Nikolai A. Chernyh

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.

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.

Isolation and characterization of Thermococcus sibiricus sp. nov. from a Western Siberia high-temperature oil reservoir

Abstract. Anaerobic organotrophic hyperthermophilic Archaea were isolated from five of eight samples from oil wells of the Samotlor oil reservoir (depth, 1,799–2,287xa0m; temperature, 60°–84°C). Three strains were isolated in pure cultures and characterized phylogenetically on the basis of comparison of the 16S rRNA gene sequences. All strains belonged to a new species of the genus Thermococcus, with Thermococcus litoralis, Thermococcus aggregans, Thermococcus fumicolans, and Thermococcus alcaliphilus being the nearest relatives (range of sequence similarity, 97.2%–98.8%). Strain MM 739 was studied in detail. The new isolate grew on peptides but not on carbohydrates. Elemental sulfur had a stimulatory effect on growth. The temperature range for growth was between 40° and 88°C, with the optimum at 78°C; the pH range was 5.8 to 9.0, with the optimum around 7.3; and the salinity range was 0.5% to 7.0%, with the optimum at 1.8%–2.0%. The doubling time at optimal growth conditions was about 43xa0min. The G+C content of the DNA was 38.4xa0mol%. The DNA–DNA relatedness between strain MM 739 and T. litoralis was 27%; between strain MM 739 and T. aggregans, it was 22%. Based on the phenotypic and genomic differences with known Thermococcus species, the new species Thermococcus sibiricus is proposed. The isolation of a hyperthermophilic archaeum from a deep subsurface environment, significantly remote from shallow or abyssal marine hot vents, indicates the existence of a subterranean biosphere inhabited by indigenous hyperthermophilic biota.

Thermincola ferriacetica sp. nov., a new anaerobic, thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction

A moderately thermophilic, sporeforming bacterium able to reduce amorphous Fe(III)-hydroxide was isolated from ferric deposits of a terrestrial hydrothermal spring, Kunashir Island (Kurils), and designated as strain Z-0001. Cells of strain Z-0001 were straight, Gram-positive rods, slowly motile. Strain Z-0001 was found to be an obligate anaerobe. It grew in the temperature range from 45 to 70°C with an optimum at 57–60°C, in a pH range from 5.9 to 8.0 with an optimum at 7.0–7.2, and in NaCl concentration range 0–3.5% with an optimum at 0%. Molecular hydrogen, acetate, peptone, yeast and beef extracts, glycogen, glycolate, pyruvate, betaine, choline, N-acetyl-d-glucosamine and casamino acids were used as energy substrates for growth in presence of Fe(III) as an electron acceptor. Sugars did not support growth. Magnetite, Mn(IV) and anthraquinone-2,6-disulfonate served as the alternative electron acceptors, supporting the growth of isolate Z-0001 with acetate as electron donor. Formation of magnetite was observed when amorphous Fe(III) hydroxide was used as electron acceptor. Yeast extract, if added, stimulated growth, but was not required. Isolate Z-0001 was able to grow chemolithoautotrophicaly with molecular hydrogen as the only energy substrate, Fe(III) as electron acceptor and CO2 as the carbon source. Isolate Z-0001 was able to grow with 100% CO as the sole energy source, producing H2 and CO2, requiring the presence of 0.2xa0gxa0l−1 of acetate as the carbon source. The G+C content of strain Z-0001T DNA G+C was 47.8 mol%. Based on 16S rRNA sequence analyses strain Z-0001 fell into the cluster of family Peptococcaceae, within the low G+C content Gram-Positive bacteria, clustering with Thermincola carboxydophila (98% similarity). DNA–DNA hybridization with T. carboxydophila was 27%. On the basis of physiological and phylogenetic data it is proposed that strain Z-0001T (=DSMZ 14005, VKM B-2307) should be placed in the genus Thermincola as a new species Thermincola ferriacetica sp. nov.

Complete Genome Sequence of the Anaerobic, Protein-Degrading Hyperthermophilic Crenarchaeon Desulfurococcus kamchatkensis

Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extrachromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis. The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS607 and IS200/IS605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis. Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organisms nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.

Fervidobacterium riparium sp. nov., a thermophilic anaerobic cellulolytic bacterium isolated from a hot spring

A novel obligately anaerobic, extremely thermophilic, organotrophic bacterium, strain 1445t(T), was isolated from a hot spring on Kunashir Island (Kuril Islands, Russia). Cells were motile rods (0.4-0.5 × 1.0-3.0 µm). The temperature range for growth at pH 7.8 was 46-80 °C, with optimum growth at 65 °C. The pH range for growth at 65 °C was pH 5.7-9.0, with optimum growth at pH 7.8. Growth was not observed at or below 40 °C, at or above 84 °C, at or below pH 5.4 or at or above pH 9.5. The isolate degraded a wide range of substrates including starch, cellulose and cellulose derivatives. Elemental sulfur stimulated growth, but sodium sulfate, sulfite and thiosulfate did not. DNA G+C content was 31 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain 1445t(T) belonged to the genus Fervidobacterium. 16S rRNA gene sequence similarities with strains of other species of the genus Fervidobacterium were 94.9-98.3 %; the type strain of Fervidobacterium gondwanense was the closest relative of strain 1445t(T). DNA-DNA hybridization of strain 1445t(T) and F. gondwanense AB39(T) revealed a relatedness value of 20 %. Based on phylogenetic data and physiological properties of the isolate, a novel species, designated Fervidobacterium riparium sp. nov., is proposed with strain 1445t(T) ( = DSM 21630(T) = VKM B-2549(T)) as the type strain.

Oligonucleotide Probes for the Detection of Representatives of the Genus Thermoanaerobacter

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection of representatives of the genus Thermoanaerobacter. To increase the specificity of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5-GCTTCCGCDYCCCACACCTA-3) detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5-TTAACTACGGCACGRAATGCTTC-3) was specific for the first group of species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5-CACTAMYGGGGTTTACAACC-3) targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5-TCCTCCATCAGGATGCCCTA-3) was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).

Complete Genome Sequence of the Hyperthermophilic Archaeon Thermococcus sp. Strain AM4, Capable of Organotrophic Growth and Growth at the Expense of Hydrogenogenic or Sulfidogenic Oxidation of Carbon Monoxide

Analysis of the complete genome of Thermococcus sp. strain AM4, which was the first lithotrophic Thermococcales isolate described and the first archaeal isolate to exhibit a capacity for hydrogenogenic carboxydotrophy, reveals a proximity with Thermococcus gammatolerans, corresponding to close but distinct species that differ significantly in their lithotrophic capacities.

PCR-based identification of hyperthermophilic archaea of the family Thermococcaceae.

ABSTRACT A method for rapid detection and identification of hyperthermophilic archaea of the family Thermococcaceae based on PCR amplification of 16S rRNA gene fragments with primers TcPc 173F (5′-TCCCCCATAGGYCTGRGGTACTGGAAGGTC-3′) and TcPc 589R (5′-GCCGTGRGATTTCGCCAGGGACTTACGGGC-3′) was developed and used for identification of new isolates.

Thermodesulfobium acidiphilum sp. nov., a new thermoacidophilic sulfate-reducing chemoautotrophic bacterium from a Kamchatkan thermal site.

An obligately anaerobic, sulfate-reducing micro-organism, strain 3127-1T, was isolated from geothermally heated soil (Oil Site, Uzon Caldera, Kamchatka, Russia). The new isolate was a moderately thermoacidophilic anaerobe able to grow with H2 or formate by respiration of sulfate or thiosulfate. The pH range for growth was 3.7-6.5, with an optimum at 4.8-5.0. The temperature range for growth was 37-65u2009°C, with an optimum at 55 °C. The G+C content of the genomic DNA was 33.7 mol%. The genome of strain 3127-1T contained two almost identical 16S rRNA genes, differing by a single nucleotide substitution. The closest 16S rRNA gene sequence of a validly published species belonged to Thermodesulfobium narugense Na82T (99.5u200a% similarity). However, the average nucleotide identity of the genomes of strain 3127-1T and T. narugense Na82T and the predicted DNA-DNA hybridization value (GGDC 2.1 blast+, formula 2) were as low as 86 and 32.5±2.5u200a%, respectively. This, together with phenotypic data, showed the new isolate to belong to a novel species, for which the name Thermodesulfobium acidiphilum sp. nov. is proposed. The type strain is 3127-1T (=DSM 102892T=VKM B-3043T).