A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria

Abstract

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium Bacillus subtilis with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect B. subtilis growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.

Keywords: bacteria; cell wall; synthetic cross-links; transpeptidases.

Fig. 1.

(A) The cell wall of Gram-positive bacteria is anchored to the outer leaflet of the membrane by lipoteichoic acids (LTAs; shown in yellow). Walled teichoic acids (WTAs; shown in orange) provide additional support between branches (36). (A, B) The peptidoglycan is composed of glycan that assembles from the disaccharide pair NAG (shown and labeled in dark green)-NAM (light green). Peptide stems (blue) connect the glycan via enzyme-catalyzed cross-linking reactions. The glycan of the cell wall is degraded by muramyl hydrolases, and to a lesser extent by lytic transglycosylases, the latter producing anhydroNAM (shown in red) as the reaction product (37).

Fig. 2.

(A) Comparative mechanisms of d,d-transpeptidase-mediated canonical cell-wall cross-linking and noncanonical cell-wall cross-linking by exogenous d-AAs in B. subtilis. (B) Structures of electrophilic noncanonical d-AAs and cognate l-AA controls. (C) Bacterial growth curves of B. subtilis untreated or treated with d-AA 1c or l-AA 1d. (D) Structure of the noncanonical 5,3-cross-linked NAG-NAM-(pentapeptide)-NAG-anhydroNAM-(tetrapeptide) formed by 1c. The gray boxes show a comparison of the synthetic cross-link and the native cross-link. For each noncanonical d-AA the primary synthetic cross-linked muropeptide formed comprises the canonical NAG-NAM-(tetrapeptide) (R′) and NAG-anhydroNAM-(tetrapeptide) (R′′), where the noncanonical d-AA is installed adjacent to the fourth-position d-Ala of the R′ stem, replacing the fifth-position d-Ala. The mass spectra corresponding to the synthetic non–cross-linked and cross-linked cell-wall species formed by (E, F) 1a, (G, H) 1b, and (I, J) 1c are shown. Compounds 2a and 2b form synthetic non–cross-linked muropeptides, but not synthetic cross-linked muropeptides. The structures and masses of each unnatural synthetic cell-wall muropeptide is provided in SI Appendix (SI Appendix, Figs. S3–S8).

Fig. 3.

(A) HPLC trace (205 nm) of isolated peptidoglycan from B. subtilis treated with 1c (4 mM). The peaks corresponding to the most abundant muropeptide species for the native non–cross-linked (nC), native cross-linked (C), synthetic non–cross-linked (SnC), and synthetic cross-linked (SC) are labeled. SnC and SC correspond to the d-AA-modified structures. The black circles denote the amidated species, the gray circles denote the nonamidated non–cross-linked species, and gold circles denote the partially amidated and nonamidated cross-linked species. (B) The percentage of synthetic non–cross-linked and synthetic cross-linked muropeptides for cell wall isolated from B. subtilis after treatment with 1c. Note, percentages are based solely on the most abundant amidated native and synthetic species. A detailed quantification is provided in SI Appendix.

Fig. 4.

B. subtilis was cultured to OD₆₀₀= 0.05 and either not treated or treated with 4 mM 1c or 1d for 2 h. The bacteria were imaged by SEM and the results are shown. SEM of untreated bacteria (A) B. subtilis wild type and (B) B. subtilis ΔdacA display a linear rod shape. The ΔdacA mutation gives slightly shortened cells. Images of B. subtilis ΔdacA treated with 1c were captured at (C) 1,500× and (D, E) 15,000× magnification. Significant cell lysis is observed as indicated by white arrows. Bacteria that survive treatment by 1c are able to successfully elongate, but cell division appears impaired. Elongated cells display a spiraled phenotype. Images of B. subtilis ΔdacA treated with 1d were captured at (F) 1,500× and (G, H) 15,000× magnification. No cell lysis was observed for bacteria treated with 1d, although cells display a subtle curl. A 1-µM white scale bar is shown in A, B, D, E, G, and H (Top Right). A 5-µM white scale bar is shown in C and F (Top Right).

Fig. 5.

(A) Simplified depiction of the proposed glycan degradation route of synthetically modified cell wall in the Gram-negative bacteria E. coli by glycan-cleaving enzymes. The noncanonical d-AAs are shown as yellow circles. The red lines indicate potential cut sites of the enzymes in each respective panel. (B) Bacteria growth curve of E. coli treated with compound 1a (1 mM). Gray circles indicate growth collection points, at which time growth was halted by pelleting and freezing of the bacterial culture. (C) HPLC trace (Abs. 205 nm) of E. coli bacterial cell wall at each collection point. (D) Quantification of SnC and SC muropeptides relative to the total muropeptide concentration of nC and SnC, and C and SC, respectively. (E) Simplified depiction of the proposed glycan degradation route of synthetically modified cell wall in the Gram-positive bacteria B. subtilis by glycan-cleaving enzymes. TAs not shown. (F) Bacteria growth curve of B. subtilis treated with compound 1a (1 mM) (G) HPLC trace (Abs. 205 nm) of B. subtilis bacterial cell wall. (H) Quantification of SnC and SC muropeptides of B. subtilis. A figure key for cell-wall structures is provided in Fig. 1.