Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum

Abstract

The UDP-MurNAc-pentapeptide is transferred to the outer face of the cell membrane by a lipid carrier and incorporated along with UDP-N-acetylglucosamine into the cell wall structure. The synthesis of other types of peptidoglycan precursors was demonstrated a few years ago in the context of several studies concerning vancomycin resistance. Vancomycin and other glycopeptide antibiotics can bind to the DAla-D-Ala terminus of pentapeptide-containing precursors by hydrogen bonding, thereby effectively blocking polymerization and preventing further cross-linking reactions (7, 41). Investigations of the molecular basis of vancomycin resistance started with strains of Enterococcus faecium and Enterococcus faecalis which showed inducible resistance to high levels of vancomycin and teicoplanin, another glycopeptide antibiotic. Examination of enzymes involved in cell wall synthesis in the resistant bacteria indicated that resistance to vancomycin was due to the synthesis of a novel type of peptidoglycan in which the terminal D-alanine residue was replaced by D-lactate, resulting in a drastic reduction of affinity for vancomycin (2,4,12,25,36). Two enzymes designated VanH and VanA are required for the synthesis of this alternative precursor (5). VanH is an ␣-ketoacid dehydrogenase that reduces pyruvate to D-lactate The synthesis of another type of peptidoglycan precursor has been described for Enterococcus gallinarum, which expresses inducible resistance to low levels of vancomycin but is susceptible to teicoplanin. In this case, the modified precursor terminates in D-serine instead of D-lactate (9). This feature results from the presence of another variant D-Ala-D-Ala ligase accepting D-serine (18).The genera Lactobacillus, Leuconostoc, and Pediococcus comprise strains and species constitutively resistant to vancomycin (15,20,33,39,46,49). Recently, peptidoglycan precursors from several of these lactic acid bacteria were analyzed. In Pediococcus pentosaceus and Lactobacillus casei (9, 26), the exclusive presence of a terminal D-lactate has been demonstrated. This presence could result from the action of a ligase which preferentially or exclusively catalyzes the synthesis of a D-Ala-D-Lac depsipeptide, as was suggested by Elisha and Courvalin (19). Analysis of Leuconostoc mesenteroides extracts identified a precursor that also terminates in D-lactate, but with an additional branched L-alanine In this paper, we report that the wild-type strain Lactobacillus plantarum NCIMB8826 is naturally resistant to high levels of vancomycin and teicoplanin and exclusively produces Dlactate-ending peptidoglycan precursors. We describe the construction of a strain defective for both D-and L-LDH, resulting in drastically reduced production of both isomers of lactate. We show that this alteration leads to the synthesis of a new type of precursor ending with D-alanine in addition to the usual muramyl depsipentapeptide observed in the wild-type strain,

Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

“…For general molecular biology techniques, we followed methods reported previously by Sambrook et al (44). The preparation of S. thermophilus chromosomal DNA and the electrotransformation of E. coli and S. thermophilus were performed as previously described (10,13,15). The primers used in this study were purchased from Eurogentec (Seraing, Belgium) and are listed in Table S1 in the supplemental material.…”

Functional and Morphological Adaptation to Peptidoglycan Precursor Alteration in Lactococcus lactis

“…Unlike their enterococcal van homologues, the genes encoding these different activities are not clustered within an operon, and they appear to have a more fundamental role in cell physiology than being simply required for vancomycin resistance (18,19,39). We, therefore, proposed that genes functionally equivalent to those of L. plantarum were initially recruited by glycopeptide producers to protect themselves against the antibiotic and were subsequently transmitted to enterococci and other bacterial species to confer resistance to vancomycin (18).…”

“…Resistance to Vancomycin; Further Adaptation to Peptidoglycan Reprogramming Is Required-Key enzymes for specific D-Lac incorporation into the cell wall of L. plantarum include the D-lactate dehydrogenase LdhD Lp , the D-Ala-D-Lac ligase ddl Lp , and the D-Ala-D-Ala dipeptidase Aad (18,19,39). Unlike their enterococcal van homologues, the genes encoding these different activities are not clustered within an operon, and they appear to have a more fundamental role in cell physiology than being simply required for vancomycin resistance (18,19,39).…”

Major Role of NAD-Dependent Lactate Dehydrogenases in Aerobic Lactate Utilization in Lactobacillus plantarum during Early Stationary Phase

“…1A). In L. plantarum, two stereospecific nLDHs are present (LdhD and LdhL), whose role in lactate production has been established (7,8). The LdhL enzyme is responsible for L-lactate production, while D-lactate can be produced in two ways: NADH-dependent reduction of pyruvate by the LdhD enzyme or racemization of L-lactate by an L-lactate-inducible lactate racemase (7,8) (Fig.…”

“…Under anaerobic conditions, Lactobacillus plantarum produces lactate as the major fermentation end product through the reduction of glycolytic pyruvate by NAD-dependent lactate dehydrogenases (nLDHs) (7,8). When grown in the presence of oxygen, L. plantarum ferments glucose into lactate until glucose becomes limiting.…”