By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus deniriJ'canj. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the S' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AMl. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPdl, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c5531 was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c_511 and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxj deletion mutant strain partly synthesized the latter two proteins, but methanol dehydrogenase activity could not be detected. The moxG deletion mutant strain lacks exclusively cytochrome c.,,,. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c_511 as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome cs_5l is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitifficans. In contrast to earlier suggestions, this cytochrome was found to be different from membranebound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.Paracoccus denitrificans is a gram-negative soil bacterium able to grow heterotrophically on a variety of multicarbon compounds, autotrophically on hydrogen and carbon dioxide, and methylotrophically on one-carbon compounds, such as methanol and methylamine. The physiology of P. denitrificans has recently been reviewed by Van Verseveld and Stouthamer (59) and by Harms and Van Spanning (19). During methyl...