Lactate racemization as a rescue pathway for supplying D-lactate to the cell wall biosynthesis machinery in Lactobacillus plantarum

Abstract

Lactobacillus plantarum is a lactic acid bacterium that produces d- and l-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for d-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production of d-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-pentadepsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for d-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires l-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by l-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production of l-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is d-lactate dependent. We also show that the growth defect can be partially restored by expression of the d-alanyl-d-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various d-2-hydroxy acids but not d-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in d-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in d-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for d-lactate production upon inactivation or inhibition of the LdhD enzyme.

FIG. 1.

Schematic representation of the connection between d-lactate production and peptidoglycan biosynthesis in the L. plantarum double ldhD lar mutant (PG6212). When both d-lactate production pathways (LdhD and Lar) are disrupted (|), production of the d-alanyl-d-lactate depsipeptide is blocked and peptidoglycan synthesis cannot occur (×). The ldhD lar mutant can be rescued in the presence of d-lactate or other d-2-hydroxy acids. Alternatively, d-alanine can replace d-lactate by heterologous expression of the d-alanyl-d-alanine ligase from L. lactis. Enzymatic reactions potentially involved in the incorporation of d-alanine in the peptidoglycan precursors of the ldhD lar suppressor mutants are depicted by dashed arrows. Regulation of the Lar activity by d- and l-lactate is indicated by curved arrows. The UDP-N-acetylmuramoyl-l-alanyl-γ-d-glutamyl-meso-diaminopimelate moiety of the peptidoglycan precursor is shown as a circle. LdhL, NAD-dependent l-lactate dehydrogenase; LdhD, NAD-dependent d-lactate dehydrogenase; Lar, lactate racemase; LpDdl, L. plantarum Ddl ligase; LcDdl, L. lactis Ddl ligase; MurF, UDP-N-acetylmuramoyl-tripeptide-d-alanyl-d-lactate ligase; PG, peptidoglycan; Van^R, resistant to high concentrations of vancomycin; Van^S, resistant to low concentrations of vancomycin.

FIG. 2.

Lactate racemization (Lar) activity in L. plantarum. (A) Lar activity of L. plantarum NCIMB8826 (wild-type, black bars), TF101 (ΔldhL, white bars), and TF102 (ldhD::cat, gray bars) at different growth stages, assayed with l-lactate as a substrate. Growth of the wild type in MRS broth at 28°C is shown as the log of the OD₆₀₀. Growth of the TF101 and TF102 mutants were comparable to that of the wild-type strain. (B) Influence of d- and l-lactate on the Lar activity of L. plantarum TF102. Cells were grown in MRS broth to an OD₆₀₀ of 0.75. Then, d- and/or l-lactate was added to the culture as indicated (bottom of panel B), and the cells were allowed to grow for 2 h. Cells were collected, and Lar activities were measured with l-lactate (▪) or d-lactate (□) as a substrate. (C) Lar activity of L. plantarum NCIMB8826, TF101, and TF102, assayed with l-lactate as a substrate. The activities were measured with (□) or without (▪) induction by l-lactate (0.2 M for 2 h at 28°C). The data shown are from one representative experiment from at least three independent repetitions.

FIG. 3.

Time course of l-lactate induction of the lactate racemase activity in L. plantarum TF101 (ΔldhL). l-lactate (0.2 M) was added to a TF101 culture at an OD₆₀₀ of 0.75. Samples were taken every 30 min for lactate racemization assay (A, white bars) and Northern blot analysis (B). The OD (A, solid line, squares) and percentage of d-lactate (A, dashed line, triangles) were monitored during induction. The Lar activity was assayed with l-lactate as a substrate. The data presented are from one of three independent experiments that gave essentially the same results. ND, Lar activity not determined. The Northern blot was carried out with a larA probe. (C) RNA electrophoresis of the Northern blot in panel B. Equivalent amounts of total RNA (8 μg) were loaded in each lane.

FIG. 4.

Structural organization of the lar operon in L. plantarum and other bacteria. Homologous genes in the different loci are colored the same. The numbers above the genes indicate the percentage of identity between the encoded protein and the corresponding protein of L. plantarum. Except for L. sakei, the LarC1-LarC2 protein homologues were compared to the complete LarC protein from L. plantarum, obtained by artificially adding an A in the frameshift region. Strains: L. plantarum, strain WCFS1 (AL935263); L. sakei, strain 23K (AY849556), P. pentosaceus, strain ATCC 25745 (AAEV000000000); C. acetobutylicum, strain ATCC 824 (AE001437); S. epidermidis, strain ATCC 12228 (AE015929); W. succinogenes, strain DSM1740 (BX571656).

FIG. 5.

Growth of the L. plantarum ldhD lar mutant (PG6212) in MRS (A) or MPL (B) broth after d-lactate starvation. The mutant was grown to an OD₆₀₀ of 0.5 in the presence of 20 mM d-lactate, washed twice in MRS (A) or MPL (B) broth, and resuspended in fresh MRS (A) or MPL (B) medium with d-lactate concentrations of 0 (⧫), 0.01 (▴), 0.05 (▵), 0.1 (⋄), 0.5 (•), or 20 (○) mM. The dl-lactate-producing wild-type NCIMB8826 strain was introduced as a control (□ [dashed line]). The data presented are from one representative experiment from three independent repetitions.

FIG. 6.

Suppression of the growth defect of the L. plantarum ldhD lar mutant (PG6212) with the d-alanyl-d-alanine forming LcDdl ligase from L. lactis. The nisin-controlled expression cassette (P_nisA::Lcddl) was stably integrated into the PG6212 chromosome (strain PG1174). The PG1174 strain was grown in MPL broth containing nisin (50 ng/ml) and d-lactate (20 mM) to an OD₆₀₀ of 0.5. Cells were then washed twice in MPL broth and resuspended in MPL containing (triangles) or not (diamonds) nisin (50 ng/ml). The parent PG6212 strain supplemented with d-lactate (20 mM) was introduced as a control (squares). The data shown are from one of three independent experiments that gave essentially the same results.