Mycofactocin Is Associated with Ethanol Metabolism in Mycobacteria

Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many assimilates cholesterol during chronic infection, and its growth in the presence of cholesterol requires most of the MFT biosynthesis genes ( , , , , , and ), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in , , and We found that the growth deficit of deletion mutants in medium containing cholesterol-a phenotypic basis for gene essentiality prediction-depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, was indispensable for ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in During ethanol treatment, mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems. Tuberculosis is caused by , and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways-e.g., cholesterol assimilation-that are critical for persistence and for pathogenesis of In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is s