Schematic representation of the high-throughput automated muropeptide analysis (HAMA) framework.

(a) The peptidoglycans of bacteria were extracted and purified, followed by mutanolysin digestion. The resulting muropeptide products are analyzed by UHPLC-MS/MS and identified using the HAMA platform. The HAMA strategy involves simplifying muropeptide structures to sequence format, which facilitates the database construction and in silico generation of b- and y- ion fragmentation spectra for matching. Muropeptide symbols: B, N-acetylglucosamine; M, N-acetylmuraminitol (without lactyl group); l, lactic acid; A, alanine; E, glutamic acid; H, diaminopimelic acid. (b) DBuilder constructs a muropeptide database containing monomers, dimers, and trimers with two types of linkage: glycosidic bonds and peptide bonds. For peptide linkages, the direct way is through a direct covalent bond between the penultimate D-Ala and mDAP residue in the acceptor peptide stem, and the indirect way is via an interpeptide bridge branching from the lysine. Donors are labeled in red, and acceptors are labeled in black. (c) The flowchart outlines the LC-MS data processing in Analyzer.

Automated identification of well-characterized peptidoglycans from E. coli and S. aureus using the HAMA platform.

(a, c) Base peak chromatograms showing the muropeptide analysis of E. coli and S. aureus. The label content includes retention time (in red), feature index, and muropeptide class (in black). (b, d) Extracted ion chromatograms of the most abundant muropeptide and their MS/MS spectra annotated with b- and y- fragments were visualized in Viewer. Muropeptide symbols: B, N-acetylglucosamine; M, N-acetylmuraminitol (without lactyl group); l, lactic acid; A, alanine; E, glutamic acid; Q, glutamine; K, lysine; G, glycine.

Muropeptides of Escherichia coli DH5α / Staphylococcus aureus SA113 analyzed by UPLC-MS/MS.

The characterized peptidoglycan types of gut bacteria used in this study.

Resolving isomeric muropeptides by in silico MS/MS fragmentation matching.

Two isomeric muropeptides with the same parent ion, m/z 471.22, were identified as two disaccharide-tripeptides: (a) B-M-l(-A-E-(N-)K eluted at 6.18 min, and (b) B-M-l(-A-Q-(D-)K eluted at 7.02 min. The sequence of each isomer was determined using in silico MS/MS fragmentation matching, with the identified sequence having the highest matching score. Muropeptide symbols: B, N-acetylglucosamine; M, N-acetylmuraminitol (without lactyl group); l, lactic acid; A, alanine; E, glutamic acid; Q, glutamine; K, lysine; N, Asparagine; D, Aspartic acid.

Muropeptide composition analysis of Bifidobacterium breve strains.

(a) Schematic representation of the two possible cross-linking types in the PGN of B. breve: Ddt-mediated 4-3 cross-link and Ldt-mediated 3-3 cross-link. Donor peptide stems are labeled in red. The arrow indicates the direction of cross-links catalyzed by transpeptidases. (b) Base peak chromatograms of muropeptide analysis of B. breve ATCC 15700, CSCC 1900, and ATCC 15698 strains. The main peaks were annotated with muropeptide symbols. (c) Heatmap showing the muropeptide compositions (% of total) of the PGN of three B. breve strains. Symbols: M, monomer; D, dimer; T, trimer (numbers following the letters indicate the number of amino acids in stem peptides). M0, disaccharide; M1, disaccharide-monopeptide; M2, disaccharide-dipeptide; M4, disaccharide-tetrapeptide; M3b, disaccharide-tripeptide with an interpeptide bridge; M3Nb, disaccharide-tripeptide with an anhydro-MurNAc and an interpeptide bridge; M4b, disaccharide- tetrapeptide with an interpeptide bridge; M4Nb, disaccharide-tetrapeptide with an anhydro- MurNAc and an interpeptide bridge; M5b, disaccharide-pentapeptide with an interpeptide bridge; D34, disaccharide-tripeptide–disaccharide-tetrapeptide with a peptide cross-link; D34N, disaccharide-tripeptide–disaccharide-tetrapeptide with a peptide cross-link and an anhydro- MurNAc; D44, disaccharide-tetrapeptide–disaccharide-tetrapeptide with a peptide cross-link; D44N, disaccharide-tripeptide–disaccharide-tetrapeptide with a peptide cross-link and an anhydro-MurNAc; D45, disaccharide-tetrapeptide–disaccharide-pentapeptide with a peptide cross-link; T335, disaccharide-tripeptide–disaccharide-tripeptide-disaccharide-pentapeptide with two peptide cross-links; T444, disaccharide-tetrapeptide–disaccharide-tetrapeptide-disaccharide- tetratapeptide with two peptide cross-links; T445, disaccharide-tetrapeptide–disaccharide- tetrapeptide-disaccharide-pentapeptide with two peptide cross-links.

Bridge length-dependent cell envelope stiffness in B. longum and B. breve.

(a) Schematic illustration of the Gram-positive PGN architecture of B. longum (orange tetrapeptide bridges) and B. breve (green monopeptide bridges). Glycan strands composed of repeating units of the β-1,4-linked disaccharides are cross-linked by interpeptide bridges, forming 3-D peptidoglycan layers. (b) Heatmap displaying the muropeptide compositions (% of total) of the PGNs in B. longum and B. breve (three strains each). Symbols: M, monomer; D, dimer; T, trimer (numbers following the letters indicate the number of amino acids in stem peptides). M0, disaccharide; M1, disaccharide-monopeptide; M2, disaccharide-dipeptide; M4, disaccharide- tetrapeptide; M5, disaccharide-pentapeptide; M3b, disaccharide-tripeptide with an interpeptide bridge; M3Nb, disaccharide-tripeptide with an anhydro-MurNAc and an interpeptide bridge; M4b, disaccharide-tetrapeptide with an interpeptide bridge; M4Nb, disaccharide-tetrapeptide with an anhydro-MurNAc and an interpeptide bridge; M5b, disaccharide-pentapeptide with an interpeptide bridge; D34, disaccharide-tripeptide–disaccharide-tetrapeptide with a peptide cross- link; D34N, disaccharide-tripeptide–disaccharide-tetrapeptide with a peptide cross-link and an anhydro-MurNAc; D44, disaccharide-tetrapeptide–disaccharide-tetrapeptide with a peptide cross-link; D44N, disaccharide-tripeptide–disaccharide-tetrapeptide with a peptide cross-link and an anhydro-MurNAc; D45, disaccharide-tetrapeptide–disaccharide-pentapeptide with a peptide cross-link; T335, disaccharide-tripeptide–disaccharide-tripeptide-disaccharide-pentapeptide with two peptide cross-links; T444, disaccharide-tetrapeptide–disaccharide-tetrapeptide-disaccharide- tetratapeptide with two peptide cross-links; T445, disaccharide-tetrapeptide–disaccharide- tetrapeptide-disaccharide-pentapeptide with two peptide cross-links; Glycan-T, trimers linked by glycosidic bonds. (c) AFM imaging of living Bifidobacterium. Topographical images in PBS buffer and the inset shows elasticity images from the top of the cell. Distribution of Young’s modulus values corresponding to the elasticity images in the inset. (d) Statistical analysis was performed for each strain, showing the distribution of two groups with the shorter interpeptide bridge corresponded to higher stiffness of cell envelope. Shown here are the mean values (cross), the median, and the 25 and 75% quartiles (boxes) obtained from N independent cells over at least three independent experiments. P values were calculated using a one-way ANOVA analysis. *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001.