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Molecular analysis of high-affinity methane-oxidizing enrichment cultures isolated from a forest biocenosis and agrocenoses

Kravchenko, I. K., Kizilova, A. K., Bykova, S. A., Men'ko, E. V., Gal'chenko, V.F.
Microbiology 2010 v.79 no.1 pp. 106-114
Methylocella, Methylocystis, Methylosinus, agricultural soils, bacteria, biocenosis, denaturing gradient gel electrophoresis, forest soils, forests, genes, greenhouse gases, methane, methanotrophs, oxidation, polymerase chain reaction, ribosomal RNA, soil sampling
Methane oxidation by microorganisms inhabiting aerobic soils is a key process involved in the regulation of the concentration of this significant greenhouse gas in the atmosphere; however, the microorganisms responsible for this process remain unknown. Three stable methane-oxidizing cultures were isolated from samples of forest soils (FS) and agricultural soils (AS) of Moscow oblast, as well as from soil samples collected from a Belgian agrocenosis (BS). The obtained enrichment cultures exhibit a high affinity for methane; their km values range from 54.2 to 176.8 nM CH₄ and are comparable to those of aerobic soils. Analysis of the fragments of the ribosomal (16S rRNA) and functional (pmoA) genes of methanotrophs by PCR— DGGE and cloning demonstrated the presence of bacteria belonging to the genera Methylocystis in FS, Methylosinus in AS and BS, and Methylocella in BS. It was established that Methylocystis and Methylosinus detected in the enrichment cultures contain the genes encoding the synthesis of the active center of two membrane-bound particulate methane monooxygenases; it is likely that one of these genes (pmoA2) is responsible for the capacity of these microorganisms for oxidation of atmospheric methane.