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Diversity of thermophilic and non-thermophilic crenarchaeota at 80 °C
FEMS Microbiology Letters 244 (2005) 61–68 www.fems-microbiology.org
Diversity of thermophilic and non-thermophilic crenarchaeota at 80 °C Thomas Kvist, Anett Mengewein, Stefanie Manzei, Birgitte K. Ahring, Peter Westermann
*
Environmental Microbiology & Biotechnology Group (EMB), BioCentrum – Danmarks Tekniske Universitet Søltofts Plads, Building 227, DK-2800 Lyngby, Denmark Received 15 November 2004; received in revised form 10 January 2005; accepted 12 January 2005 First published online 21 January 2005 Edited by C. Edwards
Abstract A hot spring in the solfataric field of Pisciarelli (Naples – Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Thermophilic; Sulfolobus; Non-thermophilic archaea; Solfatara; Clone library; 16S rRNA gene; DNA stability
1. Introduction Microbial biodiversity in extreme environments is generally considered to be scarce compared to most other environments. Although Archaea have been estimated to represent up to 20% of the biomass found on Earth [1], and thus are believed to play a major role the global ecosystem, only a few different species have so far been found and isolated from solfataric environments. Up to now, most biodiversity information about solfataric environments has been derived from isolation
*
Corresponding author. Tel.: +45 45 25 61 88; fax: +45 45 88 32 76. E-mail address: [email protected] (P. Westermann).
and cultivation studies [2]. It is, however, a general assumption that enrichment cultures underestimate the actual diversity because different organisms require different conditions to support growth [3–5]. Other studies have suggested that only as little as 0.1–1% of the prokaryotic organisms can be successfully cultivated by traditional techniques [6,7]. The kingdom of Crenarchaeota is at present divided into seven major groups: One group constituted by the cultivated thermophilic and hyperthermophilic isolates and six groups represented by sequences retrieved from low-temperature environments [8]. So far only one member of the groups from non-thermophilic environments has been cultivated (‘‘Crenarchaeum symbiosum’’) [9] but still not in axenic culture. Knowledge
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.01.021
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about non-thermophilic Crenarchaeota is, therefore, solely based on sequence data collected from various lowtemperature environments such as soil, freshwater, deep drillings, and seawater [4,10–13]. Although there are numerous examples of different monophyletic prokaryotic groups having related ecological requirements (e.g., methanogenic Archaea and anaerobic Gram positive bacteria), there are also examples of highly diverse ecological requirements within related monophyletic groups (e.g., methanogenic and halophilic Archaea). In this study we demonstrate that members previously supposed to be limited to the low temperature Crenarchaeal cluster can be found in hyperthermophilic acidic environments in similar amounts as members of the hyperthermophilic/thermophilic Crenarchaeal cluster.
2. Materials and methods 2.1. Sediment sampling A sample containing an estimated ratio of 1:1 hot spring water and surface sediment (0–2 cm) was collected by means of a sterile spoon from a hot spring in the Pisciarelli solfataric field of Naples (Italy). The sample was collected in 20 ml sterile bottles and distributed in aliquots for parallel analyses. All samples were kept on ice until the return to the laboratory (approximately 9 h), and samples for DNA analysis were afterwards frozen at 80 °C in 20% glycerol. The temperature of the hot spring was 80 °C and the pH was 3.0 (on-site measurements). 2.2. Nucleic acid extraction Extraction was carried out as described by Yu and Mohn [14] with a number of minor modifications listed below. About 1 g of sample was transferred to a bead beat screw-top tube containing 1 g of 0.2 mm silica beads (Bio Spec. Products – USA). One ml of extraction buffer [14] containing 30 ll of DEPC was added and the sample was bead beated twice for 2 min with intermediate ice cooling. The extract was centrifuged at 14,000g for 3 min, and the DNA-containing supernatant was collected. The beating procedure was repeated with fresh buffer followed by centrifugation and pooling of the two supernatants. SDS was removed by first adding ammonium acetate to a final concentration of 2 M. The mixture was then incubated 5 min on ice followed by centrifugation for 10 min at 14,000g (4 °C). The supernatant was transferred to a double volume of 7 M guanidine–HCl and mixed gently, and transferred to a spin column obtained from Genomic Mini Kit – (Aabiot – Poland). Liquid was removed from the column by applying a vacuum to the column using a spin column
compatible manifold, and washing was carried out as described by the manufacturer. DNA was eluted from the columns using 100 ll of Tris buffer (75 °C) after 5 min of incubation at room temperature. Four parallel samples were extracted from the same source, followed by subsequent pooling of the eluted DNA. 2.3. Clone library Partial 16S rRNA gene sequences were amplified using Archaea specific primers Arch21F (5 0 -TTC CGG TTG ATC CYG CCG GA-3 0 ) [15] and Ar9R (5 0 -CCC GCC AAT TCC TTT AAG TTT C-3 0 ) (906–927 Escherichia coli numbering) [10] using Ready-To-Go PCRBeads (Amersham Biosciences) in 25 ll reactions. The PCR program was initiated with 5 min denaturation at 94 °C, followed by 30 cycles of: 90 s denaturation at 94 °C – 90 s annealing at 55 °C – 90 s elongation at 72 °C. The run was terminated after a final 7 min elongation. Negative controls were prepared with both H2O and with non-target template from E. coli extracted by the method described above. The PCR products were purified by cutting out the products from a 1% agarose gel using Gel-Out Kit supplied by Aabiot (Poland) as described by the manufacturer. Cloning of the PCR product was carried out using TOPO TA cloning (Invitrogen), and successfully transformed cells were picked from LB agar plates containing 50 lg/ml kanamycin after 24 h of incubation at 37 °C. Each clone was transferred to Euro-Taq PCR mixture (Eurogentec – Belgium) and PCR with primers Arch21F and Ar9R were carried out for all clones. The presence of a PCR-product of correct size was verified by electrophoresis of the products on a 1% agarose gel. Each product from the colony PCR was cut in parallel reactions using restriction enzymes AluI and BsuRI, respectively, and the pattern from each restriction reaction was visualized by electrophoresis of the product on a 3% agarose gel. The size/migration of each fragment was measured using Gel-Pro Analyzer 3.1 (Media Cybernetics), and a representative of each pattern was chosen for sequencing. 2.4. Sequencing and phylogenetic analysis Forward and reverse sequencing were done by MWG-Biotech (Germany) using either T3/T7 or M13 uni/M13 rev sequencing primers (Invitrogen) matching sequences on the TOPO cloning vector. Contigs of forward and reverse sequences were constructed using ‘‘Cap Contig Assembly’’(BioEdit) [16]. All retrieved sequences were tested for chimeric properties using the ‘‘Chimera Check’’ [17] function and the closest relatives were detected using the ‘‘Sequence Match’’ function [17] and ‘‘Blastn’’ from NCBI [18]. Alignment of all sequences, their closest relatives and the out-group was
T. Kvist et al. / FEMS Microbiology Letters 244 (2005) 61–68
done using ClustalW for multiple sequences [19] and the alignment was subsequently checked manually for errors. A phylogenetic tree of the alignment was constructed by neighbor-joining (NJ: Jukes-Cantor/ Bootstrap100) with Methanobacterium thermoautotrophicum as outgroup using Mega2 phylogeny software [20]. The overall topology of the tree was verified using Maximum Parsimony analysis (MP: Min-Mini Heuristic – Search Factor 2) [20]. 2.5. Probe construction and fluorescent in situ hybridisation Design of an oligonucleotide probe (SoilCren750r) targeting only sequences retrieved from non-thermophilic environments was performed using the Primrose software package [21]. The probe sequence (5 0 -TTC ATC CCT CAC CGT CGA-3 0 ) was verified using Probe Match from RDPII [17]. Fluorescent in situ Hybridisation (FISH) was done as described by Ju¨rgens et al. [22] using 4% paraformaldehyde during fixation with addition of 0–40% formamide in the hybridization buffer. Sulfolobus solfataricus P2 probed with the Archaea specific probe Arch915 [23] was used as a positive control in the FISH procedure.
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3. Results In total, 201 clones were isolated and grouped according to restriction analysis (Table 1). Ten different groups were recognized and members of all groups were chosen for further analysis. Due to the high number of group-A representatives, three representatives were chosen to verify the reliability of the clone grouping. 3.1. Phylogeny of the clones The phylogenetic relationship of the aligned sequences and their closest relatives is shown in the Neighbor-joining tree in Fig. 1. The sequenced clones grouped within two clusters. One cluster, most closely related to Acidianus infernus represented 58.8% of the sequences, while 41.2% of the clone sequences remarkably clustered together with Crenarchaeal sequences previously only recognized in non-thermophilic environments. Maximum parsimony analysis verified the topology of the Neighbor-Joining tree, as all groups and subgroups fell within identical clusters using both methods. 3.2. Verification of the clone library
2.6. DNA stability under solfataric conditions About 1 g of agricultural soil was mixed with 10 ml Sulfolobus pH 3 growth medium (DSM 88), and distributed in aliquots and incubated at 80 °C. Samples were transferred to ice after 0, 5, 10, 30 and 60 min of incubation. Extraction of total nucleic acids was immediately performed as described above. PCR of the extracted products were performed as described above. Both the nucleic acid extractions and the PCR products were visualized using a 1% agarose gel. 2.7. t-RFLP for clone library verification The extracted 16S rDNA was amplified using a TETlabelled Arch21F forward primer (MWG-Biotech – Germany) and Ar9R as reverse primer, followed by a purification of the PCR-product as described above. 10 ll of the eluted PCR product was cut with restriction enzyme AluI and the fragments were analyzed on a MegaBace capillary electrophoresis analyzer with MegaBace ET900-R size standard. The analysis of the t-RFLP profile was done using Genetic Profiler software (Amersham Biosciences).
To verify that all major groups were represented in the clone library, a t-RFLP examination of the extracted DNA was included (Fig. 2). Due to the fact that not all sequences are differentiated at the 5 0 -terminal end of a restriction analysis, it is not possible to assign a peak to a specific clone, but all of the 201 clones found in the clone library can be assigned to one of the most abundant peaks. In addition, there are no peaks without clone library representatives (Table 1). A comparison between the distribution of clones and the areas of the corresponding peaks (Table 2) shows a reasonable similarity indicating that all major amplifiable archaeal 16S rRNA genes of the solfatara are represented in the clone library. 3.3. Fluorescent in situ hybridisation No detectable signal was found using this probe on the sediment sample. Positive control reactions performed on S. solfataricus served as verification of the FISH procedure using the Archaea specific probe Arch915. 3.4. DNA stability under solfataric conditions
2.8. Nucleotide sequence Accession Numbers The rRNA gene sequences have been deposited in the NCBI/GenBank using GenBank Accession Nos. AY650012–AY650023.
The nucleic acid extractions from the experiment where the non-sterile soil was added to the artificial solfatara medium are shown in Fig. 3(a). The corresponding PCR products generated with Archaea specific primers
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Table 1 Grouping of clones Group ID
Clones in this group
Relative presence (%)
t-RFa size
Peak ID groupsb
Closest relative
Similarity to closest relative (%)
Group representative Accession No.
A B C D E F G H I J
100 18 21 34 16 4 3 2 1 2
49.8 9.0 10.4 16.9 8.0 2.0 1.5 1.0 0.5 1.0
149 bp 358 bp 582 bp 558 bp 149 bp 705 bp 358 bp 358 bp 582 bp 149 bp
I II IV III I V II II IV I
Acidianus infernus (NCBI/RDP) Clone SCA1145 (RDP) Clone S15-28 (NCBI) Clone SCA1154 (RDP) Acidianus infernus (NCBI/RDP) Clone SCA1150 (RDP) Clone SCA1145 (RDP) Clone SCA1166 (RDP) Clone SCA11 (RDP) Acidianus infernus (NCBI/RDP)
97 100 99 100 99 100 98 97 99 99
AY650017 AY650012 AY650013 AY650014 AY650021 AY650015 AY650016 AY650018 AY650019 AY650020
Sum a b
201 Length of terminal restriction fragment. Referring to peaks in Fig. 2.
Nap075
99 100
clone SCA11
100
clone 660mArA8 clone Gitt-GR-39 Nap057 clone SCA1166
100 100
Nap018 clone SCA1150
58
98
clone6 60mArC10 100
100
Nap013 clone SCA1154
clone SCA1158 Nap045
100 51 100 100 100
Nap002 clone SCA1145 Nap008
78 99
Uncultured genomic fragment (AJ496176)
clone SCA1173 clone Subt-14 Nap047 100 Nap104 81
100
100 72
Nap078 Acidianus infernus
Sulfolobus Solfataricus Thermofilum pendens Methanothermobacter thermoautotroficum
0.05
Fig. 1. Phylogenetic tree of the cloned 16S rRNA genes (Bold) and their closest relatives (Normal). Bootstrap values below 50 are not shown. The bar indicates number of changes per sequence position. The SCA clones were all derived from agricultural temperate soil. The 660mAr clones were derived from a water sample while Gritt-GR-39 was derived from an uranium mining waste site.
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I 5000
Fluorescent signal
4000
3000
2000
II
III
1000
IV
VI
V
0 0
100
200
300 400 500 600 Terminal fragment size (bp)
700
800
900
Fig. 2. t-RFLP profile of the 16S rRNA genes amplified from DNA extracted from the Solfataric hot spring. The amplicons were cut with AluI. Peaks below 50–60 bp are considered as analysis-noise rather than actual positive results, as these peaks are not reproducible in parallel analyses. The small peaks between 150–250 bp have not been taken into consideration since the amount of DNA represented in these peaks is below the limit of detection for the clone-library (0.50%). Peak VI at approx. 870 bp represents uncut PCR fragments.
Table 2 Comparison of clone library and t-RFLP
4. Discussion
Peak ID groups
% of clones in this peak
% of terminal fragment in this peak
I II III IV V VI
59 11 17 11 2 0
51 19 21 3 3 3
are shown in Fig. 3(b). Neither genomic DNA nor Archaea PCR products were detected after 5 min incubation at 80 °C, pH 3.
The detection of 10 different groups among 201 clones analyzed from the harsh solfataric environment was rather unexpected, as earlier studies from similar environments have showed lower diversity [24,25]. Another unexpected result is the fact that no members of the Sulfolobus genus were found in the clone library. Selective enrichment and probing studies have previously demonstrated the presence of Sulfolobales in this environment [2,26–28]. The type strains of Metallosphaera sedula, S. solfataricus, and Acidianus infernus have also been isolated from the same location [2,27,28]. Both M. sedula and S. solfataricus were easily enriched and
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Fig. 3. (a) The fate of DNA exposed to solfataric conditions for various periods. Lane 1: marker (k/ECO91I). Lane 2: DNA from 0.1 g of soil (positive control). Lane 3, 4, 5, and 6: DNA extracted after 0, 5, 10 and 30 min incubation, respectively. (b) Products obtained from amplification of DNA exposed to solfataric conditions for various periods using archaeal 16S rRNA gene specific primers. Lane 1: marker (k/ECO91I). Lane 2: negative control. Lane 3: positive PCR control reaction (Sulfolobus solfataricus template). Lane 4, 5, 6, and 7: Archaeal PCR products after 0, 5, 10 and 30 min incubation, respectively.
isolated from our sediment samples in organic-rich medium (DSM 88). Successful DNA-extraction and subsequent PCR amplification and sequencing of the 16S RNA genes from these enrichments strongly suggested that Sulfolobus would have be found in the clone library if they were present in the sediment sample in sufficient amounts (data not shown). The in situ presence of Metallosphaera and Sulfolobus must, therefore, be below the detection limit of the clone library. Sulfolobus was not detected by the t-RFLP analysis (fragment size 423 bp.). A small peak at approx. 870 bp constituting 2.3% of the total amount and representing uncut PCR product is seen in Fig. 2. The sequence data obtained from the isolated M. sedula reveals that this organism has no site for the restriction enzyme AluI (data not shown) and could therefore be hidden in the uncut fragment.
Cultivation based phylogenetic studies have been conducted on solfataras, but so far only members of one of the thermophilic Crenarchaeal groups have been isolated. Most available information about the diversity of solfataras is consequently based on sequence data of the 16S rRNA gene. Archaeal clones grouped as nonthermophilic environmental clones have previously been detected in different and geographically distant environments [7,10,29]. A relationship between Archaeal clones found in marine environments and hot springs has previously been suggested [30], but to our knowledge this is the first time that sequences that fall within non-thermophilic clusters have been detected as an considerable part of the Archaeal 16S rRNA genes in a thermophilic environment. So far only one member of the non-thermophilic Crenarchaeota has been cultivated and the existence of this group has mainly been demonstrated by 16S rRNA gene sequences. Only one study has so far identified other genes from these organisms [31]. Our finding, that the clones closely related to non-thermophilic Crenarchaeal clones represent 42% of the total number of clones found in our library suggests that the non-thermophilic Crenarchaeota are considerably more ecologically diverse than so far anticipated. The majority of the sequences closely related to the solfatara clones (the SCA sequences) were retrieved from agricultural soil of West Madison Agricultural Research Station (USA) [7]. Solfataric fields are known to be highly dynamic areas, where hot springs frequently are created and disappear. Microorganisms inhabiting the banks and surroundings might, therefore, be included in the solfatara and lead to erroneous conclusions about the diversity. Testing this by exposing microorganisms from the adjacent soil to solfataric conditions will not solve this question since the microorganisms might be remnants from solfataric material as well as indigenous organisms. We, therefore, chose to test the stability of DNA from true non-thermophilic Crenarchaeota by incubating a temperate agricultural soil under solfataric conditions. Non-thermophilic Crenarchaeota were previously found to constitute the only amplifiable archaeal 16S rRNA genes from this environment (data not shown). The loss of amplifiable DNA after 5 min of incubation indicates that DNA from true non-thermophilic Crenarchaeota will not persist in the solfatara even if they are enclosed and protected in soil particles. Together with the high representation in the clone library, this suggests that a population of Crenarchaeota related to non-thermophilic strains is capable of withstanding the extreme conditions in a solfatara. Marteinsson et al. [32] described the findings of both thermophilic Desulfurococcus and a non-thermophilic Crenarchaeal clone in a library constructed from a subterranean hot spring (72 °C). The clone (Subt-14) also clustered together with SCA types of clones and is included in Fig. 1. Huber et al. [26] demonstrated high
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numbers of irregular cocci in the Piscarelli solfatara by the means of a Sulfolobales specific probe. Since Acidianus belongs to this order, their results are not contradicting ours. Unfortunately no probe data or references are given in their paper, and it is therefore, not possible to validate the specificity of the Sulfolobales specific probe towards the non-thermophilic Crenarchaeota. Analyzing probes from the ARB probe library v.1.2 [33], however, reveals that at least some of the suggested Sulfolobales probes target most SCA clones with only one mismatch (analysed using ‘‘Probe Match’’ [17]). To our knowledge there are so far no published results indicating any recognizable morphology of low temperature Crenarchaeal organisms that separates these organisms from known thermophilic and/or hyperthermophilic Archaea. To search for this morphology we designed a probe to target only the Archaea found in temperate environments (SoilCren750r). The probe was validated using probe Match from RDPII [17], and tested for specificity using this probe as reverse primer, resulting in products from cloned fragments. Unfortunately this probe did not yield any signal from the Pisciarelli sample after several FISH probing attempts. Since no cultivated controls are available from this group of organisms, it is impossible to evaluate whether the lack of signal was due to suboptimal fixation processes or hybridization conditions. The data presented in this study suggests that the non-thermophilic Crenarchaeota group is considerably more ecologically diverse, than previously anticipated. Similar to e.g., methanogenic Euryarchaeota the nonthermophilic Crenarchaeota appear to harbour as well true non-thermophiles as organisms capable of tolerating thermophilic conditions. A further elucidation of the functions and capabilities of the non-thermophilic Crenarchaeota awaits the isolation or genome analysis of a representative from this group of organisms.
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Acknowledgements
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This work was performed within the Danish Archaea Centre supported by a grant from the Danish Natural Science Research Council (SNF). We thank Anders Prieme´ for his excellent assistance in performing the tRFLP analysis and Thomas Ishøy and Slawomir Dabrowski for their scientific and technical assistance throughout the work and preparation of this paper.
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Diversity of thermophilic and non-thermophilic crenarchaeota at 80 °C Thomas Kvist, Anett Mengewein, Stefanie Manzei, Birgitte K. Ahring, Peter Westermann
*
Environmental Microbiology & Biotechnology Group (EMB), BioCentrum – Danmarks Tekniske Universitet Søltofts Plads, Building 227, DK-2800 Lyngby, Denmark Received 15 November 2004; received in revised form 10 January 2005; accepted 12 January 2005 First published online 21 January 2005 Edited by C. Edwards
Abstract A hot spring in the solfataric field of Pisciarelli (Naples – Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Thermophilic; Sulfolobus; Non-thermophilic archaea; Solfatara; Clone library; 16S rRNA gene; DNA stability
1. Introduction Microbial biodiversity in extreme environments is generally considered to be scarce compared to most other environments. Although Archaea have been estimated to represent up to 20% of the biomass found on Earth [1], and thus are believed to play a major role the global ecosystem, only a few different species have so far been found and isolated from solfataric environments. Up to now, most biodiversity information about solfataric environments has been derived from isolation
*
Corresponding author. Tel.: +45 45 25 61 88; fax: +45 45 88 32 76. E-mail address: [email protected] (P. Westermann).
and cultivation studies [2]. It is, however, a general assumption that enrichment cultures underestimate the actual diversity because different organisms require different conditions to support growth [3–5]. Other studies have suggested that only as little as 0.1–1% of the prokaryotic organisms can be successfully cultivated by traditional techniques [6,7]. The kingdom of Crenarchaeota is at present divided into seven major groups: One group constituted by the cultivated thermophilic and hyperthermophilic isolates and six groups represented by sequences retrieved from low-temperature environments [8]. So far only one member of the groups from non-thermophilic environments has been cultivated (‘‘Crenarchaeum symbiosum’’) [9] but still not in axenic culture. Knowledge
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.01.021
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about non-thermophilic Crenarchaeota is, therefore, solely based on sequence data collected from various lowtemperature environments such as soil, freshwater, deep drillings, and seawater [4,10–13]. Although there are numerous examples of different monophyletic prokaryotic groups having related ecological requirements (e.g., methanogenic Archaea and anaerobic Gram positive bacteria), there are also examples of highly diverse ecological requirements within related monophyletic groups (e.g., methanogenic and halophilic Archaea). In this study we demonstrate that members previously supposed to be limited to the low temperature Crenarchaeal cluster can be found in hyperthermophilic acidic environments in similar amounts as members of the hyperthermophilic/thermophilic Crenarchaeal cluster.
2. Materials and methods 2.1. Sediment sampling A sample containing an estimated ratio of 1:1 hot spring water and surface sediment (0–2 cm) was collected by means of a sterile spoon from a hot spring in the Pisciarelli solfataric field of Naples (Italy). The sample was collected in 20 ml sterile bottles and distributed in aliquots for parallel analyses. All samples were kept on ice until the return to the laboratory (approximately 9 h), and samples for DNA analysis were afterwards frozen at 80 °C in 20% glycerol. The temperature of the hot spring was 80 °C and the pH was 3.0 (on-site measurements). 2.2. Nucleic acid extraction Extraction was carried out as described by Yu and Mohn [14] with a number of minor modifications listed below. About 1 g of sample was transferred to a bead beat screw-top tube containing 1 g of 0.2 mm silica beads (Bio Spec. Products – USA). One ml of extraction buffer [14] containing 30 ll of DEPC was added and the sample was bead beated twice for 2 min with intermediate ice cooling. The extract was centrifuged at 14,000g for 3 min, and the DNA-containing supernatant was collected. The beating procedure was repeated with fresh buffer followed by centrifugation and pooling of the two supernatants. SDS was removed by first adding ammonium acetate to a final concentration of 2 M. The mixture was then incubated 5 min on ice followed by centrifugation for 10 min at 14,000g (4 °C). The supernatant was transferred to a double volume of 7 M guanidine–HCl and mixed gently, and transferred to a spin column obtained from Genomic Mini Kit – (Aabiot – Poland). Liquid was removed from the column by applying a vacuum to the column using a spin column
compatible manifold, and washing was carried out as described by the manufacturer. DNA was eluted from the columns using 100 ll of Tris buffer (75 °C) after 5 min of incubation at room temperature. Four parallel samples were extracted from the same source, followed by subsequent pooling of the eluted DNA. 2.3. Clone library Partial 16S rRNA gene sequences were amplified using Archaea specific primers Arch21F (5 0 -TTC CGG TTG ATC CYG CCG GA-3 0 ) [15] and Ar9R (5 0 -CCC GCC AAT TCC TTT AAG TTT C-3 0 ) (906–927 Escherichia coli numbering) [10] using Ready-To-Go PCRBeads (Amersham Biosciences) in 25 ll reactions. The PCR program was initiated with 5 min denaturation at 94 °C, followed by 30 cycles of: 90 s denaturation at 94 °C – 90 s annealing at 55 °C – 90 s elongation at 72 °C. The run was terminated after a final 7 min elongation. Negative controls were prepared with both H2O and with non-target template from E. coli extracted by the method described above. The PCR products were purified by cutting out the products from a 1% agarose gel using Gel-Out Kit supplied by Aabiot (Poland) as described by the manufacturer. Cloning of the PCR product was carried out using TOPO TA cloning (Invitrogen), and successfully transformed cells were picked from LB agar plates containing 50 lg/ml kanamycin after 24 h of incubation at 37 °C. Each clone was transferred to Euro-Taq PCR mixture (Eurogentec – Belgium) and PCR with primers Arch21F and Ar9R were carried out for all clones. The presence of a PCR-product of correct size was verified by electrophoresis of the products on a 1% agarose gel. Each product from the colony PCR was cut in parallel reactions using restriction enzymes AluI and BsuRI, respectively, and the pattern from each restriction reaction was visualized by electrophoresis of the product on a 3% agarose gel. The size/migration of each fragment was measured using Gel-Pro Analyzer 3.1 (Media Cybernetics), and a representative of each pattern was chosen for sequencing. 2.4. Sequencing and phylogenetic analysis Forward and reverse sequencing were done by MWG-Biotech (Germany) using either T3/T7 or M13 uni/M13 rev sequencing primers (Invitrogen) matching sequences on the TOPO cloning vector. Contigs of forward and reverse sequences were constructed using ‘‘Cap Contig Assembly’’(BioEdit) [16]. All retrieved sequences were tested for chimeric properties using the ‘‘Chimera Check’’ [17] function and the closest relatives were detected using the ‘‘Sequence Match’’ function [17] and ‘‘Blastn’’ from NCBI [18]. Alignment of all sequences, their closest relatives and the out-group was
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done using ClustalW for multiple sequences [19] and the alignment was subsequently checked manually for errors. A phylogenetic tree of the alignment was constructed by neighbor-joining (NJ: Jukes-Cantor/ Bootstrap100) with Methanobacterium thermoautotrophicum as outgroup using Mega2 phylogeny software [20]. The overall topology of the tree was verified using Maximum Parsimony analysis (MP: Min-Mini Heuristic – Search Factor 2) [20]. 2.5. Probe construction and fluorescent in situ hybridisation Design of an oligonucleotide probe (SoilCren750r) targeting only sequences retrieved from non-thermophilic environments was performed using the Primrose software package [21]. The probe sequence (5 0 -TTC ATC CCT CAC CGT CGA-3 0 ) was verified using Probe Match from RDPII [17]. Fluorescent in situ Hybridisation (FISH) was done as described by Ju¨rgens et al. [22] using 4% paraformaldehyde during fixation with addition of 0–40% formamide in the hybridization buffer. Sulfolobus solfataricus P2 probed with the Archaea specific probe Arch915 [23] was used as a positive control in the FISH procedure.
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3. Results In total, 201 clones were isolated and grouped according to restriction analysis (Table 1). Ten different groups were recognized and members of all groups were chosen for further analysis. Due to the high number of group-A representatives, three representatives were chosen to verify the reliability of the clone grouping. 3.1. Phylogeny of the clones The phylogenetic relationship of the aligned sequences and their closest relatives is shown in the Neighbor-joining tree in Fig. 1. The sequenced clones grouped within two clusters. One cluster, most closely related to Acidianus infernus represented 58.8% of the sequences, while 41.2% of the clone sequences remarkably clustered together with Crenarchaeal sequences previously only recognized in non-thermophilic environments. Maximum parsimony analysis verified the topology of the Neighbor-Joining tree, as all groups and subgroups fell within identical clusters using both methods. 3.2. Verification of the clone library
2.6. DNA stability under solfataric conditions About 1 g of agricultural soil was mixed with 10 ml Sulfolobus pH 3 growth medium (DSM 88), and distributed in aliquots and incubated at 80 °C. Samples were transferred to ice after 0, 5, 10, 30 and 60 min of incubation. Extraction of total nucleic acids was immediately performed as described above. PCR of the extracted products were performed as described above. Both the nucleic acid extractions and the PCR products were visualized using a 1% agarose gel. 2.7. t-RFLP for clone library verification The extracted 16S rDNA was amplified using a TETlabelled Arch21F forward primer (MWG-Biotech – Germany) and Ar9R as reverse primer, followed by a purification of the PCR-product as described above. 10 ll of the eluted PCR product was cut with restriction enzyme AluI and the fragments were analyzed on a MegaBace capillary electrophoresis analyzer with MegaBace ET900-R size standard. The analysis of the t-RFLP profile was done using Genetic Profiler software (Amersham Biosciences).
To verify that all major groups were represented in the clone library, a t-RFLP examination of the extracted DNA was included (Fig. 2). Due to the fact that not all sequences are differentiated at the 5 0 -terminal end of a restriction analysis, it is not possible to assign a peak to a specific clone, but all of the 201 clones found in the clone library can be assigned to one of the most abundant peaks. In addition, there are no peaks without clone library representatives (Table 1). A comparison between the distribution of clones and the areas of the corresponding peaks (Table 2) shows a reasonable similarity indicating that all major amplifiable archaeal 16S rRNA genes of the solfatara are represented in the clone library. 3.3. Fluorescent in situ hybridisation No detectable signal was found using this probe on the sediment sample. Positive control reactions performed on S. solfataricus served as verification of the FISH procedure using the Archaea specific probe Arch915. 3.4. DNA stability under solfataric conditions
2.8. Nucleotide sequence Accession Numbers The rRNA gene sequences have been deposited in the NCBI/GenBank using GenBank Accession Nos. AY650012–AY650023.
The nucleic acid extractions from the experiment where the non-sterile soil was added to the artificial solfatara medium are shown in Fig. 3(a). The corresponding PCR products generated with Archaea specific primers
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Table 1 Grouping of clones Group ID
Clones in this group
Relative presence (%)
t-RFa size
Peak ID groupsb
Closest relative
Similarity to closest relative (%)
Group representative Accession No.
A B C D E F G H I J
100 18 21 34 16 4 3 2 1 2
49.8 9.0 10.4 16.9 8.0 2.0 1.5 1.0 0.5 1.0
149 bp 358 bp 582 bp 558 bp 149 bp 705 bp 358 bp 358 bp 582 bp 149 bp
I II IV III I V II II IV I
Acidianus infernus (NCBI/RDP) Clone SCA1145 (RDP) Clone S15-28 (NCBI) Clone SCA1154 (RDP) Acidianus infernus (NCBI/RDP) Clone SCA1150 (RDP) Clone SCA1145 (RDP) Clone SCA1166 (RDP) Clone SCA11 (RDP) Acidianus infernus (NCBI/RDP)
97 100 99 100 99 100 98 97 99 99
AY650017 AY650012 AY650013 AY650014 AY650021 AY650015 AY650016 AY650018 AY650019 AY650020
Sum a b
201 Length of terminal restriction fragment. Referring to peaks in Fig. 2.
Nap075
99 100
clone SCA11
100
clone 660mArA8 clone Gitt-GR-39 Nap057 clone SCA1166
100 100
Nap018 clone SCA1150
58
98
clone6 60mArC10 100
100
Nap013 clone SCA1154
clone SCA1158 Nap045
100 51 100 100 100
Nap002 clone SCA1145 Nap008
78 99
Uncultured genomic fragment (AJ496176)
clone SCA1173 clone Subt-14 Nap047 100 Nap104 81
100
100 72
Nap078 Acidianus infernus
Sulfolobus Solfataricus Thermofilum pendens Methanothermobacter thermoautotroficum
0.05
Fig. 1. Phylogenetic tree of the cloned 16S rRNA genes (Bold) and their closest relatives (Normal). Bootstrap values below 50 are not shown. The bar indicates number of changes per sequence position. The SCA clones were all derived from agricultural temperate soil. The 660mAr clones were derived from a water sample while Gritt-GR-39 was derived from an uranium mining waste site.
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I 5000
Fluorescent signal
4000
3000
2000
II
III
1000
IV
VI
V
0 0
100
200
300 400 500 600 Terminal fragment size (bp)
700
800
900
Fig. 2. t-RFLP profile of the 16S rRNA genes amplified from DNA extracted from the Solfataric hot spring. The amplicons were cut with AluI. Peaks below 50–60 bp are considered as analysis-noise rather than actual positive results, as these peaks are not reproducible in parallel analyses. The small peaks between 150–250 bp have not been taken into consideration since the amount of DNA represented in these peaks is below the limit of detection for the clone-library (0.50%). Peak VI at approx. 870 bp represents uncut PCR fragments.
Table 2 Comparison of clone library and t-RFLP
4. Discussion
Peak ID groups
% of clones in this peak
% of terminal fragment in this peak
I II III IV V VI
59 11 17 11 2 0
51 19 21 3 3 3
are shown in Fig. 3(b). Neither genomic DNA nor Archaea PCR products were detected after 5 min incubation at 80 °C, pH 3.
The detection of 10 different groups among 201 clones analyzed from the harsh solfataric environment was rather unexpected, as earlier studies from similar environments have showed lower diversity [24,25]. Another unexpected result is the fact that no members of the Sulfolobus genus were found in the clone library. Selective enrichment and probing studies have previously demonstrated the presence of Sulfolobales in this environment [2,26–28]. The type strains of Metallosphaera sedula, S. solfataricus, and Acidianus infernus have also been isolated from the same location [2,27,28]. Both M. sedula and S. solfataricus were easily enriched and
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Fig. 3. (a) The fate of DNA exposed to solfataric conditions for various periods. Lane 1: marker (k/ECO91I). Lane 2: DNA from 0.1 g of soil (positive control). Lane 3, 4, 5, and 6: DNA extracted after 0, 5, 10 and 30 min incubation, respectively. (b) Products obtained from amplification of DNA exposed to solfataric conditions for various periods using archaeal 16S rRNA gene specific primers. Lane 1: marker (k/ECO91I). Lane 2: negative control. Lane 3: positive PCR control reaction (Sulfolobus solfataricus template). Lane 4, 5, 6, and 7: Archaeal PCR products after 0, 5, 10 and 30 min incubation, respectively.
isolated from our sediment samples in organic-rich medium (DSM 88). Successful DNA-extraction and subsequent PCR amplification and sequencing of the 16S RNA genes from these enrichments strongly suggested that Sulfolobus would have be found in the clone library if they were present in the sediment sample in sufficient amounts (data not shown). The in situ presence of Metallosphaera and Sulfolobus must, therefore, be below the detection limit of the clone library. Sulfolobus was not detected by the t-RFLP analysis (fragment size 423 bp.). A small peak at approx. 870 bp constituting 2.3% of the total amount and representing uncut PCR product is seen in Fig. 2. The sequence data obtained from the isolated M. sedula reveals that this organism has no site for the restriction enzyme AluI (data not shown) and could therefore be hidden in the uncut fragment.
Cultivation based phylogenetic studies have been conducted on solfataras, but so far only members of one of the thermophilic Crenarchaeal groups have been isolated. Most available information about the diversity of solfataras is consequently based on sequence data of the 16S rRNA gene. Archaeal clones grouped as nonthermophilic environmental clones have previously been detected in different and geographically distant environments [7,10,29]. A relationship between Archaeal clones found in marine environments and hot springs has previously been suggested [30], but to our knowledge this is the first time that sequences that fall within non-thermophilic clusters have been detected as an considerable part of the Archaeal 16S rRNA genes in a thermophilic environment. So far only one member of the non-thermophilic Crenarchaeota has been cultivated and the existence of this group has mainly been demonstrated by 16S rRNA gene sequences. Only one study has so far identified other genes from these organisms [31]. Our finding, that the clones closely related to non-thermophilic Crenarchaeal clones represent 42% of the total number of clones found in our library suggests that the non-thermophilic Crenarchaeota are considerably more ecologically diverse than so far anticipated. The majority of the sequences closely related to the solfatara clones (the SCA sequences) were retrieved from agricultural soil of West Madison Agricultural Research Station (USA) [7]. Solfataric fields are known to be highly dynamic areas, where hot springs frequently are created and disappear. Microorganisms inhabiting the banks and surroundings might, therefore, be included in the solfatara and lead to erroneous conclusions about the diversity. Testing this by exposing microorganisms from the adjacent soil to solfataric conditions will not solve this question since the microorganisms might be remnants from solfataric material as well as indigenous organisms. We, therefore, chose to test the stability of DNA from true non-thermophilic Crenarchaeota by incubating a temperate agricultural soil under solfataric conditions. Non-thermophilic Crenarchaeota were previously found to constitute the only amplifiable archaeal 16S rRNA genes from this environment (data not shown). The loss of amplifiable DNA after 5 min of incubation indicates that DNA from true non-thermophilic Crenarchaeota will not persist in the solfatara even if they are enclosed and protected in soil particles. Together with the high representation in the clone library, this suggests that a population of Crenarchaeota related to non-thermophilic strains is capable of withstanding the extreme conditions in a solfatara. Marteinsson et al. [32] described the findings of both thermophilic Desulfurococcus and a non-thermophilic Crenarchaeal clone in a library constructed from a subterranean hot spring (72 °C). The clone (Subt-14) also clustered together with SCA types of clones and is included in Fig. 1. Huber et al. [26] demonstrated high
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numbers of irregular cocci in the Piscarelli solfatara by the means of a Sulfolobales specific probe. Since Acidianus belongs to this order, their results are not contradicting ours. Unfortunately no probe data or references are given in their paper, and it is therefore, not possible to validate the specificity of the Sulfolobales specific probe towards the non-thermophilic Crenarchaeota. Analyzing probes from the ARB probe library v.1.2 [33], however, reveals that at least some of the suggested Sulfolobales probes target most SCA clones with only one mismatch (analysed using ‘‘Probe Match’’ [17]). To our knowledge there are so far no published results indicating any recognizable morphology of low temperature Crenarchaeal organisms that separates these organisms from known thermophilic and/or hyperthermophilic Archaea. To search for this morphology we designed a probe to target only the Archaea found in temperate environments (SoilCren750r). The probe was validated using probe Match from RDPII [17], and tested for specificity using this probe as reverse primer, resulting in products from cloned fragments. Unfortunately this probe did not yield any signal from the Pisciarelli sample after several FISH probing attempts. Since no cultivated controls are available from this group of organisms, it is impossible to evaluate whether the lack of signal was due to suboptimal fixation processes or hybridization conditions. The data presented in this study suggests that the non-thermophilic Crenarchaeota group is considerably more ecologically diverse, than previously anticipated. Similar to e.g., methanogenic Euryarchaeota the nonthermophilic Crenarchaeota appear to harbour as well true non-thermophiles as organisms capable of tolerating thermophilic conditions. A further elucidation of the functions and capabilities of the non-thermophilic Crenarchaeota awaits the isolation or genome analysis of a representative from this group of organisms.
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Acknowledgements
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This work was performed within the Danish Archaea Centre supported by a grant from the Danish Natural Science Research Council (SNF). We thank Anders Prieme´ for his excellent assistance in performing the tRFLP analysis and Thomas Ishøy and Slawomir Dabrowski for their scientific and technical assistance throughout the work and preparation of this paper.
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