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D-Amino Acids Govern Stationary Phase Cell Wall Remodeling in Bacteria

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Science  18 Sep 2009:
Vol. 325, Issue 5947, pp. 1552-1555
DOI: 10.1126/science.1178123

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.

In all kingdoms of life, cells predominantly use L-amino acids. In most bacteria, the only D-amino acids produced in significant quantities are D-Ala and D-Glu, which are incorporated into peptidoglycan (PG) (1). PG is a strong and elastic polymer of the bacterial cell wall that is synthesized and modified by penicillin-binding proteins (PBPs). PG counteracts the cell’s osmotic pressure, maintains cell shape, and anchors components of the cell envelope. The chemistry and structure of PG is dynamic (1, 2), but the factors that regulate alterations in the composition and architecture of PG are not well understood.

Here, we report that a mutant in Vibrio cholerae mrcA, which encodes a PBP1A homolog (12), retained its normal rod shape during exponential growth but became spherical in its stationary phase (Fig. 1A). The mutant’s shape change was due to factor(s) that accumulated in the culture media, because exponentially growing rod-shaped mrcA cells started to become spherical within 5 min after addition of cell-free supernatants from stationary phase cultures (Fig. 1B). We used the rod-to-sphere shape transition of the mrcA mutant as an indicator for such factors and examined their role in the development of PG from wild-type cells.

Fig. 1

D-Amino acids induce rod-shaped mrcA V. cholerae cells to become spheres. (A) Growth phase-dependent morphology of wild-type and mrcA V. cholerae; culture densities [optical density (absorbance) at 600 nm]. (B) Kinetics of the change in mrcA cell morphology after addition of stationary phase supernatant (Sup) + Luria-Bertani broth (LB) or LB alone. (C) Morphologic effects 45 min after addition of 1 mM L-Met or D-Met to rod-shaped mrcA cells. Scale bar, 2 μm.

Stationary phase supernatants were fractionated and assayed for their sphere-inducing activity (3). The active fractions consisted of four amino acids: Met, Leu, Val, and Ile. The D-forms, but not L-forms, of these amino acids stimulated the conversion of mrcA rods to spheres (Fig. 1C). A 1.0 mM mixture of these D-amino acids had sphere-inducing activity nearly equivalent to that of unfractionated stationary phase supernatant. Thus, the absolute stereochemistry at the alpha carbon of these amino acids determines their sphere-inducing activity.

We quantified the D- and L-forms of all amino acids in V. cholerae culture supernatants. D-Met and D-Leu were the predominant D-amino acids detected (fig. S1). Total D-amino acid concentrations were low until early stationary phase, then increased for ~8 hours and plateaued at ~1.0 mM (Fig. 2A). Thus, D-amino acid accumulation in stationary phase supernatants could account for sphere-inducing activity. D-Ala was not found in the supernatant, so this normal component of PG is not shed into the media. The abundance of particular D-amino acids, along with the ratios of the enantiomers (fig. S1), suggests a specific mechanism for creating these D-isomers in stationary phase.

Fig. 2

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

Bacteria generate the D-Glu and D-Ala found in PG by specific amino acid racemases, enzymes that convert the chiral carbon of these amino acids from L- to D-forms (4). The V. cholerae genome codes for a putative amino acid racemase (vc1312), in addition to Glu and Ala racemases. A strain lacking vc1312 (renamed here bsrV, broad-spectrum racemase Vibrio) had normal growth and morphology but produced minimal D-Met, D-Leu, D-Val, and D-Ile (Fig. 2B), which suggested that the BsrV racemase generated these four D-amino acids. Notably, BsrV was found in the periplasm (fig. S2), unlike Glu and Ala racemases (4).

Although the morphology of wild-type cells was not altered by D-amino acids (fig. S3), the metabolic expenditure of producing D-amino acids suggested they have important physiological function(s). Because exogenous D-amino acids can influence PG composition and structure (5, 6), we compared PG isolated from wild-type and bsrV V. cholerae. The bsrV mutant contained twice the amount of PG of wild-type cells in stationary phase, whereas PG levels did not differ in exponential phase (Fig. 3A). Addition of physiological amounts of D-Met and D-Leu to bsrV cultures reduced the amount of PG to wild-type levels, which confirmed that the absence of D-amino acids accounted for the increased PG in the bsrV mutant. Thus, D-amino acids negatively regulate the amount of PG in stationary phase cells. Moreover, the structure of wild-type and bsrV PG isolated from stationary phase cells differed significantly. The glycan chains in stationary phase PG from the bsrV mutant were ~80% the length of the wild type; pentapeptides were reduced by ~50%; and there was an increase in trimer muropeptides (table S1). Despite being less abundant, the PG in wild-type cells appeared to be stronger than in bsrV cells. Wild-type cells survived 20 times more than bsrV cells when subjected to an osmotic challenge (Fig. 3B). Thus, D-amino acid production by BsrV provides a cue for V. cholerae to decrease PG synthesis and to alter its cell wall in adaption to stationary phase conditions.

Fig. 3

Influence of D-amino acids 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 (PG) percentage 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 cross-linked at meso–diaminopimelic acid (DAP-DAP)], D-43 (dimer cross-linked at D-Ala-DAP), T444 (trimer cross-linked at D-Ala-DAP and D-Ala-DAP).

One mechanism by which D-amino acids could alter PG is by incorporation into the polymer. Escherichia coli grown in 20 mM D-Met incorporated this amino acid at the fourth position of the PG-peptide bridge (5). High-performance liquid chromatography (HPLC) analyses of PG isolated after the addition of physiologic D-Met (0.5 mM) to exponentially growing V. cholerae revealed two peaks that were not detected in control cultures (Fig. 3C and fig. S4, A and C). These peaks correspond to monomer and dimer muropeptides (Fig. 3D) containing D-Met, the same muropeptides observed when 20 mM (or 0.5 mM) D-Met was added to E. coli cultures (fig. S4, C and D). Therefore, physiologic production of D-amino acids by V. cholerae could influence the PG composition of nearby bacteria of different species. Furthermore, these D-Met–containing muropeptides were present in PG isolated from wild-type, but not bsrV, stationary phase cells (Fig. 3C and fig. S4, B and C). About 5% of stationary phase PG contained D-Met modified muropeptides. Thus, physiological production of D-Met by BsrV results in its incorporation into PG in stationary phase. Incorporation of D-Met probably occurs in the periplasm where it is produced, perhaps by means of the activity of a penicillin-insensitive transpeptidase (5, 7), and may directly influence the strength or flexibility of the PG owing to alterations in structure.

D-Amino acids may also have effects on PG that are not a direct consequence of their incorporation. Indeed, 2.0 mM D-Ala stimulated the conversion of rod-shaped mrcA mutant cells to spheres, even though D-Ala is already present at the site where D-Met was incorporated (fig. S5). Furthermore, exogenous D-Met (and not L-Met) prevented the binding of Bocillin-FL, a fluorescent penicillin derivative, to several V. cholerae cell envelope–associated PBPs, which suggested that D-amino acids directly bound PBPs (fig. S6). Thus, D-amino acids may be regulators, as well as substrates, of the periplasmic enzymes that synthesize and modify PG. Moreover, the periplasmic targeting of BsrV (fig. S2) efficiently links the site of D-amino acid production with the cell wall targets of their action.

Production of D-amino acids was not limited to V. cholerae. Diverse bacterial species released D-amino acids (Fig. 4A and table S2). All of these species appear to encode several putative racemases, which suggests that they could produce D-amino acids other than D-Ala and D-Glu (table S3). The particular D-amino acids identified in stationary phase supernatants varied among bacterial species (Fig. 4A).

Fig. 4

D-Amino acids are produced by diverse bacterial species and influence B. subtilis PG synthesis. (A) Concentrations of all 19 D-amino acids (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. Scale bar, 2 μm. Insets are magnified on the right of each corresponding image.

We tested if physiological concentrations of D-amino acids also influenced cell wall synthesis in Bacillus subtilis, a Gram-positive bacteria that is highly divergent from V. cholerae. To monitor synthesis, we used BodipyFL vancomycin (Van-BDP), which labels the cell wall precursor Lipid II (8). In exponential control cells grown in the presence or absence of a mixture of L-amino acids, Van-BDP predominantly stained the septum and the sidewalls in a pattern suggestive of helical and septal PG insertion (8) (Fig. 4, B and C, and fig. S7). In contrast, 30 min after the addition of a mixture of D-amino acids found in stationary phase B. subtilis supernatants (table S2), Van-BDP staining of the sidewalls was greatly reduced, which resulted in a pattern similar to that of untreated stationary phase cells (Fig. 4, B and C, and fig. S8). Thus, D-amino acids could down-regulate PG synthesis in stationary phase B. subtilis, as well as in V. cholerae. D-Amino acids released in stationary phase might influence B. subtilis PG synthesis by their incorporation into PG (as shown for D-Tyr in fig. S9) and/or by other mechanisms, such as alterations of the activity of B. subtilis PBPs. Gross changes in cell morphology occurred when B. subtilis was treated with superphysiological concentrations of D-Tyr (fig. S7), reinforcing the idea that D-amino acids regulate cell wall structure. Moreover, addition of the stationary phase D-mixture slowed the growth of exponential B. subtilis cultures (9) (fig. S10). Thus, release of D-amino acids by B. subtilis during stationary phase may be a mechanism to synchronize growth inhibition and PG synthesis when population density becomes saturating.

Here, D-amino acids were found to be released in stationary phase by diverse bacteria as agents of a process through which cell populations synchronously controlled cell wall assembly and modification. Because the accumulation of D-amino acids coincides with the transition into stationary phase and appears to down-regulate PG synthesis, D-amino acids may enable coordination of metabolic slowing in cell wall and cytoplasmic compartments when resources become scarce. Furthermore, release of D-amino acids may allow for interspecies regulation among bacteria or other organisms that occupy the same niche. D-Amino acids probably affect other aspects of bacterial physiology in addition to those described here. Indeed, D-Ser present in human urine regulates uropathogenic E. coli virulence gene expression (10), and D-amino acids induce sporulation in Myxococcus xanthus (11). Thus, bacteria have taken advantage of amino acid chirality to sense and respond to specific environmental conditions.

Supporting Online Material

www.sciencemag.org/cgi/content/full/325/5947/1552/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 to S4

References

  • * These authors contributed equally to this work.

  • Present address: Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Seoul 151-742, Republic of Korea.

  • Present address: Yale University, New Haven, CT 06520, USA.

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank B. Davis, C. Jacobs-Wagner, and D. Rudner for insightful comments on the manuscript. This work was supported by Howard Hughes Medical Institute (HHMI); NIH AI-R37-42347 (M.K.W.) and CA24487 and GM086258 (J.C.); Ministry of Education and Science, Spain (MEC) BFU2006-04574 and Fundación Ramón Areces (M.A.P.); Jane Coffin Childs Fellowship (H.L.); MEC Fellowship (F.C.); and HHMI Exceptional Research Opportunities (EXROP) (C.N.T.).
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