Substrate preference, uptake kinetics and bioenergetics in a facultatively autotrophic, thermoacidophilic crenarchaeote.

Facultative autotrophs are abundant components of communities inhabiting geothermal springs. However, the influence of uptake kinetics and energetics on preference for substrates is not well understood in this group of organisms. Here, we report the isolation of a facultatively autotrophic crenarchaeote, strain CP80, from Cinder Pool (CP, 88.7°C, pH 4.0), Yellowstone National Park. The 16S rRNA gene sequence from CP80 is 98.8% identical to that from Thermoproteus uzonensis and is identical to the most abundant sequence identified in CP sediments. Strain CP80 reduces elemental sulfur (S8°) and demonstrates hydrogen (H2)-dependent autotrophic growth. H2-dependent autotrophic activity is suppressed by amendment with formate at a concentration in the range of 20-40 µM, similar to the affinity constant determined for formate utilization. Synthesis of a cell during growth with low concentrations of formate required 0.5 µJ compared to 2.5 µJ during autotrophic growth with H2 These results, coupled to data indicating greater C assimilation efficiency when grown with formate as compared to carbon dioxide, are consistent with preferential use of formate for energetic reasons. Collectively, these results provide new insights into the kinetic and energetic factors that influence the physiology and ecology of facultative autotrophs in high-temperature acidic environments.

Vulcanisaeta distributa gen. nov., sp. nov., and Vulcanisaeta souniana sp. nov., novel hyperthermophilic, rod-shaped crenarchaeotes isolated from hot springs in Japan.

Seventeen strains of rod-shaped, heterotrophic, anaerobic, hyperthermophilic crenarchaeotes were isolated from several hot spring areas in eastern Japan, and eight representative strains were characterized further. Cells of these strains were straight to slightly curved rods, 0.4-0.6 microm in width. Occasionally, cells were branched or bore spherical bodies at the poles. They grew optimally at 85-90 degrees C and at pH 4.0-4.5. They utilized yeast extract, peptone, beef extract, Casamino acids, gelatin, starch, maltose and malate as carbon sources and sulfur and thiosulfate as possible electron acceptors. The DNA G+C contents of the novel isolates were 43.9-46.2 mol%. The lipids were mainly cyclic and acyclic tetraether core lipids. Phylogenetic analysis of the 16S rDNA sequences revealed that they represented an independent lineage in the family Thermoproteaceae. Moreover, comparison of the 16S rDNA sequences and a DNA-DNA hybridization study showed that they comprised two species, which could also be differentiated by the maximal growth temperature and degrees of NaCl tolerance. Therefore, a new genus, Vulcanisaeta gen. nov., in the family Thermoproteaceae is proposed to accommodate two novel species, Vulcanisaeta distributa sp. nov. and Vulcanisaeta souniana sp. nov. The type species is V. distributa and the type strains are V. distributa IC 017T (= JCM 11212T = DSM 14429T) and V. souniana IC-059T (= JCM 11219T = DSM 14430T).

Pyrobaculum oguniense sp. nov., a novel facultatively aerobic and hyperthermophilic archaeon growing at up to 97 degrees C.

A novel hyperthermophilic, heterotrophic, rod-shaped archaeon was isolated from a terrestrial hot spring at Oguni-cho, Kumamoto Prefecture, Japan. The new isolate, strain TE7T, grew under aerobic, microaerobic and anaerobic conditions. Isolate TE7T grew optimally at 90-94 degrees C and pH 7.0-7.5 (adjusted at 25 degrees C) under atmospheric air with vigorous shaking. Strain TE7T cells were motile rods 2-10 microm in length and covered with a surface-layer lattice. Cell yields at 90 degrees C under aerobic conditions were twice that under anaerobic conditions. Under aerobic conditions, growth was inhibited by elemental sulfur, but thiosulfate stimulated growth. Under anaerobic conditions, no growth was observed in the presence of nitrate and nitrite, but elemental sulfur, thiosulfate, L-cystine and oxidized glutathione stimulated growth. The 16S rDNA sequence of TE7T exhibited a close relationship to the sequences of Pyrobaculum aerophilum and Thermoproteus neutrophilus, which belong to the cluster of the genus Pyrobaculum. DNA-DNA hybridization analysis showed a low level of DNA similarity between TE7T and previously described Pyrobaculum species. As TE7T is phenotypically and phylogenetically different from the other members of this genus, it is described as a new species named Pyrobaculum oguniense (type strain TE7T = JCM 10595T = DSM 13380T).

Respiration of arsenate and selenate by hyperthermophilic archaea.

A novel, strictly anaerobic, hyperthermophilic, facultative organotrophic archaeon was isolated from a hot spring at Pisciarelli Solfatara, Naples, Italy. The rod-shaped cells grew chemolithoautotrophically with carbon dioxide as carbon source, hydrogen as electron donor and arsenate, thiosulfate or elemental sulfur as electron acceptor. H2S was formed from sulfur or thiosulfate, arsenite from arsenate. Organotrophically, the new isolate grew optimally in the presence of an inorganic electron acceptor like sulfur, selenate or arsenate. Cultures, grown on arsenate and thiosulfate or arsenate and L-cysteine, precipitated realgar (As2S2). During growth on selenate, elemental selenium was produced. The G+C content of the DNA was 58.3 mol%. Due to 16S rRNA gene sequence analysis combined with physiological and morphological criteria, the new isolate belongs to the Thermoproteales order. It represents a new species within the genus Pyrobaculum, the type species of which we name Pyrobaculum arsenaticum (type strain PZ6*, DSM 13514, ATCC 700994). Comparative studies with different Pyrobaculum-species showed, that Pyrobaculum aerophilum was also able to grow organotrophically under anaerobic culture conditions in the presence of arsenate, selenate and selenite. During growth on selenite, elemental selenium was formed as final product. In contrast to P. arsenaticum, P. aerophilum could use selenate or arsenate for lithoautotrophic growth with carbon dioxide and hydrogen.

Caldivirga maquilingensis gen. nov., sp. nov., a new genus of rod-shaped crenarchaeote isolated from a hot spring in the Philippines.

Two novel hyperthermophilic, rod-shaped crenarchaeotes were isolated from an acidic hot spring in the Philippines. Cells were mostly straight or slightly curved rods 0.4-0.7 micron in width. Bent cells, branched cells, and cells bearing globular bodies were commonly observed. The isolates were heterotrophs and grew anaerobically and microaerobically. The addition of archaeal cell extract or a vitamin mixture to the medium significantly stimulated growth. The isolates grew over a temperature range of 60-92 degrees C, and optimally around 85 degrees C and grew over a pH range of 2.3-6.4, and optimally at pH 3.7-4.2. The isolates utilized glycogen, gelatin, beef extract, peptone, tryptone and yeast extract as carbon sources. They required sulfur, thiosulfate or sulfate as electron acceptors. The lipids mainly consisted of various cyclized glycerol-bisdiphytanyl-glycerol tetraethers. The G+C content of the genomic DNAs was 43 mol%. The 16S rDNA contained two small introns. The comparison of the 16S rDNA exon sequences revealed that they represented an independent lineage in the family Thermoproteaceae. The two strains were included in a single species because of high levels of DNA-DNA relatedness. From these results, Caldivirga maquilingensis gen. nov., sp. nov. is proposed in the family Thermoproteaceae to accommodate these isolates. The type strain of C. maquilingensis is strain IC-167T (= JCM 10307T = MCC-UPLB 1200T = ANMR 0178T).

Thermocladium modestius gen. nov., sp. nov., a new genus of rod-shaped, extremely thermophilic crenarchaeote.

Three strains of novel, extremely thermophilic, rod-shaped crenarchaeotes were isolated from acidic hot spring areas in Japan. Cells of the three stains were straight or slightly curved rods and occasionally branched out singly or extensively, or had spherical bodies protruding at the ends of the cells. They were heterotrophs that grew anaerobically or microaerobically. The presence of CO2 in the gas phase, archaeal cell-extracts and a vitamin mixture stimulated growth of the strains. Growth occurred at 45-82 degrees C and pH 2.6-5.9 and was optimal around 75 degrees C and pH 4.0. The strains utilized glycogen, starch, gelatin and various proteinaceous complex compounds as carbon sources. They required sulfur, thiosulfate or L-cystine as possible electron acceptors. The lipids mainly consisted of various cyclic glycerol-bisdiphytanyl-glycerol tetraethers. The G+C contents of the genomic DNAs were 52 mol%. Comparison of the 16S rDNA sequences indicated that they belonged to a separate lineage in the family Thermoproteaceae. The three strains were included in a single species due to high levels of DNA-DNA hybridization values. Based upon these results, the new isolates were assigned to a new genus and species in the family Thermoproteaceae. Thermocladium modestius gen. nov., sp. nov. The type strain is Thermocladium modestius IC-125T (= JCM 10088T).