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, gamma-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(+)/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.     

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.     

A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants

In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill. cv. Moneymaker), which is a member of the intracellular NHX exchanger protein family. The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment. LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments. The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants. A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro. These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers.     

Na+/H+ antiporters,molecular devices that couple the Na+ and H+ circulation in cells

Na⁺/H⁺ antiporters are universal devices involved in the Na⁺ and H⁺ circulation of both eukaroyotes and prokaryotes, thus playing an essential role in the pH and Na⁺ homeostasis of cells. This review focuses on the major impact of the application of molecular biology tools in the study of the antiporters. These tools permit the verification of the role of the antiporters and provide insights into their unique biology. A novel signal transduction to Na⁺ involvingnhaR, a positive regulator, controls the expression ofnhaA inE. coli. A pH sensor regulates the activity of Na⁺/H⁺ antiporters, both in eukaryotes and prokaryotes. A most intricate signal transduction to pH involving phosphorylation steps controls the activity ofnhel in higher mammals. The identification of Histidine 226 in the pH sensor of NhaA is a step forward towards the understanding of the pH regulation of these proteins.     

细菌Na+/H+逆向转运蛋白的研究进展

Na+/H+逆向转运蛋白在维持细胞内pH稳态、Na+离子动态平衡和调控细胞体积方面发挥着重要作用。目前,细菌中许多参与高盐或高碱性环境压力应答的Na+/H+逆向转运蛋白得到了鉴定和功能阐释。继续挖掘高效的Na+/H+逆向转运蛋白,深入探究Na+/H+逆向转运蛋白的分子机理,将为工业菌株或农作物的改良提供新的研究思路。本文以4种模式菌株为例,简要概述细菌Na+/H+逆向转运蛋白的种类和特征,同时对其结构和功能等方面也进行探讨。     

Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

Na+/H+ exchange in the cyanobacterium Synechococcus 6311

The cyanobacterium Synechococcus 6311 adapts to grow in 0.6 M NaCl by developing an efficient system for sodium extrusion. In the present investigation cells loaded with NaC1 were subjected to a large dilution. Changes in fluorescence quenching of acridine orange as a function of transmembrane Na+ gradients provide evidence that Na+/H+ exchange activity greatly enhanced in salt-adapted cells.     

Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation

Four isoforms of the Na+/H+ exchanger (NHE6-NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid- to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.     

Na+,K+/H+ antiporters regulate the pH of endoplasmic reticulum and auxin‐mediated development

AtNHX5 and AtNHX6 are endosomal Na⁺,K⁺/H⁺ antiporters that are critical for growth and development in Arabidopsis, but the mechanism behind their action remains unknown. Here, we report that AtNHX5 and AtNHX6, functioning as H⁺ leak, control auxin homeostasis and auxin‐mediated development. We found that nhx5 nhx6 exhibited growth variations of auxin‐related defects. We further showed that nhx5 nhx6 was affected in auxin homeostasis. Genetic analysis showed that AtNHX5 and AtNHX6 were required for the function of the endoplasmic reticulum (ER)‐localized auxin transporter PIN5. Although AtNHX5 and AtNHX6 were colocalized with PIN5 at ER, they did not interact directly. Instead, the conserved acidic residues in AtNHX5 and AtNHX6, which are essential for exchange activity, were required for PIN5 function. AtNHX5 and AtNHX6 regulated the pH in ER. Overall, AtNHX5 and AtNHX6 may regulate auxin transport across the ER via the pH gradient created by their transport activity. H⁺‐leak pathway provides a fine‐tuning mechanism that controls cellular auxin fluxes.     

Monomers of the NhaA Na+/H+ antiporter of Escherichia coli are fully functional yet dimers are beneficial under extreme stress conditions at alkaline pH in the presence of Na+ or Li+

NhaA, the Na(+)/H(+) antiporter of Escherichia coli, exists in the native membrane as a homodimer of which two monomers have been suggested to be attached by a beta-hairpin at the periplasmic side of the membrane. Constructing a mutant deleted of the beta-hairpin, NhaA/Delta(Pro(45)-Asn(58)), revealed that in contrast to the dimeric mobility of native NhaA, the mutant has the mobility of a monomer in a blue native gel. Intermolecular cross-linking that monitors dimers showed that the mutant exists only as monomers in the native membrane, proteoliposomes, and when purified in beta-dodecyl maltoside micelles. Furthermore, pull-down experiments revealed that, whereas as expected for a dimer, hemagglutinin-tagged wild-type NhaA co-purified with His-tagged NhaA on a Ni(2+)-NTA affinity column, a similar version of the mutant did not. Remarkably, under routine stress conditions (0.1 m LiCl, pH 7 or 0.6 m NaCl, pH 8.3), the monomeric form of NhaA is fully functional. It conferred salt resistance to NhaA- and NhaB-deleted cells, and whether in isolated membrane vesicles or reconstituted into proteoliposomes exhibited Na(+)/H(+) antiporter activity and pH regulation very similar to wild-type dimers. Remarkably, under extreme stress conditions (0.1 m LiCl or 0.7 m NaCl at pH 8.5), the dimeric native NhaA was much more efficient than the monomeric mutant in conferring extreme stress resistance.