Evidence for Na⁺Influx via the NtpJ Protein of the KtrII K⁺Uptake System inEnterococcus hirae

Abstract

The ntpJ gene, a cistron located at the tail end of the vacuolar-type Na ؉ -ATPase (ntp) operon of Enterococcus hirae, encodes a transporter of the KtrII K ؉ uptake system. We found that K ؉ accumulation in the ntpJdisrupted mutant JEM2 was markedly enhanced by addition of valinomycin at pH 10. Studies of the membrane potential (⌬⌿; inside negative) by 3,3-dihexyloxacarbocyanine iodide fluorescence revealed that the ⌬⌿ was hyperpolarized at pH 10 in JEM2; the ⌬⌿ values of the parent strain ATCC 9790 and JEM2, estimated by determining the equilibrium distribution of K ؉ or Rb ؉ in the presence of valinomycin, were ؊118 and ؊160 mV, respectively. ⌬⌿ generation at pH 10 was accomplished by an electrogenic Na ؉ efflux via the Na ؉ -ATPase, whose levels in the two strains were quite similar. Na ؉ uptake driven by an artificially imposed ⌬⌿ (inside negative) was missing in JEM2, suggesting that NtpJ mediates Na ؉ movement in addition to K ؉ movement. Finally, the growth of JEM2 arrested in K ؉

Na+-dependent K+ Uptake Ktr System from the Cyanobacterium Synechocystis sp. PCC 6803 and Its Role in the Early Phases of Cell Adaptation to Hyperosmotic Shock

“…The system from Synechocystis sp. PCC 6803 is the third Ktr system for which Na ϩ dependence has been established (26,27). Because at present no Na ϩ -independent Ktr system is known, Na ϩ dependence may be a general feature of Ktr systems.…”

“…However, ntpJ from E. hirae has later been identified as a K ϩ -translocating subunit of the bacterial Ktr-type K ϩ uptake system (11, 21, 24 -27). Ktr from Vibrio alginolyticus is Na ϩ -dependent, and the same may apply to the Ktr system from E. hirae (26). KtrB belongs to a superfamily of K ϩ transporter proteins, with members in Archaea (KdpA, TrkH), bacteria (KdpA, TrkH, KtrB), fungi (TRK), and plants (HKT) (26 -38).…”

All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K+ Uptake by the KtrAB System from Vibrio alginolyticus

“…Ktr is present in many bacteria (1, 29 -32) and appears to always require Na ϩ ions for activity (9,10,32). It is not known whether Na ϩ activates the K ϩ transport process (32) or whether Ktr functions as a K ϩ /Na ϩ symporter (10). VaKtr consists of two types of subunits.…”

“…It belongs to the KTN (K ϩ transport, nucleotide binding) protein/domain family (PFAM2254/COG0569; GOLD genomes online database) (33) and has been proposed to regulate K ϩ transport via KtrB by a change in its binding from NAD ϩ to NADH according to a "ligand-mediated conformational switch" mechanism (33). The second subunit, the integral membrane protein VaKtrB (called NtpJ in Enterococcus hirae) (10,34), is responsible for K ϩ transport across the membrane (2,29). In this study, we mutated each of the four putative selectivity filter glycine residues in VaKtrB at positions 70, 185, 290, and 402 (see Fig.…”

Identification ofListeria monocytogenesGenes Involved in Salt and Alkaline-pH Tolerance

“…Considering the orientation of the genes and the presence of putative terminators, the phenotypes of these four mutants could not be linked to a polar effect of the mutation on downstream genes. This was not the case for sal5, in which the transposon was inserted into lmo0992, a gene of unknown function located upstream from lmo0991 which encodes a protein with similarities to NtpJ of Enterococcus hirae, a K ϩ /Na ϩ transporter (16). In these five mutants, similarity searches could not assign a putative function to the genes interrupted, but a search for transmembrane domains with the DAS program (5) revealed that FIG.…”