Dissimilatory sulfate reduction in the archaeon 'Candidatus Vulcanisaeta moutnovskia' sheds light on the evolution of sulfur metabolism.

Dissimilatory sulfate reduction (DSR)-an important reaction in the biogeochemical sulfur cycle-has been dated to the Palaeoarchaean using geological evidence, but its evolutionary history is poorly understood. Several lineages of bacteria carry out DSR, but in archaea only Archaeoglobus, which acquired DSR genes from bacteria, has been proven to catalyse this reaction. We investigated substantial rates of sulfate reduction in acidic hyperthermal terrestrial springs of the Kamchatka Peninsula and attributed DSR in this environment to Crenarchaeota in the Vulcanisaeta genus. Community profiling, coupled with radioisotope and growth experiments and proteomics, confirmed DSR by 'Candidatus Vulcanisaeta moutnovskia', which has all of the required genes. Other cultivated Thermoproteaceae were briefly reported to use sulfate for respiration but we were unable to detect DSR in these isolates. Phylogenetic studies suggest that DSR is rare in archaea and that it originated in Vulcanisaeta, independent of Archaeoglobus, by separate acquisition of qmoABC genes phylogenetically related to bacterial hdrA genes.

Genomic Analysis of Caldithrix abyssi, the Thermophilic Anaerobic Bacterium of the Novel Bacterial Phylum Calditrichaeota.

The genome of Caldithrix abyssi, the first cultivated representative of a phylum-level bacterial lineage, was sequenced within the framework of Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. The genomic analysis revealed mechanisms allowing this anaerobic bacterium to ferment peptides or to implement nitrate reduction with acetate or molecular hydrogen as electron donors. The genome encoded five different [NiFe]- and [FeFe]-hydrogenases, one of which, group 1 [NiFe]-hydrogenase, is presumably involved in lithoheterotrophic growth, three other produce H2 during fermentation, and one is apparently bidirectional. The ability to reduce nitrate is determined by a nitrate reductase of the Nap family, while nitrite reduction to ammonia is presumably catalyzed by an octaheme cytochrome c nitrite reductase εHao. The genome contained genes of respiratory polysulfide/thiosulfate reductase, however, elemental sulfur and thiosulfate were not used as the electron acceptors for anaerobic respiration with acetate or H2, probably due to the lack of the gene of the maturation protein. Nevertheless, elemental sulfur and thiosulfate stimulated growth on fermentable substrates (peptides), being reduced to sulfide, most probably through the action of the cytoplasmic sulfide dehydrogenase and/or NAD(P)-dependent [NiFe]-hydrogenase (sulfhydrogenase) encoded by the genome. Surprisingly, the genome of this anaerobic microorganism encoded all genes for cytochrome c oxidase, however, its maturation machinery seems to be non-operational due to genomic rearrangements of supplementary genes. Despite the fact that sugars were not among the substrates reported when C. abyssi was first described, our genomic analysis revealed multiple genes of glycoside hydrolases, and some of them were predicted to be secreted. This finding aided in bringing out four carbohydrates that supported the growth of C. abyssi: starch, cellobiose, glucomannan and xyloglucan. The genomic analysis demonstrated the ability of C. abyssi to synthesize nucleotides and most amino acids and vitamins. Finally, the genomic sequence allowed us to perform a phylogenomic analysis, based on 38 protein sequences, which confirmed the deep branching of this lineage and justified the proposal of a novel phylum Calditrichaeota.

Calorithrix insularis gen. nov., sp. nov., a novel representative of the phylum Calditrichaeota.

A moderately thermophilic, anaerobic bacterium designated as strain KRT was isolated from a shallow-water submarine hydrothermal vent (Kunashir Island, Southern Kurils, Russia). Cells of strain KRT were thin (0.2-0.3 µm), flexible, motile, Gram-stain-negative rods of variable length. Optimal growth conditions were pH 6.6, 55 °C and 1-3 % (w/v) NaCl. Strain KRT was able to ferment a wide range of proteinaceous substrates, pyruvate, and mono-, di- and polysaccharides. The best growth occurred with proteinaceous compounds. Nitrate significantly stimulated the growth on proteinaceous substrates decreasing H2 formation, ammonium being the main product of nitrate reduction. Strain KRT did not need the presence of a reducing agent in the medium and tolerated the presence of oxygen in the gas phase up to 3 % (v/v). In the presence of nitrate, aerotolerance of isolate KRT was enhanced up to 6-8 % O2 (v/v). Strain KRT was able to grow chemolithoheterotrophically, oxidizing H2 and reducing nitrate to ammonium. Yeast extract (0.05 g l-1) was required for growth. The G+C content of the genomic DNA of strain KRT was 47.3 mol%. 16S rRNA gene sequence analysis placed isolate KRT in the phylum Calditrichaeota where it represented a novel species of a new genus, for which the name Calorithrix insularis gen. nov., sp. nov. is proposed. The type strain of Calorithrix insularis is KRT (=DSM 101605T=VKM B-3022T).

Microbial life in Bourlyashchy, the hottest thermal pool of Uzon Caldera, Kamchatka.

Bourlyashchy is the largest and hottest pool in the Uzon Caldera, located in the territory of Kronotsky Nature Reserve, Kamchatka, Russia, with sediment surface temperatures at the margins ranging from 86 to 97 °C, and pH from 6.0 to 7.0. The microbial communities of the pool water and sediments were studied comprehensively from 2005 to 2014. Radioisotopic tracer studies revealed the processes of inorganic carbon assimilation, sulfate reduction, lithotrophic methanogenesis and potentially very active process of acetate oxidation to CO2. The total number of microbial cells in water was different in different years ranging from 5.2 to 7.0 × 10(6); in sediments, it changed from year to year between 6.3 × 10(6) and 1.75 × 10(8), increasing with a decrease in temperature. FISH with Archaea- and Bacteria-specific probes showed that the share of Bacteria differed with year, changing from 34 to 71%. According to 16S rRNA gene pyrosequencing data, lithoautotrophs (Aquificales and Thermoproteales) predominated in water samples, while in sediments they shared the niche with organotrophic Crenarchaeota, Korarchaeota, and bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). The majority of organisms in water belonged to cultivated orders of prokaryotes; the only large uncultured group was that representing a novel order in class Thermoprotei. In sediments, unclassified Aquificeae comprised a significant part of the bacterial population. Thus, we showed that the hottest of the terrestrial hot pools studied contains numerous and active microbial populations where Bacteria represent a significant part of the microbial community, and planktonic and sediment populations differ in both composition and function.

Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov., thermophilic anaerobic representatives of the phylum Planctomycetes.

Two novel strains of thermophilic planctomycetes were recovered from terrestrial and subterranean habitats. Strain R1(T) was isolated from a hot spring (Kunashir Island, Russia) and strain SBP2(T) was isolated from a deep gold mine (South Africa). Both isolates grew in the temperature range 30-60 °C and pH range 5.0-8.0. Strain R1(T) grew optimally at 60 °C and pH 6.0-6.5; for SBP2(T) optimal conditions were at 52 °C and pH 7.5-8.0. Both strains were capable of anaerobic respiration with nitrate and nitrite as electron acceptors as well as of microaerobic growth. They also could grow by fermentation of mono-, di- and polysaccharides. Based on their phylogenetic position and phenotypic features we suggest that the new isolates represent two novel species belonging to a new genus in the order Planctomycetales, for which the names Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov. are proposed. The type strain of Thermogutta terrifontis, the type species of the genus, is R1(T) ( = DSM 26237(T) = VKM B-2805(T)), and the type strain of Thermogutta hypogea is SBP2(T) ( = JCM 19991(T) = VKM B-2782(T)).

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.

Caldithrix palaeochoryensis sp. nov., a thermophilic, anaerobic, chemo-organotrophic bacterium from a geothermally heated sediment, and emended description of the genus Caldithrix.

A novel thermophilic, strictly anaerobic, chemo-organotrophic bacterium, designated MC(T), was isolated from a geothermally heated sediment of a marine hydrothermal system at Palaeochory Bay, Milos, Greece. Cells of strain MC(T) were rods of variable length (4-12 mum) and width (0.2-0.3 mum), occurring as single cells or forming large aggregates that were visible as flocs. Strain MC(T) grew optimally at pH 7.0 and 60 degrees C and with 3 % (w/v) NaCl. Strain MC(T) grew chemo-organoheterotrophically and fermented peptides and di- and polysaccharides in the presence of 0.1 g yeast extract l(-1). The DNA G+C content of strain MC(T) was 43.3 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed strain MC(T) within the genus Caldithrix. However, strain MC(T) possessed certain phenotypic features that differentiated it from the type strain of the only species of the genus Caldithrix described to date. On the basis of phylogenetic and phenotypic characteristics, it is proposed that strain MC(T) represents a novel species, Caldithrix palaeochoryensis sp. nov. The type strain is MC(T) (=DSM 21940(T) =VKM B-2536(T)). In addition, an emended description of the genus Caldithrix is presented.

Caldimicrobium rimae gen. nov., sp. nov., an extremely thermophilic, facultatively lithoautotrophic, anaerobic bacterium from the Uzon Caldera, Kamchatka.

An extremely thermophilic, strictly anaerobic, facultatively chemolithoautotrophic bacterium designated strain DS(T) was isolated from Treshchinnyi Spring, one of the hottest springs of the Uzon Caldera (Kamchatka, Russia). Cells of the novel organism were Gram-negative rods, about 1.0-1.2 microm long and 0.5 microm wide. The temperature range for growth was 52-82 degrees C, with an optimum at 75 degrees C. Growth was observed at pH 6.8-7.4, and the optimum pH was 7.0-7.2. Strain DS(T) was able to grow lithoautotrophically with hydrogen in the presence of CO(2) as a carbon source and thiosulfate or elemental sulfur as an electron acceptor. It also grew well with ethanol, fumarate, succinate or malate in the presence of thiosulfate. Yeast extract was not required for growth and did not stimulate growth. The genomic DNA G+C content was 35.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that the novel organism was a member of the family Thermodesulfobacteriaceae. On the basis of phylogenetic and physiological considerations, it is proposed that strain DS(T) represents a new genus and species, Caldimicrobium rimae gen. nov., sp. nov. The type strain of Caldimicrobium rimae is DS(T) (=DSM 19393(T) =VKM B-2460(T)).

Metabolic versatility and indigenous origin of the archaeon Thermococcus sibiricus, isolated from a siberian oil reservoir, as revealed by genome analysis.

Thermococcus species are widely distributed in terrestrial and marine hydrothermal areas, as well as in deep subsurface oil reservoirs. Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a well of the never flooded oil-bearing Jurassic horizon of a high-temperature oil reservoir. To obtain insight into the genome of an archaeon inhabiting the oil reservoir, we have determined and annotated the complete 1,845,800-base genome of T. sibiricus. A total of 2,061 protein-coding genes have been identified, 387 of which are absent in other members of the order Thermococcales. Physiological features and genomic data reveal numerous hydrolytic enzymes (e.g., cellulolytic enzymes, agarase, laminarinase, and lipases) and metabolic pathways, support the proposal of the indigenous origin of T. sibiricus in the oil reservoir, and explain its survival over geologic time and its proliferation in this habitat. Indeed, in addition to proteinaceous compounds known previously to be present in oil reservoirs at limiting concentrations, its growth was stimulated by cellulose, agarose, and triacylglycerides, as well as by alkanes. Two polysaccharide degradation loci were probably acquired by T. sibiricus from thermophilic bacteria following lateral gene transfer events. The first, a "saccharolytic gene island" absent in the genomes of other members of the order Thermococcales, contains the complete set of genes responsible for the hydrolysis of cellulose and beta-linked polysaccharides. The second harbors genes for maltose and trehalose degradation. Considering that agarose and laminarin are components of algae, the encoded enzymes and the substrate spectrum of T. sibiricus indicate the ability to metabolize the buried organic matter from the original oceanic sediment.

Ammonifex thiophilus sp. nov., a hyperthermophilic anaerobic bacterium from a Kamchatka hot spring.

A novel anaerobic, extremely thermophilic, facultatively chemolithoautotrophic bacterium designated strain SR(T) was isolated from a terrestrial hot spring in Kamchatka (Russia). The cells of the novel strain were spore-forming rods with a Gram-positive type of cell wall. The novel isolate grew at 60-82 degrees C (optimum 75 degrees C) and pH 6.0-7.5 (optimum 6.8). Molecular hydrogen and formate were used as electron donors. Thiosulfate, sulfate or elemental sulfur served as electron acceptors yielding hydrogen sulfide. No growth was observed on either substrate in the presence of nitrate as the electron acceptor. The G+C content of the DNA was 56.2 mol%. Phylogenetic analyses of the 16S rRNA gene showed that strain SR(T) was most closely related to Ammonifex degensii (96.4 % gene sequence similarity). However, the novel isolate possessed phenotypic characteristics that differed significantly from those of A. degensii, the only other recognized species of the genus Ammonifex. It is concluded that strain SR(T) (=DSM 19636(T)=VKM B-2461(T)) represents the type strain of a novel species of the genus Ammonifex, for which the name Ammonifex thiophilus sp. nov. is proposed. An emendation of the genus Ammonifex is proposed based on the phenotypic characteristics of the novel species.

Caldicellulosiruptor kronotskyensis sp. nov. and Caldicellulosiruptor hydrothermalis sp. nov., two extremely thermophilic, cellulolytic, anaerobic bacteria from Kamchatka thermal springs.

Five novel strains (2002(T), 2902, 2006, 108(T) and 117) of cellulose-degrading, anaerobic, thermophilic bacteria were isolated from terrestrial hot springs of Kamchatka (Far East, Russia). Strains 2002(T) and 108(T) were non-spore-forming bacteria with a Gram-positive type cell wall and peritrichous flagella. Optimum growth of strains 2002(T) and 108(T) occurred at pH 7.0 and at temperatures of 70 and 65 degrees C, respectively. The G+C contents of the DNA of strains 2002(T) and 108(T) were 35.1 and 36.4 mol%, respectively. Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to the genus Caldicellulosiruptor. However, DNA-DNA hybridization experiments indicated that the levels of relatedness between strains 2002(T) and 108(T) and those of recognized members of the genus Caldicellulosiruptor ranged between 32 and 54 %. Based on both phenotypic and genomic differences, strains 2002(T) and 108(T) are considered to represent two novel species of the genus Caldicellulosiruptor. The names proposed for these organisms are Caldicellulosiruptor kronotskyensis sp. nov. (type strain 2002(T)=DSM 18902(T)=VKM B-2412(T)) and Caldicellulosiruptor hydrothermalis sp. nov. (type strain 108(T)=DSM 18901(T)=VKM B-2411(T)).

Recent developments in the thermophilic microbiology of deep-sea hydrothermal vents.

The diversity of thermophilic prokaryotes inhabiting deep-sea hot vents was actively studied over the last two decades. The ever growing interest is reflected in the exponentially increasing number of novel thermophilic genera described. The goal of this paper is to survey the progress in this field made in the years 2000-2005. In this period, representatives of several new taxa of hyperthermophilic archaea were obtained from deep-sea environments. Two of these isolates had phenotypic features new for this group of organisms: the presence of an outer cell membrane (the genus Ignicoccus) and the ability to grow anaerobically with acetate and ferric iron (the genus Geoglobus). Also, our knowledge on the diversity of thermophilic bacteria from deep-sea thermal environments extended significantly. The new bacterial isolates represented diverse bacterial divisions: the phylum Aquificae, the subclass Epsilonproteobacteria, the order Thermotogales, the families Thermodesulfobacteriaceae, Deferribacteraceae, and Thermaceae, and a novel bacterial phylum represented by the genus Caldithrix. Most of these isolates are obligate or facultative lithotrophs, oxidizing molecular hydrogen in the course of different types of anaerobic respiration or microaerobic growth. The existence and significant ecological role of some of new bacterial thermophilic isolates was initially established by molecular methods.

Radioisotopic, culture-based, and oligonucleotide microchip analyses of thermophilic microbial communities in a continental high-temperature petroleum reservoir.

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 H(2)S liter(-1) day(-1) occurred at 60 and 80 degrees 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 CH(4) 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.

Caldithrix abyssi gen. nov., sp. nov., a nitrate-reducing, thermophilic, anaerobic bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent, represents a novel bacterial lineage.

A novel, moderately thermophilic, strictly anaerobic, mixotrophic bacterium, designated strain LF13T, was isolated from a deep-sea hydrothermal chimney sample that was collected at a vent site at 14 degrees 45' N, 44 degrees 59' W on the Mid-Atlantic Ridge. Cells were Gram-negative, thin, non-motile rods of variable length. Strain LF13T grew optimally at pH 6.8-7.0 and 60 degrees C with 2.5% (w/v) NaCl. It grew chemo-organoheterotrophically, fermenting proteinaceous substrates, pyruvate and Casamino acids. The strain was able to grow by respiration, utilizing molecular hydrogen (chemolithoheterotrophically) or acetate as electron donors and nitrate as an electron acceptor. Ammonium was formed in the course of denitrification. One-hundred milligrams of yeast extract per litre were required for growth of the strain. The G + C content of the genomic DNA of strain LF13T was 42.5 mol%. Neither 16S rDNA sequence similarity values nor phylogenetic analysis unambiguously related strain LF13T with members of any recognized bacterial phyla. On the basis of 16S rDNA sequence comparisons, and in combination with physiological and morphological traits, a novel genus, Caldithrix, is proposed, with strain LF13T (= DSM 13497T =VKM B-2286T) representing the type species, Caldithrix abyssi.

Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent.

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).