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Applied and Environmental Microbiology, January 2007, p. 259-270, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01570-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Communities of Archaea and Bacteria in a Subsurface Radioactive Thermal Spring in the Austrian Central Alps, and Evidence of Ammonia-Oxidizing Crenarchaeota

Gerhard W. Weidler,¹ Marion Dornmayr-Pfaffenhuemer,¹ Friedrich W. Gerbl,¹ Wolfgang Heinen,^2, and Helga Stan-Lotter^1*

Division of Molecular Biology, Department of Microbiology, University of Salzburg, Billrothstraße 11, A-5020 Salzburg, Austria,¹ Department of Microbiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands²

Received 7 July 2006/ Accepted 19 October 2006

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Scanning electron microscopy revealed great morphological diversity in biofilms from several largely unexplored subterranean thermal Alpine springs, which contain radium 226 and radon 222. A culture-independent molecular analysis of microbial communities on rocks and in the water of one spring, the "Franz-Josef-Quelle" in Bad Gastein, Austria, was performed. Four hundred fifteen clones were analyzed. One hundred thirty-two sequences were affiliated with 14 bacterial operational taxonomic units (OTUs) and 283 with four archaeal OTUs. Rarefaction analysis indicated a high diversity of bacterial sequences, while archaeal sequences were less diverse. The majority of the cloned archaeal 16S rRNA gene sequences belonged to the soil-freshwater-subsurface (1.1b) crenarchaeotic group; other representatives belonged to the freshwater-wastewater-soil (1.3b) group, except one clone, which was related to a group of uncultivated Euryarchaeota. These findings support recent reports that Crenarchaeota are not restricted to high-temperature environments. Most of the bacterial sequences were related to the Proteobacteria (, ß, , and ), Bacteroidetes, and Planctomycetes. One OTU was allied with Nitrospina sp. (-Proteobacteria) and three others grouped with Nitrospira. Statistical analyses suggested high diversity based on 16S rRNA gene analyses; the rarefaction plot of archaeal clones showed a plateau. Since Crenarchaeota have been implicated recently in the nitrogen cycle, the spring environment was probed for the presence of the ammonia monooxygenase subunit A (amoA) gene. Sequences were obtained which were related to crenarchaeotic amoA genes from marine and soil habitats. The data suggested that nitrification processes are occurring in the subterranean environment and that ammonia may possibly be an energy source for the resident communities.

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Bad Gastein, a village in the National Park "Hohe Tauern" in the Alps near Salzburg, Austria (Fig. 1), is known for its thermal mineral springs. A cluster of 17 major springs (between 962 to 1,029 m above sea level) with temperatures up to 47°C provide about 4 to 5 million liters of thermal mineral water per day (7, 9, 15). The water of the springs is collected in such a way that nearly no surface water can mix with the warm spring water, which is distributed to the spa hotels (9). Numerous medical determinations of the influence of radium 226 and radon (mainly Rn 222), which are present in the mineral water of the Bad Gastein thermal springs, on the human organism have been made (9, 15). However, almost no information about the microbial flora of these thermal springs exists (7, 18).

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FIG. 1. Geographical location of Bad Gastein (bottom right) and overview of the spring "Franz-Josef-Quelle," showing the area with water discharges numbered 21, 23, and 24. Water discharge 24 is above number 23 and cannot be seen in this picture. All discharges are collected in the little rock basin. The bottom left panel shows rock fissures with water leaking out (arrows).

One of the major springs, located at the highest altitude (1,029 m above sea level), is called "Franz-Josef-Quelle" (FJQ) (Fig. 1); it consists of a total of 27 single water discharges, issuing directly from rock fissures (18). It is located at the end of a gallery that was driven horizontally about 100 to 150 m deep into the rock. It issues from an artificial cavity from the chasms traversing the rock (7), delivering 295 m³ water per day (according to reports from 1997-1998) with a Rn 222 quantity of 296 kBq/m³ water (7, 9, 18). Its average temperature is 45.6°C, and the pH is about 8.0. The geological environment of the spring area, consisting mainly of gneiss and crystalline slate, which are defined as primary rocks, prevents rain water from trickling into the ground. As stated by Cudrigh (7), pure and unmixed thermal mineral water possesses a mineralization degree of about 350 mg/liter and a temperature of 56°C. These properties as well as the content of tritium—which should not occur in thermal water—indicated an 10% intermixture of the spring FJQ with colder ground or subsurface water, which was corroborated by measurements of d¹⁸O and deuterium (7). Furthermore, the subterranean water cycle was measured by the ¹⁴C method, which suggested a retention time of 3,600 to 3,800 years (7, 15). Currently, little is known of such subsurface environments and in particular about possible energy and carbon sources in such niches, but the interest in the microbiology of subterranean environments is clearly rising (14, 19, 20, 33, 46).

Caves harbor an especially interesting composition and variety of mineral-utilizing microorganisms, which may contribute to the formation of unusual netlike microstructures as described by Boston et al. and Northup et al. (4, 33). Very similar arrangements were detected by scanning electron microscopy in the springs of Bad Gastein (W. Heinen, H. P. M. Geurts, and A. M. Lauwers, unpublished data). Therefore, the spring FJQ was chosen for examination of its microbial composition. The inadequacy of culture-based methods for estimation of species numbers in natural environments has been well documented (1, 35), so culture-independent techniques were applied. These included 16S rRNA gene sequence analyses and restriction fragment length polymorphism (RFLP) (20). In addition, the search for crenarchaeal ammonia oxidation-related genes (48) was applied to examine this subsurface habitat, because early results in this study revealed the presence of a large number of crenarchaeal clones and, as mentioned, the energy supply for microbial cave communities is, with some exceptions, entirely unknown.

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Sampling and site description.
The samples were taken from water discharges number 21, 23, and 24, which flow into a small basin (Fig. 1). The water temperature at the point of sampling was 42.3°C with a pH value of 8.1; the air temperature was 34°C with humidity near 100%. Water samples and bacterial mats (biofilms), designated FJQF and FJQB, respectively, were taken under aseptic conditions, using sterile equipment. Water samples were collected in aluminum foil-wrapped one-liter bottles. Spatulas were used for removal of biofilm samples, which were placed into 50-ml reaction tubes. Glass slides (75 mm by 25 mm by 1 mm) were mounted into a metal rack, which held 10 slides, and were placed into the spring FJQ for up to 11 days. Following collection, the slides were placed into 50-ml reaction tubes. Samples from glass slides were designated as FJQOT. Water samples were stored at 4°C. Biofilm samples and glass slides were stored at –70°C until further use.

Some specific data for the spring FJQ are as follows (modified from reference 7): the mineralization degree of the spring water is 366 mg/liter; the O₂ content is up to 0.3 mg/kg of water; cations (in mg/kg of water) are Na⁺ (76.3) and Ca²⁺ (19.1); anions are SO₄^2– (125.3), HCO₃^– (57.3), Cl^– (25.3), and F^– (5.4). Mn²⁺ is present as a trace element in a concentration of up to 0.03 mg/kg of water. NH₃, NH₄⁺, NO₃^–, and NO₂^– are present in traces (<0.01 to 0.1 mg/kg of water).

DNA preparation from filtered water and from biofilm on glass slides.
Two liters of mineral water were passed through a 0.22-µm-pore-size autoclaved membrane filter (Durapore Filters, Millipore, Bedford, MA), using a filtration unit (SM 16201/19/20; Sartorius, Vienna, Austria) with a low-pressure vacuum. Filters were kept at –70°C until further use. Glass slides that were incubated in the spring for 11 days were broken into small pieces, and DNA extraction was performed as described by Radax et al. (38).

DNA preparation from natural biofilm samples.
During DNA extraction, presumptive organic or inorganic compounds, e.g., humic acids, phenolic compounds, and heavy metals, which can inhibit or decrease the sensitivity of PCR (10, 45), appeared as a brownish-reddish color, or as yellow color after DNA extraction. To eliminate these interfering compounds, the DNA extraction protocol described by T. Glenn (MUD-DNA extraction protocol; www.uga.edu/srel/DNA_Lab/protocols.htm) was used with the following modifications. About 2 g of brownish-white biofilm were centrifuged at 6,000 x g for 5 min at 4°C. After centrifugation, the watery supernatant was removed and 900 µl of TE buffer (100 mM Tris-Cl, pH 7.6, 10 mM EDTA) and 50 µl of lysozyme (10 mg/ml) were added. Samples were mixed for 2 min and incubated for 1 h at 37°C. Following boiling for 10 min, 100 µl of proteinase K (10 mg/ml) was added and samples were incubated for 1 h at 56°C. To each sample, guanidinium thiocyanate (Sigma, Saint Louis, MO) and diatomaceous earth (Sigma), prepared as described by Glenn (www.uga.edu/srel/DNA_Lab/protocols.htm), were added. Subsequent sample preparation was done as recommended in the protocol by Glenn. DNA was further cleaned with a Gene Clean II kit (Bio-101, Carlsbad, CA) according to the instructions of the manufacturer. The yellow color was nearly completely eliminated using this protocol.

PCR and clone library construction.
Several primer pairs were tested for PCR amplification of partial 16S rRNA gene fragments of the extracted community DNA from filtered water, glass slide, and biofilm samples by using a standard PCR approach. The primer pairs A21F (5' TTC CGG TTG ATC C[CT]G CCG GA) and A958R (5' [C/T]CC GGC GTT GA[A/C] TCC AAT T) for Archaea (11) and P797F (5' C[A/G]A A[C/T][A/C] GGA TTA GAT ACC C) and E1492R (5' TAC GG[C/T] TAC CTT GTT ACG ACT T) for Bacteria (42) were chosen, since they yielded the highest amounts of PCR product. The reactions were performed in a total volume of 50 µl with 1.25 U of Taq DNA polymerase (Fermentas, St. Leon-Rot, Germany). The PCR products were excised following separation by gel electrophoresis and recovered from the gel slices using a QIAquick gel extraction kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions. Subsequently, two separate clone libraries were created, one for Bacteria and one for Archaea, and for each sample type (water, biofilm, and glass slides). Purified PCR products were ligated into a pGEM-T vector system (Promega, Madison, WI) and transformed into competent Escherichia coli JM109 cells as recommended by the manufacturer. Positive clones were cultivated overnight in selective media, and vectors were purified using a "TENS-Mini Prep" protocol (50) with slight modifications as follows. To resuspended E. coli pellets, 300 µl of TENS buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 0.1 N NaOH, 0.5% sodium dodecyl sulfate) was added and incubated for 15 min at room temperature. Following cell lysis, 150 µl of 3 M sodium acetate, pH 5.5, was mixed with the cell lysate by inverting the reaction tubes several times. Samples were incubated on ice for 15 min and centrifuged for 10 min at 17,530 x g at 4°C and again for 15 min after precipitation of DNA. Samples were washed with 75% ethanol and air dried, and plasmid DNA was dissolved in 50 µl of double-distilled H₂O.

RFLP analysis and grouping of clones.
Purified vectors were digested for 16 h at 37°C with DdeI (Promega) for RFLP analysis, in order to separate the clones into groups according to their restriction patterns. Because DdeI cuts at four restriction sites in the pGEM-T vector, it was decided not to amplify the inserted DNA by PCR but to digest the cloned insert together with the vector. The digests were analyzed on 3% agarose gels. Inserted fragments, which were not cut by DdeI, were amplified with Archaea- or Bacteria-specific primers by a standard PCR protocol. PCR products were digested for 16 h at 37°C with HpaII (Fermentas) and separated on 3% agarose gels. Each RFLP group was preliminarily defined as an operational taxonomic unit (OTU). As representative sequences of each OTU were available, sequences were subjected to similarity matrix analysis using Similarity Matrix version 1.1 (27) of the RDP II (28, 29), to support OTU definition and RFLP grouping, respectively. The clones were clustered into operational taxonomic units at a level of sequence similarity of >99% (30, 43) in order to quantify diversity.

Sequencing and phylogenetic analysis.
At least two sequences representing each OTU group—if OTUs had more than one representative—were amplified with the primers SP6 (5' TAT TTA GGT GAC ACT ATA G) and T7 (5' TAA TAC GAC TCA CTA TAG GG). Sequences were determined by automated dideoxynucleotide methods with an ABI Prism Big-Dye Terminator cycle sequencing kit on an ABI Prism 310 genetic analyzer (Perkin-Elmer Applied Biosystems, Vienna, Austria), as recommended by the manufacturer. Sequence comparisons to identify database sequences similar to those from the clones were done using the FASTA3 (37) web interface from the European Molecular Biology Laboratory (EMBL). The potential presence of chimeric sequences was examined with the CHECK_CHIMERA program (27) available through the RDP II, release 8.1, with the chimera check program of the Bellerophon server (21), and in addition, the phylogenetic affiliations of their 5' and 3' ends were compared manually. No chimeras were recognized within archaeal sequences, but three potential chimeras were seen within bacterial sequences. Selected sequences were aligned with ClustalX (47). Subsequently the alignment was subjected to phylogenetic analysis using distance-based (22, 40) maximum parsimony and maximum likelihood methods, with programs from the phylogenetic interference package (PHYLIP) version 3.5.1c (J. Felsenstein, University of Washington, Seattle; distributed by the author) and the ClustalX software. Trees were visualized with the Treeview PPC program version 1.6.0 (36). In addition, the significance levels of interior branch points were determined by bootstrap analysis using 1,000 data resamplings. Phylogenetic trees shown in this work were constructed by using a neighbor-joining algorithm with ClustalX version 1.81 (40, 47).

Amplification of amoA (ammonia monooxygenase subunit A)-related sequences.
PCR was performed with a standard touchdown approach using the primer pair amo111F and amo643R (48), which are specific for crenarchaeal ammonia monooxygenase subunit A-related genes. The primers were a gift from Christa Schleper and coworkers, University of Bergen, Norway. PCR products from filtered water, glass slides, and biofilm material were designated as F, OT, and B, respectively, and were recovered from the gel slices using a QIAquick gel extraction kit (QIAGEN) in accordance with the manufacturer's instructions. PCR products of each sample type were cloned into the pGEM-T vector, and five randomly chosen clones of each sample were sequenced. Cloning and sequencing were performed as described above.

Statistical analysis.
The screening process was tested by statistical analyses to evaluate whether total diversity was covered by screening 415 clones. Two types of analyses were used. Coverage values were calculated by use of the following equation: C = 1 – (n/N) x 100, where n is the number of unique OTUs and N is the total number of clones examined (see reference 39 for a discussion of terms). In addition, a rarefaction analysis was performed to determine the number of unique OTUs as a proportion of the estimated total diversity. Calculations were performed using the freeware program Analytic Rarefaction version 1.3 (S. M. Holland; www.uga.edu/strata/software/Software.html).

Scanning electron microscopy.
Material for scanning electron microscopy (SEM) was taken randomly from the spring and its vicinity. A sample holder with two round 1.5-cm-diameter coverslips was placed for several days at a defined location, e.g., 2, 25, 45, and 55 cm from the rock fissures, allowing formation of a biofilm and deposition of small rock particles. The part of the coverslip that was covered by the holder was not colonized and thus served as a control. The coverslips as well as pieces of rock surface were immediately air dried and transported in a closed and dry container to the laboratory. The samples were mounted on aluminum SEM holders and coated with 1.5 nm Au/Pd (40:60) using a 208 high-resolution Cressington sputter coater (Watford, England) in order to enhance the conductance of the material and to prevent charging artifacts during scanning. Electron microscopy was carried out with a JEOL JSM-6330F field emission instrument (Tokyo, Japan) operated at 3.0 kV. This instrument is hosted by the Department of General Instrumentation of the Radboud University Nijmegen.

Nucleotide sequence accession numbers.
All small-subunit rRNA gene sequences as well as the amoA sequences were deposited in the EMBL/EBI nucleotide sequence database under the following accession numbers: AM039528 to AM039550 for partial 16S rRNA gene sequences; AM233905 and AM260487 to AM260489 for the crenarchaeal amoA sequences.

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Sampling and 16S rRNA gene analysis using RFLP.
Water samples and bacterial mats (biofilms) were designated FJQF and FJQB, respectively. The microbes, which cover the surface of the submerged boulders in the spring, seemed to exist in two different forms (18), both of which were collected as samples designated FJQB: an older colonization stage of these mats adhered strongly to the rock surfaces, whereas the second form appeared as a whitish slimy substance, which probably represented the glycocalyx of diverse microorganisms, and seemed to be less firmly attached to the rocks (18). Archaeal and bacterial 16S rRNA gene fragments were amplified with primers A21F/A958R and P797F/E1492R, respectively. A total number of 415 clones was analyzed; 283 clones were of archaeal origin, and 132 clones were of bacterial origin. RFLP analysis resulted in seven archaeal groups, designated FJQGA, and 16 bacterial groups, designated FJQGB, or FJQGBL if inserts were not cut by DdeI. This was the case for five of the 16 bacterial RFLP groups, and therefore the grouping of those five was based on the digestion of the amplified insert sequence with HpaII. When representative sequences of each group became available, sequences were compared using the Similarity Matrix version 1.1 of the RDP II. This method reduced the archaeal groups to four and bacterial groups to 14, since the similarity of some sequences was higher than 99%. These final groups were defined as one OTU each. Representative sequences of these merged groups were treated as single phylotypes included in one OTU (Table 1) and were also submitted to the EMBL/EBI nucleotide sequence database. The procedure was applied to the archaeal phylotypes FJQFA13, FJQBAA3, and FJQBAA20 (OTU FJQGA2), because these sequences were more than 99% similar to each other (data not shown), and to phylotypes FJQBAA5 and FJQFA2 (OTU FJQGA1). This procedure was also applied to bacterial phylotypes FJQOTB21, FJQFB23, and FJQBIB7 for the same reasons (FJQGBL2; Table 1).

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TABLE 1. Most similar database sequences of bacterial and archaeal 16S rRNA gene phylotypes recovered from the subsurface spring FJQ

Archaeal community composition and phylogenetic analysis.
The composition of archaeal OTUs clearly showed the abundance of crenarchaeal clones in all three sample types, filtered water, biofilm (microbial mats), and glass slide, which confirmed the common suggestion that mesophilic Crenarchaeota represent a stable and specific component in terrestrial habitats (2, 12, 34, 41). FJQGA1 and FJQGA2 consisted of clones from all three sample types, while the dominating clones of FJQGA1 originated from filtered water and FJQGA2 stemmed from the biofilm (Table 1). The database sequence that was most similar to FJQGA1 was clone SAGMA-2 of waters from a South African gold mine (46), but the most similar database sequence of FJQGA2 was an uncultured crenarchaeon, clone Gitt-GR-39 from uranium mill tailings. This result is of interest to our examinations, since the presence of radioactivity (radium, radon, and uranium) is characteristic of the thermal springs of Bad Gastein (18). Table 2 shows that crenarchaeal clones of FJQGA1 and FJQGA2 were the most frequently recovered archaeal groups in this habitat, representing 99.65% of all archaeal clones. A single clone, recovered from a glass slide, may have been of euryarchaeal origin, but was only 82.15% similar to its nearest neighbor sequence, which was from a deep-sea hydrothermal vent site (Table 1). FJQGA3, which belonged to the freshwater-wastewater-soil (1.3b) group, appeared to be a subdominant crenarchaeal group in this habitat (Table 2; Fig. 2). Four clones, recovered from glass slides, may represent this group in the FJQ subsurface thermal spring but were only 94.75% similar to its nearest neighbor sequence, which was from an oceanic crust (Table 1).

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TABLE 2. Distribution of clones from the spring FJQa

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FIG. 2. Archaeal phylogenetic tree based on 16S rRNA gene sequences, including various 16S rRNA gene clones obtained from the subsurface thermal spring FJQ. The tree was inferred by a neighbor-joining analysis of 772 corresponding positions of the 16S rRNA gene sequence (positions 107 to 879 of E. coli numbering [6]). The scale bar represents 10% nucleotide sequence difference. Four sequences from species of Bacteria constituted the outgroup. Symbols on the branches indicate bootstrap confidence values, as follows: •, >90%; , >75%. Numbering of crenarchaeal groups was chosen in accordance to references 12, 23, and 34.

Bacterial community composition and phylogenetic analysis.
Bacterial 16S rRNA gene composition and abundance of OTUs showed a higher diversity in community composition than did the archaeal representatives of this subsurface environment. Several OTUs were represented by clones of only one sample type, e.g., clones obtained from filtered water appeared in FJQGB1, FJQGB2, and FJQGB3, but FJQGB2 was represented by only one clone. FJQGB7, FJQGB8, FJQGB9, and FJQGBL4 were represented exclusively by clones from bacterial mats but only by one or two clones each. The clones obtained from glass slides represented OTUs FJQGB6 and FJQGBL3, where the latter again consisted of only one clone (Table 2). The most prominent OTUs of bacterial sequences were FJQGBL2 (25% of bacterial clones) and FJQGB5 (28% of bacterial clones), followed by FJQGBL1, FJQGB4, and FJQGB1 with about 10% of all bacterial clones each (Table 2). Other groups were represented by one to four clones (Table 2). The two OTUs accounting for the largest percentages of clones were most similar to sequences recovered from a microbial mat (FJQGBL2) and from a trichloroethene-contaminated site (FJQGB5). The less dominating groups (about 10% of the abundance of bacterial clones) were composed of OTU FJQGB1, which was only 89% similar to an uncultured strain isolated from subtropical freshwater marsh (Table 1). OTU FJQGB4 had only 87% similarity to its next relative, a clone isolated from continental shelf sediments (Antarctica). FJQGBL1 (99% sequence similarity), like OTU FJQGB7 (94% similarity), was affiliated to a cluster of thermophilic methanotrophic Bacteria and to cultured Methylocaldum-Methylococcus strains (Table 1; Fig. 3). The less frequently recovered OTUs (one to four clones) possessed sequences which were similar to those of isolates from diverse soil-like habitats, e.g., uranium-contaminated soil, uranium mine sediment waters, ANAMMOX (anaerobic ammonium oxidation) sludges, and artificial microcosms (FJQGBL3, FJQGBL4, FJQGBL5, FJQGB2, FJQGB3, FJQGB6, and FJQGB8), as well as one extreme habitat like extremely thermal soil (FJQGB9).

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FIG. 3. Bacterial phylogenetic tree based on 16S rRNA gene sequences of members of the Proteobacteria, including various 16S rRNA gene clones obtained from the subsurface thermal spring FJQ. The tree was inferred by a neighbor-joining analysis of 707 corresponding positions of the 16S rRNA gene sequence. The scale bar represents 10% nucleotide sequence difference. Four sequences from species of Archaea constituted the outgroup. Symbols on the branches indicate bootstrap confidence values, as follows: •, >90%; , >75%.

Statistical analysis.
Eighteen different OTUs were represented by two clone libraries, for Bacteria and Archaea, consisting of 415 clones. Coverage, calculated as in reference 39, reached a value of 95.66%, indicating that nearly the total diversity was covered by the two clone libraries. However, since coverage is based on the number of unique OTUs relative to total richness, evenness is not taken into account, and thus, this value should be seen as a rough estimate of diversity. The coverage value for Archaea (n = 4; N = 283) was 98.58% and for Bacteria (n = 14; N = 132) was 89.39%. As a second approach, a rarefaction analysis was done to determine if screening of 415 clones was sufficient for an estimation of diversity in the clone libraries of the spring FJQ. The expected number of the unique OTUs was plotted against the number of clones (Fig. 4). The calculated rarefaction curve of total OTUs showed a slight tendency to saturation; the curve for archaeal OTUs showed saturation, but the one for bacterial OTUs did not reach saturation (Fig. 4), indicating that diversity was not completely covered by screening of 415 clones.

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FIG. 4. Rarefaction curves for total (•) as well as for archaeal () and bacterial () OTUs of 16S rRNA gene clones. The dotted lines represent 95% confidence intervals.

Analyses of amoA (ammonia monooxygenase subunit A)-related genes.
Five clones recovered from each sample type were randomly selected and sequenced. Two sequences affiliated with amoA genes from the soil/sediment group (Fig. 5), whereas 13 clones were more similar to amoA genes from the marine water column or sediments. The sequences of amoA-related genes from Crenarchaeota showed similarities of 80.7% (clone F2), 78% (clone B2), and 77.2% (clone OT2) to "Candidatus Nitrosopumilus maritimus" (26), using FASTA Prokaryotes for the comparison. A FASTA Environmental comparison indicated 82.5% similarity of clone F2 to clone BS15.7_11 (16), 80.9% similarity of clone B2 to clone HB_A_21 (16), and 80.6% similarity of clone OT2 to clone HF130_D11 (17). All of these amoA genes were similar to clones of the marine water column or of marine sediments, which were described by Francis et al. (16) (Fig. 5). Clone F5, whose sequence affiliated to the soil Crenarchaeota, was 69.5% similar to the amoA gene of "Candidatus Nitrosopumilus maritimus," a marine Crenarchaeon, and 89.9% similar to a sequence of a terrestrial crenarchaeal amoA gene (DQ304894). Protein sequences were also analyzed and supported the DNA sequence comparisons. The peptide sequence of clone F5 showed an identity of 93.6% to clone OKR_C_5 (soil-sediment clone) (16) and a peptide similarity of 98.3% to an AmoA-like protein of soil Crenarchaeota (48). All other clones had identities between 94% and 96% and similarities of 98% to 99% to AmoA peptides of the marine water column or marine sediment origin (16).

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FIG. 5. Phylogenetic tree of amoA genes cloned from the thermal spring FJQ and closely related crenarchaeal genes from the database. The tree was inferred by a neighbor-joining analysis of 546 corresponding positions. The scale bar represents 10% nucleotide sequence difference. Bacterial amoA from Nitrosospira briensis was used as the outgroup. Designation of origin from soil/sediment or water column/sediment was as suggested by Francis et al. (16). Symbols on the branches indicate bootstrap confidence values, as follows: •, >90%; , >75%.

SEM.
SEM of small rock and artificial glass surfaces indicated a high diversity of morphological types of cells (Fig. 6). Figure 6A shows an overview of a microbial biofilm on pebbles, containing rod-shaped and coccoid cell types as well as spiral-shaped cells, and long thin and thick filaments. Figure 6B1 and B2 represent enlarged portions from Fig. 6A, which contain distinct spiral coiled cells and long filaments of various thicknesses. Figure 6C shows different morphotypes on a glass surface, which became attached following incubation in the spring for several days.

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FIG. 6. Scanning electron micrographs of natural biofilms on submerged rock surfaces in the thermal spring FJQ and microorganisms on glass surfaces, which were placed into the spring for several days. Plate A depicts an overview of a natural microbial biofilm; plates B1 and B2 show enlarged portions of plate A; plate C shows several different cell types attached to a glass surface. Different morphologies are indicated by labeled arrows (C, cocci; R, rods; S, spiral-shaped cells; F, filaments).

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Subsurface environments are very often not readily accessible and provide only limited possibilities for an investigation of the deep biosphere. However, the radioactive thermal springs in Bad Gastein, Austria, can be considered as windows to subterranean realms, since large quantities of mostly "old" thermal water (3,000 years) (7, 15) with very little admixture of cold surface water (<10%) are generated daily, and members of the rock-dwelling microbial communities are transported with the waters. There is little influence from environmental factors such as heavy rainfalls and thawing periods (7); thus the springs can be considered rather stable habitats, in dark surroundings (no sunlight), and not influenced by seasonal or weather-derived parameters.

The presence of archaeal and bacterial rRNA gene sequences was evident in water and biofilm samples recovered from the subsurface radioactive thermal spring FJQ, suggesting novel microbial communities in this subterranean location. Statistical analyses (Fig. 4) indicated that a large part of the archaeal diversity in this spring was likely included in the clone libraries and that further cloning would not lead to a higher number of archaeal OTUs, but more clones would most probably yield new bacterial sequences, as the diversity of bacteria was inadequately sampled. The presence of Crenarchaeota in this area was not too surprising since these organisms seem to appear in numerous habitats all over the world (2, 12, 34, 41). Crenarchaeota represent a significant fraction (up to 5%) of several prokaryotic communities (34); in some marine samples, Crenarchaeota constitute as much as 20% of the picoplankton (24). However, their influence on community structure and ecology is still elusive, partly because none of these organisms, with the exception of one marine Crenarchaeon (26), could be cultured until now, but recent reports show the possible importance of these organisms in the nitrogen cycle of the world (16, 41, 48). These findings were also supported by the detection of crenarchaeal amoA-related genes in the spring FJQ, albeit these "soil" Crenarchaeota seemed to possess amoA genes (13 out of 15 examined clones) which were more similar to those of marine Crenarchaeota (Fig. 5). The dominating archaeal OTUs of the environment described here belonged to the soil-freshwater-subsurface group (1.1b); a subdominant archaeal OTU belonged to the freshwater-wastewater-soil group (1.3b) of the Crenarchaeota and one belonged to the Euryarchaeota (Table 1; Fig. 2). Interestingly, all four OTUs contained isolates from glass slides, including the dominating groups, but the two subdominant OTUs were represented only by clones obtained from glass slides. This suggests that the two groups contain microorganisms that perhaps adhere to surfaces or interact with them and that sufficient DNA could only be isolated from attached cells.

The bacterial community in the spring FJQ contained two dominating groups (Table 2; Fig. 3 and 7), which comprise 9 of 14 OTUs. Sequences of these two groups affiliated with Nitrospira sp., Nitrospina sp. (-Proteobacteria) (Fig. 7), and the ß-Proteobacteria (Fig. 3). The closest relatives of all these phylotypes were uncultivated bacteria from soils, sediments, subsurface habitats, or sludges (Table 1). Nitrification, the oxidation of ammonia to nitrate, is an essential part of the nitrogen cycle. Nitrospirae are aerobic chemolithotrophs, which oxidize nitrite to nitrate (33); they were isolated from seawater, where they are ubiquitous, and from soil samples. Nitrospira moscoviensis was isolated recently from corroded iron pipes of heating systems in Moscow, Russia (13). Also, studies of the Nullarbor caves in Australia showed clones with high similarity to N. moscoviensis (20). These authors described "microbial mantles" which comprise sheets or tongues of mucoid material with embedded small crystals (20). The microbial biofilms in the spring FJQ somewhat resembled this description of the microbial mantles in Nullarbor caves. In addition, the presence of Nitrospirae is supported by the SEM pictures: Nitrospirae were described as tight or loosely coiled spirals with up to 20 turns (49), a morphology which can be seen in Fig. 6A, B1, and C). The evidence for ammonia-oxidizing Crenarchaeota (amoA-related genes; Fig. 5), nitrite-oxidizing Nitrospirae, one Nitrospina-related OTU (FJQGB4), as well as one additional OTU (FJQGBL5), which was similar to a bacterial sequence from ANAMMOX-sludge, suggested that possibly a whole nitrogen cycle takes place in the FJQ spring.

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FIG. 7. Bacterial phylogenetic tree based on 16S rRNA gene sequences of members of the Nitrospirae, Bacteroidetes, and Planctomycetes, including various 16S rRNA gene clones obtained from the subsurface thermal spring FJQ. The tree was inferred by a neighbor-joining analysis of 664 corresponding positions of the 16S rRNA gene sequence (positions 778 to 1442 of E. coli numbering [6]. Other details were as described in the legend to Fig. 3.

Most of the other analyzed OTUs were closely similar to thermotolerant or thermophilic uncultivated Bacteria, with the exceptions of clone BCM1-7B, an uncultured bacterium from subtropical freshwater marsh, and clone AKAU3482, which originated from uranium-contaminated soil (Table 1). FJQGB9 was 95% similar to an isolate from extreme thermal soil, and FJQGBL1 was affiliated with a thermophilic methanotrophic strain isolated from a greenhouse heating system at a hot spring near Szentes, Hungary (3). FJQGB7 was 94% similar to Methylococcus capsulatus strain Texas (8). Two further dominating groups of our bacterial clone library, which were represented on glass slides, were FJQGBL2 and FJQGB5. These OTUs were similar (97 and 93%, respectively) to Thiobacillus species, most of which are rod shaped and 1 to 4 µm in length, but some strains can form long filamentous aggregates (25); such morphologies also appeared in the SEM pictures (Fig. 6A and B2). Thin filaments could also stem from Leptothrix-related strains (44); one such sequence was detected in our clone library (OTU FJQGBL3).

The presence of diverse archaeal and bacterial sequences suggests that the thermal springs in the Central Alps near Bad Gastein and the underlying strata represent a novel and unique habitat for microbial life. The water of the springs may transport microbial communities from even deeper, more isolated and more extreme habitats to the surface. The results that we obtained in this study will provide the basis for further exploration of this subterranean environment.

 
We thank the personnel of the water works of the community of the village Bad Gastein, especially Johann Knoll, the technical manager of the thermal springs in Bad Gastein, for help in obtaining samples. Thanks go to H. P. M. Geurts and G. J. A. Janssen, University of Nijmegen, for assistance in maintaining and operating the scanning electron microscope; to Christa Schleper and her coworkers, University of Bergen, for providing primers for the amoA gene; and to Elisabeth Pierson, University of Nijmegen, for help with the SEM pictures.

This work was partially supported by FWF project P19250-B17 and a research fellowship from the government of the Land Salzburg.

 
* Corresponding author. Mailing address: Division of Molecular Biology, Department of Microbiology, University of Salzburg, Billrothstraße 11, A-5020 Salzburg, Austria. Phone: 43 662 8044 7210. Fax: 43 662 8044 7209. E-mail: helga.stan-lotter{at}sbg.ac.at.

Published ahead of print on 3 November 2006.

Deceased 30 June 2006.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, January 2007, p. 259-270, Vol. 73, No. 1
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