Daniel Prieur

Pyrococcus abyssi sp. nov., a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

A novel, hyperthermophilic, anaerobic, sulfurmetabolizing archaeon was isolated from a fluid sample from recently discovered hydrothermal vents in the North Fiji basin (SW Pacific), at 2000 m depth. The new organism, strain GE5, is a gram-negative, highly motile coccus. It grows between 67° and 102°C under atmospheric pressure, with an optimum at 96°C (doubling time 33 min). The upper growth temperature is extended by at least 3°C when cells are cultivated under in situ hydrostatic pressures (20 MPa). Strain GE5 is an obligate heterotroph, fermenting peptides, or mixtures of amino acids to acetate, isovalerate, isobutyrate, propionate, H2 and CO2. Hydrogen inhibits growth unless sulfur is present. In the presence of sulfur, H2S is then produced. Phylogenetic analyses of the 16 S rRNA sequence of strain GE5 places the new isolate within the Thermococcales. By its high growth temperature and physiological features the new isolate ressembles Pyrococcus sp. However it deffers by a 7% mol upper G+C-content and shows low level of DNA similarity with the two previously described species. Based on these differences the description of strain GE5 as a new species Pyrococcus abyssi (CNCM I-1302) is proposed.

An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi.

The hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re‐annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.

Extending the sub-sea-floor biosphere.

Sub‐sea-floor sediments may contain two-thirds of Earths total prokaryotic biomass. However, this has its basis in data extrapolation from ~500-meter to 4-kilometer depths, whereas the deepest documented prokaryotes are from only 842 meters. Here, we provide evidence for low concentrations of living prokaryotic cells in the deepest (1626 meters below the sea floor), oldest (111 million years old), and potentially hottest (~100�C) marine sediments investigated. These Newfoundland margin sediments also have DNA sequences related to thermophilic and/or hyperthermophilic Archaea. These form two unique clusters within Pyrococcus and Thermococcus genera, suggesting unknown, uncultured groups are present in deep, hot, marine sediments (~54� to 100�C). Sequences of anaerobic methane-oxidizing Archaea were also present, suggesting a deep biosphere partly supported by methane. These findings demonstrate that the sub‐sea-floor biosphere extends to at least 1600 meters below the sea floor and probably deeper, given an upper temperature limit for prokaryotic life of at least 113�C and increasing thermogenic energy supply with depth.

Presence and activity of anaerobic ammonium-oxidizing bacteria at deep-sea hydrothermal vents

Recent studies indicate that ammonia is an important electron donor for the oxidation of fixed nitrogen, both in the marine water column and sediments. This process, known as anammox, has so far only been observed in a large range of temperature habitats. The present study investigated the role of anammox in hydrothermal settings. During three oceanographic expeditions to the Mid-Atlantic Ridge, hydrothermal samples were collected from five vent sites, at depths ranging from 750 to 3650 m from cold to hot habitats. Evidence for the occurrence of anammox in these particular habitats was demonstrated by concurrent surveys, including the amplification of 16S rRNA gene sequences related to known anammox bacteria, ladderanes lipids analysis and measurement of a 14N15N dinitrogen production in isotope-pairing experiments at 60 and 85 °C. Together these results indicate that new deep-branching anammox bacteria may be active in these hot habitats.

Thermococcus barophilus sp. nov., a new barophilic and hyperthermophilic archaeon isolated under high hydrostatic pressure from a deep-sea hydrothermal vent.

A novel barophilic, hyperthermophilic, anaerobic sulfur-metabolizing archaeon, strain MPT (T = type strain), was isolated from a hydrothermal vent site (Snakepit) on the Mid-Atlantic Ridge (depth, 3550 m). Enrichments and isolation were done under 40 MPa hydrostatic pressure at 95 degrees C. Strain MPT was barophilic at 75, 80, 85, 90, 95 and 98 degrees C, and was an obligate barophile between 95 and 100 degrees C (Tmax). For growth above 95 degrees C, a pressure of 15.0-17.5 MPa was required. The strain grew at 48-95 degrees C under atmospheric pressure. The optimal temperature for growth was 85 degrees C at both high (40 MPa) and low (0.3 MPa) pressures. The growth rate was twofold higher at 85 degrees C under in situ hydrostatic pressure compared to at low pressure. Strain MPT cells were motile, coccoid, 0.8-2.0 microns in diameter and covered by a hexagonal S-layer lattice. The optimum pH and NaCl concentration for growth at low pressure were 7.0 and 20-30 g l-1, respectively. The new isolate was an obligate heterotroph and utilized yeast extract, beef extract and peptone for growth. Growth was optimal in the presence of elemental sulfur. Rifampicin and chloramphenicol inhibited growth. The core lipids consisted of a major archaeol and a complex lipid pattern consisting of a major phospholipid. The DNA G + C content was 37.1 mol%. Sequencing of the 16S rRNA gene revealed that strain MPT belonged to the genus Thermococcus and it is proposed that this isolate should be designated as a new species, Thermococcus barophilus.

Thermotoga subterranea sp. nov., a new thermophilic bacterium isolated from a continental oil reservoir

Abstract A thermophilic, strictly anaerobic bacterium, designated strain SL1, was isolated from a deep, continental oil reservoir in the East Paris Basin (France). This organism grew between 50 and 75°C, with an optimum at 70°C. It was inhibited by elemental sulfur and was able to reduce cystine and thiosulfate to hydrogen sulfide. The G+C content (40 mol%), the presence of a lipid structure unique to the genus Thermotoga, and the 16S rRNA sequence of strain SL1 indicated that the isolate belongs to the genus Thermotoga. Based on DNA-DNA hybridization, isolate SL1 does not show species-level similarity with the recognized species T. maritima, T. neapolitana, and T. thermarum. Based on this description of strain SL1, we propose the recognition of a new species: Thermotoga subterranea.

Desulfurobacterium thermolithotrophum gen. nov., sp. nov., a novel autotrophic, sulphur-reducing bacterium isolated from a deep-sea hydrothermal vent

A thermophilic, anaerobic, strictly autotrophic, sulphur-reducing bacterium, designated BSAT (T = type strain), was isolated from a deep-sea hydrothermal chimney sample collected at the mid-Atlantic ridge. Gram-negative cells occurred singly or in pairs as small highly motile rods. Spores were not observed. The temperature range for growth was 40 to 75 degrees C, with an optimum at 70 degrees C. The pH range for growth at 70 degrees C was from 4.4 to 7.5, with an optimum around 6.0. The sea salt concentration range for growth was 15-70 gI(-1) with an optimum at 35 gI(-1). Elemental sulphur, thiosulphate and sulphite were reduced to hydrogen sulphide. Sulphate and cystine were not reduced. The G+C content of the genomic DNA was 35 mol%. Phylogenetic analyses of the 16S rRNA gene indicated that the strain was a member of the domain Bacteria and formed a branch that was almost equidistant from members of the orders Aquificales and Thermotogales. The new organism possesses phenotypic and phylogenetic traits that do not allow its classification as a member of any previously described genus; therefore, it is proposed that this isolate should be described as a member of a novel species of a new genus, Desulfurobacterium gen. nov., of which Desulfurobacterium thermolithotrophum sp. nov. is the type species. The type strain is BSAT (= DMS 11699T).

Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent

A thermophilic, anaerobic, piezophilic, chemo-organotrophic sulfur-reducing bacterium, designated as KA3T, was isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2630 m on the East-Pacific Rise (13 degrees N). When grown under elevated hydrostatic pressure, the cells are rod-shaped with a sheath-like outer structure, motile, have a mean length of 1-1.5 microm and stain Gram-negative. They appear singly or in short chains. When grown at lower, or atmospheric, pressures, the cells elongate and become twisted. Growth is enhanced by hydrostatic pressure; the optimal pressure for growth is 40 MPa (26 MPa pressure at sampling site). The temperature range for growth is 45-70 degrees C, the optimum being around 65 degrees C (doubling time is approximately 20 min at 40 MPa). Growth is observed from pH 5 to pH 8, the optimum being at pH 6. The salinity range for growth is 10-50 g NaCl l(-1), the optimum being at 30 g l(-1). The isolate is able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates, and growth is stimulated by L-cystine and elemental sulfur. The G+C content of the genomic DNA is 29 +/- 1 mol%. According to phylogenetic analysis of the 16S rDNA gene, the strain is placed within the order Thermotogales, in the bacterial domain. On the basis of 16S rDNA sequence comparisons and morphological, physiological and genotypic characteristics, it is proposed that the isolate be described as a novel species of the genus Marinitoga, with Marinitoga piezophila sp. nov. as the type species. The type strain is KA3T (= DSM 14283T = JCM 11233T).

The manganese and iron superoxide dismutases protect Escherichia coli from heavy metal toxicity.

Superoxide dismutases (SODs) are vital components that defend against oxidative stress through decomposition of superoxide radical. Escherichia coli contains two highly homologous SODs, a manganese- and an iron-containing enzyme (Mn-SOD and Fe-SOD, respectively). In contrast, a single Mn-SOD is present in Bacillus subtilis. In E. coli, the absence of SODs was found to be associated with an increased sensitivity to cadmium, nickel and cobalt ions. Mutants lacking either sodA or sodB exhibited metal resistance to levels comparable to that of the wild-type strain. Although sod-deficient mutant cells were more resistant to zinc than their wild-type counterpart, no differences between the strains were observed in the presence of copper. In B. subtilis, the sodA mutation had no effect on cadmium and copper resistance. These results suggest that intracellular generation of superoxide by cadmium, nickel and cobalt is toxic in E. coli. They support the participation of sod genes in its protection against metal stress.

Pyrococcus CH1, an obligate piezophilic hyperthermophile: extending the upper pressure-temperature limits for life

A novel hydrothermal site was discovered in March 2007, on the mid-Atlantic ridge during the cruise ‘Serpentine’. At a depth of 4100 m, the site ‘Ashadze’ is the deepest vent field known so far. Smoker samples were collected with the ROV ‘Victor 6000’ and processed in the laboratory for the enrichment of anaerobic heterotrophic microorganisms under high-temperature and high-hydrostatic pressure conditions. Strain CH1 was successfully isolated and assigned to the genus Pyrococcus, within the Euryarchaeota lineage within the Archaea domain. This organism grows within a temperature range of 80 to 108 °C and a pressure range of 20 to 120 MPa, with optima for 98 °C and 52 MPa respectively. Pyrococcus CH1 represents the first obligate piezophilic hyperthermophilic microorganism known so far. Comparisons of growth yields obtained under high-temperature/high-pressure conditions for relative organisms isolated from various depths, showed clear relationships between depth at origin and responses to hydrostatic pressure.