Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

Abstract

Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD₆₀₀] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival.

Importance: The peptidoglycan of the bacterial cell wall is turned over steadily during growth. As peptidoglycan fragments were found in large amounts in spent medium of exponentially growing Gram-positive bacteria, their ability to recycle these fragments has been questioned. We conclusively showed recycling of the peptidoglycan component MurNAc in different Gram-positive model organisms and revealed that a MurNAc-6P etherase (MurQ or MurQ ortholog) enzyme is required in this process. We further demonstrated that recycling occurs predominantly during the transition to stationary phase in S. aureus and B. subtilis, explaining why peptidoglycan fragments are found in the medium during exponential growth. We quantified the intracellular accumulation of recycling products in MurNAc-6P etherase gene mutants, revealing that about 5% and 10% of the MurNAc of the cell wall per generation is recycled in S. aureus and B. subtilis, respectively. Importantly, we showed that MurNAc recycling and salvaging does not sustain growth in these bacteria but is used to enhance survival during late stationary phase.

FIG 1

MurQ operon (MurNAc-recycling divergon) and MurNAc catabolic pathway in E. coli (top and right, respectively), and organization of chromosomal regions of murQ orthologs of the Gram-positive bacteria S. aureus, B. subtilis, and S. coelicolor. The schematic of the organization of the E. coli K-12 murQ operon genes includes the promoters for transcription of the murQ operon (P_q) and murR (P_R) and the terminator (lollipop), according to Jaeger and Mayer (20). murQ encodes MurNAc-6P etherase, murP encodes the MurNAc-specific phosphotransferase system (PTS) transporter EII-BC domain, and yfeW encodes the low-affinity penicillin binding protein 4B (PBP4B). Upstream from the murQ gene and divergently transcribed is the murR gene, a transcriptional repressor of the MurNAc recycling divergon. The schematic for S. aureus USA300 (NCBI Reference Sequence accession no. NC_007793.1) shows putative genes for MurNAc utilization, as well as the proteins they encode. SAUSA300_0192 encodes a protein whose function is unknown, SAUSA_0193 encodes an ortholog of MurQ, SAUSA_0194 encodes an MurP-like PTS EII-BC domain protein, and SAUSA_0195 encodes a MurR-like regulator. The schematic for B. subtilis 168 (NCBI Reference Sequence accession no. NC_000964.3) shows the putative promoter P_σA in front of the recycling cluster of 6 genes, including murQ (formerly ybbI), murR (formerly ybbH), murP (formerly ybbF), encoding the MurP EII-BC domain, amiE (formerly ybbE), encoding the MurNAc-l-alanine amidase AmiE, nagZ (formerly ybbD), encoding the N-acetylglucosaminidase NagZ, and ybbC, encoding a protein whose function is unknown. The schematic for S. coelicolor A3(2)/M145 (NCBI Reference Sequence accession no. NC_003888.3) shows a putative cluster of genes for MurNAc recycling that includes SCO4308, encoding a MurR-like regulator, and SCO4307, encoding an ortholog of MurQ, as well as two open reading frames encoding proteins whose functions are unknown, SCO4305 and SCO4306. The amino acid sequence identities (%) of orthologous proteins relative to the sequences of E. coli MurQ, MurP, and MurR are shown. Catabolite-responsive elements (cre sites) were identified in the promoter regions upstream from the murQ genes of B. subtilis and S. aureus.

FIG 2

Accumulation of MurNAc-6P in ΔmurQ mutants of Gram-positive bacteria but not in the parental strains. S. aureus (Sa), B. subtilis (Bs), and S. coelicolor (Sc) wild-type strains (WT) and ΔmurQ mutants, as well as the S. aureus ΔSAUSA_0192–0195 and B. subtilis ΔmurQRP mutants (Δoperon), were cultured in LB medium for 24 h. Acetone extracts of cytosolic fractions were analyzed by LC-MS in negative-ion mode. Mass spectra of MurNAc-6P in the investigated samples are presented with the total-ion chromatograms (TIC) (×10⁵ counts per s [cps]) in gray and the extracted-ion chromatograms (EIC) (×10³ cps) (m/z⁻¹ = 372.07 and retention time on the HPLC column of 21 min) in blue.

FIG 3

Growth of S. aureus and B. subtilis wild-type strains and ΔmurQ mutants in rich medium with or without MurNAc. S. aureus (Sa) and B. subtilis (Bs) wild-type strains (WT, solid symbols) and the respective mutants (ΔmurQ, open symbols) were grown in LB medium in the absence (circles) or presence (triangles) of 0.2% MurNAc. Bacterial growth was monitored by measuring optical density at 600 nm and is presented as the mean values ± standard errors of the means (SEM) in log₁₀ scale.

FIG 4

Determination of viable cell titers of S. aureus and B. subtilis wild-type strains (WT) and their ΔmurQ mutants grown in LB medium with or without MurNAc. Wild-type and ΔmurQ mutant cells of S. aureus and B. subtilis were grown in LB medium in the absence (left two bars in each graph) or presence of 0.2% MurNAc. Bacterial cultures were diluted appropriately in 0.9% NaCl solution in 96-well plates and plated on LB agar. Viable cells were determined by counting CFU/ml (×10⁸) at mid-exponential (4 h for S. aureus and 4.5 h for B. subtilis), transition (8 h and 10 h, respectively), and stationary (24 h, 48 h, and 72 h) phases. Data are presented as the mean values ± SEM from three independent biological replicates and were analyzed for statistical significance with the nonpaired t test. A P value of <0.05 was determined as statistically significant (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, nonsignificant).