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.

Thermodesulfobium acidiphilum sp. nov., a thermoacidophilic, sulfate-reducing, chemoautotrophic bacterium from a 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 37-65 °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 (99.5 % 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±2.5 %, 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).

Desulfonatronospira sulfatiphila sp. nov., and Desulfitispora elongata sp. nov., two novel haloalkaliphilic sulfidogenic bacteria from soda lakes.

Two novel haloalkaliphilic bacteria with dissimilatory sulfidogenic metabolism were recovered from syntrophic associations obtained from anaerobic sediments of hypersaline soda lakes in Kulunda Steppe (Altai, Russia). Strain ASO3-2T was a member of a sulfidogenic syntrophic association oxidizing acetate at extremely haloalkaline conditions, and was isolated in pure culture using formate as electron donor and sulfate as electron acceptor. It was identified as representing a novel member of the genus Desulfonatronospira within the Deltaproteobacteria. In contrast to the two known species of this genus, the novel isolate was able to grow with formate as electron donor and sulfate, as well as with sulfite, as electron acceptor. Strain Acr1T was a minor component in a soda lake syntrophic association converting benzoate to methane and acetate. It became dominant in a subculture fed with crotonate. While growing on crotonate, strain Acr1T formed unusually long cells filled with polyhydroxyalkanoate-like granules. Its metabolism was limited to fermentation of crotonate and pyruvate and the ability to utilize thiosulfate and sulfur/polysulfide as electron acceptor. Strain Acr1T was identified as representing a novel member of the genus Desulfitispora in the class Clostridia. Both isolates were obligately haloalkaliphilic with extreme salt tolerance. On the basis of phenotypic and phylogenetic analyses, the novel sulfidogenic isolates from soda lakes are proposed to represent two novel species: Desulfonatronospira sulfatiphila sp. nov. (ASO3-2T=DSM 100427=UNIQEM U993T) and Desulfitispora elongata sp. nov. (Acr1T=DSM 29990=UNIQEM U994T).

Stable and Variable Parts of Microbial Community in Siberian Deep Subsurface Thermal Aquifer System Revealed in a Long-Term Monitoring Study.

The goal of this work was to study the diversity of microorganisms inhabiting a deep subsurface aquifer system in order to understand their functional roles and interspecies relations formed in the course of buried organic matter degradation. A microbial community of a deep subsurface thermal aquifer in the Tomsk Region, Western Siberia was monitored over the course of 5 years via a 2.7 km deep borehole 3P, drilled down to a Palaeozoic basement. The borehole water discharges with a temperature of ca. 50°C. Its chemical composition varies, but it steadily contains acetate, propionate, and traces of hydrocarbons and gives rise to microbial mats along the surface flow. Community analysis by PCR-DGGE 16S rRNA genes profiling, repeatedly performed within 5 years, revealed several dominating phylotypes consistently found in the borehole water, and highly variable diversity of prokaryotes, brought to the surface with the borehole outflow. The major planktonic components of the microbial community were Desulfovirgula thermocuniculi and Methanothermobacter spp. The composition of the minor part of the community was unstable, and molecular analysis did not reveal any regularity in its variations, except some predominance of uncultured Firmicutes. Batch cultures with complex organic substrates inoculated with water samples were set in order to enrich prokaryotes from the variable part of the community. PCR-DGGE analysis of these enrichments yielded uncultured Firmicutes, Chloroflexi, and Ignavibacteriae. A continuous-flow microaerophilic enrichment culture with a water sample amended with acetate contained Hydrogenophilus thermoluteolus, which was previously detected in the microbial mat developing at the outflow of the borehole. Cultivation results allowed us to assume that variable components of the 3P well community are hydrolytic organotrophs, degrading buried biopolymers, while the constant planktonic components of the community degrade dissolved fermentation products to methane and CO2, possibly via interspecies hydrogen transfer. Occasional washout of minor community components capable of oxygen respiration leads to the development of microbial mats at the outflow of the borehole where residual dissolved fermentation products are aerobically oxidized. Long-term community analysis with the combination of molecular and cultivation techniques allowed us to characterize stable and variable parts of the community and propose their environmental roles.

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.

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.

Caldanaerobacter uzonensis sp. nov., an anaerobic, thermophilic, heterotrophic bacterium isolated from a hot spring.

An anaerobic thermophilic bacterium, strain K67(T), was isolated from a terrestrial hot spring of Uzon Caldera, Kamchatka Peninsula. Analysis of the 16S rRNA gene sequence revealed that the novel isolate belongs to the genus Caldanaerobacter, with 95 % 16S rRNA gene sequence similarity to Caldanaerobacter subterraneus subsp. subterraneus SEBR 7858(T), suggesting that it represents a novel species of the genus Caldanaerobacter. Strain K67(T) was characterized as an obligate anaerobe, a thermophile (growth at 50-75 degrees capital ES, Cyrillic; optimum 68-70 degrees C), a neutrophile (growth at pH(25 degrees C) 4.8-8.0; optimum pH(25 degrees C) 6.8) and an obligate organotroph (growth by fermentation of various sugars, peptides and polysaccharides). Major fermentation products were acetate, H2 and CO2; ethanol, lactate and l-alanine were formed in smaller amounts. Thiosulfate stimulated growth and was reduced to hydrogen sulfide. Nitrate, sulfate, sulfite and elemental sulfur were not reduced and did not stimulate growth. Thus, according to the strain's phylogenetic position and phenotypic novelties (lower upper limit of temperature range for growth, the ability to grow on arabinose, the inability to reduce elemental sulfur and the formation of alanine as a minor fermentation product), the novel species Caldanaerobacter uzonensis sp. nov. is proposed, with the type strain K67(T) (=DSM 18923(T) =VKM capital VE, Cyrillic-2408(T)).

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 organism's nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.

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 degrees C with an optimum at 57-60 degrees 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 CO(2) as the carbon source. Isolate Z-0001 was able to grow with 100% CO as the sole energy source, producing H(2) and CO(2), requiring the presence of 0.2 g l(-1) of acetate as the carbon source. The G+C content of strain Z-0001(T )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-0001(T) (=DSMZ 14005, VKM B-2307) should be placed in the genus Thermincola as a new species Thermincola ferriacetica sp. nov.

Thermincola carboxydiphila gen. nov., sp. nov., a novel anaerobic, carboxydotrophic, hydrogenogenic bacterium from a hot spring of the Lake Baikal area.

A novel anaerobic, thermophilic, alkalitolerant bacterium, strain 2204(T), was isolated from a hot spring of the Baikal Lake region. The cells of strain 2204(T) were straight rods of variable length, Gram-positive with an S-layer, motile with one to two lateral flagella, and often formed aggregates of 3-15 cells. The isolate was shown to be an obligate anaerobe oxidizing CO and producing equimolar quantities of H(2) and CO(2) according to the equation CO+H(2)O-->CO(2)+H(2). No organic substrates were used as energy sources. For lithotrophic growth on CO, 0.2 g acetate or yeast extract l(-1) was required but did not support growth in the absence of CO. Growth was observed in the temperature range 37-68 degrees C, the optimum being 55 degrees C. The pH range for growth was 6.7-9.5, the optimum pH being 8.0. The generation time under optimal conditions was 1.3 h. The DNA G+C content was 45 mol%. Penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline completely inhibited both growth and CO utilization by strain 2204(T). Thus, isolate 2204(T) was found to be the first known moderately thermophilic and alkalitolerant H(2)-producing anaerobic carboxydotroph. The novel bacterium fell within the cluster of the family Peptococcaceae within the low-G+C-content Gram-positive bacteria, where it formed a separate branch. On the basis of morphological, physiological and phylogenetic features, strain 2204(T) should be assigned to a novel genus and species, for which the name Thermincola carboxydiphila gen. nov., sp. nov. is proposed. The type strain is strain 2204(T) (=DSM 17129(T)=VKM B-2283(T)=JCM 13258(T)).

Thermosinus carboxydivorans gen. nov., sp. nov., a new anaerobic, thermophilic, carbon-monoxide-oxidizing, hydrogenogenic bacterium from a hot pool of Yellowstone National Park.

A new anaerobic, thermophilic, facultatively carboxydotrophic bacterium, strain Nor1(T), was isolated from a hot spring at Norris Basin, Yellowstone National Park. Cells of strain Nor1(T) were curved motile rods with a length of 2.6-3 microm, a width of about 0.5 microm and lateral flagellation. The cell wall structure was of the Gram-negative type. Strain Nor1(T) was thermophilic (temperature range for growth was 40-68 degrees C, with an optimum at 60 degrees C) and neutrophilic (pH range for growth was 6.5-7.6, with an optimum at 6.8-7.0). It grew chemolithotrophically on CO (generation time, 1.15 h), producing equimolar quantities of H(2) and CO(2) according to the equation CO+H(2)O-->CO(2)+H(2). During growth on CO in the presence of ferric citrate or amorphous ferric iron oxide, strain Nor1(T) reduced ferric iron but produced H(2) and CO(2) at a ratio close to 1 : 1, and growth stimulation was slight. Growth on CO in the presence of sodium selenite was accompanied by precipitation of elemental selenium. Elemental sulfur, thiosulfate, sulfate and nitrate did not stimulate growth of strain Nor1(T) on CO and none of these chemicals was reduced. Strain Nor1(T) was able to grow on glucose, sucrose, lactose, arabinose, maltose, fructose, xylose and pyruvate, but not on cellobiose, galactose, peptone, yeast extract, lactate, acetate, formate, ethanol, methanol or sodium citrate. During glucose fermentation, acetate, H(2) and CO(2) were produced. Thiosulfate was found to enhance the growth rate and cell yield of strain Nor1(T) when it was grown on glucose, sucrose or lactose; in this case, acetate, H(2)S and CO(2) were produced. In the presence of thiosulfate or ferric iron, strain Nor1(T) was also able to grow on yeast extract. Lactate, acetate, formate and H(2) were not utilized either in the absence or in the presence of ferric iron, thiosulfate, sulfate, sulfite, elemental sulfur or nitrate. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. The DNA G+C content of the strain was 51.7+/-1 mol%. Analysis of the 16S rRNA gene sequence revealed that strain Nor1(T) belongs to the Bacillus-Clostridium phylum of the Gram-positive bacteria. On the basis of the studied phenotypic and phylogenetic features, we propose that strain Nor1(T) be assigned to a new genus, Thermosinus gen. nov. The type species is Thermosinus carboxydivorans sp. nov. (type strain, Nor1(T)=DSM 14886(T)=VKM B-2281(T)).

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

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.

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 degrees N), 13 enrichments of coccoid cells were obtained which grew on CO, producing H2 and CO2 at 80 degrees 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.

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.