Identification of active methylotroph populations in an acidic forest soil by stable-isotope probingc

Stefan Radajewski1, Gordon Webstera,2, David S. Reayb,3, Samantha A. Morris1, Philip Ineson4, David B. Nedwell3, James I. Prosser2 and J. Colin Murrell1

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK1
Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK2
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK3
Department of Biology, University of York, PO Box 373, YO10 5YW, UK4

Author for correspondence: J. Colin Murrell. Tel: +44 24 7652 2553. Fax: +44 24 7652 3568. e-mail: cmurrell{at}bio.warwick.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3·5) microcosms that were exposed to 13CH3OH or 13CH4. Distinct 13C-labelled DNA (13C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the 13C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the ß-subclass of the Proteobacteria. Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the 13C-DNA fractions, suggesting certain AOB assimilated a significant proportion of 13CO2, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the 13C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the ß-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the 13CH3OH and 13CH4 SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Keywords: methanotrophs, community structure, culture-independent techniques, 13C, functional genes

Abbreviations: AOB, ammonia-oxidizing bacteria; DGGE, denaturing gradient gel electrophoresis; g.d.w.,g dry weight; OTU, operational taxonomic unit; SIP, stable-isotope probing

c The GenBank accession numbers for the sequences reported in this paper are AY080911AY080961.

a Present address: Cardiff School of Biosciences, Cardiff University, Main Building, Cardiff CF10 3TL, UK.

b Present address: Institute of Ecology and Resource Management, University of Edinburgh, Darwin Building, Edinburgh EH9 3JU, UK.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Methylotrophs are micro-organisms that can use reduced one-carbon compounds as a sole source of carbon and energy (Lidstrom, 1992 ). Two important methylotrophic substrates in the terrestrial environment are methanol (CH3OH) and methane (CH4). CH3OH is produced during the decomposition of plant polymers and is probably excreted from living leaves and by CH4-oxidizing bacteria (Anthony, 1982 ), whereas CH4 is mainly produced by methanogenic Archaea in anoxic environments. Methylotrophs that grow on CH3OH and CH4 have been isolated from many environments and the characterization of pure cultures has provided information on the taxonomy, physiology, genetics and ecology of these physiologically specialized micro-organisms.

Most aerobic methylotrophs that are in laboratory culture belong to the class Proteobacteria. All of these methylotrophs use the enzyme methanol dehydrogenase to oxidize CH3OH to formaldehyde, which is the central intermediate of one-carbon metabolism. Formaldehyde can subsequently be assimilated into cell carbon (Lidstrom, 1992 ) or can be oxidized further to formate and CO2 to gain energy and to regenerate reducing equivalents (reviewed by Vorholt et al., 1999 ). CH4-oxidizing bacteria (methanotrophs) are a subgroup of methylotrophs that have been divided into types I and II ({gamma}- and {alpha}-Proteobacteria, respectively) based on phenotypic and phylogenetic characteristics (Hanson & Hanson, 1996 ). Methanotrophs oxidize CH4 to CH3OH using the enzyme methane monooxygenase, which occurs in two distinct forms that have been reviewed in detail by Murrell et al. (2000b) . A particulate, membrane-bound methane monooxygenase has been reported in all methanotrophs except Methylocella palustris (Dedysh et al., 2000 ), whereas only certain strains contain a soluble, cytoplasmic methane monooxygenase. The particulate methane monooxygenase has several similarities to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB), and it has been suggested that these two enzymes may be evolutionarily related (Holmes et al., 1995 ). Although some AOB can oxidize CH4, none are known to use it as an energy source (Bédard & Knowles, 1989 ).

Functional genes encoding key enzymes in the methylotrophic pathways of several Proteobacteria have been identified. The large subunit of methanol dehydrogenase, which contains the active site of the enzyme, is encoded by mxaF (Lidstrom et al., 1994 ; Nunn & Lidstrom, 1986 ). The putative active site subunit of particulate methane monooxygenase is encoded by pmoA, with the homologous subunit in ammonia monooxygenase encoded by amoA (reviewed by Murrell et al., 2000a ). Conserved domains within the amino-acid sequences have enabled the design of PCR primers that specifically amplify methane monooxygenase or methanol dehydrogenase genes from methylotroph, enrichment culture and environmental DNA samples (Dedysh et al., 2002 ; Dunfield et al., 1999 ; Henckel et al., 1999 ; Holmes et al., 1999 ; McDonald & Murrell, 1997 ). In cultivated strains, the phylogenetic relationships between the derived amino-acid sequences of pmoA and amoA, and to a lesser extent mxaF, reflect those obtained with 16S rDNA sequences (Holmes et al., 1995 ; McDonald & Murrell, 1997 ). Therefore, in many cases, the identity of methylotrophs can be inferred from pmoA and mxaF sequences retrieved from environmental DNA samples.

Difficulties associated with the isolation of the vast majority of micro-organisms into laboratory culture have hampered the identification of microbial populations involved in a specific process (Gray & Head, 2001 ; Hugenholtz et al., 1998 ). Several recently described techniques can, however, establish this link between identity and function under conditions approaching those in situ and without prior knowledge of the micro-organisms involved (Boschker et al., 1998 ; Lee et al., 1999 ; Orphan et al., 2001 ; Ouverney & Fuhrman, 1999 ; Radajewski et al., 2000 ). Certain phospholipid-fatty-acid- and DNA-labelling techniques have been developed using one-carbon substrates enriched with radioactive-carbon (14C) or stable-carbon (13C) isotopes; these have been subsequently applied to identify the active methylotroph communities in environmental samples directly (Boschker et al., 1998 ; Radajewski et al., 2000 ; Roslev & Iversen, 1999 ).

In a preliminary report, we described the development and application of stable-isotope probing (SIP) (Radajewski et al., 2000 ). This technique exploits the fact that certain stable isotopes (e.g. 13C and 15N) occur at low abundance in nature but can be highly enriched (i.e. >99%) in commercially available compounds. As demonstrated by Meselson & Stahl (1958) with Escherichia coli grown on , DNA synthesized during microbial growth on a substrate enriched with a heavy, stable isotope becomes labelled sufficiently to be resolved from unlabelled DNA by density-gradient centrifugation. This principle has been demonstrated with methylotroph and AOB cultures grown on 13CH3OH (Radajewski et al., 2000 ) and 13CO2 (Whitby et al., 2001 ) as a carbon source, respectively. Although the buoyant density of DNA varies with its G+C content, the incorporation of a high proportion of a naturally rare, stable isotope (2H, 15N or 13C) into DNA enhances greatly the density difference between labelled and unlabelled DNA fractions (Rolfe & Meselson, 1959 ; Vinograd, 1963 ). Indeed, SIP has been applied successfully to identify active CH3OH utilizers in an oak forest soil (Radajewski et al., 2000 ). Isolation of the 13C-labelled DNA fraction simultaneously established the function and activity of the micro-organisms and sequence analysis subsequently determined their identity. The aim of the present study was to characterize the active aerobic methylotroph populations in an acidic oak forest soil exposed to 13CH3OH or 13CH4 and to isolate certain methylotrophs detected in our preliminary study using 13CH3OH.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Soil and microcosm experimental conditions.
Microcosm experiments were used to characterize the active methylotroph communities in an oak (Quercus petraea) forest soil (0–5 cm depth sample; pH 3·5) collected from the Gisburn Forest Experiment, which was planted in 1955 on former sheep grazings (Brown & Iles, 1991 ). Microcosms consisted of a soil sample (see below) in a glass serum vial (125 ml) that was crimp-sealed with a butyl rubber stopper and incubated in the dark at 22–25 °C. Each experiment used two microcosms: one incubated with 12C-labelled substrate and the other incubated with 13C-labelled substrate. The CH3OH experiments used 12CH3OH (99·9% pure; BDH) or 13CH3OH (99·4% pure, 99·2% 13C; Isotec) and the CH4 experiments used 12CH4 (98% pure; Linde Gas) or 13CH4 (99% pure, 99% 13C; Linde Gas). The disappearance of CH3OH and CH4 was monitored at 2–7 day intervals using a GC apparatus (Pye Unicam GCD 3500) equipped with a Poropak Q column (1 mx4 mm i.d.) and a flame-ionization detector.

The first SIP experiment, which has been described in a preliminary report (Radajewski et al., 2000 ), used soil collected in November 1998 to identify the active CH3OH-assimilating methylotrophs. In brief, the microcosms contained 10 g of soil and were incubated with a starting CH3OH concentration of 0·5% (v/w). Twice during each incubation the microcosms were flushed with air (250 ml), using a 50 ml syringe to ensure that they remained aerobic. After two additions of CH3OH [day 1 and day 18 (13CH3OH) or day 24 (12CH3OH)] a total of 2·4 mmol CH3OH had been completely oxidized (44 days).

The active CH4-assimilating methylotrophs were characterized in three separate experiments using soil collected in November 1998 or January 1999. All soils were dried to a water content that was optimal for CH4 oxidation (40–50% water-holding capacity) using a pressure-plate apparatus (Reay et al., 2001b ). The dried soils were stored at 4 °C prior to use. In the first two CH4 SIP experiments, soil samples (10 g) were added to the serum vials and CH4 [0·2 mmol (5 ml), November 1998 soil; 0·4 mmol (10 ml), January 1999 soil] was injected into the headspace of the microcosms. After >95% of the initial CH4 concentration had been oxidized the vials were opened, flushed with air (500 ml) to remove any accumulated CO2, resealed and the initial CH4 concentration was re-established. The incubation was continued in the same manner until approximately 2 mmol of CH4 had been oxidized [10x0·2 mmol CH4 (69 days) or 5x0·4 mmol CH4 (54 days)]. Despite CH4 oxidation, no 13C-labelled fraction was observed following ultracentrifugation of DNA extracted from 10 g of soil from either of the 13CH4 microcosms. It was hypothesized that nutrient limitation (possibly N or P) may have prevented 13C assimilation into DNA, possibly via the uncoupling of CH4 oxidation from cell growth and/or DNA replication (Amaral & Knowles, 1995 ) or due to the assimilation of 13C into a storage product (e.g. poly-3-hydroxybutyrate; Wendlandt et al., 2001 ). Therefore, a third CH4 SIP experiment was established with slurry microcosms that consisted of soil (4 g, January 1999), dilute ammonia/nitrate mineral salts (ANMS) medium (10 ml, 0·1xANMS; Whittenbury et al., 1970 ) containing (0·9 mM), (0·5 mM), phosphate (0·4 mM) (pH 5·5) and CH4 (0·4 mmol). After repeated CH4 additions (5x0·4 mmol, as described earlier) and incubation with shaking at 80 r.p.m. for 76 days, approximately 2 mmol CH4 had been oxidized.

CH4 oxidation potentials were determined for each soil as described previously (Reay et al., 2001a ). Ammonium oxidation potentials were determined from the ratio of present in the soils using the same incubation conditions as described previously (Reay et al., 2001a ), except that 5 mM 15NH4Cl (Sigma) was supplied as a 10% solution. Nitrate was extracted from the soil samples by shaking them with 500 ml ultrahigh-purity water. The sample was then bound to an anion exchange resin (Amberlite IRA–400, 16–50 mesh; Sigma) and stored at 4 °C until analysed. Nitrate was eluted from the resin by an overnight incubation with KCl (0·80 M, 25 ml). It was then concentrated by steam distillation (Bremner & Keeney, 1966 ) and evaporated to dryness. The residue was redissolved in ultrahigh-purity water (50 µl), transferred to a 6x4 mm tin capsule containing 5 mg of a nitrogen-free electrofocussing agent (Ultrodex, Pharmacia Biotech) and dried at 75 °C. The 14N/15N ratio was determined with an elemental analyser-isotope ratio mass spectrometer (Barrie & Prosser, 1996 ).

DNA extraction and ultracentrifugation.
DNA was extracted from soil by bead beating. Soil from the CH3OH microcosms (1 g from 13C; 0·3 g from 12C) was processed in 0·3 g samples using a DNA extraction kit (FastPrep; Bio 101) and a Ribolyser cell disrupter (Hybaid), as described previously (Yeates & Gillings, 1998 ). For the CH4 microcosms (10 g soil or 10 ml slurry from 13C; 3 g soil or 3 ml slurry from 12C), this method was adapted to process larger samples (3 g soil or 3 ml slurry) with a CO2-cooled bead beater (Braun), as described by Morris et al. (2002) . CsCl was added at 1 g (ml DNA solution)-1. Ethidium bromide (100 µl, 10 mg ml-1) was added to the DNA solution, which was then transferred to a polyallomer ultracentrifuge tube (13x51 mm; Beckman). DNA fractions were resolved following centrifugation at 265000 g (55000 r.p.m. using a Beckman VTi 65 rotor) for 12–16 h at 20 °C in a CsCl density gradient (Sambrook et al., 1989 ) and visualized with UV light (365 nm).

DNA from each 12C-microcosm (12C-DNA) and the most dense fraction (approx. 0·5 ml) from each 13C-microcosm (13C-DNA) was withdrawn gently from the primary gradients (Fig. 1) using a 1 ml syringe and hypodermic needle (19 gauge). Care was taken during collection of the 13C-DNA fraction to avoid co-extraction of the 12C-DNA band. The 13C-DNA fraction was added to a new CsCl/ethidium bromide gradient and centrifuged as described for the primary gradients. No 12C-DNA fractions were observed in either of the secondary purification gradients (data not shown). The 13C-DNA fraction was re-collected in the minimum volume possible, which further removed any small amounts of 12C-DNA that may have been co-extracted during the primary collection, thereby increasing the isotopic purity of the DNA from the active methylotrophs. Ethidium bromide was extracted from the DNA with an equal volume of 1-butanol saturated with TE (10 mM Tris, 1 mM EDTA; pH 8·0) buffer. Following three extractions, the DNA was dialysed against a large volume of TE buffer, precipitated with ethanol overnight at -20 °C (Sambrook et al., 1989 ) and dissolved in 100 µl TE buffer.



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Fig. 1. Equilibrium centrifugation of DNA extracted from soil that oxidized 12C- or 13C-labelled (a) CH3OH or (b) CH4. CsCl/ethidium bromide density gradients were centrifuged at 265000 g for 12–16 h at 20 °C and visualized with UV light. The position of the 13C-DNA fraction collected for analysis is indicated by an arrow. Bar, 1 cm.

 
Characterization of the 12C- and 13C-DNA fractions.
The 12C- and 13C-DNA fractions were used as templates for PCR. Primers specific for bacterial (Eubac27F/1492R), archaeal (Arch21F/1492R) and eukaryotic (EukF/EukR) small-subunit rRNA genes (1450–1500 bp product) were used to characterize the active population at the domain level (DeLong, 1992 ). The functional genes mxaF and pmoA were also targeted using primers mxa f1003/mxa r1561 (approx. 555 bp product; McDonald & Murrell, 1997 ) and A189/A682 (approx. 525 bp product; Holmes et al., 1995 ), respectively. Reactions (50 µl) contained 1 µl of template (approx. 5–50 ng) and were performed as described previously for each primer set.

Amplification products of the correct size were cloned using the TOPO TA cloning kit (Invitrogen) and libraries of 100 clones (bacterial 16S rDNA) or 50 clones (mxaF and pmoA) were constructed. Plasmid inserts were screened by digestion with restriction endonucleases: EcoRI/RsaI for 16S rDNA from the 13CH3OH microcosm; EcoRI/RsaI and EcoRI/Sau3AI for 16S rDNA from the 13CH4 microcosm; EcoRI/HincII for mxaF; and EcoRI/RsaI and EcoRI/HincII/PvuII for pmoA. DNA fragments were resolved by electrophoresis through a 2% agarose gel and each clone was assigned to an operational taxonomic unit (OTU) that represented a unique RFLP. Complete sequence data (between the amplification primers) were obtained for one clone from each OTU by automated sequencing (model ABI 377, PE Biosystems). At least 10% of clones within each OTU were partially sequenced (>500 bp), to verify that the restriction pattern represented a single sequence type.

Targeted enrichment of methylotrophs.
In an attempt to isolate the active proteobacterial methylotrophs from the CH3OH experiment (represented by OTUs UP1–UP3), an enrichment strategy was formulated from the physiology of closely related strains [Methylocella palustris (Dedysh et al., 2000 ) and Beijerinckia indica (Becking, 1984 )] and the incubation conditions used for the microcosm. Basal medium M1 (Dedysh et al., 1998 ) was used with CH3OH (0·2%, v/v) as the sole carbon source. KNO3 was omitted from some media (M1-N), with N2 as the only nitrogen source. The initial pH of the medium was adjusted to 5·0 with phosphoric acid. Soil (5 g, November 1998) was added to 0·5 mM potassium phosphate buffer (5 ml, pH 6·8) and the sample was gently agitated on a wrist-action shaker (Stuart Scientific) for 2 h at 4  °C. The slurry (5 µl) was added to 10 ml of medium in a glass serum vial (125 ml), which was crimp-sealed with a butyl rubber stopper and incubated (20–25 °C) with shaking (80 r.p.m.). Enrichments were subcultured twice at 4–10 week intervals into the same medium, which was adjusted to pH 5·0 or pH 3·5.

DNA was extracted from 1·5 ml of each enrichment (Marmur, 1961 ) and used as a template for PCR with the bacterial 16S rDNA, mxaF and pmoA primers. PCR products were digested with the same restriction endonucleases as used previously for RFLP analyses, and patterns similar to those of the 16S rDNA and mxaF clones from the 13CH3OH SIP experiment were identified. A library of 15 clones was constructed for each PCR product from one tertiary enrichment (M1-N; pH 3·5) and analysed as described for the CH3OH SIP experiment. Complete mxaF and partial 16S rDNA sequence data (positions 375–1060; E. coli numbering) were obtained for one clone from each OTU.

Neither B. indica nor Acidobacterium capsulatum has been reported to grow on CH3OH (Becking, 1984 ; Kishimoto et al., 1991 ). However, as these bacteria were closely related to organisms detected in the 13CH3OH microcosm, B. indica subsp. indica (NCIMB 8712) and A. capsulatum (DSM 11244) were incubated at 30 °C using the respective basal medium recommended by the DSM (glucose omitted) with CH3OH (12·5 mM) as the sole carbon source. DSM, Deutsche Sammlung von Mikroorganismen; NCIMB, National Collection of Industrial, Food and Marine Bacteria.

AOB-specific analysis of DNA fractions.
As amoA sequences were detected in 13C-DNA fractions from both the 13CH3OH and 13CH4 microcosms, a more detailed analysis of these DNA fractions was undertaken using primers selective for AOB belonging to the ß-subclass of the Proteobacteria. PCR primers selective for 16S rDNA, ßamof/ßamor (McCaig et al., 1994 ) and CTO189fGC/CTO654r (Kowalchuk et al., 1997 ), were used in a nested PCR approach that yielded a 465 bp product (Webster et al., 2002 ). Primers specific for the functional amoA gene, amoA-1F/amoA-2R (approx. 490 bp product), were also used (Rotthauwe et al., 1997 ). Each microcosm DNA sample was amplified in duplicate. Ammonia oxidizer 16S rDNA PCR products were analysed by denaturing gradient gel electrophoresis (DGGE) as described previously (Webster et al., 2002 ) and the major bands were sequenced to confirm identity. Representative clones or pure cultures for each 16S rDNA cluster of ß-subgroup AOB, as designated by Stephen et al. (1996) , were included as cluster controls for the 16S rRNA gene DGGE analysis. Bands were excised, resuspended in sterile distilled water (20 µl) and re-amplified using 1 µl as template. Re-amplified PCR products were purified with a centrifugal filter (Microcon YM–100; Millipore) and resuspended in sterile distilled water (20 µl) at a final DNA concentration of 15–20 ng µl-1. Sequencing reactions were performed with primer CTO189f.

Phylogenetic analysis.
The ARB program (http://www.mikro.biologie.tu-muenchen.de) was used for sequence alignments and phylogenetic analyses. All 16S rDNA sequences were aligned using the automatic alignment tool of ARB (ALIGNER, version 2.0), and the alignments were corrected manually according to the constraints imposed by the secondary structure of 16S rRNA. To identify close relatives of the clone sequences in the ARB database, all aligned sequences were added to a tree of sequences (>1400 bp) by using a maximum-parsimony tool within ARB. Analyses used the sequences of several relatives identified in ARB and BLAST (Altschul et al., 1990 ) searches of GenBank, as well as near full-length sequences (>1350 bp) of extant methylotrophs, methanotrophs and AOB in GenBank. A filter was generated that omitted alignment positions of sequence ambiguity (N) and where sequence data were not available for all near full-length sequences.

To evaluate the tree topology, phylogenies were reconstructed with various data subsets using evolutionary distance (Jukes and Cantor model), maximum-parsimony (ARB and DNAPARS) and maximum-likelihood (default parameters for ARB and FASTDNAML) analyses of the aligned near full-length (>1350 bp) sequences (Ludwig et al., 1998 ). Aligned partial 16S rDNA sequences from the CH3OH enrichments and the AOB-specific analyses, and selected sequences from GenBank (Z73369, Z88588, AF145871 and AF358018) were inserted into each tree using a parsimony tool available within ARB and the filter generated with complete sequence data. As topologies obtained from the different analyses were generally similar, the trees presented are based on the maximum-likelihood analysis, with the confidence of branch points determined using a strict consensus rule applied to the results of the three analysis methods. Multifurcations indicate points at which the branching order was collapsed until it was supported by all three methods of analysis.

Functional-gene sequences (pmoA and mxaF) were aligned manually to related sequences extracted from GenBank. A filter was generated from the derived amino-acid sequences that excluded alignment positions containing amino-acid ambiguity (X) and missing sequence data. Phylogenies were reconstructed with evolutionary distance (Dayhoff PAM model), maximum-parsimony (ARB and PROTPARS) and maximum-likelihood (default parameters for ARB and Protein_ML) analyses, and tree topologies were evaluated as described for the 16S rDNA analysis.

Environmental 16S rDNA sequences were inspected for potential chimeras using the program CHIMERA CHECK (version 2.7; http://rdp.cme.msu.edu). Functional-gene DNA sequences were inspected for chimeras by searching for large regions of unexpected nucleotide changes when compared with sequences from reference organisms. One potential 16S rDNA chimera was identified from the CH3OH enrichment and it was not included in the analyses. It was also necessary to add a single nucleotide ambiguity (N) to the DNA sequence of the functional-gene clones P12.7, P12.9, P13.7 and M12.1 to maintain the correct reading frame for each derived amino-acid sequence. Identity between sequences was determined using the similarity matrix option available within ARB.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Soil microcosms and CsCl density gradients
Low-affinity (5%, v/v) CH4 oxidation potentials of 0·10 and 0·44 µmol (g.d.w. soil)-1 h-1 were observed for the soils collected in November 1998 and January 1999, respectively. Ammonium oxidation potentials for the soil samples were 3·88 and 4·15 nmol (g.d.w. soil)-1 h-1, respectively.

As reported previously (Radajewski et al., 2000 ), a 13C-DNA fraction was extracted from 1 g of soil from the microcosm (10 g soil) that had oxidized 2·4 mmol of 13CH3OH (Fig. 1a). However, no 13C-DNA band could be observed from 10 g of soil from two independent microcosms that had oxidized approximately 2·0 mmol of 13CH4. Consequently, the CH4 slurry experiment was established, in which a distinct and intense 13C-DNA fraction was observed (Fig. 1b). The DNA fractions analysed were collected from the soil microcosms exposed to CH3OH and the soil slurry microcosms exposed to CH4.

Identity of methylotrophs in the 13C-DNA
Using 12C-DNA from the 12CH3OH or 12CH4 microcosm as a template, specific amplification products were obtained with PCR primers that targeted the small-subunit rRNA genes of Bacteria, Archaea and Eukarya. However, when 13C-DNA from the 13CH3OH or 13CH4 microcosm was used as a template, an amplification product was only obtained with the primer set that was specific for Bacteria, indicating that the 13C-DNA fractions contained a restricted microbial community.

RFLP analysis of 100 16S rDNA clones from each 13C-DNA fraction identified six OTUs in the 13CH3OH library and eight OTUs in the 13CH4 library. Phylogenetic analysis of the 16S rDNA sequences from the 13CH3OH library identified three OTUs (UP1–UP3) that clustered with members of the {alpha}-subclass of the Proteobacteria (Fig. 2). A further three OTUs (UA1–UA3; each consisting of one clone) were most closely related (95·6–98·0% identity) to 16S rDNA sequences of members of the Acidobacterium division. The 16S rDNA sequence of UP1 (49 clones) was most closely related to that of UP8 (99·5% identity) from the CH4 library. The sequence of UP2 (47 clones) was 97·4% similar to the 16S rDNA sequence of Rhodoblastus acidophilus (formerly Rhodopseudomonas acidophila; Imhoff, 2001 ). The sequence of UP3 (one clone) was most similar to the 16S rDNA sequences of B. indica, Methylocella palustris, Methylocapsa acidiphila and clone UP1 (95·6–97·0% identity). Phylogenetic analysis of the 16S rDNA sequences from the 13CH4 library identified seven OTUs (UP4–UP9) that clustered in the {alpha}- and ß-subclasses of the Proteobacteria and one OTU (UC1; one clone) that clustered with the order Cytophagales (Fig. 2). The sequences of UP4 (92 clones), UP5 (two clones), UP6 and UP7 (each one clone) had 99·4–99·7 % identity with each other and 96·1–97·4% identity with 16S rDNA sequences from the methanotrophs Methylosinus trichosporium and Methylocystis parvus. The sequence of UP8 (two clones) was most closely related to those of UP1 and UP3 and to the 16S rDNA sequences of Methylocella palustris and Methylocapsa acidiphila. The sequence of UP9 (one clone) was most closely related to that of clone LO13.5 (96·8% identity) and to the 16S rDNA sequences of Herbaspirillum seropedicae, Janthinobacterium lividum and Paucimonas lemoignei (95·7–96·0% identity). The sequence of UC1 exhibited most similarity to that of clone LO13.4 (99·6% identity).



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Fig. 2. Maximum-likelihood tree of 16S rDNA sequences amplified from the 13C-DNA fraction of microcosms exposed to 13CH3OH ({circ}) or 13CH4 ({bullet}) and from CH3OH enrichments ({square}). Partial sequences (<1350 bp; dashed line) were added to the tree using a maximum-parsimony option within ARB. The tree was constructed with the 16S rDNA sequence of Thermotoga maritima as the outgroup and a filter (1160 nt) that excluded alignment positions with sequence ambiguity or missing data. The confidence of branch points was determined by three separate analyses (maximum-likelihood, evolutionary distance and maximum-parsimony), with multifurcations indicating branch points that were collapsed until supported in all three analyses using a strict consensus rule. Prefixes indicate sequences of uncultivated organisms of the Acidobacterium division (UA), the Proteobacteria (UP), the Cytophagales (UC) or the ß-Proteobacteria (UBP), or the sequences of clones from the CH3OH enrichment (EOH). The major phylogenetic groups are indicated at the right of the figure. The number of clones assigned to each OTU by RFLP analysis is shown in parentheses. Accession numbers for the sequences are also shown (numbers prefixed by one or two letters in parentheses). Known methylotrophs and clones retrieved from the 13C-DNA fractions of CH3OH or CH4 SIP experiments are shown in bold text.

 
Functional-gene libraries
The mxaF and pmoA primer sets amplified specific products of the correct size from both the 12C- and 13C-DNA fractions extracted from the CH3OH and CH4 microcosms. RFLP analysis of mxaF clones from the 13C-DNA fractions identified three OTUs (M13.1, 48 clones; M13.2 and M13.3, each one clone) in the 13CH3OH library and four OTUs (M13.4, 41 clones; M13.5 and M13.6, each four clones; M13.7, one clone) in the 13CH4 library. Similar mxaF sequences and proportions of clones within each OTU were identified in analyses of 12C-DNA from the 12CH3OH (M12.1, 48 clones; M12.2 and M12.3, each one clone) and 12CH4 (M12.4, 42 clones; M12.5, five clones; M12.6, three clones) microcosms. Phylogenetic analysis of derived MxaF sequences from the CH3OH libraries (M13.1–M13.3 and M12.1–M12.3), using the XoxF-like methanol dehydrogenase large-subunit homologue of Methylobacterium extorquens as the outgroup (Chistoserdova & Lidstrom, 1997 ), placed these sequences in a cluster that is distinct from all previously described methylotroph MxaF sequences. The most closely related MxaF sequences (Fig. 3) were of Methylobacterium extorquens (72·8–80·1% aa identity) and the moderately acidophilic methanotrophs Methylocella palustris and Methylocapsa acidiphila (73·8–81·0% aa identity). Phylogenetic analysis of the derived MxaF sequences from the CH4 libraries (M13.4–M13.7 and M12.4–M12.6) identified a group of sequences most similar to the MxaF sequences of the methanotrophic genera Methylosinus and Methylocystis (Fig. 3).



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Fig. 3. Maximum-likelihood tree of derived MxaF sequences detected in the 13C-DNA fraction of microcosms exposed to 13CH3OH ({circ}) or 13CH4 ({bullet}) and from CH3OH enrichments ({square}). The tree was constructed with the MxaF homologue (XoxF) of Methylobacterium extorquens (U72662) as the outgroup and a filter (170 aa) that excluded alignment positions with sequence ambiguity or missing data. The confidence of branch points was determined by three separate analyses (maximum-likelihood, evolutionary distance and maximum-parsimony), with multifurcations indicating branch points that were collapsed until supported in all three analyses using a strict consensus rule. Prefixes indicate sequences from the 13C-DNA (M13) and 12C-DNA (M12) fractions or from the CH3OH enrichment (MEOH). The number of clones assigned to each OTU by RFLP analysis is shown in parentheses. Accession numbers for the sequences are also shown (numbers prefixed by one or two letters in parentheses). Known methylotrophs and clones retrieved from the 13C-DNA fractions of CH3OH or CH4 SIP experiments are shown in bold text.

 
As many methanotrophs can also use CH3OH as a carbon source, the pmoA primer set A189/A682 was used with template DNA from both the CH4 and CH3OH microcosms. RFLP analysis of pmoA clones from the 13C-DNA fractions identified five OTUs in both the 13CH3OH (P13.1, 46 clones; P13.2–P13.5, each one clone) and 13CH4 microcosm (P13.6, 30 clones; P13.7, 13 clones; P13.8, four clones; P13.9, two clones; P13.10, one clone). In contrast to the mxaF libraries, several different sequences and OTUs (Fig. 4) were identified from the corresponding microcosms exposed to 12CH3OH (P12.1, 21 clones; P12.2, 13 clones; P12.3, seven clones; P12.4, five clones; P12.5, two clones; P12.6 and P12.7, one clone each) or 12CH4 (P12.8, 35 clones; P12.9, eight clones; P12.10, four clones; P12.11, two clones; P12.12, one clone). Phylogenetic analysis of derived amino-acid sequences from the 13CH3OH library revealed that four OTUs (49 clones) were closely related to the AmoA of Nitrosomonas europaea (Fig. 4) and only one OTU (one clone) was similar to the PmoA of Methylosinus trichosporium. Phylogenetic analysis of the derived PmoA/AmoA sequences from the 13CH4 library identified four groups of sequences. The derived PmoA sequences of P13.6 and P13.10 were most closely related to that of clone LOPA13.1, from a 13CH4 SIP experiment with peat soil (Morris et al., 2002 ), and were distinct from the PmoA of type II methanotrophs and the environmental clone RA14 (Holmes et al., 1999 ). The derived amino-acid sequence of P13.7 clustered with the PmoA of Methylocystis spp., whereas that of P13.8 grouped with the AmoA of Nitrosomonas spp. OTU P13.9 had a derived amino-acid sequence that was weakly related (58·8–61·3% aa identity) to the AmoA of ß-proteobacterial AOB. Clones from the 12C-DNA libraries contained derived amino-acid sequences that grouped with the PmoA- and AmoA-containing clones from the corresponding 13C-DNA libraries, as well as with the PmoA of type I methanotrophs (P12.6) and distant homologues of PmoA/AmoA (P12.3–P12.5 and P12.10). The differences between the 12C- and 13C-DNA pmoA libraries further demonstrated the restricted nature of the population within the 13C-DNA fractions.



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Fig. 4. Maximum-likelihood tree of derived PmoA sequences detected in the 13C-DNA fraction of microcosms exposed to 13CH3OH ({circ}) or 13CH4 ({bullet}). The tree was constructed with the derived amino-acid sequence of clone RA21 as the outgroup and a filter (161 aa) that excluded alignment positions with sequence ambiguity or missing data. The confidence of branch points was determined by three separate analyses (maximum-likelihood, evolutionary distance and maximum-parsimony), with multifurcations indicating branch points that were collapsed until supported in all three analyses using a strict consensus rule. Prefixes indicate sequences from the 13C-DNA (P13) or 12C-DNA (P12) fractions. The number of clones assigned to each OTU by RFLP analysis is shown in parentheses. Accession numbers for the sequences are also shown (numbers prefixed by one or two letters in parentheses). Known methylotrophs and clones retrieved from the 13C-DNA fractions of CH3OH or CH4 SIP experiments are shown in bold text.

 
AOB-specific analysis of DNA fractions
To investigate the unexpected observation of amoA sequences in the 13C-DNA fractions, DNA from both microcosms was used as a template for PCR amplification with AOB-specific primers. Nested PCR of the 16S rDNA of AOB within the ß-subclass Proteobacteria using 12C- and 13C-DNA fractions from both the CH3OH and CH4 SIP experiments generated specific products that were resolved by DGGE (Fig. 5). In the CH3OH microcosms, analysis of the dominant DGGE bands identified AOB sequences belonging to Nitrosomonas cluster 5 (UBP13OH1; cluster designation of Stephen et al., 1996 ) in the 13C-DNA and AOB sequences belonging to Nitrosospira clusters 1 (UBP12OH1) and 3 (UBP12OH2) in the 12C-DNA. In the CH4 microcosms, the dominant DGGE bands were identified as sequences belonging to Nitrosomonas cluster 7 (UBP13CH1) and Nitrosospira cluster 3 (UBP13CH2) in the 13C-DNA, and as sequences belonging to Nitrosospira cluster 2 (UBP12CH1) in the 12C-DNA. No PCR amplification product was obtained with the amoA-specific primer set.



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Fig. 5. DGGE of ammonia-oxidizer-selective 16S rDNA PCR products. AOB cluster controls correspond to the AOB cluster designation of Stephen et al. (1996) . Lanes (left panel): 1–4, Nitrosospira clusters 1–4; 5–7, Nitrosomonas clusters 5–7. Duplicate amplifications (right panel; A and B) were performed with the microcosm DNA samples. Lanes: 1, 13C-DNA (CH3OH); 2, 12C-DNA (CH3OH); 3, 13C-DNA (CH4); 4, 12C-DNA (CH4). DGGE bands that were sequenced are marked with an x. The prefixes 13OH or 12OH correspond to the 13C- or 12C-DNA fractions, respectively, from the CH3OH microcosm; the prefixes 13CH or 12CH correspond to the 13C- or 12C-DNA fractions, respectively, from the CH4 slurry microcosm.

 
Growth of enrichments and pure cultures on CH3OH
Pure cultures of B. indica subsp. indica and A. capsulatum grew on the recommended heterotrophic medium, but no growth was observed after 14 days incubation with CH3OH (12·5 mM) as the sole carbon source. No amplification product was obtained from either strain with the PCR primers that target mxaF.

DNA from all of the CH3OH enrichments generated an amplification product with the 16S rDNA and mxaF PCR primers, but not with the pmoA primers (A189/A682). RFLPs similar to those obtained from the 13CH3OH microcosm were identified in one tertiary enrichment (M1-N; pH 3·5). Both the 16S rDNA library and the mxaF library (15 clones each) contained three OTUs. The sequence of EOH1 (nine clones) was similar to the 16S rDNA sequence of Methylocella palustris and the sequences of clones UP1–UP3 (Fig. 2). The sequences of EOH2 (five clones) and EOH3 (one clone) clustered with the 16S rDNA sequences of members of the genera Burkholderia and Frateuria, respectively (Fig. 2). Derived MxaF sequences (MEOH1, 13 clones; MEOH2 and MEOH3, each one clone) were most similar to those of M13.1–M13.3 (Fig. 3).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
All of the SIP experiments involved long periods (40 days) of continuous exposure to 13CH3OH or 13CH4, which enabled the incorporation of 13C into the DNA of active methylotrophs. Non-methylotrophic micro-organisms could have also assimilated the 13C-labelled intermediates and by-products (e.g. 13CO2) of methylotroph metabolism during the relatively long incubation period. However, to observe a ‘heavy’ DNA fraction the major C isotope within DNA must be the 13C that was added as the labelled substrate. Thus, the turnover of 13C due to cross-feeding can be assessed qualitatively from the presence/absence and the position of a 13C-DNA fraction in a CsCl gradient. For example, the absence of any visible 13C-DNA fraction from two of the three 13CH4 microcosms, despite all three oxidizing approximately 2 mmol of 13CH4, suggests that the non-primary 13C-labelled compounds were diluted substantially by the production of 12C-labelled substrates (e.g. 12CO2) and/or by other trophic interactions within the 13C-microcosms. Similarly, the observation of very discrete 13C- and 12C-DNA fractions in the 13CH3OH soil and 13CH4 slurry microcosms rather than a smear of 13C-DNA (e.g. Morris et al., 2002 ) suggests that most cross-feeding micro-organisms did not incorporate a high proportion of 13C into their DNA. Since the separation between the 13C- and 12C-DNA fractions of a pure culture grown on 13C- or 12C-labelled substrates was approximately 1 cm in an equilibrium CsCl density gradient (Radajewski et al., 2000 ), it is estimated that the 13C-DNA fractions collected from the microcosms contained 65–100% 13C (Fig. 1). Therefore, only the DNA of micro-organisms that assimilated 13C as a primary carbon source, such as methylotrophs or organisms in a close physical and/or nutritional association with methylotrophs, would be sufficiently labelled with 13C to have been collected in the discrete 13C-DNA fractions.

Characterization of the microbial community within the 13C-DNA fractions by analysis of 16S rDNA sequences identified distinct bacterial populations involved in the assimilation of CH3OH and CH4 in the soil microcosms (Fig. 2). However, the abundance of clones in the libraries must be interpreted with caution, as limitations inherent in SIP may be compounded by biases introduced by PCR amplification (Wintzingerode et al., 1997 ). In particular, clones occurring at low frequency may represent bacteria that grow relatively slowly on the 13C-labelled substrate, bacteria which are cross-feeding on non-primary 13C-labelled compounds or sequences amplified from traces of 12C-DNA that may have been co-extracted with the 13C-DNA fraction. We noted previously that the 16S rDNA sequences recovered from the 13CH3OH microcosm were, surprisingly, confined to only two phylogenetic groups and most closely related to genera that are rarely reported as being involved in CH3OH utilization (Radajewski et al., 2000 ). The closely related strains Beijerinckia indica and A. capsulatum did not grow on CH3OH; however, both of these strains, as well as the methanotrophs Methylocella palustris and Methylocapsa acidiphila and the photoautotroph R. acidophilus, grow at acidic pH in culture. Although strains closely related by 16S rRNA phylogeny do not necessarily share physiological similarities, the acidity of the forest soil in conjunction with these phylogenetic relationships suggests that UP1–UP3 and UA1–UA3 may represent moderately acidophilic bacteria. Balancing the caveats associated with clones occurring at low frequency with the fact that the library contained three distinct, but related, OTUs in each phylogenetic group (i.e. UP1–UP3 and UA1–UA3) also suggests that these bacteria may have grown directly on 13CH3OH at the relatively high concentration (0·5%, v/w; approximately 300 mM at 40% water-holding capacity) used in the microcosm.

Most of the 16S rDNA, pmoA and mxaF clones retrieved from the acidic (pH 3·4) 13CH4 slurry microcosm were closely related to sequences of extant methanotrophs belonging to the {alpha}-Proteobacteria (type II). A recent study using fluorescent in situ hybridization and combinations of highly specific oligonucleotide probes identified Methylocystis spp. as the major component of the methanotroph population in acidic (pH 4·2) Sphagnum peat samples (Dedysh et al., 2001 ). Distinct mxaF sequences (Fig. 3, peat clones) detected in an acidic peat (pH 3·6) have also been proposed to originate from a population of acidophilic type II methanotrophs (McDonald & Murrell, 1997 ). Therefore, certain 16S rDNA (UP4–UP7) and derived MxaF (M13.4–M13.7) sequences retrieved from the 13CH4 slurry microcosm support the moderately acidophilic nature of some as yet uncultivated Methylocystis-like methanotrophs. Another OTU from the 13CH4 slurry microcosm (UP8) grouped more closely to Methylocella (Dedysh et al., 2000 ), Methylocapsa (Dedysh et al., 2002 ) and clones retrieved from a 13CH4 SIP experiment with a peat soil (LO13.10; Morris et al., 2002 ) than to other methanotrophs within the {alpha}-Proteobacteria. However, the sequence of UP8 was most similar to the sequence of UP1, indicating that it may represent bacteria that assimilated CH3OH produced by methanotrophs within the soil slurry microcosm. The combined results from these SIP experiments with 13CH3OH and 13CH4 suggest that methylotrophs closely related to Methylocella, Methylocapsa and Methylocystis are active and potentially widespread in moderately acidic environments.

The physiology of the organisms represented by UP9 (ß-Proteobacteria) and UC1 (Cytophagales) is not clear, as they did not group near any known methylotrophs. It must also be stressed that these clones occurred at low frequency and, in the absence of isolates or more definitive data, the physiology of the bacteria that are represented by these clones should be considered as speculative. It is, however, interesting to note that from a library of 100 16S rDNA clones in a 13CH4 SIP experiment, Morris et al. (2002) identified six clones (LO13.5) with 96·8% similarity to UP9, which is higher than the next nearest relative currently retrieved in a BLAST search. They also detected several Cytophaga sequences, including three clones (LO13.4) with 99·6% similarity to UC1. These culture-independent observations from two distinct 13CH4 SIP experiments raise the possibility that bacteria not previously considered to be involved in CH4 oxidation may derive a significant proportion of their carbon from products of methanotroph metabolism, or possibly even from CH4 itself.

One significant feature of the SIP approach used here is that the 13C-DNA fractions contained the entire genomes of the active methylotrophs, which enabled a parallel analysis of functional-gene sequences within these populations. As the mxaF and pmoA PCR primers were designed from sequences of extant Proteobacteria, it is likely that most of the functional-gene sequences detected in the 13C-DNA fractions were present in one or more of the Proteobacteria identified from 16S rDNA sequences (UP1–UP9). Indeed, derived MxaF sequences in the 13CH3OH microcosm were most similar to the MxaF sequences of Methylocella, Methylocapsa and Methylobacterium, whereas in the 13CH4 microcosm they were most similar to the MxaF sequences of Methylocystis and Methylosinus. Similarly, the majority of pmoA clones from the 13CH4 slurry experiment were similar to the pmoA of extant type II methanotrophs. Dunfield et al. (2002) have recently identified certain Methylocystis and Methylosinus isolates that contain two highly distinct pmoA-like sequences, which suggests that OTUs P13.6 and P13.10 may represent a second pmoA lineage possessed by some of the Methylocystis-like methanotrophs (UP4–UP7). Such potential diversity of pmoA-like sequences within a single methanotroph also excludes identification of the bacterium containing the sequence P13.9.

The pmoA primers A189/A682 can amplify pmoA, amoA and homologous sequences of unknown function from environmental samples (Henckel et al., 2000 ; Holmes et al., 1999 ; Reay et al., 2001b ). However, it was unexpected that the derived amino-acid sequence of almost all of the clones from the 13CH3OH microcosm (P13.1–P13.4) and several from the 13CH4 slurry microcosm (P13.8) grouped with the AmoA sequences of Nitrosomonas spp. (Fig. 4). DGGE of AOB-specific 16S rDNA amplification products from the 13C-DNA templates confirmed that sequences similar to Nitrosomonas spp. (UBP13OH1 and UBP13CH1) and Nitrosospira spp. (UBP13CH2) were present in the same DNA fractions that contained the amoA sequences (Fig. 2 and Fig. 4). However, the lack of an amoA-specific PCR product and the variability observed in DGGE for some duplicate amplifications (e.g. Fig. 5, lanes 3A and 3B) suggested that the AOB template DNA was not abundant in the 13C-DNA fractions. Since extant Nitrosomonas spp. are obligately autotrophic AOB and both soil samples had the capacity to oxidize [approx. 4 nmol (g.d.w. soil)-1 h-1], it seems likely that some AOB had assimilated a significant proportion of 13CO2 in the microcosms, possibly through a close physical and/or nutritional association with active methylotrophs. Interestingly, Nitrosomonas cluster 5 AOB sequences have not been previously reported in soil, nor have representatives been cultivated. Therefore, it is also possible that these AOB may have an unusual physiology when compared with other Nitrosomonas spp. Analysis of larger DNA fragments or the isolation of representative strains may help to resolve these curious observations.

An enrichment strategy to target methylotrophs represented by OTUs UP1–UP3 was only partly successful, as most mxaF and 16S rDNA clones were similar to, but distinct from, those retrieved from the 13CH3OH microcosm. Sequences detected in these enrichment libraries also suggested that acidophilic, N2-fixing, methylotrophic Burkholderia spp. may exist. Indeed, symbiotic N2-fixing Burkholderia strains have recently been isolated from the root nodules of tropical legumes (Moulin et al., 2001 ) and a N2-fixing, CH3OH utilizer with a 16S rDNA sequence identical to EOH2 (Burkholderia sp.) was isolated from the enrichments and is being characterized further. Although the strain corresponding to EOH1 could not be purified, these results demonstrate the potential use of SIP for the enrichment and isolation of novel micro-organisms with specific metabolic capabilities.

Despite the remarkable simplicity of the SIP technique, its suitability for resolving certain structure–function relationships may be limited (Radajewski et al., 2000 ). In these experiments, it was critical that sufficient 13C was incorporated into the DNA of the functionally active methylotrophs to permit collection of a 13C-DNA fraction. Factors including 13C turnover due to predation or substrate transformation, 13C dilution due to the assimilation of other carbon substrates (i.e. 12C-labelled) or 13C assimilation without DNA replication, might have influenced the community composition within the 13C-DNA fractions. In the two 13CH4 SIP experiments where a 13C-DNA band was not observed, consideration of such factors provides some insight into the CH4-oxidizing population in this soil, even though the functional component of the community was not identified. To obtain visible 13C-DNA fractions, it was thus necessary to use incubation conditions that enriched for methylotrophs in a physically, chemically and biologically complex environment, which only partly reflected that occurring in situ. Future improvements, such as the extraction of biomarkers that do not rely upon replication of the chromosome for labelling (e.g. rRNA; Gray & Head, 2001 ), the cloning of large 13C-DNA fragments to link phylogenetic and functional genes, the incubation of larger sample volumes and the use of time-course experiments, may permit a more sensitive resolution of the function and succession of methylotrophs in the environment. Furthermore, as SIP is an effective tool for the isolation of the genome of micro-organisms with previously unknown metabolic capabilities, it can provide a rational basis for the application of molecular biological techniques to investigate these uncultivated methylotroph populations in situ.


   ACKNOWLEDGEMENTS
 
This study was funded by the UK Natural Environment Research Council EDGE thematic programme (Award GST/02/1864 to J.C.M., P.I. and D.B.N. and GST/02/1837 to J.I.P.) with support from the European Union 4th Framework Programme (Grant QLRT-1999-31528 to J.C.M.). We thank Forest Enterprises for access to the study site and Peter Dunfield for sharing findings prior to their publication.


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DISCUSSION
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Received 4 February 2002; revised 18 March 2002; accepted 15 April 2002.