Thermus brevis sp. nov., a moderately thermophilic bacterium isolated from a hot spring microbial mat.

Three closely related, facultative anaerobic, Gram-stain-negative, twitching motile, short rod-shaped, non-endospore-forming, moderately thermophilic bacteria, designated strains SYSU G05001T, SYSU G05003 and SYSU G05004, were isolated from a hot spring microbial mat, collected from Rehai National Park, Tengchong, Yunnan Province, south-western China. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that these three strains were closely related to Thermus scotoductus SE-1T (97.97, 98.18, 97.90 % sequence similarity). Whole genome sequencing and polyphasic taxonomic approach were used to determine the genomic profile and taxonomic status of the novel strain SYSU G05001T. Cell growth occurred at 37-80 °C (optimum, 55 °C), pH 6.0-8.0 (optimum, pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum, 1%). Thiosulfate enhanced cell growth. MK-8 was the predominant menaquinone. The major cellular fatty acids included iso-C15 : 0, iso-C17 : 0 and anteiso-C15 : 0. The major polar lipids were consisted of aminophospholipid, glycolipid and phospholipids. The whole genome of strain SYSU G05001T consisted of 2.55 Mbp and the DNA G+C content was 64.94 mol%. The average nucleotide identity (≤94.95 %) and digital DNA-DNA hybridization (≤62.3 %) values between strain SYSU G05001T and other members of the genus Thermus were all lower than the threshold values recommended for distinguishing novel prokaryotic species. On the basis of the presented polyphasic evidence and genotypic data, it is proposed that strain SYSU G05001T (=KCTC 82627T=MCCC 1K06118T) represents a novel species of the genus Thermus, for which the name Thermus brevis sp. nov. is proposed.

Thermus thermamylovorans sp. nov., isolated from a hot spring.

A novel thermophilic bacterium, designated CFH 72773T was isolated from the enrichment of a Jinze hot spring sample which was collected from Dientan town, Tengchong county, Yunnan province, south-western PR China. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and non-sporulating. The taxonomic position of the strain was investigated by using a polyphasic approach. Growth occurred at 37-75 °C, pH 6.0-8.0 and with 0-2.0 % (w/v) NaCl. Comparison of the 16S rRNA gene sequences indicated the strain represented a member of the genus Thermus and showed close relationships to the type strains Thermus caliditerrae YIM 77925T (96.3 % similarity) and Thermus igniterrae RF-4T (96.2 % similarity). The whole genome of CFH 72773T consisted of 2.25 Mbp and the DNA G+C content was 69.5 mol%. A total of 2262 genes, including a variety of enzymes for chemolithotrophy and anerobic respiration, were predicted. The strain had a unique negative oxidase activity and could hydrolyze starch at high temperature. Furthermore, various genes related to methane, sulfur, fumarate and nitrate metabolism were found, all these indicated that it is worth studying the novel strain. The predominant menaquinone is MK-8. The predominant cellular fatty acids included iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0. The major polar lipids were comprised of aminophospholipid, glycolipid and two phospholipids. On the basis of low ANI values, different phenotypic and chemotaxonomic characters and phylogenetic analysis, we made a proposal that strain CFH 72773T represents a novel member of the genus Thermus, for which the name Thermus thermamylovorans sp. nov. is proposed. The type strain is CFH 72773T (=CCTCC AB2018244T=KCTC 43129T).

Thermus caldilimi sp. nov., a thermophilic bacterium isolated from a geothermal area.

A Gram-stain negative, aerobic bacterium, designated strain YIM 78456T, was isolated from a hot spring sediment, Ngamring county, Tibet, south-west China. The taxonomic position of the isolate was investigated by a polyphasic approach. The novel isolate was found to be aerobic and rod-shaped. Colonies were observed to be pale yellow and circular. The strain was found to grow at pH 7.0-8.0 (optimum, pH 7.0), 45-65 °C (optimum, 55 °C) and in the presence of up to 1.5% NaCl. Comparison of the 16S rRNA gene sequence of strain YIM 78456T and other members of the genus Thermus showed sequence similarities ranging from 90.3 to 97.3%, with strain YIM 78456T showing close sequence similarity to Thermus caliditerrae YIM 77925T (97.3%). The phylogenetic trees based on 16S rRNA gene sequences showed that strain YIM 78456T forms a distinct clade with T. caliditerrae YIM 77925T. The predominant menaquinone was identified as MK-8 and the DNA G+C content was determined to be 65.1 mol%. The major cellular fatty acids (> 10%) were identified as iso-C15:0, anteiso-C15:0 and iso-C17:0. The polar lipids were found to consist of an aminophospholipid, a phospholipid and glycolipids. On the basis of the morphological and chemotaxonomic characteristics, as well as genotypic data, it is proposed that strain YIM 78456T represents a novel species of the genus Thermus, for which the name Thermus caldilimi sp. nov. is proposed. The type strain is YIM 78456T (= KCTC 52948T = NBRC 113036T).

Thermus sediminis sp. nov., a thiosulfate-oxidizing and arsenate-reducing organism isolated from Little Hot Creek in the Long Valley Caldera, California.

Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0-2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (≤ 97.01%), digital DNA-DNA hybridization (≤ 32.7%), OrthoANI (≤ 87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C15:0, iso-C17:0 and anteiso-C15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by ß-galactosidase and ß-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198T (= CGMCC 1.13590T = KCTC XXX).

Physiological and genomic properties of Thermus tenuipuniceus sp. nov., a novel slight reddish color member isolated from a terrestrial geothermal spring.

Two closely related, thermophilic bacteria, designated strains YIM 76954T and YIM 76947, were isolated from the Rehai Geothermal Field, Tengchong, Yunnan province, south-west China. Polyphasic approach and whole genome sequencing were used to determine the taxonomy status and genomic profiles of the novel strains. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the two isolates were closely related to Thermus scotoductus SE-1T (97.1% sequence similarity), and T. amyloliquefaciens YIM 77409T (96.6%). The strains could be differentiated from most recognized Thermus species by their whitish to slight reddish colony color, distinct DNA fingerprinting profiles and low ANI values. Cells stained Gram-negative, rod-shaped of diameter 0.2-0.5µm and length 1.5-5.0µm. Growth occurred at 50-75°C, pH 6.0-9.0 and in the presence of up to 1.0% (w/v) NaCl concentration. Thiosulfate was found to enhance cell growth, besides improving the intensity of its colony color. Oxygen, nitrate, sulfur, and Fe(III) could be used as terminal electron acceptors for growth. MK-8 was the major respiratory menaquinone. Major fatty acids were iso-C17:0, iso-C15:0, anteiso-C17:0, and anteiso-C15:0. The genome size was 2.26Mbp with 65.5% average GC content. A total of 2374 genes was predicted, comprising 2322 protein-coding and 52 RNA genes. On the basis of the polyphasic evidence presented, it is proposed that strain YIM 76954T represents a novel species of the genus Thermus, for which the name Thermus tenuipuniceus sp. nov. is proposed. The type strain is YIM 76954T (=JCM 30350T=KCTC 4677T).

Thermus caldifontis sp. nov., a thermophilic bacterium isolated from a hot spring.

A thermophilic bacterial strain, designated YIM 73026T was isolated from a sediment sample collected from a hot spring in Tibet, PR China. The taxonomic position of the novel isolate was investigated by a polyphasic approach. The novel isolate was Gram-stain-negative, aerobic and rod-shaped. Colonies were circular, convex, opaque and yellow. The strain grew at 50-70 °C (optimum, 60 °C), pH 6.0-8.0 (optimum, pH 7.0) and in the presence of up to 1.0 % NaCl (w/v). Comparison of the 16S rRNA gene sequences of YIM 73026T and those of other members of the genus Thermus showed sequence similarities ranging from 91.2 to 97.5 %, with YIM 73026T showing closest sequence similarity to Thermus scotoductus SE-1T (97.5 %). DNA-DNA hybridization results, however, revealed that DNA-DNA reassociation values between YIM 73026T and T. scotoductus DSM 8553T (37.6 %), Thermusamyloliquefaciens YIM 77409T (34.5 %), Thermusantranikianii DSM 12462T (30.3 %), Thermuscaliditerrae YIM 77925T (28.6 %) and Thermustengchongensis YIM 77924T (27.3 %) were well below the 70 % limit for species identification. YIM 73026T contained MK-8 as the respiratory quinone, and iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and anteiso-C15 : 0 as the major cellular fatty acids (>10 %). The polar lipids consisted of one aminophospholipid, one phospholipid and two glycolipids. The genomic DNA G+C content of YIM 73026T was 65.4 mol%. On the basis of morphological, chemotaxonomic and genotypic characteristics, it is proposed that the isolate represents a novel species of the genus Thermus, for which the name Thermus caldifontis sp. nov. is proposed. The type strain is YIM 73026T (=NBRC 112415T=CCTCC AB 2016305T).

Thermus anatoliensis sp. nov., a thermophilic bacterium from geothermal waters of Buharkent, Turkey.

A Gram-stain-negative, lack of motility, catalase- and oxidase- positive bacterium (strain MT1(T)) was isolated from Buharkent hot spring in Aydin, Turkey. Its taxonomy was investigated using a polyphasic approach. The strain was able to grow at 45-80 °C, pH 5.5-10.5 and with a NaCI tolerance up to 2.0% (w/v). Strain MT1(T) was able to utilize d-mannitol and l-arabinose, not able to utilize d-cellobiose as sole carbon source. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Thermus; strain MT1(T) detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. The predominant fatty acids of strain MT1(T) were iso-C(15:0) (43.0%) and iso-C(17:0) (27.4%). Polar lipid analysis revealed a major phospholipid, one major glycolipid, one major aminophospholipid, two minor aminolipids, one minor phospholipid, and several minor glycolipids. The major isoprenoid quinone was MK-8. The DNA G+C content of MT1(T) was 69.6 mol%. On the basis of a taxonomic study using a polyphasic approach, strain MT1(T) is considered to represent a novel species of the genus Thermus, for which the name Thermus anatoliensis sp. nov. is proposed. The type strain is MT1(T) (=NCCB 100425(T) =LMG 26880(T)).

Thermus caliditerrae sp. nov., a novel thermophilic species isolated from a geothermal area.

Two thermophilic bacterial strains, designated YIM 77925(T) and YIM 77777, were isolated from two hot springs, one in the Hydrothermal Explosion (Shuirebaozhaqu) area and Frog Mouth Spring in Tengchong county, Yunnan province, south-western China. The taxonomic positions of the two isolates were investigated by a polyphasic approach. Cells of the two strains were Gram-stain-negative, aerobic and rod-shaped. They were able to grow at 50-70 °C, pH 6.0-8.0 and with a NaCl tolerance up to 0.5% (w/v). Colonies are circular, convex, non-transparent and produce yellow pigment. Phylogenetic analyses based on 16S rRNA gene sequences comparison clearly demonstrated that strains YIM 77925(T) and YIM 77777 represent members of the genus Thermus, and they also detected low-level similarities of 16S rRNA gene sequences (below 97%) compared with all other species in this genus. Their predominant menaquinone was MK-8. The genomic DNA G+C contents of strains YIM 77925(T) and YIM 77777 were 65.6 mol% and 67.2 mol%, respectively. Based on the results of physiological and biochemical tests and phylogenetic analyses, strains YIM 77925(T) and YIM 77777 could not be classified as representing any species of the genus Thermus with a validly published name. Thus the two strains are considered to represent a novel species of the genus Thermus, for which the name Thermus caliditerrae sp. nov. is proposed. The type strain is YIM 77925(T) ( = DSM 25901(T) = CCTCC 2012061(T)).

Evaluation of biomass production, carotenoid level and antioxidant capacity produced by Thermus filiformis using fractional factorial design

A fractional factorial design 2(5-1) was used to evaluate the effect of temperature, pH, and concentrations of yeast extract, tryptone and Nitsch's trace elements on the biomass, total carotenoids and protection against singlet oxygen by carotenoid extracts of the bacterium Thermus filiformis. In addition, the carotenoid composition was determined by high-performance liquid chromatography connected to a diode array and mass spectrometer detectors (HPLC-DAD-MS/MS). The production of biomass ranged from 0.113 to 0.658 g/L, the total carotenoid from 137.6 to 1,517.4 mg/g and the protection against singlet oxygen from 4.3 to 85.1 %. Results of the fractional factorial design showed that temperature had a negative effect on biomass production and a positive effect on carotenoid content and protection against singlet oxygen, besides, high levels of pH value, concentrations of yeast extract and tryptone had a positive effect on biomass production only at lower temperatures. The main carotenoids of T. filiformis were thermozeaxanthins. In the tested conditions, changes in the levels of the variables influenced the biomass, carotenoid production, and protection against singlet oxygen, although they did not influence the carotenoid profile. The results of this study provide a better understanding on the interactions among certain nutritional and cultivation conditions of a thermophile bacterium, Thermus filiformis, on biomass and carotenoid amounts, as well as on the antioxidant capacity.

Thermus composti sp. nov., isolated from oyster mushroom compost.

A Gram-stain-negative, rod-shaped bacterium (strain K-39(T)) was isolated from the thermophilic phase of the composting process for oyster mushroom substrate preparation. The strain grew at 40-80 °C (optimum, 65-75 °C), at pH 5-9 (optimum, pH 7), in media containing up to 1.5% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain K-39(T) formed a distinct lineage within the genus Thermus. Its closest cultivated relative was Thermus islandicus PRI 3838(T) (96.8% similarity). The DNA G+C content of strain K-39(T) was 71.3 mol%. The new strain could be differentiated from the related taxa by not being able to hydrolyse starch. The predominant fatty acids of strain K-39(T) were iso-C(17:0) and anteiso-C(17:0). Strain K-39(T) contained a lower amount of the fatty acid iso-C(15:0) as compared to related species of the genus Thermus. The predominant respiratory quinone of the new isolate was menaquinone MK-8. On the basis of a taxonomic study using a polyphasic approach, strain K-39(T) is considered to represent a novel species of the genus Thermus, for which the name Thermus composti sp. nov. is proposed. The type strain is K-39(T) (=DSM 21686(T)=NCAIM B 02340(T)).

Thermus arciformis sp. nov., a thermophilic species from a geothermal area.

Two aerobic, Gram-negative, non-motile, non-sporulating, yellow-pigmented bacteria, strains TH92(T) and TH91, were isolated from a hot spring located in Laibin, Guangxi, in the south-eastern geothermal area of China. The isolates grew at 40-77 degrees C (optimally at 70 degrees C) and at pH 6.0-9.5 (optimally at pH 7.5-8.0). Phylogenetic analysis of 16S rRNA gene sequences and levels of DNA-DNA relatedness together indicated that the new isolates represented a novel species of the genus Thermus with closest affinity to Thermus aquaticus, Thermus igniterrae and Thermus thermophilus. Compared with their closest relatives, strains TH92( T) and TH91 were able to assimilate a wider range of carbohydrates, amino acids and organic acids as sole carbon sources for growth, such as lactose and melibiose. The new isolates had lower combined levels of C(16 : 0 ) and iso-C(16 : 0) compared with their closest relatives. On the basis of polyphasic taxonomic characterization, strains TH92(T) and TH91 are considered to represent a single novel species of the genus Thermus, for which the name Thermus arciformis sp. nov. is proposed. The type strain is TH92(T) (=CGMCC 1.6992(T) =JCM 15153(T)).

Polyphasic analysis of Thermus isolates from geothermal areas in Iceland.

Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.

Crude-oil-degrading thermophilic bacterium isolated from an oil field.

Thermophilic bacterium strain C2, which has the ability to transform crude oils, was isolated from the reservoir of the Shengli oil field in East China. The Gram-negative, rod-shaped, nonmotile cells were grown at a high temperature, up to 83 degrees C, in the neutral to alkaline pH range. Depending on the culture conditions, the organism occurred as single rods or as filamentous aggregates. Strain C2 was grown chemoorganotrophically and produced metabolites, such as volatile fatty acids, 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl)ester, dibutyl phthalate, and di-n-octyl phthalate. It could metabolize different organic substrates (acetate, D-glucose, fructose, glycerol, maltose, pyruvate, starch, sucrose, xylose, hexadecane). The G+C content (68 mol%) and the 16S rRNA sequence of strain C2 indicated that the isolate belonged to the genus Thermus. The strain affected different crude oils and changed their physical and chemical properties. The biochemical interactions between crude oils and strain C2 follow distinct trends characterized by a group of chemical markers (saturates, aromatics, resins, asphaltenes). Those trends show an increase in saturates and a decrease in aromatics, resins, and asphaltenes. The bioconversion of crude oils leads to an enrichment in lighter hydrocarbons and an overall redistribution of these hydrocarbons.

Identification of iron-reducing Thermus strains as Thermus scotoductus.

Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S(O) as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA-DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.

Biodegradation of aliphatic and aromatic hydrocarbons at high temperatures.

In this paper, the high temperature (65-75 degrees C) biodegradation of aliphatic and aromatic hydrocarbons is investigated and kinetic parameters are derived. The shift of the physico-chemical system properties with rising temperature will be discussed in detail. For example, the solubility of naphthalene is increased by a factor of about ten if the temperature is increased from 20 to 75 degrees C. This effect is essential to increase the bioavailability of sparingly soluble hydrocarbons. It is also demonstrated in experiments that very high oxygen transfer rates can be obtained at high temperatures in the presence of hydrocarbons. It is shown that efficient phenol biodegradation is essential for high temperature hydrocarbon degradation because some microorganisms tend to transform phenols into polyphenols which are very inhibitory for microbial growth. A defined mixed culture adapted to phenol converted more than 90% of a mixture of phenol, hexadecane and pyreno and a very high maximal growth rate of 0.19 h(-1) was determined. A yield coefficient Y(X/S) of about 0.8 g (biomass)/g (hydrocarbons) was calculated in this experiment. In a separate experiment the influence of the hydrocarbon droplet size on the biodegradation is investigated at 70 degrees C using a newly isolated Thermus sp. In this case, the growth on a hexadecane/pyrene mixture was described by a model based on the Monod equation and the corresponding kinetic parameters are derived. A mixed culture was used for the bioremediation of soil in a slurry reactor. The initial contamination of 11 g/kg was lowered to about 2 g in a reactor inoculated by an immobilized culture of extreme thermophilic microorganisms, while 9 g/kg remained in a sterile control.

F-and V-ATPases in the genus Thermus and related species.

The discovery of a V-type ATPase in the gram-negative bacterium Thermus thermophilus HB8 (YOKOYAMA et al., J. Biol. Chem. 265, 21946, 1990) was unexpected, since only eukaryotic endomembranes and archaea were thought to contain this enzyme complex, and horizontal gene transfer was suggested to explain the finding. We examined membrane-associated ATPases from representatives of several groups of the genus Thermus. The enzymes were extracted with chloroform and purified by ion exchange chromatography or native gel electrophoresis. One novel Islandic isolate, T. scotoductus SE-1, as well as strain T. filiformis from New Zealand, possessed F-ATPases, as judged by the typical five subunit composition of the F1-moiety, sensitivity to azide, insensitivity to nitrate and a strong crossreaction with antibodies against the F1-ATPase from E. coli. In addition, N-terminal amino acid sequencing of the beta subunit from T. scotoductus SE-1 confirmed its homology with beta subunits from known F-ATPases. In contrast, the same extraction procedure released a V-ATPase from the membranes of T. thermophilus HB27 and T. aquaticus YT-1. The related species Meiothermus (formerly Thermus) chliarophilus ALT-8 also possessed a V-ATPase. All V-ATPases examined in this study contained larger major subunits than F-ATPases, crossreacted with antiserum against subunit A of the V-ATPase from the archaeon Halobacterium saccharovorum, and the N-terminal sequences of their major subunits were homologous to those of other V-ATPases. Sequences of the 16S rRNA gene clearly placed T. scotoductus SE-1, along with other non-pigmented Thermus strains, as a distinct species close to T. aquaticus. Our results suggested that at least two members of the genus, T. scotoductus SE-1 and T. filiformis, contain an F-ATPase, whereas several others possess a V-ATPase. These data could indicate a greater diversity of the genus Thermus than was previously thought. Alternatively, the genus may consist of species where horizontal gene transfer has occurred and others, where it has not.