Screening for soluble methane monooxygenase in methanotrophic bacteria using combined molecular and biochemical methods for hydroxylase detection. 2003

Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada. Stephan.Grosse@nrc.ca

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.

UI MeSH Term Description Entries
D008403 Mass Screening Organized periodic procedures performed on large groups of people for the purpose of detecting disease. Screening,Mass Screenings,Screening, Mass,Screenings,Screenings, Mass
D008697 Methane The simplest saturated hydrocarbon. It is a colorless, flammable gas, slightly soluble in water. It is one of the chief constituents of natural gas and is formed in the decomposition of organic matter. (Grant & Hackh's Chemical Dictionary, 5th ed)
D009281 Naphthalenes Two-ring crystalline hydrocarbons isolated from coal tar. They are used as intermediates in chemical synthesis, as insect repellents, fungicides, lubricants, preservatives, and, formerly, as topical antiseptics.
D010105 Oxygenases Oxidases that specifically introduce DIOXYGEN-derived oxygen atoms into a variety of organic molecules. Oxygenase
D004797 Enzyme-Linked Immunosorbent Assay An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed. ELISA,Assay, Enzyme-Linked Immunosorbent,Assays, Enzyme-Linked Immunosorbent,Enzyme Linked Immunosorbent Assay,Enzyme-Linked Immunosorbent Assays,Immunosorbent Assay, Enzyme-Linked,Immunosorbent Assays, Enzyme-Linked
D006899 Mixed Function Oxygenases Widely distributed enzymes that carry out oxidation-reduction reactions in which one atom of the oxygen molecule is incorporated into the organic substrate; the other oxygen atom is reduced and combined with hydrogen ions to form water. They are also known as monooxygenases or hydroxylases. These reactions require two substrates as reductants for each of the two oxygen atoms. There are different classes of monooxygenases depending on the type of hydrogen-providing cosubstrate (COENZYMES) required in the mixed-function oxidation. Hydroxylase,Hydroxylases,Mixed Function Oxidase,Mixed Function Oxygenase,Monooxygenase,Monooxygenases,Mixed Function Oxidases,Function Oxidase, Mixed,Function Oxygenase, Mixed,Oxidase, Mixed Function,Oxidases, Mixed Function,Oxygenase, Mixed Function,Oxygenases, Mixed Function
D000911 Antibodies, Monoclonal Antibodies produced by a single clone of cells. Monoclonal Antibodies,Monoclonal Antibody,Antibody, Monoclonal
D001419 Bacteria One of the three domains of life (the others being Eukarya and ARCHAEA), also called Eubacteria. They are unicellular prokaryotic microorganisms which generally possess rigid cell walls, multiply by cell division, and exhibit three principal forms: round or coccal, rodlike or bacillary, and spiral or spirochetal. Bacteria can be classified by their response to OXYGEN: aerobic, anaerobic, or facultatively anaerobic; by the mode by which they obtain their energy: chemotrophy (via chemical reaction) or PHOTOTROPHY (via light reaction); for chemotrophs by their source of chemical energy: CHEMOLITHOTROPHY (from inorganic compounds) or chemoorganotrophy (from organic compounds); and by their source for CARBON; NITROGEN; etc.; HETEROTROPHY (from organic sources) or AUTOTROPHY (from CARBON DIOXIDE). They can also be classified by whether or not they stain (based on the structure of their CELL WALLS) with CRYSTAL VIOLET dye: gram-negative or gram-positive. Eubacteria
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D012995 Solubility The ability of a substance to be dissolved, i.e. to form a solution with another substance. (From McGraw-Hill Dictionary of Scientific and Technical Terms, 6th ed) Solubilities

Related Publications

Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
January 1997, Microbial ecology,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
September 1997, Archives of biochemistry and biophysics,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
January 2007, Bioscience, biotechnology, and biochemistry,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
October 2019, Science advances,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
March 2022, Journal of microbiology and biotechnology,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
April 2004, Biochemistry,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
November 2002, Applied and environmental microbiology,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
May 2005, Journal of the American Chemical Society,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
October 2002, Current opinion in chemical biology,
Stephan Grosse, and Claudia Mueller, and Gerlind Rogge, and Karin-Dagmar Wendlandt, and Carlos B Miguez, and Hans-Peter Kleber
March 2006, Biochemistry,
Copied contents to your clipboard!