Christian Jeanthon

Radioisotopic, Culture-Based, and Oligonucleotide Microchip Analyses of Thermophilic Microbial Communities in a Continental High-Temperature Petroleum Reservoir

ABSTRACT Activity measurements by radioisotopic methods and cultural and molecular approaches were used in parallel to investigate the microbial biodiversity and its physiological potential in formation waters of the Samotlor high-temperature oil reservoir (Western Siberia, Russia). Sulfate reduction with rates not exceeding 20 nmol of H2S liter−1 day−1 occurred at 60 and 80°C. In upper horizons (AB, A, and B), methanogenesis (lithotrophic and/or acetoclastic) was detected only in wells in which sulfate reduction did not occur. In some of the wells from deeper (J) horizons, high-temperature sulfate reduction and methanogenesis occurred simultaneously, the rate of lithotrophic methanogenesis exceeding 80 nmol of CH4 liter−1 day−1. Enrichment cultures indicated the presence of diverse physiological groups representing aerobic and anaerobic thermophiles and hyperthermophiles; fermentative organotrophs were predominant. Phylogenetic analyses of 15 isolates identified representatives of the genera Thermotoga, Thermoanaerobacter, Geobacillus, Petrotoga, Thermosipho, and Thermococcus, the latter four being represented by new species. Except for Thermosipho, the isolates were members of genera recovered earlier from similar habitats. DNA obtained from three samples was hybridized with a set of oligonucleotide probes targeting selected microbial groups encompassing key genera of thermophilic bacteria and archaea. Oligonucleotide microchip analyses confirmed the cultural data but also revealed the presence of several groups of microorganisms that escaped cultivation, among them representatives of the Aquificales/Desulfurobacterium-Thermovibrio cluster and of the genera Desulfurococcus and Thermus, up to now unknown in this habitat. The unexpected presence of these organisms suggests that their distribution may be much wider than suspected.

Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents

ABSTRACT Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria(epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilonProteobacteria that are directly associated withAlvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilonProteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilonProteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.

Dissimilatory reduction of Fe(III) by thermophilic bacteria and archaea in deep subsurface petroleum reservoirs of western siberia

Abstract. Twenty-five samples of stratal fluids obtained from a high-temperature (60–84°C) deep subsurface (1700–2500 m) petroleum reservoir of Western Siberia were investigated for the presence of dissimilatory Fe(III)-reducing microorganisms. Of the samples, 44% and 76% were positive for Fe(III) reduction with peptone and H2 respectively as electron donors. In most of these samples, the numbers of culturable thermophilic H2-utilizing iron reducers were in the order of 10–100 cells/ml. Nine strains of thermophilic anaerobic bacteria and archaea isolated from petroleum reservoirs were tested for their ability to reduce Fe(III). Eight strains belonging to the genera Thermoanaerobacter, Thermotoga, and Thermococcus were found capable of dissimilatory Fe(III) reduction, with peptone or H2 as electron donor and amorphous Fe(III) oxide as electron acceptor. These results demonstrated that Fe(III) reduction may be a common feature shared by a wide range of anaerobic thermophiles and hyperthermophiles in deep subsurface petroleum reservoirs.

The first evidence of anaerobic CO oxidation coupled with H2 production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

From 24 samples of hydrothermal venting structures collected at the East Pacific Rise (13°N), 13 enrichments of coccoid cells were obtained which grew on CO, producing H2 and CO2 at 80°C. A hyperthermophilic archaeon capable of lithotrophic growth on CO coupled with equimolar production of H2 was isolated. Based on its 16S rRNA sequence analysis, this organism was affiliated with the genus Thermococcus. Other strains of Thermococcales species (Pyrococcus furiosus, Thermococcus peptonophilus, T. profundus, T. chitonophagus, T. stetteri, T. gorgonarius, T. litoralis, and T. pacificus) were shown to be unable to grow on CO. Searches in sequence databases failed to reveal deposited sequences of genes related to CO metabolism in Thermococcales. Our work provides the first evidence of anaerobic CO oxidation coupled with H2 production performed by an archaeon as well as the first documented case of lithotrophic growth of a Thermococcales representative.

Molecular ecology of hydrothermal vent microbial communities

The study of the structure and diversity of hydrothermal vent microbial communities has long been restricted to the morphological description of microorganisms and the use of enrichment culture-based techniques. Until recently the identification of the culturable fraction required the isolation of pure cultures followed by testing for multiple physiological and biochemical traits. However, peculiar inhabitants of the hydrothermal ecosystem such as the invertebrate endosymbionts and the dense microbial mat filaments have eluded laboratory cultivation. Substantial progress has been achieved in recent years in techniques for the identification of microorganisms in natural environments. Application of molecular approaches has revealed the existence of unique and previously unrecognized microorganisms. These have provided fresh insight into the ecology, diversity and evolution of mesophilic and thermophilic microbial communities from the deep-sea hydrothermal ecosystem. This review reports the main discoveries made through the introduction of these powerful techniques in the study of deep-sea hydrothermal vent microbiology.

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).

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.

Thermodesulfobacterium hydrogeniphilum sp. nov., a thermophilic, chemolithoautotrophic, sulfate-reducing bacterium isolated from a deep-sea hydrothermal vent at Guaymas Basin, and emendation of the genus Thermodesulfobacterium

A thermophilic, non-spore-forming, marine, sulfate-reducing bacterium, strain SL6T, was isolated from deep-sea hydrothermal sulfides collected at Guaymas Basin. The gram-negative-staining cells occurred singly or in pairs as small, highly motile rods. The temperature range for growth was 50-80 degrees C with an optimum at 75 degrees C. The pH range for growth at 70 degrees C was 6.3-6.8, with an optimum at 6.5. The NaCl concentration range for growth was 5-55 g l(-1), with an optimum at 30 g l(-1). H2 and CO2 were the only substrates for growth and sulfate reduction. However, growth was stimulated by several organic compounds. Sulfur, thiosulfate, sulfite, cystine, nitrate and fumarate were not used as electron acceptors. Pyruvate, lactate and malate did not support fermentative growth. Desulfoviridin was not detected. The G+C content of the genomic DNA was 28 mol%. On the basis of 16S rRNA sequence analysis, strain SL6T is related to members of the genus Thermodesulfobacterium. However, the novel organism possesses phenotypic and phylogenetic traits that differ from those of its closest relatives. Therefore, it is proposed that this isolate, which constitutes the first marine representative of this genus, should be described as the type strain of a novel species, Thermodesulfobacterium hydrogeniphilum sp. nov. The type strain is SL6T (= DSM 14290T = JCM 11239T). Because of the phenotypic characteristics of the novel species, it is also proposed that the description of the genus Thermodesulfobacterium requires emendation.

Nautilia lithotrophica gen. nov., sp. nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent.

A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525T, was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13 degrees N). This organism grew in the temperature range 37-68 degrees C, the optimum being 53 degrees C, and in the pH range 6.4-7.4, the optimum being 6.8-7.0. The NaCl range for growth was 0.8-5.0%, the optimum being 3.0%. Strain 525T grew lithoautotrophically with H2 as energy source, S0 as electron acceptor and CO2 as carbon source. Alternatively, strain 525T was able to use formate as an energy source. The G+C content of the genomic DNA was 34.7 mol%. Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525T in the epsilon-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives. On the basis of phenotypic and phylogenetic differences between strain 525T and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen. nov., sp. nov. is proposed. The type strain is strain 525T (= DSM 13520T).