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  1. Evaluation Summary:

    This manuscript tackles the important question of what proteins regulate asymmetrical cell division in Mycobacteria. This will be of interest to all individuals interested in bacterial physiology. The data are sound, but some of the conclusions need to be tempered or bolstered, in relation to the models proposed.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    In this manuscript, the authors build off prior work identifying LamA as a mycobacterial protein required for asymmetrical cell division. The authors identify PgfA as a LamA protein interaction partner. A PgfA homolog has been studied in corynebacteria where it has channel activity and is involved in lipoglycan synthesis but had not been assigned a function in mycobacteria. The authors show that PgfA is essential in mycobacteria, and interacts with MmpL3, as well as a TMM analog. The data presented are interesting, important for the field, and convincing. However, the authors also make a number of conclusions in the text for which there is no data shown.

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  3. Reviewer #2 (Public Review):

    This study from the Rego group investigates the mechanisms underlying asymmetric growth in mycobacteria. Mycobacteria are notable for their unbalanced growth pattern in which the old pole grows at a faster rate than the new pole. In previous work, Dr. Rego identified LamA as necessary for this unbalanced, or asymmetric, growth pattern although its mechanism of action remained unclear. Here they determine that a LamA interacting protein, MSMEG_317 (now PgfA), is necessary for LamA activation of asymmetric growth. PgfA is recruited to sites of cell wall synthesis, remaining at the old pole in divided cells. Localized to the mycobacterial periplasm, PgfA interacts with MmpL3 which flips mycolic acids-an essential component of the mycobacterial cell envelope-across the plasma membrane. Both PgfA and MmpL3 are essential. Significantly, overexpression of PgfA restores asymmetric growth in the absence of LamA. PgfA also interacts with an analog of the mycolic acid, trehalose monomycolate suggesting it might facilitate transport in conjunction with MmpL3. Modulating PgfA accumulation influence the abundance of other lipids suggesting a role in cell envelope homeostasis more broadly.

    Overall, I found the study to be well done and experimentally sound, although significant open questions remain. As the authors note, it is still unclear how a mycolic acid transport-related protein activates cell wall synthesis. Additionally, the means by which PgfA overexpression restores asymmetric growth in the absence of LamA when it is presumably no longer properly recruited to sites of cell wall synthesis also remains an open question. Does PgfA interact differently with the old and new pole independent of LamA? And if so, how? At the same time, there is no question that this work represents a major step forward in our understanding of the factors underlying asymmetric growth in mycobacteria and sets the stage for future work in this area.

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  4. Reviewer #3 (Public Review):

    Gupta and colleagues investigate the function of the PgfA (MSMEG_0317) protein in Mycobacterium smegmatis (Msmeg). This protein was of interest due to previous work showing that it interacts with the LamA protein involved in the asymmetric polar elongation of mycobacteria. Evidence is presented that PgfA is essential for the growth of Msmeg and that it localizes primarily to the old cell pole. This asymmetric localization as well as the asymmetric localization of the trehalose monomycolate (TMM) flippase MmpL3 was shown to be dependent on LamA. Co-immunoprecipitation was used to show the MmpL3 and PgfA interact. Moreover, cells depleted of PgfA and MmpL3 were shown to have similar terminal phenotypes - the depleted cells lost cell envelope material from their surface and lysed. PgfA depleted cells were also shown to have defective outer membrane by cryo-electron tomography. Crosslinking studies were also used to show that PgfA interacts directly with TMM. Together, these data make a strong case for the involvement of PgfA in the process of mycolic acid transport to the mycomembrane, which is a significant advance in the field of mycobacterial envelope assembly.

    Less convincing were results showing the depletion of PgfA affects the levels of TMM and its derivative TDM (trehalose dimycolate) in cells and that overexpression of PgfA can restore asymmetric polar growth to cells lacking LamA. I was also not convinced by the argument that PgfA and its homolog from related corynebacteria (NCgl2760) have different functions. There are many explanations for the failure of NCgl2760 to complement PgfA inactivation in Msmeg that do not require invoking different functions for the two proteins. Specific protein-protein interactions required for PgfA function could have diverged in the two organisms such that NCgl2760 is unable to interact with its required mycobacterial counterparts. Additionally, the lengths of mycolic acids differ between corynebacteria and mycobacteria, which may make the transporters incompatible across organisms.

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