Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays

Soil samples.Soil samples were taken from a meadow and rice fields. The meadow soil was located near Giessen, Germany (28). This soil was classified as a Gleysol with the texture of a sandy loam over clay. During the past 50 years the soil has been managed as a meadow and mowed twice a year (28). Samples were taken from the top 10 cm (pH 6.0). The rice field samples were taken from a flooded rice field (Oryza sativa, type japonica, variety Korall) at the Italian Rice Research Institute, Vercelli, Italy. Soil characteristics were described by Schütz et al. (38). Soil samples were taken at a distance of ≥20 cm from rice plants and at a depth of 5 to 10 cm.

DNA extraction from soil samples.DNA extraction and purification were performed with the Fast Spin kit (Bio 101, La Jolla, Calif.) in triplicate. The protocol was performed according to the manufacturer's instructions by using a sodium dodecyl sulfate-containing lysis buffer and bead beating. To reduce the humic acid content, DNA extracts were further purified with Sephadex G50 columns (Roche Diagnostics, Mannheim, Germany). DNA extracts were stored in Tris-EDTA (TE) buffer (pH 7.0) at −20°C for further analyses.

Spiking of meadow soil samples.A meadow soil sample from which no pmoA could be amplified was chosen. Thus, it was ensured that the measured pmoA numbers were retrieved from the cells. An aliquot (4 g) of the soil samples was homogenized in a Stomacher 80 (Sewar Medical Ltd., London, England) at high speed for 1 min. Bacterial numbers of pure cultures of Methylosinus trichosporium, Methylococcus capsulatus, and Methylomicrobium album were determined by microscopy (Axiophot; Zeiss) by analyzing undiluted and 10-fold-diluted bacterial suspensions in a Neubauer cell chamber (Glaswarenfabrik Karl Hecht KG, Sondheim, Germany). Afterwards, 6 ml of a defined mix of these cultures was added to the soil, followed by incubation at room temperature for 1 h. DNA was extracted and purified as described above in four replicates.

Cultivation and DNA extraction of bacterial strains.Bacterial strains were obtained from the National Collection of Industrial and Marine Bacteria (NCIMB), the German Culture Collection (DSMZ), or other laboratories (see below). Methanotrophic bacteria were cultivated in 25 ml of ammonium mineral salt medium as modified by Whittenbury et al. (46) in 150-ml gas-tight bottles with a headspace containing 20% (40% for Methylococcus capsulatus) CH₄. The cultures were incubated at 25°C (37°C for Methylococcus capsulatus) under continuous shaking for 5 days in darkness. Cultures were harvested by centrifugation, and the cell pellet was resuspended in 500 μl of phosphate-buffered saline (pH 7.0). DNA was extracted according to the protocol published by Henckel et al. (16). This procedure was used for Methylococcus capsulatus (NCIMB 11853), Methylocaldum gracile (NCIMB 11912), Methylobacter luteus (NCIMB 11914), Methylomicrobium album (NCIMB 11123), Methylomonas methanica (NICIMB 11130), Methylocystis parvus (NCIMB 11129), Methylosinus sporium (NCIMB 11126), and Methylosinus trichosporium (NCIMB 11131).

DNA was extracted from lyophilized cells of Methylopila helvetica (DSM 6342), Methylobacterium extorquens (DSM 1337), and the ammonia-oxidizing strain Nitrosomonas europaea (NCIMB 11850) by heating for 10 min at 100°C in 25 μl of TE buffer (pH 7.2), followed by centrifugation (for 10 min at 14,000 rpm in an Eppendorf 5417R centrifuge).

Genomic DNA solutions of the following species or strains were provided by other sources: Methylocapsa acidiphila B2 (S. Dedysh, Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia), Nitrosospira tenuis (H.-P. Koops, University of Hamburg, Hamburg, Germany), Nitrosococcus oceani strain 9 (H.-P. Koops), and Escherichia coli (Roche Diagnostics). All DNA solutions were stored at −20°C until use.

A pmoA sequence related to published forest clone sequences, MForest (AJ496664), was obtained from a deciduous forest near Marburg, Germany, as previously described by Henckel et al. (17). The pmoA gene was amplified from a soil DNA extract with the universal primers A189 forward (with GC clamp) and A682 reverse. The mixed PCR products were separated by denaturing gradient gel electrophoresis (DGGE). An individual band was excised with sterile needles and reamplified by PCR for sequencing according to the work of Henckel et al. (16). The phylogenetic affiliation of this pmoA sequence, MForest (165 amino acid residues; 495 bp), was analyzed by calculating a distance matrix. The amino acid sequence was identical to the pmoA sequence with accession number AF368372. Furthermore, MForest had high identities (96.8 and 98.7%) to the forest clones Rold5 (AF148527) and RA14 (AF148521), respectively. The closest relation to a bacterial isolate was that to Methylocapsa acidiphila B2 (70.1% identity). The reamplified PCR product of MForest was stored at −20°C.

Primer development and phylogenetic reconstructions.To design pmoA group-specific primers, the “Probe design” tool of the ARB software package (42) was used in a database consisting of pmoA and amoA nucleic acid and translated protein sequences. This database contained all publicly available pmoA sequences (updated April 2002 from GenBank) and 48 additional type II-related sequences (21), for a total of 354 sequences.

The following primers were used for the assays presented in Table 1: A189 F (GGN GAC TGG GAC TTC TGG) (23), Mb661 R (GGT AAR GAC GTT GCN CCG G) (7), II223 F (CGT CGT ATG TGG CCG AC) (this study), Mb601 R (ACR TAG TGG TAA CCT TGY AA) (this study), Mc468 R (GCS GTG AAC AGG TAG CTG CC) (this study), II646 R (CGT GCC GCG CTC GAC CAT GYG) (this study), Mcap630 R (CTC GAC GAT GCG GAG ATA TT) (this study), and Forest675 R (CCY ACS ACA TCC TTA CCG AA) (this study).

To analyze the phylogeny of MOB, quartet PUZZLE trees were calculated (41) using the PHYLIP software package (version 3.6a2; J. Felsenstein, Department of Genetics, University of Washington, Seattle; available at http://evolution.genetics.washington.edu/phylip.html). Trees were confirmed by maximum-likelihood analysis (FastDNAML, ARB software package, and PROTML, Institute Pasteur [http://bioweb.pasteur.fr/seqanal/interfaces/molphy.html]). For the PUZZLE tree based on 16S rDNA, the HKY model (Hasegawa substitution model [14a]) and a 50% base frequency filter resulting in 1,221 valid nucleotide positions were used. The pmoA- and amoA-based tree was calculated with the VT (33a) evolution model using 103 valid positions of amino acid residues. Substitution rates of each site were estimated from the data sets. In the puzzling step of both trees, 2,000 replicated trees were created and reconstructed as “unrooted.”

Sequences of the following organisms were obtained from the GenBank sequence database (http://www.ncbi.nlm.nih.gov/GenBank), aligned with the alignment tools of the ARB software package, and used for phylogenetic reconstructions (GenBank accession numbers of 16S rRNA [first number] and of pmoA or amoA DNA sequences [second number] are given in parentheses): Methylomicrobium album (X72777/U31654), Methylomicrobium pelagicum (L35540/U31652), Methylomonas methanica (AF304196/U31653), Methylobacter sp. strain BB5.1 (AF016981/AF016982), Methylocaldum szegediense (U89300/U89303), Methylocaldum gracile (U89298/U89301), Methylocaldum tepidum (U89297/U89304), Methylococcus capsulatus (X72771/L40804), Methylococcus thermophilus (X73819/not available), “Methylothermus” strain HB (U89299/not available), Methylosphaera hansonii (U67929/not available), Nitrosococcus oceani (M96395/AF047705), Nitrosococcus halophilus (none/AF272521), Methylosarcina fibrata (AF177296/AF177325), Methylosarcina quisquiliarum (AF177297/identical with pmoA sequence of Methylosarcina fibrata), Methylocystis parvus (M29026/U31651), Methylocystis sp. strain LW5 (not available/AF150791), Methylosinus sporium (M95665/AF458994), Methylosinus trichosporium Ob3b (M29024/S81887), Beijerinckia indica (M59060/no pMMO), Methylocella palustris (Y17144/has only sMMO), Methylocapsa acidiphila (AJ278726/AJ278727), pmoA DNA (forest clone) sequences of Rold5 (AF148527) and Maine6 (AF148528), Nitrosomonas europaea (AF353160/Z97861), Nitrososmonas communis (Z46981/AF2399), Nitrosomonas halophila (Z46987/AF2398), and Nitrosospira tenuis (M96405/U76552).

Real-time PCR assays.Data analysis was carried out with iCycler software (version 2.3.1370; Bio-Rad) as described by Stubner (43). The cycle at which the fluorescence of a certain target molecule number exceeded the background fluorescence (threshold cycle [C_T]) was determined from dilution series of target DNA with defined target molecule amounts. C_T was proportional to the logarithm of the target molecule number. Thus, a C_T measured in a sample could be converted to a target molecule number. More details of real-time PCR are explained by Raeymaekers (37) and Suzuki et al. (44). PCR was performed in 40-μl volumes using Thermo-fast 96 PCR plates (PeqLab, Erlangen, Germany), which were sealed with iCycler IQ optical quality tapes (Bio-Rad) on an iCycler IQ thermocycler (Bio-Rad). Each measurement was performed in four replicates.

Five microliters of DNA template was added to 35 μl of Master mix containing 4 μl of PCR buffer (Invitrogen), 3.2 μl of MgCl₂ (final concentration, 4 mM; Invitrogen), 4 μl of fatty acid-free bovine serum albumin (5 μg/μl; Sigma-Aldrich), 4 μl of mixed deoxyribonucleoside triphosphates (2 μM each; PeqLab), 0.4 μl of SybrGreen I (500-fold diluted in H₂O), 0.4 μl of each primer (MWG Biotech AG, Boersberg, Germany) (see Table 1 for final concentrations), 0.32 μl of PLATINUM DNA polymerase (5 U/μl; Invitrogen), and 18.7 μl of double-distilled water (Sigma-Aldrich). The addition of bovine serum albumin as described by Kreader (29) reduced inhibition by humic substances.

Assays were performed with a four-step thermoprofile: denaturation of DNA (25 s at 94°C), annealing of primers under stringent conditions (20 s at an assay-specific temperature) (Table 1), elongation (45 s at 72°C), and fluorescence data acquisition during an additional temperature step (10 s at a temperature above the melting point of primer dimers). The latter, assay-specific temperature was determined and verified by melting curve analysis (data not shown). As calibration standards for the real-time PCR assays, dilution series of positive-control DNA were used. DNAs from Methylococcus capsulatus (MCOC), Methylomicrobium album (MBAC and MTOT), Methylosinus trichosporium (TYPEII), Methylocapsa acidiphila (MCAP), and the DGGE band Mforest (FOREST) were the targets for pmoA-specific PCR. The positive-control DNA extracts were amplified with the assay primers (Table 1). The resulting amplicons were purified with a Qiaquick PCR purification kit as recommended by the manufacturer (Qiagen, Hilden, Germany) and cloned into pGEM-T. After reamplification with vector-specific primers according to the manufacturer's instructions (Promega, Madison, Wis.), the PCR products were purified as described above. The PCR products obtained were then quantified with the PicoGreen dsDNA quantitation kit (Molecular Probes, Leiden, The Netherlands) using fluorimetry. The measured DNA amount could be converted to target molecule numbers per microliter, and the pmoA standards were adjusted to 10¹⁰ target molecules μl⁻¹ for storage at −20°C.

Before quantification, the DNA extracts were tested for inhibitory effects of coextracted substances by determining pmoA target molecule numbers in dilution series of environmental DNA extracts according to the work of Stubner (43). The lowest dilution not inhibited was used for further measurements (also discussed in reference 43).

Statistical data analysis.A pairwise t test was performed to evaluate significant differences between target molecule and cell numbers. Calculations were performed with Excel, version 7.0 (Microsoft).

Phylogeny of methanotrophic Proteobacteria.Since additional methanotrophic genera have been described since the study of Holmes et al. (25), we compared the phylogenies of pmoA amino acid sequences and 16S rDNA sequences. For this analysis we used mainly MOB, for which both pmoA and 16S rDNA sequences are presently available in databases. The phylogenetic trees based on these two genes are very similar (Fig. 1). For Methylosphaera hansonii only 16S rDNA sequences were available. Despite the fact that Methylocella palustris is the most closely related MAB, it cannot be included in this group because it does not posses a pMMO (9). The forest clones represent a pmoA sequence cluster retrieved only from environmental studies. Therefore, no 16S rDNA sequences are known.

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FIG. 1.

Comparison of phylogenies based on 16S rDNA and pmoA and amoA. Trees A (16S rDNA) and B (pmoA) are quartet PUZZLE trees. Thick branches are those confirmed by maximum-likelihood analysis. Bars represent 10% sequence divergence. Shaded areas indicate groups defined in the text (I, Methylococcus group; II, Methylobacter/Methylosarcina group; III, Methylosinus group; IV, Methylocapsa group; V, forest clones). Names of species targeted by the real-time PCR assays are boldfaced.

The 16S rDNA tree (Fig. 1A) revealed four groups within the α and γ subclasses of Proteobacteria. To the α subclass belong the Methylosinus group (classical type II; Methylocystaceae) (14, 30) and the Methylocapsa group. The methanotrophic bacteria of the γ subclass of Proteobacteria include the Methylobacter/Methylosarcina group (Methylosarcina, Methylomonas, Methylobacter, Methylomicrobium, and Methylosphaera; classical type I methanotrophs) and the Methylococcus group (Methylococcus, Methylocaldum, and “Methylothermus” strain HB (3). Within the Methylobacter/Methylosarcina group the genera Methylobacter and Methylomicrobium could not be clearly affiliated, resulting in a tree with multiple branchings. In agreement with the description of Bowman et al. (5), Methylosphaera belongs to the Methylobacter/Methylosarcina group.

The phylogenetic analysis of pmoA amino acid sequences showed the same four groups as that of the 16SrDNA sequences (Fig. 1B). However, a fifth group comprising the forest clones was found in addition. This group affiliates with pmoA of MAB within the α subclass of Proteobacteria. It consists only of molecularly retrieved sequences from oxic forest soils (17, 25). On the basis of the combined phylogenetic analyses, all MAB can be divided into the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clone group.

pmoA targeted real-time PCR assays.Real-time PCR assays were developed to detect the phylogenetic groups defined in Fig. 1B by using pmoA as a marker gene (Table 1). For most assays the forward primer A189F, which aligns with all known pmoA sequences, was chosen. Group-specificity was achieved for most assays by designing group-specific reverse primers (Table 1). A general assay for MAB, the MTOT assay, was established using primer Mb661R (7).

The Probematch analysis in ARB showed that the MTOT assay detects almost all methanotrophic bacteria but not the sequences of Methylomonas, Methylocaldum, or the forest clones. MTOT furthermore excludes all known amoA sequences of AAOB (except for Nitrosococcus). MBAC targets all sequences within the Methylobacter/Methylosarcina group. The closest related nontarget organisms are Methylocaldum spp. (three mismatches), while for Methylosphaera hansonii no sequence data are available. The target group of MCOC is the Methylococcus group. The closest nontarget organism is a Methylosinus-affiliated sequence (one mismatch). The TYPEII assay covers all sequences affiliated with the Methylosinus group. The closest nontarget sequences belong to forest clones (two mismatches). The MCAP assay detects the pmoA sequence of Methylocapsa acidiphila strain B2. It has two mismatches to sequences of the Methylosinus group and the Methylobacter/Methylosarcina group. The FOREST assay detects sequences from the forest clone sequence group. It has at least three mismatches to sequences of the Methylococcus group and of Methylocapsa. Recently, potential paralogues of pmoA have been found in several methanotrophic strains (10). Probematch analysis in ARB revealed that the assays developed discriminate against these sequences.

For each assay, primer concentrations and annealing temperatures were experimentally optimized to obtain specific amplification (Table 1). The resulting conditions were experimentally checked by using genomic DNAs of positive- and negative-control strains. Amplification of PCR products of the correct sizes was obtained only from target organisms, not from the nontarget organisms tested (Table 2). Detection limits were determined from at least four independent measurements from dilution series of positive-control DNA (10⁷ to 10¹ target molecules per reaction). A minimum sensitivity of 10¹ to 10² target molecules per reaction for each assay was achieved.

To measure the impact of nonspecific genomic DNA on the real-time PCR measurement, target DNA was mixed with DNA of nontarget organisms. A total of 5 × 10⁶ or 5 × 10⁴ target molecules of assay MBAC were mixed with genomic DNA of E. coli (330 ng μl⁻¹) and, in a second case, with DNA of Methylococcus capsulatus (30 ng μl⁻¹). As a control the same amount of target molecules was measured without the addition of genomic DNA. It was found that genomic DNA from E. coli had no effect on the measurement of target DNA, even if there were 100-fold more genomes than target molecules in the assays (data not shown). The presence of genomic DNA of the methanotrophic nontarget organism also did not influence the measurement (data not shown).

Correlation of target molecule numbers with cell numbers.To evaluate the correlation of pmoA target molecule numbers with cell numbers, a soil was spiked with a mixed methanotrophic culture with defined numbers of cells. The DNA extracts were afterwards analyzed with assays TYPEII, MCOC, and MBAC (Table 3). We assume that at least 2 copies of pmoA per cell can be expected (39). With this assumption, cell numbers can be calculated from target molecule numbers. Measurements of four independent DNA extracts with the MCOC and MBAC assays resulted in cell numbers that were not statistically different from the numbers of cells added prior to DNA extraction (Table 3). However, the number of Methylosinus trichosporium cells determined by the TYPEII assay was equivalent to only 20% (P < 0.01) of the number of cells added (Table 3).

Analysis of the methanotrophic community in samples from a flooded rice field.Soil samples of a flooded rice field were extracted in triplicate and analyzed by real-time PCR (Table 4). The community analysis showed that bacteria of the Methylosinus group (type II MAB) and of the Methylobacter/Methylosarcina group (type I MAB) were the dominant MAB in the soil samples investigated (Table 4). MAB of the Methylococcus group, on the other hand, were less abundant (about 6% of all MAB detected). MAB of the Methylocapsa group and forest clones were below the detection limit (<1.9 × 10⁴ pmoA molecules g of soil⁻¹). The sum for the groups of MAB detected (5.0 × 10⁶ ± 1.4 × 10⁶ pmoA molecules g⁻¹) was significantly higher (P < 0.05) than that obtained with the MTOT assay (0.9 × 10⁶ ± 0.3 × 10⁶ pmoA molecules g⁻¹).