G. B. Slobodkina

Biodiversity of Thermophilic Prokaryotes with Hydrolytic Activities in Hot Springs of Uzon Caldera, Kamchatka (Russia)†

ABSTRACT Samples of water from the hot springs of Uzon Caldera with temperatures from 68 to 87°C and pHs of 4.1 to 7.0, supplemented with proteinaceous (albumin, casein, or α- or β-keratin) or carbohydrate (cellulose, carboxymethyl cellulose, chitin, or agarose) biological polymers, were filled with thermal water and incubated at the same sites, with the contents of the tubes freely accessible to the hydrothermal fluid. As a result, several enrichment cultures growing in situ on different polymeric substrates were obtained. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments obtained after PCR with Bacteria-specific primers showed that the bacterial communities developing on carbohydrates included the genera Caldicellulosiruptor and Dictyoglomus and that those developing on proteins contained members of the Thermotogales order. DGGE analysis performed after PCR with Archaea- and Crenarchaeota-specific primers showed that archaea related to uncultured environmental clones, particularly those of the Crenarchaeota phylum, were present in both carbohydrate- and protein-degrading communities. Five isolates obtained from in situ enrichments or corresponding natural samples of water and sediments represented the bacterial genera Dictyoglomus and Caldanaerobacter as well as new archaea of the Crenarchaeota phylum. Thus, in situ enrichment and consequent isolation showed the diversity of thermophilic prokaryotes competing for biopolymers in microbial communities of terrestrial hot springs.

Thermosulfurimonas dismutans gen. nov., sp. nov., an extremely thermophilic sulfur-disproportionating bacterium from a deep-sea hydrothermal vent.

An extremely thermophilic, anaerobic, chemolithoautotrophic bacterium (strain S95(T)) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Center, Pacific Ocean, at a depth of 1910 m. Cells of strain S95(T) were oval to short Gram-negative rods, 0.5-0.6 µm in diameter and 1.0-1.5 µm in length, growing singly or in pairs. Cells were motile with a single polar flagellum. The temperature range for growth was 50-92 °C, with an optimum at 74 °C. The pH range for growth was 5.5-8.0, with an optimum at pH 7.0. Growth of strain S95(T) was observed at NaCl concentrations ranging from 1.5 to 3.5% (w/v). Strain S95(T) grew anaerobically with elemental sulfur as an energy source and bicarbonate/CO(2) as a carbon source. Elemental sulfur was disproportionated to sulfide and sulfate. Growth was enhanced in the presence of poorly crystalline iron(III) oxide (ferrihydrite) as a sulfide-scavenging agent. Strain S95(T) was also able to grow by disproportionation of thiosulfate and sulfite. Sulfate was not used as an electron acceptor. Analysis of the 16S rRNA gene sequence revealed that the isolate belongs to the phylum Thermodesulfobacteria. On the basis of its physiological properties and results of phylogenetic analyses, it is proposed that the isolate represents the sole species of a new genus, Thermosulfurimonas dismutans gen. nov., sp. nov.; S95(T) (=DSM 24515(T)=VKM B-2683(T)) is the type strain of the type species. This is the first description of a thermophilic micro-organism that disproportionates elemental sulfur.

Deferrisoma camini gen. nov., sp. nov., a moderately thermophilic, dissimilatory iron(III)-reducing bacterium from a deep-sea hydrothermal vent that forms a distinct phylogenetic branch in the Deltaproteobacteria

A moderately thermophilic, anaerobic, dissimilatory iron(III)-reducing bacterium (strain S3R1(T)) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Centre in the Pacific Ocean at a depth of about 2150 m. Cells of strain S3R1(T) were ovals to short rods with a single polar flagellum, Gram-stain-negative, 0.5-0.6 µm in diameter and 0.8-1.3 µm long, growing singly or in pairs. The temperature range for growth was 36-62 °C, with an optimum at 50 °C. The pH range for growth was 5.5-7.5, with an optimum at pH 6.5. Growth of strain S3R1(T) was observed at NaCl concentrations ranging from 1.0 to 5.0 % (w/v), with an optimum at 2.0-2.5 % (w/v). The isolate used acetate, fumarate, malate, maleinate, succinate, propanol, palmitate, stearate, peptone and yeast extract as electron donors for growth and iron(III) reduction. All electron donors were oxidized completely to CO(2) and H(2)O. Iron(III) (in the form of ferrihydrite, ferric citrate or ferric nitrilotriacetate) and elemental sulfur (S(0)) were the electron acceptors that supported growth. The DNA G+C content was 64.4 mol%. Results of 16S rRNA gene sequence analysis showed that the novel bacterium was related to representatives of the orders Desulfuromonadales and Syntrophobacterales with 84-86 % sequence similarity and formed a distinct phylogenetic branch in the Deltaproteobacteria. On the basis of its physiological properties and results of phylogenetic analyses, it is proposed that the new isolate represents the sole species of a novel genus, Deferrisoma camini gen. nov., sp. nov. The type strain of Deferrisoma camini is S3R1(T) ( = DSM 24185(T)  = VKM B-2672(T)).

Complete Genome Sequence of Strain 1860, a Crenarchaeon of the Genus Pyrobaculum Able To Grow with Various Electron Acceptors

Strain 1860, a novel member of the genus Pyrobaculum, is a hyperthermophilic organotrophic crenarchaeon growing anaerobically with various electron acceptors. The complete genome sequence reveals genes for several membrane-bound oxidoreductases, the Embden-Meyerhof and Entner-Doudoroff pathways for glucose metabolism, the tricarboxylic acid cycle, the glyoxylate cycle, and the dicarboxylate/4-hydroxybutyrate cycle.

Moorella humiferrea sp. nov., a thermophilic, anaerobic bacterium capable of growth via electron shuttling between humic acid and Fe(III)

An anaerobic, thermophilic, spore-forming bacterium (strain 64-FGQ(T)) was isolated from a terrestrial hydrothermal spring from the Kamchatka peninsula, Russia. This strain utilized lactate as an electron donor, insoluble poorly crystalline Fe(III) oxide incorporated into alginate beads as a potential electron acceptor and 9,10-anthraquinone-2,6-disulfonate (AQDS) as an electron-shuttling compound. Vegetative cells of strain 64-FGQ(T) were Gram-stain-positive, peritrichously flagellated, motile, straight rods, 0.3-0.5 µm in diameter and 2.0-5.0 µm long, growing singly or forming short chains. Cells formed round refractive endospores in terminal swollen sporangia. The temperature range for growth was 46-70 °C, with an optimum at 65 °C. The pH range for growth was 5.5-8.5, with an optimum at pH 7.0. The substrates utilized by strain 64-FGQ(T) in the presence of AQDS as an electron acceptor included lactate, malate, succinate, glycerol and yeast extract. The strain fermented galactose, fructose, maltose, sucrose, pyruvate and peptone. Strain 64-FGQ(T) used AQDS, humic acid, thiosulfate, nitrate and perchlorate as electron acceptors for growth. Fe(III) was not directly reduced, but strain 64-FGQ(T) was able to grow and reduce Fe(III) oxide in the presence of small amounts of AQDS or humic acid as electron-shuttling compounds. The G+C content of the DNA of strain 64-FGQ(T) was 51 mol%. 16S rRNA gene sequence analysis placed the isolate in the genus Moorella, with the type strain of Moorella glycerini as its closest relative (97.2% similarity). Based on phylogenetic analysis and physiological characteristics, strain 64-FGQ(T) is considered to represent a novel species of the genus Moorella, for which the name Moorella humiferrea sp. nov. is proposed; the type strain is 64-FGQ(T) (=DSM 23265(T)=VKM B-2603(T)).

[Reduction of chromate, selenite, tellurite, and iron(III) by the moderately thermophilic bacterium Bacillus thermoamylovorans SKC1].

A moderately thermophilic, facultatively anaerobic bacterium capable of reducing Cr(VI) (strain SKC1) was isolated from municipal sewage. Based on the analysis of the 16S rRNA gene nucleotide sequence and DNA-DNA hybridization data, strain SKC1 was identified as a representative of the species Bacillus thermoamylovorans. B. thermoamylovorans SKC1 is capable of reducing chromate with L-arabinose as an electron donor with an optimum at 50°C and neutral pH. The culture is able to reduce Cr(VI) at its initial concentration in the medium of up to 150 mg/l. In addition to chromate, strain SKC1 is capable of reducing selenite and tellurite, as well as soluble forms of Fe(III). It was shown that Cr(VI), Te(IV), and Se(IV) exert a bacteriostatic effect on strain SKC1, and the reduction of these anions performs the detoxification function. This is the first communication on the reduction of chromate, selenite, tellurite, and soluble Fe(III) species by a culture of thermophilic bacilli.

Dissulfuribacter thermophilus gen. nov., sp. nov., a thermophilic, autotrophic, sulfur-disproportionating, deeply branching deltaproteobacterium from a deep-sea hydrothermal vent.

A thermophilic, anaerobic, chemolithoautotrophic bacterium (strain S69(T)) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Center and Valu Fa Ridge, Pacific Ocean, at a depth of 1910 m using anoxic medium with elemental sulfur as the only energy source. Cells of strain S69(T) were Gram-negative short rods, 0.4-0.6 µm in diameter and 1.0-2.5 µm in length, motile with a single polar flagellum. The temperature range for growth was 28-70 °C, with an optimum at 61 °C. The pH range for growth was 5.6-7.9, with optimum growth at pH 6.8. Growth of strain S69(T) was observed at NaCl concentrations ranging from 0.9 to 5.0%, with an optimum at 1.8-2.7 (w/v). Strain S69(T) grew anaerobically with elemental sulfur as an energy source and bicarbonate/CO2 as a carbon source. Elemental sulfur was disproportionated to sulfide and sulfate. Growth was enhanced in the presence of poorly crystalline Fe(III) oxide (ferrihydrite) as a sulfide-scavenging agent. Strain S69(T) was also able to grow by disproportionation of thiosulfate and sulfite. Sulfate was not used as an electron acceptor either with H2 or with organic electron donors. Analysis of the 16S rRNA gene sequence revealed that the isolate formed a distinct phylogenetic branch within the Deltaproteobacteria. On the basis of its physiological properties and results of phylogenetic analyses, strain S69(T) is considered to represent a novel species of a new genus, for which the name Dissulfuribacter thermophilus gen. nov., sp. nov. is proposed. The type strain of Dissulfuribacter thermophilus is S69(T) (=DSM 25762(T)=VKM B-2760(T)).

Clostridium tepidiprofundi sp. nov., a moderately thermophilic bacterium from a deep-sea hydrothermal vent

A moderately thermophilic, anaerobic bacterium (strain SG 508T) was isolated from a hydrothermal vent chimney located at 1 degrees N on the East Pacific Rise at a depth of 2650 m. Cells of strain SG 508T were straight to slightly curved rods, 0.4-0.6 microm in diameter and 2.0-3.0 microm in length. Spore formation was observed only below pH 5.5. The temperature range for growth was 22-60 degrees C, with optimum growth at 50 degrees C. The pH range for growth was 4.0-8.5, with optimum growth at pH 6.0-6.8. Growth of strain SG 508T was observed at NaCl concentrations ranging from 1.0 to 6.0 % (w/v), with optimum growth at 2.5 % (w/v). Substrates utilized by strain SG 508T included casein, peptone, tryptone, yeast extract, beef extract, starch, maltose and glucose. The products of glucose fermentation were ethanol, acetate, H2, formate and CO2. Strain SG 508T was able to reduce elemental sulfur to hydrogen sulfide. The DNA G+C content of strain SG 508T was 30.9 mol%. 16S rRNA gene sequence analysis revealed that the isolated organism belonged to cluster I of the genus Clostridium. On the basis of its physiological properties and data from phylogenetic analyses, strain SG 508T is considered to represent a novel species of the genus Clostridium, for which the name Clostridium tepidiprofundi sp. nov. is proposed. The type strain is SG 508T (=DSM 19306T =VKM B-2459T).

Microbial diversity and autotrophic activity in Kamchatka hot springs

Microbial communities of Kamchatka Peninsula terrestrial hot springs were studied using molecular, radioisotopic and cultural approaches. Analysis of 16S rRNA gene fragments performed by means of high-throughput sequencing revealed that aerobic autotrophic sulfur-oxidizing bacteria of the genus Sulfurihydrogenibium (phylum Aquificae) dominated in a majority of streamers. Another widely distributed and abundant group was that of anaerobic bacteria of the genus Caldimicrobium (phylum Thermodesulfobacteria). Archaea of the genus Vulcanisaeta were abundant in a high-temperature, slightly acidic hot spring, where they were accompanied by numerous Nanoarchaeota, while the domination of uncultured Thermoplasmataceae A10 was characteristic for moderately thermophilic acidic habitats. The highest rates of inorganic carbon assimilation determined by the in situ incubation of samples in the presence of 14C-labeled bicarbonate were found in oxygen-dependent streamers; in two sediment samples taken from the hottest springs this process, though much weaker, was found to be not dependent on oxygen. The isolation of anaerobic lithoautotrophic prokaryotes from Kamchatka hot springs revealed a wide distribution of the ability for sulfur disproportionation, a new lithoautotrophic process capable to fuel autonomous anaerobic ecosystems.

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)).