Aerobic gram-negative methylotrophs oxidize methanol to formaldehyde by using a methanol dehydrogenase that has pyrroloquinoline quinone (PQQ) as a prosthetic group. Seventy-two mutants which are unable to grow on methanol unless the growth medium is supplemented with PQQ have been isolated in the facultative methanol utilizer Methylobacterium extorquens AM1. In addition, 12 previously isolated methanol oxidation mutants of M. extorquens AM1 were shown to be able to grow on methanol in the presence of PQQ. These putative PQQ biosynthesis mutants have been complemented by using previously isolated clones containing M. extorquens AM1 DNA, which were known to contain genes necessary for oxidation of methanol to formaldehyde (mox genes (27). MDH is a tetrameric enzyme located in the periplasm that contains pyrroloquinoline quinone (PQQ) as the prosthetic group and also contains Ca2+ (37,40). PQQ was first identified as the prosthetic group of MDH and is now also known to be the prosthetic group of a few other bacterial dehydrogenases that oxidize alcohols or sugars (9). The biosynthetic pathway of PQQ has not yet been determined, but the biosynthetic precursors are known to be tyrosine and glutamate (19).

Two-hundred-and-eight new Methylobacterium extorquens AM1 methanol oxidation (Mox) mutants were isolated and placed into complementation groups. Complementation analyses identified new Mox groups in the Mxb and Mxc loci and at a new locus, Mxd. Thirty-seven mutants at the Mxb locus were divided into MxbM and MxbD complementation groups on the basis of their complementation pattern. Twenty-nine mutants at the Mxc locus fell into three complementation groups, MxcB, MxcQ and MxcE. The direction of transcription for genes at this locus could be inferred from the subclones. Eighteen of the new mutants were not complemented by previously isolated M. extorquens AM1 clones but were complemented by two new overlapping clones. This locus was called Mxd and the mutants fell into two complementation groups, MxdR and MxdS. lmmunoblots from all these mutant classes showed that all of the Mxb and Mxc strains had substantially reduced levels of MxaF (large subunit of methanol dehydrogenase) and cytochrome c,, compared to the wild-type. These mutants, particularly the Mxb mutants, also had elevated levels of cytochrome c-553. These results are consistent with a role for the MxbMD and MxcBQE complementation groups in the regulation of expression of m a F . The MxdR and MxdS mutants had normal levels of MxaF and both c-type cytochromes.