pH homeostasis and bioenergetic work in alkalophiles

Abstract A Na⁺/H⁺ antiporter catalyses coupled Na⁺ extrusion and H⁺ uptake across the membranes of extremely alkalophilic bacilli. This exchange is electrogenic, with H⁺ translocated inward > Na⁺ extruded. It is energized by the Δψ ² component of the ΔμH⁺ that is established during primary proton pumping by the alkalophile respiratory chain complexes. These complexes abound in the membranes of extreme alkalophiles. Combined activity of the respiratory chain, the antiporter, and solute transport systems that are coupled to Na⁺ re-entry, allow the alkalophiles to maintain a cytoplasmic pH that is several pH units more acidic than optimal external pH values for growth. There is no compelling evidence for a specific and necessary role for any ion other than sodium in pH homeostasis, and although there is very high cytoplasmic buffering capacity in the alkaline range, active mechanisms for pH homeostasis are crucial. Energization of the antiporter as well as the proton translocating F ₁ F ₀-ATPase that catalyses ATP synthesis in the extreme alkalophiles must accommodate the problem of the low net ΔμH⁺ and the very low concentrations of protons, per se, in the external medium. This problem is by-passed by other bioenergetic work functions, such as solute uptake or motility, that utilize sodium ions for energy-coupling in the place of protons.     

Antiporter Gene from Hordum brevisubulatum （Trin.） Link and Its Overexpression in Transgenic Tobaccos

A vacuolar Na^ /H^ antiporter cDNA gene was successfully isolated fromHordeum brevisubulatum (Trin.) Link using the rapid amplification ofcDNA ends (RACE) method. The gene was named HbNHXI and was found to consist of 1 916 bp encoding a predicted polypeptide of 540 amino acids with a conserved amiloride-binding domain. Phylogenetic tree analysis of the Na^ /H^ antiporters showed that the HbNHXI gene shares 55.3%-74.8% similarity with the vacuolar-type Na^ /H^ antiporters. Transgenic tobaccos that contain the HbNHXI gene, integrated by forward insertion into the tobacco genome, were obtained via Agrobacterium tumerfaciens and characterized for the determination of the concentration of Na^ and K^ ions, as well as proline, in the presence of 300 mmol/L NaCl. The T1 transgenic plants showed more tolerance to salt and drought than did wild-type plants. Our data suggest that overexpression of the HbNHXI gene could improve the tolerance of transgenic tobaccos to salt and drought through the function of the vacuolar Na^ /H^ antiporter.     

Evidence for a Na+ / H+ electrogenic antiporter in an alkaliphilic cyanobacterium Synechocystis

Abstract An alkaliphilic cyanobacterium characterized as a Synechocystis species was purified from a soil sample taken from a village in Java, Indonesia, by its preferential growth at elevated pH; it grew optimally at pH 9.5. Phosphorus nuclear magnetic resonance studies showed that the organism can maintain a ΔpH of over 2 pH units at an external pH of 10. It was observed that the viability of the organism in the dark was dependent on sodium ions. Evidence from experiments in which the extrusion of Na⁺ was measured from cells subjected to an alkali shock suggests that the organism possesses a Na⁺ / H⁺ electrogenic antiporter which is used for the maintenance of pH homeostasis.     

Na+/H+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the salt-sensitive Plantago media

Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na⁺ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na⁺/H⁺ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na⁺/H⁺ antiport activity with saturation kinetics and a K_m of 2.4 m M Na⁺, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na⁺ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.     

Demonstration of a K+/H+ exchange activity in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

Abstract Washed cells of Rhodopseudomonas sphaeroides forma sp. denitrificans , grown under photodenitrifying conditions, exhibited K⁺ uptake dependent on the transmembrane proton gradient (Δ pH). These cells also acidified the suspension medium in response to K⁺ pulses both aerobically and anaerobically in light and in the dark. The results indicate that the photodenitrifier has a reversible K⁺/H⁺ exchange activity which reflects its role in regulating the intracellular K⁺ concentration, as well as intracellular pH. The acidification of the external medium resulting from K⁺ pulses was inhibited by carbonyl cyanide- m -chlorophenylhydrazone (CCCP) indicating that the antiporter is energy-dependent. Addition of KCl to washed cells depolarized the membrane potential (Δψ) with a concomitant increase in ΔpH, indicating that the K⁺/H⁺ antiporter was electrogenic.     

Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl

A tomato ( Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 m M NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 m M NaCl. Analysis of callus ion content showed a strong increase in Na⁺ and Cl⁻ both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H⁺ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H⁺ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H⁺-ATPase (EC 3.6.1.35) and ATP-dependent H⁺ transport, while the stimulation of H⁺ extrusion by cells tolerant to 50 m M NaCl was correlated with an increase in plasma membrane ATP-dependent H⁺ transport. The increase of ATP-dependent H⁺ extrusion in plasma membranes isolated from 50 m M NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H⁺. Relative to NaCl-sensitive calli, plasma membrane H⁺-ATPase from calli tolerant to 50 m M NaCl showed a lower K_m for Mg²⁺-ATP. Our results indicate that tolerance of tomato calli to 50 m M NaCl increases the affinity of plasma membrane H⁺-ATPase for the substrate ATP and stimulates the H⁺-pumping activity of this enzyme without modifying its phosphohydrolytic activity.     

Two Na^＋ and Cl^- Hyperaccumulators of the Chenopodiaceae

The authors found five sodium (Na^ ) and chloride (Cl^-) hyperaccumulating halophytes in the Temperate Desert of Xinjiang, China and studied two of them (Suaeda salsa (L.) Pall. and Kalidium folium (Pall.) Moq.). K. folium and S. salsa had a NaCl content of 32.1% and 29.8%, respectively, on a dry weight basis. X-ray microanalysis of the Na in the vacuole, apoplasts and cytoplasm of the two plants indicated a ratio of 7.3:5.6:1.0 in K. folium and 7.3:6.6:1.0 in S. salsa. These data show that K. folium and S. salsa both have a high Na and Cl^- accumulating capacity, which is related to high activity of tonoplast H^ -ATPase and H^ -PPase.     

Demonstration of an Na+/H+ exchanger in mouse keratinocytes measured by the novel pH-sensitive fluorochrome SNARF-calcein

In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na⁺/H⁺ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na⁺/H⁺ exchanger nor a proliferation-associated increase in intracellular pH (pH_i) has been demonstrated.
The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na⁺/H⁺ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pH_i measurements in BALB/MK cells. Initial measurements of the pH_i using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes.
Using SNARF-calcein we were able to show proliferation to be associated with increased pH_i. However, culture conditions were critical for these measurements. Our data indicate that the Na⁺/H⁺ exchanger is involved in this process, since acid load and pH_i-recovery experiments showed the alkalization to be amiloride-sensitive.     

The involvement of a vanadate-sensitive ATPase in plasma membranes of a salt tolerant cyanobacterium

Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na⁺ and Cl⁻, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg²⁺ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H⁺-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na⁺ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H⁺-pump, which is involved in extrusion of Na⁺ and in conferring resistance to salt stress.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

ɛ-(γ-Glutamyl)lysine cross-links of spore coat proteins and transglutaminase activity in Bacillus subtilis

Abstract The capacity to transport potassium and to discriminate between the different alkali cations has been found to affect sodium tolerance in Saccharomyces cerevisiae . Mutants with a defective capacity to transport K⁺ were more sensitive to high concentrations of Na⁺ because they accumulated more Na⁺ and less K⁺ than wild-type cells which showed high discrimination between K⁺ and Na⁺.     

Modified plant plasma membrane H+-ATPase with improved transport coupling efficiency identified by mutant selection in yeast

Transport across the plasma membrane is driven by an electrochemical gradient of H⁺ ions generated by the plasma membrane proton pump (H⁺-ATPase). Random mutants of Arabidopsis H⁺-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H⁺-ATPase (PMA1). A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1. A Trp-874-Phe substitution in AHA1 was produced by site-directed mutagenesis. The modified enzymes hydrolyzed ATP at 200–500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH. AHA1 did not contribute significantly to H⁺ extrusion by transformed yeast cells. The different species of aha1, however, displayed marked differences in initial rates of net H⁺ extrusion and in their ability to sustain an electrochemical H⁺ gradient. These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H⁺-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H⁺ pumping. Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plants transporters.     

Na+-driven ATP synthesis of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

Abstract: In vivo ATP synthesis of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1, derived from endogenous respiration, was examined. ATP was synthesized at both pH 6.5 and 8.5 after the start of the endogenous respiration by supplying O₂ to the anaerobic cell suspension. The ATP synthesis at pH 6.5, but not at pH 8.5, was completely inhibited by a H⁺ conductor, carbonylcyanide m -chlorophenylhydrazone (CCCP). The CCCP-resistant ATP synthesis at pH 8.5 was strongly inhibited by an inhibitor of the respiration-dependent primary Na⁺ pump, 2- n -heptyl-4-hydroxyquinoline N -oxide, and essentially required Na⁺. These results show that this bacterium synthesizes ATP at pH 6.5 by electrochemical potentials across the membrane Δ ∼ μ _H+, whereas at pH 8.5 by Δ ∼ μ _Na+ but not Δ ∼ μ _H+.     

Cation/proton antiporter complements of bacteria: why so large and diverse?

Most bacterial genomes have five to nine distinct genes predicted to encode transporters that exchange cytoplasmic Na⁺ and/or K⁺ for H⁺ from outside the cell, i.e. monovalent cation/proton antiporters. By contrast, pathogens that live primarily inside host cells usually possess zero to one such antiporter while other stress-exposed bacteria exhibit even higher numbers. The monovalent cation/proton antiporters encoded by these diverse genes fall into at least eight different transporter protein families based on sequence similarity. They enable bacteria to meet challenges of high or fluctuating pH, salt, temperature or osmolarity, but we lack explanations for why so many antiporters are needed and for the value added by specific antiporter types in specific settings. In this issue of Molecular Microbiology, analyses of the pH dependence of cytoplasmic [Na⁺], [K⁺], pH and transmembrane electrical potential in the 'poly extremophile' Natranaerobius thermophilus are the context for assessment of the catalytic properties of 12 predicted monovalent cation/proton antiporters in the genome of this thermophilic haloalkaliphile. The results provide a profile of adaptations of the poly extremophilic anaerobe, including a proposed role of cytoplasmic buffering capacity. They also provide new perspectives on two large monovalent cation/proton antiporter families, the NhaC and the cation/proton antiporter-3 antiporter families.