Identification of a two-component regulatory system controlling methanol dehydrogenase synthesis in Paracoccus denitrificans.

Upstream of the moxFJGIR genes of Paracoccus denitrificans a regulatory region involved in methanol oxidation was identified. The nucleotide sequence of this region was determined and revealed three genes, moxZ, moxY and moxX, which are transcribed opposite to moxF and which encode proteins of 16.4, 48.2 and 24.5 kDa, respectively. Computer alignment analysis revealed that the gene products of moxY and moxX have homology with the protein histidine kinases and the response regulators, respectively, forming the two-component regulatory systems. No significant homology of the moxZ gene product with any known protein, sequenced thus far, was found. The MoxZ, MoxY and MoxX proteins were identified in Escherichia coli in a heterologous expression system. Mutants with an insertion of a kanamycin-resistance marker in moxZ, moxY and moxX were isolated. These mutant strains were unable to grow on methanol while growth on methylamine was not affected. In the moxZ mutant both subunits of methanol dehydrogenase and cytochrome c551i were not synthesized, methanol dehydrogenase activity was absent, and hardly any expression of a moxZ-lacZ transcriptional fusion was found. Complementation of the mutation was observed after addition of the three genes moxZ, Y and X, in trans. This indicates that the two-component regulatory system is involved in activation of the moxF promoter. A mutant with an unmarked deletion in moxZ was isolated. This mutant showed reduced growth on methanol relative to the wild type. Expression of the moxF-lacZ transcriptional fusion gene and methanol dehydrogenase activity in this strain were also lower than those found in the wild type. Therefore, besides the two proteins of the two-component regulatory pair, a third protein, MoxZ, appears to be involved in regulation of methanol dehydrogenase synthesis.