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Journal of Bacteriology, October 2005, p. 6750-6761, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6750-6761.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Lactate Racemization as a Rescue Pathway for Supplying D-Lactate to the Cell Wall Biosynthesis Machinery in Lactobacillus plantarum

Philippe Goffin,¹ Marie Deghorain,¹ Jean-Luc Mainardi,² Isabelle Tytgat,¹ Marie-Christine Champomier-Vergès,³ Michiel Kleerebezem,⁴ and Pascal Hols^1*

Unité de Génétique, Institut des Sciences de la Vie, Université Catholique de Louvain, Place Croix du Sud 5, B-1348 Louvain-la-Neuve, Belgium,¹ Laboratoire de Recherche Moléculaire sur les Antibiotiques INSERM E0004, 5, Rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France,² Institut National de la Recherche Agronomique, Unité Flore Lactique et Environnement Carné, Domaine de Vilvert, 78350 Jouy-en-Josas, France,³ Wageningen Centre for Food Sciences; NIZO Food Research, P.O. Box 20, 6710 BA, Ede, The Netherlands⁴

Received 26 March 2005/ Accepted 8 July 2005

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.

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* Corresponding author. Mailing address: Unité de Génétique, Université Catholique de Louvain, Place Croix du Sud 5, B-1348 Louvain-la-Neuve, Belgium. Phone: 32-10-478896. Fax: 32-10-473109. E-mail: hols{at}gene.ucl.ac.be.

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Journal of Bacteriology, October 2005, p. 6750-6761, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6750-6761.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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