Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli

Reviewer #1 (Public Review):

Summary:

Liang et. al., uses a previously devised full isotope labeling of peptidoglycan followed by mass spec to study the kinetics of Lpp tethering to PG and the hydrolysis of this bond by YafK.

Strengths:

-The labeling and mass spec analysis technique works very well to discern differentially labelled Tri-KR muropeptide containing new and old Lpp and PG.

Weaknesses:

-Only one line of experimentation using mass spec based analysis of labeled PG-Lpp is used to make all conclusions in the paper. The evidence is also not enough to fully deleanate the role of YafK.
-Only one mutant (YafK) is used to make the conclusion.
-The paper makes a lot of 'implications' with minimal proof to support their hypothesis. Other lines of experimentations must be added to fully delineate their claims.
-Time points to analyse Tri-KR isotopologues in Wt (0,10,20,40,60 min) and yafK mutant (0,15, 25, 40, 60 min) are not the same.
-Experiments to define physiological role of YafK are also missing.

Reviewer #2 (Public Review):

Summary:

The authors of this study have sought to better understand the timing and location of the attachment of the lpp lipoprotein to the peptidoglycan in E. coli, and to determine whether YafK is the hydrolase that cleaves lpp from the peptidoglycan.

Strengths:

The method is relatively straightforward. The authors are able to draw some clear conclusions from their results, that lpp molecules get cleaved from the peptidoglycan and then re-attached, and that YafK is important for that cleavage.

Weaknesses:

However, the authors make a few other conclusions from their data which are harder to understand the logic of, or to feel confident in based on the existing data. They claim that their 5-time point kinetic data indicates that new lpp is not substantially added to lipidII before it is added to the peptidoglycan, and that instead lpp is attached primarily to old peptidoglycan. I believe that this conclusion comes from the comparison of Fig.s 3A and 3C, where it appears that new lpp is added to old peptidoglycan a few minutes before new lpp is added to new peptidoglycan. However, the very small difference in the timing of this result, the minimal number of time points and the complete lack of any presentation of calculated error in any of the data make this conclusion very tenuous. In addition, the authors conclude that lpp is not significantly attached to septal peptidoglycan. The logic behind this conclusion appears to be based on the same data, but the authors do not provide a quantitative model to support this idea.

This work will have a moderate impact on the field of research in which the connections between the OM and peptidoglycan are being studied in E. coli. Since lpp is not widely conserved in gram negatives, the impact across species is not clear. The authors do not discuss the impact of their work in depth.

Author Response

We decided to address the comments of the reviewers with additional experiments and modification of the text with the aim of submitting a new version of the report.

We would like to underline that the current study is an extension of the work published in eLife (Atze et al., 2021). For this reason, and in agreement with eLife guidelines, we did not repeat all the background information on the method used to identify PG subunit isotopologues using mass spectrometry.

Reviewer #1 (Public Review):

Summary:

Liang et. al., uses a previously devised full isotope labeling of peptidoglycan followed by mass spec to study the kinetics of Lpp tethering to PG and the hydrolysis of this bond by YafK.

Strengths:

-The labeling and mass spec analysis technique works very well to discern differentially labelled Tri-KR muropeptide containing new and old Lpp and PG.

Weaknesses:

-Only one line of experimentation using mass spec based analysis of labeled PG-Lpp is used to make all conclusions in the paper. The evidence is also not enough to fully deleanate the role of YafK.

Our approach based on heavy isotope labelling and mass spectrometry has the power to identify and kinetically characterize the specific products of the reaction leading to the tethering of Lpp to PG and the hydrolysis of the corresponding bond. We therefore advocate that our experimentation is sufficient to obtain meaningful results without combining other lines of experimentation.

-Only one mutant (YafK) is used to make the conclusion.

The aim of the study is to determine the effect of the hydrolysis of the PG→Lpp bond on the dynamics of the tethering of Lpp to PG. Since YafK is the only enzyme catalyzing this reaction, it is appropriate to compare the wild-type strain to an isogenic yafK deletion mutant. Nonetheless, we carefully consider this comment and will investigate the dynamics of the tethering of Lpp to PG in mutants deficient in the production of the L,D-transpeptidases responsible for tethering Lpp to PG.

-The paper makes a lot of 'implications' with minimal proof to support their hypothesis. Other lines of experimentations must be added to fully delineate their claims.

See our answer to the first comment.

-Time points to analyse Tri-KR isotopologues in Wt (0,10,20,40,60 min) and yafK mutant (0,15, 25, 40, 60 min) are not the same.

The purpose of the experiments is to compare the kinetics of formation and hydrolysis of the PG→Lpp bond in the WT versus ΔyafK strains. Comparison of the kinetics is therefore possible even though the kinetics are not based on the exact same time points. Nonetheless, we will reproduce the kinetics experiment (see also answers to Reviewer 2) and use the same time points in these additional experiments.

-Experiments to define physiological role of YafK are also missing

We will investigate the effect of the yafK deletion on the formation of outer membrane vesicles.

Reviewer #2 (Public Review):

Summary:

The authors of this study have sought to better understand the timing and location of the attachment of the lpp lipoprotein to the peptidoglycan in E. coli, and to determine whether YafK is the hydrolase that cleaves lpp from the peptidoglycan.

Strengths:

The method is relatively straightforward. The authors are able to draw some clear conclusions from their results, that lpp molecules get cleaved from the peptidoglycan and then re-attached, and that YafK is important for that cleavage.

Weaknesses:

However, the authors make a few other conclusions from their data which are harder to understand the logic of, or to feel confident in based on the existing data. They claim that their 5-time point kinetic data indicates that new lpp is not substantially added to lipidII before it is added to the peptidoglycan, and that instead lpp is attached primarily to old peptidoglycan. I believe that this conclusion comes from the comparison of Fig.s 3A and 3C, where it appears that new lpp is added to old peptidoglycan a few minutes before new lpp is added to new peptidoglycan. However, the very small difference in the timing of this result, the minimal number of time points and the complete lack of any presentation of calculated error in any of the data make this conclusion very tenuous. In addition, the authors conclude that lpp is not significantly attached to septal peptidoglycan. The logic behind this conclusion appears to be based on the same data, but the authors do not provide a quantitative model to support this idea.

The reviewer is correct in stating that we claim that Lpp is not substantially added to lipid II before incorporation of the disaccharide-pentapeptide subunit into the expanding PG network. This conclusion is based on the paucity of PG-Lpp covalent adducts containing light PG and Lpp moieties at the earliest time points. To substantiate more thoroughly this finding, we will reproduce the kinetic experiments with more early time points. The paucity of the new→new PG-Lpp isotopologues also implies that Lpp might not be extensively tethered to septal peptidoglycan since the latter is assembled from newly synthesized PG (see our previous publication Atze et al. 2021 and references therein). Quantitatively, septal synthesis roughly accounts for one third of the total PG synthesis. It is therefore expected that tethering of Lpp to septal PG would represent one third of the total number of newly synthesized Lpp molecules tethered to PG. We therefore proposed that the paucity of new→new PG- Lpp isotopologues at early time points of the kinetics implies that Lpp is preferentially tethered to the side wall. This is only one of several conclusions that we reach in the present study and we were very careful in the wording of our results.

-This work will have a moderate impact on the field of research in which the connections between the OM and are being studied in E. coli. Since lpp is not widely conserved in gram negatives, the impact across species is not clear. The authors do not discuss the impact of their work in depth.

We respectfully disagree with this reviewer’s comment. The work reported in this article for E. coli opens the way to the analysis and comparison of the mechanisms of the tethering of proteins to PG in various bacteria. In addition, we would like to stress that the Gram-negative bacteria that produce Lpp-related proteins and tether them to the PG include other major pathogens such as Pseudomonas aeruginosa (DOI: 10.1128/spectrum.05217-22).