D-amino acids govern stationary phase cell wall remodeling in bacteria

Abstract

In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.

Figure 1

D-aa induce rod-shaped mrcA V. cholerae cells to become spheres. (A) Growth phase-dependent morphology of wild-type and mrcA V. cholerae; culture densities (OD600nm). (B) Kinetics of the change in mrcA cell morphology after addition of stationary phase supernatant (Sup) + LB or LB alone. (C) Morphologic effects 45 min after addition of 1 mM L-Met or D-Met to rod-shaped mrcA cells. Bar size = 2 μm.

Figure 2

BsrV is required for production of D-aa in V. cholerae supernatants. (A, B) Kinetics of the accumulation of the indicated D-aa in supernatants from wild-type (A) or bsrV (B) V. cholerae.

Figure 3

Influence of D-aa on the amount and composition of PG and osmotic tolerance. (A) PG quantification in wild-type or bsrV cells grown in LB or LB supplemented with D-Met and D-Leu. Murein % was normalized to wild-type in each growth phase. (B) Recovery of wild-type or bsrV V. cholerae from media with the indicated concentrations of NaCl. A representative experiment is shown. (C) Portions of the HPLC profiles of muropeptides from exponentially (Exp) growing wild-type cells ± 0.5 mM D-Met or stationary phase (Stat) wild-type or bsrV cells. D-Met-containing peaks are the monomer M4-Met and the dimer D44-Met; their structures are shown schematically in (D). Muropeptide reference peaks: D3(LD)4 (Dimer crosslinked at DAP-DAP), D-43 (Dimer crosslinked at D-Ala-DAP), T444 (Trimer crosslinked at D-Ala-DAP, D-Ala-DAP).

Figure 4

D-aa are produced by diverse bacterial species and influence B. subtilis PG synthesis. (A) Concentrations of all 19 D-aa were measured and those above 0.01 mM are displayed. Vancomycin-BDP staining of (B) exponential phase B. subtilis supplemented with a mixture of physiologic D- (or the corresponding L-) amino acids or (C) unsupplemented exponential or stationary phase B. subtilis. Bar size = 2 μm. Insets are magnified on the right of each corresponding image.