An essential periplasmic protein coordinates lipid trafficking and is required for asymmetric polar growth in mycobacteria

Abstract

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.

MSMEG_0311 is a conserved essential polar protein involved in mycobacterium cell wall metabolism

“…2B and 2C). This result is in conformity with a recently published report (Bosch et al, 2021; Gupta & Gwin, 2022). MSMEG_0319 knockdown strain showed a growth defect on solid medium, forming tiny colonies on agar plates (Fig.…”

“…Hence, we subjected the MSMEG_0311 K.D strain to the RADA pulse. A knockdown strain of mmpl served as a positive control as it is known to influence polar asymmetric growth in mycobacteria (Gupta & Gwin, 2022). It was observed that in absence of ATc, MSMEG_0311 K.D and mmpl K.D strain grew asymmetrically from one pole, as expected (Fig.…”

“…The locus comprises of MSMEG_0310, MSMEG_0311, MSMEG_0315, MSMEG_0317 and MSMEG_0319. Though, the exact function of these genes are unknown, there are reports of MSMEG_0319 and MSMEG_0317 impacting the transport of trehalose monomycolate (TMM) (Belardinelli et al, 2016; Gupta & Gwin, 2022). To the best of our knowledge, there is no literature available on the role of MSMEG_0315 and MSMEG_0311, while MSMEG_0310 is annotated as a pseudogene in M. smegmatis.…”

Defining a critical role of an essential membrane protein in mycolic acid transport in mycobacteria

“…Such function(s) may explain why removal of MSMEG_0317 also apparently affects LM/LAM biosynthesis (13). We note that a very recent report proposed a possible role for MSMEG_0317 in coordinating processes important for polar growth in mycobacteria (23). The role of MSMEG_0317 in TMM transport and/or envelope biogenesis requires further investigations.…”

Polar protein Wag31 both activates and inhibits cell wall metabolism at the poles and septum

“…Our data suggest that Wag31 may have a minor role in controlling the timing or rate of septation. Wag31 had previously been shown to localize to the cell division site after FtsZ has left ( Gupta et al, 2022 ) but before septation is completed ( Santi et al, 2013 ), and overexpression of Wag31-GFP was shown to inhibit and mis-localize septa ( Nguyen et al, 2007 ). Previous work showed that the Wag31-FtsI interaction protects FtsI from intra-membrane proteases under oxidative stress through Wag31 residues 46–48 (NSD) ( Mukherjee et al, 2009 ).…”