Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

Photoautotrophic bacteria have developed mechanisms to maintain K⁺ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K⁺ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K⁺ (Kdp). The contributions of each of these K⁺ transport systems to cellular K⁺ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K⁺ uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K⁺ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K⁺ depletion. Correspondingly, Kdp-mediated K⁺ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K⁺ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K⁺ concentration under conditions of limited K⁺ in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K⁺ availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K⁺ levels under fluctuating environmental conditions.     

Identification of a Novel Bacterial K<Superscript>+</Superscript> Channel

In an attempt to explore unknown K⁺ channels in mammalian cells, especially ATP-sensitive K⁺ (K_ATP) channels, we compared the sequence homology of Kir6.1 and Kir6.2, two pore-forming subunits of mammalian K_ATP channel genes, with bacterial genes that code for selective proteins with confirmed or putative ion transport properties. BLAST analysis revealed that a prokaryotic gene (ydfJ) expressed in Escherichia coli K12 strain shared 8.6% homology with Kir6.1 and 8.3% with Kir6.2 genes. Subsequently, we cloned and sequenced ydfJ gene from E. coli K12 and heterologously expressed it in mammalian HEK-293 cells. The whole-cell patch-clamp technique was used to record ion channel currents generated by ydfJ-encoded protein. Heterologous expression of ydfJ gene in HEK-293 cells yielded a novel K⁺ channel current that was inwardly rectified and had a reversal potential close to K⁺ equilibrium potential. The expressed ydfJ channel was blocked reversibly by low concentration of barium in a dose-dependent fashion. Specific K_ATP channel openers or blockers did not alter the K⁺ current generated by ydfJ expression alone or ydfJ coexpressed with rvSUR1 or rvSUR2B subunits of K_ATP channel complex. Furthermore, this coexpressed ydfJ/rvSUR1 channels were not inhibited by ATP dialysis. On the other hand, ydfJ K⁺ currents were inhibited by protopine (a nonspecific K⁺ channel blocker) but not by dofetilide (a HERG channel blocker). In summary, heterologously expressed prokaryotic ydfJ gene formed a novel functional K⁺ channel in mammalian cells.     

The K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter <Emphasis Type="Italic">AhNHX1</Emphasis> improved tobacco tolerance to NaCl stress by enhancing K<Superscript>+</Superscript> retention

High salinity is the one of important factors limiting plant growth and crop production. Many NHX-type antiporters have been reported to catalyze K⁺/H⁺ exchange to mediate salt stress. This study shows that an NHX gene from Arachis hypogaea L. has an important role in K⁺ uptake and transport, which affects K⁺ accumulation and plant salt tolerance. When overexpressing AhNHX1, the growth of tobacco seedlings is improved with longer roots and a higher fresh weight than the wild type (WT) under NaCl treatment. Meanwhile, when exposed to NaCl stress, the transgenic seedlings had higher K⁺/H⁺ antiporter activity and their roots got more K⁺ uptake. NaCl stress could induce higher K⁺ accumulation in the roots, stems, and leaves of transgenic tobacco seedlings but not Na⁺ accumulation, thus, leading to a higher K⁺/Na⁺ ratio in the transgenic seedlings. Additionally, the AKT1, HAK1, SKOR, and KEA genes, which are involved in K⁺ uptake or transport, were induced by NaCl stress and kept higher expression levels in transgenic seedlings than in WT seedlings. The H⁺-ATPase and H⁺-PPase activities were also higher in transgenic seedlings than in the WT seedlings under NaCl stress. Simultaneously, overexpression of AhNHX1 increased the relative distribution of K⁺ in the aerial parts of the seedlings under NaCl stress. These results showed that AhNHX1 catalyzed the K⁺/H⁺ antiporter and enhanced tobacco tolerance to salt stress by increasing K⁺ uptake and transport.     

Molecular and functional analyses of rice NHX-type Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter genes

We previously cloned a vacuolar Na⁺/H⁺ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon–intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na⁺, Li⁺, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K⁺ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter–GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter–GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na⁺ and K⁺ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.     

Trk2 transporter is a relevant player in K<Superscript>+</Superscript> supply and plasma-membrane potential control in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

In Saccharomyces cerevisiae, TRK1 and TRK2 genes encode partially redundant K⁺ transporters. Direct involvement in K⁺ uptake has been shown for Trk1p since cells growing under limiting environmental K⁺ concentrations demand its presence. The biological role of Trk2p is less understood. In our experiments, TRK2 overexpression improved the ability of trk1 cells to grow in low K⁺ and led to a higher accumulation of K⁺. Using diS-C₃(3) as a potentiometric probe, we revealed a higher hyperpolarization of trk2 cells compared to the wild type. In addition, the deletion of TRK2 in the trk1 genetic background increased the cell sensitivity to hygromycin B, spermine, and TMA. Our studies reinforced the conclusion that Trk1p is the prominent K⁺ uptake transporter and for the first time revealed that though Trk2p is much less effective, its activity contributes significantly to K⁺ supply and the maintenance of plasma-membrane potential.     

第二信使分子c-di-AMP调控细菌中钾离子转运的机制

钾离子(K~+)是维持生命体存活的必需元素。原核生物进化出一系列K~+转运系统,如Kdp系统﹑Ktr系统和Trk系统等,来维持胞内相对恒定的K~+浓度。环二腺苷酸单磷酸(cyclic diadenosine monophosphate,c-di-AMP)是新发现的第二信使分子,可以与K~+转运系统中的KdpD、KtrA和TrkA结合。当胞内c-di-AMP浓度高时,c-di-AMP会与K~+转运蛋白结合,降低其转运活性。c-di-AMP的靶标除蛋白质外,还有RNA元件,即c-di-AMP的核糖开关。高浓度的c-di-AMP与其核糖开关结合后,可抑制下游K~+转运蛋白编码基因,如kdp、ktr和trk操纵子以及kup基因的转录,从而调控K~+的转运。总之,胞内高浓度的c-di-AMP抑制细菌对K~+的吸收。c-di-AMP调控K~+转运机制的研究,不仅丰富了K~+转运的调控方式,而且也扩大了c-di-AMP的调控范围,为细菌的利用与防治提供了新思路。     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Overexpression in a non-native halophilic host and biotechnological potential of NAD<Superscript>+</Superscript>-dependent glutamate dehydrogenase from <Emphasis Type="Italic">Halobacterium salinarum</Emphasis> strain NRC-36014

Enzymes produced by halophilic archaea are generally heat resistant and organic solvent tolerant, and accordingly important for biocatalytic applications in ‘green chemistry’, frequently requiring a low-water environment. NAD⁺-dependent glutamate dehydrogenase from an extremely halophilic archaeon Halobacterium salinarum strain NRC-36014 was selected to explore the biotechnological potential of this enzyme and genetically engineered derivatives. Over-expression in a halophilic host Haloferax volcanii provided a soluble, active recombinant enzyme, not achievable in mesophilic Escherichia coli, and an efficient purification procedure was developed. pH and salt dependence, thermostability, organic solvent stability and kinetic parameters were explored. The enzyme is active up to 90 °C and fully stable up to 70 °C. It shows good tolerance of various miscible organic solvents. High concentrations of salt may be substituted with 30 % DMSO or betaine with good stability and activity. The robustness of this enzyme under a wide range of conditions offers a promising scaffold for protein engineering.     

Rhodospirillum salinarum sp. nov., a halophilic photosynthetic bacterium isolated from a Portuguese saltern

A new species of halophilic photosynthetic bacteria, Rhodospirillum salinarum, has been isolated and described. Its natural habitat are the terminal crystallization ponds of solar salt production plants. R. salinarum grows optimally at 42°C in the presence of 6–18% NaCl (w/v). Growth requirements are complex, yeast extract and peptone being required both for aerobic heterotrophic and for anaerobic phototrophic growth. Increasing concentrations of NaCl in the growth media did not give rise to any corresponding increase in intracellular concentrations of K⁺, Na⁺, polyalcohols or amino acids. Malate dehydrogenase from R. salinarum is not halophilic, being inhibited even at low concentrations of Na⁺ or K⁺. The GC mol % of DNA from R. salinarum is markedly higher than that for DNA from R. salexigens, the only previously described halophilic species of the genus Rhodospirillum.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Cloning and functional expression in <Emphasis Type="Italic">Saccharomyces cereviae</Emphasis> of a K<Superscript>+</Superscript> transporter,AlHAK, from the graminaceous halophyte, <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

High-affinity K⁺ transporters play an important role in K⁺ absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WΔ3, AlHAK performed high-affinity K⁺ uptake with a K_m value of 8 μM, and the growth of transformants was dramatically inhibited by 150 mM Rb⁺ and 150 mM Cs⁺ but less affected by 300 mM Na⁺. AlHAK may thus improve the capacity of plants to maintain a high cytosolic K⁺/Na⁺ ratio at high salinity.     

NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.     

Cloning and functional characterization of the high-affinity K<Superscript>+</Superscript> transporter HAK1 of pepper

High-affinity K⁺ uptake in plants plays a crucial role in K⁺ nutrition and different systems have been postulated to contribute to the high-affinity K⁺ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K⁺ uptake observed in roots. Pepper plants starved of K⁺ for 3 d showed high-affinity K⁺ uptake (K _m of 6 M K⁺) that was very sensitive to NH and their roots expressed a high-affinity K⁺ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast (Saccharomyces cerevisiae), CaHAK1 mediated high-affinity K⁺ and Rb⁺ uptake with K _m values of 3.3 and 1.9 M, respectively. Rb⁺ uptake was competitively inhibited by micromolar concentrations of NH and Cs⁺, and by millimolar concentrations of Na⁺.     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The High-affinity K+-translocating ATPase Complex from Clostridium acetobutylicum Consists of Six Subunits

The kdp operon of Clostridium acetobutylicum codes for the high affinity K⁺-translocating Kdp system (P-type ATPase). Beside the three large proteins KdpA, KdpB and KdpC, the kdp operon encodes the two small peptides KdpZ, KdpY and the KdpX protein. The truncation of the clostridial kdpZ and/or the kdpY gene has a significant impact on the growth of an E. coli mutant (TK2205), which is unable to grow at low potassium concentrations. These two genes together with kdpX are essential to maintain the wild-type K⁺-pump capacity of the clostridial Kdp system. Also the ATPase activity itself, the substrate specifity, and the cation specifity are determined in a major way by KdpZ, KdpY, and KdpX. Thus, this report shows the importance of the KdpZ, KdpY, and KdpX proteins for the Kdp-ATPase and therefore the corresponding operon should now be referred to as kdpZYABCX.     

Influence of ATP-dependent K<Superscript>+</Superscript>-channel opener on K<Superscript>+</Superscript>-cycle and oxygen consumption in rat liver mitochondria

The influence of the K_ATP⁺-channel opener diazoxide on the K⁺ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K_ATP⁺-channel in the range of nanomolar concentrations (50–300 nM, K _1/2 ∼ 140 nM), which results in activation of K⁺/H⁺ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K_ATP⁺-channel to the mitochondrial potassium cycle was estimated using the selective K_ATP⁺-channel blocker glibenclamide. The data show that the relative contribution of K_ATP⁺-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K⁺ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K_ATP⁺-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.     

Role of ENA ATPase in Na<Superscript>+</Superscript> efflux at high pH in bryophytes

Potassium or Na⁺ efflux ATPases, ENA ATPases, are present in all fungi and play a central role in Na⁺ efflux and Na⁺ tolerance. Flowering plants lack ENA ATPases but two ENA ATPases have been identified in the moss Physcomitrella patens, PpENA1 and PpENA2. PpENA1 mediates Na⁺ efflux in Saccharomyces cerevisiae. To propose a general function of ENA ATPases in bryophytes it was necessary to demonstrate that these ATPases mediate Na⁺ efflux in planta and that they exist in more bryophytes than P. patens. For these demonstrations (1) we cloned a third ATPase from P. patens, PpENA3, and studied the expression pattern of the three PpENA genes; (2) we constructed and studied the single and double Δppena1 and Δppena2 mutants; and (3) we cloned two ENA ATPases from the liverwort Marchantia polymorpha, MpENA1 and MpENA2, and expressed them in S. cerevisiae. The results from the first two approaches revealed that the expression of ENA ATPases was greatly enhanced at high pH and that Na⁺ efflux at high pH depended on PpENA1. The ENA1 ATPase of M. polymorpha suppressed the defective growth of a S. cerevisiae mutant at high K⁺ or Na⁺ concentrations, especially at high K⁺.