INTRODUCTION

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It is well established that bacteria conserve and transduce metabolic energy by means of an electrochemical gradient of hydrogen ions across the cytoplasmic membrane (µH⁺), in accordance with the chemiosmotic theory of Peter Mitchell (168-171). According to this theory, extrusion of protons via one primary transport system or another establishes a proton potential. A primary transport system, or primary pump, is defined as active transport directly linked to a metabolic reaction; examples include electron transport by a redox chain, a proton-translocating ATPase (Fig. 1), and a light-driven reaction such as the photosynthetic reaction center and bacteriorhodopsin (72-74). The electrochemical gradient of protons µH⁺ (proton potential, p) across the plasma membrane is the sum of two components, an electrical potential (, interior negative) and a pH gradient (pH, interior alkaline). The relationship of these parameters is described by p =   ZpH, where pH is the difference between the pH of the bulk medium and that of the cytosol and the factor Z is 2.303RT/F and is 59 mV at 25°C. The proton potential (proton motive force) can then be used by the cells to drive proton-linked energy-consuming processes. Most important, it is employed in the synthesis of ATP from ADP and inorganic phosphate by the F_OF₁-ATP synthase and in active transport by secondary transport systems which are not associated with a concurrent chemical reaction. Porters perform osmotic work by coupling the flux of one solute to that of another, for example protons. The linkage of coupled fluxes with the same direction in space is called symport, and the linkage of those with the opposite direction is called antiport (Fig. 1). Exergonic and endergonic reactions are thus coupled via the circulation of protons across the membrane (74, 78).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Chemiosmotic energy coupling. Electrogenic proton extrusion by the respiratory chain generates an electrochemical gradient of protons µH+ (proton potential), composed of a pH gradient (inside alkaline) and a membrane potential (inside negative). Proton flow into the cytoplasm via FOF1-ATP synthase energizes formation of ATP from ADP and inorganic phosphate (Pi) and, via cotransport systems, drives active uptake (symport) or extrusion (antiport) of various substrates (S).

The maintenance of a constant internal ion composition is indispensable to all living cells. Bacteria tend to maintain the cytoplasmic pH within a narrow range and to establish gradients of K⁺ and Na⁺ ions between their cytoplasm and the surrounding medium such that the cytoplasmic K⁺ concentration is higher than and the Na⁺ concentration is lower than that of the environment. It is accepted that secondary transport systems coupled to protons mediate the movements of K⁺ and Na⁺ ions. Proton movement across the membrane is the primary event not only for energy metabolism but also for performing this homeostatic work.

Microorganisms living in aquatic habitats are directly exposed to the outside world through a cell surface layer. Their habitats commonly encompass a wide range of physical conditions: oxygen, pH, salinity, temperature, light, etc. Bacteria that cannot cope with and survive in severe environments by depending on their H⁺-linked machinery alone have evolved a variety of ancillary energy conversion mechanisms. It is now recognized that Na⁺ ions supplement the role of protons in energy transduction across the bacterial membrane (154, 228). We know of diverse sodium pumps, such as (i) Na⁺-translocating membrane-bound decarboxylases in Klebsiella pneumoniae, Salmonella typhimurium, Veillonella alcalescens, Propionigenium modestum, etc. (45); (ii) Na⁺-translocating NADH oxidoreductase in various marine bacteria such as Vibrio alginolyticus (253); and (iii) the Na⁺-translocating ATPase in Enterococcus hirae, which is one of the topics of this review. All these generate an electrochemical gradient of sodium ions, which is used by the cells to drive secondary Na⁺-linked processes such as solute transport (122, 191, 254) and flagellar rotation (96, 101). In P. modestum, the Na⁺ gradient generated by the decarboxylation of organic acids is used for ATP synthesis by the Na⁺-ATPase (94, 155, 156). Alkaliphilic bacteria use an Na⁺ gradient as the driving force for solute transport and flagellar rotation at high pH (96, 101, 151, 152), when the proton concentration is too low. On the other hand, halorhodopsin functions as an electrogenic chloride pump in Halobacterium halobium, generating a membrane potential (221). These specialized energy-transducing systems are again very important for ion homeostasis in particular cases.

This article centers on cation transport in streptococci. The genus Streptococcus (70) is composed of gram-positive bacteria which occur as parasitic organisms in a wide variety of human, animal, and plant habitats (31). Streptococci are important in the dairy industry, as pathogens of animals and humans, and for their role in dental caries. Most are facultatively anaerobic, but some require additional carbon dioxide for growth and some are strict anaerobes. The metabolism of streptococci is fermentative, but nutritional requirements are complex and variable. The fundamental routes of energy metabolism run as follows. Glucose is taken up and phosphorylated to glucose-6-phosphate via the phosphoenolpyruvate-dependent phosphotransferase system (259); it is subsequently converted to pyruvate and finally to lactic acid by the glycolytic pathway. In these bacteria, which lack a respiratory chain (Fig. 1), ATP produced by substrate-level phosphorylation is hydrolyzed by the F_OF₁-ATPase with accompanying translocation of protons; the resulting proton potential is utilized for various proton-coupled transport reactions. It is noteworthy that among streptococci (Table 1), enterococci are particularly tolerant to external stresses including high temperature, high salt concentration, alkaline pH, and the presence of bile salts (70, 160). Lactococcus lactis is also known to be moderately tolerant to these factors. Facing harsh conditions such as high salinity and alkaline pH, enterococci and probably also L. lactis have evolved special energy conservation mechanisms for cation transport and homeostasis, which other streptococci may not have.

E. hirae (formerly Streptococcus faecalis), which is found in the intestine of higher animals, proved to be a useful system for unraveling the energetics of active transport, particularly the role of the F_OF₁-ATPase in chemiosmotic energy transduction (79, 80, 84-86; for reviews, see references 71 to 73). First, E. hirae, like other streptococci, lacks respiratory chains. It can generate a proton potential only by the hydrolysis of ATP, effected by the proton-translocating F_OF₁-ATPase. Second, its simple metabolic pathways allow the precise calculation of ATP yields from the few compounds that it can metabolize, such as glucose and arginine. Third, the cells are easily depleted of energy, because the organism does not make energy reserve polymers. Finally, like other gram-positive organisms, it is sensitive to ionophores and inhibitors that act on the cell membrane. Because of these advantages, research related to inorganic cation transport processes has been carried out primarily with enterococci, although work on other species such as L. lactis (formerly Streptococcus lactis and Streptococcus cremoris) has recently begun to be published. In the past several years, a number of genes encoding transport proteins for cations have been isolated from streptococci, allowing characterization of these transport systems at the molecular level. One of the major findings with E. hirae and other streptococci is that ATP plays a much more important role in membrane transport than it does in nonfermentative organisms; streptococci are unable to generate a large proton potential because they lack respiratory chains (77). Streptococci cope with their limited proton potential by expressing a variety of primary transport systems. In this article, I review chiefly the recent developments in cation transport by E. hirae and supplement this information with what is known of other streptococci.

CATION TRANSPORT SYSTEMS

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Proton ATPase

The F_OF₁-ATPase is widely distributed in bacterial cell membranes. At the level of quaternary structure, the bacterial F_OF₁-ATPase is essentially identical to the ATPases of the inner membrane of mitochondria and of photosynthetic organelles in eukaryotic cells (36, 57, 105, 222). Its importance in membrane bioenergetics continues to be emphasized, although its universal distribution in bacterial membranes has been disproven; phylogenetically related proton ATPases of the vacuolar type replace F_OF₁-ATPase in archaebacteria (217) and in one eubacterium (268) (described below). All F_OF₁-ATPases are considered to be reversible, but physiologically the enzyme operates mainly or solely in one direction or the other. In mitochondria and respiring bacteria, the enzyme functions as an ATP synthase, mediating oxidative phosphorylation energized by the electrochemical gradient of protons (proton potential) via the respiratory chain. More than two decades ago, it was reported that some streptococci synthesize a cytochrome-like respiratory chain when the medium is supplemented with haematin (212); formation of ATP from ADP and inorganic phosphate, coupled to NADH oxidation, in cell extracts was also observed (33, 202). However, in most cases the streptococcal F_OF₁-ATPase does not function as ATP synthase, because of the lack of a functional H⁺-linked electron transport system (71, 75). In this organism, the F_OF₁-ATPase functions as a hydrolase and proton movements coupled to ATP hydrolysis are used for the generation of the proton potential (42, 84).

The E. hirae enzyme is of special interest because it was the first of the bacterial membrane ATPases to be discovered (9). The physiological role of streptococcal F_OF₁-ATPase is to alkalinize the cytoplasmic pH in the acidic pH range and to establish a proton potential as the driving force for a variety of secondary H⁺-linked transport systems and for H⁺-linked flagellar rotation in motile streptococci (165).

Molecular structure and genes. As early as 1960, Abrams et al. (9) detected an ATP hydrolytic activity in E. hirae membranes. The enzyme purified from the membranes (4, 219) was initially reported to consist of 12 subunits (220). However, further biochemical examination of this enzyme established that E. hirae H⁺-ATPase belongs to the F_OF₁-ATPase synthase complex found in oxidative phosphorylation membranes (10-12, 158). The enzyme consists of two parts: F₁, the catalytic moiety, contains five subunits (, , , , and ), and the F_O membrane sector contains three subunits (a, b and c). A subunit stoichiometry for the F₁ moiety (:::: = 3:3:1:1:1), which is very similar in all F_OF₁-ATPase complexes of bacteria, is likely (3, 10-12, 158). The stoichiometry of the F_O subunits is uncertain. The F₁-ATPases in S. mutans, S. sanguis (244), and L. lactis subsp. cremoris (211) have been purified. All these purified ATPases are five-subunit enzymes with molecular sizes corresponding to those of other F₁-ATPases. Final proof that streptococcal H⁺-ATPase is an F_OF₁-ATPase was provided by obtaining the amino acid sequences of all these subunit molecules. First, a Russian group has chemically determined the amino acid sequence of the N,N'-dicyclohexylcarbodiimide (DCCD)-binding 7-kDa proteolipid extracted with chloroform-methanol from plasma membranes of E. hirae (148). This proteolipid, consisting of 71 amino acid residues, was considered to be the c subunit of the H⁺-ATPase because of the high degree of homology to the corresponding c subunit of other bacterial F_OF₁-ATPases. Inactivation of H⁺-ATPase activity by DCCD is due to the covalent modification of Glu54 in the second membrane segment of this polypeptide chain.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Gene organization of the proton ATP synthase/ATPase in some bacteria. Boxes indicate the open reading frames of the operons of E. coli (59), E. hirae (223), S. mutans (229), and a portion of the S. pneumoniae operon (55); the letters in boxes represent subunit names for comparison. The genes encoding hydrophobic subunits are shaded.

Enzymatic properties. The basic catalytic properties of the streptococcal H⁺-ATPase were worked out first for E. hirae (for reviews, see references 7 and 8) and recently for other streptococci. Each one of these streptococcal enzymes has special features. It is well known that azide is a specific inhibitor of F₁-ATPase from various sources (60). The ATP hydrolytic activity of F₁-ATPase from S. mutans and L. lactis subsp. cremoris is inhibited by azide (211, 244). By contrast, the E. hirae enzyme is insensitive to azide (3). Although the sensitivity of F₁ to azide is not understood at the structural level (60), this feature is convenient to discriminate enterococci from many other streptococci and is exploited in the SF medium marketed by Difco.

Mechanism. The streptococcal H⁺-ATPase, like other ATP synthases, is experimentally reversible; enterococcal H⁺-ATPase (260) and L. lactis H⁺-ATPase (161-163) can synthesize ATP when a large proton potential (200 mV) is artificially imposed. Because of the lack of an energy-producing proton pump, such as an electron transport system, these enzymes do not synthesize ATP under physiological conditions. Ever since Mitchell proposed the chemiosmotic concept of energy coupling (168, 169), there has been vigorous debate over the linkage between proton flow and the ATPase reaction (Fig. 1). The molecular mechanism of F_OF₁-ATP synthase is not at issue in this review. However, it is worthwhile mentioning just briefly the recent fascinating progress in studies on the mechanism of the F_OF₁-ATPase synthase with its three catalytic sites in the F₁ portion (Fig. 3) (29). A widely accepted model for energy coupling by the ATP synthase, called the binding change mechanism, has two features: (i) the major energy-requiring step is not the synthesis of ATP at the catalytic site but, rather, its release from that site, and (ii) tight binding of substrates and the release of product occur simultaneously at separate but interacting sites. It is evident from the negative cooperativity of sequential nucleotide binding that the three catalytic sites are strongly coupled. These catalytic sites may cycle in concert through the reaction. The speculation is that the required binding change is coupled to proton transport by rotation of a complex of subunits extending thorough F_OF₁: "rotation catalysis" (Fig. 3) (30). Based on the high-resolution structure of bovine F₁ (2), which verifies that each catalytic site resides in a suitably different environment, this notion was strongly supported in biochemical (49) and spectroscopic (216) studies by independent groups. Very recently, Yoshida's group directly visualized rotation of the F₁ molecule (183); attachment of a fluorescent actin filament to the  subunit served as a marker, which enabled them to observe this motion directly. Thus, the F_OF₁-ATPase is a complex that may be considered a machine, and this is best emphasized by explicit analogy to electrical motors and chemical engines; the next exciting discovery will be in the F_O sector, i.e., how rotation within the F_O subunit is coupled to H⁺ flow.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Rotating model of the FOF1-ATPase. Three pairs of F1 generate three different nucleotide binding catalytic sites. Rotation of the central shaft, a composite of the  subunit with others in FOF1, is coupled to the opening and closing of the catalytic sites. A flux of H+ (or Na+) drives the rotation of the shaft, but rotation permits the ion to flow. This model also applies to vacuolar ATPase.

Physiological work. Generation of the proton potential is one of the major functions of the streptococcal F_OF₁-ATPase; this is subsequently utilized by means of secondary H⁺-coupled transport systems, in accord with the chemiosmotic viewpoint (71-75). The magnitude of the proton potential is affected by several factors: the proton conductance of the cell membrane and the permeability of the membrane to charged molecules and to ions. Furthermore, it is also affected by cellular activities related to growth. L. lactis cells glycolyzing in a buffer at pH 6 maintained a proton potential of 160 mV, while that of growing organisms was only 140 mV (128). The difference possibly results from the partial consumption of the proton potential by various H⁺-coupled transport systems. The maximum size of the proton potential generated in enterococci is almost 130 to 150 mV at acidic pH (144). With regard to the uptake of metabolites, if we assume symport of a metabolite with one proton, a proton potential of 180 mV would suffice to sustain a concentration gradient of 10³ while 240 mV would support a gradient of 10⁴ (214); physiological gradients of transport substrates fall within this range. On the other hand, at alkaline pH, the H⁺-ATPase activity is minimal and the proton potential is too low for adequate accumulation of solutes by H⁺-linked porters (110). This leads one to expect the evolution of various primary transport pumps to supplement the H⁺-linked secondary ones.

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Growth rates of E. hirae strains as a function of medium pH. Cells were grown in medium KTY (high K+, low Na+) (A) and medium NaTY (high Na+, low K+) (B). Symbols: , strain 9790 (wild type); , strain AS25 (proton ATPase mutant); , strain Nak1 (sodium ATPase mutant); , strain 9790 in the presence of gramicidin D.

Bacteria actively extrude sodium ions and maintain the electrochemical concentration gradient of sodium directed inward. The significance of the sodium gradient in bacteria is well known (73, 74, 154, 228); the sodium current is frequently linked with cotransport systems (191) and can serve as the driving force for flagellar rotation (101). In marine bacteria, a variety of transport systems are linked to Na⁺ rather than H⁺ (122, 254); in this case, the sodium circulation is primary rather than supplemental to the proton circulation. The mechanism of sodium extrusion is generally thought to be secondary antiport of sodium ions for protons, energized by the proton potential, just as Mitchell envisaged (71, 72, 169, 171). However, the activity of the Na⁺/H⁺ antiporter is supplemented by a variety of primary transport systems energized by ATP hydrolysis, redox potential, or decarboxylation. Sodium transport in streptococci has been extensively studied in E. hirae (241). In this bacterium, the investigation of sodium transport grew out of Mitchell's antiport hypothesis but now illustrates the interplay between the primary and secondary modes of energy-linked transport (77).

Na⁺/H⁺ antiporter. Harold and his colleagues contributed fundamental information on Na⁺ transport in E. hirae. Early studies on Na⁺ transport in E. hirae were interpreted as providing support for a Na⁺/H⁺ antiporter driven by the proton potential. Sodium extrusion from the cells against the Na⁺ gradient was blocked by DCCD, an inhibitor of the proton-translocating F_OF₁-ATPase, suggesting that sodium efflux requires a proton potential. Furthermore, H⁺ influx accompanying Na⁺ efflux was observed in alkalinized Na⁺-loaded cells metabolizing in Na⁺-free buffer. In this experiment, the driving force for Na⁺ efflux was presumably the Na⁺ gradient directed outward, since DCCD prevented establishment of the proton potential (80). These findings provided the first confirmation of the proposal that bacteria contain an Na⁺/H⁺ antiporter (264). For some years, this antiporter activity was held to be an artifact connected with the newly discovered Na⁺-translocating ATPase (for details, see references 88 and 114). Briefly, the hypothesis was that the Na⁺ pump of E. hirae catalyzes an ATP-driven exchange of H⁺ for Na⁺. The antiporter activity, visible only in membrane vesicles or in a mutant deficient in the Na⁺ pump, was considered to be the antiporter moiety of a modular ATP-driven exchanger of Na⁺ for H⁺ (83, 89, 91). This interpretation, however, proved incorrect. In the wild-type strain cultured on Na⁺-limited medium, in which the Na⁺-inducible Na⁺-ATPase level was minimal, the Na⁺/H⁺ antiport activity was clearly observed (108). In response to an artificially imposed pH gradient (with exterior acid), energy-depleted cells exhibited a transient sodium extrusion which was unaffected by treatments that dissipated the membrane potential but was blocked by proton conductors. One must conclude that E. hirae has two separate Na⁺ extrusion systems: an Na⁺-ATPase and an Na⁺/H antiporter.

(i) Discovery. Although most of the early data on Na⁺ extrusion by E. hirae cells fit the sodium/proton antiport model, there was one important observation which could not be easily explained by this model: net Na⁺ movement and ²²Na⁺-Na⁺ exchange were seen only in cells capable of generating ATP (80). The search for an answer to this question led to the discovery of the sodium-translocating ATPase in E. hirae. Sodium extrusion against a concentration gradient, under conditions such that the proton potential has been totally dissipated by the presence of DCCD or protonophores and valinomycin, was readily induced by the addition of glucose or arginine. Since the energy donor common to the metabolism of glucose and arginine is ATP, Na⁺ extrusion was attributed to an ATP-driven Na⁺ pump (89). ATP-driven ²²Na⁺ uptake and Na⁺-stimulated ATP hydrolysis were observed in everted membrane vesicles in the presence of DCCD and the ionophores (90). No Na⁺-pumping activity was detected in vesicles of mutant 7683 (83); this mutant is now considered to be a double mutant defective in both Na⁺-ATPase and the Na⁺/H⁺ antiporter (114, 232). All these results suggest that a Na⁺-translocating ATPase exists in the cell membrane of E. hirae, which was the first bacterium in which a Na⁺-ATPase was discovered. Na⁺ movements unconnected to a proton potential have recently been reported in S. bovis (241); an Na⁺-ATPase is likely to be responsible.

(ii) The sodium pump is a V-ATPase. Ion-motive ATPases are divided into two categories: one which forms phosphorylated intermediates (E-P enzyme; P-ATPase) and the other which does not. P-ATPases are exemplified by the Na⁺,K⁺-ATPase, H⁺,K⁺-ATPase and Ca²⁺-ATPase of higher organisms and by a variety of ion-translocating ATPases in bacteria (194, 195). The ATPases which do not form E-P intermediates are now divided into two types: F_OF₁-ATPase (F-ATPase) and vacuolar ATPase (V-ATPase). F-ATPase functions as an ATP synthase in oxidative bacterial membranes and as the proton pump in the membranes of fermentative bacteria such as streptococci. On the other hand, the V-ATPase is known as the proton pump of acidic organelles, such as the vacuoles of fungi and plants and various endosomes of animal cells (58, 180, 181). Archaebacteria contain a V-ATPase, which is believed to mediate ATP synthesis (217). Both ATPases are quite similar multisubunit enzymes consisting of a hydrophilic catalytic portion (F₁ and V₁, respectively) and a membrane-embedded portion (F_O and V_O). The proteolipid of the membrane sector, which contains a DCCD-reactive acidic amino acid residue, is thought to be the pathway by which protons cross the membrane. However, a stretch of the sequence (about 90 amino acid residues) is commonly conserved in the N-terminal region of the V-ATPase A subunit but is not found in the sequence of the  subunit of E. coli F-ATPase. The size of the eukaryotic V-ATPase proteolipid is generally 16 to 17 kDa and is thought to have arisen by tandem duplication of the 7- to 8-kDa c subunit gene of the F-ATPase (164). The high homology between the amino acid sequences of several major subunits of these ATPases suggests a close evolutionary relationship between them (66, 164, 181, 182). The V-ATPase has been called a "big sister" of the F-ATPase (14).

View larger version (89K): [in this window] [in a new window]   FIG. 5.   Polyacrylamide gel electrophoresis of EDTA extracts of E. hirae membranes. EDTA extracts were prepared from membrane vesicles of E. hirae strains cultured in various media. The Na+-ATPase activities of each of the vesicles are shown underneath. Lanes: 1, 9790 (wild type) in low Na+; 2, 9790 in high Na+; 3, 9790 in high Na+ at high pH; 4, Nak1 (Na+-ATPase mutant) in high Na+; 5, revertant of Nak1 in high Na+. Reprinted from reference 117 with permission of the publisher.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Organization of the ntp operon and subunit structure of the E. hirae Na+-ATPase. (A) Gene organization. The arrow indicates transcriptional direction. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ATPase at different concentrations of gel. Reprinted from reference 177 with permission of the publisher.

View larger version (21K): [in this window] [in a new window]   FIG. 7.   Na+ uptake into proteoliposomes reconstituted with Na+-ATPase. Uptake was started by the addition of 5 mM ATP at 0 min. Inhibitors or ionophores were added at 10 min. (A) Symbols: , no ATP; , ATP; , ATP plus 50 mM KNO3; , ATP plus 50 µM destruxin B. (B) Symbols: , ATP; , ATP plus 25 µM valinomycin; , ATP plus 25 µM CCCP; , ATP plus 25 µM monensin. Reprinted from reference 177 with permission of the publisher.

(iii) Catalytic properties. The activity of E. hirae Na⁺-ATPase is maximal at pH 8.5 to 9.0 but not detectable at pH 6.0; the pH profile of ATPase activity fits its importance to the physiology of this bacterium at alkaline pH, as described below. This ATPase is stimulated by Na⁺ or Li⁺ ions but not by K⁺ and Ca²⁺ ions. The ATP hydrolytic activity of the purified V_OV₁ enzyme absolutely requires Na⁺; the kinetics of ATP hydrolysis showed at least two different affinities for Na⁺ (K_m values of 20 and 4 mM), and probably one more. These different Na⁺ affinities of the ATPase are definitely linked to its mechanism, but their meaning remains unsolved. The affinity of this enzyme for ATP is 0.5 mM for both the purified enzyme and the membrane-bound form (115, 177).

(iv) Mechanism. There must be common principles in the energy-transducing machinery of V-ATPase and F-ATPase molecules. It is likely that in both cases, energy transfer from ATP hydrolysis calls for three catalytic sites on the V₁ moiety and an ion-translocating pathway through the V_O proteolipid (133). The rotation catalysis mechanism, experimentally verified for the F-ATPase, is probably applicable to the V-ATPase (Fig. 3). Which subunit of the V-ATPase does rotate, as does the  subunit of F-ATPase? The most likely candidate is the D subunit, even though its sequence similarity to  is minimal. The reason is that the D subunit makes up the core of the V₁ complex, together with A and B subunits, in purified V₁ (119, 268).

(v) Linkage with K⁺ movement. Before finding the structural information on the Na⁺-ATPase by gene cloning, Harold and I suggested that this ATPase may be a Na⁺(K⁺)-ATPase (112). Potassium uptake via the KtrII K⁺ transport system is somehow linked to the Na⁺-ATPase, which, in intact cells, expels Na⁺ ions by exchange for K⁺. Moreover, the ntpJ gene, which is known to encode the K⁺ uptake capacity of the transport system, is cotranscribed with other subunits of the Na⁺-ATPase (Fig. 6A) (178). However, the NtpJ protein is separable from the Na⁺-ATPase complex by centrifugation of the solubilized fraction (177). Moreover, strain JEM2, in which the ntpJ gene has been disrupted (and which lacked K⁺ uptake), still contained normal Na⁺-ATPase (176). It therefore appears that the KtrII K⁺ uptake system is not mechanically linked with the Na⁺-ATPase complex; the K⁺/Na⁺ exchange model of this enzyme should be considered withdrawn.

(vi) Inducibility. In 1984, Kinoshita et al. (136) reported that the Na⁺-ATPase level of E. hirae is not constant. They measured the Na⁺-stimulated ATPase in mutant AS25, deficient in the H⁺-ATPase, and observed much higher levels of Na⁺-ATPase in this mutant than those found by Heefner and Harold (90) in the wild type. Moreover, both the rate of sodium transport by intact cells and the activity of Na⁺-ATPase in vesicles were altered by the culture conditions. Mutant cells grown in high-Na⁺ (0.12 M) media exhibited high activities, while those grown in low-Na⁺ (5 to 10 mM) media exhibited much lower enzymatic and transport activities. Sodium transoort and Na⁺-ATPase activity in the wild-type strain were much lower than those in the mutant strain. When the wild-type cells were grown in the presence of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), both Na⁺ transport and Na⁺-ATPase activity were elevated. Furthermore, when the wild-type cells were grown at alkaline pH, both activities increased significantly (115). The sodium ATPase is thus induced when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential, indicating that an increase in the cytoplasmic sodium level serves as the signal. Western blotting and Northern blotting experiments revealed substantial correlation between the amount of this enzyme and expression of the ntp operon (178). Even when the cells were grown on low-Na⁺ medium, the ionophores monensin and gramicidin D, which render the membrane permeable to Na⁺, significantly increased the amounts of Na⁺-ATPase and of the mRNAs for the operon (178). All these data are explained by the hypothesis that the Na⁺-ATPase is induced at the transcriptional level by an increase in the cytoplasmic Na⁺ concentration. In E. coli, transcriptional regulation of the nhaA Na⁺/H⁺ antiporter gene, responding to Na⁺ and Li⁺ ions (193), is achieved by the NhaR regulatory protein, which is a member of the LysR protein family (203). The next step for understanding the regulation of the ntp operon, in concert with that of the napA antiporter gene (238), is to discover the system that senses the Na⁺ concentration.

(vii) Comparison with other Na⁺-ATPases. Whether a vacuolar-type Na⁺-ATPase occurs in other streptococci is not clear. Based on their tolerance to salinity and alkaline pH, some related streptococci such as L. lactis and S. bovis appear to possess a Na⁺-ATPase, although it may be less prominent. We have already found that everted membrane vesicles of L. lactis contain a Na⁺-stimulated ATPase; reactions with antibodies directed against components of the E. hirae enzyme suggest the presence of homologs to NtpA and NtpB (124). An Na⁺-translocating ATPase, recently discovered in the thermophilic Clostridium fervidus (237), is very likely to be of the vacuolar type as judged by the enzymatic and biochemical properties of the purified enzyme (97). Furthermore, the enzymatic features of an Na⁺-stimulated-ATPase observed in Mycoplasma mycoides (a parasitic glycolytic organism) are virtually identical to those of the E. hirae enzyme (25), and an Na⁺-stimulated ATPase from Acholeplasma laidlawii seems to be of the V type as judged by its subunit composition (104, 159).

(viii) Distribution and evolution of V-ATPase. In the prokaryotic world, V-ATPases were first found in archaebacteria but are now known to be distributed among eubacteria such as E. hirae and Thermus thermophilus (268). Sequencing and characterization of the genes encoding the H⁺-translocating V-ATPases of other bacteria (266) suggest that all these enzymes consist of nine subunits, identical to the number for the E. hirae Na⁺-ATPase. We have found that nine open reading frames in the genome of Methanococcus jannaschii (35) correspond to individual ntp genes for the E. hirae Na⁺-ATPase subunits (248, 249), although the ion specificity of this putative V-ATPase is unknown. In these V-ATPase clusters, the order of the subunit genes is almost always the same. The Sulfolobus acidocaldarius V-ATPase operon is slightly different; the operon consists of only six genes (43). Since the whole subunit structure of Sulfolobus ATPase is unsettled, unidentified subunits may be encoded on separate genes.

Pathway of Na⁺ entry. At least one route for Na⁺ entry must exist, since a Na⁺ circulation is important for the growth of streptococci. The nature of this pathway is still unknown. The limited data at hand suggest that the Na⁺/H⁺ antiporter mentioned above may allow Na⁺ ions to enter cells whose membrane potential has been collapsed (108, 114). A different pathway was observed by Heefner and Harold (89), who showed that when a membrane potential (inside negative) was imposed by K⁺ efflux, the cells took up Na⁺ ions by exchange for K⁺. The process involved has low affinity but high capacity (K_m > 20 mM; V_max > 50 nmol/min/mg of cells) and apparently responds to both the concentration gradient and the electrical potential. Because of its low affinity, we suspect that this pathway is relatively nonspecific and reflects some kind of leakage down the electrochemical potential gradient.

Physiological work. Like all other living cells, streptococci exclude Na⁺ ions and accumulate K⁺ ions. There is no simple answer to the question why cells expend energy on generating this ion gradient. Generation of a sodium gradient is an essential aspect of any sodium circulation. The reason for the obligatory role of sodium circulation in streptococci is not evident, except for the accumulation of K⁺ at alkaline pH, which is somehow linked to Na⁺ extrusion. Na⁺-dependent secondary transport has been reported only for S. bovis (215). There is also a body of evidence to suggest that a high Na⁺ concentration in the cytoplasm is generally inhibitory to cell physiology, although specific targets have not been identified (83). On the other hand, it has long been known that K⁺ ions are the major cations specifically required for protein synthesis.

Potassium is the major intracellular cation for all living cells from animals to microorganisms. Intracellular K⁺ concentrations are kept in the range of 0.1 to 0.5 M in E. coli and Salmonella typhimurium and 0.4 to 0.6 M in E. hirae; the K⁺ concentrations remain high even when extracellular concentrations drop to starvation levels. Maintaining the intracellular K⁺ level requires several parallel potassium transport systems (17). Investigation of bacterial K⁺ transport is most advanced in E. coli and is founded on powerful genetics and molecular biology. A number of separate K⁺ transport systems are thought to occur in E. coli (51); the two three-component TrkG and TrkH systems and the one-component Kup system are constitutively expressed, while the three-component Kdp-ATPase system is derepressed under conditions of K⁺ starvation or osmotic upshock (153). In addition, there are two transport pathways for K⁺ efflux in E. coli: KefB and KefC (20, 56). These efflux systems are regulated by glutathione; whether they operate as porters or channels is currently being examined.

E. hirae possesses two K⁺ uptake systems, KtrI and KtrII, and one extrusion system, here designated Kep (K⁺ extrusion pump). KtrI appears similar to the Trk system of E. coli, but the other two systems show unique features not seen in other bacteria. The molecular characterization of these K⁺ transport systems in enterococci has just been initiated.

KtrI. The KtrI system is the major potassium uptake pathway under most conditions; it has a pH optimum near 7 and an apparent K_m of about 0.2 mM, and it attains a maximal rate of about 70 nmol of K⁺ min¹ mg of cells¹. This rate is comparable to the rate of glycolysis (100 to 150 nmol of lactate min¹ mg of cells¹), and under certain conditions cells do take up K⁺ at a rate that approaches their overall metabolic rate. KtrI also transports Rb⁺ with kinetics similar to those of K⁺, and it appears to be constitutive. An early observation that the rates of K⁺ and Rb⁺ uptake via this system increased in E. hirae grown on K⁺-deficient medium may reflect an increased activity of the F-type H⁺-ATPase in such a medium, as described above (6). There is no clear evidence for activation of KtrI activity by a change in turgor pressure or in the osmotic pressure of the medium.

KtrII (NtpJ). A second uptake system for K⁺ in E. hirae, one that is not dependent on the proton potential, was discovered by Kobayashi (139) during studies of mutant AS25, which is defective in the H⁺-ATPase and the generation of the proton potential. The small proton potential in this mutant should not support high activity of KtrI, yet the cells did accumulate K⁺ 5,000-fold. This proton potential-independent uptake system, KtrII, has a pH optimum around 9, does not transport Rb⁺ very well, and has a K_m for K⁺