Tatsunosuke Nakamura

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice

We engineered a salt‐sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar‐type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar‐type Na+/H+ antiporter in the transgenic rice plants was eight‐fold higher than that in wild‐type rice plants. Salt tolerance assays followed by non‐stress treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 300 mM NaCl for 3 days while the wild‐type rice plants could not. These results indicate that overexpression of the Na+/H+ antiporter gene in rice plants significantly improves their salt tolerance.

Overexpression of a Na+/H+ antiporter confers salt tolerance on a freshwater cyanobacterium, making it capable of growth in sea water

The salt tolerance of a freshwater cyanobacterium, Synechococcus sp. PCC 7942, transformed with genes involved in the synthesis of a Na+/H+ antiporter, betaine, catalase, and a chaperone was examined. Compared with the expression of betaine, catalase, and the chaperone, the expression of the Na+/H+ antiporter gene from a halotolerant cyanobacterium (ApNhaP) drastically improved the salt tolerance of the freshwater cyanobacterium. The Synechococcus cells expressing ApNhaP could grow in BG11 medium containing 0.5 M NaCl as well as in sea water, whereas those expressing betaine, catalase, and the chaperone could not grow under those conditions. The coexpression of ApNhaP with catalase or ApNhaP with catalase and betaine did not further enhance the salt tolerance of Synechococcus cells expressing ApNhaP alone when grown in BG11 medium containing 0.5 M NaCl. Interestingly, the coexpression of ApNhaP with catalase resulted in enhanced salt tolerance of cells grown in sea water. These results demonstrate a key role of sodium ion exclusion by the Na+/H+ antiporter for the salt tolerance of photosynhetic organisms.

K+/H+ antiporter functions as a regulator of cytoplasmic pH in a marine bacterium, Vibrio alginolyticus

The marine bacterium, Vibrio alginolyticus, regulates the cytoplasmic pH at about 7.8 over the pH range 6.0-9.0. By the addition of diethanolamine (a membrane-permeable amine) at pH 9.0, the internal pH was alkalized and simultaneously the cellular K+ was released. Following the K+ exit, the internal pH was acidified until 7.8, where the K+ exit leveled off. The K+ exit was mediated by a K+/H+ antiporter that is driven by the outwardly directed K+ gradient and ceases to function at the internal pH of 7.8 and below. The Na+-loaded cells assayed in the absence of KCl generated inside acidic delta pH at alkaline pH due to the function of an Na+/H+ antiporter, but the internal pH was not maintained at a constant value. At acidic pH range, the addition of KCl to the external medium was necessary for the alkalization of cell interior. These results suggested that in cooperation with the K+ uptake system and H+ pumps, the K+/H+ antiporter functions as a regulator of cytoplasmic pH to maintain a constant value of 7.8 over the pH range 6.0-9.0.

Mechanosensitive channel functions to alleviate the cell lysis of marine bacterium, Vibrio alginolyticus, by osmotic downshock.

The mechanosensitive channel with large conductance of Escherichia coli is the first to be cloned among stretch‐activated channels. Although its activity was characterized by a patch clamp method, a physiological role of the channel has not been proved. The marine bacterium, Vibrio alginolyticus, is sensitive to osmotic stress and cell lysis occurs under osmotic downshock. We introduced an mscL gene into Vibrio alginolyticus, and the mechanosensitive channel with large conductance functions was found to alleviate cell lysis by osmotic downshock. This is the first report to show a physiological role of the mechanosensitive channel with large conductance.

Identification of the ABC protein SapD as the subunit that confers ATP dependence to the K+-uptake systems TrkH and TrkG from Escherichia coli K-12

The activity of the two almost identical K+-uptake systems, Trk(H) and Trk(G), from Escherichia coli K-12 depends completely and partially on the presence of the trkE gene, respectively. trkE maps inside the sapABCDF operon, which encodes an ATP-binding cassette (ABC) transporter of unknown function from the subgroup of peptide-uptake systems. This study was carried out to clarify the role of sapABCDF gene products in the ATP dependence of the E. coli Trk systems. For this purpose DeltasapABCDF DeltatrkG and DeltasapABCDF DeltatrkH strains of E. coli containing plasmids with sap genes from either E. coli or Vibrio alginolyticus were used. All five plasmid-encoded E. coli Sap proteins were made in E. coli mini-cells. The presence of the ATP-binding SapD protein from either E. coli or V. alginolyticus alone was sufficient for stimulating the K+ transport activity of the Trk(H) and Trk(G) systems. K+-uptake experiments with Escherichia coli cells containing SapD variants with changes in the Walker A box Lys-46 residue, the Walker B box Asp-183 residue and the signature motif residues Gly-162 or Gln-165 suggested that adenine nucleotide binding to SapD rather than ATP hydrolysis by this subunit is required for the activity of the E. coli Trk(H) system. K+ transport via two plasmid-encoded Trk systems in a DeltasapABCDF E. coli strain remained dependent on both a high membrane potential and a high cytoplasmic ATP concentration, indicating that in E. coli ATP dependence of Trk activity can be independent of Sap proteins. These data are interpreted to mean that Trk systems can interact with an ABC protein other than SapD.

Effects of pH and monovalent cations on the potassium ion exit from the marine bacterium, Vibrio alginolyticus, and the manipulation of cellular cation contents

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

Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli

The regulation of internal Na+ and K+ concentrations is important for bacterial cells, which, in the absence of Na+ extrusion systems, cannot grow in the presence of high external Na+. Likewise, bacteria require K+ uptake systems when the external K+ concentration becomes too low to support growth. At present, we have little knowledge of K+ toxicity and bacterial outward‐directed K+ transport systems. We report here that high external concentrations of K+ at alkaline pH are toxic and that bacteria require K+ efflux and/or extrusion systems to avoid excessive K+ accumulation. We have identified the first example of a bacterial K+(specific)/H+ antiporter, Vp‐NhaP2, from Vibrio parahaemolyticus. This protein, a member of the cationu2003:u2003proton antiporter‐1 (CPA1) family, was able to mediate K+ extrusion from the cell to provide tolerance to high concentrations of external KCl at alkaline pH. We also report the discovery of two V.u2003parahaemolyticus Na+/H+ antiporters, Vp‐NhaA and Vp‐NhaB, which also exhibit a novel ion specificity toward K+, implying that they work as Na+(K+)/H+ exchangers. Furthermore, under specific conditions, Escherichia coli was able to mediate K+ extrusion against a K+ chemical gradient, indicating that E.u2003coli also possesses an unidentified K+ extrusion system(s).

Cloning and sequencing of the nhaB gene encoding an Na+/H+ antiporter from Vibrio alginolyticus

A gene has been cloned from the marine bacterium Vibrio alginolyticus that functionally complements a mutant strain of Escherichia coli, TO114, defective in three Na+/H+ antiport genes (nhaA, nhaB, chaA). The nucleotide sequence of the cloned fragment revealed an open reading frame, which encodes a protein with a predicted 528 amino acid sequence and molecular mass of 57212 Da. This gene has 62% identity to nhaB gene at the DNA level from Escherichia coli and the deduced amino acid sequence is 67% identical with E. coli NhaB. This gene is presumably the V. alginolyticus nhaB gene and will be named nhaBv.

Halotolerant Cyanobacterium Aphanothece halophytica Contains NapA-Type Na+/H+ Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH

ABSTRACT 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.

Three aspartic residues in membrane-spanning regions of Na^+/H^+ antiporter from Vibrio alginolyticus play a role in the activity of the carrier

The Na+/H+ antiporter gene from Vibrio alginolyticus restores the growth of an nhaA-defective strain of Escherichia coli, NM81, in a high NaCl medium (Nakamura, T., Komano, Y., Itaya, E., Tsukamoto, K., Tsuchiya, T. and Unemoto, T. (1994) Biochim. Biophys. Acta 1190, 465-468). This gene, named nhaAv, allowed the nhaA-defective E. coli strains, NM81(delta nhaA) and RS1 (delta nhaA, chaA-), to extrude Na+ at alkaline pH. The extrusion of Na+ occurred against its chemical gradient in the presence of membrane-permeable amine. Thus, the nhaAv gene product is functional as an electrogenic Na+/H+ antiporter in E. coli cells. The NhaAv protein has only four acidic amino acid residues in the putative membrane-spanning regions, that is, Asp-57, Asp-125, Asp-155 and Asp-156, and these Asp residues are conserved in NhaA from E. coli. Asp-111, which is predicted to be in a loop region between the transmembrane segments is also conserved in NhaA. Thus, each conserved Asp residue was replaced with asparagine by a site-directed mutagenesis. E. coli NM81 cells containing a plasmid harboring the nhaAv gene mutated at Asp-125, -155, or -156 could neither grow in a high NaCl medium nor extrude Na+ at alkaline pH against its chemical gradient. These results show that Asp-125, -155, and -156, but not Asp-57 and -111, play a role in the activity of the Na+/H+ antiporter, NhaAv.