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⁺ of 0.5 mM and a V_max of about 20 nmol of K⁺ min¹ mg of cell¹. KtrII activity is inducible, but it does not respond to K⁺ deprivation, nor is it repressed by excess K⁺; KtrII is synthesized in response to the need of the cell to expel Na⁺, because its activity is induced to a high level when mutant AS25 is grown in sodium-rich media. In the wild type, high levels of KtrII are induced when the cells are grown in sodium-rich media supplemented with a protonophore. Thus, the behavior of KtrII appears to be quite unlike that of Kdp, the K⁺-translocating ATPase of E. coli. KtrII cannot scavenge trace amounts of K⁺ from the medium, but it does permit the cells to grow under conditions that render KtrI inoperative.

View larger version (18K): [in this window] [in a new window]   FIG. 8.   KtrII K+ uptake activities of E. hirae strains. Movements of K+ (, ) and Na+ (, ) at pH 9.0 were assayed with Na+-loaded cells in the presence of protonophore; the reaction was initiated by the addition of 1 mM KCl at 10 min as indicated by the arrows. Solid symbols, 10 mM glucose added; open symbols, no glucose. (A) 9790 (parent). (B) Nak1 (sodium ATPase mutant). (C) JEM2 (mutant in which the ntpJ gene is disrupted by insertion of erythromycin resistance gene). Reprinted from reference 176 with permission of the publisher.

Potassium extrusion. K⁺ efflux in streptococci and its relation to K⁺ uptake are not yet fully understood. NEM-sensitive downhill K⁺ extrusion, analogous to the Kef system of E. coli, has been observed in several bacteria but not in streptococci or enterococci (46). Passive K⁺ efflux seen in the presence of uncouplers requires ATP (or some other energy metabolite) (18), but whether such efflux occurs through KtrI (a putative ATP-regulated K⁺/H⁺ symporter) or by some other pathways is not known. The 325B mutant, originally isolated as a strain that requires elevated concentrations of K⁺ for growth, is defective in K⁺ retention (87). The normal kinetics of K⁺ influx in this mutant suggest that the lesion affects a separate system, probably one that mediates K⁺ efflux.

View larger version (18K): [in this window] [in a new window]   FIG. 9.   Effect of diethanolamine on uphill K+ extrusion and the internal pH of E. hirae. The K+ movement (A) and the internal pH change (B) of the cells at pH 9.0 were monitored in a high-K+ buffer (250 mM K+). Solid symbols, glucose added; open symbols, no glucose. The reaction was initiated by addition of diethanolamine as indicated by the arrows. Symbols: , no addition; , 20 mM diethanolamine; , 50 mM diethanolamine; , control (in a buffer containing 50 mM diethanolamine but not glucose). Reprinted from reference 113 with permission of the publisher.

Additional potassium transport systems. Another active-transport system with low affinity and high capacity participates in K⁺ accumulation at alkaline pH. The KtrII-defective strain JEM2, in which the ntpJ gene is disrupted, did not grow at alkaline pH in K⁺-limited medium but grew well in the presence of 5 to 10 mM K⁺. Even though KtrII is inoperative, about a 20-fold concentration gradient of K⁺ is generated in this strain at alkaline pH (176). This system also recognizes Rb⁺. A novel mutant, distinct from KtrII, defective in K⁺ accumulation at alkaline pH has been isolated. The level of K⁺ accumulation in this strain is about half that in the wild type. The characteristics of this K⁺(Rb⁺) transport system, which apparently responds to the membrane potential, are under investigation (135). Potassium transport has not been extensively studied in other streptococci, but there is a report of Na⁺-dependent uptake of thallous ions (an analog of K⁺) by L. lactis (127).

Physiological work. One of the major roles of K⁺ is charge neutralization of cellular anions. One might think that other cations, such as Na⁺ or Mg²⁺, could neutralize cellular anions equally well, but in fact cells loaded with Na⁺ glycolyze and transport substrate normally but do not grow (83). The choice of K⁺ over other cations as the main intracellular cation must have some deep evolutionary reasons, which have never been quite resolved. It is generally agreed that K⁺ ions make a more "compatible" solute than Na⁺ ions, in the sense that the former are less destructive to cellular macromolecules and their associated water shells (78, 265). Indeed, Rb⁺ can completely replace K⁺ in growing cells of E. hirae. Some bacterial enzymes are activated by K⁺, but all bacteria require K⁺ for protein synthesis. A high intracellular K⁺ concentration may be required for polysome stability (50). Another important role of K⁺ transport is the regulation of the cytoplasmic pH, which is discussed below. In E. coli, turgor pressure is maintained in part by regulating intracellular K⁺ (17, 51), and the same may be true for plant cells, but it does not appear to be the case in enterococci. In E. hirae, for instance, K⁺ is the major cellular osmolite, but there is no evidence that K⁺ transport is altered by changes in cell turgor. Finally, the relatively massive K⁺ gradient directed outward (and the Na⁺ gradient directed inward) constitute a kind of energy storage that can, if necessary, drive motility and transport (228). This option may well be an extra benefit that makes the difference under some conditions.

Calcium extrusion. Calcium transport was first investigated in E. hirae. Using both intact cells and everted membrane vesicles, Kobayashi et al. demonstrated that E. hirae extrudes Ca²⁺ by a primary active transport system (146). Calcium is readily expelled from the cells in the absence of a detectable proton potential, establishing a gradient of 30:1 (out/in). In everted membrane vesicles, ATP-driven Ca²⁺ accumulation was observed in the absence of a proton potential. Autologous ⁴⁵Ca²⁺/Ca²⁺ exchange is demonstrable in both intact cells and vesicles, and it requires ATP. The K_m for the uptake activity by the vesicles varies with the pH: at the optimal pH of 7.0, the K_m is 0.1 mM, and at pH 6.0 it increases to 1.0 mM. Ca²⁺ transport is not significantly inhibited by ruthenium red, NEM, or lanthanum. The effect of ionophores on Ca²⁺ transport suggests that the process is electroneutral; probably two H⁺ ions are exchanged for each Ca²⁺ ion. Ca²⁺ transport, linked to ATP but not the proton potential, was also observed in L. lactis and S. sanguis (13, 98, 213). In all these streptococci, ATP-driven ⁴⁵Ca²⁺ uptake is inhibited by vanadate, an inhibitor of the P-ATPase, suggesting that transport is mediated by a Ca²⁺-translocating ATPase. However, Ca²⁺-stimulated ATP hydrolysis by the membranes has not been detected.

Calcium influx. No Ca²⁺-specific uptake system has been found in E. hirae. When a membrane potential, interior negative, was present, ⁴⁵Ca²⁺ uptake was proportional to the external Ca²⁺ concentration over a range of 0.1 to 10 mM (146). This potential-driven Ca²⁺ uptake probably occurs via a nonspecific leak pathway. On the other hand, an electrogenic ⁴⁵Ca²⁺ influx in ATP-depleted S. pneumoniae is sensitive to the amiloride derivative DMB at 4 µM (255, 257); in this case, Ca²⁺ movement may be mediated by a specific porter.

Physiological work. Calcium ions have often been designated a second messenger in eukaryotes (37). In prokaryotes, Ca²⁺ is also involved in a variety of cellular processes: proteolysis in sporulation of Bacillus subtilis (188), chemotaxis in E. coli (252), etc. Ca²⁺ ions activate autophosphorylation of the heat shock protein DnaK (38) and of EnvZ (206). In S. pneumoniae, about 0.15 mM calcium is required for cell growth (255, 256). When Ca²⁺ transport was inhibited by DMB at high concentrations of external Ca²⁺ (1 mM), the growth of S. pneumoniae ceased. Homeostasis of the internal calcium ion level should be important for bacterial physiology. Calcium transport systems are expected to do this work, although the details of their role are not obvious. Furthermore, high concentrations of Ca²⁺ ions induce competence of S. pneumoniae for genetic transformation in exponentially growing cultures and lysis in early stationary phase. It is also proposed that DNA uptake at competence constitutes a homeostatic response to elevated Ca²⁺ levels (255). Competence induction and autolysis are prevented by inhibition of Ca²⁺ transport with the amiloride derivative. On the other hand, E. hirae grows well in a defined medium in which the free Ca²⁺ concentration is less than 10⁸ to 10⁹ M, and no physiological role of calcium has ever been proposed.

The uptake of magnesium, a major cation in cellular physiology, has been examined in S. typhimurium and other bacteria (230, 231, 250) but not in streptococci. Trace cations are universally required as cofactors in cellular metabolism, but their uptake has not been well studied in streptococci; a few papers have suggested the existence of transport systems specific for Mn²⁺ (22) and Fe³⁺ (53). Recently, a systematic study of copper transport by E. hirae was begun by Solioz and his colleagues.

Copper ATPase. Copper is an essential heavy metal ion that serves as a cofactor for many redox enzymes, such as cytochrome c oxidase, superoxide dismutase, lysyl oxidase, and dopamine -hydroxylase. On the other hand, copper can be very toxic to both prokaryotic and eukaryotic cells through formation of free radicals. Homeostatic mechanisms have evolved to strictly balance the internal copper concentration. Regulation of the cytoplasmic copper level appears to involve influx and efflux pathways in addition to the chemical modification of copper in the cytoplasm (32). Genes involved in the control of the cytoplasmic copper level have been identified in several bacteria (225, 227). E. coli, for example, has a number of chromosomal genes, as well as a plasmid-borne one, involved in copper resistance (226). These genes have already been sequenced, but their functions remain unclear (224). On the other hand, two kinds of copper transport ATPases have been reported as the machinery for copper homeostasis of E. hirae, one directed inward and the other directed outward (234, 236).

(i) Structure. The copper ATPase in E. hirae was discovered by accident. Solioz and coworkers (187) were attempting to clone the gene encoding a vanadate-sensitive ATPase (100), which they had tentatively identified as the KtrI K⁺ transport system, by use of an antibody raised against a sample of purified ATPase (235). Since this ATPase fraction was a relatively crude preparation, they inadvertently cloned a cop operon that does not code for KtrI but is involved in conferring copper homeostasis on E. hirae (186) (Fig. 10). The cop operon consists of four genes, copYZAB (186, 239). The copY (145 amino acid residues) and copZ (69 amino acid residues) genes encode hydrophilic proteins involved in the regulation of this operon; copA and copB encode ATPases with 727 and 745 amino acid residues, respectively. CopA and CopB exhibit significant sequence similarity and are also similar to other P-ATPases. Most prominently, they resemble the human Menkes gene product (261), a copper-ATPase, with 43% similarity to CopA and 33% similarity to CopB. The sequence AspLysThrGlyThr, considered to be the phosphorylation site in all known P-ATPases, is conserved in both CopA and CopB; the aspartic acid residue that forms the acyl phosphate intermediate characteristic of these enzymes is also found (267). While CopA and CopB display extensive overall sequence similarity, their N-terminal sequences are different. The sequence GlyXCysXXCys in the N-terminal region of CopA is similar to those of various metal binding proteins, such as MerA, the mercuric reductase of Thiobacillus ferrooxidans (102), CadA, the cadmium extrusion ATPase of Staphylococcus aureus (227), and the Menkes copper ATPase. The N terminus of CopB contains three repeats of the consensus sequence MetXHisXXMetSerGlyMet, which is very similar to repeats present in the Pseudomonas syringae pv. tomato CopA protein (166), known to be a periplasmic copper binding protein (40). These conserved sequences in CopA and CopB appear to represent the binding sites for heavy metal ions, responsible for its metal specificity.

View larger version (6K): [in this window] [in a new window]   FIG. 10.   Schematic representation of the E. hirae copper ATPase (cop) operon. Boxes indicate open reading frames of the cop operon, and letters represent the gene names: copA and copB encode P-type ATPases for uptake and extrusion of copper, respectively; copY and copX (shaded) encode regulator proteins sensing copper. The black box represents the promoter/operator region.

(ii) Physiological work. Although it has not been established that copper is indispensable for the growth of E. hirae, copper probably plays a role as cofactor for some metabolic enzymes as seen in other bacteria. The growth requirement of microorganisms for copper is satisfied by less than 10 µM copper (39), which is not toxic to E. hirae and does not induce the cop operon. The cytoplasmic copper concentration may therefore be in the micromolar range, and a K_m of 1 µM for copper transport by CopB (233) appears reasonable. It is interesting that the CopB ATPase transports ⁶⁴Cu⁺ in membrane vesicles. ^110mAg⁺ was also transported by CopB ATPase at a higher rate and lower affinity, suggesting that CopB recognizes these metals in the monovalent form. Cytoplasmic copper occurs in the monovalent form, either due to the reducing environment of the cytoplasm or through the action of reductases. From the finding that CopB ATPase transports silver (Ag⁺) in membrane vesicles, it is likely that CopB plays a role in the detoxification of silver. Energy-dependent efflux of silver ions is lost in the copB-disrupted strain (239). However, the growth of the copB mutant as well as the wild type was inhibited by 5 µM Ag⁺, suggesting a minimal contribution of CopB to silver detoxification in vivo. By contrast, the copA-disrupted strain tolerates considerably higher silver concentrations than the wild-type strain does (186). Silver may be accumulated by the cells via CopA, which has a broad substrate spectrum for several metal ions.

ENERGY TRANSDUCTION

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ATP Production

Sugar metabolism. Streptococci, lacking a respiratory chain, metabolize carbohydrate without net production of reducing equivalents. A case in point is the degradation of glucose to lactic acid, resulting in the production of 2 mol of ATP per mol of glucose metabolized. Some streptococci, however, have a limited capacity to utilize exogenous electron acceptors for the degradation of substrates. Carbohydrate metabolism in L. lactis and S. thermophilus is affected by the presence of oxygen. Under aerobic conditions, L. lactis converts lactose almost quantitatively to acetic acid plus carbon dioxide, thereby producing an excess of NADH (197). These streptococci contain flavin-type NADH oxidases and NADH peroxidases, which obviates the need to reduce pyruvate to lactate or to reduce acetyl coenzyme A to ethanol. By forming acetic acid instead of lactic acid and using an oxidase to reoxidize the NADH produced, additional ATP can be formed by substrate-level phosphorylation. The metabolic end products of glucose metabolism in some other streptococci are also affected by culture conditions; the amounts of acetate and ethanol as end products increased anaerobically at alkaline pH. The glycolytic metabolism of enterococci at alkaline pH is not the same as at acidic pH, switching to a more effective production of ATP in the presence of high concentrations of carbon dioxide (67, 68). The metabolic pattern is also altered under anaerobic conditions: if fumarate reductase is present, carbohydrate metabolism by streptococci may also yield acetic acid as an end product and thus result in extra ATP. In this scheme, electrons are transferred from NADH dehydrogenase to fumarate reductase. Although these components are present in L. lactis (93), the participation of this electron transfer chain in the oxidation of NADH under anaerobic conditions has not been demonstrated.

Deiminase pathway. In addition to ATP production by the glycolytic pathway, some streptococci generate ATP by metabolism of arginine or agmatine via the corresponding deiminase pathways (41). In the initial step of these reactions, arginine or agmatine is taken up via an antiporter which catalyzes a one-for-one exchange of arginine for ornithine (198) or of agmatine for putrescine (48). No additional energy is required for translocation of these solutes. Consequently, the net yield of metabolic energy from arginine and agmatine is 1 mol of ATP per mol of substrate metabolized.

Machinery. The generation of the proton potential in streptococci is clearly the function of the F-type, H⁺-ATPase; the mutant defective in H⁺-ATPase did not generate it. Some time ago, it was reported that certain streptococci have a cytochrome-like respiratory chain (212), but nobody has written of it since. On the other hand, certain combinations of secondary transport and metabolism may contribute to the proton potential. Lactic acid bacteria produce large quantities of cytoplasmic fermentation products, building up a considerable concentration gradient directed outward. Lactic acid and other metabolic end products commonly leave the cell by way of a transport carrier, together with one or more protons. When the gradient is large enough, it can contribute significantly to the total proton potential, conserving metabolic energy by transformation of a product gradient into a proton potential. In some streptococci, the lactate porter appears to be electrogenic, with a stoichiometry of 2H⁺ per lactate (251). Net efflux of lactate plus proteins contributes to the proton gradient and thus participates in the conversion of metabolic energy into that of an ion gradient. Another mechanism for the generation of a proton potential, without participation of any proton pump, has been proposed for L. lactis (201). In this pathway, L-malate taken up by the cells is decarboxylated by malolactic enzyme to yield L-lactic acid and carbon dioxide, after which the products leave the cells. Monoanionic L-malate is taken up either by exchange for L-lactic acid or by uniport; in consequence, a negative charge is translocated from the medium into the cytoplasm, generating a membrane potential inside negative. Since charge compensation in the decarboxylation reaction requires the consumption of a proton, the cytoplasm is alkalinized relative to the outside medium and a pH gradient is generated. Thus, the free energy of decarboxylation is converted into a pH gradient simply by compartmentalization of the metabolites. It has been shown for L. lactis that L-malate utilization results in the formation of a proton potential of about 175 mV, which is high enough to drive ATP synthesis via the F-ATPase. Whether the ion gradient thus generated actually does synthesize ATP via the F-ATPase is not known. In any event, the combined action of an electrogenic transport process (uptake of substrate and excretion of product) together with the decarboxylation reaction allows a significant portion of the free energy to be conserved in the form of ion gradient.

Magnitude of the proton potential. In microorganisms of sufficient size, especially the fungus Neurospora and the cells of many algae, the internal pH and the electrical potential can be measured directly by means of microelectrodes. Bacteria are too small for this procedure, and so indirect approaches must be used; these usually involve dyes that respond to pH or electrical potential. Most of the measurements presently available are based on the principle that any ion that moves passively across the membrane should be distributed between the cytoplasm and medium in accordance with the Nernst equation. The methods used are reasonably well standardized and have been reviewed in detail (27, 129). The membrane potential is usually measured by the accumulation of radioactive lipid-soluble ions, a cation when the potential is inside negative and an anion when it is inside positive; the pH gradient is calculated from the uptake of lipid-soluble weak acids or bases, e.g., benzoate or acetylsalicylic acid (aspirin) when the internal pH is more alkaline than that of the medium and methylamine or benzylamine when it is more acidic. Nuclear magnetic resonance spectroscopy provides the most accurate measurement of the internal pH presently available. In all cases, corrections have to be applied to the data, which limit the accuracy of the determination. The method of pH measurement in bacteria and its limitations have been reviewed (27, 129).

View larger version (13K): [in this window] [in a new window]   FIG. 11.   Internal pH, membrane potential, and proton potential of E. hirae at various external pHs. E. hirae was grown in KTY (high K+, low Na+) medium. Symbols: , internal pH; , membrane potential; , proton potential.

Transport systems for most amino acids are coupled to H⁺ (197). In L. lactis, branched-chain aliphatic amino acids (Leu, Ile, and Val), neutral amino acids (Ala and Gly), aliphatic amino acids with a hydroxyl side chain (Ser and Thr), and aromatic amino acids (Phe, Tyr, and Trp) are transported by separate H⁺-linked mechanisms. In E. hirae (16) and Streptococcus pyogenes (209), the neutral amino acids, glycine, alanine, serine and threonine, are transported by a common H⁺-linked system. Proton-coupled amino acid transport has also been proposed for S. thermophilus, S. agalactiae, and S. pneumoniae, usually on the basis of inhibitor effects (199). By contrast, ATP-driven uptake accounts for the accumulation of glutamic acid, glutamine, proline, and asparagine in several organisms (81, 200); all these amino acids serve as intracellular osmolytes, involved in the regulation of turgor. In L. lactis, di- and tripeptides are taken up by a pH gradient-driven transport system (69). Proton-linked cation transport is exemplified by the Na⁺/H⁺ antiporter and possibly by KtrI in E. hirae and by the Ca²⁺/H⁺ antiporter in L. lactis subsp. cremoris. Protons also serve as the coupling ions for flagellar rotation (165); motility was observed at acidic pH but not at alkaline pH, reflecting the size of proton potential at different pHs.

There are very few examples of sodium-dependent secondary-transport systems in streptococci. Sodium-dependent uptake of amino acids has been reported in S. bovis (215), and sodium-dependent uptake of K⁺ (Tl⁺) has been reported in L. lactis (127). There is no substantial evidence for any essential functions of this kind in enterococci. In E. hirae, an Na⁺ electrochemical gradient of as much as 110 mV can be generated under certain conditions, such as alkaline pH (114, 115). I doubt that organisms fail to utilize such a powerful energy source in their metabolic economy. The mechanism of KtrII, the NtpJ-dependent K⁺ uptake system, is still under investigation, but coupled movement of Na⁺ and K⁺ is very likely (115, 176). E. hirae shows no Na⁺ requirement for growth at neutral pH, if one ignores the contamination level of less than 1 mM Na⁺. At alkaline pH and with limited K⁺, the growth of E. hirae requires the presence of sodium ions in the medium (115). Further investigation of the Na⁺-coupled secondary systems is required to clarify the significance of the Na⁺ circulation (228) in streptococci.

ROLE OF CATION TRANSPORT IN HOMEOSTASIS

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Regulation of the Cytoplasmic pH

Regulation of the cytoplasmic pH is essential for the survival of bacteria, which must be able to accommodate variations of the environmental pH, metabolic reactions that produce or consume internal protons, and the movement of acids and bases across the plasma membrane. Cellular activities demand that the cytoplasmic pH be kept within relatively narrow limits. Homeostasis of cytoplasmic pH in bacteria was first demonstrated in streptococci by Harold et al. (84). Subsequently, there have been reports dealing with other bacteria (for reviews, see references 27 and 73). Of interest is the report that the internal pH of membrane vesicles prepared from E. coli remains constant even though such vesicles have no cytoplasmic metabolic activity (205). Over the years, various mechanisms have been proposed to explain how cells maintain a constant cytoplasmic pH. Half a century ago, Gale (61) proposed that amino acid decarboxylase is induced at alkaline pH and deaminase is induced at acidic pH. In Thermoplasma acidophila, pH regulation may depend on a Donnan potential and a plasma membrane permeable to cations (99). However, the common opinion today holds that the cytoplasmic pH in bacteria is regulated mainly by H⁺-linked transport systems located in membranes (for reviews, see references 27, 73, 151, and 152). In E. coli, the respiratory proton pump and H⁺-linked transport systems are clearly involved in pH homeostasis. Acidic and alkaline external pHs call for separate and distinct mechanisms to regulate the cytoplasmic pH. In E. hirae, the H⁺-ATPase plays a role in pH homeostasis at acidic external pH whereas an independent machinery operates at alkaline pH when the magnitude of proton potential is minimal.

Acidic pH. At acidic pH (external pH below 7), proton expulsion by the F_OF₁-ATPase alkalinizes the cytoplasmic pH and contributes to its control but is not sufficient by itself. Proton extrusion by the H⁺-ATPase is electrogenic, and hence a membrane potential is generated (79, 80, 84). Because the membrane capacitance is low (about 1 µF cm²), extrusion of a substantial quantity of protons requires dissipation of the membrane potential. Cytoplasmic alkalinization is accompanied by the electrogenic accumulation of potassium ions in E. hirae (19) and L. lactis (131, 132). The cytoplasm can also be alkalinized in the presence of potassium ions plus valinomycin, an ionophore for K⁺, or of dimethyldibenzyl ammonium ion (DDA⁺), a synthetic organic cation (79, 131). Cytoplasmic alkalinization can even be achieved by the accumulation of sodium ions in mutant 7683. Since this mutant is totally defective in Na⁺ extrusion, sodium ions probably accumulate via a leak pathway in response to the membrane potential (83). To keep the cytoplasmic pH constant, the activity responsible for cytoplasmic alkalinization must be high when the cytoplasm is acidic but must diminish as the cytoplasmic pH is raised. The mechanism appears to be simple. It is attributed to a feature of the H⁺-ATPase, which has an optimal pH at 6.5; the activity is very low at an alkaline pH (132, 145). The amount of functional H⁺-ATPase in membranes varies in response to the cytoplasmic pH, as described above. Cytoplasmic pH at acidic external pH is thus kept constant by changes in both the amount and activity of the H⁺-ATPase (141). Since the cytoplasmic pH of mutant AS25 deficient in the H⁺-ATPase was not excessively acidic at pH 7 (143), the cytoplasmic pH at this external pH is not a steady state of concurrent alkalinizing and acidifying mechanisms.

Alkaline pH. Nigericin is an ionophore that exchanges K⁺ for H⁺. Its effect on the growth of E. hirae depends on the pH: in high-K⁺ medium, growth was unaffected at pH 7 (82) but was blocked at pH 9.6 (109). This appears to reflect the regulation of cytoplasmic pH. Cells growing at alkaline pH (pH 9.0 to 9.5) acidify their cytoplasm to pH 7.8 to 8.0 (Fig. 11). In the presence of nigericin, the cytoplasmic pH is identical to the external pH, and pH 9.6 is inhibitory. When the pH of the medium was brought back to 7.0, growth of the cells resumed. Cytoplasmic acidification appears to be required for the growth of E. hirae in alkaline medium.

The distribution of various transport systems for H⁺, K⁺, and Na⁺ ions described in this article is fundamental to streptococcal physiology; by these transport systems, streptococci enlarge their natural habitat, as illustrated in Fig. 12C. Cells of E. hirae flourish in K⁺-deficient media over a broad range of environmental pHs (from 5 to 11), even in the presence of high NaCl concentrations. At acidic external pH, the proton potential generated by the H⁺-ATPase, although smaller than that of aerobes, suffices to drive K⁺ accumulation by KtrI (K⁺/H⁺ symporter), sodium expulsion by the Na⁺/H⁺ antiporter, flagellar motion, and other proton-linked transport systems. The cytoplasmic pH is alkalinized to 7.5 to 8.0 for optimal growth by the interplay of electrogenic H⁺ expulsion via the H⁺-ATPase and electrogenic K⁺ influx via KtrI, but as the cytoplasmic pH rises above the pH optimum of the F-ATPase (around pH 6.5), proton translocation by the ATPase will diminish.

View larger version (15K): [in this window] [in a new window]   FIG. 12.   Circulation of H+, Na+, and K+ in bacteria. (A) E. coli. The proton current is generated by the electrogenic extrusion of protons through the respiratory chain. Protons return to the cytosol by one primary system, FOF1-ATP synthase, and several secondary systems. Among these are Trk (K+ uptake), Nha Na+/H+ antiporters (Na+ extrusion), the flagellar motor, and various proton-linked metabolite porters. Some porters are coupled to the Na+ circulation; the K+ transport systems Kup and Kef are not shown. (B) Alkaliphilic bacteria. Ion movements are similar to those of E. coli. However, flagellar motion and many secondary-transport systems are energized by Na+ circulation; K+ transport has been little studied. (C) E. hirae. At acidic pH, the proton current is generated by the FOF1-ATPase and completed by several secondary pathways that perform useful work: KtrI (K+ uptake), NapA (Na+ extrusion), the flagellar motor, and various proton-linked transport systems. At alkaline pH, there is no obvious proton circulation. Ion transport relies on ATP-driven systems including the vacuolar Na+-ATPase, Kep (K+/H+ exchange), and K+ uptake via KtrII.

High sodium concentrations pose a problem for the cells. Sodium ions leak into the cytoplasm, collapsing the membrane potential, as reported for L. lactis (21). Under these conditions, electrogenic H⁺ expulsion by the H⁺-ATPase can be accelerated. However, since the activity of the H⁺-ATPase itself is limited by the internal pH, a decrease in the membrane potential cannot be totally compensated. The internal pH remains constant, the membrane potential decreases, and the proton potential is limited. Na⁺ extrusion via the antiporter is insufficient to suppress the increase in cytoplasmic Na⁺, and K⁺ uptake via KtrI, which depends on the proton potential, comes to a halt. For all these reasons, continued growth of the cells depends on the induction of the Na⁺-ATPase together with the KtrII K⁺ uptake system; the ntp operon is responsive to an increase in the intracellular Na⁺ (178). These two transport systems were even expressed in wild-type E. hirae cultured at acidic pH in high Na⁺ medium (110, 114, 115).

Expression of the napA Na⁺/H⁺ antiporter gene is also stimulated in high-Na⁺ medium (238). Even mutants in which the Na⁺-ATPase is defective grew normally in high-Na⁺ medium at acidic pH (116). Mutants defective in the napA gene, which lack the Na⁺/H⁺ antiporter, also grew normally in high-Na⁺ medium at acidic pH, presumably thanks to the induction of the Na⁺-ATPase (134). Thus, these two sodium extrusion systems cope cooperatively with a decrease in the proton potential by high Na⁺ ions.

Growth at alkaline pH poses an even graver problem than does excess Na⁺. When the external pH rises above 8, the proton potential is drastically decreased. Acidification of the cytoplasmic pH to pH 8.2 or less is indispensable for cell physiology; with the influx of protons, the proton potential across the cell membrane goes to zero and in some cases becomes positive. Proton-linked transport systems, such as KtrI and the flagellar motor, cease to function: the Na⁺/H⁺ antiporter may reverse direction, letting Na⁺ flow inward. For the cells to regulate their cytoplasmic pH, they must accumulate K⁺ and use K⁺/H⁺ antiport to acidify the cytoplasm. Under laboratory culture conditions in a K⁺-rich medium, passive K⁺ uptake may suffice. However, when the pH is alkaline and the K⁺ concentration is limited as well, active uptake of K⁺ is required; that is the role of KtrII. Cell growth at alkaline pH depends on expression of the ntp operon; even in low-Na⁺ medium, the Na⁺-ATPase is amplified when the pH is high. Under certain conditions, growth of E. hirae at alkaline pH requires the inclusion of Na⁺ ions in the medium (115). Mutants deficient in the Na⁺-ATPase are easily selected by their inability to grow at alkaline pH (116). When the K⁺ content of the medium is low (2 mM or less) and the pH is alkaline, KtrII provides the only pathway for the efficient uptake of K⁺ (176). In the presence of more K⁺ (10 mM or more), another K⁺ transport system comes into play; this may depend on the membrane potential but has not been studied (135, 176).

Most of the other streptococci do not tolerate extremely alkaline pH, where these cation pumps are important for cell physiology. Information on cation transport systems in other streptococci is limited, but the circulations of K⁺ and Na⁺ are probably linked with proton current.

With the proton circulation playing so many roles in the bacterial energy economy, one might expect it to be an obligatory feature. This is not always the case. More than two decades ago, Harold and Van Brunt (82) showed that enterococci can grow in the presence of the powerful cation conductor gramicidin D, which makes the cell membrane permeable to all small monovalent cations including H⁺, Na⁺, and K⁺. In the presence of gramicidin D, the electrochemical potentials for all these ions go to zero, although cations still accumulate nonselectively in the cells due to their content of fixed anionic substances, such as nucleic acids. It follows that the circulation of H⁺, Na⁺, and K⁺ is dispensable for producing the architecture of the bacterial cell. However, growth in the presence of gramicidin D requires particular nutritional circumstances. The cells require a medium of high K⁺ concentration and low Na⁺ concentration; the cytoplasmic pH must not be far from 7; and high concentrations of extracellular nutrients such as amino acids are needed. In other words, the maintenance of a high cytoplasmic K⁺ level and of a slightly alkaline cytoplasmic pH would be essential to bacterial growth; the key functions of these properties are not particularly clear. The effect of ionophores on cell growth has not been extended to other streptococci. The effect of a protonophore, CCCP, on the growth of E. coli supports the conclusion that bacteria can grow in the absence of the proton potential when glucose is used as an energy source (137, 174). It is not clear how cells in the presence of CCCP secrete proteins into the periplasm, which depends on the proton potential (at least in E. coli). A variety of drug resistance systems are distributed in bacteria (149); some kinds of drug extrusion pumps have been found in L. lactis. The effectiveness of various drugs on growing cells must be considered with care, although there is so far no discrepancy in the interpretation of the effect of ionophores on enterococci.

All these things probably tell us something fundamental about cation transport. Cells require the proton/cation circulation to keep their internal environment constant and different from the external one. In the laboratory, we can arrange matters so that this function is dispensable; however, in the natural world it is essential. A diversity of cation transport systems allows bacteria to maintain homeostasis in the face of a changing external environment and affords them a broader habitat.

The essence of homeostasis in bacteria has been quite elegantly summarized by Harold in his book The Vital Force: a Study of Bioenergetics (74). "The internal environment of microorganisms, like that of other living cells, generally differs substantially from the external one in chemical composition and in physical properties. It also remains remarkably constant in the face of external fluctuations. Bacteria, unlike higher organisms, can often survive gross changes in cytosolic composition, but they reestablish the normal one before growth resumes. The reasons are simple enough. The cytoplasmic pH should not be too far from neutrality, which is optimal for most metabolism. K⁺ is always the predominant cytoplasmic cation, while the more abundant Na⁺ is excluded, although why that is so is not altogether clear. Furthermore, a number of physiological parameters must be subject to regulatory mechanisms that counter perturbations and maintain optimal values. These include the proton potential, the cytosolic pH, the phosphorylation potential of ATP, the adenine nucleotide energy change, the redox potential, and turgor pressure which supplies the driving force for wall expansion. Homeostasis is obviously an important use of biological energy, and it illustrates the interplay of the proton circulation with other modes of energy generation."

There should be a relationship between the ion species of the energy transfer reactions found in any given organism and its habitat or metabolic economy (Fig. 12). In aerobic bacteria such as E. coli, which establish a large proton potential by the respiratory chain, H⁺ serves as the coupling ion for membrane energy transduction. K⁺ uptake by the Trk system, Na⁺ extrusion by the Nha systems, and a variety of nutrient carriers are linked to H⁺ (Fig. 12A). In alkaliphilic and halphilic aerobes, Na⁺ serves as a second important coupling ion for energy transduction (74, 228); the choice of Na⁺ in addition to H⁺ as the coupling ion is sensible in their environment. The Na⁺ gradient, generated by a combination of H⁺-translocating electron transport and Na⁺/H⁺ antiporter or Na⁺-translocating electron transport in some marine bacteria (253), is used as the driving force for various kinds of nutrient uptake and for flagellar motion (Fig. 12B). A gradient of H⁺ or Na⁺ is thus utilized as the driving force for various secondary porters in most bacteria.

The same is true in enterococci and in other streptococci growing at a pH that is either neutral or acidic (Fig. 12C). However, growing streptococci maintain a significantly lower proton potential than do growing anaerobic bacteria. Some streptococci, such as enterococci, can grow at alkaline pH where the proton potential is negligible. A number of ATP-linked cation transport systems, including the Kep K⁺ transport system and the Na⁺-translocating vacuolar ATPase, unique to streptococci, surely contribute to this ability. When growing at alkaline pH, streptococci also generate a sodium electrochemical potential large enough to power secondary systems; whether they use it for that purpose is not clear. The information available is limited, and it seems premature to draw conclusions about the role of Na⁺ as a general coupling ion. The functions of the Na⁺ circulation will become clear when more is known of the distribution of sodium-linked transport systems among streptococci.

The molecular analysis of membrane transport proteins in streptococci is just beginning, with some progress being made on the genetic level. When the genome sequence of streptococci is fully determined, we shall be able to look at the evolution of cation transport systems discovered in this bacterium and perhaps find how the various primary and secondary systems are distributed and why.