DETECTION OF SOLUBLE METHANE MONOOXYGENASE PRODUCING METHYLOSINUS-TRICHOSPORIUM OB3B BY POLYMERASE CHAIN-REACTION

The soluble methane monooxygenase (sMMO) enzyme complex of methanotrop hs cometabolizes haloaliphatic compounds such as trichloroethylene. Tw o 18-mer oligonucleotides as primary primers and a nested primer of th e same length were selected to amplify specific DNA sequences of the s MMO gene cluster using polymerase chain reaction (PCR). Two DNA fragme nts of sizes 270 and 400 base pairs were obtained when purified DNA fr om the methanotroph Methylosinus trichosporium OB3b was used as templa te. 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 wer e successfully amplified in groundwater (containing native microbial p opulation) when seeded with M. trichosporium, FP1 sense (5'-ATGTCCAGCG CTCATAAC-3'), RP1 antisense (5'-TCAGATCTCGGTCAGGGC-3'), FP2 sense nest ed (5'GCCATCATCGGTCAGGGC-3'), and FP2 sense nested (5'-GCCATCATCGAGGAC ATC-3').