Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids

Abstract

Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.

Figure 1

Incorporation of NCDAAs into PG muropeptides of diverse bacteria is not linked to their synthesis. The percentage of D-Met-containing peptides was calculated relative to total muropeptide isolated. (A) Co-culture system that permits chemical communication between two strains while they are kept physically apart (top). Percentage of D-Met-containing muropeptides in PG from strain B (bsrV V. cholerae) following co-culture with either WT or bsrV V. cholerae (bottom); note that the strain A culture was inoculated 2 h before the strain B culture to permit higher NCDAAs accumulation and hence better yields of incorporation into the PG. (B) Percentage of D-Met-containing muropeptides in PG from diverse bacteria following growth in the presence of exogenous 2 mM (C. crescentus) or 5 mM (remaining species) D-Met. The data shown are representative of two independent experiments.

Figure 2

L,D-transpeptidases incorporate non-canonical D-amino acids into tetrapeptides of V. cholerae PG. Muropeptides within purified PG were identified and quantified using HPLC. (A) Schematic representation of muropeptide structures, illustrating amino acid content and peptide chain length. The canonical pentapeptide structure is shown on the left. (B) Percentage of muro4^M and muro5^M peptides, relative to total muropeptides, in PG purified from the indicated V. cholerae strains at stationary phase following growth in LB. (C) Percentage of muro4^M and muro5^M peptides, relative to total muropeptides, in PG purified from WT V. cholerae following the exponential phase growth in LB supplemented with the indicated concentration of D-Met. (D) Abundance of DAP-DAP muropeptides (muro-DAP-DAP), Lpp muropeptides (muro-Lpp) and D-Met muropeptides (muro-D-Met), relative to total amount of muropeptides from the indicated strains. Muro-Lpp includes the lipoprotein-attached muropeptides mono3-Lpp (GlcNAc-MurNAc-L-Ala-D-Glu-γ-meso-DAP-ε-L-Lys-L-Thr) and the crosslinked dimer GlcNAc-MurNAc-L-Ala-D-Glu-γ-meso-DAP-D-Ala-meso-DAP-(ε-L-Lys-L-Thr)-γ-D-Glu-L-Ala-MurNAc-GlcNAc. The Thr-Lys dipeptide corresponds to the C-terminal dipeptide of the V. cholerae homologue for E. coli Braun's lipoprotein. Digestion of sacculi with pronase E, one step of HPLC sample processing, degrades the covalently bound lipoprotein molecules leaving the C-terminal dipeptide bound to the connecting muropeptide which can therefore be easily differentiated. (E) Immunodetection of D-cysteine-labelled murein in sacculi from the indicated strains of V. cholerae. For each strain, the inset box was magnified to generate the subpanel on the right. The data shown are representative of three independent experiments.

Figure 3

In vitro characterization of LdtA. Muropeptides were identified and quantified using HPLC; no reaction products other than those shown in the figures were detected. (A) The percentage of substrate converted into product via in vitro L,D-transpeptidation by LdtA is shown for various muropeptides. Reactions were incubated for 30 min (purified muropeptides) or 2 h (sacculi). (B) The percentage of mono4 altered by in vitro LdtA transpeptidation to have a different terminal amino acid when incubated (2 h) in the presence of the indicated L- or D-amino acid is shown. The data shown in (A) and (B) are representative of three and two independent experiments, respectively.

Figure 4

Incorporation of non-canonical D-amino acids into muropeptides occurs via several Ldt-independent pathways. (A) The relative percentage of muro4^M and muro5^M peptides in PG isolated from stationary phase cultures of V. cholerae grown in the absence (control) or presence of D-cycloserine (100 μg/ml). (B) Schematic representation of the in vitro conversion of D-amino acids (D-Ala and/or D-Met) and UDP-M3 into UDP-M5 and UDP-M5^M by Ddl and MurF. (C) Yield, based on HPLC quantification of UDP muropeptides following A₂₆₂ (UDP detection), of the reaction depicted in (B), when the indicated amino acid substrates are supplied. (D) The percentage of muropeptides that contain D-Met (muro4^M or muro5^M) in various bacterial species and their ldt mutants following growth in 5 mM D-Met, or 2 mM in the case of C. crescentus. (E, F) The effects of D-cycloserine (100 μg/ml) and/or penicillin G (50 μg/ml) on the relative level of muro5^M peptides in PG isolated from C. crescentus (E) or B. subtilis (F) following growth in the presence of 2 mM or 5 mM D-Met, respectively. The data shown are representative of three independent experiments.

Figure 5

Influence of D-amino acids and Ldts on PG abundance and osmotic tolerance of stationary phase V. cholerae. (A, C) Relative abundance of PG in stationary (A) and exponential (C) V. cholerae cells. Abundance of murein from the indicated mutants was normalized to that from wild-type (WT) cells in each growth phase. Mean and standard deviation values are derived from six independent experiments. Asterisks above the error bars in the mutants represent significant P-values from comparisons of the WT strain and the corresponding mutant (^***=0.0001, ^**<0.003). (B, D) Relative survival of stationary (B) or exponential (D) V. cholerae cells following a hypo-osmotic challenge. Survival of the indicated mutants following a 15-min incubation in distilled water is shown relative to the survival of the WT strain. Asterisks represent significant P-values from comparisons of mutants with the WT strain or from comparisons of the indicated pairs; ^*<0.03, ^**<0.002, ^***<0.0001.

Figure 6

The alternative sigma factor RpoS contributes to growth phase-dependent expression of bsrV, ldtA and ldtB. (A) The abundance of Lpp muropeptides (DAP-Lpp) and DAP-DAP muropeptides (DAP-DAP) in PG from exponential and stationary phase V. cholerae. (B) The culture density (OD₆₀₀; open circles) and β-galactosidase activities (Miller units; closed symbols) of strains containing chromosomal transcription reporter fusions for bsrV, ldtA and ldtB are shown as a function of time. Note that in (B) the culture densities of all three strains overlap, and therefore it has been represented with a single growth curve. (C, D) Comparison of the β-galactosidase activities of the reporter strains shown in (B) with their ΔrpoS derivative strains in exponential (OD₆₀₀=0.4) (C) and stationary (OD₆₀₀=3.5) (D) phase. Mean and standard deviation values reflect three (A, C and D) or two (B) independent experiments; ^**=0.0012, ^***=0.0003.

Figure 7

Schematic representation of the pathways of incorporation of non-canonical D-amino acids into the V. cholerae cell wall. (1) Periplasmic BsrV racemase converts L-Met into D-Met. (2) Periplasmic L,D-transpeptidases LdtA and LdtB incorporate D-Met into the fourth position of the peptide moiety of the muropeptides. (3) Transport of D-Met into the cytoplasm through an ABC transporter, for example, a MetNIQ homologue (Kadaba et al, 2008). (4) Ddl forms D-Ala-D-Met dipeptides that (5) serve as substrates for MurF (Duncan et al, 1990) to generate a UDP-muramyl-(D-Met)-pentapeptide (UDP-M5^M). Finally, (6) cytosolic activities generate the final precursor unit (UDP-disaccharide pentapeptide) (Barreteau et al, 2008) that (7) is flipped to the outer face of the cytoplasmic membrane to be incorporated into the macromolecular murein by PBPs with transglycosylase and transpeptidase activities.