ABSTRACT The lprG -Rv1410c operon is critical for the survival of Mycobacterium tuberculosis during infection, but very little is known about the functions of its proteins. LprG is a lipoprotein, and Rv1410c encodes the major facilitator superfamily small molecule transporter P55. P55 likely exports small molecules outside of the bacterial cell, but the function of LprG is unclear. A deletion of the homologous operon in Mycobacterium smegmatis is more susceptible to ethidium bromide, and drug resistance is restored by the intact operon from M. tuberculosis . The multidrug resistance pump inhibitor reserpine inhibits resistance to ethidium bromide in both wild-type M. smegmatis and the complemented mutant, suggesting that P55-mediated transport is responsible for drug resistance and that ethidium bromide is a novel substrate for P55. In addition to hypersensitivity to ethidium bromide, cells that lack the lprG -Rv1410c operon display abnormal colony morphology and are defective for sliding motility, properties that suggest an alteration of cell wall composition. Strikingly, both ethidium bromide transport and normal cell surface properties require functional P55 and LprG, as neither alone is sufficient to restore function to the deletion mutant. Thus, P55 requires the cell surface lipoprotein for normal function.