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Applied and Environmental Microbiology, May 2003, p. 2423-2429, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2423-2429.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Steffen Kolb, Claudia Knief, Stephan Stubner, and Ralf Conrad*

Max-Planck-Institut für Terrestrische Mikrobiologie, 35043 Marburg, Germany

Received 5 August 2002/ Accepted 6 February 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 ribosomal DNA and pmoA ({alpha} subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the {alpha} and {gamma} subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. 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. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 ± 1.4) x 106 pmoA molecules g-1 for all methanotrophs. The Methylosinus group was predominant (2.7 x 106 ± 1.1 x 106 target molecules g-1). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 106 ± 0.9 x 106 target molecules g of soil-1). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 104 target molecules g of soil-1. 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.

* Corresponding author. Mailing address: Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany. Phone: 49-6421/178-800. Fax: 49-6421/178-999. E-mail: Conrad{at}staff.uni-marburg.de.

Applied and Environmental Microbiology, May 2003, p. 2423-2429, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2423-2429.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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