Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments

The 16S rRNA and pmoA genes from natural populations of methane-oxidizing b acteria (methanotrophs) were PCR amplified from total community DNA extract ed from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes , and approximately 200 clones from each library were analyzed by using res triction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on the ir RFLP patterns, and representatives of each group were sequenced and anal yzed. Studies of the 16S rRNA data obtained indicated that the existing pri mers did not reveal the total methanotrophic diversity present when these d ata were compared with pure-culture data obtained from the same environment . New primers specific for methanotrophs belonging to the genera Methylomon as, Methylosinus, and Methylocystis were developed and used to construct mo re complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pm oA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indi cated that the new primers should detect these genes over the known diversi ty in methanotrophs, In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.