Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis

Abstract

Two paralogous genes, maeE and citM, that encode putative malic enzyme family members were identified in the Enterococcus faecalis genome. MaeE (41 kDa) and CitM (42 kDa) share a high degree of homology between them (47% identities and 68% conservative substitutions). However, the genetic context of each gene suggested that maeE is associated with malate utilization whereas citM is linked to the citrate fermentation pathway. In the present work, we focus on the biochemical characterization and physiological contribution of these enzymes in E. faecalis. With this aim, the recombinant versions of the two proteins were expressed in Escherichia coli, affinity purified and finally their kinetic parameters were determined. This approach allowed us to establish that MaeE is a malate oxidative decarboxylating enzyme and CitM is a soluble oxaloacetate decarboxylase. Moreover, our genetic studies in E. faecalis showed that the citrate fermentation phenotype is not affected by citM deletion. On the other hand, maeE gene disruption resulted in a malate fermentation deficient strain indicating that MaeE is responsible for malate metabolism in E. faecalis. Lastly, it was demonstrated that malate fermentation in E. faecalis is associated with cytoplasmic and extracellular alkalinization which clearly contributes to pH homeostasis in neutral or mild acidic conditions.

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae

“…Therefore, we analyzed whether the growth of ABSA and UPSA strains would differ in a synthetic medium that contained a defined amount of malate. For this, we used SHU as defined in previous studies (56)(57)(58) supplemented with 40 mM malic acid, a concentration consistent with prior studies that have used 30 to 50 mM malic acid in vitro (51,(53)(54)(55)(59)(60)(61)(62). We observed that the growth of ABSA 834 and ABSA 729 was significantly higher than the growth of UPSA 058 and UPSA 714 in SHU (Fig.…”

“…Malic acid metabolism is typically associated with an operon comprising genes encoding a malate oxidoreductase enzyme (maeE), a per-mease/transporter (maeP), a transcriptional regulator (maeR), and/or an accessory membrane-anchored sensor kinase (maeK) (53). Bacterial MEs catalyze the oxidative decarboxylation of malate to pyruvate and CO 2 (54), which underpins malolactic fermentation. While there is some evidence that MEs contribute to energy generation and bacterial survival at low pH, a role for MEs in bacterial growth in urine has not previously been investigated.…”

“…In bacteria, a metabolic signature of L-malic acid utilization is often related to functional expression of the ME pathway, as characterized in Lactobacillus casei (55) and Enterococcus faecalis (54); however, bacteria can metabolize malic acid through different pathways; in E. coli and Bacillus subtilis, this is controlled by a two-component system (72,73); in lactic acid bacteria, including L. casei (55) and Streptococcus mutans (60,61), L-malic acid is converted to L-lactate by malolactic enzyme. E. faecalis (54,74), Streptococcus bovis (75), and L. casei (55) metabolize L-malic acid to pyruvate using the ME pathway. Oral streptococci degrade malic acid to create an alkaline environment that may protect against acid damage and oxidative and starvation stress (60)(61)(62).…”

Fine-Tuned Transcriptional Regulation of Malate Operons in Enterococcus faecalis

“…London and Meyer (27) reported in their pioneering work on malate metabolism in E. faecalis that the malate transporter and malic enzyme are induced by this substrate and apparently repressed by glucose (25). More recently, we have shown that the E. faecalis mae locus encodes the malate degradative enzymes (5). This locus is composed of two putative divergent operons, maePE and maeKR (Fig.…”

“…1A). The maeP (EF1207) (35) gene encodes a putative H ϩ /malate symporter, and the maeE (EF1206) (35) gene encodes a malic enzyme that converts malate into pyruvate (5). Pyruvate may be further metabolized via one or a combination of several pathways, depending on the energy needs and redox state of the cell, and is converted to lactate and NAD ϩ (via lactate dehydrogenase), ethanol and NAD ϩ (via alcohol dehydrogenase), or acetate and ATP (via phosphotransacetylase and acetate kinase) (25).…”