Distribution of Thermus spp. in Icelandic Hot Springs and a Thermal Gradient

The growth range in nature of bacteria belonging to the genus Thermus was investigated by sampling 55 different hot springs in Iceland. The springs ranged in temperature from 32 to 99°C, and in pH from 2.1 to 10.1. Viable counts of Thermus spp. ranging from 10 to 10⁴ CFU/100 ml of spring water were found in 27 of the springs sampled. The temperature range for these bacteria was found to be 55 to 85°C, and the pH range was from about 6.5 to above 10. Thermus spp. were found in springs containing up to 1 mM dissolved sulfide and having conductivity up to 2,000 μS/cm. The distribution of Thermus spp. in a hot spring thermal gradient was also investigated and found to agree well with the overall distribution in individual springs.     

Phylogenetic Diversity Analysis of Subterranean Hot Springs in Iceland

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.     

Properties of Thermus ruber Strains Isolated from Icelandic Hot Springs and DNA:DNA Homology of Thermus ruber and Thermus aquaticus

Seventeen pink-pigmented strains of the genus Thermus were isolated from samples collected from thermal areas of Iceland. The strains were examined by using phenotypic characterization and DNA:DNA homology and were compared with recognized strains. Visually, the strains could be divided into three groups based on their pigmentation; however, spectroscopic studies of the pigments indicated little difference among them. Most strains required a vitamin supplement for growth and used fructose, maltose, mannose, or sucrose as the sole carbon source. In the presence of nitrate, two strains were able to grow under anaerobic conditions. The optimum growth temperature was 60°C; growth did not occur at 30 or 70°C.     

Isolation and Characterization of Extremely Thermophilic Bacteria from Hot Springs

In order to investigate the mechanism of microbial growth at elevated temperatures, it was tried to isolate different thermophilic microorganisms from wide origins, such as soils, composts, manure piles and hot spring waters. As the result, 5 strains of extremely thermophilic bacteria, the maximum, the optimum and the minimum temperatures for growth of which were 80, 70~75, and 40°C, respectively, were isolated from Izu-Atagawa hot spring and Beppu hot springs. These bacteria were gram-negative, yellow-pigmented, non-motile and non-sporulating rods of 0.5~0.7 μ in diameter and 2~5 μ in length. They were heterotrophs requiring several amino acids (such as glutamate, aspartate, et al.) and vitamins (such as biotin, folic acid and p-aminobenzoic acid) and grew well at neutral to slight alkali pH. The content of GC pairs of DNAs from the 5 strains was 69~70%, and this seemed to be one of the highest values in bacteria so far known. Among the 5 strains, strain AT–62 was named as Thermus flavus sp. n. AT–62 from its morphological and physiological characteristics. Comparison between Thermus flavus and other extremely thermophilic bacteria as Thermus aquaticus and Flavobacterium thermophilum is described and discussed in reference to classification of extremely thermophilic bacteria.     

Distribution of Crenarchaeota Representatives in Terrestrial Hot Springs of Russia and Iceland

Culture-independent (PCR with Crenarchaeota-specific primers and subsequent denaturing gradient gel electrophoresis) and culture-dependent approaches were used to study the diversity of Crenarchaeota in terrestrial hot springs of the Kamchatka Peninsula and the Lake Baikal region (Russia) and of Iceland. Among the phylotypes detected there were relatives of both cultured (mainly hyperthermophilic) and uncultured Crenarchaeota. It was found that there is a large and diverse group of uncultured Crenarchaeota that inhabit terrestrial hot springs with moderate temperatures (55 to 70°C). Two of the lineages of this group were given phenotypic characterization, one as a result of cultivation in an enrichment culture and another one after isolation of a pure culture, “Fervidococcus fontis,” which proved to be a moderately thermophilic, neutrophilic (optimum pH of 6.0 to 7.5), anaerobic organotroph.     

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.     

腾冲热海七株高温厌氧菌的分离及鉴定

摘要：【目的】了解中国云南腾冲热海环境中厌氧菌的分类学特征及生理生化特性。【方法】利用Hungate厌氧操作技术,从云南腾冲热海80-93℃温泉中分离出7株高温厌氧菌,对其进行形态、生长特征及16S rRNA基因序列分析,确定菌株的系统发育地位。【结果】7株菌株细胞形态均为杆状,不产芽孢,革兰氏阴性,严格厌氧,在70℃生长良好。其中较典型的菌株RH0802能在55-80℃温度范围内生长,最适生长温度为70℃;生长pH值范围为5.5-8.5,最适pH值为7.0,能利用葡萄糖、淀粉、甘露醇、甘露糖、核糖、麦芽糖、纤维二糖、木糖、果糖、半乳糖、木聚糖、甘油,不能利用蔗糖、丙酮酸。16S rRNA基因序列相似性的比较分析表明,其中5株菌与Caldanaerobacter属菌株的最高相似性均在98%以上,而RH0804与RH0806分别为96%和93%。菌株RH0802-RH0808的序列登录号分别为FJ748766、FJ748762、FJ748761、FJ748763、FJ748765、FJ748764和FJ748767,在中国普通微生物菌种保藏中心的保藏号分别为CGMCC1.5134-1.5140。【结论】分离自腾冲热海的7株嗜热厌氧菌与Caldanaerobacter属菌株有较高相似性,可将这7株菌株鉴定为Caldanaerobacter属菌株（Caldanaerobacter. sp）,其中菌株RH0804和RH0806有成为新种的可能。     

Bacteriochlorophylls in Gliding Filamentous Prokaryotes from Hot Springs

WE have found bacteriochlorophyll-like pigments in two types of filamentous gliding prokaryotes that occur abundantly in alkaline hot springs. They are temporarily designated F-l and F-2. Representatives of F-2, found in hot springs of Iceland, Japan, New Zealand, Guatemala and the western United States, have been isolated in pure culture. They often exist as thick gelatinous mats up to about 70° C and are bright orange from the high content of carotenoids, but in North America they are normally covered by a layer of blue-green algae such as Synechococcus sp. Because of their colour and morphological characteristics, F-2 filaments were previously thought to be members of the heterotrophic, achlorophyllous flexibacteria^1,2. The trichomes are 0.5-1.0µm in diameter and of varying lengths. They are motile and glide on agar substrates at 45° C or 60° C at rates of 0.01-0.04µ/s (Fig. 1 A).     

A Thermophilic Acidophilic Bacterium from Hot Springs

A thermophilic acidophilic bacterium was isolated from Owaku-dani hot springs of Izu-Hakone National Park in Japan. This bacterium grows optimally at a temperature of 70°C and a pH of 2~3. The isolate was generally spherical in shape, with a diameter of about 0.8~1.2 μm, being gram-negative and nonmotile. The DNA base composition was 44% guanine plus cytosine. Chromatographic, chemical and infrared spectroscopic analysis of the total cellular lipid showed that the lipid constituents contained mainly ether linkages; long chain fatty acids and derivatives were absent. The data presented suggests that the thermophilic acidophilic isolate may have some relationship to the Sulfolobus.     

Resistance of Thermus spp. to Potassium Tellurite

Two members of the genus Thermus were examined for their resistance to toxic inorganic compounds. They both proved to be fairly resistant to tellurite and selenite and to many other heavy metal salts. Cell extracts of Thermus thermophilus HB8 and of T. flavus AT-62 catalyze the reduction of K₂TeO₃ in a reaction which is dependent on NADH oxidation.     

Moderately Thermophilic Magnetotactic Bacteria from Hot Springs in Nevada

Populations of a moderately thermophilic magnetotactic bacterium were discovered in Great Boiling Springs, Nevada, ranging from 32 to 63°C. Cells were small, Gram-negative, vibrioid to helicoid in morphology, and biomineralized a chain of bullet-shaped magnetite magnetosomes. Phylogenetically, based on 16S rRNA gene sequencing, the organism belongs to the phylum Nitrospirae.Magnetotactic bacteria are a metabolically, morphologically, and phylogenetically heterogeneous group of prokaryotes that passively align and actively swim along magnetic field lines (3). This behavior, called magnetotaxis, is due to the presence of intracellular, membrane-bounded, single-magnetic-domain crystals of magnetite (Fe₃O₄) and/or greigite (Fe₃S₄) (3).Most known cultured magnetotactic bacteria are mesophilic and do not grow much above 30°C (e.g., Magnetospirillum species and Desulfovibrio magneticus strains MV-1 and MC-1 [D. A. Bazylinski, unpublished data]). Uncultured magnetotactic bacteria have been observed in numerous habitats that were mostly at 30°C and below. There is only one report describing thermophilic magnetotactic bacteria despite a number of efforts to look for them (e.g., in hydrothermal vents [D. A. Bazylinski, unpublished data]). Nash (12) reported the presence of thermophilic magnetotactic bacteria in microbial mats at about 45 to 55°C adjacent to the main flow in Little Hot Creek (but not in other springs in the same area at 40 to 80°C) and in microbial mats of other springs in central California at up to 58°C, all on the east side of the Sierra Nevada mountains. Cells biomineralized bullet-shaped crystals of magnetite and were phylogenetically affiliated with the phylum Nitrospirae (12). Few additional details were provided regarding the organisms and their habitat.In this study, water and surface sediment samples were taken from the Great Boiling Springs (GBS) geothermal field in Gerlach, NV. GBS is a series of hot springs that range from ambient temperature to ∼96°C (2, 5). The geology, chemistry, and microbial ecology of the springs have been described in some detail (2, 5). The pHs of the samples ranged from 6.4 to 7.5, while the salinities were about 4 to 5 ppt, as determined with a handheld Palm Abbe PA203 digital refractometer (MISCO Refractometer, Cleveland, OH). Samples were examined for the presence of magnetotactic bacteria using the hanging drop technique on-site and in the laboratory at room temperature with and without magnetic enrichment of the sample (15). Some samples taken back to the laboratory were kept at an elevated temperature (∼62°C), while others were kept at ambient temperature. There did not appear to be a significant difference in the number of magnetotactic cells in samples taken back to the laboratory and kept at these two temperatures. Only one morphotype of magnetotactic bacteria was found in samples from nine springs whose temperatures ranged from 32 to 63°C, and we estimate their numbers to be between 10³ to 10⁵ cells ml⁻¹ in surface sediments in sample bottles. We did not observe magnetotactic cells of this type in a large number of springs or pools that were at <32°C. Only one spring positive for the presence of these magnetotactic bacteria had sediment that was partially covered with a microbial mat, while sediment at most of the springs was dark gray in color. Cells were small (1.8 ± 0.4 by 0.4 ± 0.1 μm; n = 59), Gram negative, vibrioid to helicoid in morphology, and possessed a single polar flagellum (Fig. ​(Fig.1A).1A). Magnetotactic bacteria were not observed in springs that were at 67°C and above, suggesting the maximum survival and perhaps growth temperature for the organism is about 63°C. In the lab, cells remained viable and motile in samples kept at 25 to 62°C for several months. We refer to this organism as strain HSMV-1.Open in a separate windowFIG. 1.Transmission electron microscope (TEM) images of cells and magnetosomes of strain HSMV-1. (A) TEM image of unstained cell of HSMV-1 showing a single polar flagellum and a single chain of bullet-shaped magnetosomes. The electron-dense structures at the poles were found to be phosphorus-rich based on energy-dispersive X-ray analysis (data not shown) and therefore likely represent polyphosphate granules. (B) Higher-magnification TEM image of the magnetosome chain. (C) High-magnification TEM image of magnetosomes from which a selected area electron diffraction (SAED) pattern was obtained (inset of B). The SAED pattern corresponds to the [1 0−1] zone of magnetite, Fe₃O₄: reflection o, (0 0 0); reflection a, (1 −1 1) (0.48 nm); reflection b, (1 1 1) (0.48 nm); reflection c, (2 0 2) (0.30 nm); angle a-o-b, 70.5°. (D) Iron, sulfur, and oxygen elemental maps, derived from energy-filtering transmission electron microscopy (EFTEM), showing that the positions of the magnetosome crystals correlate with increased concentrations of Fe and O, but not S, consistent with the iron oxide magnetite (Fe₃O₄).Cells of HSMV-1 biomineralized a single chain of magnetosomes that traversed the cells along their long axis (Fig. 1A to C). Selected area electron diffraction (SAED) and energy-filtering transmission electron microscopy (EFTEM) elemental maps were determined on magnetosome crystals using a Tecnai model G2 F30 Super-Twin transmission electron microscope (FEI Company, Hillsboro OR). SAED patterns of HSMV-1 magnetosome crystals (Fig. ​(Fig.1B,1B, inset) indicated that they consisted of magnetite, while EFTEM elemental maps (Fe, O and S) (Fig. ​(Fig.1D)1D) clearly showed that the crystals consisted of an iron oxide and not an iron sulfide, again consistent with the mineral magnetite. Cells contained an average of 12 ± 6 magnetosome crystals per cell (n = 15 cells) that averaged 113 ± 34 by 40 ± 5 nm in size (n = 179). A plot of the length of the crystals as a function of the shape factor (width/length ratio) is provided in Figure S1 in the supplemental material and shows that the crystals fit in the theoretical single-magnetic-domain size range (4), along with all known mature magnetosome magnetite crystals from magnetotactic bacteria (3).Whole-cell PCR amplification of the 16S rRNA gene was performed by first magnetically purifying cells of HSMV-1 using the “capillary racetrack” described by Wolfe et al. (18). Purity of the collected cells was determined by microscopic examination, and contaminating cells were never observed. The 16S rRNA gene was amplified using bacteria-specific primers 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGHTACCTTGTTACGACTT-3′ (11). PCR products were cloned into pGEM-T Easy vector (Promega Corporation, Madison, WI) and sequenced (Functional Biosciences, Inc., Madison, WI). Six of eight clones sequenced had identical inserts.Alignment of 16S rRNA gene sequences was performed using the CLUSTAL W multiple alignment accessory application in the BioEdit sequence alignment editor (7). Phylogenetic trees were constructed using MEGA version 4 (17) by applying the neighbor-joining method (14). Bootstrap values were calculated with 1,000 replicates. The 16S rRNA gene sequence of strain HSMV-1 places the organism in the phylum Nitrospirae (Fig. ​(Fig.2),2), with its closest relative in culture being Thermodesulfovibrio hydrogeniphilus (87.2% identity) (8). Two other uncultured magnetotactic bacteria are phylogenetically affiliated with the phylum Nitrospirae, including the unnamed rod-shaped bacterium strain MHB-1 (86.5% identity) (6) and the very large Candidatus Magnetobacterium bavaricum (86.4% identity) (16). Interestingly, all the magnetotactic bacteria associated with the phylum Nitrospirae thus far (e.g., Candidatus Magnetobacterium bavaricum) contain bullet-shaped magnetite crystals in their magnetosomes.Open in a separate windowFIG. 2.Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain HSMV-1 in the phylum Nitrospirae. Bootstrap values at nodes are percentages of 1,000 replicates. The magnetotactic bacteria Desulfovibrio magneticus and Candidatus Magnetoglobus multicellularis (outgroup; deltaproteobacteria) were used to root the tree. GenBank accession numbers are given in parentheses. Bar represents 2% sequence divergence.Fluorescent in situ hybridization (FISH) was used to authenticate the 16S rRNA gene sequence. A specific Alexa594-labeled probe for HSMV-1 was designed (HSMVp, 5′-CCTTCGCCACAGGCCTTCTA-3′, complementary to nucleotides 690 to 709 of the 16S rRNA molecule) based on the alignment of 10 of the most similar 16S rRNA gene sequences found in GenBank after BLAST analysis (1) and on cultivated members of the phylum Nitrospirae. FISH with the Alexa594-labeled probe was carried out after fixation of magnetically concentrated cells directly on the wells of gelatin-coated hydrophobic microscope slides with 4% paraformaldehyde. FISH was performed according to the work of Pernthaler et al. (13). The hybridization solution contained 10 ng/ml of the probe, 20% formamide, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM Na₂EDTA, and 0.01% sodium dodecyl sulfate (SDS). Cells of HSMV-1 hybridized to the HSMVp probe, while other cells in the sample did not (Fig. ​(Fig.3),3), indicating that the 16S rRNA gene sequence we obtained is from the magnetotactic bacterium under study. Strain HSMV-1 clearly represents a new genus (Fig. ​(Fig.2),2), and based on the phylogeny and what we currently know phenotypically about strain HSMV-1, we propose the name Candidatus Thermomagnetovibrio paiutensis (the GBS site was originally occupied by the Paiute Indian Tribe).Open in a separate windowFIG. 3.Fluorescent in situ hybridization (FISH) of cells of strain HSMV-1 using an HSMV-1-specific oligonucleotide rRNA probe (HSMVp). Cells used for FISH were magnetically concentrated by placing a magnet next to the side of the sample bottle for 30 min and then removed with a Pasteur pipette. This technique was used rather than the magnetic racetrack method in order to have many HSMV-1 cells as well as some other cells that could be used as a negative control. (A) Differential interference contrast (DIC) image of HSMV-1 cells (filled arrows) and other cells (negative control; empty arrows) from hot spring samples; (B) cells stained with 4′,6-diamidino-2-phenylindole (DAPI); (C) cells hybridized with the specific probe HSMVp.Nash (12) first reported thermophilic magnetotactic bacteria phylogenetically affiliated with the Nitrospirae phylum in hot springs, and it would be interesting and important to compare these organisms and their habitats. However, little can be compared at this time due to lack of information. Nash (12) reported that the one spring at Little Hot Creek was freshwater and that microbial mats were present at all springs where thermophilic magnetotactic bacteria were found. The water at our sampling sites was brackish, not freshwater, and microbial mats were not an important feature of our springs. Thus, it is difficult to determine without knowing the relationship between the organisms found by Nash (12) and strain HSMV-1 what environmental parameters are important to the growth and survival of these bacteria.It is also difficult to determine the temperature ranges for the survival and growth for strain HSMV-1 without having a pure culture. Data presented here suggest that the temperature range for both is quite wide, and this would be important for the continued presence of HSMV-1 at GBS, as temperatures in the hot springs are known to fluctuate greatly (2). Even if the maximum growth temperature of HSMV-1 is slightly lower than the maximum survival temperature (a conservative estimate) that we know of (63°C), it would still be considered a moderately thermophilic bacterium.The results presented here clearly show that some magnetotactic bacteria can be considered at least moderately thermophilic. They extend the upper temperature limit for environments where magnetotactic bacteria exist and likely grow (∼63°C) and where magnetosome magnetite is deposited, a finding that may prove significant in the study and interpretation of magnetofossils (9, 10).     

Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp.

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3'-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.     

Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.

Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of 10(-2).     

Isolation of Vibrio parahaemolyticus from Salt Springs in Florida

Vibrio parahaemolyticus was isolated from various locations in two salt springs of Florida and appears to be a normal inhabitant of these artesian waters.     

Isolation ofErwinia spp. from human sources

FiveErwinia strains were isolated from human wounds; their characteristics are reported. Their pathogenicity for man appeared doubtful.     

Phage Community Dynamics in Hot Springs

In extreme thermal environments such as hot springs, phages are the only known microbial predators. Here we present the first study of prokaryotic and phage community dynamics in these environments. Phages were abundant in hot springs, reaching concentrations of a million viruses per milliliter. Hot spring phage particles were resistant to shifts to lower temperatures, possibly facilitating DNA transfer out of these extreme environments. The phages were actively produced, with a population turnover time of 1 to 2 days. Phage-mediated microbial mortality was significant, making phage lysis an important component of hot spring microbial food webs. Together, these results show that phages exert an important influence on microbial community structure and energy flow in extreme thermal environments.     

Nonmarine Crenarchaeol in Nevada Hot Springs

Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids of the Crenarchaeota. The structurally unusual GDGT crenarchaeol has been proposed as a taxonomically specific biomarker for the marine planktonic group I archaea. It is found ubiquitously in the marine water column and in sediments. In this work, samples of microbial community biomass were obtained from several alkaline and neutral-pH hot springs in Nevada, United States. Lipid extracts of these samples were analyzed by high-performance liquid chromatography-mass spectrometry and by gas chromatography-mass spectrometry. Each sample contained GDGTs, and among these compounds was crenarchaeol. The distribution of archaeal lipids in Nevada hot springs did not appear to correlate with temperature, as has been observed in the marine environment. Instead, a significant correlation with the concentration of bicarbonate was observed. Archaeal DNA was analyzed by denaturing gradient gel electrophoresis. All samples contained 16S rRNA gene sequences which were more strongly related to thermophilic crenarchaeota than to Cenarchaeum symbiosum, a marine nonthermophilic crenarchaeon. The occurrence of crenarchaeol in environments containing sequences affiliated with thermophilic crenarchaeota suggests a wide phenotypic distribution of this compound. The results also indicate that crenarchaeol can no longer be considered an exclusive biomarker for marine species.