Screening for soluble methane monooxygenase in methanotrophic bacteria using combined molecular and biochemical methods for hydroxylase detection.

摘要

Three well known methanotrophic bacteria (Methylosinus trichosporium OB3b, Methylocystis sp. WI 14, and Methylocystis sp. GB 25) and three newly isolated methanotrophic bacteria (Methylocystis sp. WI 11, Methylocystis sp. X, and FI-9) were screened for sMMO considering the existence of hydroxylase (component A) genes as well as its gene expression. For these purposes monoclonal antibodies that specifically recognize each subunit of the hydroxylase of Methylocystis sp. WI 14 (alpha-subunit [9E5/F2], beta-subunit [4E2/G11], gamma-subunit [10G3/D7]) were produced. PCR amplification using well known primers showed that the hydroxylase encoding genes appear to be only present in M. trichosporium OB3b, Methylocystis sp. WI 11 and WI 14, and in the isolate FI-9. Western and ELISA analysis using the monoclonal antibodies revealed that all subunits of hydroxylase were present. However, in FI-9, only the alpha-subunit of the hydroxylase might be expressed. Surprisingly, in Methylocystis sp. GB 25, where no sMMO activity and no amplification with sMMO specific primers was obtained, the antibody 4E2/G11 recognized a protein band with exactly the same molecular mass as the beta-subunit of the hydroxylase. Methylocystis sp. X showed no positive reaction in any of the tests. In combination with the detection methods currently used, the described antibodies provide a powerful tool for detecting even partially expressed hydroxylase genes.