The peptidoglycan is an essential component of the bacterial cell wall, which provides a mechanical barrier against the turgor pressure of the cytoplasm thereby preventing cells from bursting and lysing (Vollmer and Bertsche, 2008). The peptidoglycan also determines the shape of bacterial cells and is intimately integrated into the cell division process since the barrier to the osmotic pressure needs to be maintained during the entire cell cycle. These functions depend upon the net-like structure of the peptidoglycan macromolecule that is made of glycan strands cross-linked by short peptides (Figure 1A). It is assembled from disaccharide-peptide subunits consisting of β,1→4 linked N-acetylglucosamine (GlcNAc) and N-acetyl muramic acid (MurNAc) and a stem peptide linked to the D-lactoyl group of MurNAc (Figure 1B; Mengin-Lecreulx et al., 1982). Polymerization of the subunit is mediated by glycosyltransferases for the elongation of glycan chains and by transpeptidases for cross-linking stem peptides carried by adjacent glycan chains.

Figure 1 with 3 supplements see all

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The last cross-linking step of peptidoglycan polymerization is catalyzed by two types of structurally unrelated transpeptidases (Biarrotte-Sorin et al., 2006), the L,D-transpeptidases (LDTs) (Mainardi et al., 2008) and the D,D-transpeptidases (Zapun et al., 2008), the latter belonging to the penicillin-binding protein (PBP) family. PBPs (Figure 1C) and LDTs (Figure 1D) use different acyl donor substrates (pentapeptide versus tetrapeptide) resulting in the formation of 4→3 versus 3→3 cross-links, respectively (Hugonnet et al., 2016; Mainardi et al., 2005). PBPs are inactivated by all classes of β-lactams, whereas LDTs are inactivated by carbapenems only (Mainardi et al., 2007). In wild-type Escherichia coli, LDTs are fully dispensable for growth, at least in laboratory conditions (Magnet et al., 2008). Accordingly, these enzymes have a minor contribution to peptidoglycan cross-linking in the exponential growth phase (Vollmer and Bertsche, 2008; Glauner, 1988). However, selection of mutants resistant to ceftriaxone, a β-lactam of the cephalosporin class, results in a full bypass of PBPs by the YcbB LDT (Hugonnet et al., 2016). The bypass requires overproduction of both the (p)ppGpp alarmone and YcbB. The peptidoglycan of the mutant grown in the presence of ceftriaxone only contains 3→3 cross-links following PBP inhibition.

D,D-carboxypeptidase (DDC) and L,D-carboxypeptidase activities sequentially remove D-Ala residues at the 5th and 4th positions of stem peptides containing a free carboxyl end, which are present in the acceptor position of dimers and in monomers. DDCs, which belong to the PBP family, hydrolyze the D-Ala⁴-D-Ala⁵ amide bond of stem pentapeptides to generate stem tetrapeptides in mature peptidoglycan (Sauvage et al., 2008). Since the reaction is nearly total in E. coli, stem pentapeptides are found in very low abundance unless DDCs are inactivated by β-lactams. The L,D-carboxypeptidase activity of LDTs hydrolyzes the DAP³-D-Ala⁴ amide bond generating tripeptide stems from tetrapeptide stems (Magnet et al., 2008). Since this reaction is incomplete the peptidoglycan contains combinations of tripeptide and tetrapeptide stems in monomers and in the acyl acceptor position of dimers. Removal of D-Ala residues by DDCs and LDTs occurs before or after cross-linking, leading to the polymorphism in the acceptor position of dimers depicted in Figure 1C and D.

By removing D-Ala⁵ from pentapeptide stems, DDCs eliminate the essential pentapeptide donor of PBPs and generate the essential tetrapeptide donor of LDTs (Mainardi et al., 2002; Hugonnet et al., 2016). By removing D-Ala⁴ from tetrapeptide stems, LDCs eliminate the essential tetrapeptide donor of LDTs thereby potentially limiting the formation of 3→3 cross-links. Thus, DDC and LDC activities control both the extent of peptidoglycan cross-linking and the relative contributions of D,D-transpeptidases and LDTs to peptidoglycan cross-linking.

The interconversion between monomers and cross-linked dimers involves not only PBPs and LDTs in the biosynthetic direction but also hydrolytic enzymes, referred to as endopeptidases, which cleave the cross-links. Endopeptidases belonging to the PBP family are specific of 4→3 cross-links whereas other endopeptidases cleave both 4→3 and 3→3 cross-links (Voedts et al., 2021). Among eight endopeptidases with partially redundant functions, at least one enzyme is essential in the context of the formation of 4→3 cross-links whereas two endopeptidases, MepM and MepK, are required in the context of 3→3 cross-links (Voedts et al., 2021; Singh et al., 2012).

Morphogenesis of E. coli cells depends upon controlled expansion of the peptidoglycan and requires scaffolding proteins, actin (MreB) and tubulin (FtsZ) homologues (den Blaauwen et al., 2008). These proteins are involved in the recruitment of peptidoglycan polymerases and in the coordination of their activities with that of hydrolases (Vollmer and Höltje, 2001). The cell cycle involves two phases, namely the MreB-dependent elongation of the side wall at a constant diameter and the FtsZ-dependent formation of the septum at mid-cell. These processes are thought to involve two distinct multienzyme complexes, the elongasome and the divisome, and specific enzymes, in particular class B PBP2 and PBP3, which mediate peptidoglycan cross-linking in the side wall and in the septum, respectively (Höltje, 1996). Accordingly, specific inactivation of PBP2 by mecillinam or of PBP3 by aztreonam results in the growth of E. coli as spheres or as filaments, respectively.

The average chemical composition of peptidoglycan is well known. Most enzymes involved in the formation and cleavage of glycosidic and amide bonds have been identified (at least one enzyme and generally several enzymes for each bond). However, the spatial arrangement of glycan strands is still a matter of debate. Most models propose a regular arrangement of glycan strands parallel to the cell surface and perpendicular to the long axis of the cell as depicted in Figure 1A; Typas et al., 2011. Alternatively, it has been proposed that the glycan strands protrude perpendicularly to the surface of the cytoplasmic membrane (Meroueh et al., 2006; Dmitriev et al., 2003). The lack of direct experimental evidence originates from the heterogeneity of the peptidoglycan macromolecule that prevents the analysis of the polymer by radiocrystallography. NMR spectroscopy also failed to determine the precise and complete structure of the polymer due to its heterogeneity and its important flexibility, as measured by solid-state NMR relaxation (Kern et al., 2008; Sharif et al., 2009). The mode of insertion of new glycan strands in the growing peptidoglycan is also not fully characterized. According to the ‘three-for-one’ model, peptidoglycan expansion involves the insertion of three neo-synthesized glycan strands to the detriment of the hydrolysis of one existing strand, which acts as a docking strand (Vollmer and Höltje, 2001). Alternatively, glycan strands may be inserted according either to a ‘one-at-a-time’ model or a ‘two-at-a-time’ model, the latter being supported by the dimeric nature of certain D,D-transpeptidases (Charpentier et al., 2002). All these models postulate that peptidoglycan expansion requires cleavage of cross-links by endopeptidases and obey to the ‘make-before-break’ principle, which proposes that cross-links in the existing peptidoglycan are hydrolyzed by endopeptidases only if the newly synthesized glycan strands are cross-linked and can sustain the osmotic pressure of the cytoplasm (Vollmer and Höltje, 2001).

At each generation, about half of the disaccharide-peptide units is released from the peptidoglycan by the combined action of endopeptidases and lytic transglycosylases (Goodell, 1985a; Johnson et al., 2013). The latter enzymes catalyze a non-hydrolytic cleavage of the β,1→4 MurNAc-GlcNAc glycosydic bond and generate GlcNAc-anhydro-MurNAc-peptides fragments via MurNAc cyclisation at positions 1 and 6. These fragments are transported into the cytoplasm by the AmpG permease and recycled according to the pathway depicted in Figure 1—figure supplement 1. The key features of the recycling pathway are that the L-Ala-γ-D-Glu-DAP tripeptide is directly recycled whereas the glucosamine moiety of GlcNAc and MurNAc reenters into the peptidoglycan biosynthesis pathway at the level of glucosamine-6-phosphate. The relationships between peptidoglycan synthesis and recycling are poorly understood. The ‘three-for-one’ growth model predicts that the docking strand is recycled; conversely, the ‘one-at-a-time’ and ‘two-at-a-time’ models do not imply that recycling is a consequence of peptidoglycan synthesis.

The peptidoglycan expansion models described above were previously investigated by pulse labeling of peptidoglycan with radioactive DAP. Indeed, the models imply specific acceptor-to-donor radioactivity ratios (ADRR) in cross-linked stem peptides (Figure 1—figure supplement 2; de Jonge et al., 1989). The ADRR may potentially vary from zero to infinite for (i) exclusive cross-linking of neo-synthesized (radioactive) donor stems to existing (unlabeled) acceptor stems (new→old; ADRR=0), (ii) the formation of cross-links between neo-synthesized (radioactive) donor and acceptor stems (new→new; ADRR=1), and (iii) cross-linking of existing (unlabeled) donor stems to neo-synthesized (labeled) acceptor stems (old→new; infinite ADRR). This method is intrinsically inaccurate since newly synthesized subunits issued from the recycling of stem peptides are mistakenly identified as material from the existing wall. Moreover, the method provides access to the average composition of dimers in labeled donor and acceptor stems rather than the labeling status of stem peptides that have directly participated in the same cross-linking reaction. Thus, an ADRR of 1 can originate either from dimers containing neo-synthesized stem peptides in the donor and acceptor positions (new→new) or, alternatively, from a combination of dimers containing a radioactive DAP either in the donor (new→old) or acceptor (old→new) position in equimolar amounts. This limitation was disregarded in previous studies since dimers containing a single labeled DAP residue in the donor position were considered to be rare, at least in wild-type E. coli grown in the absence of β-lactam. This is arguably the case for dimers generated by PBPs since pentapeptide stems are rapidly converted to tetrapeptide stems by DDCs thereby precluding existing stems from participating as donors in the formation of 4→3 cross-links by D,D-transpeptidases. Obviously, this premise does not apply to LDTs since these enzymes use tetrapeptide stems as donors (Figure 1). Here, we describe a new method based on the full labeling of the entire peptidoglycan macromolecule with stable isotopes of carbon and nitrogen combined with high-resolution mass spectrometry (MS). This method does not suffer from the limitations listed above and our results show that it enables a thorough investigation of peptidoglycan synthesis and recycling in a single experiment. We applied our method to the comparison of the modes of expansion of the peptidoglycan in bacterial strains relying on PBPs, and, for the first time, on LDT YcbB, or on both types of enzymes for peptidoglycan cross-linking. We show how previously described β-lactam-induced malfunctioning of the bacterial cell wall synthesis machinery (Cho et al., 2014) affects peptidoglycan synthesis and recycling. We also report how LDT YcbB rescues inhibition of PBPs by participating in an unprecedented mode of peptidoglycan polymerization. The results described in this report were obtained, thanks to the richness of the structural information brought by MS and MS/MS analyses of all the labeled/unlabeled sugar and amino acid residues of peptidoglycan subunits, as opposed to the radioactive signal located in the DAP moiety only. The mass data analysis pipeline automating the identification of labeled muropeptides is described in this study and may be generally applied to the screening of the mode of action of antibacterial agents acting on peptidoglycan synthesis.

Previous studies of the expansion of the peptidoglycan macromolecule relied on labeling with [³H] or [¹⁴C]DAP (de Jonge et al., 1989). Quantitatively, the number of glycan strands inserted at a time was estimated by determining the acceptor-to-donor radioactivity ratio (ADRR), that is, the radioactivity ratio in the acceptor and donor positions of dimers (Figure 1—figure supplement 2; Burman and Park, 1984). By analogy, we define here the acceptor-to-donor ratio in neo-synthesized stems (ADRNS). ADRR and ADRNS determinations rely on opposite labeling schemes involving pulse labeling with radioactive DAP versus incorporation of newly synthesized (unlabeled) subunits into the fully labeled peptidoglycan, respectively. The latter strategy was chosen because of the ease of obtaining fully labeled cells with non-radioactive heavy isotopes. Switching the medium from labeled to unlabeled was chosen, rather than the unlabeled-to-labeled switch, since this facilitates the detection of neo-synthesized isotopologues present at a low abundance at early times after the medium switch. This is due to the fact that muropeptides from the existing peptidoglycan, which are preponderant at the beginning of the kinetics, are fully labeled and hence have higher m/z values than those of muropeptides containing ¹²C and ¹⁴N light isotopes following the medium switch. Consequently, sodium adducts to the fully labeled muropeptides do not appear in the relevant region of the chromatogram that contains muropeptides of lower m/z value due to the incorporation of light isotopes.

Pulse labeling with radioactive DAP suffers from four major limitations that are not encountered in our labeling strategy. First, optimization of radioactive DAP incorporation requires mutations that prevent endogenous DAP synthesis (lysA) and conversion of DAP to L-Lys (dapF) (Burman and Park, 1984; Glauner and Höltje, 1990; Goodell, 1985a; Goodell and Schwarz, 1985b; Figure 1—figure supplement 1). Starvation prior to the addition of radiolabeled DAP was used to minimize the lag between the addition of radioactive DAP into the culture medium and its incorporation into peptidoglycan (Glauner and Höltje, 1990; Burman and Park, 1984). However, depleting the intracellular DAP pool compromised the integrity of the peptidoglycan layer (Burman et al., 1983b), affected cell morphology (Verwer and Nanninga, 1980), and reduced turnover (Burman et al., 1983b). Our labeling scheme is not affected by these confounding factors since it does not require any metabolic engineering. A second limitation of the DAP labeling procedures is their inability to discriminate between muropeptides originating from the existing peptidoglycan, from the recycled L-Ala-D-iGlu-DAP tripeptide, or from the existing UDP-MurNAc-pentapeptide pool, because all of them contain non-radioactive DAP. In contrast, our procedure easily discriminates between these three types of muropeptides since they correspond to different isotopologues, namely uniformly labeled subunits and hybrids h2 and h3, respectively (Figure 4). The latter hybrids are likely to contribute to the lag phase observed for the incorporation of radioactive DAP into peptidoglycan in previous reports (Glauner and Höltje, 1990; Burman and Park, 1984). Accordingly, a lag phase was observed in our study when h2 and h3 hybrids containing [¹³C]- and [¹⁵N]-labeled DAP were excluded from the dataset (Figure 10). A third limitation of the radioactive DAP labeling procedures for ADRR determination originates from the fact that they provide access only to the average radioactivity content of donor and acceptor stem peptides. Indeed, these procedures are based on purification of individual dimers, derivatization of their free amino group located in the acceptor stem, acid hydrolysis, and chromatography to separate DAP and derivatized DAP originating from the donor and acceptor stems, respectively (Burman et al., 1983a). Our procedure is richer in information because it provides direct access to the structure of dimers enabling identification of new→new, new→old, old→new, and old→old dimers based on differences in mass and fragmentation patterns. The fourth limitation stems from the fact that exploring peptidoglycan recycling by the radioactive DAP labeling procedures requires use of specific mutants and study designs. For example, Jacobs et al., 1994, used mutants deficient in the production of the AmpG and Opp permeases in which peptidoglycan fragments are not recycled in the cytoplasm but released in the culture medium (Jacobs et al., 1994). In contrast, our approach enables investigating peptidoglycan recycling and the mode of peptidoglycan expansion in a single experiment in the absence of any metabolic perturbation since it does not require altering the recycling pathway and is solely based on the determination of the structure of peptidoglycan fragments.

Figure 10

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Strain BW25113Δ6ldt offered the possibility to investigate the mode of insertion of new subunits by PBPs in the absence of any LDT. Aztreonam and mecillinam were used to specifically inhibit synthesis of septal and lateral peptidoglycan, respectively. In the presence of aztreonam, the ADRNS for side wall synthesis was 0.14, 5 min after the medium switch and increased to 0.20, 0.34, 0.50, and 0.64 at 20, 40, 60, 80 min, respectively (Figure 11). At this time scale, existing disaccharide-peptide units are gradually replaced by newly synthesized disaccharide-peptides accounting for the increase in new→new dimers and the increase in the ADRNS value. The extrapolation of the ADRNS at t=0 (time of the medium switch) is therefore relevant to the evaluation of the mode of insertion of new subunits into the peptidoglycan. The extrapolated ADRNS value of 0.08 (Figure 8—figure supplement 3) was similar to the ADRR value of 0.10 reported by de Jonge and collaborators based on extrapolating data to a synchronized culture exclusively containing elongating cells (de Jonge et al., 1989). In the presence of mecillinam, the ADRNS values marginally increased over time, providing an extrapolated value of 0.31 for t=0. This value is lower than the ADRR of 0.64 obtained by extrapolating data to a synchronized culture exclusively containing constricting cells (de Jonge et al., 1989). The origin of this difference is unknown but could involve the fact that blocking cell elongation by mecillinam may not fully prevent incorporation of newly synthesized subunits into the peptidoglycan since this drug does not prevent the polymerization of glycan chains by the elongasome (Uehara and Park, 2008). Together, our results are in agreement with previous analyzes (de Jonge et al., 1989) that have shown single-strand insertion of newly synthesized peptidoglycan into the side wall versus insertion of multiple strands for septal peptidoglycan synthesis. In the absence of mecillinam and aztreonam, the ADRNS value (0.14) was intermediate between the values observed in the presence of aztreonam (0.08) and mecillinam (0.31), as qualitatively expected for the combined contributions of cross-links located in the side wall and in the septum and the lower contribution of the latter to the cell surface.

Figure 11

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In the absence of β-lactam, E. coli M1.5 relied both on the D,D-transpeptidase activity of PBPs and the LDT activity of YcbB for peptidoglycan cross-linking, leading to a high peptidoglycan content in both 4→3 (56%) and 3→3 (44%) cross-linked dimers (Supplementary file 1a). The proportions of dimers containing two newly synthesized stems (new→new) or one newly synthesized and one existing stem (new→old) were similar for the two types of cross-links (Figure 7). Thus, the D,D-transpeptidase activity of PBPs and the LDT activity of YcbB were involved in similar modes of insertion of new strands into the existing peptidoglycan of strain M1.5. The ADRNS values were in the order of 0.17 and 0.22 for 4 → 3 and 3 → 3 cross-links, respectively. These values indicate that the neo-synthesized glycan strands were predominantly but not exclusively cross-linked to the existing peptidoglycan for both types of transpeptidation reactions. Peptidoglycan polymerization might involve single-strand insertion into the side wall and insertion of multiple strands into the septum, as discussed above for strain Δ6ldt. The striking similarities between dimers generated by PBPs and YcbB raise the possibility that the mode of insertion of new subunits is not determined by the transpeptidases but by other components of the peptidoglycan polymerization complexes, such as the scaffolding proteins.

The peptidoglycan of strain M1.5 grown in the presence of ampicillin was mainly (99%) cross-linked by YcbB following the inhibition of the D,D-transpeptidase activity of PBPs (Figure 9). Strikingly, the Tri→Tetra and Tri→Penta dimers were almost exclusively of the new→new type. This pattern was not found in strains M1.5 and Δ6ldt grown in the absence of β-lactam (Figures 7 and 8). The prevalence of new→new dimers may be a consequence of the inhibition of the transpeptidase activity of PBPs by ampicillin that results in the uncoupling of the cross-linking and transglycosylation reactions (Uehara and Park, 2008; Cho et al., 2014). This in turn results in the accumulation of linear glycan strands that are eventually cross-linked to each other by YcbB because they are not effectively degraded due to a mutation present in M1.5 that prevents production of lytic transglycosylase Slt70 (Voedts et al., 2021; Cho et al., 2014). The resulting new→new cross-linked material was almost exclusively connected to the existing peptidoglycan by the formation of Tri→Tri dimers because new→old isotopologues were only detected in this type of dimers. This implies that the acceptors of the cross-linking reactions in the existing peptidoglycan were tripeptides, whereas existing tetrapeptides also acted as acceptors in the absence of ampicillin (Figure 7). The nearly exclusive use of existing tripeptides as acceptors in the presence of ampicillin could be accounted for by the fact that these tripeptides originate from cleavage of 3→3 cross-links liberating the tripeptide stems present in the donor position of the existing dimers. This model implies a cooperation between YcbB and an endopeptidase that provides the tripeptide acceptor substrate of the reaction by cleaving existing 3→3 cross-links (Figure 12). This enzyme could be MepK since this endopeptidase preferentially cleaves 3→3 cross-links in vitro, is essential for bacterial growth only in strains overproducing YcbB, and is not inhibited by ampicillin (Voedts et al., 2021). According to this model, the reactions catalyzed by MepK and YcbB are concerted rather than sequential. This mode of expansion of the peptidoglycan network differs from the ‘make-before-break’ models that postulate that neo-synthesized glycan strands are cross-linked to free stem peptides of the existing peptidoglycan prior to the cleavage of existing cross-links. In the absence of ampicillin, the hydrolysis of 4→3 cross-links by endopeptidases belonging to the PBP family could generate tetrapeptide acceptors in addition to the tripeptide acceptors exclusively used in the presence of the drug.

Figure 12

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Cho et al., 2014 proposed that the antibacterial activity of β-lactams does not merely rely on a loss of function, that is, the inactivation of the D,D-transpeptidase activity of PBPs by acylation of their catalytic Ser residue, but by inducing a toxic malfunctioning of the peptidoglycan biosynthesis machinery. This involves uncoupling of transglycosylation and transpeptidation, accumulation of polymerized glycan chains, and their degradation by lytic transglycosylase Slt70 (Banzhaf et al., 2012; Cho et al., 2014; Uehara and Park, 2008). The resulting futile cycle depletes resources and prevents bacterial growth. Exposure of ΔsltY cells to mecillinam was reported to result in the polymerization of an ‘aberrant peptidoglycan’ enriched in 3→3 cross-links. In comparison to this previous report (Cho et al., 2014), our analysis revealed that overproduction of YcbB results in substantially different modifications of peptidoglycan metabolism. First, M1.5 grown in the absence of β-lactam released a high proportion of its peptidoglycan per generation (56%) (Figure 13). This indicates that formation of 3→3 cross-links by YcbB is sufficient to stimulate peptidoglycan degradation in the absence of β-lactams. Under these conditions, Slt70 was not the main lytic enzyme since this activity is impaired in M1.5 (Voedts et al., 2021). Exposure to β-lactam had an additional stimulating effect on peptidoglycan degradation that reached 78% per generation for M1.5 grown in the presence of ampicillin (Figure 13). Second, overproduction of YcbB enabled growth of M1.5 in the presence of β-lactams indicating that the ‘aberrant peptidoglycan’ described by Cho et al., 2014, can in fact sustain bacterial growth. This involved an unprecedented mode of peptidoglycan synthesis implicating polymerization of linear glycan chains, their cross-linking by YcbB, and the incorporation of the resulting neo-synthesized material into the existing peptidoglycan by the concerted action of LDT YcbB and of β-lactam-insensitive endopeptidase MepK.

Figure 13

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MS/MS spectra have been provided in Supplementary data file. The software developments required to predict and analyze the labeled/unlabeled muropeptide ions isotopic clusters either in MS or MS/MS experiments are hosted at https://gitlab.com/kantundpeterpan/masseltof, (copy archived at swh:1:rev:c45a7e66a8a89c436d41b8431938b35acfa4e53b) and published under a Free Software license.

1. 1. Adusumilli R
   2. Mallick P

   (2017) Data Conversion with ProteoWizard msConvert

   Methods in Molecular Biology (Clifton, N.J.) 1550:339–368.

   https://doi.org/10.1007/978-1-4939-6747-6_23

   • PubMed
   • Google Scholar
2. 1. Arbeloa A
   2. Hugonnet J-E
   3. Sentilhes A-C
   4. Josseaume N
   5. Dubost L
   6. Monsempes C
   7. Blanot D
   8. Brouard J-P
   9. Arthur M

   (2004) Synthesis of mosaic peptidoglycan cross-bridges by hybrid peptidoglycan assembly pathways in gram-positive bacteria

   The Journal of Biological Chemistry 279:41546–41556.

   https://doi.org/10.1074/jbc.M407149200

   • PubMed
   • Google Scholar
3. Software

   1. Atze H

   (2021) kantundpeterpan/masseltof, version swh:1:rev:c45a7e66a8a89c436d41b8431938b35acfa4e53b

   GitHub.

   https://gitlab.com/kantundpeterpan/masseltof
4. 1. Banzhaf M
   2. van den Berg van Saparoea B
   3. Terrak M
   4. Fraipont C
   5. Egan A
   6. Philippe J
   7. Zapun A
   8. Breukink E
   9. Nguyen-Distèche M
   10. den Blaauwen T
   11. Vollmer W

   (2012) Cooperativity of peptidoglycan synthases active in bacterial cell elongation

   Molecular Microbiology 85:179–194.

   https://doi.org/10.1111/j.1365-2958.2012.08103.x

   • PubMed
   • Google Scholar
5. 1. Biarrotte-Sorin S
   2. Hugonnet J-E
   3. Delfosse V
   4. Mainardi J-L
   5. Gutmann L
   6. Arthur M
   7. Mayer C

   (2006) Crystal Structure of a Novel β-Lactam-insensitive Peptidoglycan Transpeptidase

   Journal of Molecular Biology 359:533–538.

   https://doi.org/10.1016/j.jmb.2006.03.014

   • PubMed
   • Google Scholar
6. 1. Burman LG
   2. Raichler J
   3. Park JT

   (1983a) Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus

   Journal of Bacteriology 155:983–988.

   https://doi.org/10.1128/jb.155.3.983-988.1983

   • PubMed
   • Google Scholar
7. 1. Burman LG
   2. Reichler J
   3. Park JT

   (1983b) Evidence for multisite growth of Escherichia coli murein involving concomitant endopeptidase and transpeptidase activities

   Journal of Bacteriology 156:386–392.

   https://doi.org/10.1128/jb.156.1.386-392.1983

   • PubMed
   • Google Scholar
8. 1. Burman LG
   2. Park JT

   (1984) Molecular model for elongation of the murein sacculus of Escherichia coli

   PNAS 81:1844–1848.

   https://doi.org/10.1073/pnas.81.6.1844

   • PubMed
   • Google Scholar
9. 1. Charpentier X
   2. Chalut C
   3. Rémy M-H
   4. Masson J-M

   (2002) Penicillin-binding proteins 1a and 1b form independent dimers in Escherichia coli

   Journal of Bacteriology 184:3749–3752.

   https://doi.org/10.1128/JB.184.13.3749-3752.2002

   • PubMed
   • Google Scholar
10. 1. Cho H
    2. Uehara T
    3. Bernhardt TG

    (2014) Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery

    Cell 159:1300–1311.

    https://doi.org/10.1016/j.cell.2014.11.017

    • PubMed
    • Google Scholar
11. 1. Datsenko KA
    2. Wanner BL

    (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

    PNAS 97:6640–6645.

    https://doi.org/10.1073/pnas.120163297

    • PubMed
    • Google Scholar
12. 1. de Jonge BL
    2. Wientjes FB
    3. Jurida I
    4. Driehuis F
    5. Wouters JT
    6. Nanninga N

    (1989) Peptidoglycan synthesis during the cell cycle of Escherichia coli: composition and mode of insertion

    Journal of Bacteriology 171:5783–5794.

    https://doi.org/10.1128/jb.171.11.5783-5794.1989

    • PubMed
    • Google Scholar
13. 1. den Blaauwen T
    2. de Pedro MA
    3. Nguyen-Distèche M
    4. Ayala JA

    (2008) Morphogenesis of rod-shaped sacculi

    FEMS Microbiology Reviews 32:321–344.

    https://doi.org/10.1111/j.1574-6976.2007.00090.x

    • PubMed
    • Google Scholar
14. 1. Dmitriev BA
    2. Toukach FV
    3. Schaper K-J
    4. Holst O
    5. Rietschel ET
    6. Ehlers S

    (2003) Tertiary structure of bacterial murein: the scaffold model

    Journal of Bacteriology 185:3458–3468.

    https://doi.org/10.1128/JB.185.11.3458-3468.2003

    • PubMed
    • Google Scholar
15. 1. Glauner B

    (1988) Separation and quantification of muropeptides with high-performance liquid chromatography

    Analytical Biochemistry 172:451–464.

    https://doi.org/10.1016/0003-2697(88)90468-x

    • PubMed
    • Google Scholar
16. 1. Glauner B
    2. Höltje JV

    (1990) Growth pattern of the murein sacculus of Escherichia coli

    The Journal of Biological Chemistry 265:18988–18996.

    https://doi.org/10.1016/S0021-9258(17)30613-0

    • PubMed
    • Google Scholar
17. 1. Goodell EW

    (1985a) Recycling of murein by Escherichia coli

    Journal of Bacteriology 163:305–310.

    https://doi.org/10.1128/jb.163.1.305-310.1985

    • PubMed
    • Google Scholar
18. 1. Goodell EW
    2. Schwarz U

    (1985b) Release of cell wall peptides into culture medium by exponentially growing Escherichia coli

    Journal of Bacteriology 162:391–397.

    https://doi.org/10.1128/jb.162.1.391-397.1985

    • PubMed
    • Google Scholar
19. 1. Höltje JV

    (1996) A hypothetical holoenzyme involved in the replication of the murein sacculus of Escherichia coli

    Microbiology (Reading, England) 142 (Pt 8):1911–1918.

    https://doi.org/10.1099/13500872-142-8-1911

    • PubMed
    • Google Scholar
20. 1. Hugonnet J-E
    2. Mengin-Lecreulx D
    3. Monton A
    4. den Blaauwen T
    5. Carbonnelle E
    6. Veckerlé C
    7. Brun YV
    8. van Nieuwenhze M
    9. Bouchier C
    10. Tu K
    11. Rice LB
    12. Arthur M

    (2016) Factors essential for L,D-transpeptidase-mediated peptidoglycan cross-linking and β-lactam resistance in Escherichia coli

    eLife 5:e19469.

    https://doi.org/10.7554/eLife.19469

    • PubMed
    • Google Scholar
21. 1. Jacobs C
    2. Huang LJ
    3. Bartowsky E
    4. Normark S
    5. Park JT

    (1994) Bacterial cell wall recycling provides cytosolic muropeptides as effectors for beta-lactamase induction

    The EMBO Journal 13:4684–4694.

    https://doi.org/10.1002/j.1460-2075.1994.tb06792.x

    • PubMed
    • Google Scholar
22. 1. Johnson JW
    2. Fisher JF
    3. Mobashery S

    (2013) Bacterial cell-wall recycling

    Annals of the New York Academy of Sciences 1277:54–75.

    https://doi.org/10.1111/j.1749-6632.2012.06813.x

    • PubMed
    • Google Scholar
23. 1. Kern T
    2. Hediger S
    3. Müller P
    4. Giustini C
    5. Joris B
    6. Bougault C
    7. Vollmer W
    8. Simorre J-P

    (2008) Toward the Characterization of Peptidoglycan Structure and Protein−Peptidoglycan Interactions by Solid-State NMR Spectroscopy

    Journal of the American Chemical Society 130:5618–5619.

    https://doi.org/10.1021/ja7108135

    • PubMed
    • Google Scholar
24. 1. Langella O
    2. Rusconi F

    (2021) mineXpert2: Full-Depth Visualization and Exploration of MS(n) Mass Spectrometry Data

    Journal of the American Society for Mass Spectrometry 32:1138–1141.

    https://doi.org/10.1021/jasms.0c00402

    • PubMed
    • Google Scholar
25. 1. Magnet S
    2. Bellais S
    3. Dubost L
    4. Fourgeaud M
    5. Mainardi J-L
    6. Petit-Frère S
    7. Marie A
    8. Mengin-Lecreulx D
    9. Arthur M
    10. Gutmann L

    (2007) Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan

    Journal of Bacteriology 189:3927–3931.

    https://doi.org/10.1128/JB.00084-07

    • PubMed
    • Google Scholar
26. 1. Magnet S
    2. Dubost L
    3. Marie A
    4. Arthur M
    5. Gutmann L

    (2008) Identification of the L,D-transpeptidases for peptidoglycan cross-linking in Escherichia coli

    Journal of Bacteriology 190:4782–4785.

    https://doi.org/10.1128/JB.00025-08

    • PubMed
    • Google Scholar
27. 1. Mainardi J-L
    2. Morel V
    3. Fourgeaud M
    4. Cremniter J
    5. Blanot D
    6. Legrand R
    7. Frehel C
    8. Arthur M
    9. Van Heijenoort J
    10. Gutmann L

    (2002) Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium

    The Journal of Biological Chemistry 277:35801–35807.

    https://doi.org/10.1074/jbc.M204319200

    • PubMed
    • Google Scholar
28. 1. Mainardi J-L
    2. Fourgeaud M
    3. Hugonnet J-E
    4. Dubost L
    5. Brouard J-P
    6. Ouazzani J
    7. Rice LB
    8. Gutmann L
    9. Arthur M

    (2005) A novel peptidoglycan cross-linking enzyme for A beta-lactam-resistant transpeptidation pathway

    The Journal of Biological Chemistry 280:38146–38152.

    https://doi.org/10.1074/jbc.M507384200

    • PubMed
    • Google Scholar
29. 1. Mainardi J-L
    2. Hugonnet J-E
    3. Rusconi F
    4. Fourgeaud M
    5. Dubost L
    6. Moumi AN
    7. Delfosse V
    8. Mayer C
    9. Gutmann L
    10. Rice LB
    11. Arthur M

    (2007) Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem

    The Journal of Biological Chemistry 282:30414–30422.

    https://doi.org/10.1074/jbc.M704286200

    • PubMed
    • Google Scholar
30. 1. Mainardi J-L
    2. Villet R
    3. Bugg TD
    4. Mayer C
    5. Arthur M

    (2008) Evolution of peptidoglycan biosynthesis under the selective pressure of antibiotics in Gram-positive bacteria

    FEMS Microbiology Reviews 32:386–408.

    https://doi.org/10.1111/j.1574-6976.2007.00097.x

    • PubMed
    • Google Scholar
31. 1. Mengin-Lecreulx D
    2. Flouret B
    3. van Heijenoort J

    (1982) Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli

    Journal of Bacteriology 151:1109–1117.

    https://doi.org/10.1128/jb.151.3.1109-1117.1982

    • PubMed
    • Google Scholar
32. 1. Mengin-Lecreulx D
    2. van Heijenoort J
    3. Park JT

    (1996) Identification of the mpl gene encoding UDP-N-acetylmuramate: L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase in Escherichia coli and its role in recycling of cell wall peptidoglycan

    Journal of Bacteriology 178:5347–5352.

    https://doi.org/10.1128/jb.178.18.5347-5352.1996

    • PubMed
    • Google Scholar
33. 1. Meroueh SO
    2. Bencze KZ
    3. Hesek D
    4. Lee M
    5. Fisher JF
    6. Stemmler TL
    7. Mobashery S

    (2006) Three-dimensional structure of the bacterial cell wall peptidoglycan

    PNAS 103:4404–4409.

    https://doi.org/10.1073/pnas.0510182103

    • PubMed
    • Google Scholar
34. 1. Montón Silva A
    2. Otten C
    3. Biboy J
    4. Breukink E
    5. VanNieuwenhze M
    6. Vollmer W
    7. den Blaauwen T

    (2018) The Fluorescent D-Amino Acid NADA as a Tool to Study the Conditional Activity of Transpeptidases in Escherichia coli

    Frontiers in Microbiology 9:2101.

    https://doi.org/10.3389/fmicb.2018.02101

    • PubMed
    • Google Scholar
35. 1. Morè N
    2. Martorana AM
    3. Biboy J
    4. Otten C
    5. Winkle M
    6. Serrano CKG
    7. Montón Silva A
    8. Atkinson L
    9. Yau H
    10. Breukink E
    11. den Blaauwen T
    12. Vollmer W
    13. Polissi A

    (2019) Peptidoglycan Remodeling Enables Escherichia coli To Survive Severe Outer Membrane Assembly Defect

    MBio 10:e02729-18.

    https://doi.org/10.1128/mBio.02729-18

    • PubMed
    • Google Scholar
36. 1. Sauvage E
    2. Kerff F
    3. Terrak M
    4. Ayala JA
    5. Charlier P

    (2008) The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

    FEMS Microbiology Reviews 32:234–258.

    https://doi.org/10.1111/j.1574-6976.2008.00105.x

    • PubMed
    • Google Scholar
37. 1. Sharif S
    2. Singh M
    3. Kim SJ
    4. Schaefer J

    (2009) Staphylococcus aureus peptidoglycan tertiary structure from carbon-13 spin diffusion

    Journal of the American Chemical Society 131:7023–7030.

    https://doi.org/10.1021/ja808971c

    • PubMed
    • Google Scholar
38. 1. Singh SK
    2. SaiSree L
    3. Amrutha RN
    4. Reddy M

    (2012) Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

    Molecular Microbiology 86:1036–1051.

    https://doi.org/10.1111/mmi.12058

    • PubMed
    • Google Scholar
39. 1. Templin MF
    2. Ursinus A
    3. Höltje JV

    (1999) A defect in cell wall recycling triggers autolysis during the stationary growth phase of Escherichia coli

    The EMBO Journal 18:4108–4117.

    https://doi.org/10.1093/emboj/18.15.4108

    • PubMed
    • Google Scholar
40. 1. Typas A
    2. Banzhaf M
    3. Gross CA
    4. Vollmer W

    (2011) From the regulation of peptidoglycan synthesis to bacterial growth and morphology

    Nature Reviews. Microbiology 10:123–136.

    https://doi.org/10.1038/nrmicro2677

    • PubMed
    • Google Scholar
41. 1. Uehara T
    2. Park JT

    (2008) Growth of Escherichia coli: significance of peptidoglycan degradation during elongation and septation

    Journal of Bacteriology 190:3914–3922.

    https://doi.org/10.1128/JB.00207-08

    • PubMed
    • Google Scholar
42. 1. Verwer RW
    2. Nanninga N

    (1980) Pattern of meso-DL-2,6-diaminopimelic acid incorporation during the division cycle of Escherichia coli

    Journal of Bacteriology 144:327–336.

    https://doi.org/10.1128/jb.144.1.327-336.1980

    • PubMed
    • Google Scholar
43. 1. Virtanen P
    2. Gommers R
    3. Oliphant TE
    4. Haberland M
    5. Reddy T
    6. Cournapeau D
    7. Burovski E
    8. Peterson P
    9. Weckesser W
    10. Bright J
    11. van der Walt SJ
    12. Brett M
    13. Wilson J
    14. Millman KJ
    15. Mayorov N
    16. Nelson ARJ
    17. Jones E
    18. Kern R
    19. Larson E
    20. Carey CJ
    21. Polat İ
    22. Feng Y
    23. Moore EW
    24. VanderPlas J
    25. Laxalde D
    26. Perktold J
    27. Cimrman R
    28. Henriksen I
    29. Quintero EA
    30. Harris CR
    31. Archibald AM
    32. Ribeiro AH
    33. Pedregosa F
    34. van Mulbregt P
    35. SciPy 1.0 Contributors

    (2020) SciPy 1.0: fundamental algorithms for scientific computing in Python

    Nature Methods 17:261–272.

    https://doi.org/10.1038/s41592-019-0686-2

    • PubMed
    • Google Scholar
44. 1. Voedts H
    2. Dorchêne D
    3. Lodge A
    4. Vollmer W
    5. Arthur M
    6. Hugonnet J-E

    (2021) Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli

    The EMBO Journal 40:e108126.

    https://doi.org/10.15252/embj.2021108126

    • PubMed
    • Google Scholar
45. 1. Vollmer W
    2. Höltje JV

    (2001) Morphogenesis of Escherichia coli

    Current Opinion in Microbiology 4:625–633.

    https://doi.org/10.1016/s1369-5274(01)00261-2

    • PubMed
    • Google Scholar
46. 1. Vollmer W
    2. Bertsche U

    (2008) Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

    Biochimica et Biophysica Acta 1778:1714–1734.

    https://doi.org/10.1016/j.bbamem.2007.06.007

    • PubMed
    • Google Scholar
47. 1. Zapun A
    2. Contreras-Martel C
    3. Vernet T

    (2008) Penicillin-binding proteins and beta-lactam resistance

    FEMS Microbiology Reviews 32:361–385.

    https://doi.org/10.1111/j.1574-6976.2007.00095.x

    • PubMed
    • Google Scholar

Author details

1. Heiner Atze

   Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université Paris Cité., Paris, France

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1497-6373

2. Yucheng Liang

   Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université Paris Cité., Paris, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing – review and editing

   Competing interests

   No competing interests declared

3. Jean-Emmanuel Hugonnet

   Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université Paris Cité., Paris, France

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4150-0944

4. Arnaud Gutierrez

   Robustesse et Evolvabilité de la vie, Institut Cochin, Université de Paris, INSERM U1016, CNRS UMR 8104, Paris, France

   Contribution

   Data curation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Filippo Rusconi

   1. Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université Paris Cité., Paris, France
   2. PAPPSO, Université Paris-Saclay, INRAE, CNRS, Paris, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Software, Supervision, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Michel Arthur

   For correspondence

   filippo.rusconi@universite-paris-saclay.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1822-0397

6. Michel Arthur

   Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, Université Paris Cité., Paris, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Filippo Rusconi

   For correspondence

   michel.arthur@crc.jussieu.fr

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1007-636X

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