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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). 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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Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria
Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.
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- Award ID(s):
- 1654408
- PAR ID:
- 10091718
- Date Published:
- Journal Name:
- eLife
- Volume:
- 7
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.7554/eLife.37243
