Compartment-related aspects of XoxF protein functionality in Methylorubrum extorquens DM4 analysed using its cytoplasmic targeting.

The impact of periplasmic localisation on the functioning of the XoxF protein was evaluated in the well-studied dichloromethane-utilising methylotroph Methylorubrum extorquens DM4, which harbors only one paralogue of the xoxF gene. It was found that the cytoplasmic targeting of XoxF by expression of the corresponding gene without the sequence encoding the N-terminal signal peptide does not impair the activation and lanthanide-dependent regulation of the MxaFI-methanol dehydrogenase genes. Analysis of the viability of ΔxoxF cells complemented with the full-length and truncated xoxF gene also showed that the expression of cytoplasmically targeted XoxF even increases the resistance to acids. These results contradict the proposed function of the XoxF protein as an extracytoplasmic signal sensor. At the same time, the observed dynamics of growth with methanol, as well as with dichloromethane of strains expressing cytoplasmic-targeted XoxF, indicate the probable enzymatic activity of lanthanide-dependent methanol dehydrogenase in this compartment. Herewith, the only available substrate for this enzyme in cells growing with dichloromethane was formaldehyde, which is produced during the primary metabolism of the mentioned halogenated toxicant directly in the cytosol. These findings suggest that the maturation of XoxF-methanol dehydrogenase may occur already in the cytoplasm, while the factors changing affinity of this enzyme for formaldehyde are apparently absent there. Together with the demonstrated functioning of an enhancer-like upstream activating sequence in the promoter region of the xoxF gene in M. extorquens DM4, the obtained information enriches our understanding of the regulation, synthesis and role of the XoxF protein.