INTRODUCTION

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Methanotrophs are a unique group of organisms which can use methane as a sole source of carbon and energy. The ability of methanotrophs to oxidize methane is due to the possession of the enzyme methane monooxygenase. There are two distinct forms of this enzyme, the cytoplasmic soluble methane monooxygenase and the membrane-bound particulate methane monooxygenase (pMMO) (reviewed in references 21 and 22). Only the pMMO is found universally in methanotrophs and can therefore be used as a functional marker for these organisms. No genetic or structural homology is found between these two enzyme systems despite their similar functions. However, the pMMO enzyme complex shares many similarities with the ammonia monooxygenase (AMO) enzyme complex found in ammonia-oxidizing bacteria (15). These similarities include a high degree of amino acid sequence identity, similar protein complex structures, and broadly similar substrate and inhibition profiles, while each play a crucial role in cell metabolism (6, 11, 29). Methanotrophs and ammonia-oxidizing bacteria can oxidize both methane and ammonia; however, they can obtain energy only from the oxidation of methane and ammonium, respectively (3).

Oligonucleotide primers (A189f and A682r) have been designed to amplify internal fragments of the genes encoding the pMMO and AMO enzyme complexes (11). The sequence information obtained from theses genes encoding pMMO (pmoA) and AMO (amoA) has been used as phylogenetic markers for identification of methanotrophs and ammonia oxidizers (12, 19). The phylogeny of these functional genes closely reflects the 16S rRNA phylogeny of the organisms from which the gene sequences were retrieved. Therefore, retrieval of pmoA and amoA gene sequence information provides information on the diversity of these organisms in different environments (12, 19).

The A189 and A682 primers have been used extensively in environmental studies to provide a molecular profile of the methane-oxidizing community (10, 13, 20). Recently a new reverse pmoA-specific primer, mb661, used in conjunction with the A189 primer, was designed and demonstrated specificity to amplify pmoA sequences while not detecting amoA sequences (4). This new primer was used alongside 16S ribosomal DNA phylogenetic probes to determine in situ populations of methanotrophs in freshwater environments (4). The use of PCR primers to amplify the amoA genes from ammonia oxidizers has recently been critically evaluated (24), with the amoA primer set of Rotthauwe et al. (26) being recommended as the most suitable. The soluble methane monooxygenase genes have also been used to detect methanotrophs in the environment (2, 17, 18); however, this procedure detects only the subgroup of methanotrophs that contain this enzyme.

The pmoA PCR primer set A189-A682 has been adapted for denaturing gradient gel electrophoresis (DGGE) analysis as a means to study pmoA gene diversity (5, 9). The use of degenerate primers in DGGE analysis may cause the appearance of multiple bands for individual organisms, which in complex environments may cause confusion in interpretation of results. The A682r primer has four redundancies within its sequence, which is suspected to cause multiple-banding problems in DGGE analysis. This study attempted to design a new pmoA primer set containing no redundancies which could subsequently be applied in DGGE analysis of pmoA.

Aerobic soils, such as forest soils, play an important role in the global methane cycle by acting as a major sink for atmospheric methane in the atmosphere (1, 14, 30). To investigate methanotroph populations involved in methane oxidation in forest soils, pmoA clone libraries were constructed from three Danish soils, i.e., heath, oak, and sitka, and compared. The soils were obtained from a site at Hjelm Hede, Denmark. The soil types are similar. All were originally heath, but part was planted with sitka spruce over 60 years ago and part has been gradually colonized by oak woodland. The change in vegetation has effected a change in the atmospheric oxidation potentials of the soils. A plant succession from heather to oak vegetation increased methane uptake sixfold, while the introduction of sitka spruce doubled methane uptake rates relative to those of the native heathland. A detailed analysis of the physiochemical properties of the soils and their relative methane uptake potentials will be presented elsewhere (I. R. McDonald et al., unpublished data; N. Høegh et al., unpublished data). However, the major aims of this study on these heathland and forest soils were twofold: to evaluate different pmoA primer sets as tools for investigating methanotroph diversity and to investigate the methanotroph diversity in these soils, which demonstrate novel atmospheric methane oxidation potentials.

	MATERIALS AND METHODS

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Microbial strains and template DNA. The microorganisms used in this study were obtained from a culture collection of methanotrophs maintained at the University of Warwick and from the National Collection of Industrial and Marine Bacteria (Aberdeen, United Kingdom). Cultures were grown in nitrate mineral salts medium with the addition of excess methane (20% [vol/vol] in air) as the sole carbon substrate as described previously (32). DNA was extracted from cultures using the methods of Marmur (16).

Sample collection and DNA extraction. Core soil samples were taken from three sites located at Hjelm Hede in Northern Jutland, Denmark. The sampling sites were (i) native heathland (heath), (ii) established oak (oak), and (iii) sitka spruce (sitka). Core soil samples (15-cm diameter, 35-cm depth) were obtained from each of the three vegetation sites using the method of Hall et al. (7). The oak and sitka cores were extruded from the sample tube and sectioned into 5-cm sections before being placed in airtight collection bags. The heath core was sectioned into 5-cm sections down to 15 cm and then into 2-cm sections (15 to 27 cm). The soil samples were sieved (4-mm mesh) to remove stone and roots and to homogenize the soil. Total DNA was extracted from 2 g of each core section using the methods of McDonald et al. (17). High-molecular-mass DNA was excised from a 1% (wt/vol) agarose gel and purified (Geneclean II kit; Bio 101) to remove humic compounds which interfered with PCR amplification. This method yielded consistently high-quality DNA, which could be easily digested with restriction endonucleases and was suitable as a template in PCR amplification experiments. In this study, only DNA extracted from the core sections demonstrating the highest methane oxidation potentials was used (heath, 21 to 23 cm; oak, 5 to 10 cm; sitka, 20 to 25 cm) (Høegh et al., unpublished data)

Design of a new pmoA-specific primer, A650. The pmoA and amoA sequences of methanotrophs and nitrifiers presently available from the GenBank database were aligned and then scanned for conserved regions within the pmoA gene which could provide a suitable primer target site. From this analysis, a reverse primer, A650r (5' ACGTCCTTACCGAAGGT 3'), was designed. No unique region at the start of the pmoA sequence could be identified as being suitable for a new forward primer. The A650 primer was used in conjunction with the A189f primer to amplify a 478-bp internal section of the pmoA gene. The primer was tested against a range of methanotrophs and nitrifiers, including Methylococcus capsulatus (Bath), Methylococcus capsulatus (strain M), Methylomicrobium agile (A30), Methylobacter whittenburyi, Methylocaldum tepidum (LK6), Methylomonas methanica (S1), Methylomicrobium album (BG8), Methylosinus trichosporium (OB3b), Methylocystis parvus (OBBP), Methylosphaera hansonii, Nitrosomonas europaea (NCIMB 11850), Nitrosospira sp. (Np22), Nitrosococcus oceanus (NCIMB 11848), Nitrosomonas eutropha, and Nitrosospira multiformis (NCIMB 11849).

PCR amplification. PCR amplification reactions were performed in 50-µl (total volume) reaction mixtures in 0.5-ml Microfuge tubes using a DNA thermal cycler with a hot lid (Touchdown model; Hybaid, Teddington, Middlesex, United Kingdom). All PCR amplifications of the pmoA gene used the A189f primer in combination with either the A682, A650, or mb661 primer. Individual reagents and their concentrations or amounts were as follows: 1× PCR buffer, 1.5 mM MgCl₂, 0.05% W-1 (supplied with the Taq DNA polymerase), 20 µg of bovine serum albumin (Boehringer Mannheim), 200 µmol of each deoxynucleoside triphosphate, 20 pmol of each primer, 1 µl of template DNA (approximately 5 to 50 ng), and 5 U of Taq polymerase (Life Technologies). Each primer set used the same thermal profile. Taq polymerase was added after the initial denaturation step of 96°C for 5 min, followed by 30 cycles of 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min. A final extension period of 5 min at 72°C was included (11).

Construction of clone banks and restriction fragment length polymorphism (RFLP) analysis. The size and purity of each PCR product were checked on 1% (wt/vol) agarose gels (27), and the products were then ligated into the pCR 2.1 vector supplied with the TA cloning kit (Invitrogen, San Diego, Calif.) according to the manufacturer's instructions. Individual colonies containing inserts were suspended in 3 ml of nutrient broth containing ampicillin (50 µg/ml) and grown overnight at 37°C. Small-scale preparations of plasmids were performed using the methods of Saunders and Burke (28). Plasmids were digested with the restriction enzyme combinations of EcoRI-RsaI and EcoRI-PvuII-HincII. Digests were resolved on 2% (wt/vol) agarose gels and grouped manually, based on the restriction pattern obtained.

DNA sequencing and analysis. Small-scale preparations of clones from libraries were done by the method of Saunders and Burke (28), and DNA for direct sequencing of DGGE bands was prepared by purification of PCR products using a Wizard PCR purification kit (Promega, Southampton, United Kingdom). DNA sequencing reactions were carried out by cycle sequencing with the Dye Termination kit of PE Applied Biosystems (Warrington, Cheshire, United Kingdom). Phylogenetic analyses of the DNA and deduced amino acid sequences were carried out using the ARB program (http://www.mikro.biologie.tu-muenchen.de). Sequences were manually aligned with the pmoA and amoA sequences obtained from the GenBank database. Regions of sequence ambiguity and incomplete data were excluded from the analyses. Results were depicted as a consensus tree, combining the results of evolutionary distance (Dayhoff percentage of acceptable point mutations model), maximum-parsimony, and maximum-likelihood analyses of the data sets. Multifurcations indicate points where the branching order was not supported by all three methods.

DGGE analysis of the pmoA gene. A GC clamp (23) was attached to the 5' end of the pmoA-specific A650 primer. PCR amplification was performed with the GC-A650 primer and the A189 primer. A touchdown PCR program was optimized and consisted of an initial denaturation step of 5 min at 94°C, followed by 20 touchdown cycles (65 to 55°C) and 10 further cycles at 55°C for 1 min, followed by 72°C for 1 min and a final extension of 72°C for 5 min. PCR amplification with the GC-A189-A682 primer set was performed as outlined by Henckel et al. (9). PCR products were analyzed as described above.

Nucleotide sequence accession numbers. The environmental clone sequences have been deposited in the GenBank database under accession numbers AF368354 to AF368374.

	RESULTS

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Clone library construction. Three different pmoA-specific primers sets (A189-A682, A189-A650, and A189-mb661) were used to construct clone libraries from three soil samples (heath, oak, and sitka). PCR amplification products of the predicted size were obtained from each of the soils using the three primer sets. Libraries of approximately 50 clones were constructed for each soil and from each pmoA primer set (nine libraries in total). The libraries were subjected to RFLP analysis and grouped based on their representative RFLP patterns. This use of methanotroph-specific primers and operational taxonomic units (OTUs) has been shown to be effective in screening environmental clone libraries and providing an indication of methanotroph diversity (4, 12). In this study, random sequencing within classified OTU groups always demonstrated identical clone sequences, suggesting that the clone groupings based on restriction analysis were robust.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Phylogenetic analysis of the derived amino acid sequences encoded by pmoA and amoA genes retrieved from the Danish soils. Bar, 10 inferred substitutions per 100 amino acid positions. Retrieved sequences and their relationships with known pmoA sequences are grouped into established phylogenetic families A, B, C, D, E, F, and G. Uncultured methanotroph pmoA sequences RA21 and MR1 were used as the outgroup.

A189-A682 clone libraries. The clone libraries constructed with the A189-A682 primer set resulted in limited successful retrieval of methanotroph pmoA sequences. The heath and sitka libraries were dominated by the clone sequence A1 (59 and 66% of clone libraries, respectively), which demonstrated high identity (>98%) to the amoA gene of Nitrosomonas europaea. Only 4% of clones were affiliated with this sequence in the oak library. The libraries consisted of a high proportion of clone types which contained no open reading frames of significant length and which failed to show homology to other sequences after database analysis (heath, 29%; oak, 45%; and sitka, 15%). The primers appeared to retrieve a large number of hybrid nonsense amplified bands of the correct insert size, although the sequences were not identified as chimeric. A small number of clones in the libraries also failed to provide any sequence data, as indicated in Table 1 (heath, 10%; oak, 13%).

Design of primer A650. All available pmoA sequences were aligned, and a new pmoA-specific reverse primer, A650, was designed for use in both clone library construction and DGGE analysis. To facilitate DGGE analysis, the A650 primer was designed without redundancies. Designing a primer specific to all pmoA sequences was problematic; therefore, mismatches with some pmoA sequences occur. A selection of pmoA and amoA sequences showing both matching and mismatching regions compared to all three reverse primers used in this study is shown in Table 2. The A650 primer did not match any ammonia monooxygenase amoA gene sequences.

A189-A650 clone libraries. Clone libraries constructed with the A189-A650 primer set were dominated by pmoA sequences B1 and B2, showing high identity (>98%) to Methylococcus capsulatus (Bath) pmoA sequences (heath, 98%; oak, 87%; and sitka, 82% of the libraries). Limited methanotroph diversity was detected within the soils with this primer set. For example, only two OTU groups were identified in the heath and oak libraries.

mb661 clone libraries. Costello and Lidstrom (4) designed a new reverse pmoA primer (mb661) to specifically amplify pmoA sequences and not amoA sequences. The A189-mb661 primer set was found to be more useful for studying methanotroph diversity in freshwater environments.

View larger version (53K): [in this window] [in a new window]   FIG. 2.   DGGE profiles of control strains and Danish soils obtained with pmoA primers sets. (A) A189-A682 primer set. Lane 1, Methylocystis strain M; lane 2, Methylosinus trichosporium OB3b. (B) A189-A650 primer set. Lane 1, Methylococcus capsulatus (Bath); lane 2, Methylobacter whittenburyi; lane 3, Methylocystis strain M; lane 4, Methylosinus trichosporium OB3b.

DGGE analysis of soils using pmoA PCR-amplified pmoA gene fragments from a selection of control methanotroph cultures and the soils investigated in this study were analyzed by DGGE. Two different pmoA-specific primer sets were used in this study, both of which incorporated a GC clamp (GC-A189-A682 and A189-A650-GC) (23). The A650 pmoA primer was designed for application in DGGE analysis; hence, no redundancies were incorporated when designing the primer.

	DISCUSSION

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In this study, we have compared three pmoA primer sets to assess their potentials for investigating methanotroph diversity and subsequently comparing this diversity between three soil samples with contrasting cover vegetation, i.e., heath, oak, and sitka. The pmoA gene sequences retrieved in this study were used to infer methanotroph diversity, since the phylogeny of the pmoA genes reflects that of the 16S rRNAs of methanotrophs (4, 19)

Comparison of primer sets for assessing methanotroph diversity. Costello and Lidstrom (4) highlighted the disadvantage of the A189-A682 PCR primer set, in that it amplifies both amoA and pmoA sequences (11, 24). In this study the A189-A682 PCR could not be used to assess methanotroph diversity. Most sequences recovered from the libraries were homologous to the amoA gene of Nitrosomonas europaea. Also, a large number of clones in each library were nonspecific amplified PCR products exhibiting no sensible open reading frame.

Comparison of methanotroph diversity between the soil samples. A comparison of the diversity of the soils with the different primer sets is difficult, which is not surprising given the associated biases and problems caused by PCR. Also, the fact that only 50 clones were screened per clone library means that some representatives may have been missed due to the small sample size.