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^*

Hyperosmolality caused by high salinity or drought constitutes a major challenge to the growth of prokaryotes, fungi, and plants (1–5). Many bacteria, including Escherichia coli, react to such a change to hyperosmotic conditions with a biphasic response. During the first phase, E. coli cells accumulate additional K⁺ from the medium through their K⁺ uptake systems Trk and Kdp and synthesize glutamate concomitantly. Thereby, they increase the ion content of their cytoplasm and counteract plasmolysis brought about by water efflux because of the reversal of the sign of cell turgor pressure caused by external hyperosmolarity. During its second phase of the cellular adaptation, E. coli replaces this internal potassium glutamate by synthesizing high amounts of the non-ionic compatible solute trehalose (6–8). Bacillus subtilis follows a slightly different strategy in that after hyperosmotic shock, growing cells of this organism synthesize the compatible zwitterionic solute proline instead of trehalose (9, 10), and Vibrio alginolyticus KtrAB makes major contributions to net K⁺ uptake (11). Other bacteria follow similar approaches (3, 5, 12–14).

Cyanobacteria can adapt to high NaCl concentrations. Under these conditions a variety of these species synthesize high concentrations of glucosylglycerol and sucrose, which are accumulated as compatible solutes in their cytoplasm (15). During the early phase of this adaptation Synechocystis sp. PCC 6714 accumulates Na⁺. Within a few minutes, Na⁺ is replaced by K⁺. Subsequently, it takes many hours to days to replace the high K⁺ concentration in the cytoplasm by glucosylglycerol as well as minor amounts of sucrose (16). The K⁺ uptake system(s) involved in the early phases of this osmoadaptation have, however, not been identified in this early work. Since the elucidation of its genome nucleotide sequence, cyanobacterial research has focused on Synechocystis sp. PCC 6803 (//www.kazusa.or.jp/cyano/). During its long term adaptation to high NaCl concentrations, this organism also synthesizes and accumulates glucosylgylcerol (17–19). Determining from the genome sequence, strain PCC 6803 has been predicted to contain at least three types of K⁺ uptake systems, Kdp system, which is probably of minor importance (20), a Ktr system (21), which appears to play a role in salt stress by high NaCl concentrations (20) and K⁺ channels. However, the Ktr system has not been characterized any further. Recently, it has been reported that in Synechocystis sp. PCC 6803 the ntpJ gene (slr1509) is essential for both the adaptation to high NaCl concentrations and the bicarbonate transport via the SbtA system (22). SbtA-mediated bicarbonate transport was also dependent on the presence of external Na⁺ (22). Since ntpJ has been described originally as the Na⁺ transport gene J, located downstream of a cluster of ntp genes encoding the Na⁺-translocating V-type ATPase from Enterococcus hirae (23), Shibata et al. (22) interpreted their findings to mean that NtpJ functions as a Na⁺ efflux system required for the removal of Na⁺ from the cells after a hyperosmotic shock with NaCl and/or Na⁺ uptake due to symport via SbtA (22). However, ntpJ from E. hirae has later been identified as a K⁺-translocating subunit of the bacterial Ktr-type K⁺ uptake system (11, 21, 24–27). Ktr from Vibrio alginolyticus is Na⁺-dependent, and the same may apply to the Ktr system from E. hirae (26). KtrB belongs to a superfamily of K⁺ transporter proteins, with members in Archaea (KdpA, TrkH), bacteria (KdpA, TrkH, KtrB), fungi (TRK), and plants (HKT) (26–38). In particular, the comparison between bacterial KtrB and HKT systems from some plants is important, because Ktr and HKT systems are the only members of the superfamily that mediate Na⁺-dependent K⁺ transport.

Recently, Berry et al. (20) indicates that ntpJ/ktrB from Synechocystis sp. PCC 6803 has a function in K⁺ uptake. In this report, we confirm and extend their observation with a detailed study on the Ktr system from Synechocystis sp. PCC 6803. We show that NtpJ/KtrB is part of a more complex Ktr system, comprising of three kinds of subunits, KtrA, KtrB, and KtrE. In E. coli K⁺ transport via the Synechocystis KtrABE system is of moderately high affinity, is dependent on the presence of Na⁺ and on high membrane potential, but not on ATP. In this bacterium it neither transports Na⁺ inwards nor outwards. The measurement of the K⁺ content of the Synechocystis sp. PCC 6803 and the cell containing the mutation at ktrB gene revealed that the Ktr system was essential for the early phase of the adaptation of Synechocystis sp. PCC 6803 to high osmolality.

EXPERIMENTAL PROCEDURES

Growth Conditions—Synechocystis sp. PCC 6803 and mutant cells were grown at 30 °C in BG11 medium (39) containing 20 mm TES-KOH (pH 8.0 and containing 20.44 mm Na⁺ and 14.77 mm K⁺, as determined by flame photometry), and bubbled with either 3% CO₂ in air (v/v) or air alone. Solid medium contained BG11 buffered at pH 8.0, 1.5% agar, and 0.3% sodium thiosulfate. Continuous illumination was provided by fluorescent lamps (50 μmol of photons m^-2 s^-1; 400–700 nm). E. coli LB2003 (F^-, thi, lacZ, gal, rha, ΔkdpFABC5, trkD1, ΔtrkA), which lacks the three K⁺ uptake systems, Trk, Kup, and Kdp (13), and E. coli TO114 (ΔnhaA, ΔnhaB, ΔchaA), which lacks the three Na⁺/H⁺ antiporters, NhaA, NhaB, and ChaA (40), were used as host strains for growth tests to analyze K⁺ transport and Na⁺ transport, respectively. For proton motive force tests in E. coli, the strain LF12 (Δ(kdpDE)54, nagA, malT, ΔhemA) was constructed as described by Futatsugi et al. (doctoral thesis, 2001)¹ based on the strain TK2642(Δ(kdpDE)54, nagA, malT), a kind gift of Wolf Epstein. Transformants of the E. coli mutant were cultured in a synthetic medium supplemented with the required antibiotics and indicated cations as described previously (27, 40, 41). Gene expression was induced by the addition of 0.25 mm IPTG² to the medium. Osmolalities were measured using an osmometer (Advanced Osmometer, Advanced Instruments, Norwood, MA).

Reintegration of the Synechocystis ktrB Gene into the Strain PCC 6803 Genome—The strain Synechocystis ΔktrB, in which the slr1509 (ktrB) gene has been disrupted by insertion of a kanamycin resistance gene (22, 42), was used to test for rescue of KtrB activity by integration of the gene into its genome. The ktrB gene and spectinomycin resistance gene were tandemly placed under the control of the iron transporter promoter (43) and inserted into the middle of the kanamycin resistance gene of strain ΔktrB by homologous recombination using the method of Ohkawa et al. (42). Cells resistant to spectinomycin (20 μg/ml) were isolated, and the integration of the gene was confirmed by PCR with genomic DNA using the following primers: for ktrB, 5′-CCATTCTACGAACCTAAGT-3′ (primer a) and 5′-CCTTGATATCGTTGTTATT-3′ (primer b); for the kanamycin resistance gene, 5′-ACAGCGGCCGCTTGAACTTTTGCTTTGCC-3′ (primer c) and 5′-ACAGGTACCCACGGTTGATGAGAGCTT-3′ (primer d). This strain was named +ktrB.

Plasmid Construction—Synechocystis ktrA (sll0493) was amplified by PCR using the EcoRI site-containing sense primer, 5′-CAGGAATTCAGATGAAAACTAGTCATT-3′, and the PstI site-containing antisense primer, 5′-CATCTGCAGCTAAACTAGGCGGCCG-3′. Subsequently, the PCR fragment was digested with EcoRI and PstI and ligated into the corresponding sites of plasmid pSTV28 (TAKARA Co., Japan), giving plasmid pSTV28-KtrA, which was used for ktrA expression in E. coli LB2003. For ktrA expression in E. coli TO114, which is resistant to chloramphenicol, the DraI fragment containing the chloramphenicol resistance gene of pSTV28-KtrA was replaced by the ClaI-Van91I fragment containing the tetracycline resistance gene in pBR322, giving plasmid pSTV28-Tet-KtrA. Strain PCC 6803 DNA, tandemly encoding ktrE (slr1508) and ktrB (slr1509), was amplified by PCR using the XbaI site-containing sense primer, 5′-GAGTCTAGAAGGAATCTGCATGCATATTGCT-3′, and the PstI site-containing antisense primer, 5′-ATTCTGCAGTTAGCCTACCAGCAA-3′. Subsequently the product was digested with XbaI and PstI and ligated into the corresponding sites of plasmid pPAB404 (44), yielding plasmid pPAB404-KtrBE. The same DNA fragment, except for the deleted start codon ATG of ktrE, was amplified using 5′-GACTCTAGACATATTGCTTGGTTAGGAAAAAAAACGC-3′ as the sense primer (pPAB404-KtrBΔE). Strain PCC 6803 KtrB was amplified by PCR using the XbaI site, containing the sense primer, 5′-GAGTCTAGAAGGAGCTGGAATGACTATTTCC-3′, and the PstI site-containing antisense primer, 5′-ATTCTGCAGTTAGCCTACCAGCAA-3′ and subcloned into plasmid pPAB404, yielding plasmid pPAB404-KtrB. E. coli nhaA was amplified by PCR using the KpnI site containing sense primer, 5′-ATAGGTACCAGGAACTAAAATGAAACATCTGCATCGATT-3′, and the SalI site-containing antisense primer, 5′-AAAGTCGACTCAAACTGATGGACGCAA-3′, and subcloned into plasmid pPAB404, yielding plasmid pPAB404-NhaA.

Measurement of K⁺ Uptake in E. coli and Synechocystis sp. PCC 6803—K⁺ influx was essentially measured as described by Tholema et al. (27). E. coli LB2003 cells were cultured in a synthetic medium in the presence of 30 mm KCl, 0.25 mm IPTG, 25 μg/ml of ampicillin, and/or 15 μg/ml of chloramphenicol at 30 °C. E. coli LF12 were cultured in 1% tryptone, 0.5% yeast extract, 0.67% KCl, 1% glucose, 0.25 mm IPTG, and the required antibiotics at 30 °C. Synechocystis sp. PCC 6803 was cultured in BG11 medium. The cells were collected by centrifugation, resuspended in 120 mm Tris-HCl (pH 8.0) and 1 mm EDTA, shaken for 10 min at 37 °C, collected by centrifugation, and washed twice with 200 mm HEPES-NaOH (pH 7.5) (94 mm Na⁺ and 29 μm K⁺, as determined by flame photometry) or 200 mm HEPES-triethanolamine (pH 7.5) (1.9 μm Na⁺ and 0.36 μm K⁺), then resuspended in the same buffer. After shaking for 20 min at room temperature, the concentrations of cells were adjusted to an OD₅₇₈ of 3 with the same buffer. 10 mm glucose was added to the suspension 10 min prior to the start of the K⁺ uptake measurement. The net uptake of K⁺ was measured by the silicone filtration technique (21, 27, 45), the K⁺ contents of the cell pellets being determined by flame photometry (45).

Measurement of Na⁺ Uptake in E. coli—E. coli TO114 was grown in 1% tryptone, 0.5% yeast extract, 0.67% KCl, and the required antibiotics. Na⁺ uptake was measured as described above for K⁺ uptake. Na⁺ concentrations in the cells were determined by flame photometry.

Measurement of Na Efflux in E. coli—Na⁺ efflux was measured as described by Sakuma et al. (46). E. coli TO114 cells were grown in 1% tryptone, 0.5% yeast-extract, 0.67% KCl, and the required antibiotics to an OD₆₁₀ of 0.5, collected by centrifugation, resuspended in the same culture medium containing 150 mm NaCl, then kept on ice for 2 h. Na⁺ concentrations in the cells were measured as described above for Na⁺ content.

RESULTS

Three Synechocystis ktr Gene Products Are Required for K⁺ Transport Activity in E. coli—Since previous reports have suggested that Synechocystis KtrB plays a role in K⁺ uptake (20, 21, 30), rather than in Na⁺ transport (22, 47) (www.kazusa.or.jp/cyano/), we investigated the role of Synechocystis ktr genes in K⁺ transport and analyzed the Ktr gene family. We cloned the Synechocystis Ktr genes into the E. coli mutant strain LB2003. This strain carries mutations in genes encoding the three major E. coli K⁺ uptake systems, Kdp, Trk, and Kup. As a consequence, it possesses negligible K⁺ uptake activity at K⁺ concentrations in the low millimolar range and, therefore, does not grow at K⁺ concentrations ≤ 10 mm (13). In agreement with previous results in that only together the cloned ktrA and ktrB genes from V. alginolyticus enable E. coli LB2003 to transport K⁺ (21), Synechocystis ktrB alone did not restore K⁺ transport in the strain (Fig. 1). The Synechocystis genome contains a ktrA orthologue (sll0493) at a site quite distant from that of the ktrB (slr1509) gene. However, coexpression of ktrA and ktrB in E. coli LB2003 also did not lead to growth at low K⁺ concentrations (Fig. 1). The ktrB gene (slr1509) forms a cistron with gene slr1508 (47). Coexpression of slr1508 with ktrB and ktrA in E. coli LB2003 resulted in growth at low K⁺ concentrations (Fig. 1). To exclude the possibility of a polar effect on the expression of ktrB, the initiation codon of slr1508 was removed from the plasmid containing the slr1508-slr1509 cistron, giving plasmid pPAB404-KtrBΔE. Together with the ktrA-containing plasmid, pSTV28-KtrA, it failed to restore growth of the LB2003 strain at low K⁺ concentrations (Fig. 1). These results showed that three Synechocystis ktr genes, ktrA, ktrB, and slr1508 are essential for K⁺ transport activity in E. coli. Gene product KtrA is a peripheral membrane protein thought to regulate K⁺ transport via KtrB by NAD⁺/NADH binding (21, 48). KtrB is the K⁺-translocating subunit discussed above, and slr1508 is a novel type of ktr gene, which we have named ktrE rather than ktrC or ktrD, because in B. subtilis orthologues of its ktrA and ktrB genes have been named ktrC and ktrD (11). The subunit of ktrE has not been found in any other Ktr/Trk/HKT/Kdp transporter. Amino acid sequence alignment studies showed sequence similarity between KtrE and glycosyl transferases (Fig. 1D). KtrE is 25 and 23% identical to WbbP of Shigella dysenteriae (49) and WbgM of E. coli (50), respectively.

Na⁺ Activates KtrABE-mediated K⁺ Uptake in E. coli—To further characterize the Ktr system, we examined the ion transport activity of plasmid-containing, K⁺-depleted E. coli LB2003 cells suspended in 200 mm HEPES-NaOH buffer (94 mm Na⁺ and 29 μm K⁺, as measured by flame photometry) in the presence of glucose. Addition of 1 mm KCl led to rapid and extensive net K⁺ uptake only when all three Synechocystis ktrABE were coexpressed in the cells (Fig. 2A), confirming the results of the growth assays (Fig. 1, B and C).

The K⁺ transport activity of both wheat HKT1 and V. alginolyticus KtrAB is enhanced by Na⁺ (27, 29). To test whether this was also the case for Synechocystis KtrABE, the LB2003 cells were suspended in low Na⁺ buffer (200 mm HEPES-triethanolamine buffer; 1.9 μm Na⁺ and 0.36 μm K⁺, as measured by flame photometry). Addition of 1 mm KCl to the cells with the empty plasmid led to a small amount of K⁺ uptake, which was not enhanced by the subsequent addition of 1 mm NaCl (Fig. 2B). Cells expressing ktrABE took up a similar amount of K⁺ after the addition of the KCl. However, the subsequent addition of NaCl to these cells led to continuous and extensive K⁺ uptake, indicating that in E. coli K⁺ uptake via Synechocystis KtrABC depends on the presence of Na⁺. In addition, the reversal sequence of the addition of 1 mm Na⁺ and 1 mm K⁺ showed K⁺ uptake transport in the cells by addition of 1 mm KCl (Fig. 2C). The stimulatory effect was Na⁺-specific, since addition of 1 mm LiCl, RbCl, CsCl, or CaCl₂ failed to stimulate K⁺ uptake (Fig. 2D). Because in Fig. 5, we show that KtrABE in Synechocystis sp. PCC 6803 is involved in adaptation to higher osmolalities of both NaCl and sorbitol, we tested the effect of osmolality on the transport process. When either 200 mm sorbitol or 100 mm NaCl (of equal osmolality) was added to the low Na⁺ buffer (200 mm HEPES-triethanolamine buffer) before the addition of 1 mm KCl, K⁺ was taken up by cells preincubated with NaCl, but not by those preincubated with sorbitol (Fig. 2, E and F), showing that the stimulation of KtrABE transport in E. coli was because of the ionic effect of Na⁺ and not to its osmotic effect.

Further Characterization of KtrABE in E. coli—Na⁺-dependent K⁺ uptake had a pH optimum at pH 7.5 and did not occur at pH 5.5 (Fig. 2G), arguing against the active participation of protons (H⁺) in this process. In the presence of Na⁺, the K_m of KtrABE for K⁺ was ∼60 μm and its V_max 110 nmol·min^-1 mg^-1 of cell protein (Fig. 2H). This K_m-value for K⁺ is similar to those of 50 μm K⁺ for V. aliginolyticus KtrAB in E. coli (21). These data indicate that in E. coli Synechocystis KtrABE functions as a rapid K⁺ uptake system with a moderately high affinity for K⁺. The affinity of KtrABE for Na⁺ was also measured in an experiment where Na⁺ (0.2–50 mm) was added in the presence of 1 mm K⁺. The K_m value for Na⁺ in the stimulation of K⁺ uptake was 2.1 mm (Fig. 2I).

Mode of Energy Coupling to K⁺ Transport in E. coli—Next, we examined the mode of energy coupling to KtrABE-mediated K⁺ uptake in E. coli. Omission of the energy source, glucose, from the standard assay medium led to severe inhibition of net K⁺ uptake (Fig. 3A), suggesting that ATP and/or the transmembrane proton motive force (pmf) or one of its components, the membrane potential (ΔΨ) or the transmembrane pH difference (ΔpH), is involved in KtrABE transport activity. The protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), which abolishes the pmf, inhibited K⁺ uptake (Fig. 3B), suggesting that KtrABE activity depends on the pmf. However, in F₁F_oATPase-containing E. coli, such as strain LB2003, CCCP is known to decrease cellular ATP levels because of enhanced ATP hydrolysis via this enzyme (e.g. Ref. 51). To clarify this point, we used E. coli strain H500 (ΔhemA), which is deficient in heme synthesis and so lacks a functional respiratory electron transport chain (Futatsugi doctoral thesis, 2001).¹ When grown on glucose in the absence of δ-aminolevulinate, this strain did not show the cytochrome Soret (γ) absorption bands in the 410–440 nm region, but these appeared when δ-aminolevulinate was added. In the absence of δ-aminolevulinate, neither ΔΨ nor ΔpH could be detected in the mutant and it grew at a slightly slower rate than the wild type on glucose. These results suggest that in these cells, the pmf is absent, but glycolysis provides enough ATP for cell growth. In E. coli, Kdp is the only K⁺ uptake system active in the absence of a pmf (52, 53). We therefore constructed the mutant strain LF12, which is both ΔhemA and ΔkdpDE. As shown in Fig. 3C, LF12 cells containing empty vector grown in the presence of δ-aminolevulinate showed some K⁺ uptake activity, presumably due to the other two K⁺ uptake systems, Kup and Trk, still present in this strain, but uptake was enhanced at least 5-fold when the KtrABE were also present. No detectable K⁺ uptake was observed in LF12 containing KtrABE grown in the absence of δ-aminolevulinate. These results show that KtrABE requires the pmf for activity and does not use ATP as the sole source of energy. At pH 7.5, ΔΨ is the major component of the pmf, because at this pH value, the difference in pH between the cytosol and the external space is very small. Thus, ΔΨ appears to couple energy to K⁺ transport via the KtrABE system.

KtrABE Does Not Have Na⁺ Transport Activity—Since it has been suggested that NtpJ/KtrB functions as a Na⁺-efflux system (22), we tested whether we could detect Na⁺ transport activity of Synechocystis KtrABE in E. coli. For this purpose we used the E. coli TO114 as a host, which possesses negligibly low Na⁺ extrusion activity (40). As shown in Fig. 4A, E. coli TO114 cells transformed with a plasmid encoding the E. coli Na⁺/H⁺ antiporter, NhaA, extruded Na⁺, whereas, under the same conditions, cells expressing KtrABE did not. This is confirmed by the growth test of the cells (Fig. 4B). When 100 mm NaCl was incorporated into the solid medium, cells expressing KtrABE did not grow, whereas cells expressing NhaA grew well (Fig. 4B). This failure to rescue the deficiency in Na⁺ extrusion activity in E. coli supported the notion that KtrABE does not alleviate Na⁺ toxicity in E. coli. We then tested whether KtrABE mediates Na⁺ uptake. KtrABE failed to stimulate Na⁺ uptake by E. coli TO114 (Fig. 4C). It should be noted that this experiment was done in the same HEPES-triethanolamine buffer in which KtrABE exhibited Na⁺-dependent K⁺ uptake (Fig. 2B), showing that this buffer does not inactivate KtrABE. Taken together, the data presented in Fig. 4 show that, in E. coli, Synechocystis KtrABE neither mediated Na⁺ efflux nor Na⁺ uptake.

Hypersensitivity to High Osmolality of Synechocystis sp. PCC 6803 Conferred by ktrB Mutation—The data from Figs. 1, 2, 3, 4 indicate that the Ktr system is a Na⁺ dependent and rapid K⁺ uptake system and is unlikely to contribute to removal of Na⁺ toxicity under conditions of hyperosmotic stress. To understand the precise physiological role of the Ktr system in Synechocystis sp. PCC 6803, we performed cell growth and K⁺ transport experiments with the wild type, the ΔktrB strain, generated by insertion of a kanamycin resistance cassette into ktrB (22), and a newly constructed +ktrB strain, which caries a copy of ktrB under the control of an iron-inducible promoter, in the middle of the kanamycin resistance gene of the ΔktrB strain (Fig. 5, A and B). In particular, because the study using the ΔktrB strain (20, 22) did not check that only the ktrB gene was affected, we constructed the +ktrB strain. On plates (Fig. 5C) and in liquid medium (Fig. 5D) growth of the ΔktrB strain was completely inhibited by the addition of 300 mm sorbitol or 300 mm NaCl to the BG11 medium (20.44 mm Na⁺ and 14.77 mm K⁺, as determined by flame photometry). Both wild-type and the +ktrB strain continued to grow under these conditions (Fig. 5), showing that it is only the lack of the Ktr system that gave rise to the osmosensitive phenotype. Since the ΔktrB strain was also sensitive to high concentrations of non-ionic compound, sorbitol, we conclude that KtrB plays a role in the response to hyperosmotic stresses in general.

KtrB-mediated K⁺ Uptake by Synechocystis sp. PCC 6803 Has Similar Properties to KtrABE in E. coli—Before exploring the role of the Ktr system in osmoadaptation of Synechocystis sp. PCC 6803 further, we examined whether this system behaved similarly in its native organism as in the E. coli hosts. For this purpose we measured net K⁺ uptake by K⁺-depleted energized Synechocystis cells. At low external Na⁺ concentrations (1.9 μm), addition of 5 mm KCl hardly led to K⁺ uptake; however, the subsequent addition of 5 mm NaCl caused a rapid and substantial, KtrB-mediated net K⁺ uptake (Fig. 6A). This effect was due to the addition of 5 mm NaCl but not because of the small increase in osmolality (from 312 to 334 mOsm), since it was not seen using an equivalent osmolality of sorbitol (10 mm) in the absence of Na⁺ (Fig. 6B). Moreover, addition of 200 mm sorbitol, which increased the osmolality from 312 to 549 mOsm, did not lead to net K⁺ uptake in the absence of Na⁺ (Fig. 6C). This indicates that as in E. coli (Fig. 2B), KtrABE requires Na⁺ for K⁺ uptake activity in Synechocystis sp. PCC 6803.

Second, we examined the energy dependence of KtrB-mediated K⁺ transport in Synechocystis sp. PCC 6803 (Fig. 7). As shown in E. coli (Fig. 3B), this process was prevented by the presence of the protonophore CCCP (Fig. 7B), suggesting a role of the pmf in the coupling of energy to the K⁺ transport via the Ktr system. Taken together these data show that the properties of the Ktr system in Synechocystis sp. PCC 6803 are similar to those in E. coli, in which a more extensive characterization of the system was possible.

The Role of the Ktr System in Osmoadaptation of Synechocystis sp. PCC 6803—We then examined the role of a functional Ktr system on the K⁺ content of cells after hyperosmotic shock. The effect of the addition of 100 mm NaCl or 200 mm sorbitol to HEPES-NaOH buffer (pH 7.5, 94 mm NaCl), on KtrB-mediated transport in Synechocystis sp. PCC 6803 was tested (Fig. 8). The ΔktrB strain did not accumulate much K⁺ under these conditions, and some of their K⁺ was lost rapidly after a hyperosmotic shock with either 100 mm NaCl (Fig. 8A) or 200 mm sorbitol (Fig. 8B). Wild-type and +ktrB cells behaved similarly in that after an initial net loss of K⁺ after the hyperosmotic shock, they either accumulated an extensive amount of additional K⁺ by hyperosmotic shock with NaCl (Fig. 8A) or returned their K⁺ to a level slightly higher than that before the osmotic shock (Fig. 8B).

To further evaluate the role of the Ktr system in the osmoadaptation of Synechocystis sp. PCC 6803, the cells were hyperosmotically treated for 10 min with addition of 200 mm sorbitol before K⁺ addition (Fig. 9). Both the wild type and the ΔktrB strain lost a considerable amount of K⁺ after the shock. After the addition of K⁺ the wild type reaccumulated K⁺ to a level clearly exceeding that before the osmotic upshock (Fig. 9A). By contrast the ΔktrB cells did not reaccumulate any K⁺ under these conditions (Fig. 9B). Fig. 9C shows initial K⁺ uptake rates after the addition of 5 mm KCl to wild type, ΔktrB cells, and +ktrB cells. After a hypertonic shock, the Ktr system enabled the cells to take up K⁺ much faster. From the results of Figs. 8 and 9, we conclude that the role of the Ktr system in osmoadaptation of Synechocystis sp. PCC 6803 lies in its protection of the cells against K⁺ loss immediately after the hyperosmotic shock and in the subsequent readjustment of the cell turgor pressure by additional accumulation of K⁺ under these conditions. The Ktr system can fulfill this role as long as enough Na⁺ for the activation of the Ktr system and enough K⁺ for uptake is present in the medium.

DISCUSSION

Previous work on the function of slr1509 (ktrB/ntpJ) in Synechocystis sp. PCC 6803, has given conflicting results. On the one hand, inactivation of this gene results in both a high NaCl-sensitive phenotype of the organism and inhibition of its cotransport activity via SbtA. These observations have been explained by assuming that slr1509 extrudes Na⁺ from the cells (22). On the other hand, amino acid alignment studies have shown that slr1509 encodes a protein belonging to the KtrB family of K⁺ translocator proteins superfamily (20, 21), and preliminary studies have shown that KtrB plays a role in K⁺ uptake by Synechocystis sp. PCC 6803 (20). One of the aims of our study was to find out the function and physiological role of KtrB. Our experiments expressing the Synechocystis ktr genes in E. coli demonstrated that KtrABE is a K⁺ uptake system dependent on Na⁺ but is neither involved in Na⁺ uptake nor Na⁺ extrusion (Figs. 2, B and C and 4). The KtrB-mediated K⁺ transport with respect to Na⁺ dependence and protonophore-sensitivity was also analyzed in Synechocystis sp. PCC 6803 (Figs. 2B,3,6A, and 7). Hence we consider it unlikely that in Synechocystis sp. PCC 6803 the KtrB-mediated system extrudes Na⁺ as predicted by Shibata et al. (22), and we propose to modify their hypothesis as follows: In Synechocystis sp. PCC 6714, the earliest phase of adaptation to a hyperosmotic shock with NaCl consists of extensive Na⁺ uptake into the cells (16). Cells lacking an active Ktr system would not be able to carry out the subsequent phase of adaptation consisting of the exchange of the high cytoplasmic Na⁺ concentration against K⁺ (16). If the same applies to strain PCC 6803, the ΔktrB strain would continue to contain high Na⁺ concentrations in its cytoplasm. The Na⁺/H⁺ antiporters, for which the Synechocystis sp. PCC 6803 genome contains several genes (47, 54) would be unable to remove the Na⁺ from the cytoplasm, because Na⁺/H⁺ antiport activity leads to overacidification of the cytoplasm. Only when this Na⁺/H⁺-antiport process is coupled to a combination of electrogenic H⁺ extrusion by the H⁺ pumps of the cells (thereby counteracting overacidification of the cytoplasm, but also leading to the formation of a large ΔΨ and thereby to inhibition of further H⁺ extrusion) and electrogenic K⁺ uptake (via the KtrB-dependent system, which will decrease ΔΨ, thereby allowing the cells to extrude more protons), will the cells be able to remove their Na⁺ effectively from the cytoplasm (55, 56) (Fig. 10). Failure to extrude Na⁺ in the ΔktrB cells gives the cells low Na⁺ motive force across the cytoplasmic membrane. Since the force is believed to be the driving force for uptake via SbtA, low Na⁺ motive force in the ΔktrB cells explains the inhibition of transport in the ΔktrB cells observed by Shibata et al. (22). If Na⁺ binds to the Ktr system, and this stimulates the K⁺ uptake, which is proposed in plant Ktr/HKT-type transporters (57), there is an alternative explanation on the requirement of Na⁺ and the Ktr system for SbtA-mediated uptake. It is possible that SbtA-mediated uptake is coupled to K⁺ uptake by KtrABE and that the dependence of uptake on the presence of Na⁺ is a result of the Na⁺ requirement for the function of KtrABE (Figs. 6, 8, and 9). CO₂ uptake is driven by the pmf generated from photosynthetic PSI electron transport in Synechococcus sp. PCC 7942 (58). Assuming that SbtA and KtrABE would form the coupling system for and K⁺, the pmf may serve as an energy source for Na⁺-dependent K⁺ uptake by KtrABE in Synechocystis sp. PCC 6803.

The results in this study indicate that the primary role of the Ktr system in osmoadaptation of Synechocystis sp. PCC 6803 to high osmolality occurs during the early phase of this process. We showed for the first time that this system is not only essential for the adaptation of the cells to osmotic stress caused by ionic compounds like NaCl, but also to that by the non-ionic compound sorbitol (Fig. 5). Both the results reported by Shibata et al. and this study show in growth experiments on plates that the Ktr system is also required for long term osmoadaptation, since the ΔktrB strain do not grow at high concentrations of either NaCl or sorbitol (Fig. 5). The loss of K⁺ by the addition of 100 mm NaCl (Fig. 8A) or 200 mm sorbitol (Figs. 8B and 9) was the unexpected because we thought that only water loss occurred by osmotic upshock without K⁺ loss. The loss of K⁺ induced by hyperosmotic stress may have an adverse effect on cell growth, and may lead to the cessation of cell growth as shown in Fig. 5, C and D. The basis of the K⁺ loss in the ΔktrB cells may be similar to that observed in ΔktrAB cells of B. subtilis, which lost all of K⁺ about 0.4 h after a hyperosmotic shock with NaCl (11). In this case, although 4 mm K⁺ was present in the medium, the mutant failed to accumulate K⁺ in the cells. Taken together, we predict that one of the roles of the Ktr system is to refill K⁺, which is lost because of hyperosmotic shock. Interestingly, other K⁺ uptake systems in the ΔktrB cells, like Kdp or putative K⁺ channels, could not take over the function of the Ktr system with respect to K⁺ uptake (Figs. 5, C and D and 9). During the late phase of the osmoadaptation, the cells synthesize mainly the osmolyte glucosylglycerol, which replaces the previously accumulated Na⁺ and K⁺ in the cytoplasm and accumulates slowly to high concentrations in many cyanobacteria, including Synechocystis sp. PCC 6803 and Synechocystis sp. PCC 6714 (15–19). The other indication of the involvement of the KtrB system in long term osmoadapatation (Fig. 5 and data by Shibata et al., Ref. 22) is that the reaccumulation of K⁺ to the original or higher concentration level by the Ktr system in the cells after K⁺ loss by hyperosmotic shock is needed for the shift in metabolism of the cells to glucosylglycerol synthesis during the later phases of osmoadaptation. Such a process occurs in E. coli, for which a mutant strain, which is defective in additional K⁺ uptake after hyperosmotic shock has been shown to be impaired in the synthesis of its compatible solute trehalose as well (6). Very recently, this intriguing effect has been explained by the demonstration of the release of transcriptional suppression of osmoregulated genes by high potassium glutamate concentrations in the cytoplasm of E. coli in the early phase of osmoadaptation (59). It remains to be elucidated in Synechocystis sp. PCC 6803.

The second goal of this work was to characterize the KtrB-mediated K⁺ uptake system from Synechocystis sp. PCC 6803. We wanted to know whether in this organism KtrB acts alone, or whether it requires additional gene products for its activity. For this purpose, we cloned putative ktr genes from the Synechocystis sp. PCC 6803 into an E. coli K⁺ uptake negative strain, and examined its growth at low K⁺ concentrations. Unlike the Ktr system from V. alginolyticus, which requires both ktrA and ktrB for K⁺ uptake activity in E. coli (21), Synechocystis Ktr system needs besides these two ktr genes, a third gene, ktrE for the activity (Fig. 1). We confirmed that this result was not an artifact caused by the polar effect of ktrE on ktrB expression in the ΔktrE strain by use of the construct with deletion of only the start codon from ktrE (Fig. 1). The ktrE gene encodes a product with sequence similarity to glycosyl transferases (Fig. 1D). This is the first report that KtrE is involved in a function in K⁺ transport, but it is not known how it exerts this function. Dependence of ΔΨ component of the pmf for activity for the Ktr system suggests that the Ktr system functions coordinately with the energy generation system like photosynthesis and/or respiratory chain (Figs. 3 and 7). In this study, we found several requirements for exertion of the Ktr system, which are cellular subunits of KtrA and KtrE, ΔΨ component, and Na⁺. This indicates that the Ktr system is highly regulated in Synechocystis sp. PCC 6803.

Because the instant activation of KtrB-mediated K⁺ uptake occurs by the addition of Na⁺ in Synechocystis sp. PCC 6803 (Fig. 6), and the Ktr system is the main K⁺ uptake system in Synechocystis sp. PCC 6803 (Figs. 5, 8, and 9), we predict that a major reason that Synechocystis sp. PCC 6803, and possibly also other cyanobacteria require Na⁺ for their growth, lies in the Na⁺ dependence of their Ktr system. The system from Synechocystis sp. PCC 6803 is the third Ktr system for which Na⁺ dependence has been established (26, 27). Because at present no Na⁺-independent Ktr system is known, Na⁺ dependence may be a general feature of Ktr systems. Our data suggest that the Ktr system does not transport Na⁺ (Fig. 4). However HKT systems from plants, which are close relatives of KtrB, all transport Na⁺ (29, 33–38, 60–63). Moreover, under certain conditions KtrB proteins from both E. hirae and V. alginolyticus transport Na⁺ as well (Ref. 26).³ Hence we could not still rule out the possibility that the Ktr system mediates coupled K⁺/Na⁺ cotransport. Because of the lack of a proper mutant (which should be inactivation of both the activities of Na⁺/H⁺ antiport and of K⁺ uptake), we could not test this property on the Synechocystis KtrABE system in E. coli.

The genome of Synechocystis sp. PCC 6803 contains 43 genes that encode putative histidine kinases. A histidine kinase has been identified as a sensor of osmotic stress (64), using deletions in each of these genes (65). We have screened the corresponding mutant strains for growth under hyperosmotic conditions because of either 300 mm NaCl or 300 mm sorbitol and found that all of the mutants grew under high osmolyte conditions at which the ΔktrB strain did not survive. We have not identified any cross connection between the pathways of KtrB-dependent osmoadaptation and a two-component system (data not shown).