MftG is crucial for alcohol metabolism of mycobacteria by linking mycofactocin oxidation to respiration

Mycofactocin is a redox cofactor essential for the alcohol metabolism of Mycobacteria. While the biosynthesis of mycofactocin is well established, the mftG gene, which encodes an oxidoreductase of the glucose-methanol-choline superfamily remained functionally uncharacterized. Here, we show that MftG enzymes strictly require mft biosynthetic genes, and are found in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ?mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin re-oxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria. Overall design: In order to access the impact of ethanol and glucose on the mutant strain lacking the mftG gene on Mycolicibacterium smegmatis genome MC2 155, transcriptomic data of the WT strain and mutant growing until exponential phase in each carbon source was obtained. Biological triplicates of M. smegmatis WT and ?mftG strains were incubated in the two different conditions (glucose and ethanol). The wt grown on each condition (carbon source) was defined as control condition.