Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

Antibiotics of the β-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate β-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this undocumented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a β-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

Tandem mass spectrometry analysis of the labeled disaccharide-tripeptide. The observed m/z obs and calculated m/z cal values of the parental ion, [M+H] + , as determined in the absence of fragmentation (MS1), are indicated. The m/z calc value was used to select the ion for fragmentation. The difference between the m/z obs and m/z calc values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively. The peaks corresponding to the list of fragments appearing in the table are highlighted by orange dots in the mass spectrum. In the mass spectrum, peaks differing by the loss of H 2 0 are connected by a dashed line. The tables only contain the m/z values for the fragments containing H 2 0. An interactive report of the MS 2 analysis is available in Supplementary File F1.1 .

Precursor ion (MS1)
m/zobs m/zcalc ppm GlcN(-Ac)-GlcN Red (-Ac)-Lac-Ala- Glu Supplementary Data 1.2: Tandem mass spectrometry analysis of the h1-type hybrid of the disaccharide-tripeptide. The molecule is fully unlabeled except for one of the two glucosamine moieties present in GlcNAc or MurNAc leading to the presence of two isotopomers. The observed m/z obs and calculated m/z calc values of the parental ion, [M+H] + , as determined in the absence of fragmentation (MS 1 ), are indicated. The m/z cal value was used to select the ion for fragmentation. The difference between the m/z obs and m/z cal values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively. The fragmentation led to two series of fragments specific of the isotopomers containing the unlabeled glucosamine moiety in the GlcNAc (h1G) or MurNAc Red (h1M) residues, respectively (discriminatory fragments  Supplementary Data 2.2: Tandem mass spectrometry analysis of the h1-type hybrid of the disaccharide-tetrapeptide. The molecule is fully unlabeled except for one of the two glucosamine moieties present in GlcNAc or MurNAc leading to the presence of two isotopomers. The observed m/z obs and calculated m/z calc values of the parental ion, [M+H] + , as determined in the absence of fragmentation (MS 1 ), are indicated. The m/z cal value was used to select the ion for fragmentation. The difference between the m/z obs and m/z cal values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively. The fragmentation led to two series of fragments specific of the isotopomers containing the unlabeled glucosamine moiety in the GlcNAc (h1G) or MurNAc Red (h1M) residues, respectively (discriminatory fragments). The fragments specific of h1G and of h1M are highlighted by orange and green dots in the mass spectrum. The other fragments are common to the fragmentation patterns of h1G and of h1M. The corresponding peaks are highlighted by blue dots in the mass spectrum. The mass spectrum indicates the presence of both isotopomers as expected from the recycling and synthesis pathways since UDP-MurNAc exclusively derives from UDP-GlcNAc. In the mass spectrum, peaks differing by the loss of H  The m/z calc value was used to select the ion for fragmentation. The difference between the m/z obs and m/z calc values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (  Supplementary Data 3.3: Tandem mass spectrometry analysis of the Tri→Tri dimer containing a uniformly labeled disaccharide-tripeptide cross-linked to a uniformly unlabeled disaccharide-tripeptide (3→3 cross-link). The observed m/z obs and calculated m/z cal values of the parental ion, [M+2H] 2+ , as determined in the absence of fragmentation (MS1), are indicated. The m/z calc value was used to select the ion for fragmentation. The difference between the m/z obs and m/z calc values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively. The fragmentation potentially leads to two series of fragments (discriminatory fragments) specific of the isotopomers containing the fully labeled disaccharide subunit in the donor (all heavyall light) or acceptor position (all light-all heavy). These fragments are highlighted by green and orange dots in the mass spectrum, respectively. The other fragments are common to the fragmentation of the two isotopomers (highlighted by blue dots in the mass spectrum). The mass spectrum indicates the presence of the all light-all heavy isotopomers (orange dots). The peak at m/z obs 681.358 (green dot) can also be accounted for by the loss of H 2 0 from the peak at 699.367 indicating that all peaks may be accounted for by the presence of the all light-all heavy isotopomer.
In the mass spectrum, peaks differing by the loss of H The m/z calc value was used to select the ion for fragmentation. The difference between the m/z obs and m/z calc values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively. The peaks corresponding to the list of fragments appearing in the table are highlighted by orange dots in the mass spectrum. In the mass spectrum, peaks differing by the loss of H  The m/z calc value was used to select the ion for fragmentation. The difference between the m/z obs and m/z calc values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively. The peaks corresponding to the list of fragments appearing in the table are highlighted by orange dots in the mass spectrum.
In the mass spectrum, peaks differing by the loss of H Supplementary Data 5.3: Tandem mass spectrometry analysis of the Tri→Tetra dimer containing a uniformly labeled disaccharide-tetrapeptide cross-linked to a uniformly unlabeled disaccharide-tripeptide (3→3 cross-link). The observed m/z obs and calculated m/z cal values of the parental ion, [M+2H] 2+ , as determined in the absence of fragmentation (MS1), are indicated. The m/z calc value was used to select the ion for fragmentation. The difference between the m/z obs and m/z calc values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively.
In the mass spectrum, peaks differing by the loss of H 2 0 are connected by a dashed line. The arrows indicate the position of the 3→3 and 4→3 crosslinks. Residues in the acceptor are underlined. An interactive report of the MS2 analysis is available in Supplementary File F5.3.
Supplementary Data 6.3: Tandem mass spectrometry analysis of the Tetra→Tetra dimer containing a uniformly labeled disaccharide-tripeptide cross-linked to a uniformly unlabeled disaccharide-tripeptide (4→3 cross-link). The observed m/z obs and calculated m/z cal values of the parental ion, [M+2H] 2+ , as determined in the absence of fragmentation (MS1), are indicated. The m/z calc value was used to select the ion for fragmentation. The difference between the m/z obs and m/z calc values is indicated in ppm. The GlcNAc residue is indicated in two moieties, glucosamine (GlcN) and the acetyl group (Ac), since these moieties are potentially differentially labeled. For the same reason, the reduced (Red) MurNAc residue is indicated in three moieties, reduced glucosamine (GlcN Red ), the acetyl group (Ac), and the d-lactoyl group. Labeled and unlabeled moieties are represented in red and purple, respectively. The fragmentation potentially leads to two series of fragments (discriminatory fragments) specific of the isotopomers containing the fully labeled disaccharide subunit in the donor (all heavy-all light) or acceptor position (all light-all heavy). These fragments are highlighted by green and orange dots in the mass spectrum, respectively. The other fragments are common to the fragmentation of the two isotopomers (highlighted by blue dots in the mass spectrum). The mass spectrum indicates the presence of the all light-all heavy isotopomers (orange dots). The peak at m/z obs 1128.568 (green dot) can also be accounted for by the loss of H 2 0 from the peak at 1142.567 indicating that all peaks may be accounted for by the presence of the all light-all heavy isotopomer.
In the mass spectrum, peaks differing by the loss of H 2 0 are connected by a dashed line. The arrows indicate the position of the 3→3 and 4→3 crosslinks. Residues in the acceptor are underlined. An interactive report of the MS2 analysis is available in Supplementary File F6.3.