Detection of methane oxidizing bacteria in forest soil by monooxygenase PCR amplification

Atmospheric methane oxidation by a spruce forest soil from Norway at 15 deg rees C was found to be maximal at a depth of ca 7 cm. Examination of the ki netics 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 sugges ted the presence of active methane oxidizing bacteria with a high affinity for methane. DNA was extracted from the 5-10 cm horizon, purified, and subj ected to PCR amplification with primers directed toward the monooxygenase g enes pmoA and amoA, which are essential for methane oxidation. Hybridizatio n analysis of the clone library subsequently constructed revealed that 49% of the 76 cloned PCR fragments were putative methanotroph pmoA sequences an d 16% were putative ammonium oxidizing nitrifier amoA sequences. Sequencing of 28 clones identified three major groups showing homology to pmoA from M ethylococcus capsulatus, beta-subdivision ammonia-oxidizers (amoA), and a n ew group of monooxygenase pmoA/amoA sequences.