Molecular ecology of methanotrophs in a forest soil

Upland soils are a significant sink of atmospheric methane, but the organisms responsible for methane consumption have yet to be identified. The ecology of methanotrophs was investigated in a beech forest soil which exhibited atmospheric-methane-uptake. Maximal methane-oxidation was observed in the upper mineral layer between the organic and inorganic horizons at a rate of 3.1 +/- 0.3 nmoles CH 4 g [fresh wt soil]-1 d-1. A clone bank of the methanotroph pmoA gene was constructed by PCR amplification from soil DNA extracts. The PCR primers used coamplify the related amoA gene of ammonia-oxidizers. The clones recovered grouped into three clusters: Nitrosospira-like sequences, a group somewhat related to alpha-Proteobacteria methanotrophs and previously referred to as the RA14 group, and a cluster which could not be characterized as either amoA or pmoA sequences. No pmoA genes closely related to genera of cultured methanotrophs were obtained. The 16S rDNA was also targeted using eubacterial and methanotroph-specific primers. 16S rDNA sequence analysis revealed the presence of organisms distantly related to known methanotrophs. Methanotroph enrichment cultures were established by inoculating mineral salts medium with soil and incubating under an atmosphere of 10% methane in air. Gene sequencing from the enrichments indicated the presence of organisms belonging to the genera Methylosinus and Methylocystis. The results suggest that the dominant methanotrophs in the forest soil are not related to known organisms and do not grow under conditions typically used to isolate methanotrophs.