Introduction

Methylocella spp. are aerobic facultative methanotrophs (Alphaproteobacteria), with three validated species, M. palustris, M. silvestris and M. tundrae, isolated from acidic soils (Dunfield et al., 2003; Dedysh et al., 2000, 2004). M. silvestris BL2, which can grow on a variety of multicarbon substrates (Dedysh et al., 2005; Theisen et al., 2005), oxidizes methane using the soluble methane monooxygenase encoded by the operon mmoXYBZDC. Methylocella spp. lack the particulate methane monooxygenase encoded by the pmoCAB operon, which is present in all other methanotrophs (Dedysh et al., 2000, 2004; Dunfield et al., 2003). Although a few studies have detected Methylocella-related 16S rRNA genes in different environments (for example, Dunfield et al., 2003; Dedysh et al., 2000, 2004; Chen et al., 2008a, 2008b; Cockell et al., 2009; Fabiani et al., 2009; Han et al., 2009), there is little information on the distribution and abundance of Methylocella. Current molecular techniques for example, pmoA microarray (Bodrossy et al., 2003) and real-time quantitative PCR (qPCR) (Kolb et al., 2003; Tuomivirta et al., 2009), that are commonly applied to study the diversity and abundance of methanotrophs, would not detect Methylocella, as these methods target pmoA. Therefore, we aimed in this study (1) to develop Methylocella 16S rRNA gene-targeting PCR-based assays to assess the environmental distribution of Methylocella, and (2) to design Methylocella mmoX primers and develop a mmoX-targeted qPCR assay to enumerate Methylocella in environmental samples.

Results and discussion

Methylocella-related 16S rRNA gene-targeting PCR assays were developed using the primers Type IIF (Chen et al., 2007) and Mcell-1445 (Dedysh et al., 2005). Primer Type IIF was originally designed to detect 16S rRNA genes of type II methanotrophs, including the genus Methylocella, whereas primer Mcell-1445 was originally designed as a Methylocella 16S rRNA gene fluorescent in situ hybridization probe. PCR conditions were optimized and validated with DNA from pure cultures of known methanotrophs (for methodology, see Supplementary Information). Specificity of these primers to detect Methylocella 16S rRNA genes in DNA extracted from environmental samples was verified by constructing clone libraries using seven samples that were found to be positive for the 16S rRNA gene PCR products of Methylocella either by a direct and/or by a nested PCR approach (see Table 1 for samples analyzed by clone libraries). These seven samples were chosen to cover a wide range of pH values ranging from moderately acidic to very alkaline. Over 82% of the 16S rRNA gene sequences from these clone libraries were closely related (>97% identity) to Methylocella, and the remainder were related to 16S rRNA genes from Alphaproteobacteria and Gammaproteobacteria (Hyphomicrobium, Methylobacter) or Bacteroidetes (Flexibacter) (data not shown). The distribution of these non-Methylocella sequences was random across these samples. DNA extracted from various environmental samples was PCR-screened for Methylocella-related 16S rRNA genes (Table 1). Among 23 samples originating from diverse locations, Methylocella-related 16S rRNA gene PCR products were detected in 15 samples. Of these, six were positive for Methylocella-related 16S rRNA genes using a direct PCR approach. All these six samples were acidic, suggesting a higher abundance of Methylocella in these environments compared with other samples that were more neutral or alkaline in nature and which required a nested PCR approach for detection of Methylocella. In fact Methylocella-related 16S rRNA genes were detected not only from acidic and neutral environments, but also from alkaline environments such as Lonar Lake sediment, India (pH 10.0) (Table 1). These findings suggest that Methylocella is not limited to acidic pH in nature, despite being originally isolated from an acidic soil. Recently Fabiani et al. (2009) and Han et al. (2009) detected Methylocella-related 16S rRNA gene sequences in DNA extracted from an alkaline waste-treatment plant soil (pH 9), Italy, and an alkaline Chinese coal mine soil (pH 9), respectively. On the basis of our survey and the findings of these authors, it is evident that Methylocella are more widely distributed in nature than previously thought. It is interesting to note that no sequences were detected from high-salinity environments. To the best of our knowledge, there are no previously published reports of marine Methylocella sequences, suggesting that salinity may restrict the diversity of this genus. We found Methylocella in soils associated with a wide diversity of vegetation (Table 1), therefore these data do not permit any conclusions to be drawn regarding particular Methylocella–plant associations.

Table 1 PCR-based survey for Methylocella in the environmental samples
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To enumerate Methylocella populations in environmental samples, a Methylocella mmoX gene-targeting SYBR-Green based qPCR assay was developed targeting mmoX specific for Methylocella spp. Functional gene primers mmoXLF (5′-GAAGATTGGGGCGGCATCTG-3′) and mmoXLR (5′-CCC AATCATCGCTGAAGGAGT-3′) were designed based on mmoX sequences available in GenBank using the ARB program (Ludwig et al., 2004). Although activity cannot be inferred from its presence, the use of mmoX rather than the 16S rRNA gene, by restricting the investigation to the functional group being studied, enables a much higher sensitivity of detection in complex environments (McDonald et al., 2008). PCR conditions were optimized and validated with DNA from pure cultures of known methanotrophs (for methodology, see Supplementary Information). Clone library analysis of mmoX PCR products retrieved from five environmental samples (Table 1) indicated that over 84% of the mmoX sequences were closely related (>97% identity) to Methylocella spp. and the remainder were related to mmoX genes of other methanotrophs, for example Methylobacter, Methylocystis or Methylococcus (data not shown). Detection of a certain level of non-target sequences by primers designed to target a particular bacterial genus has earlier been observed (Fierer et al., 2005). The qPCR assay was validated by spiking Ufton landfill cover soil (United Kingdom) with known amounts of M. silvestris BL2 cells (ranging from 103 to 105 cells g−1 soil), and by detecting the mmoX copies from the spiked soil (Figure 1b). Ufton landfill cover soil was chosen for the spiking study because we did not detect any Methylocella-related 16S rRNA genes from this soil, which supports the earlier observation of Héry et al. (2008), who also did not detect Methylocella spp. from this location. The detection limit of the qPCR assay was 103 cells g−1 of soil. The amplification efficiency of the reactions was between 80% and 104% (slope −3.92 to −3.27). Extraction and detection efficiency from the spiked soil was 88–111% (Figure 1b). The presence of Methylocella in amplified qPCR products was confirmed by clone library analysis (data not shown). The abundance of Methylocella detected in selected environmental samples varied from 0.9 (±0.2) × 106 (Colne Estuary sediment, Essex, UK) to 3.3 (±0.6) × 106 cells g−1 (Hornavan, Sweden) (Figure 1a). Methylocella in Lonar Lake sediment (India) and Arctic soil (Svalbard, Arctic) were below the limits of detection. However, it was surprising to see that the abundance of Methylocella in the Svalbard Arctic soil was below the limits of detection, as we detected Methylocella 16S rRNA gene PCR products with these samples using the direct PCR approach. Failure to detect Methylocella-related mmoX genes in Arctic soil (Svalbard, Arctic) might be accounted for by the fact that there is one copy of mmoX in the genome of M. silvestris compared with two copies of the 16S rRNA gene. There are no previous qPCR data on the abundance of Methylocella in the environment; however, using fluorescent in situ hybridization, Dedysh et al. (2001) showed that the numbers of Methylocella spp. in Sphagnum peat was ∼ 106 cells g−1 of wet soil. The abundance of Methylocella that we detected in peat soil (Moor House peat) was 2.3 (±0.6) × 106 copies g−1 of wet soil (Figure 1a), which is in good agreement with the data of Dedysh et al. (2001). In contrast, the total number of pmoA copies in DNA extracted from these selected environmental samples varied from 1.8 (±0.1) × 107 (Colne Estuary sediment, Essex, UK) to 3.8 (±0.5) × 109 pmoA copies g−1 (Hornavan, Sweden) (Supplementary Table 1). The qPCR protocol developed for Methylocella spp., in parallel with investigations into the activity of these organisms, can now be applied to study the effects of environmental parameters, for example availability of alternative carbon sources, salinity, vegetation type or pH, on the distribution and abundance of Methylocella in many environments.

Figure 1
figure 1

(a) The number of mmoX copies detected by qPCR in DNA extracted from soil and sediment. (b) Detection of mmoX copies from the spiked soil. The dotted line shows the theoretical 100% detection efficiency. Error bars indicate the s.d. of triplicate measurements.

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