Halotolerant Cyanobacterium Aphanothece halophytica Contains a Betaine Transporter Active at Alkaline pH and High Salinity

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow in media of up to 3.0 M NaCl and pH 11. This cyanobacterium can synthesize betaine from glycine by three-step methylation using S-adenosylmethionine as a methyl donor. To unveil the mechanism of betaine uptake and efflux in this alkaliphile, we isolated and characterized a betaine transporter. A gene encoding a protein (BetT_{A. halophytica}) that belongs to the betaine-choline-carnitine transporter (BCCT) family was isolated. Although the predicted isoelectric pH of a typical BCCT family transporter, OpuD of Bacillus subtilis, is basic, 9.54, that of BetT_{A. halophytica} is acidic, 4.58. BetT_{A. halophytica} specifically catalyzed the transport of betaine. Choline, γ-aminobutyric acid, betaine aldehyde, sarcosine, dimethylglycine, and amino acids such as proline did not compete for the uptake of betaine by BetT_{A. halophytica}. Sodium markedly enhanced betaine uptake rates, whereas potassium and other cations showed no effect, suggesting that BetT_{A. halophytica} is a Na⁺-betaine symporter. Betaine uptake activities of BetT_{A. halophytica} were high at alkaline pH values, with the optimum pH around 9.0. Freshwater Synechococcus cells overexpressing BetT_{A. halophytica} showed NaCl-activated betaine uptake activities with enhanced salt tolerance, allowing growth in seawater supplemented with betaine. Kinetic properties of betaine uptake in Synechococcus cells overexpressing BetT_{A. halophytica} were similar to those in A. halophytica cells. These findings indicate that A. halophytica contains a Na⁺-betaine symporter that contributes to the salt stress tolerance at alkaline pH. BetT_{A. halophytica} is the first identified transporter for compatible solutes in cyanobacteria.     

Halotolerant cyanobacterium Aphanothece halophytica contains an Na+-dependent F1F0-ATP synthase with a potential role in salt-stress tolerance

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H(+)-ATP synthase, Aphanothece halophytica contains a putative F(1)F(0)-type Na(+)-ATP synthase (ApNa(+)-ATPase) operon (ApNa(+)-atp). The operon consists of nine genes organized in the order of putative subunits β, ε, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa(+)-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa(+)-ATPase exhibited Na(+)-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na(+) ion protected the inhibition of ApNa(+)-ATPase by N,N'-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na(+)-loaded inverted membrane vesicles. Expression of the ApNa(+)-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na(+)-dependent F(1)F(0)-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.     

Halotolerant cyanobacterium Aphanothece halophytica contains NapA-type Na+/H+ antiporters with novel ion specificity that are involved in salt tolerance at alkaline pH

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na+/H+ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na+/H+ and Li+/H+ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K+ uptake-deficient E. coli mutant and exhibited K+ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.     

BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti

Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of betS in E. coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with K(m)s of 16 +/- 2 microM and 56 +/- 6 microM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na(+) driven. In an S. meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. beta-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed. Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S. meliloti subjected to osmotic upshock.     

Pseudomonas syringae BetT is a low-affinity choline transporter that is responsible for superior osmoprotection by choline over glycine betaine

The plant pathogen Pseudomonas syringae derives better osmoprotection from choline than from glycine betaine, unlike most bacteria that have been characterized. In this report, we identified a betaine/carnitine/choline family transporter (BCCT) in P. syringae pv. tomato strain DC3000 that mediates the transport of choline and acetylcholine. This transporter has a particularly low affinity (K(m) of 876 microM) and high capacity (V(max) of 80 nmol/min/mg of protein) for choline transport relative to other known BCCTs. Although BetT activity increased in response to hyperosmolarity, BetT mediated significant uptake under low-osmolarity conditions, suggesting a role in transport for both osmoprotection and catabolism. Growth studies with mutants deficient in BetT and other choline transporters demonstrated that BetT was responsible for the superior osmoprotection conferred to P. syringae by choline over glycine betaine when these compounds were provided at high concentrations (>100 microM). These results suggest that P. syringae has evolved to survive in relatively choline-rich habitats, a prediction that is supported by the common association of P. syringae with plants and the widespread production of choline, but genus- and species-specific production of glycine betaine, by plants. Among the three putative BCCT family transporters in Pseudomonas aeruginosa and six in Pseudomonas putida, different transporters were predicted to function based on similarity to Escherichia coli BetT than to P. syringae BetT. Functional P. putida and P. aeruginosa transporters were identified, and their possession of a long C-terminal tail suggested an osmoregulatory function for this tail; this function was confirmed for P. syringae BetT using deletion derivatives.     

Halotolerant cyanobacterium Aphanothece halophytica contains an Na(+)/H(+) antiporter, homologous to eukaryotic ones, with novel ion specificity affected by C-terminal tail

Recently, a cyanobacterium Synechocystis sp. PCC 6803 has been shown to contain an Na(+)/H(+) antiporter gene homologous to plants (SOS1 and AtNHX1 from Arabidopsis) and mammalians (NHEs from human) but not to Escherichia coli (nhaA and nhaB). Here, we examined whether a halotolerant cyanobacterium Aphanothece halophytica has homologous genes. It turned out that A. halophytica contains an Na(+)/H(+) antiporter homologous to plants, mammalians, and some bacteria (nhaP from Pseudomonas and synnhaP from Synechocystis) but with novel ion specificity. Its gene product, ApNhaP (Na(+)/H(+) antiporter from Aphanothece halophytica), exhibited the Na(+)/H(+) antiporter activity over a wide pH range between 5 and 9 and complemented the Na(+)-sensitive phenotype of the antiporter-deficient E. coli mutant. The ApNhaP had virtually no activity for the Li(+)/H(+) antiporter but showed high Ca(2+)/H(+) antiporter activity at alkaline pH. The ApNhaP complemented the Ca(2+)-sensitive phenotype of the E. coli mutant but not the Li(+)-sensitive phenotype. The replacement of a long C-terminal tail of ApNhaP with that of Synechocystis altered the ion specificity of the antiporter. These results suggest that the ion specificity of an Na(+)/H(+) antiporter is partly determined by the structural properties of the C-terminal tail, which was well exemplified in the case of A. halophytica.     

The BCCT family of carriers: from physiology to crystal structure

Increases in the environmental osmolarity are key determinants for the growth of microorganisms. To ensure a physiologically acceptable level of cellular hydration and turgor at high osmolarity, many bacteria accumulate compatible solutes. Osmotically controlled uptake systems allow the scavenging of these compounds from scarce environmental sources as effective osmoprotectants. A number of these systems belong to the BCCT family (betaine-choline-carnitine-transporter), sodium- or proton-coupled transporters (e.g. BetP and BetT respectively) that are ubiquitous in microorganisms. The BCCT family also contains CaiT, an L-carnitine/γ-butyrobetaine antiporter that is not involved in osmotic stress responses. The glycine betaine transporter BetP from Corynebacterium glutamicum is a representative for osmoregulated symporters of the BCCT family and functions both as an osmosensor and osmoregulator. The crystal structure of BetP in an occluded conformation in complex with its substrate glycine betaine and two crystal structures of CaiT in an inward-facing open conformation in complex with L-carnitine and γ-butyrobetaine were reported recently. These structures and the wealth of biochemical data on the activity control of BetP in response to osmotic stress enable a correlation between the sensing of osmotic stress by a transporter protein with the ensuing regulation of transport activity. Molecular determinants governing the high-affinity binding of the compatible solutes by BetP and CaiT, the coupling in symporters and antiporters, and the osmoregulatory properties are discussed in detail for BetP and various BCCT carriers.     

Nitrate uptake in the halotolerant cyanobacterium Aphanothece halophytica is energy-dependent driven by DeltapH

The energetics of nitrate uptake by intact cells of the halotolerant cyanobacterium Aphanothece halophytica were investigated. Nitrate uptake was inhibited by various protonophores suggesting the coupling of nitrate uptake to the proton motive force. An artificially-generated pH gradient across the membrane (DeltapH) caused an increase of nitrate uptake. In contrast, the suppression of DeltapH resulted in a decrease of nitrate uptake. The increase of external pH also resulted in an enhancement of nitrate uptake. The generation of the electrical potential across the membrane (Deltapsi) resulted in no elevation of the rate of nitrate uptake. On the other hand, the valinomycin-mediated dissipation of Deltapsi caused no depression of the rate of nitrate uptake. Thus, it is unlikely that Deltapsi participated in the energization of the uptake of nitrate. However, Na(+)-gradient across the membrane was suggested to play a role in nitrate uptake since monensin which collapses Na(+)-gradient strongly inhibited nitrate uptake. Exogenously added glucose and lactate stimulated nitrate uptake in the starved cells. N, N'-dicyclohexylcarbodiimide, an inhibitor of ATPase, could alsoinhibit nitrate uptake suggesting that ATP hydrolysis was required for nitrate uptake. All these results indicate that nitrate uptake in A. halophytica is ATP-dependent, driven by DeltapH and Na(+)-gradient.     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

Metabolic engineering for betaine accumulation in microbes and plants

Plants accumulate a variety of osmoprotectants that improve their ability to combat abiotic stresses. Among them, betaine appears to play an important role in conferring resistance to stresses. Betaine is synthesized via either choline oxidation or glycine methylation. An increased betaine level in transgenic plants is one of the potential strategies to generate stress-tolerant crop plants. Here, we showed that an exogenous supply of serine or glycine to a halotolerant cyanobacterium Aphanothece halophytica, which synthesizes betaine from glycine by a three-step methylation, elevated intracellular accumulation of betaine under salt stress. The gene encoding 3-phosphoglycerate dehydrogenase (PGDH), which catalyzes the first step of the phosphorylated pathway of serine biosynthesis, was isolated from A. halophytica. Expression of the Aphanothece PGDH gene in Escherichia coli caused an increase in levels of betaine as well as glycine and serine. Expression of the Aphanothece PGDH gene in Arabidopsis plants, in which the betaine synthetic pathway was introduced via glycine methylation, further increased betaine levels and improved the stress tolerance. These results demonstrate that PGDH enhances the levels of betaine by providing the precursor serine for both choline oxidation and glycine methylation pathways.     

Characterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na(+)-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12 degrees C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and gamma-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

Salt stress enhances choline uptake in the halotolerant cyanobacterium Aphanothece halophytica

The uptake of [14C]choline by a suspension of exponential-phase Aphanothece halophytica under various conditions has been studied. Salt stress was found to enhance the uptake of choline. The kinetics of choline transport followed the Michaelis-Menten relationship with apparent K(m) values of 272 and 286 microM, maximum rates of transport (V(max)) of 18 and 37 nmol/min/mg protein for unstressed and salt-stressed cells, respectively. Choline uptake under salt stress was significantly reduced in chloramphenicol-treated cells, suggesting that the activation by salt stress occurred via an inducible transport system. This was corroborated by the existence of the periplasmic choline binding protein, whose content was higher in cells grown under salt-stress condition. Exogenously provided choline significantly increased the growth rate of cells grown under salt stress, although less efficiently than glycine betaine. The presence of 1 mM choline in the growth medium conferred tolerance to high salinity on A. halophytica with the maintenance of high growth up to 1.5 M NaCl. The uptake of choline was Na(+)-dependent, sensitive to various metabolic inhibitors as well as thiol-reactive agents. The results of competition studies suggested that N-methyl on one end of molecule and on the other end either an aldehyde, an alcohol or a neutral group were important features for substrate recognition.     

Presence of a Na+-stimulated P-type ATPase in the plasma membrane of the alkaliphilic halotolerant cyanobacterium Aphanothece halophytica

Aphanothece cells could take up Na(+) and this uptake was strongly inhibited by the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells preloaded with Na(+) exhibited Na(+) extrusion ability upon energizing with glucose. Na(+) was also taken up by the plasma membranes supplied with ATP and the uptake was abolished by gramicidin D, monensin or Na(+)-ionophore. Orthovanadate and CCCP strongly inhibited Na(+) uptake, whereas N, N'-dicyclohexylcarbodiimide (DCCD) slightly inhibited the uptake. Plasma membranes could hydrolyse ATP in the presence of Na(+) but not with K(+), Ca(2+) and Li(+). The K(m) values for ATP and Na(+) were 1.66+/-0.12 and 25.0+/-1.8 mM, respectively, whereas the V(max) value was 0.66+/-0.05 mumol min(-1) mg(-1). Mg(2+) was required for ATPase activity whose optimal pH was 7.5. The ATPase was insensitive to N-ethylmaleimide, nitrate, thiocyanate, azide and ouabain, but was substantially inhibited by orthovanadate and DCCD. Amiloride, a Na(+)/H(+) antiporter inhibitor, and CCCP showed little or no effect. Gramicidin D and monensin stimulated ATPase activity. All these results suggest the existence of a P-type Na(+)-stimulated ATPase in Aphanothece halophytica. Plasma membranes from cells grown under salt stress condition showed higher ATPase activity than those from cells grown under nonstress condition.     

Roles of three transporters, CbcXWV, BetT1, and BetT3, in Pseudomonas aeruginosa choline uptake for catabolism

Pseudomonas aeruginosa uses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline in P. aeruginosa physiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, in P. aeruginosa growth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ΔbetT1 and ΔbetT1 ΔbetT3 mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdR-mediated cbcXWV induction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.     

Identification of an ATP-driven, osmoregulated glycine betaine transport system in Listeria monocytogenes.

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na(+)-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na(+) for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na(+) is an indication that only the ATP-dependent transporter and the Na(+)-glycine betaine symporter occur in L. monocytogenes.     

Partial deletion of a loop region in the high affinity K+ transporter HKT1 changes ionic permeability leading to increased salt tolerance

HKT1 is a high affinity K(+) transporter protein that is a member of a large superfamily of transporters found in plants, bacteria, and fungi. These transporters are primarily involved in K(+) uptake and are energized by Na(+) or H(+). HKT1 is energized by Na(+) but also mediates low affinity Na(+) uptake and may therefore be a pathway for Na(+) uptake, which is toxic to plants. The aim of this study was to identify regions of HKT1 that are involved in K(+)/Na(+) selectivity and alter the amino acid composition in those regions to increase the ionic selectivity of the transporter. A highly charged loop was identified, and two deletions were created that resulted in the removal of charged and uncharged amino acids. The functional changes caused by the deletions were studied in yeast and Xenopus oocytes. The deletions improved the K(+)/Na(+) selectivity of the transporter and increased the salt tolerance of the yeast cells in which they were expressed. In light of recent structural models of members of this symporter superfamily, it was necessary to determine the orientation of this highly charged loop. Introduction of an epitope tag allowed us to demonstrate that this loop faces the outside of the membrane where it is likely to facilitate the interaction with cations such as K(+) and Na(+). This study has identified an important structural feature in HKT1 that in part determines its K(+)/Na(+) selectivity. Understanding the structural basis of the functional characteristics in transporters such as HKT1 may have important implications for increasing the salt tolerance of higher plants.