Defining a critical role of an essential membrane protein in mycolic acid transport in mycobacteria

A major feature of the mycobacterial outer membrane (OM) is the presence of long, branched chain mycolic acids (MAs), which render the OM hydrophobic and impervious against various noxious substances, including antibiotics. While the biosynthesis of MA is well studied, the mechanisms governing its transport from the inner membrane to the OM remain largely elusive. In this study, we characterized the function of MSMEG_0317 in Mycobacterium smegmatis , a membrane protein encoded within a conserved genetic locus that has been implicated in MA metabolism and/or transport. Using a conditional knockout mutant, we demonstrate that msmeg_0317 is essential for mycobacterial growth. Depleting msmeg_0317 from cells blocks the formation of MA species found at the OM, establishing a critical function in MA transport across the cell envelope. We further reveal that MSMEG_0317 exists as stable dimers in vitro that require the presence of its N- and C-terminal transmembrane helices, both of which are important for functionality in cells. Our work defines the essential role of MSMEG_0317 in MA metabolism and/or transport, and offers new insights into cell envelope biogenesis in mycobacteria.