Investigation of Methane Oxidizers Community in Soil by Using Denaturant Gradient Gel Electrophoresis

 Methanotrophs in soil serves as a major atmospheric methane sink, and soil management has a direct impact on the diversity of microorganisms in soil. This study was set to identify methanotrophs in soil under different management systems. The study was conducted at the Ohio Agricultural Research and Development Center (OARDC), Ohio State University (OSU), Wooster, Ohio, US as part of their long continuous research in No-Till production. Twenty-four samples were collected during winter (February, 2012) from different ecological sites; (1) no-tillage, (2) tillage, (3) grassland, and (4)
forest. Direct DNA extraction enabled specific amplification of pmoA, encoding a subunit of the particulate methane monooxygenase encoding gene, and the 16S rRNA genes of methanotrophs for direct identification.
 Primers targeting pmoA and 16S rRNA genes of both type-I and type-II methanotrophs were all tested via in silico polymerase chain reaction (PCR). The primers which gave best results and were chosen for this study are the A189f-A682r and A189f-mb661 primer sets for specific amplification of pmoA; and type IF-type IR and type IIF-type IIR for the amplification of the 16S rRNA
genes of type-I and type-II methanotrophs, respectively. PCR was successful in amplifying all targeted genes. The utilized primers along with the thermocycling conditions were optimized for analysis by denaturant gradient gel electrophoresis (DGGE). A semi-nested approach proved to be more efficient in obtaining better amplicons. Bands from DGGE profile were all purified from gel, re-amplified, re-resolved in gel to assess bands purity. The resultant bands were purified and sent for sequencing.  The retrieved sequences were all aligned with matched database sequences, and by utilizing bioinformatics tools, the sequences were grouped based on bootstrapping method. Profile of the functional gene were able to retrieve diverse groups of methanotrophs, including Methylobacter,
Methylomonas, Methylocystis, Methylomicrobium, Methylococcus, in addition to a number of uncultured methanotrophs. The primers used to amplify the 16S rRNA genes were able to detect; Methylomonas, Methylomicrobium, Methylosarcina, Methylobacter, Methylococcus, and Methylocaldum within type-I category, and Methylosinus, Methylocystis, Methylocapse, and Methylocella within type-II category, respectively. These results were all depicted as phylogenetic trees.  Further analysis of methanotrophs using terminal restriction fragment length polymorphism (T-RFLP) technique by targeting pmoA gene was attempted. Electropherograms were successfully generated with four four-base cutter enzymes. The lack of a functional gene-specific database has led to not getting meaningful informations regarding those data. However, these data, along with other collected data, is considered valuable resources toward creating a database for this gene.