Figures and data
Alanine racemase activity counters acetate intoxication
(A) The Nebraska Transposon Mutant library was screen against 20 mM acetic acid, pH 6.0 to identify mutants with altered growth phenotypes. The WT strain and transposon mutants were grown for 24 h in TSB ± 20 mM acetic acid. The bacterial growth at 24 h was measured spectrophotometrically (OD600) and normalized to WT growth. The X and Y-axis on the plot represent normalized growth values for each mutant in the presence or absence of acetate. (B) The growth of the WT, alr1 mutant, and alr1 complemented strain in TSB supplemented with 20 mM acetic acid. (C) Aerobic growth of WT, alr1, citZ, citZalr1 mutants in TSB media lacking glucose but supplemented with 20 mM acetic acid. (D) LC-MS/MS analysis was performed to quantify the intracellular D-Ala-D-Ala pool in strains cultured for 3 h (exponential phase) in TSB ± 20 mM acetic acid. (E) The growth of strains was monitored following D-Ala supplementation (5 mM) in TSB + 20 mM acetate, (n=3, mean ± SD). Ac, acetate.
Translational coupling of dat to pepV limits the alr1 mutant from countering acetate intoxication
(A) Schematic representation of various engineered mutations in the pepV-dat locus.SD, Shine-Dalgarno motif; TSS, transcriptional start site. (B)-(D) Growth of engineered mutants was monitored spectrophotometrically (OD600) in TSB supplemented with 20 mM acetate (n=3, mean ± SD). (E) RT-qPCR to determine dat transcription in various mutants relative to the WT strain.
Reaction orientation and fluxes through Alr1 and Dat
(A) Schematic representation of various isotopologues of D-Ala-D-Ala and D-Glu generated from 13C 15N 3 1 labeled L-Ala. Metabolites in blue mainly arise from Alr1, red, through the Ald1/2-Dat pathway and yellow are unlabeled intermediates within cells. The mass isotopologue distribution of (B) D-Ala-D-Ala and (C) D-Glu were determined by LC-MS/MS following the growth of S. aureus in chemically defined media supplemented with 13C 15N1 L-Ala (n=3, mean ± SD). Isotopologues of D-Ala-D-Ala shown in grey color are minor species and are noted in Table S9. (D) The mean competitive fitness (w) was determined by co-culturing the WT strain with an isogenic mutant that contained either the empty pAQ59 vector integrated into the SaPI chromosomal site (WTEV) or the pAS8 vector containing dat under the control of its native promoter (WTdat) (n=18, the dotted lines indicate the median and quartiles).
Acetate intoxication impacts soluble PG precursor pools and cell wall cross-linking.
(A) The intracellular pool of PG intermediates in exponential phase cultures of S. aureus was estimated using LC-MS/MS analysis. cps, counts per second (B) ddl and murF transcription in the exponential growth phase was determined by RT-qPCR analysis (n=3, mean ± SD). (C) Cell wall muropeptide analysis of the WT, alr1 and dat mutants was determined following growth in TSB ± 20 mM acetate for 3 h. Cell wall cross-linking was estimated as previously described (49). Ac, acetate.
Acetate anion inhibits Ddl activity.
(A) The intracellular D-Ala was determined by LC-MS/MS analysis. (B) The ddl gene was overexpressed in S. aureus using a cadmium inducible expression system (pSP36). CdCl2, 0.312 µM. (C) Inhibition of recombinant His-tagged Ddl activity in the presence of 300 mM sodium acetate (D) IC50 curve of the inhibition of rDdl by acetate. Michaelis-Menten kinetics of rDdl in varying concentrations of (E) D-Ala, and (F) ATP in the presence of acetate to assess the inhibition mechanism. (G) Structure of the acetate bound Ddl (PDB:8FFF). (H) Acetate bound to the ATP binding site of Ddl (I) Acetate bound to the second D-Ala binding site of Ddl. The calculated Fo-Fc omit maps are contoured to 3σ and the mesh is shown in blue. (J) Superimposed structure of acetate bound Ddl (slate blue) with StaDdl apo structure (PDB:2I87, beige) and StaDdl-ADP complex structure (PDB:2I8C, grey) showing a shift of ω loop (red) to ATP binding site. The D-Ala-D-Ala was modeled at the D-Ala binding site using Thermos thermophius HB8 Ddl structure (PDB:2ZDQ). The bound ADP (grey) of PDB:2I87 and modeled D-Ala-D-Ala (light blue) indicates the positioning of Ac at ATP and second D-Ala binding sites respectively. Ac, acetate; V, velocity.
Biologically relevant weak acids inhibit growth of the alr1 mutant.
Molecular docking of (A) lactate (B) propionate and (C) itaconate to the ATP binding site of Ddl. (D) The relative positions and poise of different organic anions in relation to acetate in the D-Ala binding site of Ddl was determined using Schrödinger Glide. The growth (OD600) of the WT and alr1 mutant in TSB containing (E) lactic acid (40 mM) (F) propionic acid (20 mM) and (G) itaconic acid (20 mM) in the presence or absence of 5 mM D-Ala.
Model depicting the role of Alr1 in countering organic acid anion-mediated inhibition of Ddl.
During its growth, S. aureus (WT) maintains a substantial intracellular pool of D-Ala through the activity of Alr1. Any excess D-Ala is subsequently converted into D-Glu by the action of the Dat enzyme. The high concentration of D-Ala is crucial for the optimal functioning of Ddl and serves to prevent the inhibition of Ddl by acetate (Ac-) and other organic acid anions. This process generates sufficient D-Ala-D-Ala, which is rapidly incorporated into the PG tripeptide precursor UDP-NAM-AEKAA to form UDP-NAM-AEKAA, which ultimately contributes to a robust cross-linked PG (murein) sacculus. In the alr1 mutant, the Dat reaction orientation is switched to preserve intracellular D-Ala. Nevertheless, this change is inadequate to maintain sufficient D-Ala pool to shield Ddl from inhibition by Ac-, due to tight control of dat translation. This results in an excess of UDP-NAM-AEK, which competes effectively with UDP-NAM-AEKAA for PG incorporation. The absence of a terminal D-Ala-D-Ala in the PG hinders crosslinking and leads to impaired growth following acetate intoxication.
Alr1 and Dat are the primary routes of D-Ala production in S. aureus.
(A) Schematic of the predicted D-Ala-D-Ala generating pathways in S. aureus. Growth curves of various S. aureus strains grown in the (B) absence or (C) presence of 5 mM D-Ala (n=3, mean ± SD).
Overexpression of dat rescues the growth defect of the alr1 mutant
(A) dat was cloned in a multicopy vector (pSP4) controlled by its native promoter. The S. aureus strains containing pSP4 and pLI50 (empty vector) were grown in TSB supplemented with 20 mM acetate (B) RT-qPCR analysis of dat expression in the alr1 mutant in the presence or absence of 20 mM acetic acid (n=3, mean ± SD).
Muropeptide analysis.
Representative chromatograms of muropeptide extracts from (A-C) WT, alr1 and dat mutants, and following (D-F) acetate intoxication. A unique peak corresponding to NAG-NAM-AEK (M3, m/z, Da: 826.4080) was identified in the alr1 mutant. The peak area of M3 was normalized to the total area of peaks observed in the chromatogram and expressed as percent (see inset figure in B, n=3, mean ± SD). M, monomer; D, dimer; T, trimer, Tt, tetramer.
Overexpression of ddl rescues the growth defect of the alr1 mutant
The growth (OD600) of the WT and alr1 mutants overexpressing Ddl (pSP36; cadmium inducible expression of ddl) in TSB supplemented with (A) lactic acid (40 mM), (B) propionic acid (20 mM) and (C) Itaconic acid (20 mM) in the presence or absence of 5 mM D-Ala (n=3, mean ± SD).
Effect of acetate on Ddl activity
Impact of acetate on Ddl stability assessed by DSF
Refinement statistics of Ddl/Acetate structure
Glide scores from molecular docking studies of organic anions
Strains used in this study
Plasmids used in this study
Primers used in this study
Table of Multiple Reaction Monitoring (MRM) transitions
