Tolerance of low pH in Schizosaccharomyces pombe requires a functioning pub1 ubiquitin ligase

A strain of Schizosaccharomyces pombe carrying a disrupted Na⁺/H⁺ antiporter gene (sod2::sup3-5), in addition to the common auxotrophic mutations, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9. Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongated and failed to form colonies at pH 3.5. Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence of two independently segregating mutations. One, pub1 ( protein ubiquitin ligase 1), has recently been reported as an E3 protein ubiquitin ligase involved in cdc25 turnover. The second has been named elp3-1 (elongated at low pH). Genetic dissection of the original strain revealed that poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the function of permeases responsible for uptake of the necessary nutrients. Suppression of the high pH sensitivity required the presence of both the pub1-1 and elp3-1 mutations. While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of LiCl (3 mM) in liquid culture, it was capable of partially suppressing the extreme Li⁺ sensitivity caused by the sod2 disruption. Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was improved over that of either pub1-1 or sod2::ura4 cells. The elp3-1 mutation had no effect on the Li⁺ tolerance in either wild-type or sod2::ura4 backgrounds. pub1-1 cells are elongated and incapable of colony formation at pH 3.5. In contrast, elp3-1 cells are elongated at pH 3.5 and pH␣5.5 (the normal pH of minimal medium) but can form colonies under both conditions. J227 cells are significantly longer than either single mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5. Complementation cloning in the J227 background yielded a genomic clone of pub1, allowing us to define the intron-exon structure of the gene. Sequences with high homology to the predicted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd4). Based on the nature of our mutant selection, the pH-sensitive phenotype of the strains selected, and the known involvement of RSP5/NPI1 in membrane permease turnover in S. cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transport processes. This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at high and low external pH, and in the presence of toxic levels of Li⁺. Received: 12 September 1996 / Accepted: 19 December 1996     

Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli

Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal N a⁺/H⁺ antiporters, the result was a Li⁺ sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low external pH. Sod2 was then expressed in E. coli MH1 which is devoid of endogenous Na⁺/H⁺ antiporters. These cells became more resistant to external LiCl, but only in Na⁺ containing media. In the absence of external Na⁺, the presence of sod2 reduced growth. The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na⁺/H⁺ antiporter in conjunction with different housekeeping systems of E. coli host cells.     

Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4

Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.     

Revealing the Ligand Binding Site of NhaA Na+/H+ Antiporter and Its pH Dependence

pH and Na⁺ homeostasis in all cells requires Na⁺/H⁺ antiporters. In most cases, their activity is tightly pH-regulated. NhaA, the main antiporter of Escherichia coli, has homologues in all biological kingdoms. The crystal structure of NhaA provided insights into the mechanism of action and pH regulation of an antiporter. However, the active site of NhaA remained elusive because neither Na⁺ nor Li⁺, the NhaA ligands, were observed in the structure. Using isothermal titration calorimetry, we show that purified NhaA binds Li⁺ in detergent micelles. This interaction is driven by an increase in enthalpy (ΔH of −8000 ± 300 cal/mol and ΔS of −15.2 cal/mol/degree at 283 K), involves a single binding site per NhaA molecule, and is highly specific and drastically dependent on pH; Li⁺ binding was observed only at pH 8.5. Combining mutational analysis with the isothermal titration calorimetry measurements revealed that Asp-163, Asp-164, Thr-132, and Asp-133 form the Li⁺ binding site, whereas Lys-300 plays an important role in pH regulation of the antiporter.     

The <Emphasis Type="Italic">alm1</Emphasis><Superscript><Emphasis Type="Italic">+</Emphasis></Superscript> gene from <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> encodes a coiled-coil protein that associates with the medial region during mitosis

We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 ⁺ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 ⁺ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 ⁺ / alm1::ura4 ⁺ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe.     

E3 Ubiquitin Ligase Pub1 Is implicated in Endocytosis of a GPI-Anchored Protein Ecm33 in Fission Yeast

We previously identified three glycosylphosphatidylinositol (GPI)-anchored proteins including Ecm33, as multicopy suppressors of the phenotypes of a mutant allele of cis4⁺ that encodes a zinc transporter in fission yeast. Here, we further identified two multicopy suppressor genes, ubi1 ⁺ and ubc4 ⁺, encoding ubiquitin-ribosomal fusion protein and ubiquitin conjugating enzyme E2, respectively. In addition, Ubi1 or Ubc4 overexpression failed to suppress the phenotypes of the double deletion of cis4 ⁺ and pub1 ⁺ gene, which encodes a HECT-type ubiquitin ligase E3. During exponential phase GFP-Ecm33 localized at the growing cell tips of the cell surface and the medial region in wild-type cells. Notably, during the post-exponential and stationary phase, GFP-Ecm33 in wild-type cells was internalized and mostly localized to the Golgi/endosomes, but it was still stably localized at the cell surface in Δpub1 cells. The Δpub1 cells showed osomoremedial phenotypes to various drugs indicating their defects in cell wall integrity. Altogether, our findings reveal a novel role for Pub1 in endocytosis of Ecm33 and regulation of cell wall integrity in fission yeast.     

Kinetics of charge translocation in the passive downhill uptake mode of the Na/H antiporter NhaA of Escherichia coli

The Na⁺/H⁺ antiporter NhaA is the main Na⁺ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li⁺ and Na⁺ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na⁺ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent K_m values were 11 mM for Na⁺ and 7.3 mM for Li⁺ at basic pH and 180 mM for Na⁺ and 50 mM for Li⁺ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent K_m as well as turnover (V_max).     

Li+ effect on the cell wall of the yeast Saccharomyces cerevisiae as probed by FT-IR spectroscopy

The effect of Li⁺ ions as a transformation inducing agent on the yeast cell wall has been studied. Two Saccharomyces cerevisiae strains, p63-DC5 with a native cell wall, and strain XCY42-30D(mnn1) which contains structural changes in the mannan-protein complex, were used. Fourier transform infrared (FT-IR) spectroscopy has been used for the characterization of the yeast strains and for determination of the effect of lithium cations on the cell wall. A comparison of the carbohydrate absorption band positions in the 970–1185 cm^?1 range, of Na⁺ and Li⁺ treated yeast cells has been estimated. Absorption band positions of the cell wall carbohydrates of p63-DC5 were not influenced by the studied ions. On the contrary, the treatment of XCY42-30D(mnn1) cells with Li⁺ ions shifted glucan band positions, implying that the cell wall structure of strain XCY42-30D(mnn1) is more sensitive to Li⁺ ion treatment.     

Sodium-dependent succinate uptake in purple bacterium Ectothiorhodospira shaposhnikovii

Succinate, malate and fumarate uptake in purple sulfur bacterium Ectothiorhodospira shaposhnikovii, strain 1 K MSU, obligatorily depends on the presence of Na⁺. Other monovalent cations such as K⁺, Li⁺, NH₄⁺ could not replace Na⁺. Experiments with energy-depleted cells have shown that succinate uptake against its concentration gradient can be energized by artificially imposed sodium gradients (ΔpNa).An artificial membrane potential (inside negative) inhibited ΔpNa-driven succinate uptake at pH 7.0 but stimulated it at pH 9.0.The results confirm the suggestion that succinate uptake in E. shaposhnikovii is carried out in symport with Na⁺.     

Biochimica et biophysica acta,vol. 644 (1981)

In the presence of an iso-osmotic concentration (0.4 M) of LiCl, the exit of cellular K⁺ and concomitant entry of Li⁺ in the marine bacterium, Vibrio alginolyticus, were enhanced by an increase in the medium pH, with an optimum at about pH 9.6. In addition to alkaline pH, the K⁺ exit in the NaCl medium required the presence of a weak base such as diethanolamine, ethanolamine or methylamine, which is permeable to the membrane in its unprotonated form. No net entry of Na⁺ was detected in this case and the amine accumulated in exchange for K⁺. The K⁺ exit observed at alkaline pH could be explained by the function of a K⁺/H⁺ antiporter. Once the cells were loaded with the amine, their exposure to the NaCl medium in the absence of loaded amine induced the entry of Na⁺. In RbCl or CsCl medium, fast entry of Rb⁺ or Cs⁺ and exit of K⁺ were observed at neutral pH (7.5), and the rate of K⁺ exit increased with the medium pH. From these results, we established a simple method for the replcement of cellular cations with a desired cation (Li⁺, Na⁺, K⁺, Rb⁺ or Cs⁺). The present method was found to be applicable also to Escherichia coli.     

Effect of temperature,salts, pH and other factors on the development of peritrichous flagella in Vibrio alginolyticus

The development of peritrichous flagella and, consequently, swarming of Vibrio alginolyticus depend on a complex relationship between temperature, salt concentrations and pH. At temperatures above 28°C V. alginolyticus did not develop peritrichous flagella unless certain minimal concentrations of NaCl are present: the higher the temperature, the higher the NaCl concentrations required for peritrichous flagella synthesis. This requirement for NaCl at high temperatures is much more pronounced at pH 9 than at pH 6. High temperatures and low concentrations of NaCl also inhibited swarming of cells already armed with peritrichous flagella. Other cations, such as Li⁺, K⁺ and Mg²⁺, replaced NaCl only at temperatures below 28°C.     

The alm1+ gene from Schizosaccharomyces pombe encodes a coiled-coil protein that associates with the medial region during mitosis

We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 ⁺ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 ⁺ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 ⁺ / alm1::ura4 ⁺ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe. Received: 9 April 1999 / Accepted: 22 October 1999     

Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis

The novel species Halomonas zhaodongensis NEAU-ST10-25^T recently identified by our group is a moderate halophile which can grow at the range of 0–2.5 M NaCl (optimum 0.5 M) and pH 6–12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25^T in this study for Na⁺(Li⁺)/H⁺ antiporter genes by selection in Escherichia coli KNabc lacking three major Na⁺(Li⁺)/H⁺ antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.     

Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic <Emphasis Type="Italic">Halomonas</Emphasis> sp. Y2

Na⁺/H⁺ antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na⁺/H⁺ antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na⁺ and Li⁺ translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0–8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na⁺ and Li⁺, while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.     

Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver

Although both amiloride- and phloretin-sensitive Na⁺/Li⁺ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li⁺ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li⁺ by hepatocytes, preloaded with Na⁺ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K _m 1.2 mm Li⁺, V _max 40 μmol · (kg dry wt · min)⁻¹, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H⁺ was curvilinear suggesting a modifier role of [H⁺] _i . This system persisted in Na⁺-depleted cells, although with apparent K _m 3.8 mm. (b) A phloretin-sensitive component, with K _m 1.2 mm, V _max 21 μmol · (kg · min)⁻¹, was unaffected by pH but was inactive in Na⁺-depleted cells. Phloretin inhibited Li⁺ uptake and Na⁺ efflux in parallel. (c) A residual uptake increased linearly with the external Li⁺ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li⁺ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H⁺]. Received: 8 April 1999/Revised: 19 July 1999     

Involvement of the Spliceosomal U4 Small Nuclear RNA in Heterochromatic Gene Silencing at Fission Yeast Centromeres

prp13-1 is one of the mutants isolated in a screen for defective pre-mRNA splicing at a nonpermissive temperature in fission yeast Schizosaccharomyces pombe. We cloned the prp13⁺ gene and found that it encodes U4 small nuclear RNA (snRNA) involved in the assembly of the spliceosome. The prp13-1 mutant produced elongated cells, a phenotype similar to cell division cycle mutants, and displays a high incidence of lagging chromosomes on anaphase spindles. The mutant is hypersensitive to the microtubule-destabilizing drug thiabendazole, supporting that prp13-1 has a defect in chromosomal segregation. We found that the prp13-1 mutation resulted in expression of the ura4⁺ gene inserted in the pericentromeric heterochromatin region and reduced recruitment of the heterochromatin protein Swi6p to that region, indicating defects in the formation of pericentromeric heterochromatin, which is essential for the segregation of chromosomes, in prp13-1. The formation of centromeric heterochromatin is induced by the RNA interference (RNAi) system in S. pombe. In prp13-1, the processing of centromeric noncoding RNAs to siRNAs, which direct the heterochromatin formation, was impaired and unprocessed noncoding RNAs were accumulated. These results suggest that U4 snRNA is required for the RNAi-directed heterochromatic gene silencing at the centromeres. In relation to the linkage between the spliceosomal U4 snRNA and the RNAi-directed formation of heterochromatin, we identified a mRNA-type intron in the centromeric noncoding RNAs. We propose a model in which the assembly of the spliceosome or a sub-spliceosome complex on the intron-containing centromeric noncoding RNAs facilitates the RNAi-directed formation of heterochromatin at centromeres, through interaction with the RNA-directed RNA polymerase complex.     

Li/Na exchange and Li active transport in human lymphoid cells U937 cultured in lithium media

Lithium transport across the cell membrane is interesting in the light of general cell physiology and because of its alteration during numerous human diseases. The mechanism of Li⁺ transfer has been studied mainly in erythrocytes with a slow kinetics of ion exchange and therefore under the unbalanced ion distribution. Proliferating cultured cells with a rapid ion exchange have not been used practically in study of Li⁺ transport. In the present paper, the kinetics of Li⁺ uptake and exit, as well as its balanced distribution across the plasma membrane of U937 cells, were studied at minimal external Li⁺ concentrations and after the whole replacement of external Na⁺ for Li⁺. It is found that a balanced Li⁺ distribution attained at a high rate similar to that for Na⁺ and Cl^? and that Li⁺/Na⁺ discrimination under balanced ion distribution at 1–10 mM external Li⁺ stays on 3 and drops to 1 following Na, K-ATPase pump blocking by ouabain. About 80% of the total Li⁺ flux across the plasma membrane under the balanced Li⁺ distribution at 5 mM external Li⁺ accounts for the equivalent Li⁺/Li⁺ exchange. The majority of the Li⁺ flux into the cell down the electrochemical gradient is a flux through channels and its small part may account for the NC and NKCC cotransport influxes. The downhill Li⁺ influxes are balanced by the uphill Li⁺ efflux involved in Li⁺/Na⁺ exchange. The Na⁺ flux involved in the countertransport with the Li⁺ accounts for about 0.5% of the total Na⁺ flux across the plasma membrane. The study of Li⁺ transport is an important approach to understanding the mechanism of the equivalent Li⁺/Li⁺/Na⁺/Na⁺ exchange, because no blockers of this mode of ion transfer are known and it cannot be revealed by electrophysiological methods. Cells cultured in the medium where Na⁺ is replaced for Li⁺ are recommended as an object for studying cells without the Na,K-ATPase pump and with very low intracellular Na⁺ and K⁺ concentration.     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.     

Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake

In this study, metagenomic DNA was screened for the Na⁺/H⁺ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na⁺/H⁺ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na⁺/H⁺ antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6 %) with a putative NhaD-type Na⁺/H⁺ antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na⁺/H⁺ antiporters from Halomonas elongata (20.8 %), Alkalimonas amylolytica (19.0 %), Vibrio parahaemolyticus (18.9 %) and Vibrio cholerae (18.7 %). pH-dependent Na⁺(Li⁺)/H⁺ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na⁺(Li⁺)/H⁺ antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na⁺/H⁺ antiporters. Also, the apparent K _m values of Hb_NhaD for Na⁺ and Li⁺ at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na⁺/H⁺ antiporter.