Localization of a Pyrroloquinoline Quinone Biosynthesis Gene Near the Methanol Dehydrogenase Structural Gene in Methylobacterium organophilum DSM 760

Unité de Régulation de l'Expression Génétique, Département de Biochimie et Génétique Moléculaire, Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, France ABSTRACT Summary: A partial Sau 3 AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF , the structural gene for methanol dehydrogenase, was isolated. In M. organophilum , pSUP106 behaves as a suicide plasmid. This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF , by marker exchange. Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R" hybrid plasmids in Escherichia coli HB101. Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M. organophilum. The gene involved, pqqA , was subcloned and localized. Present address: Unité de Génie Microbiologique, Département des Biotechnologies, Institut Pasteur.