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J. Biol. Chem., Vol. 281, Issue 29, 19822-19829, July 21, 2006

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Potassium/Proton Antiport System of Escherichia coli^*

Martha V. Radchenko, Kimihiro Tanaka, Rungaroon Waditee^¶, Sawako Oshimi, Yasutomo Matsuzaki, Masahiro Fukuhara, Hiroshi Kobayashi^||, Teruhiro Takabe^¶, and Tatsunosuke Nakamura¹

From the Department of Microbiology, Niigata University of Pharmacy and Applied Life Sciences, Niigata 950-2081, Japan, Graduate School of Environmental and Human Sciences, Meijo University, Nagoya 468-8502, Japan, ^¶Research Institute of Meijo University, Tenpaku-ku, Nagoya, Aichi 468-8502, Japan, and ^||Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8675, Japan

Received for publication, January 12, 2006 , and in revised form, May 8, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The intracellular level of potassium (K⁺) in Escherichia coli is regulated through multiple K⁺ transport systems. Recent data indicate that not all K⁺ extrusion system(s) have been identified (15). Here we report that the E. coli Na⁺ (Ca²⁺)/H⁺ antiporter ChaA functions as a K⁺ extrusion system. Cells expressing ChaA mediated K⁺ efflux against a K⁺ concentration gradient. E. coli strains lacking the chaA gene were unable to extrude K⁺ under conditions in which wild-type cells extruded K⁺. The K⁺/H⁺ antiporter activity of ChaA was detected by using inverted membrane vesicles produced using a French press. Physiological growth studies indicated that E. coli uses ChaA to discard excessive K⁺, which is toxic for these cells. These results suggest that ChaA K⁺/H⁺ antiporter activity enables E. coli to adapt to K⁺ salinity stress and to maintain K⁺ homeostasis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Potassium is the major monovalent intracellular cation of Escherichia coli as well as of other prokaryotic and eukaryotic cells. Potassium has four major roles in E. coli; it is an osmotic solute, an activator of intracellular enzymes, a regulator of intracellular pH, and a second messenger to stimulate accumulation of compatible solutes (1, 2). Cytoplasmic pools of K⁺ are tightly regulated in bacteria by a number of different transport systems that vary with regard to kinetics, energy coupling, and regulation (3).

Intracellular K⁺ concentrations are regulated in E. coli through a variety of systems, including Kdp-, Trk-, and Kup (formerly TrkD)-related uptake systems (4–7), KefB- and KefC-related efflux systems (8), Kch K⁺ channels (9), MscL-, MscS (formerly YggB)-, and MscK (formerly KefA)-related mechanosensitive channels that transport small molecules (including K⁺) across the membrane (10–12), and MdfA multidrug-resistance transporter (13, 14).

The list of E. coli K⁺ transport systems is incomplete with respect to potassium efflux. These K⁺ efflux systems may not have been studied extensively because potassium is a major monovalent intracellular cation and because of the belief that living cells accumulate K⁺ and extrude the smaller Na⁺ ion. However, recent data indicate that excessive amounts of K⁺ can be toxic for living cells and that cells regulate K⁺ levels via K⁺ efflux systems (15–17). The only well studied K⁺ efflux systems in E. coli utilize KefB and KefC. The activity of these systems is manifested by rapid but reversible loss of K⁺ when cells are treated with N-ethylmaleimide (18, 19). However, KefB and KefC act to acidify the cytoplasm only when activated by toxins and are probably not involved in regulating intracellular pH under other conditions (20). The kch gene of E. coli encodes a protein homologous to eukaryotic K⁺ channels (9). However, despite the possibility that physiological roles of Kch involve bulk K⁺ uptake or membrane potential regulation (21), to date no Kch channel activity has been detected. Deletion of kch has no detectable effect on bacterial growth in various media or on their survival under various applied stresses (22). Moreover, wild-type and kch mutant bacteria do not differ in their cytoplasmic K⁺ concentrations (23).

Energetic and physiologic considerations suggest the existence of unrecognized K⁺ efflux system(s) in E. coli, and K⁺/H⁺ antiporters are ideal candidates for these unrecognized systems. The energetic argument is based on the high electrical membrane potential (interior negative) maintained by bacteria growing in neutral or alkaline pH. A membrane potential of –120 mV would concentrate K⁺ 100-fold. For example, in a situation where the external K⁺ concentration is 500 mM, the bacterial cytoplasm would be expected to contain 50 M K⁺ at equilibrium, a situation clearly not compatible with bacterial growth. Potential reasons for the growth of E. coli in Luria Bertani (LB) medium containing 500 mM K⁺ are the presence of an energy-coupled efflux system to prevent excessive accumulation of K⁺ (17) and inhibition of K⁺ uptake systems by the high concentration of K⁺ in the environment or cytoplasm (24, 25).

The physiologic argument for the existence of new K⁺ efflux systems is based on the requirement of bacteria to reduce intracellular K⁺ when turgor pressure is dangerously high. Mechanosensitive channels can mediate rapid efflux of K⁺, which lowers the cytoplasmic osmolarity. However, gradual K⁺ efflux was seen when medium osmolarity was progressively diluted (26). This suggested the existence of a pathway more specific than the mechanosensitive channels, and K⁺/H⁺ antiporters are possible candidates for mediating this type of K⁺ efflux.

K⁺/H⁺ antiporter activity has been demonstrated by the effect of monovalent cations on the pH gradient formed by oxidation of lactate in inverted membrane vesicles from E. coli (27). Regulation of cytoplasmic pH by K⁺/H⁺ exchange was postulated, where protons that were returned to the cytoplasm by the K⁺/H⁺ antiporter prevented cytoplasmic alkalinization during proton extrusion associated with the formation of a proton motive force or during growth at alkaline pH (28). A similar K⁺/H⁺ antiporter activity has been demonstrated in Vibrio algoinolyticus (29, 30). Despite the indirect evidence for the existence of the K⁺/H⁺ antiporter systems in bacteria, the candidate genes remain elusive. However, we recently cloned the first bacterial K⁺(specific)/H⁺ antiporter, which is encoded by the gene nhaP2, and demonstrated that proteins NhaA and NhaB are Na⁺(K⁺)/H⁺ antiporters of Vibrio parahaemolyticus (17). In addition, recent study showed that MdfA multidrug resistance transporter is able to mediate alkaline pH tolerance by Na⁺/H⁺ or K⁺/H⁺ exchange (13, 14). The affinity of MdfA to Na⁺ and K⁺ ions is very low (in the range of 50 mM) (13).

Because of the lack of knowledge about genes encoding K⁺/H⁺ antiporter systems of E. coli described by Rosen and coworkers (27, 28), we attempted to identify K⁺/H⁺ antiporter(s) of this bacterium. Our previous data indicated that E. coli strain TO114, which lacks the genes nhaA, nhaB, and chaA, cannot mediate K⁺ efflux under diethanolamine (DEA)² pressure, whereas E. coli strain LB2003, which carries nhaA, nhaB, and chaA, extrudes K⁺ under the same conditions (17). Therefore, we studied the genes nhaA, nhaB, and chaA as possible candidates to mediate K⁺ efflux from E. coli. Our data indicate that the protein encoded by chaA is not only a Na⁺(Ca²⁺)/H⁺ antiporter but also a K⁺/H⁺ antiporter.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bacterial Strains and Growth Conditions—E. coli strains W3110 (F^{– –} IN (rrnD-rrnE)1) (31), TO110 (W3110 chaA::Cm^r), TO112 (W3110 nhaA::Km^r chaA::Cm^r), TO113 (W3110 nhaA::Km^r nhaB::Em^r), TO114 (W3110 nhaA::Km^r nhaB::Em^r chaA::Cm^r), TO116 (W3110 nhaB::Em^r chaA::Cm^r) (32), LB2003 (F^– kup1 kdpABC5 trkA rpsL metE thi rha gal) (3), and LB2005 (LB2003 chaA::Cm^r) (this study) were used to study chaA. These strains were grown in solid or liquid LBK medium containing 1% tryptone, 0.5% yeast extract, 25 mM Tris, and KCl (final concentration of K⁺ varied from 10 to 800 mM and was determined by atomic absorption spectrophotometry), at pH 6.5 or 8.5 adjusted by HCl. Media containing 10, 30, 100, 300, 500, 600, 700, and 800 mM K⁺ are referred to here as LBK10, LBK30, LBK100, LBK300, LBK500, LBK600, LBK700, and LBK800, respectively. No KCl was added to LBK10, but residual amounts of K⁺ (10 mM) were detected by flame spectrophotometry, and therefore we do not refer to this medium as LBK0. E. coli K-12 strain DH5 (recA1 gyrA (Nal) (lacIZYAargF) (80dlac [lacZ]M15), pir RK6) (33), and E. coli SY327 pir ((lac pro) argE(Am) rif nalA recA56) (34) were used for routine cloning procedures and were grown and harvested in LB medium (35).

Construction of E. coli LB2005—A mutation strategy was designed to replace the entire open reading frame of the target gene chaA with the gene cat-1 (Fig. 1). QIAamp DNA Mini kit (Qiagen Sciences) was used to purify chromosomal DNA of E. coli LB2003. Using the E. coli genome sequence as a template, two pairs of restriction site-containing sense and antisense PCR primers (LSf, 5'-ATGAGCTCCCCGCCAGGATCGGTGCC-3'; LSr, 5'-CGGGATCCCAGTATATCTCCTCCG-3'; RSf, 5'-GCGGATCCTATGGTTATCCCTTTGCA-3'; RSr1, 5'-GCGAATTCTCTCTTCTGGCAGTCGAT-3') were designed to amplify two fragments of 1000 base pairs (bp) flanking chaA. Plasmid pSTV28 was used as a template to amplify a 1000-bp fragment containing cat-1 (CMf, 5'-CCGGATCCATGGAGAAAAAAATCACT-3'; CMr, 5'-ATGGATCCTTACGCCCCGCCCTGCCCA-3'). PCR was performed according to the instructions accompanying iTaq^TM DNA Polymerase (Bio-Rad Laboratories). Using the DNA ligation kit Ver.2.1 (TaKaRa Bio, Inc., Shiga, Japan), all PCR products were inserted into pT7Blue T-Vector (Novagen) by TA cloning (Fig. 1). The resulting recombinant plasmids were named pLS (containing the 1000-bp PCR product flanking the left shoulder of chaA), pRS (containing the 1000-bp PCR product flanking the right shoulder of chaA), and pCAT-1 (containing the 1000-bp PCR fragment of cat-1) (Fig. 1). Plasmid pRS was digested with the enzymes BamHI and EcoRI to obtain a 1000-bp PCR product. This product was purified from agarose gels with QFX PCR DNA and Gel Purification kit (Amersham Biosciences) and subcloned into the BamHI/EcoRI restriction sites of pLS to obtain the pLSRS recombinant plasmid. Using a SacI-containing sense primer (LSf, 5'-ATGAGCTCCCCGCCAGGATCGGTGCC-3'), a SalI-containing antisense primer (RSR2, 5'-GCGTCGACTCTCTTCTGGCAGTCGAT-3'), and pLSRS as a template, a 2-kbp fragment flanking chaA was amplified (creating a PCR product with the entire chaA gene deleted). This 2-kbp PCR fragment was cloned into pT7Blue T-Vector and then subcloned into the multiple cloning site of the pCVD442 suicidal vector (36) using the unique SacI and SalI restriction sites. The resulting plasmid was named pCLR (Fig. 1). Plasmids pCLR and pCAT-1 were digested with BamHI, and a 1-kbp fragment containing cat-1 was cloned into the 2-kbp PCR fragment of pCLR. Thus, cat-1 in the plasmid was flanked by the same 1-kbp sequences as chaA in the E. coli genome. This ligation mixture was introduced into E. coli strain SY327 pir, and the resulting plasmid was named pCLR-1 (Fig. 1). Using the calcium chloride technique, recombinant plasmid pCLR-1 was transformed into E. coli LB2003, and the cells were plated on LB agar medium containing ampicillin (100 µg/ml) and chloramphenicol (50 µg/ml) to obtain partial merodiploid cells containing the integrated pCLR-1 (35). To select for second recombination events, the overnight LB/ampicillin and chloramphenicol culture was plated in LB. Serial dilutions were plated on LB and modified LB plates in which NaCl was replaced with 5% sucrose and further incubated for 24 –32 h at 30 °C. Sucrose-resistant colonies were checked for ampicillin sensitivity and chloramphenicol resistance. Colonies that proved to be ampicillin sensitive and sucrose/chloramphenicol resistant were checked by PCR to determine whether they retained chaA or whether it was replaced with cat-1. To purify vectors and recombinant plasmids during construction of the LB2005 strain, the Quantum Prep® Plasmid miniprep kit (Qiagen Sciences) was used.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Construction scheme of recombinant strain E. coli LB2005. LS, left shoulder; RS, right shoulder; cat-1, chloramphenicol resistance gene.

The filtration was performed with a vacuum/pressure pump (Millipore Corp., Bedford, MA). The filters were immersed into 4 ml of 5% trichloroacetic acid, and the K⁺ contents were immediately determined using an atomic absorption spectrophotometer (AA-6200; Shimadzu Corp., Kyoto, Japan). A standard K⁺ solution (K⁺ 1000 ppm) (Nacalai Tesque, Inc., Kyoto North Office, Japan) was used as a control. Control samples were diluted in trichloroacetic acid (5%), and concentrations of K⁺ were 0, 0.125, 0.25, 0.5, 1, and 2 ppm, respectively. The intracellular concentrations of K⁺ in E. coli were calculated as described previously (29) assuming 5.4 µl of internal water space/mg of cell protein.

Measurement of DEA-dependent K⁺ Efflux Against a K⁺ Chemical Gradient—This method was similar to the method described above, except the reaction buffer contained 20 mM N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid, 0.4 M KCl at pH 9.0 adjusted by KOH. In this buffer, K⁺ efflux under DEA pressure went against an outward K⁺ chemical gradient.

Measurement of K⁺/H⁺ Exchange Activities Detected by the Acridine Orange Fluorescence Quenching Method—All activities were examined on inverted membrane vesicles prepared from cells grown in LBK300 as previously described (37). E. coli cells were harvested by centrifugation at 3,100 x g for 10 min at 4 °C, washed, and resuspended in 10 ml of TCDS buffer containing 10 mM Tris-HCl, pH 7.5, 0.14 M choline chloride, 0.5 mM dithiothreitol, and 0.25 M sucrose. The cells were applied to a French Pressure cell (4000 psi), and then the solution was centrifuged at 110,000 x g for 60 min at 4 °C and the pellet was resuspended in 300 µl of TCDS buffer. The antiporter activity was based upon the establishment of a transmembrane pH gradient (pH) by the addition of salt to the reaction mixture that contained 10 mM Tris-HCl (titrated with HCl to the indicated pH), 5 mM MgCl₂, 0.14 M choline chloride, 1 µM acridine orange, and membrane vesicles (100 µg of protein) in a final volume of 2 ml. The pH was monitored at 25 °C with acridine orange as a probe at an extinction wavelength of 492 nm (bandwidth 1.5 nm) and emission wavelength of 525 nm (bandwidth 3.0 nm) using a Shimadzu RF-5300PC spectrophotometer. Before the addition of salt, Tris-DL-lactate (2 mM) was added to initiate fluorescence quenching due to respiration. Lactate energizes the vesicle and accumulates H⁺ within vesicles, which causes the accumulation of dye and fluorescence quenching. Upon the addition of salt (10 –100 mM), fluorescence increases because of the excretion of H⁺ by antiporters and therefore allows monitoring of dequenched fluorescence. Then, NH₄Cl (25 mM) was added to dissipate pH. The K⁺/H⁺ antiporter activity is expressed as percent dequenching, when quenching is equal to 100%. Data represent the average of six separate determinations ± S.D. Assays were conducted at a pH range from 7.0 to 9.0.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Effect of diethanolamine on K+ efflux from wild-type E. coli W3110 cells (A) and E. coli TO114 (B). Cells were suspended in 0.4 M NaCl and 10 mM CAPSO at pH 9.0 (closed symbols). 50 mM diethanolamine-HCl, pH 8.5, was added to the buffer after 2 min (open symbols). All data are the means ± S.D. of six independent determinations.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Measurement of K⁺ Efflux From E. coli Cells by the DEA-dependent Method Using Na⁺ Buffer—To examine the K⁺ efflux activity of wild-type E. coli strain W3110 and mutants TO112, TO113, TO114, and TO116, the DEA-dependent method was used as described under "Experimental Procedures." Fig. 2 shows the effect of DEA on K⁺ efflux from E. coli W3110 and E. coli TO114. In buffer containing 0.4 M NaCl and 10 mM CAPSO at pH 9.0, cells maintained 150 –200 nmol K⁺/mg of protein. Upon the addition of 50 mM DEA, W3110 cells released 150 –230 nmol K⁺/mg of protein (Fig. 2A), whereas TO114 cells were unable to mediate K⁺ efflux (Fig. 2B). These data suggest that genes nhaA, nhaB, and chaA are possible candidates for K⁺/H⁺ exchange in E. coli. To establish which gene was responsible for K⁺ efflux activity, we studied strains TO110, TO112, TO113, and TO116. TO113 cells, containing the chaA gene, released 114 –120 nmol K⁺/mg of protein upon addition of 50 mM DEA (Fig. 3C). Negligible K⁺ efflux (10 –30 nmol K⁺/mg of protein) was observed with TO116 cells, containing nhaA, and TO112 cells, containing nhaB (Fig. 3, A and B, respectively). The release of K⁺ from TO110 cells was similar to the K⁺ release from TO114 cells described in Fig. 2B (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Effect of diethanolamine on K+ efflux from normal cells of E. coli TO112 (B), TO113 (C), and TO116 (A). Cells were suspended in 0.4 M NaCl and 10 mM CAPSO at pH 9.0 (closed symbols). 50 mM diethanolamine-HCl, pH 8.5, was added to the buffer after 2 min (open symbols). All data are the means ± S.D. of six independent determinations.

Measurement of K⁺ Extrusion from E. coli by the DEA-dependent Method Using K⁺ Buffer—To determine whether ChaA can mediate K⁺ exchange against, as well as with, a K⁺ concentration gradient, we examined DEA-dependent K⁺ efflux from cells in K⁺ buffer. Experimental conditions were established whereby any release of K⁺ had to go against a K⁺ concentration gradient (see "Experimental Procedures"). The TO113 (chaA-positive) cells and TO114 (chaA-negative) cells maintained 250 –300 nmol K⁺/mg of protein in buffer containing 0.4 M KCl, 20 mM N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid, 0.2% glucose, pH 9.0 (Fig. 4, A and B). After 16 min under these conditions, only negligible (i.e. 5–15 nmol K⁺/mg of protein) K⁺ efflux was observed. Upon addition of 50 mM DEA, TO113 cells released 115–145 nmol K⁺/mg of protein (Fig. 4A), whereas TO114 showed no significant K⁺ efflux (Fig. 4B). The experiment was repeated six times, and each time TO113 cells released <145 nmol K⁺/mg of protein into K⁺ buffer. The moderate release of K⁺ may have been due to the presence of K⁺ uptake systems that interfered with K⁺ efflux systems in TO113 cells.

LB2003 and LB2005 cells were used to determine whether ChaA can maintain K⁺ efflux against a K⁺ concentration gradient. Strain LB2003 has deficient K⁺ uptake systems, and thus K⁺ uptake probably would not interfere with K⁺ efflux in these cells. chaA was deleted from the LB2003 strain to derive the LB2005 strain, as described under "Experimental Procedures." In K⁺ buffer, LB2003 and LB2005 cells maintained 250 –300 nmol K⁺/mg of protein (Fig. 4, C and D). Upon addition of 50 mM DEA, LB2003 cells released 205–240 nmol K⁺/mg of protein (Fig. 4C), whereas LB2005 cells did not significantly release K⁺ (Fig. 4D).

K⁺/H⁺ Exchange Activities of E. coli-inverted Membrane Vesicles and Effects of pH and KCl Concentrations on K⁺/H⁺ Exchange—The data described above suggested that chaA encodes a protein responsible for K⁺ efflux under DEA pressure. Next, the K⁺/H⁺ exchange activity of ChaA was studied by using inverted membrane vesicles generated from LB2003 and LB2005 cells. Exchange activities were monitored by measuring acridine orange fluorescence quenching upon the addition of D-lactate, with subsequent dequenching upon addition of KCl as described under "Experimental Procedures." The K⁺/H⁺ exchange activity was detected in inverted membranes made from LB2003 cells but not in those from LB2005 cells (Fig. 5A). Furthermore, the extent of dequenching increased with increasing KCl concentration (Fig. 5B). Addition of 10 mM KCl yielded 14 –15% dequenching, whereas addition of 50 and 100 mM KCl yielded 33–38 and 48 –50% dequenching, respectively (Fig. 5, A and B). When LB2005 inverted membranes were used, only negligible (2–3%) dequenching was observed, irrespective of KCl concentration (Fig. 5, A and B). The K⁺/H⁺ exchange activities of inverted membranes were pH dependent and increased with increasing pH, with prominent dequenching of fluorescence at pH 9.0 upon the addition of KCl (Fig. 5C). These results suggested that ChaA has K⁺/H⁺ exchange activity with an estimated K_m >10 mM for K⁺ (Fig. 5).

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Effect of diethanolamine on K+ efflux from E. coli TO113, TO114, LB2003, and LB2005. Cells were suspended in 0.4 M KCl and 20 mM N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid, 0.2% glucose, pH 9.0, (closed symbols). 50 mM diethanolamine-HCl, pH 8.5, was added to the buffer after 2 min (open symbols). All data are the means ± S.D. of six independent determinations.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. K+/H+ exchange activity of the ChaA antiporter in E. coli LB2003 (A) and E. coli LB2005 (B) measured at pH 9.0 by the acridine orange fluorescence quenching method as described under "Experimental Procedures." C, evaluation of ChaA affinity for K+, measured by fluorescence quenching (%) using different concentrations of KCl. All data are the means ± S.D. of three independent determinations.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our previous work (17) indicated that nhaA, nhaB, and nhaP2 from Vibrio are plausible candidates for maintaining K⁺ efflux under DEA pressure and that E. coli had similar transporter(s). The present study revealed that chaA encodes an antiporter with the ability to mediate K⁺ efflux and K⁺/H⁺ exchange (Figs. 3C, 4A and C, and 5A and C) and therefore represents a potential K⁺/H⁺ antiporter. Previous (14) and present studies showed that nhaA and nhaB appear not to be involved in K⁺ efflux from E. coli cells. This reveals a difference in the physiologies of E. coli and V. parahaemolyticus. NhaA and NhaB of V. parahaemolyticus are Na⁺(K⁺)/H⁺ antiporters, although the K⁺/H⁺ exchange activity of these proteins appears to be secondary to the Na⁺/H⁺ antiporter activity (17).

Previous studies of chaA indicate that it has a physiological role in sodium (40) and calcium (32, 41) ion extrusion. The apparent K⁺/H⁺ antiport activity was not detected in the initial characterization of this antiporter, probably due to the fact that ChaA was not assayed at high enough pH and [KCl]. The present study reveals that chaA encodes a K⁺/H⁺ antiporter. ChaA extrudes K⁺ against both outwardly (Fig. 3C) and inwardly (Fig. 4, A and C) directed K⁺ concentration gradients. The K⁺/H⁺ exchange activity of ChaA is pH dependent and active at alkaline pH. We did not detect exchange activities of inverted membrane vesicles at pH lower than 8.0, but the activity became prominent at pH 9.0 (Fig. 5). Furthermore, the ChaA affinity for K⁺ increased with increasing KCl concentration (Fig. 5C). These results indicate that ChaA is involved in KCl salinity tolerance. A similar K⁺/H⁺ exchange activity has been described in plants (42, 43) and humans (44).

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Effects of different KCl concentrations and pH 6.5 (A and B) or pH 8.5 (C and D) on bacterial strains after 9 h of incubation in media containing residual amounts of NaCl (A and C) or 100 mM NaCl (B and D) at 37°C. Two-way analysis of variance was used, and the differences were considered significant at p < 0.0001. All data are the means ± S.D. of three independent determinations.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Effects of different KCl concentrations and alkaline pH 8.5 on bacterial strains after 24 h of incubation at 37 °C. Two-way analysis of variance was used, and the differences were considered significant at p < 0.001. All data are the means ± S.D. of three independent determinations.

Our physiological study of E. coli strains revealed another interesting finding. Concentrations of KCl higher than 500 mM in medium were found to be toxic (Figs. 6 and 7). KCl toxicity was more prominent at alkaline pH (Figs. 6 and 7). KCl toxicity may not have been studied previously because of the generally accepted idea that bacteria use K⁺ as a primary solute for osmotic regulation. However, most bacteria, with the exception of halophilic species, use K⁺ only under conditions of low osmolarity and utilize polar molecules under conditions of higher osmolarity. This is consistent with the current finding that environments containing excessive amounts of K⁺ are toxic and that a K⁺/H⁺ antiporter is required to remove excess K⁺ under such conditions.

Our results indicate the importance of chaA for cell survival and adaptation (Figs. 6 and 7). E. coli strains containing chaA were able to adapt to conditions of high KCl concentration and alkalinity more easily and were able to grow under these conditions at least two times faster than strains lacking chaA (Figs. 6 and 7). In summary, these findings indicate that chaA encodes a physiologically significant K⁺/H⁺ antiporter. Antiporter activation is triggered by alkaline pH and high external or internal KCl concentration. Under these conditions, ChaA is able to maintain K⁺ efflux from E. coli against a K⁺ concentration gradient.

	FOOTNOTES

 
^* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (14572051) and by a grant from the Promotion and Mutual Aid Corporation for Private Schools in Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Microbiology, Niigata University of Pharmacy and Applied Life Sciences, 5-13-2 Kamishin'ei-cho, Niigata 950-2081, Japan. Tel. and Fax: 81-25-268-1210; E-mail: tnak{at}niigata-pharm.ac.jp.

² The abbreviations used are: DEA, diethanolamine; LB, Luria Bertani; CAPSO, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid.

	ACKNOWLEDGMENTS

 
We thank Evert P. Bakker for providing E. coli strain LB2003, Xiaoling Wang for providing plasmid pCVD442 and E. coli strain SY327 pir, and Nobuyuki Uozumi and Evert P. Bakker for helpful discussions.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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