A cell wall synthase accelerates plasma membrane partitioning in mycobacteria
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eLife assessment
This paper addresses an important question: the relationship between the cell wall and other, primarily lipid, based components of the cell envelope. Building on previous work, the authors provide data suggesting that the activity of a PonA2, non-essential peptidoglycan synthase, promotes membrane partitioning through its role in cell wall synthesis. While the data are consistent with this model, the reviewers felt additional experiments are necessary to fully support the authors' conclusions.
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Abstract
Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced both by bilayer-intrinsic factors like molecular composition and by bilayer-extrinsic factors such as interactions with other membranes and solid supports. While cellular membranes can departition in response to bilayer-intrinsic or -extrinsic disruptions, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis spatially and biochemically departitions in response to the fluidizing agent benzyl alcohol, then repartitions upon fluidizer washout. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth during recovery from benzyl alcohol exposure. PonA2’s role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. Active cell wall polymerization promotes de novo membrane partitioning and the completed cell wall polymer helps to maintain membrane partitioning. Our work highlights the complexity of membrane–cell wall interactions and establishes a facile model system for departitioning and repartitioning cellular membranes.
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eLife assessment
This paper addresses an important question: the relationship between the cell wall and other, primarily lipid, based components of the cell envelope. Building on previous work, the authors provide data suggesting that the activity of a PonA2, non-essential peptidoglycan synthase, promotes membrane partitioning through its role in cell wall synthesis. While the data are consistent with this model, the reviewers felt additional experiments are necessary to fully support the authors' conclusions.
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Reviewer #1 (Public Review):
The manuscript by Kado and coworkers investigates the mechanism underlying the partitioning of the cytoplasmic domain of Mycobacteria in specific domains. Earlier work from the authors' laboratories has shown that the membrane consists of two different domains, the intracellular membrane domain (IMD) which is enriched at the pole, and the 'conventional' plasma membrane (termed PM-CW). In work published last year (García-Heredia et al., 2021) the authors described experiments that implicated both an intact cell wall and the polar localizing protein DivIVA in the promotion of membrane compartmentalization in M. smegmatis. The current work provides insight into what it is about the intact cell wall that is really required to facilitate partitioning, by looking at the recovery of membrane partitioning after …
Reviewer #1 (Public Review):
The manuscript by Kado and coworkers investigates the mechanism underlying the partitioning of the cytoplasmic domain of Mycobacteria in specific domains. Earlier work from the authors' laboratories has shown that the membrane consists of two different domains, the intracellular membrane domain (IMD) which is enriched at the pole, and the 'conventional' plasma membrane (termed PM-CW). In work published last year (García-Heredia et al., 2021) the authors described experiments that implicated both an intact cell wall and the polar localizing protein DivIVA in the promotion of membrane compartmentalization in M. smegmatis. The current work provides insight into what it is about the intact cell wall that is really required to facilitate partitioning, by looking at the recovery of membrane partitioning after disruption the membrane fluidizer benzyl alcohol.
In a clever Tn-seq experiment, various genes are identified that are associated with recovery, notably PonA2, a non-essential peptidoglycan synthase. This experiment, and the subsequent characterization of PonA2 as a factor that is important for membrane partitioning, is convincing.
The paper describes an important finding with implications for microbiologists interested in the interplay between peptidoglycan and the membrane, and membrane biologists in general. As the authors state, the study of physical connections between the membrane and matrix or cell wall in live cells is complicated and this study shows how a minimal disruption in peptidoglycan synthesis can affect membrane organization.
However, given the additional role of DivIVA in this process, the claims that the cell wall polymer is "critical" (summary) or "initiates" (title) membrane partitioning are not sufficiently supported by the data.
Strengths:
- The Tn seq experiment and the subsequent confirmation that PonA2 is required for recovery of cells after treatment with benzylalcohol.
- The genetic dissection of PonA2's functionalities, showing that the transglycosylase activity is required for the synthesis of peptidoglycan that allows repartitioning is convincing.
Weakness:
- Since the authors have previously shown a contribution of DivIVA to membrane partitioning (García-Heredia et al., 2021), the authors should consider this in the current work. It is very well possible that, as the authors stated previously, both the PonA2 synthesized cell wall and DivIVA contribute to effective recovery. Given that DivIVA is essential, the Tn-seq experiment will not identify it as a factor contributing to recovery. It is clear that over time, dPonA2 cells recover from the benzyl-alcohol treatment (Fig. 3 - it would also be interesting to see a 12h polarity calculation for dPonA2 as for wild-type in F4C). It is also clear that PonA2 is not the only factor contributing to membrane partitioning, as dPonA2 cells show partitioning before disruption. Importantly, DivIVA does not delocalize upon benzyl alcohol treatment so it is also present at the right location from the moment of wash out to act as a reorganizing factor for membrane partitioning. A recovery experiment in a strain with a DivIVA depletion allele could be very informative and should be included.
- On the one hand, the authors argue that it is "the pre-existing cell wall polymer, rather than active cell wall polymerization" (line 335-6) that is required for membrane partitioning, on the other hand, the authors argue that "PonA2 does not affect membrane-cell wall interaction under basal conditions" (line 215, 378-9). This is counterintuitive - if a priori PonA2 synthesized cell wall is required for recovery (active cell wall polymerization is not needed) one would expect that in a PonA2 knockout strain the interaction between wall and membrane is also (slightly) altered. In fact, several experiments in the paper provide, in my view, an indication for the latter:
o The propidium iodide experiment clearly shows that dPonA2 cells have a membrane that is much more susceptible to damage upon benzyl alcohol treatment. Also, when examining the data, it may be that the TG domain of PonA2 plays a critical role in this process: although the difference in PI positive cells in the population of dponA2 cells compared to TP- ponA2 is non-significant, the difference between the wild-type complementation and the TP- ponA2 allele should also be tested for significance. The distribution in the TP- ponA2 population between the six experiments is rather large and one could argue, looking at the graph, that there is at least a subpopulation in the TP- ponA2 allele that does complement. If so, this would be important as this experiment was done immediately after benzyl alcohol washout and thus does not depend on ongoing activity by the ponA2 mutants.
o There is no comparison between the polarity of marker proteins between wild-type and dponA2 cells. The single cell image in Fig. 4A suggests that Ppm1 is more distributed throughout the dPonA2 cell, whereas in the wild type cell PPm1 is really focused. This would be similar to the slight distribution change in peptidoglycan synthesis (F6C) between wild-type and dPonA2 cells, which is significant.
o The PM-CW fraction (Fig4D) of wild-type cells before benzyl alcohol treatment shows a more focused zone compared to the dPonA2 sample - the latter more resembles the PM-CW fractions of cells treated with benzyl-alcohol.
o The hyperosmotic shock experiment (Fig 4 supplement 2) - it seems that wild-type cells have a larger number of cells that have two plasmolysis bays - 9% with 'subpolar & midcell' bays vs 1% in dPonA2 cells. Also, there is a fraction of cells that have no plasmolysis bay. To me, it would seem better to determine first the % of cells with one or more bays vs cells with no bays, and then compare the number of bays per cell only for the population of cells that show plasmolysis. -
Reviewer #2 (Public Review):
In this work Kado and colleagues analyzed cell membrane partitioning in Mycobacterium smegmatis. Based on the membrane fluidizing effect of benzyl alcohol they did a transposon sequencing that are sensitive to the treatment. Among a group of genes that code for antiporter, they identify the bifunctional PBP PonA2 to be involved in benzyl alcohol sensitivity. Membrane partitioning in domains with higher and lower fluidity seems to depend on the peptidoglycan cell wall. In particular, de novo partitioning depends on preexisting cell wall, but not on the active synthesis. The authors use a variety of techniques to support their claims.
The authors claim that the membrane in Msmeg is partitioned in IMDs (intracellular membrane domain) and a PM-CW (apparently a more rigid membrane domain). I know that the term …
Reviewer #2 (Public Review):
In this work Kado and colleagues analyzed cell membrane partitioning in Mycobacterium smegmatis. Based on the membrane fluidizing effect of benzyl alcohol they did a transposon sequencing that are sensitive to the treatment. Among a group of genes that code for antiporter, they identify the bifunctional PBP PonA2 to be involved in benzyl alcohol sensitivity. Membrane partitioning in domains with higher and lower fluidity seems to depend on the peptidoglycan cell wall. In particular, de novo partitioning depends on preexisting cell wall, but not on the active synthesis. The authors use a variety of techniques to support their claims.
The authors claim that the membrane in Msmeg is partitioned in IMDs (intracellular membrane domain) and a PM-CW (apparently a more rigid membrane domain). I know that the term IMD has been used before, but I find this misleading. Intracellular means that something is within the cell. Here we are talking about different fluidities within the 2D space of the membrane. I do not think that this term is meaningful and should be used.
The authors suggest that PonA2 regulates the density (or heterogeneity - I assume the authors mean degree of crosslinking?) of the peptidoglycan, thereby influencing membrane partitioning (lines 371-372). This claim would require a PG analysis and a comparison of the cross-linking degree. The influence of PonA2 on membrane partitioning remains somewhat unclear. While the authors claim that PonA2 was also shown to provide a protective effect against other stresses, such as heat, it is not certain that this has to do with membrane partitioning. Although increase in temperature has certainly an effect on membrane dynamics, heat also triggers unfolded protein response. Bacteria furthermore adapt their membranes quickly to changes in temperature and likely adaption also takes place when other stressors influence membrane fluidity. Also, only the TG defective PonA2 led to the phenotype and not the TP mutation, which would argue against a change in crosslinking.
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