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  1. Evaluation Summary:

    The authors describe the innovative use of a heavy-isotope labeling strategy combined with mass spectrometry analysis to investigate the role of peptidoglycan biosynthesis by an L,D-transpeptidase and penicillin binding proteins in Escherichia coli. They use isotopic labeling of the peptidoglycan following by a chase experiment with label to study how new peptidoglycan is assembled into pre-existing peptidoglycan. The data suggests that new material is inserted one strand at the time on the lateral wall while it appears to be inserted as multiple strands at the division septum. The data are novel and provide important insights, together with notable methodological advances. The study will be of interest to microbiologists studying bacterial cell wall turnover and for drug discovery efforts.

    (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. Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    The authors describe an innovative use of heavy-isotope labeling strategy combined with MS analysis to investigate the role of peptidoglycan biosynthesis by YcbB (l,d-transpeptidase) and PBP to understand the spatial organization and insertion of the polymerized peptidoglycan in E. coli. The strain M1.5 was uniformly 15N and 13C-labeled using M9 minimal medium with labeled glucose and ammonium chloride as sole carbon and nitrogen sources, respectively. The LCMS confirmed that heavy-isotope labeled muropeptide fragments from mutanolysin-digested isolated cell walls of E. coli are resolved in the m/z dimension from the unlabeled with distinct isotopic distribution from the labeling. The use of tandem MS/MS analysis to differentiate the isomers of dimers (tri-tetra and tetra-tri) corresponding to 3-3 and 4-3 crosslinkage types in PG dimers, mixed labeling due to PG recycling of the old muropeptides, and incorporation of the nascent PG to old PG. Hence, combined use of heavy-isotope labeling with MS/MS enabled identification and quantification of newnew, newold, and oldold dimers from cell walls of E. coli. The absence of oldnew in 3-3 CL was surprising since tetrapeptide stem from old PG can be used as a donor by YcbB (LDT). A similar absence of oldnew in 4-3 CL indicated that the mode of insertion of PG depends on the structure of the acceptor stem, not the type of transpeptidase (LDT or PBP). Furthermore, the use of PBP-specific antibiotics and analyzing PG composition as a function of time, the changes in the observed muropeptide composition (PG dimers) provided the kinetic information. The similarities in the kinetics of PG incorporation by PBPs and LDTs suggest that the insertion event is independent of the transpeptidases but may depend on other factors such as scaffolding proteins. In addition, accurate identification and quantification of isotope-labeled PG dimers determined the acceptor-to-donor ratio for neo-synthesized stems from E. coli that were grown in the presence of antibiotic provided the key evidence for the difference in the mechanisms of nascent PG insertion into the sidewall and multiple strands for septal PG synthesis in E. coli. The manuscript is insightful and highlights the innovative use of a heavy-isotope labeling strategy combined with MS analysis to investigate the mechanisms of PG recycling and strand insertion in bacteria.

  3. Reviewer #2 (Public Review):

    The insertion of new peptidoglycan in the pre-existing peptidoglycan layer has been the object of intense research during the last decades and led to several competing models. The authors tried to discriminate among the several models by using a chase experiment that first isotopically labeled the peptidoglycan then chased it with unlabeled material. The use of high-resolution mass spectrometry provided with an unprecedented definition on the evolution of the different substructures of the peptidoglycan. By focusing on the isotopic label of dimers which reflect the cross-linking of two peptidoglycan strands, the authors were able to follow the rate of incorporation of novel material. By calculating the ratio of labeled versus unlabeled monomers participating in the building of dimers, the authors were able to conclude that new peptidoglycan strands are inserted into the pre-existing peptidoglycan a strand at the time on the lateral wall of bacteria while during cell division, multiple peptidoglycan strands are inserted simultaneously.

    The data presented by the authors based on mass spectrometry analysis of the peptidoglycan isolated at different time points during a chase experiment are consistent with the authors conclusions. The methodology is very elegant and even allows to discriminate whether the new material is de novo synthesized or results from recycling of the old peptidoglycan. The data supports previous data from the literature using radioactive labeling of peptidoglycan followed by also a chase experiment. The experimental design has been validated previously and the innovation is brought by the use of mass spectrometry that allows to track the exact origin of each peptidoglycan fragment rather than measure averages as with radioactive labeling. Finally, the authors were able by using mutants and selective beta-lactams to show that the mode of insertion of new peptidoglycan strands does not depend on the type of transpeptidase since the authors obtain similar results whether transpeptidation is accomplished by penicillin-binding proteins or by the alternative L,D-transpeptidases.

    I only have two major comments to be addressed by the authors.

    1. While the data on the one by one strand insertion in the lateral wall is supported by all the presented data, there seems to be a conflict in the data presented for the insertion of new peptidoglycan strands during cell division. The calculation of the ratio of new vs old peptidoglycan presented in Supplementary figure 12 for bacteria treated with mecillinam (that grow as cocci and perform only cell division) indeed suggests that multiple peptidoglycan strands are inserted since the beginning of the chase. However, this data is based on abundance of labeled, hybrid and unlabeled peptidoglycan fragments presumably presented in Figure 8. And the data on panel D, left graph does not show any immediate presence in the peptidoglycan of unlabeled peptidoglycan fragments. In fact, these are as low in abundance as in none treated or aztreonam treated cells (that elongate but do not divide). Maybe I'm missing something but I cannot reconcile these two figures.

    2. As indicated in several figure legends, the data presented is from two independent experiments. Does this mean that the authors only performed twice the chase experiments? The experiment itself is rather simple to perform and it's the downstream analysis that is extremely time consuming. How confident are the authors of the reproducibility of the data?

  4. Reviewer #3 (Public Review):

    This is a very interesting and well written paper on a new approach to analyse the kinetics of peptidoglycan (PG) synthesis. Originally this was done by giving the cells a pulse of radioactive DAP, after which the PG was isolated and digested with muramidases to isolate disaccharide variants and crosslinked dimers of disaccharides by HPLC. This method has some disadvantages as is outlined in the manuscript. The new method is to label the entire sacculus with heavy atoms, while the cells are growing in M9 medium with 13C and 15N and then dilute the cells in unlabelled medium while following the kinetics of incorporation of new material in the existing PG. The manuscript first details the reliability of the method, proving that the PG is 99.9% labelled after growth in heavy medium, The PG is for 0.01% labelled in light medium and that shifting from heavy to light does not change the composition of PG. The identification by mass spectrometry is shown to be reliable by comparing the predicted masses with the observed masses. The data are statistically sound. In the introduction the status of the research is step by step well explained, and arguments are given why it would be useful to have another method. Analysis of the PG resulted in labelled, unlabelled or hybrid material. The hybrids could be discriminated in h1, h2 and h3 of which the first two could be explained by the PG recycling pathway and the last by the combination of unlabelled UDP-murNacpp form the existing cellular pool and the newly synthesized GlcNac. In this manuscript, only the major muropeptides have been identified thoroughly by MS. Due to the absence of LPP TPases that couple LPP to PG, derivatives of this connection were absent. The purpose of the manuscript was not to determine the composition of PG but to determine the kinetics of insertion of newly synthesized PG. PG was isolated and analysed at various timepoints up to one mass doubling time after the chase with unlabelled medium. The ration of labelled muropeptides and unlabelled muropeptides was determined. In the used strain, only new donor > new acceptor, new > old and old > old crosslinks are found. No old > new, indicating that the old strains do not have any donor peptides left as they are mostly converted to tetrapeptides by the D,D-carboxypeptidases. This result supports multistrand as well as single strand insertion. A delta6 LD-TPase mutant strain and the addition of aztreonam that inhibits cell division to this mutant converted all insertion to new>old indicating single strand insertion during length growth. Inhibition of length growth did still give a mixture of single and multi-strand insertion. Ampicillin which inhibits all PBPs in the presence of the LD-TPase YhcB caused mostly new>new and old>old insertion, suggesting that the new polymerized material by non-PBP GTases is not degraded (as Slt70 is absent in this strain) but crosslinked through 3>3 bonds. Under these conditions only tri acceptors in the old PG were used to make new>old crosslinks, suggesting that endopeptidase activity is needed to insert the glycan strands. The authors proposed a model for PG synthesis in which endopeptidases and TPases work together to insert new single PG strands.

    The cells are grown in mecillinam and aztreonam, which inhibit length growth and division, respectively. The cells are expected to have a round and filamentous morphology, respectively, but this is not shown in the manuscript, where a very non wild-type strain is used for the experiments. The concentration of the antibiotics is perhaps based on the literature, but maybe this strain has difference susceptibility to the antibiotics?

    In the absence of LD-TPases the insertion of new PG is predominantly new>old for the first 20 minutes, whereas in the M1.5 strain, this is 50% new>old and 50% new>new. Their mass doubling times are 67 and 90 min, respectively. Both strains should contain dividing cells directly after the medium change. If septum formation is multistrand insertion, one would expect new>new in both strains. Then aztreonam is added to the delta 6 LD-TPases strain and the mode of insertion remains the same (new>old), suggesting that length growth is single strand insertion. However, I do not think that this can be concluded because with division it was also single stranded. Then in the presence of mecillinam the authors claim that they see new>old and new>new insertion and therefore, claim that division requires multiple strand insertion. But when I look at the graph of the mecillinam cells, 1) it looks as if the PG synthesis is considerably slowed down in comparison to the other two situations. This is unexpected given the that the mass doubling time did not change according to Table S2. 2. If you extrapolate the lines in the graph, they are not that different from the strains without antibiotics or with aztreonam. 3. The new>old dominates, which one does not expect from cells that enlarge predominantly through division. The explanation that the futile cycle of PG TGase activity may result in insertion of old>new strand by PBP1b and 1a is plausible, but it does not prove that septation is multistrand. Would it be possible, to calculate the amount of surface generated through septation and elongation per min and predict the expected increase in old>new and new>new in the M1.5 strain to see if the observed data would fit the proposed model?

    In general, it is a pity that no wild-type strain is used for the new method. The conclusion of the paper is very important, i. e. new PG is inserted strand by strand likely by the combined action of an TPase and an endopeptidase. The absence of data on a WT strain diminishes the soundness of the conclusion. Since it can be expected that this paper will be cited for a long time by many people, it is important that it is complete.