Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene.
Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Bath also produces a soluble MMO.
Gray Freshwater Biological Institute, University of Minnesota, Navarre 55392.
H C
HC
Tsien
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Hanson
D001483:Base Sequence; D001673:Biodegradation, Environmental; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D015342:DNA Probes; D004269:DNA, Bacterial; D016521:Electrophoresis, Gel, Pulsed-Field; D008772:Methylococcaceae / Q000201:enzymology / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D012995:Solubility; D014241:Trichloroethylene / Q000378:metabolism*
D015342:DNA Probes; D004269:DNA, Bacterial; D014241:Trichloroethylene; D010105:Oxygenases; C021555:methane monooxygenase