Dissecting the contribution of Kup and KimA to Enterococcus faecalis potassium homeostasis

In previous studies, we highlighted the remarkable diversity of potassium transport systems in Enterococcus faecalis. This microorganism encodes five distinct potassium transport systems: the Kdp complex (KdpFABC and KdpED), the Ktr systems (KtrAB and KtrAD), and transporters from the Kup family, specifically Kup and KimA. Research focused on the E. faecalis JH2-2 strain, which, unlike E. faecalis V583, lacks the Kdp complex and exhibits an altered expression of the kup gene due to the insertion of the IS6770 sequence. Here, we characterize the kimA-kup locus of the JH2-2 strain, which harbors genes encoding a metal transport protein (MntE), the Opu osmoresistance system (OpuCA-CB-CC-CD), and a Ca²⁺-ATPase (CaaT). It was determined that the kimA gene is monocistronic and transcribed from a consensus vegetative promoter featuring conserved -10 (TAgcAT) and -35 (TTGACt) boxes. RT-qPCR analysis revealed that kimA expression was significantly upregulated under acidic stress. Furthermore, both kimA and kup expression are increased under alkaline conditions in the presence of NaCl. To dissect functional roles, growth parameters of mutant strains KimA⁺ (ΔktrAΔkup) and Kup⁺ (ΔktrAΔkimA) were analyzed. Kinetic assays demonstrated that the Kup⁺ strain exhibited higher potassium transport affinity than KimA⁺. Furthermore, differential responses to pH and osmotic stress highlighted distinct physiological contributions of KimA and Kup systems. KimA seems to be involved in K⁺ uptake at high concentrations (> 0.5 mM) while Kup played a dominant role at low potassium concentrations (> 0.2 mM) and under alkaline-osmotic stress. All these results delineate the functional divergence of the two transporters and our understanding of the ion homeostasis and stress adaptation mechanisms in E. faecalis.