Georges Barbier

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.

Phylogenetic characterization of the bacterial assemblage associated with mucous secretions of the hydrothermal vent polychaete Paralvinella palmiformis.

As part of an ongoing examination of microbial diversity associated with hydrothermal vent polychaetes of the family Alvinellidae, we undertook a culture-independent molecular analysis of the bacterial assemblage associated with mucous secretions of the Northeastern Pacific vent polychaete Paralvinella palmiformis. Using a molecular 16S rDNA-based phylogenetic approach, clone libraries were constructed from two samples collected from active sulfide edifices in two hydrothermal vent fields. In both cases, clone libraries were largely dominated by epsilon-Proteobacteria. Phylotypes belonging to the Cytophaga-Flavobacteria and to the Verrucomicrobia were also largely represented within the libraries. The remaining sequences were related to the taxonomic groups Fusobacteria, Green non-sulfur bacteria, Firmicutes, gamma- and delta-Proteobacteria. To our knowledge, this is the first report of the presence of Verrucomicrobia, Fusobacteria and green non-sulfur bacteria on hydrothermal edifices. The potential functions of the detected bacteria are discussed in terms of productivity, recycling of organic matter and detoxification within the P. palmiformis microhabitat.

Thermosipho melanesiensis sp. nov., a New Thermophilic Anaerobic Bacterium Belonging to the Order Thermotogales, Isolated from Deep-Sea Hydrothermal Vents in the Southwestern Pacific Ocean

A new thermophilic, anaerobic rod-shaped bacterium, strain BI429T was isolated from the gills of a deep-sea vent hydrothermal mussel, Bathymodiolus brevior, from the Lau Basin (Southwestern Pacific Ocean). Phenotypically, this isolate exhibited characteristics similar to those described for members of the order Thermotogales. This organism was identified as a member of the genus Thermosipho on the basis of the presence of the typical outer sheath-like structure (toga), its 16S rRNA sequence, and its ability to grow on carbohydrates (sucrose, starch, glucose, maltose, lactose, cellobiose, and galactose). The cells of this organism were gram negative and rod shaped and generally occurred singly or in pairs, rarely occurring as chains with a maximum of five rods. At the optimum temperature for growth (70 degrees C), optimum pH (6.5), and optimum salinity (30 g of NaCl per liter), the doubling time was 100 min. In spite of the high percentage of similarity of its 16S rRNA sequence with that of Thermosipho africanus (98.6%), the weak level of DNA-DNA reassociation with this strain (2%) and particular physiological characteristics allowed us to differentiate this new organism from the sole species of the genus Thermosipho previously described (T. africanus). On the basis of these observations, we propose that the new organism should be described as a new species, Thermosipho melanesiensis. The type strain of T. melanesiensis is BI429.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

Abstract The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERSs collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30°C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170 kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Extraction and purification of DNA from organic rich subsurface sediments (ODP Leg 169S)

Abstract Molecular biology offers many new tools for the characterisation of mixed communities of microorganisms. Approaches that require the extraction and purification of bulk community DNA from sediments and soils must contend with contaminants such as humic acids and heavy metals that can interfere with subsequent genetic analysis. This paper reports on the adaptation of DNA extraction and purification techniques to samples of organic rich sediments collected during the Ocean Drilling Program Leg 169S in Saanich Inlet, British Columbia. In an extraction time series, DNA yield increased up to 48xa0h (at 37°C), after which there were negligible increases in yield and signs of degradation. Resulting extracts, rich in humic substances, blocked the DNA polymerase enzyme even at high dilution. Standard purification procedures (phenol/chloroform extraction followed by silica-based DNA binding or agarose gel electrophoresis) proved ineffective in removing PCR inhibitors. The inhibitory effect was eliminated by cesium chloride density gradient centrifugation with eukaryote DNA added as a carrier, permitting amplification and cloning of SSU (small subunit) rRNA genes. A detailed extraction and purification protocol is presented. These procedures, although time-consuming, may be applicable to other sediment types where microbial DNA is particularly difficult to extract or purify.

Microbial diversity in smoked salmon examined by a culture-independent molecular approach--a preliminary study.

Microbial biodiversity in sliced vacuum-packed cold smoked salmon was investigated using culture-independent molecular biology techniques. Sliced smoked salmon was stored for 25 days after being packed at 4 degrees C. DNA was extracted from sliced vacuum-packed cold smoked salmon. PCR DNA amplification were carried out using universal eubacterial primers corresponding to Escherichia coli 16S rRNA gene. 16S rRNA genes were amplified, cloned in E. coli and compared using Amplification Ribosomal DNA Restriction Analysis (ARDRA). 106 clones were studied and classified into 13 Operational Taxonomic Units (OTUs). Sequences obtained to describe those 13 OTUs were compared to GenBank data. They indicated the presence of Vibrio species. Enterobacteraceae and also marine psychrophilic clones related to Alteromonas macleodii, which were not encountered within cultures, but no Gram-positive species have been obtained. Those results indicate that bias in description of microbial diversity may be encountred in both molecular and cultural techniques.

Thermostable amylolytic enzymes of thermophilic microorganisms from deep-sea hydrothermal vents

Resume Des microorganismes isoles de sources hydrothermales sous-marines, se developpant au-dela de 60 °C, ont ete cribles pour detecter lactivite amylolytique. Sur 269 isolats testes, 70 etaient positifs. Neuf archaea (incluant Thermococcus hydrothermalis AL662 et T. fumicolans ST557) et une bacterie thermophile ont ete choisies pour la determination de la thermostabilite et des optimums de temperature et de pH de leurs enzymes amylolytiques. Les activites α-amylase, pullulanase et α-glucosidase ont ete detectees pour quatre archaea du genre Thermococcus , dont les souches AL662 et ST557. Larchaeon hyperthermophile anaerobie T. Hydrothermalis fut selectionne pour letude de lactivite α-glucosidase. La caracterisation preliminaire de lenzyme a ete realisee. Le petit nombre d α-glucosidases hautement thermostables decrites a ce jour et les proprietes tres interessantes de cette enzyme font que ces travaux presentent un interet certain pour des applications biotechnologiques.

Purification, molecular properties and specificity of a thermoactive and thermostable proteinase from Pyrococcus abyssi, strain st 549, hyperthermophilic archaea from deep-sea hydrothermal ecosystem

A protease was isolated and purified from the supernatant of a culture of hyperthermophilic archaebacteria: Pyrococcus abyssi strain st 549. Purification consisted of three chromatographic steps. The enzyme purification yield was 4% and the purification factor 890. This protease is a seryl‐protease hydrolyzing proteins and peptides with a preference for cleavage at the aromatic and hydrophobic residues in P1 and P′1 positions. Its activity is optimal at 95°C and at pH 9. The electrophoretic mobility of the protease observed by zymogram suggests that it can adopt several oligomer forms. Three of them predominate displaying apparent molecular masses of 150, 105 and 60 kDa. Interdependence of the observed bands was revealed by changing the denaturation conditions of the samples (temperature, SDS concentration) before electrophoresis.

Isolation and characterization of extremely thermophilic archaebacteria related to the genus Thermococcus from deep-sea hydrothermal Guaymas Basin

During the “Guaynaut” oceanographic cruise performed by IFREMER in November 1991, sediment cores were collected from high-temperature and petroleum-rich deposits in an active hydrothermal zone, at the Guaymas basin (Central gulf of California). Those samples were collected by the French deep-sea manned submersible “Nautile” at a depth of 2000 meters. Four sediment cores of 20–40-cm length were drilled at the bottom of a block assemblage of active smokers inside sediments whose temperatures were 3.5°C on the top to 105°C at 20 cm depth. They were subsampled in 22 slices of 5-cm thickness and used for isolation of heterotrophic hyperthermophilic microorganisms, after inoculation in sulfur-free SME liquid medium. From those enrichments 18 isolates were obtained, 2 growing at 95°C and 16 at 80°C, and their taxonomic characterization was undertaken. Lipid analysis indicated the presence of diethers and tetraethers in the cell walls and membranes, characteristic of Archaebacteria. Examinations by scanning electron microscopy showed that isolates were cocci of heterogeneous sizes (diameter from 0.1 to 0.5 micrometers) or thick, piled-up discs 0.5 μm thick and 1 μm in diameter. Both forms were embedded in a dense fiber network. Physiologically they were found to be anaerobic, heterotrophic, and hyperthermophilic (80°–95°C). Determination of the DNA base composition resulted in G + C mol % values ranging from 36 to 57. Qualitative hybridizations of the 18 isolate DNAs with hyperthermophilic Archaebacteria reference strain DNAs showed that hybridizations occurred neither with the two species of Pyrococcus nor with the two species of Desulfurococcus, nor with Staphylothermus marinus. On the other hand, all isolates hybridized with at least one of the three species of Thermococcus tested (T. celer, T. stetteri, T. litoralis). Restriction polymorphism on a PCR-amplified fragment of the rrn operon showed that 12 isolates had the same profile as T. celer and T. stetteri, 4 isolates had the same profile as T. litoralis, and 2 had new profiles, suggestive that they are new species.

Diversity of anaerobic heterotrophic thermophiles isolated from deep‐sea hydrothermal vents of the Mid‐Atlantic Ridge

Abstract During the MARVEL oceanographical cruise performed in September 1997, samples were collected from the deep-sea vents of hydrothermal sites on the Mid-Atlantic Ridge. Eighty-four thermophilic and hyperthermophilic heterotrophic microorganisms were isolated using different culture media containing cellobiose, xylan, starch, lipidic or proteic substrates. These isolates were obtained in anaerobic conditions, at 65 degrees C, 85 degrees C and 95 degrees C. Fifty of them were classified using amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA and 16S rDNA sequencing. The strains classified have been assigned to the archaeal order Thermococcales and to the bacterial orders Thermotogales and Clostridiales. Variations in growth temperature and carbon sources were efficient enough to generate taxonomic diversity within enrichment cultures. Presumptive new genera and new species were isolated. Two isolates were confirmed as type strains of new species of new genera recently described: Marinitoga camini and Caloranaerobacter azorensis.