Glycerol is metabolized in a complex and strain-dependent manner in Enterococcus faecalis

Abstract

Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism.

FIG. 1.

Aerobic (A) and anaerobic (B) growth of different E. faecalis strains on glycerol. For anaerobic growth, 0.26% (wt/vol) fumarate (22.4 mM) was added to the ccM17MOPS growth medium. Symbols: white triangles, TX0104; white squares, JH2-2; white circles, MMH594; black triangles, V583; white diamonds, HH22; black squares, OG1RF; and multiplication signs, ATCC 19433.

FIG. 2.

Operon structures containing genes potentially implicated in glycerol metabolism in E. faecalis V583. The genes with assigned putative functions encode the following proteins: glpF, glycerol uptake facilitator; glpO, glycerol-3-P oxidase; glpK, glycerol kinase; glpD, glycerol-3-P dehydrogenase; gtaB, UTP glucose-1-phosphate uridylyltransferase; lgt, prolipoprotein transferase; hprK, HPr kinase/phosphatase; adh, alcohol dehydrogenase; gldA, glycerol dehydrogenase; and dhaK and dhaL, the two subunits of dihydroxyacetone kinase. Genes related to glycerol metabolism are highlighted in bold in each operon structure. hyp indicates genes encoding hypothetical proteins.

FIG. 3.

Aerobic growth of the JH2-2 wild-type strain (squares), the ΔgldA1 mutant (circles), the ΔglpK mutant (triangles), and the ΔgldA1 ΔglpK double mutant (diamonds) on glycerol. When used, catalase was added to growing cultures after 2 h of incubation. Growth in the presence of catalase led to similar results for the JH2-2 wild-type strain, the ΔgldA1 mutant, and the ΔglpK mutant. For clarity, these combined results are represented by one growth curve (multiplication signs). Catalase addition had no effect on the growth of the double mutant.

FIG. 4.

Anaerobic growth of the JH2-2 wild-type strain (squares), the ΔgldA1 mutant (circles), the ΔglpK mutant (triangles), and the ΔgldA1 ΔglpK double mutant (diamonds) on 0.3% (wt/vol) glycerol in the presence of 0.26% (wt/vol) fumarte.

FIG. 5.

Proposed model for aerobic glycerol catabolism in strains JH2-2, TX0104, and MMH594. Strains TX0104 and MMH594 utilize preferentially the dhaK and glpK pathways, respectively, for glycerol dissimilation. In the JH2-2 strain, glycerol flows through both pathways. In the dhaK pathway, glycerol is first oxidized to dihydroxyacetone (DHA) by glycerol dehydrogenase (G-dehydrogenase) and then phosphorylated to DHAP by dihydroxyacetone kinase. In the glpK pathway, glycerol is first phosphorylated to glycerol-3-P by glycerol kinase (G-kinase) and then oxidized to DHAP by glycerol-3-P oxidase (G3P-oxidase).