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The humus samples were taken from a mixed forest in Northern Finland (65° 15´ N, 28° 50´ E) with a podzol on moraine soil type. The sampling area was covered with Norway Spruce and Scots Pine and consisted of two treatments: clear-cutting (A) and clear-cutting followed by prescribed burning two years later (B). The samples collected one year after the prescribed burning included a control soil (C) from an untreated standing forest. Each of the three bulk “composite samples” consisted of a mixture of twenty subsamples (twenty individual cores sampled with a 72-mm-diameter soil corer) (Pietikainen and Fritze, 1995). The composite samples were collected from the different sites on the same day. The area of each site was 50 m x 50 m, and the minimal distance between any of the 20 subsamples was 2 m. Crude DNA was isolated from a soil sample using proteinase K, cetyltrimethyl ammonium bromide and chloroform, and then applied to a Wizard DNA Clean-Up System Minicolumn (Promega, USA) with isopropanol purification (Saano et al., 1995).
Lake water was taken from mesohumic boreal lake Valkea Kotinen which is located in the Evo forest area of southern Finland (61°24` N, 24°07` E). Water was sampled using a 2.5 liter Limnos sampler from the surface (0.1 m) down to the deepest water layers (7 m) at 0.5 m intervals from the pelagial.
For microbial DNA extraction, integrated water samples (0-7 m) were taken from the pelagial in July and stored in pre-cleaned, sterile Nalgene bottles. The samples were then kept on crushed ice and in the dark prior to filtration. Cells from 250 ml of water were collected by filtration onto hydrophilic polycarbonate filters (2-µm pore size, 47 mm diameter (Whatman, UK)) and frozen immediately at -25°C until nucleic acids extraction.
Extraction began with the addition of 5 ml of lysis buffer (1 mg/ml lysozyme, 40 mM EDTA, 50 mM TrisHCl, 0,75 M sucrose, pH 8.0) and incubation at 37°C for 30 min (Massana et al., 1997). Proteinase K (0.5 mg/ml) and sodium dodecyl sulfate (1% w/v) were then added to the filter and incubated at 55°C for 2 h. The lysate was recovered from the filter and placed into a fresh tube. The filter was then rinsed with an additional 2 ml lysis buffer and incubated at 55°C for 10 min before pooling the lysates. To the pooled lysate, 5 M NaCl (final concentration 0.7 M) and hexadecyltrimethyl ammonium bromide (final concentration 1% w/v in the presence of 0.7M NaCl) were added. This mixture was incubated at 65°C for 20 min before extraction with chloroform-isoamyl alcohol (24:1). The upper aqueous-DNA phase was removed and placed into a fresh tube and DNA precipitated after the addition of 0.6 volumes of isopropanol. The pellet was washed with 70% (v/v) ethanol, dried and dissolved in 100 µl of water. This crude DNA sample was then further purified using a Wizard DNA clean up kit (Promega, USA) (Saano et al., 1995) before use as template DNA for PCR.
Sediment cores of 25 cm (length) by 5 cm (diameter) were collected at several sampling sites along the intertidal bank of the river Douro (Portugal) estuary (41º 08' N, 08º 40' W), where the salinity ranged from 1 to 5 practical salinity units. They were composed mainly of coarse sand (dominant particle size > 500 µm). One of the cores showed a clearly visible 2 cm-thick black layer of thin, compact sediment 10 cm from the surface, and a 3 cm-thick orange layer 19 cm from the surface. The cores were kept in the dark at 4ºC and processed within 1 hour. Core slice samples (4-5 mm) were taken at different depths of each core, including the top aerobic region of all samples and the 10 cm and 19 cm layers of the above mentioned core.
The samples were suspended in 4 ml SET-buffer (20% sucrose, 50 mM EDTA, 50 mM Tris-HCl pH 8.0) before the addition of 150 ml lysozyme (5 mg/ml SET), 400 ml SDS (10% w/v) and 100 ml proteinase K (20 mg/ml in SET-buffer). The samples were incubated at 60ºC for 30 min before centrifugation at 10,000 x g for 10 min. The supernatant fluids were then extracted twice with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). To precipitate the DNA, 0.1 volumes of 3 mol/l sodium acetate (pH 5.2) and 2 volumes of 100% ethanol were added. The DNA was collected by centrifugation (14,000 x g, 10 min), washed in 70% ethanol, dried and resuspended in sterile distilled water.
Fragments of 16S rRNA gene (positions 7-927, Escherichia coli numbering) were amplified using a nested PCR approach with primers described in Table 3. First round PCR was performed using two sets of primers: (i) Ar4F - Un1492R and (ii) Ar4F - Ar958R, producing amplicons approximately 1500 and 1000 bp in length, respectively. Both sets of first round PCR products were then used as template in a nested PCR using primer set Ar3F - Ar9R. These Archaea-specific primers hybridized at positions internal to those targeted by primers used in both first round PCR reactions, and generated fragments approximately 900 bp in length. DNA extracted from soil samples was amplified using a single round of PCR with the "internal" primer set Ar3F - Ar9R primers.
Reaction mixtures of 20 ml contained 1-5 ng of DNA template and 100 pmol of each primer. PCR was carried out using the following reaction conditions:
for soil and water samples: 94°C for 4 min; 40 cycles of 94°C for 1 min, 55°C for 1 min, 73°C for 3 min.
for sediment samples: 94ºC for 4 min, 42 cycles of 94ºC for 1 min, 45ºC for 1 min, and 73ºC for 2 min.
PCR products were only accepted for further analysis when a simultaneous negative control PCR (water instead of DNA) showed no amplification. PCR products were subject to agarose gel electrophoresis, and DNA of appropriate size was extracted from the gel and cloned into pGEM-T vector plasmid (Promega, USA). The archaeal origin of the inserts was checked by Southern blot hybridization using an Archaea-specific probe (the corresponding region of 16S rRNA from Halobacterium salinarum DSM 668 without primers). For hybridization conditions, see paper II. The inserts of the clones (74 clones, paper I; 138 clones, paper II; 235 clones, paper III; 160 clones, paper IV) were divided into groups according RFLP patterns using: AvaII (paper III); AvaII and MspI (paper IV); AvaII, MspI and RsaI (papers I and II) restriction enzymes.
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Several clones from each RFLP group (or just one when represented by a single clone) were chosen for sequence analysis. Altogether, the following were sequenced:
from soil samples - 15 clones (accession numbers X96688 - X96696, Y08984, Y08985, and AJ006919 - AJ006922);
from water samples - 28 clones (accession numbers AJ131263 - AJ131278 and AJ131311 - AJ131322);
from sediment samples - 14 clones (accession numbers AF201355 - AF201368).
Samples for fluorescent in situ hybridization were collected during a period from March to September from three depths: the epilimnion (0-1 m depth), the metalimnion (1-3 m depth) and the hypolimnion (3-7 m depth, near the bottom sediment). Water samples were taken at 0.5 m intervals with a one-liter Limnos sampler in the pelagial of the lake at the deepest point. Replicate lake water samples were taken from the epi-, meta- and hypolimnetic strata, pooled and stored in sterile 5 liter Nalgene bottles. Samples were kept on crushed ice and in the dark during transportation to the laboratory. Within 2-3 hours after sampling, the microbial cells were concentrated from 15-20 ml of water by filtration onto polycarbonate filters (0.2 µm pore size, 47 mm diameter) (Whatman, UK) by vacuum filtration at < 100 P m-2. The filters were prepared and fixed for FISH as described previously (Amann, 1996; Glöckner et al., 1996) and stored at -25°C until further processing.
Fluorescent in situ hybridization followed the protocol of Amann, 1996 and Glöckner et al., 1996. Oligonucleotides labeled with the dye carbocyanine CY3 were purchased from INTERACTIVA (Germany). The specificity, nucleotide sequence and respective hybridization buffer formamide concentration for each probe is summarized in Table 3.
To detect Archaea in the samples, previously described 16S rRNA gene probes EURY498, CREN499 (Burggraf et al., 1994) and ARCH915 (Stahl and Amann, 1991) were used with new probes designed using the ARB "Probe_design" program (Strunk and Ludwig, 2000). In addition to the EURY498, three new probes for the Euryarchaeota (EURY514, EURY496 and EURY499) were designed taking into account new Archaea sequences including the VAL clones (paper IV). New probes CREN512 and EURY514 targeted sequences at the kingdom level and were specific to the Cren- and Euryarchaeota, respectively. New probe CREN569 was specific to most environmental sequences of Crenarchaeota. The optimal formamide concentration for hybridization with new probes CREN512, EURY499 and EURY514 were determined using pure cultures of Crenarchaeota (Pyrobaculum neutrophilum, Sulphobococcus zilligii, Thermoproteus tenax) and Euryarchaeota (Methanosarcina barkery, Methanobacterium formicium). Pure cultures of Bacteria (Escherichia coli and Sinorhizobium) were used as negative controls for all Archaea probes. Probe-specific cell counts were calculated taking into consideration counts obtained with negative control NON338 (Wallner et al., 1993).
For hybridization, a section of the filter was placed on a glass slide, overlaid with 20 ml of hybridization solution (0.9 M NaCl, 20 mM Tris/HCl (pH 7.4), 0.01% SDS w/v, 0-20% (v/v) formamide (Table 3) and 50 ng of probe) and incubated at 46°C for 2 h in an equilibrated humid chamber (Glöckner et al., 1996). Optimal formamide concentrations for the new probes were determined empirically using a formamide series from 0% to 50% (v/v) at 10% intervals. The filters were transferred to a pre-warmed (48°C) vial containing 50 ml of washing solution (70 mM NaCl, 20 mM Tris/HCl (pH 7.4), 5 mM EDTA, and 0.01% SDS (w/v)) and incubated floating freely at 48°C for 15 min. Filters were dried on filter paper (Whatman, UK) and overlaid with 50 ml of DAPI solution (1 µg/ml in water) for 5-10 min at room temperature in the dark. The filters were briefly washed in 70% (v/v) ethanol and then in distilled water, and dried on filter paper before mounting on slides with Citifluor AF1 (Citifluor Ltd., Canterbury, UK). Epifluorescence microscopy was performed as described previously (Glöckner et al., 1996) using a Zeiss Axioplan microscope equipped with specific filter sets (DAPI: Zeiss01 and CY3: Chroma HQ41007, Chroma Tech. Corp., Brattleboro, VT, USA).
Programs CHECK_CHIMERA, RANK_SIMILARITY and SUGGEST_TREE from the RDP (Ribosomal Database Project) (Maidak et al., 2001; Maidak et al., 1996) were applied to the studied sequences in order to detect possible chimeric artifacts, to find the most similar sequences from the RDP database, as well as place sequences on an existing phylogenetic RDP tree.
In the case of the FFSB soil sequences (paper I), reference 16S rRNA sequences were retrieved from GenEMBL and RDP databases and aligned manually according to their primary and secondary structures using Gelassembler sequencing editor (WISCONSIN PACKAGE, Version 8.1) (Wisconsin Package Software, 1994). Sites of uncertain alignment were excluded from the analysis. Three methods of phylogenetic analysis from PHYLIP package version 3.5c (Felsenstein, 1993) were used; neighbor-joining (using distances calculated with Jukes and Cantor correction), parsimony and maximum likelihood. Bootstrap support (Felsenstein, 1985) (Phylip package) was calculated to check the topology perturbation in the neighbor-joining and parsimony methods.
In the case of all water, sediment and all other soil sequences (paper II) phylogenetic analysis was performed using the package ARB (Strunk and Ludwig, 2000). Only sequences with sufficient length for phylogeny (min. 400 nucleotides) were included in the analysis. The ARB_EDIT tool was used first for automatic sequence alignment. The sequence alignment were then corrected manually. Regions of ambiguous alignment were excluded from the analysis. A 50% invariance criterion for the inclusion of individual nucleotide sequence position in the analysis was used to avoid possible treeing artifacts. During tree construction, several trees differing in the set of alignment positions as well as reference sequences were used. Phylogenetic trees were inferred by performing maximum likelihood (fastDNAml) (Olsen et al., 1994), maximum parsimony (Felsenstein, 1993) and neighbor-joining (with Jukes-Cantor distance correction (Jukes and Cantor, 1969)) analysis included in the ARB package.
