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Kolb, S; Knief, C; Stubner, S; Conrad, R: Quantitative Detection of Methanotrophs in Soil by Novel pmoA-Targeted Real-Time PCR Assays, Applied and Environmental Microbiology, 69(5), 2423-2429 (2003)
Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S rDNA and PmoA (a subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation, and revealed 5 distinct groups of methanotrophs within the a- and g- subclass of Proteobacteria, i.e. the Methylococcus group, Methylobacter/Methylosarcina group, Methylosinus group, Methylocapsa group and forest clones group (cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen™ for each of these 5 groups and for all methanotrophic bacteria targeting the pmoA gene. The detection limits were between 101 and 102 target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. The analysis of the methanotrophic community structure in a flooded rice field soil showed 5.0±1.4 ´106 pmoA molecules g-1 of all methanotrophs. The Methylosinus group was predominant (2.7±1.1 ´106 target molecules g-1). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0±0.9 ´106 target molecules g-1 soil). On the other hand, pmoA affiliated to the forest clones and the Methylocapsa group was below the detection limit of 1.9 ´104 target molecules g-1 soil. Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.
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