Small proteins CrgA and Rv0207c modulate the essential mycolic acid transporter MmpL3

MmpL protein complexes are major contributors to lipid/metabolite and drug transport in Mycobacterium tuberculosis and related mycobacteria and are important targets in current drug discovery efforts. At the core of these complexes are proton-motive force driven transporters belonging to the Resistance-Nodulation-cell Division superfamily of proteins that enable export of various substrates across complex bacterial cell envelopes. This study focuses on MmpL3, which transports trehalose monomycolate in M. tuberculosis and related bacterial species and plays an essential role in the assembly of the mycobacterial outer membrane. Association of MmpL3 with proteins involved in biosynthesis of mycolic acids, cell wall glycans and cell division pathways placed this transporter at the center of a complex crosstalk between various cell envelope biosynthetic processes. Here, we characterized functional interaction of MmpL3 with CrgA, a key transmembrane cell division regulatory protein, and Rv0207c, a cytoplasmic conserved mycobacterial protein. We analyzed how inactivation of CrgA and Rv0207c in vivo and their interactions with MmpL3 in vitro affect the MmpL3-dependent lipid transport activities. We found that the two proteins modulate the activities of MmpL3 and its interactions with substrates and inhibitors in a similar manner by preventing ligands access to their binding sites in the transporter. Our results provide biochemical evidence that CrgA and Rv0207c function in the same pathway and coordinate the transport activity of MmpL3 with polar cell growth and division.