Gaël Erauso

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.

Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon Pyrococcus abyssi

ABSTRACT Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (102 to 103 transformants per μg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

Insights into Dynamics of Mobile Genetic Elements in Hyperthermophilic Environments from Five New Thermococcus Plasmids

Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

Observation of virus-like particles in high temperature enrichment cultures from deep-sea hydrothermal vents

A systematic search was carried out on samples collected in various geographically distant hydrothermal sites located on the East Pacific Rise (EPR 9 degrees N and 13 degrees N) and Mid-Atlantic Ridge (MAR 36 degrees N and 37 degrees N) to investigate the diversity of virus-like particles (VLPs) from deep-sea vents. Eighty-nine positive enrichment cultures were obtained from one hundred and one crude samples at 85 degrees C. VLPs were detected by electron microscopy in fifteen different enrichments. Among the different morphotypes observed, the lemon-shaped type prevailed but rods and novel pleomorphic morphologies were also observed. Several observations strongly suggested that host strains of the novel VLPs belong to the hyperthermophilic euryarchaeal order Thermococcales.

Phenotypic Characterization, DNA Similarities, and Protein Profiles of Twenty Sulfur-Metabolizing Hyperthermophilic Anaerobic Archaea Isolated from Hydrothermal Vents in the Southwestern Pacific Ocean

We performed phenotypic and physiological studies with 20 hyperthermophilic microorganisms isolated from hydrothermal vents located in the North Fiji Basin (southwestern Pacific Ocean) at a depth of 2,000 m. These isolates were strict anaerobes that were regular to irregular coccoids and used elemental sulfur in their metabolism. Growth was observed at temperatures ranging from 50 to 101°C. The DNA base compositions varied from 43 to 60 mol%. All of these organisms were heterotrophs and fermented peptides to acetate, isovalerate, isobutyrate, and propionate. They contained both diether and tetraether lipids in their membranes, which indicates that they belong to the domain Archaea. DNA-DNA hybridization experiments revealed that there were two distinct homology groups, which correlated well with results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of soluble whole-cell proteins, and these groups corresponded to the genera Pyrococcus and Thermococcus. Five isolates exhibited levels of DNA-DNA relatedness with Pyrococcus abyssi ranging from 71 to 100% and produced almost identical protein patterns. The remaining isolates formed a weakly homogeneous group based on DNA-DNA similarity data and protein patterns; the results of unweighted pair group cluster analyses suggested that these isolates were members of five new species of the genus Thermococcus.

Spatial distribution of microbial communities in the shallow submarine alkaline hydrothermal field of the Prony Bay, New Caledonia

The shallow submarine hydrothermal field of the Prony Bay (New Caledonia) discharges hydrogen- and methane-rich fluids with low salinity, temperature (< 40°C) and high pH (11) produced by the serpentinization reactions of the ultramafic basement into the lagoon seawater. They are responsible for the formation of carbonate chimneys at the lagoon seafloor. Capillary electrophoresis single-strand conformation polymorphism fingerprinting, quantitative polymerase chain reaction and sequence analysis of 16S rRNA genes revealed changes in microbial community structure, abundance and diversity depending on the location, water depth, and structure of the carbonate chimneys. The low archaeal diversity was dominated by few uncultured Methanosarcinales similar to those found in other serpentinization-driven submarine and subterrestrial ecosystems (e.g. Lost City, The Cedars). The most abundant and diverse bacterial communities were mainly composed of Chloroflexi, Deinococcus-Thermus, Firmicutes and Proteobacteria. Functional gene analysis revealed similar abundance and diversity of both Methanosarcinales methanoarchaea, and Desulfovibrionales and Desulfobacterales sulfate-reducers in the studied sites. Molecular studies suggest that redox reactions involving hydrogen, methane and sulfur compounds (e.g. sulfate) are the energy driving forces of the microbial communities inhabiting the Prony hydrothermal system.

Isolation of new plasmids from hyperthermophilic Archaea of the order Thermococcales

A collection of 57 strains of hyperthermophilic Archaea from the order Thermococcales was screened for the presence of plasmids; 9 plasmids present in six of these strains were isolated and characterized in terms of size and cross-hybridization. The Notl macrorestriction patterns of genomic DNA of strains harbouring these plasmids were obtained. Pyrococcus abyssi strains GE27 and GE23 as well as Thermococcus sp. GE31 contained a single plasmid of 3.5 kb (pGN27), 16.8 kb (pGN23) and 5.3 kb (pGN31), respectively, whilst the three strains I559, I560 and I690 all contained two plasmids of 3.5 kb (pSN559, pSN560, pSN690) and 24 kb (pLN559, pLN560, pLN690), respectively. Plasmid pGN27 strongly cross-hybridized with the previously described plasmid pGT5 from P. abyssi strain GE5, whilst plasmids pGN23 and pGN31 did not cross-hybridize with each other, nor with any other plasmid. The three small plasmids of strains I559, I560 and I690 cross-hybridized, as well as their three large plasmids. Macrorestriction pattern analysis and the results of plasmid cross-hybridization experiments indicated that these three strains were different but closely related, and likely belonged to the genus Thermococcus. This study shows that plasmids are widespread in hyperthermophilic archaea, and significantly increases the number and diversity of plasmids available for laboratory work.

The catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi, a new deep-sea hyperthermophilic archaeobacterium

The catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi were studied in the GE5 strain isolated from a deep-sea hydrothermal vent located in the North-Fiji Basin in the SW Pacific Ocean. The enzyme from this hyperthermophilic archaeobacterium shows homotropic cooperative interactions between catalytic sites for the utilization of its two substrates, carbamoylphosphate and aspartate. The activity of this enzyme is subject to allosteric regulation. It is feed-back inhibited by the end-product cytidine triphosphate independently of temperature. In contrast, its sensitivity to the feed-back inhibitor uridine triphosphate and to the activator adenosine triphosphate disappears at high temperature. The unusual response of this aspartate transcarbamoylase to carbamoylphosphate analogues suggests a particular mode of binding of this substrate to the catalytic site as compared to the homologous enzymes of other organisms. Aspartate transcarbamoylase of Pyrococcus abyssi exhibits a remarkable stability towards high temperature and pressure.

Microbial diversity in a submarine carbonate edifice from the serpentinizing hydrothermal system of the Prony Bay (New Caledonia) over a 6-year period

Active carbonate chimneys from the shallow marine serpentinizing Prony Hydrothermal Field were sampled 3 times over a 6 years period at site ST09. Archaeal and bacterial communities composition was investigated using PCR-based methods (clone libraries, Denaturating Gel Gradient Electrophoresis, quantitative PCR) targeting 16S rRNA genes, methyl coenzyme M reductase A and dissimilatory sulfite reductase subunit B genes. Methanosarcinales (Euryarchaeota) and Thaumarchaea were the main archaeal members. The Methanosarcinales, also observed by epifluorescent microscopy and FISH, consisted of two phylotypes that were previously solely detected in two other serpentinitzing ecosystems (The Cedars and Lost City Hydrothermal Field). Surprisingly, members of the hyperthermophilic order Thermococcales were also found which may indicate the presence of a hot subsurface biosphere. The bacterial community mainly consisted of Firmicutes, Chloroflexi, Alpha-, Gamma-, Beta-, and Delta-proteobacteria and of the candidate division NPL-UPA2. Members of these taxa were consistently found each year and may therefore represent a stable core of the indigenous bacterial community of the PHF chimneys. Firmicutes isolates representing new bacterial taxa were obtained by cultivation under anaerobic conditions. Our study revealed diverse microbial communities in PHF ST09 related to methane and sulfur compounds that share common populations with other terrestrial or submarine serpentinizing ecosystems.

Vallitalea pronyensis sp. nov., isolated from a marine alkaline hydrothermal chimney

A novel thermotolerant, anaerobic, Gram-stain-positive, spore-forming bacterium was isolated from a hydrothermal chimney in Prony Bay, New Caledonia. This strain, designated FatNI3(T), grew at 15-55 °C (optimum 30 °C) and at pH 5.8-8.9 (optimum 7.7). It was slightly halophilic, requiring at least 0.5 % NaCl for growth (optimum 2.5-3.0 %), and was able to grow at up to 6 % NaCl. Sulfate, thiosulfate, elemental sulfur, sulfite, nitrate and nitrite were not used as terminal electron acceptors. Growth of strain FatNI3(T) was inhibited in the presence of sulfite (2 mM) or nitrite (2 mM). Strain FatNI3(T) fermented cellobiose, glucose, mannose, maltose, sucrose, galactose, lactose, ribose, fructose, rhamnose, raffinose, xylose, yeast extract, peptone and biotrypticase. The main fermentation products from glucose metabolism were acetate, ethanol, H2 and CO2. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The main polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, and unknown glycolipids and phospholipids. The G+C content of the genomic DNA was 36.6 mol%. On the basis of phylogenetic and physiological properties, strain FatNI3(T) ( = DSM 25904 = JCM 18391) belonging to the phylum Firmicutes, class Clostridia, order Clostridiales, is proposed as the type strain of a novel species of the genus Vallitalea, for which the name Vallitalea pronyensis sp. nov. is proposed.