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Archaeal diversity in Icelandic hot springs

Thomas Kvist , Birgitte K. Ahring , Peter Westermann
DOI: http://dx.doi.org/10.1111/j.1574-6941.2006.00209.x 71-80 First published online: 1 January 2007

Abstract

Whole-cell density gradient extractions from three solfataras (pH 2.5) ranging in temperature from 81 to 90°C and one neutral hot spring (81°C, pH 7) from the thermal active area of Hveragerði (Iceland) were analysed for genetic diversity and local geographical variation of Archaea by analysis of amplified 16S rRNA genes. In addition to the three solfataras and the neutral hot spring, 10 soil samples in transects of the soil adjacent to the solfataras were analysed using terminal restriction fragment length polymorphism (t-RFLP). The sequence data from the clone libraries in combination with 14 t-RFLP profiles revealed a high abundance of clones clustering together with sequences from the nonthermophilic I.1b group of Crenarchaeota. The archaeal diversity in one solfatara was high; 26 different RFLP patterns were found using double digestion of the PCR products with restriction enzymes AluI and BsuRI. The sequenced clones from this solfatara belonged to Sulfolobales, Thermoproteales or were most closest related to sequences from uncultured Archaea. Sequences related to group I.1b were not found in the neutral hot spring or the hyperthermophilic solfatara (90°C).

Keywords
  • Crenarchaeota
  • solfataras
  • t-RFLP
  • clone library analysis
  • gradient centrifugation

Introduction

Cultivable Archaea are divided into Euryarchaeota and Crenarchaeota based on phylogenetic and phenotypic characters. The cultivated euryarchaeotes harbour a large group of diverse mesophilic and thermophilic anaerobes and halophiles, whereas the cultivated representatives of the crenarchaeotes mainly constitute hyperthermophilic sulphur-dependent thermophilic species. In recent years several studies have, however, expanded the ecological range of the crenarchaeotes. Crenarchaeotal 16S rRNA gene sequences have been detected in various nonthermophilic environments such as freshwater (Jürgens et al., 2000; Keough et al., 2003), marine sediments (Vetriani et al., 1999), seawater (Delong 1992; Crump & Baross 2000; Ochsenreiter et al., 2003), and different soil environments (Jürgens et al., 1997; Jürgens & Saano 1999; Furlong et al., 2002). Molecular studies suggest that crenarchaeotal sequences from nonthermophilic environments can be divided into four groups (Dawson et al., 2000). Group I, which is most commonly found in temperate environmental samples, is divided into three subgroups. Although the phylogeny of the subgroups is derived from 16S rRNA gene sequences, there has been some agreement between the different environments from which the sequences have been found and the three subgroups: marine group 1a, freshwater aquatic sediment and terrestrial soil group 1b, and forest soil group 1c. This correspondence has been weakened by recent studies as sequences falling within subgroup 1b have been detected in hot springs and solfataric environments (Marteinsson et al., 2001; Kanokratana et al., 2004; Kvist et al., 2005).

Most studies have described these organisms at the genetic level captured as cloned fragments from different environments. Details of the nutritional and growth requirements that could support the growth of these uncharacterized organisms have been presented (Grosskopf et al., 1998; Simon et al., 2000; Kaeberlein et al., 2002), and recently an autotrophic ammonia-oxidizing member of the marine group 1a Crenarchaeota was isolated in pure culture (Konneke et al., 2005). Simon et al. (2005) showed that mesophilic soil crenarchaeotes of the I.1b group could be maintained in an undefined coculture for several months, but no members of this group have to our knowledge been isolated.

Solfataric fields are highly dynamic environments where solfataras continuously emerge and disappear. Thus, the 16S rRNA gene sequences belonging to group I.1b crenarchaeotes detected in solfataras might be from nongrowing allochthonous organisms from the temperate soils of the solfataric field. We have, however, previously demonstrated that DNA from nonthermophilic soil crenarchaeotes cannot be detected by specific PCR after 5 min of incubation under solfataric conditions (Kvist et al., 2005). Here we compare the genetic diversity of Archaea from different hot springs and adjacent soils in a solfataric field near Hveragerði, Iceland, to elucidate the presence of subgroup I.1b crenarchaeotes in these environments. In order to minimize the possibility that group I.1b crenarchaeotes detected in the solfataras simply are products of DNA from the surrounding environment, rather than from intact cells from the sample, we separated cells from the samples by density gradient centrifugation (Priemé, 1996) before DNA extraction. By combining terminal restriction fragment length polymorphism (t-RFLP) analysis and clone libraries from a series of sample sites within a solfatara, we demonstrate that group I.1b crenarchaeotes detected in solfataras are probably not present as a consequence of newly transferred DNA from the surrounding soils, but most likely are microorganisms growing within the solfataras.

Materials and methods

Sampling

Fourteen samples were collected from the Hveragerði area (Iceland) from four different hot springs (A, B, Cs and Cn) ranging in temperature from 81 to 90°C. Additionally, 10 samples from transects perpendicular to the hot springs were collected. All four hot springs were localized within an area of c. 750 × 1500 m. The relative positions of all sampling sites are shown in Fig. 1. A mixture of surface sediment and water (1 : 1) was collected with a scoop from the solfataras and the hot spring. Transect samples from adjacent soils were collected in sterile 50-mL syringes with cut-off tips. The small soil columns were transferred to sterile 50-mL tubes (Falcon). The samples were transported from the site to the laboratory in an isolated cooling box at c. 4°C. All sample parameters are given in Table 1.

1

Map showing the sampling locations within the geothermally active area, near Hveragerði (Iceland).

View this table:

Sample parameters

SampleTransect distance to solfatara/hot spring (cm)Temperature
(°C)pH
Solfatara A812.5
TA130303.0
TA2100444.5
TA3200N/A4.5
Solfatara B812.5
TB170283.5
TB2120223.0
TB3200203.5
Solfatara Cs902.5
TCs1100233.5
TCs2200334.0
Hot Spring Cn817.0
TCn1100243.5
TCn2200374.0

Whole-cell extraction

Cells were separated from the sediment and soil using Nycodenz gradient centrifugation as described by Burmolle et al. (2003). Briefly, the soil sample was mixed with Tween 80/tetra sodium pyrophosphate buffer (TSPP) and gently sonicated to release the cells from the soil matrix. The mixture was then placed on top of a Nycodenz solution (1.3 g mL−1), and during centrifugation the soil particles passed through the Nycodenz leaving the cells at the interphase. The cell-containing liquid phase was then removed and washed in TSPP buffer.

Nucleic acid extraction and PCR

Extraction of nucleic acids from the whole-cell fraction of the samples was carried out as previously described (Kvist et al., 2005). Briefly, the samples were bead beaten in 1 mL of extraction buffer, and the released DNA was bound to a silica membrane in a Genomic Mini kit (Aabiot). The DNA was washed twice and subsequently eluted in 100 μL TE-buffer (pH 8). 16S rRNA genes present in the nucleic acid extractions were used as templates in PCR reactions, followed by cloning and t-RFLP analysis. The PCRs for the cloning reactions were performed using Ready-to-Go Beads (Amersham) with Archaea 16S rRNA gene-specific primers A21f (5′-TTCCGGTTGATCCYGCCGGA-3′) (DeLong 1992) and Ar9r (5′-CCCGCCAATTCCTTTAAGTTTC-3′) (Jürgens et al., 1997). Thirty PCR cycles were carried out starting with an initial denaturation step at 94°C for 2 min. Cycle conditions were: 94°C (30 s), 55°C (30 s) and 72°C (60 s). The PCR was terminated after 7 min elongation.

t-RFLP

PCR for the t-RFLP reaction was carried out as described for the cloning reaction, using TET-labelled forward primer A21f (MWG-Biotech) in combination with reverse primer Ar9r. PCR products were cut with BsuRI (Fermentas) after extraction from a 1% agarose gel. t-RFLP analyses were performed on a MegaBace capillary sequenator (Amersham Biosciences) using MegaBace ET900-R size standard (Amersham Biosciences). The data obtained were analysed using the software ‘Genetic Profiler’ version 1.5 (Molecular Dynamics). Predictions of the expected fragment sizes of the t-RFLP were determined after retrieving all available crenarchaeotal sequences from the ribosomal database project (RDPII) (Maidak et al., 1999). The sequences lacking information in the area of the A21f primer were aligned to complete sequences using ClustalW multiple sequence alignment. After having aligned the sequences and determined the size of the missing sequence information, all gaps were removed from the alignment, and the terminal restriction fragments were calculated as the size measured from the 5′-end of the forward primer to the restriction site of BsuRI. In order to evaluate peaks of the t-RFLP analysis, and thereby estimate possible candidates for identification, the TAP-t-RFLP analysis program from the RDPII database (Maidak et al., 1999) was used.

Terminal restriction fragment lengths and corresponding peak areas were exported from Genetic Profiler into a percentage matrix excluding peaks representing less than 0.5% of terminal DNA fragments. A Bray–Curtis similarity matrix was generated and used as basis for a CLUSTER similarity dendrogram (square root transformation/group average) using the multivariate statistical software Primer5 (PRIMER-E Ltd).

Cloning

The cloning procedure was carried out using TOPO-TA cloning kit (Invitrogen) as described by the manufacturer. Successfully cloned fragments were transformed into electro competent Escherichia coli (TOP10) (Invitrogen). Clones were grown and selected on LB agar supplemented with kanamycin (50 μg mL−1), ampicillin (100 μg mL−1) and X-gal (40 μg mL−1) as described in the cloning manual. Colony-forming clones without β-gal activity were selected for further analysis. Selected clones were grown in Terrific Broth (Difco) supplemented with kanamycin (50 μg mL−1) and ampicillin (100 μg mL−1) for 16 h at 37°C with shaking. Plasmids were then purified from 2 mL of culture using Plasmid Mini kit (Aabiot).

RFLP

To avoid problems regarding fragment orientation in the cloning vector during RFLP analysis, primers A21f and Ar9r were used for colony PCR during preparation of PCR products from each clone. PCR products were double digested with enzymes AluI and BsuRI in (× 1) Y+/Tango Yellow buffer (Fermentas). Enzymatic digestion was performed at 37°C and the reaction was terminated at 80°C after 3 h of incubation. All reactions were analysed and grouped in restriction patterns after electrophoresis on 15% acryl amide gels in a Hoefer gel electrophoresis equipment. Acryl amide gels were run for 90 min at 300 V.

Sequencing and phylogenetic analysis

Representatives from all restriction patterns were sequenced by Macrogen Inc. using m13-forward (5′-GTAAAACGACGGCCA GT-3′) and m13-reverse (5′-GCGGATAACAATTTCACACAGG-3′) primers, and assembly of the partial forward and reverse fragments was performed using CapContig in BioEdit (Hall 1999). Alignment of the assembled sequences including the closest matching sequence data obtained from the RDPII (Maidak et al., 1999) and NCBI/GenBank was carried out using ClustalW (Chenna et al., 2003). Neighbour-joining trees were created using the Jukes–Cantor algorithm in the phylogenetic software Mega2.1 (Kumar et al., 2001) with 100 bootstrap calculations.

Results

The t-RFLP analysis was used to group the samples with respect to the length of the terminal fragments. The profiles (Table 2, Figs 2 and 3) showed that solfataras A and B had a high similarity, as all terminal fragments were present in both samples. Consequently, only solfatara A was chosen for further phylogenetic analysis. By contrast, the solfatara ‘Cs’ and the neutral hot spring ‘Cn’ were less than 10% similar to any of the other sample sites, reflecting the different temperature of solfatara Cs and different pH of neutral spring Cn (Fig. 3).

View this table:
2

RFLP data from all samples

TRF size (bp)Sample site
ATA1TA2TA3BTB1TB2TB3CsTCs1TCs2CnTCn1TCn2
464.0
665.0
692.73.54.9
7515.0
782.94.0
835.1
8410.2
913.57.717.19.7
928.725.0
9323.612.729.3
958.43.4
1069.0
10925.021.3
11328.319.3
1146.2
1152.94.313.365.811.8
1163.610.7
14743.0
1516.7
1549.07.610.42.96.48.013.07.69.34.9
1707.0
17415.13.7
17818.010.9
1792.0
18514.414.0
18626.7
19435.042.638.850.752.217.721.539.955.926.231.332.8
2154.03.04.32.52.12.83.03.82.21.5
2184.0
2191.9
2212.1
226
2345.21.7
2396.015.68.513.26.35.18.811.715.59.99.012.3
24111.06.26.9
2435.03.034.2
2445.83.28.04.99.54.4
2464.74.7
2621.6
2632.2
2683.0
  • The values shown in the table represent the relative amount (%) of the specific terminal restriction fragment (TRF) in each sample.

2

Terminal restriction fragment length polymorphism profiles of the three solfataric hot springs (A, B and Cs) and the neutral hot spring (Cn). Peaks are numbered and the corresponding clones are listed in Tables 3 and 4, together with terminal fragment sizes in the four samples (A, B, Cs and Cn).

3

Dendrogram of the similarity of all hot spring and transect samples differentiated by the relative amount and the size of their terminal restriction fragment. The respective transect samples have the prefix ‘T’.

Qualitatively and quantitatively most of the t-RFLP patterns only varied less than 50% among and along the transects compared with the variation between the neutral spring and the solfataras (Table 2, Fig. 3). All transect samples contained a peak at 194 bp, and in most samples this was the dominating peak. Except for the neutral spring transect sample TCn2, which harboured several unique peaks, peaks corresponding to the Sulfolobales (241, 243 bp) and the Thermoproteales (109, 178 bp) were only detected in the solfataric springs (Table 2).

When comparing the t-RFLP profiles of solfatara A and B with the samples in transects perpendicular to the solfataras, the most abundant peak found in all profiles is that at 194 bp corresponding to group I.1b crenarchaeotes when analysed in the TAP-t-RFLP analysis program of the RDPII database.

In total, 152 clones from solfatara A, 160 clones from hot spring Cn and 21 clones from solfatara Cs were analysed. Twenty-six different RFLP clone restriction patterns were found in solfatara A, 25 in hot spring Cn and five in solfatara Cs. The clones analysed from solfatara A fell within three groups (Fig. 4): Sulfolobales, Thermoproteales and group I.1b Crenarchaeota. The I.1b group constituted 18% of the clones in the clone library and 35% of the t-RFLP peak area, while the thermophilic groups constituted 72 and 65%, respectively (Table 3).

4

Phylogenetic tree of the cloned 16S rRNA genes and their closest relatives. Solfatara A. The scale bar indicates the number of changes per sequence position. Bootstrap values below 40% are not shown. GenBank accession numbers of the deposited sequences are DQ441477DQ441530.

View this table:
3

Summary of t-RFLP results and the corresponding clones from solfatara A (HverdxxxA) together with relevant cultured and uncultured crenarchaeotes

t-RFLP size (bp)Peak ID*Sequence name/IDPercentage t-RFLPPercentage
109AIHverd198A, Thermocladium modestius2517
178AIIHverd006A, Hverd020A, Hverd043A, Hverd156A, Hverd174A, Hverd194A, Hverd226A, Thermoproteus tenax1836
194AIIIHverd004A, Hverd005A, clone Nap013, clone SCA1145, clone SCA1151, clone SCA1154, clone SCA1158, clone SCA1166, clone SCA11703518
195Not detectedHverd190A1
196Not detectedHverd026A1
239AIVHverd007A62
241AVHverd002A, Hverd065A, Hverd148A, Hverd168A, Hverd169A, Hverd175A, Hverd210A, Hverd224A, Hverd227A, Stygiolobus azoricus, Metallosphaera sedula1117
243AVIHverd166A, Hverd223A, Hverd228A, Sulfolobus solfataricus, Acidianus infernus57
244Not detectedHverd206A1
  • * Identification of the peak in Fig. 2– solfatara A.

  • Relative amount of the population represented in this peak.

  • Relative number of clones with this terminal fragment size.

Sequences belonging to five different crenarchaeotal groups were detected in the neutral hot spring (Fig. 5): Thermoproteales, Desulfurococcales, group I.2 crenarchaeotes and two thermophilic groups. No sequences related to group I.1b were detected in either clone library or t-RFLP analysis (Table 4).

5

Phylogenetic trees of the cloned 16S rRNA genes and their closest relatives. Hot Spring Cn. The scale bar indicates the number of changes per sequence position. Bootstrap values below 40% are not shown. GenBank accession numbers of the deposited sequences are DQ441477DQ441530.

View this table:
4

Summary of t-RFLP results and the corresponding clones from hot spring Cn (HverdxxxN) together with relevant cultured and uncultured crenarchaeotes

t-RFLP size (bp)Peak IDSequence name/IDPercentage t-RFLP*Percentage
46NIHverd019N42
75NIIHverd002N, Hverd018N, Hverd025N, Hverd124N, Hverd147N, Thermofilum pendens159
78NIIIHverd118N48
106NIVHverd066N99
107Not detectedHverd014N, clone pSL121
147NVHverd003N, Hverd012N, Hverd037N, Hverd091N, Hverd146N, Thermoproteus neutrophilus4351
170NVIHverd023N, Hverd031N, Hverd096N711
179NVIIHverd034N, Hverd104N, Hverd108N, Vulcanisaeta distributa22
218NVIIIUnknown4
239NIXHverd033N, Hverd044N, clone HAuD-LA4296
268NXHverd050N31
  • * Identification of the peak in Fig. 2– solfatara A.

  • The relative amount of the population represented in this peak.

  • The relative number of clones with this terminal fragment size.

Analysis of the clone library of solfatara Cs resulted in five closely related clones, all belonging to the Sulfolobales. All clones were found to have the anaerobic Stygioglobus azoricus as closest cultivated relative (Fig. 6).

6

Phylogenetic tree of the cloned 16S rRNA genes and their closest relatives. Solfatara Cs. The scale bar indicates the number of changes per sequence position. Bootstrap values below 40% are not shown. GenBank accession numbers of the deposited sequences are DQ441477DQ441530.

Discussion

Diversity studies of solfataras are so far scarce and current knowledge of these extreme environments is mainly based on cultivation studies. The present study and other recent studies based upon molecular analyses of the 16S rRNA gene have demonstrated a rather impressive diversity given the high temperature and low pH. Up to 65 different phylotypes of crenarchaeotes have been described from one solfatara (Kvist et al., 2005; Siering et al., 2006). In most of the studies of these environments, many and often most sequences are only distantly related to cultivated relatives. Here we have focused on the group I.1b crenarchaeotes, which previously were considered to be an environmentally uniform group belonging to temperate soil environments (Dawson et al., 2000). Several studies have, however, demonstrated the presence of considerable quantities of group I.1b crenarchaeotes in thermophilic environments. In the present study, 18% of the total number of clones found in solfatara A belonged to group I.1b. In the study by Kanokratana et al. (2004), 17 different RFLP archaeal patterns were found in the Bor Khlueng hot spring, Thailand (55°C, pH 6.6), but in contrast to our study they did not find any sequences related to cultivated Archaea. Two of the clones from their study showed 88–91% 16S rRNA gene sequence similarity to the two most closely related clones SCA1166 and SCA1173, previously found in temperate soil (Bintrim et al., 1997). These two clones represented 29.8% of the total number of clones analysed. In an Icelandic subterranean hot spring (71.8°C, pH unknown) two clones representing 19% of the sequenced clones were most closely related to the temperate soil clone SCA 1173 (74–84% 16S rRNA gene sequence similarity) (Marteinsson et al., 2001). In a previous study of a solfatara in Pisciarelli (Naples, Italy) 42% of the 201 analysed clones clustered within the I.1b group (Kvist et al., 2005). The many observations therefore indicate that the group I.1b crenarchaeotes previously limited to low-temperature environments must be widened to comprise thermophilic members. As previously demonstrated, nonthermophilic soil I.1b crenarchaeotes can only be detected by PCR for less than 5 min after they have been subjected to solfataric conditions (Kvist et al., 2005). The possibility of finding considerable amounts of I.1b nonthermophilic crenarchaeotes in a solfatara as a result of transfer from the surrounding soil are, therefore, considered to be minimal.

Owing to the dynamic nature of solfataric fields, mineral residues from extinct solfataras form a considerable part of the solfataric soils. The peak at 194 bp detected in all the soil samples might, therefore, comprise group I.1b crenarchaeotes derived from extinct solfataras, but could also include true mesophilic crenarchaeotes, which secondarily have colonized the residues. Only cultivation-based studies of these so far uncharacterized organisms can clarify this issue.

Despite the limited geographical area of the investigated Hveragerði solfataric field, the chemical and physical variation within this field is vast, and is clearly reflected in the microbial diversity. The t-RFLP analysis showed similar populations in solfataras A and B, but also illustrated that this was not a universal distribution in the whole area, as sequences related to group I.1b were not detected in solfatara Cs or in the hot spring Cn from the clone library and t-RFLP analysis. Solfatara Cs was found to harbour a lower archaeal diversity compared with the other solfataras. This difference is probably due to the high temperature (90°C) compared with the 81°C measured in solfataras A and B. The absence of crenarchaeotal sequences related to I.1b in solfatara Cs might be explained if the solfatara had recently been formed and was not yet fully colonized. However, data retrieved from the transect samples 30 cm from the solfataras, where the 194-bp peak is abundant, indicate that I.1b organisms probably could be present in solfatara Cs if the conditions in the solfatara had supported their growth.

The t-RFLP peak at 194 bp is found in nearly all of the analysed samples except for those from solfatara Cs and hot spring Cn. These two sites are less than 4 m apart, and the samples taken between Cs and Cn showed a high abundance of the I.1b peak. This observation supports that I.1b crenarchaeotes found in solfataras A and B do not constitute a ubiquitous nongrowing background present as a result of transfer of nonthermophilic organisms from the surrounding environment but rather represent actively growing organisms.

This study and the few other studies of solfataric microbial diversity have revealed that solfataras are extremely variable with respect to diversity among different solfataric fields, but also within a single field. Given that the extremophiles inhabiting these environments constitute potential sources of new bioactive compounds, and because the geothermal areas harbouring the solfataric fields are potential suppliers of geothermal energy, it is important to continue with diversity mapping and culturing to avoid conflicts between exploitation and conservation.

Acknowledgements

This study was performed within the Danish Archaea Centre supported by the Danish Natural Science Research Council. Anders Priemé (University of Copenhagen) is acknowledged for assisting in the preparation of t-RFLP samples and analysis of t-RFLP data.

Footnotes

  • Editor: Gary King

References

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