Redundant potassium transporter systems guarantee the survival of Enterococcus faecalis under stress conditions

Abstract

Enterococcus is able to grow in media at pH from 5.0 to 9.0 and a high concentration of NaCl (8%). The ability to respond to these extreme conditions requires the rapid movement of three critical ions: proton (H⁺), sodium (Na⁺), and potassium (K⁺). The activity of the proton F₀F₁ ATPase and the sodium Na⁺ V₀V₁ type ATPase under acidic or alkaline conditions, respectively, is well established in these microorganisms. The potassium uptake transporters KtrI and KtrII were described in Enterococcus hirae, which were associated with growth in acidic and alkaline conditions, respectively. In Enterococcus faecalis, the presence of the Kdp (potassium ATPase) system was early established. However, the homeostasis of potassium in this microorganism is not completely explored. In this study, we demonstrate that Kup and KimA are high-affinity potassium transporters, and the inactivation of these genes in E. faecalis JH2-2 (a Kdp laboratory natural deficient strain) had no effect on the growth parameters. However, in KtrA defective strains (ΔktrA, ΔkupΔktrA) an impaired growth was observed under stress conditions, which was restored to wild type levels by external addition of K⁺ ions. Among the multiplicity of potassium transporters identify in the genus Enterococcus, Ktr channels (KtrAB and KtrAD), and Kup family symporters (Kup and KimA) are present and may contribute to the particular resistance of these microorganisms to different stress conditions. In addition, we found that the presence of the Kdp system in E. faecalis is strain-dependent, and this transporter is enriched in strains of clinical origin as compared to environmental, commensal, or food isolates.

Keywords: Enterococcus faecalis; KUP/HAK/KT K+ transporters; Kdp system; Ktr family; potassium transport.

Figure 1

Schematic representation of the mechanisms involved ion fluxes in Enterococcus faecalis.

Figure 2

KimA and Kup of Enterococcus faecalis are potassium transporters. (A) pWH844 derived carrying full copy of Kup (pWH-kup) or KimA (pWH-kimA) used in this study. (B) Escherichia coli LB650 strains harboring plasmids pWH844, pWH-kup, and pWH-kimA were grown in M9-mod solid medium with or without supplementation of KCl (1, 5, or 50 mM). (C) Growth rate parameters determined at different KCl concentrations of LB650 derived strains, pWH844 (blue dot), pWH-kup (red dot), and pWH-kimA (green dot). Inhibition of Kup activity by c-di-AMP (D) pBAD derived carrying a copy of cdaA^lmo (pBP370) or cdaA^lmo* (pBP373) used in this study. (E,F) Escherichia coli LB2003 harboring the plasmid combinations pWH844/pBP370 (blue dot), pWH-kup/pBP370 (empty red dot), and pWH-kup/pBP373 (full red dot), was cultivated in minimal salt M9mod medium, and supplemented without arabinose (E) or with arabinose 0.05% (F).

Figure 3

Growth curves of Enterococcus faecalis JH2-2 mutants under different conditions. Wild type (black dot), ΔktrA (light blue square), ΔkupΔktrA (yellow circle), and ΔktimAΔktrA (orange triangle) strains were grown in LBG (A,D,G), mLBG (B,E,H), or mLGB medium supplemented with 10 mM KCl (C,F,I). Initial pH was fixed at 9.0 units (A–C), 7.0 (D–F), or 5.0 (G–I).

Figure 4

Inhibition of growth rate of Enterococcus faecalis JH2-2 mutants by NaCl. Wild type (black dot), ΔktrA (light blue square), ΔkupΔktrA (yellow circle), and ΔktimAΔktrA (orange triangle) strains were grown in LBG (A,C) and mLBG medium (B,D) at initial pH 9.0 (top) or 7.0 (low) in the presence of increasing concentrations of NaCl.

Figure 5

KtrA contributes to the osmotic response in Enterococcus faecalis. Wild type (black dot), ΔktrA (light blue square), ΔkupΔktrA (yellow circle), and ΔktimAΔktrA (orange triangle) strains were grown in mLBG at alkaline initial pH and supplemented with 3% NaCl (A) or 750 mM sorbitol (C). In both growth conditions, the addition of 10 mM KCl (B and D, respectively) completely restored defective strains growth to near wild-type levels. (E) Schematic representation of the plasmid pBV-ktrA used for complementation of a full copy of the wild type ktrA gene. (F) Expression of ktrA from a pBV153 fully complemented the growth defects of the KtrA-mutant strains in mLBG at alkaline initial pH and supplemented with 3% NaCl, and in all conditions tested in this study (data not shown).

Figure 6

(A) Heatmap showing the presence of K⁺ transporters and related ATPases in Enterococcus species. A presence-absence matrix was constructed and Distance-based clustering was applied. Dark blue indicates presence of the genes analyzed. (B) Analysis of the presence of kdp genes and the phylogenomic relationships among E. faecalis members. 34 common ancestral genes were individually aligned, concatenated, and trimmed resulting in a final alignment containing a total of 4,726 residues. The evolutionary history of the 1,510 E. faecalis members was inferred with RAxML algorithm (Stamatakis, 2014) and displayed and annotated using iTOL (Letunic and Bork, 2011). The inner circles colors indicate the source of isolation; F, food (orange); C, commensal (yellow); E, environmental (light blue); and H, clinical (green). The red outside circles indicate the presence of the kdp genes within the strains.