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Nine thermophilic strains of aerobic, non-sporulating, heterotrophic bacteria were isolated after enrichment of chimney material sampled from a deep-sea hydrothermal field at a depth of 2634 m on the East-Pacific Rise (1 °N). The bacteria stained Gram-negative. They were rod-shaped and measured approximately 0.5 μm in width and 1.5–3.5 μm in length. They grew at 55–80 °C, pH 6–8 and 1–6 % NaCl. Optimal growth was observed at 70–75 °C, pH 7.0 and 1–3 % NaCl. The organisms were identified as members of the genus Rhodothermus, having a 16S rRNA gene similarity of 98.1 % with Rhodothermus marinus DSM 4252T. The novel isolates differed morphologically, physiologically and chemotaxonomically from R. marinus, e.g. in lack of pigmentation, response to hydrostatic pressure, maximum growth temperature and DNA G+C content. DNA–DNA hybridization revealed a reassociation value of 37.2 % between strain PRI 2902T and R. marinus DSM 4252T, which strongly suggested that they represent different species. Furthermore, AFLP fingerprinting separated the novel strains from R. marinus reference strains. It is therefore concluded that the strains described here should be classified as representatives of a novel species for which the name Rhodothermus profundi sp. nov. is proposed; the type strain is PRI 2902T (=DSM 22212T =JCM 15944T).

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).

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.

An in situ sampler that encloses individual blocks of thermally stable solid medium made with Gelrite was successfully deployed at a deep-sea hydrothermal vent environment. A mixed population of microorganisms was enriched on the surface of the Gelrite blocks. Thermophilic Bacilli spp. and bacteria belonging to the Thermotogales were isolated.Key words: in situ enrichment, sampler, hydrothermal vent.

The physiology of the deep-sea hyperthermophilic, anaerobic vent archaeon Pyrococcus abyssi, originating from the Fiji Basin at a depth of 2,000 m, was studied under diverse conditions. The emphasis of these studies lay in the growth and survival of this archaeon under the different conditions present in the natural habitat. Incubation under in situ pressure (20 MPa) and at 40 MPa increased the maximal and minimal growth temperatures by 4(deg)C. In situ pressure enhanced survival at a lethal high temperature (106 to 112(deg)C) relative to that at low pressure (0.3 MPa). The whole-cell protein profile, analyzed by one-dimensional sodium dodecyl sulfate gel electrophoresis, did not change in cultures grown under low or high pressure at optimal and minimal growth temperatures, but several changes were observed at the maximal growth temperature under in situ pressure. The complex lipid pattern of P. abyssi grown under in situ and 0.1- to 0.5-MPa pressures at different temperatures was analyzed by thin-layer chromatography. The phospholipids became more complex at a low growth temperature at both pressures but their profiles were not superimposable; fewer differences were observed in the core lipids. The polar lipids were composed of only one phospholipid in cells grown under in situ pressure at high temperatures. Survival in the presence of oxygen and under starvation conditions was examined. Oxygen was toxic to P. abyssi at growth range temperature, but the strain survived for several weeks at 4(deg)C. The strain was not affected by starvation in a minimal medium for at least 1 month at 4(deg)C and only minimally affected at 95(deg)C for several days. Cells were more resistant to oxygen in starvation medium. A drastic change in protein profile, depending on incubation time, was observed in cells when starved at growth temperature.

International audience; During the Integrated Ocean Drilling Program (IODP) Expedition 331 at the Iheya North hydrothermal system in the Mid-Okinawa Trough by the D/V Chikyu, we conducted microbiological contamination tests of the drilling and coring operations. The contamination from the drilling mud fluids was assessed using both perfluorocarbon tracers (PFT) and fluorescent microsphere beads. PFT infiltration was detected from the periphery of almost all whole round cores (WRCs). By contrast, fluorescent microspheres were not detected in hydrothermally active core samples, possibly due to thermal decomposition of the microspheres under high-temperature conditions. Microbial contamination from drilling mud fluids to the core interior subsamples was further characterized by molecular-based evaluation. The microbial 16S rRNA gene phylotype compositions in the drilling mud fluids were mainly composed of sequences of Beta- and Gammaproteobacteria, and Bacteroidetes and not archaeal sequences. The phylotypes that displayed more than 97% similarity to the sequences obtained from the drilling mud fluids were defined as possible contaminants in this study and were detected as minor components of the bacterial phylotype compositions in 13 of 37 core samples. The degree of microbiological contamination was consistent with that determined by the PFT and/or microsphere assessments. This study suggests a constructive approach for evaluation and eliminating microbial contamination during riser-less drilling and coring operations by the D/V Chikyu.

A numerical taxonomic study has been carried out with 80 strains, newly isolated, from three geographically separated deep-sea hydrothermal vents (Mid-Atlantic Ridge, Guaymas Basin and Lau Basin) and eleven thermophilic reference strains representing 11 Bacillus species. The deep-sea isolates were all halotolerant spore-forming rods and grew aerobically above 65°C. Results from unweighted average linkage cluster analysis of a similarity matrix derived from the simple matching coefficient, showed formation of nine major phena, which were defined at the 83% similarity level or above. Seven phena were composed exclusively of strains isolated from the same site (4 from Mid-Atlantic Ridge, 1 from Guaymas Basin and 2 from Lau Basin). The majority of the Lau Basin isolates clustered with 6 of the reference strains in one phenon, while isolates from Mid-Atlantic Ridge and Guaymas Basin were found separated from this phenon at the 69% similarity level. The other reference strains showed less than 69% similarity with the deep-sea isolates.

We examined cell morphology, ploidy level, cell size, pigment composition, and genome size in 16 cultured strains of Phaeocystis Lagerheim. Two strains originated from the Antarctic, 3 from the tropical Western Atlantic, and 11 from temperate regions (Eastern Atlantic, English Channel, North Sea, and Mediterranean Sea). Thirteen strains made colonies morphologically similar to P. glo-bosa Scherffel, whereas three never formed colonies under any circumstances. Five-rayed star-like structures with filaments were observed in 11 strains. In several strains, two ploidy levels were observed, one (haploid) linked to flagellates and one (diploid) linked to colonies. Cell size did not appear to be a very good criterion for distinguishing strains since size distributions overlapped. Pigment analysis by reversed-phase-high-performance liquid chroma-tography allowed the strains to be grouped into three clusters that differed from each other mainly by the relative proportions of three carotenoids: fucoxanthin, 19′-hex-anoyloxyfucoxanthin, and diadinoxanthin. All strains contained low levels of 19′-butanoyloxyfucoxanthin. Differences in genome size measured by flow cytometry delimited at least five groups. On the basis of both pigment composition and genome size, six clusters were defined, one corresponding to an Antarctic species (possibly P. antarc-tica), one to P. globosa, and the rest probably to several yet-undescribed species or subspecies. Two main conclusions emerge from this study. First, the taxonomy of the genus Phaeocystis needs to be clarified through a combination of morphological, biochemical, and molecular studies. Second, sexuality is a prevalent phenomenon in Phaeocystis, but controls of the sexual cycle are most likely strain-dependent.

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).

A novel thermophilic, anaerobic and organotrophic bacterium, designated strain MC3T, was isolated from a coastal thermal spring on Île Saint-Paul in the Southern Indian Ocean. Cells of strain MC3T were motile rods, 0.8–1.0 μm wide and 1.0–2.4 μm long during exponential phase and up to 7.0 μm long during stationary phase. Strain MC3T was an anaerobic organotroph able to use diverse organic compounds. It was also able to reduce sulfur to sulfide. Growth was observed at temperatures ranging from 45 to 70 °C (optimum at 60 °C), between pH 5.5 and 7.5 (optimum at pH 6) and from 8 to 46 g NaCl l−1 (optimum at 26 g l−1). The total G+C content of the genomic DNA was 26.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain MC3T was affiliated with the genus Marinitoga within the order Thermotogales. It shared 94.4–95.7 % 16S rRNA gene sequence similarity with strains of other Marinitoga species; Marinitoga hydrogenitolerans was found to be the most closely related organism. Based on the data from the phylogenetic analysis and the physiological properties of the novel isolate, strain MC3T should be classified as a representative of a novel species, for which the name Marinitoga litoralis sp. nov. is proposed; the type strain is MC3T (=DSM 21709T =JCM 15581T).

The original publication is available at www.springerlink.com; International audience; Prokaryotic diversities of 12 geothermal hot springs located in Northern, Central and Southern Tunisia were investigated by culture-based and molecular approaches. Enrichment cultures for both aerobic and anaerobic microorganisms were successfully obtained at temperatures ranging from 50 to 75 C. Fourteen strains including four novel species were cultivated and assigned to the phyla Firmicutes (9), Thermotogae (2), Betaproteobacteria (1), Synergistetes (1) and Bacteroidetes (1). Archaeal or universal oligonucleotide primer sets were used to generate 16S rRNA gene libraries. Representative groups included Proteobacteria, Firmicutes, Deinococcus-Thermus, Thermotogae, Synergistetes, Bacteroidetes, Aquificae, Chloroflexi, candidate division OP9 in addition to other yet unclassified strains. The archaeal library showed a low diversity of clone sequences belonging to the phyla Euryarchaeota and Crenarchaeota. Furthermore, we confirmed the occurrence of sulfate reducers and methanogens by amplification and sequencing of dissimilatory sulfite reductase (dsrAB) and methyl coenzyme M reductase a-subunit (mcrA) genes. Altogether, we discuss the diverse prokaryotic communities arising from the 12 geothermal hot springs studied and relate these findings.

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 degrees C and a pressure range of 20 to 120 MPa, with optima for 98 degrees 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.

International audience; 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 (14)N(15)N dinitrogen production in isotope-pairing experiments at 60 and 85 degrees C. Together these results indicate that new deep-branching anammox bacteria may be active in these hot habitats.

Summary The 3561 m Vostok ice core sample originating from the subglacial Lake Vostok accretion (frozen lake water) ice with sediment inclusions was thoroughly studied by various means to confirm the presence of the thermophile bacterium Hydrogenophilus thermoluteolus reported earlier in the 3607 m accretion ice sample. PCR and molecular-phylogenetic analyses performed in two independent laboratories were made using different 16S rRNA gene ( rrs ) targeted primers. As a result, rrs -targeted PCR permitted to recover several very closely related clones with a small genetic distance to Hydrogenophilus thermoluteolus ( < 1%). In addition, RubisCO ( cbbL or rbcL ) and NiFe-Hydrogenase (hoxV or hupL ) targeted PCR have also allowed to recover sequences highly related to Hydrogenophilus thermoluteolus . All these results point to the presence of thermophilic chemoautotrophic microorganisms in Lake Vostok accretion ice. They presumably originate from deep faults in the bedrock cavity containing the lake in which episodes of seismotectonic activity would release debris along with microbial cells.

A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0–120 g l−1, with the optimum at 10–20 g l−1. The temperature range for growth at pH 7·0 was 4–50 °C, with the optimum at 37–40 °C. The optimum pH for growth was 7·0–8·0. The optimum pressure for growth was 0·1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2T (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA–DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

The whole-cell protein inventory of the deep-sea barophilic hyperthermophile Thermococcus barophilus was examined by one-dimensional SDS gradient gel electrophoresis when grown under different pressure conditions at 85 degrees C (Topt). One protein (P60) with a molecular mass of approximately 60 kDa was prominent at low pressures (0.3 MPa hydrostatic pressure and 0.1 MPa atmospheric pressure) but not at deep-sea pressures (10, 30, and 40 MPa). About 17 amino acids were sequenced from the N-terminal end of the protein. Sequence homology analysis in the GenBank database showed that P60 most closely resembled heat-shock proteins in some sulfur-metabolizing Archaea. A high degree of amino acid identity (81%-93%) to thermosome subunits in Thermococcales strains was found. Another protein (P35) with molecular mass of approximately 35.5 kDa was induced at 40 MPa hydrostatic pressure but not under low-pressure conditions. No amino acid sequence homology was found for this protein when the 40 amino acids from the N-terminal end were compared with homologous regions of proteins from databases. A PTk diagram was generated for T. barophilus. The results suggest that Phabitat is about 35 MPa, which corresponds to the in situ pressure where the strain was obtained.

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA-DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75 degrees C, pH 8.0; and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8T, however, showed slower growth under these conditions.

A sensitive variant of the method detecting hemolytic plaque-forming cells (PFC) was used to study the possibility of production of antibody of two different specificities by single cells. 2,4,6-Trinitrophenyl (TNP), conjugated pigeon erythrocytes (PRBC) were given in a single immunizing injection to mice, and their spleen cells were plated on days 3, 4, and 5 after this injection with both PRBC and TNP-sheep erythrocytes (SRBC). A significant number of PFC lysing both erythrocytes (3.24 × 106 cells) was found on the 3rd day after immunization. On the 4th day, out of 13.39 × 106 cells examined in all experiments, 6.260 (468 × 106 cells) monospecific PFC and 137 (10.2 × 106 spleen cells) bispecific PFC were found, whereas on the 5th day, only 0.74 × 106 cells were bispecific. Studies on cross-reactivity between PRBC and SRBC or TNP-SRBC showed that only 0.11 to 0.2 × 106 cells produce cross-reacting antibody. Furthermore, inhibition studies with TNP-bovine albumin or TNP-ε-aminocaproic acid showed that these inhibitors did not influence anti-pigeon PFC at all, but sharply reduced the number of anti-TNP-SRBC PFC and provoked a disappearance of bispecific PFC. On the other hand, short term incubation with puromycin also caused disappearance of bispecific PFC. The same proportion of double PFC was found after immunization of mice with native PRBC and SRBC. It can be inferred, therefore, that shortly (on the 3rd and 4th day) after primary immunization with two unrelated antigens, a low but significant percentage of cells simultaneously produce and secrete antibody of two different specificities.