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Remodeling the notoriously impenetrable mycobacterial outer membrane with synthetic lipids enhances cellular permeability, sensitizing bacteria to the clinically used antibiotic rifampicin. 

Mycobacteria, including the human pathogen Mycobacterium tuberculosis , grow by inserting new cell wall material at their poles. This process and that of division are asymmetric, producing a phenotypically heterogeneous population of cells that respond non-uniformly to stress (Aldridge et al., 2012; Rego et al., 2017). Surprisingly, deletion of a single gene – lamA – leads to more symmetry, and to a population of cells that is more uniformly killed by antibiotics (Rego et al., 2017). How does LamA create asymmetry? Here, using a combination of quantitative time-lapse imaging, bacterial genetics, and lipid profiling, we find that LamA recruits essential proteins involved in cell wall synthesis to one side of the cell – the old pole. One of these proteins, MSMEG_0317, here renamed PgfA, was of unknown function. We show that PgfA is a periplasmic protein that interacts with MmpL3, an essential transporter that flips mycolic acids in the form of trehalose monomycolate (TMM), across the plasma membrane. PgfA interacts with a TMM analog suggesting a direct role in TMM transport. Yet our data point to a broader function as well, as cells with altered PgfA levels have differences in the abundance of other lipids and are differentially reliant on those lipids for survival. Overexpression of PgfA, but not MmpL3, restores growth at the old poles in cells missing lamA . Together, our results suggest that PgfA is a key determinant of polar growth and cell envelope composition in mycobacteria, and that the LamA-mediated recruitment of this protein to one side of the cell is a required step in the establishment of cellular asymmetry. 

Abstract Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green‐emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose‐based fluorogenic probe featuring a molecular rotor turn‐on fluorophore with bright far‐red emission (RMR‐Tre). RMR‐Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no‐wash, low‐background fluorescence detection of live mycobacteria. Aided by the red‐shifted molecular rotor fluorophore, RMR‐Tre exhibited up to a 100‐fold enhancement inM. tuberculosislabeling compared to existing fluorogenic trehalose probes. We show that RMR‐Tre reports onM. tuberculosisdrug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.