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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; Richardson et al., 2016). 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.

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No abstract

Protein export pathways are important for bacterial physiology among pathogens and non-pathogens alike. This includes the Twin-Arginine Translocation (Tat) pathway, which transports fully folded proteins across the bacterial cytoplasmic membrane. Some Tat substrates are virulence factors, while others are important for cellular processes like peptidoglycan remodeling. Some bacteria encode more than one copy of each Tat component, including the Gram-negative soil isolate Acinetobacter baylyi. One of these Tat pathways is essential for growth, while the other is not. We constructed a loss-of-function mutation to disrupt the non-essential tatC2 gene and assessed its contribution to cell growth under different environmental conditions. While the tatC2 mutant grew well under standard laboratory conditions, it displayed a growth defect and an aberrant cellular morphology when subjected to high temperature stress including an aberrant cellular morphology. Furthermore, increased sensitivities to detergent suggested a compromised cell envelope. Lastly, using an in vitro co-culture system, we demonstrate that the non-essential Tat pathway provides a growth advantage. The findings of this study establish the importance of the non-essential Tat pathway for optimal growth of A. baylyi in stressful environmental conditions.