Detection of soluble methane monooxygenase producing Methylosinus trichosporium OB3b by polymerase chain reaction

Abstract

The soluble methane monooxygenase (sMMO) enzyme complex of methanotrophs cometabolizes haloaliphatic compounds such as trichloroethylene. Two 18-mer oligonucleotides as primary primers and a nested primer of the same length were selected to amplify specific DNA sequences of the sMMO gene cluster using polymerase chain reaction (PCR). Two DNA fragments of sizes 270 and 400 base pairs were obtained when purified DNA from the methanotroph Methylosinus trichosporium OB3b was used as template. The primers were specific for sMMO sequences of M. trichosporium, since none of the 13 bacterial isolates screened yielded the expected length of PCR-amplified DNA fragments. The detection limit of the PCR method was 5 x 10(2) cells of M. trichosporium. The sMMO sequences were successfully amplified in groundwater (containing native microbial population) when seeded with M. trichosporium, FP1 sense (5'-ATGTCCAGCGCTCATAAC-3'), RP1 antisense (5'-TCAGATGTCGGTCAGGGC-3'), FP2 sense nested (5'GCCATCATCGGTCAGGGC-3'), and FP2 sense nested (5'-GCCATCATCGAGGACATC-3').

Detection of Methanotrophs in Groundwater by PCR

“…The genes encoding sMMO (mmo) have been cloned and sequenced (6,7,26). The sequence information obtained has been used to design primers for PCR that can be used to detect a portion of mmoX in environmental samples (15,17,19). Primers for amplification of a portion of the gene encoding methanol dehydrogenase have been designed and used with environmental samples, but sequencing is required to distinguish products amplified from methanotrophs from products amplified from other methylotrophs (16).…”