A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies.
Mechanistic details of methanol oxidation catalyzed by the periplasmically-located pyrroloquinoline quinone-dependent methanol dehydrogenase of methylotrophs can be elucidated using site-directed mutants. Here, we present an in situ colony assay of methanol dehydrogenase, which allows robotic screening of large populations of intact small colonies, and regrowth of colonies for subsequent analysis.
Department of Bioenergetics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany; Microbial Culture Collection, National Centre for Cell Science, Pune, Maharashtra 411021, India.
Venkata Ramana
VR
Vemuluri
Department of Bioenergetics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany.
Shreya
S
Shaw
Department of Bioenergetics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany.
Caroline
C
Autenrieth
Department of Bioenergetics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany. Electronic address: robin.ghosh@bio.uni-stuttgart.de.
Robin
R
Ghosh
D000429:Alcohol Oxidoreductases / Q000032:analysis*; D001419:Bacteria / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D008403:Mass Screening; D000432:Methanol / Q000378:metabolism; D008828:Microbiological Techniques / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045542:PQQ Cofactor / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism* / Q000382:microbiology; D011809:Quinones
D001426:Bacterial Proteins; D011809:Quinones; D045542:PQQ Cofactor; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D000432:Methanol
Dye-linked assay; Methanol dehydrogenase; Methylotrophs; PQQ; Pyrroloquinoline quinone; Robotic screening