Detection of Methane Oxidizing Bacteria in Forest Soil by Monooxygenase PCR Amplification

Atmospheric methane oxidation by a spruce forest soil from Norway at 15 degrees C was found to be maximal at a depth of ca 7 cm. Examination of the kinetics of this methane oxidation revealed an apparent K(m) of 403.1 nM and a V(max) of 2.2 nmol g(-1) dry weight soil h(-1). The low apparent K(m) suggested the presence of active methane oxidizing bacteria with a high affinity for methane. DNA was extracted from the 5-10 cm horizon, purified, and subjected to PCR amplification with primers directed toward the monooxygenase genes pmoA and amoA, which are essential for methane oxidation. Hybridization analysis of the clone library subsequently constructed revealed that 49% of the 76 cloned PCR fragments were putative methanotroph pmoA sequences and 16% were putative ammonium oxidizing nitrifier amoA sequences. Sequencing of 28 clones identified three major groups showing homology to pmoA from Methylococcus capsulatus, beta-subdivision ammonia-oxidizers (amoA), and a new group of monooxygenase pmoA/amoA sequences.