipids.

Biological cells are fascinating systems of inconceivable complexity, which fulfil various functions. Among others, cells are able to execute motions, to produce heat, to breathe, to subsist, to grow wand proliferate and to die. Science aims at deciphering the different functionalities and activities of, and inside, the cells and how their different components participate to them. The methods employed are also versatile, as optical approaches by microscopies, modelling and simulations, spectroscopies, thermodynamic measurements and much more, each procuring some pieces of the puzzle. Although the different investigations are laborious and time consuming, research is the only way to disentangle the world at the microscopic level surrounding us. In the present study, we cite a few examples of studies on whole cells and cell components by different neutron scattering techniques to illustrate the modern possibilities. As neutrons are not charged, they have interactions directly with the atomic nuclei and give access to structural as well as dynamical information through coherent and incoherent neutron scattering. These techniques can be applied to the same samples and under identical experimental conditions so that we can gain knowledge on the correlations between structural and dynamical functions. Here, we present applications of neutron experiments to decipher the behaviour of complex biological samples, which study was not possible by other probes. The first example focuses on the molecular basis of the adaptation of cells living under extreme conditions, such as Archaea from the deep sea hydrothermal vents which experience both high temperature and high pressure stresses. Molecular dynamics seems to play a key role for adaptation as it is increased for the proteome of cells from such environment, in contrast to common expectation. In the second example, we exposed endogenous nanoparticles, low density lipoproteins, to high hydrostatic pressure, to shed light on the flexibility and stability of such particles under extreme conditions. Here we found that the native particle was surprisingly resistant to pressure application, concerning both dynamics and structure, while a modified form thereof was not.

International audience; In live cells, high concentrations up to 300-400 mg/mL, as in Eschericia coli (Ellis, R J. Curr. Opin. Struct. Biol. 2001, 11, 114) are achieved which have effects on their proper functioning. However, in many experiments only individual parts of the cells as proteins or membranes are studied in order to get insight into these specific components and to avoid the high complexity of whole cells, neglecting by the way the influence of crowding. In the present study, we investigated cells of the order of Thermococcales, which are known to live under extreme conditions, in their intact form and after cell lysis to extract the effect of crowding on the molecular dynamics of the proteome and of water molecules. We found that some parameters characterizing the dynamics within the cells seem to be intrinsic to the cell type, as flexibility typical for the proteome, others are more specific to the cellular environment, as bulk water's residence time and some fractions of particles participating to the different motions, which make the lysed cells' dynamics similar to the one of another Thermococcale adapted to live under high hydrostatic pressure. In contrast to studies on the impact of crowding on pure proteins we show here that the release of crowding constraints on proteins leads to an increase in the rigidity and a decrease in the high pressure sensitivity. In a way similar to high pressure adaptation in piezophiles, the hydration water layer is decreased for the lysed cells, demonstrating a first link between protein adaptation and the impact of crowding or osmolytes on proteins.

Copper isotopic composition is altered in cancerous compared to healthy tissues. However, the rationale for this difference is yet unknown. As a model of Cu isotopic fractionation, we monitored Cu uptake in Saccharomyces cerevisiae, whose Cu import is similar to human. Wild type cells are enriched in 63Cu relative to 65Cu. Likewise, 63Cu isotope enrichment in cells without high-affinity Cu transporters is of slightly lower magnitude. In cells with compromised Cu reductase activity, however, no isotope fractionation is observed and when Cu is provided solely in reduced form for this strain, copper is enriched in 63Cu like in the case of the wild type. Our results demonstrate that Cu isotope fractionation is generated by membrane importers and that its amplitude is modulated by Cu reduction. Based on ab initio calculations, we propose that the fractionation may be due to Cu binding with sulfur-rich amino acids: methionine and cysteine. In hepatocellular carcinoma (HCC), lower expression of the STEAP3 copper reductase and heavy Cu isotope enrichment have been reported for the tumor mass, relative to the surrounding tissue. Our study suggests that copper isotope fractionation observed in HCC could be due to lower reductase activity in the tumor.

Six bacterial strains were isolated from the oak-Scleroderma citrinum ectomycorrhizosphere in acidic and nutrient-poor forest soil for their high efficacy to weather minerals. Four of the six isolates, PML1(12)T and PML1(4), PML1(14) and PML1(16), were further characterized extensively. They were Gram negative, obligate aerobic, motile, non spore forming and rod-shaped. The major fatty acids of strain PML1(12)T were cyclo-C17:0, cyclo-C19:0-ω8c, C16:0 and C18:1-ω7c. The GC content of the DNA was 60.8%. The 16S rRNA and GyrB analyses showed that the four PML strains formed a distinct phylogenetic lineage within the genus Caballeronia, most closely related to Caballeronia udeis. This result was confirmed by whole-genome phylogeny analyses done on strain PML1(12)T. The results of digital DNA–DNA relatedness further supported the separation of the new isolates from closely related species. Morphological, chemotaxonomic properties were also consistent with the description of the genus Caballeronia. It is therefore proposed that strains PML1(12)T and PML1(4), PML1(14) and PML1(16) be recognized as a novel species, for which the name Caballeronia mineralivorans sp. nov. is proposed. The type strain is PML1(12)T (= DSM 104028 and LMG 2991).

International audience; A novel hyperthermophilic, piezophilic, anaerobic archaeon, designated NCB100(T), was isolated from a hydrothermal vent flange fragment collected in the Guaymas basin at the hydrothermal vent site named 'Rebecca's Roost' at a depth of 1997 m. Enrichment and isolation were performed at 100 degrees C under atmospheric pressure. Cells of strain NCB100(T) were highly motile, irregular cocci with a diameter of -1 mu m. Growth was recorded at temperatures between 70 and 112 degrees C (optimum 105 degrees C) and hydrostatic pressures of 0.1-80 MPa (optimum 40-50 MPa). Growth was observed at pH 3.5-8.5 (optimum pH 7) and with 1.5-7% NaCl (optimum at 2.5-3 %). Strain NCB100(T) was a strictly anaerobic chemo-organoheterotroph and grew on complex proteinaceous substrates such as yeast extract, peptone and tryptone, as well as on glycogen and starch. Elemental sulfur was required for growth and was reduced to hydrogen sulfide. The fermentation products from complex proteinaceous substrates were CO2 and H-2. The G+C content of the genomic DNA was 41.3 %. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain NCB100(T) belongs to the genus Pyrococcus, showing 99% similarity with the other described species of the genus Pyrococcus. On the basis of physiological characteristics, DNA G+C content, similarity level between ribosomal proteins and an average nucleotide identity value of 79 %, strain NCB100(T) represents a novel species for which the name Pyrococcus kukulkanii sp. nov. is proposed. The type strain is NCB100(T) (=DSM 101590(T) =Souchotheque de Bretagne BG1337(T)).

This review examines recent applications of stable copper, zinc and sulfur isotopes to medical cases and notably cancer. The distribution of the natural stable isotopes of a particular element among coexisting molecular species varies as a function of the bond strength, the ionic charge, and the coordination, and it also changes with kinetics. Ab initio calculations show that compounds in which a metal binds to oxygen- (sulfate, phosphate, lactate) and nitrogen-bearing moieties (histidine) favor heavy isotopes, whereas bonds with sulfur (cysteine, methionine) favor light isotopes. Oxidized cations (e.g., Cu(II)) and low coordination numbers are expected to favor heavy isotopes relative to their reduced counterparts (Cu(I)) and high coordination numbers. Here we discuss the first observations of Cu, Zn, and S isotopic variations, three elements closely related along multiple biological pathways, with emphasis on serum samples of healthy volunteers and of cancer patients. It was found that heavy isotopes of Zn and to an even greater extent Cu are enriched in erythrocytes relative to serum, while the difference is small for sulfur. Isotopic variations related to age and sex are relatively small. The 65Cu/63Cu ratio in the serum of patients with colon, breast, and liver cancer is conspicuously low relative to healthy subjects. The characteristic time over which Cu isotopes may change with disease progression (a few weeks) is consistent with both the turnover time of the element and albumin half-life. A parallel effect on sulfur isotopes is detected in a few un-medicated patients. Copper in liver tumor tissue is isotopically heavy. In contrast, Zn in breast cancer tumors is isotopically lighter than in healthy breast tissue. 66Zn/64Zn is very similar in the serum of cancer patients and in controls. Possible reasons for Cu isotope variations may be related to the cytosolic storage of Cu lactate (Warburg effect), release of intracellular copper from cysteine clusters (metallothionein), or the hepatocellular and biosynthetic dysfunction of the liver. We suggest that Cu isotope metallomics will help evaluate the homeostasis of this element during patient treatment, notably by chelates and blockers of Cu trafficking, and understand the many biochemical pathways in which this element is essential.

International audience; A novel strictly anaerobic, hyperthermophilic archaeon, designated strain CDGST, was isolated from a deep-sea hydrothermal vent in the Cayman Trough at 4964m water depth. The novel isolate is obligate anaerobe and grows chemoorganoheterotrophically with stimulation of growth by sulphur containing compounds. Its growth is optimal at 75 ◦C,pH6.0 and under a pressure of 50 MPa. It possesses the broadest hydrostatic pressure range for growth that has ever been described for a microorganism. Its genomic DNA G+ C content is 51.11 mol%. The novel isolate belongs to the genus Thermococcus. Phylogenetic analyses indicated that it is most closely related to Thermococcus barossii DSM17882T based on its 16S rRNA gene sequence, and to ‘Thermococcus onnurineus’ NA1 based on its whole genome sequence. The average nucleotide identity scores with these strains are 77.66% for T. barossii and 84.84% for ‘T. onnurineus’, respectively. Based on the draft whole genome sequence and phenotypic characteristics, strain CDGST is suggested to be separated into a novel species within the genus Thermococcus, with proposed name Thermococcus piezophilus (type strain CDGST = ATCC TSD-33T =UBOCC 3296T).

We report the genome sequence of Thermococcus superprofundus strain CDGS(T), a new piezophilic and hyperthermophilic member of the order Thermococcales isolated from the world's deepest hydrothermal vents, at the Mid-Cayman Rise. The genome is consistent with a heterotrophic, anaerobic, and piezophilic lifestyle.

We report here the complete sequence and fully manually curated annotation of the genome of strain Ch5, a new member of the piezophilic hyperthermophilic species Thermococcus barophilus .

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer, with increasing incidence worldwide. The unrestrained proliferation of tumour cells leads to tumour hypoxia which in turn promotes cancer aggressiveness. While changes in the concentration of copper (Cu) have long been observed upon cancerization, we have recently reported that the isotopic composition of copper is also altered in several types of cancer. In particular, we showed that in hepatocellular carcinoma, tumour tissue contains heavier copper compared to the surrounding parenchyma. However, the reasons behind such isotopic signature remained elusive. Here we show that hypoxia causes heavy copper enrichment in several human cell lines. We also demonstrate that this effect of hypoxia is pH, HIF-1 and -2 independent. Our data identify a previously unrecognized cellular process associated with hypoxia, and suggests that in vivo tumour hypoxia determines copper isotope fractionation in HCC and other solid cancers.

We have established a defined growth medium for the piezophilic hyperthermophilic archaeon Thermococcus barophilus , which allows growth yields of ca. 108 cells/ml under both atmospheric and high hydrostatic pressure . Our results demonstrate a major impact of hydrostatic pressure on amino acid metabolism , with increases from 3 amino acids required at atmospheric pressure to 17 at 40 MPa. We observe in T. barophilus and other Thermococcales a similar discrepancy between the presence/absence of amino acid synthesis pathways and amino acid requirements, which supports the existence of alternate, but yet unknown, amino acid synthesis pathways, and may explain the low number of essential amino acids observed in T. barophilus and other Thermococcales. T. barophilus displays a strong metabolic preference for organic polymers such as polypeptides and chitin, which may constitute a more readily available resource of carbon and energy in situ in deep-sea hydrothermal vents. We hypothesize that the low energy yields of fermentation of organic polymers, together with energetic constraints imposed by high hydrostatic pressure, may render de novo synthesis of amino acids ecologically unfavorable. Induction of this metabolic switch to amino acid recycling can explain the requirement for non-essential amino acids by Thermococcales for efficient growth in defined medium.

The deep biosphere is composed of very different biotopes located in the depth of the oceans, the ocean crust or the lithosphere. Although very different, deep biosphere biotopes share one common feature, high hydrostatic pressure. The deep biosphere is colonized by specific organisms, called piezophiles, that are able to grow under high hydrostatic pressure. Bacterial piezophiles are mainly psychrophiles belonging to five genera of γ-proteobacteria, Photobacterium, Shewanella, Colwellia, Psychromonas and Moritella, while piezophilic Archaea are mostly (hyper)thermophiles from the Thermococcales. None of these genera are specific for the deep biosphere. High pressure deeply impacts the activity of cells and cellular components, and reduces the activity of numerous key processes, eventually leading to cell death of piezosensitive organisms. Biochemical and genomic studies yield a fragmented view on the adaptive mechanisms in piezophiles. It is yet unclear whether piezophilic adaptation requires the modification of a few genes, or metabolic pathways, or a more profound reorganization of the genome, the fine tuning of gene expression to compensate the pressure-induced loss of activity of the proteins most affected by high pressure, or a stress-like physiological cell response. In contrast to what has been seen for thermophily or halophily, the adaptation to high pressure is diffuse in the genome and may concern only a small fraction of the genes.

Microbes often face contrasted and fluctuating environmental conditions, to which they need to adapt or die. Because membranes play a central role in regulating fluxes inward and outward from the cells, maintaining the appropriate structure of the membrane is crucial to maintain cellular integrity and functions. This is achieved in bacteria and eucarya by a modification of the membrane lipid compositions, a strategy termed homeoviscous adaptation. We review here evidence for homeoviscous adaptation in Archaea, and discuss the limits of this strategy and our knowledge in this very peculiar domain of life.

It is now clear that active methanogens are present in the deep-subsurface. This paper reviews microbial population structures and the biodegradation of organic compounds to methane in situ within oil reservoirs and coal deposits. It summarizes our current knowledge of methanogenes and methanogenesis, fermenters, synthrophs and microbial metabolism of complex organic compounds in these two widely occurring organic-rich subsurface environments. This review is not intended to be an exhaustive report of microbial diversity. Rather, it illustrates the similarities and differences between the two environments with specific examples, from the nature of the organic molecules to the methanogenic metabolic pathways and the structure of the microbial populations to demonstrate that widely diverging microbial populations show surprisingly similar metabolic capabilities.

Analysis of the 16S rRNA gene sequences generated from Xerocomus pruinatus and Scleroderma citrinum ectomycorrhizospheres revealed that similar bacterial communities inhabited the two ectomycorrhizospheres in terms of phyla and genera, with an enrichment of the Burkholderia genus. Compared to the bulk soil habitat, ectomycorrhizospheres hosted significantly more Alpha -, Beta -, and Gammaproteobacteria .

7 pages; International audience; We have isolated a novel Siphoviridae phage (named Sol-P11) morphotype from the surface sands of the Sahara Desert with a ribbon-like structure at the tail extremity. Sol-P11 was found to grow on a Bacillus subtilis strain isolated from the same environment and to contain a double stranded DNA genome of approximately 120 kb in length incapable of being hydrolysed by a wide variety of restriction endonucleases. The major constituent proteins of CsCl-purified Sol-P11 virions were 65, 50, 30, and 24 kDa in size, with the 30 kDa polypeptide being the major protein of the 85 nm diameter icosahedral capsid, and the other three proteins comprising the major polypeptides of the tail (320 nm in length) and ribbon-like structure. Moreover, different sized phages displaying a Sol-P11 morphology were observed in phage preparations from the Death Valley and Namib deserts. Sol-P11-like phage morphotypes have been previously described, including PBPI, a flagellum-specific phage that infects B. pumilis and phage BcP15 infecting the marine bacterium, Burkholderia cepacia DR11. We thus propose that Sol-P11 represents a member of a novel morphotype of Siphoviridae phages that use a ribbon-like structure, instead of caudal fibers, to attach to their host cell.

Analysis of the complete genome of Thermococcus sp. strain AM4, which was the first lithotrophic Thermococcales isolate described and the first archaeal isolate to exhibit a capacity for hydrogenogenic carboxydotrophy, reveals a proximity with Thermococcus gammatolerans , corresponding to close but distinct species that differ significantly in their lithotrophic capacities.

International audience; Unlike farmland, forests growing on acidic soils are among the terrestrial ecosystems that are the least influenced or amended by man. Forests which developed on acidic soils are characterized by an important stock of inorganic nutrients entrapped in poorly weatherable soil minerals. In this context, the mineral-weathering process is of great importance, since such minerals are not easily accessible to tree roots. To date, several bacterial genera have been noted for their ability to weather minerals and, in the case of some of them, to improve tree nutrition. Nevertheless, few studies have focused their analyses on mineral-weathering bacterial communities in relation to geochemical cycles and soil characteristics, their ecological origin, associated tree species and forest management practices. Here we discuss the heterogeneity of the mineral-weathering process in forest soils and present what is known concerning the taxonomic and functional characteristics of mineral-weathering bacteria, as well as the different locations where they have been isolated in forest soils. We also discuss the biotic and abiotic factors that may influence the distribution of these bacteria, such as the effect of tree species or forest management practices.

Thermococcus barophilus is a hyperthermophilic, anaerobic, mixed heterotrophic, and carboxydotrophic euryarchaeon isolated from the deep sea hydrothermal vent Snakepit site on the mid-Atlantic ridge at a depth of 3,550 m. T. barophilus is the first true piezophilic, hyperthermophilic archaeon isolated, having an optimal growth at 40 MPa. Here we report the complete genome sequence of strain MP, the type strain of T. barophilus . The genome data reveal a close proximity with T hermococcus sibiricus , another Thermococcus isolated from the deep biosphere and a possible connection to life in the depths.

International audience; X-ray absorption spectroscopy is a well-established method for probing local structural and electronic atomic environments in a variety of systems. We used X-ray absorption near-edge structure (XANES) spectroscopy for monitoring in real-time conditions selenium reduction in situ in live cultures of Shewanella oneidensis MR-1 under high hydrostatic pressure. High-quality XANES data show that Shewanella oneidensis MR-1 reduces selenite Se(IV) to red elemental selenium Se(0) up to 150 MPa without any intermediate redox state. MR-1 reduces all selenite provided (5-10 mm) between 0.1 and 60 MPa. Above 60 MPa the selenite reduction yield decreases linearly with pressure and the activity is calculated to stop at 155 +/- 5 MPa. The analysis of cultures recovered after in situ measurements showed that the decrease in activity is linked to a decrease in viability. This study emphasizes the promising potential of XANES spectroscopy for real-time probing in situ microbial redox transformations of a broad range of metal and metalloid elements in live samples, including under high hydrostatic pressure.

An obligate piezophilic anaerobic hyperthermophilic archaeon, designated strain CH1T, was isolated from a hydrothermal vent site named ‘Ashadze’, which is located on the Mid-Atlantic Ridge at a depth of 4100 m. Enrichment and isolation of the strain were carried out at 95 °C under a hydrostatic pressure of 42 MPa. Cells of strain CH1T were highly motile irregular cocci with a diameter of ~1–1.5 µm. Growth was recorded at 80–108 °C (optimum 98 °C) and at pressures of 20–120 MPa (optimum 52 MPa). No growth was observed under atmospheric pressures at 60–110 °C. Growth was observed at pH 6.0–9.5 (optimum 7.5–8.0) and in 2.5–5.5 % (w/v) NaCl (optimum 3.5 %). Strain CH1T was strictly anaerobic and grew on complex proteinaceous substrates, such as yeast extract, Peptone, and casein, as well as on sucrose, starch, chitin, pyruvate, acetate and glycerol without electron acceptors. The G+C content of the genomic DNA was 49.0±0.5 mol%. Analysis of 16S rRNA gene sequences revealed that strain CH1T belongs to the genus Pyrococcus. Based on its physiological properties and similarity levels between ribosomal proteins, strain CH1T represents a novel species, for which the name Pyrococcus yayanosii sp. nov. is proposed. The type strain is CH1T ( = JCM 16557). This strain is also available by request from the Souchothèque de Bretagne (catalogue LMBE) culture collection (collection no. 3310).

En libre-accès sur Archimer : http://archimer.ifremer.fr/doc/00027/13797/11043.pdf; International audience; The current paper reviews strategies employed by microorganisms from the deep biosphere, especially piezophiles (from the greek piezo = to press and philo = love), to cope with high hydrostatic pressure (HHP) prevailing in these biotopes. The aim of this review is not to constitute an exhaustive report of our current knowledge on the physiology of piezophiles, as recent reviews have covered part of this subject in detail (Abe, 2007; Lauro and Bartlett, 2008; Michiels et al., 2008; Simonato et al., 2006). Rather, we illustrate here, via a few chosen examples, where we stand in our understanding of the mechanisms employed by microorganisms from the depths of our planet to cope with HHP.

International audience; Aqueous solutions equilibrated with supercritical CO(2) (150 degrees C and total pressure of 150 bar) were investigated in order to characterize their respective conditions of carbonation. Dissolution of olivine and subsequent precipitation of magnesite with a net consumption of CO(2) were expected. A quantified pure mineral phase (powders with different olivine grain diameter [20-80 mu m], [80-125 mu m], [125-200 mu m] and [>200 mu m]), CO(2) (as dried ice) were placed in closed-batch reactors (soft Au tubes) in the presence of solutions. Different salinities (from 0 to 3400 mM) and different ratios of solution/solid (mineral phase) (from 0.1 to 10) were investigated. Experiments were performed over periods from 2 to 8 weeks. Final solid products were quantified by the Rock-Eval 6 technique, and identified using X-ray diffraction, Raman spectroscopy, electron microprobe and scanning electron microscopy. Gaseous compounds were quantified by a vacuum line equipped with a Toepler pump and identified and measured by gas chromatography (GC). Carbon mass balances were calculated. Olivine reacted completely with CO(2), trapping up to 57 +/- 2% (eqC of initial CO(2)) as magnesite; some amorphous silica also formed. Olivine grain diameter and solution/mineral ratios appeared to be the primary controls on the reaction, salinity acting as a second order parameter. During the experiments, fluid analyses may not be performed with approach adopted but, geochemical modelling was attempted to give information about the solution composition. This showed an interesting mineral matrix evolution. Under the experimental conditions, olivine appeared to be a good candidate for CO(2) trapping into a geologically stable carbonate, magnesite. The possible use of mafic and ultramafic rocks for CO(2) sequestration is discussed.

Conference on Goldschmidt 2010 - Earth, Energy, and the Environment ; Knoxville, TN ; June 13-18, 2010; International audience

X-ray spectroscopies are very powerful tools to determine the chemistry of complex dilute solutes in abiotic and biotic systems. We have assayed their suitability to monitor the chemistry of complex solutions in a live biotic system. The impact of the probe on cells was quantified for 4 different cellular organisms differing in their resistance level to environmental stresses. We show that none of the organisms tested can survive the radiation doses needed for the acquisition of meaningful spectroscopic data. Therefore, on one hand, X-ray spectroscopy cannot be applied to the monitoring of single cells, and cellular damages have to be taken into account in the interpretation of the evolution of such systems. On the other hand, due to the limited extension of X-ray induced cellular damages in the culture volume, it is possible to probe a population of live cells provided that the culture to beam probe ratio is large enough to minimize the impact of mortality on the evolution of the biological system. Our results suggest that it could be possible to probe the volume in the close vicinity of a cell without affecting its activity. Using this setup we could monitor the reduction of selenite by the X-ray sensitive bacterium, Agrobacterium tumefaciens strain C58, for 24 h. This method has a great potential to monitor the respiration of various metals, such as iron, manganese and arsenic, in situ under relevant environmental conditions by live microorganisms.

We monitored alcoholic fermentation in Saccharomyces cerevisiae as a function of high hydrostatic pressure. Ethanol production from 0.15 M glucose was measured by Raman spectroscopy in situ in a diamond-anvil cell. At 10 MPa, fermentation proceeds three times faster than at ambient pressure and the fermentation yield is enhanced by 5% after 24 h. Above 20 MPa, the reaction kinetics slows down with increasing pressure. The pressure above which no more ethanol is produced is calculated to be 87 +/- 7 MPa. These results indicate that the activity of one or several enzymes of the glycolytic pathway is enhanced at low pressure up to 10 MPa. At higher pressures, they become progressively repressed, and they are completely inhibited above 87 MPa. Although fermentation was predicted to stop at ca. 50 MPa, due to the loss of activity of phosphofructokinase, the present study demonstrates that there is still an activity of ca. 30% of that measured at ambient pressure at 65 MPa. This study also validates the use of Raman spectroscopy for monitoring the metabolism of living microorganisms.

Life on Earth can be traced back to as far as 3.8 billion years (Ga) ago. The catastrophic meteoritic bombardment ended between 4.2 and 3.9 Ga ago. Therefore, if life emerged, and we know it did, it must have emerged from nothingness in less than 400 million years. The most recent scenarios of Earth accretion predict some very unstable physico-chemical conditions at the surface of Earth, which, in such a short time period, would impede the emergence of life from a proto-biotic soup. A possible alternative would be that life originated in the depth of the proto-ocean of the Hadean Earth, under high hydrostatic pressure. The large body of water would filter harmful radiation and buffer physico-chemical variations, and therefore would provide a more stable radiation-free environment for pre-biotic chemistry. After a short introduction to Earth history, the current tutorial review presents biological and physico-chemical arguments in support of high-pressure origin for life on Earth.

The Rhodococcus erythropolis strain W2 has been shown previously to degrade the N-acylhomoserine lactone (AHL) quorum-sensing signal molecule N-hexanoyl-l-homoserine lactone, produced by other bacteria. Data presented here indicate that this Gram-positive bacterium is also capable of using various AHLs as the sole carbon and energy source. The enzymic activities responsible for AHL inactivation were investigated in R. erythropolis cell extracts and in whole cells. R. erythropolis cells rapidly degraded AHLs with 3-oxo substituents but exhibited relatively poor activity against the corresponding unsubstituted AHLs. Investigation of the mechanism(s) by which R. erythropolis cells degraded AHLs revealed that 3-oxo compounds with N-acyl side chains ranging from C8 to C14 were initially converted to their corresponding 3-hydroxy derivatives. This oxidoreductase activity was not specific to 3-oxo-AHLs but also allowed the reduction of compounds such as N-(3-oxo-6-phenylhexanoyl)homoserine lactone (which contains an aromatic acyl chain substituent) and 3-oxododecanamide (which lacks the homoserine lactone ring). It also reduced both the d- and l-isomers of n-(3-oxododecanoyl)-l-homoserine lactone. A second AHL-degrading activity was observed when R. erythropolis cell extracts were incubated with N-(3-oxodecanoyl)-l-homoserine lactone (3O,C10-HSL). This activity was both temperature- and pH-dependent and was characterized as an amidolytic activity by HPLC analysis of the reaction mixture treated with dansyl chloride. This revealed the accumulation of dansylated homoserine lactone, indicating that the 3O,C10-HSL amide had been cleaved to yield homoserine lactone. R. erythropolis is therefore capable of modifying and degrading AHL signal molecules through both oxidoreductase and amidolytic activities.

The culture of opine-producing transgenic Lotus plants induces the increase in the rhizosphere of bacterial communities that are able to utilize these molecules as sole carbon source. We used transgenic Lotus plants producing two opines, namely mannopine and nopaline, to characterize the microbial communities directly influenced by the modification of root exudation. We showed that opine-utilizers represent a large community in the rhizosphere of opine-producing transgenic Lotus. This community is composed of at least 12 different bacterial species, one third of which are able to utilize the opine mannopine and two thirds the opine nopaline. Opine utilizers are diverse, belonging to the Gram-positive and -negative bacteria. We described two novel mannopine-utilizing species, Rhizobium and Duganella spp., and five novel nopaline-utilizing species, Duganella, Afipia, Phyllobacterium, Arthrobacter, and Bosea spp. Although opine utilizers mostly belong to the α-Proteobacteria, Rhizobiaceae family, there is little overlap between the populations able to utilize each of the two opines produced by the plants. Noticeably, in the rhizosphere of transgenic Lotus, only the opine mannopine favors the growth of Agrobacterium tumefaciens, the bacterium from which opines have been characterized. The diversity of opine utilizers from the rhizosphere of Lotus plants is greater than that observed from any other environment. Therefore, transgenic plants with engineered exudation constitute an excellent tool to isolate and characterize specific microbial populations.

Bacteria degrading the quorum-sensing (QS) signal molecule N-hexanoylhomoserine lactone were isolated from a tobacco rhizosphere. Twenty-five isolates degrading this homoserine lactone fell into six groups according to their genomic REP-PCR and rrs PCR-RFLP profiles. Representative strains from each group were identified as members of the genera Pseudomonas, Comamonas, Variovorax and Rhodococcus. All these isolates degraded N-acylhomoserine lactones other than the hexanoic acid derivative, albeit with different specificity and kinetics. One of these isolates, Rhodococcus erythropolis strain W2, was used to quench QS-regulated functions of other microbes. In vitro, W2 strongly interfered with violacein production by Chromobacterium violaceum, and transfer of pathogenicity in Agrobacterium tumefaciens. In planta, R. erythropolis W2 markedly reduced the pathogenicity of Pectobacterium carotovorum subsp. carotovorum in potato tubers. These series of results reveal the diversity of the QS-interfering bacteria in the rhizosphere and demonstrate the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates.

Several tactics exist to improve the survival of an introduced microorganism of interest in the plant environment. One, derived from studies on the Agrobacterium-plant interaction and the role of opines in this interaction, proposes to promote growth of the inoculant in the plant environment via the establishment of a bias in the rhizosphere. It is supported by the occurrence of natural biases, such as those generated by opine-like molecules, by calestegins, or by mimosine. Opine-mediated biases have allowed several investigators to favor the growth of opine-degrading bacteria or communities under sterile or axenic environments or in microcosms mimicking near field conditions. Another way to favor a given microbe consists in impeding growth of competing microorganisms. Experiments performed using detergent or bacteriostatic agents as amendments under field or near field conditions yielded promising results. Research perspectives for engineering plant-microbe interactions also include specific engineering of predation and strategies designed to interfere with some of the signals perceived by the microbes, provided these signals control the expression of functions central to microbial fitness. In this respect, quorum-sensing signal molecules, such as N-acyl-homoserine lactones, may be valuable targets for the development of biocontrol agents and procedures.

Conjugal transfer of Ti plasmids from Agrobacterium spp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon. Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84. In this study we showed that this plasmid contains two copies of traR , and each is associated with a different opine-regulated operon. One copy, traR noc , is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B. Mutating traR noc abolished induction of transfer by nopaline but not by the agrocinopines. A mutation in ocd , an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy, traR acc , is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in both traR genes was not induced for transfer by either opine. However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer. The association of traR noc with nox is unique, but the operon containing traR acc is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies of traR , each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types.

Publisher Summary This chapter presents strategies and protocols for studying the quorum-sensing system used by Agrobacterium. tumefaciens to regulate conjugal transfer of its Ti plasmids from one bacterium to another as well as the applications of these protocols to quorum-sensing systems of other bacteria. The Ti plasmids code for the uptake and catabolism of the opines produced by the crown gall tumors. These plasmids code for two independent transfer systems; one, called vir, is responsible for processing and transfer of the T-region from the bacterium to host plant cells. The quorum-sensing system of the Ti plasmid conjugal transfer apparatus, as well as the components of this system, has provided broadly applicable models and tools for the study of this gene regulatory paradigm in other bacteria. The chapter describes the physiological, genetic, biochemical, and structural studies of TraR and the target genes it regulates by quorum sensing have contributed to our understanding of the nature of these systems as well as the molecular mechanisms by which acylhomoserine lactone signals are translated to gene regulation through LuxR-like transcription factors. The chapter describes a number of these tools and assays. In each case the chapter presents a brief rationale for the assay or technique, and then outlines in general form the method. It indicates the general applicability of, and, where appropriate, the strengths, limitations, and pitfalls of each test.

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes , and A. vitis . The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn 5 -induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn 5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn 5 was located in the mutant. Sequence analysis showed that the Tn 5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound.

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadori opines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 coinherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by two hierarchical signalling systems. Transfer is dependent on a subset of opines produced by the plant tumours induced by the bacterium. Induction also requires an acyl-homoserine lactone signal, called AAI, that is produced by the bacteria themselves. AAI is the co-inducer for TraR, the transcriptional activator required for expression of the tra regulon. Octopine induces conjugation of the octopine-mannityl opine-type Ti plasmids by regulating the expression of traR via OccR, the octopine-dependent activator of the opine regulon. We have discovered a second traR-like gene, trlR, on the octopine-mannityl opine-type Ti plasmids pTi15955 and pTiR10. This gene is located in an operon coding for a mannopine transport system and is expressed as part of the mannityl opine regulon. Sequence analysis indicated that trlR is a frameshift allele of traR, and the resulting protein lacks the carboxy-terminal domain thought to constitute the DNA-binding region of TraR. Expression of trlR inhibited octopine-induced conjugation of pTi15955 and pTiR10 by suppressing the TraR-mediated transcription of the tra and trb operons. Although TrlR had no effect on the expression of traR, TraR activated the expression of trlR. Southern hybridizations indicated that several other Ti and opine-catabolic plasmids contain more than one copy of genes homologous to traR. We propose that trlR is a dominant negative allele of traR and that TrlR inhibits conjugation by forming inactive heteromultimers with TraR.

Most of Earth’s biotopes are under extreme environmental conditions, e.g. distant from the optimal life conditions for humans. Nevertheless, a large biological diversity of organisms, the extremophiles, inhabit such environments. For instance, organisms living at deep-sea hydrothermal vents experience temperatures above 100°C, high concentrations of reduced metals, the absence of oxygen and high hydrostatic pressures. To date, there is no understanding of the molecular mechanisms enabling them to sustain such extreme conditions. The cell membrane is particularly sensitive to external conditions, but at the same time must maintain specific physical properties, such as fluidity and permeability, to preserve the integrity and functionality of the cell. This thesis work seeks to understand how the lipid bilayer can remain functional at high temperatures and high pressures and thus can allow life under extreme conditions. This work establishes novel membrane components, apolar lipids from the polyisoprenoid’s family, which play the role of membrane regulators and confer stability to the lipid bilayer, along with dynamism and heterogeneity, which are essential properties for an optimal function of the membrane. Apolar lipids are localized in the midplane of the lipid bilayer and adjust membrane curvature and permeability. Because of such specific localization, apolar lipids reduce proton membrane permeability at high hydrostatic pressures. They establish functional domains with differentiated functions that could favour the insertion of specific membrane proteins and hence could enable essential cell functions that require highly curved membrane domains, such as fusion and fission. All these results demonstrate experimentally a novel cell membrane architecture for extremophiles in which the presence and the quantity of polyisoprenoids play a key role in cell membrane adaptation and constitute a novel adaptation route to extreme conditions applicable to the origin of life., La majorité des biotopes terrestres se trouve sous des conditions environnementales dites extrêmes, c'est-à-dire éloignées des conditions optimales de vie des humains. Néanmoins, une grande variété biologique d’organismes biologiques y habite, i.e. les extrémophiles. Par exemple, il existe des organismes vivants adaptés aux conditions des sources hydrothermales de l'océan profond: températures supérieures à 100°C, fortes concentrations en métaux réduits, absence d'oxygène, hautes pressions hydrostatiques, sans que l'on comprenne les mécanismes moléculaires leur permettant de résister à de telles conditions. La membrane cellulaire est un des constituants cellulaires le plus sensible aux conditions externes, mais en même temps, elle doit maintenir des caractéristiques physicochimiques très spécifiques, en terme de rigidité et de perméabilité, pour rester fonctionnelle. Ce travail de thèse cherche à comprendre comment la bicouche lipidique peut demeurer fonctionnelle sous hautes températures et hautes pressions, et permettre la vie en conditions extrêmes. Ce travail identifie de nouveau constituants de la membrane, les lipides apolaires de type polyisoprènes, qui jouent le rôle de régulateurs membranaires, et confèrent à la bicouche lipidique la stabilité, mais aussi le dynamisme et l'hétérogénéité, indispensables pour une fonctionnalité optimale de la membrane. Ces lipides apolaires se placent au centre de la bicouche lipidique et modifient la courbure et la perméabilité de la membrane. En raison de leur emplacement, ils diminuent la perméabilité aux protons sous hautes pressions hydrostatiques. Ils créent des îlots fonctionnels différenciés pouvant faciliter l’insertion de certaines protéines membranaires ou l’accomplissement des fonctions essentielles au cycle cellulaire comme la fusion et la fission qui requièrent une forte courbure. L'ensemble des résultats valide expérimentalement une nouvelle architecture membranaire chez les extrémophiles où la présence et la quantité de lipides apolaires jouent un rôle fondamental, et représente une voie nouvelle d'adaptation aux conditions extrêmes applicable à l'origine de la vie.

Most of Earth’s biotopes are under extreme environmental conditions, e.g. distant from the optimal life conditions for humans. Nevertheless, a large biological diversity of organisms, the extremophiles, inhabit such environments. For instance, organisms living at deep-sea hydrothermal vents experience temperatures above 100°C, high concentrations of reduced metals, the absence of oxygen and high hydrostatic pressures. To date, there is no understanding of the molecular mechanisms enabling them to sustain such extreme conditions. The cell membrane is particularly sensitive to external conditions, but at the same time must maintain specific physical properties, such as fluidity and permeability, to preserve the integrity and functionality of the cell. This thesis work seeks to understand how the lipid bilayer can remain functional at high temperatures and high pressures and thus can allow life under extreme conditions. This work establishes novel membrane components, apolar lipids from the polyisoprenoid’s family, which play the role of membrane regulators and confer stability to the lipid bilayer, along with dynamism and heterogeneity, which are essential properties for an optimal function of the membrane. Apolar lipids are localized in the midplane of the lipid bilayer and adjust membrane curvature and permeability. Because of such specific localization, apolar lipids reduce proton membrane permeability at high hydrostatic pressures. They establish functional domains with differentiated functions that could favour the insertion of specific membrane proteins and hence could enable essential cell functions that require highly curved membrane domains, such as fusion and fission. All these results demonstrate experimentally a novel cell membrane architecture for extremophiles in which the presence and the quantity of polyisoprenoids play a key role in cell membrane adaptation and constitute a novel adaptation route to extreme conditions applicable to the origin of life.; La majorité des biotopes terrestres se trouve sous des conditions environnementales dites extrêmes, c'est-à-dire éloignées des conditions optimales de vie des humains. Néanmoins, une grande variété biologique d’organismes biologiques y habite, i.e. les extrémophiles. Par exemple, il existe des organismes vivants adaptés aux conditions des sources hydrothermales de l'océan profond: températures supérieures à 100°C, fortes concentrations en métaux réduits, absence d'oxygène, hautes pressions hydrostatiques, sans que l'on comprenne les mécanismes moléculaires leur permettant de résister à de telles conditions. La membrane cellulaire est un des constituants cellulaires le plus sensible aux conditions externes, mais en même temps, elle doit maintenir des caractéristiques physicochimiques très spécifiques, en terme de rigidité et de perméabilité, pour rester fonctionnelle. Ce travail de thèse cherche à comprendre comment la bicouche lipidique peut demeurer fonctionnelle sous hautes températures et hautes pressions, et permettre la vie en conditions extrêmes. Ce travail identifie de nouveau constituants de la membrane, les lipides apolaires de type polyisoprènes, qui jouent le rôle de régulateurs membranaires, et confèrent à la bicouche lipidique la stabilité, mais aussi le dynamisme et l'hétérogénéité, indispensables pour une fonctionnalité optimale de la membrane. Ces lipides apolaires se placent au centre de la bicouche lipidique et modifient la courbure et la perméabilité de la membrane. En raison de leur emplacement, ils diminuent la perméabilité aux protons sous hautes pressions hydrostatiques. Ils créent des îlots fonctionnels différenciés pouvant faciliter l’insertion de certaines protéines membranaires ou l’accomplissement des fonctions essentielles au cycle cellulaire comme la fusion et la fission qui requièrent une forte courbure. L'ensemble des résultats valide expérimentalement une nouvelle architecture membranaire chez les extrémophiles où la présence et la quantité de lipides apolaires jouent un rôle fondamental, et représente une voie nouvelle d'adaptation aux conditions extrêmes applicable à l'origine de la vie.

The use of marine or terrestrial microorganisms for long-term sequestration of CO2 is a possible solution to reduce the CO2 content in atmosphere. This thesis work focuses on calcifying organisms, in particular the cyanobacteria, which can fix CO2 as biomass and calcium carbonate. The latter is insoluble in water; precipitates may therefore constitute a long term sink. Understanding of the calcification mechanisms induced by cyanobacteria and the possibility of controlling these processes are necessary to develop a technology for CO2 sequestration. This biotechnology could be an alternative technology to CO2 capture and geological storage. Synechococcus strain PCC8806, marine cyanobacteria purified by the Institute Pasteur de Paris is used during the experimental work in this thesis. The first important result of this work is to develop an analytical strategy that allowed access to a mass balance of carbon and calcium in a cyanobacteria culture on hydrogencarbonate. The implementation of this strategy in various tests of this work has also allowed to accurately quantify the production of organic carbon (biomass) and inorganic carbon (CaCO3) according to the calcium and Ci introduced (hydrogencarbonate) in the medium. We then studied the calcite precipitation during growth of Synechococcus PCC8806 in the presence of calcium. For that, culture conditions were varied in order to understand the occurrence of precipitation events and the influence of nucleation sites. The development of crystals has also been carefully studied by scanning electron microscopy. Another part of this work has identified the inorganic carbon source used by Synechococcus PCC8806 for photosynthesis. This was an opportunity to rewrite the equations related to transfers between atmospheric CO2 and the carbonate medium, as well as the equations of photosynthesis depending on the conditions of availability of two sources of inorganic carbon (CO2 and hydrogencarbonate). In addition, we have revealed the effects of diurnal and nocturnal phases of the growth of cyanobacteria on the carbonate system balance and pH. This work also allowed estimating the cyanobacteria growth rates and thus calculating growth yields per unit area. This will ultimately optimize biomass and calcite production in an industrial process, L’utilisation des microorganismes marins ou terrestres pour la séquestration à long terme du CO2 est une des solutions envisagées pour diminuer la teneur en CO2 dans l'atmosphère. Le travail de cette thèse se concentre sur les microorganismes calcifiants, et notamment les cyanobactéries, qui peuvent fixer du CO2 sous forme de biomasse et carbonate de calcium. Ce dernier, insoluble dans l’eau, précipite et peut donc constituer un puits à long terme. La compréhension des mécanismes de calcification induits par les cyanobactéries et la possibilité de contrôler ces processus sont nécessaires pour développer une technologie de séquestration du CO2. Cette biotechnologie pourrait constituer une alternative à la technologie de capture et stockage géologique du CO2. Synechococcus PCC8806 une souche marine de cyanobactérie purifiée à l'Institut Pasteur de Paris est utilisée comme organisme au cours du travail expérimental réalisé dans le cadre de cette thèse. Le premier résultat important de cette thèse est le développement d'une stratégie analytique ayant permis d'accéder à un bilan de masse carbone et calcium au cours d'une culture de cyanobactérie sur hydrogénocarbonate. La mise en œuvre de cette stratégie au cours de différents essais réalisés dans le cadre de ce travail a permis par ailleurs de quantifier avec précision la production de carbone organique (biomasse) et de carbone inorganique (CaCO3) en fonction du calcium et du carbone inorganique présent dans les milieux de culture. Nous avons ensuite étudié la précipitation de la calcite au cours de la croissance de Synechococcus PCC8806 en présence de calcium. Pour cela les conditions de culture ont été variées de telle sorte que la survenue des évènements de précipitations a pu être comprise ainsi que l'influence de sites de nucléation mis en évidence. Le grossissement des cristaux a également été étudié attentivement par microscopie électronique à balayage. Une autre partie de ce travail a permis d'identifier la source de carbone inorganique utilisée par Synechococcus PCC8806 pour la photosynthèse. Cela a été l'occasion de réécrire les équations liées aux transferts entre le CO2 atmosphérique et le système carbonaté, ainsi que les équations de photosynthèse en fonction des conditions de disponibilité des deux sources de carbone inorganique (CO2 et hydrogénocarbonate). De plus ont pu être mis en évidence, les effets des phases diurne et nocturne de la croissance de cyanobactéries sur les équilibres du système carbonaté et le pH. Ce travail a également permis de déterminer les vitesses de croissance des cyanobactéries et donc de calculer des rendements de croissance par unité de surface. Cela permettra à terme d'optimiser la production de biomasse et de calcite dans un procédé industriel